Associations between smoking, GST genotypes and N7-methylguanine levels in DNA extracted from bronchial lavage cells

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Abstract

N7-Methylguanine (N7-MeG) DNA adducts are markers of human exposure to methylating agents including tobacco-specific nitrosamines (TSNAs). Repair of this adduct is poor, so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism. N7-MeG adducts in lung DNA from bronchial lavage samples were measured to determine whether levels were higher in smokers than non-smokers, and if levels were modified by genetic variation in carcinogen-metabolising enzymes. Adducts were detected in 38 out of 44 DNA samples by 32P post-labelling of the N7-methyldeoxyguanosine-3′-monophosphate (N7-MedGp) isolated from DNA digests by two-stage HPLC. N7-MeG adduct levels were higher in smokers than in never smokers ((9.99±20.3)×10−7 versus (0.58±0.50)×10−7 N7-MedGp/deoxyguanosine-3′-monophosphate (dGp); P=0.02) and intermediate in ex-smokers ((5.59±15.6)×10−7 N7-MedGp/dGp). Adduct levels tended to be higher in individuals with GSTM1 null, GSTT1 null or GSTP1 ile/ile genotypes. When genotypes were combined, N7-MedGp levels among GSTM1 null/GSTT1 null individuals (n=6) were higher than among those having at least one wild-type allele of these two genes ((26.1±38.0)×10−7 versus (2.73±4.07)×10−7 N7-MedGp/dGp), although the results were not statistically significant (P=0.13). Adduct levels were highest in individuals with three unfavourable genotypes (GSTM1 null/GSTT1 null and GSTP1 ile/ile) compared with others ((74.5±13.1)×10−7 versus (2.64±3.89)×10−7 N7-MedGp/dGp, P=0.02). N7-MeG adduct levels in DNA isolated from lung tissue thus reflect exposure to cigarette smoke, and genetic variation in carcinogen-metabolising enzymes may modify these levels.

Introduction

Human exposure to tobacco-specific nitrosamines (TSNAs) is possibly greater than that of any other potent carcinogen [1]. Formed by nitrosoation of nicotine (a tertiary amine) and the secondary amines nornicotine, anabasine and anatabine, during curing and smoking of tobacco [2], they are the only group of compounds specifically associated with tobacco and tobacco products [3] and have been shown to induce adenocarcinomas of the lung in laboratory animals [4]. The yield of TSNA in tobacco is positively correlated with nitrate content of the cigarette [5]. Activation of TSNAs in the lung can result in the formation of DNA adducts, of which N7-methylguanine (N7-MeG) is produced in the greatest abundance. This adduct is relatively innocuous, but it may serve as a good biomarker of DNA damage caused by TSNAs and other methylating agents in tobacco smoke. Indeed, N7-MeG has been shown to be correlated with levels of other pro-mutagenic and carcinogenic adducts resulting from TSNA exposure such as O6-MeG [6]. Furthermore, repair of N7-MeG is poor [7], so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism.

There have been relatively few studies that have measured N7-MeG levels in human lung tissue (Table 1). 32P post-labelling has been used to measure N7-methyldeoxyguanosine-3′-monophosphate (N7-MedGp) levels in lung tissue but all studies except that of Kato et al. [12] have involved small numbers of patients and only two have reported a relationship between N7-MeG adduct levels and smoking [10], [11]. Polymorphisms exist in the genes encoding carcinogen-metabolising enzymes, which result in variations in their expression and catalytic properties. Such variations may result in differences in the ability of lung tissue to metabolise TSNA. N7-MeG levels have been associated with polymorphisms in the genes CYP2E1 and CYP2D6 [12]. An increase in N7-MeG levels was found in CYP2D6 extensive metabolisers and heterozygotes compared with poor metabolisers, and an increase was also observed in CYP2E1 CD individuals compared with CYP2E1 DD individuals. These differences were, however, only evident in persons with low serum cotinine levels. GST enzymes are involved in the detoxification of many carcinogens, and adduct levels have been reported in some but not all studies to be higher among GSTM1-null and GSTT1-null individuals [14]. Further, O6-MedGp levels were found to be higher in colon tumours of GSTT1-null individuals than of GSTT1 wild-type individuals [15]. NAD(P)H:quinone oxido-reductase (NQO) has been show to be present in lung epithelium and this enzyme is able to metabolise carcinogens found in tobacco smoke [16]. We have reported an increase in small-cell lung cancer risk among carriers of a rare NQO allele in this study population [17].

We have carried out the following study to test the hypothesis that N7-MeG levels in lung DNA are higher in smokers than in non-smokers, and that these levels are modified by genetic variation in carcinogen-metabolising enzymes.

Section snippets

Study population

The subjects were enrolled in a study of susceptibility to lung cancer, details of which have been published previously [17]. Patients were recruited from bronchoscopy clinics at the North West lung centre, South Manchester University NHS Trust, Manchester, UK. Eligible cases were all patients diagnosed as having, or with a history of, cancers of the lung, trachea or bronchus between March 1996 and April 1997. Controls were selected from all other patients who were free of benign and malignant

Differences in N7-MedGp levels between smokers, ex-smokers and never smokers

N7-MedGp was detected in 9 out of 10 self-reported smokers, 25 out of 28 ex-smokers and 4 out of 6 never smokers; there was no significant difference in alkylation frequency (P=0.32). Detected adduct levels ranges are: (0.26–83.7)×10−7 N7-MedGp/dGp. Including samples with no detectable N7-MedGp meant that level ranges are: (0.05–65.2)×10−7, (0.05–83.7)×10−7 and (0.05–1.17)×10−7 N7-MedGp/dGp in smokers, ex-smokers and never smokers, respectively. Inter-quartile ranges were: (1.24–4.44)×10−7,

Discussion

In this study we find a clear association between N7-MedGp levels in DNA isolated from bronchial lavage cells and exposure to cigarette smoke. Surprisingly, given the high levels of tobacco-specific nitrosoamines present in cigarette smoke and their metabolism to methylating agents, there appears to be only two other studies that have reported an association between N7-MedGp levels and smoking status. Large inter-individual variation in adduct levels were identified, which may impact on the

Acknowledgements

We would like to thank the staff and patients at the Bronchoscopy Unit of the North West Lung Centre, Wythenshawe Hospital. The work was funded by a bequest fellowship for S.J.L. from the University of Manchester and in part by Cancer Research UK.

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