Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue

^a Department of Health Risk Analysis and Toxicology, Maastricht University, 6200 MD Maastricht, The Netherlands
^b Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland
^c Department of Cardiothoracic Surgery
^d Department of Cardiology, Academical Medical Center, University of Amsterdam, Amsterdam, The Netherlands
^e Laboratory of Biochemical Risk Analysis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA

	ABSTRACT

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Cancer and cardiovascular diseases share risk factors such as smoking, and the onset of both diseases have been suggested to have a common mechanistic basis. The binding of carcinogens to DNA (carcinogen-DNA adducts), genetic polymorphisms in carcinogen-detoxifying enzymes glutathione S-transferases (GSTs), and genetic polymorphisms in the vitamin D receptor (VDR) are among the candidates for modifiers of cancer risk. We determined whether these biomarkers could be related to individual characteristics of patients suffering from cardiovascular diseases. For that purpose, DNA from the right atrial appendage of 41 patients who underwent open heart surgery was analyzed for smoking-related DNA adducts and polymorphisms in GSTM1, GSTT1, and VDR genes. Statistical analysis was used to identify any patient's characteristics associated with these molecular markers. Our results showed that heart tissue of cigarette smokers contained a variety of aromatic DNA adducts in significantly elevated levels compared to ex-smokers (P<0.01) or nonsmokers (P<0.001). A linear relationship was observed between DNA adduct levels and daily cigarette smoking (r_s=0.73; P=0.0003). Since cardiac myocytes are terminally differentiated cells that have lost their ability to divide and seemingly have limited DNA repair capacities, their levels might accumulate with time and thereby affect heart cell function or viability. Substantial interindividual differences between DNA adduct levels were observed, and persons with severe coronary artery disease (CAD), as assessed by coronary angiography, had higher DNA adduct levels than persons with no or mild CAD (P=0.04). As polymorphisms in GST genes have been shown to modulate DNA adduct levels and risk for lung cancer in smokers, we explored for the first time whether the GST polymorphisms could also explain deviating heart DNA adduct levels and CAD risk. However, no relation could be found between these covariants. In contrast, a VDR genotype, which has been associated with decreased serum levels of the active hormonal form of vitamin D and increased risk for certain cancers, seemed to be related to severity of CAD (P=0.025). Our findings support the hypothesis that smoking-related DNA damage may be involved in the onset of cardiovascular diseases and suggest that VDR genotype may be a useful susceptibility marker of CAD.—van Schooten, F. J., Hirvonen, A., Maas, L. M., de Mol, B. A., Kleinjans, J. C. S., Bell, D. A., Durrer, J. D. Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue. FASEB J. 12, 1409–1417 (1998)

Key Words: smoking-related DNA adducts • genetic polymorphisms • CAD

	INTRODUCTION

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TOBACCO SMOKING HAS AN IMPORTANT role in the etiopathogenesis of diseases such as cardiovascular disease and cancer of the respiratory tract. The more than 3800 chemicals identified in tobacco smoke include at least 40 known carcinogens such as tobacco-specific nitrosamines, aromatic amines, and tars (1). Even though the smoking-associated cancer risk has been attributed to exposure to these hazardous compounds, the mechanistic relationship between smoking and cardiovascular diseases has remained unclear. It has been suggested that somatic mutations may play a role in the ontogenesis of cardiovascular diseases (2). First, the proliferated smooth muscle cells in human atherosclerotic plaques have been shown to be of monoclonal origin (3). Second, it has been shown that DNA from atherosclerotic plaques has transforming capacity in NIH3T3 cells, making them capable of inducing tumors in nude mice (4). With respect to smoking, polycyclic aromatic hydrocarbons (PAHs)² found in the tar fraction of cigarette smoke are of particular interest. Several studies have shown that PAHs can initiate and accelerate atherosclerosis in experimental animals (2). Mutagenic PAHs, of which benzo(a)pyrene (B[a]P) is the most studied, exert their DNA damaging action after metabolic activation (5). The generation of reactive metabolites, predominantly epoxides, and the subsequent formation of DNA adducts are believed to be an essential step in the process by which many carcinogens mediate their biological effects. DNA adducts have been detected in various tissues, and the levels seem to be quantitatively related to the cancer risk (6–11). Studies have shown high adduct levels in cardiac tissue, and there has been much attention to the relation between DNA damage in heart and aorta and the onset of cardiomyopathies and cardiovascular diseases (reviewed in ref 12). Recently, a study by De Flora et al. (13) showed DNA adduct levels in smooth muscle cells of human atherosclerotic lesions to be related to known atherogenic risk factors.

Historically, a range of conventional markers such as cholesterol and more specific lipoproteins have been used in epidemiological studies to evaluate the risk for coronary artery disease (CAD). More recently, genetic markers such as restriction fragment length polymorphisms (RFLPs) in genes involved in lipid metabolism have been associated with susceptibility to CAD (14). Similarly, cancer research has revealed potential susceptibility markers to identify persons with a particular high cancer risk after chemical exposure. The genetic polymorphisms in glutathione S-transferases (GSTs) may be especially important: glutathione (GSH) and glutathione-related enzymes play an important role in the cellular metabolism and detoxification of xenobiotics, including PAHs. The GSTs consist of a broad range of isoenzymes (15,16). Two of the genes encoding for these enzymes—GSTM1 and GSTT1—have been shown to exhibit null alleles (lack of the gene) in humans. GSTM1 is expressed in only about 50% of the Caucasian population (17), and people lacking the GSTM1 gene have been suggested to be at a significantly increased risk of developing smoking-related cancers (18–20). This is thought to be related to the important role of GSTM1 in the detoxification of reactive compounds, particularly epoxides. Comparably, GSTT1 detoxifies potential carcinogens that are present in cigarette smoke, including ethylene oxide, and homozygous absence of the GSTT1 gene has been shown to be relatively common (15–30%) in the Caucasian populations studied so far (21). Individuals lacking both GSTM1 and GSTT1 genes may be at a particularly high risk of developing smoking-related cancers (22). Since the DNA damaging effects by reactive mutagens in respective target tissues may be a common event in the initiation of the cancer process and the onset of cardiovascular diseases, it is of interest to examine whether persons with a concurrent deficiency of both of the genes are more prone to CAD than those with the genes.

Recently, it has been shown that common allelic variations in the vitamin D receptor locus (VDR) can be used to predict bone turnover rate and risk of osteoporosis (23) and prostate cancer (24). Since vitamin D functions as a potent regulator of bone and calcium homeostasis as well as of cellular differentiation and proliferation in many target tissues, it may also play an important role in chronic degenerative diseases such as cancer and cardiovascular diseases. Vitamin D acts via its dihydroxylated metabolite (calcitriol) through the highly specific VDR, which is known to be present in most cells, including heart (25). Antiproliferative activity in vitro of calcitriol has been documented in osteosarcoma, melanoma, colon cancer, prostate cancer, and breast cancer cells, and data from epidemiological studies have suggested an inverse relationship between circulating levels of calcitriol and the risk of prostate, breast, and colon cancer (reviewed in ref 26). There is also epidemiological evidence that the risk for CAD is inversely associated with plasma calcitriol levels (27). Moreover, a frequent BsmI polymorphism of the vitamin D receptor has been shown to be highly correlated with serum calcitriol levels (23, 28).

In view of the suggestion that some processes leading to cancer and cardiovascular diseases may have a common mechanistic basis, we studied the above-mentioned molecular biomarkers used in cancer research in relation to severity of CAD.

	SUBJECTS AND METHODS

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Patients
Samples of the right atrial appendage were obtained during right atrial cannulation from 41 consecutive patients undergoing open heart surgery for the first time because of coronary artery disease (n=32; 78%), valvular heart disease (n=7; 17%), myxoma cordis (n=1; 2%), and atrial septal defect (n=1; 2%). The right atrial appendage is a small muscular extension of the right atrium that overlaps the anterior surface of the aortic root as it projects upward and to the left. The internal lengths varied between 0.3 and 2.5 cm, and the mean (±SE) weight of the samples was 150 ± 17 g (n=41), which ranged from 52 to 399 g. The atrial appendages were routinely amputated at the time of surgery and had a normal, healthy macroscopic appearance. The reason of examining the right atrial appendage was its availability whenever right atrial cannulation for open-heart surgery was performed and because, in several studies, DNA adduct levels in cardiac tissue from smokers were found to be high, particularly in right coronary arteries (reviewed in ref 12).

Patients were asked by their cardiologist to participate and were recruited 1 day before surgery after appropriate permission was obtained. The participation rate was 100%. Informed consent was obtained in all cases. All patients were of European origin and classified as white. The study was approved by the medical ethical commission of the Academical Medical Center of the University of Amsterdam. During medical examination before surgery, information concerning the diagnosis of hypertension, function of the left ventricle, and valvular defects was obtained. Blood cholesterol levels and triglyceride levels were measured by the hospital's clinical chemistry department using standard enzymatic methods. We obtained each patient's medical history by using a questionnaire with standardized items. The presence or absence (yes/no) of a history of hypertension requiring treatment, of diabetes, and of agina pectoris was recorded. Furthermore, current medications, smoking habits, occupation, and living conditions (urban/rural), and the presence or absence of CAD among first degree relatives were recorded. Questions about smoking habits included whether or not they smoked cigarettes, how many cigarettes they smoked per day, and for how many years they had been smoking. Nonsmokers were defined as patients who had never smoked, ex-smokers were those who had smoked for a period during their lives and stopped smoking at least 1 year before the surgery, and current smokers smoked at the time of sampling. Nonsmokers were also asked whether or not they lived with a smoker (passive smoking).

We used coronary angiography to assess the severity of CAD. Quantification of the degree of coronary stenosis was done visually. We categorized the patients according to the severity of coronary artery disease in the right coronary artery, the circumflex branch, the left descending anterior artery, and/or the left main stem. This was done as follows: normal coronary arteries or wall irregularities (category 0); one stenosed coronary artery of 50% or more (category 1); two stenosed coronary arteries of 50% or more (category 2); three stenosed coronary arteries of 50% or more (category 3); and three stenosed coronary arteries of 50% or more with a left main stem stenosis of 50% or more (category 4). Table 1 summarizes patients' characteristics and smoking histories.

DNA adduct analysis
DNA was isolated from a representative part of the tissue samples by using standard phenol-chloroform extraction procedures. DNA was analyzed for DNA adducts by the ³²P-postlabeling technique: hydrolysis of the DNA to 3' mononucleotides, 5'-labeling with high specific activity [-³²P]ATP, and analysis by thin-layer chromatography (29, 30). The detection and measurement of the DNA adducts relies on differences in chromatographic mobility between normal and carcinogen- modified nucleotides. To enhance the sensitivity of the assay, we used nuclease P1 (NP1) to cleave the normal 3'-monophosphates to deoxyribonucleosides, which do not serve as substrates for polynucleotide kinase in the labeling reaction (29). The modified nucleotides, however, are resistant to cleavage by NP1 and will subsequently be labeled. With this procedure, levels as low as 1 in 10⁹–10¹⁰ nucleotides can be detected for aromatic and other hydrophobic adducts. DNA (10 µg) was digested by using micrococcal endonuclease (0.4 U) and spleen phosphodiesterase (2.8 µg) for 3 h at 37°C. Subsequently, half of the digest was treated with NP1 (6.3 µg) for 40 min at 37°C. The modified nucleotides were labeled with [-³²P]ATP (50 µCi/ sample) by incubation with T4-polynucleotide kinase (5.0 U) for 30 min at 37°C. [-³²P]ATP was synthesized in the laboratory by using carrier-free -³²P (Dupont, Brussels). NP1 efficiency and ATP excess were checked with an aliquot of the enriched fraction by 1-dimensional chromatography on poly(ethyleneimine) (PEI) -cellulose sheets from Merck (Darmstadt, Germany) (solvent: 0.12 M NaH₂PO₄, pH 6.8). Radiolabeled adduct nucleotide biphosphates were separated by chromatography on PEI-cellulose sheets from Machery Nagel (Germany). The following solvent systems were used: D1, 1 M NaH₂PO₄ pH 6.5; D2, 8.5 M urea, 5.3 M lithium formate pH 3.5; D3, 1.2 M lithium chloride, 0.5 M Tris, 8.5 urea pH 8.0; D4, 1.7 M NaH₂PO₄, pH 6.0. In each experiment, three standards of [³H]BPDE-modified DNA with known modification levels (1 per 10⁷, 10⁸, and 10⁹ nucleotides) were run in parallel for quantitation purposes. Quantification was performed by using phosphor-imaging technology (Molecular Dynamics, Sunnyvale, Calif.). The remaining half of the digest was used to determine the final amount of DNA in the assay; the normal nucleotides were labeled with [-³²P]ATP (20 µCi/sample) by incubation with T4-polynucleotide kinase (2.5 U) for 30 min at 37°C. Nucleotides were separated by 1-dimensional chromatography on PEI-cellulose sheets from Merck (solvent: 0.12 M NaH₂PO₄, pH 6.8); samples with apparent protein or RNA contamination were discarded. A dAp standard (27.5 pmol/µl) was labeled in each experiment for quantification purposes.

Genotyping analysis
GSTM1 genotype was determined using a polymerase chain reaction (PCR) -based method, described in detail elsewhere (20). Briefly, the GSTM1 null genotype was identified on the basis of the absence of the GSTM1-specific fragment after electrophoresis on ethidium-bromide-stained 3% 3:1 NuSieve/agarose gel (FMC Bioproducts, Rockland, Maine). The consistent presence of another fragment, amplified by using ß-globin primers, acted as an internal control, excluding the possibility of a false interpretation due to failure in the PCR reaction. Similarly, GSTT1 specific primers were as described (21) and used together with the ß-globin primers to detect the GSTT1 genotype.

VDR genotype was also determined by a PCR based method described by Morrison and co-workers (23). After amplification, 5 µl of the PCR product was digested with Bsm I restriction enzyme (New England Biolabs, Beverly, Mass.) and then subjected to electrophoresis as described above. The single undigested PCR fragment identified the homozygous BB genotype whereas the homozygous bb genotype was identified by observing two digested fragments estimated at 650 bp and 170 bp. Heterozygotes had both digested and undigested fragments.

Statistical analysis
The levels of aromatic DNA adducts among groups were subjected to nonparametrical statistical analysis: Kruskal Wallis or Mann Whitney U. Correlation coefficients and two-tailed significances were calculated using of the nonparametric Spearman's rank correlation analysis. Multiple regression analysis was carried out using the level of DNA adducts as the dependent variable. DNA adduct values were log-transformed (log 10) to obtain a more close-to-normal distribution. Independent variables comprised: age, smoking habits (current smoker, ex-smoker, and nonsmoker), total number of cigarettes smoked per lifetime, degree of CAD (stage 0, 1, or 2/stage 3 or 4), and GSTM1 and GSTT1 genotype (positive/negative).

To get an impression about the relative association between several variables and the degree of CAD, crude odds ratios (OR) were calculated from contingency tables. Statistical significance was determined by 95% confidence intervals following Mantel-Haenszel's approach. All statistical evaluations were made using SPSS 6.1 (SPSS Inc., Chicago, Ill.).

	RESULTS

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DNA adduct analysis
Phosphorimages of the chromatograms of ³²P-postlabeled digests of DNA from smokers revealed so-called diagonal radioactive zones (DRZ) ( Fig. 1C). Generally, these DRZ are typical for DNA damage induced by complex mixtures such as cigarette smoke, and contain a number of diffuse spots representing aromatic DNA adducts: by adjusting the sensitivity of the phosphorimager, two predominant spots could be detected within the DRZ of smoker's heart DNA. Similar DRZ-like patterns with two predominant spots were also found in heart DNA from ex- smokers ( Fig. 1B; profile of a person who quit 8 years earlier). In this respect, no clear qualitative differences in DNA patterns between smokers and ex-smokers could be observed, but only differences in intensities. As shown in Fig. 1A, generally no DRZ were observed in the nonsmokers; the spot in the example of the nonsmoker was also seen in control samples, such as blank DNA, and was therefore considered as a background spot.

View larger version (98K): [in this window] [in a new window]   Figure 1. DNA adduct profiles of 32P- postlabeled digests of human right atrial appendages from a nonsmoker (A), an ex- smoker (B), and a smoker (C). NA was isolated from right atrial appendages and analyzed by nuclease-P1 enrichment prior to postlabeling for aromatic DNA adducts. Resolution of the 32P-labeled adducts was carried by chromatography in two dimensions on polyethylenimine-cellulose TLC sheets (10 x 10 cm). The origin on the bottom left-hand corner of the chromatograms was excised after chromatography. Adduct profiles on the chromatograms were visualized and quantitated by using a PhosphorImager (Molecular Dynamics, Sunnyvale). For comparison, panel D shows the adducts resulting from labeling calf thymus DNA reacted in vitro with (+)-anti-benzo(a)pyrene diol epoxide (BPDE), which is the reactive metabolite of B[a]P. In each experiment, (3H)-BPDE-DNA standards with known modification levels (1 adduct per 109–107 nucleotides) were run for quantitation purposes.

Relationships between various variables and DNA adduct levels
In most right atrial appendages, aromatic DNA adduct levels were above the detection limit of the ³²P-postlabeling assay ( Fig. 2A). Mean adduct levels in heart tissue of current cigarette smokers were approximately twofold higher than those in ex-smokers and three times higher than those in nonsmokers ( Fig. 2A and Table 1). Although not statistically significant, adduct levels in ex-smokers were somewhat higher than in nonsmokers. In ex-smokers, no relation was seen between DNA adduct levels and the number of years after quitting smoking. DNA adduct levels thus appeared to be predominantly determined by present smoking habits ( Fig. 2B, Spearman's rank r_s=0.73, P=0.0003) rather than by the lifetime smoking dose (r_s=0.25, P=0.11).

View larger version (19K): [in this window] [in a new window]   Figure 2. A) Levels of aromatic DNA adducts in right atrial appendages of operable heart patients. B) Relationship between cigarettes smoked per day and aromatic DNA adduct levels. Each dot represents a different patient; filled symbols mean GSTM1 positive and open symbols mean GSTM1 negative subjects. DNA was assayed at least three times for aromatic DNA adducts in independent assays performed on different days. The variability between the assays was around 20%. Mean adduct levels in smokers are significantly enhanced compared with ex-smokers (P0.05) and nonsmokers (P0.01). Linear regression showed a correlation between cigarettes smoked per day and DNA adduct levels (correlation coefficient 0.55, P=0.003). Ex-smokers were excluded from this analysis.

Although nonsmokers had never been actively exposed to tobacco smoke, they had detectable DNA adduct levels in their heart tissue. As shown in Fig. 2A, three persons in the group of nonsmokers had relatively high adduct levels; two lived with smokers and were therefore exposed to environmental tobacco smoke. Among nonsmokers, the mean DNA adduct level in passive smokers (4.0±3.1, n=5) was twice that detected in nonexposed persons (2.0±2.7, n=6), but this difference was not statistically significant (Mann-Whitney; P=0.1).

We did not observe any clear influence of GST genotypes on DNA adduct levels in the total study group: the adduct levels among GSTM1 null individuals (5.9±6.4, n=22) were similar to those among GSTM1 positive individuals (6.9±4.5, n=19). Somewhat lower adduct levels were observed for those lacking the GSTT1 gene (4.3±4.3, n=8) compared to those with the gene (6.8±5.8, n=33) (Mann-Whitney; P=0.3). Persons lacking both genes had lower adduct levels (4.8±5.3, n=5) than those having one (5.8±6.4, n=20) or both of the genes (7.5±4.5, n=16) (Kruskal Wallis; P=0.2). Exclusion of nonsmokers from the analysis did not change the trend: GSTM1 positive (7.9±4.2, n=15) vs. null (7.3±7.1, n=15); and GSTT1 positive (8.2±5.8, n=25) vs. null (4.9±4.9, n=5) (Mann-Whitney; P=0.3). Current smokers lacking the GSTM1 gene had similar DNA adduct levels (11.3±7.9, n=7) compared to current smokers with the gene (9.6±3.9, n=8) (Mann-Whitney; P=0.6).

Persons with severe CAD (categories 3 and 4) had higher heart DNA adduct levels (7.5±6.0, n=27) than those having no or mild CAD (categories 0, 1 and 2) (4.0±3.9, n=13) (Mann-Whitney; P=0.039). Spearman's rank correlation (r_s) between DNA adduct levels and categorized CAD (categories 0 to 4) was 0.27 (P=0.09). An association was also found between lifetime smoking and CAD (categorized 0 to 4) (r_s=0.36, P=0.025).

Table 2 shows the results from multiple regression analysis on DNA adduct levels in human heart. Only smoking was significantly associated with DNA adduct levels, although some correlation was also found between CAD and DNA adduct levels. Age or GST genotype were not associated with the DNA adduct levels. Neither were any of the other individual parameters studied, i.e., sex, diet, medication, hypertension, functioning left ventricle, valve defects, alcohol consumption, occupational exposure to PAH, or site of residence (urban and rural).

Relationship between various parameters and stenosis of coronary arteries
Table 3 shows the crude OR of several variables for CAD. The OR for the well-established risk factor cholesterol was 6.9 (confidence interval, 1.4–33.3; P<0.05), with 6.5 mM as a cutoff point for hypercholesterolemia. Other established risk factors such as family history, hypertension, and smoking tended to correlate with the degree of CAD, but these associations were not statistically significant. Similar nonsignificant correlation was observed between DNA adduct levels and the CAD severity when the geometric mean value of the total group was used as a cutoff point. As for the potential genetic susceptibility, no involvement of the GST genotypes was found. Moreover, although patients with severe stenosed arteries were more likely to have the VDR genotype bb, this association only approached the significance threshold (OR of 4.2, 95% CI 0.8–22.5, P=0.09; Armitage Doll trend, P=0.06).

	DISCUSSION

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We examined the relation between smoking, DNA damage in human heart, genetic polymorphisms in GSTs and VDR, and coronary artery disease. Considerable amounts of DNA adducts were found in right atrial appendages, the levels being highly related to smoking habits. Patients suffering from severe CAD tended to have higher adduct levels in their heart tissue than those having no or mild CAD. The VDR genotype associated with decreased serum levels of the active form of vitamin D appeared to be predictive of CAD.

The autoradiographs of chromatograms of ³²P-postlabeled DNA digests showed a diagonal zone of various spots characteristic of DNA damage induced by cigarette smoke constituents, such as aromatic compounds. Similar patterns have been reported in DNA from mouse skin and from organs of animals treated with cigarette tar, with the highest levels found in heart and lung tissue (31). Previous studies of humans have shown similar DRZs of smoking-related adducts in DNA of the respiratory tract, smooth muscle layer of sclerotic lesions, and heart autopsy material (9, 10, 13, 32–35). DNA adduct levels in heart tissue appear to be comparable, or even higher, than those in lung tissue. In the present study, mean heart adduct levels in smokers were 10.4 ± 5.9 adducts per 10⁸ nucleotides. In comparison, earlier studies by Van Schooten et al. (9, 30) using the same procedure showed mean adduct levels in smokers of 1.9 in peripheral blood lymphocytes, 6.4 in pulmonary alveolar macrophages, and 10.6 in lung tissue. Both lung and heart are highly exposed to chemical compounds present in cigarette smoke, due to `first pass' effects, and subsequent adduct formation depends on metabolic activation and deactivation pathways. The formation of DNA adducts in heart may be caused by metabolism in heart tissue itself (36) or, alternatively, by circulating reactive metabolites (37). Moreover, adducts may persist in heart because of inefficient DNA repair processes and low cell turnover rate (12, 38).

Proliferation of lung cells may result in mutations in the DNA adduct sites. If these mutations occur in genes relevant for carcinogenesis, such as proto-oncogenes or tumorsuppressor genes, this may lead to initiation of tumorigenesis. The idea of the involvement of such genes in cardiovascular diseases arose from the observation that c-H-ras proto-oncogene is overexpressed in rat aortic smooth muscle cells after B[a]P exposure (39). Recently it was shown that the p53 protein, which exhibits tumor suppressor activity (40), is abnormally accumulated in some cases of coronary restenosis (an enhanced proliferation of smooth cells that may occur in patients who underwent coronary angioplasty) (41). In a subsequent study, no changes in the p53 gene, which is the most commonly altered gene in human cancer, were found in the smooth muscle cells of human atherosclerotic lesions (42). This may be explained by the fact that cell division is a requisite to convert DNA adducts into mutations and proliferation of arterial smooth muscle cells seems to be low (43). It can, however, be promoted by lipoprotein[a] (44), and it is noteworthy that cholesterol associated with the atherosclerotic lesions and surrounding cells can act as a tumor promotor (45). Although smoking-related DNA damage in nondividing heart muscle cells rarely leads to tumor formation (46), it can conceivably cause alterations in gene expression and production of aberrant proteins that may contribute to other degenerative diseases, such as cardiomyopathies (11, 38, 47).

The presence of DNA adducts in heart of nonsmokers may be the result of other possible sources of exposure to aromatic compounds such as passive smoking, polluted inner city air, and contaminated or smoked food; occupational exposure may also play a role. Epidemiological findings have shown an association between environmental exposure to tobacco smoke and the occurrence of cardiovascular diseases (48, 49). Although the lifetime cigarette consumption of ex-smokers was equal to current smokers, they had adduct levels nearly similar to those persons who never smoked. Giving up smoking thus leads to reduction of adduct levels, although it is not clear how fast this happens; no relation was found between adduct levels and time after quitting smoking.

In the present study, we evaluated biomarkers associated with the severity of CAD as judged by angiography. It has already been shown that angiography is a useful tool in case control studies involving individuals with and without CAD (50). In this study, the DNA adduct levels were associated with the degree of CAD. Although no causal relation can be inferred from our results, the differences in DNA adduct formation might be one of the genetic components explaining individual differences in CAD susceptibility. Total serum cholesterol level has been demonstrated by a variety of epidemiological methods, including angiographic evaluation (51), to be a well-validated biomarker of CAD risk (14). Hypercholesterolemia, a well-known risk factor for CAD (52–54), was clearly associated with the severity of CAD also in the present study.

Among this small group of patients, we found that individuals with severe CAD were more likely to contain the VDR genotype bb. Although this observation could be due to chance, such a result would be in agreement with previous findings of an inverse association between circulating calcitriol and CAD (27, 55). The bb genotype has been shown to be associated with low levels of circulating calcitriol (23). The protective activity of vitamin D in cardiovascular diseases and cancer may result from a common mechanistic basis. In vitro experiments have shown an action of calcitriol on myocardial cells and on intimal cells of large vessels. Therefore, any effect of vitamin D in the etiology of cardiovascular disease could merely be mediated through calcitriol itself rather than through its action via the classical homeostasis system (56). Our findings should encourage researchers to unravel the general function of calcitriol, which may lead to a more mechanistic answer for the role of vitamin D in the complex etiology of degenerative disease processes.

	FOOTNOTES

 
¹ Correspondence: E-mail: f.vanschooten{at}grat.unimaas.nl

² Abbreviations: GSTs, S-transferases; GSH, glutathione; VDR, vitamin D receptor locus; PAHs, polycyclic aromatic hydrocarbons; B[a]P, benzo(a)pyrene; CAD, coronary artery disease; RFLPs, restriction fragment length polymorphisms; NP1, nuclease P1; PEI, poly(ethyleneimine); DRZ, diagonal radioactive zones; PCR, polymerase chain reaction; OR, odds ratios.

Received for publication January 22, 1998. Accepted for publication June 16, 1998.

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