Omega-3 polyunsaturated fatty acids and their - Research ...

Omega-3 polyunsaturated fatty acids and their

impact upon the biosynthesis of

endocannabinoids and N-acylethanolamines in

human skin cells in the presence and absence of

ultraviolet radiation

A thesis submitted to The University of Manchester for

the degree of PhD in the Faculty of Medical and Human

Sciences

2015

Abdalla F. Mohammed Almaedani

Manchester Pharmacy School

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CONTENTS………………………………………………………………..2

LIST OF TABLES ............................................................................ 12

LIST OF FIGURES .......................................................................... 15

LIST OF ABBREVIATIONS ............................................................ 21

Abstract .......................................................................................... 24

Declaration ..................................................................................... 25

Copy right statement ..................................................................... 25

Acknowledgement ......................................................................... 26

CHAPTER 1: Introduction ............................................................. 28

1.1. Introduction to human skin biology ...................................... 28

1.1.1. Epidermis ........................................................................................................... 29

1.1.1.1. Epidermal layers ....................................................................................... 30

1.1.1.1.1 Stratum basale .................................................................................. 30

1.1.1.1.2. Stratum spinosum .......................................................................... 30

1.1.1.1.3. Stratum granulosum ...................................................................... 30

1.1.1.1.4. Stratum corneum ............................................................................ 31

1.1.1.2. Epidermal cells .......................................................................................... 31

1.1.1.2.1. Keratinocytes ................................................................................... 31

1.1.1.2.1.1. Immortalized HaCaT keratinocytes .................................... 33

1.1.1.2.2. Melanocytes ..................................................................................... 33

1.1.1.2.3. Langerhans cells ............................................................................. 34

1.1.1.2.4. Merkel cells ...................................................................................... 35

1.1.2. Dermis................................................................................................................. 35

1.1.2.1. Dermal cells ............................................................................................... 36

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1.1.2.1.1. Fibroblasts ........................................................................................ 36

1.1.2.1.2. Mast cells .......................................................................................... 37

1.1.2.1.3. Subcutaneous layer ....................................................................... 37

1.2. Ultraviolet radiation (UVR) and its effects on cells .............. 38

1.2.1. Effects of ultraviolet radiation on human skin ............................................... 39

1.3. Omega-3 polyunsaturated fatty acids (n-3 PUFA) ............... 40

1.3.1. Metabolism of essential fatty acids in human skin ....................................... 41

1.3.2. Polyunsaturated fatty acids and impact upon eicosanoid production ....... 44

1.3.3. Omega-3 PUFA and inflammatory gene expression ................................... 46

1.3.4. The protective effect of n-3 PUFA in UVR induced skin inflammation ...... 47

1.4. The endocannabinoid system ............................................... 47

1.4.1. Biosynthesis of N-arachidonoylethanolamide (AEA, anandamide) ........... 49

1.4.1.1. N-acyltransferases .................................................................................... 50

1.4.1.2. N-acyl phosphatidylethanolamine phospholipase D ............................ 50

1.4.1.3. Alternate pathways of anandamide biosynthesis ................................. 51

1.4.2. Biosynthesis of 2-arachidonoyl glycerol (2-AG) ........................................... 53

1.4.3. Catabolism of AEA and 2-AG .......................................................................... 55

1.4.3.1. Fatty acid amide hydrolase ...................................................................... 56

1.4.3.2. N-acylethanolamine hydrolysing acid amidase .................................... 57

1.4.4. N-acylethanolamines ........................................................................................ 58

1.4.5. Endocannabinoids and their impact on the skin ........................................... 59

1.5. Aims and objectives of the study........................................................................ 62

CHAPTER 2: Materials and Methods ............................................ 64

2.1. Cell culture and maintenance ................................................ 64

2.1.1. Cell lines ............................................................................................................. 64

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2.2. Cell culture .............................................................................. 64

2.2.1. Materials ............................................................................................................. 64

2.2.2. Equipment .......................................................................................................... 65

2.2.3. Cell thawing ....................................................................................................... 65

2.2.4. Cell line maintenance ....................................................................................... 65

2.2.5. Cell treatment with fatty acids ......................................................................... 66

2.2.6. Cell irradiation .................................................................................................... 67

2.2.7. Collection of cell pellets and culture medium ................................................ 67

2.2.8. Cell counting ...................................................................................................... 68

2.2.9. Cryogenic storage of the cells ......................................................................... 68

2.3. Western blotting ..................................................................... 69

2.3.1 Chemicals ............................................................................................................ 69

2.3.2. Primary and Secondary Antibodies ................................................................ 70

2.3.3. Various consumables and small items .......................................................... 71

2.4. Sample preparation ................................................................ 71

2.4.1. Protein Extraction .............................................................................................. 71

2.4.2. Determination of protein concentration .......................................................... 72

2.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

(SDS-PAGE).................................................................................... 74

2.5.1 SDS-PAGE: Gel Preparation ............................................................................ 74

2.5.2 SDS-PAGE: Gel Electrophoresis ..................................................................... 75

2.5.3. Wet Transblotting .............................................................................................. 76

2.5.4. Blocking and Immunoblotting .......................................................................... 77

2.5.5. Protein visualization .......................................................................................... 79

2.5.6. Membrane stripping and re-probing ............................................................... 79

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2.6. Densitometry analysis ............................................................ 80

2.7. Antibody optimization ............................................................ 80

2.7.1. COX-2 and GAPDH Antibodies ...................................................................... 80

2.7.2. Optimization of 12-Lipoxygenase (murine leukocyte) Polyclonal Antiserum

.......................................................................................................................... ..82

2. 8. Analysis of endocannabinoids by LC-MS/MS ..................... 83

2.8.1. Materials ............................................................................................................. 83

2.8.2. Experimental description of LC-MS/MS analysis ......................................... 83

2.8.3. Extraction Protocol ............................................................................................ 84

2.8.4. LC-MS/MS analysis of endocannabinoids and NAE.................................... 84

2.8.5. Standards for quantification and calibration lines ......................................... 86

2.9 Clinical study ........................................................................... 87

2.9.1. Experimental design ......................................................................................... 87

2.9.2. Simulated summer sunlight exposures .......................................................... 88

2.9.3. Irradiation Protocol ............................................................................................ 89

2.9.4. Skin colour measurements .............................................................................. 89

2.9.5. Blood collection and serum separation .......................................................... 90

2.9.6. Lipid Extraction .................................................................................................. 91

2.10. Statistical Analysis ............................................................... 92

CHAPTER 3: The effect of n-3 PUFA on the formation of

endocannabinoids and N-acylethanolamines in HaCaT

keratinocytes and 46BR.IN fibroblasts in response to ultraviolet

radiation ......................................................................................... 93

3.1 Introduction .............................................................................. 93

6

3.2 Aims of the study ..................................................................... 94

3.3 Materials and Methods ............................................................ 95

3.3.1. Materials ............................................................................................................. 95

3.3.2. Experimental description of LC-MS/MS analysis ......................................... 95

3.3.3. Extraction protocol ............................................................................................ 95

3.3.4. LC-MS/MS analysis of endocannabinoids and N-acylethanolamines ....... 95

3.3.5. Standards for quantification and calibration lines ......................................... 95

3.3.6. Statistical analysis (refer to section 2.10. chapter 2) ................................... 95

3.4. Results ..................................................................................... 95

3.4.1. Quantification of endocannabinoids and N-acylethanolamines in irradiated

and non-irradiated HaCaT keratinocytes treated with oleic acid........................... 95


and non-irradiated HaCaT keratinocytes treated with docosahexaenoic acid .... 99


and non-irradiated HaCaT keratinocytes treated with eicosapentaenoic acid 103

3.4.4. Quantification of endocannabinoids and N-acylethanolamines in culture

medium from irradiated and non-irradiated HaCaT keratinocytes treated with oleic

acid ............................................................................................................................... 107

3.4.5. Quantification of endocannabinoids and N-acylethanolamines in cell culture

medium from irradiated and non-irradiated HaCaT keratinocytes treated with

docosahexaenoic acid ............................................................................................... 110

3.4.6. Quantification of endocannabinoids and N-acylethanolamines in cell culture

medium from irradiated and non-irradiated HaCaT keratinocytes treated with

eicosapentaenoic acid ............................................................................................... 113

3.4.7. Quantification of the endocannabinoids and N-acylethanolamines in

irradiated and non-irradiated 46BR.IN fibroblasts treated with oleic acid. ......... 118

7

3.4.8. Quantification of the endocannabinoids and other N-acylethanolamines in

irradiated and non-irradiated 46BR.IN fibroblasts treated with docosahexaenoic

acid ............................................................................................................................... 122

3.4.9. Quantification of the endocannabinoids and N-acylethanolamines in

irradiated and non-irradiated 46BR.IN fibroblasts treated with eicosapentaenoic

acid. .............................................................................................................................. 126

3.4.10. Quantification of the endocannabinoids and N-acylethanolamines in culture

medium from irradiated and non-irradiated 46BR.IN fibroblasts treated with oleic

acid ............................................................................................................................... 130

3.4.11. Quantification of the endocannabinoids and N-acylethanolamines in culture

medium from irradiated and non-irradiated 46BR.IN fibroblasts treated with

docosahexaenoic acid ............................................................................................... 133

3.4.12. Quantification of the endocannabinoids and other N-acylethanolamines in

culture medium from irradiated and non-irradiated 46BR.IN fibroblasts treated with

eicosapentaenoic acid ............................................................................................... 136

3.5. Discussion ............................................................................. 141

3.5.1. Determination the effect of oleic acid and ultraviolet radiation on the

intracellular and extracellular levels of endocannabinoids and N-acylethanolamines

in HaCaT Keratinocytes and 46BR.IN fibroblasts. ................................................ 141

3.5.2. Determination the effects of n-3 PUFA on the intracellular and extracellular

levels of the endocannabinoids and N-acylethanolamines in HaCaT Keratinocytes

and 46BR.IN fibroblasts in response to ultraviolet radiation. ............................... 141

CHAPTER 4: The effect of n-3 PUFA on endocannabinoid

metabolizing enzymes in HaCaT keratinocytes and 46BR.IN

fibroblasts in response to ultraviolet radiation. ........................ 147

4.1. Introduction ........................................................................... 147

4.1.1. Cyclooxygenase-2 .......................................................................................... 147

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4.1.2. Lipoxygenases ................................................................................................. 148

4.2. Aims of this study ................................................................. 149

4.3 Materials and Methods .......................................................... 150

4.3. Western blotting ................................................................................................. 150

4.3.1. Materials ........................................................................................................... 150

4.3.2. Experimental Design ...................................................................................... 150

4.3.3. Protein extraction ............................................................................................ 150

4.3.4. SDS-PAGE ..................................................................................................... 150

4.4. Results ................................................................................... 150

4.4.1. The effect of UVR and n-3 PUFA on COX-2 protein levels in HaCaT

keratinocytes and 46BR.IN fibroblasts. ................................................................... 150

4.4.1.1. The effect of docosahexaenoic acid and ultraviolet radiation ........ ..150

4.4.1.2. The effect of oleic acid and ultraviolet radiation ................................. 154

4.4.1.3. The effect of eicosapentaenoic acid and ultraviolet radiation .......... 154

4.5. The effect of UVR and n-3 PUFA on LOX protein levels in

HaCaT keratinocytes and 46BR.IN fibroblasts .......................... 159

4.6. The effect of UVR and n-3 PUFA on N-

acylphosphatidylethanolamine phopholipase-D (NAPE-PLD) and

fatty acid amide hydrolase (FAAH) protein levels in HaCaT

keratinocytes and 46BR.IN fibroblasts ...................................... 163

4.6.1. Effect of oleic acid and UVR on FAAH and NAPE-PLD in HaCaT

keratinocytes ............................................................................................................... 163

4.6.2. Effect of eicosapentaenoic acid and UVR on FAAH and NAPE-PLD in HaCaT

keratinocytes cell lines. ............................................................................................. 164

4.6.3. Effect of docosahexaenoylethanolamide and UVR on FAAH and NAPE-PLD

in HaCaT keratinocytes ............................................................................................. 164

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4.6.4. Effect of oleic acid and UVR on FAAH and NAPE-PLD protein level in

46BR.IN fibroblasts .................................................................................................... 171

4.6.5. Effect of eicosapentaenoic acid and UVR on FAAH and NAPE-PLD in

46BR.IN fibroblasts .................................................................................................... 171

4.6.6. Effect of docosahexaenoylethanolamide and UVR on FAAH and NAPE-PLD

in 46BR.IN fibroblasts ................................................................................................ 172

4.7 Discussion .............................................................................. 179

4.7.1. The effect of omega-3 polyunsaturated fatty acids and UVR on COX-2

protein expression in HaCaT keratinocytes and 46BR.IN fibroblasts ................ 179

4.7.2. The effect of omega-3 polyunsaturated fatty acids and UVR on NAPE-PLD

and FAAH protein expression in HaCaT keratinocytes and 46BR.IN fibroblasts

................................................................................................................................. …..182

CHAPTER 5: Investigation the effect of ultraviolet radiation on

endocannabinoids and N-acylethanolamines in White Caucasians

of skin phototype II and South Asians of skin phototype V..... 186

5.1. Introduction ........................................................................... 186

5.2. Aim of the study .................................................................... 187

5.3. Materials and Methods ......................................................... 187

5.3.1. Statistical Analysis .......................................................................................... 187

5.4. Results ................................................................................... 187

5.4.1. Effect of UVR on serum endocannabinoids and N-acylethanolamines .. 187

5.4.1.1 Effect of UVR on serum palmitoylethanolamide .................................. 187

5.4.1.2. Effect of UVR on serum linoleoyalethanolamide ................................ 189

5.4.1.3. Effect of UVR on serum oleoylethanolamide ...................................... 190

5.4.1.4. Effect of UVR on serum stearoylethanolamide................................... 191

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5.4.1.5. Effect of UVR on serum docosahexaenoylethanolamide ................. 192

5.4.1.6. Effect of UVR on serum N-arachidonoylethanolamide ...................... 193

5.4.1.7. Effect of UVR on serum 2-arachidonoyl glycerol ............................... 194

5.4.1.8. Effect of UVR on serum 1-arachidonoyl glycerol ............................... 196

5.4.1.9. Effect of UVR on docosapentaenoylethanolamide ............................ 197

5.4.1.10. Effect of UVR on serum Myristoylethanolamide ............................... 198

5.4.1.11. Effect of UVR on serum dihomo gamma linoleoyalethanolamide . 199

5.4.1.12. Effect of UVR on serum eicosapentaenoylethanolamide ............... 200

5.5. Discussion ............................................................................. 201

CHAPTER 6: General discussion and future studies ............... 204

6.1. Introduction ........................................................................... 204

6.2. General discussion ............................................................... 205

6.3. Future directions .................................................................. 209

References ................................................................................... 211

Appendices .................................................................................. 237

Appendix 1 ................................................................................... 237

1. Fatty acid stock solutions ..................................................................................... 237

1.1. Oleic Acid (OA) stock solution (50mM) ...................................................... 237

1.2. Eicosapentaenoic Acid (EPA) stock solution (50mM) .............................. 237

1.3. Docosahexaenoic Acid (DHA) stock solution (50mM) ............................. 237

Appendix 2 ................................................................................... 237

2.1. Preparation of BSA standards .......................................................................... 237

2.2. Genesis software (Version 2) ........................................................................... 238

2.3. Sample calculation for adjusting the protein content of the cell lysate ....... 238

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Appendix 3 ................................................................................... 239

3.1 Preparation of buffers and stains ...................................................................... 239

3.1.1. Tank (running) buffer: (PH 8.4-8.5) 1 litre ............................................... 239

3.1.2. Transblotting buffer: (PH 8.4-8.5) 1 litre.................................................. 239

3.1.3. Lower (separating) gel Tris buffer (PH 8.8) - 0.5 litre ........................... 239

3.1.4. Upper (stacking) gel Tris buffer (PH 6.9) - 0.5 litre ............................... 240

3.1.5. Sample buffer 25 ml ................................................................................... 240

3.1.6. Coomassie blue stain for SDS ................................................................. 241

3.1.7. Destain for SDS .......................................................................................... 241

3.1.8. Fast green 0.1% for PVDF membrane .................................................... 241

3.1.9. ECL solution ................................................................................................ 241

Appendix 4 ................................................................................... 242

4.1. Membrane stripping ........................................................................................... 242

4.1.1. Mild stripping buffer .................................................................................... 242

4.2. Protocol of stripping western blots for re-probing .......................................... 242

Appendix 5 ................................................................................... 243

5.1. Chloroform/methanol (2:1 ratio) ....................................................................... 243

5.2. Internal standards (1 ng/µl AEA-d8 and 1 ng/µl 2-AG-d8) ........................... 243

5.3 Endocannabinoid cocktail for calibration line (400 pg/µl) .............................. 243

5.4. Mobile Phase A for endocannabinoid analysis .............................................. 244

5.5. Mobile Phase B for endocannabinoid analysis .............................................. 244

5.6. Seal wash ............................................................................................................ 244

5.7. Needle Wash ....................................................................................................... 245

5.8. Shutdown solution I ............................................................................................ 245

5.9. Shutdown solution II ........................................................................................... 245

Appendix 6 ................................................................................... 246

63232 words

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LIST OF TABLES

Table 2.1: Composition of the sample buffer used for protein extraction. ..... 72

Table 2.2: Reagents used for the preparation of the acrylamide gel. .............. 75

Table 2.3: multiple reaction monitoring (MRM) transitions for the LC/ESI-MS/MS

assay of endocannabinoids and their congeners .............................. ………….85

Table 2.4: Guidelines for the generation of standards of different concentrations

to be used to construct an endocannabinoid calibration line ................... …..86

Table 2.5. Schematic pattern showing the time points of the UV radiation and

blood samples collection. ................................................................................... .88

Table 2.6. Sample Code: The highlighted days showing the days of UVR exposure

and/or the blood samples collection during the study……………….................88

Table 3.1. Summary of the significant findings in HaCaT keratinocytes cells and

their cell culture medium in response to DHA and/or UVR…………………….116

Table 3.2. Summary of the significant findings in HaCaT keratinocytes cells and

their cell culture medium in response to EPA and/or UVR……………………..117

Table 3.3. Summary of the significant findings in 46 BR.IN fibroblasts cells and

their cell culture medium in response to DHA and/or UVR……………………..139

Table 3.4. Summary of the significant findings in 46 BR.IN fibroblasts cells and

their cell culture medium in response to EPA and/or UVR……………………..140

Table 6.1. Comparison between palmitoylethanolamide levels in White

Caucasians of skin phototype II and South Asians of phototype V pre and post

UVR. .......................................................................................................... ……….246

Table 6.2. Comparison between linoleoyalethanolamide levels in White


UVR. ..................................................................................................................... .246

13

Table 6.3. Comparison between oleoylethanolamide levels in White Caucasians

of skin phototype II and South Asians of phototype V pre and post UVR. ... 247

Table 6.4. Comparison between stearoylethanolamide levels in White


UVR. ...................................................................................................................... 247

Table 6.5. Comparison between docosahexaenoylethanolamide levels in White


UVR. ...................................................................................................................... 248

Table 6.6. Comparison between N-arachidonoylethanolamide levels in White


UVR. ...................................................................................................................... 248

Table 6.7. Comparison between 2-arachidonoyl glycerol levels in White


UVR. ...................................................................................................................... 249

Table 6.8. Comparison between 1-arachidonoyl glycerol levels in White


UVR. ...................................................................................................................... 249

Table 6.9. Comparison between docosapentaenoylethanolamide levels in White


UVR. ...................................................................................................................... 250

Table 6.10. Comparison between Myristoylethanolamide levels in White


UVR. ...................................................................................................................... 250

Table 6.11. Comparison between dihomo gamma linoleoyalethanolamide levels

in White Caucasians of skin phototype II and South Asians of phototype V pre

and post UVR....................................................................................................... 250

14

Table 6.12. Comparison of different time point of UVR exposure on

eicosapentaenoylethanolamide levels in White Caucasians of skin phototype II.

.............................................................................................................................. 251

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LIST OF FIGURES

Figure 1.1: Skin Structure .................................................................................... 28

Figure 1.2: Epidermal differentiation. ................................................................. 29

Figure 1.3: UV radiation spectrum ...................................................................... 38

Figure 1.4: Metabolism of n-3 and n-6 polyunsaturated fatty acids ................ 43

Figure 1.5: Chemical structures of the tetrahydrocannabinol, anandamide (AEA)

and 2-arachidonoyl glycerol (2-AG) .................................................................... 49

Figure 1.6: Schematic showing the variuos pathways of anandamide (AEA)

biosynthesis . ....................................................................................................... 52

Figure 1.7: Biosynthesis of 2-arachidonoyl glycerol (2-AG) ............................ 54

Figure 1.8: Catabolism of anandamide (AEA) and 2-arachidonoyl glycerol (2-

AG). ......................................................................................................................... 56

Figure 2.1. Side view of the haemocytometer slide........................................... 69

Figure 2.1. A. The haemocytometer chamber cell ............................................. 69

Figure 2.2: 96-wells microplate............................................................................ 73

Figure 2.3: Template showing how the samples were loaded into the 96-wells

microplate .............................................................................................................. 73

Figure 2.4: typical calibration line plotted from BSA standards ...................... 73

Figure 2.5: Schematic representation of western blotting and detection

procedure ............................................................................................................... 74

Figure 2.6: Protein denaturation using heat and SDS....................................... 76

16

Figure 2.7: Diagram showing the position of the blotting pads inside the blotting

module during the transblotting process ........................................................... 78

Figure 2.8: Diagram showing the position of PVDF membrane in the blotting

module. .................................................................................................................. 78

Figure 2.9: schematic summarizing the protein visualization process .......... 80

Figure 2.10: Optimization of COX-2 and GAPDH antibodies. ........................... 81

Figure 2.11: Optimization of 12-LOX (murine leukocyte) Polyclonal antiserum.

.............................................................................................................................. ..82

Figure 2.12: Philips HB588 cabinet ..................................................................... 89

Figure 2.13: Spectrophotometer.......................................................................... 90

Figure 3.1 Effect of oleic acid treatment on endocannabinoids and N-

acylethanolamines levels in HaCaT keratinocytes in response to UV radiation.

................................................................................................................................ 98

Figure 3.2 Effect of docosahexaenoic acid treatment on endocannabinoids and

N-acylethanolamines levels in HaCaT Keratinocytes in response to UV radiation.

.............................................................................................................................. 102

Figure 3.3 Effect of eicosapentaenoic acid treatment on endocannabinoids and

N-acylethanolamines levels in HaCaT Keratinocytes in response to UV radiation.

.............................................................................................................................. 106


acylethanolamines N-acylethanolamines levels in HaCaT keratinocytes culture

medium in response to UV radiation. ............................................................... 109


N-acylethanolamines levels in HaCaT keratinocytes culture medium in response

to UV radiation..................................................................................................... 112

17


N-acylethanolamines levels in HaCaT keratinocytes culture medium in response

to UV radiation..................................................................................................... 115


acylethanolamines levels in 46BR.IN fibroblasts in response to UV radiation..

............................................................................................................................. .121


N-acylethanolamines levels in 46BR.IN fibroblasts in response to UV radiation

.............................................................................................................................. 125


N-acylethanolamines levels in 46BR.IN fibroblasts in response to UV radiation

.............................................................................................................................. 129


acylethanolamines levels in 46BR.IN fibroblasts culture medium in response to

UV radiation. ........................................................................................................ 132


N-acylethanolamines levels in 46BR.IN fibroblasts culture medium in response

to UV radiation..................................................................................................... 135


N-acylethanolamines levels in 46BR.IN fibroblasts culture medium in response

to UV radiation..................................................................................................... 138

Figure 4.1 COX-2 protein levels in docosahexaenoic acid and UVR treated

46BR.IN fibroblasts. (A) ...................................................................................... 152

Figure 4.2 COX-2 protein levels in docosahexaenoic acid and UVR treated

HaCaT Keratinocytes. ......................................................................................... 153

18

Figure 4.3 COX-2 protein levels in oleic acid and UVR treated in 46BR.IN

fibroblasts. ........................................................................................................... 155

Figure 4.4 COX-2 protein levels in oleic acid and UVR treated in HaCaT

Keratinocytes. ..................................................................................................... 156

Figure 4.5 COX-2 protein levels in eicosapentaenoic acid and UVR treated in

46BR.IN fibroblasts. ............................................................................................ 157

Figure 4.6 COX-2 protein levels in eicosapentaenoic acid and UVR treated in


Figure 4.7 12-LOX protein levels in oleic acid and UVR treated in in 46BR.IN

fibroblasts. ........................................................................................................... 160

Figure 4.8 12-LOX protein levels in docosahexaenoic acid and UVR treated in


Figure 4.9 12-LOX protein levels in oleic acid and UVR treated in HaCaT

keratinocytes. ...................................................................................................... 162

Figure 4.10: FAAH protein levels in oleic acid and UVR treated in HaCaT

Keratinocytes. ..................................................................................................... 165

Figure 4.11: NAPE-PLD protein levels in oleic acid and UVR treated in HaCaT

Keratinocytes. ..................................................................................................... 166

Figure 4.12: FAAH protein levels in eicosapentaenoic acid and UVR treated in


Figure 4.13: NAPE-PLD protein levels in eicosapentaenoic acid and UVR treated

in HaCaT Keratinocytes. ..................................................................................... 168

Figure 4.14: FAAH protein levels in docosahexaenoic acid and UVR treated in


19

Figure 4.15: NAPE-PLD protein levels in docosahexaenoic acid and UVR treated

in HaCaT Keratinocytes ...................................................................................... 170

Figure 4.16: FAAH protein levels in oleic acid and UVR treated in 46BR.IN

fibroblasts. ........................................................................................................... 173

Figure 4.17: NAPE-PLD protein levels in oleic acid and UVR treated in 46BR.IN

fibroblasts ............................................................................................................ 174

Figure 4.18: FAAH protein levels in eicosapentaenoic acid and UVR treated in


Figure 4.19: NAPE-PLD protein levels in eicosapentaenoic acid and UVR treated

in 46BR.IN fibroblasts. ........................................................................................ 176

Figure 4.20: FAAH protein levels in docosahexaenoic acid and UVR treated in


Figure 4.21: NAPE-PLD protein levels in docosahexaenoic acid and UVR treated

in 46BR.IN fibroblasts. ........................................................................................ 178

Figure 5.1. Palmitoylethanolamidelevels in serum before and after multiple UVR

exposures ............................................................................................................ 188

Figure 5.2. Linoleoyalethanolamide levels in serum before and after multiple

UVR exposures.................................................................................................... 189

Figure 5.3. Oleoylethanolamide levels in serum before and after multiple UVR

exposures.. .......................................................................................................... 190

Figure 5.4. Stearoylethanolamide levels in serum before and after multiple UVR

exposures ............................................................................................................ 191

Figure 5.5. Docosahxaenoylethanolamide levels in serum before and after

multiple UVR exposures. .................................................................................... 193

20

Figure 5.6. N-arachidonoylethanolamide levels in serum before and after

multiple UVR exposures.. ................................................................................... 194

Figure 5.7. 2-arachidonoyl glycerol levels in serum before and after multiple

UVR exposures.................................................................................................... 195

Figure 5.8. 1-arachidonoyl glycerol levels in serum before and after multiple

UVR exposures.................................................................................................... 196

Figure 5.9. Docosapentaenoylethanolamide levels in serum before and after

multiple UVR exposures. .................................................................................... 197

Figure 5.10. Myristoylethanolamide levels in serum before and after multiple

UVR exposures.................................................................................................... 198

Figure 5.11. Dihomo gamma linoleoyalethanolamide levels in serum before and

after multiple UVR exposures. ........................................................................... 199

Figure 5.12. Eicosapentaenoylethanolamide levels in serum before and after

multiple UVR exposures ..................................................................................... 200

21

LIST OF ABBREVIATIONS

1-AG 1-arachidonoyl-glycerol 2-AG 2-arachidonoyl-glycerol 2-AG-d8 2-Arachidonoyl glycerol-deuterium 8 2-AGMT 2-AG membrane transporter 46BR.1N Human fibroblast cell line AA Arachidonic acid AEA-d8 Anandamide-deuterium 8 ALA α-Linolenic acid AMT AEA membrane transporter APS Ammonium persulfate CB1 Cannabinoid receptor type-1 CB2 Cannabinoid receptor type-2 cDNA Complementary Deoxyribonucleic acid CHCl3 Chloroform COX-2 Cyclooxygenase-2 COXs Cyclooxygenases CPD Cyclobutane pyrimidine dimmers DAGL Diacylglycerol lipase DGLA Dihomo γ linolenic acid DGLEA Dihomo γ linolenoylethanolamide DHA Docosahexaenoic acid DHEA Docosahxaenoylethanolamide DHET Dihydroxyeicosatrienoic acid DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DT Delayed tanning DTT Dithiothreitol ECACC European Collection of Cell Culture ECL Enhanced Chemilluminescent EDTA Ethylenediaminetetraacetic acid EPA Eicosapentaenoic acid EPEA Eicosapentaenoylethanolamide ES+ Positive ionization mode FAAH Fatty acid amide hydrolase FBS Foetal bovine serum FCS Foetal calf serum GAPDH Glyceraldehyde 3-phosphate dehydrogenase GLA γ linolenic acid GP-AEA Glycerophospho-arachidonoyl ethanolamide GPCR G-protein-coupled receptors HaCaT Human adult, calcium, temperature HBSS Hank’s Balanced Salt Solution HCl Hydrochloric acid HETE Hydroxy eicostetraeneic acid HODEs Hydroxyoctadecadienoicacids HPEPE Hydroperoxy eicosapentaenoic acid

22

HPETE Hydroperoxy eicosatetraenoic acid HRP Horseradish peroxide iPLA2 Independent cytosolic Ca2+ phospholipase A2 LA Linolenic acid LC-MS/MS Liquid chromatography tandem mass spectrometry LEA Linoleoylethanolamide LOXs Lipoxygenases LT Leukotriene LTA4 Leukotriene A4 LTB4 Leukotriene B4 LXA4 Lipoxin A4 LXB4 Lipoxin B4 Lyso-NAPE Lyso-N-arachidonoyl phosphatidylethanolamine Lyso-PA Lysophosphatidic acid Lyso-PLD Lysophospholipase D MAGL Monoacylglycerol lipase MAR Maresins MED Minimal erythema dose MEME Minimum essential medium eagle MeOH Methanol MRM Multiple reaction monitoring n -3 PUFA Omega -3 polyunsaturated fatty acid n -6 PUFA Omega 6 polyunsaturated fatty acid N2 Nitrogen NAAA N-acylethanolamine hydrolysing acid amidase NADA N-arachidonoyl-dopamine NAEs N-acylethanolamines NAPE N-arachidonoyl phosphatidylethanolamine NAPE-PLD N-acyl phosphatidylethanolamine phospholipase D NAT N-acyltransferase NHAK Normal human adult keratinocytes NHEKs Cultured normal human epidermal keratinocytes NO Nitric oxide OA Oleic acid OEA Oleoylethanolamide PA Phosphatidic acid PBS Phosphate buffered saline PC Phosphatidylcholine PDs Protectins PE Phosphatidylethanolamine PEA N-palmitoylethanolamide PG Prostaglandin PGDS Prostaglandin D synthases PGE2 Prostaglandin E2 PGES Prostaglandin E synthase PGFS prostaglandin F synthase PGG2 Prostaglandin G2 PGH2 Prostaglandin H2 PIC Protease inhibitor cocktail PL Phospholipase PLA2 Phospholipase A2 PLC Phospholipase C

23

PLD Phospholipase D PS Penicillin streptomycin PtdCho Phosphatidylcholine PtdIns Phosphatidylinositol PTPN22 Protein tyrosine phosphatase, non-receptor type 22 PVDF Plyvinylidene difluoride ROS Reactive oxygen species RvD D-series resolvins RvE E- resolvins series SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate-Polyacrylamide gel

electrophoresis SED Standard erythemal dose SPE Solid phase extraction sPLA2 Secretary phospholipases A2 STEA N-stearoylethanolamine TEMED Tetramethylenediamine THC Tetrahydrocannabinol TNF-α Tumor necrosis factor-alpha TRPV1 Transient receptor potential channel type V1 TXAS Thromboxane A synthase UCA Urocanic acid UVR Ultraviolet radiation Abhd 4 αβ-hydrolase 4

24

The University of Manchester

Abdalla F. Mohammed Almaedani

Doctor of Science (DSc)

Omega-3 polyunsaturated fatty acids and their impact upon the

biosynthesis of endocannabinoids and N-acylethanolamines in human skin

cells in the presence and absence of ultraviolet radiation.

March 2015

Abstract

Endocannabinoids are endogenous lipid mediators involved in various biological

processes, and have immunomodulatory and anti-inflammatory activities.

Anandamide (arachidonoyl ethanolamine, AEA) and 2-arachidonoyl glycerol

(2-AG) are the main representatives of this group. The endocannabinoid

receptors CB1 and CB2 with AEA have been found in human HaCaT

keratinocytes and fibroblasts, but the metabolic pathway leading to

endocannabinoid production in the skin has not been fully elucidated. This study

aimed to investigate the profile of endocannabinoids and their main metabolizing

enzymes in human skin cells and assess whether omega-3 polyunsaturated fatty

acids (n-3 PUFA) altered these profiles. In addition, an investigation was carried

out to check whether UV radiation could stimulate the production of

endocannabinoids and N-acylethanolamines (NAE) in human skin cells. For this

purpose HaCaT keratinocytes and 46RB.1N fibroblast cells were treated with 10

and 50µM of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or

oleic acid (OA) in the presence or absence of UVR (15mJ/cm2). Data suggest

that n-3 PUFA may both directly (by up-regulating NAPE-PLD levels) and

indirectly (by decreasing FAAH levels) increased endocannabinoid and NAE

levels in HaCaT keratinocytes and 46BR.IN fibroblasts. DHA treatment

significantly decreased COX-2 expression in the absence of UVR and inhibited

UVR-induced COX-2 overexpression in 46BR.IN fibroblasts. In contrast, DHA

appeared to induce COX-2 up-regulation in the absence of UVR and did not

prevent UVR induced COX-2 up-regulation in HaCaT keratinocytes. EPA

appeared to induce COX-2 down-regulation in the absence of UVR and did not

prevent UVR induced COX-2 up-regulation in both HaCaT keratinocytes and

46BR.IN fibroblasts. UVR did not have any significant effect on

endocannabinoid and NAE biosynthesis. However, UVR induced

endocannabinoid production in some experiments of this study. A clinical study

was carried on 16 volunteers from two different ethnic groups and two different

skin types. The purpose was to assess the effect of UVR on the serum

endocannabinoids and NAE, therefore, the volunteers were subjected to multiple

doses (1.3, SED/ 6 min) of UVR for 6 weeks. Data showed that UVR did not

have major effect on human serum NAE in both skin phototypes II and V but

increased 2-AG in human serum in both skin types but the more pronounced

effect was evident in skin phototypes V rather than in skin phototypes II. Human

serum docosahxaenoylethanolamide levels were found to be higher in White

Caucasians group (skin phototypes II). Based on these it can be concluded that

n-3 PUFA and UVR alter the endocannabinoids and NAE profile in HaCaT

keratinocytes and 46BR.IN fibroblasts. In addition, results of the clinical study

indicated that UVR has no major effects on serum endocannabinoids or NAE

therefore, further studies are required to address this question in vivo.

25

Declaration

I declare that no portion of the work referred to in the thesis has been submitted in

support of an application for another degree or qualification of this or any other university

or other institute of learning.

Copy right statement

i. The author of this thesis (including any appendices and/or schedules to this thesis)

owns certain copyright or related rights in it (the “Copyright”) and s/he has given The

University of Manchester certain rights to use such Copyright, including for

administrative purposes.

ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic

copy, may be made only in accordance with the Copyright, Designs and Patents Act

1988 (as amended) and regulations issued under it or, where appropriate, in

accordance with licensing agreements which the University has from time to time. This

page must form part of any such copies made.

iii. The ownership of certain Copyright, patents, designs, trademarks and other

intellectual property (the “Intellectual Property”) and any reproductions of copyright

works in the thesis, for example graphs and tables (“Reproductions”), which may be

described in this thesis, may not be owned by the author and may be owned by third

parties. Such Intellectual Property and Reproductions cannot and must not be made

available for use without the prior written permission of the owner(s) of the relevant

Intellectual Property and/or Reproductions.

iv. Further information on the conditions under which disclosure, publication and

commercialisation of this thesis, the Copyright and any Intellectual Property and/or

Reproductions described in it may take place is available in the University IP Policy (see

http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis

restriction declarations deposited in the University Library, The University Library’s

regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The

University’s policy on Presentation of Theses.

26

Acknowledgement

First and foremost, I must acknowledge my limitless thanks to Allah, the Ever-

Magnificent; the Ever-Thankful, for His help and bless. I am totally sure that this work

would have never become truth, without His guidance.

I owe a deep gratitude to my supervisors who were more than generous with their

expertise and precious time. I would like to sincerely thank Prof. Anna Nicolaou, the

main supervisor and Dr. Costas Demonacos, the co-supervisor for their countless

hours of reflecting, reading, encouraging, and most of all patience throughout the entire

process. Also I would like to thank Dr. Steven Britland and Dr. Sobia Kauser for their

help and guidance through all the time they were engaged in this study.

I wish to thank Dr. Paula Urquhart and Dr. Alexandra Kendall for their willingness to

provide any assistance requested at any time during this research. Also, I would like to

express my thanks to Dr. Sharon Murphy, Dr. Naser Alaasswed, Dr. Naser Derbal and

Magdalena Kiezel and to all people who took part in making this thesis real. Also, I

would like to express my thanks to Manchester School of Pharmacy for giving me an

opportunity to complete this work. Special thanks goes to Dr. Jeffery Penny.

Finally I cannot end without conveying a deepest thanks my whole family especially my

wife Najah for her patience, support and for helping me in any possible way to

accomplish my degree.

27

This thesis is dedicated to my brother

Ibrahim for his endless support,

generosity and encouragement

28

CHAPTER 1: Introduction

1.1. Introduction to human skin biology

The skin is the largest organ of the body, it is forms the physical barrier that protect the

body from environmental stress and regulates water inward and outward (Hunter, 2003)

Skin also has a protection role against microorganisms, chemical agents and ultraviolet

radiation (Madison, 2003). Skin is composed of three structural layers including: the

epidermis, the dermis and the subcutaneous layer (Figure 1.1). Human skin also

contains various appendages including sebaceous glands, which secrete an oily or

waxy matter, called sebum, to lubricate and waterproof the skin and hair of mammals

(Hunter, 2003) . Sweat glands are found in almost every part of the skin and produce

sweat that works to maintain the homeostasis of the body. Widespread vasculature in

the dermis helps to regulate body temperature, to deliver oxygen and nutrients, and to

remove toxins and waste products (Hunter, 2003).

Figure 1.1. Skin Structure. Adapted from (www.MayoClinic.com), December, 2014

http://www.mayoclinic.com/

29

1.1.1. Epidermis

The epidermis is the outermost skin layer contains epidermal cells such as,

keratinocytes, Merkel cells, melanocytes and Langerhans cells (Figure 1.1).

Keratinocytes are the main cells of the epidermis, these cells are connected to each

other by proteins called desmosomes. The epidermis layer is formed during

keratinocytes differentiation (Green et al., 1982, Eckert, 1989). These are four layers

arranged as rows started from the stratum basale layer which lies above the basement

membrane of the skin (Figure 1.2). Stratum spinosum is the second layer situated above

the basale layer. Stratum granulosum is the third layer in the skin named based on the

granules that form it. These granules supports the terminal stage in keratinocyte

differentiation to the stratum corneum which is the outermost layer in the epidermis

(Figure 1.2). Furthermore, the stratum lucidum is a thin layer found in the thick

epidermis. This layer represents a conversion from the stratum granulosum and stratum

corneum and is not usually seen in thin epidermis (Figure 1.1).

Figure 1.2. Epidermal differentiation. Adapted from (www.jcs.biologists.org),

December, 2014.

http://www.jcs.biologists.org/

30

1.1.1.1. Epidermal layers

1.1.1.1.1 Stratum basale

Stratum basale is the innermost layer of the epidermis (Figures 1.1 and 1.2), located

next to the dermis. Keratinocytes are the main cells in this layer where they are attached

to the basement membrane by hemidesmosomes (Heyden et al., 1992). This layer also

contains on another cells such as Langerhans, Merkel cells and melanocytes.

Melanocytes are responsible for melanin production. Melanin is a pigment that provides

protection against ultraviolet radiation (UVR) (Prota, 1997, Kobayashi et al., 1998a). It

is well known that the black skin is less susceptible to UVR than the white skin because

the distribution and rate of production of melanin is different in white and black skin

(reviewed in (Brenner and Hearing, 2008)).

1.1.1.1.2. Stratum spinosum

The stratum spinosum is found on top of the basal layer (Figures 1.1 and 1.2) and

consists of two to six rows of keratinocytes. Keratins produced by keratinocytes in this

layer are serves to form cytoplasmic protein termed tonofilaments (Mottaz and

Zelickson, 1975). This protein is required for the formation of the desmosomes that

connect the cell membranes of adjacent keratinocytes together. The continuation of

epidermal differentiation makes the cells in the upper layers of stratum spinosum to

become flat and long as step toward the stratum granulosum formation.

1.1.1.1.3. Stratum granulosum

Stratum granulosum is formed after the stratum spinosum layer during keratinocytes

differentiation (Figures 1.1 and 1.2.). Stratum granulosum is a thin layer in the epidermis

contains on protein called keratohyalin assists to bind the keratin filaments together. As

the keratinocytes continue in their differentiation, they lose the nuclei and organelles to

form non-viable corneocytes in the stratum corneum. Also, during this time

keratinocytes secrete lamellar granules that containing lipids and proteins into the

31

extracellular space to form hydrophobic lipid envelope works as the skin barrier

(Freinkel, 2001). Finally, the cornified cells are formed as the keratinocytes lose their

ability to differentiate in this layer (Ishida-Yamamoto and Iizuka, 1998).

1.1.1.1.4. Stratum corneum

Epidermal differentiation is end up with the formation of the stratum corneum layer

(Figures 1.1 and 1.2). The stratum corneum functions as a barrier to protect the skin

from environmental insults or any harmful substances or organisms. The stratum

corneum in addition regulates water loss from the body and the water plays an important

role in the integrity of stratum corneum barrier. The barrier nature of the stratum

corneum depends on the proteins and lipids in the lamellar bodies secreted from the

keratinocytes. According to Wilkes, (1973) the stratum corneum is composed of about

75-80% protein, 5-15% lipid (Wilkes et al., 1973). The protein is found in the

keratinocytes and it is about 70% alpha-keratin and 10% beta-keratin while, 5% is

proteinaceous cells envelop and the 15 % remaining are formed of enzymes and other

proteins. While, phospholipids do not exist in the stratum corneum layer (Lampe et al.,

1983), fatty acids, cholesterol, cholesterol sulfate and sterol/wax esters represents the

main lipids in this layer.

1.1.1.2. Epidermal cells

1.1.1.2.1. Keratinocytes

Keratinocytes are the major type of cell in the epidermis. They constitute approximately

95% of all cells found in the epidermis (Eckert, 1989) . In the stratum basale

keratinocytes exist as either stem cell-like cells i.e. they generate progeny by mitosis

some of which retain stem cell characteristics, or transiently amplifying cells i.e. they

replicate with a higher frequency than the stem cell-like keratinocytes but are only

capable of limited population doublings (Eckert, 1989). Once a keratinocyte becomes

fully committed to differentiation it will detach from the basement membrane,

32

differentiate and ultimately cease to proliferate as it migrates toward the skin surface,

where it will be sloughed off as a fully differentiated dead cornified cell (Figure 1.1)

(Eckert, 1989). As the keratinocyte migrates it expresses a progressive array of

different keratins, which are a family of tough and insoluble structural proteins that help

the skin protect from the external environment and help maintain its structure (Heyden

et al., 1992). Keratinocytes in the basale layer of the epidermis produce keratin type 5

and type 14 (Heyden et al., 1992) and throughout the epidermis they are joined to one

another by intercellular junctions known as desmosomes.

As the keratinocyte differentiate to the stratum spinosum layer its replicative potential is

reduced as its ability to produce keratin increases and its ability to produce different

types of keratin changes (Heyden et al., 1992). In addition to the production of keratin,

the keratinocytes also produce keratinosomes. At this stage of terminal differentiation

the keratinocytes mainly produce keratin types 1 and 10 (Heyden et al., 1992) (Figure

1.1.). Once the keratinocyte reaches the stratum granulosum it can no longer undergo

cell division and instead undergoes cornification (Eckert, 1989). Cornification is due to

the presence of keratohyalin granules and filaggrins (Candi et al., 2005). The epidermis

is between 5mm and 150mm thick (depending on body site) and as mentioned before,

it takes about 30 days for a keratinocyte to differentiate from the stratum basale to the

stratum corneum (Eckert, 1989). Calcium is required for a keratinocyte to differentiate

from the stratum spinosum and from the stratum granulosum to the stratum corneum

(Bikle, 2004), while protein kinase-C (PKC) is needed for the keratinocytes to

differentiate from the stratum spinosum to the stratum granulosum (Denning, 2004)

(Figure 1.1.). The intracellular concentration of calcium increases as keratinocytes

differentiate from the stratum basale to the stratum corneum, allowing the cells to

increase the number of their intracellular connections (Denda et al., 2003).

Keratinocytes have been reported to participate in immunological and inflammatory

processes (Forslind et al., 2004). It is well known that keratinocytes produce cytokines

such as interleukins (IL) and tumour necrosis factor-alpha (TNF-α), which modulate the

inflammatory response to UVR exposure (Tsatsanis et al., 2006). After UV radiation,

TNF-α and IL-1α are up-regulated in keratinocytes, activates NF-κB transcription factor.

33

This transcription factor controls expression of around 90 genes involved in

inflammation including cyclooxygenase-2 (COX-2) (Tsatsanis et al., 2006). In contrast,

UVR increases expression of the anti-inflammatory cytokine; interleukin-10 (IL-10)

which in turn decrease other pro-inflammatory cytokines such interleukin-1 (IL-1) and

interleukin-6 (IL-6) (Rivas and Ullrich, 1992).

1.1.1.2.1.1. Immortalized HaCaT keratinocytes

Life of normal human somatic cells is limited and their growth is ceased irreversibly after

certain number of divisions through a process called cellular senescence, or replicative

senescence. However, cellular immortality or unlimited proliferation can be induced

under culture conditions. Cellular immortality can be reproducibly induced by certain

DNA viruses such as simian virus 40, adenovirus types 5 and 12, and human papilloma

virus types 16, 18, 33 (Rhim et al., 1985, Rhim et al., 1986).

The HaCaT line is a spontaneously immortalized human keratinocyte cell line which

developed through long-term culture of normal human adult skin keratinocytes at

reduced calcium concentration and elevated temperature (Boukamp et al., 1985). It was

designated as HaCaT (Ha = human adult, Ca = calcium, T = temperature) to indicate

its origin and the initial culture conditions. Although, this cell lines has genetic

abnormalities, such as mutations in p53 (Lehman et al., 1993), it is still widely used and

important for many studies of skin biology and the pathogenesis of skin-related diseases.

1.1.1.2.2. Melanocytes

In addition to keratinocytes, the stratum basale contains other specialised cell types

such as the melanocytes (Figure 1.2.) (De Luca et al., 1988). Melanin pigment is

synthesized within the melanocytes and then transferred to surrounding keratinocytes

(Prota, 1997). Melanin absorbs ultraviolet light and protects skin from solar radiation

(Prota, 1997). In addition to its photoprotective role, epidermal melanin also regulates

cytokines activity where synthetic melanin was found to inhibit pro-inflammatory

cytokines include TNF, IL-6 and IL-10 (Mohagheghpour et al., 2000). Moreover, human

melanocytes serves as phagocytes against microorganisms (Le Poole et al., 1993).

34

Also, epidermal melanocytes were found to produce PGE2 under basal condition

(Nicolaou et al., 2004). Whereas, this eicosanoid was increased after UVB exposure

(Gledhill et al., 2010).

Two forms of melanin are found: eumelanin is the more common brown/black form,

whereas the less common phaeomelanin is red or yellow. Melanocytes make surface

contact with adjacent keratinocytes through dendritic connections, and this allows the

pigment granules to pass from the melanocytes to the keratinocytes (Kobayashi et al.,

1998b). Melanocyte presence appears to be inducible with chronic exposure to light

increasing the relative proportion of the pigment-forming cells within the basal layer.

There are equal numbers of melanocytes in a given body site in darker and lighter skin

types, but darker-skinned people have more active and efficient melanocytes (De Luca

et al., 1988).

1.1.1.2.3. Langerhans cells

Langerhans cells (LCs) are the main antigen presenting cells in the epidermis (Steinman

et al., 1995), and it has been reported to be involved in the skin’s immune responses

(Steinman et al., 1995). In addition, LCs reported to have a role in inflammatory

conditions (Kripke et al., 1990, Hemmi et al., 2001). LCs are also found within the

stratum basale (Figure 1.2) and in hair follicles (Steinman et al., 1995). They were

described in 1868 by Paul Langerhans and they are derived from bone marrow. They

are dendritic cells and through these processes connect to keratinocytes (Steinman et

al., 1995). Although LCs are not efficiently phagocytic, they may present the antigens

to lymphocytes in the lymph nodes (Steinman et al., 1995).

When compared to other membranes of the body, the skin comes into contact with many

potential antigens and hence the LCs play an important role in conditions such as

allergic contact dermatitis. As they are not linked to other cells by desmosomes, they

can move and migrate from the skin to the immune system (lymph nodes) (Kripke et al.,

1990). It is thought that they are able to internalise and process foreign antigens and

transport them to the skin lymph nodes where cutaneous immune responses are

35

initiated (Cumberbatch et al., 2003). Upon exposure to UVR, LCs migrate to the lymph

nodes where they are scavenge by cells of the immune system mainly Th cells for the

presence of antigen (Allan et al., 2006, Dandie et al., 2001). LCs migration and

maturation is mainly controlled by cytokines that are secreted by LCs end those

secreted by surrounding keratinocytes (Kripke et al., 1990, Griffiths et al., 2005).

1.1.1.2.4. Merkel cells

They are large cells found in low number in the stratum basale layer of the epidermis

(Sidhu et al., 2005) (Figure 1.2.). Merkel cells first described in 1875 by F. S. Merkel.

They are found in greatest numbers around the touch- sensitive sites of the body, such

as the lips and fingertips (Iggo and Muir, 1969). Merkel cells are associated with an

afferent nerve terminal, forming a structure known as a Merkel cell-neuron complex

(Saxod, 1996). Merkel cells also contain sensory receptors called merkel receptors

found in touch-sensitive areas of the skin surfaces such as hands and feet (Halata et

al., 2003).

1.1.2. Dermis

Dermis (Figure 1.2) is the second layer of the skin contains intensive connective tissues

and the dermal fibroblasts formed the majority in this layer (Fleischmajer et al., 1995).

It also contains lymphatic vessels, hair follicles, sebaceous, sweat glands sensory

neurones, motor neurons and a tough, supportive cell matrix (Wilkes et al., 1973, Sorrell

et al., 1996). Two layers comprise the dermis: thin papillary layer and thicker reticular

layer. The papillary dermis lies below and connects with the epidermis (Sorrell et al.,

1996). It contains thin loosely arranged collagen fibres, thick bundles of collagen run

parallel to the skin surface in the deeper reticular layer, which extends from the base of

the papillary layer to the subcutis tissue (Sorrell et al., 1996). Fibroblasts in this layer

produce collagen, elastin and structural proteoglycans, together with immunocompetent

mast cells and macrophages (Fleischmajer et al., 1993). Collagen fibres make up 70%

of the dermis provide its strength and toughness whereas elastin maintains normal

36

elasticity and flexibility and proteoglycans provide viscosity and hydration (Sorrell et al.,

1996).

1.1.2.1. Dermal cells

1.1.2.1.1. Fibroblasts

The presence of dermal fibroblasts within the dermal tissues is vital, as dermal

fibroblasts are responsible for the production and structuring of extracellular matrix

components, or ECM, within the skin (Ross et al., 1970). Further, dermal fibroblasts

communicate among themselves and with different cells to regulate the physiology of

the skin (Ross et al., 1970). Dermal fibroblasts constantly produce substances which

are then used to make up ECM, including various types of collagen; fibronectin and

types of elastin, integrin, proteoglycan and laminin, thus supporting the connective

tissues in the skin (Ross et al., 1970, Fleischmajer et al., 1993, Sorrell et al., 1996). Due

to this function, the dermal fibroblast is viewed as a central element in recovery from

wounds (Ross et al., 1970). Dermal fibroblasts are found both in the reticular dermis

and papillary dermis, and culturing these two subgroups has revealed variation in the

features of each (Azzarone and Macieira-Coelho, 1982, Schafer et al., 1985, Sorrell et

al., 1996, Sorrell et al., 2004).

The area available for molecules which display solubility to be transferred to the

epidermal layer, as well as for the occurrence of epithelial–mesenchymal interaction is

expanded by a large degree by the dermal papillae epidermis (Schafer et al., 1985,

Sorrell et al., 1996). The dermal reticular layer is located between the superficial and

the deep vascular plexi, and separates the hypodermis and dermis (Azzarone and

Macieira-Coelho, 1982, Schafer et al., 1985). The reticular dermis is penetrated by hair

follicles as well as the cells of the dermis with which they are linked, which frequently

end within the adipocyte-rich hypodermal layer (Azzarone and Macieira-Coelho, 1982).

Division takes place at greater pace in papillary fibroblasts compared with their reticular

counterparts (Azzarone and Macieira-Coelho, 1982, Schafer et al., 1985, Sorrell et al.,

1996, Sorrell et al., 2004). Biological study concerning the skin frequently utilises

46BR.IN fibroblasts, and are produced for this end by taking them from a subject who

37

has hypo-gammaglobulinemia before immortalizing them via plasmid pSV3neo,

transfection to express the T-antigen SV40, as described by Arlett et al. (1988).

The skin’s epidermal layer is bound with the dermis beneath through means of a

basement membrane composed of multiple molecules structured in a complex manner

(Burgeson and Christiano, 1997, Aumailley and Rousselle, 1999). Fibroblasts and

keratinocytes work together to create this membrane, which is distinguishable from

other tissues of the skin (Fleischmajer et al., 1993, Marinkovich et al., 1993, Smola et

al., 1998, Moulin et al., 2000). At the dermal-epidermal junction or DEJ, collagens IV

and VII are excreted from keratinocytes and fibroblasts, while laminin-1 and laminin-5

are excreted from fibroblasts and keratinocytes respectively, to form a linear array

(Marinkovich et al., 1993).

1.1.2.1.2. Mast cells

The mast cells of the skin lie deeper within the tissues, being found in the dermis, where

they are grouped around blood vessels and nerves (Eady et al., 1979). Mast cells have

an important role in inflammatory and allergic reactions such as allergic dermatitis, and

they secrete bioactive mediators such as histamine, cytokines and eicosanoids such as

prostaglandin D2 (PGD2) (Dawicki and Marshall, 2007, Wedemeyer et al., 2000). In

addition they have been reported to control local blood flow and angiogenesis (Marks

et al., 1986). These cells are affected by UVR which induces mast cells migration from

skin to draining lymph nodes (Byrne et al., 2008).

1.1.2.1.3. Subcutaneous layer

The subcutaneous tissue (Figure 1.2) is the layer between the dermis and the fascia

(Cross and Roberts, 1993). The fat tissue acts to preserve neutral fat, cushion against

external physical pressure, retain moisture and generate heat (Cross and Roberts,

1993). The subcutaneous tissue is largely composed of fat cells, accumulated fat cells

separated by the connective fibroid fat septum are called fat lobules (Wilkes et al.,

38

1973). Fibre bundles produced in the dermis and firmly connected with the fascia and

periostea through the subcutaneous tissue are found throughout this region (Cross and

Roberts, 1993). These fibre bundles are called retinaculae cutis, and they strengthen

the connection between the dermis and deeper tissues (Cross and Roberts, 1993).

1.2. Ultraviolet radiation and its effects on cells

The wavelength of the sunlight radiation is divided into three main regions of

wavelengths (Figure 1.3). Ultraviolet (UV), visible, and infrared. UV radiation includes

the wavelengths from 200 to 400 nm, these wavelengths are just shorter than those of

visible light (400-700 nm). It is then characterized further UVA (315-400 nm), UVB (280-

315 nm) and UVC (100-280 nm) reviewed in (Matsumura and Ananthaswamy, 2004).

UVC from sunlight is virtually completely screened out by the earth's atmosphere ozone

layer. UV light is essential for normal skin health and a suitable dose of UVB radiation

(280–320 nm) is necessary for processes including synthesis of vitamin D synthesis in

the skin. However, UVR has also harmful effects on skin cells, these effects will be

discussed in more details below.

Figure 1.3: UV radiation spectrum (Adapted from www.globalspec.com) December

2014.

http://www.globalspec.com/

39

1.2.1. Effects of ultraviolet radiation on human skin

Excessive UV radiation penetrates the epidermis and dermis and interferes with normal

skin function, causing a number of health disorders. UVB is the most harmful type of

UVR, it has been reported to be responsible for erythema (sunburn), which is the visual

effect of UVR on the skin, usually characterised by dilatation of the dermal blood vessels

and then increases the blood flow, consequently leading to skin redness (Diffey, 1991).

This is apparent from about 4 hours after exposure and is maximal at about 24 hours

(Young et al., 1996, Rhodes et al., 2009). An immediate tan is produced at between 5

and 10 minutes of the skin being exposed to ultraviolet radiation, and this type of tan

fades after twelve hours. By contrast, a delayed tan appears as the result of melanin

excreted by stimulated melanocytes and may last from several weeks to several months

(Diffey, 1991). Erythemal reactions vary based on the amount of UV radiation exposure,

the spectrum within which it is emitted and the individuals’ skin. Potential for burning

from sun exposure is frequently measured through observing the subject’s minimal

erythema dose or MED. In addition, multiple-occasion UVR exposure can lead to

epidermal hyperplasia causing the cells of the epidermis to hyperproliferate and this

layer to thicken. At between 1 and 3 weeks, the stratum corneum becomes three times

thicker, and at 7 weeks, may be up to 7 times as thick as originally. However, between

4 and 8 weeks after sun exposure ends, thickening is completely reversed. The thicker

skin produced with frequent ultraviolet exposure protects against damage from that

further exposure (Diffey, 1991, Soter, 1990).

For certain instances, burning from sun exposure is linked to skin alterations, which may

include for example the presence in the dermis of inflammatory cells (Gilchrest et al.,

1983, Hawk et al., 1988) the occurrence of the protein p53 (Burren et al., 1998), and

the presence of burned apoptotic cells in the epidermal layer (Sheehan and Young,

2002). Further, skin which was exposed to UVB radiation could display

immunosuppression (Novakovic et al., 2001) as well as melanogenesis (Sheehan et al.,

1998, Sheehan et al., 2002). In general, two pathways have been proposed for damage

caused by cytotoxicity disrupting cells’ regular functioning and linking UVB exposure to

carcinogenesis in the skin’s tissues. Exposure to UVB can produce damage directly

40

incurred by DNA through the forming of atypical covalent bonds joining pyrimidine bases

next to each other within molecules of DNA in an excited state (Young et al., 1998).

Where these anomalies are not destroyed via the pathway for nucleotide excision repair

prior to the DNA being further replicated (Young et al., 1996, Bykov et al., 1999, Budden

and Bowden, 2013), they disturb base pairs, and produce mutations which cause

melanomagenesis (Budden and Bowden, 2013).

Secondly, UVB exposure produces oxidative stress for normal cells within the skin,

through leading to production of reactive oxygen species (ROS) (Lee et al., 2013a),

which cause harm to structural elements within cells, such as to membranes, lipids,

DNA and proteins (Waster and Ollinger, 2009). The eventual result of this includes

photoaging (Pillai et al., 2005) and photodamage (Marrot and Meunier, 2008), as well

as photocarcinogenesis (Halliday, 2005).

Similar to UVB, UVA can also cause DNA damage primarily through the generation of

ROS (Kvam and Tyrrell, 1997). However, UVA and UVB have been found to stimulate

the production of melanin and induce apoptosis of melanocytes and keratinocytes an

effect that has been recognised as a protective mechanism in the skin (Bowen et al.,

2003). UVB radiation is mostly absorbed by the epidermis and upper dermis whereas,

UVA, with its longer wavelength can penetrate deeper through the epidermis to reach

the dermis (Costin and Hearing, 2007).

1.3. Omega-3 polyunsaturated fatty acids

Any fatty acid composed with over three or more double bonds is known as a

polyunsaturated fatty acid, PUFA, or long-chain polyunsaturated fatty acid, and includes

more than 17 atoms of carbon. For omega-3 polyunsaturated fatty acids, the omega (n)

element of the name represents the methyl terminal, while 3 denotes the position of the

final double-bond at the same end. There are 2 principal omega-3 polyunsaturated fatty

acids, namely C22:6 n-3 docosahexaenoic acid or DHA, and C20:5 n-3

eicosapentaenoic acid or EPA. These are generally contained in the diet through oils

from fish: for example from salmon, sardine or mackerel and sardines. Meanwhile, C18

41

n-3 PUFA α-linolenic acid or ALA, which is the parent to omega-3, can be obtained from

oils of vegetal origin and humans can use this to synthesise both DHA and EPA. The

advantages of these fatty acids for health are considered various (Wall et al., 2010).

Research suggests that omega-3 PUFA enhance the health of the heart and circulatory

system (De Caterina, 2011) as well as improving rheumatoid disorders (Bhangle and

Kolasinski, 2011). Further, recent research suggests an effect of these fatty acids

against cancer: specifically in combatting colorectal cancers or CRCs (CRC) (Cockbain

et al., 2012).

Polyunsaturated fatty acids act in the regulation of a variety of processes, such as

clotting of the blood and blood pressure, as well as nervous and brain development and

function (Das, 2006). Further, these fatty acids are an element in inflammation response

regulation via the creation of eicosanoids, which are powerful in terms of their mediating

function (Das, 2006, Calder, 2006). Neither linoleic acid (LA, C18:2n-6), which is used

in building n-6 UPFA, nor α-linolenic acid (ALA, C18:3n-3), which is necessary to create

omega-3 polyunsaturated fatty acids, can be produced in humans, and thus must be

provided by dietary means (Burr and Burr, 1929).

1.3.1. Metabolism of essential fatty acids in human skin

In 1929 Burr and Burr stated that there are two fatty acids that cannot be produced by

mammalian cells. These are Linoleic acid (LA; 18; 2n-6) and α-linolenic acid (ALA; 18:

3n-3), thus, termed essential fatty acids (Burr and Burr, 1929). LA is found in plant oils

such as sunflower, safflower, and corn oils, cereals, animal fat, and wholegrain bread,

while ALA is abundant in green leafy vegetables, flaxseed, and rapeseed oils. Although

mammalian cells cannot synthesize LA and ALA, they can metabolize them into more

physiologically active compounds by the introduction of further double bonds

(desaturation) via Δ5 and Δ6 desaturases, and by lengthening the acyl chain

(elongation) via elongases (Figure 1.4). LA has been demonstrated to be the most

abundant PUFA in human skin (Chapkin et al., 1986, Wertz, 1992). LA has reported to

play a role in the epidermal water barrier function (Hansen and Jensen, 1985). LA also

42

undergoes metabolism to produce different metabolites, for example, incubation of 15-

lipoxygenase (15-LOX) prepared from skin epidermis, metabolizes LA to 13-

hydroxyoctadecadienoic acid (13-HODE) which is the major metabolite of LA in the

epidermis (Ziboh et al., 2000). Elongation and desaturation of LA and ALA produces

eicosapentaenoic acid (EPA; 20: 5n-3), arachidonic acid (AA; 20: 4n-6), and

docosahexaenoic acid (DHA; 22: 6n-3). Elongation of gamma-linolenic acid (GLA; 18:

3n-6) forms dihomo-gamma-linolenic acid (DGLA; 20: 3n-6). At high concentrations

DGLA is metabolized by epidermal cyclooxygenases (COX) into prostaglandins of 1-

series such as PGE1 and also by 15-LOX to 15-hydroxyeicosatrienoic acids such as 15-

HETrE (Ziboh et al., 2000). However, EPA and DHA have also been shown to be

metabolized by epidermal 15-LOX to predominantly monohydroxylated metabolites

such as 15-hydroxyeicosapentaenoic acid (15-HEPE) and 17-hydroxydocosahexaenoic

acid (17-HoDHE) respectively (Miller et al., 1990, Miller et al., 1991). The n-6 PUFA AA

represents about 9% of the total fatty acids in humans skin (Vroman et al., 1969). AA

metabolised in the epidermis by the COX pathway to produce different types of

prostaglandins (PGs) such as PGE2, PGF2α and PGD2 (Vroman et al., 1969). Also, AA

metabolised by 15-LOX to generate 15-hydoxyeicosatetraenoic acid (15-HETE) (Ziboh

et al., 2000). In contrast, 5-LOX activity in the epidermis has reported to be insufficient

to transform AA to leukotriene (LTB4) (Iversen et al., 1993). Finally, it is well documented

how the essential fatty acids are important in the epidermis, thus any deficiency of them

will have an effect on skin health (Hansen and Jensen, 1985).

43

Figure 1.4: Metabolism of omega-9, 6 and 3 fatty acids. Adapted from (Wall et al.,

2010). The essential fatty acids Linoleic and alpha-Linolenic acids are not synthesized

by mammalian cells and need to be obtained through diet.

44

1.3.2. Polyunsaturated fatty acids and impact upon eicosanoids production

The eicosanoid group encompasses prostaglandins or PG, leukotrienes or LI,

prostacyclin or PGI2 and thromboxanes or TX. These find their precursor in 20-carbon

PUFA; principally AA. They are significant in mediating and regulating functions for

inflammatory processes (Tilley et al., 2001), and in modulation of inflammation as a

response in terms of length of time for which inflammation persists, and how strong

these processes are (Tilley et al., 2001, Kinsella et al., 1990). Despite this importance,

generally, the impact physiologically which follows inflammation is based upon which

cells are located in that area, how the response is caused, timing for production of

eicosanoids and numbers of the various eicosanoids as related to the area involved, as

well as how sensitive the region is where the eicosanoids are produced is at tissue and

cellular level (Calder, 2008). Polyunsaturated fatty acids of the n-3 and n-6 varieties

compete PUFA in generating eicosanoids via COX enzymes and LOX enzymes:

however, as inflammatory cells generally have a significant percentage of AA, this

generally forms the principal substrate in synthesising eicosanoids (Calder, 2001,

Simopoulos, 2002). Within the membranes of cells, phospholipases release AA, and in

particular phospholipase A2 with free AA thereafter providing substrates for LOX and

COX. As COX metabolises AA, 2-series PGs as well as 2-series thromboxanes or TXs

are produced. Significant quantities of prostaglandin F2α (PGF2α) and prostaglandin E2

(PGE2) are generated by monocytes and macrophages, while some PGE2 is created by

neutrophils and prostaglandin D2 or PGD2 is generated by mast cells.

When AA is metabolised via the 5-LOX path, this leads to production of derivative of

hydroxy hydroperoxy. These include 5-HPETE and 5-HETE, as well as 4-series LTs

and, leukotrienes A4 (LTA4), B4 (LTB4), C4 (LTC4), D4 (LTD4) and E4 (LTE4). LTB4 is

generated by monocytes, neutrophils and macrophages. Meanwhile, mast cells,

eosinophils and basophils generate LTC4, LTE4 and LTD4. The Arachidonic acid gives

the basis for so-called proinflammatory eicosanoids (Bagga et al., 2003), which include

interleukin-6 (IL-6) as generated by macrophage cells in response to PGE2, and the

functions of which include dilation of vascular channels and generation of pain (Tilley et

al., 2001, Bagga et al., 2003). Neutrophils meanwhile can be activated by LTB4, which

45

also impacts leukocytes by acting strongly as a chemotactic agent, as well as

stimulating macrophage generation of cytokines involved in inflammation including

tumour necrosis factor alpha, or TNFα, IL-1 beta, or IL-1β and IL-6 (Tilley et al., 2001).

While the status of proinflammatory mediator is frequently applied to eicosanoids

derived from AA however, these substances are also significant in modulation of

immune responses because of their ability to interact with leukocytes in a manner which

displays considerable complexity the initial inflammatory processes (Tilley et al., 2001,

Simopoulos, 2002). Despite these benefits, where such substances are present in

excess, the tissue local to their production may be harmed and conditions involving

inappropriate inflammatory response, as well as thrombosis, are partially caused by this

mechanism (Simopoulos, 2003).

LOX metabolises DHA to produce docosanoids, as well as resolvins (Hong et al., 2003).

Resolvins have a mediatory function, act locally and are endogenous: they act against

inflammation and help to regulate immune response (Serhan et al., 2002, Serhan et al.,

2007, Schwab et al., 2007). As concerns the cell, functions encompass reduction in

neutrophils infiltrating as well as regulating reactive oxygen species and the cytokine-

chemokine axis, and effecting reduction in inflammation-response (Serhan et al., 2000,

Serhan et al., 2002).

Both LOX and COX metabolise EPA, creating disparate eicosanoid groups, namely

HEPE, 3-series TXs and TXs, and 5-series LTs. As contrasted with AA-derived

eicosanoids, those of EPA may not be so, or may act against inflammation (Bagga et

al., 2003, Robinson and Stone, 2006). Variation in inflammatory effect between AA- and

EPA-derived eicosanoids is exemplified by LTB4 in comparison with LTB5. LTB4 is

stronger in producing inflammatory response than LTB5, due to its action as a

chemotactic agent for neutrophils, which is greater by a factor of between 10 and 100

(Goldman et al., 1983, Lee et al., 1984). In addition, resolvin E1, which derives from

EPA, is protective against colitis, as evidenced by studies of animals (Weylandt et al.,

2007). Further, PGE2 is described in the literature as more powerful in inducing

macrophage IL-6 generation in comparison with PGE3 (Bagga et al., 2003). Greater

amounts of n-3 PUFA, including DHA and EPA ingested leads to increased

46

concentration of these substances within inflammatory cell membranes, as reported by

Endres et al. (1989) and Yaqoob et al. (2000). Both DHA and EPA make up

inflammatory cells in humans in dose-response relation to their intake (Healy et al.,

2000, Rees et al., 2006) with this make-up able to reduce AA to some extent.

1.3.3. Omega-3 PUFA and inflammatory gene expression

In addition to their effects on the eicosanoids, n-3 PUFA exert their anti-inflammatory

effect by changing the inflammatory gene expression through their effects on

transcription factors such as NFκB and peroxisome proliferator-activated receptors

(PPARs) (Calder, 2006, Calder, 2002). NFκB is a transcription factor that plays an

important role in various inflammatory signalling pathways. It controls several cytokines

such as IL-1, IL-2, IL-6, IL-12, TNF-α, chemokines like IL-8, monocyte chemoattractant

protein-1, adhesion molecules and inducible enzymes such as the inducible nitric oxide

synthase (iNOS) and COX-2 (Ghosh and Karin, 2002). EPA has been shown to block

the activity of NFκB through decreased degradation of the inhibitory subunit of NFκB,

IκB, in cultured pancreatic cells and human monocytes (Lo et al., 1999, Novak et al.,

2003, Zhao et al., 2004).This has also been supported by the finding that transgenic

mice that endogenously biosynthesize n-3 PUFAs from n-6 PUFAs are protected from

colitis through a decrease in NFκB activity (Hudert et al., 2006).

PPARs are ligand-activated nuclear transcription factors that play important roles in

cellular differentiation, cancer, inflammation, insulin sensitization, atherosclerosis and

several metabolic diseases (Chawla et al., 2001). Ligands for PPARs are PUFAs,

especially those of the n-3 family and their eicosanoid derivatives (Kota et al., 2005).

When activated, PPARs bind to the PPAR-response element and repress or induce the

transcription of target genes. PPARs have been shown to inhibit NFκB and therefore

play an important role in several inflammatory processes (Poynter and Daynes, 1998,

Ricote et al., 1999). EPA and DHA for instance inhibit lipopolysaccharide-induced

activation of NFκB via a PPAR-γ-dependent pathway in human kidney-2 cells (HK-2) (Li

et al., 2005).

47

1.3.4. The protective effect of n-3 PUFA in UVR induced skin inflammation

The harmful effects of UVR in the skin, include acute effects such as erythema,

inflammatory and immune modulatory changes (Young, 2006). Chronic effects

eventually lead to photoaging and photocarcinogenesis (Taylor et al., 1990). Although,

there are some strategies to protect against UVR-induced skin damage such as topical

sunscreens (Rhodes, 1998), these strategies have been found to be inadequate. The

n-3 PUFA, EPA and DHA are considered as a dietary component with a promising

photoprotective effect against the deleterious effects of UVR. They are mainly present

in fish oil, and have been shown to have a protective effect against photocarcinogenesis

in mice (Black et al., 1992). Supplementation with fish oil also reduces UVB-erythemal

sensitivity in humans (Rhodes et al., 1994), by inhibition of UVB-induced PGE2 levels in

the skin (Rhodes et al., 1995). Additionally, n-3 PUFA have been reported to improve

some inflammatory diseases, including psoriasis (Mayser et al., 1998) and rheumatoid

arthritis (Kremer, 2000), and can inhibit tumor formation (De Vries and van Noorden,

1992).

1.4. The endocannabinoid system

The major psychotropic component of the cannabis sativa plant, Δ9-

tetrahydrocannabinol (THC) was discovered in 1964 (Gaoni and Mechoulam, 1964).

After more than 20 years the first THC receptor was identified in the mammalian brain

and named cannabinoid receptor type-1 (CB1) (Devane et al., 1988). Two years later

and then the second G-protein-coupled receptor (CB2) was cloned (Matsuda et al.,

1990). Whilst CB1 was shown to be extremely abundant in the brain, and hence

suggested to be responsible for THC psychoactivity, CB2 was expressed in its highest

levels in immune cells. The cloning of the cannabinoid receptors opened the way for

more research to identify endogenous lipid ligands termed endocannabinoids. The first

endocannabinoid to be discovered was N-arachidonoyl ethanolamide (AEA,

anandamide) (Devane et al., 1992), soon after that followed by the detection of the 2-

arachidonoyl-glycerol (2-AG) which shows high affinity for CB1 and CB2 receptors

(Mechoulam et al., 1995, Sugiura et al., 1995). Furthermore, more endocannabinoids

48

have been found in the last decade, including 2-arachidonyl-glycerol ether (noladin

ether) (Hanus et al., 2001), N-arachidonoyl-dopamine (NADA) (Bisogno et al., 2000)

and virodhamine (Porter et al., 2002). However, their pharmacological activity and

metabolism has not yet been thoroughly investigated. Therefore, the N-

acylethanolamine anandamide and the monoacylglycerols 2-AG are still the most

studied endocannabinoids (Figure 1.5).

The endocannabinoids exert their biological functions by binding to or activating CB1

and CB2 receptors (Howlett et al., 2002). However, they may have different responses

for example, AEA behaves as a partial agonist at both CB1 and CB2 receptors, but has

higher affinity for the CB1 receptor (Howlett et al., 2002). Thus, AEA has been reported

to have the same pharmacological functions to cannabinoids such as Δ9-THC, by

activating the CB1 receptors in the brain (Smith et al., 1994). In addition, AEA shows

potent immunomodulatory and anti-inflammatory activities by interacting peripherally

with CB receptors (Stefano et al., 1996).

AEA is transported into cells by the AEA membrane transporter (AMT) and its action is

terminated by the enzyme fatty acid amide hydrolase (FAAH). Whereas, 2-AG uptake

occurs through the 2-AG membrane transporter (2-AGMT) (Hermann et al., 2006), 2-

AG can be hydrolysed by FAAH but the main enzyme responsible for 2-AG degradation

is monoacylglycerol lipase (MAGL) (Cravatt et al., 1996, Dinh et al., 2002).

49

Figure 1.5: Chemical structures of the Δ9-tetrahydrocannabinol (THC), N-

arachidonoylethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG). Adapted

from (Bari et al., 2011).

1.4.1. Biosynthesis of N-arachidonoylethanolamide (AEA, anandamide)

Anandamide or AEA is produced from membrane phospholipids, having the precursor

N-arachidonoyl phosphatidylethanolamine or NAPE. These phospholipid undergo

hydrolysis and form AEA via an enzyme: N-acyl phosphatidylethanolamine

phospholipase D (NAPE-PLD) as shown in Figure 1.6 (Di Marzo et al., 1994, Guo et al.,

2005). This may be simplistic however, since synthesis of AEA appears to encompass

a variety of pathways, including AEA produced independently from NAPE-PLD, as

concentrations of the end substance were unaffected by knockout of NAPE-PLD in a

study involving mice (Liu et al., 2006). Studies in mice also revealed NAPE-AEA

conversion from NAPE-PLD (Leung et al., 2006).

50

1.4.1.1. N-acyltransferases

AA gives the derivative AEA as ethanolamine, derivatives of N-acylethanolamine (NAE)

produced from metabolising different fatty acids and include a large number with

endocannabinoid functions, this includes DHEA. In biosynthesising NAE, initially

phosphatidylethanolamines (PE) are N-acylated to created ethanolamine phospholipids

which are N-acylated, including NAPE regulated via the Ca2+ dependent N-

acyltransferase or NAT (Hansen et al., 2000, Cadas et al., 1997, Jin et al., 2007).

Phosphatidylcholine (PC), 1-acyl-lyso-PtdCho and PE are utilised by NAT to form

donors in terms of substrate, while the enzyme selects acyl groups from these located

at sn-1 for extraction.

1.4.1.2. N-acyl phosphatidylethanolamine phospholipase D

N-acyl phosphatidylethanolamine-phospholipase D, or NAPE-PLD is a member of the

zinc metallohydrolase group from the β-lactamase, being separate in functional and

structural terms from PLD enzymes generating phosphatidic acid or PA by hydrolysing

glycerophospholipids, including PC, which acts between cells as a signalling molecule

(Liscovitch et al., 2000, Okamoto et al., 2004). The principle pathway for synthesising

AEA as well as various NAEs is NAPE-PLD (Schmid, 2000, Hansen et al., 2000, Schmid

and Berdyshev, 2002, Nakane et al., 2002). NAE being released from NAPE via NAPE-

PLD forms stage two in the process of synthesising NAE (Okamoto et al., 2007). It is

suggested that zinc is a component of NAPE-PLD which is vital to catalysis, while

purified recombinant enzyme correlates specifically to NAPE, is virtually non-reactive

when considering significant glycerophospholipids including PE and PC (Wang et al.,

2006). From animal tissue, there is only one enzyme which controls direct generation

of NAE as released by NAPE: NAPE-PLD. In terms of regulating transcripts, the Sp1

transcription factor is identified as contributing to baseline expression of NAPE-PLD in

a regulatory capacity (Zhu et al., 2011).

51

1.4.1.3. Alternate pathways for anandamide biosynthesis

A range of enzymes are identified as having roles in biosynthesising AEA, such as

secretory phospholipase A2 or sPLA2. This functions to hydrolyse NAPE, forming N-

arachidonoyl lysophosphatidylethanolamine or lyso-NAPE, and this is capable of

hydrolysation via lysophospholipase D (lyso-PLD), giving AEA independently of Ca2+

(see Figure 1.6) (Sun et al., 2004). Furthermore, αβ-hydrolase 4 or Abhd 4 may be

capable of affecting lyso-NAPE or NAPE and thus creating glycerophospho-

arachidonoyl ethanolamide or GP-AEA. This substance then acts on metal-reliant

phosphodiesterases, resulting in the formation of AEA (see Figure 1.6) (Leung et al.,

2006). Further, phospholipase C PLC can act in hydrolysation of NAPE, forming

phosphoanandamide, and this in turn undergoes dephosphorylation via phosphatases

including tyrosine phosphatase PTPN22, and AEA is produced in this manner, as show

in Figure 1.6 (Liu et al., 2006). Further, in macrophages treated with LPS, there was no

alteration in the level of AEA found for siRNA knockdown NAPE-PLD, PTPN22 and

Abhd 4 (Liu et al., 2008). The formation of AEA via avenues other than those discussed

above is considered to occur through free AA condensing alongside ethanolamine

within a FAAH reversed reaction (Arreaza et al., 1997, Kurahashi et al., 1997), which

may take place given adequate quantities of ethanolamine and AA (Katayama et al.,

1999). Generation of AEA in this manner was achieved on a study on live mice in which

part of their livers was removed (Mukhopadhyay et al., 2011). Furthermore, McCue et

al. (2009) state that spontaneous generation of AEA is possible based on ethanolamine

and arachidonoyl-CoA.

52

Figure 1.6. Schematic showing the various pathways of anandamide biosynthesis.

Adapted from (Bisogno and Maccarrone, 2014).

53

1.4.2. Biosynthesis of 2-arachidonoyl glycerol

In using phospholipids to produce AA, a principle avenue was viewed as producing

diacylglycerol lipase (DAGL) via the action of phospholipase C or PLC upon

phosphatidylinositol (Prescott and Majerus, 1983). The same avenue is also recognised

as central to 2-AG synthesis (Sugiura et al., 2006, Ueda et al., 2011), which takes place

within postsynaptic neurons as a reaction to Gq/11-coupled receptors being stimulated

and depolarised. Kano et al. (2009) report that the release of the resultant 2-AG allows

mediation of retrograde synaptic suppression via CB1 receptors located in presynaptic

terminals. The path described includes use of PLC in hydrolysing 2-arachidonoyl-

phosphatidylinositol or PtdIns, forming diacylglycerol (DAG), which contains arachidonic

acid, and subsequently undergoes hydrolysation via DAGL, forming 2-AG, as shown in

Figure 1.7. The most abundant PtdIns molecule in mammalian tissues is 1-stearoyl-2-

arachidonoyl-PtdIns is the most frequently encountered Ptdlns within the tissue of

mammals and allows 2-AG to be created in preference to different MAGs. The β subtype

or PLC may be stimulated via Gq/11-coupled receptors (Fukami et al., 2010), with the

implication that PLC is important for generating neurotransmitter-reliant 2-AG with

mediation through Gq/11-coupled receptors within postsynaptic neurons. However, it is

also possible to convert PtdCho to 2-arachidonoyl-DAG (Oka et al., 2005) and the same

is demonstrated for PA by Bisogno et al. (1999b) with this forming 2-AG precursor.

PtdIns also sets in motion a path in which lyso-PtdIns production via PLA1 leads to lyso-

PtdIns-specific PLC releasing 2-AG (Ueda et al., 1993, Tsutsumi et al., 1994) (Figure

1.6), as shown in Figure 1.6. Moreover, arachidonoyl-lysophosphatidic acid or Lyso-PA

can also generate 2-AG (Nakane et al., 2002).

DAGL is an enzyme linked with membranes and which hydrolyses DAG in a preferential

manner at locus sn-1 (Okazaki et al., 1981, Chau and Tai, 1981). Cloning via cDNA

demonstrates two genes, α and β, which have a high degree of relation to each other,

as involved in DAGL in humans (Bisogno et al., 2003). A broad distribution pattern has

been found for DAGLα within the tissues of both mice and humans: it is found in

particularly strong concentrations within the nervous system of both animals, and in

human pancreatic tissue. Further localisation to PLCβ, Gq/11-coupled receptors and

54

Gq/11α within certain surfaces of synapses and neurons (Kano et al., 2009). Mice in

which DAGLα-was present in insufficient quantities display significantly lowered 2-AG

within the brain, which suggests a high degree of involvement for DAGLα as opposed

to DAGLβ with the production of 2-AG (Gao et al., 2010, Tanimura et al., 2010).

Figure 1.7. Schematic showing the main steps in the biosynthesis of the 2-

Arachidonoylglycerol. Adapted from (Bisogno and Maccarrone, 2014)

55

1.4.3. Catabolism of AEA and 2-AG

Subsequent to their generation endocannabinoids rapidly become inactive, as shown in

Figure 1.8. AEA within the brain returns to neurons before undergoing hydrolysation via

FAAH to form ethanolamine and AA (Glaser et al., 2005, Fowler, 2007). As well as this

process, LOX, COX-2 and cytochrome P450 may also generate prostaglandin

ethanolamines (PG-EA), or prostamides, by metabolising AEA (Karsak et al., 2007).

Further, COX-2 is capable of metabolising 2-AG through PGH2-GE as an intermediate

in the production of prostaglandin glycerol esters including glyceryl prostaglandins and

PG-GE (Kozak et al., 2000). Transportation of 2-AG to cells is carried out either through

diffusion or via 2-AG transporters (Hermann et al., 2006). The majority of 2-AG

hydrolysation taking place in brain tissues is conducted by monoacylglycerol lipase

(MAGL). This substance allows 2-monoglycerides and 1-monoglycerides to be

converted to glycerol and fatty acid (Dinh et al., 2002, Blankman et al., 2007). Further,

it is possible that 2-AG is inactivated in a process which involves phosphorylating it with

monoacylglycerol kinase (Nakane et al., 2002). As transportation, metabolic pathways

and biosynthesis differ for 2-AG and AEA, it is suggested that each has its own

mechanisms for regulation. Further, direct inhibition of 2-AG by AEA is described by De

Petrocellis and Di Marzo (2009). Additionally, Fezza et al. (2008) report higher

concentration of AEA within the striatum as effecting reduction in 2-AG’s impact upon

GABAergic transmission.

56

Figure 1.8: Catabolism of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG);

scheme adapted from (Ueda et al., 2013)).

1.4.3.1. Fatty acid amide hydrolase

FAAH is an amidohydrolase categorised within the amidase group of serine hydrolases,

and has an association with membranes. FAAH is responsible for hydrolysing NAEs,

including AEA, creating ethanolamine and specific fatty acids (Deutsch et al., 2002,

57

McKinney and Cravatt, 2005). Research involving FAAH−/− mice showed that FAAH

was important in the degrading process of several NAEs, including AEA (Cravatt et al.,

2001, Clement et al., 2003). It is also involved in hydrolysation of oleamide and various

fatty acid amides (Cravatt et al., 1996), as well as N-acyltaurine, as reported by

Saghatelian et al. (2004). Hydrolysation of 2-AG occurs rapidly via FAAH (Goparaju et

al., 1998), although studies have not considered FAAH to have a central importance in

degradation of 2-AG within brain tissue (Goparaju et al., 1999, Blankman et al., 2007).

Unlike rodent species studied, human tissues show expression of a FAAH isozyme

which has approximately 20% sequence identity when viewed at the level of amino

acids (Wei et al., 2006). The isozyme is categorised as FAAH-2, as distinct from FAAH-

1, or the first FAAH to be identified. FAAH-2 shows a different localisation to FAAH-1,

being found on droplets of lipids as opposed to the endoplasmic reticulum. Further,

Kaczocha et al. (2010) report that for FAAH-2, the N-terminal hydrophobic area of

FAAH-2 is shown to be a sequence for localising the isozyme on lipid droplets, and this

implies a distinct function of FAAH-2 in comparison with FAAH-1. Moreover a range of

symptoms are suspected to include FAAH involvement, among which are neuropathic

pain as well as depressed and anxious states. This has led to the expectation that

substances which inhibit FAAH may present drugs which have an effect in these

scenarios (Petrosino and Di Marzo, 2010). Thus, development of a number of

specifically targeted FAAH inhibitors has occurred, such as for OL-135 (Lichtman et al.,

2004), URB597(Kathuria et al., 2003), PF-3845 (Ahn et al., 2009) and PF-04457845

(Ahn et al., 2011).

1.4.3.2. N-acylethanolamine hydrolysing acid amidase

The initial identification of the lysosomal enzyme N-acylethanolamine hydrolysing acid

amidase or NAAA took place in humans in CMK megakaryoblastic cells, as reported by

Ueda et al. (1999). This was followed by its identification in rat subjects within various

tissues including those of the lung (Ueda et al., 2001). When the enzyme was cloned

via cDNA, it was identified as cysteine hydrolase from the N-terminal nucleophile

hydrolase supergroup (Tsuboi et al., 2005, Tsuboi et al., 2007a, Ueda et al., 2010). The

58

enzyme was considered a protein similar to the lysosomal enzyme acid ceramidase in

sequence homology (Hong et al., 1999). Acid ceramidase acts in hydrolysing ceramide,

forming fatty acids and sphingosine. Despite this, sequence homology has not been

observed for FAAH and NAAA. Based on the fact that NAAA is localised within

lysosomes, it shows activity solely when pH is in the acid area of the spectrum,

hydrolysing a range of NAEs but favouring PEA as shown in vitro. The NAAA

glycoprotein in humans has 4 loci of N-glycosylation (Zhao et al., 2007, West et al.,

2011). West et al. (2011) report that a recombinant form of NAAA is generated to form

a proenzyme which is not active, undergoing conversion through autocatalytic cleavage

of Phe125 and Cys126, forming a heterodimer based upon α and β elements and which

displays catalytic activity. Expression of NAAA can be found within a variety of tissues

in both human and rodent subjects, and is principally expressed within macrophage

cells (Sun et al., 2005, Tsuboi et al., 2007b). NAAA is expressed to the greatest degree

in humans within prostate tissue (Wang et al., 2008). Stimulation of NAAA has been

achieved in vitro via non-ionic detergent, including Nonidet P-40 Triton X-100, as well

as through dithiothreitol, which is an agent in reducing thiol. Nonidet P-40 may be

replaced by phospholipids which contain choline or ethanolamine, such as

sphingomyelin, PtdCho and PtdEtn, in endogenous stimulation, whereas dithiothreitol

can be replaced with dihydrolipoic acid, which is α-lipoic acid in reduction (Tai et al.,

2012).

1.4.4. N-acylethanolamines

N-acylethanolamines, or NAEs, are substances derived from fatty acids, and these

present themselves within the body tissues of mammals (Schmid, 2000, Hansen et al.,

2000, Schmid and Berdyshev, 2002, Nakane et al., 2002). Certain NAEs are shown to

fulfil particular roles, including for example N-stearoylethanolamide (STEA), which is

pro-apoptotic (van der Stelt et al., 2002). while N-palmitoylethanolamide (PEA) acts

against inflammation, as well as having neuroprotective and analgesic properties, and

being an agonist for peroxisome proliferator-activated receptor-α as opposed to CB1

and CB2 (Calignano et al., 1998, Smart et al., 2002, LoVerme et al., 2005). Further, N-

59

oleoylethanolamide or OEA is suggested to be anorexic by Rodriguez de Fonseca et

al. (2001). Further, OEA is an agonist for a range of receptors, such as; G protein-

coupled receptor GPR119, peroxisome proliferator-activated receptor-α and transient

receptor potential vanilloid 1 (Pavón et al., 2010). Each of these receptors derive from

membrane phospholipids. Phosphatidylethanolamine (PE) is converted to N-acyl

phosphatidylethanolamine (NAPE) by means of Acyltransferases, and NAPE

undergoes hydrolysation via NAPE-PLD, producing N-acylethanolamine (Hansen et al.,

2000), which is further elaborated in Section 1.4.1., and as an instance of this,

hydrolysing N-pamitoylphosphatidylethanolamide, a phospholipid precursor, is

considered via NAPE-PLD is considered to be a principle avenue in biosynthesising N-

palmitoylethanolamide, as proposed by Schmid et al. (1983). Various N-acyl chains for

NAE are considered to be biosynthesised via transacylation–phosphodiesterase.

Further, Sugiura et al. (1996) did not identify N-acyl species when catalysing reactions

via N-acyltransferase, and nor did Wang et al. (2006) when using NAPE-PLD.

1.4.5. Endocannabinoids and their impact on the skin

The endocannabinoid receptors CB1 and CB2 have been found in human and murine

skin cells such as cutaneous nerve fibres, mast cells, epidermal keratinocytes and cells

of the adnexal tissues (Stander et al., 2005, Ibrahim et al., 2005, Blazquez et al., 2006,

Telek et al., 2007, Casanova et al., 2003). In particular, CB1 is expressed on different

types of the cutaneous nerves such as the large myelinated nerve fibres in the papillary

dermis, also on the small nerve fibres that associate with hair follicles and on the nerve

fibres in the epidermis (Stander et al., 2005). In addition, the presence of CB1 receptor

has been reported in all dermal mast cells (Stander et al., 2005). CB2 has been found

in the skin on large myelinated nerve fibre bundles of the superficial and deep reticular

dermis, small unmyelinated nerves of the papillary dermis and occasionally on nerves

of the epidermis (Stander et al., 2005), as well as in human SZ95 sebocytes (Dobrosi

et al., 2008).

60

The CB receptors have been suggested to be involved in the differentiation of

keratinocytes (Casanova et al., 2003). For instance, endocannabinoids were found to

regulate human epidermal differentiation, probably via CB1-dependent mechanisms

.Therefore, local administration of AEA inhibited the differentiation of cultured NHEK

and HaCaT keratinocytes, as evidenced by the transcriptional downregulation of keratin

1, keratin 5, involucrin and transglutaminase-5 and suppression of the formation of

cornified envelopes (Maccarrone et al., 2003b). In addition, activation of the CB

receptors either by phytocannabinoids or these from synthetic sources has been

demonstrated to inhibit the proliferation process in cultured transformed human

epidermal keratinocytes (HPV-16 E6/E7) (Casanova et al., 2003). However, cellular

growth of cultured NHEKs and non-tumorigenic human HaCaT and murine MCA3D

keratinocytes was not altered by synthetic CB1 and CB2 agonists (Casanova et al.,

2003).

AEA and 2-AG have been found in murine epidermal cells (Berdyshev et al., 2000) and

in rodent skin (Calignano et al., 1998). In human skin, AEA and 2-AG were detected in

organ-cultured hair follicles (Telek et al., 2007) and SZ95 sebocytes (Dobrosi et al.,

2008). Endocannabinoids also have been suggested to play a regulatory role in the

human pilosebaceous unit; which control a wide range of the biological functions of the

skin from stem-cell supply through immunomodulation to cytokine production

(Roosterman et al., 2006, Paus et al., 2006a, Paus et al., 2006b). For example;

endocannabinoids might act as a regulators of human hair growth. Consistently with this

idea, CB1 receptor antagonists have been shown to induce hair growth in mice

(Srivastava et al., 2009).

As it was mentioned above endocannabinoids are involved in the regulation of the skin

cells growth in vivo. Local administration of synthetic CB1 and CB2 agonists induce

growth inhibition of malignant skin tumors generated by intradermal inoculation of

tumorigenic PDV.C57 mouse keratinocytes into nude mice (Casanova et al., 2003). This

growth inhibition was accompanied by enhanced intra-tumor apoptosis and impaired

tumor vascularization. Conversely, CB receptors and the related signalling pathways

have been suggested to be involved in the promotion of in vivo skin carcinogenesis

61

(Zheng et al., 2008). By using CB1/CB2 double gene-deficient mice, the study

demonstrated that absence of CB1 and CB2 receptors results in a marked decrease in

UVB-induced skin carcinogenesis. They also found marked attenuation of UVB-induced

activation of MAPKs and nuclear factor-κB was associated with CB1 and CB2 receptors

deficiency (Zheng et al., 2008).

Endocannabinoids exert a protective role in allergic inflammation of the skin (Karsak et

al., 2007). Also the same study found that mice lacking CB1 and CB2 or treated with

antagonists of these receptors exhibited exacerbated allergic inflammatory response

(Karsak et al., 2007). The existence of the endocannabinoid-mediated protection was

also supported by a reduced allergic response in the skin of FAAH-deficient mice that

has increased levels of AEA (Karsak et al., 2007). However, 2-AG levels were increased

in skin with acute and chronic contact dermatitis (Oka et al., 2006) and the symptoms

of skin inflammation were significantly reduced by CB2 antagonists (Oka et al., 2006).

Moreover, the cutaneous inflammation of contact dermatitis was found to be decreased

in CB2-deficient mice (Ueda et al., 2007), and similar suppression of the inflammatory

response by orally administered CB2 antagonists was also observed (Ueda et al., 2007,

Ueda et al., 2005).

Most of these studies focused on the existence of endocannabinoids and their CB1 and

CB2 receptors in human skin cells and the role of these receptors on skin health and

skin disorders. However, the role of these endocannabinoids in skin disorders, in

particular these related to UVR is still waiting to be investigated. Specifically, it would

be interesting to investigate the effects of UVR on endocannabinoid production by

human skin cells. As the n-3 PUFA, EPA and DHA are considered as a dietary

component with a promising photoprotective effect against the deleterious effects of

UVR, the formation of n-3 PUFA, NEA is also interest.

62

1.5. Aims and objectives of the study

Exposure to UV radiation has a range of effects and can cause skin disorders such as

erythema, sunburn, photoaging and photocarcinogenesis (Taylor et al., 1990, Young,

2006). It is clear that the predominant exposure to UVR may be occurs inadvertently

under everyday circumstances (Godar et al., 2003). There are some protection

strategies to save the skin from the harmful effects that may be initiated by UVR during

times of intense exposure (Wingerath et al., 1998). For example, avoidance of sun

exposure, wearing protective clothing or using the topical application of sunscreens.

Although the use of topical protection sunscreens is considered as a part of skin cancer

prevention plans (Moloney et al., 2002), this method of protection may affect the

synthesis of vitamin D in the skin leading to disorders related to vitamin D deficiency

such as reduced bone strength (Moloney et al., 2002). In addition to its action on vitamin

D synthesis, sunscreens should follow standardized methods to apply its protection

effect against UVB. For example, each cm2 of skin should be covered by 2 mg of

sunscreen (Ferguson, 1997). In reality, sunscreens are employed under

nonstandardized conditions, and often topical application may be inadequate to confer

optimal protection against UV radiation (Bech-Thomsen and Wulf, 1992, Krutmann,

2001, Diffey, 1996). Therefore, finding more effective protection approaches is still

required.

Photoprotection by endogenous compounds provided from the diet is became one of

the most promising approach in this field. In general, n-3 PUFA EPA and DHA have

been considered as a dietary component with a promising photoprotective effect against

the deleterious effects of UVR (Black et al., 1992, Rhodes et al., 1994, Rhodes et al.,

1995). They exert their anti-inflammatory effects through competition with AA as

substrates for COXs and LOXs enzymes, resulting in the formation of less active

prostaglandins and leukotrienes (Lands et al., 1992). n-3 PUFA also reduces UVB-

erythemal sensitivity in humans (Rhodes et al., 1994), by inhibition of UVB-induced

PGE2 levels in the skin (Rhodes et al., 1995). N-3 PUFA can also alter the levels of

other PUFA acting as substrates for endocannabinoids and other NAEs (Kozak et al.,

2002a, Kozak et al., 2002b, Kozak et al., 2004). n-3 PUFA therefore, could have an

63

effect on the endocannabinoid pathways in the skin cells and may lead to production of

NAE such as N-stearoylethanolamine (STEA) which has been reported to have pro-

apoptotic activity (van der Stelt et al., 2002) and/or N-palmitoylethanolamine (PEA) that

showed anti-inflammatory activity, analgesic and neuroprotective effects (Calignano et

al., 1998, Smart et al., 2002, LoVerme et al., 2005). Manipulation of the

endocannabinoid pathways through n-3 PUFA supplementation could be the major

defense mechanism behind the protective effect of n-3 PUFA against UVR in the skin

cells. Therefore, we will examine the hypothesis that n-3 PUFA particularly EPA and

DHA may have a biological role in protecting human skin cells against the harmful

effects of UVR through their effect on the formation of endocannabinoid and NAE or

through their effect on the endocannabinoid metabolizing enzymes such as FAAH,

NAPE-PLD, COX-2 and LOX. To address these questions we used two cell lines:

HaCaT keratinocytes and 46BR.1N fibroblasts. Cells will be treated DHA and EPA in

the presence and absence of UVR. AEA, 2-AG, NAE, NAPE-PLD, FAAH, COX-2 and

12-LOX and 15 LOX were investigated in nonirradiated and irradiated cells using

Western blot for protein experiment and LC-MS/MS for lipid analysis. Moreover, through

our collaboration in a clinical study, we will examine the effect of UVR on

endocannabinoids and NAE found in circulation. Therefore, the specific objectives of

this project were:

1. To study the effect of UVR and n-3PUFA on the formation of endocannabinoids

and their congeners in HaCaT keratinocytes and 46BR.1N fibroblasts.

2. To explore the effect of UVR and n-3PUFA on the endocannabinoid

metabolizing enzymes.

3. To assess the effect of UVR on the circulating levels of endocannabinoids and

their congeners through analysis of serum samples.

64

CHAPTER 2: Materials and Methods

2.1. Cell culture and maintenance

2.1.1. Cell lines

The HaCaT human keratinocyte cell line was provided by Dr S. Britland, University of

Bradford and the 46BR.1N human fibroblast cell line was purchased from The European

Collection of Cell Culture (ECACC).

2.2. Cell culture

2.2.1. Materials

Hank’s balanced salt solution (HBSS, Cat No. C-40390) was purchased from Promo

Cell, (Heidelberg, Germany). Dulbecco’s Modified Eagle’s Medium - high glucose

(DMEM, Cat No. D6429-6X500ML), minimum essential medium eagle (MEME, Cat No.

M2279-6X500ML), foetal bovine serum heat inactivated (FBS, Cat No F9665-500ML),

non-essential amino acid solution (Cat No. M7145-100ML), L-glutamine (Cat No.

G7513-100ML), dimethyl sulfoxide (DMSO, Cat No. D2438-50ML), Dulbecco’s

phosphate buffered saline, (Cat No. D8662-6x500ML), 0.4% trypan blue solution (Cat

No.T8154-100ML), sodium pyruvate (Cat No. S8636-100ML), oleic acid (OA; 99%

purity, Cat No. O1008), cis-5,8,11,14,17-eicosapentaenoic acid (EPA; 99% purity, Cat

No. E2011-25MG), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA; 98% purity, Cat

No. D2534-25MG), penicillin (10,000U/ml) streptomycin (10mg/ml) (Cat No. P4333-

100ML), amphotericin B (2.5ng/ml, Cat No. A2942-100ML) and phenol red free trypsin-

EDTA solution (1X), (Cat No 59430C-500ML) were all purchased from Sigma (UK).

0.25% trypsin-EDTA solution (1X) and phenol red (Cat No. 25200072-500ML) were

purchased from Invitrogen (USA). 15ml centrifugation plastic tubes, 50 ml self-standing

centrifugation plastic tubes, T-25, and T-75 culture flasks, 6, 12, and 24 well cell culture

cluster and 96-well microplate were all purchased from Corning, (Amsterdam,

Netherlands). 25ml plastic tubes, Tissue culture dishes (100/20mm) and serological

plastic pipettes 10ml were from Sarstedt (Leicester, UK).

65

2.2.2. Equipment

Microscope: CK40-F200, (Olympus, Japan). UV lamp: UV 236 B, (Waldmann,

Germany). Light meter, (Waldmann, Germany). Centrifuge: Rolofix 32 Ilettich

entrifugen, (Scientific Laboratory supplies, Germany). Shaker: MS1 Minishaker, (IKA-

Labortachnik, Brasil). Incubator: Heraeus CO2 Incubator BBD 622, Thermo Scientific,

UK. Microplate reader: ELx800, (BioTek Instruments), USA. Aspirator: Vacumsafe

confort, (IBS Integra Biosciences, Switzerland) and Thermocouple, Improved Neubauer

haemocytometer, (Gallencamp, UK). Philips HB588 Sunstudio irradiation cabinet

(Eindhoven, The Netherlands), fitted with a combination of 11 Arimed B (Cosmedico

GmbH, Stuttgart, Germany) and 13 Cleo Natural (Philips, Eindhoven, The Netherlands)

fluorescent tubes. Bentham DTM300 spectroradiometer (Bentham, Reading, UK),

Ocean Optics S2000 spectroradiometer (Ocean Optics, Dunedin, FL, USA) and Minolta

CM-2500d hand-held spectrophotometer (Konica Minolta, Tokyo, Japan).

2.2.3. Cell thawing

A cryovial was removed from liquid nitrogen and immediately place in water bath at

37°C for 2 min. When about 80% of the cryovial’s content has thawed, the cells were

pipetted out into a T75 flask, topped up with 8ml of pre-warmed complete medium and

then placed in the incubator. After 24 hours, cells were investigated to insure that they

were attached. At the same time the culture medium was changed to remove any non-

adherent cells, replenish nutrients, and remove any dimethyl sulfoxide (DMSO) residue.

2.2.4. Cell line maintenance

Cells were maintained in 100% humidified incubator at 37oC with 5% (v/v) CO2. All

culturing procedures were carried out in a class II microbiological safety cabinet to

provide a sterile cell culturing condition. HaCaT keratinocytes were cultured in DMEM

medium supplemented with 10% foetal bovine serum (FBS), 1% of the antibiotic

penicillin streptomycin (PS) and 1% of the antifungal amphotericin B. 46BR.1N cells

66

were cultured in MEME medium supplemented with 15% foetal bovine serum (FBS),

1% of non-essential amino acids, 1:100 of sodium pyruvate (100mM), 1:100 of L-

glutamine (200mM), 1% penicillin streptomycin (PS) and 1% amphotericin B. All cells

were grown in T-75 culture flasks with vented caps.

Cells were observed every day under a microscope to ensure that they continued to

grow. When the cells had reached about 90% confluence they were passaged. The

medium was aspirated and 5ml of pre-warmed (37°C) HBSS solution was added. In

order to wash the cells, the HBSS solution was gently swirled inside the flask and after

it discarded, 4 ml of warm (37°C) 0.25% trypsin-EDTA solution was added and the cells

were incubated at 37°C for 15-20 minutes for HaCaT and 2-5 min for 46BR.IN, to detach

them from the wall of the flask. Cells were then transferred into 15 ml centrifuge tube.

The flask was then rinsed with 4-5 ml appropriate complete cell culture medium and

added to the cells in the centrifuge tube to neutralise the trypsin. The tube was spun

down (2,000 rpm, 5 min), the supernatant was removed and replaced by 4 ml of fresh

complete medium. The cells were then re-suspended by gently pipetting the medium up

and down. Then, the cells were counted (section 2.2.8) and an appropriate volume of

cell suspension was transferred into new cell culture flasks or cell culture dishes, as

needed. Fresh medium was added to cover the surface of the flask (8 ml added toT-75

and 10 ml added to100/20 mm Petri dish). All flasks and or dishes were then placed in

the incubator and used for further work.

2.2.5. Cell treatment with fatty acids

All fatty acids (EPA, DHA and OA) were dissolved in DMSO to prepare stock solutions

of 50mM. These solutions were aliquoted to 50µl and stored at -20°C until further use

(appendix 1 section 1.1 – 1.3). Cells were treated with either 10µM or 50µM (2µl or 10µl

from the 50mM stock solution was added to 100/20 mm Petri dish containing of 10ml of

the medium and 70% confluent cells). Control treatment was carried out by adding 10µl

DMSO into 10ml of medium. All cells were then incubated for 72 h before UV irradiation.

67

2.2.6. Cell irradiation

After 72 h incubation period, the medium was removed and the cells were washed with

5ml/dish pre-warmed sterile PBS (37°C) to remove any remaining media and floating

cells. Cells were then covered with a small volume (5ml/dish) of pre-warmed sterile PBS

before the UVR treatment. The UV lamp was switched on and left for 10 min to stabilize.

Light meter was used to measure the irradiance of the lamp (mW/cm2) to make sure

that the lamp had stabilized. Typically, this value was at 0.33 mW/cm2. The cover of

the Petri dish was removed and cells were placed at the base of the UV box, 30 cm

under the UV lamp and irradiated for the required duration. The exposure time was

calculated as follows: Exposure time (sec) = Dose (mJ/cm2)/UVR intensity (mW/cm2).

This corresponds approximately to 45 sec for 15mJ/cm2. After irradiation, PBS was

aspirated, discarded and replaced with 10ml of serum free media. The cells were further

incubated for 24 hours at 37oC with 5% (v/v) CO2.

2.2.7. Collection of cell pellets and culture media

24 pre-labelled sterile 15 ml centrifuge tubes were prepared before starting the

collection of cells pellets and culture media. In each experiment 12 petri dishes were

used and each experiment was performed in duplicate (A and B). Experiment generated

samples were as follows: Control, Control with UVR, Fatty acid at 10 µM, Fatty acid at

10 µM with UVR, Fatty acid at 50 µM and Fatty acid at 50 µM with UVR. The fatty acids

used were either OA or DHA or EPA as indicated in the figure legends.

The culture medium from each dish was collected 24 hours post UVR by a serological

pipette and transferred into sterile pre-labelled tube. The medium was stored at -80°C

for further use. To collect the cells, HaCaT and 46BR.IN were incubated in 4 ml of warm

(37°C) 0.25% trypsin-EDTA solution for 15-20 minutes or 2-5 minutes respectively.

Trypsin was neutralised by adding 4 ml of warm complete medium; the content of the

dish was then transferred to a pre-labelled 15 ml centrifuge tube. The tube was

centrifuged using 2,000 rpm, for 5 min. The supernatant was removed and replaced by

5ml of PBS. The cells were then re-suspended by gently pipetting the PBS up and down.

68

In order to collect and store the cells; the cell suspension was spun down again at 2,000

rpm, for 5 min at room temperature, then the supernatant was removed and the cell

pellet was stored at -80°C to be used for analysis.

2.2.8. Cell counting

In order to count the cells, 100µl of the cell suspension was transferred into an

Eppendroff® tube. A dilution 1:1 was done when the cell suspension was mixed with

100µl of 0.4% trypan blue solution. The haemocytometer slide was cleaned using 70%

ethanol, the face of the slide was moistened with exhaled breath and a glass coverslip

was affixed (Figure 2.1). 10µl of the cell suspension was transferred into the chamber.

The cells were counted in 4 grids per chamber (Figure 2.1) under the microscope, using

the 10x focus. The average cells of 4 grids were calculated and this number was used

to count the total number of cell as follows:

Number of cells/ml = [average cell number] x [dilution factor (2)] x [104/ml]. Eq. (1)

Total number of cells = [Cells/ml] x [volume of original cell suspension (ml)]. Eq. (2)

2.2.9. Cryogenic storage of the cells

Cells were stored in liquid nitrogen following the procedure described below: 4ml of

trypsin-EDTA was added into the culture flask to detach the cells. The detached cells

were pelleted by centrifugation at 2,000 rpm, for 5 min. The supernatant was aspirated

and the cells re-suspended in 2 ml cryogenic solution consisting of 90% (v/v) FBS and

10% DMSO, 1ml of the cell stock was then transferred into each cryogenic vial (Nunc)

and cells were stored in - 80oC for 48 hours as a protective step to minimize damage

and death; before they were transferred into liquid nitrogen (-196oC) for long time

storage.

69

Figure 2.1. Side view of the haemocytometer slide. Adapted from

(www.MicrobeHunter.com).

Figure 2.1. A. The haemocytometer chamber cell. (A) The arrow in the square on the

top right corner indicated how the cells were counted (adapted from Veterinary

Hematology and Cytology, SCIE 19207). (B) Showed how the cells were counted at the

edge of each square. Adapted from (www.pheculturecollections.org.uk).

2.3. Western blotting

2.3.1 Chemicals

Bromophenol blue (Cat No. B5525), Tween® 20 (Cat No. P1379), phosphate buffered

saline tablets (Cat No. P4417), ethylenediaminetetraacetic acid disodium salt dehydrate

(EDTA) (Cat No. E5513), glycerol (Cat No. G8773), Sodium dodecyl sulfate (Cat No.

70

L3771), Trizma® base (Cat No. T4661), acetic acid (Cat No. 34245), glycine (Cat No.

G8898), methanol (Cat No. 494437), ammonium persulfate (Cat No. A3678), TEMED

(Cat No. T9281), Fast Green FCF (Cat No. F7252), ponceau S solution (Cat No. P7170-

1L), protease inhibitor cocktail (PIC) (Cat No. P8340), Coomassie® brilliant blue (Cat

No. B0149), Luminol (Cat No. 123072), P-Coumaric acid (Cat No. C9008), GBX fixer

(Cat No. P7167 – 5GA), and GBX developer (cat No. P7042 - 5GA) all were obtained

from Sigma (UK). Novex® ECL HRP chemilluminescent substrate reagents 2x125 ml

(Cat No. WP 20005) was from Invitrogen (UK). AcrylaFLOWGel 30% (Cat No. H17344)

and BisacrylaFLOWGel 2% (Cat No. H17356) were supplied by Flowgen Bioscience

(UK). DC Protein Assay Reagents (Cat No. 500-0116), lyophilized bovine serum (Cat

No. 500-0007), dithiothreitol (DTT) (Cat No. 161-0611), 10x Tris/glycine buffer (Cat No.

161-0734-1L) and 10x Tris/glycine/SDS buffer (Cat No. 161-0772-5L) were from BioRad

(USA).

2.3.2. Primary and Secondary Antibodies

COX-2 (human) polyclonal antibody (72KDa), from rabbit (Cat No. 160107), COX-2

(mouse) monoclonal antibody (72KDa), (Cat No. 160112), Fatty Acid Amide Hydrolase

polyclonal antibody, from rabbit (63KDa), (Cat No. 101600), NAPE-PLD (6-20)

polyclonal antibody (46KDa), from rabbit (Cat No. 10306), Fatty Acid Amide Hydrolase

Western Ready Control (Cat No. 10010182) and COX-2 (human) Western Ready

Control (Cat No. 10009624) were obtained from Cayman (USA). Anti-biotin, HRP-linked

Antibody from goat (Cat No. 7075S) and Biotinylated Protein Ladder Detection (Cat No.

7727S) were from Cell Signaling (USA). Anti-GAPDH (6C5) mouse monoclonal

antibody (Cat No. AB8245) and brain: cerebellum (right) (Human) whole cell lysate -

adult normal tissue (Cat No. AB30069) were provided by AbCam (UK). ECL Rabbit

IgG, HRP-Linked Whole Ab (from donkey) (Cat No. NA934) and ECL Mouse IgG, HRP-

Linked Whole Ab (from sheep) (Cat No. NA931) were from GE Healthcare (UK).

71

2.3.3. Various consumables and small items

Syringes PP/EP with removable needle (Cat No. Z116890), Whatman® filter paper (Cat

No. Z146382), Parafilm® M (Cat No. P7793), exposure cassette Kodak® BioMax™ (Cat

No. C4729) and Kodak® X-Omat LS film - size 5 in. (13 cm) × 7 in. (18 cm) (Cat No.

F1274-50EA) were from Sigma (UK). Immobilon Blottimg Filter Paper, 7x8.4 cm sheet

(Cat No. IBFP0785C) and Immobilon-P Membrane, PVDF, 0.45 µm, 15 x 15 cm sheet

(Cat No. IPVH15150) were purchased from Millipore (USA). Mini incubation trays (Cat

No. 170-3902) were from BioRad. Cassettes, 1.0 mm (Cat No. NC2010), Combs, 1.0

mm 10 well (Cat No. NC3010), Invitrolon™ PVDF⁄Filter Paper Sandwiches (Cat No.

LC2005) and Xcell SureLock Mini-Cell mark (Cat No. EI0002) were purchased from

Invitrogen (USA). Mini-PROTEAN® TGX precast gels 10% and 10-well comb, 50µl/well

(Cat No. 456-1034) were from BioRad (USA).

2.4. Sample preparation

2.4.1. Protein extraction

Cell pellets were defrosted in ice and then re-suspended initially in 200µl of ice cold

freshly prepared sample buffer (Table 2.1). (Appendix 3 section 3.1.5). 1:100 ice cold

protease inhibitor cocktail (PIC), that contained 4-(2-aminoethyl) benzenesulfonyl

fluoride (AEBSF), pepstatinA, E-64, bestatin, leupeptin and aprotinin, was then added

and the suspension was mixed and kept in ice for 10 minutes. The suspension was

homogenized by mixing repeatedly using a 5 ml syringe for 10 times to lyse cellular

membranes. If foam was produced during lysis the suspension was centrifuged 3 times

to reduce it. The suspension was then incubated on ice for 15 minutes, and then mixed

and centrifuged at 13,000 rpm for 5 min. The resulting supernatant that contained the

cellular protein was transferred to a new Eppendrof tube and kept on ice waiting for the

protein assay to be done or kept in -20 oC for further use.

72

Table 2.1: Composition of the sample buffer used for protein extraction.

Ingredients Concentration Final volume or weight

Glycerol 10% 2.5ml

EDTA 0.02M 0.19g

SDS 6% 1.5g

Tris base 62.4mM 0.19g

dH2O 25ml

2.4.2. Determination of protein concentration

The measurement of the protein concentration was carried out using the BIO RAD DC

protein assay kit. This colorimetric assay for the protein concentration is based on the

Lowry assay (Lowry et al., 1951). The protein reacts with alkaline copper solution

(reagent A) and then reacts with folin reagent (reagent B) and forms a blue colour with

maximum absorbance at 750nm and minimum at 450nm.

A calibration line (Figure 2.4) was constructed using six dilutions of bovine serum

albumin (BSA) (0.1, 0.2, 0.4, 0.6, 0.8 and 1 (mg/ml)) (appendix 2 section 2.1). As they

left for the blank, 5µl of the sample buffer (appendix 3 section 3.1.5) were added into

the first three wells of the 96-well microplate (Figure 2.2). Then, 5µl of each BSA

dilutions were added in triplicate into the 96-well microplate (Figure 2.3). Three dilutions

(1:2, 1:5 and 1:10) were prepared from each test sample. 1ml of reagent A was mixed

with 20µl of the surfactant solution (Reagent S) before adding it into every well of a 96-

well microplate. Then, 25µl and 200µl of reagent A and B were also added to each well.

The microplate was then covered with foil and left for 15 min at room temperature. The

absorbance was read at 650nm using a microplate reader, run by the genesis software

version 2 (appendix 2 section 2.2). The protein content was calculated based on the

equation corresponding to the calibration curve and the concentration was estimated

for all test samples (appendix 2 section 2.3).

73

Figure 2.2: 96-wells microplate (Adapted from www. Edgebio.com)

Figure 2.3: Template showing how the samples were loaded into the 96-wells

microplate

Figure 2.4: typical calibration line plotted from BSA standards

74

2.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Electrophoresis is a system used to separate large molecules, for example, proteins or

DNA which as charged atoms can be determined in an electric field as indicated by their

atomic weight. Protein movement in the gel relies on upon its charge and atomic weight.

Additionally, protein mobility dependent on the pH of the medium encompassing the

protein and on the gel itself. The movement rate of the proteins is also dependent on

gel concentration.

Figure 2.5: Schematic representation of western blotting and detection

procedure (Adapted from http://missinglink.ucsf.edu/lm/molecularmethods).

2.5.1 SDS-PAGE: Gel preparation

The lower resolving gel was prepared as described in the Table 2.2; (appendix 3 section

3.1.3). Gel was poured into a disposable gel cassette approximately 1 mm above the

first division line. The gel was poured slowly into the cassette to avoid formation of air

bubbles. In order to prevent oxygenation of the stacking gel during the polymerization,

300-500 µl of distilled water was added to the top of the gel. Polymerization of the lower

separating gel occurred in about 30-45 minutes at room temperature. During this time

the upper stacking gel was made (appendix 3 section 3.1.3) (without adding 10%

75

ammonium persulfate, (APS). When the lower gel polymerization was completed

(usually after 30 - 40 minutes), the distilled water was removed and 10% APS was

added to the upper stacking gel which applied on top of the solidified resolving gel.

1.5mm well combs were inserted into the gel cast for the formation of loading wells in

the gel. The upper stacking gel was then left for about 15-20 min for solidification. SDS-

PAGE was performed in 8% gel as described below in Table 2.2.

Table 2.2 Reagents used for the preparation of the acrylamide gel.

Reagents Separating (lower) gel 8%

Stacking (upper) gel 4.5%

Acrylamide 7.8 ml 2.2 ml

Bis-Acrylamide 3.12 ml 900 µl

Gel-Tris buffer 7.5 ml 3.75 ml

dH2O 11.6 ml 8.15 ml

ΤΕΜΕD 30 µl 30 µl

10% APS 100 µl 90 µl

2.5.2 SDS-PAGE: Gel electrophoresis

When preparing the SDS-PAGE, the samples to be analysed were denatured. SDS

loading buffer was added to the cell lysates and then boiled to unfolding protein into

primary (linear) form (Figure 3.7). SDS is an anionic detergent responsible only for the

disruption of non-covalent bonds in the proteins. Therefore the reducing agent;

dithiothreitol (DTT) was added to disrupt the disulfide covalent bonds which were not

affected by SDS (Figure 3.7). SDS also, confers negative charge in protein samples to

allow protein migration during SDS-PAGE. DTT was added at a concentration of 50mM;

an equal volume was added to the protein sample in 1:1 dilution. 0.08% of the tracking

agent bromophenol blue was then added to the protein sample and the mixture was

boiled for 5 min. The molecular weight markers were boiled at the same time for 3 min.

A biotinylated protein ladder was used as molecular weight marker on Western blots

when using the horseradish peroxidase (HRP). The molecular weight ladders are a

mixture of purified proteins covalently coupled to biotin that resolve to 10 bands that

have a size range of 9-200 kDa. The electrophoresis chamber was then assembled and

the running buffer was made (appendix 3 section 3.1.1). The comb was removed after

the gel was polymerized. The gel cassette was placed into the electrophoresis chamber

76

the tank was filled with running buffer and the protein samples were loaded into the

wells on the gel. Electrophoresis was carried out at a constant voltage of 125V for

approximately 90 minutes or until the bromophenol blue stain reached the end of the

cassette. The electrophoresis chamber was then assembled and the running buffer was

prepared (appendix 3 section 3.1.1). The comb was removed after the gel was

polymerized. The gel cassette was placed into the electrophoresis chamber the tank

was filled with running buffer and the protein samples were loaded into the wells on the

gel.

Figure 2.6: Protein denaturation using heat and SDS; circles indicate that the fold

caused by disulfide covalent bonds were not affected by heating and SDS. DTT

eliminated any tertiary or quaternary structures. Adapted from (www.ruf.rice.edu).

2.5.3. Wet transblotting

The PVDF membrane was activated in 100% methanol for 30 seconds and rinsed with

distilled water for about 2 min until hydrophobicity (apparent by beading of water on the

surface) disappears. In order to equilibrate the membrane, the membrane was

incubated in 15ml of the transblot buffer (appendix 3 section 3.1.2) for about 5 min.

When the electrophoresis was completed, the gel cassette was removed from the

electrophoresis tank, opened and the upper stacking gel was removed. The lower gel

was placed in a blotting module (Figure 2.7) which allowed the protein in the gel to be

transferred to the PVDF membrane; the position of the PVDF membrane inside the

blotting module is shown in Figure 2.8 A and B. The blotting module was then placed in

77

the electrophoresis chamber and run at 100 V for 2 hours. When the transblotting

finished, the PVDF membrane was placed in a plastic square dish containing fast green

stain (appendix 3 section 3.1.8) for about 2 min, after that the stain was poured off and

destain was added (appendix 3 section 3.1.7). When green bands were apparent on

white background, destain was removed. Next, the membrane was placed in another

plastic dish containing PBS and left for 2 min to be washed.

2.5.4. Blocking and Immunoblotting

The membrane was replaced in 5% non-fat milk in PBS for 1 hour at room temperature

to reduce non-specific antibody binding on non-target proteins. The milk solution was

changed after 30 min. Finally, the membrane was washed with PBS for 3 times and

submitted to primary antibody incubation overnight in the cold room under agitation.

After the incubation with the primary antibody; the membranes were washed with PBST

for 3 time 10 min each and 1 time with PBS for 5 min on a shaker. After that, the

membrane was incubated with the secondary antibody for 2 hours at room temperature

under agitation; the biotinylated protein ladder was added one hour later. The

membrane was then washed again as before to prepare for the protein visualization

step.

78

Figure 2.7: Diagram showing the position of the blotting pads inside the blotting

module during the transblotting process (Adapted from Invitrogen user manual).

Figure 2.8: Diagram showing the position of PVDF membrane in the blotting module. A

and B show the single and double membrane transblotting process respectively

(Adapted from; Invitrogen user manual).

79

2.5.5. Protein visualization

After the final washing step, the membrane was placed in a square dish filled with PBS.

PBS was then poured off; the membrane was dried by soft tissue or filter paper and

transferred into another clean square dish. 10 ml of freshly made ECL solution

(Appendix 3 section 3.1.9) was added into the clean dish covered with foil and left for 5

min at room temperature allowing the reaction between the ECL and HRP antibody

(Figure 2.9). The ECL solution was decanted and the PVDF membrane was removed

and dried off any excess moisture using tissue paper with the membrane’s protein side

up. Then the membrane was placed with the protein side down onto wrinkle free cling

film which was then folded over carefully. The cling film was cut and the membrane was

transferred to the exposure cassette with the protein side up and the exposure cassette

closed.

The exposure cassette was opened in the dark room; a photographic film was placed

on the top of the wrapped membrane; then the exposure cassette was closed and some

weights put on it for 5 min. During that time, the developing (Developer 50 ml in 200 ml

dH2O) and fixing (fixer 50 ml in 200 ml dH2O) solutions were prepared. After exposing

the film for the required time the cassette was opened and the film was bended at the

top left hand corner. The film was then placed in the developing solution tray until black

bands appeared (this was visualized by holding up to the light). Subsequently, the film

was put in the H2O tray with gently agitation for 2-3 min. Then, the film was placed in

the fixing solution tray with gently agitation for 2-3 min, it was then rinsed under tap

water to remove any residue. Finally, the film was left for 10 min to dry either in the

drying cabinet or standing up vertically on the bench.

2.5.6. Membrane stripping and re-probing

The PVDF membrane was incubated in a square petri dish with mild stripping buffer

(appendix 4 section 4.1.1) for 10 min at room temperature. The incubation was repeated

for another 10 min with fresh stripping buffer and the membrane was then washed with

PBS for 10 min 2 times, at room temperature and 2 times with PBS containing 0.05%

80

Tween 20, for 5 min each. Finally, the membrane was incubated with PBS only for 5

min before starting the blocking step (appendix 4 section 4.2).

Figure 2.9: schematic summarizing the protein visualization process. Adapted from

(www.gelifesciences.com)

2.6. Densitometry analysis

Densitometric analysis of Western blots (Schmidt et al., 1987, Gassmann et al., 2009)

was carried out using ImageJ software (Schneider et al., 2012). The intensity of the

protein band in each treatment was normalized to that of the corresponding GAPDH

protein band under the same conditions. The ratio of the intensity of the protein band

divided with the intensity of the GAPDH band in the untreated cells was arbitrarily set to

1. The ratio of the intensity of the protein versus the intensity of the GAPDH band in the

remaining samples was calculated accordingly.

2.7. Antibody optimization

2.7.1. COX-2 and GAPDH Antibodies

Two different concentrations of the antibodies against COX-2 and GAPDH were used

as shown in Figure 2.10. Cellular extract from the fibroblasts cells 46 BR.1N containing

300 µg protein subjected to SDS-PAGE and Western blot analysis as described above.

81

The PVDF membrane was then cut into three strips. The membrane was incubated

overnight with primary antibodies of COX-2 and GAPDH in the cold room followed by

incubation with the secondary antibodies for 2 hours at room temperature. The

biotinylated protein ladder was added one hour later. Figure 2.11 shows that the loading

control GAPDH (36 kDa) appeared clearly in strip 1 with dilution of 1:10,000 indicating

that further dilution was necessary and the exposure time (5 min) had to be reduced.

The COX-2 band (72 kDa) at a dilution of 1:200 shown in strip 2 was better than that in

strip 3 incubated with the primary antibody at dilution of 1:400.

After several experiments, the optimal dilution for the GAPDH antibody was found to be

1:30,000. Also, good quality of bands was achieved when the imaging time was reduced

from 5 min to 30 – 60 sec. However, the optimal dilution of COX-2 antibody was 1:200

and the bands required 3 to 5 min exposure time to be seen clearly. Moreover, all the

secondary antibodies used in this study were used at dilution range 1:500 – 1:2000.

Figure 2.10: Optimization of COX-2 and GAPDH antibodies. Aumont of protein was

300 µg per gel. (A) Showed GAPDH band after the PVDF membrane was incubated

with GAPDH (Mouse) monoclonal antibody at dilution of 1:10,000. (B) Showed COX-2

band after the membrane was incubated with COX-2 (Human) polyclonal antibody at

dilution of 1:200. (C) Showed COX-2 band after the membrane was incubated with

COX-2 (Human) polyclonal antibody at dilution of 1:400. HRP- (Anti-Mouse) 1:1500,

HRP- (Anti-Rabbit) at dilution of 1:1500.

82

2.7.2. Optimization of 12-Lipoxygenase (murine leukocyte) Polyclonal Antiserum

Cells from the fibroblastic cell lines 46 BR.1N were suspended in the lysis buffer (10%

Glycerol, 0.02M EDTA, 62.4mM Tris base, 6% SDS and dH2O) and the protein was

extracted as described in section 2.5.1 The protein obtained content was diluted to

2µg/µl. In order to have 30 µg/well, 15 µl of this solution were loaded into each well.

After transblotting (section 2.6.2), the PVDF membrane was blocked and then

incubated with the primary antibodies of 12-LOX overnight in the cold room. This was

followed by incubation with the secondary antibodies in the next day for 2 hours at

room temperature. The biotinylated protein ladder was added one hour later. This

experiment indicated that a dilution 1:1000 of 12-LOX antibody was slightly high but

still acceptable.

Figure 2.11: Optimization of 12-LOX (murine leukocyte) Polyclonal antiserum.

Dilution = 1:1000. HRP (Anti rabbit) secondary antibody, dilution = 1:1000. 12-LOX

expression after OA treatment in 46BR.IN fibroblasts in the presence and absence of

UVR. Protein dilution was 30 µg/well.

83

2. 8. Analysis of endocannabinoids by LC-MS/MS

2.8.1. Materials

Acetonitrile (HPLC grade), methanol (HPLC grade), chloroform (HPLC grade) and

glacial acetic acid (HPLC grade), all were purchased from, Fisher scientific,

Loughborough, UK. 50, 100, 250 and 500 µl glass syringes were purchased from SGE,

Australia. Glass tubes and glass Pasteur pipettes were purchased from Fisher,

Loughborough, UK. Amber glass vial, 100 µl insert vials, crew caps, and septa all were

purchased from Kinesis, Bedfordshire, UK.

2.8.2. Experimental description of LC-MS/MS analysis

Each pellet and medium from HaCaT keratinocytes and 46BR.IN fibroblasts was given

a code. The purpose of this was to blind the running of the samples in LC-MS/MS

analysis. Pellets and medium represent the same experimental design described in

section 2.2.7.

All endocannabinoids were analysed by multiple reaction monitoring (MRM) in the

positive ionization mode (ES+). Quantification is based on the construction of calibration

lines using commercially available standards and deuterated internal standards (AEA-

d8 and 2-AG-d8).

In this experiment the following compounds were analysed: AEA, 2-AG, PEA, ALEA,

LEA, OEA, STEA, DHEA, MEA, PDEA, POEA, HEA, DGLEA, DPEA, DEA, NEA, LGEA

and 1-AG. Chloroform/methanol 2:1(v/v) mixture (appendix 5 section 5.1) was added

into the cell pellets or the medium to extract polar and non-polar compounds. Water was

then added to form the bilayer phase, where the lipids in the lower chloroform layer and

the non-lipid components in the top methanol/water layer. Chloroform was removed by

evaporation under nitrogen (N2) and the lipid residue was then reconstituted in 100µl

ethanol (HPLC grade). Finally, the extracts were stored at -20 ºC before LC/ESI-MS/MS

analysis.

84

2.8.3. Extraction Protocol

All samples (pellets or culture medium) were defrosted in ice before start. 3 ml of ice

cold chloroform (CHCl3): methanol (MeOH) (2:1v/v all HPLC grade) (appendix 5 section

5.1) was added to the cell pellets and mixed well. However, 2 ml of the treated medium

were mixed with 9 ml of ice cold chloroform (CHCl3): methanol (MeOH). After that, 20ng

(20µl x 1ng/µl) of the internal standards anandamide-d8 (AEA-d8) and 40ng (40µl x

1ng/µl) 2-Arachidonoyl glycerol-d8 (2-AG-d8) (appendix 5 section 5.2) were added to

each sample (pellet or medium) using a Hamilton glass syringe. The syringe was wiped

with a clean tissue in between addition of the standard to the sample and refilling the

syringe sample. The samples were vortexed and left on ice for 30 min. During that time

the refrigerated centrifuge was switch on to cool down to 4 oC and the centrifuge was

set to 5 x 1000 RPM. 0.5 ml of Mill Q water was added to each cell pellets; but not to

the medium, to generate the two phase layers. The sample was mixed well and

centrifuged for 5 min at 4 oC, 5000 RPM.

The bottom organic phase was transferred into a clean glass 12ml vial using a glass

Pasteur pipette. After that the organic phase was dried down under nitrogen and the

extract reconstituted in 100µl of ethanol (HPLC grade) using a Hamilton glass syringe.

The syringe was wiped with a clean tissue in between addition. The ethanol was dripped

around the inside of the tube to collect dried lipids. Then, the collection was mixed well

and centrifuged for 10 seconds. At that point all the extracts were transferred to an insert

in a labelled amber vial with septa and lid. A parafilm was wrapped around the lid to

stop evaporation of solvent and stored on ice until finished. The syringe was washed

with 3x ethanol washes 1 and 2 and the extracts were kept in -20 oC while awaiting LC-

MS/MS analysis.

2.8.4. LC-MS/MS analysis of endocannabinoids and NAE

AEA, 2-AG, PEA, ALEA, LEA, OEA, STEA, DHEA, MEA, PDEA, POEA, HEA, DGLEA,

DPEA, DEA, NEA, LGEA and 1-AG were separated using an isocratic system

composed of two solvents (A and B) mixed at constant ratio of 30:70 (v/v).

85

Solvent A was acetonitrile: water: acetic acid, 2:98:0.5 (v/v/v) and solvent B was

acetonitrile: water: acetic acid, 98: 2: 0.5 (v/v/v).

The run time was set at 69 min. Separation was performed on a C18 (Luna 5µl, 150 x

2.0 mm) column. The injection volume was 3µl for standards and the biological extracts.

Details of MRM transitions, as well as indicative retention times, for 12

endocannabinoids and their congeners were shown in Table 2.3. The instrument is

operated in the positive ionisation mode, and for all compounds, the MS/MS settings

are as follows: capillary voltage 4500 V, source temperature 100 ºC, desolvation

temperature 400 ºC, cone voltage 35 Dwell time 0.2 s. The transitions should be split at

10 min so that those that are eluted before 10 min and those that are eluted after 10

min have a more focussed analysis. LC/ESI-MS/MS analysis was performed on a

Waters Alliance 2695 HPLC pump with a Waters 2690 autosampler coupled to an

electrospray ionisation (ESI) triple quadruple Quattro Ultimo mass spectrometer. The

Mass LynxTM V 4.0 was used as operating software to control the instrument and data

acquisition.

Table 2.3 multiple reaction monitoring (MRM) transitions for the LC/ESI-MS/MS

assay of endocannabinoids and their congeners

Compound MRM Collision energy (eV)

Retention time* (min)

PEA 300>62 13 32.57

ALEA 322>62 14 20.53

LEA 324>62 15 25.89

OEA 326>62 16 38.92

STEA 328>62 15 51.46

AEA 348>62 15 26.30

AEA-d8 356>63 16 25.92

DHEA 372>62 15 25.51

2-AG 379>287 18 35.01

2-AG-d8 387>295 20 34.13

1-AG 379>287 18 37.63

MEA 272>62 12 4.76

PDEA 286>62 12 5.42

POEA 298>62 14 5.07

HEA >62 12 7.27

DGLEA 350>62 14 6.11

DPEA 374>62 14 5.79

86

2.8.5. Standards for quantification and calibration lines

The following standards were used to generate calibration lines: AEA, 2-AG, PEA,

ALEA, LEA, OEA, STEA, DHEA, MEA, PDEA, POEA, HEA, DGLEA, DPEA, DEA, NEA,

LGEA and 1-AG (appendix 5 section 5.3). These calibration lines were used to calculate

the quantity of each one of the endocannabinoid compound of interest. At least 5

concentrations each calibrate should be used for each calibration line and

concentrations ranged from 1-200 pg/µl (Table 2.5). However, the range of these

calibration lines can be adjusted depending on the amount of compound found in the

samples. Standards are made up in amber vials with 100 µl inserts, sealed with Parafilm

and stored at -20 ºC until analysis. By integrating the chromatograms from each

standard concentration, the area under the peak for each compound from the first and

second injections can be determined. Additionally, as each injection also contains an

internal standard representative of each type of endocannabinoid, these peak-area

values can be normalized as a peak-area to internal standard ratio. The mean peak-

area to internal standard ratio for each concentration of standard can be used to

construct a calibration line, which can be analyzed by the least-squares linear

regression method to calculate its gradient.

Table 2.4: Guidelines for the generation of standards of different concentrations

to be used to construct an endocannabinoid calibration line

Final concentration (pg/µl)

200

160

120

100

80 50 40 20 10 4 2 1

Ethanol (µl) 10 20 30 35 40 47.5

50 55 57.5

59 59.5

59.75

400 pg/µl cocktail (µl)

50 40 30 25 20 12.5

10 5 2.5 1 0.5 0.25

1 ng/µl 2-AG-d8 (µl)

40 40 40 40 40 40 40 40 40 40 40 40

1 ng/µl AEA-d8 (µl)

40 40 40 40 40 40 40 40 40 40 40 40

Final volume (µl)

100*

100*

100*

100*

100*

100*

100*

100*

100*

100*

100*

100*

*Final volume does not include 40 µl of 1 ng/µl 2-AG-d8 as this internal standard is dried

down before the addition of other reagents, so it does not contribute to the final volume.

87

2.9 Clinical study

The clinical study was conducted by the group of Professor LE Rhodes at the Salford

Royal Photodermatology Unit. All samples were collected by Sarah Felton (OXFORD

UNIVERSITY HOSPITALS NHS TRUST). This work is part of a research collaboration

between the two labs.

2.9.1. Experimental design

Prof LE Rhodes and Dr Sarah Felton designed and undertook this study. Sixteen

healthy volunteers aged 23-59 years from Greater Manchester, UK, were engaged in

this study: 10 white Caucasians of skin phototype II and 6 South Asians of skin

phototype V. Subjects were excluded from participation if pregnant or breastfeeding,

taking vitamin D supplements or photoactive medication, if they had a personal history

of skin cancer, photosensitivity or systemic lupus erythematosus, or if they had used a

sunbed/sunbathed in the 3 months prior to commencement of the study, or during the

study period. All received and read the study information leaflets at least a week prior

to their decision to take part. They subsequently attended for an initial assessment visit

where the study was discussed and they were given the opportunity to ask questions

before providing their consent to participate. The Tables (2.5 and 2.6) below illustrates

the time points of UV radiation and blood samples collection throughout the study.

Volunteers were given short, sub-erythemal exposures to UV radiation three times

weekly for 6 weeks, whilst wearing informal summer clothing.

88

WEEK 1 WEEK2 WEEK3 WEEK4 WEEK5 WEEK6

UVR BS UVR BS UVR BS UVR BS UVR BS UVR BS

MONDAY - + + - + - + - + - + -

TUESDAY - - - - - - - - - - - -

WEDNESDAY + + + - + - + - + - + -

THURSDAY - - - - - - - - - - - -

FRIDAY + + + + + + + + + + + +

Table 2.5. Schematic pattern showing the multiple time points of UVR exposure and

blood sample (BS) collection days

WEEK 1 WEEK2 WEEK3 WEEK4 WEEK5 WEEK6

UVR BS UVR BS UVR BS UVR BS UVR BS UVR BS

MONDAY - D 0 + D 7 + D 14 + D 21 + D 28 + D 35

TUESDAY - - - - - - - - - - - -

WEDNESDAY + D 2 + - + - + - + - + -

THURSDAY - - - - - - - - - - - -

FRIDAY + D 4 + - + - + - + - + D 39

Table 2.6. Sample code: The highlighted days showing the days of UVR exposure

and/or blood sample collection

2.9.2. Simulated summer sunlight exposures

A Philips HB588 irradiation cabinet (Figure 2.13) was used to deliver whole body UV

exposure. This cabinet was fitted with fluorescent tubes in an alternating pattern, to

provide an UV emission spectrum as close as possible to summer sunlight (95% UVA:

320–400 nm, 5% UVB: 290–320 nm). Emission from the radiation cabinet was

characterised using a Bentham DTM300 spectroradiometer and monitored using an

Ocean Optics S2000 spectroradiometer by Dr R. Kift (School of Earth, Atmospheric and

Environmental Sciences, University of Manchester).

89

Figure 2.12: Philips HB588 cabinet. Adapted from (www.philips.com). December, 2014.

2.9.3. Irradiation Protocol

Wearing protective eye goggles, standardised T-shirts and knee-length shorts,

volunteers lay prone (i.e. face down) on the sunbed with the canopy closed.

Approximately 35% of their skin surface area was exposed. A 6-week course of

exposures was selected to be concordant with the length of the summer school holiday

period when the population is most exposed to sunlight. The course of simulated

summer sunlight was given three times a week in January and February when ambient

UVB is negligible at UK latitudes and people have their lowest vitamin D status (Webb

and Engelsen, 2006). An exposure of 1.35 standard erythemal dose (SED) (Diffey et

al., 1997) was given to each subject at every visit. The time required to deliver this dose

was found to be 6.5 min after accurate measurement of cabinet UV irradiance (Taylor

et al., 2002); a constant UV dose was maintained throughout the study by adjusting for

any decrease in irradiance by increasing delivery time.

2.9.4. Skin colour measurements

All participants had assessments of their buttock skin colour non-invasively at baseline

(i.e. prior to sunbed exposure) using a Minolta CM-2500d hand-held spectrophotometer

(Figure 2.14). Readings were taken when the volunteers had been placed prone for 3

min prior to their irradiation, to eliminate heat from the sunbed and postural variations

http://www.philips.com/

90

in circulation as confounding factors. UV-exposed and UV-protected buttock skin

subsequently underwent weekly spectrophotometric measurements. Data were

recorded in standard three-dimensional L*A*B* format, as recommended by the

Commission International de le’Eclairage (Robertson, 1978). L*represents white-black

differentiation, whereby an L* of 100 is pure white and conversely L* of 0 is pure black,

A* values reflect the balance between green (negative) and increasing redness

(positive) whilst B* represents the differentiation between blue (negative) and yellow

(positive). The spectrophotometer was ‘Zero calibrated’ at study commencement by Dr

D Allan, in addition to being calibrated using its ‘White Calibration’ system each time the

power was turned on prior to each participant’s measurements, to guarantee accuracy

of readings. Readings were made in triplicate at each site and the mean calculated.

Figure 2.13: Spectrophotometer. Adapted from. (www.konicaminolta.com). December,

2014.

2.9.5. Blood collection and serum separation

Each volunteer had a venous blood sample taken at baseline for measurement of

endocannabinoids (anandamide and 2-arachidonoyl glycerol). Samples were repeated

at the beginning of each week of the study. Samples were centrifuged in a centrifuge

at 2400rpm for 15 minutes and once the serum had separated it was aspirated using a

pipette and stored in a glass sample vial in a -20°C freezer. The samples were then

transferred to the University of Manchester on dry ice and stored at -80°C awaiting LC-

MS/MS analysis.

http://www.konicaminolta.com/

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2.9.6. Lipid Extraction

The total number was 153 serum samples, 147 samples had the same volume (400µl),

and the other 16 samples were in the range from 350µl to 1000µl. All the samples were

defrosted in ice. 3 ml of ice cold chloroform (CHCl3): methanol (MeOH) (2:1v/v all HPLC

grade) (appendix 5 section 5.1) was added to the serum and mixed well. After that, the

whole volume of each sample was transferred into a new 12 ml glass tube. 20ng (20µl

x 1ng/µl), 20µl of the internal standards Anandamide-d8 (AEA-d8) and 40ng (40µl x

1ng/µl), 40µl of 2-Arachidonoyl glycerol-d8 (2-AG-d8) (appendix 5 section 5.2) was

added to each sample using a Hamilton glass syringe. The syringe was wiped with a

clean tissue in between addition of the standard to the sample and refilling the syringe

sample. The samples were mixed well and left on ice for 30 min (Samples were mixed

each 10 min). During that time the refrigerated centrifuge was switch on to cool down to

4 oC and the centrifuge was set to 5 x 1000 RPM. 0.5 ml or 1 ml of Mill Q water was

added to each sample, to generate the two phase layers. The samples were mixed well

and centrifuged for 5 min at 4 oC, 5000 RPM. The bottom organic phase was transferred

into a clean glass 12ml vial using a glass Pasteur pipette. After that the organic phase

was dried down under nitrogen and the extract reconstituted in 100µl of ethanol (HPLC

grade) using a Hamilton glass syringe. The syringe was wiped with a clean tissue in

between addition. The ethanol was dripped around the inside of the tube to collect dried

lipids. Then, the collection was mixed well and centrifuged for 10 seconds. At that point

all the extracts were transferred to an insert in a labelled amber vial with septa and lid.

A parafilm was wrapped around the lid to stop evaporation of solvent and stored on ice

until finished. The syringe was washed with 3x ethanol washes 1 and 2 and the extracts

were kept in -20 oC awaiting for LC-MS/MS analysis as described in sections 2.8.4 and

2.8.5.

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2.10. Statistical Analysis

Data are presented as mean ± standard deviation where appropriate. In order to

determine that our data are parametric or non-parametric, all the data were subjected

to Shapiro-wik’s test (P>0.05) and their skewness and standard error were measured.

The histogram of all the data was inspected visually. Regarding to the Z value (which

was calculated manually by dividing the skewness value on the standard error), some

of our data have a small skew but it does not differ significantly from normality.

Therefore, it was assumed that the data are approximately normally distributed.

Comparisons between groups in chapters 3, 4 and 5 were made with one-way analysis

of variance (ANOVA) followed by Tukey post hoc test. In chapter 5 the comparison was

carried out between the different time points of UVR exposure in each skin phototype

separately. All statistical analyses were done using SPSS Statistics software version

22. P < 0.05 was considered to be a significant level of difference.

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CHAPTER 3: The effect of n-3 PUFA on the formation of endocannabinoids and N-acylethanolamines in HaCaT keratinocytes and 46BR.IN fibroblasts in response to ultraviolet radiation

3.1 Introduction

The endocannabinoid system has been identified in different types of skin cells and

tissues as reported in several studies (Mechoulam et al., 1998; Howlett et al., 2002;

Pacher et al., 2006; Di Marzo, 2008). These studies reported the existence of AEA and

2-AG, in human skin along with the enzymes that are involved in their synthesis and

metabolism (Calignano et al., 1998; Berdyshev et al., 2000; Maccarrone et al., 2003;

Karsak et al., 2007). Moreover, the cannabinoid receptors CB1 and CB2 were identified

in many types of human and murine skin cells such as organ-cultured hair follicles, SZ95

sebocytes, cutaneous nerve fibres, mast cells and epidermal keratinocytes (Ständer et

al., 2005, Ibrahim et al., 2005, Blázquez et al., 2006, Telek et al., 2007b, Telek et al.,

2007a, Casanova et al., 2003). Endocannabinoids are also involved in the regulation of

human epidermal homeostasis. AEA, for instance has been found to inhibit the

differentiation of cultured normal human epidermal keratinocytes (NHEKs) and HaCaT

keratinocytes (Maccarrone et al., 2003; Paradisi et al., 2008). Endocannabinoids also

exert a protective role in skin allergic inflammation (Karsak et al., 2007).

The endocannabinoids AEA is an N-acylethanolamines (NAE). Other NAE, such as N-

palmitoylethanolamide (PEA), N-oleoylethanolamide (OEA) and N-

stearoylethanolamide (SEA), show different receptor preference, including affinity for

GPR55, GPR18, GPR119, TRPV1 (transient receptor potential channel type V1) or

PPARa, while often showing less or no affinity for CB1 or CB2 receptors (Alexander and

Kendall, 2007; Di Marzo et al., 2007; Farrell and Merkler, 2008; Hansen and Diep,

2009). Therefore, they were not always classified as endocannabinoids because they

lack the affinity to the endocannabinoid receptors. The group of NAE contains more

than 80 different conjugates of long-chain fatty acids with amino acids (lipoamino acids;

elmiric acids) or neurotransmitters (Burstein and Zurier, 2009; Connor et al., 2010; Tan

et al., 2010). NAE can be rapidly synthesized in membranes, released on demand by

the same biosynthesis pathway of AEA and degraded by the enzyme FAAH (Bisogno,

94

Ligresti et al. 2005, Bisogno 2008). In addition, NAE can also be produced through the

direct conversion of the n-3 PUFA to yield for example palmitoylethanolamide (PEA)

which has anti-inflammatory properties (Klein, Newton et al. 2003, Klein 2005,

O'Sullivan 2007, Hoareau, Buyse et al. 2009). Based on all this information we believe

that n-3 PUFA have a role in the endocannabinoids biosynthesis. Furthermore, n-3

PUFA, especially EPA and DHA, are considered a dietary component with a promising

photoprotective action against the deleterious effects of UVR (Black et al., 1992,

Rhodes et al., 1994, Rhodes et al., 1995). They exert their anti-inflammatory effects by

their competition with AA as substrates for COXs and LOXs, resulting in the formation

of less active prostaglandins and leukotrienes (Lands et al., 1992). N-3 PUFA also

reduces UVB-erythemal sensitivity in humans (Rhodes et al., 1994), by inhibition of

UVB-induced PGE2 levels in the skin (Rhodes et al., 1995).

3.2 Aims of the study

The aim of this study was to explore the hypothesis that EPA and DHA may play a role

in protecting human skin cells against the harmful effects of UVR through their effect on

the formation of endocannabinoids and other NAE. This study was carried out using a

model system including HaCaT keratinocytes and 46BR.1N fibroblasts. The specific

objective was to investigate the effect of n-3 PUFA on the formation of

endocannabinoids and other NAE in HaCaT and 46BR.1N cells pellets (intracellular)

and cell culture medium (extracellular) in response to UVR.

95

3.3 Materials and Methods

3.3.1. Material (refer to section 2.8.1 chapter 2)

3.3.2. Experimental description of LC-MS/MS analysis (refer to section 2.8.2

chapter 2)

3.3.3. Extraction protocol (refer to section 2.8.3 chapter 2)

3.3.4. LC-MS/MS analysis of endocannabinoids and NAE (refer to section 2.8.4

chapter 2)

3.3.5. Standards for quantification and calibration lines (refer to section 2.8.5

chapter 2)

3.3.6. Statistical analysis (refer to section 2.10. chapter 2)

3.4. RESULTS

The concentration of fatty acids and UVR dose used for this work were not toxic to the

cells. This was established by another research worker in the lab and the data is

included in his PhD thesis (Al-Aasswad 2013).

3.4.1. Quantification of endocannabinoids and NAE in irradiated and non-

irradiated HaCaT keratinocytes treated with OA

Treatment with OA 10µM or 50µM decreased STEA levels to be 369 pg/mg and 289

pg/mg respectively, in concentration dependent manner compared to the non-irradiated

control, 422 pg/mg (Figure 3.1A, compare bar 1 to bars 3 and 5). When these cells were

submitted to UV radiation, their STEA levels were 393 pg/mg and 273 pg/mg

respectively in OA 10µM and 50µM (Figure 3.1, compare bar 2 to bars 4 and 6).This

result indicated that UVR could not reverse OA effect on this mediator. However, STEA

level was increased in the irradiated HaCaT control cells, 522 pg/mg, compared to the

nonirradiated HaCaT cells (Figure 3.1, compare bar 1 with bar 2).

96

PEA levels estimated in the untreated cells were 1027pg/mg (Figure 3.1B, bar 1). Small

reduction (836 pg/mg and 900 pg/mg) of the PEA levels was evident in the cells treated

with either OA10µM or 50µM, respectively (Figure 3.1B, compare bar 1 to bars 3 and

5). The highest level of PEA was found in the irradiated control sample, 1374 pg/mg

(Figure 3.1, compare bar 2 to bar 1). Submitted OA treated cells 10µM or 50µM to UVR

resulted in a dose dependent decrease in PEA levels 982 pg/mg and 827pg/mg

compared to the irradiated control cells (Figure 3.1B, compare bar 2 to bars 4 and 6).

The OEA levels in the control untreated cells were assessed to be 1471 pg/mg (Figure

3.1C, bar 1). This level decreased in a dose dependent manner to 1140 pg/mg and 1099

pg/mg in HaCaT cells treated with OA 10µM or 50µM respectively (Figure 3.1C, bar 3

and bar 5). Exposing the OA treated cells (10µM and 50µM) to UVR also decreased the

OEA level in a dose dependent fashion, 1240 pg/mg and 1113 pg/mg compared to the

irradiated control cells 1632 pg/mg (Figure 3.1C, compare bar 2 to bars 4 and 6).

Treatment with OA10µM or 50µM did not induce significant changes in LEA levels, 51

pg/mg and 43 pg/mg compared to the untreated control cells 59 pg/mg (Figure 3.1D,

compare bar 1 to bars 3 and 5). In the irradiated cells LEA level was estimated to be 58

pg/mg indicating that UVR did not affect the LEA levels in these cells compared to the

nonirradiated control cells (Figure 3.1D, compare bar 1 to bar 2). UV radiation of OA

10µM or 50µM treated cells induced fluctuated levels of LEA, 64 pg/mg and 39 pg/mg

respectively, compared to the irradiated control cells 58 pg/mg (Figure 3.1D, compare

bar 2 to bar 4 and 6).

DHEA levels in HaCaT cells were estimated to be 480 pg/mg (Figure 3.1E, bar 1).

Treatment of HaCaT cells with 10µM OA alone induced a minor increase in the DHEA

level 559 pg/mg compared to the untreated cells (Figure 3.1E, compare bar 1 to bar 3)

whereas increase of the OA dose to 50µM decreased the DHEA level 265 pg/mg

compared to those observed in the untreated cells (Figure 3.1E, compare bar 1 to bar

5). The highest DHEA level was found in the irradiated control sample, which was

estimated to be 643 pg/mg (Figure 3.1E, bar 2). This level decreased in a dose

97

dependent fashion to 469 pg/mg and 226 pg/mg when the OA 10µM or 50µM treated

cells were irradiated (Figure 3.1E, compare bar 2 to bars 4 and 6).

AEA levels were estimated to be 433 pg/mg in the control untreated HaCaT cells (Figure

3.1F, bar 1). Treatment with OA 10µM decreased the AEA level to 295 pg/mg (Figure

3.1F, compare bar 1 to bar 3). In addition, further reduction in AEA level 195 pg/mg was

seen when the OA concentration was increased to 50µM (Figure 3.1F, compare bar 1

to bar 5). In the irradiated control HaCaT cells AEA level reduced to 371 pg/mg

compared to the nonirradiated control cells (Figure 3.1F, compare bar 1 to bar 2).

Exposed OA 10µM and 50µM treated cells to UV light dose dependently decreased the

AEA level to 263 pg/mg and 173pg/mg (Figure 3.1F, compare bar 2 to bars 4 and 6).

This indicated that the OA and UVR synergistically down-regulated AEA levels.

2-AG level was recorded to be 2732 pg/mg in the untreated HaCaT control cells. (Figure

3.1G bar, 1). Concentration dependent decrease, 2255 pg/mg and 1668 pg/mg was

observed in 2-AG levels when HaCaT cells were treated with OA 10µM or 50µM (Figure

3.1G compare bar 1 to bars 3 and 5). In the irradiated HaCaT control cells 2-AG level

was slightly reduced to 2520 pg/mg compared to the nonirradiated control cells (Figure

3.1G compare bar 1 to bar 2). Minor increase in 2-AG level was seen in HaCaT

irradiated cells treated with OA 10µM (Figure 3.1G compare bar 2 to bar 4). However,

this level was decreased again when these cells were treated with OA 50µM (Figure

3.1G compare bar 2 to bar 6).

1-AG level was slightly lower in the untreated control HaCaT cells 2208pg/mg compared

to the irradiated control cells 2504 pg/mg (Figure 3.1H compare bar 1 and bar 2). The

level of 1-AG was also decreased in a dose dependent manner, 2040 pg/mg and 1550

pg/mg respectively in OA 10µM or 50µM treated cells compared to the untreated (Figure

3.1 compare bar 1 to bars 3 and 5). Similar results were observed when OA 10µM or

50µM (2487pg/mg and 1504pg/mg) treated cells were irradiated compared to the

irradiated control (Figurer 3.1 compare bar 2 to bars 4 and 6).

98

Figure 3.1 Effect of OA treatment on endocannabinoids and NAE levels in HaCaT

keratinocytes in response to UV radiation. As described in Material and Methods,

cells were treated either with 10 or 50 µM OA for 72h and then they were irradiated with

15J/cm2 UVB. Cells were harvested with 0.25% trypsin-EDTA solution 24h post UVR.

Quantification of endocannabinoids and NAE in HaCaT keratinocytes was carried out

using LC-MS/MS analysis. Data shown as mean of n = 2 experiments. No statistical

analysis was performed.

99


irradiated HaCaT keratinocytes treated with DHA

Pellets from three independent experiments were extracted as described in materials

and Methods (Section 2.7.3) and analyzed for endocannabinoids and N-

acylethanolamines levels in irradiated and non-irradiated HaCaT Keratinocyte cells

treated with DHA.

The STEA level was estimated to be 483pg/mg in the untreated control HaCaT cells.

Treatment with DHA 10µM or 50µM for 72h induced dose dependent small reduction,

473pg/mg and 455pg/mg in the STEA levels compared to the untreated cells (Figure

3.2A compare bar 1 to bars 3 and 5). Compared to the irradiated control cells, STEA

level was slightly increased respectively to 505pg/mg and 492pg/mg in cells treated with

DHA 10µM or 50µM plus UVR (Figure 3.2A compare bar 2 with bars 4 and 6). No

significant differences were seen between groups in this experiment.

Figure 3.2B presented that the treatment with DHA either 10µM or 50µM moderately

increased the PEA levels in a dose dependent manner, 2350pg/mg and 2402pg/mg

compared to the untreated control cells which showed PEA level at1926pg/mg (Figure

3.2B compare bar 1 to bars 3 and 5). UVR-treated control showed the lowest PEA level

in this experiment (1476pg/mg) (Figure 3.2B bar 2). Compared to the irradiated control

cells, PEA levels also increased respectively to 2032pg/mg and 1776pg/mg in HaCaT

cells treated with DHA10µM or 50µM and submitted to UV radiation (Figure 3.10

compare bar 2 to bars 4 and 6). Statistically, there was a significant difference between

DHA 50µM and DHA 50µM plus UV radiation (P = 0.035).

OEA level was assessed to be 4030pg/mg in the untreated control cells (Figure 3.2C,

bar 1). Treatment with DHA 10µM or 50µM for 72h significantly decreased the OEA

levels, in a dose dependent fashion, respectively to 2504pg/mg (P = 0.001) and

2460pg/mg (P = 0.001) compared to the untreated control cells (Figure 3.2C compare

bar 1 to 3 and 5). Likewise, 2 fold decreases was seen in OEA level in the irradiated

control cells (1855pg/mg), compared to the non-irradiated control HaCaT cells (Figure

3.2C compare bar 2 to bar 1). Moreover, OEA levels were insignificantly decreased

100

when the irradiated cells were treated with DHA 50µM, 1794pg/mg (Figure 3.2C, bar 6).

However, this level was slightly increased, 2261pg/mg when the irradiated cells were

treated with DHA 10µM (Figure 3.2C, bar 4).

LEA levels in the nonirradiated control cells were assessed to be 310pg/mg (Figure

3.2D bar 1) while, after the UVR stimulation of control cells, this level significantly

decreased 2 fold to 190 pg/mg (P = 0.002) (Figure 3.2D bar 2). Treatment with DHA

either 10µM or 50µM for 72h also significantly decreased the LEA levels to 79pg/mg (P

= 0.001) and 90pg/mg (P = 0.001) respectively, compared to the untreated HaCaT cells

(Figure 3.2D compare bar 1 to bars 3 and 5). Similar effect was seen, 69pg/mg (P =

0.002) and 94pg/mg (P = 0.01) when the DHA 10µM or 50µM treated cells were

irradiated compared to the irradiated control cells (Figure 3.2D compare bar 2 to bars 4

and 6).

DHEA levels in untreated HaCaT control cells were estimated to be 676pg/mg (Figure

3.2E, bar 1). DHEA level was slightly higher in the irradiated control 704pg/mg,

compared to the nonirradiated control cells (Figure 3.2E compare bar 1 to bar 2). DHEA

level significantly increased in nonirradiated HaCaT cells treated with DHA 50µM

2045pg/mg (P = 0.001), compared to control cells (Figure 3.2E compare bar 1 to bar 5).

While, DHA 10µM induced only minor increase in DHEA level, 912pg/mg compared to

its control cells (Figure 3.2E compare bar 1 to bar 5). However, DHEA level was

significantly increased when HaCaT cells treated with DHA 50µM, 2045pg/mg,

compared to those treated with 10µM, 912pg/mg (P = 0.001) (Figure 3.2E compare bar

3 to bar 5). In cells treated with DHA 50µM plus UVR DHEA level was also significantly

increased to 2382pg/mg compared to the irradiated cells treated with 10µM, 1340pg/mg

and to the irradiated control cells as well (P = 0.001) (Figure 3.2E compare bar 4 to bar

6 and compare bar 2 to bar 6).

AEA levels in untreated HaCaT cells were estimated to be 321pg/mg, whereas, this

level was slightly increased after UV radiation in control cells to be 353pg/mg (Figure

3.2 F, bar 1 and 2). AEA levels was dose dependently increased when HaCaT cells

were treated with DHA 10µM or 50µM to 397pg/mg and 421pg/mg respectively (Figure

101

3.2 F compare bar 3 to bar 5). Similar effect was seen when these treated cells were

irradiated (DHA10µM or 50µM, 413pg/mg and 447pg/mg respectively) (Figure 3.2 F

compare bar 4 to bar 6). However significant differences were only observed between

50µM and irradiated and nonirradiated control cells (P = 0.03 and 0.02 respectively)

(Figure 3.2 F compare bar 1 to bar 5 and compare bar 2 to 6).

2-AG level in the control sample was 4827 pg/mg, whereas, its level was almost the

same; 4836pg/mg after UVR exposure (Figure 3.2 G bar 1 and bar 2). Treatment with

DHA 10µM or 50µM for 72h, decreased the 2-AG level respectively to 3822pg/mg and

3651pg/mg, compared to the untreated control cells (Figure 3.2 G compare bar 1 to

bars 3 and 5). Furthermore, 2-AG levels in UVR-treated cells combined with DHA either

10µM or 50µM were also reduced to 3600pg/mg and 4277pg/mg respectively,

compared to the irradiated control cells (Figure 3.2 G compare bar 2 to bars 4 and 6).

1-AG level was estimated to be 5133 pg/mg in the untreated control cells (Figure 3.2 H

bar 1) No major change was seen in 1-AG level 5345pg/mg, after UV irradiation (Figure

3.2 H compare bar 1 to bar 2). Whereas, treatment with DHA 10µM or 50µM for 72h,

significantly decreased 1-AG levels to 3341pg/mg (P = 0.01) and 2782pg/mg (P =

0.002), compared to the untreated control cells (Figure 3.2 H compare bar 1 to 3 and

5). Additionally, 1-AG level was also significantly decreased in a dose dependent

manner, 3700pg/mg (P = 0.02) and 2692pg/mg (P = 0.001) when the UVR-stimulated

cells were treated with DHA 10µM or 50µM compared to the irradiated control cells

(Figure 3.2 H compare bar 2 to bars 4 and 6).

102

Figure 3.2 Effect of DHA treatment on endocannabinoids and NAE levels in HaCaT


cells were treated either with DHA10 or 50µM for 72h and then they were irradiated with


Quantification of endocannabinoids and NAE in HaCaT keratinocytes was carried out

using LC-MS/MS analysis. Data shown as mean ± SD of n = 3 experiments. *P<0.05,

**P<0.01, ***P<0.001, comparing data to control. Figure B, * = compare to non-

irradiated cells treated with DHA 50µM, $. Figure E, § = compared to non-irradiated cells

treated with DHA 10µM while, ≠ = compared to irradiated cells treated with DHA 10µM.

103


irradiated HaCaT keratinocytes treated with EPA

Pellets from three independent experiments were extracted as described in Materials

and Methods (Section 2.7.3) and analyzed for endocannabinoids and N-

acylethanolamines levels in irradiated and non-irradiated HaCaT keratinocyte cells

treated with EPA. Data are presented as mean ± standard deviation where appropriate.

Comparisons between groups were made with one-way analysis of variance (ANOVA)

followed by Tukey test using SPSS Statistics software. P < 0.05 was considered as

significant level of difference.

STEA levels in HaCaT control cells were assessed to be 389pg/mg (Figure 3.3 A bar

1). Treatment with EPA 10µM or 50µM for 72h did not induce significant changes in

STEA levels which was estimated between 382pg/mg and 438pg/mg compared to the

untreated control cells (Figure 3.3 A, compare bar 1 to 3 and 5). However, UVR induced

minor decreases in STEA level either in control cells or in the treated cells (316pg/mg,

347pg/mg and 357pg/mg compare bar 2 with bars 4 and 6 respectively).

PEA level was calculated to be 2699 in untreated control cells (Figure 3.3 B, bar 1).

Treatment with EPA 10 µM or 50 µM resulted in small decreases in PEA levels in these

cells, 2162pg/mg and 2288pg/mg respectively compared to untreated control cells

(Figure 3.3 B, compare bar 1 to bars 3 and 5). However, PEA level was significantly

reduced in irradiated cells to 1935pg/mg compared to nonirradiated HaCaT cells (P =

0.035) (Figure 3.3 B, compare bar 1 to bar 2). Whereas, when the EPA treated cells

were subjected to UVR there were no significant changes in PEA levels (1953pg/mg

and 2001pg/mg) compared to irradiated control cells (Figure 3.3 B, compare bar 2 to

bars 4 and 6).

Untreated HaCaT cells showed OEA level at 2069 pg/mg (Figure 3.3 C, bar 1).

Treatment of the HaCaT Keratinocyte cell with two different concentrations of EPA

(10µM or 50µM) for 72h increased the OEA levels to 2184 pg/mg and 2564 pg/mg,

respectively in dose dependent manner (Figure 3.3 C compare bar 1 to 3 and 5).

Likewise, OEA level was also increased in irradiated control cells to 2429 pg/mg.

104

However, this effect of UVR was reversed in dose dependent way in EPA10µM or 50µM

treated cells to 2085 pg/mg and 1807pg/mg, respectively) when they were irradiated

(Figure 3.3 C, compare bar 2 to bars 4 and 6).

LEA levels in HaCaT control cells were assessed to be 334 pg/mg (Figure 3.3 D, bar 1)

while, after the UVR exposure this level significantly fallen to 158 pg/mg (P = 0.001)

(Figure 3.3 D, bar 2). Treatment with EPA 10µM or 50µM for 72h decreased the LEA

levels to 242 pg/mg and 214 pg/mg, respectively, in dose dependent manner (Figure

3.3 D compare bar 1 to 3 and 5). However, the difference was only significant (P =

0.012) between untreated control and EPA 50 µM treated cells (Figure 3.3 D, compare

bar 1 with bar 5). In irradiated control cells LEA level was dropped to 158 pg/mg (Figure

3.3 D, bar 2), but raised in EPA10µM treated cells to 198pg/mg (Figure 3.3 D, bar 4)

and then decreased further to 120 pg/mg in the EPA 50µM treated cells. (Figure 3.3 D,

bar 6).

DHEA levels in the untreated control cells were found to be 1061pg/mg (Figure 3.3 E,

bar 1). Treatment with EPA either 10 µM or 50 µM significantly reduced DHEA levels to

783 pg/mg and 754 pg/mg respectively in dose dependent manner compared to the

untreated control cells (P = 0.001)(Figure 3.3 E, compare bar 1 to bars 3 and 5).

Moreover, UVR induced significant down-regulation in DHEA level to 821pg/mg

compared to the non-irradiated control cells (P = 0.001) (Figure 3.3 E, compare bar 2

to bar 1). These effect also continued when EPA 10µM (733 pg/mg) or 50µM (684

pg/mg) treated HaCaT cells were subjected to UV radiation compared to the irradiated

control cells (Figure 3.3 E, compare bar 2 to bars 4 and 6).

Figure 3.3 F showed that treatment with EPA at two different concentrations; 10 µM or

50 µM did not induce any noteworthy changes in AEA levels which was estimated to

be 93 pg/mg and 112 pg/mg respectively, compared to untreated control cells,

107pg/mg (Figure 3.3 F, compare bar 1 to bars 3 and 5). However, AEA level was

increased in irradiated HaCaT cells treated with EPA 10µM or 50µM to 116pg/mg and

119pg/mg compared to the irradiated control cells that its AEA level was estimated to

be 95pg/mg (Figure 3.3 F, compare bar 2 to bars 4 and 6). Statistical analysis showed

105

significant difference only between irradiated cells threated with EPA 50µM and

irradiated control cells (P = 0.037).

2-AG levels in the untreated control cells were estimated to be 4099pg/mg (Figure 3.3

G, bar 1). Treatment with EPA 10µM or 50µM for 72h, dose dependently decreased 2-

AG levels to 4002pg/mg and 3412pg/mg, compared to the untreated control cells

(Figure 3.3 G, compare bar 1 to bars 3 and 5). Similar effect was also observed in the

irradiated cells treated with EPA 10µM or 50µM (3091pg/mg and 3023pg/mg) compared

to the irradiated control cells (3132pg/mg) (Figure 3.3 G, compare bar 2 to bars 4 and

6).

1-AG levels in the untreated control cells were assessed to be 3967pg/mg (Figure 3.3

H, bar 1), whereas, UV radiation significantly down-regulated 1-AG level to 2481pg/mg

(P = 0.003) (Figure 3.3 H, bar 2). Similarly, treatment with EPA 50µM significantly

decreased 1-AG level to 2359pg/mg compared to the untreated control HaCaT cells (P

= 0.002) (Figure 3.3 H, compare bar 1 to bar 5). Treatment with EPA 10µM also

decreased 1-AG level to 3133pg/mg (Figure 3.3 H, compare bar 1 to bar 3) compared

to the untreated control cells. Although, there was decrease effect after UV radiation in

EPA 10µM or 50µM treated cells, no significant differences were noted in 1-AG levels

(2247pg/mg and 3089pg/mg, respectively) (Figure 3.3 H, compare bar 2 to bars 4 and

6).

106

Figure 3.3 Effect of EPA treatment on endocannabinoids and NAE levels in HaCaT


cells were treated either with EPA10 or 50 µM for 72h and then they were irradiated

with 15J/cm2 UVB. Cells were harvested with 0.25% trypsin-EDTA solution 24h post

UVR. Quantification of endocannabinoids and NAE in HaCaT keratinocytes was carried

out using LC-MS/MS analysis. Data shown as mean ± SD of n = 3 experiments.

*P<0.05, **P<0.01, ***P<0.001, comparing data to control.

107

3.4.4. Quantification of endocannabinoids and NAE in culture medium from

irradiated and non-irradiated HaCaT keratinocytes treated with OA

Supernatant from HaCaT Keratinocyte cells treated with OA was analysed for the OEA

level. Figure 3.4 A, showed that the OEA level in the untreated medium was assessed

to be 369pg/ml (Figure 3.4 A, bar 1). Treatment with OA 10µM and 50µM decreased

this level to 295pg/ml and 318pg/ml respectively compared to untreated control medium

(Figure 3.4 A, bar 1 to bars 3 and 5). Radiation of control medium up-regulated OEA

level up to 421pg/ml compared to the nonirradiated control medium (Figure 3.4 A, bar

1 to bar 2). However, this level decreased to 308pg/ml and 373pg/ml respectively when

OA 10µM or 50µM treated HaCaT culture medium were irradiated (Figure 3.4 A, bar 2

to bars 4 and 6).

In the supernatant of untreated control cells STEA levels were found to be 2501pg/ml

(Figure 3.4 B, bar 1). Treatment with 10µM or 50µM of OA for 72h did not induce major

changes in STEA levels (2501pg/ml and 2250pg/ml) compared to the untreated control

culture medium (Figure 3.4 B, bar 1 to bars 3 and 5). On the other hand, UVR increased

the STEA level to 3501pg/ml in the irradiated control culture medium compared to the

nonirradiated control culture medium (Figure 3.4 B, compare bar 1 to bar 2). This level

was similar in the irradiated culture medium treated with OA 10µM or 50µM (3501pg/ml

and 3751pg/ml) compare to irradiated control culture medium (Figure 3.4 B, compare

bar 2 to bars 4 and 6).

In cell cultural medium of untreated control cells DHEA level was estimated to be

152pg/ml (Figure 3.4 C, bar 1), whereas, there was no much difference in DHEA level

in irradiated control culture medium (165pg/ml) compared to the non-irradiated control

(Figure 3.4 C, compare bar 1 to bar 2). In addition, treatment with OA either 10 µM or

50 µM did not change DHEA levels (156pg/ml and 154pg/ml, respectively) compared to

the untreated control culture medium (Figure 3.4 C, compare bar 1 to bars 3 and 5).

However, a small dose dependent decrease was observed in DHEA levels when OA

10µM or 50µM (148pg/ml and 141pg/ml) treated culture medium were irradiated

compared to the irradiated control culture medium (Figure 3.27 compare bar 2 to bars

4 and 6).

108

2-AG levels in the supernatant of the untreated control cells were assessed to be

1193pg/ml (Figure 3.4 D, bar 1). After UVR stimulation, 2-AG level was almost the same

as its level in untreated control culture medium, 1182pg/ml (Figure 3.4 D, compare bar

1 to bar to bar 2). Furthermore, treatment with OA 10 µM slightly increased 2-AG to

1245pg/ml, whereas at a concentration 50µM, 2-AG level was approximately the same,

1158pg/ml compared to nonirradiated control culture medium (Figure 3.4 D, compare

bar 1 to bars 3 and 5). However, when OA10 µM or 50 µM treated culture medium were

irradiated, 2-AG levels increased to 1201pg/ml and 1296pg/ml in dose dependent

manner compared to irradiated control culture medium (Figure 3.4 D, compare bar 2 to

bars 4 and 6).

1-AG levels in the supernatant of untreated control medium were measured to be

3025pg/ml (Figure 3.4 E, bar 1). A very small decrease in 1-AG level was observed after

UV radiation compared to nonirradiated control in dose dependent manner medium

(Figure 3.4 E, compare bar 1 to bar to bar 2). Treatment with OA either 10 µM or 50 µM

did not show any remarkable changes in 1-AG levels (3054pg/ml and 3354pg/ml)

compared to the untreated control in dose dependent manner medium (Figure 3.4 E,

compare bar 1 to bars 3 and 5). UVR-stimulation of these treated in dose dependent

manner medium increased 1-AG protein level in dose dependent manner to 3049pg/ml

and 3257pg/ml compared to irradiated control medium (Figure 3.4 E, compare bar 2 to

bars 4 and 6).

109

Figure 3.4 Effect of OA treatment on endocannabinoids and NAE levels in HaCaT

keratinocytes culture medium in response to UV radiation. As described in Material

and Methods, cells were treated either with OA10 or 50 µM for 72h and then they were

irradiated with 15J/cm2 UVB. Medium was collected 24h post UVR. Quantification of

endocannabinoids and NAE in HaCaT keratinocytes was carried out using LC-MS/MS

analysis. Data shown as mean of n = 2 experiments. No statistical analysis was

performed.

110

3.4.5. Quantification of endocannabinoids and NAE in cell culture medium from

irradiated and non-irradiated HaCaT keratinocytes treated with DHA.

Figure 3.5 A, showed that the base line of OEA in untreated medium was estimated to

be 541pg/ml (Figure 3.5 A, bar 1). DHA 10µM or 50µM treatment dose dependently

increased OEA level in HaCaT cultural medium to 624pg/ml and 681pg/ml compared to

untreated control medium (Figure 3.5 A, compare bar 1 to bar 3 and 5). OEA level was

significantly increased in irradiated control medium cells to 767pg/ml (P = 0.005)

compared to the nonirradiated control medium (Figure 3.5 A, compare bar 1 to bar 2).

Similar effect but, no significant difference was seen when DHA 10µM (840pg/ml) or

50µM (830pg/ml) treated medium were subjected to UVR (Figure 3.5 A, compare bar 2

to bars 4 and 5) compared to irradiated control medium.

STEA levels in untreated control medium were found to be 5168pg/ml (Figure 3.5 B, bar

1). UV light significantly reduced STEA level in irradiated control medium to 2501pg/ml

(P = 0.001) compared to nonirradiated control medium (Figure 3.5 B, compare bar 1 to

bar 2). Treatment with DHA 10µM also significantly decreased STEA level to 2667pg/ml

(P = 0.001) compared to untreated control medium (Figure 3.5 B, compare bar 1 to bar

3). However, DHA 50µM induced only small decrease (4668pg/ml) in STEA level

compared to untreated control medium (Figure 3.5 B, compare bar 1 to bar 5). Both

concentrations of DHA (10µM or 50µM) increased STEA protein levels to 3334pg/ml

when they were irradiated compared to the irradiated control medium (Figure 3.5 B,

compare bar 2 to bars 4 and 6).

DHEA levels in untreated control medium were measured to be 136pg/ml (Figure 3.5

C, bar 1), and this level was quite similar in the irradiated control medium, 130pg/ml

(Figure 3.5 C, bar 2). However, no significant differences were noticed in DHEA level in

non-irradiated medium treated with DHA 10 µM (130pg/ml) or 50 µM (144pg/ml) (Figure

3.5 C, compare bar 1 to bars 3 and 5) compared to untreated control medium. DHA

10µM (142pg/ml) and 50µM (144pg/ml) also have no effect on the irradiated medium

compared to the irradiated control medium (Figure 3.5 C, compare bar 2 to bars 4 and

6).

111

2-AG levels in untreated control medium were assessed to be 1161pg/ml (Figure 3.5 D,

bar 1) whereas, slight increase in 2-AG levels were observed in culture medium treated

with DHA 10µM or 50µM, 1207pg/ml and 1212pg/ml respectively, compared to

untreated control medium (Figure 3.5 D, compare bar 1 to bars 3 and 5). Moreover, 2-

AG level was also increased after UVR stimulation up to 1336pg/ml compare to

nonirradiated control medium (Figure 3.5 D, compare bar 1 to bar 2). However, 2-AG

level was dropped to 1094pg/ml in UVR-stimulated medium treated with DHA 10µM and

then increased again to 1363pg/ml with DHA 50µM compared to irradiated control

medium (Figure 3.5 D, compare bar 2 to bars 4 and 6).

1-AG levels in the untreated control medium were considered to be 3072pg/ml (Figure

3.5 E, bar 1) whereas, in cell culture medium treated with 10µM or 50µM DHA, 1-AG

levels were increased to 3153pg/ml and 3635pg/ml compared to untreated control

medium (Figure 3.5 E, compare bar 1 to bars 3 and 5). After UVR stimulation, 1-AG was

elevated up to 3712pg/ml but, this level was then dropped to 3414pg/ml and 3348pg/ml

UVR-stimulated medium treated with DHA 10µM or 50µM respectively (Figure 3.5 E,


112

Figure 3.5 Effect of DHA treatment on endocannabinoids and NAE levels in HaCaT


and Methods, cells were treated either with DHA10 or 50 µM for 72h and then they were



analysis. Data shown as mean ± SD of n = 3 experiments. *P<0.05, **P<0.01,

***P<0.001, comparing data to control.

113

3.4.6. Quantification of endocannabinoids and NAE in cell culture medium from

irradiated and non-irradiated HaCaT keratinocytes treated with EPA.

OEA levels in HaCaT cultural medium of untreated control cells were found to be

425pg/ml (Figure 3.6 A, bar 1). This level slightly increased in a dose dependent manner

to 407pg/ml and 437pg/ml in cell culture medium treated with EPA 10µM or 50µM

respectively, compared to untreated control HaCaT medium (Figure 3.6 A, compare bar

1 to bars 3 and 5). However, UVR significantly increased OEA level in irradiated control

up to 555pg/ml compared to nonirradiated control medium (P = 0.001) (Figure 3.6 A,

compare bar 1 to bar 2). This effect was significantly enhanced when EPA 10µM

(407pg/ml) (P = 0.002) or 50µM (500pg/ml) (P = 0.008) treated medium were irradiated

compared to irradiated control medium (Figure 3.6 A, compare bar 2 to bars 4 and 6).

STEA levels in untreated control medium were calculated to be 2667pg/ml (Figure 3.6,

B bar 1). Treatment with EPA10µM or 50µM increased STEA level to 3001pg/ml and

2834pg/ml respectively, compared to untreated control medium (Figure 3.6 B, compare

bar 1 to bars 3 and 5). Compared to nonirradiated control medium, UV light increased

STEA level in the irradiated control medium to 3167pg/ml (Figure 3.6 B, Compare bar

1 to bar 2). STEA level was then down-regulated to 2167pg/ml in irradiated medium

treated with EPA 10µM, but increased again to 3667pg/ml as EPA 50µM was tested

against UVR (Figure 3.6 B, compare bar 2 to bars 4 and 6).

DHEA levels in untreated control medium were estimated to be 140pg/ml (Figure 3.6 C,

bar 1), this level was slightly reduced in irradiated control medium to 132pg/ml (Figure

3.6 C, bar 2). However, no significant changes were noticed in DHEA level at EPA 10

µM (137pg/ml) or 50 µM (132pg/ml) treated groups compared to untreated control

medium (Figure 3.6 C, compare bar 1 to bars 3 and 5). Also, no major changes were

observed in DHEA levels, 132pg/ml and 137pg/ml respectively, when EPA 10µM and

50µM treated medium were examined against UVR compared to irradiated control

medium (Figure 3.6 C, compare bar 2 to bars 4 and 6).

2-AG levels in untreated control medium were assessed to be 1258pg/ml and there was

no noticeable change in its level in culture medium treated with EPA 10µM or 50µM,

114

1171pg/ml and 1282pg/ml, respectively (Figure 3.6 D, compare bar 1 to bars 3 and 5).

UVR stimulation did not induce any major change in 2-AG (1148pg/ml) compared to

nonirradiated control culture medium (Figure 3.6 D, compare bar 1 to bar 2). However,

2-AG levels increased to 1201pg/ml and 1213pg/ml in a dose dependent manner in

EPA 10µM or 50µM treated culture medium with UV radiation compared to the irradiated

control culture medium (Figure 3.6 D, compare bar 2 to bars 4 and 6).

1-AG levels in untreated control medium were estimated to be 3058pg/ml (Figure 3.3 E,

bar 1). Whereas, in HaCaT culture medium treated with EPA 10µM or 50µM 1-AG levels

increased to 3498pg/ml and significantly to 3833pg/ml (P = 0.022) respectively, in a

dose dependent manner compared to untreated control culture medium (Figure 3.6 E,

compare bar 1 to bar 3 and 5). After UV radiation, 1-AG level in irradiated control cells

was calculated to be 3111pg/ml, which is slightly higher than its level in nonirradiated

control culture medium (Figure 3.6 E, compare bar 1 to bar 2). However, this level

increased up 3688pg/ml and to 3521pg/ml respectively in UVR-stimulated culture

medium treated with EPA 10µM or 50µM (Figure 3.6 E, compare bar 2 to bars 4 and 6).

115

Figure 3.6 Effect of EPA treatment on endocannabinoids and NAE levels in HaCaT


and Methods, cells were treated either with EPA10 or 50 µM for 72h and then they were



analysis. Data shown as mean ± SD of n = 3 experiments. *P<0.05, **P<0.01,

***P<0.001, comparing data to control.

116

Table 3.1. Summary of the significant findings in HaCaT keratinocytes cells and their

cell culture medium in response to DHA and/or UVR. Data shown as mean ± SD of n =

3 experiments. *P<0.05, **P<0.01, ***P<0.001, comparing data to control using one way

(ANOVA) followed by Tukey post hoc test.

117

Table 3.2. Summary of the significant findings in HaCaT keratinocytes cells and their

cell culture medium in response to EPA and/or UVR. Data shown as mean ± SD of n =

3 experiments. *P<0.05, **P<0.01, ***P<0.001, comparing data to control using one way


118

3.4.7. Quantification of the endocannabinoids and other NAE in irradiated and

non-irradiated 46BR.IN fibroblasts treated with OA.

In general, because of N= 2 in OA experiments (Figures 3.25-3.31), statistically it is not

possible to assess if there are any significant differences in endocannabinoid congeners

level between the different groups in those experiments.

Compared to untreated control cells STEA levels in nonirradiated control group cells

were measured to be 2151pg/mg (Figure 3.7 A, bar 1). Compared to the untreated

control cells, STEA level was increased up to 5835pg/mg in nonirradiated cells treated

with 10µM OA and to 3494 pg/mg in the cells that were treated with 50µM of the same

fatty acid (Figure 3.7 A compare bar 1 to bars 3 and 5). However, a 2-fold increase was

seen in STEA after UVR exposure in the control cells (4757pg/mg) (Figure 3.7 A bar 2).

Whereas, treatment of the irradiated cell with OA 10µM or 50µM increased STEA level

up to 4238 pg/mg and 6251pg/mg respectively, compare to irradiated control cells

(Figure 3.7 A, compare bar 2 to bars 4 and 6).

Figure 3.7 B, presented some changes in PEA levels when untreated control cells were

compared with OA and UVR treated cells. PEA level in nonirradiated control group was

2958 pg/mg (Figure 3.7 B bar 1). However, this level was increased in irradiated control

cells up to 5123pg/mg, (Figure 3.7 B, bar 2). More elevation in PEA level (7522 pg/mg)

was noticed after the incubation with 10µM OA alone (Figure 3.7 B, bar 3). While, this

level was dropped to 2809 pg/mg (Figure 3.7 B, bar 5) when the OA concentration was

increased to 50µM. These increases in PEA level were also seen when the treated

samples were combined with UVR; 6527pg/mg in bar 4 and 8596 pg/mg in bar 6

compared to the irradiated control in bar 2.

OEA levels in untreated cells were 2259 pg/mg. Incubation of the 46BR.IN fibroblasts

with OA 10µM for 72h increased OEA level slightly to 3612 pg/mg, whereas, this level

was decreased to 2124 pg/mg with OA 50µM (Figure 3.7 C, compare bar 1 to bars 3

and 5). Likewise, UVR elevated OEA level in irradiated control cells to 5367pg/mg

compare to nonirradiated control cells (Figure 3.7 C, compare bar 1 to bar 2). However,

119

this effect was reversed when OA10µM (2712 pg/mg) or 50µM (3126 pg/mg) treated

cells were irradiated (Figure 3.7 C, compare bar 2 to bars 4 and 6).

DHEA levels in untreated control cells were estimated to be 323 pg/mg (Figure 3.7 D,

bar 1). Whereas, DHEA level dramatically increased in irradiated control cells up to 732

pg/mg (Figure 3.7 D, bar 2). Moreover, incubation of OA 10µM or 50µM treated cells

also raised DHEA levels to 695 pg/mg and 411pg/mg respectively, compared to

nonirradiated control cells (Figure 3.7 D, compare bar 1 to bars 3 and 5). However,

DHEA was decreased to 339 pg/mg and 625 pg/mg respectively, when OA10µM or

50µM treated cells were irradiated compared to irradiated control cells (Figure 3.7 C,


AEA levels were found to be 323 pg/mg in untreated control cells, however, in irradiated

control cells UVR increased this level up to 488 pg/mg (Figure 3.7 E, compare bar 1 to

bar 2).Treatment with OA10µM increased AEA level up to 556 pg/mg while, in OA 50µM

AEA level was decreased to 343 pg/mg, compared to untreated control cells (Figure 3.7

E, compare bar 1 to bars 3 and 5). AEA level was also decreased to 339 pg/mg in

irradiated treated with OA10µM, in contrast, OA 50µM in irradiated cells, raised AEA up

to 625 pg/mg compared to irradiated control cells (Figure 3.7 E, compare bar 2 to bars

4 and 6).

2-AG levels in untreated control cells were estimated to be 753 pg/mg (Figure 3.7 F, bar

1). UVR stimulation sharply increased 2-AG level up to 2562 pg/mg (Figure 3.7 F, bar

2). When these cells were incubated with 10µM or 50µM OA for 72h, 2-AG levels were

increased to 1389 pg/mg and 1096 pg/mg respectively compared to untreated control

cells (Figure 3.7 F, compare bar 1 to bars 3 and 5). However, 2-AG level was reduced

in UVR-stimulated cells treated with OA 10µM to 1780 pg/mg but, when the same cells

were treated with OA 50µM 2-AG level increased up to 3126 pg/mg compared to the

irradiated control cells (Figure 3.7 F, compare bar 2 to bars 4 and 6).

In untreated control cells 1-AG levels were found to be 753pg/mg whereas, this level

increased to 2684pg/mg in irradiated control cells (Figure 3.7 G, compare bar 1 to bar

2). Treatment with OA 10µM or 50µM in addition, increased 1-AG levels to 1667pg/mg

120

and 1096pg/mg compared to untreated control cells (Figure 3.7 G, compare bar 1 to

bars 3 and 5). However, 1-AG level was reduced to 1865pg/mg in UVR-stimulated cells

treated with OA 10µM compared to irradiated control cells but, when the OA 50µM was

used 1-AG level increased up to 3126 pg/mg in these cells compared to their control

cells (Figure 3.7 G, compare bar 2 to bars 4 and 6).

121

Figure 3.7 Effect of OA treatment on endocannabinoids and NAE levels in 46BR.IN

fibroblasts in response to UV radiation. As described in Materials and Methods, cells

were treated either with OA10 or 50 µM for 72h and then they were irradiated with


Quantification of endocannabinoids and NAE in 46BR.IN fibroblasts was carried out



122


non-irradiated 46BR.IN fibroblasts treated with DHA

STEA levels were calculated to be 3741pg/mg in untreated control cells (Figure 3.8 A,

bar 1). In addition, when these cells were incubated with DHA 10µM or 50µM for 72h,

STEA levels were increased to 5459pg/mg and 5984pg/mg, respectively (Figure 3.8 A,

compare bar 1 to 3 and 5). STEA level in the irradiated control was slightly lower

(3287pg/mg) compared to its level in nonirradiated control cells (Figure 3.8 A, compare

bar 1 to bar 2). In UVR-stimulated cells treated with DHA 10µM or 50µM STEA levels

were significantly increased in dose dependent manner to 7881pg/mg (P = 0.001) or

7153pg/mg (P = 0.002), respectively compared to the irradiated control (Figure 3.8 A,


Figure 3.48 showed that there were no significant differences in PEA levels between

nonirradiated (2276pg/mg) and irradiated (2343pg/mg) control cells (Figure 3.8 B,

compare bar 1 to bar 2). PEA level was slightly increased up to 3237pg/mg after the

incubation with 10µM DHA alone (Figure 3.8 B, bar 3), while, this level dropped to

2844pg/mg (Figure 3.8 B, bar 5) when DHA concentration was increased to 50µM. The

same trend was also seen when the treated cells were exposed to UVR. PEA level was

significantly increased up to 4658pg/mg (P = 0.001) and 3646pg/mg (P = 0.03) with

DHA10µM and 50µM respectively, compared to the irradiated control (Figure 3.8 B,

compare bar 2 to bar 4 and 6).

Figure 3.8 C showed that OEA levels in untreated control cells were assessed to be

1791pg/mg (Figure 3.8 C, bar 1). However, this level was decreased to 1407pg/mg in

irradiated control cells (Figure 3.8 C, bar 2). Treatment with DHA10µM or 50µM

significantly increased OEA levels up to 3394pg/mg (P = 0.001) and 2942pg/mg (P =

0.01) respectively, compared to untreated control cells (Figure 3.8 C, compare bar 1 to

bars 3 and 5). This effect was in addition, enhanced when DHA10µM or 50µM treated

cells were irradiated, OEA levels were significantly further increased up to 4587pg/mg

(P = 0.001) and 4107pg/mg (P = 0.001) respectively, compared to irradiated control

cells (Figure 3.8 C, compare bar 2 to bars 4 and 6).

123

DHEA levels in untreated control cells were estimated to be 345 pg/mg (Figure 3.8 D,

bar 1). Whereas, in irradiated control cells this level was dropped to 309 pg/mg (Figure

3.8 D, bar 2). Treatment with DHA 10 µM or 50 µM significantly up-regulated DHEA

protein levels in dose dependent manner up to 631pg/mg (P = 0.016) and 1085pg/mg

(P = 0.001) compared to the untreated control cells (Figure 3.8 D compare bar 1 to bars

3 and 5). Likewise, similar trend was observed when the treated groups combined with

UVR, OEA levels were significantly increased to 1165 pg/mg (P = 0.001) and 1184

pg/mg (P = 0.001) in DHA 10µM or 50µM, respectively compared to irradiated control

cells (Figure 3.8 D, compare bar 2 to bars 4 and 6).

Figure 3.8 E showed AEA levels at 517 pg/mg in untreated control cells, this level was

slightly decreased after UVR to 494 pg/mg in irradiated control cells (Figure 3.8 E,

compare bar 1 and bar 2). Treatment with DHA10µM or 50µM increased AEA level up

to 663 pg/mg and 632 pg/mg, respectively compared to untreated control cells (Figure

3.8 E, compare bar 1 to bars 3 and 5). Furthermore, AEA levels were significantly further

increased up to 981pg/mg (P = 0.001) and 841pg/mg (P = 0.001) respectively in

irradiated cells treated with DHA 10µM or 50µM compared to irradiated control cells

(Figure 3.8 E, compare bar 2 to bars 4 and 6).

2-AG levels in the untreated control cells were found to be 1680 pg/mg (Figure 3.8 F,

bar 1). Whereas, UVR stimulation significantly increased this level up to 3544pg/mg (P

= 0.001) in the irradiated control cells (Figure 3.8 F, bar 2). Also, when these cells were

treated with 10µM or 50µM DHA for 72h, 2-AG levels were increased to 2290 pg/mg

and 2037pg/mg, respectively compared to the untreated control cells (Figure 3.8 F,

compare bar 1 to bars 3 and 5). However, in comparison to the irradiated control cells,

2-AG level was dramatically increased up to 5009 pg/mg (P = 0.002) in UVR-stimulated

cells treated with DHA 10µM (Figure 3.8 F, compare bar 2 to bar 4). However, 2-AG

was decreased to 3223 pg/mg, when DHA 50µM was used in combination with UVR

compared to the irradiated control cells (Figure 3.8 F, compare bar 2 to bar 6).

1-AG levels in the untreated control cells were measured to be 1236 pg/mg, whereas,

UVR significantly increased 1-AG level to 2985 pg/mg (P = 0.001) (Figure 3.8 G,

124

compare bar 1 to bar 2). When these cells were treated with 10µM or 50µM DHA for

72h, 1-AG level increased to 1700 pg/mg and 1433 pg/mg respectively compared to the

untreated control cells (Figure 3.8 G, compare bar 1 to bars 3 and 5). Moreover, 1-AG

levels were additional increased to 3573 pg/mg and 3517pg/mg in UVR-stimulated cells

treated with DHA 10µM or 50µM respectively compared to the irradiated control cells

(Figure 3.8 G, compare bar 2 to bars 4 and 6).

125

Figure 3.8 Effect of DHA treatment on endocannabinoids and NAE levels in

46BR.IN fibroblasts in response to UV radiation. As described in Material and

Methods, cells were treated either with DHA10 or 50 µM for 72h and then they were

irradiated with 15J/cm2 UVB. Cells were harvested with 0.25% trypsin-EDTA solution

24h post UVR. Quantification of endocannabinoids and NAE in 46BR.IN fibroblasts was

carried out using LC-MS/MS analysis. Data shown as mean ± SD of n = 3 experiments.


126


non-irradiated 46BR.IN fibroblasts treated with EPA.

STEA levels in the control cells were calculated to be 5427 pg/mg, whereas, this level

was slightly higher, 5868 pg/mg in the irradiated control cells (Figure 3.9 A, compare

bar 1 to bar 2). Incubation of these cells with EPA 10µM or 50µM for 72h reduced STEA

levels to 4063 pg/mg and 4864 pg/mg, respectively compared to the untreated control

cells (Figure 3.9 A, compare bar 1 to 3 and 5). However, treatment with EPA 10µM or

50µM did not induce any remarkable changes in STEA levels in UV irradiated cells,

compared to the irradiated control cells (Figure 3.9 A, compare bar 2 to bars 4 and 6).

Figure 3.9 B, illustrated that PEA levels in the control cells were measured to be 3336

pg/mg, while, this level was slightly decreased to 3174 pg/mg in the irradiated control

cells (Figure 3.9 B, compare bar 1 to bar 2). Two different concentrations of EPA (10µM

or 50µM) were used. PEA levels decreased to 2340 pg/mg and 2378 pg/mg,

respectively in dose dependent manner compared to the untreated control cells (Figure

3.9 B, compare bar 1 to bars 3 and 5). Whereas, in the irradiated cells treated with EPA

10µM or 50µM, PEA levels increased to 3402 pg/mg and 3860 pg/mg respectively, in a

dose dependent manner compared to the irradiated control cells (Figure 3.9 B, compare

bar 2 to bars 4 and 6)

OEA levels in the untreated cells were estimated to be 3484 pg/mg, while UV radiation

down-regulated this level to 2591 pg/mg (Figure 3.9 C, compare bar 1 to bar 2).

Incubation of 46BR.IN fibroblastic cells with EPA 10µM or 50µM for 72h decreased the

OEA level significantly to 1886 pg/mg (P = 0.015) and marginally to 2782 pg/mg

respectively compared to the untreated control cells (Figure 3.9 C, compare bar 1 to

bars 3 and 5) However, OEA level was increased to 3070 pg/mg in irradiated cells

treated with EPA 10µM (Figure 3.9 C, bar 4), but with EPA 50µM this level was dropped

to 1940 pg/mg compared to the irradiated control cells (Figure 3.9 C, compare bar 2 to

bars 4 and 6).

DHEA levels in the untreated control cells were considered to be 255 pg/mg, whereas,

this level was raised up to 327 pg/mg in the irradiated control cells (Figure 3.9 D,

127

compare bar 1 to bar 2). Treatment with EPA 10µM decreased DHEA level to 214 pg/mg

whilst, EPA 50µM significantly increased DHEA level to 452pg/mg (P = 0.008) compare

to the untreated control cells (Figure 3.9 D, compare bar 1 to bars 3 and 5). This figure

also illustrated that DHEA significantly increased to 656 pg/mg (P = 0.001) and 604

pg/mg (P = 0.001), respectively after EPA 10µM or 50µM treated cells were irradiated

compared to the irradiated control cells (Figure 3.9 D, compare bar 2 to bars 4 and 6).

Figure 3.9 E showed AEA level at 32pg/mg in the untreated control cells (Figure 3.9 E,

bar 1). UVR stimulation, did not induce any major change in AEA level in the irradiated

control cells (30 pg/mg) (Figure 3.9 E, bar 2). Moreover, treatment of the nonirradiated

cells with EPA, either 10µM or 50µM also did not produce any noticeable change in

AEA level, 30 pg/mg and32 pg/mg, respectively compared to the untreated control cells

(Figure 3.9 E, compare bar 1 to bars 3 and 5). In addition, when the UV irradiated cells

were treated with the same fatty acids either 10µM or 50µM no significant changes were

seen in AEA level in this experiment (32pg/mg and 33pg/mg) (Figure 3.9 E compare

bar 2 and bars 4 and 6).

2-AG levels in nonirradiated control cells were estimated to be 3144 pg/mg however, in

the irradiated control cells this level significantly increased about 3-fold to 10501 pg/mg

(P = 0.001) (Figure 3.9 F, compare bar 1 to bar 2). No significant differences were seen

when these cells were incubated with EPA 10µM or 50µM for 72h, 3518 pg/mg and

3453 pg/mg respectively (Figure 3.9 F, compare bar 1 to bars 3 and 5). In addition,

when these cells were treated with EPA 10µM, 2-AG level was approximately similar to

its level in the irradiated control cells but, when EPA 50µM was used 2-AG level slightly

decreased to 9025 pg/mg (Figure 3.9 F, compare bar 2 to bars 4 and 6).

1-AG levels in the untreated control cells were assessed to be 3059 pg/mg (Figure 3.9

G, bar 1). Treatment with EPA 10µM or 50µM for 72h decreased 1-AG level to 2835

pg/mg and 3853 pg/mg respectively (Figure 3.9 G, compare bar 1 to bars 3 and 5). UVR

stimulation significantly increased 1-AG level to 10377 pg/mg (P = 0.001) compared to

nonirradiated control cells (Figure 3.9 G, compare bar 1 to bar 2). However, 1-AG level

was slightly increased to 11251 pg/mg in UVR-stimulated cells treated with EPA 10µM

128

(Figure 3.9 G, compare bar 2 to bar 4) and then reduced to 7818 pg/mg when these

cells treated with EPA 50µM (Figure 3.9 G, compare bar 2 to bar 6).

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Figure 3.9 Effect of EPA treatment on endocannabinoids and NAE levels in

46BR.IN fibroblasts in response to UV radiation. As described in Material and

Methods, cells were treated either with EPA10 or 50 µM for 72h and then they were

irradiated with 15J/cm2 UVB. Cells were harvested with 0.25% trypsin-EDTA solution

24h post UVR. Quantification of endocannabinoids and NAE in 46BR.IN fibroblasts was

carried out using LC-MS/MS analysis. Data shown as mean ± SD of n = 3 experiments.


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3.4.10. Quantification of the endocannabinoids and other NAE in culture medium

from irradiated and non-irradiated 46BR.IN fibroblasts treated with OA

Because of N=2 in the OA experiments, statistically it is not possible to assess if there

are any significant differences in endocannabinoids congeners level between the

different groups in those experiments.

STEA levels in control medium were 3001 pg/ml (Figure 3.10 A, bar 1). Adding OA

10µM or 50µM to cell culture medium decreased STEA level to 2000 pg/ml and 2501

pg/ml respectively compared to the untreated control (Figure 3.10 A, compare bar 1 to

bars 3 and 5). Furthermore, STEA level was decreased to 2501 in the irradiated control

culture medium compared to the nonirradiated control medium (Figure 3.10 A, compare

bar 1 to bar 2). Treatment with OA either 10µM increased STEA level in cell culture

medium of the irradiated cells, whereas, 50µM did not induce any significant change

compare to their control (Figure 3.10 A, compare bar 2 to bars 4 and 6).

Figure 3.10 B, presented DHEA level at 473 pg/ml in the untreated control medium

(Figure 3.10 B, bar 1). This level was dropped to 359 pg/ml after UVR stimulation

compared to nonirradiated culture medium (Figure 3.10 B, compare bar 1 to bar 2).

Using two different concentrations of OA, either 10µM or 50µM also decreased DHEA

level to 2257 pg/ml and 402 pg/ml respectively in the nonirradiated culture medium

compared to their control (Figure 3.10 B, compare bar 1 to bars 3 and 5). In contrast,

DHEA levels were dose dependently increased in the culture medium of irradiated cells

after treatment with OA 10µM or 50µM compared to the irradiated control culture

medium (Figure 3.10 B, compare bar 2 to bars 4 and 6).

2-AG levels in the untreated control medium were calculated to be 2749 pg/ml (Figure

3.10 C, bar 1) In addition, minor downregulation in 2-AG level was noticed when the

culture medium of the nonirradiated cells was incubated with OA 10µM (1952 pg/ml) or

50µM (2495 pg/ml) compared to their control (Figure 3.63 compare bar 1 to bars 3 and

5). No significant changes were seen in 2-AG level in the culture medium of irradiated

cells compared to nonirradiated cell culture medium (Figure 3.10 C, compare bar 1 to

bar 2). However, treatment with OA 10µM or 50µM did not induce any major changes

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in 2-AG levels, 2580 pg/ml and 2527 pg/ml respectively compare to the medium of the

irradiated control cells (Figure 3.10 C, compare bar 2 to bars 4 and 6).

1-AG levels in the untreated control culture medium were 34507 pg/ml whereas, this

level was decreased to 25505 pg/ml in the cell culture medium of the irradiated cells

(Figure 3.10 D, compare bar 1 to bar 2). Adding OA 10µM to the cell culture medium

decreased 1-AG levels to 28506 pg/ml while in those treated with 50µM 1-AG level was

increased to 35007 pg/ml (Figure 3.10 D, compare bar 1 to bar 3 and bar 5).

Furthermore, using the UVR in combination either with 10µM or 50µM OA increased1-

AG level in dose dependent manner to 33007 pg/ml and 400008 pg/ml respectively

compared to the irradiated control culture medium (Figure 3. 10 D, compare bar 2 to

bar 4 and bar 6).

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Figure 3.10 Effect of OA treatment on endocannabinoids and NAE levels in

46BR.IN fibroblasts culture medium in response to UV radiation. As described in

Materials and Methods, cells were treated either with OA10 or 50 µM for 72h and then

they were irradiated with 15J/cm2 UVB. Medium was collected 24h post UVR.




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from irradiated and non-irradiated 46BR.IN fibroblasts treated with DHA.

Figure 3.11 A, showed STEA level at 5001 pg/ml in cell cultural medium of untreated

control cells. However, after UVR stimulation this level was significantly reduced to 2334

pg/ml in cell culture of the irradiated control cells compared to the nonirradiated medium

(P = 0.001)(Figure 3.11 A, compare bar 1 to bar 2). Treatment with DHA, either 10µM

or 50µM also significantly decreased STEA level in dose dependent manner to 2834

pg/ml (P = 0.001) and 2334 pg/ml (P = 0.001) compared to untreated culture medium

(Figure 3.11 A, compare bar 1 to bars 3 and 5). In contrast, the combination between

the DHA 10µM or 50µM and UVR increased STEA level to 3001 pg/ml and significantly

to 4001 pg/ml (P = 0.008), respectively compared to cell culture medium of irradiated

control cells (Figure 3.65 compare bar 2 to bars 4 and 6).

DHEA levels were estimated to be 352 pg/ml in the untreated control culture medium.

This level was however, increased to 504 pg/ml after UVR stimulation in the irradiated

control culture medium (Figure 3.11 B, Compare bar 1 to bar 2). DHA 10µM slightly

reduced DHEA level to 320 pg/ml while, DHA 50µM, increased it to 462 pg/ml compared

to the culture medium of nonirradiated cells (Figure 3.11 B, compare bar 1 to bars 3 and

5). Similar effect was observed when DHA 10µM (431 pg/ml) or 50µM (611 pg/ml)

treated culture medium were irradiated, compared to the irradiated control culture

medium (Figure 3.11 B, compare bar 2 to bars 4 and 6).

In untreated control medium, 2-AG levels were assessed to be 2283 pg/ml whereas,

when this culture medium was treated with 10µM or 50µM DHA, marginally increases

were seen in 2-AG levels, 2699 pg/ml and 2486 pg/ml, respectively compared to the

untreated control culture medium (Figure 3.11 C, compare bar 1 to bars 3 and 5). In cell

culture of the irradiated control cells 2-AG level was slightly increased to 2766 pg/ml

compared to the nonirradiated medium (Figure 3.11 C, compare bar 1 to bar 2). In

addition, when the treated medium were irradiated, 2-AG levels increased to 3215 pg/ml

and 3965 pg/ml with DHA 10µM and 50µM, respectively compared to the cell cultural of

the irradiated control cells (Figure 3.11 C, compare bar 2 to bars 4 and 6). Figure 3.11

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D, showed 1-AG levels at 40341 pg/ml in the untreated medium (Figure 3.11 D, bar 1).

UVR stimulation increased this level to 59012 pg/ml compared to the nonirradiated

medium (Figure 3.11 D, compare bar 1 to bar 2). Treatment with DHA10µM and 50µM

decreased this level to 36341 pg/ml and 38508 pg/ml, respectively compared to the

untreated medium (Figure 3.11 D, compare bar 1 to bars 3 and 5). DHA 10µM or 50µM

1-AG also decreased 1-AG level to 37674 pg/ml and 44342 pg/ml, respectively when

the DHA treated medium was irradiated, compared to the irradiated control medium

(Figure 3.11 D, compare bar 2 to bars 4 and 6).

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Figure 3.11 Effect of DHA treatment on endocannabinoids and NAE levels in


Material and Methods, cells were treated either with DHA10 or 50 µM for 72h and then




**P<0.01, ***P<0.001, comparing data to control.

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from irradiated and non-irradiated 46BR.IN fibroblasts treated with EPA.

STEA levels were found to be 3001 pg/ml in cell cultural medium of the untreated control

cells (Figure 3.12 A, bar 1). However, this level was reduced after UVR stimulation to

2167 pg/ml in the irradiated control medium (Figure 3.12 A, compare bar 1 to bar 2).

Treatment with EPA, either 10µM or 50µM decreased STEA level to 2167 pg/ml (Figure

and 2667 pg/ml, respectively. In contrast, the combination between the EPA and UVR

increased the STEA level in dose dependent way to 2334 pg/ml and 3001 pg/ml,

respectively compare to the irradiated control medium (Figure 3.12 A, compare bar 2 to

bars 4 and 6).

Figure 3.12 B, showed DHEA levels at 223 pg/ml in the cell culture of untreated control

cells. After UVR stimulation, this level was almost the same, 247 pg/ml in the irradiated

control medium (Figure 3.12 B, compare bar 1 to bar 2). In compare to the untreated

culture medium, treatment with EPA 10µM or 50µM did not produce any remarkable

change in DHEA levels, 247 pg/ml and 226 pg/ml respectively (Figure 3.12 B, compare

bar 1 to bars 3 and 5). In addition, the combination between EPA and UVR also did not

show any significant changes, as DHEA levels were 258 pg/ml and 303 pg/ml after EPA

10µM and 50µM treatment, respectively (Figure 3.12 B, compare bar 2 to bars 4 and

6).

2-AG levels were estimated to be 2298 pg/mg in cell culture medium of untreated control

cells (Figure 3.12 C, bar 1). However, UVR stimulation significantly increased this level

to 4004 pg/ml in the irradiated control culture medium (P = 0.026) (Figure 3.12 C,

compare bar 1 to bar 2). EPA10µM or 50µM in addition, increased 2-AG protein level to

3309 pg/ml and significantly to 4311 pg/ml (P = 0.009), respectively compared to the

untreated culture medium (Figure 3.12 C, compare bar 1 to bars 3 and 5). Compared to

the irradiated control culture medium, 2-AG level decreased to 3319 pg/mg but

increased again to 4499 pg/ml in cell culture medium of irradiated cells treated with EPA

10µM and 50µM, respectively (Figure 3.12 C, compare bar 2 to bars 4 and 6).

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In untreated control medium, 1-AG levels were measured to be 47343 pg/ml, but in

irradiated control medium this level was slightly decreased to 36841pg/ml (Figure 3.12

D, compare bar 1 to bar 2). Adding EPA10µM and 50µM into cell culture medium of the

nonirradiated cells, reduced 1-AG levels to 31173 pg/ml and 37174 pg/ml, respectively.

In contrast, UVR in combination with 10µM or 50µM EPA increased 1-AG levels to

46676 pg/ml and 41008 pg/ml, respectively, compared to the irradiated control medium

(Figure 3.12 D, bar 2 to bars 4 and 6).

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Figure 3.12 Effect of EPA treatment on endocannabinoids and NAE levels in


Materials and Methods, cells were treated either with EPA10 or 50 µM for 72h and then




**P<0.01, ***P<0.001, comparing data to control.

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Table 3.3. Summary of the significant findings in 46 BR.IN fibroblasts cells and their cell

culture medium in response to DHA and/or UVR. Data shown as mean ± SD of n = 3

experiments. *P<0.05, **P<0.01, ***P<0.001, comparing data to control using one way


140

Table 3.4. Summary of the significant findings in 46 BR.IN fibroblasts cells and their cell

culture medium in response to EPA and/or UVR. Data shown as mean ± SD of n = 3

experiments. *P<0.05, **P<0.01, ***P<0.001, comparing data to control using one way


141

3.5. Discussion

3.5.1. Determination of the OA and UVR effects on the intracellular and

extracellular levels of endocannabinoids and NAE in HaCaT Keratinocytes and

46BR.IN fibroblasts.

As non-essential monounsaturated fatty acid rich in cell membrane, lacking the ability

to be metabolized to DHA or EPA (Grammatikos et al., 1994), OA was selected to be a

control fatty acid in the present study. Our results showed that the UVR induced up-

regulation in STEA, PEA, OEA and DHEA was inhibited when the irradiated HaCaT

keratinocytes were treated with OA. In 46BR.IN fibroblasts the most noticeable effect

was that the intracellular levels of STEA, PEA, OEA, DHEA, AEA, 2-AG and 1-AG were

increased after UVR stimulation. However, this effect was inhibited and enhanced with

OA 10µM and 50µM respectively.

Only a few mediators were detected in the culture medium from HaCaT keratinocytes

and 46BR.IN fibroblasts, including STEA, (OEA HaCaT keratinocyte culture medium

only), DHEA and 2-AG. Fluctuated effects ranging from minor decrease or increase

were observed on the extracellular levels of these mediators after OA treatment and or

UVR exposure. In general, the fluctuation effects of OA treatment on the levels of the

above mentioned mediators could be attributed to the inability of OA to form these

mediators in HaCaT keratinocytes or 46BR.IN fibroblasts. Apart from this, the above

mentioned results related to OA treatment were obtained from experiments carried out

only twice; therefore concrete conclusions cannot be drawn from these observations.

3.5.2. Determination of the n-3 PUFA effects on the intracellular and extracellular

levels of the endocannabinoids and NAE in HaCaT Keratinocytes and 46BR.IN

fibroblasts in response to ultraviolet radiation.

Data from this study showed that DHA treatment increased intracellular levels of PEA,

DHEA and AEA in HaCaT keratinocytes, while mediators such OEA, LEA and 1-AG

were found to be decreased after DHA treatment in these cells. However, DHA has no

major effects on intracellular levels of STEA and 2-AG in HaCaT keratinocytes.

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Furthermore, DHA increased the extracellular levels of OEA, 2-AG and 1-AG in HaCaT

keratinocytes and has no major effects on STEA and DHEA extracellular levels.

However, EPA treatment was found to reduce the intracellular levels of PEA, LEA,

DHEA, 2-AG and 1-AG in HaCaT keratinocytes. EPA treatment increased only OEA

level in HaCaT cells and has no remarkable effects on intracellular levels of STEA and

AEA.

In 46BR.IN fibroblasts, the intracellular levels of STEA, PEA, OEA, LEA, DHEA, AEA,

2-AG and 1-AG was found to be increased after DHA treatment. DHA also increased

the extracellular levels of OEA, 2-AG and 1-AG in these cells, whereas, the extracellular

levels of mediators such STEA and DHEA did not change by DHA. In contrast to DHA,

EPA treatment decreased STEA, PEA, OEA, DHEA and 1-AG intracellular levels in

46BR.IN fibroblasts; EPA had no effects on AEA and 2-AG intracellular levels in

46BR.IN fibroblasts. Similar to DHA, EPA increased the extracellular levels of OEA, 2-

AG and 1-AG in the media of 46BR.IN fibroblasts and had no major effects on STEA

and DHEA extracellular levels.

UV radiation decreased the intracellular levels of STEA, PEA and LEA in HaCaT

keratinocyte and 46BR.IN fibroblasts both in the presence and absence of DHA or EPA

treatments. Fluctuated effects on DHEA, AEA, 2-AG and 1-AG were observed post UV

radiation but in general the intracellular levels of these mediators tended to increase

after UVR exposure in HaCaT keratinocyte and 46BR.IN fibroblasts. Moreover, UVR

induced up-regulation in the extracellular levels of OEA, 2-AG and 1-AG in cell cultural

medium of HaCaT keratinocyte and 46BR.IN fibroblasts. However, the extracellular

levels of STEA and DHEA did not change post UV radiation in the media of these cells.

Overall, the trend shown in these results is consistent with another study carried out on

rodents which showed that a fish oil diet containing high levels of EPA and DHA lead to

a decrease in the intracellular formation of the 2-AG, PEA, STEA and OEA but not AEA

and DHEA (Artmann et al., 2008) in the liver of the test animals. Furthermore, AEA and

2-AG also have been reported to decrease in 3T3-F442A adipocytes after 72h

incubation with 100µM DHA (Matias et al., 2008). One possible interpretation of these

143

results is that DHA can reduce cell and tissue levels of AA and AA - derived downstream

metabolites, including AEA and/or AG, potentially due to substrate competition among

biosynthetic enzymes (Artmann et al., 2008, Connor et al., 1990, Rapoport, 2008,

Watanabe et al., 2003, Lim and Suzuki, 2001, Matias et al., 2008, Batetta et al., 2009).

This competition among lipid biosynthetic enzymes utilizing fatty acids as substrates

could represent an important influence on the formation of downstream lipid

metabolites, including their derived endocannabinoids metabolite components.

Furthermore, the acylation of endocannabinoid precursors such as membrane

phospholipids NAPE and DAG by DHA could be a potential explanation for the changes

observed toward DHA-derived (DHEA) in HaCaT keratinocytes and 46BR.IN

fibroblasts. Alternatively, the results of the current study showed that AEA, DHEA and

PEA intracellular levels increased as DHA concentration increased in the irradiated and

nonirradiated HaCaT keratinocytes. Moreover, AEA and DHEA intracellular levels were

significantly increased in a dose dependent manner after the irradiated and non-

irradiated 46BR.IN fibroblasts were treated with DHA. These results are in agreement

with another study in which it has been demonstrated that brain levels of AEA and DHEA

in piglets increased with DHA treatment (Berger et al., 2001). In addition, other studies

in different species have confirmed an increased formation of AEA, 2-AG and DHEA in

the jejunum of the rat after fish oil diet providing high level of EPA and DHA (Artmann

et al., 2008). DHA has been found in the brain, gut, retina and liver in concentrations

similar or higher than those of AEA (Berger et al., 2001, Wood et al., 2010), therefore,

increase its derivative DHEA level is a normal consequence. Hence, many studies

demonstrated that the DHEA can be synthesized from its precursor in different types of

tissues (Balvers et al., 2010, Kim et al., 2011). Moreover, another study suggested the

N-docosahxaenoyl-PE as a possible precursor of DHEA (Bisogno et al., 1999). The

effect of the DHA and EPA treatment on STEA, OEA, LEA and 2-AG was similar in both

HaCaT keratinocytes and 46BR.IN fibroblasts after UV radiation. In general the changes

recorded in their intracellular levels were minor. UV radiation in this study caused the

DHEA intracellular levels to be significantly increased in DHA treated cells of both used

models (AEA level was increased in 46BR.IN fibroblasts only). DHEA intracellular level

144

was also significantly increased after UV radiation in 46BR.IN fibroblasts treated with

EPA. Moreover, the intracellular levels of 2-AG and its isomer, 1-AG were found to be

significantly increased after UVR exposure either in EPA treated or untreated 46BR.IN

fibroblasts.

All these findings showed that the UV radiation might have an effect on the formation of

endocannabinoids and NAE. These effects can be attributed to phospholipase A2

(PLA2) activity that induced after UVR exposure (Chen et al., 1996, Cohen and DeLeo,

1993, Hanson and DeLeo, 1990), as PLA2 has been reported to be one of the possible

biosynthetic routes of NAEs via N-acyl-lysoPE (Sun et al., 2004). Moreover, PLA2 has

been reported to induce an increase in NAEs levels including AEA (Berdyshev et al.,

2000). Moreover, UVB can phosphorylate ERK (Chang et al., 2011) and p38 mitogen-

activated kinases (Bachelor et al., 2005, Kim et al., 2005) which then triggers NF-kB

(Chun and Surh, 2004). Therefore, UVR could impact FAAH and NAPE-PLD proteins

levels through their transcriptional aspects and gene expression. NF-kB is a translation

factor that perform a key part in cellular reaction to ecological pressure such as UVR by

causing or controlling the expression of particular gene (Herrlich et al., 2008). UVR-

induced NF-kB initial has been revealed in several research. For example, NF-kB initial

in epidermis fibroblasts has been confirmed after UVA radiation (Vile et al., 1995, Reelfs

et al., 2004). Also, UVB was discovered to generate NF-kB initial in human epidermis

keratinocytes (Adhami et al., 2003) and NF-kB could control FAAH gene expression

(Maccarrone et al., 2003a). Furthermore, Liu et al., has recommended NF-kB as the

primary translation factor manages LPS-induced AEA features (Liu et al., 2003).

Moreover, NF-kB was discovered to communicate with specific proteins 1 (SP 1); the

translation factor of NAPE-PLD gene (Sancéau et al., 1995). For example, p65 a subunit

of NF-kB has been found to interact with SP1 (Perkins et al., 1994, Pazin et al., 1996).

Therefore, UVR could regulates the expression of endocannabinoids and their

congeners throughout the transcriptional factors of their genes. All these findings

support our findings. These data could be the first step for future studies to define

potentially beneficial relationships between the effects of n-3 PUFA on

endocannabinoids and their related lipids. Also endocannabinoids modification due to

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UVB exposure could be one of the defense mechanism performance by human skin

cells against UVB. Therefore, the biochemical role of n-3 PUFA in respect to the

interaction with the biosynthesis of endocannabinoids and NAE should be further

investigated.

Apart of UVB, UVA (320-400 nm) is a possible environmental stress has obtained less

attention, even though extreme sun visibility is also associated with extreme contact

with UVA (320-400 nm). The UVA transfer (80 nm) is twice that of UVB (40 nm), and

90% to 95% of solar UV radiation energy that gets to the surface of our planet is UVA;

only 5% to 10% of it is UVB. UVA has a longer period wavelengths than UVB and thus

spreads throughout further into skin. Because its short wavelength, UVB can penetrate

only the full epidermis and only 10 to 15% of it can reach the upper layer of the dermis;

papilla. However, 50% of UVA are absorbed by the epidermis, while the rest penetrate

the dermis up to 2mm depth (Césarini, 1996). The thickness of the epidermis is

increasing with direct consequence on the UVB absorption, making the basal layer of

the epidermis out of reaction with UVB radiations (Césarini, 1996). The UVA sunburn is

more violaceous with prominent vasodilation, therefore, UVA-induced erythema is not

representation of pigmentation or melanogenesis. In the UVB, the induced division of

the keratinocytes increases the thickness of the epidermis and so the total content in

melanin, whereas, in the UVA, the multiplication of keratinocytes is provided directly by

stimulation of melanogenesis (McKinlay and Diffey, 1987). UVA has been proven to

cause mutations in mammalian cells. For example, research in murine cell lines have

confirmed that UVA can generate mutations (Hitchins et al., 1986, Lundgren and Wulf,

1988). Moreover, UVA at 365nm could be mutagenic to cultured human fibroblasts

(Enninga et al., 1986). The prospective dangerous impact of UVA has also been

confirmed in cultured human melanocytes. UVA (320-400 nm) was found to induce DNA

single-strand breaks in cultured human melanocytes (Wenczl et al., 1998, Marrot and

Meunier, 2008). UVB are essentially inducing DNA dimers which are most of the time,

error free repaired. However, UVA induce DNA strand breaks much less frequently than

UVB, but the strand breaks are error prone lesions (Césarini, 1996). As a consequence.

After long tern exposure the genetic code is altered equally by UVA and UVB (Césarini,

146

1996). UVR also has an effect on lipids in human skin where they subjected to

peroxidation through indirect mechanisms after UV radiation (Césarini, 1996). UVB and

UVA leading to activated molecules responsible for the production of several cytokines,

mitosis inducer, vascular changes and systemic distance effects (Césarini, 1996).

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CHAPTER 4: The effect of n-3PUFA on endocannabinoid metabolizing enzymes in HaCaT keratinocytes and 46BR.IN fibroblasts in response to ultraviolet radiation.

4.1. Introduction

4.1.1. Cyclooxygenase-2

COX-2 is a stress inducible enzyme, expressed in different human skin cells such as

fibroblasts, macrophages and keratinocytes (Gilroy et al., 2001). In response to different

stimuli such as UVB, phorbol 12-myristate 13-acetate (PMA) and interleukin-1β (IL-1β),

the expression of COX-2 in these cells is stimulated. This in turn plays an important role

in inflammatory and tissues injury (Gilroy et al., 2001). Moreover, UVB has been

demonstrated to activate the cytosolic phospholipase A2 (cPLA2), which mediates

prostaglandin synthesis in human keratinocytes during the first 6h of UVB exposure

(Gresham et al., 1996). COX-2 mRNA and COX-2 protein levels have been detected in

human keratinocytes following 24 h of UVB irradiation irrespectively of the presence of

serum (Buckman et al., 1998). Also, COX-2 expression is significantly increased in

human dermis and epidermis 24 h after UVB exposure (Rhodes et al., 2009). Moreover,

a recent study demonstrated the up-regulation of COX-2 expression and protein levels

in cultured HaCaT cells and in mice subjected to 30mJ/cm2 UVB (Lee et al., 2013b).

COX-2 is upregulated in response to induction by multiple mitogenic and pro-

inflammatory stimuli, including growth factors, cytokines, hormones, serum, hypoxia,

bacterial endotoxin, and carcinogens (Smith et al., 2000, Trifan and Hla, 2003, Buckman

et al., 1998). Furthermore, COX-2 is involved in pathophysiological conditions such as

inflammation, pain, fever wound repair, angiogenesis, vasodilation and increased

vascular permeability (Smith et al., 2000, Gilroy et al., 2001, Funk, 2001).

While AA is the normal substrate for COX-1 and COX-2 isoforms, other fatty acids can

be oxygenated by both of them (Laneuville et al., 1995, Wada et al., 2007, Yuan et al.,

2009). COX-2 for example, has up to 30% efficiency to oxygenate the fatty acid

eicosapentaenoic acid (EPA) (20:5 n-3) compared with AA. Whereas, COX-1 efficiency

to oxygenate the same fatty acid is less than 5% (Wada et al., 2007, Malkowski et al.,

148

2001). COX-2 can oxygenate a more extensive array of substrates compared with COX-

1, presumably because of an approximately 20% larger active site (Kurumbail et al.,

1996). Also, it has been established that COX-2, but not COX-1, oxygenates the

endocannabinoids 2-AG and AEA to produce respectively prostaglandin glycerol esters

(PG-Gs) and prostaglandin ethanolamines (PG-EAs) (Kozak et al., 2000, Kozak et al.,

2001b). 2-AG and AEA are widely distributed in mammalian tissues including human

skin (Bisogno, 2008). COX-2 oxygenates endocannabinoid substrates using the same

mechanism employed for AA, generating prostaglandin, thromboxane, and prostacyclin

ethanoloamides, glycerol esters, and glycine (Kozak et al., 2000, Kozak et al., 2002a,

Kozak et al., 2001a). The ability of COX-2 to metabolize endocannabinoid substrates

suggests that this isoform may be involved in the endogenous endocannabinoid

signalling system. Therefore, our study aimed to investigate the effects of UVR and n-3

PUFAs on COX-2 in relation to endocannabinoids.

4.1.2. Lipoxygenases

5, 12, and 15 Lipoxygenases (LOX), are related to the dioxygenase family. They are

involved in the polyunsaturated fatty acid metabolism in human skin. Some of these

LOX are involved in skin disorders. 15-LOX was found to exist in two isoforms, 15-LOX-

1 and 15-LOX-2. 15-LOX-1 was reported to be the main enzyme metabolizing linoleic

acid into 13-hydroxyoctadecadienoic acid (13-HODE) (Baer et al., 1991), while 15-LOX-

2 metabolize AA into 15-HETE (Brash et al., 1997). 12-LOX and 15-LOX expression in

human keratinocyte cell line has been demonstrated to be affected by UV radiation.

UVR decrease the 12-LOX and increase the 15-LOX levels in these cells line (Yoo et

al., 2008). In addition, Rhodes et al. found that LOX metabolites such as 11-HETE, 12-

HETE and 15-HETE were increased at, 4h, 18h and 24h respectively, after UVB

irradiation of human skin (Rhodes et al., 2009)

12-LOX has been reported to metabolise the endocannabinoid AEA to produce the

ethanolamide derivative of 12(S)-HETE-EA in rat pineal gland (Hampson et al., 1995,

Ueda et al., 1995). Purified 12-LOX from porcine leukocytes also has been reported to

149

oxygenate AEA to produce 12(S)-HETE-EA (Hampson et al., 1995). This study has also

showed that the porcine 12-LOX enzyme did not discriminate between the AEA and AA.

Ueda et al. reported that the 12-LOX from porcine leukocytes and the 15-LOX-1 from

rabbit reticulocytes could oxygenate AEA at rates comparable to those for AA (Ueda et

al., 1995). However, human platelet 12-LOX was only marginally active, and porcine

leukocyte 5-LOX was inactive with AEA as the substrate (Ueda et al., 1995).

Furthermore, another study demonstrated that 12-LOX from porcine leukocytes, could

efficiently oxygenate 2-AG to produce the glycerol ester of 12(S)-HETE (12(S)-HETE-

G) (Moody et al., 2001). Kozak et al. showed that human 15-LOX-1, and human 15-

LOX-2 metabolized 2-AG efficiently, whereas human leukocyte 5-LOXs showed no

activity with this substrate (Kozak et al., 2002b).

4.2. Aims of this study

Since n-3 PUFA alter the pathways of COX and LOX enzymes that are involved in the

metabolism of endocannabinoids and NAE (Kozak et al., 2002a, Kozak et al., 2002c,

Kozak et al., 2004), they could have an effect on the endocannabinoids pathways in

skin cells. This may lead to increased NAE production such as N-stearoylethanolamide

which has been reported to have pro-apoptotic activity (Maccarrone et al., 2002) and/or

N-palmitoylethanolamide that showed anti-inflammatory activity, analgesic and

neuroprotective effects (Calignano et al., 1998, Lambert et al., 2002, LoVerme et al.,

2005). This manipulation of the endocannabinoid pathways through n-3 PUFA could be

the major defense mechanism behind the protective effect of n-3 PUFA against UVR in

the skin cells. Accordingly, the hypothesis of this part of our study was based on that

the n-3 PUFA particularly EPA and DHA, may play more protective role saving human

skin cells from the harm of UVR through their effect on the endocannabinoids

metabolizing enzymes such as COX-2 and LOXs. Therefore, this study aimed to

investigate the effect of n-3 PUFA and UVR on protein levels of the inducible isoforms

of COX-2, 12-LOX and 15-LOX. The effect of n-3 PUFA and UVR on the protein levels

of enzymes, respectively NAPE-PLD and FAAH that control formation and degradation

of endocannabinoids was also examined.

150

4.3 Materials and Methods

4.3.1. Western Blotting (refer to section 2.3 chapter 2)

4.3.2. Materials (refer to section 2.3.1 chapter 2)

4.3.3. Primary and secondary antibodies (refer to section 2.3.2 chapter 2)

4.3.4. Protein extraction (refer to section 2.4.1 chapter 2)

4.3.5. Determination of protein concentration (refer to section 2.4.2 chapter 2)

4.3.6. SDS-PAGE (refer to section 2.5. chapter 2)

4.3.7. Statistical analysis (refer to section 2.10. chapter 2)

4.4. RESULTS

4.4.1. The effect of UVR and n-3 PUFA on COX-2 protein levels in HaCaT

keratinocytes and 46BR.IN fibroblasts.

4.4.1.1. The effect of DHA and UVR

The results shown in Figure 4.1 suggested that UVR does not significantly up-regulate

COX-2 protein level in 46BR.IN fibroblasts compared to non-irradiated control cells

(Figure 4.1 A and B). However, DHA treatment at concentration10μM or 50μM

significantly decreased COX-2 level (P = 0.007 and 0.002, respectively) compared to

the untreated control cells (Figure 4.1 A and B). Although these effects were reversed

by UVR, DHA treatments were able to retain COX-2 in lower levels compared to the

non-irradiated and irradiated control cells, especially DHA 50μM which showed stronger

inhibitory effect (P = 0.03) against UVR-induced COX-2 up-regulation (Figure 4.1 A and

B).

The results shown in Figure 4.2 indicate some changes in COX-2 protein level among

the differentially treated groups but all the below discussed changes in Figure 4.2 were

not enough to be accounted as significant differences. This data indicated that COX-2

protein level was increased in UVR irradiated HaCaT keratinocytes compared to the

151

non-irradiated cells (Figure 4.2 A and B). In the treated HaCaT cells, COX-2 level was

slightly increased in cells treated with DHA (10μM or 50μM) compared to untreated

control. This effect was enhanced with UVR radiation when combined with treated cells

(Figure 4.2 A and B). This data also showed that COX-2 levels were higher than any

other cells when the DHA 10μM or 50μM treated cells were exposed to UV radiation.

152

Figure 4.1 COX-2 protein levels in DHA and UVR treated 46BR.IN fibroblasts. (A)

Western blot analysis of COX-2 protein levels in 46BR.IN fibroblasts treated with10μM

or 50μM DHA and irradiated with UVR or left untreated as indicated. GAPDH was used

as loading control. One representative out of 3 independent experiments is shown. (B)

Densitometry analysis of Western blots was carried out using ImageJ software. The

intensity of the COX-2 protein band was normalized to that of the GAPDH protein band.

The ratio of the intensity of the COX-2 band with the intensity of the GAPDH band in the

non-treated cells was arbitrarily set to 1. The ratio of the intensity of the COX-2 versus

the intensity of the GAPDH band in the other groups was calculated accordingly. Data

shown as mean ± SD of n = 3 experiments. *P<0.05, **P<0.01, ***P<0.001, comparing

data to control.

153

Figure 4.2 COX-2 protein levels in DHA and UVR treated HaCaT keratinocytes.

A. Western blot analysis of COX-2 protein levels in HaCaT keratinocytes treated

with10μM or 50μM DHA and irradiated with UVR or left untreated as indicated. GAPDH

was used as loading control. One representative out of 3 independent experiments is

shown. B. Densitometry analysis of Western blots was carried out using ImageJ

software. The intensity of the COX-2 protein band was normalized to that of the GAPDH

protein band. The ratio of the intensity of the COX-2 band with the intensity of the

GAPDH band in the non-treated cells was arbitrarily set to 1. The ratio of the intensity

of the COX-2 versus the intensity of the GAPDH band in the other groups was calculated

accordingly. Data shown as mean ± SD of n = 3 experiments. Comparing data to control.

154

4.4.1.2. The effect of OA and UVR

The effect of OA and UVR on COX-2 protein level was followed in 46BR.IN fibroblasts

untreated or treated with 10μM or 50μM OA in the presence or absence of UVR

irradiation using Western blot analysis. COX-2 protein level was not found to be

significantly increased after UV radiation compared to the non-irradiated 46BR.IN

fibroblasts (Figure 4.3 A and B). However, treatment with OA using 10μM concentration

kept COX-2 level at the same range of untreated control cells. A similar effect was seen

when the OA was used with UVR compared to the irradiated control cells (Figure 4.3 A

and B). Whereas, increasing the concentration of OA up to 50μM caused the COX-2

level to be slightly upregulated compared to untreated control cells. However, no change

was seen when these cells were treated with UVR compared to the irradiated control

cells (Figure 4.3 A and B).The same result was seen in the data shown in Figure 4.4

where the effect of OA and UVR on COX-2 protein level in HaCaT keratinocytes is

depicted. These data (Figure 4.4 A and B) also indicated that the effect of 10μM or 50μM

OA alone or in combination with UVR on COX-2 protein level was similar to that seen

in 46BR.IN fibroblasts under the same conditions.

4.4.1.3. The effect of EPA and UVR

The effect of EPA and UVR on COX-2 protein level was investigated in 46BR.IN

fibroblasts and HaCaT keratinocytes, untreated or treated with10μM or 50μM EPA in

the presence or absence of UVR irradiation using western blot analysis. COX-2 protein

level was observed to be increased in UVR irradiated cells in both 46BR.IN fibroblasts

and HaCaT keratinocytes compared to their untreated control cells (Figure 4.5 A and B

and Figure 4.6 A and B). Dose dependent effect appeared as a down-regulation of COX-

2 protein level was recorded in 46BR.IN fibroblasts and HaCaT keratinocytes treated

with EPA 10μM or 50μM compared to the untreated control cells. Nevertheless the

statistical analysis did not show any significant differences (Figure 4.5 A and B and

Figure 4.6 A and B). However, COX-2 protein levels were noted to be increased after

155

those groups of EPA treatment were submitted to UVR exposure in both cells models;

46BR.IN fibroblasts and HaCaT keratinocytes (Figure 4.5 A and B and Figure 4.6 A and

B).

Figure 4.3 COX-2 protein levels in OA and UVR treated in 46BR.IN fibroblasts. A.

Western blot analysis of COX-2 protein levels in 46BR.IN fibroblasts treated with 10μM

or 50μM OA and irradiated with UVR or left untreated as indicated. GAPDH was used

as loading control. One representative out of 3 independent experiments is shown. B.


intensity of the COX-2 protein band was normalized to that of the GAPDH protein band.

The ratio of the intensity of the COX-2 band with the intensity of the GAPDH band in the

non-treated cells was arbitrarily set to 1. The ratio of the intensity of the COX-2 versus


shown as mean ± SD of n = 3 experiments. Comparing data to control.

156

Figure 4.4 COX-2 protein levels in OA and UVR treated in HaCaT keratinocytes. A.

Western blot analysis of COX-2 protein levels in HaCaT keratinocytes treated with 10μM


as loading control. Data are representative of 3 experiments. B. Densitometry analysis

of Western blots was carried out using ImageJ software. The intensity of the COX-2

protein band was normalized to that of the GAPDH protein band. The ratio of the

intensity of the COX-2 band with the intensity of the GAPDH band in the non-treated

cells was arbitrarily set to 1. The ratio of the intensity of the COX-2 versus the intensity

of the GAPDH band in the other groups was calculated accordingly. Data shown as

mean ± SD of n = 3 experiments. Comparing data to control.

157

Figure 4.5 COX-2 protein levels in EPA and UVR treated in 46BR.IN fibroblasts. A.

Western blot analysis of COX-2 protein levels in 46BR.IN fibroblasts treated with10μM

or 50μM EPA and irradiated with UVR or left untreated as indicated. GAPDH was used

as loading control. Data are representative of 3 experiments. B. Densitometry analysis

of Western blots was carried out using ImageJ software. The intensity of the COX-2

protein band was normalized to that of the GAPDH protein band. The ratio of the

intensity of the COX-2 band with the intensity of the GAPDH band in the non-treated

cells was arbitrarily set to 1. The ratio of the intensity of the COX-2 versus the intensity

of the GAPDH band in the other groups was calculated accordingly. Data shown as

mean ± SD of n = 3 experiments. Comparing data to control.

158

Figure 4.6 COX-2 protein levels in EPA and UVR treated in HaCaT Keratinocytes.

A. Western blot analysis of COX-2 protein levels in HaCaT Keratinocytes treated

with10μM or 50μM EPA and irradiated with UVR or left untreated as indicated. GAPDH



software. The intensity of the COX-2 protein band was normalized to that of the GAPDH

protein band. The ratio of the intensity of the COX-2 band with the intensity of the

GAPDH band in the non-treated cells was arbitrarily set to 1. The ratio of the intensity

of the COX-2 versus the intensity of the GAPDH band in the other groups was calculated


159

4.5. The effect of UVR and n-3 PUFA on LOX protein levels in HaCaT keratinocytes

and 46BR.IN fibroblasts.

LOX mediated metabolism of AEA and 2-AG has been discussed before in this chapter

(see section 4.2.1). Several Western blots experiments were carried out to assess the

effect of UVR and n-3 PUFA on LOX protein in HaCaT keratinocytes and 46BR.IN

fibroblasts. Since it is difficult to detect LOX protein levels possibly because of low levels

of expression of this enzyme in human skin cells three representative experiments will

be presented in this section.

Data in Figure 4.7 showed that 12-LOX protein level was increased after UVR exposure

in the irradiated 46BR.IN fibroblasts cells compared to non-irradiated control cells.

However a small decrease in its level was observed in cells treated with OA 10μM. On

the other hand, in cells treated with OA 10μM plus UVR, 12-LOX level was as same as

its level in the irradiated control cells (Figure 4.7 A and B). Also, cells that treated with

OA 50μM and OA 50μM plus UVR showed similar level of 12-LOX to that in untreated

and irradiated control cells respectively (Figure 4.7 A and B). Results in Figure 4.8 also

showed that 12-LOX level was increased in the irradiated control cells compared to non-

irradiated. Cells treated with DHA10μM presented minor decreases effect in 12-LOX

level compared to untreated 46BR.IN fibroblasts (Figure 4.8 A and B). This effect was

increased when a higher DHA concentration was used (50μM). UVR failed to increase

12-LOX level in the DHA treated (10μM or 50μM) cells (Figure 4.8 A and B). As in

46BR.IN fibroblasts, HaCaT keratinocytes cells treated with UV increased12-LOX level

compared to the non-irradiated cells (Figure 4.9 A and B). No changes in 12-LOX levels

were seen in cells treated with DHA 10μM or 50μM compared to untreated control cells

(Figure 4.9 A and B). Combined with UVR, DHA 10μM or 50μM also did not induce any

change in 12-LOX protein level (Figure 4.9 A and B).

160

Figure 4.7 12-LOX protein levels in OA and UVR treated in in 46BR.IN fibroblasts.

A. Western blot analysis of 12-LOX protein levels in 46BR.IN fibroblasts treated with

10μM or 50μM OA and irradiated with UVR or left untreated as indicated (n = 1). GAPDH

was used as loading control. B. Densitometry analysis of Western blots was carried out

using ImageJ software. The intensity of the 12-LOX protein band was normalized to that

of the GAPDH protein band. The ratio of the intensity of the 12-LOX band with the

intensity of the GAPDH band in the non-treated cells was arbitrarily set to 1. The ratio

of the intensity of the 12-LOX versus the intensity of the GAPDH band in the other

groups was calculated accordingly.

161

Figure 4.8 12-LOX protein levels in DHA and UVR treated in in 46BR.IN fibroblasts.

A. Western blot analysis of 12-LOX protein levels in 46BR.IN fibroblasts treated with

10μM or 50μM DHA and irradiated with UVR or left untreated as indicated (n = 1).

GAPDH was used as loading control. B. Densitometry analysis of Western blots was

carried out using ImageJ software. The intensity of the 12-LOX protein band was

normalized to that of the GAPDH protein band. The ratio of the intensity of the 12-LOX

band with the intensity of the GAPDH band in the non-treated cells was arbitrarily set to

1. The ratio of the intensity of the 12-LOX versus the intensity of the GAPDH band in

the other groups was calculated accordingly.

162

Figure 4.9 12-LOX protein levels in OA and UVR treated in HaCaT keratinocytes.

A. Western blot analysis of 12-LOX protein levels in HaCaT keratinocytes treated with

10μM or 50μM OA and irradiated with UVR or left untreated as indicated (n = 1). GAPDH

was used as loading control. B. Densitometry analysis of Western blots was carried out

using ImageJ software. The intensity of the 12-LOX protein band was normalized to that

of the GAPDH protein band. The ratio of the intensity of the 12-LOX band with the


of the intensity of the 12-LOX versus the intensity of the GAPDH band in the other

groups was calculated accordingly.

163

4.6. The effect of UVR and n-3 PUFA on NAPE-PLD and FAAH protein levels in

HaCaT keratinocytes and 46BR.IN fibroblasts

NAPE-PLD and FAAH are the enzymes responsible for endocannabinoid biosynthesis

and degradation, respectively. Protein levels of these enzymes were investigated in

HaCaT keratinocytes and 46BR.IN fibroblasts. In three independent experiments, these

cells were treated with DHA, EPA and OA at two doses: 10μM or 50μM. Each

experiment was divided into two groups; control with and without UVR and n-3 PUFAs

treated with and without UVR (see sections 2.2.5 and 2.2.6 for more details). NAPE-

PLD and FAAH protein levels were estimated by Western blot analysis.

4.6.1. Effect of OA and UVR on FAAH and NAPE-PLD in HaCaT keratinocytes

The effect of OA and UVR on FAAH protein level in HaCaT keratinocyte cell line was

assessed using Western blot analysis (Figure 4.10 A). Insignificant down-regulation

effect was seen in FAAH protein level in the irradiated compared to non-irradiated

HaCaT cells (Figure 4.10 A and B). Data in this figure showed that OA treatment at dose

10μM or 50μM increased FAAH levels compared to the untreated control cells (Figure

4.10 A and B). However, a combination of OA 10μM or 50μM treated groups with UVR

did not affect FAAH levels significantly compared to the irradiated controls cells (Figure

4.10 A and B). NAPE-PLD levels were slightly increased after UVR exposure compared

to the non-irradiated control cells. Furthermore, treatment either with OA 10μM or 50μM

insignificantly decreased NAPE-PLD compared to the non-irradiated. Moreover, NAPE-

PLD levels also were insignificantly reduced with OA 10μM or 50μM plus UVR

compared to the irradiated control cells. In general, NAPE-PLD protein levels were

almost similar in all treated groups either with or without UV radiation (Figure 4.11 A

and B).

164

4.6.2. Effect of EPA and UVR on FAAH and NAPE-PLD in HaCaT keratinocytes cell

lines.

FAAH protein level was reduced in the irradiated control HaCaT keratinocytes

compared to the non-irradiated control cells (Figure 4.12 A and B). Dose dependently

increase was observed in HaCaT keratinocytes treated with EPA 10µM or 50µM

compared to untreated HaCaT cells (Figure 4.12 A and B). However, this effect was

reversed by UVR in both, EPA10µM and 50µM treated groups. In this case FAAH levels

were reduced to the same level as in the irradiated control cells (Figure 4.12 A and B).

Data presented in Figure 4.13 suggested that UV radiation increased NAPE-PLD

protein level in HaCaT cells compared to the non-irradiated control. Furthermore,

NAPE-PLD protein levels also increased in cells treated with EPA 10µM and this effect

was enhanced with EPA 50µM (P = 0.002) compare to the untreated HaCaT cells

(Figure 4.13 A and B). However, combination of EPA 10µM and UVR induced

insignificant decreases in NAPE-PLD level compared to the irradiated control cells but

this effect was reversed in cells treated with EPA 50µM plus UVR compared to the

irradiated control HaCaT cells (Figure 4.13 A and B).

4.6.3. Effect of DHA and UVR on FAAH and NAPE-PLD in HaCaT keratinocytes

Data in Figure 4.14 indicated that FAAH protein level was reduced after HaCaT cells

were submitted to UV radiation compared to the non-irradiated control cells. However,

this level was increased in cells treated with treated with DHA 10µM compared to

untreated cells (Figure 4.14 A and B). This effect was then prevented by UVR which

reduced FAAH levels but to range upper than in the irradiated control cells. Likewise,

treatment with DHA 50µM decreased FAAH to its lowest level in this experiment and

this effect was however terminated by UV radiation which brought FAAH level to be as

same as in the irradiated cells treated with DHA 10µM (Figure 4.14 A and B). NAPE-

PLD protein level was decreased in HaCaT keratinocytes submitted to UVR compared

to non-irradiated control cells (Figure 4.15 A and B). Furthermore, its level was also

decreased in cells treated with DHA10µM or 50µM compared to untreated cells.

165

However, when this group was exposed to UVR these effects were reversed (Figure

4.15 A and B).

Figure 4.10: FAAH protein levels in OA and UVR treated in HaCaT keratinocytes.

A. Western blot analysis of FAAH protein levels in HaCaT keratinocytes treated

with10μM or 50μM OA and irradiated with UVR or left untreated as indicated. GAPDH



software. The intensity of the FAAH protein band was normalized to that of the GAPDH

protein band. The ratio of the intensity of the FAAH band with the intensity of the GAPDH

band in the non-treated cells was arbitrarily set to 1. The ratio of the intensity of the

FAAH versus the intensity of the GAPDH band in the other groups was calculated


166

Figure 4.11: NAPE-PLD protein levels in OA and UVR treated in HaCaT

keratinocytes. A. Western blot analysis of NAPE-PLD protein levels in HaCaT

keratinocytes treated with10μM or 50μM OA and irradiated with UVR or left untreated

as indicated. GAPDH was used as loading control. One representative out of 3

independent experiments is shown. B. Densitometry analysis of Western blots was

carried out using ImageJ software. The intensity of the NAPE-PLD protein band was

normalized to that of the GAPDH protein band. The ratio of the intensity of the NAPE-

PLD band with the intensity of the GAPDH band in the non-treated cells was arbitrarily

set to 1. The ratio of the intensity of the NAPE-PLD versus the intensity of the GAPDH

band in the other groups was calculated accordingly. Data shown as mean ± SD of n =

3 experiments. Comparing data to control.

167

Figure 4.12: FAAH protein levels in EPA and UVR treated in HaCaT keratinocytes.

A. Western blot analysis of FAAH protein levels in HaCaT keratinocytes treated with

10μM or 50μM EPA and irradiated with UVR or left untreated as indicated. GAPDH was

used as loading control. One representative out of 3 independent experiments is shown.

B. Densitometry analysis of Western blots was carried out using ImageJ software. The

intensity of the FAAH protein band was normalized to that of the GAPDH protein band.

The ratio of the intensity of the FAAH band with the intensity of the GAPDH band in the

non-treated cells was arbitrarily set to 1. The ratio of the intensity of the FAAH versus



168

Figure 4.13: NAPE-PLD protein levels in EPA and UVR treated in HaCaT


keratinocytes treated with 10μM or 50μM EPA and irradiated with UVR or left untreated








3 experiments. **P<0.01, comparing data to control.

169

Figure 4.14: FAAH protein levels in DHA and UVR treated in HaCaT keratinocytes.

A. Western blot analysis of FAAH protein levels in HaCaT keratinocytes treated with

10μM or 50μM DHA and irradiated with UVR or left untreated as indicated. GAPDH was








170

Figure 4.15: NAPE-PLD protein levels in DHA and UVR treated in HaCaT


keratinocytes treated with10μM or 50μM DHA and irradiated with UVR or left untreated








3 experiments. Comparing data to control.

171

4.6.4. Effect of OA and UVR on FAAH and NAPE-PLD protein level in 46BR.IN

fibroblasts

The effect of OA and UVR on FAAH protein level in 46BR.IN fibroblasts was studied

using western blot analysis. Our findings demonstrated that there were on significant

changes in FAAH protein level in UVR irradiated 46BR.IN cells compared to non-

irradiated control cells (Figure 4.16 A and B). However, no changes were observed in

FAAH levels in OA 10μM or 50μM treated cells compared to the untreated control cells.

Compared to the irradiated control cells, minor increases were noticed in FAAH levels

when those treated cells exposed to UVR.

NAPE-PLD protein level showed similar patterns to that of FAAH protein levels in

46BR.IN fibroblasts where its level was also insignificantly decreased after UVR

exposure compared to non-irradiated control cells. This minor effect was enhanced

when the OA (10μM or 50μM) treatment was combined with UV radiation (Figure 4.17

A and B). Data also showed a slight reduction in NAPE-PLD level in cells treated with

OA 10μM. In contrast, OA at concentration 50μM caused the level of NAPE-PLD to be

increased compared to untreated control cells (Figure 4.17 A and B).

4.6.5. Effect of EPA and UVR on FAAH and NAPE-PLD in 46BR.IN fibroblasts

FAAH protein level was reduced after UV radiation compared to the non-irradiated

control cells. EPA treatment in both doses used (10µM and 50µM) also induced minor

decrease in FAAH level compared to the untreated cells (Figure 4.18 A and B). A similar

effect was observed when those treated cells were submitted to UV radiation (Figure

4.18 A and B).

The data shown in Figure 4.19 showed that there were minor increases in NAPE-PLD

levels in the irradiated cells compared to non-irradiated control cells (Figure 4.19 A and

B). In addition, both doses used (10µM or 50µM) of EPA induced dose dependently

minor increases compared to the untreated cells (Figure 4.19 A and B). This effect was

enhanced when those treated cells were exposed to UV radiation.

172

4.6.6. Effect of DHA and UVR on FAAH and NAPE-PLD in 46BR.IN fibroblasts

FAAH protein levels were found to be slightly decreased in the irradiated 46BR.IN

fibroblasts compared to the non-irradiated control cells (Figure 4.20 A and B). Also, a

similar effect was observed in DHA 10µM or 50µM and in DHA 50µM plus UVR (Figure

4.20 A and B). However, a small increase in FAAH level was seen in cells treated with

DHA 10µM combined with UV radiation (Figure 4.20 A and B). Data in Figure 4.21

indicated that NAPE-PLD protein level also was reduced after UVR compared to non-

irradiated cells (Figure 4.21 A and B). Comparing to the untreated control cells, DHA

10µM also showed the same effect. On the other hand, treatment with DHA 50µM and

DHA 10 µM or 50 µM plus UVR increased NAPE-PLD levels to the same range

compared to the irradiated and non-irradiated control cells (Figure 4.21 A and B).

173

Figure 4.16: FAAH protein levels in OA and UVR treated in 46BR.IN fibroblasts. A.

Western blot analysis of FAAH protein levels in 46BR.IN fibroblasts treated with 10μM


as loading control. One representative out of 3 independent experiments is shown. B.







174

Figure 4.17: NAPE-PLD protein levels in OA and UVR treated in 46BR.IN

fibroblasts. A. Western blot analysis of NAPE-PLD protein levels in 46BR.IN fibroblasts

treated with 10μM or 50μM OA and irradiated with UVR or left untreated as indicated.

GAPDH was used as loading control. One representative out of 3 independent

experiments is shown. B. Densitometry analysis of Western blots was carried out using

ImageJ software. The intensity of the NAPE-PLD protein band was normalized to that

of the GAPDH protein band. The ratio of the intensity of the NAPE-PLD band with the


of the intensity of the NAPE-PLD versus the intensity of the GAPDH band in the other

groups was calculated accordingly. Data shown as mean ± SD of n = 3 experiments.

Comparing data to control.

175

Figure 4.18: FAAH protein levels in EPA and UVR treated in 46BR.IN fibroblasts.

A. Western blot analysis of FAAH protein levels in 46BR.IN fibroblasts treated with

10μM or 50μM EPA and irradiated with UVR or left untreated as indicated. GAPDH was








176

Figure 4.19: NAPE-PLD protein levels in EPA and UVR treated in 46BR.IN


treated with 10μM or 50μM EPA and irradiated with UVR or left untreated as indicated.









177

Figure 4.20: FAAH protein levels in DHA and UVR treated in 46BR.IN fibroblasts.

A. Western blot analysis of FAAH protein levels in 46BR.IN fibroblasts treated with

10μM or 50μM DHA and irradiated with UVR or left untreated as indicated. GAPDH was








178

Figure 4.21: NAPE-PLD protein levels in DHA and UVR treated in 46BR.IN


treated with10μM or 50μM DHA and irradiated with UVR or left untreated as indicated.









179

4.7 Discussion

4.7.1. The effect of omega-3 polyunsaturated fatty acids and UVR on COX-2

protein expression in HaCaT keratinocytes and 46BR.IN fibroblasts

UVR radiation can cause skin disorders like sunburn, DNA damage and carcinogenesis.

It is well documented that COX-2 overexpression post UVR is involved in skin

inflammation and skin cancer and it may be the key factor behind all the detrimental

effects that UVR induces. However, n-3 PUFA are well known for their beneficial role

as anti-inflammatory compounds which may be a promising component that may play

a crucial role against the detrimental effects of UVR on skin cells. Therefore, in this

study we sought to examine the effect of n-3 PUFA on UVR-induced up-regulation in

COX-2 protein expression in HaCaT keratinocytes and 46BR.IN fibroblasts.

COX-2 is expressed in human keratinocytes and fibroblasts and involved in keratinocyte

differentiation (Leong et al., 1996) and human foreskin fibroblastic (HFF) cells (Gilroy et

al., 2001). Many factors may be involved in COX-2 expression such as multiple

mitogenic and pro-inflammatory stimuli (Maldve and Fischer, 1996, Feng et al., 1995).

Among these factors, growth factors (Feng et al., 1995), cytokines (Feng et al., 1995),

bacterial endotoxin (Xie et al., 1992) and carcinogens (Kujubu et al., 1991). Also, as

mentioned before during the differentiation process in human keratinocytes (Leong et

al., 1996) or fibroblastic cells (Gilroy et al., 2001). According to the Gilroy study, COX-2

expression is increased in fibroblasts cells when they were exposed to phorbol 12-

myristate 13-acetate (PMA) or interleukin-1β (IL-1β), this happened during the

quiescent G0 phase. Whereas when the cells entered the committed G1 phase of the

cell cycle, COX-2 expression was suppressed. Also it was reported that COX-2 mRNA

levels were reduced by 53% and 70% when a majority of the cells had entered the S

and G2/M phases of cell cycle, respectively. Gilroy et al., also suggested that COX-2

expression in cycling cells is firmly controlled to avoid excessive oxidative DNA damage.

Therefore, any dysregulation in this system could lead to detrimental effects.

Results from our study showed that COX-2 levels were up-regulated in the UVR

irradiated cells in levels slightly higher than those that were not irradiated in both cell

180

models. These results are in agreement with other previous studies showing up-

regulation of COX-2 protein levels following UVB irradiation in HaCaT keratinocytes

(Buckman et al., 1998, Tang et al., 2001, Kim et al., 2007). COX-2 protein expression

was also found to be up-regulated in human keratinocytes in volunteers subjected to

UVB radiation up to 4 times (Buckman et al., 1998). COX-2 was also overexpressed in

the skin of hairless mice upon UVB radiation (Kim et al., 2007). Moreover, Bachelor et

al., reported an increase in COX-2 mRNA after UVB radiation in SKH-1 mouse

epidermis (Bachelor et al., 2005). Findings from all of the above mentioned studies were

in agreement with our results.

It has been suggested that UVB induces COX-2 expression throughout increased

activation of transcription factors, p38 mitogen activated protein kinase (MAPK) and

phosphatidylinositol 3 kinase (PI3K) which will then phosphorylate the cyclic AMP-

responsive element binding protein (CREB) which is needed for the COX-2 to be

expressed (Bachelor et al., 2005). In addition, Tang et al., (2001) demonstrated that

CREB is essential for both basal and UVB induced COX-2 expression in HaCaT

keratinocytes (Tang et al., 2001). Furthermore, NF-kB is another transcription factor

which may play a key role in the UVB-induced COX-2 expression. Accordingly, UVB

has been found to lead to phosphorylation of ERK (Chang et al., 2011) and p38 mitogen-

activated kinases (Bachelor et al., 2005, Kim et al., 2005) which then activates NF-kB

and induces COX-2 expression (Chun and Surh, 2004). However, Chen et al.,

demonstrated that the ERK was not essential for UVB-induced COX-2 gene expression

in HaCaT keratinocytes (Chen et al., 2001). Moreover, it has been suggested that UVR

induced cytoplasmic expression of human antigen R (HuR) which in turn increases

COX-2 mRNA stability (Sengupta et al., 2003, Tong et al., 2007).

The effect of the n-3 PUFA DHA and EPA on COX-2 expression was also examined.

Our findings indicated that DHA treatment significantly decreased COX-2 expression in

46BR.IN fibroblasts in dose dependent manner (Figure 4.1). UVR-induced COX-2

expression was also dose dependently inhibited by 10μM or 50μM DHA in the same

cells. Similar results in different cell lines have been demonstrated by previous study

where DHA reported to block COX-2 expression in UVR stimulated keratinocytes

181

(Zhang and Bowden, 2008). Moreover, DHA has been also shown to revert UVR-

induced COX-2 expression in nr-HaCaT cells (Sengupta et al., 2003, Zhang and

Bowden, 2008) In this case DHA reduced the expression of COX-2 mRNA stabilizer

HuR in many cells including keratinocytes (Sengupta et al., 2003, Zhang and Bowden,

2008). Moreover, topical application of DHA has been also reported to inhibit UVB-

induced COX-2 expression in mouse skin (Rahman et al., 2011). According to Rahman

et al., (2011) this effect was attributed to the ability of DHA to attenuate UVB-induced

DNA binding of NF-kB through the inhibition of the phosphorylation of IKKα/β and

phosphorylation and degradation of IKBα and nuclear localization of p50 and p65

proteins. However, our results showed an opposite effect, when HaCaT keratinocytes

were treated with DHA 10μM or 50μM, DHA did not inhibit UVR induced COX-2

expression in the irradiated cells (Figure 4.2). Our results here are also in consistent

with another study that used a different stimulus and different type of cells. They were

found that the DHA enhanced phorbol ester or interleukin 1β-induced COX-2 expression

in rat vascular endothelial cells (Machida et al., 2005).The authors attributed this effect

to the ability of DHA induced p44/42 MAPK activation. Similar effect of DHA was also

reported by Hirafuji et al., and Diep et al., who suggested the role of DHA induced

p44/42 or p38 MAPK activation (Diep et al., 2000, Hirafuji et al., 2002).

Data from the present study also showed that EPA 10µM or 50µM decreased COX-2

protein levels in concentration dependent manner in both HaCaT keratinocytes and

46BR.IN fibroblasts cells. These results are in line with what another study indicated

that COX-2 expression was increased in young skin human after EPA treatment (Kim

et al., 2006). EPA and DHA also reduced COX-2 expression in HT-29 human colorectal

cells (Calviello et al., 2004). Furthermore, COX-2 expression was found to be decreased

in rats treated with flaxseed oil which is a high source of n-3 PUFA (Bommareddy et al.,

2006). Similar to DHA, EPA may exert its effect on COX-2 expression through NFkB.

EPA has been reported to inhibit NFkB expression in cultured pancreatic and human

monocytes via inhibition of IkB degradation, the inhibitory subunit of NFkB (Lo et al.,

1999, Novak et al., 2003, Zhao et al., 2004). This finding has been shown by another

study that suggested that endogenously biosynthesized n-3 PUFA protect transgenic

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mice from colitis through a decrease in NFkB activity (Hudert et al., 2006). However,

our results showed that both EPA treatment and UV radiation synergistically induced

COX-2 expression in both cells types. Our data also showed that UVR significantly

increased COX-2 level in EPA treated HaCaT keratinocytes. In another study, a

synergistic action has also been reported between UVB and EPA to induce TNF-α

expression (Pupe et al., 2002). Indeed, TNF-α has been suggested to stimulate gene-6

(TSG-6) which in turn up-regulate COX-2 expression in macrophages which

preferentially produce PGD2 and its metabolite 15-deoxy-12,14-prostaglandinJ2

(15dPGJ2) (Mindrescu et al., 2005). 15dPGJ2 and n-3 PUFA both may play a key role

in the activation of the anti-inflammatory peroxisome-proliferator-activated-receptor

gamma (PPAR gamma) (Jump and Clarke, 1999, Mindrescu et al., 2005). In addition,

EPA and DHA has also been found to up-regulate COX-2 expression in HaCaT

keratinocytes (Chene et al., 2007). The effect could be essential to increase the

production of 3-series PGs (i.e. PGD3, PGI3 and PGE3) through EPA and DHA-induced

COX-2 expression (Fischer et al., 1988). It should be noted that the 3-series PGs may

play an important role as an anti-inflammatory eicosanoids as they are weaker

inflammatory than 2-series PGS (Bagga et al., 2003).

4.7.2. The effect of omega-3 polyunsaturated fatty acids and UVR on NAPE-PLD

and FAAH protein expression in HaCaT keratinocytes and 46BR.IN fibroblasts

Here we explore for the first time the effect of the UVB on the NAPE-PLD and FAAH,

the two enzymes that responsible for AEA biosynthesis and degradation respectively.

Although, there were no significant differences, our results showed some trends

indicating that UVR may reduce FAAH levels while, mixed effects observed on NAPE-

PLD levels in HaCaT keratinocytes and 46BR.IN fibroblasts. These were supported by

other observations presented in this study that AEA and 2-AG synthesis was found to

be slightly increased after UVR exposure of both types of cells models used in this study

(Figures 3.2 F, 3.7 F, 3.8 F and 3.9 F. Chapter 3).

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DHA and EPA treatment decreased FAAH levels and increased NAPE-PLD levels in

both HaCaT keratinocytes and 46BR.IN fibroblasts. This has lent support to other

results in this study showing that DHA increased AEA production in HaCaT

keratinocytes (Figure 3.2 F. Chapter 3).

In an attempt to explain our findings, we examined many aspects. Presumably, UVR

could affect FAAH and NAPE-PLD protein levels through their transcriptional factors

and gene expression. NF-kB is a transcription factor that plays a key role in cellular

response to environmental stress such as UV radiation by inducing or suppressing the

expression of specific gene (Herrlich et al., 2008). UVR-induced NF-kB activation has

been reported in several studies. For example, NF-kB activation in human skin

fibroblasts has been demonstrated after UVA radiation (Vile et al., 1995, Reelfs et al.,

2004). Also, UVB was found to induce NF-kB activation in human epidermal

keratinocytes (Adhami et al., 2003). Apart from this, Leptin was demonstrated to

stimulate the activity and expression of FAAH throughout FAAH gene by activating the

promoter region through a STAT3 (signal transduction and activator of transcription 3)

element (Maccarrone et al., 2003a). Moreover, Waleh et al., has reported that FAAH

enzyme has estrogen response element (ERE) in its FAAH gene in mouse (Waleh et

al., 2002). Thus, sex hormones have been reported to down-regulate FAAH gene

expression (Maccarrone et al., 2000). Progesterone for example was found to up-

regulate the FAAH gene in human T-cells via the Ikaros transcription factor (Maccarrone

et al., 2003a). In contrast, endogenous steroid 17β-estradiol (E2) has been suggested

to regulate many transcription factors and may be enhance FAAH gene expression at

transcriptional and translational level (Rossi et al., 2007). It is noteworthy noting that

estrogen receptors (ER) has been found to interact with some transcription factors to

regulate or repress transcription (Cheng et al., 2003). NF-kB is one of these transcription

factors that were found to interact with ER (Frasor et al., 2009). Therefore, an interaction

between NF-kB and ER may be one of the mechanisms behind UVR-induced FAAH

down-regulation observed in our results. This is in line with previous observation that

suggested NF-kB could regulate FAAH expression (Maccarrone et al., 2003b).

184

Furthermore, Liu et al., has suggested NF-kB as the main transcription factor that

regulates LPS-induced AEA synthesis (Liu et al., 2003).

Data from the present study also showed that UV radiation affected NAPE-PLD protein

levels in a fluctuated manner. However, the general trend was that UVR suppressed

NAPE-PLD expression. These data are in line with another research reported that LPS

produce an inhibition effect on NAPE-PLD expression (Zhu et al., 2011). In fact, these

observations may be due to many reasons, the effect of UV radiation on gene

expression of this enzyme could be one of them. NAPE-PLD gene was cloned in rodent

and human (Okamoto et al., 2004). The expression of this gene has been found to be

controlled by the transcription factor SP1 (Zhu et al., 2011). An environmental stress

such as UV radiation may affect gene expression through up- or downregulation of their

transcriptional factors. These may occur via the interaction of transcriptional factors with

each other as mentioned earlier. For instance, a functional interaction between SP1

and NF-kB has been shown to induce IL-6 gene expression (Sancéau et al., 1995).

Also, p65 which is subunit of NF-kB has been found to synergistically activate HIV-1

transcription with SP1 (Perkins et al., 1994, Pazin et al., 1996). Therefore, interaction of

NF-kB with SP1 can act synergistically to activate transcription factor of many target

genes. Therefore, this is may be the case behind UVR-induced changes in NAPE-PLD

levels in our study, especially with the notion of that the environmental stress induced

genes expression was found to be controlled by the transcription factor, NF-kB (Hayden

and Ghosh, 2012).

NF-kB itself could be controlled by n-3 PUFA which then regulates cellular signalling

and genes expression, in particular those related to inflammatory signalling and lipid

metabolism (Sampath and Ntambi, 2005, Schmitz and Ecker, 2008). In addition, n-3

PUFA could affect the membrane phospholipids and as a result, the production of

eicosanoids (Sampath and Ntambi, 2005). As an example, n-3 PUFA have been found

to affect eicosanoids production through their inhibitory effect on leptin and then the

expression of lipogenic gene in adipose tissue (Jump and Clarke, 1999, Raclot et al.,

1997), This may explain the down-regulation of FAAH by EPA and DHA observed in our

study, as leptin was found to stimulate the activity of FAAH gene expression

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(Maccarrone et al., 2003a). As a matter of interest, the effect of n-3 PUFA on genes

expression seems to be very complicated. For example, they could exert positive and

negative effects on NF-kB (Sampath and Ntambi, 2005). n-3 PUFA can initiate the

phosphorylation of the IkB and then allow NF-kB nucleus translocation and as a results

the transcription of different genes such as COX-2 and IL-6 (Calder, 1997, Khalfoun et

al., 1997). In contrast, n-3 PUFA was reported to decrease the phosphorylation of the

IkB and then inhibit NF-kB translocation and target gene transcription (Camandola et

al., 1996, Zhao et al., 2004). All these could explain the effects of n-3 PUFA on the

FAAH and NAPE-PLD protein levels observed in the current study.

Taken together these results, it suggest that n-3 PUFA can induce endocannabinoid

production through their effect on endocannabinoid related enzymes. UVR-induced

endocannabinoid biosynthesis could suggest a defense mechanism of skin cells

followed UV radiation. However, further studies are still required.

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CHAPTER 5: Investigation the effect of ultraviolet radiation on endocannabinoids and N-acylethanolamines in White Caucasians of skin phototype II and South Asians of skin phototype V

5.1. Introduction

The endocannabinoids AEA and 2-AG and NAE are widely distributed in many cells and

tissues (Devane et al., 1988, Devane et al., 1992, Matsuda et al., 1990, Mechoulam et

al., 1995, Schmid, 2000). They are involved in many physiological and pathological

functions including anti-nociception, anti-inflammatory effects, reproduction, modulation

of vascular tone, obesity, cancer, schizophrenia, sleep and drug addiction (Di Marzo,

1998, Piomelli, 2003, Pacher et al., 2006).

Ultraviolet radiation (UVR) is an environmental insult that can cause several health

disorders to human beings ranging from skin inflammation to skin cancer. It is well

known that UVR exerts its harmful effects on the human body through their interaction

with many biological processes such as genes expression and lipid biosynthesis and

metabolism pathways. Endocannabinoids and NAE have been suggested to play a key

role as anti-inflammatory and anti-cancer bioactive lipids. However, the effect of UVR

on their biochemical pathways has not been fully studied. The effect of UVR on

endocannabinoids and their congeners on human skin cells was examined in chapters

3 and 4 of this study. Here we examine the hypothesis that UVR may affect

endocannabinoid levels in the circulation and we examined this using volunteers of two

different ethnicities. Therefore, the specific objective here was to investigate the effect

of UVR on serum endocannabinoids and NEA. Volunteers of two skin types were used

to measure the levels of serum endocannabinoids and NAE in response to multiple

doses of UVR.

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5.2. Aim of the study

To explore the effect of UVR on the levels of endocannabinoids and N-

acylethanolamines in human serum from White Caucasians of skin phototype II and

South Asians of phototype V.

5.3. Materials and Methods (For more details about the volunteers, experimental

design and lipid extraction please refer to Chapter 2, sections 2.9.1, 2.9.2, 2.9.3, 2.9.4,

2.9.5 and 2.9.6).

5.3.1. Statistical Analysis (refer to chapter 2 section 2.10).

5.4. RESULTS

Volunteers were from two different ethnic groups; 10 White Caucasians of skin

phototype II and 6 South Asians of skin phototype V. They were subjected to 8 doses

of UVR each time receiving the same dose (1.3 SED) from day 4, 7, 14, 21, 28, 35 and

day 39 of the study, the baseline was coded as day 0.

5.4.1. Effect of UVR on serum endocannabinoids and N-acylethanolamines

5.4.1.1 Effect of UVR on serum palmitoylethanolamide

Palmitoylethanolamide (PEA) level in White Caucasians was estimated to be 3160

pg/ml on day 0 which represents the baseline of the PEA, pre UVR. On day 4, PEA level

was increased to 4048 pg/ml and then from the day 7 to day 39, PEA levels were almost

as same as the baseline level (Figure 5.1, compare day 0 to days 7, 14, 21, 28, 35 and

39 ). PEA baseline level in South Asians participants was found to be 2814 pg/ml on

day 0. Compared to this baseline, PEA levels were slightly decreased to 2571 pg/ml,

2473 pg/ml, 2533 pg/ml and 2474 pg/ml on day 4, 7, 21 and 39 respectively. Whereas,

on day 28 which is the time point for the fifth dose of the UVR, PEA level was slightly

increased up to 3023 pg/ml compared to the baseline (Figure 5.1, compare day 0 to day

28). While, on day 14 and 35 the PEA levels were approximately similar to the baseline

level (Figure 5.1, compare day 0 to day 14 and 35). Overall, One-Way ANOVA followed

188

by Tukey post hoc test did not show any significant differences in PEA levels between

the different time points of UVR exposure either in the White Caucasians of skin

phototype II or in the South Asians of skin phototype V. As a result, it was concluded

that exposure to UVR for 6 weeks did not cause significant changes in PEA levels in

the human serum samples from White Caucasians of skin phototype II and South

Asians of skin phototype V.

Figure 5.1. PEA levels in serum before and after multiple UVR exposures (1.35

standard erythemal dose) in White Caucasians (skin phototype II, n=10 volunteers) and

South Asians (skin phototype V, n = 6 volunteers). Data shown as mean ± SD of n = 3

experiments. Comparing data to control.

189

5.4.1.2. Effect of UVR on serum linoleoyalethanolamide

Linoleoyalethanolamide (LEA) baseline levels were measured to be 870pg/ml and

1015pg/ml in skin phototype II and skin phototype V respectively. The comparison

between the different time points of UVR exposure was done by One-way ANOVA

following by Tukey Post hoc tests. There were no significant changes in LEA levels in

both skin phototype between all-time points of UVR exposures. LEA baseline in White

Caucasians of skin phototype II was assessed to be 870pg/ml. UVR exposure from day

4 to day 21 did not induce any noteworthy changes in LEA levels compared to the

baseline on day 0. However, in day 28 LEA level was increased up to 1004 pg/ml and

then decreased again to 947 pg/ml, 885 pg/ml in day 35 and 39 respectively. In addition,

the LEA baseline in South Asians of skin phototype V was calculated to be 1015 pg/ml.

The repeated doses of UVR on days 4, 7, 21 and 39 decreased the LEA level to 916

pg/ml, 920 pg/ml, 928 pg/ml and 969 pg/ml respectively. However, on day 14, 28 and

35 there were no important changes in LEA level compared to the baseline (Figure 5.2,

compare day 0 to day 14, 28 and 35). Consequently, One-Way ANOVA showed that 6

weeks of UVR exposure did not bring about any significant changes in LEA levels

between the different time points in the examined groups

Figure 5.2. LEA levels in serum before and after multiple UVR exposures (1.35




190

5.4.1.3. Effect of UVR on serum oleoylethanolamide

OEA baseline levels were calculated to be 1412pg/ml and 798pg/ml in skin phototype

II and skin phototype V respectively. The comparison between the OEA levels in each

time points of UVR exposure was done by One-way ANOVA following by Tukey Post

hoc tests. Accordingly, there were no significant changes in OEA levels either in skin

phototype II or phototype V (Figure 5.3).

OEA baseline level in White Caucasians of skin phototype II was calculated to be

1412pg/ml while, this level slightly increased to 1513pg/ml, 1700pg/ml and 1589pg/ml

on day 4, day 28 and day 39 respectively. However, there were no intense changes in

OEA levels among the other time points of UVR exposure representative on day 7, 14,

21 and 35 (Figure 5.3). In South Asians of phototype V, OEA baseline level was

estimated to be 798pg/ml on day 0. Compared to this baseline level, no significant

changes were seen in OEA level especially on day 4, 14 and 28 (Figure 5.3, compare

day 0 to day 4, 14 and 28). However, on day 7, 21, 35 and 39 OEA levels insignificantly

decreased to 684pg/ml, 598pg/ml, 675pg/ml and 681pg/ml respectively (Figure 5.3,

compare day 0 to day 7, 21, 35 and 39).

Figure 5.3. OEA levels in serum before and after multiple UVR exposures (1.35




191

5.4.1.4. Effect of UVR on serum stearoylethanolamide

Stearoylethanolamide (STEA) baseline levels were considered to be 713 pg/ml and 671

pg/ml in skin phototype II and skin phototype V respectively. The comparison between

the STEA levels in each time points of UVR exposure was done by One-way ANOVA

following by Tukey post hoc test. As a result, there were no significant changes in STEA

levels in both skin phototype between all-time points of UVR exposures. STEA baseline

level in the White Caucasians group was estimated to be 713 pg/ml (Figure 5.4).

However, STEA level was 943 pg/ml after the first dose of the UVR on day 4. In the

South Asians group STEA baseline was assessed to be 671 pg/ml (Figure 5.4).

However, STEA levels in volunteers from South Asia were decreased on day 4 and 7

after the first two doses of the UVR to 436 pg/ml and 457 pg/ml respectively. Whereas,

on day 14, 21, 28, 35 and 39, STEA levels were increased to 548 pg/ml, 493 pg/ml, 591

pg/ml, 560 pg/ml and 557 pg/ml respectively (Figure 5.4, compare day 0 to days 4, 7,

14, 21, 28, 35 and 39). Generally, different time points of UVR exposure did not induce

any significant changes in STEA levels in both tested groups.

Figure 5.4. STEA levels in serum before and after multiple UVR exposures (1.35




192

5.4.1.5. Effect of UVR on serum docosahxaenoylethanolamide

Docosahxaenoylethanolamide (DHEA) baseline levels were considered to be 2797

pg/ml and 792 pg/ml in skin phototype II and skin phototype V respectively. The

comparison between the DHEA levels in each time points of UVR exposure was done

by One-way ANOVA following by Tukey Post hoc tests. There were no significant

differences in DHEA levels of both tested groups of skin phototypes (Figure 5.5). DHEA

baseline level was considered to be 2797 pg/ml (day 0). Compared to this baseline,

DHEA level was decreased to 2282 pg/ml after the first dose of UVR on day 4 (Figure

5.5, compare day 0 to day 4). However, no significant differences were recorded

between these time points. Regardless of DHEA levels on day 4, there were no dramatic

changes in the DHEA levels between the other time points representative on day 7, 14,

21, 28, 35 and 39 (Figure 5.5). In addition, Serum samples from six South Asians of

skin phototype V were extracted and analyzed for DHEA. Baseline levels were

assessed to be 792 pg/ml (day 0). DHEA levels were increased to 849 pg/ml, 1081

pg/ml, 1103 pg/ml and 1127 pg/ml on days 4, 14, 28 and 35 respectively. (Figure 5.17,

compare day 0 to days 4, 14, 28 and 35). However, on days 7, 21 and 39 DHEA levels

were decreased to 707 pg/ml, 626 pg/ml and 755 pg/ml respectively, compared to the

baseline level on day 0 (Figure 5.17, compare day 0 to days 7, 21 and 39). Accordingly,

we can conclude that UVR exposure for 6 weeks did not end in any significant changes

in DHEA levels in the White Caucasians and South Asians groups when the UVR effect

was examined in each group separately

193

Figure 5.5. DHEA levels in serum before and after multiple UVR exposures (1.35




5.4.1.6. Effect of UVR on serum arachidonoylethanolamide

Arachidonoylethanolamide (AEA) baseline levels were considered to be 326 pg/ml and

280 pg/ml in skin phototype II and skin phototype V respectively. The comparison

between the AEA levels in each time points of UVR exposure was done by One-way

ANOVA following by Tukey post hoc test (Figure 5.6). White Caucasians volunteers

were extracted and analyzed for AEA. AEA level was estimated to be 326 pg/ml (day 0,

Figure 5.6). In general, AEA levels were similar between all the time points of UVR

exposure compared to the baseline on day 0. (Figure 5.6, compare day 0 to day 4, 7,

14, 21, 28, 35 and 39). Thus, no significant differences in AEA levels were found in this

experiment. AEA baseline level in South Asians volunteers was considered to be 280

pg/ml (day 0, Figure 5.6). AEA levels were decreased to 150 pg/ml, 196 pg/ml and 145

pg/ml on days 4, 7 and day 21 respectively, compared to day 0 (Figure 5.6, compare

194

day 0 to days 4, 7 and 21). In addition, AEA levels were also slightly decreased to

272pg/ml, 202pg/ml and 237pg/ml on days 14, 35 and 39 respectively. On the other

hand, on day 28 AEA level was increased to 322pg/ml compared to the baseline level

(Figure 5.6, compare day 0 to day 28). At the end, no significant differences were found

in this experiment.

Figure 5.6. AEA levels in serum before and after multiple UVR exposures (1.35




5.4.1.7. Effect of UVR on serum 2-arachidonoyl glycerol

2-arachidonoyl glycerol (2-AG) baseline levels were considered to be 993 pg/ml and

1786 pg/ml in skin phototype II and skin phototype V respectively. The comparison

between the 2-AG levels in each time points of UVR exposure was done by One-way

ANOVA following by Tukey Post hoc tests. Based on our data, there were no significant

differences in 2-AG levels in all time points. 2-AG baseline in White Caucasians group

was calculated to be 993 pg/ml on day 0 (Figure 5.7). Compared to its baseline, 2-AG

level was markedly increased up to 2364 pg/ml after the first exposure of UVR on day

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4. In day 7, 2-AG level was decreased to 1101pg/ml recording the nearest level to the

baseline on day 0 (Figure 5.7, compare day 0 to day 7). After that, 2-AG level was

increased again to 1732 pg/ml, 1575 pg/ml, 1474 pg/ml, and 1527 pg/ml on day 14, 21,

28 and 35 respectively. However, on day 39, 2-AG level decreased to 1231 pg/ml but

still more than its baseline level.

2-AG baseline in South Asians group was considered to be 1786 pg/ml on day 0 (Figure

5.7). Compared to its baseline, 2-AG level was increased up to 2088pg/ml, 2487pg/ml

and 2560pg/ml on day 4, 21 and 28 respectively (Figure 5.7, compare day 0 to days 4,

21 and 28). Conversely, on day 7, 14, 35 and 39, 2-AG levels was slightly decreased to

1800 pg/ml, 1699 pg/ml, 1682 pg/ml and 1185 pg/ml respectively, compared to the

baseline level (Figure 5.7, compare day 0 to days 7, 14, 35 and 39). Finally, our results

did not show any significant changes in 2-AG levels between the different time points of

UVR exposure either in White Caucasians or South Asians group.

Figure 5.7. 2-AG levels in serum before and after multiple UVR exposures (1.35




196

5.4.1.8. Effect of UVR on serum 1-arachidonoyl glycerol

1-arachidonoyl glycerol (1-AG) baseline in White Caucasians was considered to be 742

pg/ml on day 0 (Figure 5.8). Compared to the baseline, UVR did not alter the 1-AG

levels in day 4, 7, 14, 28 and 35. Whereas, 1-AG levels were slightly decreased on day

22 compared to day 0 (Figure 5.8, compare day 0 to days 4, 7, 14, 21, 28 and 35). The

highest level of 1-AG was seen on day 39 (865pg/ml) (Figure 5.8, compare day 0 to day

39). 1-AG baseline in South Asians was measured to be 847 pg/ml on day 0 (Figure

5.8). 1-AG level was decreased to 726 pg/ml, 508 pg/ml, 651 pg/ml, 618 pg/ml, 713

pg/ml and 467 pg/ml on days 4, 7, 14, 28, 35 and 39 respectively (Figure 5.8, compare

day 0 to days 4, 7, 14, 28, 35 and 39). On the contrary, 1-AG level was increased to

895 pg/ml on day 21 compared to its baseline level on day 0 (Figure 5.8, compare day

0 to day 21). Overall, there were no significant changes in 1-AG level between all the

time points of UVR exposure in the data obtained from both groups this experiment.

Figure 5.8. 1-AG levels in serum before and after multiple UVR exposures (1.35




197

5.4.1.9. Effect of UVR on docosapentaenoylethanolamide

Docosapentaenoylethanolamide (DPEA) level in White Caucasians was measured to

be 76pg/ml (day 0, Figure 5.9). Overall, DPEA levels were similar among all the time

points of UVR exposure compared to the baseline on day 0. (Figure 5.9, compare day

0 to day 4, 7, 14, 21, 28, 35 and 39). DPEA baseline level in South Asians was assessed

to be 48 pg/ml (day 0, Figure 5.9). Also, there were no important changes in DPEA

levels, they were similar between all the time point of UVR exposures compared to the

baseline on day 0. (Figure 5.9, compare day 0 to day 4, 7, 14, 21, 28, 35 and 39). Hence,

no significant differences in DPEA levels were found in this experiment according to

One-Way ANOVA test used for both groups.

Figure 5.9. DPEA levels in serum before and after multiple UVR exposures (1.35




198

5.4.1.10. Effect of UVR on serum Myristoylethanolamide

Myristoylethanolamide (MEA) level in White Caucasians was considered to be 465pg/ml

on day 0, whereas on day 4, 7 and day 28, MEA levels were increased to 723pg/ml,

599pg/ml and 793pg/ml respectively (Figure 5.10, compare day 0 to day 4, 7 and day

28). After that, MEA levels were decreased on day 14, 21, 35 and 39 to 374pg/ml,

541pg/ml, 449pg/ml and 403pg/ml respectively, compared to the baseline level (Figure

5.10, compare day 0 to day 14, 21, 35 and 39). MEA baseline level in South Asians was

considered to be 335pg/ml on day 0. Compared to the baseline, MEA levels were

increased to 337pg/ml, 449pg/ml, 773pg/ml, 477pg/ml and 492pg/ml on days 4, 7, 14,

28 and 35 respectively (Figure 5.10, compare day 0 to days 4, 7, 14, 28 and 35). The

highest level of MEA was recorded at day 15 (773pg/ml). However, MEA levels were

decreased only on day 21 and 39 to 299 pg/ml and 297 pg/ml respectively, compared

to the baseline level (Figure 5.10, compare day 0 to day 21 and day 39). Yet, One-Way

ANOVA did not show any significant differences in MEA levels between all the time

points of UVR exposure in both groups examined. Therefore, this result conclude that

exposure to UVR for 6 weeks did not elicit significant changes in MEA level compared

to the MEA baseline.

Figure 5.10. MEA levels in serum before and after multiple UVR exposures (1.35




199

5.4.1.11. Effect of UVR on serum dihomo gamma linoleoyalethanolamide

Dihomo gamma linoleoyalethanolamide (DGLEA) level in White Caucasians was

assessed to be 25 pg/ml (day 0, Figure 5.11). In general, DGLEA levels were similar

between all the time points of UVR exposure compared to the baseline on day 0. (Figure

5.11, compare day 0 to day 4, 7, 14, 21, 28, 35 and 39). DGLEA baseline level in South

Asians was calculated to be 20pg/ml on day 0. Compared to the baseline, there were

no major changes in DGLEA levels between all the time points of UVR exposure (Figure

5.11, compare day 0 to day 4, 7, 14, 21, 28, 35 and 39). Consequently, no significant

differences were seen in DGLEA levels in this experiment. One-Way ANOVA followed

by Tukey did not show any significant differences in the DGLEA levels between different

time points of UVR exposure in all groups.

Figure 5.11. DGLEA levels in serum before and after multiple UVR exposures (1.35




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5.4.1.12. Effect of UVR on serum eicosapentaenoylethanolamide

Eicosapentaenoylethanolamide (EPEA) was found in 6 out of 10 White Caucasians of

skin phototype II. EPEA baseline level was calculated to be 44 pg/ml. this level did not

change after the first dose of UVR on day 4 (43 pg/ml). Compared to the baseline on

day 0, EPEA levels however increased to 69pg/ml, 72 pg/ml, 70 pg/ml, 88 pg/ml and 79

pg/ml from the second dose of UVR on day 7 until day 35 respectively (Figure 5.12,

compare day 0 to days 7, 14, 21, 28 and 35). On day 39, EPEA level decreased to the

baseline level (45pg/ml). These data were analyzed using One-Way ANOVA to

investigate the differences in the EPEA levels between different time points of UVR

exposure. Statistically, there were no significant differences in EPEA levels after 6

weeks of UVR exposure, compared to the EPEA baseline.

Figure 5.12. EPEA levels in serum before and after multiple UVR exposures (1.35




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5.5. Discussion

The aim of this study was to determine the effect of UVR on human serum

endocannabinoids and NAE using volunteers with two different skin phototypes. We

wanted to see if there are any differences in serum endocannabinoids and NAE in

response to multiple doses of UVR. The study was carried out for 6 weeks to mimic

UVR exposure during summer time. All clinical work was undertaken by Prof Rhodes’

group.

Based on our data, there were differences in the baseline levels of the

endocannabinoids and NAEs in the White Caucasians and South Asians. However,

findings from this study showed that UVR had no effect on PEA, LEA, STEA and DGLEA

levels in either skin phototypes. Regardless of the differences in the baseline between

the two tested skin types, multiple doses of UVR approximately induced similar

fluctuating effects on PEA, LEA, STEA and DGLEA levels in both skin types.

In general, NEA levels were found at higher concentrations in White Caucasians of skin

phototype II than South Asians of phototype V. PEA for example was at the highest level

among the detected NAE then DHEA, OEA, LEA, STEA, MEA, AEA, DP-EA, and

DGLEA. Moreover, DGLEA was at the lowest level in all the NAEs found. Its levels were

under 30 pg/ml in both groups. On the other hand, EPEA was found in 6 out of 10

volunteers of the White Caucasian group while, it was not found in the South Asian

group. UVR increased 2-AG in the serum of both groups. However, 2-AG levels were

higher in the South Asian group compared to the White Caucasian group. In all the time

points including the day 0 (baseline), no significant differences were recorded between

those time points in both examined groups. Therefore, based on these observations it

was concluded that UV radiation at the multiple doses used in this clinical study has no

effect on the serum endocannabinoids and their congeners in both skin phototype used.

Therefore, further study is still needed.

Sample stability could be another issue behind the negative results showed here in our

study. The human serum samples for the clinical study were stored at -80oC. Although,

all endocannabinoids and NAEs were detected by LC-MS/MS analysis, the samples

202

may be affected by storage. Especially, with NAEs which have very low levels in intact

cells and tissue (Berdyshev et al., 2000). Furthermore, it is well known that the

endocannabinoids and NEA are usually produced on demand rather than being stored

in cells (Schreiber et al., 2007) Also, the levels of such of these NAE are significantly

reduced with samples frizzing and thawing (Schreiber et al., 2007). Although this was

not an issue in this study, since the samples were only defrosted once, for the analysis.

Apart from this, endocannabinoid and NAE levels were higher in skin phototype II than

skin phototype V.

As it was mentioned before, the baseline of these endocannabinoids and NAE were

different thus, the differences in their baseline levels may attributed to other factors such

as the dietary habits of the participants but not to the UVR itself. It is well known that

diet has a large effect on endocannabinoid and NAE levels in our body. Since the

examined groups in this study were from different ethnic groups, they could have

different life styles especially in the way of food consumption.

The current study showed that the levels of OEA, DHEA, AEA and DPEA were higher

in the White Caucasians of skin phototype II than the South Asians of phototype V. This

result may be due to the diet of the White Caucasians volunteers, especially food that

contains omega-3 PUFA such as DHA and EPA. Dietary fatty acids can exert multiple,

indirect influences on lipid metabolism across n-3, n-6, n-9 pathways through processes

including tissue fatty acid uptake, distribution, and oxidation, fatty acid circulation and

elimination rates (Rapoport, 2008, Rapoport et al., 2007, Madsen et al., 1999, Polozova

et al., 2006, Emken et al., 1993). Further support for this suggestion comes from the

observation that DHA has been reported to induce an increase in plasma concentrations

of its NAE derivative DHEA (Wood et al., 2010). In addition, another study, reported

that two week supplementation with DHA affected brain and plasma NAE and

endocannabinoid metabolites to favour the formation of DHA and EPA derivatives.

Specifically, DHEA and EPG concentrations increased in both plasma and brain,

whereas AEA decreased in brain and OEA, AG, OG and AA levels decreased in plasma

(Wood et al., 2010). All these data suggest that competition among lipid biosynthetic

enzymes utilizing fatty acids as substrates could represent an important influence on

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the formation of downstream lipid metabolites, including their derived endocannabinoid

metabolome constituents (Wood et al., 2010).

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CHAPTER 6: General discussion and future studies

6.1. Introduction

The skin is the largest organ of the human body. It forms the physical barrier that protect

the body from environmental stress and regulates the inward and outward passage of

water (Hunter, 2003). Skin has also a protective role against microorganisms, chemical

agents and UVR. It is well known that UV radiation is a dangerous environmental

stressor on the skin. Repeated UVR exposure especially due to higher levels of UVR

reaching the earth, makes the skin impairs its protective function and as a consequence,

skin inflammation, DNA damaging and skin cancer may develop. Therefore, finding an

alternative way to protect our skin from harmful UV radiation is important. Based on

several investigations dietary n-3 PUFA could be a reliable way offering protection

against an array of health disorders. N-3 PUFA are now some of the most studied fatty

acids that have been thought to play a key role as anti-inflammatory agent. N-3 PUFA

can regulate a wide range of functions in the body, including blood pressure, blood

clotting, and correct development and functioning of the brain and nervous systems

(Das, 2006). Furthermore, they have a role in regulating inflammatory responses

through the production of inflammatory mediators such as the eicosanoids (Das, 2006,

Calder, 2006). EPA and DHA are believed to have several health benefits (Wall et al.,

2010). There is evidence that n-3 PUFAs improve cardiovascular outcomes (De

Caterina, 2011) and that they have efficacy in rheumatoid conditions (Bhangle and

Kolasinski, 2011).There is now emerging evidence that n-3 PUFAs may also have anti-

cancer activities (Cockbain et al., 2012). All these discoveries suggest that n-3 PUFA

could play a role in protecting the human body from the harmful effects of environmental

stressors like UV radiation.

N-3 PUFA can also generate endocannabinoids which are a group of lipid mediators,

derived from fatty acid precursors linked to an ethanolamine moiety. They can also

stimulate various physiological effects including hypothermia, anti-nociception,

vasodilation and anti-inflammatory effects (Di Marzo, 1998, Zygmunt et al., 1999) and

have been implicated in several physiological functions and conditions ranging from

analgesia, reproduction, modulation of vascular tone, obesity, cancer, schizophrenia,

205

and multiple sclerosis (Pacher et al., 2006). Endocannabinoid congeners such as the

NAEs Include palmitoylethanolamide (PEA), oleoylethanolamide (OEA) and

stearoylethanolamide (STEA). There are also involved in several physiological and

pathophysiological processes, such as, inflammation, pain sleep, reproduction and drug

addiction (Piomelli, 2003, Lo Verme et al., 2005, Fu et al., 2003). Therefore, our study

was set out to explore the effect of n-3 PUFA on endocannabinoids and their

metabolizing enzymes in human skin cells exposed to UV radiation. The study has also

sought to know whether the n-3 PUFA and UVR could have an effect on the formation

of endocannabinoids and their related fatty acids. In order to address these questions,

this study was carried out on two types of human skin cells lines, HaCaT keratinocytes

and 46BR.IN fibroblasts. Furthermore, a clinical study has taken place to investigate the

effect of UV radiation on serum endocannabinoids and other NAE.

6.2. General discussion

Generally, our results show that the DHA treatment increased the intracellular levels of

endocannabinoid and NAEs such as AEA, PEA and DHEA in HaCaT keratinocytes.

Whereas, in 46BR.IN fibroblasts DHA was more effective and increased all the detected

endocannabinoid and their congeners including STEA, PEA, OEA, DHEA, AEA and 2-

AG. These results are in the same line with other studies which demonstrated that DHA

supplementation increased AEA and DHEA levels (Berger et al., 2001, Artmann et al.,

2008). On the other hand, EPA treatment decreased OEA, PEA, LEA and DHEA in

HaCaT keratinocytes and 46BR.IN fibroblasts. Whereas, the intracellular levels of AEA

and 2-AG did not change in both cells model but 2-AG level may increase in 46BR.IN

fibroblasts. These results are also supported with the observations in Artmann study

showed that EPA and DHA decreased the intracellular levels of the 2-AG, PEA, STEA

and OEA but not AEA and DHEA (Artmann et al., 2008). Furthermore, AEA and 2-AG

also have been reported to be decreased in 3T3-F442A adipocytes after DHA treatment

(Matias et al., 2008).

206

UV radiation was found to decrease the intracellular of STEA, PEA and LEA in both

HaCaT keratinocytes and 46BR.IN fibroblasts in the presence and absence of n-3 PUFA

supplementations. However, the intracellular levels of DHEA, AEA and 2-AG were

observed to fluctuate in both cells lines post UV radiation. The extracellular levels of

OEA and 2-AG were found to be increased whereas, STEA and DHEA levels did not

change in the irradiated HaCaT keratinocytes and 46BR.IN fibroblasts. All these finding

showed that UV radiation may be affect the formation of the endocannabinoids and their

related compounds. These findings may attributed to phospholipase A2 (PLA2) activity

that is induced after UVR exposure (Chen et al., 1996, Cohen and DeLeo, 1993, Hanson

and DeLeo, 1990), as PLA2 has been reported to be one of the possible biosynthetic

routes of NAEs via N-acyl-lysoPE (Sun et al., 2004). Moreover, UVR has been reported

to stimulate N-acyl-PE and NAEs including AEA (Berdyshev et al., 2000).

Data from a second study showed that UVR-induced COX-2 up-regulation. In fact, this

observation is well documented, for example, several studies suggested the up-

regulation of COX-2 protein levels mRNA and following UVB irradiation in HaCaT

keratinocytes, in human keratinocytes in volunteers subjected to UVB radiation, in

mouse epidermis and in the skin of hairless mice (Buckman et al., 1998, Tang et al.,

2001, Bachelor et al., 2005, Kim et al., 2007).

Our results also showed that DHA treatment significantly decreased COX-2 expression

in 46BR.IN fibroblasts in dose dependent manner. UVR-induced COX-2 expression was

also dose dependently inhibited by DHA treatment in 46BR.IN fibroblasts. Similarly, but

in different cells, DHA was reported to block UVR induced COX-2 expression (Sengupta

et al., 2003, Zhang and Bowden, 2008, Rahman et al., 2011). In contrast, DHA did not

inhibit UVR-induced COX-2 expression in HaCaT keratinocytes in our study. These

results are also in agreement with another study used different stimulus and different

type of cells. They found that DHA enhanced phorbol ester or interleukin 1β-induced

COX-2 expression in rat vascular endothelial cells (Machida et al., 2005).

Data from our study showed that EPA decreased COX-2 protein levels in a

concentration dependent manner in both HaCaT keratinocytes and 46BR.IN fibroblasts

207

cells. These results are in agreement with another studies where EPA induced down-

regulation in COX-2 expression (Calviello et al., 2004, Kim et al., 2006). In addition, our

results suggest that EPA did not prevent UVR-induced COX-2 expression in either cell

line.

Back to endocannabinoid, our results revealed that UVR induced down-regulation in

FAAH levels while, there were no consistent effects on NAPE-PLD in HaCaT

keratinocytes and 46BR.IN fibroblasts. However, AEA and 2-AG synthesis was found

to be increased after UVR exposure in both cell lines used in this study.

DHA and EPA treatment decreased FAAH level and increased NAPE-PLD levels in both

HaCaT keratinocytes and 46BR.IN fibroblasts. This was consistent with our results

showing that DHA increased AEA production in HaCaT keratinocytes as observed in

another part of this study.

Data from the present study also indicated that UV radiation could inhibit NAPE-PLD

expression. These data are in line with another research reported that LPS inhibits

NAPE-PLD expression (Zhu et al., 2011). In fact, these observations may be due to

many reasons, such as gene expression changes induced due to UV radiation.

In the clinical study, our findings showed that UVR has no significant effects on PEA,

LEA, STEA, PEA, LEA and DGLEA levels in either skin phototype (II or V). However, 2-

AG levels were found increased in both skin phototypes post UV radiation. In general,

NEA levels were higher in the White Caucasian of skin phototype II than the South Asian

of phototype V. PEA for example was the most abundant NAE among the detected

NAEs while, DGLEA was at the lowest level between all the found NAEs. EPEA was

found in 6 out of 10 volunteers of the White Caucasian group. 2-AG level was higher in

the South Asian group while, DHEA levels were much higher in White Caucasians group

than South Asians group. All these observations suggest that these differences between

two groups of volunteers can be attributed to diet and not UVR because there were

huge difference in the baseline levels of endocannabinoid and NAE. Overall, the main

findings of this project are:

208

1. N-3 PUFA increased NAPE-PLD levels in HaCaT keratinocytes and 46BR.IN

fibroblasts and as a result increased the production of endocannabinoids and

NAE.

2. N-3 PUFA reduced FAAH levels in HaCaT keratinocytes and 46BR.IN

fibroblasts which could be an indirect way to increase the concentration of

endocannabinoids and NAE.

3. N-3 PUFA induced AEA and 2-AG biosynthesis in HaCaT keratinocytes and

46BR.IN fibroblasts

4. DHA treatment significantly decreased COX-2 expression in 46BR.IN

fibroblasts

5. DHA inhibited UVR-induced COX-2 overexpression in 46BR.IN fibroblasts

6. DHA induced COX-2 up-regulation in HaCaT keratinocytes

7. DHA did not prevent UVR induced COX-2 up-regulation in HaCaT

keratinocytes.

8. EPA induced COX-2 down-regulation in HaCaT keratinocytes and 46BR.IN

fibroblasts.

9. EPA did not prevent UVR induced COX-2 up-regulation in both HaCaT

keratinocytes and 46BR.IN fibroblasts.

10. UVR did not have any effect on endocannabinoid and NAE biosynthesis.

However, UVR induced endocannabinoids production in some experiments and

this could suggest a defense mechanism of skin cells followed UV radiation.

11. UVR had no major effect on human serum NAE in both skin phototypes II and

V

12. UVR increased 2-AG in human serum NAE in both skin phototypes II and V

13. Human serum NAE levels were found to be higher in White Caucasian group

(skin phototypes II).

14. Human serum 2-AG level was higher in the South Asian group (skin phototypes

V).

209

6.3. Future directions

This study has shown that n-3 PUFA increased endocannabinoids and NAE in human

skin cells. Also our results showed that n-3 PUFA increased NAPE-PLD expression and

decreased FAAH in the human cell lines HaCaT keratinocytes and 46BR.IN fibroblasts.

These results indicated that n-3 PUFA may alter endocannabinoid biosynthesis

pathways in two ways; (a) directly by increasing NAPE-PLD which in turn increases the

biosynthesis of endocannabinoids and NAE. (b) Indirectly by decreasing FAAH, as a

result increasing the intracellular levels of endocannabinoids. However, it will be of

interesting to elucidate the effect of these n-3 PUFA on the 2-AG synthesizing enzyme

diacylglycerol lipase (DAGL) and the 2-AG degrading enzyme monoacylglycerols lipase

(MAGL) in HaCaT keratinocytes and 46BR.IN fibroblasts in the presence and absence

of UVR. Furthermore, it will be essential to investigate the effect of n-3 PUFA in NAPE-

PLD, FAAH, DAGL and MAGL in primary HaCaT keratinocytes and in 46BR.IN

fibroblasts in the presence and absence of UVR. Also, it is importance to assess the

effect of UVR on the biosynthesis of endocannabinoids and NAE through measuring

Ca2+ intracellular levels per and post UVR.

Data from this study also showed that DHA supplementation significantly decreased

COX-2 expression and inhibited UVR-induced COX-2 up-regulation in 46BR.IN

fibroblasts. In contrast, in HaCaT keratinocytes, DHA exerts an opposite effect by

increasing COX-2 expression and did not prevent UVR-induced COX-2 expression in

the irradiated HaCaT keratinocytes. Moreover, our results showed that EPA induced

COX-2 down-regulation in HaCaT keratinocytes and 46BR.IN fibroblasts. However,

EPA did not inhibit UVR-induced COX-2 expression in the HaCaT keratinocytes and

46BR.IN fibroblasts. Therefore, further study is still required to assess the effect of DHA

and EPA on COX-2 expression in the presence and absence of UVR in primary HaCaT

keratinocytes and in 46BR.IN fibroblasts.

Our study did not produce results that could help us to assess the effect of n-3 PUFA

on 12- and 15-LOX in HaCaT keratinocytes and 46BR.IN fibroblasts. Therefore, more

work is needed to determine the effect of n-3 PUFA on 12- and 15-LOX in these cells

and in the presence and absence of UVR. Furthermore, it will be interesting to clarify

210

the effect of exogenous AEA and 2-AG on the expression of COX-2, 12-LOX and 15-

LOX in HaCaT keratinocytes and 46BR.IN fibroblasts in the presence and absence of

UVR.

Several studies reported that n-3 PUFA and UVR have an effect on gene expression on

different cells and tissues. Further work aiming to examine NAPE-PLD, FAAH, DAGL

and MAGL gene expression in HaCaT keratinocytes and 46BR.IN fibroblasts, in the

presence and absence of n-3 PUFA and UVR is needed.

Finally, our results showed that UVR has no major effects on circulating levels of

endocannabinoids and NAE as measured in human serum samples. We believed that

the differences observed in endocannabinoids and NAE may be attributed to dietary

habits of the volunteers engaged in the clinical study. Therefore, it will be essential to

assess the effect diet on endocannabinoid and NAE in the human circulation.

211

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237

Appendices

Appendix 1

1. Fatty acid stock solution

1.1. Oleic Acid (OA) stock solution (50mM)

1. OA 25 mg

2. Dimethyl sulfoxide (DMSO) 1.8 ml

3. Aliquot to 50µl each and store at -20°C.

1M of OA = 282.46 g, so 14.123g are needed to prepare OA at concentration of 50mM.

1.2. Eicosapentaenoic Acid (EPA) stock solution (50mM)

1. EPA 25 mg



1M of EPA = 302.45 g, so 15.123g are needed to prepare EPA at concentration of

50mM

1.3. Docosahexaenoic Acid (DHA) stock solution (50mM)

1. Docosahexaenoic acid 25 mg



1M of DHA = 328.49 g, so 16.424g are needed to prepare DHA at concentration of 50m.

Appendix 2

2.1. Preparation of BSA standards

Prepare BSA solutions in 100 µl

1. The stock solution of BSA is 1.54 mg/ml

2. The required concentrations are 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/ml

238

Example: (0.1mg/ml) / (1.54 mg/ml) = 0.06 x 100µl = 6 µl

6 µl BSA+ 94 µl of sample buffer (Appendix 3, section 3.1.5) = 100µl

3. Do this calculation of the rest of required concentrations

4. Put in eppendroff tubes and vortex

5. Store in -20 oC to be used for the protein assay

2.2. Genesis software (Version 2)

1. In a 96-well microplate add 5 µl of each BSA standard and 5 µl of each test

sample all in triplicate

2. Add 25 µl Protein Assay Reagent A (Bio-Rad) in each well

3. Add 200 µl Protein Assay Reagent B (Bio-Rad) in each well

4. Leave the plate to stand in the dark for 15 min

5. Read the absorbance of all wells using a plate reader at 650 nm

6. To estimate the protein content, launch Genesis software (Version 2).

7. Once opened, click on ‘Protocol’ at the menu bar and then ‘Open’.

8. From the list, choose ‘protein.prt’ and confirm the selection ‘OK’.

9. Once set up, gently place the micro-plate onto the reader holder and run

samples (running man icon at the task bar).

10. Type any relevant comments and confirm by ‘RUN’. Note: Filter must be set at

650 nm.

11. Save data.

2.3. Sample calculation for adjusting the protein content of the cell lysate

Example: if the sample volume is 200 µl and the protein concentration of the sample is

3.7µg/µl, then a dilution to a concentration of 2µg/µl is needed.

So: C1V1=C2V2

V2 = (3.7µg/µlx200µl )/(2µg/µl ) =370µl

370 µl - 200 µl = 170 µl.

239

Therefore; 170 µl of sample buffer (Appendix 3, section 3.1.5) should be added to the

original lysate to generate a sample of: volume 370 µl, protein concentration 2µg/µl.

Appendix 3

3.1 Preparation of buffers and stains

3.1.1. Tank (running) buffer: (PH 8.4-8.5) 1 litre

1. Tris base 30 g

2. Glycine 144 g

3. SDS 10 g

4. Add dH2O to adjust the volume to 1 litre

Do not adjust PH

In case of preparing 1 L, mix all solids together, add dH2O up 1000 ml using a stirrer

mix very gently to dissolve all ingredients.

3.1.2. Transblotting buffer: (PH 8.4-8.5) 1 litre

1. Tris base 30 g

2. Glycine 144 g

3. dH2O to adjust the volume to 1 litre. Do not adjust PH

In case of preparing 1 L, mix all solids together, add dH2O up 1000 ml using a stirrer

mix very gently to dissolve all ingredients.

3.1.3. Lower (separating) gel Tris buffer (PH 8.8) - 0.5 litre

1. Tris base 90.8 g

2. SDS 2 g

3. dH2O 500 ml

Adjust PH using concentrated HCl

240

3.1.4. Upper (stacking) gel Tris buffer (PH 6.9) - 0.5 litre

1. Tris base 30.3 g

2. SDS 2 g

3. dH2O 500 ml

Adjust pH using concentrated HCl (Add few drops until pH adjusted)

3.1.5. Sample buffer 25 ml

10% Glycerol, 2.5 ml

0.02M EDTA, 0.185 g

MW = 372.24 g

1M = 372.24 g/L

Quantity required is 0.02 M in 100 ml

(0.02M x 372.24 g ) / 1M = 7.44 g

(7.44 g x 100 ml) / (1000 ml) = 0.74 g

To dissolve EDTA add few drops of NaOH 1M. 1M = 40 g/1000ml

(1mM = 4 g/100 ml)

6% SDS, 1.5g

62.4mM Tris base, 0.19g

MW = 121.14 g

1M = 121.14 g/L

Quantity required is 62 mM in 100 ml

1M = 121.14 g

(0.062M x 121.14 g) / 1M = 7.51 g

(7.51 x 100 ml) / (1000 ml) = 0.76 g

241

Bromophenol blue 0.08% (omit this step and do it when adding samples)

dH2O to adjust the volume to 25ml

3.1.6. Coomassie blue stain for SDS

1. 125 ml dH2O

2. 100 ml methanol

3. 25 ml acetic acid

4. 0.1% w/v coomassie blue

Filter before use with Whatman filter paper and funnel.

3.1.7. Destain for SDS

1. 250 ml dH2O

2. 200 ml methanol

3. 50 ml glacial acetic acid

3.1.8. Fast green 0.1% for PVDF membrane

1. 0.5 g fast green

2. 500 ml dH2O Filter with Whatman filter paper and funnel

3.1.9. ECL solution

1. Tris base 2.5 ml (pH 8.5)

2. Luminal 130 µl

3. P-coumaric acid 60 µl

4. H2O2 28 µl

5. dH2O 22.32 ml.

The mixture was protected from the light by mixed in 50 ml universal tube wrapped in

foil.

242

Appendix 4

4.1. Membrane stripping

4.1.1. Mild stripping buffer

1. Glycine1.5 g

2. SDS 0.1 g

3. Tween 20 (1ml)

Adjust pH to 2.2 by using concentrated HCl (Add few drops until pH adjusted) Bring

volume up to 100 ml with dH2O.

4.2. Protocol of stripping western blots for re-probing

1. Put the PVDF membrane in a 10cm square petri dish contains of an enough

volume of mild stripping buffer that will cover the PVDF membrane, usually use

15 – 20 ml.

2. Incubate at room temperature for 10 min.

3. Discharge mild stripping buffer.

4. Incubate PVDF membrane again at room temperature for 10 minutes with fresh

stripping buffer.

5. Discharge buffer.

6. Incubate PVDF membrane at room temperature for 10 min with PBS

7. Pour off the PBS

8. Incubate PVDF membrane again at room temperature for 10 min with PBS

9. Pour off the PBS

10. Incubate PVDF membrane at room temperature for 5 min with PBS/Tween20

(0.05%)

11. Pour off the PBS/Tween20 (0.05%)

12. Incubate PVDF membrane again at room temperature for 5 min with

PBS/Tween20 (0.05%)

13. Pour off the PBS/Tween20 (0.05%)

14. Incubate PVDF membrane at room temperature for 5 min with PBS

243

15. Ready for blocking stage.

Appendix 5

5.1. Chloroform/methanol (2:1 ratio)

1. Add 100 ml methanol (HPLC grade) into 500 measuring flask.

2. Add 300 ml of chloroform (HPLC grade) to step number 1

3. Store at room temperature for use, this solution can stand up to one month

5.2. Internal standards (1 ng/µl AEA-d8 and 1 ng/µl 2-AG-d8)

1. In an amber vial, mix 100 µl of 100 ng/µl stocks with 900 µl ethanol (HPLC grade)

to make a 10 ng/µl stock

2. In another amber vial, mix 100 µl 10 ng/µl stocks and mix with 900 µl ethanol

(HPLC grade) to make a 1 ng/µl stock

3. Seal both 1 ng/µl stock and remaining 10 ng/µl stocks with Parafilm and store at -

20 ºC for up to three months

5.3 Endocannabinoid cocktail for calibration line (400 pg/µl)

1. Using a Hamilton syringe, measure 40 µl of the 10 ng/µl AEA standard into an

amber vial

2. Add 40 µl each of the 10 ng/µl 2-AG, ALEA, DHEA, OEA, SEA, PEA, EPEA

and LEA standards, rinsing the Hamilton syringe between standards

3. Add 640 µl ethanol (HPLC grade) to make up to 1 ml

4. Mix thoroughly

244

5.4. Mobile Phase A for endocannabinoid analysis

1. Measure 20 ml acetonitrile (HPLC grade) into a 1 L measuring cylinder

2. Add 5 ml glacial acetic acid (HPLC grade)

3. Make up to 1 L with deionized water

4. Vacuum filter

5. Transfer to 1 L bottle

Should be made fresh at the beginning of each run

5.5. Mobile Phase B for endocannabinoid analysis

1. Measure 20 ml deionized water into a 1 L measuring cylinder


3. Make up to 1 L with acetonitrile (HPLC grade)

4. Vacuum filter



5.6. Seal wash

1. Measure 100 ml acetonitrile (HPLC grade) into a 1 L measuring cylinder


3. Vacuum filter

4. Transfer to 1L bottle

245


5.7. Needle Wash

1. Measure 300 ml deionized water into a 1 L measuring cylinder

2. Make up to 1 L with acetonitrile (HPLC grade)

3. Vacuum filter



5.8. Shutdown solution I

1. Measure 500 ml methanol (HPLC grade) into a 1 L measuring cylinder



4. Vacuum filter



5.9. Shutdown solution II

1. Measure 500 ml methanol (HPLC grade) into a 1 L measuring cylinder


3. Vacuum filter


246


Appendix 6

Table 6.1. Comparison between PEA levels in White Caucasians of skin phototype

II and South Asians of phototype V pre and post UVR.

Skin phototype II Skin phototype V

Days of UVR exposure PEA (pg/ml) PEA (pg/ml)

Day 0 3160 2814

Day 4 4048 2571

Day 7 3085 2473

Day 14 3049 2892

Day 21 3041 2533

Day 28 3311 3023

Day 35 3184 2871

Day 39 3251 2474

Table 6.2. Comparison between LEA levels in White Caucasians of skin phototype



Days of UVR exposure LEA (pg/ml) LEA (pg/ml)

Day 0 870 1015

Day 4 896 916

Day 7 893 920

Day 14 863 1101

Day 21 810 928

Day 28 1004 1235

Day 35 947 1115

Day 39 885 969

247

Table 6.3. Comparison between OEA levels in White Caucasians of skin

phototype II and South Asians of phototype V pre and post UVR.

Table 6.4. Comparison between STEA levels in White Caucasians of skin



Days of UVR exposure STEA (pg/ml) STEA (pg/ml)

Day 0 713 671

Day 4 943 436

Day 7 755 457

Day 14 709 548

Day 21 699 493

Day 28 789 591

Day 35 731 560

Day 39 781 557


Days of UVR exposure OEA (pg/ml) OEA (pg/ml)

Day 0 1412 798

Day 4 1513 731

Day 7 1324 684

Day 14 1471 731

Day 21 1390 598

Day 28 1700 739

Day 35 1437 675

Day 39 1589 681

248

Table 6.5. Comparison between DHEA levels in White Caucasians of skin



Days of UVR exposure DHEA (pg/ml) DHEA (pg/ml)

Day 0 2797 792

Day 4 2282 849

Day 7 2902 707

Day 14 2643 1081

Day 21 2517 626

Day 28 3023 1103

Day 35 2703 1127

Day 39 2605 755

Table 6.6. Comparison between AEA levels in White Caucasians of skin phototype



Days of UVR exposure AEA (pg/ml) AEA (pg/ml)

Day 0 326 280

Day 4 358 150

Day 7 315 196

Day 14 331 272

Day 21 298 145

Day 28 347 322

Day 35 339 202

Day 39 383 237

249

Table 6.7. Comparison between 2-AG levels in White Caucasians of skin



Days of UVR exposure 2-AG (pg/ml) 2-AG (pg/ml)

Day 0 993 1786

Day 4 2364 2088

Day 7 1101 1800

Day 14 1732 1699

Day 21 1575 2487

Day 28 1474 2560

Day 35 1527 1682

Day 39 1231 1185

Table 6.8. Comparison between 1-AG levels in White Caucasians of skin



Days of UVR exposure 1-AG (pg/ml) 1-AG (pg/ml)

Day 0 742 847

Day 4 771 726

Day 7 765 508

Day 14 690 651

Day 21 564 895

Day 28 710 618

Day 35 684 713

Day 39 865 467

250

Table 6.9. Comparison between DP-EA levels in White Caucasians of skin



Days of UVR exposure DP-EA (pg/ml) DP-EA (pg/ml)

Day 0 76 43

Day 4 67 48

Day 7 73 47

Day 14 73 53

Day 21 71 43

Day 28 81 66

Day 35 75 63

Day 39 73 47

Table 6.10. Comparison between Myristoyl-EA levels in White Caucasians of skin



Days of UVR exposure Myristoyl-EA (pg/ml) Myristoyl-EA (pg/ml)

Day 0 465 335

Day 4 723 337

Day 7 599 449

Day 14 374 773

Day 21 541 299

Day 28 793 477

Day 35 449 492

Day 39 403 297

Table 6.11. Comparison between DGLEA levels in White Caucasians of skin



Days of UVR exposure DGLEA (pg/ml) DGLEA (pg/ml)

Day 0 25 20

Day 4 25 25

Day 7 26 19

Day 14 26 26

Day 21 26 18

Day 28 26 25

Day 35 29 18

Day 39 22 15

251

Table 6.12. Comparison of different time point of UVR exposure on EPEA levels

in White Caucasians of skin phototype II.

Days of UVR exposure EPEA (pg/ml)

0 44

4 43

7 69

14 72

21 70

28 88

35 79

39 45

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