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* Molecular Epidemiology Unit and
Molecular Vascular Medicine Unit, School of Medicine, University of Leeds, LS2 9JT, U.K.
1Correspondence: Molecular Epidemiology Unit, Algernon Firth Building, School of Medicine, University of Leeds, LS2 9JT, U.K. E-Mail: c.p.wild{at}leeds.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Key Word: epoxide hydrolase • acute myocardial infarction • GSTM1 • RFLP • case-control study
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Unrepaired DNA damage can result in the induction of
somatic mutations in genes regulating cell growth, thus providing a
mechanism for carcinogenesis. Furthermore, the spectrum of mutations
observed in the p53 tumor suppressor gene in tobacco-induced lung
tumors is consistent with the DNA adducts formed by tobacco smoke
constituents (2
, 3)
. In contrast, the pathogenic effects
of tobacco smoke on the development of vascular disease are less well
defined. One possibility is that, in a process parallel to
carcinogenesis, tobacco smoke-induced DNA damage causes smooth muscle
cell proliferation in the intima of arteries, thereby contributing to
atherosclerotic plaque formation (4)
. The damage could
result from direct chemical binding to DNA or be a consequence of
inflammation and oxidative stress consistent with a response-to-injury
model (5)
. The hypothesis that DNA damage plays a role in
vascular disease has received support from observations in animal
models and in humans (4
, 6)
. In experimental animals,
chemicals in tobacco smoke (e.g., benzo[a]pyrene, 1,3-butadiene) and
environmental tobacco smoke have been reported to induce and stimulate
atherosclerotic plaque formation (7
, 8)
. In addition, in
both humans and animals tobacco carcinogens induce DNA adducts in cells
of the vasculature at high levels (6
, 9
10
11)
. Aromatic DNA
adducts are present in smooth muscle cells of human atherosclerotic
lesions of the abdominal aorta (6
, 9)
and in heart tissue
(11)
. Finally, a recent ecological correlation study in
five European countries suggested a gradient of oxidative DNA adduct
levels in peripheral blood lymphocytes that correlated with mortality
rates from premature coronary heart disease in men (12)
.
The majority of genotoxic chemicals in tobacco smoke require metabolism
in order to bind to cellular macromolecules. Enzymes, including the
multigene family of glutathione S-transferases (GST) and microsomal
epoxide hydrolase (EPXH), detoxify these reactive metabolites to more
water-soluble and readily excretable forms. Their expression therefore
modulates the amount of chemical binding to DNA, and polymorphisms in
these genes have been associated with modified risk of tobacco-related
cancers, including lung, in smokers (13)
. A number of
common polymorphisms occur that affect enzyme activity; these include
gene deletions in the GSTM1 and GSTT1 genes, which result in
individuals lacking in the corresponding enzyme activity. In the case
of GSTP1 and EPXH, there are single base pair polymorphisms that also
affect enzyme activity (14
, 15)
. Each enzyme is implicated
in the detoxification of carcinogens present in tobacco smoke and
consequently polymorphisms in these genes may confer susceptibility to
cardiovascular disease if DNA damage is important in the disease
process. Therefore, we examined this question in a case-control study
of subjects characterized for coronary atheroma by angiography and for
a past history of acute myocardial infarction (AMI).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), dNTPs, agarose, and the 100 bp DNA marker ladder were supplied by
Gibco BRL/Life Technologies (Paisley, U.K.). BsmA I
restriction enzyme and 10x NE buffer 3 were purchased from New England
Biolabs (Beverly, Mass.). EcoRV, buffer D, and bovine serum
albumin (BSA) were supplied by Promega Corporation (Madison, Wis.). PCR
reactions were performed on a Hybaid Omn-E thermal cycler (Hybaid,
Ashford, U.K.).
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DNA samples
As described previously (16)
, 398 patients admitted
for routine angiography for investigation of suspected coronary artery
disease were recruited at two centers over an 18 month period. Of
these, DNA was available for analysis for 367 patients in the current
study (Table 2
). A control cohort of 196 healthy, age- and race-matched Caucasian
control subjects, with no history of angina or AMI, was recruited from
local Family Health Services Authority general practice registers.
Clinical histories were taken for each patient and control, and all
subjects gave informed consent according to a protocol approved by
United Leeds Teaching Hospitals NHS Trust and Pinderfields Health Trust
Ethics Committees. Venous blood (10 ml) was taken before 9:00 a.m after
an overnight fast, and genomic DNA was extracted as described
(17)
. The patient group was further subdivided into two
groups on the basis of the occurrence or not of a prior AMI (according
to WHO criteria) determined from the patients’ clinical history.
Clinical and biochemical data on these subjects have been published
previously (16)
. Briefly, mean cholesterol levels (6.26
mmol/l ± 1.14) were significantly higher in patients compared to
controls (5.82±1.13) as were triglyceride levels (2.25 mmol/l ±
1.55 compared to 1.70±0.99).
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Results of angiography were graded as normal, single, double, or triple
vessel disease based on the presence of 
50% stenosis in a major
coronary artery or one of their branches as determined by
ultrasonography. Results were reported by a cardiologist blind to
patient status or genotype.
Smoking status was determined from interviews with patients and controls at the time of blood sampling. Patients were asked whether they were smokers at the time of recruitment (‘current smoker’) or had ever been a regular smoker (‘ever smoker’).
GSTT1/M1 multiplex PCR
Analysis of the GSTT1 and GSTM1 genes was conducted using a
multiplex PCR reaction modified from Abdel-Rahman et al.
(18)
, with the ubiquitous ß-globin gene as an internal
control. Briefly a PCR reaction was carried out in a 20 µl volume
containing ~100 ng of genomic DNA, 10 mM Tris-HCl pH8.3, 50 mM KCl,
1.5 mM MgCl2, 200 µM of each dNTP, and 0.5 U of
AmpliTaq Gold DNA polymerase, with 10 pmol of both GSTT1-A/B
and GSTM1-A/B, and 20 pmol of ß-globin-A/B primers (Table 1)
. The PCR
conditions were 15 min preincubation step at 95°C, 36 cycles of 1 min
at 95°C, 1 min at 60°C, 1 min at 72°C, and a final postcycling 10
min extension step at 72°C. Five microliters of PCR product was
analyzed electrophoretically on a 2% agarose gel stained with ethidium
bromide (250 ng/ml) and the presence or absence of the GSTT1 (480 bp)
and GSTM1 (215 bp) amplicons was determined in the presence of the
control ß-globin gene (268 bp) (Fig. 1a
).
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GSTP1 and epoxide hydrolase PCR-RFLP
The determination of allele distribution for GSTP1 was carried
out using a modified PCR-restriction fragment length polymorphism
(RFLP) (14)
. Briefly, a PCR reaction was conducted
essentially identical to the GSTT1/M1 multiplex, but with 20 pmol of
GSTP1-A/B primers, and the annealing temperature was altered to 56°C.
The 176 bp amplified fragment of the GSTP1 gene was subjected to
restriction digest in a 15 µl reaction volume containing 7.5 µl of
PCR product, 100 mM NaCl, 50 mM Tris-HCl pH7.9, 10 mM
MgCl2, 1 mM dithiothreitol, and 5 U of
BsmA I, at 55°C for 16 h. The digest product was
analyzed electrophoretically on a 3% agarose gel stained with ethidium
bromide (250 ng/ml) and the genotype was determined by analysis of the
bands on the gel (Fig. 1b
): homozygous for isoleucine one
band (176 bp), homozygous for valine two bands (91 bp and 85 bp), and
heterozygous three bands (176 bp, 91 bp, and 85 bp).
The allele determination for the EPXH exon 3 mutation was adapted
from Smith and Harrison (19)
and proceeded according to a
reaction scheme essentially similar to the GSTP1 RFLP, except that the
primers EPXH-A/B were used in the amplification reaction at a
concentration of 20 pmol. The 162 bp product was digested in a 20 µl
reaction volume containing 10 µl of PCR product, 6 mM Tris-HCl pH7.9,
150 mM NaCl, 6 mM MgCl2, 1 mM dithiothreitol, 0.1
mg/ml BSA, and 10 U of EcoRV at 37°C for 2 h. The
products were analyzed on a 3% agarose gel stained with ethidium
bromide (250 ng/ml) and the genotype was determined by analysis of the
bands observed (Fig. 1c
): homozygous for tyrosine one band
(140 bp), homozygous for histidine one band (162 bp), and heterozygous
two bands (140 and 162 bp).
Statistical analysis
Results obtained for the GST and EPXH genotypes were analyzed
with reference to current and past smoking status using Pearson
2 contingency tables and Fisher’s one-tailed
exact test. Logistic regression was used to examine the relationship
between genotype and disease, allowing for other variables. All
statistical analysis were performed using SPSS v8.00 (SPSS Inc.)
statistical analysis software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The mean age and the age range were similar
across groups, but the patient group had an excess of males compared to
females and a higher body:mass index as reported previously
(16)
. Among the patients, 73% had stenosis 50% in at
least one major coronary artery or one of their branches as diagnosed
by angiography. The remaining 27% of this series of consecutive
patients had no evidence of stenosis based on the criteria used, but
were recruited based on chest pains and clinical indications of
coronary artery disease. Almost half the patients had had a prior
incident of AMI; among these, the degree of stenosis was greater than
in those patients with no history of AMI (P=<0.0001; Table 2
).
The main finding of this study is that the GSTM1 homozygous null
genotype occurred at a significantly lower frequency in the AMI patient
group (48%) compared both to the patients with no history of AMI
(59%) and to the control group (57.2%) (see Table 3
). The GSTM1 null frequency did not differ between the latter two
groups. When subjects were stratified for smoking status, the
association between GSTM1 genotype and AMI was observed only in
smokers, suggesting that the polymorphism is more significant in the
presence of tobacco smoke exposure. The association between GSTM1
genotype and AMI remained significant after adjusting for age, sex, and
stenosis (presence or absence) by logistic regression. The frequency of
GSTM1 null genotype did not vary with age, indicating that the change
in the AMI group was not simply a result of selective mortality, nor
was there any relation between GSTM1 genotype and the biochemical
parameters (e.g., cholesterol and triglyceride levels) (data not
shown).
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In contrast to the data with GSTM1, no significant associations were
observed between the GSTT1 null, GSTP1, or EPXH polymorphisms and
either of the two cardiovascular disease groups
(X2 test; P>0.1; Table 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nevertheless, the hypothesis receives support from
recent observations that DNA damage, as reflected by both
carcinogen-DNA adducts and oxidative DNA damage, is observed in
vascular tissue and indeed occurs at higher levels than commonly
observed in other tissues (6
, 9)
. Furthermore, DNA adduct
levels were higher in patients with severe coronary artery disease
(three stenosed coronary arteries) than in patients with lower degrees
of stenosis (11)
. Although these studies are informative
in demonstrating that DNA damage is occurring in the cells of the
vessel wall, they have limitations in establishing a causal role in
disease. This is partly because it is difficult to make comparisons
between diseased and healthy vascular tissue due to sampling
limitations, but also because of the inherent risk of reverse causation
with the presence of disease resulting in higher adduct levels. An
alternative and complementary approach taken in the current study was
to examine the influence of genetic polymorphisms in xenobiotic
metabolizing enzymes on risk of cardiovascular disease, with the
hypothesis that if DNA damage is an important step in the natural
history of the disease, then these polymorphisms would be important
modulators of the exposure-disease association. This approach is
further supported by the observations that GST enzymes are expressed in
cells of the vasculature and could therefore influence the levels of
DNA damage in these target cells (20
, 21)
.
This study indicates that the GSTM1 genotype may be a significant
factor in the pathogenesis of AMI. Possession of the GSTM1 null
genotype appears to be protective against AMI, an effect that was most
marked in smokers. This association between genotype and disease was
specific to GSTM1 even though GSTT1, GSTP1, and EPXH genotypes have
also been associated with risk of tobacco-related cancers
(13)
. Furthermore, the GSTM1-associated risk was limited
specifically to AMI rather than coronary artery disease in general.
Development of AMI involves complex phenotypes including smooth muscle
hyperplasia and plaque formation, plaque instability, and clot
formation leading to vessel occlusion and tissue death. It is well
recognized that plaque rupture is the final antecedent to the
development of AMI and that plaque instability is not necessarily
related to the size of the atheromatous lesion(s). The beneficial
effects of cessation of cigarette smoking on the associated risk of AMI
are so rapid (22)
, they are probably related to increased
plaque stability rather than to atheroma regression. One possible
interpretation of the current data is that GSTM1 null causes a relative
increase in plaque stability in the general population, leading to a
slight decrease in AMI risk, whereas in smokers, a group with enhanced
plaque instability, this effect is magnified.
Given the specific association between tobacco smoke and risk of
vascular disease and the role of GST enzymes in the detoxification of
tobacco smoke carcinogens (23)
, our hypothesis was that
the effect of the genotype on risk would be most marked among smokers.
In the case of GSTM1 null genotype and lung cancer, an increased risk
has been observed in heavier compared to light smokers
(24)
. Our hypothesis is supported by the observation that
when the GSTM1 data were analyzed by smoking status, the association
with AMI was restricted to smokers. The information on smoking status
was relatively crude, comprising self-reported data, but was consistent
in both categories of either current or past smoking history. It is
notable that there are relatively few current smokers, which may
reflect underreporting and recent decisions to quit smoking in the
patient groups.
In the current study, the GSTM1 null genotype was less frequent in the
AMI group than in the other patient group and controls. This is in
contrast to observations of an increased risk of lung cancer in
individuals with GSTM1 null genotype (25)
, which
presumably reflects the higher levels of aromatic DNA adducts found in
lung tissue in association with the null genotype (26
, 27)
. In contrast, there are almost no data with respect to GSTM1
and cardiovascular disease. Van Schooten and colleagues
(11)
examined polycyclic aromatic hydrocarbon-DNA adducts
in samples of the right atrial appendage of patients undergoing open
heart surgery. Although these authors found no significant difference
in adduct levels by GSTM1 genotype, it is interesting in the light of
our data that the mean adduct level was lower in the GSTM1 null
individuals, and this effect was more marked in individuals null for
both GSTM1 and GSTT1 genes. In a previous study of atherosclerosis,
individuals with advanced vessel disease (50% stenosis in iliac
and/or femoral arteries and patients with severe lower extremity
atherosclerosis) had higher GSTM1 enzymatic activity (28)
.
It is possible, therefore, that the effect of the GSTM1 genotype
differs in the development of cancer and cardiovascular disease. The
association between GSTM1 genotype and AMI was not affected by age,
sex, or degree of stenosis, as revealed by logistic regression
analysis, suggesting that the difference in these parameters between
the patient and control groups did not explain that association.
Mechanistic explanations for the association require study, but one
possibility is that GSTM1 could produce a metabolite that promotes
atherogenesis or plaque instability in a way parallel to the recognized
ability of GSTT1 to lead to formation of reactive metabolites with
halogenated hydrocarbons (29)
. Alternatively, the null
genotype could result in up-regulation of another enzyme more effective
at detoxification of atherogenic compounds; the coordinated expression
of GSTM1 and GSTM3 has been suggested from studies in human lung tissue
(30)
and CYP1A2 activity was higher in individuals null
for GSTM1 (31)
. However, it should be stressed that to
date no direct evidence for these proposed mechanisms is available.
In conclusion, the finding of a significant association between GSTM1 genotype, smoking status, and AMI suggests a pathway by which smoking could alter risk of cardiovascular disease via DNA damage. These results imply that further studies of the precise mechanisms by which DNA damage influences the natural history of atherothrombotic disease progression are merited.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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