dna oxidation and base excision repair in lung and …
TRANSCRIPT
DNA OXIDATION AND BASE EXCISION REPAIR IN LUNG AND
LIVER OF 4-(METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-
BUTANONE TREATED MICE
by
Neeraj Gupta
A thesis submitted to the Department of Pharmacology and Toxicology
In conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
(April, 2011)
Copyright © Neeraj Gupta, 2011
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Abstract
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary
carcinogen found in unburned tobacco and tobacco smoke. To exert its carcinogenic effect, NNK
is metabolically activated to reactive intermediates that can damage DNA by alkylation or
pyridyloxobutylation. NNK also has the ability to induce DNA oxidation and alter DNA repair
activities that can result in deficient repair and potentially exacerbate carcinogenesis. Base
excision repair (BER) is a ubiquitous DNA repair system that mainly repairs oxidative DNA
damage. The goal of this study was to determine the effect of NNK on DNA oxidation status and
BER activity in A/J mouse lung and liver. Female mice were treated with 10 mol of NNK i.p.
and lung and liver were isolated 1, 2 and 24 hours post administration. DNA was isolated from
lung and liver, and the formation of 8-hydroxydeoxyguanosine (8-OHdG, a biomarker of DNA
oxidation) was assessed by high-performance liquid chromatography with electrochemical
detection. At 1, 2 and 24 hours in both murine lung and liver, there was no statistically
significant difference in 8-OHdG levels (n = 4, P > 0.05) between control and NNK-treated mice.
To assess BER, cell-free whole tissue nuclear protein extracts from liver and lung were prepared
and incubated with a plasmid substrate containing oxidative DNA damage. In vivo treatment
with NNK did not alter BER activity in lung or liver compared to control mice (n=3 or 4, P >
0.05). These experiments indicate that acute treatment with a tumourigenic dose of NNK does
not significantly stimulate oxidative DNA damage or significantly alter BER activity in murine
lung and liver.
Keywords: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, oxidative DNA damage, base
excision repair
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Co-Authorship
This research was conducted by the candidate, Neeraj Gupta, under the supervision of Dr.
Thomas E. Massey.
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Acknowledgements
First and foremost, I would like to thank my supervisor, Dr. Thomas Massey, for his
guidance and support during this project. I would also like to thank current and former lab
members for their advice, assistance and friendship: Ms. Sandra Graham, Mr. Mason Curtis, Dr.
Pamela Brown, and Dr. Katherine Guindon. Additionally, I would like to thank the faculty,
administrative staff and students of the Department of Pharmacology and Toxicology for their
assistance and advice. I would like to thank my thesis committee members, Dr. Louise Winn and
Dr. James Reynolds, for giving this project additional direction and focus. I would also like to
thank Dr. Donald Maurice for his helpful advice on preparing plasmid.
This research would not be possible without the financial support from the Canadian
Institute of Health Research, the Department of Pharmacology and Toxicology, and the School of
Graduate Studies and Research.
Finally I would especially like to thank my family for their constant love, support and
encouragement during the entire course of this project.
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Table of Contents
Abstract ............................................................................................................................................ ii
Co-Authorship ................................................................................................................................ iii
Acknowledgements ......................................................................................................................... iv
Table of Contents ............................................................................................................................. v
List of Figures ................................................................................................................................ vii
List of Abbreviations .................................................................................................................... viii
Chapter 1 GENERAL INTRODUCTION ....................................................................................... 1
1.1 Statement of research problem ............................................................................................... 1
1.2 Multi-stage process of carcinogenesis ................................................................................... 1
1.2.1 Tumour initiation ............................................................................................................ 2
1.2.2 Tumour promotion .......................................................................................................... 3
1.2.3 Tumour progression ........................................................................................................ 4
1.3 Lung cancer and smoking ...................................................................................................... 4
1.4 Human lung cancer classification .......................................................................................... 7
1.4.1 Modelling lung cancer in mouse ..................................................................................... 8
1.5 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) ...................................................... 9
1.5.1 NNK and lung cancer .................................................................................................... 10
1.5.2 Metabolic pathways of NNK ........................................................................................ 14
1.5.3 Cytochrome P450 enzymes and bioactivation of NNK ................................................ 17
1.5.4 NNK and DNA adducts ................................................................................................ 17
1.6 Base excision repair and oxidative stress ............................................................................. 19
1.6.1 Genetic damage induced by ROS ................................................................................. 22
1.6.2 NNK and DNA oxidation ............................................................................................. 23
1.7 Base excision repair (BER) pathway ................................................................................... 25
1.7.1 Decision between short and long patch repair in BER ................................................. 29
1.7.2 BER and carcinogenicity .............................................................................................. 30
1.8 NNK and DNA repair systems ............................................................................................ 32
1.9 Research hypothesis and objectives ..................................................................................... 34
Chapter 2 MATERIALS AND METHODS .................................................................................. 35
2.1 Reagents ............................................................................................................................... 35
2.2 Animal Treatments............................................................................................................... 35
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2.3 Isolation of DNA from lung and liver .................................................................................. 36
2.4 DNA digestion ..................................................................................................................... 37
2.5 Determination of 8-OHdG and 2’-dG levels by high-performance liquid chromatography
with electrochemical detection .................................................................................................. 38
2.6 Cell-free whole tissue nuclear protein extract preparation .................................................. 38
2.7 Preparation of 8-OHdG adducted plasmid ........................................................................... 40
2.8 In vitro base excision repair assay ....................................................................................... 41
2.9 Data analysis ........................................................................................................................ 44
Chapter 3 RESULTS...................................................................................................................... 45
3.1 Levels of 8-OHdG following in vivo treatment with NNK.................................................. 45
3.2 Characterization of 8-OHdG adducted plasmid DNA substrate for in vitro repair assay .... 48
3.3 Optimization of nuclear protein extract amount for in vitro repair assay ............................ 51
3.4 Optimization of incubation time for in vitro repair assay .................................................... 51
3.5 Effect of in vivo treatment of mice with NNK on BER in lung and liver ............................ 53
Chapter 4 GENERAL DISCUSSION ............................................................................................ 57
4.1 In vivo treatment with NNK does not induce oxidative DNA damage ................................ 57
4.2 In vivo treatment with NNK does not alter overall BER activity ........................................ 60
4.3 Conclusions .......................................................................................................................... 65
4.4 Future directions .................................................................................................................. 65
REFERENCES .............................................................................................................................. 68
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List of Figures
Figure 1.1 Formation of tobacco specific nitrosamines (TSNA) from major (nicotine) and minor
(nornicotine, anabasine and anatabine) tobacco alkaloid precursors ..................................... 11
Figure 1.2 Pathways of NNK metabolism ..................................................................................... 15
Figure 1.3 Proposed mechanism of generation of ROS by uncoupling of CYP during NNK
metabolism ............................................................................................................................. 26
Figure 1.4 Mechanisms of BER pathway depicting short patch and long patch repair ................. 27
Figure 2.1 Chromatograms demonstrating resolution of 2’-dG and 8-OHdG, isolated from lung
DNA of mice following in vivo treatment with saline ........................................................... 39
Figure 2.2 Schematic of in vitro DNA repair synthesis assay on damaged plasmid DNA by
nuclear extracts isolated from whole tissue. .......................................................................... 42
Figure 2.3 UV-illuminated ethidium bromide stained agarose gel of linearized plasmid DNA and
phosphor image demonstrating the ability of cell-free nuclear protein extracts to catalyze in
vitro DNA repair synthesis .................................................................................................... 43
Figure 3.1 Effect of in vivo treatment with NNK on 8-OHdG levels in lung DNA ...................... 46
Figure 3.2 Effect of in vivo treatment with NNK on 8-OHdG levels in liver DNA ...................... 47
Figure 3.3 Characteristics of 8-OHdG adducted pBluescript SK+ plasmid by photo-oxidation of
methylene blue ....................................................................................................................... 49
Figure 3.4 Optimization of 8-OHdG adducted plasmid for in vitro BER assay ............................ 50
Figure 3.5 Optimization of protein amount for in vitro BER assay ............................................... 52
Figure 3.6 Optimization of incubation time for in vitro BER assay .............................................. 54
Figure 3.7 Effect of in vivo treatment with NNK on in vitro BER in mouse lung extracts towards
8-OHdG adducted plasmid .................................................................................................... 55
Figure 3.8 Effect of in vivo treatment with NNK on in vitro BER in mouse liver extracts towards
8-OHdG adducted plasmid .................................................................................................... 56
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List of Abbreviations
A adenine
AC adenocarcinoma
ad libitum free feeding
ADP adenosine diphosphate
amol atto (10-18
) moles
AP apurinic/apyrimidinic site
BER base excision repair
bp basepair
BP benzo[ ]pyrene
C cytosine
oC degree(s) Celsius
Ci curie(s)
cm centimetre(s)
CYP cytochrome P450
dATP deoxyadenosine triphosphate
dCTP deoxycytidine triphosphate
DFO deferoxamine mesylate
2’-dG 2’-deoxyguanosine
dGTP deoxyguanosine triphosphate
DNA deoxyribonucleic acid
5’dRP 5’-deoxyribose-5-phosphate
dTTP deoxythymidine triphosphate
EDTA ethylenediaminetetraacetic acid
ix
e.g. exempli gratia (for example)
EtBr ethidium bromide
Fe iron
Fen1 flap endonuclease I
g gram(s)
g relative centrifugal force
G guanine
GC-MS gas chromatography-mass spectrometry
HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
H2O2 hydrogen peroxide
HPB 4-hydroxy-1-(3-pyridyl)-1-butanone
HPLC-ECD high-performance liquid chromatography with
electrochemical detection
hr hour(s)
i.e. id est (that is)
in vitro outside the living body
in vivo inside the living body
i.p. intraperitoneal
iso-NNAC 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid
iso-NNAL 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol
kb kilobasepair
kg kilogram(s)
K-ras Kirsten-ras oncogene
Lig III DNA ligase III
A microampere(s)
x
Ci microcurie(s)
g microgram(s)
L microlitre(s)
M micromolar
mol micromole(s)
M molar
min minute(s)
mg milligram(s)
7-mG 7-methylguanine
MGMT O6-methylguanine-DNA methyltransferase
mL millilitre(s)
mm millimetre(s)
mM millimolar
mRNA messenger ribonucleic acid
MUTYH adenine-DNA glycosylase
mV millivolt(s)
nA nanoampere(s)
NAB N'-nitrosoanabasine
NAT N'-nitrosoanatabine
NER nucleotide excision repair
ng nanogram(s)
nm nanometre(s)
NMWL nominal molecular weight limit
NNA 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanal
NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
xi
NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
NNN N'-nitrosonornicotine
NO2- nitrite
NO3- nitrate
NSCLC non-small cell lung cancer
1O2 singlet oxygen
O2- superoxide radical anion
OGG1 8-oxoguanine glycosylase 1
OH hydroxyl radicals
8-OHdG 8-hydroxydeoxyguanosine
O6-mG O
6-methylguanine
O4-mT O
4-methylthymine
OPB 4-oxo-4-(3-pyridyl)butanal
OPBA 4-oxo-4-(3-pyridyl)butyric acid
32P phosphorous-32
P < probability less than
P > probability greater than
p16 p16ink4a
tumour suppressor gene
PCNA proliferating cell nuclear antigen
POB pyridyloxobutyl
Pol DNA polymerase beta
RF-C replication factor C
RGS regulator of G-protein signaling
RNA ribonucleic acid
ROS reactive oxygen species
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SCC squamous cell cancer
SCLC small cell lung cancer
SD standard deviation
SDS sodium dodecyl sulfate
SEM standard error of the mean
T thymine
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
TSNA tobacco specific nitrosamines
U enzymatic unit(s)
UV ultraviolet
v/v volume per volume
v/v/v volume per volume per volume
W watt(s)
WHO World Health Organization
x times
XRCC1 X-ray repair cross-complementing protein
2xYT yeast extract tryptone
~ approximately
> greater than
< less than
plus or minus
1
Chapter 1
GENERAL INTRODUCTION
1.1 Statement of research problem
Lung cancer is currently the leading cause of cancer death worldwide. The widespread
use of cigarettes and tobacco products was responsible for the rise in lung cancer incidence
during the 20th century. A potent pulmonary carcinogen that most likely plays a major role in
tobacco-induced lung cancer is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is
found in cigarettes and tobacco smoke and a substantial amount of experimental data has shown
NNK to induce lung tumours in a variety of laboratory animals. Since cigarettes are widely
available and consumed by so many, understanding the mechanism of action of major tobacco-
specific carcinogens like NNK, is critical for developing novel preventative or treatment therapies
for tobacco-induced lung cancer. The current goal of the research is to investigate the mechanism
of action of NNK and in particular; 1) to assess the extent of DNA oxidation in murine lung and
liver after in vivo treatment with NNK; 2) to determine if in vivo treatment with NNK can alter
the activity of base excision repair (BER), a major DNA repair system.
1.2 Multi-stage process of carcinogenesis
Cancer can be defined as the loss of regulatory mechanisms that manage homeostasis and
normal growth of cells. The transformation of a healthy cell into a cancerous one is dependent on
the accumulation of a number of genetic and epigenetic changes. Abnormalities in the genome
and epigenome (changes in gene expression caused by mechanisms that do not involve the DNA
sequence) can alter the way a cell functions in its microenvironment and can result in cancer.
These changes in cell function, dubbed the hallmarks of cancer, include upregulation of growth
signals and receptors, insensitivity to growth inhibition, evasion of apoptosis, limitless replication
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potential, sustained angiogenesis, tissue invasion and metastasis (Hanahan and Weinberg, 2000).
The development of cancer typically follows a multistage process and can be described by a
model involving three stages; initiation, promotion and progression.
1.2.1 Tumour initiation
The initiation stage of carcinogenesis involves the transformation of a healthy cell into a
preneoplastic cell. During initiation, the molecular structure of DNA is irreversibly altered by an
initiating agent (either chemical, physical or biological in nature) in a dose dependent manner
(Pitot et al., 1981). The resulting products are initiated cells that are difficult to detect due to
similar histology and phenotype to normal cells. Initiating agents and their metabolites introduce
non-lethal damage to the genome and/or epigenome by covalently binding to DNA, inducing
strand breaks, distorting the helix structure or modifying components such as nitrogenous bases
or sugar-phosphate backbone (Pitot et al., 1981). It is well established that the DNA binding
capacity of a carcinogen is correlated to its carcinogenic potency. Initiated cells require one
round of DNA replication for the damage to be permanent (Bertram, 2000). An initiated cell will
continue to exist and replicate unless the DNA damage is repaired or the cell is eliminated.
Abnormal alterations in DNA can be attributed to endogenous factors or exogenous
agents such as UV light, ionizing radiation, and chemical carcinogens. Endogenous damage to
DNA can arise from reactive molecular byproducts from normal cellular processes. Metabolism
of oxygen during aerobic respiration is a major source of reactive oxygen species that can oxidize
DNA and cause genetic mutations (Klaunig and Kamendulis, 2004). DNA also undergoes
frequent spontaneous damage due to the inherent instability of the DNA helix structure. For
example, in a given cell, depurination occurs 104 times a day and is caused by the breakage of the
N-glycosidic bond connecting nitrogenous base to deoxyribose sugar (Bertram, 2000). Apurinic
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sites in DNA can result in random base insertion, potentially giving rise to mutations during
replication or transcription.
Chemicals are of major importance in causing damage to DNA during the initiation stage
of carcinogenesis. It is estimated that over 80% of all cancer deaths in industrialized nations are
attributed to exogenous chemical factors found in diet, tobacco, alcohol, and occupational hazards
(Luch, 2005). Most chemical carcinogens are not themselves directly carcinogenic. A few
carcinogens like ethylene oxide, bis(chloromethyl) ether and nitrogen mustard gas are considered
“direct carcinogens” as they can directly interact with biological macromolecules without
metabolic activation (Luch, 2005). Most chemical carcinogens require in vivo metabolic
activation to form reactive electrophilic intermediates that covalently bind to nucleophilic
macromolecules including DNA, RNA and proteins (Heidelberger, 1977; Luch, 2005; Miller and
Miller, 1981). These chemicals include aromatic and heterocyclic amines, aminoazo dyes,
polyaromatic hydrocarbons, N-nitrosamines and halogenated olefins (Luch, 2005).
1.2.2 Tumour promotion
The promotion stage of carcinogenesis involves the selective clonal expansion of the
initiated cell by long term exposure to promoting agents. This stage is considered to be reversible
and dose-dependent (Klaunig and Kamendulis, 2004). Compared to initiation, direct changes to
DNA structure or genetic code are not observed during promotion (Pitot et al., 1981). Promoting
agents (including hormones, drugs, metabolites or other chemicals) bind to surface, cytoplasmic
or nuclear receptors of the initiated cell, induce changes in transcription and alter gene expression
(Pitot et al., 1981). For example, chemical compounds such as benzo[a]pyrene (BP) and 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) can act as tumour promoters through arylhydrocarbon
receptor-mediated signal transduction. Both chemicals can bind arylhydrocarbon receptors in the
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cytoplasm, dimerize and translocate to the nucleus where the complex bind DNA response
elements and regulate expression of several key proteins. Genes that promote growth and
differentiation are upregulated while genes that promote apoptosis are downregulated (Luch,
2005). BP and TCDD can also alter expression patterns of cell cycle proteins such as cyclins via
arylhydrocarbon receptors. Taken together, transcriptional changes in factors that control growth,
differentiation, apoptosis and cell cycle ultimately result in the clonal expansion of the initiated
cells.
1.2.3 Tumour progression
Progression is the final stage of carcinogenesis and occurs when the preneoplastic cells
are converted into cancer cells. The progression stage is irreversible and cells undergo karyotypic
change characterized by aneuploidy and chromosomal instability that can be observed by light
microscopy (Pitot et al., 1981). A loss of genetic integrity results in increased growth rate,
increased invasiveness, and changes in biochemical and morphological characteristics that drives
formation of the primary tumour.
1.3 Lung cancer and smoking
Globally lung cancer is the leading cause of cancer death among men and second leading
cause of cancer death among women, accounting for 1.5 million new cases and 1.3 million deaths
annually (World Health Organization, 2010). In Canada, lung cancer is the leading cause of
cancer-related death and the second most diagnosed cancer among men and women (Canadian
Cancer Society, 2010). Lung cancer was diagnosed in 24, 000 Canadians and claimed over 20,
000 lives in 2010 (Canadian Cancer Society, 2010). Once diagnosed, the 5-year prognosis of
lung cancer is poor with only 10 to 15% of patients surviving (Sekido et al., 1998; Wood et al.,
2004).
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An extensive amount of epidemiologic data collected in the past 50 years has clearly
established the link between cigarette smoking and lung cancer. It is estimated that smoking and
exposure to tobacco smoke cause 85 to 90% of lung cancer cases in men and 75 to 80% of lung
cancer cases in women (Hecht, 1999; Subramanian and Govindan, 2007). Cigarette smoke is
chemically complex and currently contains over 4000 chemicals, 50 that are known carcinogens
including polycyclic aromatic hydrocarbons, aromatic amines and tobacco specific nitrosamines
(Health Canada, 2010). The particulate phase of cigarette smoke contains over 3500 compounds
including carcinogens (Hecht, 1999). Unburned tobacco contains at least 16 known carcinogens
(Hecht, 2003) and smokeless tobacco products that are rising in popularity and are perceived as a
safer alternative to cigarettes, contain 30 known carcinogens (Boffetta et al., 2008). It should be
noted that cigarettes and tobacco smoke can also increase the risk for other serious health
problems including heart disease, stroke and chronic lung disease. They have also been linked to
cancer of the larynx, oesophagus, mouth, bladder, cervix, pancreas, and kidneys (World Health
Organization, 2010).
Since the mid 1950s there has been a decline in smoking prevalence in Canada and other
developed countries like the US and UK (Giovino, 2002). In 1985, 35% of Canadians aged 15
and older were considered current smokers, but by 2008 the rate dropped to 15% (Health Canada,
2010). The decline in smoking prevalence in Canada is due to a combination of factors including
tax increases, advertising restrictions, prominent health warnings, smoking restrictions in public
places and educational campaigns over the past 60 years (Cunningham, 1996).
Even though smoking rates have declined, in recent years the drop has started to level off
suggesting that a proportion of the population (approximately 4.9 million Canadians) will
continue to smoke cigarettes despite increased efforts to curb the behaviour (Health Canada,
2010). These individuals are at an increased risk of developing lung cancer compared to former
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smokers and never-smokers (Sekido et al., 1998). In addition, individuals who are never-smokers
but are exposed to tobacco smoke from direct smoking exhalation (mainstream smoke) or an idle
burning cigarette (sidestream smoke) have an increase risk of lung cancer (Hecht, 1999). Never-
smokers are also exposed to carcinogens and particulate matter from cigarette smoke that have
been trapped in hair, skin, and fabrics (Health Canada, 2010). Former smokers are also at risk of
lung cancer as smoking cessation reduces the risk, but cancer risk will never return to levels of
never-smokers (Sekido et al., 1998). Although the decline in smoking rates in developed
countries like Canada are encouraging, the global statistics offer a stark reality of tobacco use.
The World Health Organization (WHO) has termed the global use of cigarettes an
epidemic. There are currently more than 1 billion smokers with nearly 80% of them living in low-
and middle-income countries (World Health Organization, 2010). Global tobacco consumption is
currently increasing 3.4% each year. Cigarettes are widely available and accessible with 5.5
trillion cigarettes being produced and consumed annually (World Health Organization, 2010).
More than 5 million people die per year from tobacco use and by 2030 this number is expected to
rise to 8 million people (World Health Organization, 2010). Hence, tobacco use is the leading
cause of preventable premature death. Large epidemics of lung cancer are expected in China and
India where approximately 40% of all smokers reside (Hecht, 1999). In China it is estimated that
several million cases of lung cancer will arise by 2050 based on current smoking prevalence and
population growth (Liu et al., 1998).
Although smoking and exposure to tobacco smoke are responsible for the majority of
lung cancer cases, genetic variation likely plays a role in pulmonary carcinogenesis. Only 20% of
smokers actually develop lung cancer, suggesting that some individuals may have genetic
susceptibility towards carcinogenesis (Wei and Spitz, 1997). A likely gene candidate that may
contribute to familial lung cancer and lung cancer susceptibility is RGS17 found on chromosome
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6q23-25 (You et al., 2009). The RGS17 protein is a member of the regulator of G-protein
signaling (RGS) family that can indirectly enhance or inhibit the activities of downstream
proteins (e.g. adenylate cyclase) and secondary messengers (e.g. cyclic AMP) (You et al., 2009).
Lung tumours from cancer patients have been shown to accumulate high levels of RGS17 mRNA
transcripts compared to normal lung tissue, suggesting a potential role in carcinogenesis (You et
al., 2009). Other factors such as genetic variation in an individual’s ability to metabolize
carcinogens into genotoxic intermediates, detoxify those intermediates, and repair damaged DNA
can also determine susceptibility to carcinogenesis. Polymorphisms in genes encoding proteins
that participate in such reactions are associated with lung cancer risk (Schwartz, 2004).
1.4 Human lung cancer classification
The lung is comprised of over 40 unique cell types that perform a variety of functions
such as gas exchange at the alveoli, blood-barrier at the alveolar junction, immunity against
pathogens and removal of particulate matter from the respiratory tract. In 98% of lung cancer
cases the epithelial cells lining the bronchiolar airway of the lung are transformed into cancerous
cells called carcinomas (Travis et al., 2004). Lung cancer has heterogeneous tumour biology
compared to other common cancers such as breast, colon and prostate (Borczuk et al., 2003). It is
histologically categorized into two major classes; small cell lung cancer (SCLC) and non-small
cell lung cancer (NSCLC). Other rare tumour types include sarcomatoid carcinoma, salivary
gland tumours and carcinoid tumours (Travis et al., 2004).
SCLC represent 15 to 25% of all lung cancer cases with tumours centrally located in
larger airways such as the primary or secondary bronchi (Sekido et al., 1998). Compared to
normal cells of the bronchi, SCLC cells are small and have scant cytoplasm, ill defined cell
borders, finely granular chromatin, and inconspicuous or absent nucleoli (Jackman and Johnson,
8
2005). SCLC is an aggressive form of cancer that has rapid replication rate and undergoes early
metastasis (Wood et al., 2004). Although most individuals’ SCLC initially respond to
chemotherapy and radiation therapy, prognosis is poor with 3% survival over 3 years (Chute et
al., 1999). SCLC occurs in individuals with smoking history and has a strong association with
tobacco use (Hanrahan and Glisson, 2010).
Non-small cell lung cancer (NSCLC) consist of 75 to 85% of lung cancer cases and are
further divided into major subtypes; squamous cell carcinoma (SCC) (30% of all lung cancer
cases), adenocarcinoma (AC) (40%) and large cell carcinoma (9%) (Sekido et al., 1998).
Development of SCC and AC are commonly associated with smoking. SCC originate in the
central bronchus while AC are found in peripheral lung, in distal airways and alveoli of the lung
(Corvalan and Wistuba, 2010).
1.4.1 Modelling lung cancer in mouse
Lung cancers in both mice and humans share morphologic, histogenic and molecular
features. Some strains of mice are particularly more sensitive to chemical induction of lung
cancer from tobacco specific carcinogens or cigarette smoke (Meuwissen and Berns, 2005). The
A/J and SWR mice are the most sensitive mouse strains, while others range from intermediate
sensitivity (O20, BALB/c) to more resistant (CBA, C3H) and highly resistant (C57BL/6 and
DBA) (Meuwissen and Berns, 2005). Several pulmonary adenoma susceptibility loci have been
mapped to oncogenes and tumour suppressor genes and can be attributed to sensitivity between
strains of mice. For example, polymorphisms in intron 2 of the K-ras oncogene are found in most
susceptible strains of mice and are thought to induce major increases in K-ras expression as well
as alter sensitivity to respiratory carcinogens (You et al., 1993; Chen et al., 1994). The K-ras
protein is a GTPase that activates and recruits other proteins in signal transduction pathways to
9
facilitate growth of cells. Polymorphisms also occur at higher frequency in sensitive mice at a
susceptibility locus on chromosome 4 that encodes the tumour suppressor gene p16ink4a
(p16)
(Zhang et al., 2002; Herzog et al., 1999). The p16 protein is critical in regulating progression in
the cell cycle and in particular G1/S cell cycle transition. Mutations in K-ras and p16 gene have
also been well documented in human SCLC and NSCLC, suggesting development of lung
cancers between mice and humans could occur in a similar fashion (Sekido 1998).
The development of AC typically involves a series of morphologically distinct
preneoplastic changes (Horio et al., 1996). They include the sequential stages of hyperplasia,
adenoma, carcinoma in situ and finally metastatic carcinoma in both mice and humans (Stoner,
1998; Malkinson, 1998). In mouse models, the induction of lung tumours with chemical
carcinogens typically results in adenomas and ACs. One way in which mice and humans differ is
that malignant AC rarely metastasize in mouse models, as mice usually die from respiratory
distress caused by carcinoma masses in lung that obstructs gas exchange (Malkinson, 1998). The
cause of death in human AC patients is usually related to consequences of metastasis (Malkinson,
1998).
1.5 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
N-nitrosamines (referred to nitrosamines in this thesis) are present in many common
consumer items including tobacco, cosmetics, beer and bacon (Bertram, 2000). Over 200
nitrosamines and their carcinogenic activity have been confirmed in laboratory animals (Hecht,
1999). Among consumer products, cigarettes and smokeless tobacco contain levels of
carcinogenic nitrosamines 100 to 1000 times greater than other products intended for
consumption (Hecht, 2003). A major class of nitrosamines found in cigarettes are tobacco
10
specific nitrosamines (TSNA) (Figure 1.1). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a
nicotine derived nitrosaminoketone (NNK) and considerable research indicates that it is likely to
be an important contributor to tobacco-induced cancers. Among TSNA characterized, NNK,
NNN and NAT are found in greatest quantities in cigarettes and NNK, NNN and NNAL are the
most carcinogenic TSNA (Hecht, 1998; Hecht, 2003). Typical cigarettes contain 1 to 2 g of
NNK for every gram of unburned tobacco (Hecht, 2002). Every gram of commonly used
smokeless tobacco contains 1 to 5 g of NNK and NNN (Boffetta et al., 2008).
TSNA are formed from nitrosation of different alkaloids during curing, aging,
fermentation and burning of tobacco (Spiegelhalder and Bartsch, 1996; Brunnemann et al., 1996).
Nicotine, the addictive component of cigarettes, is the major alkaloid found in tobacco
comprising 85 to 90% of total alkaloid content (Boffetta et al., 2008). NNK, NNAL and NNN
are specifically formed when nicotine undergoes a nitrosation reaction. The formation of TSNA
is dependent on nitrosating agents, such as nitrites (NO2-), that can originate from different
sources. Nitrate (NO3-), a common constituent in tobacco, can be reduced to nitrite which can
then specifically react with tobacco alkaloids forming TSNA (Spiegelhalder and Bartsch, 1996).
The formation of some TSNA has also been shown to occur in vivo in humans and experimental
animals (Osterdahl and Slorach, 1988; Carmella et al., 1997).
1.5.1 NNK and lung cancer
NNK is a potent systemic lung carcinogen found in unburned tobacco and tobacco smoke
which induces lung tumours in commonly used rodent models regardless of route of
administration. Tumours of the respiratory system have been observed in multiple studies using
NNK-treated mice, rats and hamsters. For example, female A/J mice treated with a total dose of
11
Figure 1.1 Formation of tobacco specific nitrosamines (TSNA) from major (nicotine) and minor (nornicotine, anabasine and anatabine) tobacco
alkaloid precursors (modified from Hecht, 1998). Currently, 7 TSNA have been identified including 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB), N'-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanol (NNAL), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL), and 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso-NNAC).
With the exception of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanal (NNA), all have been detected in tobacco. Note, NNA has only been
synthesized and detected in vitro, and therefore technically is not a TSNA (Hecht et al., 1978; Hecht, 1998).
12
0.11 mmol of NNK i.p., induced an average of 36.7 lung tumours per mouse (Castonguay et al.,
1983). In contrast, a similar dose of NNN and NNAL induced only 1.6 and 26.3 lung tumours
per mouse respectively. Similarly, among strain A mice treated with different TSNA, NNK
induced more lung adenomas per mouse than either NNN or NNA (Hecht et al., 1978). F344
rats treated subcutaneously with a total dose of 3.4 mmol NNK over 20 weeks developed tumours
in the nasal cavity, liver and lungs, while the same dose of NNN only gave rise to tumours in the
nasal cavity (Hecht et al., 1980). Additionally, Syrian golden hamsters treated with low dose of
NNK (5 mol) induced respiratory tract tumours in 6 of 20 animals treated (Hecht et al., 1983).
The accumulation of animal data indicate that NNK is one of the most potent pulmonary
carcinogens among TSNA identified.
NNK has organoselectivity for lung tissue in experimental animals, mainly inducing
adenomas and ACs regardless of route of administration (i.e. topically, intraperitoneally,
intravenously, subcutaneously, intravesically (into the urinary bladder), or administered through
drinking water) (Hoffmann et al., 2001; LaVoie et al., 1987; Prokopczyk et al., 1991; Lijinsky et
al., 1991). In addition to being a potent pulmonary carcinogen, treatment with NNK results in
cancers of other tissues and organs. Tumours of the nasal cavity, oral cavity and liver occur in a
variety of rodents treated with NNK (Hecht and Hoffmann, 1988; Hecht, 2003). Furthermore,
NNK and NNAL are the only known pancreatic carcinogens in unburned tobacco and tobacco
smoke. Male F344 rats treated with NNK or NNAL in drinking water significantly induced ACs
of acinar or ductal origin in the exocrine pancreas (Rivenson et al., 1988). Pancreatic lesions are
spontaneous in control animals and rarely observed (Hecht, 2003). NNK could also be
responsible for tobacco-induced cervix cancer. Levels of NNK and its metabolites were elevated
in the cervix of women who smoke (Prokopczyk et al., 1997). Cervical cells and subcellular
13
fractions of the cervix have the capacity to bioactivate NNK and could generate reactive
intermediates that damage DNA (Prokopczyk et al., 2001).
NNK’s capacity to specifically induce lung adenoma and AC may be important in
tobacco-induced lung cancers in humans. The tumours induced by NNK in rodent species are
morphologically and topographically similar to human bronchiolar and bronchioalveolar AC
(Wynder and Hoffmann, 1994). Among smokers, the incidence of AC has increased over the past
5 decades, surpassing SCC as the most common form of lung cancer. The analysis of lung cancer
cases from the late 1960s to the late 1980s has shown an increase in the prevalence and rate of
AC cases compared to SCC cases (Devesa et al., 1991; Auerbach and Garfinkel, 1991). Changes
in cigarette design and smoking behaviour have likely contributed to the shift in AC. The
introduction of filter tips to cigarettes traps the particulate phase of tobacco smoke but also
impedes the delivery of nicotine into the body. To compensate for decreased levels of nicotine,
cigarette users tend to inhale more deeply, and smoke more intensely to increase pulmonary
absorption of nicotine. This practice also disperses tobacco smoke carcinogens farther into the
respiratory tract and exposes the distal bronchi and bronchioalveolar junction to relatively high
levels of smoke carcinogens, including polyaromatic hydrocarbons, volatile aldehydes and
TSNA. Reduction of nicotine levels in low tar cigarettes has also resulted in similar changes in
smoking behaviour. Furthermore, the chemical components of cigarettes have been altered and
may have contributed to increased AC. Between 1959 and 1997, tobacco blends have
incorporated more air cured tobacco, resulting in cigarettes and tobacco products that have higher
nitrate and TSNA content, including NNK, while lowering the amount of polyaromatic
hydrocarbons (Hoffmann et al., 2001). More intense smoking, deeper inhalation and increased
TSNA content in cigarettes are considered to be major contributors in increasing lung AC among
smokers (Wynder and Hoffmann, 1994).
14
1.5.2 Metabolic pathways of NNK
The main NNK metabolism pathways are illustrated in Figure 1.2. To exert
carcinogenicity, NNK requires metabolic activation that generates reactive metabolites that can
adduct DNA. Bioactivation or metabolic activation refers to the process where an inactive
compound is converted into active metabolites. NNK undergoes major reactions including
carbonyl reduction, pyridine oxidation, and -hydroxylation. Denitrosation and ADP adduct
formation of NNK have only been observed in vitro using rat liver microsomes (Castonguay et
al., 1991; Peterson et al., 1994). The carbonyl reduction of NNK, catalyzed by 11 -
hydroxysteroid dehydrogenase, cytosolic carbonyl reductase and aldo-keto reductase, result in the
major metabolite NNAL (Maser, 2004). In general, the reduction of NNK to NNAL occurs in
many tissues including lung and liver. Upon NNK administration, NNAL is rapidly formed and
is the predominate metabolite in blood (Hecht, 1998). NNAL is not a final detoxified product,
since it can undergo bioactivation and has carcinogenic activity (Rivenson et al., 1988;
Castonguay et al., 1983). The reconversion of NNAL to NNK via oxidation does not occur as
extensively as reduction, however it may be of interest as it could play a role in NNAL
carcinogenicity.
The -hydroxylation reactions of NNK and NNAL are of major interest as they result in
reactive intermediates that can covalently bind DNA, RNA and proteins. Like most carcinogens,
NNK and its metabolites require metabolic activation to exert carcinogenic effect. The -
hydroxylation occurs on the methyl or methylene carbons that are adjacent to the N-nitroso group.
Hydroxylation of the methyl carbon of NNK forms 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-
1-butanone (1), that decomposes to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (2) and
formaldehyde. Compound (2) is highly reactive and can form bulky pyridyloxobutyl (POB)
adducts on DNA, RNA and protein. Compound (2) can also react with water forming 4-hydroxy-
15
Figure 1.2 Pathways of NNK metabolism. Metabolites I and II are detoxified products produced by pyridine oxidation. Metabolites III to VII are
endpoints of -hydroxylation and are generated by reactive metabolites that pyridyloxobutylate , methylate and pyridylhydroxybutylate
DNA (modified from Smith et al., 2003).
16
1-(3-pyridyl)-1-butanone (keto alcohol or HPB) (III). HPB can be quantified to determine the
extent of hydroxylation at NNK’s methyl carbon.
Like NNK, NNAL also undergoes similar -hydroxylation reactions to form electrophilic
metabolites. The α-hydroxylation of the methyl carbon of NNAL forms an intermediate (3),
which decomposes to formaldehyde and the DNA pyridylhydroxybutylating species, 4-hydroxy-
4-(3-pyridyl)-1-butanediazohydroxide (4). Compound (4) can react with water to form 4-
hydroxy-1-(3-pyridyl)-1-butanol (Diol) (VI) or cyclizes to 2-(3-pyridyl)tetrahydrofuran (pyridyl-
THF) (VII).
Hydroxylation of the methylene carbon of NNK results in the formation of 4-hydroxy-4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (5) that spontaneously decomposes to methane
diazohydroxide and 4-oxo-4-(3-pyridyl)butanal (keto aldehdye or OPB) (6). Methane
diazohydroxide is a methylating agent and interacts with DNA at several bases. OPB can be
trapped as a bisulfite adduct to quantify -methylene hydroxylation or undergo further oxidation
to form 4-oxo-4-(3-pyridyl)butyric acid (IV) (keto acid of OPBA). Hydroxylation at the
methylene carbon of NNAL (7), ultimately yields the methylating species, methane
diazohydroxide (8) and a hydroxy aldehyde which can cyclize to 5-hydroxy-2-(3-
pyridyl)tetrahydrofuran (lactol) and could be further oxidized to 4-hydroxy-4-(3-pyridyl)butyric
acid (hydroxy acid) (V).
The major pathway of detoxification of NNK and NNAL occurs via pyridine oxidation.
Pyridine N-oxidation of NNK and NNAL generates 4-(methylnitrosamino)-1-(3-pyridyl-N-
oxide)-1-butanone (NNK-N-oxide) (I) and 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-
butanol (NNAL-N-oxide) (II) respectively. These N-oxide products are readily excreted through
the urine. Glucuronidation of NNAL is another important detoxification route and can occur at
the carbinol or the pyridine nitrogen of NNAL (Not shown in Figure 1.2). In addition O-
17
glucuronidation of compound 2 has been observed in rats and is a potential detoxified product
(Murphy et al., 1995).
1.5.3 Cytochrome P450 enzymes and bioactivation of NNK
The metabolic activation of NNK is catalyzed mainly by cytochrome P450 (CYP)
enzymes. In particular, the CYP1, CYP2 and CYP3 families of enzyme are primarily responsible
for catalyzing oxidation of xenobiotics, generating polar metabolites that can either be excreted or
remain within the body for further biotransformation. The liver is the major site of xenobiotic
metabolism, containing an abundance of CYP enzymes that are easily accessible for study. The
respiratory tract also contains CYPs and can be a site of metabolism for therapeutics and
xenobiotics that are inhaled or in the systemic circulation. The pharmacological and toxicological
effects of agents depend on the lung’s ability to activate or deactivate these agents via
biotransformation and in fact, the carcinogenicity of NNK depends on metabolic activation in the
respiratory system. Although human lung tissue contains similar hepatic CYPs involved in
xenobiotic metabolism, low level expression of pulmonary CYPs make it difficult to fully
characterize their identity and absolute levels. The levels of pulmonary CYPs are not uniform
throughout the lung and may vary depending on cell type and region. Enzymes that likely play a
major role in NNK hydroxylation in human lung and liver include CYP1A1, 1A2, 2A6, 2A13 and
2B6 (Nademanee, 1992; Smith et al., 1995; Smith et al., 1996; Patten et al., 1996; Su et al., 2000;
Jalas et al., 2003; Jalas et al., 2005).
1.5.4 NNK and DNA adducts
The metabolic activation of NNK and NNAL results in reactive metabolites that can
methylate and pyridyloxobutylate DNA. The methane diazohydroxide metabolite generated from
NNK metabolism, methylates DNA at several bases producing 7-methylguanine (7-mG), O6-
18
methylguanine (O6-mG), and O
4-methylthymine (O
4-mT) (Hecht, 1998). NNK-induced
pyridyloxobutyl (POB) adducts are bulky DNA adducts and are detected at the N7-, N
2- and O
6-
positions of guanine and O2- positions of cytosine and thymine (Jalas et al., 2005). The extent of
NNK-induced DNA methylation and POB adduction are dependent on a number of factors
including the site of hydroxylation (the methyl or methylene carbon of NNK), levels of
bioactivation and detoxification, and ability to repair DNA adducts. NNK’s ability to induce both
methyl and POB adducts may be important for carcinogenicity as most other nitrosamines just
induce one type of DNA damage. Treatment with either NNN or N-nitrosodimethylamine, agents
that can only pyridyloxobutylate or methylate DNA respectively, have lower pulmonary
carcinogenicity compared to NNK which induce both types of adducts (Hoffmann et al., 1984;
Hecht et al., 1986).
Methyl and POB DNA adducts have been detected in the lungs of human smokers. DNA
isolated from peripheral lung and tracheobronchial tissue from smokers had significantly higher
levels of POB adducts compared to non-smokers (Foiles et al., 1991; Holzle et al., 2007). The 7-
mG adduct was found at higher levels in smokers compared to non-smokers (Mustonen et al.,
1993). Similarly, O6-mG was also detected in the lungs of smokers (Wilson et al., 1989). It
should be noted that methyl and POB adducts detected in smokers could be due to reactive
metabolites generated from NNK, but also from other nitrosamines including NNN and N-
nitrosodimethylamine found in cigarettes and tobacco smoke (Hecht, 2003).
The O6-mG adduct may be of particular importance in pulmonary carcinogenesis in
mice. O6-mG is strongly correlated with tumour multiplicity in mice treated with NNK or
acetoxymethyl-methylnitrosamine, an analog of NNK that exclusively methylates DNA (Peterson
et al., 2001). In addition, O6-mG adducts that are persistent and escape DNA repair, induce
GA transition mutations in DNA. GGTGAT mutations have been found in codon 12 of the
19
K-ras oncogene in a high proportion of lung AC induced by NNK in A/J mice, suggesting a
possible role of O6-mG in tumour induction (Belinsky et al., 1989). Persistent POB adducts can
also induce mutations including GA and GT in codon 12 of K-ras gene in lung tumours of
NNK-treated mice (Ronai et al., 1993). Mutations at codons 12, 13 and 61 of K-ras can result in
uncontrolled growth and potentially develop into cancers. K-ras mutations are detected in 20 to
30% of human lung ACs and 15 to 20% of all non-small cell lung cancers, with frequent
mutations at codon 12 (Sekido et al., 2003). Lung AC from smokers frequently had more K-ras
mutations compared to ACs from former smokers and never-smokers (Slebos et al., 1991; Westra
et al., 1993).
NNK has also been shown to induce epigenetic changes by altering methylation status of
the p16 tumour suppressor gene. The specific methylation of cytosine residues in CG
dinucleotides that are found near the promoter region of genes, can silence expression via
chromatin remodelling. For example, in vivo treatment with NNK in rats induced
hypermethylation in CG dinucleotides in the promoter region of the p16 gene in 94% of lung AC
(Belinsky et al., 1998). Methylation of the promoter region was associated with reduced p16
protein levels that could impair cell cycle checkpoints and potentially contribute to carcinogenesis
(Belinsky et al., 1998). In vivo treatment with NNK is likely altering the activity of DNA
methyltransferases that are responsible for methylating cytosine residues in CG dinucleotides
(Belinsky, 1998).
1.6 Base excision repair and oxidative stress
An organism’s genome is vulnerable to a wide array of DNA damaging agents of
endogenous and exogenous origin. DNA repair systems are required to maintain genomic
integrity by identifying and removing damage from the DNA helix structure to prevent
20
mutagenicity and cytotoxicity. Several types of DNA repair have been identified including base
excision repair (BER), mismatch repair, nucleotide excision repair (NER) and direct reversal
repair. Each type of repair is responsible for fixing specific types of DNA damage.
The BER pathway recognizes and replaces a wide array of damaged bases from DNA. A
major function of BER is to remove nucleotides that have been oxidized by reactive oxygen
species (ROS). ROS is a collective term that can refer to a free radical oxygen species that have
an unpaired electron in its valence shell; it also refers to certain non-radicals that are either strong
oxidizing reagents or easily converted to free radicals (Wiseman and Halliwell, 1996). The major
ROS generated in biological systems include superoxide radical anions (O2-), hydroxyl radicals
( OH), peroxyl radicals (RO2 ), alkoxyl radicals (RO ), hydrogen peroxide (H2O2) and singlet
oxygen (1O2) (Wiseman and Halliwell, 1996). Production of ROS can arise from both
endogenous and exogenous sources. Different environmental agents including tobacco smoke
and air pollutants contain ROS and can generate ROS. Chemical carcinogens (such as
benzo[ ]pyrene, aflatoxin and benzene), ionizing radiation and UV light can also induce ROS
(Loft and Poulsen, 1996).
In biological systems, a major source of endogenous ROS is the complete reduction of
oxygen via the electron transport chain in mitochondria during cellular respiration.
Approximately 4 to 5% of molecular oxygen that undergoes one-electron reduction is converted
to superoxide radical anions (Klaunig and Kamendulis, 2004). Mitochondrial superoxide
dismutase, an antioxidant enzyme, can catalyze the conversion of excess superoxide radical
anions to hydrogen peroxide (Klaunig and Kamendulis, 2004) while catalase and glutathione
peroxidase convert hydrogen peroxide to water. Excess hydrogen peroxide can leak from the
21
mitochondria and into the cytosol (Yu, 1994). In the presence of ferrous iron, hydrogen peroxide
can decompose to hydroxyl radicals via the Fenton reaction:
Fe2+
+ H2O2 Fe3+
+ OH + OH-
Hydroxyl radicals are also formed in a similar manner when reduced forms of other transition
metals such as copper come into contact with hydrogen peroxide. Iron and copper are
sequestered in proteins including ferritin, transferrin, caeruloplasmin and metallothionein.
Oxidative stress has been shown to release transition metals from proteins and the released metals
can then participate in Fenton reactions to produce hydroxyl radicals (Halliwell and Aruoma,
1991).
Numerous enzymes that catalyze redox reactions can also contribute to the generation of
ROS. During CYP catalyzed metabolism, the generation of ROS can arise via different
processes. Redox cycling in the presence of molecular oxygen and uncoupling can result in the
generation of superoxide anion radical and hydrogen peroxide (Klaunig and Kamendulis, 2004).
Several CYPs have the ability to generate ROS during metabolism. CYP2E1 produces a
prolonged burst of ROS near the site of substrate oxidation during ethanol metabolism (Ekstrom
and Ingelman-Sundberg, 1989). Similarly, the metabolism of phenobarbital by CYP2B can
uncouple resulting in release of superoxide radical anion (Rice et al., 1994). Other enzymes can
also produce ROS when catalyzing reactions. Organelles derived from endoplasmic reticulum
called peroxisomes are responsible for breaking down fatty acid chains and contain high levels of
oxidase enzymes that generate hydrogen peroxide (Yu, 1994). The immune response can
generate excess ROS in a process called the respiratory burst. Activated macrophages and
hepatic Kupffer cells elicit rapid and transient increase in oxygen uptake to generate superoxide
anion radicals and hydrogen peroxide (Klaunig and Kamendulis, 2004). Release of ROS is
22
important for a host’s defense and results in localized tissue inflammation and induction of
genotoxicity and cytotoxicity in foreign microbes (Yu, 1994).
1.6.1 Genetic damage induced by ROS
Excess production of ROS can shift cells into an oxidative state with deleterious
consequences to DNA. In a given cell, an estimated 105 oxidative DNA lesions are formed per
day (Fraga et al., 1990). A wide variety of modifications on pyrimidine and purine bases as well
as the sugar-phosphate backbone in DNA have been observed in the oxidative state. In addition,
ROS can directly and indirectly result in single or double stranded DNA breaks and abasic sites.
Although over 20 base lesions have been identified, the most abundant lesion detected is the
hydroxylation of C-8 in guanine residues known as 8-hydroxydeoxyguanosine (8-OHdG) (Cooke
et al., 2003). Oxidation of guanine can also result in the ring opened product 2,6-diamino-4-
hydroxy-5-formamidopyrimidine. Other abundant lesions of DNA oxidation include 8-
oxoadenine, 2-hydroxyadenine, 5-hydroxyctosine, cytosine glycol and thymine glycol (Loft and
Poulsen, 1996). The type of oxidative lesion generated depends on the type of ROS interacting
with DNA. Superoxide anion radical and hydrogen peroxide have low reactivity with DNA
directly (Wiseman and Halliwell, 1996). However, hydroxyl radicals are highly reactive and can
induce almost any type of oxidized DNA modification (Loft and Poulsen, 1996). Singlet oxygen
generated by UV light has high specificity for guanine residues resulting in 8-OHdG.
If left unrepaired, oxidative DNA damage can induce miscoding mutations in DNA. The
vast majority of oxidative lesions result in base substitutions such as transition or transversion
mutations. A transition mutation is a point mutation that changes purine nucleotides into the
opposite purine (A G) or pyrimidine nucleotides into the opposite pyrimidine (C T).
Conversely, a transversion mutation refers to substitution of a pyrimidine for a purine or vice
23
versa. Base deletions and insertions are seldom observed as a result of oxidized DNA (Loft and
Poulsen, 1996).
Although ROS can modify all DNA bases, modifications of GC pairs are common in the
oxidized state. Hydroxyl radicals from ionizing radiation can induce potential GCCG or AT
base pair substitutions (Loft and Poulsen, 1996). Excess singlet oxygen can react with guanine
and induce GCAT base pair substitutions (McBride et al., 1992; Epe, 1991). ROS can also
interact with the deoxynucleotide pool resulting in oxidized nucleotides that can be
misincorporated in DNA during synthesis. For example, oxidized deoxyguanosine triphosphate
(dGTP) from the nucleotide pool can misincorporate opposite adenine (Cheng et al., 1992).
8-OHdG, a major oxidative lesion, is mutagenic in bacterial and mammalian cells that
can produce a GT transversion in oncogenes and tumour suppressor genes (Klaunig and
Kamendulis, 2004; Loft and Poulsen, 1996). 8-OHdG has been shown to induce mutations in
codon 12 of the K-ras oncogene in murine fibroblast cells (Jackson, 1994). Human fibroblasts
exposed to ROS generating systems induced GT and CA transversions at codon 249 of the
p53 tumour suppressor gene, an important mutation commonly seen in human tumours (Hussain
et al., 1994). Oxidative DNA damage has also been shown to induce transformation of normal
cells. Syrian hamster embryos treated with ROS generating systems developed 8-OHdG lesions
and underwent cellular transformation (Zhang et al., 2000). Antioxidants inhibited the formation
of 8-OHdG and cellular transformation in cells that were treated with ROS.
1.6.2 NNK and DNA oxidation
NNK has been shown to induce oxidative DNA damage in rodent species. F344 rats
treated with a single i.p. dose of NNK (20 mg/rat) had significantly higher 8-OHdG levels in lung
compared to controls (Chung and Xu, 1992). Treatment of female A/J mice with multiple doses
24
of NNK (0.25 mg/mouse, 3 times weekly for 3 weeks by gavage) caused significantly elevated
levels of 8-OHdG in lung and liver DNA (Chung and Xu, 1992). Similarly, female A/J mice
treated with multiple doses of NNK via gavage (0.5 mg/mouse, 3 times per week for 3 weeks) or
a single dose of NNK i.p. (2 mg/mouse) had elevated levels of 8-OHdG in lung DNA (Rosa et al.,
1998). The use of antioxidant agents in conjunction with NNK, also suggests NNK can induce
oxidative DNA damage. Concomitant treatment with (–)-epigallocatechin-3-gallate, an
antioxidant found in green teas, significantly reduced the number of lung adenomas per mouse
and 8-OHdG levels in lungs of A/J mice chronically treated with NNK via gavage (Xu et al.,
1992). In the same study, (–)-epigallocatechin-3-gallate exerted little effect on levels of O6-mG
in NNK-treated mice, indicating a potential role of oxidation in carcinogenesis (Xu et al., 1992).
Oxidation of DNA can also result in other forms of genetic damage, such as DNA strand
breaks. Human lung fibroblasts incubated with NNK resulted in a dose dependent increase in
DNA strand breaks (Weitberg and Corvese, 1993). Co-treatment with NNK and
hypoxanthine/xanthine oxidase, enzymes that generate ROS, further increased the levels of strand
breaks compared to NNK alone. Conversely, concomitant treatment with NNK and oxygen
radical scavengers such as mannitol, catalase and superoxide dismutase protected DNA from
strand breaks, suggesting NNK can potentiate its effects through ROS (Weitberg and Corvese,
1993). Treatment with NNK can also induce micronuclei in rat fibroblast skin cells in a dose
dependent manner. Formation of micronuclei occurs when DNA fails to divide equally between
daughter cells during cell division due to DNA strand breaks by exogenous agents such as
chemicals or radiation. Superoxide dismutase, an antioxidant enzyme, reduced micronuclei
formation in rat skin fibroblasts treated with NNK, suggesting involvement of NNK mediated
ROS production in DNA strand breakage (Kim and Wells, 1996).
25
The mechanism by which NNK induces oxidative DNA damage has not been fully
characterized. It is hypothesized that ROS can be generated during CYP metabolism of NNK.
During the first step of oxidation, iron(III) of the protoporphyrin-IX ring of a CYP creates a bond
with triplet oxygen. The iron(III)-oxygen complex is relatively stable; however superoxide anion
radicals (O2-) can be released, resulting in the breakdown or uncoupling of the oxidation
reactions catalyzed by CYP (Meunier et al., 2004). Additional superoxide anion radicals and
electrons can also be released during electron transfers in the CYP catalytic cycle. Microsomes
containing human CYP1A1, CYP1A2, CYP2B6 and CYP3A4 have been shown to produce
superoxide anion radicals during substrate metabolism (Puntarulo and Cederbaum, 1998). A
proposed mechanism of generation of ROS during CYP metabolism of NNK is outlined in Figure
1.3. This involves uncoupling during CYP metabolism of NNK, resulting in release of
superoxide anion radical. Although the mechanism has not been fully characterized, it appears
that NNK can generate ROS that can oxidize DNA.
1.7 Base excision repair (BER) pathway
Repair of oxidative damage to the genome by BER is carried out in three major steps:
recognition and excision of the damaged DNA bases, insertion of nucleotides and finally ligation
(Figure 1.4). The first step of BER is carried out by DNA glycosylases that recognize and
remove damaged or incorrect bases by hydrolyzing the N-glycosidic bond between base and
deoxyribose sugar (Christmann et al., 2003). In human cells, 11 different glycosylases have been
identified and characterized based on the types of modified bases removed (Christmann et al.,
2003). For example, 8-oxoguanine glycosylase 1 (OGG1) recognizes and releases 8-OHdG
paired with cytosine. Adenine-DNA glycosylase (MUTYH), identifies and removes 8-OHdG
lesions that have incorrectly mispaired with adenine during DNA replication in humans
26
Figure 1.3 Proposed mechanism of generation of ROS by uncoupling of CYP during NNK
metabolism (modified from Yang and Smith, 1996). During -hydroxylation of NNK, the initial
oxidation step is believed to form an -nitrosamino radical and superoxide anion at the CYP
iron(III) protoporphyrin-IX ring (initial radical species not shown in figure). In some cases both
radical species recombine, in a process called oxygen rebound to form the alcohol intermediate
(1). In other cases uncoupling could occur and release superoxide anion from the CYP active site.
The resulting -nitrosamino radical (2) could fragment into nitric oxide (3) and an imine (4).
Nitric oxide could then be oxidized to nitrite (NO2-) while the imine could be hydrolyzed to an
aldehyde (5) and methylamine (6).
27
Figure 1.4 Mechanisms of BER pathway depicting short patch and long patch repair (Christmann
et al., 2003). AP: apurinic/apyrimidinic; 5’dRP: 5’-deoxyribose-5-phosphate; Polβ, Polδ/ε:
DNA polymerases; Lig I, Lig III: DNA ligases; XRCC1: X-ray repair cross-complementing
protein 1; RF-C: replication factor C; Fen1: flap endonuclease 1; PCNA: proliferating cell
nuclear antigen.
28
(Cooke et al., 2003). Glycosylases have been further divided into 2 major subtypes; type I and
type II. Type I glycosylase identify and remove modified bases leaving an apurinic or
apyrimidinic site (AP) in DNA (Christmann et al., 2003). Type II glycosylases recognize and
remove damaged bases, but can also cleave the DNA phosphodiester backbone by endogenous
3’-endonuclease activity, giving rise to a single strand break (Christmann et al., 2003). In the
case of type I glycosylases, the phosphodiester backbone of DNA is cleaved by a separate AP
endonuclease that can result in 5’-deoxyribose-5-phosphate (5’dRP) and 3’-hydroxyl DNA
termini (Fortini and Dogliotti, 2007).
After removal of the damaged base, depending on the type of BER, a single nucleotide or
series of nucleotides will be inserted. In short patch repair, DNA polymerase (Pol ) exerts
lyase activity and catalyzes the release of the hemiacetal form of 5’dRP formed at the DNA
terminus of the AP site (Christmann et al., 2003). The resulting gap is filled by a single
nucleotide by Pol . DNA ligase III (Lig III) participates in short patch repair and seals the DNA
backbone (Christmann et al., 2003). Both Lig III and Pol have been shown to bind with the
scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1) for added stability and
function (Caldecott et al., 1994; Robertson et al., 2009). The net result of short patch repair is
replacement of the damaged base by a single nucleotide.
In long patch repair, Pol first inserts a single nucleotide at the AP site (Fortini and
Dogliotti, 2007). After Pol dissociation, further DNA synthesis is done by DNA polymerase
and , resulting in a longer repair patch (Christmann et al., 2003). Proliferating cell nuclear
antigen (PCNA) and replication factor C (RF-C) are recruited to stabilize DNA polymerase and
during nucleotide insertion (Stucki et al., 1998). The original 5’dRP flap structure is cleaved by
flap endonuclease I (FenI) (Klungland and Lindahl, 1997). PCNA forms a complex with FenI to
29
stimulate its endonuclease activity. Finally, DNA ligase I interacts with PCNA and Pol to seal
the DNA backbone (Srivastava et al., 1998; Christmann et al., 2003). The net result of long patch
repair is replacement of the damaged base by a series of nucleotides, 2 to 15 nucleotides in
length.
Other accessory proteins may have a role during BER. The addition of purified p53
protein to cell free extracts or reconstituted BER pathway stimulates BER in vitro. Conversely,
immunodepletion of p53 from cell extracts and deficient p53 cell lines have decreased BER
activity (Zhou et al., 2001; Achanta and Huang, 2004). The p53 protein has been shown to
directly bind and stabilize AP endonuclease and OGG1 glycosylase to enhance BER activity
(Achanta and Huang, 2004). Furthermore, p53 has been shown to stabilize the interaction
between Pol and DNA termini at AP sites (Zhou et al., 2001).
1.7.1 Decision between short and long patch repair in BER
The decision to undergo short or long patch BER is not fully characterized. Several
hypotheses exist attempting to elucidate the switch between short and long patch repair. The
capacity to cleave the 5’dRP terminus by Pol plays an important role in determining which
pathway BER performs. Pol can cleave the 5’dRP terminus in hemiacetal form and completes
BER via the short patch pathway. Oxidized or reduced AP sites, 3’-unsaturated aldehydes or 3’-
phosphates are resistant to Pol lyase activity and result in further processing through the long
patch repair pathway (Nakamura et al., 2000; Robertson et al., 2009; Christmann et al., 2003).
The type of DNA termini produced at the AP site depend on the oxidative DNA lesion present as
well as the glycosylase used for repair (Fortini et al., 1999). Other factors such as relative
concentration of ATP and the stage of the cell cycle may also play a role in determining whether
long or short patch BER proceeds (Petermann et al., 2003; Fortini and Dogliotti, 2007).
30
Although short and long patch BER most likely occur concurrently, the degree to which
each occurs has not been fully characterized. In vitro assays using cell free extracts suggest that
repair of 8-OHdG adducts is carried out predominantly by short patch repair (Dianov et al., 1998;
Fortini et al., 1999). However, in intact cells transfected with a plasmid containing an 8-OHdG
lesion, repair patches longer than one nucleotide were observed in 55 to 80% of plasmids,
suggesting that long patch repair of 8-OHdG also plays a role in intact cell systems (Sattler et al.,
2003).
1.7.2 BER and carcinogenicity
Deficient DNA repair can result in persistent DNA damage. If replication occurs before
the lesion is repaired, a permanent change in DNA may be introduced, which in turn could
potentially contribute to carcinogenesis. Knockout mice have been used to test the effects of
deficient BER on oxidative DNA damage and cancer susceptibility. Mice deficient in major
glycosylases had no drastic changes on phenotype and marginal effects on spontaneous
tumourigenesis. For example, OGG1 knockout mice developed a significantly higher number of
spontaneous lung adenocarcinoma (AC) and accumulated higher levels of 8-OHdG lesions in
liver compared to wild type mice (Sakumi et al., 2003). Mice deficient in MUTYH glycosylase
had increased incidence of spontaneous intestinal tumours (Sakamoto et al., 2007). Knockout
mice deficient in both OGG1 and MUTYH accumulated 8-OHdG lesions in lung and small
intestine, and had increased frequency of lung and ovarian tumours and lymphomas (Xie et al.,
2004; Russo et al., 2004). Although these studies have shown moderate effect on spontaneous
tumourigenesis, exposing knockout mice to pro-oxidants can exacerbate carcinogenesis.
Treatment with the pro-oxidant dimethylarsinic acid in OGG1-deficient mice resulted in a greater
number of lung tumours (Kinoshita et al., 2007). These types of experiments demonstrate that
BER can function as a protective mechanism during carcinogenesis.
31
Human cancers have also been analyzed for mutations in major proteins of the BER
pathway. Low OGG1 activity is associated with higher risk of some cancers. Squamous cell
carcinoma (SCC) of the head and neck was significantly associated with low OGG1 activity in
peripheral blood mononuclear cells (Paz-Elizur et al., 2006). Furthermore, leukocytes and
peripheral blood mononuclear cells obtained from lung cancer patients had reduced capacity to
repair 8-OHdG lesions in DNA constructs compared to healthy controls (Paz-Elizur et al., 2003).
Smokers with low OGG1 activity also had higher risk of developing lung cancer (Paz-Elizur et
al., 2003). Low OGG1 activity in cancer patients could be due to polymorphisms, differences in
expression levels, protein stability, post-translational modifications, or presence or absence of
natural activators and inhibitors.
Deficiency in the MUTYH glycosylase has been linked to rare forms of familial
colorectal cancer in humans. A British family with a history of colorectal cancer had a germline
biallelic missense mutation in the MUTYH gene that resulted in 2 variants, both with a single
amino acid substitution (David et al., 2007). An in vitro study using the human MUTYH variants
in E. coli deficient in MutY (homologue of human MUTYH), found decreased repair of 8-OHdG
mispaired with adenine compared to wild type MUTYH (Chmiel et al., 2003). Tumours from the
family contained GT transversion mutations in the tumour suppressor gene adenomatous
polyposis coli, likely due to the accumulation of oxidative DNA lesions that could not be repaired
as a result of deficient BER (David et al., 2007). Thus far, this is the only BER germ line
mutation to be directly implicated in carcinogenesis (Paz-Elizur et al., 2008).
While deficient BER can result in elevated oxidative DNA damage and incidence of
cancer, conflicting evidence suggests that the mere presence of oxidative DNA damage may not
be necessary or sufficient to induce carcinogenesis in vivo (Cooke et al., 2003). Elevated
oxidative DNA damage is seen in several human pathological conditions (e.g. rheumatoid
32
arthritis, systemic lupus, and neurodegenerative conditions) without increase in cancer
susceptibility (Cooke et al., 2003) and glycosylase knockout mice did not show increased
incidence of spontaneous carcinogenesis in several organ systems (Klungland et al., 1999;
Minowa et al., 2000; Paz-Elizur et al., 2008). Other DNA repair systems and redundant
glycosylases may be able to mitigate the effect of deficient BER. Mismatch repair and nucleotide
excision repair have the limited ability to repair 8-OHdG adducts which could act as a backup
repair mechanism in glycosylase knockout mice (Colussi et al., 2002; Reardon et al., 1997).
Epidemiologic studies have also shown polymorphisms of major BER genes (OGG1 glycosylase,
AP endonuclease, XRCC1, etc.) are weakly associated with cancer and may only play a minor
role in carcinogenesis in humans (Hung et al., 2005a; Hung et al., 2005b; Vogel et al., 2004; Paz-
Elizur et al., 2008). Further studies need to be conducted to elucidate the specific contribution of
BER and oxidative DNA damage in different organ systems of experimental animals and humans,
particularly during oxidative and carcinogenic challenge.
1.8 NNK and DNA repair systems
Studies on the effects of NNK on DNA repair systems are limited. To date, only 2 types
of DNA repair have been shown to be affected by treatment with NNK; nucleotide excision
repair (NER) and direct reversal repair. The NER pathway recognizes and removes bulky
chemical adducts that can induce helix distortion in DNA. NER is also responsible for repairing
cyclobutane pyrimidine dimers and photoproducts, two major kinds of lesions generated from UV
light (de Laat et al., 1999). Using an in vitro NER assay, mice treated with 10 mol of NNK i.p.,
had reduced activity for NER of POB adducted plasmid in extracts prepared from lung tissue
(Brown and Massey, 2009). Interestingly, the same NNK treatment increased NER in liver
extracts, suggesting inter-organ differences in susceptibility of NNK tumourigenicity.
33
Immunoblots showed an alteration in levels of key NER proteins, indicating that treatment with
NNK can also alter protein levels of NER.
Direct reversal repair is the direct removal of a specific DNA adduct by a specialized
class of proteins (Lindahl and Wood, 1999). For example, O6-methylguanine-DNA
methyltransferase (MGMT) irreversibly binds and removes alkyl groups from O6-alkylguanine.
In vivo treatment with NNK reduced MGMT activity in different lung cell types including type II
cells and non-ciliated bronchiolar epithelial cells (Belinsky et al., 1988). In addition, MGMT
activity was inhibited in cultured rat hepatocytes by OPB, a reactive intermediate generated by α-
hydroxylation of NNK (Liu et al., 1992). The decline of MGMT activity could be due to direct
adduct formation on the repair protein by reactive NNK metabolites (Mijal et al., 2004).
Treatment with NNK could also induce alkyl or POB adducts in DNA which can act as
competitive substrates during MGMT repair (Peterson et al., 2001). Some bulky POB adducts
like O6-[4-oxo-4-(3-pyridyl)-butyl]guanine can compete with O
6-alkylguanine for repair by
MGMT, resulting in reduced removal of O6-alkyl guanine by MGMT (Wang et al., 1997;
Peterson et al., 2001). These results demonstrate the possibility for NNK to alter the activity and
levels of protein of DNA repair pathways in lung and liver.
With respect to BER, in vivo treatment with NNK has been studied in female OGG1
knockout mice (Igarashi et al., 2009). This study found NNK-treated knockout mice had
significantly higher incidence and multiplicity of lung tumours compared to NNK-treated wild
type mice. Antioxidants inhibited enhanced lung carcinogenesis in NNK-treated knockouts and
NNK-treated wild type mice. These results suggest a potential role for an intact BER pathway in
protecting against NNK-induced pulmonary carcinogenesis.
34
1.9 Research hypothesis and objectives
Lung cancer is the leading cause of cancer death worldwide. Although cigarettes are by
far the largest cause of lung cancer cases among men and women, the mechanism by which
cigarettes induce pulmonary carcinogenesis are not fully characterized. A potent pulmonary
carcinogen found at significant levels in tobacco products is the tobacco specific nitrosamine,
NNK. Treatment with NNK induced lung ACs in a variety of animal species. The metabolism of
NNK by CYP enzymes results in the formation of reactive intermediates that can methylate and
pyridyloxobutylate DNA and induce damage in major tumour suppressor genes and oncogenes.
In addition, NNK has the potential to induce oxidative DNA damage. DNA repair systems are
critical for protecting genomic integrity from chemical agents like NNK. Alterations in repair
activities can result in persistent DNA lesions that can potentially exacerbate carcinogenesis. The
overall objective of this research was to determine the ability of NNK to cause DNA oxidation in
an established mouse model of NNK lung tumourigenicity, as well as to assess potential effects
of NNK on BER activity in murine lung and liver. Treatment with NNK has been shown to alter
NER and MGMT repair systems and may have a similar effect on BER.
Hypothesis 1: In vivo treatment with NNK induces oxidative DNA damage in murine lung and
liver.
Objective 1: Determine the effect of in vivo treatment with NNK on levels of 8-OHdG in A/J
mouse lung and liver.
Hypothesis 2: In vivo treatment with NNK alters BER activity in murine lung and liver.
Objective 2: Determine the effect of in vivo treatment with NNK on overall BER activity in A/J
mouse lung and liver.
35
Chapter 2
MATERIALS AND METHODS
2.1 Reagents
To evaluate oxidative DNA damage, chemicals and reagents were obtained as follows:
NNK from Toronto Research Chemicals (North York, ON); 8-OHdG, 2’-deoxyguanosine (2’-
dG), alkaline phosphatase, nuclease S1, nuclease P1 from Sigma (St. Louis MO); Ultrafree-MC
10,000 nominal molecular weight limit (NMWL) filter units from Millipore (Bedford, MA);
Wako DNA Extractor TIS kit from Wako Chemicals USA, Inc. (Richmond, VA). To assess BER
activity, chemicals and reagents were obtained as follows: proteinase K from Sigma (St. Louis,
Mo); Slide-A-Lyzer dialysis cassettes 3500 molecular weight cutoff from Thermo Scientific
(Rockford IL); EcoR1 from New England Biolabs (Beverley, MA); [32
P] dGTP from Perkin
Elmer (Waltham, MA). All other chemicals were reagent grade and were obtained from common
commercial suppliers.
2.2 Animal Treatments
Female A/J mice, aged 7 to 8 weeks (Jackson Laboratories, Bar Harbour, ME) were
housed with a 12 hour light/dark cycle and provided food and water ad libitum. Mice were
treated with a single tumourigenic dose of 10 mol NNK (~118 mg/kg NNK for mice that had an
average weight of ~17 g) in saline (0.1 mL, i.p.) or 0.9% saline control (0.1 mL, i.p.). This NNK
treatment protocol has been shown to induce 7 to 12 lung adenomas per A/J mouse 16 weeks later
(Hecht et al., 1989). To avoid the use of anesthetics that could interfere with biochemical
processes in lung or liver, mice were sacrificed by cervical dislocation 1, 2 or 24 hours following
NNK administration. The time points were selected to assess BER activity and levels of
oxidative DNA damage since NNK can induce substantial DNA damage in lung after the first
36
few hours after NNK administration (Peterson and Hecht, 1991; Chung and Xu, 1992; Tjaelve,
1991). Lungs and livers were perfused with TE buffer (10 mM Tris, 1 mM EDTA, pH 7.9) and
excised. Organs were snap frozen in liquid nitrogen and stored at –80oC until nuclear extract
preparation or DNA isolation. For each time point, 20 mice were treated with NNK and another
20 mice with saline. To determine BER activity, 4 sets of lungs were pooled together to produce
1 lung nuclear protein extract while 1 liver was used to produce 1 liver nuclear protein extract.
To assess oxidative DNA damage, 1 set of lungs and 1 liver were required. A total of 120 mice
were required for conducting 4 independent experiments at 1, 2 and 24 hour time points.
2.3 Isolation of DNA from lung and liver
DNA was isolated from lung and liver tissue using a Wako DNA TIS Extractor kit,
according to the manufacturer’s recommendations with minor modifications: 0.1 mM
deferoxamine mesylate (DFO) was added to the lysis solution, enzyme reaction solution, and TE
buffer, while 0.3 mM DFO was added to the sodium iodide solution. DFO is an iron-chelating
agent that prevents Fenton reactions and subsequent production of hydroxyl radicals that could
artifactually oxidize DNA during sample preparation (Ravanat, 2005b). Briefly, lung and liver
tissue (~0.15 g and ~0.6 g respectively) was pulverized into powder using a mortar and pestle in
liquid nitrogen. Lysis solution was added to the tissue powder and homogenized to disrupt
cellular membranes. The whole tissue lysate was centrifuged (600 x g for 10 minutes at 4oC) and
RNAse, proteinase and an unspecified oxidation inhibitor were added and incubated at 37oC for 1
hour. The lysate was centrifuged (10,000 x g for 5 minutes at 4oC) and the supernatant kept.
DNA was precipitated with 300 L sodium iodide solution and washed with alcohol solutions.
The resulting DNA was dried and dissolved in TE buffer containing 0.1 mM DFO.
Concentration and purity of DNA were determined by spectrophotometry, using absorbance at
37
260 nm and 260 nm/280 nm absorbance ratio respectively. For purity of DNA, absorbance ratios
between 1.8 and 2.0 were accepted. DNA was stored at –80oC until digestion steps.
2.4 DNA digestion
Thorough digestion of genomic DNA is necessary for accurate measurement of genomic
8-OHdG levels (Huang et al., 2001). Calf thymus DNA and pBluescript SK+ plasmid was used
to optimize DNA digestion to ensure accuracy in the measurement of 8-OHdG levels. Nuclease
P1 and S1 enzymes and incubation times were tested with calf thymus DNA, and selected based
on which combinations produced the lowest 8-OHdG levels (data not shown). Preliminary results
suggested that DNA digestion with nuclease P1 had the lowest levels of 8-OHdG. Incubating calf
thymus DNA with nuclease P1 and alkaline phosphatase for different incubation times (ranging
from total times of 15 minutes to 2 hours) did not seem to have an effect on lowering 8-OHdG
levels. The final digestion method was performed using the method described by Lan et al. with
minor modifications (Lan et al., 2000). Briefly, DNA was diluted with TE buffer containing 0.1
mM DFO to a final concentration of 0.15 mg/mL in 100 L volume. Ten L of 0.5 M sodium
acetate (pH 5.1) and 1 L of 1 M MgCl2 were added to the DNA. To denature genomic DNA,
samples were heated at 100oC for 5 minutes and immediately placed on ice for 5 minutes. One
L of nuclease P1 (4 mg/mL) was added to DNA and incubated at 37oC for 1 hour. Solution pH
was adjusted to 7.8 by adding 1.25 L of 1 M Tris (pH 10.5). Porcine alkaline phosphatase was
prepared by dissolving in water with 0.1 mM DFO (0.1 U/ L). Ten L of alkaline phosphatase
was added to the sample and incubated at 37oC for 1 hour. Enzymes were precipitated by adding
2 L of 5.8 M acetic acid. Samples were transferred to 10,000 NMWL filter units and
centrifuged (12,000 x g for 20 minutes at 4oC), separating digested nucleosides from enzymes.
38
Samples were stored at –80oC until analysis. Gel electrophoresis was performed to check for
comprehensive DNA digestion.
2.5 Determination of 8-OHdG and 2’-dG levels by high-performance liquid
chromatography with electrochemical detection
To assess the levels of 8-OHdG and 2’-dG in genomic DNA, high-performance liquid
chromatography with multichannel electrochemical detector (HPLC-ECD) (ESA Inc.,
Chelmsford, MA) was used. Compounds were separated on a Waters S-3 4.6 150 mm column
with 0.1% methanol/99.9% 50 mM phosphate buffer (pH 5.5) at a flow rate of 1.0 mL/min (Bolin
et al., 2004; Guindon et al., 2007). The four electrochemical detector channels were set to 0, 250,
475 and 875 mV. The ratio of 8-OHdG to 2’-dG was determined by comparing the concentration
of each nucleoside in a digested DNA sample. The concentration of 8-OHdG and 2’-dG was
quantified by determining the area under the curve (Figure 2.1) and interpolated to standard
curves of 8-OHdG and 2’-dG. It should be noted that DNA isolation, digestion and HPLC
analysis was carried out in one week, in order to prevent prolonged storage and artifactual
oxidation (Kasai, 1997).
2.6 Cell-free whole tissue nuclear protein extract preparation
Cell-free whole tissue nuclear protein extracts were prepared from ~1.0 to 1.5 g of total
tissue from livers of individual mice and from pooled lungs of 4 mice. These extracts, active in
DNA repair synthesis, were prepared as described previously (Wood et al., 1988; Wood et al.,
1995; Bedard et al., 2005). Extracts were stored at –80oC until repair reactions were carried out.
Protein concentration of extracts were determined by the Lowry assay using bovine serum
albumin as a standard (Lowry et al., 1951). Extracts from lung and liver typically contained ~5
and ~15 mg/mL protein, respectively.
39
Figure 2.1 Chromatograms demonstrating resolution of A) 2’-dG and B) 8-OHdG, isolated from
lung DNA of mice following in vivo treatment with saline. Isocratic chromatography with
electrochemical detection was performed using a Waters S-3 4.6 x 150 mm column and a mobile
phase of 0.1% methanol/99.9% 50 mM phosphate buffer (v/v) (pH 5.5) at 1.0 mL/min at room
temperature. The nucleoside 2’-dG was detected at 875 mV and 8-OHdG at 250mV. The area
under the curve was calculated to determine the concentration of 2’-dG and 8-OHdG in samples.
A = microampere; nA = nanoampere; 2’-dG = 2’-deoxyguanosine; 8-OHdG = 8-
hydroxydeoxyguanosine.
40
2.7 Preparation of 8-OHdG adducted plasmid
A 2961 bp plasmid derived from pBluescript SK+ (Stratagene, La Jolla, CA) was grown
in E. Coli DH5 cells in 2xYT broth and isolated using a Qiagen Plasmid Mega Kit (Qiagen,
Valencia, CA), as per manufacturer’s instructions with minor modifications. Instead of growing
E. Coli in 500 mL liquid culture, a total of 1 L was grown in 2xYT liquid culture and eventually
applied to a single anion-exchange column to isolate plasmid. The method to prepare DNA
containing 8-OHdG adducts was adapted from Floyd et al. and Schneider et al. (Floyd et al.,
1989; Schneider et al., 1990). The BER pathway can repair 8-OHdG adducts and its activity can
be assessed using the in vitro DNA repair assay. Briefly, 50 L of pBluescript SK+ plasmid (0.4
mg/mL in TE buffer) was placed on a parafilm-covered petri dish on ice. In the dark, 50 L of
methylene blue was added to the plasmid and exposed to white light from a 100 W tungsten lamp
at a distance of 10 cm for 5 minutes. When irradiated with white light, methylene blue generates
singlet oxygen that specifically reacts with guanine residues resulting in 8-OHdG adducts
(Schneider et al., 1990). A range of concentrations of methylene blue (final concentration of 1, 2,
5, 10 and 20 M) was used to optimize the production of oxidized plasmid. Immediately after
light exposure, irradiated DNA was precipitated with 20 L of 5 M NaCl and 300 L 100%
ethanol. DNA was washed with 500 L of 80% ethanol twice, dried down and re-dissolved in TE
buffer. To determine the extent of 8-OHdG adduction, plasmid was thoroughly digested and
subjected to HPLC-ECD. Gel electrophoresis was performed on a 1% agarose gel to determine
extent of nicking damage in methylene blue damaged plasmid. DNA densitometry of the
photograph of the UV-illuminated ethidium bromide gel (ChemImager 4000, Alpha Innotech
Corporation, San Leandro, CA) was performed to quantify the percentage of nicked plasmid (~4
kb band) and intact supercoiled plasmid (~2 kb band) used in the in vitro repair assay.
41
2.8 In vitro base excision repair assay
The repair synthesis assay (Figure 2.2) was performed as described (Wood et al., 1988;
Wood et al., 1995; Bedard et al., 2005) with slight modifications. The 50 L reaction mixture
contained 400 ng of 8-OHdG damaged plasmid DNA or plasmid containing baseline DNA
oxidation, 40 mM HEPES-KOH (pH 7.8), 0.5 mM dithiothreitol, 5.0 μM dGTP, 20 M of each
dATP, dCTP and dTTP, 23 mM phosphocreatine, 18 g bovine serum albumin (nuclease-free,
Sigma Aldrich, St. Louis, MO), 2.5 g creatine phosphokinase, 2.0 mM ATP, 5.0 mM MgCl2, 0.4
mM EDTA, 100 mM KCl, 7.1 Ci [α32
P] dGTP, and 10 g of lung or 25 g of liver protein
extract (concentrations listed are final concentrations). Untreated plasmid was used as a negative
control and contained background levels of DNA oxidation. Samples were incubated for 3 hours
at 30°C unless otherwise indicated. Reactions were terminated by adding EDTA (final
concentration, 20 mM). Following incubation with 7.3 mg/mL RNase (final concentration, at
37°C for 10 minutes) and 925 g/mL proteinase K with 0.5% SDS (final concentration, at 65°C
for 30 minutes), plasmid DNA was purified by extraction with 250 L of
phenol/chloroform/isoamyl alcohol (25:24:1; v/v/v), precipitated with 1 mL of 100% ice-cold
ethanol, washed in 1 mL of 80% ethanol, dried and linearized with 1 U of EcoR1. Gel
electrophoresis was performed with the linearized plasmid on a 1% agarose gel. Plasmid DNA
recovery was normalized by densitometry of the photograph of the UV-illuminated ethidium
bromide gel (ChemImager 4000, Alpha Innotech Corporation, San Leandro, CA). The extent of
[α32
P] dGTP incorporation was determined by phosphor imaging (Cyclone Plus Phosphor Imager,
Perkin Elmer, Waltham, MA) of the dried gel (Figure 2.3). To determine damage-specific BER
activity in lung and liver of saline control and NNK-treated mice, repair synthesis activity found
in untreated plasmid containing baseline levels of oxidation was subtracted from repair synthesis
42
Figure 2.2 Schematic of in vitro DNA repair synthesis assay on damaged plasmid DNA by
nuclear extracts isolated from whole tissue. Squares on plasmid represent oxidative DNA lesions.
8-OHdG: 8-hydroxydeoxyguanosine; dGTP: deoxyguanosine triphosphate; EtBr: ethidium
bromide (modified from Salles et al., 1995).
43
Figure 2.3 UV-illuminated ethidium bromide stained agarose gel of linearized plasmid DNA and
phosphor image demonstrating the ability of cell-free nuclear protein extracts to catalyze in vitro
DNA repair synthesis. DNA in lanes 1 through 4 were incubated with nuclear protein from saline
control lung. Lanes 1 and 2 are replicates and demonstrate background repair synthesis activity
towards untreated plasmid DNA that contains background level of oxidation. Lanes 3 and 4 are
replicates and show repair of 8-OHdG adducted plasmid.
1 2 3 4
Untreated plasmid + + - -
8-OHdG damaged plasmid - - + +
3 kb
2 kb
1kb
44
activity found in plasmid containing oxidative DNA damage. The in vitro repair assay was
performed in duplicate for each protein extract.
In addition, to optimize the in vitro repair assay, nuclear protein extracts from lungs and
livers of control female A/J mice were prepared and tested. The level of oxidative damage in
plasmid substrate, protein amount and incubation time were investigated to determine the linear
range of each parameter in the in vitro repair assay. Each specific parameter of the BER assay
was tested using separate aliquots from a single liver extract (prepared from 1 animal) or a single
lung extract (prepared from 4 animals).
2.9 Data analysis
Statistically significant differences in levels of DNA oxidation and BER activity between
NNK-treated and control groups were determined by unpaired Student’s t-test (GraphPad Prism 5
software). If heterogeneity of variance was present, data were transformed using the log-log
function prior to statistical analysis. P < 0.05 was considered significant in all cases.
45
Chapter 3
RESULTS
3.1 Levels of 8-OHdG following in vivo treatment with NNK
The background levels of 8-OHdG in calf thymus DNA and freshly isolated pBluescript
SK+ plasmid were on average 21.5 and 10.1 8-OHdG/105 2’-dG respectively. Gel
electrophoresis revealed thorough digestion of calf thymus DNA and plasmid.
DNA oxidation was assessed in lungs of NNK-treated and saline control mice at each
time point (Figure 3.1). No changes in 8-OHdG levels were observed between control mice (19.3
± 3.9 8-OHdG/105 2’-dG) and NNK-treated mice (19.5 ± 3.3 8-OHdG/10
5 2’-dG) at the 1 hour
time point (n=4, P = 0.928). At the 2 hour time point, there was an apparent 14% increase in 8-
OHdG levels in the lungs of NNK-treated mice compared to control lungs; however, this was not
statistically significant (n=4, P = 0.238). Lungs of control mice appeared to have a 15% increase
in 8-OHdG levels compared to treated mice at 24 hours, but statistical significance was not
attained (n=4, P = 0.119).
In liver, the levels of 8-OHdG were not statistically different between NNK-treated mice
and control mice at any of the time points examined (Figure 3.2). An apparent 16% increase in 8-
OHdG levels was observed in NNK-treated mice compared to controls at 1 hour, but was not
statistically significant (n=4, P = 0.253). There appeared to be a 27% increase in 8-OHdG levels
in NNK-treated mice compared to control mice at 2 hours; however this was not statistically
significant (n=4, P = 0.166). At 24 hours, control mice (24.8 ± 6.7 8-OHdG/105 2’-dG) had
similar levels of 8-OHdG compared to mice treated with NNK (23.9 ± 6.7 8-OHdG/105 2’-dG)
and was not statistically significant (n=4, P = 0.864).
46
Lung 1 hr
Saline NNK
0
10
20
30
40
A
Ratio 8
-OH
dG
/10
5 2
'-dG
Lung 2 hr
Saline NNK
0
10
20
30
40
B
Ratio 8
-OH
dG
/10
5 2
'-dG
Lung 24 hr
Saline NNK
0
10
20
30
40
C
Ratio 8
-OH
dG
/10
5 2
'-dG
Figure 3.1 Effect of in vivo treatment with NNK on 8-OHdG levels in lung DNA. DNA was
isolated from mice treated with 10 mol NNK or saline and sacrificed 1 (A), 2 (B) or 24 (C) hr
post-treatment. Results are presented as mean SD of 4 animals performed in triplicate and are
expressed as the ratio of the concentration of 8-OHdG to 2’-dG. No statistically significant
differences were found between NNK and saline control (P > 0.05, Student’s t-test).
47
Liver 1 hr
Saline NNK
0
10
20
30
40
A
Ratio 8
-OH
dG
/10
5 2
'-dG
Liver 2 hr
Saline NNK
0
10
20
30
40
B
Ratio 8
-OH
dG
/10
5 2
'-dG
Liver 24 hr
Saline NNK
0
10
20
30
40
C
Ratio 8
-OH
dG
/10
5 2
'-dG
Figure 3.2 Effect of in vivo treatment with NNK on 8-OHdG levels in liver DNA. DNA was
isolated from mice treated with 10 mol NNK or saline and sacrificed 1 (A), 2 (B) or 24 (C) hr
post-treatment. Results are presented as mean SD of 4 animals performed in triplicate and are
expressed as the ratio of the concentration of 8-OHdG to 2’-dG. No statistically significant
differences were found between NNK and saline control (P > 0.05, Student’s t-test).
48
3.2 Characterization of 8-OHdG adducted plasmid DNA substrate for in vitro repair
assay
Photoactivated methylene blue was used to induce 8-OHdG lesions in pBluescript SK+
plasmid (Figure 3.3 A). An increase in 8-OHdG levels was observed between untreated plasmid
and plasmid treated with 1, 2, 5, 10 and 20 M of methylene blue. Untreated plasmid contained a
baseline level of oxidation of 16.1 3.6 8-OHdG/105 2’-dG (n=4, mean SEM), while a
maximum concentration of 20 M methylene blue induced an average of 167 16.4 8-OHdG/105
2’-dG (n=4, mean SEM). Plasmid was also assessed for nicking using gel electrophoresis and
DNA densitometry (Figure 3.3 B). An increase in nicked plasmid was observed when increasing
concentrations of methylene blue was used to induce oxidative DNA damage.
To choose an appropriate substrate for the in vitro repair assay, untreated plasmid and
plasmid treated with 1, 2, 5, 10 and 20 M of methylene blue were incubated with nuclear protein
extracts prepared from control lung and liver to determine extent of repair activity (Figure 3.4).
Oxidative DNA damage in the form of 8-OHdG ranged from ~16 8-OHdG/105 2’-dG lesions in
untreated plasmid to ~170 8-OHdG/105 2’-dG in plasmid treated with 20 M methylene blue.
BER was assessed by normalizing the amount of [32
P] dGTP incorporated to plasmid recovery
( g). As anticipated, incubation of 10 g of lung extract with untreated plasmid containing
baseline oxidation resulted in the lowest levels of BER activity (Figure 3.4 A). Plasmid
containing higher levels of 8-OHdG had increased levels of repair activity compared to untreated
plasmid. A pronounced plateau in repair activity was observed in plasmid containing ~75 to
~175 8-OHdG/105 2’-dG. The incubation of 25 g liver extract with untreated plasmid also
produced the lowest levels of repair activity (Figure 3.4 B). An increase in repair activity was
observed in plasmid containing up to ~75 8-OHdG/105 2’-dG and seemed to plateau at higher
levels of oxidative DNA damage.
49
0 5 10 15 20 250
50
100
150
200
A
Methylene Blue ( M)
8-O
HdG
/10
5 2
'-dG
0 5 10 15 20 250
10
20
30
40
B
% o
f n
icke
d p
lasm
id
Methylene Blue ( M)
Figure 3.3 Characteristics of 8-OHdG adducted pBluescript SK+ plasmid by photo-oxidation of
methylene blue. Plasmid was grown in and isolated from E. coli DH5 cells. Levels of 8-OHdG
are presented as mean SEM of four independent experiments performed in duplicate and are
expressed as the ratio of the concentration of 8-OHdG to 2’-dG (A). The percentage of nicked
plasmid are expressed as mean SEM of three or four independent experiments (B).
50
0 25 50 75 100 125 150 175 2000
1000
2000
3000
4000
5000
A Lung, 10 g
8-OHdG/105 2'-dG
am
ol[
32P
]dG
TP
/g D
NA
0 25 50 75 100 125 150 175 2000
2000
4000
6000
8000
10000
12000
B Liver, 25 g
8-OHdG/105 2'-dG
am
ol[
32P
]dG
TP
/g D
NA
Figure 3.4 Optimization of 8-OHdG adducted plasmid for in vitro BER assay. Protein extract
from control lung (10 g, A) or liver (25 g, B) was incubated with plasmid containing varying
amounts of 8-OHdG for 3 hr. Results are presented as mean of duplicates and are expressed as
amol[32
P] dGTP incorporated per g of DNA. Plasmid treated with 5 M methylene blue induced
~40 8-OHdG/105 2’-dG and was chosen to assess NNK’s effect on BER in lung and liver.
51
Plasmid treated with 5 M methylene blue was selected to assess the effect of NNK on
repair activity in both lung and liver. With this concentration of methylene blue, repair activity
was ~5-fold higher compared to that in untreated plasmid in repair reactions containing lung or
liver extract. This plasmid substrate contained ~40 8-OHdG/105 2’-dG (Figure 3.3) and was used
in subsequent in vitro repair assays.
3.3 Optimization of nuclear protein extract amount for in vitro repair assay
Saline control lung and liver extract was tested with the in vitro repair assay to select an
optimal protein amount to assess BER activity. A range of concentrations of both nuclear protein
extracts were incubated with plasmid treated with 5 M methylene blue for 3 hours. Repair
reactions containing neither lung nor liver extracts resulted in no incorporation of [32
P] dGTP
and thus no BER activity. Incubation reactions containing a maximum of 25 g of lung extract
resulted in an increase in repair activity (Figure 3.5 A). BER activity reached a plateau in
reactions incubated with higher amounts of lung extract. Similarly, repair reactions containing a
maximum of 50 g of liver extract appeared to increase BER activity in a linear fashion (Figure
3.5 B). Higher amounts of liver extract resulted in a plateau of BER activity. Ten g of lung
extract and 25 g of liver extract were chosen to assess BER activity in organs of control and
NNK-treated mice as increases and decreases in BER activity were detectable with these amounts
of protein.
3.4 Optimization of incubation time for in vitro repair assay
To select an optimal incubation time for the repair assay, repair activity was assessed
over time with control lung and liver nuclear protein extracts. Repair reactions contained plasmid
oxidized with 5 M methylene blue and 10 g of lung extract or 25 g of liver extract. An
52
0 10 20 30 40 50 60 70 800
2000
4000
6000
8000
10000
12000
LungA
Protein ( g)
am
ol[
32P
]dG
TP
/g D
NA
0 10 20 30 40 50 60 70 800
2000
4000
6000
8000
10000
12000
LiverB
Protein ( g)
am
ol[
32P
]dG
TP
/g D
NA
Figure 3.5 Optimization of protein amount for in vitro BER assay. Varying amounts of protein
extract from control lung (A) or liver (B) were incubated with plasmid damaged by 5 M
methylene blue for 3 hr. This parameter was tested using a single lung extract (A) or separate
aliquots from a single liver extract (B). Results are presented as mean of duplicates (A) or mean
SEM of three independent experiments performed in duplicate (B). Ten g of lung protein and 25
g of liver protein was chosen to assess the effect of NNK on BER.
53
increase in repair activity was observed in repair reactions containing 10 g of lung extract or 25
g of liver extract across all incubation times (Figure 3.6 A and B). The levels of BER activity
appeared to be linear at a maximum incubation time of 4 hours in both lung and liver. An
incubation time of 3 hours was picked to assess BER in lung and liver of control and NNK-
treated mice.
3.5 Effect of in vivo treatment of mice with NNK on BER in lung and liver
Damage-specific BER activity was assessed in lung and liver of mice following in vivo
treatment with NNK using the optimized parameters. No statistically significant differences were
observed between lung extracts prepared from NNK-treated mice and control mice at each time
point (Figure 3.7). At the 1 hour time point, mice treated with NNK appeared to have a 34%
reduction in BER activity in lung tissue compared to controls, however statistical significance
was not attained (n=4, P = 0.114). An apparent 13% decrease in BER activity was observed in
lungs of NNK-treated mice at 2 hours, but was not statistically significant (n=4, P = 0.535). At
24 hours lungs of NNK-treated mice appeared to have a 21% decline in repair activity, but was
not statistically significant (n=4, P = 0.146).
In liver, no statistically significant differences were observed at the 1 hour time point as
repair activities of NNK treatment and control groups were similar (n=4, P = 0.818) (Figure 3.8).
At 2 hours NNK-treated mice had an apparent 16% reduction in BER activity in liver; however
this was not statistically significant (n=3 or 4, P = 0.163). BER activity was slightly reduced in
livers of NNK-treated mice at the 24 hour time point but statistical significance was not attained
(n=4, P = 0.675).
54
Lung
0 1 2 3 4 50
1000
2000
3000
A
Time (hr)
am
ol[
32P
]dG
TP
/g D
NA
Liver
0 1 2 3 4 50
2000
4000
6000
8000
B
Time (hr)
am
ol[
32P
]dG
TP
/g D
NA
Figure 3.6 Optimization of incubation time for in vitro BER assay. Protein extracts from control
lung (10 g, A) or liver (25 g, B) were incubated for varying amounts of time with plasmid
damaged by 5 M methylene blue. This parameter was tested using a single lung extract (A) or
separate aliquots from a single liver extract (B). Results are presented as mean of duplicates (A)
or mean SEM of three independent experiments performed in duplicate (B). A 3 hr incubation
time was chosen to assess NNK’s effect on BER in lung and liver.
55
Lung 1 hr
Control NNK 0
1000
2000
3000
4000
5000
n=4 n=4
A
am
ol[
32P
]dG
TP
/g D
NA
Lung 2 hr
Control NNK 0
1000
2000
3000
4000
5000
n=4 n=4
B
am
ol[
32P
]dG
TP
/g D
NA
Lung 24 hr
Control NNK 0
2000
4000
6000
8000
n=4 n=4
C
am
ol[
32P
]dG
TP
/g D
NA
Figure 3.7 Effect of in vivo treatment with NNK on in vitro BER in mouse lung extracts towards
8-OHdG adducted plasmid. Extracts were prepared from mice treated with 10 mol NNK or
saline and sacrificed 1 (A), 2 (B) or 24 (C) hr post-treatment. Results are presented as mean SD
and expressed as damage-specific BER. No statistically significant differences were found
between NNK and saline control at each time point (P > 0.05, Student’s t-test).
56
Liver 1 hr
Control NNK 0
1000
2000
3000
4000
5000
n=4 n=4
A
am
ol[
32P
]dG
TP
/g D
NA
Liver 2 hr
Control NNK 0
1000
2000
3000
4000
5000
n=3 n=4
B
am
ol[
32P
]dG
TP
/g D
NA
Liver 24 hr
Control NNK 0
2000
4000
6000
8000
n=4 n=4
C
am
ol[
32P
]dG
TP
/g D
NA
Figure 3.8 Effect of in vivo treatment with NNK on in vitro BER in mouse liver extracts towards
8-OHdG adducted plasmid. Extracts were prepared from mice treated with 10 mol NNK or
saline and sacrificed 1 (A), 2 (B) or 24 (C) hr post-treatment. Results are presented as mean SD
and expressed as damage-specific BER. No statistically significant differences were found
between NNK and saline control at each time point (P > 0.05, Student’s t-test).
57
Chapter 4
GENERAL DISCUSSION
In the present study, female A/J mice were treated with a tumourigenic dose of NNK and
the level of DNA oxidation and BER activity was determined in lung and liver of control and
NNK-treated mice.
4.1 In vivo treatment with NNK does not induce oxidative DNA damage
NNK-induced carcinogenesis typically involves metabolic activation to generate reactive
intermediates that methylate or pyridyloxobutylate DNA. In vivo treatment with NNK in rodents,
induces DNA damage in oncogenes and tumour suppressor genes that play a critical role in
carcinogenesis (Hecht, 1999). In addition, in vivo treatment with NNK has been reported to
induce oxidative DNA damage in the form of 8-OHdG in tissues such as lung and liver in mice
(Chung and Xu, 1992; Rosa et al., 1998; Weitberg and Corvese, 1993). The consequences of
excess 8-OHdG, a major oxidative DNA lesion, include GT transversion mutations in
oncogenes and tumour suppressor genes that could contribute to carcinogenesis (Klaunig and
Kamendulis, 2004).
Reported basal levels of DNA oxidation in biological systems have been inconsistent,
with values of 8-OHdG differing by ~2 orders of magnitude (Collins et al., 2004; Ravanat,
2005a). The large variation in 8-OHdG levels can largely be attributed to different methods and
techniques used to isolate and assess DNA oxidation. For example, the levels of 8-OHdG in pig
liver DNA ranged from 0.21 to 44.1 8-OHdG per 105 2’-dG using HPLC-ECD and gas
chromatography-mass spectrometry (GC-MS) (Collins et al., 2004). Guanine is readily
oxidizable during sample preparation and some techniques are conducive to spurious oxidation
that can overestimate the levels of DNA oxidation. GC-MS is prone to high levels of artifactual
58
oxidation during derivatization which require high temperature and a silylating agent to make
DNA samples volatile for separation (Cadet et al., 1998). Conversely, the lowest levels of basal
8-OHdG have been observed when using enzymes specific for 8-OHdG (Ravanat, 2005a). In
enzymatic nicking assays such as the comet assay, levels of 8-OHdG can range from < 1 to 10 8-
OHdG residues per 106 2’-dG. It is unclear whether enzymes bind all 8-OHdG sites in genomic
DNA. Failure to do so would underestimate the levels of 8-OHdG (Halliwell and Whiteman,
2004; Ravanat, 2005a). When measuring background levels of oxidative DNA damage in control
tissues and cells, HPLC-ECD has generated values as low as several 8-OHdG per 106 2’-dG in
human lymphocytes and as high as 20 8-OHdG per 105 2’-dG in untreated murine liver (Zhang et
al., 2004; ESCODD et al., 2005; Guindon et al., 2007).
In the present study, DNA of lung and liver was isolated, digested and assessed for the
ratio of 8-OHdG per 105 2’-dG using HPLC-ECD. For chromatographic methods including
HPLC-ECD, the addition of metal chelators and antioxidants during sample preparation is
standard practice to reduce spurious oxidation (European Standards Committee on Oxidative
DNA Damage, 2003; Ravanat, 2005a). DFO, an iron chelator that prevents spurious oxidation
via Fenton reactions, and an additional unspecified antioxidant (included in the Wako DNA
Extractor TIS kit) were used when isolating DNA. In addition, a sodium iodide extraction
method was utilized to avoid the use of phenol and chloroform extraction which can artificially
enhance 8-OHdG levels (Claycamp, 1992; Ravanat, 2005a). DNA digestion was also optimized
for thorough digestion to reduce spurious oxidation (Huang et al., 2001). Gel electrophoresis of
the DNA digests revealed absence of high molecular weight DNA, indicating that genomic DNA
was thoroughly digested. DNA isolation, digestion and HPLC analysis of control and NNK-
treated samples were carried within one week to minimize artifactual DNA oxidation. Samples
59
from control and NNK-treated animals for a given time point were processed at the same time to
minimize the potential impact of inter-day assay variability (Kasai, 1997).
The levels of 8-OHdG were comparable to a number of studies found in the literature.
The background levels of 8-OHdG in calf thymus DNA were 21.5 8-OHdG per 105 2’-dG and
were comparable to optimal levels determined by the European Standards Committee on
Oxidative DNA Damage which ranged from 1.9 to 21.3 8-OHdG per 105 2’-dG when using
HPLC-ECD (Lunec, 1998). These results suggest that artifactual oxidation was minimal when
using the current protocol in the present study. In addition, control levels of oxidative DNA
damage in murine lung and liver were not variable at each time point, and comparable to other
studies that assessed 8-OHdG in such tissues using HPLC-ECD (Zhang et al., 2004; Guindon et
al., 2007).
In the present study, 8-OHdG was assayed in lungs and livers of mice treated with a
tumourigenic dose (10 mol) of NNK after 1, 2 and 24 hours. No statistically significant effects
in 8-OHdG levels were observed in lung or liver of mice treated with NNK at each time point,
indicating in vivo treatment with NNK did not induce oxidative DNA damage. However, a trend
towards elevated 8-OHdG levels was observed in liver and less so in lungs from NNK-treated
mice in the first 2 hours. It is important to recognize that these trends were non-significant, as an
apparent 27% increase in 8-OHdG levels in liver (n=4, P = 0.166) and apparent 14% increase in
8-OHdG levels in lung (n=4, P = 0.238) was observed at 2 hours. Although not statistically
significant this trend towards elevated 8-OHdG levels is similar to other studies that reported an
increase in 8-OHdG levels in lung and liver 2 hours after NNK administration, with a decrease to
basal levels after 24 hours (Chung and Xu, 1992; Xu et al., 1992; Rosa et al., 1998).
60
Although some studies have reported oxidative DNA damage in experimental animals
after NNK treatment, this is not always the case. In some studies, in vivo treatment with NNK
failed to cause statistically significant changes in DNA oxidation. Treatment with 19.3 mol of
NNK i.p. appeared to increase the levels of 8-OHdG in A/J mouse lung and liver 2 hours after
administration, but the effect was not statistically significant (Chung and Xu, 1992).
Furthermore, treatment of A/J mice with multiple doses of NNK (11.65 mg/kg body weight, via
gavage, 3 times weekly for 10 weeks) did not significantly affect 8-OHdG levels in liver 2 hours
post-treatment (Xu et al., 1992). In such studies, NNK could have altered oxidative DNA
damage in specific cell types of lung and liver but these effects may not have been detected when
analyzing oxidative DNA damage from whole lung and liver. Similar trends were observed in the
current study, with statistically non-significant increases in 8-OHdG levels in lung and liver
following NNK treatment, suggesting NNK may actually have limited effect on DNA oxidation
status.
4.2 In vivo treatment with NNK does not alter overall BER activity
DNA repair systems are critical for protecting and maintaining the genome from
exogenous and endogenous damaging agents. Several DNA repair systems have been identified
and categorized based on the type of damage repaired. A major function of the BER pathway is
to identify and remove bases that have been damaged by ROS. The effects of NNK have
previously been studied on nucleotide excision repair (NER) and direct reversal repair systems,
but have not on BER.
In the present study, an in vitro repair assay was optimized to assess BER in lung and
liver of mice treated with NNK. To assess BER activity, a plasmid substrate containing 8-OHdG
lesions was incubated with cell-free nuclear protein extracts prepared from murine lung and liver.
61
Methylene blue irradiated with white light was used to induce oxidative DNA damage in the
plasmid substrate. During photo-oxidation, methylene blue is activated into a transitory excited
state which in turn can react with stable oxygen to generate reactive singlet oxygen (Floyd et al.,
1989). When exposed to DNA, singlet oxygen has high specificity for guanine residues and
usually generates 8-OHdG lesions (Epe, 1991). An increase in 8-OHdG levels was observed
when using a range of methylene blue concentrations to induce oxidative damage. The levels of
8-OHdG were dependent on the final concentration of methylene blue during exposure to light.
These results are consistent with literature that showed increasing 8-OHdG levels in DNA treated
with increasing concentrations of methylene blue irradiated with light (Floyd et al., 1989;
Schneider et al., 1990).
Plasmid was also assessed for nicking status using gel electrophoresis and DNA
densitometry. Methylene blue was selected as an oxidizing agent as it preferentially induces 8-
OHdG over DNA strand breaks that could potentially be repaired by systems other than BER
(Schneider et al., 1990). In the current study, only 3% of the untreated plasmid was nicked,
leaving 97% in supercoiled form. Plasmid treated with 5 M methylene blue and subsequently
chosen to assess BER in lung and liver was 12% nicked, leaving 88% in supercoiled form.
Although a small percentage of both types of plasmid were nicked, DNA single strand nicks have
been shown to stimulate very little incorporation of radiolabelled nucleotides when using a
similar in vitro repair assay that assessed NER (Wood et al., 1988; Brown et al., 2007).
Similarly, no significant differences were observed in NER towards nicked and supercoiled
plasmid that contained POB adducts, indicating no artifactual increase in damage-specific repair
activity due to nicks (Brown et al., 2007). These findings suggest that the limited amounts of
nicked DNA present in the plasmid substrate and used in the current study likely had minimal
impact on the BER activities measured.
62
When choosing an appropriate plasmid substrate to assess BER, several plasmid
substrates were prepared with varying levels of DNA oxidation. BER activity was dependent on
the amount of 8-OHdG induced by methylene blue within the plasmid substrate. Untreated
plasmid containing background levels of 8-OHdG had the lowest activity compared to plasmid
substrates treated with methylene blue. An upper limit of BER activity was reached in plasmid
substrate treated with high concentrations of methylene blue that induced high levels of 8-OHdG.
The repair system provided by the lung and liver extract could have been limiting, resulting in a
plateau in repair activity. Plasmid treated with 5 M methylene blue induced ~40 8-OHdG per
105 2’-dG and was selected for the assay since repair in this plasmid was ~5-fold higher
compared to untreated plasmid.
BER activity was also protein concentration-dependent and repair duration-dependent.
When assessing protein concentration in the assay, nuclear extract was required for repair activity
as repair reactions that contained no protein had no incorporation of [32
P] dGTP. A plateau in
BER activity was observed when incubations contained > 30 g of lung extract or > 50 g of
liver extract, presumably due to limiting amounts of damaged plasmid substrate. It should be
noted that at higher protein amounts (ranging from 100 to 200 g of liver extract in particular), a
decrease in repair activity was sometimes observed (data not shown). The presence of excessive
amount of proteins may have interfered with recognition and binding of 8-OHdG lesions,
reducing overall efficiency and repair activity. When assessing incubation time, an apparent
linear increase in repair activity was observed in murine lung and liver over 4 hours. Repair
reactions that were terminated immediately had some repair activity. BER proteins may begin to
initiate repair processes upon addition of oxidized plasmid substrate followed by reaction
termination. These results were similar to studies that demonstrated BER activity in extracts
from rat brain was dependent on protein concentration and incubation time (Lan et al., 2003;
63
Chen et al., 2000). In those studies, an upper limit in BER activity was also observed at protein
amounts > 30 g and incubation times > 8 hours.
When assessing the effects of NNK on BER in liver and lung, selection of an oxidized
plasmid substrate, protein concentration and incubation time was chosen based on the ability to
detect increases and decreases in BER activity. Plasmid treated with 5 M methylene blue, a 3
hour incubation time and 10 g of lung or 25 g of liver extract were chosen for the present in
vitro repair assay. No statistically significant differences in BER were observed in lung or liver
between control and NNK-treated mice at 1, 2 or 24 hours. Hence, administration of a
tumourigenic dose of NNK did not affect BER activity up to 24 hours post treatment. In previous
studies, NNK was found to inhibit NER and O6-methylguanine-DNA methyltransferase (MGMT)
in lung, and increase NER in liver (Belinsky et al., 1988; Liu et al., 1992; Brown and Massey,
2009) but failed to alter BER activity in the current study. As a result, it appears that NNK could
selectively modify DNA repair pathways like NER and MGMT, while sparing others like BER.
However, it is also possible that the previously reported effectiveness of NNK at altering non-
BER DNA repair systems could be due to differences in: doses of NNK; experimental designs;
species and strains of animals; and time points. For example, MGMT inhibition by NNK was
demonstrated in specific lung cell types of rats treated with multiple doses of NNK (Belinsky et
al., 1988). Furthermore, rat hepatocytes treated with OPB, a reactive metabolite of NNK, had
inhibited MGMT activity (Liu et al., 1992). Nonetheless, NNK’s inhibition of NER in lung was
studied using the same dose and strain of mouse and a time course similar to that of the current
BER study (Brown and Massey, 2009).
One mechanism by which NNK could modify specific repair pathways is by forming
specific protein adducts. Treatment with NNK has been shown to pyridyloxobutylate proteins
64
including hemoglobin and MGMT (Murphy et al., 1990; Mijal et al., 2004). Electrophilic
intermediates generated during NNK metabolism may preferentially bind to major proteins of
NER or MGMT systems modifying their overall repair activity, but may react less with BER
proteins, perhaps due to lower affinity or lack of an available specific amino acid binding motif.
It should be noted that an apparent decreasing trend in BER (by 34% and 21%, at 1 and
24 hours post administration respectively) was observed in lungs of NNK-treated mice, although
it was not statistically significant. A potential explanation for the lack of significant effect of
NNK on repair of the oxidized plasmid is repair of 8-OHdG by additional mechanisms.
Treatment with NNK could have inhibited BER activity in lung, but this inhibition could have
been mitigated by other functional BER glycosylases or non-BER DNA repair mechanisms
present in the nuclear extracts. Humans and mice both have several BER glycosylases that have
overlapping function and redundancy to ensure extra protection against frequently occurring
oxidative DNA lesions, like 8-OHdG (Hazra et al., 2003; Cooke et al., 2003). For example, BER
glycosylases such as OGG1 and Nei-like 1 both repair 8-OHdG adducts in DNA and either
glycosylase could act as a backup in case one has compromised activity (Hazra et al., 2003). In
some cases mismatch repair and NER systems have also been shown to repair 8-OHdG lesions in
DNA, and could alleviate the effect of decreased BER (Reardon et al., 1997; Colussi et al., 2002;
Patel et al., 2007). In the present study, these additional repair mechanisms could have also
played a role in reducing the effect of NNK on BER and protecting the genome from oxidative
DNA in lungs of mice.
A tumourigenic dose of NNK (10 mol) given i.p. also had no significant effect on BER
activity in liver. Interestingly, the same dose and route of administration of NNK increased NER
activity and levels of specific NER proteins in liver of A/J mice 24 hours post administration
(Brown and Massey, 2009). Hence, NNK increases NER activity in liver while having no effect
65
on BER. These results are consistent with the fact that NER and BER are regulated differently.
For example, post-translational modifications such as phosphorylation, ubiquitylation and
sumoylation occur to varying degrees to proteins in the NER and BER pathways and could affect
enzyme recruitment, DNA-protein interactions, protein-protein interactions and catalytic function
of each DNA repair system (Huang and D'Andrea, 2006; Branzei and Foiani, 2008).
4.3 Conclusions
The research presented in this thesis has demonstrated that a single in vivo administration
of NNK did not induce oxidative DNA damage in A/J mouse lung or liver at 1, 2 or 24 hours.
Only a slight increasing trend in oxidative DNA damage was observed in lung and liver that was
not statistically significant. Additionally, this was the first study to test the effects of NNK on
BER. Using an optimized in vitro repair assay, NNK did not alter overall BER activity in lung
and liver at each time point. A decreasing trend in BER activity was observed in lung that was
not statistically significant.
4.4 Future directions
Although not statistically significant, an increasing trend in 8-OHdG and decreasing trend
in BER activity in lung and liver of mice treated with NNK was observed and should be further
investigated. This was the first study to test the effect of NNK on BER and supplementary
studies should be carried out to verify the present findings. Additional experiments could be
conducted to determine if NNK has effect on BER and DNA oxidation over a daily, weekly or
monthly time course. The current research investigated potential effects of NNK in lung and liver
acutely at 1, 2 and 24 hours post administration. The dose of NNK employed in the current study
(10 mol) has been shown to cause induction of lung adenomas in mice 16 weeks later (Hecht et
al., 1989). The progression of lung adenomas to malignant adenocarcinomas was observed in
66
lungs 50 weeks post NNK administration (Hecht, 1998). Assessing NNK’s effect on DNA
oxidation and BER in lung and liver over a longer period of time could further elucidate its role
during carcinogenesis. Furthermore, the current research used a single i.p. dose of NNK that does
not accurately model chronic smoking behaviour in humans. While a single 10 mol dose given
i.p. will most likely induce pulmonary carcinogenesis in mice, chronic administration of NNK at
lower doses over several weeks more closely resembles smoking behaviour.
Investigating the effects of NNK is important for understanding tobacco-induced
carcinogenesis. Substantial experimental data has shown NNK to be one of the most potent
pulmonary carcinogens found in cigarettes and tobacco products and most likely plays a role in
tobacco-induced human cancers. Studying NNK in isolation from a chemical agent as complex
as tobacco smoke is helpful in specifically characterizing the molecular mechanism by which a
major component of cigarettes affects DNA repair processes. However, to expand the scope of
this project, a second approach should also be taken that investigates the effect of cigarette smoke
on repair activity in animal models. NNK is only one component of cigarettes and tobacco
smoke that contain over 4000 chemicals including 55 known carcinogens. The current study is
limited by only investigating the effects of NNK on DNA damage and repair. Exposing
experimental animals to cigarette smoke would represent human smoking more accurately by
having inhalation as the primary route of administration as well as treating with agents that have
the same chemical composition as tobacco smoke. BER and other repair processes could be
assessed in lung and liver in these studies.
If statistically significant differences in BER activity were observed in future studies, the
next logical step would be to determine where and how changes were occurring in the pathway.
A modified in vitro oligonucleotide repair assay would be carried out to ascertain if changes
occurred during the recognition and excision step, or nucleotide insertion and ligation step of the
67
BER pathway. If changes in activity occurred at both or either steps, immunoblots of key BER
proteins would be performed on control and NNK-treated animals to determine if changes in
repair activity were due to variation in protein levels. Real-time reverse transcription polymerase
chain reaction could be performed to determine if potential changes in levels of BER proteins
were due to changes in mRNA transcript levels. A nuclear run-on assay could also be used to
determine if NNK alters transcription rates of particular BER genes. Reactive intermediates
generated by metabolism of NNK have been shown to adduct proteins such as hemoglobin
(Murphy et al., 1990) and could methylate or pyridyloxobutylate key BER proteins that could
change protein activity or stability. Adducted BER proteins could be separated using SDS-PAGE
and following immunoblotting, excised, digested and subjected to mass spectrometry for
identification.
Potential changes in the activity of BER proteins could also be due to changes in post-
translational modifications. Phosphorylation, sumoylation and ubiquitylation of repair proteins
are important for regulating several types of DNA repair (Branzei and Foiani, 2008; Huang and
D'Andrea, 2006). For example, sumoylation of certain BER glycosylases can alter catalytic
activity for specific substrates, as well as reduce affinity for the AP site which allows access to
DNA for further processing by additional BER proteins (Hardeland et al., 2002; Steinacher and
Schär, 2005). Enzymatic techniques and mass spectrometric analysis could be used to determine
if NNK-induced changes in BER activity were due to alterations in post-translational
modifications on specific proteins. These types of experiments would ultimately investigate
components of NNK’s mechanism of action and could be helpful in identifying susceptible
individuals and future development of novel preventative therapies or treatments for tobacco-
induced cancer.
68
REFERENCES
Achanta G and Huang P (2004) Role of p53 in sensing oxidative DNA damage in response to
reactive oxygen species-generating agents. Cancer Res 64:6233-6239.
Auerbach O and Garfinkel L (1991) The changing pattern of lung carcinoma. Cancer 68:1973-
1977.
Bedard LL, Alessi M, Davey S and Massey TE (2005) Susceptibility to aflatoxin B1-induced
carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in
rat. Cancer Res 65:1265-1270.
Belinsky SA (1998) Role of the cytosine DNA-methyltransferase and p16INK4a genes in the
development of mouse lung tumors. Exp Lung Res 24:463-479.
Belinsky SA, Devereux TR, Maronpot RR, Stoner GD and Anderson MW (1989) Relationship
between the formation of promutagenic adducts and the activation of the K-ras protooncogene in
lung tumors from A/J mice treated with nitrosamines. Cancer Res 49:5305-5311.
Belinsky SA, Dolan ME, White CM, Maronpot RR, Pegg AE and Anderson MW (1988) Cell
specific differences in O6-methylguanine-DNA methyltransferase activity and removal of O6-
methylguanine in rat pulmonary cells. Carcinogenesis 9:2053-2058.
Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E, Baylin SB
and Herman JG (1998) Aberrant methylation of p16(INK4a) is an early event in lung cancer and
a potential biomarker for early diagnosis. Proc Natl Acad Sci USA 95:11891-11896.
Bertram JS (2000) The molecular biology of cancer. Mol Aspects Med 21:167-223.
Boffetta P, Hecht S, Gray N, Gupta P and Straif K (2008) Smokeless tobacco and cancer. Lancet
Oncology 9:667-675.
Bolin C, Stedeford T and Cardozo-Pelaez F (2004) Single extraction protocol for the analysis of
8-hydroxy-2'-deoxyguanosine (oxo8dG) and the associated activity of 8-oxoguanine DNA
glycosylase. J Neurosci Methods 136:69-76.
Borczuk AC, Gorenstein L, Walter KL, Assaad AA, Wang L and Powell CA (2003) Non-small-
cell lung cancer molecular signatures recapitulate lung developmental pathways. Am J Pathol
163:1949-1960.
Branzei D and Foiani M (2008) Regulation of DNA repair throughout the cell cycle. Nat Rev Mol
Cell Biol 9:297-308.
Brown PJ, Bedard LL and Massey TE (2007) Repair of 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone-induced DNA pyridyloxobutylation by nucleotide excision repair. Cancer Lett.
69
Brown PJ and Massey TE (2009) In vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) induces organ-specific alterations in in vitro repair of DNA
pyridyloxobutylation. Mutat Res 663:15-21.
Brunnemann KD, Prokopczyk B, Djordjevic MV and Hoffman D (1996) Formation and analysis
of tobacco-specific N-nitrosamines. Critical Reviews in Toxicology 26:121-137.
Cadet J, D'Ham C, Douki T, Pouget JP, Ravanat JL and Sauvaigo S (1998) Facts and artifacts in
the measurement of oxidative base damage to DNA. Free Rad Res 29:541-550.
Caldecott KW, McKeown CK, Tucker JD, Ljungquist S and Thompson LH (1994) An interaction
between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol Cell Biol 14:68-
76.
Canadian Cancer Society. (2010). Canadian Cancer Statistics. www.cancer.ca. Accessed: March
2010.
Carmella SG, Borukhova A, Desai D and Hecht SS (1997) Evidence for endogenous formation of
tobacco-specific nitrosamines in rats treated with tobacco alkaloids and sodium nitrite.
Carcinogenesis 18:587-592.
Castonguay A, Lin D, Stoner GD, Radok P, Furuya K, Hecht SS, Schut HA and Klaunig JE
(1983) Comparative carcinogenicity in A/J mice and metabolism by cultured mouse peripheral
lung of N'-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and their
analogues. Cancer Res 43:1223-1229.
Castonguay A, Pepin P and Briere N (1991) Modulation of 4-(methylnitrosamino)-1-(3-pyridyl)-
1 butanone demethylation and denitrosation by rat liver microsomes. Cancer Lett 59:67-74.
Chen B, Johanson L, Wiest JS, Anderson MW and You M (1994) The second intron of the K-ras
gene contains regulatory elements associated with mouse lung tumor susceptibility. Proc Natl
Acad Sci USA 91:1589-1593.
Chen D, Lan J, Pei W and Chen J (2000) Detection of DNA base-excision repair activity for
oxidative lesions in adult rat brain mitochondria. J Neurosci Res 61:225-236.
Cheng KC, Cahill DS, Kasai H, Nishimura S and Loeb LA (1992) 8-Hydroxyguanine, an
abundant form of oxidative DNA damage, causes G---- T and A----C substitutions. J Biol Chem
267:166-172.
Chmiel NH, Livingston AL and David SS (2003) Insight into the functional consequences of
inherited variants of the hMYH adenine glycosylase associated with colorectal cancer:
complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding
mutated E.coli enzymes. Journal of Molecular Biology 327:431-443.
Christmann M, Tomicic MT, Roos WP and Kaina B (2003) Mechanisms of human DNA repair:
an update. Toxicology 193:3-34.
70
Chung FL and Xu Y. Increased 8-oxodeoxyguanosine levels in lung DNA of A/J mice and F344
rats treated with the tobacco-specific nitrosamine 4-(methyhiitrosamine)-l-(3-pyridyl)-1-
butanone. 1992. Oxford University Press.
Chute JP, Chen T, Feigal E, Simon R and Johnson BE (1999) Twenty years of phase III trials for
patients with extensive-stage small-cell lung cancer: perceptible progress. Journal of Clinical
Oncology 17:1794-1801.
Claycamp HG (1992) Phenol sensitization of DNA to subsequent oxidative damage in 8-
hydroxyguanine assays. Carcinogenesis 13:1289-1292.
Collins AR, Cadet J, Moller L, Poulsen HE and Viňa J (2004) Are we sure we know how to
measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Archives of Biochemistry and
Biophysics 423:57-65.
Colussi C, Parlanti E, Degan P, Aquilina G, Barnes D, Macpherson P, Karran P, Crescenzi M,
Dogliotti E and Bignami M (2002) The mammalian mismatch repair pathway removes DNA 8-
oxodGMP incorporated from the oxidized dNTP pool. Current Biology 12:912-918.
Cooke MS, Evans MD, Dizdaroglu M and Lunec J (2003) Oxidative DNA damage: mechanisms,
mutation, and disease. FASEB J 17:1195-1214.
Corvalan A and Wistuba II (2010) Molecular pathology of lung cancer, in Lung Cancer:
Prevention, Management, and Emerging Therapies (Stewart DJ ed) pp 1-25.
Cunningham R (1996) A Prescription for Health, in Smoke and mirrors The Canadian Tobacco
War pp 247-265, IDRC International Development Research Centre.
David SS, O'Shea VL and Kundu S (2007) Base-excision repair of oxidative DNA damage.
Nature 447:941-950.
de Laat WL, Jaspers NG and Hoeijmakers JH (1999) Molecular mechanism of nucleotide
excision repair. Genes Dev 13:768-785.
Devesa SS, Shaw GL and Blot WJ (1991) Changing patterns of lung cancer incidence by
histological type. Cancer Epidem Biomark Prev 1:29-34.
Dianov G, Bischoff C, Piotrowski J and Bohr VA (1998) Repair pathways for processing of 8-
oxoguanine in DNA by mammalian cell extracts. J Biol Chem 273:33811-33816.
Ekstrom G and Ingelman-Sundberg M (1989) Rat liver microsomal NADPH-supported oxidase
activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1).
Biochem Pharmacol 38:1313-1319.
Epe B (1991) Genotoxicity of singlet oxygen. Chem Biol Interact 80:239-260.
71
ESCODD, Gedik CM and Collins A (2005) Establishing the background level of base oxidation
in human lymphocyte DNA: results of an interlaboratory validation study. The FASEB Journal
19:82-84.
European Standards Committee on Oxidative DNA Damage (2003) Measurement of DNA
oxidation in human cells by chromatographic and enzymic methods. Free Radic Biol Med
34:1089-1099.
Floyd RA, West MS, Eneff KL and Schneider JE (1989) Methylene blue plus light mediates 8-
hydroxyguanine formation in DNA. Arch Biochem Biophys 273:106-111.
Foiles PG, Akerkar SA, Carmella SG, Kagan M, Stoner GD, Resau JH and Hecht SS (1991)
Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and
nonsmokers. Chem Res Toxicol 4:364-368.
Fortini P and Dogliotti E (2007) Base damage and single-strand break repair: mechanisms and
functional significance of short- and long-patch repair subpathways. DNA Repair 6:398-409.
Fortini P, Parlanti E, Sidorkina OM, Laval J and Dogliotti E (1999) The type of DNA glycosylase
determines the base excision repair pathway in mammalian cells. J Biol Chem 274:15230-15236.
Fraga CG, Shigenaga MK, Park JW, Degan P and Ames BN (1990) Oxidative damage to DNA
during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci USA
87:4533-4537.
Giovino GA (2002) Epidemiology of tobacco use in the United States. onc 21:7326-7340.
Guindon KA, Bedard LL and Massey TE (2007) Elevation of 8-hydroxydeoxyguanosine in DNA
from Isolated Mouse Lung Cells following In Vivo Treatment with aflatoxin B1. Toxicol Sci.
Halliwell B and Aruoma OI (1991) DNA damage by oxygen-derived species: Its mechanism and
measurement in mammalian systems. FEBS Lett 281:9-19.
Halliwell B and Whiteman M (2004) Measuring reactive species and oxidative damage in vivo
and in cell culture: how should you do it and what do the results mean? British Journal of
Pharmacology 142:231-255.
Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70.
Hanrahan EO and Glisson B (2010) Small cell carcinoma, in Lung Cancer: Prevention,
Management, and Emerging Therapies (Stewart DJ ed) pp 395-434.
Hardeland U, Steinacher R, Jiricny J and Schar P (2002) Modification of the human thymine-
DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover. EMBO J 21:1456-
1464.
72
Hazra TK, Izumi T, Kow YW and Mitra S (2003) The discovery of a new family of mammalian
enzymes for repair of oxidatively damaged DNA, and its physiological implications.
Carcinogenesis 24:155-157.
Health Canada. (2010). Tobacco Health Concerns. www.hc-sc.gc.ca/hc-ps/tobac-tabac/index-
eng.php. Accessed: October 2010.
Hecht SS (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines.
Chem Res Toxicol 11:559-603.
Hecht SS (1999) Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 91:1194-1210.
Hecht SS (2002) Cigarette smoking and lung cancer: chemical mechanisms and approaches to
prevention Lancet Oncol 3:461-469.
Hecht SS (2003) Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev
Cancer 3:733-744.
Hecht SS, Chen CB, Hirota N, Ornaf RM, Tso TC and Hoffmann D (1978) Tobacco-specific
nitrosamines: formation from nicotine in vitro and during tobacco curing and carcinogenicity in
strain A mice. J Natl Cancer Inst 60:819-824.
Hecht SS and Hoffmann D (1988) Tobacco-specific nitrosamines, an important group of
carcinogens in tobacco and tobacco smoke. Carcinogenesis 9:875-884.
Hecht SS, Morse MA, Amin S, Stoner GD, Jordan KG, Choi CI and Chung FL (1989) Rapid
single-dose model for lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-
1-butanone and the effect of diet. Carcinogenesis 10:1901-1904.
Hecht SS, Trushin N, Castonguay A and Rivenson A (1986) Comparative tumorigenicity and
DNA methylation in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-
nitrosodimethylamine. Cancer Res 46:498-502.
Hecht SS, Adams JD, Numoto S and Hoffmann D (1983) Induction of respiratory tract tumors in
Syrian golden hamsters by a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) and the effect of smoke inhalation. Carcinogenesis 4:1287-1290.
Hecht SS, Chen Ch, Ohmori T and Hoffmann D (1980) Comparative Carcinogenicity in F344
Rats of the Tobacco-specific Nitrosamines, N Ǧ-Nitrosonornicotine and 4-(N-Methyl-N-
nitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res 40:298-302.
Heidelberger C (1977) Chemical carcinogenesis. Cancer 40:Suppl-3.
Herzog CR, Noh S, Lantry LE, Guan KL and You M (1999) Cdkn2a encodes functional variation
of p16INK4a but not p19ARF, which confers selection in mouse lung tumorigenesis. Mol
Carcinog 25:92-98.
73
Hoffmann D, Hoffmann I and El Bayoumy K (2001) The less harmful cigarette: a controversial
issue. a tribute to Ernst L. Wynder. Chem Res Toxicol 14:767-790.
Hoffmann D, Rivenson A, Amin S and Hecht SS (1984) Dose-response study of the
carcinogenicity of tobacco-specific N-nitrosamines in F344 rats. J Cancer Res Clin Oncol
108:81-86.
Holzle D, Schlobe D, Tricker AR and Richter E (2007) Mass spectrometric analysis of 4-
hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung. Toxicology 232:277-
285.
Horio Y, Chen A, Rice P, Roth JA, Malkinson AM and Schrump DS (1996) Ki-ras and p53
mutations are early and late events, respectively, in urethane-induced pulmonary carcinogenesis
in A/J mice. Mol Carcinog 17:217-223.
Huang TT and D'Andrea AD (2006) Regulation of DNA repair by ubiquitylation. Nat Rev Mol
Cell Biol 7:323-334.
Huang X, Powell J, Mooney LA, Li C and Frenkel K (2001) Importance of complete DNA
digestion in minimizing variability of 8-oxo-dG analyses. Free Radic Biol Med 31:1341-1351.
Hung RJ, Brennan P, Canzian F, Szeszenia-Dabrowska N, Zaridze D, Lissowska J, Rudnai P,
Fabianova E, Mates D, Foretova L, Janout V, Bencko V, Chabrier A, Borel S, Hall J and Boffetta
P (2005a) Large-scale investigation of base excision repair genetic polymorphisms and lung
cancer risk in a multicenter study. J Natl Cancer Inst 97:567-576.
Hung RJ, Hall J, Brennan P and Boffetta P (2005b) Genetic polymorphisms in the base excision
repair pathway and cancer risk: a HuGE review. Am J Epidemiol 162:925-942.
Hussain SP, Aguilar F, Amstad P and Cerutti P (1994) Oxy-radical induced mutagenesis of
hotspot codons 248 and 249 of the human p53 gene. onc 9:2277-2281.
Igarashi M, Watanabe M, Yoshida M, Sugaya K, Endo Y, Miyajima N, Abe M, Sugano S and
Nakae D (2009) Enhancement of lung carcinogenesis initiated with 4-(N-
hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone by Ogg1 gene deficiency in female, but not
male, mice. Journal of Toxicological Sciences 34:163-174.
Jackman DM and Johnson BE (2005) Small-cell lung cancer. [Review] [159 refs]. Lancet
366:1385-1396.
Jackson JH (1994) Potential molecular mechanisms of oxidant-induced carcinogenesis. Environ
Health Perspect 102:Suppl-7.
Jalas JR, Ding X and Murphy SE (2003) Comparative metabolism of the tobacco-specific
nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanol by rat cytochrome P450 2A3 and human cytochrome P450 2A13. Drug Metab
Dispos 31:1199-1202.
74
Jalas JR, Hecht SS and Murphy SE (2005) Cytochrome P450 enzymes as catalysts of metabolism
of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Chem Res
Toxicol 18:95-110.
Kasai H (1997) Analysis of a form of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, as a
marker of cellular oxidative stress during carcinogenesis. Mutation Research/Reviews in Mutation
Research 387:147-163.
Kim PM and Wells PG (1996) Genoprotection by UDP-glucuronosyltransferases in peroxidase-
dependent, reactive oxygen species-mediated micronucleus initiation by the carcinogens 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene. Cancer Res 56:1526-1532.
Kinoshita A, Wanibuchi H, Morimura K, Wei M, Nakae D, Arai T, Minowa O, Noda T,
Nishimura S and Fukushima S (2007) Carcinogenicity of dimethylarsinic acid in Ogg1-deficient
mice. Cancer Sci 98:803-814.
Klaunig JE and Kamendulis LM (2004) The role of oxidative stress in carcinogenesis. Annu Rev
Pharmacol Toxicol 44:239-267.
Klungland A and Lindahl T (1997) Second pathway for completion of human DNA base
excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).
EMBO Journal 16:3341-3348.
Klungland A, Rosewell I, Hollenbach S, Larsen E, Daly G, Epe B, Seeberg E, Lindahl T and
Barnes DE (1999) Accumulation of premutagenic DNA lesions in mice defective in removal of
oxidative base damage. Proc Natl Acad Sci USA 96:13300-13305.
Lan J, Henshall DC, Simon RP and Chen J (2000) Formation of the base modification 8-
hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic
kainic acid in the rat. Journal of Neurochemistry 74:302-309.
Lan J, Li W, Zhang F, Sun FY, Nagayama T, O'Horo C and Chen J (2003) Inducible repair of
oxidative DNA lesions in the rat brain after transient focal ischemia and reperfusion. J Cereb
Blood Flow Metab 23:1324-1339.
LaVoie EJ, Prokopczyk G, Rigotty J, Czech A, Rivenson A and Adams JD (1987) Tumorigenic
activity of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine
(NNN) on topical application to Sencar mice. Cancer Lett 37:277-283.
Lijinsky W, Thomas BJ and Kovatch RM (1991) Local and Systemic Carcinogenic Effects of
Alkylating Carcinogens in Rats Treated by Intravesicular Administration. Cancer Science
82:980-986.
Lindahl T and Wood RD (1999) Quality control by DNA repair. Science 286:1897-1905.
75
Liu BQ, Peto R, Chen ZM, Boreham J, Wu YP, Li JY, Campbell TC and Chen JS (1998)
Emerging tobacco hazards in China: 1. Retrospective proportional mortality study of one million
deaths. BMJ 317:1411-1422.
Liu L, Castonguay A and Gerson SL (1992) Lack of correlation between DNA methylation and
hepatocarcinogenesis in rats and hamsters treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone. Carcinogenesis 13:2137-2140.
Loft S and Poulsen HE (1996) Cancer risk and oxidative DNA damage in man. Journal of
Molecular Medicine 74:297-312.
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the folin
phenol reagent. J Biol Chem 193:265-275.
Luch A (2005) Nature and nurture - lessons from chemical carcinogenesis. Nature Reviews
Cancer 5:113-125.
Lunec J (1998) ESCODD: European Standards Committee on Oxidative DNA Damage. Free
Radic Res 29:601-608.
Malkinson AM (1998) Molecular comparison of human and mouse pulmonary adenocarcinomas.
Exp Lung Res 24:541-555.
Maser E (2004) Significance of reductases in the detoxification of the tobacco-specific
carcinogen NNK. Trends in Pharmacological Sciences 25:235-237.
McBride TJ, Schneider JE, Floyd RA and Loeb LA (1992) Mutations induced by methylene blue
plus light in single-stranded M13mp2. Proc Natl Acad Sci USA 89:6866-6870.
Meunier B, de Visser SlP and Shaik S (2004) Mechanism of Oxidation Reactions Catalyzed by
Cytochrome P450 Enzymes. Chemical Reviews 104:3947-3980.
Meuwissen R and Berns A (2005) Mouse models for human lung cancer. Genes Dev 19:643-664.
Mijal RS, Thomson NM, Fleischer NL, Pauly GT, Moschel RC, Kanugula S, Fang Q, Pegg AE
and Peterson LA (2004) The Repair of the Tobacco Specific Nitrosamine Derived Adduct O6-[4-
Oxo-4-(3-pyridyl)butyl]guanine by O6-Alkylguanine-DNA Alkyltransferase Variants. Chem Res
Toxicol 17:424-434.
Miller EC and Miller JA (1981) Mechanisms of chemical carcinogenesis. Cancer 47:Suppl-64.
Minowa O, Arai T, Hirano M, Monden Y, Nakai S, Fukuda M, Itoh M, Takano H, Hippou Y,
Aburatani H, Masumura K, Nohmi T, Nishimura S and Noda T (2000) Mmh/Ogg1 gene
inactivation results in accumulation of 8-hydroxyguanine in mice. Proc Natl Acad Sci USA
97:4156-4161.
76
Murphy SE, Spina DA, Nunes MG and Pullo DA (1995) Glucuronidation of 4-
((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone, a metabolically activated form of 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone, by phenobarbital-treated rats. Chem Res Toxicol
8:772-779.
Murphy SE, Palomino A, Hecht SS and Hoffman D (1990) Dose-Response Study of DNA and
Hemoglobin Adduct Formation by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 Rats.
Cancer Res 50:5446-5452.
Mustonen R, Schoket B and Hemminki K (1993) Smoking-related DNA adducts: 32
P-
postlabeling analysis of 7-methylguanine in human bronchial and lymphocyte DNA.
Carcinogenesis 14:151-154.
Nademanee K (1992) The amiodarone odyssey. J Am Coll Cardiol 20-5:1063-1065.
Nakamura J, La DK and Swenberg JA (2000) 5'-nicked apurinic/apyrimidinic sites are resistant to
beta-elimination by beta-polymerase and are persistent in human cultured cells after oxidative
stress. J Biol Chem 275:5323-5328.
Osterdahl BG and Slorach S (1988) Tobacco-specific N-nitrosamines in the saliva of habitual
male snuff dippers. Food Additives & Contaminants 5:581-586.
Patel PR, Bevan RJ, Mistry N and Lunec J (2007) Evidence of oligonucleotides containing 8-
hydroxy-2'-deoxyguanosine in human urine. Free Radical Biology and Medicine 42:552-558.
Patten CJ, Smith TJ, Murphy SE, Wang M-H, Lee J, Tynes RE, Koch P and Yang CS (1996)
Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by
heterologously expressed human P450 enzymes and the effect of P450-specific chemical
inhibitors on this activation in human liver microsomes. Arch Biochem Biophys 333:127-138.
Paz-Elizur T, Ben-Yosef R, Elinger D, Vexler A, Krupsky M, Berrebi A, Shani A, Schechtman E,
Freedman L and Livneh Z (2006) Reduced repair of the oxidative 8-oxoguanine DNA damage
and risk of head and neck cancer. Cancer Res 66:11683-11689.
Paz-Elizur T, Krupsky M, Blumenstein S, Elinger D, Schechtman E and Livneh Z (2003) DNA
repair activity for oxidative damage and risk of lung cancer. J Natl Cancer Inst 95:1312-1319.
Paz-Elizur T, Sevilya Z, Leitner-Dagan Y, Elinger D, Roisman LC and Livneh Z (2008) DNA
repair of oxidative DNA damage in human carcinogenesis: potential application for cancer risk
assessment and prevention. Cancer Lett 266:60-72.
Petermann E, Ziegler M and Oei SL (2003) ATP-dependent selection between single nucleotide
and long patch base excision repair. DNA Repair 2:1101-1114.
Peterson LA and Hecht SS (1991) O6-Methylguanine is a critical determinant of 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenesis in A/J mouse lung. Cancer Res
51:5557-5564.
77
Peterson LA, Thomson NM, Crankshaw DL, Donaldson EE and Kenney PJ (2001) Interactions
between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis. Cancer Res 61:5757-
5763.
Peterson LA, Ng DK, Stearns RA and Hecht SS (1994) Formation of NADP(H) Analogs of
Tobacco-Specific Nitrosamines in Rat Liver and Pancreatic Microsomes. Chem Res Toxicol
7:599-608.
Pitot HC, Goldsworthy T and Moran S (1981) The natural history of carcinogenesis: implications
of experimental carcinogenesis in the genesis of human cancer. Journal of Supramolecular
Structure & Cellular Biochemistry 17:133-146.
Prokopczyk B, Cox JE, Hoffmann D and Waggoner SE (1997) Identification of tobacco-specific
carcinogen in the cervical mucus of smokers and nonsmokers. J Natl Cancer Inst 89:868-873.
Prokopczyk B, Rivenson A and Hoffmann D (1991) A study of betel quid carcinogenesis IX.
Comparative carcinogenicity of 3-(methylnitrosamino)propionitrile and 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone upon local application to mouse skin and rat oral mucosa. Cancer Lett
60:153-157.
Prokopczyk B, Trushin N, Leszczynska J, Waggoner SE and El Bayoumy K (2001) Human
cervical tissue metabolizes the tobacco-specific nitrosamine, 4- (methylnitrosamino)-1-(3-
pyridyl)-1-butanone, via alpha-hydroxylation and carbonyl reduction pathways. Carcinogenesis
22:107-114.
Puntarulo S and Cederbaum AI (1998) Production of Reactive Oxygen Species by Microsomes
Enriched in Specific Human Cytochrome P450 Enzymes. Free Radical Biology and Medicine
24:1324-1330.
Ravanat JL (2005a) Measuring Oxidized DNA Lesions as Biomarkers of Oxidative Stress : An
Analytical Challenge. FABAD Journal of Pharmaceutical Sciences 30:100-113.
Ravanat JL (2005b) Oxidized DNA Lesions as Biomarkers of Oxidative Stress : An Analytical
Challenge. FABAD Journal of Pharmaceutical Sciences 30:100-113.
Reardon JT, Bessho T, Kung HC, Bolton PH and Sancar A (1997) In vitro repair of oxidative
DNA damage by human nucleotide excision repair system: possible explanation for
neurodegeneration in xeroderma pigmentosum patients. Proc Natl Acad Sci U S A 94:9463-9468.
Rice JM, Diwan BA, Hu H, Ward JM, Nims RW and Lubet RA (1994) Enhancement of
hepatocarcinogenesis and induction of specific cytochrome P450-dependent monooxygenase
activities by the barbiturates allobarbital, aprobarbital, pentobarbital, secobarbital and 5-phenyl-
and 5-ethylbarbituric acids. Carcinogenesis 15:395-402.
Rivenson A, Hoffmann D, Prokopczyk B, Amin S and Hecht SS (1988) Induction of lung and
exocrine pancreas tumors in F344 rats by tobacco-specific and Areca-derived N-nitrosamines.
Cancer Res 48:6912-6917.
78
Robertson AB, Klungland A, Rognes T and Leiros I (2009) DNA repair in mammalian cells:
Base excision repair: the long and short of it. Cellular & Molecular Life Sciences 66:981-993.
Ronai ZA, Gradia S, Peterson LA and Hecht SS (1993) G to A transitions and G to T
transversions in codon 12 of the Ki-ras oncogene isolated from mouse lung tumors induced by 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related DNA methylating and
pyridyloxobutylating agents. Carcinogenesis 14:2419-2422.
Rosa JG, Prokopczyk B, Desai DH, Amin SG and el-Bayoumy K (1998) Elevated 8-hydroxy-2'-
deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-
phenylenebis(methylene)selenocyanate. Carcinogenesis 19:1783-1788.
Russo MT, De Luca G, Degan P, Parlanti E, Dogliotti E, Barnes DE, Lindahl T, Yang H, Miller
JH and Bignami M (2004) Accumulation of the Oxidative Base Lesion 8-Hydroxyguanine in
DNA of Tumor-Prone Mice Defective in Both the Myh and Ogg1 DNA Glycosylases. Cancer
Res 64:4411-4414.
Sakamoto K, Tominaga Y, Yamauchi K, Nakatsu Y, Sakumi K, Yoshiyama K, Egashira A, Kura
S, Yao T, Tsuneyoshi M, Maki H, Nakabeppu Y and Tsuzuki T (2007) MUTYH-null mice are
susceptible to spontaneous and oxidative stress induced intestinal tumorigenesis. Cancer Res
67:6599-6604.
Sakumi K, Tominaga Y, Furuichi M, Xu P, Tsuzuki T, Sekiguchi M and Nakabeppu Y (2003)
Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption.
Cancer Res 63:902-905.
Salles B, Frit P, Provot C, Jaeg JP and Calsou P (1995) In vitro eukaryotic DNA excision repair
assays: an overview. Biochimie 77:796-802.
Sattler U, Frit P, Salles B and Calsou P (2003) Long-patch DNA repair synthesis during base
excision repair in mammalian cells. EMBO Reports 4:363-367.
Schneider JE, Price S, Maidt L, Gutteridge JM and Floyd RA (1990) Methylene blue plus light
mediates 8-hydroxy 2'-deoxyguanosine formation in DNA preferentially over strand breakage.
Nucleic Acids Research 18:631-635.
Schwartz AG (2004) Genetic predisposition to lung cancer. Chest 125:Suppl-9S.
Sekido Y, Fong KM and Minna JD (1998) Progress in understanding the molecular pathogenesis
of human lung cancer. Biochim Biophys Acta 1378:F21-F59.
Sekido Y, Fong KM and Minna JD (2003) Molecular genetics of lung cancer. Annual Review of
Medicine 54:73-87.
Slebos RJC, Hruban RH, Dalesio O, Mooi WJ, Offerhaus GJA and Rodenhuis S (1991)
Relationship between K-ras oncogene activation and smoking in adenocarcinoma of the human
lung. J Natl Cancer Inst 83:1024-1027.
79
Smith GBJ, Bend JR, Bedard LL, Reid KR, Petsikas D and Massey TE (2003) Biotransformation
of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in peripheral human lung microsomes.
Drug Metab Dispos 31:1134-1141.
Smith TJ, Guo Z, Guengerich FP and Yang CS (1996) Metabolism of 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone (NNK) by human cytochrome P450 1A2 and its inhibition by phenethyl
isothiocyanate. Carcinogenesis 17:809-813.
Smith TJ, Stoner GD and Yang CS (1995) Activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) in human lung microsomes by cytochromes p450, lipoxygenase, and
hydroperoxides. Cancer Res 55:5566-5573.
Spiegelhalder B and Bartsch H (1996) Tobacco-specific nitrosamines. Eur J Cancer Prev
5:Suppl-8.
Srivastava DK, Berg BJ, Prasad R, Molina JT, Beard WA, Tomkinson AE and Wilson SH (1998)
Mammalian abasic site base excision repair. Identification of the reaction sequence and rate-
determining steps. J Biol Chem 273:21203-21209.
Steinacher R and Schär P (2005) Functionality of Human Thymine DNA Glycosylase Requires
SUMO-Regulated Changes in Protein Conformation. Current Biology 15:616-623.
Stoner GD (1998) Introduction to mouse lung tumorigenesis. Exp Lung Res 24:375-383.
Stucki M, Pascucci B, Parlanti E, Fortini P, Wilson SH, Hubscher U and Dogliotti E (1998)
Mammalian base excision repair by DNA polymerases delta and epsilon. onc 17:835-843.
Su T, Bao Z, Zhang QY, Smith TJ, Hong JY and Ding X (2000) Human cytochrome P450
CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic
activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone.
Cancer Res 60:5074-5079.
Subramanian J, and Govindan R (2007) Lung cancer in never smokers: a review. J Clin Oncol
25:561-70.
Tjaelve H (1991) The tissue distribution and the tissue specificity of bioactivation of some
tobacco-specific and some other N- nitrosamines. CRC Crit Rev Toxicol 21:265-294.
Travis WD, Brambilla E, Muller-Hermelink HK and Harris CC (2004) Pathology and genetics of
tumours of the lung, pleura, thymus and heart . Lyon : IARC Press, Oxford : Oxford University
Press .
Vogel U, Nexo BA, Wallin H, Overvad K, Tjonneland A and Raaschou-Nielsen O (2004) No
association between base excision repair gene polymorphisms and risk of lung cancer.
Biochemical Genetics 42:453-460.
80
Wang L, Spratt TE, Liu X-K, Hecht SS, Pegg AE and Peterson LA (1997) Pyridyloxobutyl
adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-
pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase.
Chem Res Toxicol 10:562-567.
Wei Q and Spitz MR (1997) The role of DNA repair capacity in susceptibility to lung cancer: a
review. Cancer Metastasis Rev 16:295-307.
Weitberg AB and Corvese D (1993) Oxygen radicals potentiate the genetic toxicity of tobacco-
specific nitrosamines. Clin Genet 43:88-91.
Westra WH, Slebos RJ, Offerhaus GJ, Goodman SN, Evers SG, Kensler TW, Askin FB,
Rodenhuis S and Hruban RH (1993) K-ras oncogene activation in lung adenocarcinomas from
former smokers. Evidence that K-ras mutations are an early and irreversible event in the
development of adenocarcinoma of the lung. Cancer 72:432-438.
Wilson VL, Weston A, Manchester DK, Trivers GE, Roberts DW, Kadlubar FF, Wild CP,
Montesano R, Willey JC and Mann DL (1989) Alkyl and aryl carcinogen adducts detected in
human peripheral lung. Carcinogenesis 10:2149-2153.
Wiseman H and Halliwell B (1996) Damage to DNA by reactive oxygen and nitrogen species:
role in inflammatory disease and progression to cancer. Biochem J 313 ( Pt 1):17-29.
Wood AJJ, Spira A and Ettinger DS (2004) Multidisciplinary Management of Lung Cancer. New
Eng J Med 350:379-392.
Wood RD, Biggerstaff M and Shivji MKK (1995) Detection and Measurement of Nucleotide
Excision Repair Synthesis by Mammalian Cell Extracts in Vitro. Methods: A Companion to
Methods in Enzymology 7:163-175.
Wood RD, Robins P and Lindahl T (1988) Complementation of the xeroderma pigmentosum
DNA repair defect in cell- free extracts. Cell 53:97-106.
World Health Organization. (2010). Tobacco Free Initiative. www.who.int/tobacco/en/.
Accessed: March 2010.
Wynder EL and Hoffmann D (1994) Smoking and lung cancer: scientific challenges and
opportunities. Cancer Res 54:5284-5295.
Xie Y, Yang H, Cunanan C, Okamoto K, Shibata D, Pan J, Barnes DE, Lindahl T, McIlhatton M,
Fishel R and Miller JH (2004) Deficiencies in mouse Myh and Ogg1 result in tumor
predisposition and G to T mutations in codon 12 of the K-ras oncogene in lung tumors. Cancer
Res 64:3096-3102.
Xu Y, Ho CT, Amin SG, Han C and Chung FL (1992) Inhibition of tobacco-specific nitrosamine-
induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants.
Cancer Res 52:3875-3879.
81
Yang CS and Smith TJ (1996) Mechanisms of nitrosamine bioactivation and carcinogenesis.
Advances in experimental medicine and biology 387:385-394.
You M, Wang D, Liu P, Vikis H, James M, Lu Y, Wang Y, Wang M, Chen Q, Jia D, Liu Y, Wen
W, Yang P, Sun Z, Pinney SM, Zheng W, Shu XO, Long J, Gao YT, Xiang YB, Chow WH,
Rothman N, Petersen GM, de Andrade M, Wu Y, Cunningham JM, Wiest JS, Fain PR, Schwartz
AG, Girard L, Gazdar A, Gaba C, Rothschild H, Mandal D, Coons T, Lee J, Kupert E, Seminara
D, Minna J, Bailey-Wilson JE, Amos CI and Anderson MW (2009) Fine Mapping of
Chromosome 6q23-25 Region in Familial Lung Cancer Families Reveals RGS17 as a Likely
Candidate Gene. Clinical Cancer Research 15:2666-2674.
You M, Wang Y, Nash B and Stoner GD (1993) SHORT COMMUNICATION: K-ras mutations
in benzotrichloride-induced lung tumors of A/J mice. Carcinogenesis 14:1247-1249.
Yu BP (1994) Cellular defenses against damage from reactive oxygen species. Physiological
Reviews 74:139-162.
Zhang H, Xu Y, Kamendulis LM and Klaunig JE (2000) Morphological transformation by 8-
hydroxy-2'-deoxyguanosine in Syrian hamster embryo (SHE) cells. Toxicological Sciences
56:303-312.
Zhang YT, Zheng QS, Pan J and Zheng RL (2004) Oxidative damage of biomolecules in mouse
liver induced by morphine and protected by antioxidants. Basic Clin Pharmacol Toxicol 95:53-
58.
Zhang Z, Wang Y, Herzog CR, Liu G, Lee HW, DePinho RA and You M (2002) A strong
candidate gene for the Papg1 locus on mouse chromosome 4 affecting lung tumor progression.
onc 21:5960-5966.
Zhou J, Ahn J, Wilson SH and Prives C (2001) A role for p53 in base excision repair. EMBO J
20:914-923.