CYP1A1 and GSTM1 genotypes affect benzo[a]pyrene DNA adducts in smokers' lung: comparison with aromatic/hydrophobic adduct formation
Kroum Alexandrov1,
Ingolf Cascorbi2,
Margarita Rojas1,
Guy Bouvier3,
Erik Kriek4 and
Helmut Bartsch1,5
1 German Cancer Research Center (DKFZ), Division of Toxicology and Cancer Risk Factors, P.O. Box 101949, D-69009 Heidelberg, Germany
2 Ernst Moritz Arndt University of Greifswald, Medical Faculty, Friedrich-Loeffler-Strasse 23d, D-17487 Greifswald, Germany
 |
Abstract
|
---|
Benzo[a]pyrene diol epoxide (BPDE)DNA adducts are involved in the induction of p53 mutations and probably in the causation of human lung cancer associated with cigarette smoking. The ratio between CYP1A1 and GST enzyme activities is a critical determinant of the target dose of carcinogenic BPDE and other DNA-reactive PAH metabolites. In this review, we summarize the published data on modulation of (+)-anti-BPDEDNA adduct levels in smokers' lungs by CYP1A1*2 genotypes alone or in combination with GSTM1 polymorphism and compare these results with those reported for aromatic/hydrophobic (bulky) DNA adducts. The data published so far show only a trend for a non-significant increase in bulky DNA adduct levels in subjects with GSTM1*0 or the CYP1A1*2GSTM1*0 genotype combination. In contrast, a clear dependence of (+)-anti-BPDEDNA adduct levels was found as a function of the CYP1A1 and GSTM1 genotypes: In lung parenchyma, this adduct was more pronounced in persons with the GSTM1*0 genotype, and CYP1A1*2GSTM1*0 carriers had higher (+)-anti-BPDEDNA adduct levels than those with CYP1A1*1/*1-GSTM1*0. The homozygous CYP1A1*2/*2 carriers in the GSTM1*0 group had the highest (+)-anti-BPDEDNA adduct levels. Our analysis leads to the conclusion that the risk-modifying effects of metabolic genotypes and of gene interactions might be more easily identifiable if specific markers of structurally defined adducts were used, such as the (+)-anti-BPDEDNA adduct. These results are also consistent with the hypothesis that BP (PAH) induce G:C to T:A transversion mutations in the hotspot codons of the p53 tumor suppressor gene and are thus involved in malignant transformation of the lung tissue of smokers.
Abbreviations: BP, benzo[a]pyrene; BPDE, benzo[a]pyrene-diolepoxide; BPDE-DNA adduct, (+)-anti-benzo[a]pyrene-diol epoxide-DNA adduct; CYP, cytochrome P450; GST, glutathione S-transferase; HPLC-FD, high-performance liquid chromatography with fluorescence detection; NAT, N-acetyltransferase; PAH, polycyclic aromatic hydrocarbons; nt, nucleotide
 |
Introduction
|
---|
The probability that a smoker will develop lung cancer is related to both the dose of tobacco carcinogens and the individual's genetic background. This geneenvironment interaction offers a possibility for defining individual genetic risk profiles, which would be important for identifying subgroups at highest risk for disease. Polycyclic aromatic hydrocarbons (PAH)DNA adducts are relevant in the initiation of PAH-related carcinogenesis, and their levels have been found to vary considerably among persons with similar ambient or environmental exposure to PAH (1), implying that the effect of these adducts is related to inherited differences in host susceptibility. Genetically determined host factors may thus modify the extent of DNA damage in smokers' lungs and therefore modulate cancer risk.
Benzo[a]pyrene (BP), considered to be the bullet of the `smoking gun', is the most extensively studied (2) carcinogen in cigarette smoke and has been known for a long time to be involved in the causation of lung cancer (3,4). The distribution of adducts formed by BP within DNA (5,6) corresponds closely to the sites in the p53 gene with the highest mutation frequencies in lung tumor tissue from smokers, but not in that from non-smokers (7). In lung cancer patients, cigarette smoking has been associated in a dose-related fashion with an increase in p53 mutations, which are predominantly G
T transversions. These findings provide substantial evidence that BP (and similar PAH)DNA adducts are involved in induction of mutations in p53 and in human lung cancer causation. Similar relationships could not be established for other DNA adducts formed in human lung, as their structures have not been elucidated (8).
Cytochrome P450 (CYP)-related enzymes activate PAH by producing highly reactive DNA-damaging metabolites. This is best exemplified by the metabolism of BP, which in human lung tissue undergoes two successive oxygenation reactions mediated predominantly by CYP1A1, ultimately leading to the highly mutagenic BP diol epoxide (BPDE) (9). Further, high CYP1A1 activity generates intracellular oxidative stress, leading subsequently to the production of reactive oxygen species (10).
Elevated CYP1A1 activity correlates with adverse effects in humans: high CYP1A1 inducibility in lymphocytes has been related to a high lung cancer risk (1114). The fact that CYP1A1 expression can vary 50-fold in human lung tissue (1517) explains the large interindividual differences in CYP1A1-mediated BP metabolism (1820) and in BPDEDNA adduct levels in lung tissue samples after incubation with BP in vitro (21). A positive correlation was found between CYP1A1 activity and pulmonary PAH (BPDE)-associated DNA adduction (22,23). This variation in enzyme inducibility could be due to genetic polymorphisms in CYP1A1 or in genes involved in the control of its expression. However, most studies have not addressed whether such polymorphisms play a role in CYP1A1-related enzyme activity (2432). Some studies showed effects of glutathione S-transferase (GST) M1 (31,33) and aryl hydrocarbon receptor polymorphisms (34) on CYP1A1 activity, while others found no key role of these polymorphisms in CYP1A1 inducibility (3537). When BP metabolism was measured in CYP1A1 allelic variants expressed in cell lines, similar BP oxidation rates (38) but different enzyme kinetics have been reported (39,40). The diol epoxides of PAHs are detoxified predominantly by GSTs, the Mu class showing the highest activity towards most PAH epoxides such as (+)-anti-BPDE (4144 and references therein), followed by GSTP1-1 and GSTA1-1 (44). About 50% of the white population lacks the GSTM1 gene, and this deletion has been linked to a moderately increased risk for lung cancer in smokers in some studies (45). It has been hypothesized that persons homozygous for the GSTM1 deletion should have higher PAH(BPDE)DNA adduct levels. Thus, the ratio between CYP1A1 and GSTM1 enzyme activities is critical for the tissue dose of carcinogenic BP diol epoxide and other reactive PAH intermediates available for reaction with DNA. Extensive studies have been performed on the possible associations between polymorphisms in BP metabolizing genes, such as CYP1A1 and GSTM1, and cancer susceptibility (45,46 and references therein). Although some of these polymorphisms have been shown to be risk modifiers, many of the results are controversial.
Large inter-individual differences have been observed in the levels of aromatic/hydrophobic (bulky) PAHDNA adducts in human lung autopsy samples (47) and in bronchial biopsy specimens (4750), whereby smokers have higher adduct levels than ex-smokers and non-smokers. The relationship between CYP1A1 and GSTM1 gene polymorphism and the level of bulky PAHDNA adducts in human lungs has been the subject of many investigations. At present, the evidence for a role of single metabolic genotypes and their combinations on the formation of these adducts is weak and usually non-significant (5156). The conflicting results of these studies may be due to differences in race, sample size, smoking status or other confounding factors. A major problem was that these studies addressed the relationship between a defined metabolic genotype and DNA adducts, the structures of which are largely unknown, and use of an exposure marker for a chemical with a defined structure is clearly more appropriate for this kind of study. The implication of BPDEDNA adducts in G
T mutations in p53 and their plausible role in lung cancer (57) makes them an attractive marker for studying the relationship between genotype and DNA damage.
In this review, we summarize published results on modulation of BPDEDNA adduct levels in smokers' lungs, mainly by the CYP1A1*2 genotype in combination with GSTM1 polymorphism (5760). We compare these results with those for aromatic/ hydrophobic (bulky) DNA adducts (5156). The method used for measuring these adducts is evaluated critically. Finally, the role of various CYP and GST genotypes in modulating DNA adduct levels in smokers' lungs in relation to lung cancer risk is discussed.
 |
Methods for detection of DNA adducts in human lung
|
---|
Immunological methods and 32P-postlabelling assays have been most often used to quantify carcinogenDNA adducts in human lung; however, each has some limitations. Both assays can be used to detect a broad spectrum of PAHDNA adducts. Antibodies designed to recognize anti-BPDEDNA adducts showed various degrees of cross-reactivity with structurally related congeners. 32P-Postlabelling of adducts results in a number of spots, often unidentified, and overlapping of spots on a chromatogram is liable to interfere with adduct identification and eventually with quantification. The adduct levels in a given sample measured by both methods differ considerably (see ref. 1 for details). The precise chemical structures of most adducts have not been identified with immunological methods or 32P-postlabelling assays, and these adducts are known as `PAHDNA', `hydrophobic DNA', `aromatic DNA' or `bulky DNA' adducts, without unequivocal identification. In some studies, these adducts are considered to be `carcinogenDNA' adducts even when the carcinogens involved are not properly identified. When a mixture of different adducts is present in the `bulky DNA' adduct fraction, the effect of the single metabolic genotype would be difficult to determine.
Consequently, studies of DNA adducts of a known carcinogen such as (+)-anti-BPDE could define the role of genotype in adduct formation more precisely. Shields et al. (57) and Andreassen et al. (61) used immunoaffinity chromatography coupled with high-performance liquid chromatography (HPLC)synchronous fluorescence detection (FD) to monitor specific BPDEDNA adducts in human lung. However, the recovery of the method was low. Our laboratory developed a HPLCFD method to enhance the specificity of PAHDNA adduct detection in human lung tissue (62,63), and this method has allowed us to examine the correlation between the CYP1A1 and GSTM1 genotypes and BPDEDNA adduct levels (60,64). This assay was validated and subsequently used by others to measure BPDEDNA adducts in human and rodent tissues and cells (44,6567). The recovery was high and reproducible, with a detection limit of 1 adduct per 109 unmodified nucleotides from 1 mg of DNA.
 |
Aromatic/hydrophobic (bulky) DNA adducts
|
---|
Studies on DNA adducts in smokers' lungs are summarized in Table I
. Study #1 reported interim results on bulky DNA adducts in the lung parenchyma of 86 smokers (51). Smokers with the GSTM1*0 genotype had a slight, non-significant increase in adduct levels in comparison with smokers with the GSTM1*1 genotype.
In study #2, the level of bulky DNA adducts was determined in lung tissue from 70 current smokers (52). In relation to the GSTM1 genotype, a higher (mean) level was found in patients with GSTM*0 than those with GSTM1*1 (12.5 ± 8.5 vs. 9.4 ± 6.0 per 108 nt; P = 0.088). The number of cigarettes smoked per day had only a minor effect on adduct levels.
Study #3 showed that CYP1A1*2 carriers had no effect on bulky DNA adducts in the lungs of smokers (53). The adduct levels were very similar in each of the four CYP1A1 and GSTM1 genotype combinations. After adjustment for either smoking status or malignancy, there was no statistically significant effect of the `risk' combination (CYP1A1*2GSTM1*0) on DNA adduct levels. A further study #4 (54) confirmed the results of study #3 (53) and showed that CYP1A1*2 polymorphism did not significantly affect DNA adduct levels in human bronchus. The adduct level was, however, 34% higher in CYP1A1*2 heterozygotes than in CYP1A1*1/*1 [11.4 ± 5.4 (n = 7) vs. 8.54 ± 4.0 per 108 nt (n = 93); P value not available] in the combined group of current smokers and short-term ex-smokers.
Study #5 (55) showed the effect of genetic polymorphisms on DNA adduct levels in lung tissue from 165 patients with non-small-cell lung cancer (142 smokers and 23 non-smokers). CYP1A1*2 carriers had a higher level of adducts than CYP1A1*1/*1 homozygotes, but the difference was not significant (9.96 ± 5.60 vs. 7.94 ± 5.12 per 108 nt; P = 0.11). Patients with the GSTM1*1 gene intact had slightly more adducts than those with the GSTM1*0 (8.78 ± 5.35 vs. 7.71 ± 5.10 per 108 nt; P = 0.072). Among individuals with the GSTM1*0 phenotype, the number of adducts was increased with borderline significance in carriers of the CYP1A1*2 allele (10.35 ± 5.86 per 108 nt; n = 16) over that in CYP1A1*1/*1 carriers (7.11 ± 4.73 per 108 (n = 71); P = 0.043) (subgroup F). No significant associations were found between adduct level and genotype among the adenocarcinoma cases. A significantly higher frequency of the combined CYP1A1*2GSTM1*0 genotype in patients with elevated adduct levels was found as compared to the reference group (7/12 = 17% vs. 16/325 = 5%; P = 0.009).
The results of study #6 (56) showed at least six times more formation of hydrophobic DNA adducts, than found in studies #15 (5155). Study #6 included 32 smokers, 38 non-smokers and three persons who had been exposed to high concentrations of PAH. No correlations were reported between smoking-associated DNA adduct levels in lung tissues and genetic polymorphisms in CYP1A1 or GSTM1 or their four combinations. Although persons with the CYP1A1*2/*2GSTM1*0 combination had twice as many adducts as those with the CYP1A1*1/*1GSTM1*1 combination, the difference was not significant.
 |
BPDEDNA adducts
|
---|
Using an immunoaffinity chromatography32P-postlabelling assay which should be capable of detecting BPDEDNA adducts, the authors of study #7 (57) analysed 38 human lung samples (autopsy material). Adduct levels were categorized as present (six males, one female) or not detected (25 males, six females), and the relationships with the CYP1A1*1/*1 genotype (32), heterozygous CYP1A1*2 (n = 2) and homozygous n = CYP1A1*2/*2 alleles (n = 4) were analysed. The six persons with adducts were of the CYP1A1*1/*1 phenotype (means not given). BPDEDNA adduct formation was not associated with the presence of a mutant CYP1A1*2 allele. Of the 38 human lung samples, 20 were from persons with the GSTM1*1 phenotype and 18 with GSTM1*0. Six of seven persons with BPDEDNA adducts were of the GSTM1*0 genotype, and a statistically significant relationship with this genotype was found. The mean BPDEDNA adduct level was 6.4 adducts in persons with a GSTM*0 phenotype and 1.2 adducts in those with GSTM*1. When the data were adjusted for serum cotinine concentration and log-transformed for age, linear regression analysis revealed that the BPDEDNA adduct levels were associated only with the GSTM1*0 phenotype.
Study #8 (58) gave the results for 90 human autopsy specimens of cancer-free lung tissue, including data already reported by Shields et al. (57; study #7). Only nine specimens had detectable adducts, and seven were from persons with a wild CYP1A1*1/*1 genotype. When BPDE-DNA adduct levels were categorized as positive or not detected, they were not associated with the CYP1A1*2 (*2/*2) genotype. The GSTM1*0 genotype was found for 47 of the 90 specimens and for eight of the nine with detectable adducts. When the data were adjusted for serum cotinine content and log-transformed for age, linear regression analysis showed that only the GSTM1*0 genotype was associated with BPDEDNA adduct levels.
An HPLCFD assay (62,63) was used to determine BPDEDNA adduct levels in human bronchus samples from the Institute of Occupational Health, Helsinki, Finland (study #9; interim results were reported in ref. 59). All samples were found to be from persons with the CYP1A1 `inducible' phenotype, determined by immunohistochemical staining (antibodies against CYP1A1). As the quantity of DNA was small, the DNA from all the bronchial specimens was pooled according to GSTM1 status (*1 or *0). Samples with the combination of `inducible' CYP1A1 and GSTM1*0 genotype had about 100 times more BPDEDNA adducts at a smaller smoking `dose' than those with the combination of `inducible' CYP1A1 and GSTM1*1. A similar large difference in adduct levels was not seen in lung parenchyma from the same patients.
In 1998, Rojas et al. (60) first reported a clear effect of CYP1A1 and GSTM1 polymorphism on the formation of BPDEDNA adducts in lungs from lung cancer patients (study #10). None of the patients with the GSTM1*1 genotype had detectable BPDEDNA adducts (limit of detection, <0.2 per 108 nt in 0.5 mg DNA), independently of the CYP1A1 genotype, whereas all patients with GSTM1*0 had detectable adducts. When the adduct levels were categorized into not detectable (all 14 samples with GSTM1*1) and detectable (all six samples with GSTM1*0),
2 < 0.00005.
In the group with the GSTM1*0 genotype, four carriers of the wild CYP1A1*1/*1 genotypes had low levels of adducts formation (0.50.8 per 108 nt), but the two with the rare combination of CYP1A1*2/*2 had six-fold higher BPDEDNA adduct levels. The level of adducts was significantly different between individuals who carried the CYP1A1*1/*1GSTM1*1 and the CYP1A1*1/*1GSTM1*0 combinations (P < 0.001) and between those with the CYP1A1*2/*2GSTM1*0 and the CYP1A1*1/*1GSTM1*0 combinations (P < 0.01; Wilcoxon rank sum test). The combination of homozygous mutated CYP1A1*2/*2 and GSTM1*0 led to a greater increase in BPDEDNA adduct level than in persons with CYP1A1*2 and GSTM1*1.
By using a specific, sensitive method to measure a defined exposure marker, the (+) anti-BPDEDNA adduct, these authors showed that (i) none of the patients with the GSTM1*1 genotype had detectable BPDEDNA adducts (detection limit, 0.2 per 108 nt); (ii) those with the combined CYP1A1*1/*1 (or heterozygous for CYP1A1*2) and GSTM1*0 genotype had low levels of BPDEDNA adducts; and (iii) those with the rare CYP1A1*2/*2 mutant allele and GSTM1*0 had the highest BPDEDNA adduct levels. These results also provide evidence that the CYP1A1 genotype leads to elevated adduct levels when combined with GSTM1*0, implying elevated or induced CYP1A1 activity dependent on the polymorphisms described (31,33).
The correlation between GSTM1 genotype and susceptibility to lung cancer or increased BPDEDNA adducts in the lung from GSTM1*0 smokers appears to be perplexing on the basis of very low or no detectable level of GSTM1 in human lung (68). There are two biologically plausible explanations that have been postulated until now in relation to the carcinogenesis process. Firstly, GSTM1 is in linkage disequilibrium with GSTM3 which expression in human lung linearly correlated with those of the most highly expressed GST, GSTP1 (69), and thus may be relevant to BPDE detoxification. Secondly, BP is metabolized by the liver, the major site of its metabolism and independently of the site of its uptake. Accordingly, BP derived adducts found in the lung, result in part from leakage of proximal (BP-7,8-diol) and/or ultimate (BPDE) DNA-binding intermediates from the liver into the systematic circulation and subsequent transport and DNA adduct formation at the lung as, has been demonstrated in the rat (70).
 |
Relationship of DNA adduct levels with CYP1A1- and GSTM1-related enzyme expression in lung tissue of smokers
|
---|
Positive correlations have been observed between CYP1A1-related enzyme activity and bulky BPDE adduct levels in human lung tissue (62,71,72). Geneste et al. (71) measured pulmonary DNA adducts and BP hydroxylase activity in non-neoplastic surgical samples of lung parenchyma from smokers. A positive linear correlation was found in smokers between DNA adduct levels and BP hydroxylase activity (r = 0.69; P < 0.001; n = 19). In another study (62), a highly significant correlation was found between pulmonary microsomal BP hydroxylase activity and the level of BPDEDNA adducts (r = 0.91; P < 0.01; n = 13). Hydrophobic DNA adduct levels were found to be significantly related to lung CYP1A1 expression, measured by quantitative reverse transcriptasepolymerase chain reaction, and large inter-individual differences were observed in both parameters (72). Immunohistochemical analysis of lung tissue specimens from lung cancer patients showed that bulky DNA adducts were significantly associated with the expression of CYP1A1 protein but not with that of GSTM1 protein or CYP1A1/GSTM1 polymorphisms (56). These results suggest that, with high environmental exposure to PAH, CYP1A1 protein inducibility might be more important for DNA adduct formation than that of its polymorphic variants.
In view of the rapid turnover of CYP1A1 mRNA and the half-life of ~1.7 years for DNA adducts in bronchial tissue of ex-smokers, ~25% of the difference in the level of DNA adducts can be explained by differences in CYP1A1 expression (72). Other factors, such as exposure to PAH, phase II enzyme activity, DNA repair processes and cell turnover, could also affect the level of DNA adducts.
 |
Relationships of other CYP and GST genotypes with aromatic hydrophobic DNA adducts
|
---|
Relatively few studies have been published on the relationships between aromatic DNA adducts in lung and metabolic genotypes other than CYP1A1 and GSTM1. Studies of the levels of bulky DNA adducts in lung tissue showed no statistically significant relationship between polymorphisms in CYP1B1, CYP2C9 and CYP2D6 alone or in combination (54,55,73).
Interactions were found between the GSTM1 and N-acetyltransferase (NAT) 2 genotypes on the levels of aromatic DNA adducts in lung cancer patients and in population controls (74). Among controls who smoked, the slow NAT2 phenotype, in particular in combination with GSTM1*0, was strongly associated with high adduct levels. In contrast, an antagonistic genegene interaction was seen among cases who smoked, but only in current smokers, in whom the combined GSTM1*0 and rapid NAT2 genotype resulted in an increase in adduct level as a function of both age and daily cigarette use. The authors concluded that slow NAT2 genotype, in particular when combined with the GSTM1*0 genotype, confers increased susceptibility to adduct formation, gene mutation and lung cancer when the smoking `dose' is low.
The GSTP1Val105 allelic variant of the enzyme was found to have greater catalytic activity for carcinogenic diol epoxides of PAH, such as BPDE, than the Ile105 variant (43,44,69), and thus should better protect DNA from damage by these diol epoxides. In study #2 (52), 70 lung cancer patients who were current smokers were grouped according to their GSTP1 genotype. Significantly higher mean hydrophobic (PAH)DNA adduct levels were found in patients with GSTP1*B and /or 1*C alleles than in those with the GSTP1*A genotype. Patients with the 1*C genotype had significantly higher adduct levels than patients with 1*A (15.64 ± 10.23 vs. 7.9 ± 5.1 per 108 nt; P = 0.006). The effect of the combined genotypes of GSTP1 and GSTM1 was also examined. The lowest adduct level was found among patients with GSTM1 active and GSTP11*A genotypes (6.68 ± 3.57 adducts per 108 nt), and patients with GSTM1*0 and GSTP 1*B or 1*C had significantly higher adduct levels than those with all other genotype combinations (9.35 ± 5.98 per 108 nt; P = 0.011). The highest level was found in the group with the combination GSTM1*0 and GSTP1 *C(19.03 ± 11.04 per 108 nt). In this study therefore, the GSTP1 *A genotype partially protected DNA against reactive hydrophobic (PAH) compounds, but the results are contrary to those that would be predicted on the basis of the catalytic activity of the allelic variants (43,69).
In another group of patients consisting of 142 smokers and 23 non-smokers, GSTP1 polymorphism did not affect the level of bulky DNA adducts in lung parenchyma: 7.96 ± 4.96 per 108 adducts for GSTP1*A (n = 77), 8.52 ± 5.73 for GSTP1*B (n = 72) and 8.14 ± 4.17 for GSTP *C (n = 10) (55). Similar results were obtained when this patient group was analysed according to histological subtype (squamous-cell carcinoma and adenocarcinoma). Schoket et al. (54) reported contrasting results to those described above (52,55): in the group with the GSTM1*0 phenotype (n = 19), GSTP1*B (n = 7) and GSTP1*C (n = 4) carriers had significantly lower adduct levels (by 5060%) than GSTP1*A homozygotes.
The results obtained in these three studies on the relationship between aromatic/hydrophobic (bulky) DNA adducts and GSTP1 polymorphism (with 32P-postlabelling) are conflicting and require further investigation, preferably with other methods of adduct detection.
 |
Conclusions
|
---|
Aromatic/hydrophobic (bulky) DNA adducts
GSTM1 polymorphism alone did not affect the levels of aromatic/hydrophobic (bulky) DNA adducts. A trend to an increasing adduct level was observed in persons with the CYP1A1*2GSTM1*0 genotype combination. While studies on the occurrence of bulky DNA adducts in smokers' lungs showed doseresponse relationships with smoking `dose', they could not relate specific genotypes or the adduct(s) involved in the initiation of lung cancer. The 32P-postlabelling method which is used to determine the level of these adducts is non-specific. Thus, the lack of chemical specificity in adduct measurement may have hampered identification of any correlation with specific metabolic polymorphism.
BPDEDNA adduct
The effects of metabolic genotypes and geneenvironmental interactions were better to identify by use of specific exposure markers with defined structures, as exemplified by the (+)-anti-BPDEDNA adduct. In lung parenchyma, the BPDEDNA adduct level was found to correlate with the GSTM1*0 genotype. The CYP1A1*2GSTM1*0 combination was associated with a higher BPDEDNA adduct level than wild type CYP1A1. Persons with the CYP1A1*2/*2GSTM1*0 combination had the highest level of BPDEDNA adduct formation. Thus, GSTM1*0 together with the mutant CYP1A1 allele can affect BPDEDNA adduct levels in smokers' lungs. These results (i) provide a mechanistic understanding of the results of epidemiological studies in which these `at risk' genotypes in Japanese, who have a much higher allele frequency than whites, correlated with a higher risk for smoking-related lung cancers (24,29,35,45); (ii) are consistent with the prevalence of G:C to T:A transversion mutations in the P53 gene in lung tumours of smokers, a point mutation that is indicative of PAH-related mutational damage; and (iii) show that the presence of a high frequency of G:C to T:A transversion mutations in codons 157 and 249 of P53 in non-malignant lung tissue from patients with smoking-associated lung cancer is consistent with the hypothesis that bulky tobacco carcinogens such as BP and PAH induce G:C to T:A transversions at the hotspot codons (75) and are thus probably involved in malignant transformation of lung tissue in smokers (5,6).
This analysis should provide guidance for future molecular epidemiological studies, as it strongly supports the hypothesis that the risk-modifying effect of metabolic genotypes and genegene interactions is more easily identifiable if specific, sensitive markers for structurally defined carcinogenDNA adducts are used. This article is dedicated to Harold zur Hausen on the occasion of his retirement as head of the German Cancer Research with gratitude and appreciation for 20 years of leadership.
 |
Notes
|
---|
3 Current addresses: 682 chemin de St Julien 06410 Biot, France and 
4 Straat van Gibraltar 32, 1183 GW Amstelveen, The Netherlands 
5 To whom correspondence should be addressed Email: h.bartsch{at}dkfz.de 
 |
Acknowledgments
|
---|
The authors particularly acknowledge the financial support given by the DKFZ, Heidelberg to Dr M.Rojas. Mrs Susanna Fuladdjusch is thanked for her skilled secretarial help and Mrs Elisabeth Heseltine for the editing.
*This article is dedicated to Harold zur Hausen on the occasion of his retirement as head of the German Cancer Research with gratitude and appreciation of 20 years of leadership.
 |
References
|
---|
- Kriek,E., Rojas,M., Alexandrov,K. and Bartsch,H. (1998) Polycyclic aromatic hydrocarbonDNA adducts in humans: relevance as biomarkers for exposure and cancer risk. Mutat. Res., 400, 215231.[ISI][Medline]
- Osborn,M.R. and Crosby,N.T. (1987) Benzopyrenes. In Cambridge Monographs on Cancer Research. Cambridge University Press, Cambridge, pp. 1323.
- Wynder,E.L. and Graham,E.A. (1950) Tobacco smoking as a possible etiologic factor in bronchogenic carcinoma. J. Am. Med. Assoc., 143, 329336.[ISI]
- Kreyberg,L. (1954) The significance of histological typing in the epidemiology of the primary epithelial lung tumours: a study of 446 cases. Br. J. Cancer, 8, 199208.[ISI]
- Denissenko,M.F., Pao,A., Rang,M. and Pfeifer,G.P. (1996) Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hotspots in P53. Science, 274, 430432.[Abstract/Free Full Text]
- Smith,L.E., Denissenko,M., Bennett,W.P., Li,H., Amin,S., Tang,M. and Pfeifer,G.P. (2000) Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons. J. Natl Cancer Inst., 92, 803810.[Abstract/Free Full Text]
- Hollstein,M. Sidransky,D., Vogelstein,B. and Harris,C.C. (1991) p53 mutations in human cancers. Science, 253, 4953.[ISI][Medline]
- Hussain,S.P. and Harris,C.C. (1998) Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumour suppressor genes. Cancer Res., 58, 40234037.[ISI][Medline]
- Gelboin,H. (1980) Benzo(a)pyrene metabolism, activation and carcinogenesis: role of regulation of mixed-function oxidases and related enzymes. Physiol. Rev., 60, 11071165.[Free Full Text]
- Park,J.-Y.K., Shigenaga,M.K. and Ames,B.N. (1996) Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indol (3,2-b)carbazole is associated with oxidative DNA damage. Proc. Natl Acad. Sci. USA, 93, 23222327.[Abstract/Free Full Text]
- Kellerman,G., Luyten-Kellerman,M. and Shaw,C.R. (1973) Genetic variation of aryl hydrocarbon hydroxylase in human lymphocytes. Am. J. Hum. Genet., 25, 327331.[ISI][Medline]
- Kouri,R.E., McKinney,C.E., Slomiany,D.J., Snodgrass,D.R., Wray,N.P. and McLemore,T.L. (1982) Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes. Cancer Res., 45, 50305037.
- Karki,N.T., Pokela,R., Nuutinen,L. and Pelkonen,O. (1987) Aryl hydrocarbon hydroxylase in lymphocytes and lung tissue from lung cancer patients and controls. Int. J. Cancer, 39, 565570.[ISI][Medline]
- McLemore,T.L., Adelberg,S., Liu,M.C., McMahon,N.A., Yu,S.J. and Hubbard,W.C. (1990) Expression of CYP1A1gene in patients with lung cancer: evidence for cigarette smoke-induced gene expression in normal lung tissue and for altered gene regulation in primary pulmonary carcinomas. J. Natl Cancer Inst., 82, 13331339.[Abstract]
- Petruzzelli,S., Camus,A.M., Carrozzi,L., et al. (1988) Long-lasting effects of tobacco-smoking on pulmonary drug-metabolizing enzymes: a casecontrol study on lung cancer patients. Cancer Res., 48, 46954700.[Abstract]
- Anttila,S., Hietanen,E., Vainio,H., Camus,A.M. Gelboin,H.V., Park,S.S., Heikkilae,L., Karjalainen,A. and Bartsch,H. (1991) Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients. Int. J. Cancer, 47, 681685.[ISI][Medline]
- Anttila,S., Vainio,H., Hietanen,E., et al. (1992) Immunohistochemical detection of pulmonary cytochrome P450IA and metabolic activities associated with P450IA1 and P450IA2 isozymes in lung cancer patients. Environ. Health Perspect., 98, 179182.[ISI][Medline]
- Cohen,G.M., Mehta,R. and Brown,M. (1979) Large interindividual variations in metabolism of benzo(a)pyrene by peripheral lung tissue from lung cancer patients. Int. J. Cancer, 24, 129133.[ISI][Medline]
- Prough,R.A., Sipal,Z. and Jakobsson,S.W. (1977) Metabolism of benzo(a)pyrene by human lung microsomal fractions. Life Sci., 21, 16291636.[ISI][Medline]
- Rojas,M., Camus,A.M., Alexandrov,K., Husgafvel-Pursianen,K., Antilla,S., Vainio,H. and Bartsch,H. (1992) Stereoselective metabolism of ()-benzo[a]pyrene-7,8-diol by human lung microsomes and peripheral blood lymphocytes: effect of smoking. Carcinogenesis, 13, 929933.[Abstract]
- Harris,C., Autrup,H., Connor,R., Barrett,L.A., McDowell,E.M. and Trump,B.F. (1976) Interindividual variation in binding of benzo(a)pyrene to DNA in cultured human bronchi. Science, 194, 10671069[ISI][Medline]
- Bartsch,H., Castegnaro,M., Rojas,M., Camus,A.M., Alexandrov,K. and Lang,M. (1992) Expression of pulmonary cytochrome P4501A1 and carcinogen-DNA adduct formation in high risk subjects for tobacco-related lung cancer. Toxicol. Lett., 64, 477483.
- Bartsch,H., Petruzzelli,S., De Flora,S., et al. (1992) Carcinogen metabolism in human lung tissues and the effect of tobacco smoking: results from a case-control multicenter study on lung cancer patients. Environ. Health Perspect., 98, 119124.[ISI][Medline]
- Kawajiri,K., Nakachi,K., Imai,K., Yoshii,A., Shinoda,N. and Watanabe,J. (1990) Identification of genetically high risk individuals to lung cancer by DNA polymorphism of the cytochrome P4501A1 gene. FEBS Lett., 263, 131133.[ISI][Medline]
- Petersen,D.D., McKinney,C.E., Ikeya,K., Smith,H.H., Bale,A.E. and McBride,O.W. (1991) Human CYP1A1 gene: cosegregation of the enzyme inducibility phenotype and an RFLP. Am. J. Hum. Genet., 48, 720725.[ISI][Medline]
- Cosma,G., Crofts,F., Currie,D., Wirgin,L., Toniolo,P. and Garte,S.J. (1993) Racial differences in restriction fragment length polymorphisms and messenger RNA inducibility of human CYP1A1 gene. Cancer Epidemiol. Biomarkers Prev., 2, 5357.[Abstract]
- Cosma,G., Crofts,F., Currie,D., Toniolo,P. and Garte,S.J. (1993) Relationship between genotype and function of the human CYP1A1 gene. J. Toxicol. Environ. Health, 40, 309316.[ISI][Medline]
- Crofts,F., Taioli,E., Tranchman,J., Cosma,G.N., Currie,D., Toniolo,P. and Garte,S.J. (1994) Functional significance of different human CYP1A1 genotypes. Carcinogenesis, 15, 29612963.[Abstract]
- Kiyohara,C., Nakanishi,Y., Inutsuka,S., Takayama,K., Hara,N., Motohiro,A., Tanaka,K., Kono,S. and Hirohata,T. (1998) The relationship between CYP1A1 aryl hydrocarbon hydroxylase activity and lung cancer in a Japanese population. Pharmacogenetics, 8, 315323.
- Jacquet,M., Lambert,V., Baudoux,E., Muller,M., Kremers,P. and Gielen,J. (1996) Correlation between P450CYP1A1 inducibility, MspI genotype and lung cancer incidence. Eur. J. Cancer, 32A, 17011706.
- Stucker,I., Jacquet,M., Waziers,I., Cénée,S., Beaune,P., Kremers,P. and Hémon,D. (2000) Relation between inducibility of CYP1A1,GSTM1 and lung cancer in a French population. Pharmacogenetics, 10, 617662.[ISI][Medline]
- Wedlund,P.J., Kimura,S., Gonzales,F.J. and Nebert,D.W. (1994) 1462V mutation in the human CYP1A1 gene: lack of correlation with either the MspI kb (M2) allele or CYP1A1 inducibility in a three-generation family of East-Mediterranean descent. Pharmacogenetics, 4, 2426.
- Vaury,C., Laine,R., Noguiez,P., Coppet,P., Jaulin,C., Praz,F., Pompon,D. and Amor-Gueret,M. (1995) Human glutathione S-transferase M1 null genotype is associated with high inducibility of cytochrome P4501A1 gene transcription. Cancer Res., 55, 55205523.[Abstract]
- Smart,J. and Daly,A.K. (2000) Variation in induced CYP1A1 levels: relationship to CYP1A1, Ah receptor and GSTM1 polymorphisms. Pharmacogenetics, 10, 1124.[ISI][Medline]
- Smith,G.B.J., Harper,P.A., Wong,J.M.Y., Lam,M.S.M., Reid,K.R., Petsikas,D. and Massey,T.E. (2001) Human lung microsomal cytochrome P4501A1 (CYP1A1) activities: impact of smoking status and CYP1A1, arylhydrocarbon receptor and glutathione S-transferase M1 genetic polymorphisms. Cancer Epidemiol. Biomarkers Prev., 10, 839853.[Abstract/Free Full Text]
- Cauchi,S., Stücker,I., Solas,C., et al. (2001) Polymorphisms of human aryl hydrocarbon receptor (AhR) gene in a French population: relationship with CYP1A1 inducibility and lung cancer. Carcinogenesis, 22, 18191824.[Abstract/Free Full Text]
- Anttila,S., Lei,X-D., Elovaara,E., Karjalainen,A., Sun,W., Vainio,H. and Hankinson,O. (2000) An uncommon phenotype of poor inducibility of CYP1A1 in human lung is not ascribed to polymorphisms in the AHR, ARHT, or CYP1A1 genes. Pharmacogenetics, 10, 741751.[ISI][Medline]
- Zhang,Z.-Y., Fasco,M.J., Huang,L., Guengerich,F.P. and Kaminski,L.S (1996) Characterisation of purified human recombinant cytochrome P4501A1-Ile462 and -Val462: assessment of a role for the rare allele in carcinogenesis. Cancer Res., 56, 39263933.[Abstract]
- Persson,I., Johansson,I. and Ingelman-Sundberg,M. (1997) In-vitro kinetics of two human CYP1A! variant enzymes suggested to be associated with interindividual differences in cancer susceptibility. Biochem. Biophys. Res. Commun., 231, 227230.[ISI][Medline]
- Schwarz,D., Kisselev,P., Cascorbi,I., Schunck,W.H. and Roots,I. (2001) Differential metabolism of benzo(a)pyrene and benzo(a)pyrene-7,8-diol by human CYP1A1 variants. Carcinogenesis, 22, 453459.[Abstract/Free Full Text]
- Mannervik,B. and Danielson,H. (1988) Glutathione S-transferases structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283337.
- Hayes,J.D. and Strange,R.C. (2000) Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology, 61, 154166.[ISI][Medline]
- Sundberg,K., Johansson,A.-F., Stenberg,G., Widersten,M., Seidel,A., Mannervik,B. and Jernstrom,B. (1998) Differences in the catalytic efficiencies of allelic variants of glutathione S-transferase P1-1 towards carcinogenic diol-epoxides of polycyclic aromatic hydrocarbons. Carcinogenesis, 19, 433436.[Abstract]
- Sundberg,K., Dreij,K., Seidel,A. and Jernström,B. (2002) Glutathione conjugation and DNA adduct formation of dibenzo(a,l)pyrene and benzo(a)pyrene diol-epoxides in V79 cells stably expressing different human glutathione transferases. Chem. Res. Toxicol., 15, 170179.[ISI][Medline]
- Bartsch,H., Nair,U., Risch,A., Rojas,M., Wikman,H. and Alexandrov,K. (2000) Genetic polymorphism of CYP genes, alone or in combination, as a risk modifier of tobacco-related cancers. Cancer Epidemiol. Biomarkers Prev., 9, 328.[Abstract/Free Full Text]
- Autrup,H. (2000) Genetic polymorphisms in human xenobiotic-metabolizing enzymes as susceptibility factors in toxic response. Mutat. Res., 464, 6576.[ISI][Medline]
- Cuzick,J., Routledge,M.N., Jenkins,D. and Garner,R.C. (1990) DNA adducts in different tissues of smokers and non-smokers. Int. J. Cancer, 45, 673678.[ISI][Medline]
- Phillips,D.H., Schoket,B., Hewer,A., Bailey,E., Kostic,S. and Vincze,I. (1990) Influence of cigarette smoking on the levels of DNA adducts in human bronchial epithelium and white blood cells. Int. J. Cancer, 46, 569575.[ISI][Medline]
- Dunn,B.P., Vedal,S., San,R.H.C., Kwan,W.-F., Nelems,B., Enarson,D.A. and Stich,H.F. (1991) DNA adducts in bronchial biopsies. Int. J. Cancer, 48, 485492.[ISI][Medline]
- Van Schooten,F.J., Hillebrand,M.J.X., van Leeuwen,F.E., Lutgerink,J.T., van Zandwijk,N., Jansen,H.M. and Kriek,E. (1990) Polycyclic aromatic hydrocarbon-DNA adducts in lung tissue from lung cancer patients. 11, 16771681.
- Bartsch,H. and Hietanen,E. (1996) The role of individual susceptibility in cancer burden to environmental exposure. Environ. Health Perspect., 104, 569577.[ISI][Medline]
- Ryberg,D., Skaug,V., Hewer,A., et al. (1997) Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk. Carcinogenesis, 18, 12851289.[Abstract]
- Schoket,B., Phillips,D.H., Poirier,M.C., Kostic,S. and Vincze,I. (1998) Smoking-associated bulky DNA adducts in bronchial tissue related to CYP1A1 and GSTM1 genotypes in lung patients. Carcinogenesis, 19, 841846.[Abstract]
- Schoket,B., Papp,G., Lévay,K., Mrackova,G., Kadlubar,F.F. and Vincze,I. (2001) Impact of metabolic genotypes on levels of biomarkers of genotoxic exposure. Mutat. Res., 482, 5769.[ISI][Medline]
- Butkiewicz,D., Cole,K.L., Phillips,D.H., Harris,C.C. and Chorazy,M. (1999) GSTM1, GSTP1, CYP1A1 and CYP2D6 polymorphism in lung cancer patients from an environmentally polluted region of Poland: correlation with lung DNA adduct levels. Eur. J. Cancer Prev., 8, 315323.[ISI][Medline]
- Cheng,Y.-W., Chen,C.Y., Lin,P., Chen,C.-P., Huang,K.H., Lin,T.-S., Wu,M.-H. and Lee,H. (2000) DNA adduct level, in lung tissue may act as a risk biomarker of lung cancer. Eur. J. Cancer, 36, 13811388.[ISI][Medline]
- Shields,P.G., Bowman,E.D., Harrington,A.M., Doan,V.T. and Weston,A. (1993) Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes. Cancer Res., 53, 34863492.
- Kato,S., Bowman,E.D., Harrington,A.M., Blomeke,B. and Shields,P. (1995) Human lung carcinogen-DNA adducts levels mediated by genetic polymorphisms in vivo. J. Natl Cancer Inst., 87, 902907.[Abstract]
- Bartsch,H. (1996) DNA adducts in human carcinogenesis: Etiological relevance and structure-activity relationship. Mutat. Res., 340, 6779.[ISI][Medline]
- Rojas,M., Alexandrov,K., Cascorbi,I., et al. (1998) High benzo(a)pyrene diol-epoxide DNA adduct levels in lung and blood cells from individuals with combined CYP1A1MspI/MspI-GSTM1*0/*0 genotypes. Pharmacogenetics, 8, 109118.[ISI][Medline]
- Andreassen,A., Kure,E.H., Nielsen,P.S., Autrup,H. and Haugen,A. (1996) Comparative synchronous fluorescence spectrophotometry and 32P-postlabelling analysis of PAH-DNA adducts in human lung and relationship to TP53 mutations. Mutat. Res., 368, 275282.[ISI][Medline]
- Alexandrov,K., Rojas,M., Geneste,O., Castegnaro,M., Camus,A.-M., Petruzzelli,S., Giuntini,C. and Bartsch,H. (1992) An improved fluorometric assay for dosimetry of benzo[a]pyrene diol-epoxide-DNA adducts in smokers lung: comparison with total bulky adducts and aryl hydrocarbon hydroxylase activity. Cancer Res., 52, 62486253.[Abstract]
- Rojas,M., Alexandrov,K., van Schooten,F.-J., Hillebrand,M., Kriek,E. and Bartsch,H. (1994) Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparison with 32P-postlabelling and ELISA. Carcinogenesis, 15, 557560.[Abstract]
- Rojas,M., Cascorbi,I., Alexandrov,K., Kriek,E., Auburtin,G., Mayer,L., Koop-Schneider,A., Roots,I. and Bartsch,H. (2000) Modulation of benzo(a)pyrene diol-epoxide-DNA adduct levels in human white blood cells by CYP1A1, GSTM1 and GSTT1 polymorphism. Carcinogenesis, 21, 3541.[Abstract/Free Full Text]
- Lodovici,M., Akpan,V., Giovannini,L., Migliani,F. and Dolara,P. (1998) Benzo[a]pyrene diol-epoxide DNA adducts and levels of polycyclic aromatic hydrocarbons in autoptic samples from human lungs. Chem.-Biol. Interact., 116, 199212.[ISI][Medline]
- Sticha,R.K., Staretz,E., Wang,M., Liang,H., Kenney,M.J. and Hecht,S.S. (2000) Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse. Carcinogenesis, 21, 17111719.[Abstract/Free Full Text]
- Schwerdtle,T., Seidel,A. and Hartwig,A. (2002) Effect of soluble and particulate nickel compounds on the formation and repair of stable benzo[a]pyrene DNA adducts in human lung cells. Carcinogenesis, 23, 4753.[Abstract/Free Full Text]
- Anttila,S.,Hirvonen,A.,Vainio,H.,Husgafvel-Pursiainen,K.,Hayes,J.D. and Ketterer,B. (1993) Immunohistochemical localisation of glutathione S-transferases in human lung. Cancer Res., 53, 56435648.[Abstract]
- Coles,B., Yang,M., Lang,N.P. and Kadlubar,F.F. (2000) Expression of hGSTP1 alleles in human lung and catalytic activity of the native protein variants towards 1-chloro-2,4-dinitrobenzene, 4-vinylpyridine and (+)-anti benzo[a]pyrene-7,8-diol-9,10-oxide. Cancer Lett., 156, 167175.[ISI][Medline]
- Wall,K.L., Gao,W.S., te Koppele,J.M., Kwei,G.Y., Kaufman,F.C. and Thurman,R.G. (1991) The liver plays a central role in the mechanism of chemical carcinogenesis due to polycyclic aromatic hydrocarbons. Carcinogenesis, 12, 783786.[Abstract]
- Geneste,O., Camus,A.-M., Castegnaro,M., Petruzelli,S., Macchiarini,P., Angeletti,C.A.A. and Bartsch,H. (1991) Comparison of pulmonary DNA adduct levels, measured by 32P-postlabelling and aryl hydrocarbon hydroxylase activity in lung parenchyma of smokers and ex-smokers. Carcinogenesis, 12, 13011305.[Abstract]
- Mollerup,S., Ryberg,D., Hewer,A., Phillips,D.H. and Haugen,A. (1999) Sex differences in lung CYP1A1 expression and DNA adduct levels among lung cancer patients. Cancer Res., 59, 33173320.[Abstract/Free Full Text]
- Ozava,S., Schoket,B., McDaniel,L.P., Tang,Y.-M., Ambrasone,C.B, Kostic,S., Vincze,I. and Kadlubar,F.F. (1999) Analyses of bronchial bulky DNA adduct levels and CYP2C9,GSTP1 and NQO1 genotypes in a Hungarian study population with pulmonary diseases. Carcinogenesis, 20, 991995.[Abstract/Free Full Text]
- Hou,S.-M., Fält,S., Yang,K., Nyberg,F., Pershagen,G., Hemminki,K. and Lambert,B. (2001) Differential interactions between GSTM1 and NAT2 genotypes on aromatic DNA adduct level and HPRT mutant frequency in lung cancer patients and population controls. Cancer Epidemiol. Biomarkers Prev., 10, 133140.[Abstract/Free Full Text]
- Hussain,S.P., Amstad,P., Raja,K., et al. (2001) Mutability of p53 hotspot codons to benzo(a)pyrene diol-epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung. Cancer Res., 61, 63506355.[Abstract/Free Full Text]
Received June 12, 2002;
revised July 31, 2002;
accepted August 15, 2002.