Genetic polymorphism in the conjugating enzyme UGT1A1 and the risk of head and neck cancer.

Abstract

UDP-glucuronosyltransferase 1A1 (UGT1A1) is an enzyme which catalyses not only the glucuronidation of tobacco smoke carcinogens like benzopyrene, but also of the endogenous substrate bilirubin. Bilirubin for a long time was considered to be only a toxic waste product of hemoglobin degradation, but recent findings have shown that bilirubin is a potent antioxidant, which may play a protective role against cancer. We investigated whether a genetic polymorphism in UGT1A1 (UGT1A1*28), associated with a reduced UGT1A1 enzyme activity, may have a risk-modifying effect on head and neck carcinogenesis. Blood samples from 421 patients with oral, pharyngeal or laryngeal carcinoma, and 417 healthy controls were investigated for the UGT1A1*28 polymorphism. On the basis of the occurrence of this polymorphism, patients and controls were divided according to predicted UGT1A1 enzyme activity (low, intermediate, high). Logistic regression analysis showed a significant increased distribution of predicted high activity UGT1A1*1 polymorphisms among the patients (OR: 1.37; 95% CI: 1.02-1.83). Stratified analyses demonstrated that predicted high activity UGT1A1 polymorphisms were present even more significantly in patients with laryngeal cancer, older patients, heavy smokers and heavy drinkers. In conclusion, the predicted high activity UGT1A1*1 polymorphism, which results in lower serum levels of the endogenous antioxidant bilirubin, was associated with an increased risk of head and neck cancer.

Full text

Page 1

Genetic polymorphisms in some
biotransformation enzymes and their impact
on head and neck cancer susceptibility

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© 2011 M. Lacko, Maastricht, The Netherlands

ISBN 978 94 6159 059 6
Production: Datawyse bv | Universitaire Pers Maastricht
Cover painting: Jan Steen (1625-1679), “Soo voer gesongen, soo na gepepen” (c.1665)

Publishing of this thesis was financially supported by:
Dutch sponsors: Atos Medical BV, Glaxo Smith Kline BV, Meditop Medical Products BV, Stallergenes BV
Slovak sponsors: NovaMed spol. s r.o., European Club - Slovakia

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Genetic polymorphisms in some
biotransformation enzymes and their impact
on head and neck cancer susceptibility



PROEFSCHRIFT

Ter verkrijging van de graad van doctor
aan de Universiteit Maastricht,
op gezag van de Rector Magnificus,
Prof. mr. G.P.M.F. Mols.
volgens het besluit van het College van Decanen,
in het openbaar te verdedigen
op donderdag 23 juni 2011 om 16.00 uur

door

Martin Lacko
Geboren op 18 april 1966 te Banska Bystrica (Slowakije)

UNIVERSITAIRE
PERS MAASTRICHT
U
P
M

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Promotores:
Prof. dr. J.J. Manni
Prof. dr. B. Kremer

Copromotor:
Dr. W.H.M. Peters (Universitair Medisch Centrum St. Radboud, Nijmegen)

Beoordelingscommissie:
Prof. dr. F.C.S. Ramaekers (voorzitter)
Prof. dr. A.J.M. Balm (Nederlands Kanker Instituut-AvL ziekenhuis, Amsterdam)
Prof. dr. M.F. von Meyenfeldt
Prof. dr. F.J. van Schooten

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To my wife Esther,
to my children Lukas, Nicolai, Lara
and to my parents

Page 6

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Contents
Chapter 1
General introduction and outline of this thesis...........................................................9

Chapter 2
Microsomal epoxide hydrolase genotypes and the risk for head and neck cancer..29

Chapter 3
COX-2 polymorphisms and the risk for head and neck cancer in white patients......45

Chapter 4
Genetic polymorphisms in the tobacco smoke carcinogens detoxifying enzyme
UGT1A7 and the risk of head and neck cancer..........................................................57

Chapter 5
Genetic polymorphism in the conjugating enzyme UGT1A1 and the risk of
head and neck cancer................................................................................................71

Chapter 6
Combined effect of genetic polymorphisms in phase I and II biotransformation
enzymes on head and neck cancer risk ....................................................................85

Chapter 7
Summary, future implications and perspectives of this research...........................107
Nederlandse samenvatting (Summary in Dutch) ....................................................115
Slovenský súhrn (Summary in Slovak) ....................................................................123

Acknowledgment ....................................................................................................131
Curriculum vitae .....................................................................................................135

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9

Chapter 1
General introduction and outline of
this thesis

Part of this chapter is published as:
Genetic polymorphisms of smoking-related carcinogen detoxifying enzymes and
head and neck cancer susceptibility

Martin Lacko
Michael B. Oude Ophuis
Wilbert H.M. Peters
Johannes J. Manni









Anticancer Research 2009; 29: 753–761.

Page 10

Chapter 1

10
Introduction
Squamous cell carcinoma of the head and neck (SCCHN) including cancer of the oral
cavity, pharynx and larynx, worldwide accounts for about 650,000 new cases annu-
ally. Recent estimates have indicated that SCCHN is the fifth most common cancer,
resulting in approximately 300,000 deaths annually.1 The incidence and mortality of
SCCHN vary with geographical location, race and gender.

Exposure to tobacco and tobacco smoke, consumption of alcohol and infection by
oncogenic serotypes of Human Papilloma Virus (HPV), are considered to be the
most important etiological factors for the development of SCCHN.2–6 Poor oral hy-
giene, betel nut or shamma chewing, occupational exposure to carcinogenic chemi-
cals as well as infection with Human Immunodeficiency Virus (HIV) or Ebstein-Barr
Virus (EBV) are also associated with an increased risk of head and neck cancer.7–15

With 2346 new cases in 2006 in The Netherlands, SCCHN occupied the sixth place in
the total cancer incidence ranking for men and the tenth place for women. SCCHN is
responsible for 4.1% of the cancer incidence in this country.16
Tobacco smoke induced carcinogenesis and genetic susceptibility
The incidence of SCCHN in tobacco and alcohol consumers is significantly higher
compared to non-consumers. More than 60 carcinogens have been identified in
tobacco smoke and at least 16 in unburned tobacco.17 Polycyclic aromatic hydrocar-
bons (PAHs) such as benzo(a)pyrene (BaP) together with tobacco specific nitrosa-
mines and aromatic amines are the most important tobacco related carcinogens.17

Although the chance of developing SCCHN increases with the level of tobacco smok-
ing and alcohol consumption, it is obvious that not every (heavy) smoker and/or
(excessive) alcohol consumer develops head and neck cancer. Not only the level of
smoking, but also whether the (pro)carcinogens in tobacco smoke are activated or
detoxified by phase I and phase II biotransformation enzymes present in the epithe-
lial cells of the upper aerodigestive tract (UADT), will influence the extent of expo-
sure of the UADT tract to carcinogens. The risk for an individual to develop SCCHN
after exposure to tobacco carcinogens may therefore also depend on alterations in
the activity of the biotransformation enzymes, (either increasing or decreasing)

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Introduction

11
which may be the result of sequence variations in the genes (genetic polymor-
phisms) coding for these enzymes. This implies, that not only the exposure to the
potential carcinogens, but also other factors such as genetically determined inter-
individual differences in the metabolism and excretion of tobacco smoke carcino-
gens as well as variation in the activity of other important enzymes involved in pro-
tection against cancer, may play an important role in the development of SCCHN.
The presence of genetic susceptibility in the pathogenesis of SCCHN is strongly sug-
gested by the higher incidence of these cancers in first-degree relatives of patients
with SCCHN.18

Carcinogens and activated procarcinogens in tobacco smoke may react with the
DNA of exposed tissues, such as the epithelial cells of the UADT. This can lead to the
formation of DNA adducts and subsequently to mutations in crucial genes such as
oncogenes or tumor suppressor genes, ultimately resulting in the development of
cancer.19 However, as mentioned above, these processes may be under the influ-
ence of biotransformation or detoxification enzymes, which are essential for the
metabolism and subsequent excretion of carcinogens. Detoxification of tobacco
smoke (pro)carcinogens, together with DNA repair and apoptotic pathways for cells
with deformed DNA, probably are the most important rescue pathways in prevent-
ing the development of tobacco induced SCCHN,17,20 (see Figure 1).

Tobacco
(pro)carcinogens
Metabolic
activation
Persistence of
miscoding
Excretion
Phase I
Phase II
DNA adducts
Normal DNA
Mutation of
crucial genes
SCCHN
Lung cancer
etc
Apoptosis
Biotransformation
DNA Repair

Figure 1. Simplified and modified scheme of tobacco smoke-related carcinogenesis.17

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Chapter 1

12
The biotransformation of many tobacco smoke (pro)carcinogens such as BaP takes
place in two phases: transformation of the mostly lipophilic compounds into more
polar molecules (phase I), followed by conjugation reactions (phase II). The latter
conversion into more water-soluble compounds in general makes them less biologi-
cally active and facilitates the excretion of the toxins and carcinogens from the
body, which diminishes the exposure of the tissues to these compounds.

However, the phase I reactions, mediated by enzymes such as cytochrome P-450
(CYP) and microsomal epoxide hydrolase (mEH), often result in the formation of
phenols, epoxides and other reactive intermediates, that eventually can be trans-
formed into highly carcinogenic electrophilic compounds such as BaP-diol-
epoxides.21,22 These compounds, in the absence of a rapid further intervention by
phase II conjugating enzymes such as glutathione S-transferases (GSTs) and uridine
5’-diphosphate (UDP)-glucuronosyltransferases (UGTs), may form DNA adducts and
subsequently initiate carcinogenesis (see Figure 2).

Since the UADT is in direct contact with potentially toxic and/or carcinogenic agents
present in tobacco smoke, the mucosa of the UADT acts as a first-line barrier. En-
zymes of the phase I and II biotransformation pathways present in the epithelial
cells of the UADT therefore play an important role in the metabolism and the excre-
tion of tobacco smoke (pro)carcinogens23 and protect this first-line barrier cells
against these (pro)carcinogens.

The activity of the enzymes involved in biotransformation may differ between indi-
viduals. It is well-known now, that genetic polymorphisms like single nucleotide
polymorphisms (SNPs) in the above mentioned enzymes occur, resulting in func-
tional abnormalities, which may be one of the explanations for the differences in
inter-individual susceptibility for the development of SCCHN.24–26

Genetic polymorphisms in these enzymes, which potentially might modify the sus-
ceptibility for head and neck cancer, are discussed below.

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Introduction

13


Figure 2. Scheme of biotransformation pathways and metabolic activation of six important tobacco smoke carcinogens.
(Reprinted with permission from Macmillan Publishers LtD: Nature Review Cancer17, copyright 2003, http://www.nature.com/nrc/index.html)
a: Benzo[a] pyrene (BaP) pathway; b: 4-(Methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK) pathway; c: N-Nitrosodimethylamine (NDMA) pathway;
d: N-Nitrosonornicotine (NNN) pathway; e: Ethylene oxide pathway; f: 4-Aminobiphenyl (4-ABP) pathway; Phase I enzymes: Cytochrome P450 (P450),
Epoxide hydrolase (EH) Phase II enzymes: Glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT), N-acetyl transferase (NAT)

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Chapter 1

14
Microsomal epoxide hydrolase (mEH)
The mEH is one of the phase I enzymes involved in biotransformation and
(de)toxification of potential tobacco smoke (pro)carcinogens. mEH is present in
microsomes derived from the endoplasmatic reticulum and is highly expressed in
most tissues, among them also the mucosa of the UADT.27,28 This enzyme has a role
in both activation and detoxification of environmental (pro)carcinogens. mEH cata-
lyzes the hydrolysis of reactive epoxide intermediates, in preparation for conjuga-
tion reactions (phase II detoxification) and excretion. However, in collaboration with
CYP enzymes, mEH may activate the PAHs present in tobacco smoke, such as BaP,
leading to highly reactive carcinogenic diol-epoxides.22,29

The gene coding for mEH (EPHX1) covers nine exons and is located on chromosome
1q42.1. There are two known amino acid-altering polymorphisms in the EPHX1
gene, which may lead to changes in mEH enzyme activity. The exon 3 polymor-
phism, with corresponding substitution of histidine for tyrosine at position 113 of
the enzyme, is associated with a 40–50% decrease in mEH activity.30 The exon 4
polymorphism, with substitution of arginine for histidine at position 139, may in-
crease mEH activity by approximately 25%.30, 31 According to Benhamou et al. the
expected mEH activity can be classified as low, intermediate or high, depending on
the combinations of the exon 3 and exon 4 polymorphisms on the two alleles,32 (see
Table 2, Chapter 2). Because a higher activity of mEH can be associated with higher
concentrations of carcinogenic diol-epoxides in the mucosa of the UADT, a subpopu-
lation of tobacco smokers with the predicted high activity mEH polymorphisms,
might have a higher risk of SCCHN compared to the subpopulations with intermedi-
ate or low mEH activity polymorphisms.

Several studies have investigated the role of mEH polymorphisms in head and neck
carcinogenesis,26,33–36 (see Table 4, Chapter 2). However, only Jourenkova-Mironova
et al. found a significant increased risk of oral, pharyngeal and laryngeal cancer in
their study population of French Caucasian smokers with predicted high and inter-
mediate mEH activity polymorphisms, as compared to the predicted low mEH activ-
ity subpopulation.26 Park et al. found that the predicted high mEH activity polymor-
phisms were significantly associated with an increased risk for oral and laryngeal
cancer only in heavy smoking (>35 packyears) Caucasians, but not in African-
American subjects.33 Unfortunately, they did not report on the predicted mEH activ-

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Introduction

15
ity (low, intermediate, high) and oropharyngeal cancer risk in their population.
Amador et al. observed a higher incidence of the predicted high activity mEH
Tyr/Tyr genotype in patients with oropharyngeal cancer as compared to a control
population.35 Wenghoefer et al. found no association between the mEH polymor-
phisms with predicted high enzyme activity and an increased risk for SCCHN.34 To-
Figueras et al. reported an increased risk of laryngeal cancer among a Spanish-
Caucasian study population with predicted high mEH activity genotypes in combina-
tion with the 105Ile/105Ile variant of glutathione S-transferase P1 (GSTP1).36 How-
ever, none of the mEH polymorphisms alone were associated with an altered risk of
laryngeal cancer.

Baxter et al.37 recently suggested that the PCR-restriction fragment length polymor-
phism (PCR-RFLP) assay, which was often used in the exon 3 mEH genotyping re-
search, might be unreliable. Due to the possible presence of an additional polymor-
phism in codon 119, the use of a primer adhering to the region containing codon
119 might falsely lead to an apparent 113 His/His genotype instead of the 113
His/Tyr variant. The methods applied by Jourenkova-Mironova et al.,26 Park et al.33
and Amador et al.35 may be inaccurate because of the use of a primer covering
codon 119, whereas the methods for estimating the exon 3 polymorphism applied
by Wenghoefer et al.34 and To-Figueras et al.36 were not potentially inaccurate,
since no primer adhering to codon 119 was used here.
Glutathione and glutathione S-transferases (GSTs)
Glutathione (GSH) is an intracellular thiol that neutralizes (pro)carcinogenic and
highly reactive electrophilic compounds, a process catalyzed by GSTs. GSTs are a
family of cytosolic enzymes, involved in phase II biotransformation.38,39 GSH is pro-
duced mainly in the liver (hepatocytes), by coupling of the amino acids glycine, cys-
teine and glutamic acid.40 Hepatic glutathione is transported to most other tissues
via the blood.40 High levels of glutathione have been demonstrated in mucosal cells
of oral/oropharyngeal and laryngeal tissues.41 When the GSH production is reduced
or GSH is depleted, reactive electrophilic compounds may freely circulate and may
cause damage of DNA or other important biomolecules. Since detoxification by GSH
is strongly dependent on the GST enzymes, a reduction or deficiency of GST iso-
forms may also result in more DNA damage.38,40,42 Any factor that may disturb the