MODIFICATION OF NUCLEOTIDE EXCISION REPAIR ... - QSpace

MODIFICATION OF NUCLEOTIDE EXCISION REPAIR

ACTIVITY BY 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-

BUTANONE (NNK) AND SULFORAPHANE

by

Christopher Matthew Harris

A thesis submitted to the Department of Biomedical and Molecular Sciences

In conformity with the requirements for

the degree of Doctor of Philosophy

Queen’s University

Kingston, Ontario, Canada

(January, 2017)

Copyright ©Christopher Matthew Harris , 2017

ii

Abstract

The studies described in this thesis investigated the relationship between the DNA repair

pathway nucleotide excision repair (NER, which is critical for protecting against carcinogenesis),

and two exogenous chemicals, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone (NNK) and the nutraceutical sulforaphane.

NER activity in female A/J mouse liver nuclear extracts decreased by 46% 12 h post

NNK treatment and increased by 48% 24 h post NNK. These changes in NER activity were not

attributed to alterations in levels of the NER proteins XPC, XPA, XPB and p53. However, the

binding of hepatic XPA and XPB to damaged DNA at both timepoints was increased, while the

binding of XPC to damaged DNA was unchanged at 12 h and decreased at 24 h.

A 63% decrease in NER activity in female A/J mouse lung nuclear protein extracts 24 h

post NNK treatment was not attributed to changes in levels of XPC, XPA, XPB or p53.

However, the binding of lung XPA and XPB to damaged DNA was decreased and the binding of

XPC to damaged DNA was increased at 24 h. Hence, NNK-induced NER changes are time and

organ dependent and are associated with changes in the binding activities, but not the levels, of

specific recognition and early excision NER proteins.

A dose (100 mg/kg) and timepoint (6 h) of maximal effect of sulforaphane on female

CD-1 mouse hepatic and pulmonary mRNA levels of two genes regulated by Nrf2, a major

signalling pathway of sulforaphane mediated-effects, were found. A 50% increase in NER

activity was observed in liver nuclear extracts 12 h post sulforaphane treatment, while lung NER

at 12 h and liver NER at 6 h were not affected by sulforaphane. The increase in hepatic NER

activity at 12 h was not associated with changes in levels of XPC, XPA, XPB or p53 or in the

binding of hepatic XPC, XPA and XPB to damaged DNA. These results suggest that the impact

of sulforaphane on repair-associated processes is time and organ dependent and is not associated

iii

with changes in the levels or activities of these specific recognition and early excision NER

proteins.

iv

Co-Authorship

This research was conducted by the candidate Christopher M. Harris, under the

supervision of Dr. Thomas E. Massey. Natalie Chow provided the qRT-PCR work contained in

chapter 4.

v

Acknowledgements

I would like to begin by thanking Dr. Thomas Massey, my supervisor, for his guidance

and support throughout the work that is presented here. I would like to thank you for your

patience and encouragement through this whole process; without you I would be lost. Also thank

you to Dr. Kanji Nakatsu for helping me learn how to write scientifically.

Thank you to Natalie Chow, who provided much of the hard work comprising Chapter 4.

Your work is so appreciated, and rewards you every day.

I would also like to thank the inimitable Sandra Graham for technical assistance on this

project, and Drs. Jeanne Mulder and Pamela Brown for helpful suggestions. Jeanne and Sandy,

thanks to you especially for your positivity and your kindness in dealing with my flaws and me.

Thanks to my friends and family in Toronto, London, New York and Kingston- Jessi,

David, Roz, Jon, Kelcey, Gab and all of the Queen’s people- you know who you are. I love you

all; you are my bottomless well of support and motivation. Mia and James- I love every moment

I get to spend with you. I love seeing you grow and learn, I can’t wait to see you fix this world.

Mia- I am sure there is a typo in here, I will be blaming that on you.

Thank you to my parents and in-laws: you are always understanding about the things I

say and do that you don’t understand. I am so fortunate to have two families that only want for

me what I want for myself. My gift to you is this is the only section of this book I expect you to

read.

Finally, thanks to my peerless partner, Erica. My life would be so quiet without you, in

so many ways. You are the reason our life together is colourful. You are my good spirit, my

better self; I love jumping over bad times, holding your hand.

This work was supported by the Canadian Institutes of Health Research and the Natural

Sciences and Engineering Research Council of Canada.

vi

Table of Contents

Abstract ............................................................................................................................................ ii

Co-Authorship ................................................................................................................................. iv

Acknowledgements .......................................................................................................................... vList of Figures .................................................................................................................................. x

List of Abbreviations .................................................................................................................... xiv

Chapter 1 General Introduction ....................................................................................................... 1 Statement of the research problem ......................................................................................... 11.1

Chemical carcinogenesis ........................................................................................................ 31.2

DNA repair and nucleotide excision repair ........................................................................... 51.3

1.3.1 DNA repair ...................................................................................................................... 51.3.2 Nucleotide excision repair .............................................................................................. 6

1.3.2.1 Triple-check recognition .......................................................................................... 8

1.3.2.2 Incision/excision ...................................................................................................... 91.3.2.3 Gap filling .............................................................................................................. 10

1.3.3 Changes in NER activity ............................................................................................... 101.3.3.1 Rate limiting NER proteins .................................................................................... 11

1.3.3.2 p53 and NER .......................................................................................................... 121.3.3.3 Conditions that increase NER activity. .................................................................. 131.3.3.4 Conditions that decrease NER activity .................................................................. 14

NNK ..................................................................................................................................... 151.4

1.4.1 Occurrence and source of NNK .................................................................................... 151.4.2 Biotransformation of NNK ........................................................................................... 181.4.3 Detoxification of NNK ................................................................................................. 20

1.4.4 Carcinogenicity of NNK in experimental animals ........................................................ 20

1.4.5 NNK and human cancers .............................................................................................. 21

1.4.6 DNA repair and NNK ................................................................................................... 24

Sulforaphane ........................................................................................................................ 261.5

1.5.1 Glucosinolates ............................................................................................................... 26

1.5.2 Sulforaphane properties and kinetics ............................................................................ 291.5.3 Sulforaphane as a cancer chemopreventive, cytotoxic and cytoprotective chemical ... 30

1.5.4 Nrf2 ............................................................................................................................... 321.5.5 The actions of Nrf2 ....................................................................................................... 35

vii

1.5.6 Sulforaphane mechanisms not associated with Nrf2 .................................................... 36

1.5.7 Sulforaphane and DNA repair ...................................................................................... 37 Research hypotheses and objectives .................................................................................... 391.6

Chapter 2 Time course of effects of NNK on nucleotide excision repair activity on A/J mouse

liver and possible mechanisms of change ...................................................................................... 42 Introduction .......................................................................................................................... 422.1

Materials and methods ......................................................................................................... 462.2

2.2.1 Animal treatments ......................................................................................................... 462.2.2 Preparation of cell-free whole tissue nuclear protein extract ........................................ 46

2.2.3 Preparation of damaged plasmid DNA ......................................................................... 47

2.2.4 In vitro DNA repair synthesis assay ............................................................................. 482.2.5 Immunoblot analysis of NER protein levels in mouse liver nuclear protein extracts .. 49

2.2.6 Analysis of binding of NER incision proteins to damaged DNA ................................. 492.2.7 Data analysis ................................................................................................................. 51 Results .................................................................................................................................. 512.3

2.3.1 Time course of effects of NNK treatment on mouse liver NER activity ...................... 512.3.2 The effect of in vivo treatment of mice with NNK on levels of key NER proteins in

mouse liver nuclear protein extracts ...................................................................................... 532.3.3 Effect of in vivo treatment of mice with NNK on binding of liver NER proteins to

damaged DNA ....................................................................................................................... 56 Discussion ............................................................................................................................ 592.4

Chapter 3 Investigation of the mechanisms by which NNK decreases nucleotide excision repair

activity in mouse lung .................................................................................................................... 63 Introduction .......................................................................................................................... 633.1


3.2.1 Animal treatments ......................................................................................................... 66

3.2.2 Preparation of cell-free whole tissue nuclear protein extract ........................................ 67

3.2.3 Preparation of damaged plasmid DNA ......................................................................... 673.2.4 In vitro DNA repair synthesis assay ............................................................................. 67

3.2.5 Immunoblot analysis of NER protein levels in mouse lung nuclear protein extracts ... 673.2.6 Analysis of binding of NER recognition proteins to damaged DNA ........................... 67

3.2.7 Data analysis ................................................................................................................. 67

Results .................................................................................................................................. 683.3

viii

3.3.1 Effect of treatment of mice with NNK on repair synthesis of mouse lung nuclear

protein extracts ....................................................................................................................... 683.3.2 Effect of in vivo treatment of mice with NNK on levels of key NER proteins in mouse

lung nuclear protein extracts .................................................................................................. 70

3.3.3 Effect of in vivo treatment of mice with NNK on key NER protein DNA binding in

mouse lung nuclear protein extracts ...................................................................................... 72

Discussion ............................................................................................................................ 743.4

Chapter 4 Investigation into the effects of in vivo sulforaphane treatment on nucleotide excision

repair activity and nucleotide excision repair recognition proteins ............................................... 77

Introduction .......................................................................................................................... 774.1


4.2.1 Animal treatment for dose response and time course of sulforaphane effects ............. 81

4.2.2 Analysis of mRNA from Nrf2-regulated genes ............................................................ 814.2.3 Preparation of cell-free whole tissue nuclear protein extracts ...................................... 824.2.4 Preparation of damaged plasmid DNA ......................................................................... 82

4.2.5 In vitro DNA repair synthesis assay ............................................................................. 824.2.6 Immunoblot analysis of NER incision protein levels in mouse liver nuclear protein

extracts ................................................................................................................................... 824.2.7 Analysis of binding of NER recognition proteins to damaged DNA ........................... 83

4.2.8 Data analysis ................................................................................................................. 83 Results .................................................................................................................................. 834.3

4.3.1 Response of two Nrf2 regulated genes to in vivo treatment with sulforaphane ............ 83

4.3.2 Timecourse of responses of two Nrf2 regulated genes to 100 mg/kg of sulforaphane . 854.3.3 Effect of in vivo treatment of mice with sulforaphane on repair synthesis activity in

mouse liver and lung nuclear protein extracts ....................................................................... 87

4.3.4 Effects of in vivo treatment of mice with sulforaphane on levels of NER proteins in

mouse liver nuclear protein extracts ...................................................................................... 89

4.3.5 Effect of in vivo treatment of mice with sulforaphane on key NER protein DNA

binding in mouse liver nuclear protein extracts ..................................................................... 91

Discussion ............................................................................................................................ 934.4

Chapter 5 General Discussion ........................................................................................................ 97

Overall conclusions .............................................................................................................. 975.1

Future directions .................................................................................................................. 995.2

ix

5.2.1 Post-translational modifications of NER proteins as a basis for observed changes in

GG-NER activity ................................................................................................................... 995.2.1.1 XPC binding to damaged DNA ........................................................................... 101

5.2.1.2 XPA binding to damaged DNA ........................................................................... 101

5.2.1.3 TFIIH binding and activity .................................................................................. 1025.2.2 Effects of sulforaphane and NNK on other NER proteins .......................................... 103

5.2.2.1 RPA ...................................................................................................................... 103

5.2.2.2 PCNA ................................................................................................................... 1045.2.2.3 Post-translational modifications of p53 ............................................................... 105

5.2.3 Protein adducts ............................................................................................................ 106

5.2.4 The effects of sulforaphane are mediated through HDAC inhibition and Nrf2 activation

.............................................................................................................................................. 107

5.2.5 The effects of cigarette smoking on NER activity in humans .................................... 107References ....................................................................................... Error! Bookmark not defined.Appendix A The effect of DMSO on nucleotide excision repair activity in A/J mouse lung and

liver .............................................................................................................................................. 131

x

List of Figures

Figure 1.1 Simplified mechanism of global genome nucleotide excision repair. Adapted from Li

et al. (2015b) and Marteijn et al. (2015). ......................................................................................... 7

Figure 1.2 Structures of the three most common tobacco-specific nitrosamines formed by the

nitrosation of nicotine. Abbreviations in text. .............................................................................. 17

Figure 1.3 Pathways of NNK metabolism to reactive metabolites that * methylate,☆

pyridyloxobutylate or ★ pyridylhydroxybutylate (© Brown, 2008). ........................................... 19

Figure 1.4 The hydrolytic conversion of glucoraphanin to sulforaphane by myrosinase. ............. 28

Figure 1.5 Model of Nrf2 activation mechanism. In the absence of stressors, Nrf2 is bound to

dimeric Keap1 and serves as a substrate for ubiquitin ligase to target Nrf2 for ubiquitination and

proteasomal degradation. Activation occurs via oxidant or electrophilic reaction with cysteine

residues on Keap1, leading to stabiliziation of Nrf2 through an unclear mechanism (reviewed by

Baird & Dinkova-Kostova, 2011) and eventual translocation into the nucleus where Nrf2

dimerizes with small Mafs and activates ARE-dependent transcription of several cytoprotective

enzymes. NAD(P)H:quinone oxidoreductase 1 (NQO1), phosphoglycerate dehydrogenase

(PGD), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), ferritin

light chain (FTL). ........................................................................................................................... 34

Figure 2.1. Effects of in vivo NNK or saline treatment on in vitro DNA repair activity of mouse

liver extracts towards NNKOAc-induced pyridyloxobutyl DNA damage. Extracts were prepared

from mice treated with either 10 µmol NNK or saline and killed 2, 4, 12, 24 or 72 h post-dosing.

Data are presented as mean + SD. * significantly different from control (p<0.05; n=4 pools of 4

mouse livers each.) ......................................................................................................................... 52

Figure 2.2 a) Relative density of each key NER protein in mouse liver nuclear protein extracts

isolated from saline and NNK-treated mice 12 h post-dosing, relative to a constant internal

standard extract. Data are presented as mean ± SD. b) Representative immunoblots of the

xi

relative levels of each key NER protein in mouse liver extracts. S= nuclear protein extracts from

saline treated mice, N=nuclear protein extracts from NNK treated mice; n=4 pools of 4 mouse

livers each. ..................................................................................................................................... 54

Figure 2.3 a) Relative density of each key NER protein in mouse liver nuclear protein extracts

isolated from saline and NNK-treated mice 24 h post-dosing, relative to a constant internal

standard extract. Data are presented as ean + SD. b) Representative immunoblots of the relative

levels of each key NER protein in mouse liver extracts. S= nuclear protein extracts from saline

treated mice, N=nuclear protein extracts from NNK treated mice; n=4 pools of 4 mouse livers

each. ............................................................................................................................................... 55

Figure 2.4 Effects of in vivo NNK treatment on in vitro NER protein binding to NNKOAc-

induced pyridyloxobutyl DNA damage 12h post treatment. Data are presented as mean + SD. *

Significantly different from control (p<0.05; n=4 pools of 4 mouse livers each.) ........................ 57


induced pyridyloxobutyl DNA damage 24h post treatment. Data are presented as mean + SD. *

Significantly different from control (p<0.05; n=4 pools of 4 mouse livers each.) ........................ 58

Figure 3.1 Effects of in vivo NNK or saline treatment on in vitro DNA repair activity of A/J

mouse lung extracts towards NNKOAc-induced pyridyloxobutyl DNA damage. Extracts were

prepared from mice treated with either sterile saline or 10 µmol NNK and sacrificed 24h post-

dosing. Data are presented as mean + SD. (*) Significantly different from control (n=4 pools of 4

mice per pool, p<0.05). .................................................................................................................. 69

Figure 3.2 a) Relative density of each key NER protein in A/J mouse lung nuclear protein extracts

isolated from saline or NNK-treated mice 24h post-dosing, relative to a constant internal standard

extract. Data are presented as the mean + SD presented. b) Representative immunoblots of the

relative levels of each key NER protein in mouse lung extracts. S= nuclear protein extracts from

saline treated mice, N=nuclear protein extracts from NNK treated mice; ; n=4 pools of lungs of 4

mice per pool. ................................................................................................................................. 71


derived pyridyloxobutyl DNA damage. The data are presented as mean + SD. (*) Significantly

xii

different from control (XPA n=5 pools of lungs of 4 mice per pool; XPC and XPB n=6 pools of

lungs of 4 mice per pool, p<0.05). ................................................................................................. 73

Figure 4.1 mRNA levels for dose-response relationship of two Nrf2 regulated genes relative to

control, GCLC and HO-1; 3 h after treatment with sulforaphane (SF; n=4). The data are

presented as mean + SD. (*) Significantly different from control (p<0.05). ................................. 84

Figure 4.2 Timecourse of effects for two Nrf2 regulated genes HO-1 and GCLC in lung and liver

after treatment with 100mg/kg of sulforaphane (SF) or corn oil (n=4). The data are presented as

mean + SD. (*) Significantly different from control (p<0.05). ..................................................... 86

Figure 4.3 Effects of in vivo sulforaphane treatment on in vitro DNA repair activity of mouse

liver and lung nuclear protein extracts towards NNKOAc-induced pyridyloxobutyl DNA damage.

Extracts were prepared from mice treated with either 100mg/kg sulforaphane or corn oil and

killed 6 h or 12 h post-dosing (n=4 pools of lungs of 4 mice per pool or n=4 livers). Data are

presented as mean + SD. (*) Significantly different from control (p<0.05). ................................ 88

Figure 4.4 a) Relative density of each NER protein in mouse liver extracts isolated from corn oil

or sulforaphane-treated mice 12 h post-dosing, relative to a constant internal standard extract

(CTL). The data are presented as mean + SD. b) Representative immunoblots of the relative

levels of each key NER protein in mouse liver extracts. S= nuclear protein extracts from

sulforaphane treated mice, C=nuclear protein extracts from corn oil treated mice; all experiments

n=4. ................................................................................................................................................ 90

Figure 4.5 Effect of in vivo sulforaphane treatment on in vitro NER protein binding to

pyridyloxobutyl-adducted DNA. All data are presented as mean + SD. (all n=4, p>0.05) .......... 92

Figure A.1 Effects of in vivo DMSO treatment on in vitro DNA repair activity of mouse lung or

liver extracts towards NNKOAc-induced pyridyloxobutyl DNA damage. Extracts were prepared

from mice treated with either 0.1mL sterile saline or 0.04mL DMSO and killed 2 h post-dosing.

Data are presented as mean + SD. (*) Significantly different from control (n=4 pools of liver or

lungs of 4 mice per pool, p<0.05). ............................................................................................... 134

xiv

List of Abbreviations

AGT O6-alkylguanine-DNA alkyltransferases

ARE antioxidant response element

ATP adenosine triphosphate

ATR ataxia-telangiectasia mutated and Rad3-related

BAX bcl-2-like protein 4

BER base excision repair

°C degree(s) Celsius

CAK cyclin-dependent kinase-activating kinase

cDNA complementary DNA

CYP cytochrome p450

dATP deoxyadenosine triphosphate

dCTP deoxycytidine triphosphate

DDR direct damage reversal

dGTP deoxyguanosine triphosphate

DNA deoxyribonucleic acid

dsDNA double-stranded DNA

DTT dithiothreitol

dTTP deoxythymidine triphosphate

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

EpRE electrophile response elements

et al. et alia (and others)

FTL ferritin light chain

g gram(s)

xv

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GCLC glutamate-cysteine ligase catalytic subunit

GG-NER global genome nucleotide excision repair

GSH glutathione

GST glutathione-S-transferase

h hour(s)

HDAC histone deacetylase

HEPES-KOH 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-potassium hydroxide

HO-1 heme oxygenase 1

HPB 4-hydroxy-1-(3-pyridyl)-1-butanone

HPLC high performance liquid chromatography

HRP horseradish peroxidase

i.e. id est (that is)

in vitro outside the living body

in vivo inside the living body

i.p intraperitoneal injection

ITC isothiocyanate

KEAP1 kelch-like ECH-associated protein 1

L litres

M molar

mRNA messenger RNA

mM millimolar

mg milligram

mL millilitre(s)

µCi microCurie(s)

xvi

µg microgram

µmol micromoles

µL microlitres

NAB 1-nitrosoanabasine

NAT N’-nitrosoanatabine

NER nucleotide excision repair

ng nanograms

NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

NNKOAc 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone

NNN N’-nitrosonornicotine

NQO1 NAD(P)H:quinone oxidoreductase 1

Nrf2 NF-E2-related factor 2

p53 tumour suppressor protein p53

PAH polycyclic aromatic hydrocarbons

PBS phosphate-buffered saline

PCR polymerase chain reaction

PCNA proliferating cell nuclear antigen

PGD phosphoglycerate dehydrogenase

PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

PheT r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene

POB pyridyloxobutyl

PVDF polyvinylidene fluoride

RNA ribonucleic acid

ROS reactive oxygen species

xvii

RPA replication protein A

SD standard deviation

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SF sulforaphane

SF-GSH SF-glutathione

SF-NAC SF-N-acetylcysteine

SF-Cys SF-cysteine

SIRT1 sirtuin 1

ssDNA single stranded DNA

SUMO small ubiquitin-like modifiers

TC-NER transcription coupled nucleotide excision repair

TFIIH transcription factor II human

TGF-β1 Transforming growth factor β-1

TSNA tobacco specific nitrosamines

U enzymatic unit(s)

UV ultra-violet

UV-DDB UV-damaged DNA-binding protein

V volt(s)

XP xeroderma pigmentosum

XPA xeroderma pigmentosum class A protein

XPB xeroderma pigmentosum class B protein

XPC xeroderma pigmentosum class C protein- human homologue of RAD23B

XPD xeroderma pigmentosum class D protein

xviii

XPF-ERCC1 xeroderma pigmentosum class F protein- excision repair cross-complementing

repair class 1 protein

XPG xeroderma pigmentosum class G protein

1

Chapter 1

General Introduction

Statement of the research problem 1.1

Cancer is a major cause of morbidity and mortality worldwide. In Canada, cancer was

responsible for 30.2% of all deaths in 2012, with an average annual per patient cost of care

estimated at over $39,000 (Boyle & Levin, 2009). These direct financial costs represent only a

small portion of the impact of cancer on Canadian society. This class of diseases decreases

quality of life for patients and their families, places emotional and financial burdens on the

families of those afflicted with the diseases (e.g. loss of income, medical expenditures), and

represents a major demand on the medical system. Indirect and direct costs accrue in spite of the

application of contemporary surgical, radiological and chemotherapeutic practices. Clearly, it is

essential that biomedical research characterizes the mechanisms involved in cancer genesis and

progression in order to develop novel strategies to decrease morbidity and mortality associated

with this class of diseases.

Generally, cancer is characterized by unrestricted cell growth through the loss of

regulatory mechanisms that manage homeostasis and normal growth of cells. This transformation

to an abnormal, cancerous cell is dependent on the accumulation of a number of genetic and

epigenetic changes. Genomic integrity is constantly under threat from both exogenous and

endogenous chemicals and processes. Environmental agents and reactive chemicals generated by

cellular metabolic processes can attack DNA, causing various types of damage, including bulky

adducts and single-nucleotide alterations. Persistence of DNA damage can lead to mutations that

can be replicated and these alterations can change gene function or regulation of gene expression

2

and possibly lead to progression to a carcinogenic phenotype. DNA repair processes exist in

order to ensure genomic stability through excision or direct repair of damaged sequences.

Conversion of environmental carcinogens into their DNA-damaging forms (i.e. metabolic

activation or bioactivation) and the ensuing DNA damage have been the subjects of considerable

investigation, but the effects of environmental carcinogens on the DNA repair pathways have

only more recently been studied in detail. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

(NNK) is one of the most potent carcinogens tested in experimental animals and is present in

large quantities in burned and unburned tobacco. Once in the body, NNK is bioactivated into

several highly reactive DNA-binding species that form DNA adducts. Genomic stability after

genetic insult such as DNA adduction is achieved through several forms of DNA repair: direct

reversal, excision repair, post-replication repair and recombination repair. Nucleotide excision

repair (NER) and base-excision repair (BER) pathways respond to NNK-induced DNA damage.

Of particular relevance to NNK-induced DNA damage is NER, which recognizes and removes

bulky, helix-distorting genetic damage, and is modulated after treatment of experimental animals

with NNK. NER comprises three overlapping stages: recognition, incision/excision and gap

filling. Recently, regulation of these three stages has been shown to be complex and multiple

compounds appear to affect the regulation of their activities. Particular focus has been on ‘early

phase’ NER, comprising the recognition and early incision stages. However, the mechanisms of

NNK-induced NER activity changes are still not understood.

The development of novel chemopreventive strategies and effective cancer treatments

that affect NER necessitates a thorough understanding of the mechanisms governing NER

activity. In the absence of a comprehensive model for NER activity, adverse outcomes can result

from treatment. For example, preconditioning a tumour cell line with a nonlethal dose of UV-

radiation enhances NER following damage induced by cisplatin treatment (Choi et al., 2015).

3

However, UV-radiation causes DNA damage on its own; therefore, the optimal approach to

enhancing NER would be to increase NER without causing toxicity including additional DNA

damage. One promising compound in this regard is sulforaphane, a phytochemical found in high

concentrations in broccoli sprouts and other cruciferous vegetables. Sulforaphane has multiple

beneficial actions including the activation and enhancement of detoxification pathways

(Tortorella et al., 2015). Sulforaphane may have an effect on NER (Abassi Joozdani et al., 2015;

Piberger et al., 2014), although no studies have investigated whether these effects occur in vivo.

This thesis investigates the effects of in vivo treatment of mice with NNK or sulforaphane

on pulmonary and hepatic NER activities.

Chemical carcinogenesis 1.2

Carcinogenesis is the genesis of cancer, whereby healthy cells transform into abnormal

cells characterized by unrestricted growth and the potential to spread to other tissues. There are

four classes of carcinogens: chemical, physical (radiation), biological, and genetic (Pitot &

Dragan, 1991). The most diverse and well-studied class of carcinogens is the chemical

carcinogens, compounds that, after exposure to living tissue, result in neoplasms, or new and

abnormal growth of tissue (for review, see Loeb & Harris, 2008). The 18th century surgeon

Percival Pott published the first link between chemical exposure and carcinogenesis when he

reported a high incidence of scrotal cancer in chimney sweeps (Oliveira et al., 2007). It took over

200 years of incremental research before the connection between the chemicals in soot and

carcinogenesis were understood, from confirmation of the role of coal tar in the experimental

induction of cancer in rabbits to identification of benzo[a]pyrene-induced adducts in DNA and

that damage-induced DNA mutations were important in understanding the mechanisms of

carcinogenesis (Loeb & Harris, 2008). It is often the case that the link between carcinogenesis

and chemical exposure is described well before the mechanism of carcinogenesis is understood.

4

In fact, many mechanisms still have not been described or have only recently been discovered

(Pitot & Dragan, 1991; Loeb & Harris, 2008).

In the most widely accepted model of carcinogenesis, cancers develop in three distinct

stages: initiation, promotion and progression (Pitot & Dragan, 1991). Initiation via a genetic

carcinogen occurs when DNA damage alters the expression of genes involved in key regulatory

pathways. In order for the damage to result in a neoplasm it must be converted to a permanent

mutation through replication and persistence of the mutated gene (Pitot & Dragan, 1991). One

study estimated 3 to 10 instances of genetic damage were necessary to allow a cell to progress to

the promotion stage (Barrett, 1993). Promotion increases the proliferation rate at the

preneoplastic foci by altering gene expression, and results in the formation of a neoplastic

growth; there is no direct genetic mutation at this stage (Pitot & Dragan, 1991; Oliveira et al.,

2007). Promotion is the only stage of carcinogenesis that is reversible. There is no direct

structural genetic mutation in this stage; rather there is an alteration in gene expression.

Progression is the last stage of carcinogenesis and is irreversible. Neoplastic cells develop a

malignant phenotype that is largely driven by a stimulation of cell growth pathways and

inhibition of cell death pathways. Progression is characterized by permanent changes to the

genome and epigenome that dysregulate cellular metastatic capability, growth rate and

invasiveness of the cell genome (Oliveira et al., 2007). Pathologists identify differences in these

parameters between healthy and cancerous cells as part of the diagnostic process.

Mechanistically, carcinogens induce either structural modification of DNA or alteration

of cell metabolism, growth or death pathways in order to initiate neoplastic development (Pitot &

Dragan, 1991). Changes to DNA structure occur most often and cause mutations by perturbing

normal copying and replication of DNA and as discussed above, these mutations can be important

for both initiation and progression (Perera et al., 1982). DNA damage can be repaired through

5

one of six DNA repair pathways, and these pathways are imperative for reduction of carcinogenic

risk.

DNA repair and nucleotide excision repair 1.3

1.3.1 DNA repair

DNA damage ranges from mismatched deoxynucleotides to single or double strand

breaks and to the addition of bulky adducts. Organisms have evolved six processes to repair

damaged DNA (Branzei & Foiani, 2008): homologous recombination, non-homologous end-

joining, mismatch repair, base excision repair (BER), direct damage reversal (DDR) and

nucleotide excision repair (NER). Damage of endogenous origin caused by metabolic and other

biochemical reactions is repaired by BER, DDR, mismatch repair and strand-break repair

pathways (De Bont, 2004; Branzei & Foiani, 2008; Bates, 2010), while single and double strand

breaks are repaired through homologous recombination or non-homologous end-joining. The

DDR and BER pathways involve the removal and repair of small single-nucleotide adducts such

as oxidized DNA bases formed by endogenous reactive oxygen species (ROS), while mistakes in

DNA replication resulting in mismatched base-pairings are repaired by mismatch repair. Some

forms of DNA damage caused by exposure to exogenous agents are repaired by BER, DDR,

mismatch repair and strand-break repair pathways. However, helix-distorting DNA lesions

caused by bulky DNA adducts derived from a number of exogenous agents and pyrimidine

dimers caused by ultra-violet (UV) irradiation, are repaired largely by NER (Branzei & Foiani,

2008). NER is a multi-step process that involves multiple proteins working in concert to excise

large bulky adducts and patch the excised region with new nucleotides. NER plays an important

role in preventing cancer and attenuating the effects of chemical carcinogens; this pathway is

described in detail below.

6

1.3.2 Nucleotide excision repair

NER progresses through three steps: recognition, wherein the lesion is recognized and

repair begins; incision/excision, wherein several proteins work in concert to cut and remove the

single DNA strand with the lesion; and gap filling, wherein DNA replication machinery fills in

the gap left by the excised strand. NER is divided into two sub-pathways, distinguished by how

the damage is recognized: global genome NER (GG-NER) and transcription coupled NER (TC-

NER). In GG-NER, damage is recognized by the complex xeroderma pigmentosum (XP) class C

protein-human homologue of RAD23B (XPC; Sugasawa et al., 1998). It is only this recognition

step that differs between the two sub-pathways; in fact, apparently the only difference between

GG-NER and TC-NER is the lack of the recognition protein XPC in TC-NER. However,

mutations repaired by GG-NER are a better predictive marker for carcinogen-induced

tumourigenesis than mutations repaired through TC-NER, suggesting GG-NER plays a more

important role in protection against carcinogenesis (Balajee & Bohr, 2000). Figure 1.1 displays

the three overlapping steps of GG-NER, which are described below.

7

Figure 1.1 Simplified mechanism of global genome nucleotide excision repair. Adapted

from Li et al. (2015b) and Marteijn et al. (2015).

8

1.3.2.1 Triple-check recognition

The rate-limiting step of GG-NER is lesion recognition, which proceeds through three

checkpoints, as discussed by Marteijn et al. (2015). The recognition protein XPC binds to DNA

with helix-distorting lesions or thermodynamically destabilized sites such as base-base

mismatches. XPC can often bind indiscriminately leading to false positives, making additional

recognition checkpoints important (Li et al., 2015b; Sugasawa et al., 1998). Additionally, XPC

poorly recognizes UV-induced cyclobutane pyrimidine dimers, and an additional protein, UV-

damaged DNA-binding protein (UV-DDB; also known as XPE) binds to these adducts first and

facilitates XPC loading (Sugasawa et al., 2001; Sugasawa et al., 2005). The final two recognition

checks are transcription factor II H (TFIIH) and XP class A protein (XPA). TFIIH is a multi-

subunit complex comprised of a three-subunit cyclin-dependent kinase (CDK)-activating kinase

(CAK) module and Core7, a seven subunit complex made up of two ATP-dependent DNA

helicases (3’ to 5’ XP class B protein (XPB) and 5’ to 3’ XP class D protein (XPD)) p62, p52,

p44, p34 and p8 (Park et al., 1995; Li et al., 1998; Li et al., 2015b; Habraken et al., 1996;

Bradsher et al., 2000). DNA bubble-bound XPC recruits TFIIH, which then scans for lesions

through bi-directional helicase strand threading of XPB and XPD in which TFIIH moves along

the DNA strand with XPB scanning the 3’-5’ strand and XPD scanning the 5’-3’ strand (Li et al.,

2015b). XPA binds altered nucleotides in a single-strand DNA (ssDNA) context, confirming

damage, but also is integral to stimulating the release of the CAK subcomplex from TFIIH which

inhibits the helicase function (Camenisch et al., 2006; Coin et al., 2008; Li et al., 2015b). Thus,

the triple check system proceeds via: (1) sensing of base-pairing disruptions by XPC; (2) TFIIH-

mediated, XPA-accelerated strand threading for translocation-inhibiting injuries; and (3)

recognition of chemically altered nucleotide by XPA (Li et al., 2015b; Marteijn et al., 2015;

Sugasawa et al., 1998). TC-NER is initiated when a lesion blocks RNA polymerase II during

9

elongation (Fousteri & Mullenders, 2008; Hanawalt & Spivak, 2008), TFIIH and XPA are

recruited to the site of damage as above, and initiate the incision/excision step (Fousteri &

Mullenders, 2008; Damsma et al., 2007; Sarker et al., 2005). In GG-NER progression into the

incision/excision phase is marked by the binding of replication protein A (RPA) and the

uncoupling of XPC.

1.3.2.2 Incision/excision

At the beginning of the incision/excision phase, the helicases XPB and XPD have halted

at the lesion and their action has opened up the area around the lesion forming a

‘bubble’(Bradsher et al., 2000; Evans et al., 1997). Herein, XPA and RPA act as scaffolds,

keeping the complementary strands from re-annealing, protecting ssDNA from degradation and

act to recruit the other necessary proteins (Missura et al., 2001; de Laat et al., 1998; Park et al.,

1995; Shivji et al., 1995; Rademakers et al., 2003; Yokoi, 2000). The release of XPC allows for

the loading of XP class G protein (XPG) followed by XP class F protein- excision repair cross-

complementing repair class 1 protein (XPF-ERCC1) to the lesion-carrying strand (Araújo et al.,

2001; O’Donovan et al., 1994; Riedl et al., 2003). These two proteins are nucleases that incise

the damaged strand; XPG cuts at the 3’ end of the lesion strand, and XPF-ERCC1 attaches to and

cuts at the 5’ end of the strand; the double cut allows for displacement and excision of the

damaged strand (Araújo et al., 2001; O’Donovan et al., 1994). RPA continues to protect the

undamaged ssDNA from degradation, and the damaged strand is released by the helicases

(Matsunaga et al., 1995; Moggs et al., 1996; Mu et al., 1996; Constantinou et al., 1999;

O’Donovan et al., 1994).

10

1.3.2.3 Gap filling

The final step of NER, gap filling, begins before XPF-ERCC1 has excised the damaged

strand; DNA polymerase δ or ε attaches to the undamaged strand and begins replication with the

help of proliferating cell nuclear antigen (PCNA), and the newly synthesized patch of DNA is

ligated to preexisting DNA through DNA ligase I (Shivji et al., 1995; Moser et al., 2007; Ogi &

Lehmann, 2006).

1.3.3 Changes in NER activity

Most of the NER repair proteins are named for the rare genetic disorder they were

discovered in, xeroderma pigmentosum (XP). XP is typified by an inability to repair DNA

damage caused by UV light, resulting in severe sunburn when exposed to only small amounts of

sunlight, and often followed by skin malignancies at a very young age. There are several types of

XP; the most common types are attributable to a mutation in one of seven necessary NER genes:

XPC, XPA, XPB, XPD, ERCC4 (XPF), RAD2/ERCC5 (XPG) or a gene required to replicate

DNA with unrepaired damage POLH (XPV; DNA polymerase eta). The disease can have various

degrees of severity, dependent on the mutation type and the protein mutated (Lehmann et al.,

2011). XPC and XPE are only required for GG-NER and not TC-NER, so individuals who are

deficient in one of these two genes do not have as strong a sunburn response as individuals with

another polymorphism, likely due to the activity of TC-NER (Lehmann et al., 2011). The effects

shown by XP patients are exemplary of the importance of each of these NER proteins, and

inhibition of each can have effects on NER activity.

There are examples of XP-like phenotypes in cells as well. Testicular germ cell tumours

are especially susceptible to cisplatin treatment, due to a significant reduction in the levels of

XPA protein (Köberle et al., 1999). In counterpoint, several cancer cell types have developed

resistance to DNA-damaging chemotherapeutics associated with upregulation of certain NER

11

proteins including upregulation of DDB2 and XPC in malignant melanoma cells (Barckhausen et

al., 2014) and XPF- ERCC1 upregulation in ovarian cancer cells (Reed, 1998). Research has

shown there are many ways NER activity can be affected, but in each case the alterations are

attributable to effects on a single protein involved in the NER pathway.

1.3.3.1 Rate limiting NER proteins

XPC was previously identified as the rate-limiting protein of NER activity, due to its

importance in lesion recognition; however the binding affinity of XPC for lesions does not always

fully explain the difference in excision activity for cellular extracts (Lee et al., 2014; Li et al.,

2015b). Also, an overabundance of XPC protein is actually detrimental to cell viability, likely

due to false-positive binding (Aboussekhra et al., 1995). Recent understanding of the lesion

recognition process discussed above (“triple check recognition”) may partially explain these

phenomenon, with XPC functioning as a “blunt tool”, binding to many false-positives sites, with

TFIIH and XPA acting as the final confirmation for NER to proceed (Li et al., 2015b; Marteijn et

al., 2015). These rate-limiting proteins are important, as it has been shown that exposure to

genotoxic carcinogens can cause significant changes in NER activity through changes to specific

proteins.

Changes to NER activity appear to occur through the change of NER protein levels in the

nucleus, through post-translational modifications that change NER protein binding to damaged

DNA and through mutations to genes encoding for the NER proteins (Coin et al., 2004; Fan &

Luo, 2010; Kang et al., 2011; Lehmann et al., 2011; Li et al., 2015b; Li et al., 2011b; van Cuijk

et al., 2015). Defective XP protein functioning causes a cancer-prone phenotype, with increased

DNA mutation loads and tumour incidences (Lehmann et al., 2011). The causes of these changes

are not necessarily mutually exclusive, as can be observed in the circadian rhythm of XPA, which

appears to be caused both by changes to NER protein levels and post-translational modifications

12

(Kang et al., 2010b; Kang et al., 2011). As discussed in subsection 1.3.2, the protein XPA

provides support and additional confirmation in lesion recognition and structural support. At

homeostasis, XPA protein level exhibits circadian rhythmicity, achieved by transcriptional

control and degradation via post-translational modification by HERC2 E3 ubiquitin ligase (Kang

et al., 2010b; Kang et al., 2011). In addition to control of protein level in the nucleus,

protein/protein interactions of XPA involve both phosphorylation by the checkpoint kinase

ataxia-telangiectasia mutated and Rad3-related (ATR) and deacetylation by SIRT1 which control

binding activity of XPA to damaged DNA (Fan & Luo, 2010; Wu et al., 2006). Together, these

lead to diurnal variation in overall NER activity (Kang et al., 2010b; Kang et al., 2011).

1.3.3.2 p53 and NER

In response to genotoxic stresses, tumour suppressor protein p53 (p53) has multiple

effects that serve to enhance cancer prevention; these include promotion of apoptosis, cell cycle

arrest, and DNA repair activity increases (Seo & Jung, 2004). Although p53 is not essential for

NER when replicating the pathway in vitro using purified protein, there is still some unclear link

between NER and p53 (Aboussekhra et al., 1995; Adimoolam & Ford, 2003). p53 appears to

affect both TC-NER and GG-NER in several cell types (Dregoesc et al., 2007); however, Li-

Fraumani human fibroblasts with decreased p53 function are deficient in GG-NER with no effect

observed in TC-NER (Ford & Hanawalt, 1995). One theory is that p53 stimulates chromatin

decondensation prior to lesion recognition, but the most likely role p53 plays within NER is

regulation of lesion recognition (Rubbi & Milner, 2003; Adimoolam & Ford, 2003).

After UV-induced damage, p53 is translocated from the cytosol to the nucleus, where it

increases the expression of genes integral to NER (DDB2, XPC and XPA; Chang et al., 2008;

Hwang et al., 1999; Barckhausen et al., 2014; Liu et al., 2012). p53 is also involved in the

translocation of XPA into the nucleus through the ATR kinase activity in vitro, and this action

13

may explain why both TC-NER and GG-NER are increased by p53 induction (Li et al., 2011a).

p53 can also bind directly to damaged DNA (Chang et al., 2008) and p53 enhances repair of UV-

induced damage, probably by direct recruitment of TFIIH factors XPB and XPD. Deletion of the

C-terminus of p53 leads to decreased recruitment of TFIIH for repair of UV-induced adducts

(Léveillard et al., 1996; Chang et al., 2008; Hoffmann et al., 1996). Additionally, p53 is

apparently necessary for repair of adducts of the tobacco carcinogen metabolites benzo[a]pyrene

diol-epoxide and benzo(g)chrysene diol-epoxide, since these adducts persisted in p53 null human

fibroblast cells (Lloyd & Hanawalt, 2000; Wani et al., 2002). Also, deficient p53 significantly

decreases NER of UV-induced DNA damage and benzo[a]pyrene-induced DNA adducts (Zhu et

al., 2000; Ikehata et al., 2010; Wani et al., 2002). However, there appear to be some

compensatory mechanisms that are yet to be understood, as DNA damage responses appear to be

mechanistically different between p53 wildtype and deficient cells (Li et al., 2011b). Overall,

p53 is integral to the response of cells to injury by exogenous chemicals, and in vitro it appears to

have a role in NER but there is no universal understanding of this role.

1.3.3.3 Conditions that increase NER activity.

DNA-damage induced changes in NER activity were first observed in cell extracts which

gain a period of increased NER activity after treatment with UV light (Protić et al., 1988). This

increase is mediated by Sirt1 deacetylation and ATR-p53 import of XPA into the nucleus (Fan &

Luo, 2010; Ming et al., 2010; Donninger et al., 2015; Choi et al., 2015). Additionally, XPC and

DDB2 may induce phosphorylation of XPA through recruitment of ATR kinase to the damaged

site (Ray et al., 2013). This phosphorylation of XPA also appears to be UV-induced, and

inhibiting XPA phosphorylation decreases cell viability after UV-damage (Wu et al., 2006). A

low level dose of UV light has even been used for chemoprevention against cisplatin exposure in

lung cancer cells (Choi et al., 2015). Although not fully understood, it appears p53 is important

14

for the increase in NER activity after exposure to several carcinogens. Mulder et al. (2014)

showed that mouse liver p53 deficiency resulted in attenuation of the NER increased activity

observed after treatment with the carcinogen aflatoxin B1 (Bedard et al., 2005). Additionally, it

is possible that p53 is responsible for the chemopreventive effects selenium compounds have

against colon and mammary cancers in rodent models (Fischer et al., 2006; Seo et al., 2002). In

human and mouse fibroblasts, selenium in the form of selenomethionine significantly induces

NER, which likely involves interaction with p53 induction (Seo et al., 2002; Fischer et al., 2006).

The nicotine-derived carcinogen NNK also significantly increases NER activity specifically in

the livers of A/J mice (Brown & Massey, 2009), and its effects are one of the topics of this thesis.

1.3.3.4 Conditions that decrease NER activity

Decreases in NER activity are observed after treatment with certain chemicals. In human

skin fibroblasts, arsenic significantly decreases the expression of XPC and XPE, leading to a

significant decrease in repair of 6-4 photoproducts, which are bulky DNA adducts induced by

UVC exposure (Nollen et al., 2009). In human alveolar epithelial cells, hypochlorous acid, a

byproduct of the inflammatory response, also decreases the transcription of XPC, leading to a

significant decrease in NER (Güngör et al., 2007). Cyclosporin A causes a significant decrease in

XPA and XPG expression, leading to a significant decrease in NER in human fibroblasts

(Kuschal et al., 2011). However, some of the causes of NER activity decreases are still not all

understood, including the inhibition of NER by the plant toxin arecoline in HEp-2 squamous

carcinoma cells, and particulate matter in human lung adenocarcinoma cells (Mehta et al., 2008;

Huang et al., 2016). Several metals including nickel (in human fibroblasts), cadmium and

chromium (in chinese hamster ovary cells) cause significant decreases in NER (Hu et al., 2004b;

Fatur et al., 2003; Hu et al., 2004c). The mechanisms of the effects of these metals on NER are

not yet understood, but it appears that they inhibit NER in the lesion recognition-early incision

15

step. The nicotine-derived carcinogen NNK also significantly decreases NER activity

specifically in the lungs of A/J mice (Brown & Massey, 2009), and its effects are one of the

topics of this thesis.

NNK 1.4

1.4.1 Occurrence and source of NNK

According to the World Health Organization, tobacco kills 50% of its users,

approximately 6 million people a year worldwide; 5 million users and 1 million due to second

hand smoke inhalation (Organization, 2012). Tobacco use is declining in most industrialized

countries, but overall consumption is increasing worldwide due in part to population growth and

economic development (Jatoi et al., 2009). Tobacco products contain a diverse array of

chemicals, including nicotine and several carcinogens. These carcinogens are derived from

various chemicals including polycyclic aromatic hydrocarbons (PAH), tobacco-specific

nitrosamines (TSNAs), aromatic amines, heterocyclic aromatic amines, aldehydes, aza-arenes and

other organic and inorganic compounds (Hecht, 1999). Nicotine is the addictive component of

tobacco and tobacco smoke; it is not a complete carcinogen, but it is known to activate various

signaling pathways related to tumour promotion (Hukkanen et al., 2005; Warren & Singh, 2013).

Nicotine also undergoes chemical conversions into carcinogenic TSNAs such as NNK, 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), iso-NNAL, 1-nitrosoanabasine (NAB), 4-

(methylnitrosamino)-4-(3-pyridyl)butyric acid, N’-nitrosonornicotine (NNN) and N’-

nitrosoanatabine (NAT). TSNAs are formed through the nitrosation of nicotine and related

tobacco alkaloids. Nitrosation reactions occur by replacement of N-H with N-N=O in the case of

secondary amines or via oxidative cleavage of carbon-nitrogen bonds of tertiary amines (Figure

1.2). Of the TSNAs, NNN, NNK and NAT are found in the highest quantities in both burned and

16

unburned tobacco (Appleton et al., 2013; Borgerding et al., 2012; Counts et al., 2004; Stanfill et

al., 2011). The complexity of tobacco smoke prevents definitive assignment of cause and effect,

but NNK, NNAL and NNN appear to play a significant role in cancer induction (Hecht et al.,

1993a; Hecht, 1998; Hecht, 1999). TSNAs are also present in significant amounts in unburned

tobacco, and NNK and NNN are likely to play a major role in oral cancer induction (Hecht et al.,

1993a; Hecht, 1998; Hecht, 1999). NNK is also present in many e-cigarette preparations, but at

much lower concentrations than in cigarettes (Farsalinos et al., 2015; Goniewicz et al., 2014).

NNK is the most potent TSNA in terms of tumourigenicity in experimental animals (Hecht et al.,

1993a; Hecht, 1998). TSNAs induce tumours in the esophagus, lung, liver, pancreas and bladder,

but the major target organ of effect is substantially affected by chemical structure and animal test

species (Hecht et al., 1993a; Hecht, 1998; Hecht, 1999). The major organ of NNK carcinogenesis

is the lung, and a single dose of NNK can cause lung and respiratory tract cancers in experimental

animals (Hecht & Hoffmann, 1989).

17

Figure 1.2 Structures of the three most common tobacco-specific nitrosamines formed by

the nitrosation of nicotine. Abbreviations in text.

18

1.4.2 Biotransformation of NNK

NNK is a procarcinogen, and it has multiple metabolic pathways as illustrated in Figure

1.3. Carbonyl reduction and α-carbon hydroxylation are the two most important reactions leading

to the ultimate carcinogenic metabolites (Hecht, 1998). Carbonyl reduction of NNK results in the

formation of NNAL, which can undergo bioactivation similar to that of NNK. NNAL can be

oxidized to NNK, and this NNK reconversion may be important in NNK carcinogenicity (Hecht,

1998). Both NNK and NNAL can be α-carbon hydroxylated, which form DNA-reactive species

that are mutagenic through methylation and pyridyloxobutylation of DNA.

NNK α-methyl carbon hydroxylation generates 4-(hydroxymethylnitrosamino)-1-(3-

pyridyl)-1-butanone (1), which decomposes to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (2),

which can pyridyloxobutylate DNA. Reaction of 2 with water produces 4-hydroxy-1-(3-pyridyl)-

1-butanone (Keto Alcohol; III). NNAL α-methyl carbon hydroxylation generates a reactive

metabolite (3) which then produces 4-hydroxy-4-(3-pyridyl)-1-butanediazohydroxide (4) which is

further metabolized to 4-hydroxy-1-(3-pyridyl)-1-butanol (Diol, IV) ;4 can

pyridylhydroxybutylate DNA. NNK α-methylene carbon hydroxylation leads to formation of 4-

hydroxy-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (5), which decomposes to methane

diazohydroxide (6), which can methylate DNA, and keto aldehyde which is further oxidized to

for 4-oxo-4-(3-pyridyl) butyric acid (keto acid; IV). NNAL α-methylene carbon hydroxylation

goes through similar metabolism forming methane diazohydroxide (8) and hydroxy aldehyde

which is further oxidized to 4-hydroxy-4-(3-pyridyl) butyric acid (hydroxy acid; V).

In the laboratory, DNA can be pyridyloxobutylated (POB) by a chemically activated

form of NNK, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), and

NNKOAc has been used previously to show the importance of POB damage (Brown et al., 2008;

Peterson et al., 2001).

19

Figure 1.3 Pathways of NNK metabolism to reactive metabolites that * methylate,☆

pyridyloxobutylate or ★ pyridylhydroxybutylate (© Brown, 2008).

N

NCH3

O NO

N

NCH3

NO

OH

NN CH2OHN O

OH

N

N CH3

N OOH

OH

N

NCH3

NO

O

O

NN CH2OHN O

O

NN CH3

NO

O

OH

N

NCH3

NO

OH

O

N

NO

NOH

CHOH

CO2

N

OHO

N

HO

OCH3N NOH

CH3OH

N

OHO

O

NO OH CH3N NOH

CH3OH

N

OH

O

OH

N

N NOHOH

CHOH

CO2

N

OHOH

OH2 OH2 OH2OH2

NNK NNAL

NNK-N-Oxide NNAL-N-Oxide

Reduction

+

Keto Alcohol

+

Keto Aldehyde

Keto Acid

Lactol

+

Hydroxy Acid

+

Diol

Detoxification Detoxification

α -HydroxylationMetabolic Activation

Pathways

+ + + +

I II

III IV V VI

α α

(1)

(2) (4)

(3)(5)

(6)

(7)

(8)

* *

20

1.4.3 Detoxification of NNK

The detoxification of NNK and NNAL occurs mainly through N-oxidation, resulting in

the formation of excretable N-oxides (Figure 1.3; Hecht, 1998). In addition, NNAL is

glucuronidated to form both β-O-[4-(methylnitrosamino)-1-(3-pyridyl)-1-but-1-yl]-D-

glucosiduronic acid and β-N-[4-(methylnitrosamino)-1-(3-pyridyl)-1-but-1-yl]-D-glucosiduronic

acid, depending on whether the glucuronidation occurs at the carbinol group or the pyridine

nitrogen (Morse et al., 1990; Hecht et al., 1993b; Hecht et al., 2008). These appear to be

important detoxification products of NNK as they are readily excreted. 4-Oxo-4-(3-pyridyl)-1-

butanediazohydroxide undergoes O-glucuronidation in rats and may be another important

detoxification product of NNK.

DNA damage caused by NNK is removed via several DNA repair pathways, discussed in

subsection 1.4.6.

1.4.4 Carcinogenicity of NNK in experimental animals

In experimental animals, NNK is the most potent carcinogenic TSNA, and in rodents it

causes almost exclusively lung adenocarcinomas regardless of route of exposure (Hecht, 1998;

Hoffmann et al., 1996). NNAL, an NNK metabolite and a carcinogen itself, has approximately

30-70% the potency of NNK, and also leads largely to lung tumours when administered directly

to animals (Hecht et al., 1990; Hoffmann et al., 1996). F344 rats develop lung tumours after

exposure to NNK in drinking water, or via subcutaneous injection, gavage, oral swab or

intravenous administration (Hecht et al., 1980; Hecht, 1998; Hecht, 1999). Lung tumours are

always predominant over local (e.g. esophageal) tumours, with the nasal cavity being the second

most common site. Lung tumours are always adenomas or adenocarcinomas. Liver tumours are

observed only in high subcutaneous doses, and these are hepatocellular carcinomas and

hemangiosarcomas (Hecht, 1998; Hecht, 1999). In hamster, NNK causes lung adenomas and

21

adenocarcinomas predominantly, with some squamous and adenosquamous carcinomas (Hecht,

1998). Tracheal tumours are multiple papillomas or NNK administration obliterates the tracheal

lumen, while nasal cavity cancers and liver tumours are not observed. Both sensitive and

resistant mouse strains develop lung tumours after NNK treatment; liver and forestomach

tumours can also develop but are infrequent (Hecht, 1998; Hecht, 1999). Overall, NNK-induced

tumour incidence and multiplicity are usually lower in resistant mouse strains, and the time to

neoplasms depends on the resistance of the strain (Hecht, 1998).

A/J mice have been used extensively in studies of lung tumour induction by NNK. In the

most common assay, a single i.p. dose of 10 µmol of NNK per mouse results in 7-12 lung

adenoma or adenocarcinoma tumours per mouse after 16 weeks (Hecht & Hoffmann, 1989).

This model is championed for its ease of use, and the obvious separation of initiation, promotion

and progression. In a second assay, oral NNK administration in drinking water over 7 weeks

(total dose 44 µmol) also leads to lung tumours, but forestomach tumours also develop.

1.4.5 NNK and human cancers

The TSNA NNK is present in high concentration in cigarette and smokeless tobacco

(Appleton et al., 2013; Ashley et al., 2003; IARC Working Group on the Evaluation of

Carcinogenic Risks to Humans, 2007). In animal studies, NNK is not only the most potent

individual TSNA, but also one of the most potent individual carcinogens in tobacco. The over 60

carcinogens found in cigarette smoke make the connection difficult, but research in experimental

animals has implicated NNK as a carcinogen likely to be important in human lung cancer (Hecht,

2014; Hecht et al., 2016).

NNK levels in mainstream smoke have a direct association with total urinary NNAL

levels; as discussed in section 1.4.1, NNAL is a carcinogenic NNK metabolite (Ashley et al.,

2010). Smokers and nonsmokers exposed to secondhand tobacco smoke have NNAL in their

22

urine, and total NNAL has been widely used as a biomarker of NNK exposure (Ashley et al.,

2010; Hecht et al., 2016). In fact, NNAL is the only tobacco carcinogen biomarker consistently

elevated in nonsmokers exposed to secondhand smoke (Hecht, 2014). Several studies have

shown a statistically significant association between total NNAL level and lung cancer

(Hoffmann et al., 1996; Yuan et al., 2011; Yuan et al., 2014; Appleton et al., 2014). They

illustrate the relationship between NNK and lung cancer, and the importance of the study of

NNK-induced carcinogenesis. The first case-controlled study looked at three biomarkers of

cigarette smoking: cotinine, a metabolite of nicotine, NNAL, a biomarker of NNK exposure, and

r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), a biomarker of PAH exposure,

using 100 randomly selected lung cancer cases and 100 controls from the Prostate, Lung,

Colorectal and Ovarian Cancer Screening Trial (Church et al., 2009). They found a significant

association between lung cancer risk and total urinary NNAL with no association between lung

cancer risk and any other tobacco constituent biomarker tested, including PAH, and volatile

organic hydrocarbons acrolein, benzene, 1,3-butadiene, crotonaldehyde and ethylene oxide (Yuan

et al., 2014). Additionally, adenocarcinoma risk was significantly associated with total urinary

NNAL, but not non-adenocarcinoma lung tumours. In larger cohort studies, total urinary NNAL

has consistently been associated with lung cancer, specifically adenocarcinoma risk. Using the

18,244 person Shanghai cohort, Stephanov et al. (2014) and Yuan et al. (2011). showed total

urinary NNAL is strongly associated with lung, but not esophageal cancers after correction for

NNN exposure again, there were no associations between lung cancer risk and all other tobacco

urinary biomarkers tested. These results are consistent with carcinogenicity studies in

experimental animals treated with NNK as discussed above, making NNAL the most important

tobacco-associated cancer risk biomarkers available (Hecht et al., 2016).

23

TSNA levels are highly variable between tobacco brands and tobacco-derived smoke

worldwide (Appleton et al., 2013; Ashley et al., 2003; 2007). In a study across 35 years, NNK in

mainstream smoke ranged from 8.7 to 868 ng/cigarette with the highest concentrations coming

from cigarettes manufactured in the 1980’s (Appleton et al., 2013). NNK levels are highest in

U.S.-branded cigarettes, when compared to all other local brands, while Canadian brands have

some of the lowest levels (Ashley et al., 2003; Hecht, 2014). The level of TSNAs in e-cigarette

replacement liquids and smoke are considerably lower than that of tobacco cigarettes (Kim &

Shin, 2013; Farsalinos et al., 2015; Goniewicz et al., 2014). One study found that NNK was

present only in trace amounts in vapour, undetectable to 28.3 ng per e-cigarette (Goniewicz et al.,

2014). Replacement of cigarette smoking with e-cigarette use may be an effective harm

reduction strategy by smokers, but the presence of TSNAs in e-cigarette vapour suggests that the

risk is not completely abrogated. TSNAs NNK, NNN, NNAL and NAB are all found in

smokeless tobacco, and are considered the most harmful toxicants present in those products

(IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2007; Hoffmann &

Djordjevic, 1997). There is some debate on the amounts, but since 1980, TSNA levels in

cigarettes have decreased substantially through different curing, processing and storing

procedures; however they are still found in high concentrations (Hecht, 2014; Gunduz et al.,

2016). A global survey of smokeless tobacco products found NNK in concentrations between 4.5

and 516,000 ng/g product (Stanfill et al., 2011), and NNK has historically been found in

concentrations of 7,870,000 ng/g of product (2007). Additionally, urinary NNAL levels are

higher in smokeless tobacco users than in persons using smoked tobacco.

Worldwide, incidence of adenocarcinomas has been gradually increasing in number over

a 20 year period, and it is possible that NNK plays a significant role in this increase (Devesa et

al., 2005; Houston et al., 2014). Between 2004 and 2009 in a U.S. census, adenocarcinoma rose

24

1.3% in men and 2.8% in women while overall incidence rates of lung cancer have decreased

(Houston et al., 2014). This is despite declining cigarette use in most Western countries and

shifts to filtered/low tar cigarettes worldwide. Two explanations have been proposed for these

increases, one concerning cigarette design and a second concerning NNK concentrations in

tobacco (Burns et al., 2011; Hoffmann et al., 1996). The introduction of filters and other design

features resulted in deeper inhalation by smokers and delivery of smoke constituents to the distal

lung, leading to an increase in adenocarcinomas which are peripheral lung tumours (Burns et al.,

2011; Hoffmann et al., 1997). As discussed above, NNK induces adenocarcinomas in

experimental animals and the second explanation revolves around a relative increase in NNK

exposure. There is some debate about whether the trends of NNK levels in mainstream smoke,

smokeless and cigarette tobacco have decreased worldwide (Hecht, 2014). However, the levels

of benzo[a]pyrene and other PAHs have significantly decreased since the 1950s (Counts et al.,

2004; Hoffmann et al., 1997). PAHs tend to induce squamous cell carcinomas of the lung

(Humans, 2010) which have decreased in frequency, and it is possible that the increase in

adenocarcinomas is due to an increase in the ratio of NNK to benzo[a]pyrene, as discussed by

Hecht et al. (2014).

1.4.6 DNA repair and NNK

The genotoxic effects of NNK are mediated through mutations created due to the

formation of pyridyloxobutylated and methyl DNA adducts. Methyl adducts are repaired by

alkyltransferases, enzymes that catalyze single-step reactions whereby the methyl group is

transferred to an amino acid residue on the transferase itself. Pyridyloxobutylation occurs at the

N7- and O6- positions of guanine, and the O2- position of cytosine and thymine. O6-

Pyridyloxobutylated guanine and O2-pyridyloxobutylated thymine are the most stable of these

four adducts, and make up most of the damage caused after NNK exposure (Peterson, 2010).

25

Under hydrolytic conditions, unstable POB adducts decompose, returning the DNA to its normal

state (Hecht et al., 1993a; Peterson et al., 1990). O6-Methylguanine, the most common methyl

adduct, is repaired by O6-alkyl-guanine-DNA-alkyltransferase (Peterson & Hecht, 1991). O6-

Pyridyloxobutylated guanine is primarily repaired by O6-alkyl-guanine-DNA-alkyltransferase

(AGT); however, knockdown or depletion of AGT does not appear to significantly increase cell

death in vitro or tumour incidence in vivo, meaning NER may play an important additional role

(Peterson, 2010; Urban et al., 2012). The other POB adducts are repaired primarily by NER

(Wang et al., 1997; Brown et al., 2008; Hecht et al., 1993a; Peterson & Hecht, 1991). Treatment

of A/J mice with the pyridyloxobutylating agent, NNKOAc, showed the lack of a clear

relationship between a single POB adduct and the incidence of lung tumour formation (Urban et

al., 2012). Three adducts persisted in mouse lung DNA in significant levels (O6-

pyridyloxobutylated guanine, 7-pyridyloxobutylated guanine and O2-pyridyloxobutylated

thymine), indicating multiple adducts may contribute to the tumourigenic properties of NNK

(Urban et al., 2012).

As discussed above, no single DNA damage repair pathway can deal with all of the

diverse types of DNA adducts formed after most carcinogenic exposure. Repair of the damage

caused by NNK proceeds by NER, BER and DDR, which have been discussed previously in

section 1.3.1. Without these DNA repair mechanisms, cell death or the formation of neoplasms is

inevitable. Therefore, any change in DNA repair activity will have an effect on the potential for

carcinogenesis. So, it is of note that NNK treatment of A/J mice results in a significant change

AGT and NER activities in the liver and lung (Brown & Massey, 2009; Peterson et al., 2001).

The results showed significant decreases in AGT and NER activities of lung extracts, significant

increase of NER activity in liver extracts and no effect on AGT activity in liver extracts (Brown

& Massey, 2009; Peterson et al., 2001). The increase of NER activity in the liver could be

26

explained as a homeostatic response to alleviate the damage caused by the carcinogens on DNA,

and an increase in DNA repair capacity has been observed in several situations discussed above.

However, the mechanism behind the changes to NER activity after treatment with NNK is still

inconclusive. The decrease of NER activity in the lungs is especially important, as adducts

caused by the carcinogens may persist in the lungs, thereby increasing the probability of

neoplastic growth. Again, a decrease in NER activity after treatment with a chemical has been

observed in the situations described above, but the mechanism causing NNK-mediated change in

activity has yet to be revealed.

Sulforaphane 1.5

1.5.1 Glucosinolates

Epidemiological studies in several countries have shown an apparent protective effect of

consumption of cruciferous vegetables leading to reduction of cancer risk (Higdon et al., 2007;

Royston & Tollefsbol, 2015). They are among the dominant food crops worldwide and include

such diet staples as cauliflower, Brussels sprouts, cabbage, bok choy and broccoli (Verkerk et al.,

2009). Cruciferous vegetables are high in a number of vitamins, nutrients, and phytochemicals

and are especially high in a group of sulfur-containing phytochemicals, the glucosinolates

(Verkerk et al., 2009).

Glucosinolates are responsible for the odour and spicy or bitter taste of cruciferous

vegetables. Over 100 glucosinolates have been identified in the cruciferae, many of which form

biologically active compounds after hydrolysis (Fahey et al., 2001). The glucosinolate molecule

comprises two parts: a variable side chain and a sulfur-linked β-D-glucopyranose moiety (Figure

1.4; Fahey et al., 2001). This hydrolysis is most frequently catalyzed by myrosinase, an enzyme

found in the cruciferae which is released and brought in contact with the glucosinolates when the

27

plants are cut or chewed; although there appears to be some myrosinase activity by human gut

bacteria as well (Bones & Rossiter, 2006). Myrosinase catalyzes the hydrolysis of the glucosidic

bond, yielding glucose and an unstable thiohydroxamate-o-sulfonate which undergoes

spontaneous rearrangement to form a number of possible products dependent on the original

glucosinolate side chain structure, presence of metal ions and reaction conditions such as pH

(Bones & Rossiter, 2006). At a pH of 6 to7, the major products are isothiocyanates (ITCs; Bones

& Rossiter, 2006). Over 120 ITCs have been described, but relatively few are found in the

cruciferae. Of the ITCs, one with a high concentration in broccoli sprout extract, sulforaphane,

has become a focus of interest for its anti-carcinogenic effects.

28

Figure 1.4 The hydrolytic conversion of glucoraphanin to sulforaphane by myrosinase.

29

1.5.2 Sulforaphane properties and kinetics

Significant amounts of the glucosinolate glucoraphanin, the precursor to sulforaphane,

are found in Brassicaceae, and it is found in extremely high amounts in broccoli sprouts (16.6

µmol/g in young sprouts; 1.08 µmol/g in mature broccoli; Fahey et al., 1997; Fahey et al., 2001).

Glucoraphanin is relatively stable, and hydrolysis to sulforaphane is mainly mediated by

myrosinase after mastication/blending or conversion by human microbiota in the colon (Fahey et

al., 2001). Method of preparation of cruciferous vegetables can also have a major impact on

sulforaphane formation: boiling, steaming and microwaving can inactivate myrosinase (Pérez et

al., 2014; Li et al., 2013a). Raw florets can be eaten and 60-80% of the glucosinolate content is

converted to sulforaphane-nitrile, not the ITC, due to combined effects of a non-catalytic protein

cofactor (epithiospecifer protein) and myrosinase (Juge et al., 2007; Matusheski et al., 2006).

However, mild cooking can denature the cofactor while preserving myrosinase, resulting in

almost 100% conversion of glucoraphanin into sulforaphane (Juge et al., 2007). Consequently,

several different preparations of broccoli sprouts have been developed to increase availability of

sulforaphane (Li et al., 2013a). In humans, sulforaphane is rapidly absorbed, metabolized and

excreted, with 80% of parent compound and metabolites found in the urine 12-24 h after

administration (Atwell et al., 2015; Shapiro et al., 1998; Ye et al., 2002; Hu et al., 2004a; Clarke

et al., 2011; Li et al., 2013a). In humans and mice, peak plasma concentrations are found 0.5-1 h

after consumption of sulforaphane or broccoli sprout extract (Petri et al., 2003; Ye et al., 2002; Li

et al., 2013a). However, the conversion to sulforaphane by the gut microflora can take more

time, leading to a secondary peak 2 h after consumption of a broccoli sprout extract (Li et al.,

2013a).

Sulforaphane is the most hydrophobic of all dietary ITCs and rapidly diffuses into the

intestinal epithelium. Sulforaphane then undergoes metabolism conjugation with glutathione

30

(SF-GSH), catalyzed by glutathione-S-transferase (GST) enzymes, followed by subsequent

formation of SF-N-acetylcysteine (SF-NAC) and SF-cysteine (SF-Cys). An autoradiographic

whole-body study showed sulforaphane-derived radioactivity in the GI tract, liver, kidney and

blood (Tortorella et al., 2015). In CD-1 and ICR mice, the greatest concentrations of

sulforaphane, SF-GSH, SF-Cys and SF-NAC were measured between 0.5-2 h in the liver, kidney,

lung, heart and muscle (Clarke et al., 2011; Li et al., 2013a). In F344 rats, the plasma

concentration of sulforaphane reaches its peak at 4 h after oral dosing (Hu et al., 2004a).

There is general consensus that sulforaphane can cause biologically opposite effects

depending on its concentration (Sestili & Fimognari, 2015). At low doses, it exerts

chemopreventative, indirect antioxidant and cytoprotective effects, whereas at high doses it exerts

cytotoxic and antitumour effects (Sestili & Fimognari, 2015; Zanichelli et al., 2012a; Zanichelli

et al., 2012b). After treatment with sulforaphane, gene expression profiles in mice, rats and

cancer cell lines show effects at a wide range of timepoints, with significant changes from 3-24 h

(Bhamre et al., 2009; Hu et al., 2006). In a gene activity profile studying 39,000 genes in CD-1

livers from mice treated with 90 mg/kg sulforaphane, Hu et al. (2006) found significantly

elevated gene expression at both 3 h and 12 h, with the greater number of individual gene

inductions occurring at 12 h.

1.5.3 Sulforaphane as a cancer chemopreventive, cytotoxic and cytoprotective chemical

As reviewed by Tortorella et al. (2015), sulforaphane has gained particular interest as a

cancer chemopreventative chemical, particularly with respect to inhibition of phase I enzymes

and production of DNA adducts, induction of phase II enzymes, and anti-inflammatory and anti-

oxidant activities.

Sulforaphane can inhibit phase I enzymes, significantly reducing the burden of many

procarcinogens through inhibiting the enzymes that bioactivate them. Thus, inhibition of phase I

31

enzymes is an important mechanism of blocking chemical carcinogenesis. Sulforaphane can

inhibit phase I metabolism through direct interactions with cytochrome P450 enzymes (CYP) or

by regulating transcript levels (Juge et al., 2007). Specifically, sulforaphane inhibits CYP1A1

and 2B1/2 in a concentration dependent manner in rat hepatocytes and CYP3A4 in human

hepatocytes (Mahéo et al., 1997). However, most research has focused on the effect of

sulforaphane on phase II enzymes and production of carcinogen detoxification products.

Recent evidence illustrates the protective effects of sulforaphane against a wide range of

cytotoxic and carcinogenic chemicals in several cell lines and animal models. Prior treatment

with sulforaphane and several other ITCs caused decreases in lung adenocarcinoma incidences

and malignant lung tumour multiplicity in A/J mice treated with a mixture of benzo[a]pyrene and

NNK (Conaway et al., 2005). Co-treatment of sulforaphane with 14C-labelled 2-amino-1-methyl-

6-phenylimidazo[4,5-b]pyridine (PhIP) caused a significant decrease in PhIP-generated DNA

adducts, associated with increases in phase II detoxification enzymes (Bacon et al., 2003).

Sulforaphane treatment increases total GST activity, which acts to detoxify the hepatocarcinogen

aflatoxin B1-8,9-epoxide (Gao et al., 2010). Sulforaphane inhibits acetaminophen-induced

hepatotoxicity, apparently at least partially through induction of heme oxygenase 1 (HO-1)

induced antioxidant effects (Noh et al., 2015). A similar mechanism likely reduces doxorubicin-

induced oxidative stress (Li et al., 2015a; Singh et al., 2015). In addition, sulforaphane can

protect fibroblasts from ionizing radiation (Mathew et al., 2014). UV-radiation induced skin

carcinogenesis in SKH-1 hairless mice was substantially inhibited by topical treatment with a

broccoli sprout extract containing sulforaphane (Dinkova-Kostova & Abramov, 2015). Few

clinical sulforaphane studies exist; however, pretreatment of human skin with sulforaphane

causes a significant decrease in UV radiation-induced apoptosis and a significant increase in the

antioxidant enzyme catalase (Kleszczyński et al., 2013; Knatko et al., 2015).

32

Research on sulforaphane has focussed largely on its effects after concurrent treatment

with a chemical or other damaging agents, but sulforaphane can have multiple other effects in

models of disease. For example, diabetes-induced testicular cell death and aortic damage can be

abrogated through the antioxidant effects of sulforaphane (Wu et al., 2015; Wang et al., 2014b;

Wang et al., 2014a). Additionally, sulforaphane pretreatment causes a decrease in blood brain

barrier disruption and neurological dysfunction following ischemic stroke in Sprague-Dawley rats

(Alfieri et al., 2013). Sulforaphane can also have cytotoxic and antiproliferative effects in certain

cancer cells; for example, colon cancer cell counts were significantly reduced after sulforaphane

treatment (Rajendran et al., 2013). Sulforaphane attenuates the proteolysis and mRNA

degradation of programmed cell death 4, a tumour suppressor protein in HepG2 cells (Cho et al.,

2014). Sulforaphane acts as an antiproliferative agent in mammary cancer cells, apparently by

perturbation of mitotic microtubules thereby halting cell cycle exit from metaphase (Jackson &

Singletary, 2004). Many of these actions, anti-cancer and other are mediated through

sulforaphane activation of the transcription factor NF-E2-related factor 2 (Nrf2).

1.5.4 Nrf2

Nrf2 is a transcription factor involved in regulation of several cytoprotective genes in

response to oxidative and electrophilic stresses (Itoh et al., 1997; Kwak & Kensler, 2010;

Tortorella et al., 2015). The hundreds of target genes of Nrf2 are activated through antioxidant

response elements (ARE) or electrophile response elements (EpRE) in their promoter regions

(Itoh et al., 2004). Unstressed, Nrf2 is regulated through a ubiquitin-proteasome pathway via

kelch-like ECH-associated protein 1 (Keap1) association and CR6-interacting factor 1 promoted

ubiquitin degradation (Figure 1.5;Kang et al., 2010a). Inactivated Nrf2 is held in the cytoplasm

through binding of Keap1 dimer (Itoh et al., 2004; Kang et al., 2010a). Nrf2 is released by

Keap1 in the presence of ROS or electrophiles through one of two pathways (Baird & Dinkova-

33

Kostova, 2011). In the first pathway, reactive cysteines in Keap1 dimers are modified after

reaction with electrophiles, thereby releasing the bound Nrf2 (Wakabayashi et al., 2004). In the

second pathway, secondary sensor proteins activate protein kinase signaling pathways,

phosphorylating Nrf2 causing release of Keap1 (Huang et al., 2002). After release of Keap1 and

translocation into the nucleus, Nrf2 dimerizes with multiple small Maf proteins and binds to ARE

or EpRE promoter regions, inducing target gene expression (Motohashi et al., 2004; Itoh et al.,

2004). Further control of Nrf2 effects have also been discovered: alcohol can decrease Nrf2

induction through an increase in transforming growth factor β-1 (TGF-β1) expression, which

appears to impair Nrf2-ARE signalling (Sueblinvong et al., 2014). There is also further control

of Nrf2-mediated transcription of target genes: cyclic adenosine monophosphate-response

element-binding protein can enhance transcription, whereas Nrf2 target genes can be negatively

regulated through the actions of p53 and CCAAT enhancer-binding protein alpha (Faraonio et al.,

2006; Ikeda et al., 2006; Shen et al., 2004).

34

Figure 1.5 Model of Nrf2 activation mechanism. In the absence of stressors, Nrf2 is bound

to dimeric Keap1 and serves as a substrate for ubiquitin ligase to target Nrf2 for

ubiquitination and proteasomal degradation. Activation occurs via oxidant or electrophilic

reaction with cysteine residues on Keap1, leading to stabiliziation of Nrf2 through an

unclear mechanism (reviewed by Baird & Dinkova-Kostova, 2011) and eventual

translocation into the nucleus where Nrf2 dimerizes with small Mafs and activates ARE-

dependent transcription of several cytoprotective enzymes. NAD(P)H:quinone

oxidoreductase 1 (NQO1), phosphoglycerate dehydrogenase (PGD), glutamate-cysteine

ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), ferritin light chain (FTL).

35

1.5.5 The actions of Nrf2

Nrf2 increases the synthesis of GST and other electrophile conjugating enzymes, direct

acting antioxidants and reducing equivalents as well as the 20S proteasome catalytic core causing

the ubiquitin-independent degradation of oxidized proteins (Itoh et al., 2010; Jaramillo & Zhang,

2013; Kwak & Kensler, 2010). Creation of the Nrf2 knockout mouse allowed for an

understanding of the wide range of effects displayed in Figure 1.5 (Ramos-Gomez et al., 2001;

Ramos-Gomez et al., 2003). Microarray data has shown Nrf2 is involved in the induction of

antioxidant proteins, NADPH-generating enzymes, drug metabolizing enzymes, drug

transporters, metal binding proteins and stress response proteins as reviewed by Hayes et al.

(2010). Nrf2 is integral for protection against chemical carcinogenesis as many genotoxic

carcinogens are either directly DNA reactive or are bioactivated to electrophilic DNA-reactive

species. The importance of Nrf2 signaling in reduction of electrophilic species was first shown

with acetaminophen toxicity which, although not genotoxic, produces toxicity via formation of a

protein-binding electrophile (Chan et al., 2001; Enomoto et al., 2001). An increase in Nrf2

signalling increases the synthesis of GSH through increased expression of glutamate-cysteine

ligase catalytic subunit (GCLC) and phosphoglycerate dehydrogenase (PGD). Other detoxifying

molecules such as NAD(P)H:quinone oxidoreductase 1 (NQO1) increase the conjugation and

excretion of reactive metabolites. Ferritin light chain (FTL) is also induced and prevents

uncontrolled surges in the intracellular free concentration of the highly reactive, poorly soluble

ferric iron (Kensler et al., 2007). In addition, metallothionein, a potent antioxidant, is induced

with Nrf2 and results in the reduction of DNA damage, and can decrease diabetic neuropathy

(Chubatsu & Meneghini, 1993; Wu et al., 2015). Knockout or reduction of Nrf2 results in the

infiltration of inflammatory cells, increase in pro-inflammatory cytokines and an increase in

oxidative damage. Active Nrf2 decreases the development of aberrant crypt foci, thought to be

36

an early marker of colorectal cancer, in a model of colitis-associated colorectal cancer

(Paszkowska-Szczur et al., 2015). Oxidative stress is implicated in multiple disease states linked

to exposure of environmental toxicants. The role of Nrf2 in protecting against oxidative stress

appears to occur at multiple levels. During periods of non-stress, the effect of Nrf2 on basal

levels of reactive oxygen species (ROS) is minimal, but this effect is integral to mitochondrial

membrane potential, fatty acid oxidation, availability of substrates for respiration and ATP

synthesis (Dinkova-Kostova & Abramov, 2015). During periods of high oxidative stress, Nrf2

signalling becomes vital for the decrease of ROS within mitochondria and in the rest of cell

(Dinkova-Kostova & Abramov, 2015; Gong & Cederbaum, 2006). However, the overall action

of Nrf2 remains to be understood, as several studies have indicated results in which Nrf2 action

was associated with pro-carcinogenic activity; one study using urethane to initiate lung tumours

in mice showed resistance to tumour growth in Nrf2 deficient mice (Satoh et al., 2013).

Additionally, heterozygous Nrf2 mice treated with dimethylhydrazine had a reduced tumour

burden when compared to wildtype mice (Rajendran et al., 2015).

1.5.6 Sulforaphane mechanisms not associated with Nrf2

In addition to the downstream effects of Nrf2 induction discussed above, sulforaphane is

associated with several actions independent of Nrf2 induction including inhibition of phase I

biotransformation and acts as a histone deacetylase (HDAC) inhibitor. HDACs remove acetyl

groups on nuclear histones, leading to condensed chromatin and repressed transcription. The

action of sulforaphane as an HDAC inhibitor leads to an increase in acetylated histones and hence

overall transcriptional activation. Importantly, HDAC inhibition occurs in cells lacking Nrf2

expression, indicating an action independent of Nrf2 (Dashwood & Ho, 2007; Ho et al., 2009).

Whereas the Nrf2 effects and phase I biotransformation effects are largely due to direct

37

interaction with sulforaphane, the HDAC inhibitor effects appear to largely be mediated by SF-

NAC and SF-Cys (Myzak et al., 2006).

Sulforaphane treatment can inhibit both class I and class II HDACs, and can affect a wide

range of genes that are neutral, beneficial or detrimental for chemoprevention (Baier et al., 2014).

An increase in HDAC activity is observed in a number of cancer types, and can result in the

repression of cell cycle and apoptotic mechanisms, among others. The HDAC inhibitory activity

of sulforaphane was first reported by Myzak et al. (2004). Sulforaphane also significantly

increased acetylated H3 and H4 histones (Myzak et al., 2006). Acetylated H3 and H4 appear to

have opposing effects, and the extent to which HDAC inhibition affects transcriptional control is

still not fully understood (Gansen et al., 2015). However, research in tumour cell lines showed

that H4 acetylation is tied to the transcriptional regulation of important chemopreventive proteins

p21 and bcl-2-like protein 4 (BAX) (Myzak et al., 2006). BAX is a pro-apoptotic protein, while

the effects of p21 are variable; it has roles in cell cycle progression, motility, apoptosis and DNA

repair (Murphy et al., 2002). BAX and p21 are repressed in several tumour cells, and their

derepression after treatment with sulforaphane has added to the growing literature on its

chemopreventive and chemotherapeutic effects (Dashwood & Ho, 2007; Ho et al., 2009).

1.5.7 Sulforaphane and DNA repair

Sulforaphane can have direct and indirect effects on DNA repair. Several studies have

shown that pretreatment with sulforaphane can lead to a decrease in the effect of genotoxic

carcinogens; however the mechanisms of the beneficial effects of sulforaphane are not fully

understood. Several examples of the anti-genotoxic effect of sulforaphane have shown effects

largely due to phase II detoxification. For example, the decrease in aflatoxin B1-induced DNA

adducts with sulforaphane pretreatment appears to be largely mediated through the Nrf2-

induction of GST (Techapiesancharoenkij et al., 2015). In HepG2 human hepatoma cells, co-

38

treatment of sulforaphane with PhIP leads to a significantly reduced amount of PhIP-DNA

adducts (Bacon et al., 2003). However, treatment with sulforaphane after treatment with PhIP

does not lead to any significant reduction in PhIP-DNA adducts, leading the authors to conclude

that sulforaphane does not have a significant effect on DNA repair activities (Bacon et al., 2003).

On the other hand, benzo[a]pyrene- and 1,6-dinitropyrene-DNA adducts are significantly reduced

in human mammary epithelial cells after treatment with sulforaphane, but the reduction could not

be explained by increased detoxification (Singletary & MacDonald, 2000). Furthermore, Nrf2

inhibition caused a significant decrease in UV-B induced homologous recombination repair in

A549 cells (Jayakumar et al., 2015). Also, sulforaphane-enhanced H3 histone acetylation may

facilitate DNA repair, as relaxation of chromatin allows for binding of repair proteins (Duan &

Smerdon, 2014).

Jayakumar et al. (2015) showed that Nrf2 activation is directly involved in homologous

recombination repair, the first example of a direct action in DNA repair. p21, a downstream

target of p53 that is believed to exert a number of the actions of p53 including regulation of DNA

repair pathways, has been implicated with Nrf2 induction and stability (Chen et al., 2009). On

the other hand, p53 is directly and indirectly involved in many DNA repair pathways, and it is

known to be inversely related to the action of Nrf2 (Faraonio et al., 2006). A hypothesis for these

actions was presented by Jaramillo and Zheng, whereby strong induction of p53 inhibits Nrf2 by

an undefined mechanism, decreasing antioxidant defense and cell survival pathways; promoting

apoptosis (2013). Mild stress only weakly induces p53, but induces downstream effector proteins

including p21, stalling the cell cycle at G1/S-phase checkpoint, inducing DNA repair pathways

and the Nrf2 mediated cytoprotective response (Jaramillo & Zhang, 2013).

As of yet, there has not been a report of in vivo data on the effect of sulforaphane

treatment on NER activity. However, an in vitro experiment showed that sulforaphane

39

pretreatment caused a significant decrease in repair of (+)-anti-benzo[a]pyrene-7,8-diol-9,10-

epoxide- induced adducts in HCT 116 cells (Piberger et al., 2014). This decrease appeared to be

caused by a direct interaction between sulforaphane and repair protein XPA through a release of

the XPA zinc-finger domain. This resulted in an inactivated protein complex and a decrease in

XPA- damaged DNA binding (Piberger et al., 2014).

Research hypotheses and objectives 1.6

As discussed, genetic instability caused by persistence of bulky DNA adducts can lead to

apoptosis and carcinogenesis. Treatment with exogenous chemicals not only cause bulky DNA

adducts, but can also have significant effects on NER activity. However, the mechanisms of

these NER changes are not fully understood. In addition, the treatment with an exogenous

chemical that can increase NER activity without causing DNA damage would be important for

the modification of carcinogenic risk after exposure to a DNA damaging agent. However, no

agent that causes an increase in NER activity without concomitant DNA damage has been

discovered. In this thesis, the effects of the DNA damaging carcinogen NNK and the

chemoprotective phytochemical sulforaphane on the mechanisms of changes to NER activity are

studied.

Rationale: Studies using NNK showed that administration to a mouse resulted in a change to the

NER activity in the liver and lung (Brown & Massey, 2009). These effects were organ-

dependent; there was a decrease in lung NER activity, but an increase in liver NER activity

(Brown & Massey, 2009). It was hypothesized that the increase in NER activity protected the

liver from carcinogenesis, while the decrease in NER activity in the lung makes that organ more

susceptible to carcinogenesis; however, the mechanism(s) involved in these changes in activity

are still not understood. Also, the time course of effects on NER activity in the liver after

treatment with NNK has not been described.

40

Hypothesis 1: The effects of in vivo NNK treatment on in vitro NER activity of liver nuclear

extracts occur at times other than 24 h after treatment and are associated with alterations in levels

of specific proteins in the NER pathway.

Objective 1. To determine the effects of NNK treatment on NER activity in female A/J mouse

liver at 2, 4, 12, 24 and 72 h post treatment.

Objective 2. To determine the effects of NNK treatment on nuclear levels of the recognition and

early-incision NER proteins XPC, XPA, XPB and p53 as well as on binding activity to damaged

DNA in female A/J mouse liver.

Hypothesis 2: Decreased NER activity in lung from NNK-treated female A/J mice is associated

with effects on specific proteins in the NER pathway.

Objective 1. To determine the effects of NNK treatment on nuclear protein levels of the

recognition and early-incision NER proteins XPC, XPA, XPB and p53 as well as on binding

activity to damaged DNA in female A/J mouse lung.

Rationale: Sulforaphane treatment leads to activation of multiple genes involved in

chemopreventative pathways. Pre-treatment with sulforaphane before several types of DNA-

damaging carcinogens also significantly decreases the amount of DNA adducts in a sample and

sulforaphane causes significant changes to proteins associated with NER. However, recent work

indicates that in vitro exposure of human colon cancer (HCT 116) cells to sulforaphane causes a

decrease in NER activity through inhibition of XPA binding activity (Piberger et al., 2014). NER

is integral to the chemoprotective response to genotoxic chemicals, and sulforaphane is a current

candidate for use as a chemoprotective agent; thus it is important to understand the effect of

sulforaphane on NER.

Hypothesis 3: Sulforaphane treatment causes changes to NER activity in female CD-1 mouse

liver and lung that are through changes to specific NER proteins.

41

Objective 1. To determine a dose of sulforaphane that affects expression of Nrf-2 regulated genes

in female CD-1 mouse liver and lung.

Objective 2. To determine a time point that affects Nrf-2 regulated genes HO-1 and GCLC in

female CD-1 mouse liver and lung.

Objective 3. To determine whether NER is affected at this time point and dose of sulforaphane.

Objective 4. To determine the effects of sulforaphane treatment on nuclear levels of the

recognition and early-incision NER proteins XPC, XPA, XPB and p53 as well as on binding

activity to damaged DNA.

42

Chapter 2

Time course of effects of NNK on nucleotide excision repair activity on

A/J mouse liver and possible mechanisms of change

After in vivo treatment with the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanone (NNK), mouse liver has higher levels of DNA adducts than does mouse

lung, but mouse lung is the major target of NNK-induced carcinogenesis. One potential

explanation for the lack of liver carcinogenesis is an adaptive increase in nucleotide excision

repair (NER) activity in mouse liver. However, the timecourse and mechanisms of this increase

are not well understood. A 46% decrease in NER activity was observed in liver nuclear extracts

12 h post NNK treatment and a 48% increase was observed at 24 h post NNK. These changes in

NER activity were not attributed to changes in levels of the key recognition and early incision

NER proteins XPC, XPA, XPB and p53. However, the binding of hepatic XPA and XPB to

damaged DNA at both timepoints was increased. The binding of XPC to damaged DNA was

unchanged at 12 h and decreased at 24 h. The impact of NNK on mouse liver NER -associated

processes is time dependent and associated with changes in the binding activities of specific

recognition and early excision NER proteins to damaged DNA.

Introduction 2.1

Nicotine-derived nitrosamino ketone (NNK) or 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone is one of the tobacco-specific nitrosamines which play an important role in

carcinogenesis. NNK is one of the most potent cancer-causing tobacco-specific nitrosamines in

all animal species tested, and is believed to be a major contributor to the induction of lung

43

adenocarcinoma in humans and animals (Hecht et al., 1993a; Hecht, 1998). NNK is present in

unburned tobacco and tobacco smoke but must be metabolically activated to be carcinogenic. α-

Methylene carbon hydroxylation leads to formation of 4-hydroxy-4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanone, which decomposes to methanediazohydroxide, which methylates DNA

bases (Figure 1.3). α-Methyl carbon hydroxylation generates 4-(hydroxymethylnitrosamino)-1-

(3-pyridyl)-1-butanone, which decomposes to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide,

which can pyridyloxobutylate DNA (Figure 1.3). Pyridyloxobutylation (POB) occurs at the N7-

and O6- positions of guanine, and the O2- position of cytosine and thymine.

Bioactivation and formation of DNA adducts are critical determinants of mutagenicity

and the maintenance of genetic integrity depends on the ability to repair damaged DNA. DNA

methylation is repaired by direct reversal by O6-alkylguanine-DNA alkyltransferases (AGT)

while POB adducts are repaired primarily by nucleotide excision repair (NER; Brown et al.,

2008; Kotandeniya et al., 2013; Li et al., 2009; Peterson et al., 2001; Peterson, 2010; Urban et al.,

2012). Previous reports have shown that treating mice with a tumourigenic dose of NNK causes

a 290% increase in liver NER activity in an in vitro assay (Brown & Massey, 2009). It is possible

the significant increase in NER activity causes a chemopreventive effect in mouse liver, which is

resistant to carcinogenesis after NNK exposure (Hecht et al., 1993a). However, no study has yet

examined the timecourse of effects of NNK exposure on liver NER activity.

NER can be divided into two subpathways: global genome repair (GG-NER) and

transcription coupled repair (TC-NER). TC-NER removes lesions from transcribed strands of

active genes, whereas GG-NER removes lesions from non-transcribed strands of transcribed

genes and non-transcribed regions of the genome. NER proceeds through three sequential steps:

recognition, incision/excision and DNA resynthesis (Figure 1.1). The damage recognition step is

the distinctive difference between GG-NER and TC-NER, whereas the two subpathways employ

44

a common set of proteins for the remaining steps. We have focused our work on GG-NER

because mutations in nontranscribed genes or genes on the nontranscribed strand of active genes

are better predictive markers for carcinogen-induced tumourigenesis than mutations in actively

transcribed genes (Balajee & Bohr, 2000). Recent focus in this field has been on the regulation of

“early stage repair” which encompasses the recognition and early incision stages of NER.

Lesion recognition is a ‘triple-check’ process, where a bulky adduct is first recognized by

the xeroderma pigmentosum (XP) group C (XPC) complex, then verified by the multi-subunit

transcription factor II H (TFIIH) and XP group A (XPA) (Marteijn et al., 2015). XPC does not

bind directly to the bulky adduct, but to the undamaged ssDNA opposite the lesion (Trego &

Turchi, 2006). XPC recruits TFIIH, which then scans for lesions bidirectionally via the two

helicases XPB and XPD (Li et al., 2015b). XPA further verifies damage, sensing chemically

altered nucleotides and displaces the cyclin-dependent kinase-activating kinase (CAK)

subcomplex from TFIIH (Camenisch et al., 2006; Coin et al., 2008). The release of XPC and

binding of replication protein A (RPA) are the events which lead to conclusion of early stage

repair; XPA and RPA then form stable structures for downstream NER factors. Tumour

suppressor protein 53 (p53) also appears to be involved in early stage NER; functional p53 is

required for NER but its exact role remains elusive (Lakin & Jackson, 1999; Zhu et al., 2000).

One theory is that p53 stimulates chromatin decondensation prior to lesion recognition, but the

most likely role p53 plays within NER is regulation of lesion recognition (Rubbi & Milner, 2003;

Adimoolam & Ford, 2003). p53 is known to interact directly with TFIIH in repair of UV light-

induced cyclobutane-pyrimidine dimers and pyrimidine-pyrimidone photoproducts (Chang et al.,

2008) and can transcriptionally regulate XPA nuclear import (Li et al., 2011a) and levels of XPC

(Adimoolam & Ford, 2003).

45

Previous research showed that the presence or absence of each of the more than 30 NER

proteins could increase or decrease NER activity (Nocentini et al., 1997; Araújo et al., 2001;

Hoogervorst et al., 2004; Koberle et al., 2006; Coin et al., 2008; Brown & Massey, 2009; Brown

et al., 2010; Kang et al., 2010b; Fan & Luo, 2010; Melis et al., 2011; Li et al., 2011a; Fadda,

2013; Rossner et al., 2013; Li et al., 2013b; Ziani et al., 2014; Mulder et al., 2014; Choi et al.,

2015; Donninger et al., 2015; Li et al., 2015b). Only recently have specific proteins been

identified that induce changes in NER activity without changes in protein levels, generally

through post-translational modifications involving ubiquitin or ubiquitin-like compounds (Huang

& D’Andrea, 2006; Kang et al., 2010b; Gali et al., 2012; van Cuijk et al., 2015). Early stage

NER, that is the recognition and early incision stages, has recently been identified as a key stage

for induction of NER after carcinogen exposure (Marteijn et al., 2015; Choi et al., 2015;

Donninger et al., 2015; Li et al., 2015b).

It was previously shown there is a significant increase in NER activity in mouse liver

tissue after a tumourigenic dose of NNK (Brown & Massey, 2009). This response is not unique,

in that several other studies have shown that an increase in NER activity is caused by treatment of

cell lines with a DNA damaging agent, either UV light (Choi et al., 2015; Tomicic et al., 2011),

or mitomycin C (Protić et al., 1988), and in vivo with aflatoxin B1 (Bedard et al., 2005). The

upregulation of NER is consistent with an adaptive response that protects cells from mutagens,

and a recent study showed that pre-treatment with a nonlethal dose of UV-light caused a

significant increase in NER of subsequent cisplatin damage in lung cancer cells (Choi et al.,

2015). However, these studies have been carried out in cell culture and/or are all shown at one or

two timepoints following carcinogen treatment, so persistence of the effect in vivo has not been

established.

46

In this study, there is a significant change in NER activity at two time points in mouse

liver after treatment with NNK. These effects are associated with changes in the binding

activities of NER proteins XPC, XPA, XPB or p53 in liver nuclear protein extracts to damaged

DNA and are not attributable to a change in the nuclear levels of these proteins.

Materials and methods 2.2

2.2.1 Animal treatments

Female A/J mice, aged 8-11 weeks (Taconic Labs, Hudson, NY) were housed with a 12 h

light/dark cycle and provided food and water ad libitum. A/J mice were chosen for this study

since NNK carcinogenicity studies have been carried out predominantly in this strain (Hecht,

1998). Mice were treated with saline (0.1 mL, i.p.) or a single dose of 10 µmol NNK (Toronto

Research Chemicals, Toronto, ON) in saline (0.1 mL, i.p.), a dose of NNK that induces a

significant increase in multiplicities of lung adenocarcinomas in A/J mice (Hecht et al., 1989).

Due to concerns over mouse NER activity diurnal variation (Kang et al., 2010b), treatments were

carried out at the same time every day, starting at 0900 h. Two, 4, 12, 24, or 72 h following NNK

administration, mice were killed by cervical dislocation. Livers were perfused with 10mM

Tris/1mM EDTA (pH 7.9), excised, finely chopped, frozen in liquid nitrogen, and stored at -80°C

until extract preparation. All animals were cared for in accordance with the guidelines of the

Canadian Council on Animal Care and the experimental procedures involving animals were

approved by the Queen’s University Animal Care Committee.

2.2.2 Preparation of cell-free whole tissue nuclear protein extract

Nuclear protein extracts from 1.0 to 1.3 g of tissue were prepared from pooled livers of

four mice as described (Wood et al., 1988; Wood et al., 1995) with modifications (Bedard et al.,

2005). Briefly, chopped tissue pieces frozen in liquid nitrogen were pulverized into a fine

47

powder with mortar and pestle. Hypotonic lysis of pulverized tissue was done for 30 minutes on

ice under gentle rotation in 4 mL hypotonic lysis buffer (10 mM Tris-HCl (pH 7.9), 1 mM EDTA,

5 mM dithiothreitol). Following the addition of protease inhibitors (0.5 mM phenyl methyl

sulfonyl fluoride, 4.7 mM leupeptin, 1.6 mM aprotinin and 1.5 mM pepstatin), lysate was

homogenized using a Dounce homogenizer (20 strokes). With gentle stirring on ice, 5 mL of

sucrose-glycerol solution (50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 2 mM dithiothreitol, 25 %

sucrose, 50 % (v/v) glycerol) and 1 mL saturated ammonium sulfate (neutralized with NaOH)

were added dropwise, and homogenate was gently stirred for an additional 30 minutes.

Homogenate was centrifuged for 3 h at 100,000 g at 4°C and the supernatant was collected,

leaving 1 mL above pellet. With gentle stirring on ice, supernatant was adjusted to 0.33 g/mL

with ammonium sulfate, 10 µL of 1 M NaOH per gram of ammonium sulfate added and

supernatant was stirred for an additional 30 min. Precipitate was collected by centrifugation at

15,000 g at 4°C for 20 min. Pellet was dissolved in 200 µL of dialysis buffer (20 mM HEPES-

KOH (pH 7.9), 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 17 % (v/v) glycerol, 2 mM

dithiothreitol) and dialysis was performed against three changes of 200mL (per Slide-A-Lyzer

cassette, ThermoFisher, Waltham, MA, USA) of buffer for 18 h and changed after 1 and 16 h of

dialysis. The dialysate was clarified by centrifugation at 10,000 g for 3 min, and stored in

aliquots at -80°C. Protein contents of extracts were determined by the Lowry method as modified

by Peterson et al. (1977). Extracts from livers contained between 10 to 15 mg/mL protein.

Extracts were frozen in liquid nitrogen and stored at -80°C.

2.2.3 Preparation of damaged plasmid DNA

Pyridyloxobutylated plasmid DNA was prepared as described (Brown et al., 2008). In

the laboratory, DNA can be pyridyloxobutylated by a chemically activated form of NNK, 4-

(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). Briefly, NNKOAc (20mM,

48

Toronto Research Chemicals, North York, ON, Canada) was reacted with 1.04 mg/mL

pBluescript SK+ plasmid DNA (Stratagene, La Jolla, CA, USA) in a 250 µL reaction mixture

containing sodium citrate buffer (final concentration, 15 mM sodium citrate, pH 7.0, 1 mM

EDTA) and 4 U/mL porcine liver esterase (Sigma-Aldrich Co., St. Louis, MO, USA) for 1 h at

room temperature. Plasmid DNA damaged by this method contained approximately 10-12

adducts/plasmid as determined by HPLC analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone release

with equal proportions of the four major POB adducts, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine,

O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine, O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine and O6-[4-

(3-pyridyl)-4-oxobut-1-yl]-2´-deoxyguanosine as determined by mass spectrometry (Brown et al.,

2008).

2.2.4 In vitro DNA repair synthesis assay

The repair synthesis assay was performed as described (Brown & Massey, 2009). With

minor changes: each 50 µL reaction mixture contained 400 ng of pyridyloxobutylated plasmid

DNA or undamaged plasmid DNA, 40 mM HEPES-KOH (pH 7.8), 0.5 mM DTT, 4.0 µmol/L

dATP, 20µM of each of dGTP, dCTP, and dTTP, 23 mM phosphocreatine, 18 µg bovine serum

albumin (nuclease-free, Sigma-Aldrich Co., St. Louis, MO, USA), 2.5 µg creatine

phosphokinase, 2.0 mM ATP, 5.0 mM MgCl2, 0.4 mM EDTA, 100mM KCl, 100µg protein

extract and 2.0 µCi [α32P]dATP (Perkin Elmer, Waltham, MA, USA). The repair synthesis assay

was performed in duplicate for each nuclear protein extract and results are presented as the

mean± SD (n=4). To determine damage-specific repair activity, radioisotope incorporation into

undamaged plasmid DNA was subtracted from repair synthesis activity calculated with POB

damaged DNA.

49

2.2.5 Immunoblot analysis of NER protein levels in mouse liver nuclear protein extracts

Rabbit polyclonal antibodies anti-human XPA (1/2,000; ab10905), anti-human XPB

(1/4,000; ab53198), anti-human XPC (1/2,000; ab78064) and mouse monoclonal antibody anti-

human p53 (1:1,000; ab28) were purchased from Abcam Inc. (Cambridge, MA, USA). HRP-

conjugated polyclonal goat anti-rabbit IgG (1/5,000; sc-2004) and HRP-conjugated polyclonal

donkey anti-mouse IgG (1/5,000; sc-2314) were purchased from Santa Cruz Biotechnology Inc.

(Santa Cruz, CA, USA).

Nuclear protein extracts (5µg) were subjected to sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes

(Millipore, Billerica, MA, USA). Membranes were incubated in 10% skim milk overnight at 4°C

and proteins immunoblotted with above antibodies for 1 h at room temperature. After incubating

membranes with secondary antibodies conjugated to horseradish peroxidase for 1 h at room

temperature, specific proteins were visualized as immunoreactive bands by the enhanced

chemiluminescence detection system (Perkin Elmer, Waltham, MA, USA). To normalize for

protein loading, α-actinin was used as a housekeeping protein (1/5,000; sc-15335; Santa Cruz

Biotechnology, Santa Cruz, CA, USA) as a loading control. Films were scanned and processed

with Licor® Image Studio™ Lite (Mandel Scientific Company, Guelph, ON, Canada). Total

values were converted to relative densities using a standard that was prepared by pooling tissues

from 16 animals. Immunoblots were performed in duplicate for each nuclear protein extract and

results are presented as the relative densities ± SD (n=4).

2.2.6 Analysis of binding of NER incision proteins to damaged DNA

An assay to assess the binding of NER proteins to damaged DNA was performed as

described (Frit et al., 1998; Li et al., 1998; Salles et al., 1995a; Salles et al., 1995b; Trego &

Turchi, 2006) with slight modifications. Briefly, 50 ng of POB-adducted or undamaged plasmid

50

DNA in 10 mM phosphate buffer (pH 7.0) were adsorbed on Microlite II Microtitre 96 well

plates (Thermo Scientific, Waltham, MA, USA) that were pre-treated with 100 µL poly-L-lysine

hydrochloride (1 mg/mL) in phosphate-buffered saline (10mM phosphate buffer (pH 7.2)/ 150

mM NaCl) at 30°C for 30 min with gentle shaking. Picogreen dsDNA quantitation kit

(Invitrogen Molecular Probes, Carlsbad, CA, USA) was used to verify the amount of plasmid

DNA adsorbed to wells with a microplate reader. The protein binding reaction was carried out in

50 µL per well and contained mouse lung (100 µg) or liver (120 µg) nuclear protein extract in 100

mM KCl, 40 mM HEPES-KOH (pH 7.6), 7.0 mM MgCl2, 2.0 mM ATP, 0.5 mM DTT, 10 mM

phosphocreatine, 2.5 µg creatine phosphokinase, 2.0 mM EGTA, and 18 µg bovine serum

albumin (nuclease-free, Sigma-Aldrich Co., St. Louis, MO, USA). After 3 h at 30 ºC, wells were

washed three times with 50 µL of PBS plus 0.01 % Tween-20, and proteins bound to immobilized

DNA were eluted with 25 µL of sample buffer (62.5 mM Tris-HCl (pH 6.8), 4 M urea, 10 % (v/v)

glycerol, 2 % (w/v) SDS, 5 % (v/v) β-mercaptoethanol, 0.003 % (w/v) bromophenol blue) for 30

min at 30 ºC with shaking, followed by 15 min at 85 ºC. The proteins were then resolved by

SDS-PAGE and analyzed as described above (Subsection 2.2.5) for immunoblot analyses with

minor modifications. Primary antibodies were incubated overnight and at higher concentrations

(XPC 1/1,000; XPA 1/2,000; XPB 1/4000). Each DNA bound protein sample was comprised of

reactions pooled from 3 wells. Protein binding reaction samples were conducted in duplicate for

each nuclear protein extract. Films and stained blots were scanned and processed with Adobe

Photoshop 6.0 Software, and densitometry of the scanned image was performed using Li-Cor

Biosciences Image Studio Lite 5.2.5 software. Relative density for undamaged-DNA- and

damaged-DNA-bound NER proteins was calculated and protein levels were normalized by total

amount of protein bound based on Amido Black staining. Binding factor was calculated by

51

subtracting the average relative protein level of undamaged DNA bound protein from the average

relative protein level of damaged-DNA-bound protein.

2.2.7 Data analysis

Statistically significant differences in repair activity and NER protein levels between

treatment groups were determined by unpaired Student’s t-tests (Graphpad Prism 6 software).

Due to inter-experiment variability, Mann-Whitney U test was used comparing DNA-associated

NER protein binding (Subsection 2.2.6). P<0.05 was considered significant in all cases.

GraphPad Prism 6 was used to graph all results.

Results 2.3

2.3.1 Time course of effects of NNK treatment on mouse liver NER activity

To observe the effect of NNK treatment over time, A/J mice were treated with a

tumourigenic dose of NNK and sacrificed at 2, 4, 12, 24, or 72 h. NNK treatment caused a 46%

decrease in activity at 12 h and a 48 % increase in NER activity at 24 h (Figure 2.1). The 2 h

timepoint had higher control and treated NER activities than other timepoints, this is possibly

attributable to diurnal variation in in vivo NER activity (Kang et al., 2010b; Kang et al., 2011).

This variation was not the focus of this study and was not pursued further.

52

Figure 2.1. Effects of in vivo NNK or saline treatment on in vitro DNA repair activity of

mouse liver extracts towards NNKOAc-induced pyridyloxobutyl DNA damage. Extracts

were prepared from mice treated with either 10 µmol NNK or saline and killed 2, 4, 12, 24

or 72 h post-dosing. Data are presented as mean + SD. * significantly different from

control (p<0.05; n=4 pools of 4 mouse livers each.)

2h 4h 12h 24h 72h0

5000

10000

15000

Mouse Liveramol

[α32

P] d

ATP

inco

rpor

ated

/µg

DN

ASalineNNK

*

*

53

2.3.2 The effect of in vivo treatment of mice with NNK on levels of key NER proteins in

mouse liver nuclear protein extracts

The effect of NNK on levels of key NER proteins in mouse liver extracts was examined.

At 12 h (Figure 2.2) and 24 h (Figure 2.3) post-treatment liver extracts showed no significant

changes in key NER protein levels.

54

Figure 2.2 a) Relative density of each key NER protein in mouse liver nuclear protein

extracts isolated from saline and NNK-treated mice 12 h post-dosing, relative to a constant

internal standard extract. Data are presented as mean ± SD. b) Representative

immunoblots of the relative levels of each key NER protein in mouse liver extracts. S=

nuclear protein extracts from saline treated mice, N=nuclear protein extracts from NNK

treated mice; n=4 pools of 4 mouse livers each.

55

Figure 2.3 a) Relative density of each key NER protein in mouse liver nuclear protein

extracts isolated from saline and NNK-treated mice 24 h post-dosing, relative to a constant

internal standard extract. Data are presented as ean + SD. b) Representative immunoblots

of the relative levels of each key NER protein in mouse liver extracts. S= nuclear protein

extracts from saline treated mice, N=nuclear protein extracts from NNK treated mice; n=4

pools of 4 mouse livers each.

56

2.3.3 Effect of in vivo treatment of mice with NNK on binding of liver NER proteins to

damaged DNA

To determine if NNK affects binding of NER proteins to damaged DNA, an in vitro assay

was used that measures the binding of repair proteins to damaged DNA. There was a significant

increase in the binding of XPB (12 h: 2,137% increase; 24 h: 98% increase) and XPA (12 h:

461% increase; 24 h: 962% increase) after NNK treatment at both 12 h (Figure 2.4) and 24 h

(Figure 2.5), whereas there was no significant change in XPC binding at 12 h (Figure 2.4) and a

significant decrease in XPC binding at 24 h (45% decrease; Figure 2.5).

57


induced pyridyloxobutyl DNA damage 12h post treatment. Data are presented as mean +

SD. * Significantly different from control (p<0.05; n=4 pools of 4 mouse livers each.)

XPC XPB XPA0.0

0.5

1.0

1.5

2.0

2.5

12h Liver Extracts

Bin

ding

Fac

tor

SalineNNK

*

*

58


induced pyridyloxobutyl DNA damage 24h post treatment. Data are presented as mean +

SD. * Significantly different from control (p<0.05; n=4 pools of 4 mouse livers each.)

XPC XPB XPA0.0

0.5

1.0

1.5

2.0

2.5

24h Liver Extracts

Bin

ding

Fac

tor

SalineNNK

*

*

*

59

Discussion 2.4

This study examined the effect of NNK treatment on liver NER activity at several

timepoints post exposure. Previously, NNK had been shown to increase NER activity in mouse

liver after 4 h and 24 h (Brown & Massey, 2009). Here, we expanded to a time course of effects

of NNK treatment on NER activity, encompassing 2, 4, 12, 24 and 72 h post-treatment. We

showed that NNK affects the repair of POB-DNA adducts and begin to understand how it effects

NER regulation.

We confirm a significant increase in liver NER activity at 24 h post-treatment the

timepoint of maximal hepatic POB adduct levels in A/J mouse lung (Peterson & Hecht, 1991).

Although the timecourse of hepatic NNK-derived DNA adducts for A/J mice has not been

reported, POB adduct levels are consistently greater in the liver than in the lung (Hecht et al.,

1993a; Peterson & Hecht, 1991). We did not confirm the significant effect at 4 h, and we see a

smaller effect than that observed previously; Brown and Massey showed a 200% increase in NER

activity 4 h post-treatment and 290% induction 24 h post-treatment (2009). Here, we found no

significant effect on NER activity (p = 0.4) 4 h post NNK treatment, but a 48% increase in

activity 24 h post-NNK (Figure 2.1). The reason for these inconsistencies with the previous study

is not readily apparent. We made two changes to the protocol used previously. In the previous

study, livers from 4 different mice were used for NER assays, while in the present study, we used

a pool of livers from four NNK- treated A/J mice for each of four samples, in order to be

consistent with the lung study, where a pool of 4 lungs is required for a sufficient nuclear protein

extract concentration for NER activity assays (Chapter 3). Additionally, the manufacturer no

longer produces the radiolabelled nucleotide used in the previous study, [α32P]dTTP, and this was

replaced in this study with [α32P]dATP. It is unlikely these changes affected the observations at 4

60

h. Several years have transpired between the studies, and it is possible that genetic drift has

resulted in a somewhat blunted NNK response in the A/J mice.

Notably, we found that NNK treatment inhibited NER at 12 h post-treatment. This is the

first time we have found a carcinogen-mediated decrease in NER activity after carcinogen

treatment in mouse liver. Since there were no significant changes in NER activity at other

timepoints, subsequent mechanistic experiments focused on the 12 and 24 h timepoints.

Dysregulation of many of the more than 30 proteins involved in NER can change repair

activity (Araújo et al., 2001; Chang et al., 2008; Li et al., 1998; Melis et al., 2011; Ray et al.,

2013). Of particular importance are the early stage repair recognition and early incision phase

proteins XPC, XPA, TFIIH and p53. Several in vitro studies have shown that alterations in these

proteins appear to cause a change in NER activity after exposure to other DNA-damaging agents.

p53 may contribute to the binding of XPA to damaged DNA (Li et al., 2011a), and the

increase in NER activity caused by chronic low doses of aflatoxin B1 was diminished in mice that

were heterozygous for defective p53 (Mulder et al., 2014). Although p53 status is important in

NNK-mediated carcinogenesis (Ellinger-Ziegelbauer et al., 2004), the lack of effect of NNK on

levels of the proteins XPC and XPA, whose expression is regulated by p53, or on XPB (a subunit

of TFIIH) suggests that the changes in NER activity we have observed with NNK treatment are

not attributable to p53-mediated changes in expression of these components of NER.

A recent study has shown that exposure of cultured human lung cancer cells to UV light

increases NER activity without a significant increase in repair protein levels, but with an increase

in XPA binding activity (Choi et al., 2015). The binding of NER proteins XPC, XPA and TFIIH

(XPB) has previously been used to predict the activity of NER (Frit et al., 1998; Li et al., 1998;

Salles et al., 1995a; Koberle et al., 2006). In partial agreement, we observed that the binding of

XPA and XPB to damaged DNA was altered by NNK treatment at 12 h (Figure 2.4) and XPA,

61

XPB and XPC at 24 h (Figure 2.5) post-treatment with NNK. It was expected that the binding of

XPA and XPB to damaged DNA would closely reflect NER activity; this was the case at 24h, but

at 12 h there was a decrease in NER activity with NNK treatment, which was opposite to effects

on XPA and XPB binding.

The decrease in XPC binding to damaged DNA at 24h post NNK may have been a

consequence of the increase of TFIIH (XPB) and XPA, as after these proteins and RPA bind

within the repair complex, XPC is displaced to allow it to sense other DNA damage (Li et al.,

2015b; Melis et al., 2011). In fact, failure of XPC release has been associated with a decrease in

NER activity (Li et al., 2015b; Long et al., 2015). It is also possible that inhibition of NER

activity seen at 12 h with NNK is due to effects on late stage repair. Late-stage NER protein such

as RPA, XPG, XPF or PCNA might be inhibited, either through covalent binding of an NNK

metabolite or a decrease in protein synthesis. Previously, it has been theorized that NNK causes

covalent modifications to key NER proteins (Brown & Massey, 2009) and recent research has

shown that RPA is particularly susceptible to damage (Guven et al., 2015). It is possible that

NNK inhibits NER at 12 h through damage or covalent binding to RPA. In future studies it may

be valuable to assess the binding of RPA, as this protein stays bound as the linkage between early

and late stage NER (Gourdin et al., 2014). Furthermore, perturbation of either XPF or XPG

inhibits NER (Araújo et al., 2001; McNeil & Melton, 2012; Su et al., 2012; Tomicic et al., 2011).

However, the cycle of synthesis and degradation of these proteins is not well understood, so the

potential ability of XPF and XPG activities to recover quickly after insult, is not known.

Alternatively, the importance of post-translational modifications of key NER proteins in

response to bulky DNA damage is now recognized (Beli & Jackson, 2015; Dou et al., 2010; Gali

et al., 2012; Hoege et al., 2002; Huang & D’Andrea, 2006; Kang et al., 2010b; Poulsen et al.,

2013; van Cuijk et al., 2015). In UV-induced NER, regulation of XPC, XPA and TFIIH

62

damaged-DNA binding is controlled by these post-translational modifications. The lack of any

significant NNK-mediated changes in XPC, XPB or XPA protein levels at 12 h (Figure 2.2) or 24

h (Figure 2.3) suggests that the observed changes in NER activity could be caused by post-

translational modifications, and it may be of value to examine such modifications to NER

proteins in the future.

Overall, we show the effects of NNK treatment on NER activity across several

timepoints. At the two timepoints with significant and opposite changes of NER activity, 12 h

and 24 h, we also evaluated changes in pre-incision NER protein levels and binding to damaged

DNA. We found that NNK treatment caused an increase in binding of XPA and XPB at both

timepoints, no change in binding of XPC at 12 h and a significant decrease of XPC binding at 24

h. It is likely that NNK increases the activity of early-stage NER at 24 h and, while early stage

NER is upregulated at 12 h, the transition step between early and late stage could be inhibited, or

another protein downstream of XPC release could be inhibited, causing the decrease in NER

activity. Further research to determine the specific mechanism responsible for this phenomenon

is required and will help provide an understanding of the processes by which NER protects

against genotoxic insults.

63

Chapter 3

Investigation of the mechanisms by which NNK decreases nucleotide

excision repair activity in mouse lung

After in vivo exposure to the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanone (NNK), mouse lung is the major target of NNK-induced carcinogenesis.

One explanation for this susceptibility to carcinogenesis is a decrease in nucleotide excision

repair (NER) activity in mouse lung. However, the mechanisms of this decrease are not well

understood. A 63% decrease in NER activity was observed in lung nuclear protein extracts 24 h

post NNK. This change in NER activity were not attributed to changes in levels of key

recognition and early incision NER proteins XPC, XPA, XPB or p53. However, the binding of

lung XPA and XPB to damaged DNA was decreased and the binding of XPC to damaged DNA

was increased at 24 h. These results suggest that the impact of NNK on repair-associated

processes are associated with changes in the activities of specific recognition and early excision

NER proteins.

Introduction 3.1

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-

specific nitrosamines that play an important role in smoking-induced lung carcinogenesis. NNK

is one of the most potent cancer-causing tobacco-specific nitrosamine in all animal species tested,

and is has been correlated with the induction of lung adenocarcinoma in both humans and animals

(Hecht et al., 1993a; Hecht, 1998). NNK is found in unburned tobacco and tobacco smoke but

must be metabolically activated as described in subsection 1.4.2. Metabolic activation results in

the formation of methyl and pyridyloxobuylated DNA.

64

Genetic integrity depends on the ability to repair damaged DNA, bioactivation and

formation of DNA adducts are critical determinants of mutagenicity. DNA methylation is

repaired by O6-alkylguanine-DNA alkyltransferases (AGT), while POB adducts are repaired

largely by nucleotide excision repair (NER) (Peterson et al., 2001). Previous reports have shown

that treating mice with a tumourigenic dose of NNK causes a significant decrease in NER activity

in mouse lung nuclear extracts 24 h after treatment (Brown & Massey, 2009). A/J mice treated

with NNK form a large number of lung adenomas (Hecht et al., 1993a), and it is possible that the

decrease in NER activity is a significant contributor to lung tumourigenicity after NNK exposure.

However, the mechanism of the decrease in NER is not yet understood.

As discussed in previous chapters, NER can be divided into two subpathways: global

genome repair (GG-NER) and transcription coupled repair (TC-NER). TC-NER removes lesions

from transcribed strands of active genes, whereas GG-NER removes lesions from non-transcribed

strands of transcribed genes and non-transcribed regions of the genome. NER can be divided into

three steps: recognition, incision/excision and DNA resynthesis. The damage recognition protein

xeroderma pigmentosum (XP) group C (XPC) complex is the distinctive difference between GG-

NER and TC-NER, and they employ a common set of proteins for the remaining steps. Balajee

and Bohr displayed mutations in genes on the nontranscribed strand or nontranscribed genes may

be better predictive markers than genes that are actively transcribed, thus we focussed our work

on GG-NER (2000).

Removal of many of the more than 30 GG-NER proteins will lead to disruption of the

NER pathways, however, changes in NER activity appear to be mediated largely by the proteins

that carry out lesion recognition and early incision (Li et al., 2015b; Marteijn et al., 2015).

Lesion recognition is a ‘triple-check’ process, where a bulky adduct is first recognized by XPC,

then verified by XP group A (XPA) and the multi-subunit transcription factor II Human (TFIIH)

65

(Marteijn et al., 2015). XPC does not bind directly to the bulky adduct, but rather to the

undamaged single stranded DNA opposite the lesion. XPA further verifies damage, binding to

chemically altered nucleotides, and its presence displaces the cyclin-dependent kinase-activating

kinase -subcomplex (CAK) from TFIIH (Coin et al., 2008). The presence of XPC attracts TFIIH,

which then scans for lesions bidirectionally via the two helicases XP group B (XPB) and XP

complementation group D (XPD), and this action is significantly increased after CAK

disassociation (Li et al., 2015b). The binding of an additional scaffolding protein, replication

protein A (RPA), signals the end of the recognition stage; XPA and RPA form stable structures

for downstream NER factors and protect the DNA strand from degradation. Tumour suppressor

protein 53 (p53) also appears to be involved in early stage NER, and functional p53 is required

for NER (Lakin & Jackson, 1999; Zhu et al., 2000) but its exact role in NER remains elusive

(Momot et al., 2014; Loewer et al., 2013; Li et al., 2011b; Ikehata et al., 2010). Studies have

shown that p53 is involved both directly and indirectly in NER; it is known to interact directly in

repair of UV-DNA adducts (carried out by NER) likely through interaction with the XPB subunit

of TFIIH(Chang et al., 2008), and it can induce XPC protein synthesis (Adimoolam & Ford,

2003) and can transcriptionally regulate nuclear import of XPA (Li et al., 2011a).

Classical understanding of NER regulation dealt with increasing or decreasing nuclear

protein levels of key NER proteins in order to increase or decrease NER activity (Aboussekhra et

al., 1995). Many of the NER proteins are named for xeroderma pigmentosum, a class of genetic

diseases wherein the protein is inactivated, thereby inhibiting NER activity. Additionally, diurnal

variation in NER appears to be caused by diurnal variation in degradation of XPA protein (Kang

et al., 2010b; Kang et al., 2011). More recently, research on NER activity after UV-induced

DNA damage and NER factor post-translational modifications have added another layer on to

NER regulation (Huang & D’Andrea, 2006; Dou et al., 2010; Kang et al., 2011; van Cuijk et al.,

66

2015; Li et al., 2015b). The binding of XPC (Ming et al., 2010), XPA (Fan & Luo, 2010; Kang

et al., 2011) and TFIIH (Coin et al., 2008) to the NER complex is all mediated by post-

translational modifications. Induction of these pathways is implicated in the induction of NER

activity without the increase of nuclear levels of the key NER proteins (Choi et al., 2015; Fan &

Luo, 2010; Li et al., 2015b). Disruption of any of these pathways has also been implicated in the

inhibition of NER activity (Choi et al., 2015; Gaddameedhi et al., 2015; Li et al., 2011a; Marteijn

et al., 2015; Ray et al., 2013).

There is a decrease in NER activity in mouse lung tissue after a tumourigenic dose of

NNK (Brown & Massey, 2009). This effect is not unique to NNK several other studies have

shown a decrease in NER activity after chemical treatment with the anticancer drug STI571

(Sliwinski et al., 2008), toposiomerase I/II inhibitor F11782 in vitro (Kruczynski et al., 2004) and

the mycotoxin aflatoxin B1 in vivo(Bedard et al., 2005). The combination of an NNK-mediated

decrease in NER activity and NNK-mediated DNA damage is hypothesized to make the mouse

lung especially susceptible to carcinogenesis.

In this study, we show that the decrease in mouse lung NER activity that occurs at 24 h

after treatment with NNK is not due to a change in the amounts of key early phase NER proteins

in the nuclear protein extracts, but we demonstrate changes in their binding activities to damaged

DNA.


3.2.1 Animal treatments

Animals were treated as described in section 2.2.1.

67

3.2.2 Preparation of cell-free whole tissue nuclear protein extract

Nuclear protein extracts were prepared from pooled lungs of four mice as described in

section 2.2.2. Extracts from lungs contained between 5 to 8 mg/mL protein. Extracts were frozen

in liquid nitrogen and stored at -80°C.


Pyridyloxobutylated plasmid DNA was prepared as described in section 2.2.3.


The repair synthesis assay was performed as described in section 2.2.4.

3.2.5 Immunoblot analysis of NER protein levels in mouse lung nuclear protein extracts

The immunoblot analysis of NER protein levels in mouse lung nuclear protein extracts

was performed as described in section 2.2.5.

3.2.6 Analysis of binding of NER recognition proteins to damaged DNA

An assay to assess the binding of NER proteins to damaged DNA was performed as

described in section 2.2.6 using mouse lung nuclear protein extracts.

3.2.7 Data analysis

Statistically significant differences in repair activity and NER protein levels between

treatment groups were determined by unpaired Student’s t-test (Graphpad Prism 6 software). Due

to inter-experiment variability Mann-Whitney U test was used comparing DNA-associated NER

protein binding (section 3.2.6). P<0.05 was considered significant in all cases. GraphPad Prism 6

was used to graph all results.

68

Results 3.3

3.3.1 Effect of treatment of mice with NNK on repair synthesis of mouse lung nuclear

protein extracts

At 24 h following in vivo NNK treatment, repair synthesis activity of mouse lung extracts

toward POB damage was decreased by 63% (Figure 3.1). This is consistent with a previous result

from Brown and Massey (2009).

69

Figure 3.1 Effects of in vivo NNK or saline treatment on in vitro DNA repair activity of A/J

mouse lung extracts towards NNKOAc-induced pyridyloxobutyl DNA damage. Extracts

were prepared from mice treated with either sterile saline or 10 µmol NNK and sacrificed

24h post-dosing. Data are presented as mean + SD. (*) Significantly different from control

(n=4 pools of 4 mice per pool, p<0.05).

Saline NNK0

1000

2000

3000

24h Lung Extractsamol

[α32

P] d

ATP

inco

rpor

ated

/µg

DN

A

*

70

3.3.2 Effect of in vivo treatment of mice with NNK on levels of key NER proteins in mouse

lung nuclear protein extracts

The effect of NNK on levels of key NER proteins in mouse lung nuclear protein extracts

(the same extracts as were used for NER assays) was examined (Figure 3.2). Twenty-four hours

post NNK, no significant changes in specific NER protein levels were observed, relative to

control at the same time point.

71

Figure 3.2 a) Relative density of each key NER protein in A/J mouse lung nuclear protein

extracts isolated from saline or NNK-treated mice 24h post-dosing, relative to a constant

internal standard extract. Data are presented as the mean + SD presented. b)

Representative immunoblots of the relative levels of each key NER protein in mouse lung

extracts. S= nuclear protein extracts from saline treated mice, N=nuclear protein extracts

from NNK treated mice; ; n=4 pools of lungs of 4 mice per pool.

72

3.3.3 Effect of in vivo treatment of mice with NNK on key NER protein DNA binding in

mouse lung nuclear protein extracts

To determine if NNK was affecting the interactions between key NER proteins and

damaged DNA, an in vitro assay was used that measures the binding of repair proteins to

damaged DNA. There was a significant decrease in the binding of XPB (53% decrease) and XPA

(83% decrease) after NNK treatment at 24 h (Figure 3.3), whereas there was a significant increase

in XPC binding at 24 h (115% increase; Figure 3.3).

73


derived pyridyloxobutyl DNA damage. The data are presented as mean + SD. (*)

Significantly different from control (XPA n=5 pools of lungs of 4 mice per pool; XPC and

XPB n=6 pools of lungs of 4 mice per pool, p<0.05).

XPC XPA XPB0.0

0.5

1.0

1.5

2.0

24h Lung Extracts

Bin

ding

Fac

tor

SalineNNK

* * *

74

Discussion 3.4

In this study, we examined the effect of NNK treatment on lung NER activity at 24 h post

exposure, the timepoint of maximal POB adduct levels in A/J mouse lung (Peterson & Hecht,

1991). We confirm the previously described decrease in NER activity at 24 h (Brown & Massey,

2009); this inhibition of NER may be a major contributor to the high incidence of A/J mouse lung

tumours observed after NNK treatment. Previously, reports have shown that mutation or removal

of many of the more than 30 proteins involved in NER can decrease repair activity (Araújo et al.,

2001; Chang et al., 2008; Li et al., 1998; Melis et al., 2011; Ray et al., 2013). Of particular

importance are the early stage repair recognition and early incision phase proteins XPC, XPA,

TFIIH and p53 and we examined these proteins in NNK-treated mouse lung nuclear protein

extracts first via immunoblotting for protein levels, as well as by the ability to bind to damaged

DNA.

NNK had no significant effect on levels of p53, XPC, XPA or XPB proteins (Figure 3.2).

As discussed above, the contributions of p53 to NER has been under investigation for a number

of years (Vélez-Cruz & Johnson, 2012; Zhu et al., 2000); functional p53 is required for NER but

its exact role in NER has not yet been fully elucidated (Momot et al., 2014; Loewer et al., 2013;

Li et al., 2011b; Ikehata et al., 2010). Studies have shown that p53 acts both directly and

indirectly in NER, and NER activity induction caused by exposure to low-doses of aflatoxin B1

was abrogated with heterozygous mutant p53 (Mulder et al., 2014). Although p53 status is

important in NNK-mediated lung tumourigenesis (Ellinger-Ziegelbauer et al., 2004), it appears

that this is largely due to processes other than NER. UV light exposure increased NER activity in

A549 and H460 human lung cancer cells without a significant increase in levels of NER proteins

XPC, XPA, XPB, XPD, XPE, XPF and XPG (Choi et al., 2015). Rather, the elevated NER was

associated with increased binding of XPA to damaged DNA (Choi et al., 2015), linked to post-

75

translational modifications of key NER proteins in response to UV-light exposure (Marteijn et al.,

2015; Dou et al., 2010; Gali et al., 2012; Hoege et al., 2002; Huang & D’Andrea, 2006; Kang et

al., 2010b; Poulsen et al., 2013; van Cuijk et al., 2015). Here, we show a significant decrease in

the binding activity of XPA and XPB in lung nuclear extracts 24 h post-treatment with NNK,

which coincides with the decrease in NER activity in the same extracts (Figure 3.1 and Figure

3.3).

XPC, XPB and XPA all bind together to the damaged DNA strand, and XPC only

increases the likelihood of XPB and XPA binding, so it is unlikely that the significant increase in

XPC binding is the cause of the significant decrease in XPA and XPB binding (Figure 3.3). The

binding of XPA and TFIIH, however, are intricately tied together, as XPA catalyzes the release of

the CAK subcomplex of TFIIH, which increases TFIIH binding activity to damaged DNA (Coin

et al., 2008; Li et al., 2015b). XPA binding activity is affected not only by protein levels in the

nuclear extract, but also through deacetylation via sirtuin 1 (SIRT1). Recently, it has been shown

that, after UV exposure, induction of SIRT1 via the Ras association domain-containing protein 1

tumour suppressor appears to decrease the level of acetylated XPA in several tumour cell lines

(Donninger et al., 2015). Deacetylated XPA is required for efficient repair of bulky adducts

(Donninger et al., 2015; Kang et al., 2011), and knockdown of SIRT1 significantly decreases the

NER activity in vitro (Fan & Luo, 2010). SIRT1 levels are lowered in smokers (Rajendrasozhan

et al., 2008), and in A/J mouse lung extracts treated with NNK plus nicotine (Iskandar et al.,

2013). The lack of any significant change in XPC, XPB or XPA protein levels following NNK

treatment (Figure 3.2) would be consistent with effects on NER being caused by the post-

translational modifications discussed above.

The observed NNK-induced increase in XPC binding could be due to a damage response

in the lung; after UV light damage, XPC is ubiquitylated, which increases its affinity for damaged

76

DNA in vitro (Sugasawa et al., 2005). It is probable that NNK causes a similar effect, leading to

an increase in the binding activity of XPC. Additionally, XPC release is mediated by

SUMOylation of the ubiquitylated XPC in vitro (van Cuijk et al., 2015). NER is inhibited if XPC

is not released before XPG and XPF/ERCC1 can bind to the repair complex, so it is possible that

disruption of this SUMOylation of XPC contributes to the decrease in NER activity observed here

(Riedl et al., 2003; Long et al., 2015; van Cuijk et al., 2015). However, the role of these

modifications in NNK-induced XPC binding has yet to be confirmed.

It is possible that the activity of an NER protein such as XPG or XPF, whose activity we

did not assess, is significantly inhibited by NNK treatment, either through covalent binding of an

NNK metabolite or a decrease in protein synthesis. Indeed, decreased levels or modified

structures of either XPF or XPG can inhibit repair (Araújo et al., 2001; McNeil & Melton, 2012;

Su et al., 2012; Tomicic et al., 2011). However, both XPG and XPF are reliant on the release of

XPC and the binding of TFIIH and would be expected to be significantly inhibited by the changes

in binding activities we have shown here (Araújo et al., 2001; van Cuijk et al., 2015; Ziani et al.,

2014).

We found that NNK treatment caused a significant decrease in binding activity of XPA

and XPB and a significant increase in binding of XPC at 24 h in mouse lung. Previously, similar

alterations in the binding activities of XPC and XPA have been connected to decreases in NER

activity (Choi et al., 2015; Donninger et al., 2015; Li et al., 2015b; Liu et al., 2005; Saijo et al.,

2004; van Cuijk et al., 2015). It is possible the change in NER activity is due exclusively to the

changes in protein binding observed here, but further research is required to elucidate the

mechanisms that lead to the changes in binding activity.

77

Chapter 4

Investigation into the effects of in vivo sulforaphane treatment on

nucleotide excision repair activity and nucleotide excision repair

recognition proteins

The phytochemical sulforaphane has gained interest recently for its apparent connection

with the reduction of cancer risk and other cytoprotective properties. Nucleotide excision repair

(NER) is an important repair pathway for mitigating carcinogenesis from exogenous DNA

damaging agents; however, it is unclear whether sulforaphane affects NER in vivo. We first

found a dose and time point of apparent maximal effect of sulforaphane in treated mouse lung and

liver using two genes regulated by Nrf2, one of the major signalling pathways of sulforaphane

mediated-effects. A 99% increase in NER activity was observed in liver nuclear extracts 12 h

post sulforaphane treatment. This change in NER activity was not attributed to changes in levels

of key recognition and early incision NER proteins XPC, XPA, XPB or p53. Additionally, the

binding of hepatic XPC, XPA and XPB to damaged DNA was not affected by sulforaphane

treatment. These results suggest that the impact of sulforaphane on repair-associated processes is

time and organ-dependent and is not associated with changes in the levels or damaged-DNA

binding activities of these specific recognition and early excision NER proteins.

Introduction 4.1

Epidemiological studies in several countries have shown a correlation between high

consumption of cruciferous vegetables and reduction of cancer risk (Higdon et al., 2007;

Tortorella et al., 2015). Cruciferous vegetables are major food crops worldwide and include such

diet staples as cauliflower, Brussels sprouts, cabbage, bok choy and broccoli (Verkerk et al.,

78

2009). They are high in a number of vitamins, nutrients, phytochemicals and are especially high

in a group of sulfur-containing phytochemicals, the glucosinolates (Bones & Rossiter, 2006;

Fahey et al., 2001; Verkerk et al., 2009).

Glucosinolates are responsible for the odour and spicy or bitter taste of these vegetables,

and have gained interest due to their various functions after they are activated. Glucosinolates are

hydrolyzed into biologically active compounds by the enzyme myrosinase, which is found in a

separate cellular compartment from the glucosinolate; the glucosinolates and myrosinase come

into contact after the plant is crushed or chewed (Bones & Rossiter, 2006). One of the best

characterized glucosinolates is glucoraphanin which is hydrolyzed to sulforaphane (Figure 1.4).

Sulforaphane is the most hydrophobic of all dietary ITCs and thus is highly lipophilic and is

rapidly diffused into the intestinal epithelium. After diffusion, sulforaphane undergoes

metabolism via the mercapturic acid pathway through conjugation with glutathione (SF-GSH).

Subsequent steps result in the generation of sulforaphane-cysteine (SF-Cys) followed by

sulforaphane-N-acetylcysteine (SF-NAC). Sulforaphane and its metabolites have shown anti-

inflammatory, antibiotic, antioxidant and anticarcinogenic actions (Alumkal et al., 2015; Atwell

et al., 2015; Clarke et al., 2008; Hunakova et al., 2014; Knatko et al., 2015; Li et al., 2014; Noh

et al., 2015).

The many actions of sulforaphane are mediated largely by activation of the Kelch-like

ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling

pathway (Baier et al., 2014; Hu et al., 2006). However, sulforaphane metabolites SF-Cys and

SF-NAC can also have direct actions as HDAC inhibitors. Sulforaphane treatment is associated

with increased apoptosis and decreases in metastasis, proliferation and angiogenesis in tumour

cells and decreases in DNA lesions after treatment with carcinogens in experimental animals and

79

tumour cell lines (Alumkal et al., 2015; Aoki et al., 2001; Clarke et al., 2008; Gao et al., 2010;

Kim et al., 2015; Noh et al., 2015). Nrf2 null mice have increased tumour incidence (Aoki et al.,

2001; Ramos-Gomez et al., 2001; Ramos-Gomez et al., 2003; Saw et al., 2014) and many of the

actions of sulforaphane are abrogated in Nrf2 null mice (Fahey et al., 2001; Kwak & Kensler,

2010; Ramos-Gomez et al., 2001). The HDAC inhibitory effects are not well characterized, but

have been linked to the acetylation of histones H3 and H4 and tied to the induction of important

tumour suppressor and pro-apoptosis proteins p21 and Bax (Gansen et al., 2015; Myzak et al.,

2006)

Sulforaphane appears to decrease the number of DNA adducts after pretreatment with

several genotoxic carcinogens: diesel exhaust (Aoki et al., 2001), 2-amino-1-methyl-6-

phenylimidazo(4,5-b)pyridine (Bacon et al., 2003), benzo[a]pyrene (Ramos-Gomez et al., 2003)

and aflatoxin B1 (Gao et al., 2010). However, its use is contraindicated with certain DNA-

damaging chemotherapeutic drugs as it can protect tumours from the chemotherapeutics

(Alumkal et al., 2015; Hunakova et al., 2014; Kim et al., 2015; Li et al., 2015a; Li et al., 2014).

Multiple studies have shown that sulforaphane-mediated induction of phase II biotransformation

pathways contributes to the decrease of reactive metabolites causing DNA adducts, but few

studies thus far have looked at sulforaphane effect on DNA repair (Jayakumar et al., 2015;

Piberger et al., 2014). In HepG2 human hepatoma cells, co-treatment of sulforaphane with PhIP

leads to a significantly reduced amount of PhIP-DNA adducts (Bacon et al., 2003). However,

treatment with sulforaphane after treatment with PhIP does not lead to any significant reduction

in PhIP-DNA adducts, leading the authors to conclude sulforaphane does not have a significant

effect on DNA repair activities (Bacon et al., 2003). Benzo[a]pyrene- and 1,6-dinitropyrene-

DNA adducts are significantly reduced in human mammary epithelial cells after treatment with

sulforaphane, but the reduction could not be explained by increased detoxification (Singletary &

80

MacDonald, 2000). Additionally, Nrf2 inhibition caused a significant decrease in UV-B induced

homologous recombination repair in A549 cells (Jayakumar et al., 2015). Also, sulforaphane-

induced H3 histone acetylation may facilitate DNA repair, as relaxation of the chromatin allows

for binding of repair proteins (Duan & Smerdon, 2014).

DNA damage caused by exposure to exogenous chemicals is repaired largely by

nucleotide excision repair (NER; Rechkunova et al., 2011). This multi-step process involves over

30 proteins which excise and replace adducted nucleotides. Persistence of adducts may lead to

mutations and apoptosis or carcinogenesis. NER can be induced by many exogenous chemicals

through multiple pathways via increased expression of key proteins (Kang et al., 2011; Lefkofsky

et al., 2015) or by post-translational modifications such as acetylation, ubiquitylation and

SUMOylation (Beli & Jackson, 2015; Choi et al., 2015; Coin et al., 2004; Donninger et al., 2015;

Kang et al., 2011; Li et al., 2015b; Li et al., 2011a; Li et al., 2013b; Poulsen et al., 2013; Silver et

al., 2011; van Cuijk et al., 2015). Decreased NER activity can be caused by decreases in key

NER proteins through adducts or mutations in regulatory genes (Lefkofsky et al., 2015). There

has yet to be a report of sulforaphane’s effects on NER activity per se in vivo. However, in vitro,

sulforaphane directly displaced the zinc-finger domain of a key NER protein, XPA, inhibiting its

ability to bind to damaged DNA (Piberger et al., 2014). Sulforaphane interacts with many

proteins that are involved in NER regulation including p53 (Kim et al., 2015; Li et al., 2011a;

Piberger et al., 2014; Saw et al., 2014) and p21 (Atwell et al., 2015; Colton et al., 2006; Dutto et

al., 2016; Mocquet et al., 2008).

To our knowledge, this is the first study to examine the in vivo effect of sulforaphane

treatment on NER activity, and it includes an assessment of levels and activities of several key

proteins in the NER pathway. First we found the dose and time point of maximal sulforaphane

81

effect on Nrf2 regulated gene induction. We then measured the change in NER activity using an

in vitro NER activity assay. Finally, we measured the change in key NER protein levels and the

ability of key NER proteins to bind to damaged DNA.


4.2.1 Animal treatment for dose response and time course of sulforaphane effects

Female CD-1 mice, aged 6-9 weeks (Charles River Canada Inc., St. Constant, PQ,

Canada), were housed with a 12-hour light/dark cycle. CD-1 mice were chosen for this study due

to their use in a previous NER activity study (Bedard et al., 2005), and their use in sulforaphane

kinetics and in vivo studies on Nrf2 regulated genes (Li et al., 2013a; Philbrook & Winn, 2014).

Animals were provided AIN-76A purified diet (BIOSERV, Flemington, New Jersey, USA) and

water ad libitum. Dose of 0, 10, 20, 50, or 100 mg/kg sulforaphane was given in a volume of 200

µL corn oil by gavage. After 3 hours, mice were killed by cervical dislocation, and livers and

lungs were removed and flash frozen in liquid nitrogen. After the dose was determined, for the

time course, mice were treated with 100 mg/kg sulforaphane in 200 µL corn oil or 200 µL corn

oil by gavage. In the timecourse, mice were killed after 1, 3, 6, 12 or 24 h; livers and lungs were

removed and flash frozen in liquid nitrogen. All animals were cared for in accordance with the

guidelines of the Canadian Council on Animal Care and the experimental procedures involving

animals were approved by the Queen’s University Animal Care Committee.

4.2.2 Analysis of mRNA from Nrf2-regulated genes

Total RNAs from mouse lungs or livers of control and sulforaphane-treated mice were

isolated from 20-30 mg tissue with the RNeasy mini kit (Qiagen, Mississauga, ON, Canada).

Total RNA quality was confirmed by formaldehyde-agarose gel electrophoresis. cDNAs were

synthesized from 5 µg of total RNA following the protocol for TaqMan High Capacity cDNA

82

Reverse Transcription (Applied Biosystems, Grand Island, NY, USA). PCR reactions were

carried out using 200 ng cDNA product, each tube containing 7 µL RNase-free water, 10 µL

TaqMan Universal PCR Master Mix and 1µL TaqMan Gene Expression assay kit for each of the

genes of interest (Cat# 4331182; HO-1: Mm00516005_m1; GCLC: Mm01253561_m1; GAPDH:

Mm99999915_g1). Levels of reverse transcription product were measured using fluorescein

amidite fluorescence collected during real-time PCR on a BIORAD CFX96 thermal cycler

(Hercules, CA, USA). Relative mRNA levels were quantified by normalization with reference

gene GAPDH and expressed using the delta-delta Ct method.

4.2.3 Preparation of cell-free whole tissue nuclear protein extracts

Nuclear protein extracts from 1.0 to 1.3 g of tissue were prepared from livers or pooled

lungs of four mice as described in section 2.2.2. Extracts from lungs or livers contained between

5 to 20 mg/mL protein. Extracts were frozen in liquid nitrogen and stored at -80°C.





4.2.6 Immunoblot analysis of NER incision protein levels in mouse liver nuclear protein

extracts

The immunoblot analysis of NER protein levels in mouse liver nuclear protein extracts

was performed as described in section 2.2.5.

83

4.2.7 Analysis of binding of NER recognition proteins to damaged DNA

An assay to assess the binding of NER repair proteins to damaged DNA was performed

as described in section 2.2.6 using mouse liver nuclear protein extracts.

4.2.8 Data analysis

For dosage study, Kruskal-Wallis test was used to analyze qRT-PCR activity. In the time

point study, multiple Mann-Whitney U tests were used to analyze qRT-PCR activity data at the

individual time points. Statistically significant differences in repair activity and NER protein

levels between treatment groups were determined by unpaired Student’s t-test. Due to inter-

experiment variability, Mann-Whitney U test was used comparing DNA-associated NER protein

binding (Subsection 4.2.7). P<0.05 was considered significant in all cases. GraphPad Prism 6

was used to graph all results.

Results 4.3

4.3.1 Response of two Nrf2 regulated genes to in vivo treatment with sulforaphane

To assess activation of Nrf2 signaling, expression of two Nrf2 regulated genes, heme

oxygenase-1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC), was measured.

mRNA levels for each gene were normalized to values obtained for glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) mRNA. At three hours post treatment, GCLC mRNA levels were

elevated with 100 mg/kg sulforaphane in lung tissue, and were increased with 50 and 100mg/kg

sulforaphane in liver tissue (Figure 4.1). HO-1 mRNA levels were elevated with 50 and 100

mg/kg sulforaphane in liver only (Figure 4.1). It was decided that 100 mg/kg sulforaphane would

be used for the timecourse, as it most consistently increased mRNA levels of these Nrf2 reporter

genes.

84

Figure 4.1 mRNA levels for dose-response relationship of two Nrf2 regulated genes relative

to control, GCLC and HO-1; 3 h after treatment with sulforaphane (SF; n=4). The data are

presented as mean + SD. (*) Significantly different from control (p<0.05).

GCLC lung

0 10 20 50 100

0

2

4

6

8

Dose mg SF/kg mouse

GC

LC m

RN

A (re

lativ

e to

con

trol)

*

GCLC liver

0 10 20 50 100

0

2

4

6

8

Dose mg SF/kg mouse

GC

LC m

RN

A (re

lativ

e to

con

trol)

* *

HO-1 lung

0 10 20 50 100

0

2

4

6

8

Dose mg SF/kg mouse

HO

-1 m

RN

A (re

lativ

e to

con

trol)

HO-1 liver

0 10 20 50 100

0

2

4

6

8

Dose mg SF/kg mouse

HO

-1 m

RN

A (re

lativ

e to

con

trol)

**

85

4.3.2 Timecourse of responses of two Nrf2 regulated genes to 100 mg/kg of sulforaphane

Sulforaphane (100mg/kg) in 200 µL corn oil was administered to female CD-1 mice that

were killed at 1, 3, 6, 12, or 24 h later. In mouse lung, there was a significant increase in HO-1

mRNA at 3 h (Figure 4.2; p<0.05) and an apparent trend at 6 h (Figure 4.2; p>0.05), as well as a

significant increase of GCLC at 3 and 6 h (Figure 4.2; p<0.05). In mouse liver, there was

significant increase of HO-1 at 6 h (Figure 4.2; p<0.05) and a trend at 3 h (Figure 4.2; p>0.05);

and a significant increase of GCLC at 1, 3 and 6 h (Figure 4.2; p<0.05).

86

Figure 4.2 Timecourse of effects for two Nrf2 regulated genes HO-1 and GCLC in lung and

liver after treatment with 100mg/kg of sulforaphane (SF) or corn oil (n=4). The data are

presented as mean + SD. (*) Significantly different from control (p<0.05).

HO-1 lung

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF0

2

4

6

8

10

Treatment

Rel

ativ

e Ex

pres

sion

1hr

3hr

6hr

12hr

24hr*

HO-1 liver

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF0

2

4

6

8

10

Treatment

Rel

ativ

e Ex

pres

sion

1hr

3hr

6hr

12hr

24hr

*

GCLC lung

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF0

2

4

6

8

10

Treatment

Rel

ativ

e Ex

pres

sion

1hr

3hr

6hr

12hr

24hr

*

*GCLC liver

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF

corn

oil SF0

2

4

6

8

10

Treatment

Rel

ativ

e Ex

pres

sion

1hr

3hr

6hr

12hr

24hr***

87

4.3.3 Effect of in vivo treatment of mice with sulforaphane on repair synthesis activity in

mouse liver and lung nuclear protein extracts

The timepoints 6 h and 12 h post-treatment were chosen for NER activity studies because

protein synthesis can sometimes lag behind increases in mRNA transcripts (Greenbaum et al.,

2003). At 6 h following in vivo sulforaphane treatment, repair synthesis activity of mouse liver

extracts toward POB damage showed no significant change relative to control (Figure 4.3).

However, repair activity increased by 99% at 12 h post in vivo dosing in mouse liver (Figure 4.3).

Due to the concentration of nuclear protein extract required for the repair assay, and the amount

of lung tissue needed for nuclear protein extracts, we were unable to treat sufficient animals to

test 6 h lung NER activity. There was no significant change in NER activity in 12 h mouse lung

nuclear extract (Figure 4.3).

88

Figure 4.3 Effects of in vivo sulforaphane treatment on in vitro DNA repair activity of mouse

liver and lung nuclear protein extracts towards NNKOAc-induced pyridyloxobutyl DNA

damage. Extracts were prepared from mice treated with either 100mg/kg sulforaphane or

corn oil and killed 6 h or 12 h post-dosing (n=4 pools of lungs of 4 mice per pool or n=4

livers). Data are presented as mean + SD. (*) Significantly different from control (p<0.05).

6h liver 12h liver 12h lung0

5000

10000

15000

20000am

ol [α

32P

] dAT

P in

corp

orat

ed/µ

g D

NA

Corn Oil

SF

*

89

4.3.4 Effects of in vivo treatment of mice with sulforaphane on levels of NER proteins in

mouse liver nuclear protein extracts

Since sulforaphane had a significant effect on NER activity in mouse liver extracts, levels

of recognition NER proteins in mouse liver extracts following sulforaphane treatment were

examined (Figure 4.4). At 12 h post-treatment, liver extracts showed no significant changes in

early phase NER recognition and incision proteins XPC, XPA, XPB and p53.

90

Figure 4.4 a) Relative density of each NER protein in mouse liver extracts isolated from

corn oil or sulforaphane-treated mice 12 h post-dosing, relative to a constant internal

standard extract (CTL). The data are presented as mean + SD. b) Representative

immunoblots of the relative levels of each key NER protein in mouse liver extracts. S=

nuclear protein extracts from sulforaphane treated mice, C=nuclear protein extracts from

corn oil treated mice; all experiments n=4.

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4.3.5 Effect of in vivo treatment of mice with sulforaphane on key NER protein DNA

binding in mouse liver nuclear protein extracts

To determine if sulforaphane affects the interactions between key NER proteins and

damaged DNA, an assay was used that measures the binding of NER repair proteins to damaged

DNA during the NER reaction. Binding of the three recognition and incision NER proteins was

not significantly different between liver extracts from sulforaphane treated and control animals at

12 h after treatment (Figure 4.5).

92

Figure 4.5 Effect of in vivo sulforaphane treatment on in vitro NER protein binding to

pyridyloxobutyl-adducted DNA. All data are presented as mean + SD. (all n=4, p>0.05)

XPCXPA

XPB0

1

2

3

4

12h Liver Extracts

Rel

ativ

e B

indi

ng

Corn OilSulforaphane

93

Discussion 4.4

In this study, it was confirmed that sulforaphane increases expression of two genes

known to be induced by Nrf2 activation: GCLC and HO-1. Furthermore, at 100 mg/kg,

sulforaphane increased NER activity by 99% in liver tissue in an in vitro NER activity assay 12 h

after treatment. Recently, sulforaphane has been under investigation for its multiple effects

against cancer (Lenzi et al., 2014). Sulforaphane is known to inhibit cancer initiation after

treatment with a carcinogen, as well as promotion and progression of tumour cells (Clarke et al.,

2008; Kwak & Kensler, 2010). Sulforaphane has been used prophylactically to decrease DNA

adducts after treatment with several DNA damaging agents (Aoki et al., 2001; Bacon et al., 2003;

Gao et al., 2010; Ramos-Gomez et al., 2003). In many cases, it was theorized that sulforaphane

induces detoxification pathways and clears metabolites before they can have a genotoxic effect,

as sulforaphane induces phase II biotransformers and phase III transporters (Atwell et al., 2015;

Clarke et al., 2008; Knatko et al., 2015; Rajendran et al., 2013). Here, we describe another

mechanism that can decrease genotoxicity—upregulation of NER, thereby enhancing removal of

adduct-damaged DNA regions. This is the first report of an effect of in vivo treatment of

sulforaphane on NER activity.

The induction of established Nrf2 regulated genes with 100 mg/kg sulforaphane at 3 to 6

h post-treatment is consistent with previous work (Clarke et al., 2011; Hu et al., 2006; Keum,

2011; Li et al., 2013a). Sulforaphane treatment of C57BL/6J mice showed a similar effect- a

significant increase of HO-1 at 3 h and no significant increase at 12 h in treated mouse liver (Hu

et al., 2006). Additionally, Philbrook and Winn showed a significant increase in GCLC mRNA

transcripts in mouse liver of pregnant and non-pregnant CD-1 mice only after 6 h of sulforaphane

treatment (Philbrook & Winn, 2014). Initially, only the liver tissue could be used for the NER

activity experiments due to the amount of tissue required for lung nuclear protein extracts (n of 1

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requires 4 whole mouse lungs). As a result, we chose the 6 h timepoint for the NER activity

assay, as both liver HO-1 and GCLC are significantly increased (Figure 4.2). Metabolism and

clearance of sulforaphane are rapid in experimental animals and humans (Clarke et al., 2011;

Petri et al., 2003; Shapiro et al., 2006). Female CD-1 mice receiving a large bolus dose of

sulforaphane-enriched broccoli extract (approximately 2.5 mg/g) excreted the sulforaphane by 24

h (Li et al., 2013a). Small concentrations of sulforaphane appear to have important effects on

Nrf2 regulation and HDAC inhibition as sulforaphane can have different and opposite effects at

high and low concentrations (Alfieri et al., 2013; Zanichelli et al., 2012a). As a result, we also

chose to look at the effect of sulforaphane treatment on NER activity at the 12 h timepoint, which

is after the effects of Nrf2 on HO-1 and GCLC have subsided.

There are more than 30 proteins involved in NER, and multiple studies have shown these

proteins can influence NER activity through post-translational ubiquitylations, SUMOylations

and increased or decreased abundance (Kang et al., 2010b; Kang et al., 2011; Huang &

D’Andrea, 2006; Poulsen et al., 2013; Silver et al., 2011; van Cuijk et al., 2015). Recently, three

key proteins have been identified as the triple check, rate-limiting step in NER (discussed in

section 1.3.2.1): xeroderma pigmentosum (XP) class C (XPC), XP class A (XPA) and

transcription factor II H (TFIIH) (Marteijn et al., 2015; Li et al., 2015b). As discussed in

Marteijn et al., NER progresses sequentially after initial sensing of the damage by XPC (2015).

XPC recruits XPB-containing TFIIH and XPA, which both verify the DNA damage and protect

the DNA strand. Other research has shown a significant change in the ability of XPC, XPA and

XPB to bind to damaged DNA in an in vitro, damaged-DNA NER protein binding assay after

exposure to the carcinogen NNK (Chapter 2 and Chapter 3). Here, we show a significant

sulforaphane-induced increase in liver NER activity at 12 h without a significant change in levels

or damaged DNA binding activities of specific NER proteins (Figure 4.3-Figure 4.5). The

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increased NER activity may be due to a mechanism occurring downstream of XPA and TFIIH

binding of the damaged DNA strand (Figure 1.1). There are other, less understood NER proteins

downstream of TFIIH that may be involved in the increased NER activity, including proliferating

cell nuclear antigen (PCNA; Cazzalini et al., 2014) and XPF-ERCC1 (Su et al., 2012; Tomicic et

al., 2011). PCNA is recruited to the DNA strand after DNA unwinding by TFIIH, and binds to

RPA, which acts to stabilize the replication complex (Overmeer et al., 2011). XPF-ERCC1 is a

nuclease that makes the incision to the 5’ of the DNA lesion, and binds to XPA (Fadda, 2013;

Matsunaga et al., 1995). Thus, these are important proteins to examine in the future.

Sulforaphane has the potential to activate antioxidant response elements and multiple

pathways that are involved in NER regulation (Rajendran et al., 2013). p53 is important for cell

cycle arrest allowing for prolonged repair before replication of damaged DNA and presence of

p53 appears to affect NER activity in some in vitro repair assays (Chang et al., 2008). However,

p53 was not a cellular target of sulforaphane in HCT116 and XP12RO cells (Piberger et al.,

2014) and we show there is no significant effect on p53 protein amount in sulforaphane-treated

liver nuclear protein extracts (Figure 4.4). Therefore, it is unlikely the significant increase in

NER activity in sulforaphane treated mouse liver at 12 h (Figure 4.4) is due to p53.

Another of the proteins induced by sulforaphane is p21, which has multiple roles as a

cell-cycle regulator and a regulator of transcription, cell motility, apoptosis and DNA repair. This

protein is likely induced via the HDAC inhibitory actions of sulforaphane metabolites SF-NAC

and SF-Cys and these effects are sustained (Myzak et al., 2004; Myzak et al., 2006). In fact,

Myzak et al. (2006) showed the HDAC inhibitory effects of sulforaphane persisted to 48h in the

colonic mucosa of Apcmin mice after a single bolus dose of either sulforaphane or SF-NAC.

Specifically, p21 appears to have an effect on NER; however there are multiple reports that p21

has positive, neutral or negative effects on total NER activity (Dutto et al., 2016).

96

One previous study reported a significant decrease in TC-NER activity after in vitro

treatment of HCT116 and XP12RO human colon cancer and fibroblast cells with sulforaphane

(Piberger et al., 2014). That study concluded sulforaphane caused an uncoupling of the zinc-

finger domain of XPA which abrogates damaged-DNA binding of XPA (Piberger et al., 2014).

Here, rather than an impairment of in vitro NER activity, we observed a 99% increase in NER

activity in liver tissue 12 h post in vivo treatment with sulforaphane (Figure 4.3). We also see no

significant change in the binding of XPA to damaged DNA at 12 h post-treatment (Figure 4.5).

Piberger et al. concluded that only early-stage repair (TC-NER) was affected by sulforaphane in

these cell lines, whereas our in vitro repair assay is optimized to measure effects on late-stage

repair (global-genome NER (GG-NER)). We focus on GG-NER because nontranscribed genes or

genes on the nontranscribed strand of active genes (both repaired by GG-NER) are better

predictive markers for carcinogen-induced tumourigenesis than mutations in actively transcribed

genes (Balajee & Bohr, 2000).

It is known that carcinogens can increase NER activity (Bedard et al., 2005; Brown &

Massey, 2009; Choi et al., 2015; Protić et al., 1988). As a chemopreventive strategy, it could be

valuable to identify a non-genotoxic chemical that can cause similar effects. Although it is

unclear whether sulforaphane is completely non-genotoxic (Baasanjav-Gerber et al., 2011), our

findings show it may be a candidate for this purpose.

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Chapter 5

General Discussion

Overall conclusions 5.1

Endogenously formed metabolic products as well as environmental chemicals and

radiation constantly threaten DNA integrity. DNA damage loads may total 104-105 DNA lesions

per mammalian cell per day, and it is this damage that can initiate carcinogenesis (Swenberg et

al., 2011). It is therefore imperative that the damage is removed and the DNA repaired in a

timely and error-free manner through one of several DNA repair pathways (Bates, 2010). It has

been established experimentally that there are several endogenous pathways and exogenous

agents that can affect the activity of DNA damage repair pathways (Bates, 2010). Additionally,

defects in DNA repair pathways predispose individuals to cancer; for example, individuals with

xeroderma pigmentosum have a high incidence of skin cancer. GG-NER is one DNA damage

repair pathway whose activity is changed with exposure to certain exogenous agents (Bedard et

al., 2005; Brown & Massey, 2009; Cho, 2002; Choi et al., 2015; Fatur et al., 2003; Li et al.,

2015b; Marteijn et al., 2015; Mulder et al., 2014; Nollen et al., 2009). Several successive steps

are important for error-free repair through GG-NER, and only recently has our understanding of

the successive steps of GG-NER and their regulation expanded. Additionally, this research into

GG-NER regulation has exclusively been done in vitro. In this thesis, we focussed on one part of

the GG-NER pathway, the recognition and early incision phase, and the effects of in vivo

treatment with NNK and sulforaphane on specific protein levels and activities.

Earlier studies showed that treatment of mice with tumourigenic doses of NNK or

aflatoxin B1increased NER activity in mouse liver and decreased it in lung (Bedard et al., 2005;

Brown & Massey, 2009). Additionally, p53 status altered aflatoxin B1 mediated change of NER

98

(Mulder et al., 2014). However, the specific role of NER recognition proteins, p53 and the

change of NER activity across a 72 h period had not been investigated after treatment with NNK.

There was a significant decrease in NER activity at 12 h post NNK treatment and a

significant increase in NER activity at 24 h in mouse liver (Chapter 2) and a significant decrease

in NER activity at 24 h in mouse lung (Chapter 3). NNK treatment caused no significant change

in levels of NER proteins XPC, XPA, XPB and p53 in mouse liver or lung nuclear protein

extracts at 12 and 24 h (Chapter 2, liver; Chapter 3, lung). At 12 h in liver, there was no change

in XPC binding to damaged DNA, but decreases in XPA and XPB binding (Chapter 2).

Conversely, at 24 h in mouse liver, there was a decrease in XPC binding and increases in XPA

and XPB binding (Chapter 2). In comparing the damaged-DNA binding activities of recognition

proteins at 24 h in mouse lung, there was a significant increase in XPC binding, and significant

decreases in XPA and XPB binding (Chapter 3). These results indicated NNK can have time-

and tissue-dependent effects on NER and the ability of specific NER proteins to bind to damaged

DNA, and it is likely this effect is due in part to changes in recognition-protein damaged-DNA

binding activity rather than altered levels of the proteins.

Following a single dose of DMSO, there was a significant decrease in NER activity at 2 h

in mouse liver relative to an i.p. saline treatment (Appendix A). DMSO is often used as a vehicle

for in vivo and in vitro administration of chemicals, but its effects are often not described. The

mechanism by which DMSO affects NER activity was not pursued further as DMSO was not

used in this thesis, but it shows the importance of understanding the vehicle used in future studies.

Sulforaphane has been under investigation for multiple effects leading to a decrease in

DNA adducts, including those repaired by NER (Gerhauser, 2013; Singletary & MacDonald,

2000; Topè & Rogers, 2009). However, the effect of in vivo treatment with sulforaphane on NER

activity had not been studied previously. In order to gauge the effect of sulforaphane treatment

99

on Nrf2, mRNA levels of two Nrf2 regulated genes were assayed in a dose and time course study.

We found only the 100 mg/kg sulforaphane dose and the 6 h time point consistently caused an

increase in GCLC and HO-1 mRNA levels in mouse liver and lung (Chapter 4). There was no

effect of sulforaphane on NER activity at 6 h in mouse liver or at 12 h in mouse lung nuclear

protein extracts, but there was a significant increase in NER activity in mouse liver at 12 h

(Chapter 4). Sulforaphane treatment caused no significant change in levels of NER proteins

XPC, XPA, XPB and p53 in 12 h mouse liver nuclear protein extract and there was no significant

change in the damaged-DNA binding activities of recognition proteins XPC, XPA and XPB

(Chapter 4). This indicates that, despite increasing NER activity at 12 h, sulforaphane doesn’t

appear to do so by increasing the ability of these recognition proteins to bind to damaged DNA,

and the cause of the increase of NER activity remains to be elucidated. Suggestions for possible

mechanisms are discussed below.

Future directions 5.2

5.2.1 Post-translational modifications of NER proteins as a basis for observed changes in

GG-NER activity

Only recently have specific proteins been identified that induce changes in DNA repair

activity without changes in protein levels, and this generally occurs through post-translational

modifications involving ubiquitin or ubiquitin-like compounds (Gali et al., 2012; Huang &

D’Andrea, 2006; Kang et al., 2010b; van Cuijk et al., 2015). The covalent attachment of the 76

amino acid residue protein ubiquitin and the approximately 100 residue small ubiquitin-related

modifier (SUMO) is the most common. Although ubiquitylation and SUMOylation were initially

discovered as mechanisms targeting the degradation of proteins by the proteasome, their actions

more recently have been recognized to include regulation of protein activity, localization and

interactions (Bergink & Jentsch, 2009; Komander & Rape, 2012; Jackson & Durocher, 2013).

100

Other post-translational modifications such as phosphorylation and acetylation can also affect

DNA repair (Cazzalini et al., 2014; Donninger et al., 2015; Duan & Smerdon, 2014; Fan & Luo,

2010; Gansen et al., 2015). In fact, all DNA repair pathways appear to be regulated in some way

by phosphorylation, acetylation, ubiquitylation and/or SUMOylation, and recent work has shown

that several steps in the regulation of the GG-NER pathway are dependent on acetylation,

ubiquitylation and SUMOylation (Rüthemann et al., 2016).

Early stage NER has recently been identified as a key stage for NER activity changes

after carcinogen exposure (Choi et al., 2015; Donninger et al., 2015; Li et al., 2015c; Marteijn et

al., 2015), and recent in vitro work has indicated the importance of post-translational

modifications of key NER proteins in response to bulky DNA damage (Beli & Jackson, 2015;

Dou et al., 2010; Gali et al., 2012; Hoege et al., 2002; Huang & D’Andrea, 2006; Kang et al.,

2010b; Poulsen et al., 2013; van Cuijk et al., 2015). The cascade of effects after carcinogen

exposure is still being revealed, but recent research into the effects of exposure of cultured cells

to UV light has allowed for a better understanding of the sequence of events and regulation of

these events (Marteijn et al., 2015). However, until now this research has not been expanded

with other agents of DNA damage, or after in vivo exposure. It is possible that the differences in

NER protein damaged-DNA binding observed after treatment with NNK (Chapters 2 and 3)

could be caused by changes to post-translational modifications of XPC, XPA and TFIIH (XPB).

Additionally, it is possible the modification of another component of the NER pathway causes the

increase in NER activity observed after treatment with sulforaphane (Chapter 4). Expansion of

our understanding of the NER regulation events after in vivo exposure sulforaphane or NNK will

be an important priority in the future.

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5.2.1.1 XPC binding to damaged DNA

Recognition protein XPC can be ubiquitylated at multiple sites and SUMOylated (van

Cuijk et al., 2015). After UV exposure, XPC is both ubiquitylated at lysine 48 and SUMOylated,

which increase XPC damaged-DNA binding affinity and protect XPC from degradation,

respectively (Sugasawa et al., 2005; Wang et al., 2005). XPC release from damaged DNA is then

mediated by RNF111, a ubiquitin ligase that ubiquitylates XPC at lysine 63 and is a critical

component of the TGF-β signaling pathway (Miyazono & Koinuma, 2011; Poulsen et al., 2013;

van Cuijk et al., 2015). Release of the XPC protein allows the incorporation of the endonucleases

XPG and ERCC1/XPF into the repair complex, and removal of RNF111 stalls in vitro NER of

UV-induced DNA lesions (Poulsen et al., 2013; van Cuijk et al., 2015). No study has examined

the effect of NNK on RNF111, but abrogation of the TGF-β signalling pathway significantly

increases NNK-induced lung tumour frequency (Kanehira et al., 2005). Additionally, it is

unclear whether XPC binding to POB DNA damage is affected by these post-translational

modifications as has been observed for UV-induced DNA damage. XPC binding to damaged

DNA increased in lung nuclear protein extracts 24 h post NNK, but decreased in liver extracts 24

h post NNK (Chapter 2 and Chapter 3). It is possible the ubiquitylation/ SUMOylation of XPC is

important in the organ and time dependent changes in XPC binding activity after NNK treatment,

and this should be a focus of future research.

5.2.1.2 XPA binding to damaged DNA

The recognition protein XPA can be ubiquitylated and acetylated (Donninger et al., 2015;

Kang et al., 2011). In mice, XPA degradation is controlled by ubiquitylation in a diurnal cycle

and as a result NER activity varies throughout the day (Kang et al., 2010b; Kang et al., 2011).

XPA binding to damaged DNA, however, is regulated by deacetylation (Fan & Luo, 2010; Kang

et al., 2011). After UV exposure, induction of sirtuin 1 (SIRT1) via the RASSF1A tumour

102

suppressor appears to decrease the level of acetylated XPA (Donninger et al., 2015). SIRT1 is an

important mammalian deacetylase with roles in the regulation of gene expression, cell survival,

proliferation, differentiation, metabolism, immune response and carcinogenesis (Wang & Chen,

2013). Deacetylated XPA is required for efficient repair of bulky adducts (Donninger et al.,

2015; Kang et al., 2011), and knockdown of the SIRT1 gene significantly decreases NER activity

in vitro (Fan & Luo, 2010). There was an increase in XPA binding in liver extracts 12 h and 24 h

post NNK treatment, and a decrease in XPA binding in lung extracts 24 h post NNK treatment

(Chapter 2 and Chapter 3). It is possible that the acetylation of XPA is important in the observed

changes in binding activity after NNK treatment, and this should be a focus of future research.

SIRT1 can be detected using an antibody against SIRT1.

5.2.1.3 TFIIH binding and activity

TFIIH binding activity is regulated by the binding of XPC and XPA to damaged DNA

(Coin et al., 2008; Choi et al., 2015; Donninger et al., 2015; Fan & Luo, 2010; Li et al., 2015b).

Whereas the presence of XPC recruits TFIIH to the damaged site, the presence of XPA stimulates

the release of the CAK subunit from the TFIIH subunit (Coin et al., 2008) which significantly

increases the binding efficiency of TFIIH to the area near the lesion (Choi et al., 2015; Donninger

et al., 2015; Fan & Luo, 2010). It appears that the presence of XPA and the release of the CAK

subunit are more important than XPC presence for TFIIH binding activity, so any change to XPA

binding can cause a change in TFIIH binding (Li et al., 2015b). Additionally, the lack of any

significant change in XPB or XPA protein levels (Chapters 2 and 3) suggests that the changes in

XPB binding activity could be the result of the change in XPA binding activity. Also, XPB and

XPD are helicases, and it is unclear whether protein binding is concomitant with helicase activity.

TFIIH helicase activity is closely tied with CAK release, which is another important connection

between TFIIH and XPA (Coin et al., 2008; Li et al., 2015b). However, there is no information

103

on whether NNK or sulforaphane affect the helicase activity of XPB or XPD. It is possible to

quantify the activity of XPB and XPD as helicases in an assay (Shin et al., 2005), but this assay

has never been optimized for use with whole tissue protein extracts. Therefore, it is also

important for future studies to examine the effects of NNK and sulforaphane on TFIIH binding

and XPB and XPD helicase activity.

5.2.2 Effects of sulforaphane and NNK on other NER proteins

5.2.2.1 RPA

In NER, RPA forms the linkage between pre-incision factors such as XPC, XPA and

TFIIH and post-incision factors such as proliferating cell nuclear antigen (PCNA), DNA

polymerases and ligases, protecting the single stranded DNA after damage excision (Overmeer et

al., 2011; Gourdin et al., 2014; Riedl et al., 2003). Like other NER proteins, RPA is subject to

multiple post-translational modifications, including phosphorylation, poly-ADP ribosylation,

ubiquitylation and SUMOylation (Dou et al., 2010; Maréchal & Zou, 2015; Hass et al., 2012;

Binz et al., 2004) and is involved in both DNA repair and replication. After cellular DNA

damage such as UV-induced damage, RPA is hyper-phosphorylated, which down-regulates its

role in DNA replication and appears to promote its activity in DNA repair (Binz et al., 2004).

However, the binding activity of RPA after DNA damage has never been quantified. In the

future, the damaged-DNA NER protein binding assay used in this thesis could be used to probe

for RPA binding activity. The other post-translational modifications appear to regulate the

activation of the ATR checkpoint, which controls cell cycle arrest after DNA damage, and

promote largely homologous recombination DNA repair; however, their roles in NER specifically

are not yet understood (Gourdin et al., 2014; Hass et al., 2012; Maréchal & Zou, 2015). RPA is

integral for NER, and any damage or dysregulation of RPA can have a detrimental effect on NER

104

activity (Guven et al., 2015). Therefore, it is important to understand the role of post-

translational modifications on RPA binding to NER proteins and ssDNA in NER.

5.2.2.2 PCNA

During NER, PCNA is recruited to the DNA strand after DNA unwinding by TFIIH, and

binds to RPA, which acts to stabilize the replication complex (Overmeer et al., 2011). PCNA

binds the dually incised DNA and recruits polymerases for DNA elongation. PCNA can be

ubiquitylated, SUMOylated and acetylated (Cazzalini et al., 2014; Gali et al., 2012; Kumar &

Saha, 2015; Mocquet et al., 2008; Riedl et al., 2003). Ubiquitylation and SUMOylation were the

first post-translational modifications to be unambiguously identified for PCNA (Hoege et al.,

2002), and these modifications are important for DNA replication and maintaining genomic

integrity (Gali et al., 2012; Hoege et al., 2002; Kannouche et al., 2004; Kumar & Saha, 2015;

Kubota et al., 2013). On the other hand, acetylation appears to be important in NER regulation

(Cazzalini et al., 2014). PCNA acetylation occurs in proliferating cells and after UV damage, and

may be partially controlled by p21 (Cazzalini et al., 2014). p21 has multiple roles as a cell-cycle

regulator, a regulator of transcription, and in cell motility, apoptosis and DNA repair (Cazzalini et

al., 2010; Colton et al., 2006; Dutto et al., 2016; Lee et al., 2009). Specifically, p21 appears to

have an effect in NER; however, different studies have reported positive, neutral or negative

effects on total NER activity (Cazzalini et al., 2010; Dutto et al., 2016). Many of these effects

may be due to varied experimental conditions and cell types, and a current hypothesis is that in

vivo p21 has positive effects on NER activity through its interactions with PCNA (Dutto et al.,

2016).

The results in Chapter 4 show that there is an increase in DNA repair activity in mouse

liver 12 h after sulforaphane exposure, but this was not accompanied by changes in levels of

XPC, XPA, XPB or p53 or in the binding of XPC, XPA or XPB to damaged DNA. The

105

explanation for upregulated NER activity in the absence of changes in these NER proteins

remains to be elucidated, but PCNA acetylation could conceivably play a role. The effect of

sulforaphane on PCNA acetylation has not been reported, but the protein p21 is affected by

sulforaphane treatment (Chen et al., 2009; Cho et al., 2014; Ho et al., 2009). Future experiments

should examine the role of p21 in the sulforaphane-mediated NER activity increase reported in

Chapter 4.

5.2.2.3 Post-translational modifications of p53

As discussed in previous chapters, p53 has multiple effects on NER including indirect

effects on the transcription of NER proteins DDB2 and XPC and direct effects through binding to

damaged DNA and to NER proteins (Adimoolam & Ford, 2003; Bhana et al., 2008; Chang et al.,

2008; Dregoesc et al., 2007; Lakin & Jackson, 1999). Additionally, p53 is frequently mutated in

human tumours (Rodin & Rodin, 2000). In this thesis, treatment with NNK or sulforaphane

caused no significant change in levels of p53 protein in nuclear protein extracts (Chapters 2-4).

However, p53 is known to be controlled by many post-translational modifications including

phosphorylation, dephosphorylation and acetylation, which mediate its cell cycle arrest, apoptosis

and DNA damage repair effects (Lakin & Jackson, 1999; Ashcroft et al., 1999). TFIIH is likely

involved in the effects mediated by p53; both XPB and XPD appear to bind to p53 (Chang et al.,

2008; Hou et al., 2003; Léveillard et al., 1996). p53 presence and XPB/XPD binding appear to

enhance NER activity (Gillet & Scharer, 2006; Léveillard et al., 1996; Wang, 2003), but the

mechanism is not clear. Also, there is no report of whether post-translational modifications of

p53 are important for the effects of p53 on NER activity. In addition, the decreases observed in

NER activity (Chapters 2 and 3) could be due to covalent modifications of p53 either in the C-

terminal amino acid of p53, or in the DNA binding region, thereby inhibiting the increased NER

efficiency provided by p53. Protein modifications can be observed after protein fractionation by

106

2-D SDS-PAGE and mass-spectrometry after immunoblotting. Western blotting with antibodies

for p53 phosphorylation and acetylation can be used to test whether these post-translational

modifications have any role in the significant changes to NER activity after sulforaphane and

NNK treatment.

The research in this thesis does not entirely rule out a role for p53 as a cause of the

effects observed after treatment with NNK or sulforaphane. Unfortunately, the binding of p53 to

damaged DNA could not be assessed reliably using the nuclear protein extracts isolated in this

thesis. Optimization of p53 in the damaged DNA-NER protein binding assay could be an

important next step to fully explain the importance of p53 in NNK- and sulforaphane-mediated

NER activity changes.

5.2.3 Protein adducts

It has previously been theorized that NNK causes covalent modifications to key NER

proteins (Brown, 2008). HPB-releasing hemoglobin adducts have been detected in smokers and

in rats treated with NNK, but it is not known if NNK also forms adducts with NER proteins

(Hecht et al., 1993a; Peterson et al., 1990). It is possible that the effects shown in mouse lung

after treatment with NNK are due to a covalent adduction of NER proteins. There are known

differences in bioactivation between A/J mouse lung and liver, and the organs may differ in their

abilities to respond to adducted proteins or it is possible that NER proteins may be adducted only

in the lung (Zheng & Takano, 2011; Zhou et al., 2012). Covalent protein adducts in any of the

binding regions of NER proteins XPC, XPA or XPB would affect their abilities to bind to

damaged DNA. Other NER proteins such as RPA, XPF or XPG could be affected in a similar

way by covalent modifications. RPA is particularly susceptible to damage (Guven et al., 2015),

and work has shown that modified structures of both XPF and XPG can inhibit repair (Araújo et

al., 2001; McNeil & Melton, 2012; Su et al., 2012; Tomicic et al., 2011). Potentially adducted

107

NER proteins can be separated by 2D SDS-PAGE, identified by immunoblotting and subjected to

mass spectrometric analysis.

5.2.4 The effects of sulforaphane are mediated through HDAC inhibition and Nrf2

activation

It is unclear whether the Nrf2 downstream effects or the histone deacetylase inhibitor

effects of sulforaphane mediate the increase in hepatic NER activity reported in Chapter 4. The

time point of maximal effect for our chosen Nrf2 genes was before 12 h; however, effects of

sulforaphane can occur at various timepoints in vivo (Hu et al., 2004a; Hu et al., 2006). In the

future, repeating the experiment in the Nrf2 knockout mouse will allow for better understanding

of the effects of sulforaphane on NER. If the effect of sulforaphane on NER is mediated

exclusively by the activation of Nrf2, then it is expected the effect on NER will not be observed

in the Nrf2 knockout mouse. Whereas, if the effect of sulforaphane on NER activity is due to

HDAC inhibition, then the effects observed in this thesis will persist in the Nrf2 knockout mouse.

The sulforaphane metabolites, SF-Cys and SF-NAC, are the major HDAC inhibitors (Myzak et

al., 2004; Myzak et al., 2006), and these can be investigated if it appears the NER effect is due to

the HDAC inhibitory effect of sulforaphane.

5.2.5 The effects of cigarette smoking on NER activity in humans

NNK is a known carcinogen, and it appears to be important in smoking-induced human

lung cancers with increased urinary NNK metabolites linked to increased risk for lung

adenocarcinomas (Hecht et al., 2016; Stepanov et al., 2014; Yuan et al., 2011; Yuan et al., 2014).

However, no study has been reported regarding the effects of smoking on NER activity. It has

been established that NNK inhibits NER in mouse lung (Brown, 2008), and it is possible smoking

can cause a similar effect in human lung. Nuclear protein can be extracted from human lung

108

tissue with a similar protocol to the one used in this thesis, and these extracts can be used to

assess DNA repair activities on POB adducts using the same NER activity assay used in this

thesis. If there is an effect of smoking on NER, approaches used and described in this thesis

could be used to determine mechanisms.

109

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Appendix A

The effect of DMSO on nucleotide excision repair activity in A/J mouse

lung and liver

Introduction

Dimethyl sulfoxide (DMSO) is an organic solvent with a rich pharmacological history

(Santos et al., 2003). Initially known in the 19th century as a byproduct of the wood industry, it is

frequently used for clinical and laboratory purposes (Santos et al., 2003). In the laboratory it is a

utility solvent, as it can dissolve both polar and nonpolar compounds, whereas DMSO received

approval from the US Food and Drug Administration for treatment of interstitial cystits in 1978

(Parkin et al., 1997). It has been used most successfully in treatment of leukemia, and proven to

have multiple anticancer properties in cells and in vivo (Camici et al., 2006; Jia et al., 2010; Pal et

al., 2012). In invasive lung adenocarcinoma cells, DMSO stimulated the tumour suppressor gene

HLJ1, thereby inhibiting cell invasion, migration and proliferation (Wang et al., 2012). DMSO

appears to cause retinal cell death at low doses in vivo, inducing apoptosis by inhibiting

mitochondrial respiration (Galvao et al., 2014). It is also known to be mutagenic in two Ames

test strains (Hakura et al., 1993).

Damage caused by endogenous reactive metabolites and exogenous factors can cause

multiple kinds of damage to a cell. Several methods of clearing and repairing DNA damage exist,

depending on the structure or type of damage (Branzei & Foiani, 2008). Repair of bulky adducts,

most commonly associated with damage caused by exposure to exogenous chemicals, is

performed by nucleotide excision repair (Branzei & Foiani, 2008). No less than 30 proteins are

involved in the multi-step process of lesion excision and repair in nucleotide excision repair

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(Gillet & Scharer, 2006; Wood et al., 1988), and their importance can be understood by the lack

of viability of cells lacking active proteins (Nouspikel, 2009; Yasuda et al., 2007).

In the laboratory, many compounds important in DNA repair studies are dissolved

exclusively in DMSO. Studies on AFB1 are of specific interest in DNA repair studies, as

previously described a tissue-specific alteration of nucleotide excision repair (NER) activity after

intraperitoneal dosage of AFB1 (Bedard et al., 2005). Briefly, after treating mice with AFB1,

Bedard et al. found treated liver tissue extracts had increased nucleotide excision repair activity,

whereas treated lung tissue extracts had decreased nucleotide excision repair activity (2005).

Here, we were interested in comparing two commonly used solvents, sterile saline and DMSO.

In this experiment, we observed a decrease of DMSO treated liver NER activity, and no change in

lung NER activity.

Materials and methods

Animal treatments

Female A/J mice (JAX Laboratories), aged 7-10 weeks, were housed in a 12 h light/dark

cycle and provided with food and water ad libitum. Mice were treated with saline (0.1mL, i.p.) or

DMSO (0.04 mL, i.p.). Two hours following this administration, mice were killed by cervical

dislocation. Lungs and livers were perfused with 10mM Tris/1mM EDTA (pH 7.9), excised,

finely chopped, frozen in liquid nitrogen, and stored at -80ºC until extract preparation. All

animals were cared for in accordance with the guidelines of the Canadian Council on Animal

Care. Experimental procedures involving animals and animal care were approved by the Queen’s

University Animal Care Committee.

Preparation of cell-free whole tissue nuclear protein extract

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Nuclear protein extracts were prepared from pooled lungs or pooled livers of four mice as described in section 2.2.2.

Preparation of damaged plasmid DNA


In vitro DNA repair synthesis assay


Results

Effect of in vivo treatment of mice with DMSO on DNA repair synthesis of mouse liver and

lung nuclear protein extracts

Following in vivo DMSO treatment after 2 h, repair synthesis activity of mouse lung

extracts toward POB DNA damage showed no significant change relative to control (Figure A.1).

However, DNA repair activity decreased by 39% at 2 h post in vivo dosing in mouse liver (Figure

A.1).

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Figure A.1 Effects of in vivo DMSO treatment on in vitro DNA repair activity of mouse lung

or liver extracts towards NNKOAc-induced pyridyloxobutyl DNA damage. Extracts were

prepared from mice treated with either 0.1mL sterile saline or 0.04mL DMSO and killed 2

h post-dosing. Data are presented as mean + SD. (*) Significantly different from control

(n=4 pools of liver or lungs of 4 mice per pool, p<0.05).

Lung Liver0

5000

10000

15000am

ol [α

32P

] dA

TP in

corp

orat

ed/µ

g D

NA

SalineDMSO

*

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Conclusions

DMSO is an important laboratory solvent, and has found use in several pharmacological

actions. In this study, we show DMSO has a significant negative effect on NER activity in

treated liver after 2 hours, but not in treated lungs at the same timepoint. We have previously

shown the effects of exogenous chemicals on NER activity can at least partially be explained by

effects on specific NER proteins (Chapter 2 and Chapter 3). Considering how prevalent the use

of DMSO is in the laboratory, investigations into the molecular causes of this significant decrease

in NER activity are warranted.

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