ORIGINAL PAPER

Enhanced phospholipase B activity and alterationof phospholipids and neutral lipids in Saccharomycescerevisiae exposed to N-nitrosonornicotine

Panneerselvam Vijayaraj • Jayaraja Sabarirajan •

Vasanthi Nachiappan

Received: 13 July 2010 / Accepted: 22 October 2010

� Springer Science+Business Media B.V. 2010

Abstract A tobacco-specific nitrosamine (TSNA),

N-nitrosonornicotine (NNN), is a potent carcinogen

present in cigarette smoke, and chronic exposure to it

can lead to pulmonary cancer. NNN causes changes in

phospholipid metabolism and the mechanism is yet to

be elucidated. Exposure of Saccharomyces cerevisiae

to 50 lM NNN leads to a substantial decrease in

phosphatidylserine (PS) by 63%, phosphatidylcholine

(PC) by 42% and phosphatidylethanolamine (PE) by

36% with a concomitant increase in lysophospholipids

(LPL) by 25%. The alteration in phospholipid content

was dependent on increasing NNN concentration.

Reduced phospholipids were accompanied with

increased neutral lipid content. Here we report for the

first time that NNN exposure, significantly increases

phospholipase B (PLB) activity and the preferred

substrate is PC, a major phospholipid responsible for a

series of metabolic functions. Furthermore, NNN also

promotes the alteration of fatty acid (FA) composition;

it increases the long chain fatty acid (C18 series) in

phospholipids specifically phosphatidylethanolamine

(PE) and PS; while on the contrary it increases short

chain fatty acids in cardiolipin (CL). NNN mediated

degradation of phospholipids is associated with

enhanced PLB activity and alteration of phospholipid

composition is accompanied with acyl chain

remodelling. Understanding the altered phospholipid

metabolism produced by NNN exposure is a worth-

while pursuit because it will help to understand the

toxicity of tobacco smoke.

Keywords Tobacco specific nitrosamines �N-Nitrosonornicotine � Phospholipid �Phospholipase B � Neutral lipid � Free fatty acid

Abbreviations

TSNA’s Tobacco specific nitrosamines

NNN N-Nitrosonornicotine

PC Phosphatidylcholine

PE Phosphatidylethanolamine

PS Phosphatidylserine

PI Phosphatidylinositol

PA Phosphatidic acid

PG Phosphatidylglycerol

CL Cardiolipin

LPC Lysophosphatidylcholine

LPL Lysophospholipid

PLB Phospholipase B

PLA2 Phospholipase A2

FA Fatty acid

MAG Monoacylglycerol

DAG Diacylglycerol

TAG Triacylglycerol

FFA Free fatty acid

SE Sterol esters

ROS Reactive oxygen species

DMSO Dimethyl sulphoxide

Present Address:P. Vijayaraj � J. Sabarirajan � V. Nachiappan (&)

Department of Biochemistry, Bharathidasan University,

Tiruchirappalli 24, Tamilnadu, India

e-mail: vasanthibch@gmail.com

123

Antonie van Leeuwenhoek

DOI 10.1007/s10482-010-9526-1

Introduction

Phospholipids are major components of cellular

membranes that take part in a series of metabolic

events including maintenance of cellular permeability,

regulation of proteins associated with the membrane

and regulation of intracellular signalling by providing

signalling molecules (Yamashita et al. 1997; Carman

and Zeimetz 1996; Exton 1994). The functions of

membrane is inherently linked to the type of phos-

pholipids present in the cell membrane, and their

interactions with each other. Environmentally induced

alterations in phospholipid composition can lead to

changes in membrane integrity, permeability, mem-

brane cell injury and even death (Stenger et al. 2009).

This is especially true with toxic substances because

they easily generate reactive oxygen species (ROS)

which in turn interact with cell membrane (Nachiap-

pan et al. 1994). Cigarette smoke is one of the potent

environmental pollutants, because smoking is highly

addictive and a habit that is difficult to break.

Tobacco-specific nitrosamines (TSNAs) are the main

toxic compounds present in cigarette smoke and

tobacco products (Hecht and Hoffmann 1988).

Specifically N-nitrosonornicotine (NNN) is a potent

pulmonary carcinogen, reported by the International

Agency for Research on Cancer as a human carcinogen

(IARC Monogr 2007) and exerts a wide spectrum of

biological effects on the lung and lung airways,

including oxidative stress, inflammation and DNA

damage (Hecht and Hoffmann 1988; Lin et al. 2010).

Smoking is associated with reduced surfactant phos-

pholipids in the bronchoalveolar lavage particularly

phosphatidylcholine (PC) (Oulton et al. 1991; Finley

and Ladman 1972). NNN primarily targets the lung and

alters the composition of lipid-protein complex specif-

ically in the surfactant and alters its biological functions.

More interestingly interaction of NNN to the membrane

mainly depends on the phospholipid composition and its

fatty acid composition as well (Schuster et al. 1992,

1995; Scott 2004). Carcinogenicity of NNN is well

documented; however impact on lipids particularly

phospholipid metabolism is yet to be elucidated.

The objective of this study was to analyze in detail

the influence of NNN on metabolism of neutral lipids

and phospholipids and to depict the underlying mech-

anisms using yeast as a model system. Assessment of

chemicals and their toxicological data usually requires

a large number of animal species, hence a microbial

system is a useful and an alternative approach for our

objectives. The Saccharomyces cerevisiae, a well

studied eukaryotic model system is used to characterise

the effect of NNN on lipid metabolism. Investigations

were also under taken to study the alterations in the

fatty acid composition of the neutral and phospholip-

ids. Overall results suggest that reduced phospholipid

content is due to activated PLB, resulting in increased

lysophospholipid (LPL) content and also adaptation of

membrane by altered fatty acid composition. Our

studies, depicting the changes in phospholipids and

neutral lipids by NNN, would contribute to a better

understanding of the molecular mechanisms underly-

ing its toxicity.

Materials and methods

Materials

Wild-type strain S. cerevisiae BY4741 [MATa his3D1

leu2D0 met15D0 ura3D0] was procured from Open

Biosystems (AL, USA) and its isogenic plb1D, plb2Dand plb3D were gifted by CIMAP, India. [32P]

orthophosphate (5000 Ci/mmol) was obtained from

Bhabha Atomic Research Centre (Mumbai, India) and

[1-14C] acetate (1000 Ci/mmol) was obtained from

PerkinElmer Life Sciences (USA). Yeast medium,

lipid and fatty acid standards were obtained from

Sigma (India). Silica gel 60F254 TLC plates were

from Merck (India). All chemicals and solvents were

purchased from Sigma unless specifically mentioned.

NNN was obtained from Sigma (India). We performed

purity assessment by liquid chromatography electro-

spray ionization tandem mass spectrometry (LC/ESI-

MS/MS) for NNN and it was about 99% pure (data not

shown). Stock solution was prepared in dimethyl

sulphoxide (DMSO) and stored at -20�C till use.

Maximum DMSO in growth media was 0.05% (v/v), a

concentration that had no detectable effect on yeast

growth (data not shown).

Growth conditions and sensitivity assay

Yeast cells were grown in YPD (1% yeast extract, 2%

peptone, and 2% dextrose) medium (pH 7.0) with

aeration at 30�C. The cells were grown in 5 ml of

YPD medium for 12 h and harvested, transferred to

Antonie van Leeuwenhoek

123

25 ml of YPD media with indicated concentrations of

NNN. Cells were grown at 30�C at 180 rpm and cell

growth was studied by measuring the A600 of the

cultures at frequent time intervals. The sensitivity of

the yeast cells to NNN was observed on solid medium

supplemented by spotting experiments. For this the

cells were serially diluted and spotted on YPD plates

containing 2% agar with or without NNN and

incubated for 48 h at 30�C. The dilutions that are

used are 10-1, 10-2, 10-3, 10-4 and 10-5.

Labelling of yeast lipids

Phospholipid alterations were analysed by [32P] ortho-

phosphate labelling (Ghosh et al. 2008) and neutral

lipids were analysed by [1-14C] acetate labelling

(Rajakumari and Daum 2010). The cells were grown

at 30�C in YPD medium with or without different

concentrations of NNN. To this either 100 lCi of [32P]

orthophosphate (5000 Ci/mmol) or 0.5 lCi of [1-14C]

acetate (1000 Ci/mmol) was added. Cells were grown

for 12 h and harvested by centrifugation. Unused radio

labelled phosphate and acetate were removed by

washing with 2% phosphoric acid and equal numbers

of cells were taken, subjected to lipid extraction.

Lipid extraction and Identification

Total lipids from yeast cells were extracted by the

method of Bligh and Dyer (1959). Briefly, to the cell

pellet 400 ll of methanol and 200 ll of chloroform

were added and vortexed. To this, 400 ll of acidified

water (2% orthophosphoric acid) was added and

vigorously vortexed. Chloroform extracts containing

total lipids were subjected to two-dimensional thin

layer chromatography (TLC) with Silica Gel. In TLC

plates phospholipid separation was done by using the

following solvents, for first dimension chloroform/

methanol/ammonia (65:25:5, v/v) and for the second

dimension chloroform/methanol/acetone/acetic acid/

water (50:10:20:15:5, v/v). Neutral lipids were sep-

arated by using petroleum ether: diethyl ether: acetic

acid (70:30:1, v/v) as the solvent system. Individual

lipids were located by comparing the Rf values of the

unknown with the Rf values of the standard. The

radioactive spots were developed by autoradiography

for 12 h. The spots were scraped from the TLC plate,

and the incorporation was counted in liquid scintil-

lation counter (PerkinElmer Life Sciences, USA).

Fatty acids analysis

Fatty acids of both neutral lipids and phospholipids

were analyzed by gas chromatography. Lipids were

separated by TLC and individual lipids were

extracted from the silica gel with chloroform/meth-

anol (2:1 v/v), and subjected to methanolysis using

BF3/methanol for conversion to methyl esters (Mor-

rison and Smith 1964). Fatty acid methyl esters were

separated by gas chromatography and quantification

was referred to heptadecanoic methyl ester, as an

internal standard.

Preparation of phospholipid substrates

Radioactively labelled phospholipids were prepared

biosynthetically by incubating yeast cells with [32P]

orthophosphate for 12 h, followed by extraction of

lipids and separation of phospholipid by 2D-TLC

(Wagner and Paltauf 1994). Purity was typically

[95%, as checked by TLC. Ethanol-solubilized

phospholipid micelles were prepared by sonication

(Gheriani-Gruszka et al. 1988).

Phospholipase assays

Phospholipase activity was assayed using ethanol-

solubilized [32P] orthophosphate labelled phospho-

lipids. The concentration of PC was 2 mM and the

specific radioactivity was 5000 Ci/mmol (Fisher et al.

1992). The buffer (200 ll) contained 50 mM Tris–

HCl pH 8.0/1 mM-EGTA/10 mM-CaCl2. The reac-

tion was initiated by addition of enzyme sources

(50 lg) from corresponding yeast lysate (wild type

and plbD strains treated with or without NNN) and

incubated at 37�C for 60 min. The assay was also

performed in the presence or absence of calcium. The

reaction were stopped by adding chloroform: meth-

anol: 2% phosphoric acid (1:2:1, v/v) and lipids were

extracted, separated by TLC and quantified. Protein

was determined by the method of Bradford (1976).

Results

Effect of NNN on the lipid composition

Cells were treated with NNN and phospholipid

alterations were monitored. We performed the study

Antonie van Leeuwenhoek

123

with different concentrations of NNN (0–400 lM)

and the incorporation of [32P] orthophosphate into

phospholipids was decreased significantly when

compared with control. Figure 1a depicted the con-

centration dependent reduction of total phospholipid

up to 400 lM of NNN; Even though the maximum

reduction (*57%) was in 400 lM of NNN-treated

cells, when compared to control, it was only *17%

greater than 50 lM of NNN treated (*40% reduc-

tion vs control). Hence 50 lM was chosen for further

studies. The impact of 50 lM NNN on individual

phospholipids were analysed (Fig. 1b). There was a

significant (P \ 0.001) reduction in phosphatidylser-

ine (PS) (63%) when compared to control. Apart

from PS, profound decrease was observed in PC

(42%) and phosphatidylethanolamine (PE) (36%).

This might be as a consequence of PS reduction,

since in yeast PS is a precursor for both PC and PE

(Vance and Steenbergen 2005). Observed reduction

of PG leads to the decreased content in CL (37%)

(Chicco and Sparagna 2006). Reduction of phospho-

lipid was accompanied with a simultaneous increase

in LPL (Fig. 1b). Based on the above observations,

we suggest that NNN significantly (P \ 0.001)

decreases the overall phospholipid content.

Declined phospholipid synthesis suggested that

there might be a rapid metabolic switchover from

phospholipid synthesis to neutral lipid leading to its

accumulation. [14C] acetate incorporation into neutral

lipid was increased approximately 30–50% in NNN

treated cells when compared to control (Fig. 1c).

Cells treated with 50 lM of NNN showed a signif-

icant (P \ 0.001) increase in diacylglycerol (DAG)

(threefold) and an increase in TAG (51%) when

compared to control. Increased DAG content might

be due to enhanced hydrolysis of phospholipid. In

addition, both MAG (51%) and sterol (31%) content

were also increased with NNN when compared to

control cells. Similar trend was observed with sterol

esters (SE) also. There was also a significant

(P \ 0.001) increase in FFA (90%) content in NNN

treated cells.

Labelling of yeast cells in the presence of NNN

clearly showed a significant (P \ 0.001) reduction in

phospholipids and a significant increase (P \ 0.001)

in neutral lipids associated with an increase in LPL

(Fig. 1b) and FFA (Fig. 1c). These results have led us

to investigate the involvement of NNN on the

phospholipid degrading enzyme, PLB.

Phospholipase B activity enhanced by NNN

To provide evidence for NNN induced hydrolysis of

phospholipid, we analyzed phospholipase activity using

[32P] orthophosphate labelled phospholipid as substrate

and NNN-treated or untreated yeast lysate as an enzyme

source from both wild type and all three PlbDmaintained uniformity throughout. Figure 2 depicts

the effect of the NNN on hydrolysis of phospholipids by

phospholipase. PC was the preferred substrate and

hydrolysis of PC was substantially (58%) higher with

50 lM NNN treated cells followed by PS (22%) when

compared to other phospholipids (Fig. 2a).

To assess the effect of NNN on hydrolysis of

phospholipid by different PLBs, we used wild type and

three plbD strains (plb1D, plb2D and plb3D) cell lysate

as enzyme source. When the cells were exposed

to NNN, concentration-dependent hydrolysis of

phospholipid was observed in both wild type and

all three mutants (Fig. 2b). The activity was greatly

enhanced by NNN in wild type (*64%), whereas

mutant cells showed diminished activity with

plb1D\ plb2D\ plb3D\ wild type. Both plb2Dand plb3D responded similar to wild type in presence

of NNN and plb1D showed significantly lower enzyme

activity (*16%) as compared to wild type. From these

results we suggest that enhanced phospholipase activ-

ity may be due to plb1 stimulated by NNN exposure.

It is worth assessing the effect of Ca2? on NNN

mediated phospholipid hydrolysis. We performed the

enzyme assay in the presence or absence of Ca2? with

or without NNN (Fig. 2c). NNN enhances both Ca2?

dependent as well as independent phospholipase

activity. The activity was greatly enhanced by the

addition of 10 mM Ca2? (*1.4-fold increase) whereas

in the absence of Ca2? *1.2-fold increase was

observed when compared to their respective controls

(Fig. 2c). Our result suggests that Ca2? partially

activates NNN mediated phospholipid hydrolysis.

Involvement of TSNA’s in phospholipid

molecular species alteration

Table 1 shows the changes in FA composition in both

phospholipids and neutral lipids of NNN treated cells.

Cells during adaptation to NNN exposure; showed an

overall decrease in C16 series of fatty acids (C16:0

and C16:1) and an overall increase in C18 series fatty

acids (C18:0 and C18:1), particularly in PE and PS.

Antonie van Leeuwenhoek

123

In this study, alteration of the molecular species in

the presence of NNN were significantly (P \ 0.001)

higher in phospholipids when compared with neutral

lipids (Table 1). In PE monounsaturated FA (C18:1)

was increased significantly where as saturated C16

FA was decreased in NNN treated cells. NNN

exposed cells seemed to increase the saturated FA

chain length in PI. Among the phospholipids, short

chain FA content (C12:0 and C14:0) was increased in

CL in NNN treated cells (Table 1). In sterol there was

a marked increase in C18 series FAs.

Cell counts

NNN treated cells yielded a lower (*80%) number

of cells than control (Fig. 3a), and it can be due to

cell damage and it was shown in Fig. 3b.

Discussion

Reduction of phospholipid content with NNN exposure

was fully corroborated, using S. cerevisiae as a model

system for investigating membrane lipid homeostasis.

In order to understand the impact of NNN on lipid

metabolism, S. cerevisiae cells were treated over a wide

range of NNN concentration. The alterations in total

phospholipid levels were determined in presence of

NNN and a concentration dependent reduction in total

phospholipid content (Fig. 1a) was observed. Reported

evidences showed that NNN exposure alters the lipid

composition of the membrane during adaptation and it

serves as an important factor for NNN binding

(Schuster et al. 1995; Sauk and Norris 1988).

During the last two decades, carcinogenicity of

TSNA compounds and its associated problems has

Fig. 1 Exposure to NNN alters the lipid composition. Equal

number of cells was taken in each group and at the end of the

experiment, lipids were extracted and resolved on silica TLC

and quantified. a Effect of NNN on incorporation of [32P]

orthophosphate into yeast phospholipid. The cells were grown

in YPD medium containing 100 lCi of [32P] orthophosphate

for 12 h in the absence or presence of different concentrations

(0–400 lM) of NNN as described in ‘‘Materials and Methods’’.

b, c Cells were grown in the absence or presence of optimal

concentration of NNN (50 lM) in the medium. b Phospholipid

content was observed by labelling phospholipids with 100 lCi

of [32P] orthophosphate. c Neutral lipid content was observed

by labelling neutral lipids with 0.5 lCi of [14C] acetate. Values

are the mean of three separate experiments, each performed in

duplicate. Data are means ± SD, �P \ 0.05, #P \ 0.01 and

*P \ 0.001 versus control

Antonie van Leeuwenhoek

123

been the subject of intensive investigation and

tremendous progress has been made. However under-

standing membrane remodelling, associated with

carcinogenicity, particularly phospholipid alteration,

is yet to be studied. Pathophysiological mechanisms

of TSNA compounds depend on membrane organi-

sation/composition. One of the first steps of carcino-

genesis by TSNAs is by inhalation of tobacco smoke

and the generation of reactive oxygen species within

the cells (Alavanja 2002). We have earlier reported

that increased membrane damage occurs through

reactive oxygen species (ROS) during NNN exposure

(Nachiappan et al. 1994). Interaction of NNN with

targeted cells mainly depends on lipid and fatty acid

composition (Schuster et al. 1992). A moderate

reduction in phospholipid content and alteration in

fatty acid pattern was also observed in presence of

NNN by others (Schuster et al. 1986, 1990; Sauk and

Norris 1988). Reported evidence suggests that NNN

may contribute to the alteration in the structure and

function of membrane lipids and also to its remod-

elling enzyme activity. Lands proposed that a deac-

ylation–reacylation cycle was involved in the

remodelling system. The Lands cycle consists of

phospholipid cleavage by phospholipases to produce

a lysophospholipid, ATP-driven activation of another

FFA by acyl-CoA synthetase, and transfer of the fatty

acid from the acyl-CoA to the lysophospholipid by

acyl-CoA:lysophospholipid acyltransferases (Lands

and Crawford 1976).

Interaction of NNN with targeted cells mainly

depends on lipid and fatty acid composition (Schuster

et al. 1992). NNN is a potent pulmonary carcinogen

and phospholipids are important components required

Fig. 2 NNN enhances PLB activity. The cells were grown for

12 h in the absence or presence of NNN and the cell lysate was

used as an enzyme source. The obtained cell lysate was incubated

with [32P] orthophosphate labelled phospholipid at 37�C for

60 min and the enzyme activity was measured. a Effect of NNN

(50 lM) on hydrolysis of various phospholipids. b The rate of

phospholipid hydrolysis was measured in wild type and plbDstrains (plb1, plb2 and plb3) exposed to various concentration of

NNN. c Effect Ca2? on hydrolysis of PC. The values are the

means of three separate experiments, each performed in

duplicate. Data are means ± SD, �P \ 0.05, #P \ 0.01 and

*P \ 0.001 versus control

Antonie van Leeuwenhoek

123

Ta

ble

1E

ffec

to

fN

NN

on

alte

rati

on

of

fatt

yac

idco

mp

osi

tio

nin

yea

stp

ho

sph

oli

pid

and

neu

tral

lip

id

Fat

tyac

id(%

)C

10

:0C

12

:0C

14

:0C

16

:0C

16

:1C

18

:0C

18

:1C

18

:2

Ph

osp

hat

idy

lch

oli

ne

Co

ntr

ol

0.0

6±

0.0

10

.69

±0

.01

2.3

7±

0.5

24

0.0

5±

1.1

41

9.3

3±

1.2

11

.18

±0

.32

36

.27

±1

.55

–

NN

N0

.08

±0

.01�

0.7

6±

0.0

8*

2.0

4±

0.4

2*

42

.46

±1

.51

*1

1.8

1±

1.5

7*

1.3

6±

0.4

54

1.4

6±

3.2

*–

Ph

osp

hat

idy

leth

ano

lam

ine

Co

ntr

ol

0.2

3±

0.0

15

.36

±0

.13

5.6

3±

0.5

27

0.1

4±

1.1

71

1.4

7±

1.4

35

.50

±0

.34

1.6

4±

1.5

5–

NN

N–

1.6

4±

0.0

5*

2.4

1±

0.9

2*

50

.64

±1

.51

*6

.62

±0

.44

*1

.92

±0

.48

*3

6.7

4±

3.2

3*

–

Ph

osp

hat

idy

lser

ine

Co

ntr

ol

1.7

7±

0.1

42

1.7

9±

1.2

71

4.1

0±

0.5

24

9.2

0±

3.1

38

.10

±1

.32

3.2

2±

0.4

61

.77

±0

.73

–

NN

N1

.73

±0

.32

#1

6.4

0±

1.4

7*

10

.48

±0

.41

*3

9.8

0±

1.5

2*

4.5

5±

1.5

4*

18

.25

±1

.65

*7

.20

±1

.45

*1

.54

±0

.53

*

Ph

osp

hat

idy

lin

osi

tol

Co

ntr

ol

–5

.61

±0

.51

11

.90

±0

.53

41

.24

±1

.53

7.8

3±

1.4

21

9.4

1±

1.5

11

2.2

1±

0.3

21

.77

±0

.56

NN

N–

0.8

1±

0.0

8#

5.7

4±

0.4

2*

49

.03

±2

.30

*3

.43

±1

.55

*2

7.8

2±

2.4

3*

9.5

2±

0.8

4*

3.6

2±

0.9

2*

Ph

osp

hat

idic

acid

Co

ntr

ol

4.6

7±

0.4

21

7.0

4±

0.5

31

5.3

3±

0.5

12

1.0

4±

0.4

21

0.4

0±

0.7

51

4.4

7±

0.4

51

3.5

3±

0.3

73

.48

±0

.28

NN

N5

.20

±0

.82

*1

7.5

4±

1.2

4*

15

.59

±0

.81�

21

.01

±1

.32

9.8

4±

1.2

4*

15

.00

±0

.57

*1

2.5

5±

0.4

2*

3.2

3±

0.3

7#

Car

dio

lip

in

Co

ntr

ol

0.5

1±

0.0

95

.84

±0

.32

5.4

4±

0.7

23

4.8

6±

2.1

17

.00

±0

.74

4.9

7±

3.2

03

0.3

3±

1.5

4–

NN

N3

.14

±0

.70

*1

3.0

4±

1.6

0*

12

.28

±1

.02

*2

2.9

1±

2.1

2#

14

.28

±1

.52

*1

2.6

7±

1.0

0*

21

.64

±1

.98

*–

Ste

rol

Co

ntr

ol

–1

0.1

1±

0.6

21

4.0

8±

0.9

43

1.7

4±

2.0

74

.85

±1

.18

23

.41

±3

.22

11

.42

±0

.92

4.3

7±

2.0

7

NN

N0

.02

±0

.00

97

.13

±1

.01

#8

.45

±1

.73

#3

0.5

3±

2.9

61

0.8

4±

1.4

2*

26

.26

±5

.43

#7

.78

±1

.32

#8

.96

±2

.01

*

Tri

gly

ceri

de

Co

ntr

ol

0.1

1±

0.0

08

18

.73

±1

.01

14

.66

±0

.81

30

.79

±4

.31

3.8

1±

1.3

32

0.4

4±

3.2

18

.46

±1

.12

2.9

7±

0.0

3

NN

N0

.11

±0

.01

17

.61

±2

.33

15

.57

±3

.22

31

.41

±1

.24

*4

.13

±1

.94

*1

9.3

0±

4.2

18

.33

±3

.20

�3

.49

±1

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to maintain the normal pulmonary function. Decreased

secretion of PC (*50%) was observed in rat alveolar

type II cells by cigarette smoke exposure (Wirtz and

Schmidt 1996) and similar observation was also

reported by Giammona et al. (1971). Clinical reports

showed decreased surfactant phospholipids in the

lavage fluid of smokers (Hohlfeld et al. 1997; Finley

and Ladman 1972). Hence, we monitored the quan-

titative and qualitative content of both phospholipids

and neutral lipids in S. cerevisiae that were treated

with NNN. Cells exposed to NNN, approximately

40% (Fig. 1a) decrease in the total phospholipid was

observed in [32P] orthophosphate labelling and a

significant reduction were noted in PC, PS and PE

content (Fig. 1b). The changes in these phospholipids

may lead to alteration in the membrane composition of

the cells. According to de Kruijff (2006), the cells

adjust their membrane properties to environmental

conditions, among other mechanisms, by changing the

ratio of PC/PE. Decreased PC content results in the

loose packing, as found in disordered membranes

(Juresic et al. 2009). In addition, altered acyl chain

composition was observed during PC depletion. PC

and PE have a different influence on the conformation

of transmembrane proteins and consequently on the

membrane organization and function (Boumann et al.

2006; Kruijff 2006; Storey et al. 2001). We observed a

substantial reduction in PS and it is an important cause

for depletion of major phospholipids, since PS is a key

intermediate in the synthesis of PC and PE in yeast

(Birner et al. 2001, 2003; Carman and Zeimetz 1996).

The above reports strengthen our results and the

formation of LPL (24%) in this study is higher than

reported by Schuster et al. (1995).

Each phospholipid has a vital role in the normal

function of the cells and its alteration leads to

membrane dysfunction (Ghosh et al. 2008) Regulation

of phospholipid and neutral lipid synthesis are inter-

related (Carman and Henry 1999). Phosphatidic acid

(PA) is a vital phospholipid that acts as a biosynthetic

precursor for the formation of both phospholipids and

neutral lipids (Rajakumari et al. 2008; Carman and

Henry 2007; Athenstaedt and Daum 1999). The

increased neutral lipids content may be due to NNN-

treated cells utilizes PA for neutral lipid synthesis. In

this study, depletion of phospholipids was observed

with [32P] orthophosphate labelling and the [1-14C]

acetate labelling depicted increased neutral lipid

synthesis in the presence of NNN. Here we have

shown that there was a substantial increase in DAG,

TAG, FFA and sterol, levels with NNN treated cells

(Fig. 1c). Our data are in accordance with the obser-

vation that defective phospholipid metabolism leads to

accumulation of neutral lipids (Malanovic et al. 2008).

Increased TAG in presence of NNN might be due

to increased activity of either DAG-acyltransferase

(Cases et al. 1998) or phospholipid: diacylglycerol

acyltransferase (PDAT) (Dahlqvist et al. 2000). There

are several phospholipases namely phospholipases B,

C, and D (Merkel et al. 2005; Flick and Thorner 1993;

Mayr et al. 1996). Phospholipase C produces DAG

(Flick and Thorner 1993) whereas phospholipase D

produces PA (Mayr et al. 1996; Mendonsa and

Engebrecht 2009). In addition, phospholipid depletion

may be due to dephosphorylation of PA by PA-

phosphatase to produce DAG, which is subsequently

converted to TAG (Gaspar et al. 2008).

Among the deacylating phospholipases, PLB cat-

alyzed reactions are having vital role in the regulation

of lipid synthesis, with potential implications for lipid

pathologies (Hurley and McCormick 2008). The

activation of this enzyme results in the generation

of lysophospholipids and fatty acids that can directly

affect other cellular processes (Schweizer 2004). In

this present study, we observed a significant decrease

in the phospholipid content and a simultaneous

increase in LPL (Fig. 1b) and FFA (Fig. 1c).

Reported evidences suggest that cigarette smoke

Fig. 3 Growth and viability—effect of NNN on the growth

and viability of cells was monitored in liquid medium at 30�C.

a Cell growth was studied in the absence or presence of NNN

(50 lM), by measuring the A600 of the cultures at frequent

intervals. b Aliquots of 1:10 serial diluted Cells (A600 = 1.0)

were spotted to solid YPD-agar plate with or without NNN and

incubated for 48 h at 30�C

Antonie van Leeuwenhoek

123

reduces the phospholipid content (Scott 2004) by

activated phospholipase A2 (Oulton et al. 1991).

Reduction of phospholipids may be due to increased

degradation and this was confirmed by enzymatic

hydrolysis of phospholipids by phospholipase assay.

We found that the enhanced phospholipase activity in

the presence of NNN, and the hydrolysis of PC was

significantly higher when compared to other phos-

pholipids (Fig. 2a). Current study suggested that the

reduction of phospholipids is mainly the result of

NNN mediated phospholipase activity and PC is a

preferred substrate. Apart from PC, marked hydroly-

sis was also observed in PS, PE, and PG in the

presence of NNN (Fig. 2a).

In S. cerevisiae PLBs are encoded by plb1, plb2,

and plb3 and are associated with the plasma mem-

brane, cell wall, and periplasmic space, respectively.

plb1 and plb2 preferred to hydrolyse PC, PE, PS and PI

whereas Plb3 hydrolyzes only PI and PS (Merkel et al.

2005). To identify the role NNN on these enzyme

activities, we performed phospholipase assay with all

three mutants and wild type cells. Our results suggest

that NNN significantly enhances the plb1 activity as

compared to plb2 and plb3 (Fig. 2b). PC hydrolysis

was markedly decreased when compared with wild-

type cells. This reveals the existence of other PC

degrading enzyme(s) other than plb1 that are activated

(Merkel et al. 2005). plb1 appears to be primarily

responsible for the degradation of mainly PC and to

some extent PI (Lee et al. 1994; Henry and Patton-

Vogt 1998) and we also observed similar results.

Interestingly, over expression of Plb2 does not result

in degradation of PC or PI, while Plb3 hydrolysed PI,

but not PC (Merkel et al. 2005). In addition, increased

plb1 activity leads to a significant increase in TAG and

a concomitant decrease in cellular phospholipids

(Merkel et al. 2005), suggesting that fatty acids used

for TAG synthesis are derived partly from PLB

catalysed phospholipid degradation.

Phospholipases are activated either at low Ca2?

concentrations or at high concentrations. To check

whether Ca2? is required for NNN mediated

phospholipid breakdown in vitro, we tested for

phospholipase assay with or without Ca2? in wild

type cells treated with or without NNN. In the

presences of NNN, the phospholipase activity was

greatly enhanced by the addition of Ca2?. In addition,

we have observed the increased enzyme activity in

NNN-treated cell even in the absence of Ca2?. It may

be due to PC degrading enzyme(s) other than Ca2?

dependent phospholipase that are activated under

certain conditions. Reported evidence showed that

PLB from yeast can be either Ca2? or other cations

dependent (Merkel et al. 2005; Fyrst et al. 1999; De

Kroon 2007). Overall results suggest that NNN

decreases the phospholipid content by enhancing

PLB activity and is associated with an increase in

LPL and free fatty acid content.

Decreased C16 series fatty acids (C16:0 and C16:1)

and increased C18 series fatty acids (C18:1) were

observed in NNN treated cells. The changes were more

predominant in PE the most abundant phospholipid

during PC depletion. Our results were strengthened by

Ferreira et al. (2004) who also found that the excess

saturated FAs are stored mainly in specific phospho-

lipids and not in TAG and steryl esters. The acyl chain

composition of CL is important for its function and we

investigated the CL profile in the presence of NNN.

The average length of the fatty acids is decreased,

specifically with increase in C12:0 and C14:0 and a

decrease in C16 series. The increase in myristic acid

might have a vital role in protein modification, an

essential step in signaling reactions such as the Ras

pathway, thus fatty acid modification alters cellular

proliferation and function (Tehlivets et al. 2007). Cell

viability was assessed with 50 lM of NNN and a 20%

reduction in growth was depicted. Possibly, the low PC

levels interfere with the diauxic shift; leading to

increased cell death (Boumann et al. 2006).

To summarize, we demonstrated the changes in

lipids by NNN in S. cerevisiae. Our results suggest

the decreased phospholipid content may be due to

enhanced PLB activity associated with increased

generation of lysophospholipids and free fatty acids.

We also observed accumulation of neutral lipids and

substantial changes in fatty acid composition. PLB/

PLA2 is a key enzyme involved in the pathology of

many clinically distinct lipid dysfunctions (Hurley

and McCormick 2008). Further studies using mutants

are required to clarify the mechanisms of the NNN in

both phospholipid and neutral lipid metabolism.

Acknowledgments The financial support from Bharathidasan

University, Tiruchirappalli, India is gratefully acknowledged.

We also acknowledge Department of Biochemistry, Indian

Institute of Science, Bangalore, India for helping us to conduct

the radioactive study and also providing the radioactive material.

Conflict of interest None.

Antonie van Leeuwenhoek

123

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