Modulation of benzo[a]pyrene diolepoxide–DNA adduct levels in human white blood cells by CYP1A1, GSTM1 and GSTT1 polymorphism

Margarita Rojas, Ingolf Cascorbi2, Kroum Alexandrov, Erik Kriek4, Guy Auburtin3, Lucienne Mayer3, Annette Kopp-Schneider1, Ivar Roots2 and Helmut Bartsch5

Division of Toxicology and Cancer Risk Factors and
1 Division of Biostatistics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany,
2 Institute of Clinical Pharmacology, University Clinic Charité, Humboldt University of Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany,
3 Institut National de l'Environnement Industriel et des Risques, F-60550 Verneuil en Hallate, France and
4 Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The modulation of benzo[a]pyrene diolepoxide (BPDE)–DNA adduct levels by polymorphisms in the CYP1A1, GSTM1 and GSTT1 genes was assessed in leukocytes of Caucasian males. Eighty-nine coke oven workers (35 smokers, 36 ex-smokers and 18 non-smokers) were recruited from job categories with different exposure levels to polycyclic aromatic hydrocarbons (PAH), together with 44 power plant workers (all smokers) not exposed to PAH. BPDE–DNA adducts were detected in 69 of 133 (52%) DNA samples with a 100-fold variation (range 0.2–44 adducts/108 nt) and a median of 1.6 adducts/108 nt. All samples with the GSTM1 active genotype (n = 59) and five out of 74 samples with GSTM1*0/*0 (7%) showed non-detectable adducts (<0.2 adducts/108 nt) and 69 of 74 subjects with GSTM1*0/*0 (93%) had detectable adducts (>0.2 adducts/108 nt). The difference in adduct level between the GSTM1*0/*0 and GSTM1 active genotypes was highly significant (P < 0.0001). No significant difference in adduct level between the GSTT1*0/*0 and GSTT1 active genotypes was seen. All heterozygotes (CYP1A1*1/*2) from subjects of GSTM1 active type did not have detectable adducts. Among the GSTM1-deficient individuals (n = 69), 42 with the CYP1A1*1/*1 genotype showed a lower adduct level (median 1.3, range 0.2–4.1 adducts/108 nt) compared with 26 individuals with heterozygous mutated CYP1A1*1/*2 genotypes (median 2.5, range 0.4–6.1 adducts/108 nt, P < 0.015). One individual with low PAH exposure and the rare combination CYP1A1*2A/*2AGSTM1*0/*0 showed an extremely high level of 44 adducts/108 nt. Significant differences in detectable adduct levels were found between the CYP1A1*1/*1 and CYP1A1*1/*2 genotypes in the exposed group low + medium (P = 0.01) and for all adduct levels, detectable and non-detectable (set at a fixed value), in highly exposed individuals and in ex-smokers (P = 0.03), whereas no such differences were observed in the control group. Mutated CYP1A1*1/*2 increased the adduct level in non-smokers from the exposed group (1.4 versus 2.2 adducts/108 nt), but had no effect on the smokers from the exposed group (2.3 versus 2.8 adducts/108 nt). When all variables were dichotomized, statistical evaluation showed that CYP1A1 status (P = 0.015), PAH exposure (P = 0.003) and smoking (P = 0.006) had significant effects on adduct levels which increased in the order: CYP1A1*1/*1 < CYP1A1(*1/*2 or *2A/*2A); environmental exposure < occupational exposure; non-smokers < smokers, whereby adducts increased with cigarette dose and the duration of smoking. Higher levels of BPDE–DNA adducts in individuals with the combined CYP1A1(1/*2 or *2A/*2A)–GSTM1*0/*0 genotype suggest that these genotype combinations are at increased risk for contracting lung cancer when exposed to PAH.

Abbreviations: B[a]P, benzo[a]pyrene; BPDE, (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; CYP1A1, CYTOCHROME P450 1A1 gene; CYP1A1*1/*1, wild-type; CYP1A1*1/*2 (*1/*2A, 2A/4, 2B/4), heterozygotes; CYP1A1*2/*2 (*2/*2A, 2B/ 2B), homozygotes; GSTM1, glutathione S-transferase M1 gene; GSTT1, glutathione S-transferase T1 gene; HPLC-FD, HPLC with fluorometric detection; PAH, polycyclic aromatic hydrocarbons; PAQ, number of packs of cigarettes smoked per day multiplied by the number of years of smoking.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is increasing evidence that individual susceptibility to lung cancer is modulated by host factors which affect metabolism of environmental lung carcinogens such as benzo[a]pyrene (B[a]P). B[a]P can be activated to (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) in human tissues by the drug metabolizing enzymes CYP1A1, CYP2C9 and CYP3A4 (13), as well as by prostaglandin H synthases (4,5). BPDE reacts directly with DNA forming covalent adducts, which seem to be a critical step in cancer induction (6). Enzymes crucial in preventing the formation of BPDE–DNA adducts include GSTM (1 or 3) and GSTP1 (7,8).

Although single polymorphic genes have been reported to be associated with increased lung cancer risk (reviewed in ref. 9), the effect of genotype combinations should be more pronounced. The formation of polycyclic aromatic hydrocarbon (PAH)–DNA adducts as a result of PAH exposure has clearly been demonstrated, but large inter-individual variations (10- to 100-fold) in PAH–DNA adduct levels at similar exposure levels have been reported in most studies (reviewed in ref. 10). The impact of different cytochrome P450 1A1 gene (CYP1A1) and glutathione S-transferase M1 gene (GSTM1) genotypes on the formation of bulky PAH–DNA adducts in leukocytes is controversial at present (9,10,1127). Elevated PAH–DNA adduct levels were observed with the GSTM1*0/*0 genotype, but no association was found with the homozygous CYP1A1 `at risk' genotype (MspI or exon 7 mutation) (2831). However, in bronchial tissue of smokers with highly active CYP1A1 and deleted GSTM1, BPDE–DNA levels were 100-fold higher than in subjects with active GSTM1 (32). In contrast, another study denied effects of the CYP1A1 (MspI or exon 7 mutation) and GSTM1*0/*0 genotypes on bulky aromatic DNA adduct levels in human bronchial tissue using the 32P-post-labelling method (33). Studies on leukocytes isolated from occupationally and environmentally exposed subjects, e.g. chimney sweeps, coke oven and foundry workers, soldiers, coal tar-treated psoriasis patients and fire-fighters, showed no or an insignificant increase in PAH–DNA adduct levels dependent on GSTM1 genotype (1127). Positive associations were resolved from studies considering the CYP1A1 and GSTM1 genotypes (9,13,24,26). Moreover, the impact of CYP1A1 and GSTM1 genotype on DNA adduct levels dependent on smoking habit is currently unclear (15,19,2127). In a recent study, using 32P-post-labelling analysis for DNA adducts in healthy volunteers, individuals with the GSTM1*0/*0 genotype had significantly elevated adduct levels in mononuclear blood cells compared with GSTM1 active (26). Additionally, DNA adduct levels in CYP1A1*2B/*2B (n = 3) individuals were higher than those in CYP1A1*1/*2B individuals among moderate smokers. The CYP1A1(*1/*2B or *2B/*2B)–GSTM1*0/*0 combinations showed the highest adduct level among moderate smokers (24). No relevant information exists in the published literature concerning the relation between glutathione S-transferase T1 gene (GSTT1) genotype and bulky aromatic DNA adducts.

The aim of this study was to examine whether BPDE–DNA adducts in leukocytes vary with CYP1A1, GSTM1 or GSTT1 genotype, alone or in combination. We selected coke oven workers because these individuals have long-term exposure to coal tar-derived products such as PAH, particularly B[a]P, which have been shown to cause respiratory tract cancers in certain occupations (34).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and sample collection, B[a]P in air and questionnaire data
Blood samples were obtained from 89 male coke oven workers and from 44 power plant workers during the summer of 1996 (Houillères du Bassin de Lorraine, France). All individuals were male French Caucasians. Ethical clearance and informed consent were obtained from the participants through the collaborating physicians. All participants completed a self-administered questionnaire.

Among the two occupationally or environmentally exposed groups 79 individuals were current smokers and 36 ex-smokers. Coke oven workers were exposed to PAH at the time of blood collection. The selection was based on our previous findings that non-smoking workers exposed occupationally to PAH had low BPDE–DNA adduct levels in their leukocytes, whereas current smoking markedly increased the formation of these adducts in leukocytes (35). A small group of 18 non-smokers (occupationally exposed) was included as a control. Peripheral blood samples were withdrawn by venipuncture into tubes containing EDTA and were collected between June and September 1996. Samples were coded, placed on ice and transported immediately to the medical centre of the plant. Total leukocytes were prepared the same day and the cells were frozen at –80°C before DNA extraction. All samples were transported to the German Cancer Research Center in Heidelberg for further analysis.

Air concentrations of B[a]P at the workplace were determined as an annual average by industrial hygienists from the data of personal sampling devices. Workers were exposed for 6–8 working h/day for at least 4–6 months prior to blood collection. The workers were divided into three groups based on the level of exposure: low <1.5 µg B[a]P/m3 (n = 49); medium 1.5–4 µg B[a]P/m3 (n = 13); high >4 µg B[a]P/m3 (n = 27). Workers with other occupational exposures were excluded from the study.

DNA isolation
Leukocytes were collected by centrifugation after incubation with 0.12 M NH4Cl to lyse the erythrocytes and DNA was isolated as described (9). The concentration of DNA was determined spectrophotometrically by measuring the UV absorbance at 260 nm and the purity was ascertained by the ratio at 260/280 and 260/230 nm.

BPDE–DNA adduct determination by HPLC/fluorometric assay
DNA (100 µg) was used for analysis of B[a]P tetrols, allowing quantification of BPDE–DNA adducts. Analysis of B[a]P tetrols by HPLC with fluorometric detection (HPLC–FD) was carried out as previously described (36,37). The levels of BPDE adducts per 108 nt was calculated using 1 pmol/mg DNA: 3.125 = 1 adduct/106 nt. B[a]P tetrol standards were obtained from the NCI Carcinogen Standard Repository (Midwest Research Institute, Kansas City, MO).

CYP1A1, GSTM1 and GSTT1 genotyping
PCR–restriction fragment length DNA polymorphism-based analysis of CYP1A1 gene polymorphisms and GSTM1 gene deletion was as previously described (38,39). An allele carrying only a T->C transition 1194 bp downstream of exon 7 in the 3'-flanking region, leading to a MspI restriction site (m1), was termed *2A. An allele with m1 plus a mutation in exon 7, leading to an Ile/Val exchange at codon 462 (m2) due to an A->G transition at nt 4889 was termed *2B. An allele carrying a C->A transversion at position 4887 in exon 7 of CYP1A1 leading to a Thr/Asn exchange at codon 461 (close to the known codon 462 Ile/Val mutation) was termed *4 (38) The corresponding deficient phenotype of the GSTM1*0/*0 genotype was termed the GSTM1 null phenotype. The GSTM1 active genotype comprises the following allele configurations: GSTM1*A/*A, GSTM1*A/*0, GSTM1*B/*B and GSTM1*B/*0.

Statistical analysis
Differences in adduct levels between the various groups were assessed with the Wilcoxon rank sum test. Differences in proportions of adduct levels present between different groups were assessed with Fisher's exact test or with the {chi}2 test where appropriate. The presence or absence of DNA adducts was modelled as a function of CYP1A1, GSTM1 and GSTT1 status, smoking, exposure and age using logistic regression. Detectable adduct levels were included in a covariance analysis to investigate the influence of GSTT1, CYP1A1, smoking and exposure on adduct level, controlling for age, excluding one individual with an outlying adduct level of 44 adducts/108 nt. All analyses were performed with SAS software (v.6.12; SAS Institute Inc., Cary, NC).


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The mean age of non-smokers, ex-smokers and smokers in the groups with low, medium and high exposure and smokers from the non-exposed group was similar, ranging from 39.3 to 42.8 years. The mean (± SD) of number of packs of cigarettes smoked per day multiplied by the number of years of smoking (PAQ) was almost twice as low in ex-smokers (12.3 ± 8.2) compared with current smokers (22.6 ± 11.9). The CYP1A1, GSTM1 and GSTT1 genotype frequencies did not differ between coke oven workers and the power plant group and agreed with the frequencies described in the literature for Caucasian populations (Table IGo) (3847). The low frequency of GSTT1*0/*0 in the power plant workers is a chance finding in this small group of subjects (n = 44).


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Table I. Distribution of CYP1A1, GSTM1 and GSTT1 genotypes in coke oven workers and in power plant workers
 
BPDE–DNA adducts in leukocytes
Preliminary experiments on several workers showed that after two samplings over a 5 day working period no significant increase in DNA adduct levels was detectable. For this reason we performed only one sampling per individual during the 2 month period of blood collection (35,48). BPDE–DNA adducts in leukocyte DNA samples from 89 coke oven workers, including smokers and non-smokers, and from 44 unexposed power plant workers who were smokers were analysed. BPDE–DNA adducts were detected in 69 of 133 DNA samples (52%), with a 100-fold variation (range 0.2–44 adducts/108 nt) and a median of 1.6 adducts/108 nt (Table IIGo). Large intra- and inter-individual differences between adduct values were found. The average adduct level of 45 workers with the GSTM1*0/*0 genotype from the exposed group (median 2.0 adducts/108 nt, range 0.4–44 adducts/108 nt) was significantly enhanced compared with 24 subjects with the GSTM1*0/*0 genotype from the power plant group (median 0.8 adducts/108 nt, range 0.2–6.1 adducts/108 nt, P < 0.01).


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Table II. BPDE–DNA levels in leukocytes from coke oven workers and power plant workers
 
Coke oven workers exposed occupationally to PAH were divided into three groups: low exposure with 21/49 positive adduct samples (<0.15 µg B[a]P/m3); medium exposure with 9/13 positive samples (0.15–4 µg B[a]P/m3); high exposure with 15/27 positive samples (>4 µg B[a]P/m3). The average personal exposure differed by about one order of magnitude between each group. Nevertheless, the median BPDE–DNA adduct levels did not increase from the low to the high exposure groups (P = 0.5). In the total PAH-exposed population, the average BPDE–DNA adduct value of smokers (2.6 adducts/108 nt) was 1.5-fold higher than that of non-smokers (1.7 adducts/108 nt) (P < 0.01). Evaluation including also adduct levels below the detection limit (set as a fixed value) showed no significant difference (P = 0.1). In our previous study on the lymphocyte plus monocyte fraction, this difference was more pronounced (35), which is in agreement with a recent study showing that bulky adduct levels in lymphocyte DNA were significantly higher in smokers as compared with non-smokers, whereas no such difference was observed in leukocyte DNA adduct levels (14). The difference in adduct levels between exposed and non-exposed smokers was 3.2-fold (Table IIGo). We did not determine adduct formation in environmentally PAH-exposed non-smokers, because we anticipated that adduct levels would be below the detection limit (35).

Effect of CYP1A1, GSTM1 and GSTT1 genotype on BPDE–DNA adducts
Individuals with the GSTM1 active genotype (n = 59) did not show detectable BPDE–DNA adducts (<0.2 adduct/108 nt), independent of the CYP1A1 genotype, whereas 69 of 74 individuals with GSTM1*0/*0 had adduct levels with a median of 1.6 adducts/108 nt (range 0.2–44) (Table IIIGo). Categorizing the adduct levels into non-detectable (all 59 samples with GSTM1 active) and detectable (69 samples with GSTM1*0/*0) and testing this correlation with Fisher's exact test yielded P < 0.0001. The five subjects with GSTM1*0/*0 and no detectable adducts were CYP1A1*1/*1 carriers, with three having GSTT1 active and two having GSTT1*0/*0. All had a history of low exposure and were ex-smokers or had no exposure but were smokers. Among the GSTM1*0/*0 group, 42 individuals with the CYP1A1*1/*1 genotype showed low levels of adduct formation (median 1.3 adducts/108 nt, range 0.2–4.1) compared with 26 individuals with heterozygous mutated CYP1A1 genotypes (median 2.5 adducts/108 nt, range 0.4–6.1, P < 0.015) (Table IIIGo). Interestingly, one individual with low PAH exposure and the rare combination CYP1A1*2A/*2A and GSTM1*0/*0 showed an extremely high adduct level of 44 adducts/108 nt, as verified by a duplicate assay.


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Table III. BPDE–DNA adducts in leukocytes from occupationally and environmentally exposed workers grouped according to CYP1A1, GSTM1 and GSTT1 genotype and their combination
 
In the group with GSTT1 active, 59/111 samples showed detectable adducts and 10/22 GSTT1*0/*0 samples showed the presence of adducts. Excluding the subject with an extremely high adduct level (44 adducts/108 nt), there was no clear difference in adduct level between the GSTT1*0*0 and GSTT1 active genotypes (Table IIIGo).

Table IVGo shows the effect of CYP1A1 polymorphism on adduct levels in subjects exposed to different levels of B[a]P and its relation to smoking. An increase in adduct level in mutated CYP1A1 can be observed compared with wild-type CYP1A1 in different subgroups. This increase was mostly not statistically significant in the univariate analysis for both evaluations (all adduct levels including those below the detection limit). However, a significant increase was observed for detectable adduct levels in the subgroup of low and medium exposure (P < 0.01) as well as for all adduct levels (detectable and below the detection limit) in the high exposure group (P = 0.04) and in ex-smokers (P = 0.03).


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Table IV. Detectable levels of BPDE–DNA adducts in leukocytes from occupationally and environmentally exposed workers with respect to exposure and smoking status, grouped according to CYP1A1 polymorphism
 
Multivariate analysis of BPDE–DNA adduct levels
Using logistic regression analysis for the presence or absence of detectable adducts with the variables exposure, smoking and CYP1A1, GSTM1 and GSTT1 status revealed that GSTM1 had a significant effect (P < 0.0001), but no other factor could be found which influenced the presence/absence of detectable adducts. Analysis of covariance was used to investigate the combined influence of CYP1A1, smoking and exposure on DNA adduct levels, controlling for age in those subjects with detectable adduct levels, excluding the individual with 44 adducts/108 nt. Variables were dichotomized as follows: CYP1A1 versus CYP1A1 (*1/*2 or *2A/*2A), environmental exposure (power plant workers) versus occupational exposure (coke oven workers) and non-smokers and ex-smokers versus smokers. This analysis revealed significant effects for CYP1A1 status (P = 0.015), exposure (P = 0.003) and smoking (P = 0.007).

Another covariance model was used to incorporate the quantitative effect of smoking in a model with CYP1A1 status and exposure dichotomized and PAQ as a quantitative variable (n = 54). In this analysis, age (P = 0.009), exposure (P = 0.001) and PAQ (P = 0.015) had significant effects, but the effect of CYP1A1 status was of only marginal significance (P = 0.07).


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BPDE–DNA adducts in leukocytes: effect of exposure and smoking
During the last 15 years a large number of reports have appeared that describe the determination of DNA adducts in leukocytes from individuals occupationally exposed to PAH, i.e. in workers employed on coke ovens, in metal foundries and in the production of aluminium. An equally large number of studies have focused on PAH–DNA adducts related to tobacco smoking. In the majority of these reports, the 32P-post-labelling assay and immunochemical methods have been employed for adduct detection. Large variations were found with these methods, with adduct levels ranging from 0.1 to 35 adducts/108 nt (summarized in ref. 10). In the present study, using leukocytes and HPLC–FD, we detected BPDE–DNA adducts in 45 of 89 blood samples (range 0.6–44 adducts/108 nt). In the group of power plant workers BPDE–DNA adducts were detected in 24 of 44 blood samples (range 0.2–6.1 adducts/108 nt). Several relevant biological and methodological aspects were discussed in our recent review (10) that could explain the discrepancies in results between the different published studies. Moreover, the available data from human studies indicate that lymphocytes are a better surrogate than total white blood cells (10).

We found higher BPDE–DNA adduct levels in the exposed group compared with the non-exposed group (2.0 versus 0.8 adducts/108 nt). Previously, using lymphocytes plus monocytes, we found this difference to be seven times greater than in the present study using leukocytes (35). We did not find differences in adduct levels between the low, medium and high exposure groups. Data from other studies indicated, however, that increased PAH concentrations in ambient air are associated with higher levels of blood cell PAH–DNA adducts (reviewed in ref. 10).

Coke oven workers who smoked had significantly higher BPDE–DNA adduct values in their leukocytes than non-smoking coke oven workers and smoking power plant workers. Data from recent studies showed an enhanced effect of smoking on PAH–DNA adduct levels in workers exposed to high PAH concentrations. The increased formation of and large variability in PAH–DNA adducts in smoking aluminium and coke oven workers (14,35,48) and of BPDE–DNA adducts in smoking coke oven workers (9) suggest that variations in genetically controlled PAH metabolism, modulated by CYP/GST polymorphism, may lead to synergistic effects in DNA binding and, possibly, increased lung cancer risk.

BPDE–DNA adducts in leukocytes: effect of CYP1A1, GSTM1 and GSTT1 genotype
Our study was designed to evaluate the influence of three metabolic susceptibility genes, CYP1A1, GSTM1 and GSTT1, on BPDE–DNA adduct levels in total peripheral leukocytes from coke oven workers and matched non-PAH-exposed workers (controls). We studied the association between CYP1A1, GSTM1, GSTT1 and their combinations and BPDE–DNA adduct levels in peripheral leukocytes in 133 subjects. The impact of PAH exposure and smoking was evaluated.

GSTM1 null subjects had detectable BPDE–DNA adducts, whereas GSTM1 active subjects did not, confirming similar results from a previous study analysing human lung and leukocytes (9). In this study subjects with the GSTM1 active genotype did not have detectable BPDE–DNA adducts, whereas they were detectable in subjects with the GSTM1 null genotype (9). This is in agreement with the findings of Kato et al. (30) and Shields et al. (31), who found a positive association between the GSTM1 null genotype and PAH–DNA adduct levels in human lung tissue. In a study on PAH–DNA adducts in smokers using mononuclear cells, Grinberg-Funes et al. (25) found that the mean adduct levels were higher in individuals with the GSTM1 null genotype, but the difference compared with GSTM1 active was not significant. Butkiewicz et al. (26) found high adduct levels in granulocytes from healthy smokers with the GSTM1 null genotype. A recent study showed a significant increase in DNA adduct levels in leukocytes from lung cancer patients (49), similar to that found previously in a subset of DNA samples from the same patients (36). Smoking lung cancer patients with the genotype combination GSTM1 null–GSTP1(AG or GG genotype) had significantly higher PAH–DNA adduct levels in lung tissue compared with all other genotype combinations (28). Other studies, however, did not report differences in PAH–DNA adduct levels (lymphocytes or leukocytes) between subjects with the GSTM1 null and GSTM1 active genotypes analysing occupationally or environmentally PAH-exposed subjects and smokers (11,14,17,21,23) or lung cancer patients (50).

In this study we have shown that subjects with a heterozygous mutated CYP1A1 genotype (*1/*2 group) had a 2-fold increase in the median adduct levels compared with the wild-type (*1/*1). One individual with the very rare homozygous mutated genotype (*2A/*2A) showed an extremely high adduct level of 44 adducts/108 nt. While smokers with the mutated *1/*2 genotype from the non-exposed group showed a trend towards increased adduct levels, the effect of the mutated *1/*2 genotype in the exposed group was not clear. When several confounding factors, such as B[a]P exposure or cigarette dose (PAQ), were taken into account in an analysis of covariance, subjects with the CYP1A1*1/*2 genotype showed higher adduct levels, and these adduct levels increased with smoking (PAQ).

Mooney et al. (21) reported that PAH–DNA adducts in heavy smokers were 2-fold higher in samples from all subjects with the CYP1A1 exon 7 variant polymorphism than in those without. In contrast, chimney sweeps and fire-fighters with the CYP1A1*1/*1 genotype had significantly higher bulky DNA adduct levels in white blood cells than workers with a Msp1 mutation, measured by 32P-post-labelling (11,17); other studies showed no difference in adduct levels dependent on CYP1A1 genotype (13,50).

While neither the CYP1A1 Msp1 heterozygous genotype alone nor the GSTM1*0/*0 genotype alone were associated with a significant increase in lung cancer risk, the presence of both genetic traits was associated with a 2-fold increase in risk (43). Rothman et al. (17) suggested that the combination of wild type CYP1A1 and deleted GSTM1 was not associated with an increased trend to form adducts; PAH–DNA adduct levels were determined by ELISA assay. Moreover, the only individual in this study (17) who had the rare CYP1A1*2A/*2AGSTM1*0/*0 combination had undetectable levels of PAH–DNA adducts in leukocytes. In summary, the above studies (11,16,17,21,25) failed to find a correlation between the presence of detectable adducts, GSTM1 and/or mutated CYP1A1(*1/*2, *2A/*2A) combination, which is in contrast to the results of our study, which used a sensitive and specific method for BPDE adduct detection.

The ethnic distribution of the GSTT1 polymorphism differs from that of GSTM1 and the two are clearly not linked. Caucasians have a prevalence of ~50% for GSTM1 null (40) and 20.4% for GSTT1 null (41). In our study we found prevalence figures for the GSTT1 null genotype of 20.2% in coke oven workers and 9.1% in power plant workers. The latter frequency seems to be a chance finding in this small group. The homozygous null genotype of the human GSTT1 gene might be a potential candidate for cancer risk. It is not yet known whether BPDE is as good a substrate for GSTT1 as it is for GSTM1. Our data indicate that GSTT1 null is not a critical factor in mediating BPDE–DNA adducts in leukocytes. Fifty-nine out of 111 subjects (51.8%) with GSTT1 active showed detectable adducts.

Association between the CYP1A1 and GSTM1*0/*0 genotypes, BPDE–DNA adducts and risk for smoking-related lung cancer
The association between the GSTM1*0/*0 genotype and an increased risk for developing smoking-related lung cancer is still controversial (reviewed in refs 9,46–50). The results of a meta-analysis of all studies reported supports the view that GSTM1 null alone increases susceptibility to lung cancer among smokers (relative risk 1.4) (47).

Since the GSTM1*0/*0CYP1A1*1/*2A(*2B) combined genotype has been closely linked to lung cancer risk in oriental populations, we focused on the formation of BPDE–DNA adducts. In our study the effect of this genetic combination on DNA adduct levels was statistically significant. We also noted the absence of adducts in subjects with the protective CYP1A1*1/*1GSTM1 active genotype and a gradation from lowest DNA adduct levels in GSTM1*0/*0 subjects with CYP1A1*1/*1 to those with CYP1A1*1/*2. The highest DNA adduct level was observed in the most susceptible GSTM1*0/*0CYP1A1(*1/*2, *2A/*2A) combination.

Taken together, our results show a clear effect of the combination of CYP1A1 and GSTM1 genotypes on the formation of BPDE–DNA adducts in human leukocytes. Using the defined marker compound B[a]P and a specific, sensitive method for B[a]P–DNA adduct detection (HPLC–FD), we conclude that: (i) individuals with the GSTM1 active genotype did not show detectable BPDE–DNA adducts; (ii) those with CYP1A1*1/*1 or heterozygous for CYP1A1*2A or *2B and with GSTM1*0/*0 in combination showed low levels of BPDE–DNA adducts; (iii) one individual with a CYP1A1*2A/*2A mutant allele from the GSTM1*0/*0 group showed an extremely high BPDE–DNA adduct level. These results confirm our previous results (9) and demonstrate that the combination of CYP1A1(*2A/*2A or *2B) and GSTM1*0/*0 appears to be a host factor that can modulate the level of BPDE–DNA adducts in human leukocytes. These results provide a mechanistic understanding of previous studies that correlated these `at risk' genotypes in Japanese with increased smoking-related lung cancer. Our findings are also consistent with the prevalence of G·C->T·A transversion mutations in the p53 gene of lung tumours, indicative of PAH-related mutational damage, which occurs more frequently in persons who are GSTM1*0/*0 (52). Furthermore, the coincidence of mutational hotspots in p53 and BPDE adduct hotspots suggests that B[a]P metabolites, or structurally related compounds, are involved in the transformation of human lung tissue in smokers (6).

In conclusion, the results of this study revealed that genetic polymorphism of carcinogen-metabolizing enzymes affects BPDE–DNA binding in human leukocytes and that cigarette smoking and exposure to PAH have a synergistic effect. Higher levels of BPDE–DNA adducts in leukocytes from individuals with the combined CYP1A1(*1/*2 or *2A/*2A)–GSTM1*0/*0 genotype suggests that carriers of these genotype combinations are at increased risk for contracting lung cancer when exposed to PAH.


    Acknowledgments
 
We are indebted to G.Bielefeldt for skilled secretarial assistance. This work was supported by a DKFZ visiting scientist award to M.R.


    Notes
 
5 To whom correspondence should be addressed Email: h.bartsch{at}dkfz-heidelberg.de. Back


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 Abstract
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 References
 

  1. Shimada,T., Yun,C.H., Yamasaki,H., Gautier,C., Beaune,P.H. and Guengerich,P. (1992) Characterisation of human lung microsomal cytochrome P450 1A1 and its role in the oxidation of chemical carcinogens. Mol. Pharmacol., 41, 856–864.[Abstract]
  2. Bartsch,H., Petruzzelli,S., De Flora,S., Hietanen,E., Camus,A.-M., Castegnaro,M., Alexandrov,K., Rojas,M., Saracci,C. and Giuntini,C. (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, 119–124.[ISI][Medline]
  3. London,S.J., Daly,A.K., Leathart,J.B.S., Navidi,W.C. and Idle,J.R. (1996) Lung cancer risk in relation to the CYP2C9*1/CYP2CP*2 genetic polymorphism among African-Americans and Caucasians in Los Angeles County, California. Pharmacogenetics, 6, 527–533.[ISI][Medline]
  4. Kargman,S., Charleson,S., Cartwright,M., Frank,J., Riendeau,D., Mancini,J., Evans,J. and O'Neil,G. (1996) Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey, and human gastrointestinal tracts. Gastroenterology, 111, 445–454.[ISI][Medline]
  5. Sivarajah,K., Lasker,J.M. and Eling,T.E. (1981) Prostaglandin synthetase-dependent cooxidation of (±)-benzo[a]pyrene-7,8-dihydrodiol by human lung and other mammalian tissues. Cancer Res., 41, 1834–1839.[ISI][Medline]
  6. Denissenko,M.F., Pao,A., Tang,M.S. and Pfeifer,G.R. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science, 274, 430–432.[Abstract/Free Full Text]
  7. Ketterer,B., Harris,J.M., Talaska,G., Meyer,D.J., Pemble,S.E., Taylor,J.B., Lang,N.P. and Kadlubar,F.F. (1992) The human glutathione S-transferase supergene family, its polymorphism, and its effects on susceptibility to lung cancer. Environ. Health Perspect., 98, 87–94.[ISI][Medline]
  8. Sundberg,K., Johansson,A.-S., Stenberg,G., Widersten,M., Seidel,A., Mannervik,B. and Jernström,B. (1998) Difference in the catalytic efficiencies of allelic variants of glutathione transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons. Carcinogenesis, 19, 433–436.[Abstract]
  9. Rojas,M., Alexandrov,K., Cascorbi,I., Brockmöller,J., Likhachev,A., Pozharisski,K., Bouvier, G., Auburtin,G., Mayer,L., Kopp-Schneider,A., Roots,I. and Bartsch,H. (1998) High benzo[a]pyrene diol-epoxide DNA adduct levels in lung and blood cells from individuals with combined CYP1A1 MspI/MspI-GSTM1*0/*0 genotypes. Pharmacogenetics, 8, 109–118.[ISI][Medline]
  10. Kriek,E., Rojas,M., Alexandrov,K. and Bartsch,H. (1998) Polycyclic aromatic hydrocarbon–DNA adducts in humans: relevance as biomarkers for exposure and cancer risk. Mutat. Res., 400, 215–231.[ISI][Medline]
  11. Ichiba,M., Hagmar,L., Rannug,A., Högstedt,B., Alexandrie,A.K., Carstensen,U. and Hemminki,K. (1994) Aromatic DNA adducts, micronuclei and genetic polymorphism for CYP1A1 and GSTM1 in chimney sweeps. Carcinogenesis, 15, 1347–1352.[Abstract]
  12. Poirier,M.C., Rothman,N., Schoket,B., Pan,C.E., Shamkhani,H., Scott,B.G. and Weston,A. (1997) Biomarkers of susceptibility and polycyclic aromatic hydrocarbon (PAH) exposure in U.S. Army soldiers [abstract]. Proc. Am. Assoc. Cancer Res., 38, 460.
  13. Hemminki,K., Dickey,C., Karlsson,S., Bell,D., Hsu,Y., Tsai,W.-Y., Mooney,L.A., Savela,K. and Perera,F.P. (1997) Aromatic DNA adducts in foundry workers in relation to exposure, life style and CYP1A1 and glutathione transferase M1 genotype. Carcinogenesis, 18, 345–350.[Abstract]
  14. Binková,B., Topinka,J., Mrácková,G., Gajdosová,D.,Vidová,P., Stávková,Z., Peterka,V., Pilcik,T., Rimar,V., Dobiás,L., Farmer,P.B. and Srám,R.J. (1998) Coke oven workers study: the effect of exposure and GSTM1 and NAT2 genotypes on DNA adduct levels in white blood cells and lymphocytes as determined by 32P-postlabelling. Mutat. Res., 416, 67–84.[ISI][Medline]
  15. Ichiba,M., Wang,Y., Oichi,H., Iyadomo,M., Shomo,N. and Tomokuni,K. (1996) Smoking-related DNA adducts and genetic polymorphism for metabolic enzymes in human lymphocytes. Biomarkers, 1, 211–214.[ISI]
  16. Santella,R.M., Perera,F.P., Young,T.L., Zhang,Y.-J., Chiamprasert,S., Tang,D., Wang, L.W., Beachman,A., Lin,J.-H. and DeLeo,V.A. (1995) Polycyclic aromatic hydrocarbon–DNA and protein adducts in coal tar treated patients and controls and their relationship to glutathione S-transferase genotype. Mutat. Res., 334, 117–124.[ISI][Medline]
  17. Rothman,N., Shields,P.G., Poirier,M.C., Harrington,A.M., Ford,P. and Strickland,P.T. (1995) The impact of gluthatione S-transferase and cytochrome P450 1A1 genotypes on white blood cell polycyclic aromatic hydrocarbon–DNA adduct levels in humans. Mol. Carcinog., 14, 63–68.[ISI][Medline]
  18. Hou,S.-M., Lambert,B. and Hemminki,K. (1995) Relationship between hprt mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers. Carcinogenesis, 16, 1913–1917.[Abstract]
  19. Nielsen,P.S., de Pater,N., Okkels,H. and Autrup H. (1996) Environmental air pollution and DNA adducts in Copenhagen bus drivers, effect of GSTM1 and NAT2 genotypes on adduct levels. Carcinogenesis, 17, 1021–1027.[Abstract]
  20. Binková,B., Lewtas,J., Misková,I., Rössner,P., Cerná,M., Mrácková,G., Peterková,K., Mumford,J., Meyer,S. and Srám,R.J. (1996) Biomarker studies in Northern Bohemia. Environ. Health Perspect., 104, 591–597.[ISI][Medline]
  21. Mooney,L.V., Bell,D.A., Santella,R.M., Van Bennekum,A.M., Ottman,R., Paik,M., Blaner,W.S., Lucier,G.W., Covey,L., Young,T.-L., Cooper,T.B., Glassman,H. and Perera,F.P. (1997) Contribution of genetic and nutritional factors to DNA damage in heavy smokers. Carcinogenesis, 18, 503–509.[Abstract]
  22. Dallinga,J.W., Pachen,D.M.F.A., Wijnhoven,S.W.P., Breedijk,A., Van't Veer,L., Wigbout,G., Van Zandwijk,N., Maas,L.M., Van Agen,E., Kleinjans,J.C.S. and Van Schooten,F.J. (1998) The use of 4-aminobiphenyl–hemoglobin adducts and aromatic DNA adducts in lymphocytes of smokers as biomarkers of exposure. Cancer Epidemiol. Biomarkers Prev., 7, 571–577.[Abstract]
  23. Nielsen,P.S., Okkels,H., Sigsgaard,T., Kyrtopoulos,S. and Autrup,H. (1996) Exposure to urban and rural air pollution. DNA and protein adducts and the effect of glutathione S-transferase genotype on adduct level. Int. Arch. Occup. Environ. Health, 68, 170–176.[ISI][Medline]
  24. Ichiba,M., Wang,Y.P., Oichi,H., Zhang,J.S., Iyadomo,M., Minagawa,M. and Tomokuni,K. (1998) Lymphocytes, DNA adducts and genetic polymorphism for metabolic enzymes in low dose cigarette smokers. Biomarkers, 3, 63–71.[ISI]
  25. Grinberg-Funes,R.A., Singh,V.N., Perera,F.P., Bell,D.A., Young,T.L., Dickey,C., Wang,L.W. and Santella,R.M. (1994) Polycyclic aromatic hydrocarbon–DNA adducts in smokers and their relationship to micronutrients levels and the glutathione S-transferase M1 genotype. Carcinogenesis, 15, 2449–2454.[Abstract]
  26. Butkiewicz,D., Grzybowska,E., Hemminki,H., Øvrebø,S., Haugen,A., Motykiewicz,G. and Chorazy,M. (1998) Modulation of DNA adduct levels in human mononuclear white blood cells and granulocytes by CYP1A1, CYP2D6 and GSTM1 genetic polymorphisms. Mutat. Res., 415, 97–108.[ISI][Medline]
  27. Soni,M., Madurantakan,M. and Krishnaswamy,M. (1998) Glutathione S-transferase mu (GSTmu) deficiency and DNA adducts in lymphocytes of smokers. Toxicology, 126, 155–162.[ISI][Medline]
  28. Ryberg,D., Skaug,V., Hewer,A., Phillips,D.H., Harries,L.W., Wolf,C.R., Ogreid,D., Ulvik,A., Vu,P. and Haugen,A. (1997) Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk. Carcinogenesis, 18, 1285–1289.[Abstract]
  29. Shields,P.G., Caporaso,N.E., Falk,R.T., Sugimura,H., Trivers,G.E., Trump,B.F., Hoover,R.N., Weston,A. and Harris,C.C. (1993) Lung cancer, race, and a CYP1A1 genetic polymorphism. Cancer Epidemiol. Biomarkers Prev., 2, 481–485.[Abstract]
  30. 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, 902–907.[Abstract]
  31. 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, 3486–3492.[Abstract]
  32. Bartsch,H. (1996) DNA adducts in human carcinogenesis: etiological relevance and structure–activity relationship. Mutat. Res., 340, 67–79.[ISI][Medline]
  33. 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, 841–846.[Abstract]
  34. International Agency for Research on Cancer (1984) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 34, Polynulear Aromatic Compounds, Part 3. IARC, Lyon, pp. 37–64.
  35. Rojas,M., Alexandrov,K., Auburtin,G., Wastiaux-Denamur,A., Mayer,L., Mahieu,B., Sebastien,P. and Bartsch,H. (1995) Anti-benzo[a]pyrene diolepoxide–DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking. Carcinogenesis, 16, 1373–1376.[Abstract]
  36. 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, 557–560.[Abstract]
  37. 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 diolepoxide–DNA adducts in smoker's lung: comparison with total bulky adducts and aryl hydrocarbon hydroxylase activity. Cancer Res., 52, 6248–6253.[Abstract]
  38. Cascorbi,I., Brockmöller,J. and Roots,I. (1996) A C4887A polymorphism in exon-7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility. Cancer Res., 56, 4965–4969.[Abstract]
  39. Brockmöller,J., Kerb,R., Drakoulis,N., Nitz,M. and Roots I. (1993) Genotype and phenotype of glutathione S-transferase class µ isoenzymes in lung cancer patients and controls. Cancer Res., 53, 1004–1011.[Abstract]
  40. Bell,D.A., Taylor,J.A., Paulson,D.F., Robertson,C.N., Mohler,J.L. and Lucier,G.W. (1993) Genetic risk and carcinogenic exposure: a common inherited defect of the carcinogen metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J. Natl Cancer Inst., 85, 1150–1164.
  41. Nelson,H.H., Wiencke,J.K., Christiani,D.C., Cheng,T.J., Zuo,Z.-F., Schwartz,B.C., Lee,B.-K., Spitz,M.R., Wang,M., Xu,X. and Kelsey,K.T. (1995) Ethnic differences in the prevalence of the homozygous deleted genotype of glutathione S-transferase theta. Carcinogenesis, 1243–1245.
  42. Seidegård,J., Pero,R., Markowitz,M., Roush,G., Miller,D. and Beattie,E.J. (1990) Isoenzymes of glutathione transferase (class Mu) as a marker for susceptibility to lung cancer: a follow up study. Carcinogenesis, 11, 33–36.[Abstract]
  43. Nazar-Stewart,V., Motulski,A.G., Eaton,D.L., White,E., Hornung,S.K., Leng,Z.T., Stapleton,P. and Weuss,N.S. (1993) The glutathione S-transferase mu polymorphism as a marker for susceptibility to lung carcinoma. Cancer Res., 53, 2313–2318.[Abstract]
  44. Hirvonen,A., Husgafvel-Pursiainen,K., Anttilla,S. and Vainio,H. (1993) The GSTM1 null genotype as a potential risk modifier for squamous cell carcinoma of the lung. Carcinogenesis, 14, 1479–1481.[Abstract]
  45. Zhong,S., Howie,A.F., Ketterer,B., Taylor,J., Hayes,J.D., Beckett,G.J., Wathen,C.G., Wolf,C.R. and Spurr,N.K. (1991) Glutathione S-transferase µ locus: use of genotyping and phenotyping assays to assess association with lung cancer susceptibility. Carcinogenesis, 12, 1533–1537.[Abstract]
  46. Brockmöller,J., Cascorbi,I., Kerb,R. and Roots,I. (1996) Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 1, glutathione S-transferase M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res., 56, 3915–3925.[Abstract]
  47. McWilliams,J.E., Sanderson,B.J.S., Harris,E.L., Richert-Boe,K.K. and Henner,W.D. (1995) Glutathione S-transferase M1 (GSTM1) deficiency and lung cancer risk. Cancer Epidemiol. Biomarkers Prev., 4, 589–594.[Abstract]
  48. Van Schooten,F.J., Van Leeuwen,F.E., De Looff,A.J.A., Dijkmans,A.P.G., Van Rooij, J.G.M., Den Engelse,L. and Kriek,E. (1995) Polycyclic aromatic hydrocarbon–DNA adducts in white blood cell DNA and 1-hydroxypyrene in the urine from aluminum workers: relation with job category and synergistic effect of smoking. Cancer Epidemiol. Biomarkers Prev., 4, 69–77.[Abstract]
  49. Tang,D.L., Rundle,A., Warburton,D., Santella,R.M., Tsai,W.-Y., Chiamprasert,S., Hsu,Y.Z. and Perera,F.P. (1998) Association between both genetic and environmental biomarkers and lung cancer: evidence of a greater risk of lung cancer in women smokers. Carcinogenesis, 19, 1949–1953.[Abstract]
  50. Pastorelli,R., Guanci,M., Cerri,A., Negri,E., LaVecchia,C., Fumagalli,F., Mezzeti,M., Cappelli,R., Panigalli,T., Fanelli,R. and Airoldi,L. (1998) Inpact of inherited polymorphisms in glutathione S-transferase M1, microsomal epoxide hydrolase, cytochrome P450 enzymes on DNA, and blood protein adducts of benzo[a]pyrene diolepoxide. Cancer Epidemiol. Biomarkers Prev., 7, 703–709.[Abstract]
  51. Garcia-Closas,M., Kelsey,K.T., Wiencke,J.K., Xu,X., Wain,J.C. and Christiani,D.C. (1997) A case–control study of cytochrome P4501A1, glutathione S-transferase M1, cigarette smoking and lung cancer susceptibility. Cancer Causes Control, 8, 544–553.[ISI][Medline]
  52. Ryberg,D., Kure,E., Lystad,S., Skaug,V., Stangeland,L., Mercy,I., Borresen,A.-L. and Haugen,A. (1994) p53 mutations in lung tumors: relationship to putative susceptibility markers for cancer. Cancer Res., 54, 1551–1555.[Abstract]
Received April 6, 1999; revised July 5, 1999; accepted September 21, 1999.