DNA adducts associated with tobacco smoking could provide a marker of biologically effective dose of tobacco carcinogens and improve individual cancer risk prediction. A significant number of clinical and epidemiologic studies have reported associations of increased DNA adduct levels with the occurrence of the prevalent tobacco related cancers including cancer of the lung, head and neck, and bladder. The inducibility of DNA adducts following in vitro treatments using blood lymphocytes also appears to be a risk factor in the development of lung and head and neck cancer. Corroborative evidence pointing to the importance of DNA adducts in tobacco carcinogenesis include numerous studies showing associations of tobacco smoke exposure with the induction of DNA adducts in humans in vivo. Further effort is necessary, however, to more fully characterize the dose–response relationship between smoking and DNA adducts in exposed target and surrogate tissues. The relationship between gene polymorphisms thought to modify tobacco-related cancer risk and DNA adduct levels is complex. Results of some DNA adduct studies (both in vitro and in vivo) appear inconsistent with the epidemiologic findings. This is evident for polymorphisms involving both carcinogen metabolism (e.g. GSTP1) and DNA repair (e.g. XRCC1). Molecular studies of human tumors suggest associations of p53 mutation with DNA adducts and have revealed correlations of DNA adduct levels with somatic alterations (e.g. 3p21 LOH) that are thought to occur at the very earliest stages of tobacco carcinogenesis. More research is needed to assess the relationship between endogenous sources of DNA adducts and tobacco smoke exposure and the relative oncogenic effects of chemically stable versus unstable DNA adducts. Many potentially fruitful new avenues of cancer research are emerging that integrate DNA adduct analyses with assessments of smoking, genetics, diet and ambient air quality. These investigations aim to understand the multifactorial nature of interindividual variability in response to tobacco carcinogens. As these trends continue a variety of innovative study designs and approaches will become important in human populations.
DNA adducts are physical complexes formed between reactive chemical species and sites within the DNA molecule. DNA adducts have been proposed as potential markers of ‘biologically effective dose’ from exposure to tobacco carcinogens that may help to provide an integrated measure of carcinogen exposure relevant to individual cancer risk assessment. The emphasis of this review is on human in vivo studies except where model in vitro or animal studies are relevant to a specific issue. This review emphasizes literature appearing since 1998; below are cited reviews of earlier literature. The field of investigation that addresses DNA modifications in human cancer is expansive. This review is restricted to epidemiologic and clinical studies that address five areas bearing on the etiologic role of DNA adducts in tobacco carcinogenesis:
The evidence that genetic polymorphisms implicated in tobacco related cancers modify DNA adduct concentrations in human tissues;
The evidence that DNA adducts are risk factors in the development of tobacco related cancers;
The association of DNA adducts with molecular alterations involving oncogenes and tumor suppressor genes in human tumors;
The potential importance of endogenous and chemically unstable DNA adducts in tobacco carcinogenesis; and
Methodologic issues that affect the validity of investigations into the role of DNA adducts in tobacco carcinogenesis.
Several recent reviews have addressed the use of DNA adducts in risk assessment and toxicology studies (van Delft et al., 1998a; Vainio, 1998; Timbrell, 1998; Kriek et al., 1998; Garner, 1998; Carmichael, 1998; Hemminki et al., 2001). Other reviews emphasize the chemistry of adduct formation and laboratory methodology (Phillips et al., 2000; Reddy, 2000; Szeliga and Dipple, 1998). Although most recent studies have employed only a few analytical approaches (i.e. 32P-postlabeling or immunologically based assays), new technologies and improved applications have also been reviewed (Turteltaub and Dingley, 1998, accelerated mass spectrometry); (Marzilli et al., 2001, capillary-electrophoresis-mass spectrometry); (Shinozaki et al., 1998, flow cytometry); (Ni et al., 1998, electrospray tandem mass spectrometry); (Petruzzelli et al., 1998, serum BPDE-DNA antibodies by ELISA); (Bucci et al., 1998, competitive ELISA); (Xing et al., 2001, capillary electrophoresis); (Tan et al., 2001, immunoelectrophoresis with laser induced fluorescence).
Previous reviews of genetic polymorphisms have assessed the potential for adducts to provide an intermediary tool for genotyping studies (Hemminki et al., 2000). The relationship of genotype to adduct formation in determining risk for lung, esophagus and oral cancer have been evaluated (Bartsch et al., 1998). An overall weight of evidence approach concluded that under conditions giving rise to detectable PAH–DNA adducts levels in tissues, most studies reviewed reported a significant role for GSTM1 deletion either alone or in combination with CYP1A1 variants on variations in adduct levels and urinary metabolites of genotoxic substances (Pavanello and Clonfero, 2000).
Glutathione S-transferases and cytochrome P4501A1
Although early studies suggested a role of GSTM1 deficiency in aromatic-hydrophobic DNA adduct levels (Ryberg et al., 1994; Grinberg-Funes et al., 1994; Shields et al., 1993; Kato et al., 1995) these studies were criticized (Cuzick, 1995) for not defining cigarette smoking exposure adequately and for not having sufficient size to adjust for potential statistical artifacts. Continued work on the GSTs has appeared (Table 1) and more recent studies have included polymorphism of the GSTT1 and GSTP1 genes. The GSTP1 isoforms could be particularly relevant in lung carcinogenesis as GSTP1 is highly expressed in lung and shows high activity in detoxifying diol epoxides of PAHs. Human GSTP1 expression in cell culture has been shown to be very effective in preventing the formation of BPDE–DNA adducts produced in vitro (Fields et al., 1998). Two common polymorphisms in GSTP1 give rise to four genotypic variants in human populations. Although allelic variants (i.e. A313G; Ile105Val) show reduced enzyme activity toward the model substrate 1-chloro-2,4-dinitrobenzene (Watson et al., 1998) when transfected into human HepG2 cells these variants were shown to provide greater protection against the formation of (+)-anti-BPDE–DNA adducts (Hu et al., 1999) compared with the wildtype GSTP1 isoform. The wildtype GSTP1 contains isoleucine at position 105. Catalytic properties of both recombinant and native variants of GSTP1 enzymes were compared with wildtype forms and the variants were shown to display greater catalytic efficiency towards carcinogenic PAHs (Coles et al., 2000a). Lung, bladder and esophageal cancer risk, however, have been associated with GSTP1 genotypes presumed to result in greater not less PAH detoxification (Ryberg et al., 1997; Kihara and Noda, 1999; Harries et al., 1997; van Lieshout et al., 1999).
DNA adduct studies thus far have not fully resolved this apparent anomaly. In studies of newborns, PAH–DNA adducts were marginally higher among subjects with the GSTP1 Ile/Val and Ile/Ile genotypes compared with the GSTP1 Val/Val genotype (Whyatt et al., 2001). Similarly, the lowest levels of DNA adducts were observed among non-smoking women carrying the GSTP1 Val/Val genotype (Grzybowska et al., 2000) compared to others, but in occupationally exposed populations no effect of the GSTP1 polymorphism by itself has been reported on variations in PAH–DNA adduct levels (Zhang et al., 2000; Viezzer et al., 1999; Grzybowska et al., 2000; Schoket et al., 2001). The GSTP1 polymorphism was also not found to be not predictive of PAH–DNA adduct levels in bronchial tissues of lung cancer patients (Ozawa et al., 1999).
There have been suggestions of interactions between GSTP1 and other polymorphisms, but again the picture is far from clear. Whereas PAH–DNA adducts in occupationally exposed workers were elevated in GSTM1 null subjects and in persons carrying a combined GSTM1 null and GSTP1 variants (Schoket et al., 2001), another study of cigarette smokers found evidence of the GSTM1 non-null genotype interacting with GSTP1 variants and being associated with elevated DNA adducts (Butkiewicz et al., 2000). A suggested interaction of the MspI variant of CYP1A1 with the GSTP1 Val/Val genotype on adduct levels in newborns was reported in one study (Whyatt et al., 2001). It has been proposed (Coles et al., 2000b) that GSTP1 like GSTM1 is coordinately regulated with the GSTM3 locus. However, GSTM3 genotypes were not found to affect the relationship of GSTP1 with lung cancer in a recent study of 389 lung cancer cases and 353 controls in Germany (Risch et al., 2001).
A re-examination of GSTP1 genotype–phenotype relationships (Sundberg et al., 2002) has suggested that the GSTP1 enzyme containing valine at position 105 may actually have less than, or at most equal activity in reducing, PAH–DNA adduct formation compared with the wildtype GSTP1 Ile105/Ala114 isoform. The authors caution against extrapolating catalytic information from pure enzymes to the complex situation within the intact cell. Notably, they found only about 1–2% of the expected rate of BPDE conjugation to be observed in vivo in cells, which the authors postulate may be a result of the reduced concentrations of lipophilic substrates available for conjugation by the soluble GSTs (Sundberg et al., 2002).
Among coke oven workers the GSTM1 null genotype was associated with adducts among highly PAH exposed groups but GSTT1 null subjects, who are deficient in GSTT1 mediated GSH conjugation, demonstrated lower not higher adduct burdens (Viezzer et al., 1999). Negative studies have also appeared in which adduct levels in non-tumorous lung or blood MNCs were not modified by GSTM1 (Wiencke et al., 1999). Although several analytic factors have been discussed in regards to discordant results (Phillips and Castegnaro, 1999), some studies point to factors in the diet that may influence these types of comparisons. For example, GSTM1 null smokers were reported to have higher adduct levels compared with non-null smokers, and these associations were found to be modified by plasma antioxidants (Wang et al., 1998b). DNA adducts were found higher in GSTM1 null subjects in the EPIC–Italy study, and were inversely associated with the consumption of fresh fruit, vegetables, olive oil and antioxidants (Palli et al., 2000). Earlier studies suggested an important role for dietary antioxidants on DNA adduct results in smokers (Grinberg-Funes et al., 1994; Mooney et al., 1997). Given the significant role dietary constituents are now thought to play in lung cancer (Williams and Sandler, 2001) further work in this area is needed. Such information may help to improve preventive strategies involving anti-oxidants and dietary interventions (Saha et al., 2001; Jacobson et al., 2000).
Expression of CYP1A1 as assessed by mRNA levels in lung tissue was shown to be positively correlated with variations in hydrophobic-DNA adduct levels in currently smoking lung cancer patients (Mollerup et al., 1999). Earlier work showed a strong correlation of microsomal aryl hydrocarbon hydroxylase activity in lung with BPDE–DNA adduct levels (Alexandrov et al., 1992). The importance of CYP1A1 gene polymorphisms and other CYP related gene polymorphisms in adduct formation is more ambiguous. Polymorphisms at the CYP1A1 locus were not associated with variations in adduct levels in occupationally exposed workers (Schoket et al., 2001; Grzybowska et al., 2000; Zhang et al., 2000; Pan et al., 1998; Motykiewicz et al., 1998), nor in lung cancer patients (Schoket et al., 1998; Wiencke et al., 1999). One small study of adducts in lung cancer patients, however, did suggest an interaction of the GSTM1 null genotype with homozygosity of the CYP1A1 MspI variant (Rojas et al., 1998). Several studies have reported CYP1A1 variants to be associated with higher adduct levels in newborn blood and placental tissues (Whyatt et al., 1998, 2001).
Despite years of study, the role of CYP1A1 gene polymorphisms in PAH activation remains unclear. The notion that DNA adduct studies in vivo among smokers and occupationally exposed populations or patients with smoking related cancers could help to elucidate the toxicologic functionality of CYP polymorphisms has not yet been proven valid. The unexpected and paradoxical finding of increased hepatic DNA adducts of BP in CYP1A1 knockout mice also suggests that further study is warranted on the kinetics of metabolic clearance of PAHs and its impact on DNA adduct formation (Uno et al., 2001).
N-acetylation and aromatic amines
After cancer of the lung and bronchi, bladder cancer is the most prevalent tobacco related malignancy. A series of earlier studies suggested that differences in carcinogen-adduct levels can correlate closely to differences in bladder cancer risks that are determined both by carcinogen exposure intensity and metabolic genotype (Vineis and Martone, 1996; Vineis and Ronco, 1992). Generally these studies support the idea that adduct levels correlate with smoking related risk, particularly those differences in risk associated with smoking black (air cured) versus blonde (flue cured) tobacco. Protein–carcinogen adduction has been the focus of most of the research. Some potential inconsistencies were raised, however, in studies of DNA adducts. For example, no evidence of case control differences in urothelial cell DNA adduct levels were recorded in bladder biopsies (Talaska et al., 1994), nor were associations observed between 4-aminobiphenyl-DNA adduct levels and p53 mutations in bladder tumors (Martone et al., 1998). In urine, the major adduct forming species associated with smoking black tobacco was suggested to be PhIP (Peluso et al., 1991). More relevant to the more widely consumed flu cured tobacco is the identification of the putative N-(deoxyguanosin-8-yl)-4-aminobiphenyl DNA adduct in exfoliated bladder cells, the concentration of which was found to be related to the number of cigarettes smoked per day (Talaska et al., 1991).
Recently, in a case control study of bladder cancer, NAT2 slow acetylator genotypes were associated with increased aromatic DNA adducts in white blood cells from buffy coats obtained from cancer cases, but no such association was evident among the controls (Peluso et al., 2000). Researchers also reported that dietary fruits and vegetables were inversely related to adduct levels. Curiously, smoking was not found to be associated with the DNA adducts levels measured (Peluso et al., 2000). In the aforementioned case series of bladder cancer biopsies no association of NAT2 genotypes was observed with 4-ABP DNA adduct measurements although current smoking and smoking air cured tobacco were positively associated with increased adduct levels (Martone et al., 1998). NAT2 genotypes associated with slow acetylator phenotypes were associated with higher aromatic-DNA adduct levels (Hou et al., 2001; Matullo et al., 2001a). No effect of either NAT1 or NAT2 polymorphisms was found in an analysis of aromatic amine-DNA adducts in another study (Godschalk et al., 2001). These mixed results relating adduct measurements and NAT2 genotypes should be considered in light of the overall modest increase in risk for bladder cancer associated with polymorphisms at this locus indicated in a recent meta-analysis of epidemiologic studies (Marcus et al., 2000).
Quinone reductases and oxidative DNA damage
Only a few studies have examined DNA adducts in pancreatic tissues of smokers and non-smokers. One of these concluded that neither GSTM1 nor NQO1 modified DNA adducts related to oxidative stress (Kadlubar et al., 1998). This study must be considered preliminary with regard to the less prevalent genotypes (e.g. NQO1 and GSTT1 homozygous variant individuals), and more studies could be valuable. Cigarette smoking is one of only a few identified environmental risk factors for pancreatic cancer. A recent study (Duell et al., 2002) indicated an increased risk of GSTT1 null subjects among heavy smokers and perhaps a higher risk among women who were GSTT1 null and who smoked heavily.
A strong mechanistic rationale implicates metabolic polymorphisms in the formation of smoking related DNA adducts, however, the evidence supporting genetic modification of DNA adducts from cigarette smoking in vivo in man must be considered equivocal. Strong associations of CYP1A1 expression and enzyme activity with PAH–DNA adducts in vivo and negative or inconsistent findings with CYP1A1 genetic variants argues against the functional importance of the known CYP1A1 polymorphisms. Indeed, the relationships between the common genetic variations and enzyme inducibility is still unclear. Extensive characterization of the catalytic properties of GSTP1 variants and the weak or inconsistent relationships in vivo require further evaluation. Hemoglobin carcinogen adducts appear a better marker of biological effective dose for bladder cancer compared with DNA adducts.
DNA repair polymorphisms
XRCC1, XCRCC3, XPD, ERCC2, hOGG1, p53
While several epidemiological studies have implicated polymorphisms in DNA repair enzymes in cancers of the lung (Ratnasinghe et al., 2001; Park et al., 2002; Divine et al., 2001; David-Beabes and London, 2001; Butkiewicz et al., 2001; Cheng et al., 2000a; Sugimura et al., 1999), head and neck (Olshan et al., 2002; Sturgis et al., 1999) and bladder (Stern et al., 2001), several of these reports have indicated protective effects of alleles that are presumed to compromise DNA repair activities (David-Beabes and London, 2001; Olshan et al., 2002; Stern et al., 2001). One possible explanation for this apparent inconsistency, drawn from recent investigations of ultraviolet radiation related basal and squamous skin cancer, is that there are different effects of DNA damage in cells with intact apoptotic mechanisms versus those that have already lost the apoptotic response to DNA damage stimuli. Lower repair capacity could thus be protective for cancer, in that deficient cells would be targeted for cell death rather than survive and harbor deleterious mutations (Nelson et al., 2002). Such a model predicts interactions of DNA repair polymorphisms with tobacco carcinogen dose and/or dose intensity. Further study is obviously required, as both positive (Park et al., 2002) and negative interactions (David-Beabes and London, 2001) of heavy smoking and the XRCC1 Gln allele have already been recorded for lung cancer risk.
Although these epidemiologic observations make it problematic to formulate a priori hypotheses regarding DNA adducts and repair polymorphisms, several reports indicate increased adduct burdens in individuals who carry DNA repair variants. In a study of blood MNC DNA adducts with chromatographic properties similar to adducts detected in cells treated with simple phenols and quinones, researchers observed a significant association of the XRCC1 Gln allele with detectable DNA adduct levels. Adduct levels also showed a positive interaction with blood donor age but appeared unrelated to cigarette smoking (Duell et al., 2000). No association was observed for polymorphisms in the ERCC2 gene and phenol related adducts. Higher levels of bulky hydrophobic–DNA adducts were observed in white blood cells of non-smokers who were homozygous for the XRCC1 399Gln allele (Matullo et al., 2001b). In another study, XRCC3 variant alleles were associated with higher adduct levels in non-smokers (Matullo et al., 2001a). Among traffic exposed workers the XPD 751Gln allele was associated with higher adduct levels (Palli et al., 2001).
A polymorphism involving the endonuclease 8-oxo-guanine DNA glycosylase I (hOGG1;[C1245G;Exon7,Ser326Cys]) was not found to affect aromatic DNA adducts in pancreatic tissues (Li et al., 2002). No association of 8-oxo-dG levels and hOGG1 were found in lung tumor and normal lung tissue (Hardie et al., 2000). Recent work has questioned the functional importance of the hOGG1 Ser326Cys variant in repair of 8-oxo-dG lesions (Janssen, 2001, #399) and in lung cancer pathogenesis itself (Wikman et al., 2000). Finally, PAH–DNA adduct levels in white blood cells were suggested to be associated with the occurrence of a P53 polymorphism (Zhang et al., 2000).
These early studies suggest that DNA repair polymorphisms may help us to understand endogenous or age related DNA adduct formation and removal. More comprehensive studies of smokers are required to address the role of these genetic variants in smoking-induced DNA damage. Studying interactions among gene loci and carcinogen dose will require more sophisticated models and statistical analyses. At this time it is difficult to predict whether high risk genotypes will be associated with increased or decreased adduct level. The relationship may be different at early phases of a chronic exposure compared with later times and the establishment of genetically altered tissues.
New candidate gene polymorphisms
Additional polymorphic candidate genes with potential to modify tobacco related cancer risk via modulation of DNA adduct levels, include the phenol sulfotransferases such as SULT1A1 (Nowell et al., 2000), myeloperoxidase (Petruska et al., 1992; London et al., 1997; Le Marchand et al., 2000; Schabath et al., 2000; Misra et al., 2001; Rojas et al., 2001; Kantarci et al., 2002), dihydronicotinamide riboside (NRH)-quinone oxidoreductase 2 (NQQ2) (Jaiswal et al., 1999; Long and Jaiswal, 2000), UDP-glucuronosyl transferases (Ren et al., 2000; Grant and Bell, 2000; Yueh et al., 2001), and the transcription factor Nrf2, which is essential for inducible and constitutive expression of a group of detoxification and antioxidant enzymes (Aoki et al., 2001).
DNA adducts and cancer risk
Three lines of epidemiologic evidence implicate DNA adducts in the etiology of smoking related cancers. These include: case–control and cohort studies showing associations of cancer occurrence with smoking related DNA adducts, cancer risk associated with the induction of DNA adducts in biopsied cells, and correlative studies of adduct levels with exposures to known smoking carcinogens in target or surrogate tissues. The latter indirectly support a causal relationship of DNA adduct formation with cancer risk, Table 2.
DNA adducts associated with lung cancer
Studies of DNA adducts in lung cancer patients have indicated higher adduct levels in lung tissue of cancer cases (Cheng et al., 2000b, 2001) and in their peripheral white blood cells (Vulimiri et al., 2000; Perera et al., 1989) compared with controls. Higher adduct levels were reported in lung tissue from women compared with men (Cheng et al., 2001). In a prospective study, researchers reported that increased adduct levels in white blood cells were associated with lung cancer risk among those who were current smokers at the time of blood sampling (Tang et al., 2001). A small cohort study of heavy smokers reported that high adduct levels in bronchoalveolar cells were associated with higher cancer mortality (Bonassi et al., 2001), though not specifically lung cancer. DNA adducts induced in vitro have been associated with lung cancer risk in case–control studies (Li et al., 2001b; Wei et al., 2000).
DNA adducts and cancers of the bladder, head and neck, pancreas and uterine cervix
DNA adduct levels in white blood cells were significantly associated with bladder cancer risk (Peluso et al., 1998, 2000). A series of studies have shown that smoking is associated with smoking related adducts in the oral cavity and larynx (Banaszewski et al., 2000; Szyfter et al., 1999a,b; Nath et al., 1998). In a case–control study, induced DNA adduct levels were found to be associated with risk of head and neck cancer (Li et al., 2001a). Particularly striking was the finding of specific adducts in gingival tissue derived from unsaturated aldehydes in cigarette smoke (Nath et al., 1998). A small study of pancreatic cancer patients reported higher levels of several DNA adduct species in nontumorous pancreatic tissues from cases compared with control tissues (Wang et al., 1998a). Smoking is a risk factor in cervical cancer and DNA adducts related to smoking have been observed in cervical cells of smokers (Melikian et al., 1999a). Interestingly, HPV infection appeared to enhance induction of DNA adducts by BPDE in cervical epithelial cells (Melikian et al., 1999b) suggesting a mechanism for the interaction of HPV and smoking in cervical cancer risk.
DNA-adducts and exposure to lung carcinogens
Studies of highly occupationally exposed populations have generally reported DNA adducts to be related to airborne PAH exposure but sorting out the independent effects of occupational and smoking related contributions to total PAH burdens has been problematic. Some exposures postulated to pose an increased risk for lung cancer have failed to lead to detectable increases in adduct levels. For example, among workers exposed to PAHs in carbon electrode manufacturing no correlations were found between DNA adducts and occupational PAH exposures (Arnould et al., 1999; van Delft et al., 1998b). In another study, and one of the only population based investigations on DNA adducts, researchers did not find that PAH–DNA adducts in blood lymphocytes correlated with differences in PAH exposure related to urban air pollution (Kyrtopoulos et al., 2001).
As indicated above, adduct levels in white blood cells have been associated with tobacco related cancer risk. Direct correlations among surrogate tissue and target tissues, however, are few in number. An early important observation was that smoking effects on adducts were stronger when measurements were made using DNA isolated from blood MNC compared with the DNA from the short-lived granulocyte fraction of WBC (Savela and Hemminki, 1991). In a subsequent study, hydrophobic-DNA adducts in blood MNCs were found to be a reliable surrogate of lung DNA adduct levels (Wiencke et al., 1995). Another researcher used induced sputum to assess smoking related DNA adducts (Nia et al., 2000). White blood cell adduct levels were shown to provide a suitable surrogate for DNA adducts in bronchoalveolar cells (Godschalk et al., 1998). The lung retains PAHs, and higher concentrations can be found in men compared with women, and perhaps among high risk ethnic minority groups (Seto et al., 1993; Goldman et al., 2001). Interestingly, concentrations of PAHs measured directly in lung tissue at autopsy were found to correlate well with PAH–DNA adduct levels measured in the same tissues (Lodovici et al., 1998). This observation suggests that exposed lung may constitute a tissue reservoir of unmetabolized PAHs. Immune cells from the peripheral blood or bronchoalveolar cells that migrate and circulate through lung tissue may be exposed to such accumulated PAHs. This may help explain the correlation of peripheral blood or bronchoalveolar cell DNA adduct levels with those measured in lung parenchyma and airways.
Dose response for DNA adducts and cigarette smoking
The nature of the dose–response for cigarette smoking and DNA adduct formation has significant implications in assessing cancer risks associated with tobacco use (Vineis et al., 2000). It is significant, therefore, that it is still somewhat unclear which measures of tobacco smoke exposure are most highly correlated with smoking related DNA adduct levels. Several studies now support the idea that current smokers have higher adduct levels compared with former smokers, and that daily cigarette consumption (cigarettes per day) is the most predictive measure of variations in adduct levels in target (Wiencke et al., 1999; Szyfter et al., 1999a; Banaszewski et al., 2000; Romano et al., 1999) and surrogate tissues of current smokers (Nia et al., 2000; Hou et al., 2001). In one study the daily cigarette tar consumed, but not the number of cigarettes, was found to be predictive of adduct levels in blood MNCs (Godschalk et al., 1998). Daily cigarette consumption in current smokers also predicted PAH–DNA adduct levels in cardiac tissue from patients undergoing open heart surgery (van Schooten et al., 1998). Nonetheless some investigations have reported higher levels of aromatic-DNA adducts in former smokers (Vulimiri et al., 2000). Earlier suggestions (Phillips et al., 1988) that DNA adducts may reflect lifetime cumulative tobacco smoke exposure as estimated by the pack–year variable do not appear to have been substantiated.
Few studies have examined predictors of adduct levels among former smokers specifically. One of the earliest studies of lung PAH–DNA adducts in former smokers reported that adduct levels were similar to those observed among never smokers after about 5 years of smoking cessation (Phillips et al., 1988). Later, a biphasic loss of PAH–DNA adducts was proposed that included a rapid initial phase followed by a second slower phase of adduct loss (Schoket et al., 1993). Studies of subjects in a smoking cessation program indicated an estimated half-life of PAH–DNA adducts of about 9–13 weeks in blood leukocytes (Mooney et al., 1995). The half-life of PAH–DNA adducts in the lung is probably much more protracted. One study of lung cancer patients indicated a half-life of approximately 1.7 years (Schoket et al., 1998) among short-term quitters. In a study of lung tissues from former smokers that included long-term quitters (>10 years), several statistical models were applied that suggested an approximate 10% decline in adducts per year for about 9 years and a much slower residual decline after 9 years (Thurston et al., 2000). Among these former smokers early age of smoking initiation was the most important predictor of interindividual variations in lung DNA adduct levels after adjusting for other measures of cigarette consumption (Wiencke et al., 1999). Aromatic-DNA adducts were also found to be increased in smokers who started smoking at an early age in another study (Vulimiri et al., 2000). Time elapsed since quitting smoking would seem a logical inverse predictor of DNA adducts in former smokers but this has not been reported. Although animal models have been used to address these questions (Helleberg et al., 2001; Izzotti et al., 1999) they do not take into account the protracted exposures typical in humans, nor the cellular and physiologic changes induced in tissues undergoing chronic exposure, which may modify adduct induction and repair. As indicated below, widespread accumulations of genetically altered clones of cells is known to occur in smokers chronically exposed to tobacco carcinogens (Franklin et al., 1997).
DNA adducts and somatic mutation in tumors
DNA adducts and p53
A review several years ago cited the need for human clinical studies of DNA adducts and the mutational spectra of cancer-relevant genes, and noted the paucity of studies in which mutations as a consequence of DNA damage at specific genes had been investigated in vivo (Kriek et al., 1998). This conclusion is still relevant today. The important mechanistic hypotheses on p53 mutational spectrum and smoking (see Hussain in this volume) raise significant challenges for clinical and epidemiological investigators to translate these insights into human populations. Early relatively small studies suggested that aromatic-DNA adduct levels were increased in tissues from patients with p53 mutant lung cancer compared with cases bearing non-p53 mutant tumors (Kure et al., 1996; Ryberg et al., 1997). Another study reported only a weak association between PAH–DNA adduct levels in lung tissue and p53 mutations (Andreassen et al., 1996). Our group found that lung cancer patients with high hydrophobic–DNA adduct levels in non-tumorous lung tissue (above the median adduct level) were threefold more likely to have a tumor containing a p53 mutation (Wiencke et al., 1998). This association was significant, even after adjusting for smoking status. In contrast, in a study of bladder cancer, 4-aminbiphenyl–DNA adducts were not found to be associated with the p53 mutational status of tumors (Martone et al., 1998). Larger studies with carefully collected smoking and lifestyle histories are necessary to define the relationship of DNA adduct burden in vivo and the p53 mutational status and spectra of human lung cancer.
DNA adducts and LOH at 3p21
An event more common than p53 mutation in lung cancer is the LOH and deletion of regions of the short-arm of chromosome 3 (e.g. 3p14, 3p12). LOH in these regions is strongly associated with smoking history (Nelson et al., 1998; Hirao et al., 2001; Zienolddiny et al., 2001) and is thought to be among the earliest events in lung carcinogenesis (Wistuba et al., 1999, 2000; Park et al., 1999; Endo et al., 2000; Mitsudomi et al., 1996). These early somatic events may be the molecular basis for what Slaughter first termed ‘field cancerization’. According to this idea, grossly normal epithelium can be ‘preconditioned’ by carcinogen exposure. Such preconditioning may result in early tobacco-induced somatic mutations that are propagated by enhanced cell replication into a clonal ‘field’ of genetically altered progenitor cells.
Consistent with these observations, we found a strong correlation of early age of smoking initiation with LOH at a loci near the hMLH1 gene on chromosome 3p21 in surgically resected lung cancer patients (Hirao et al., 2001). Interestingly, our investigations also revealed a link between these events and increased hydrophobic–DNA adduct levels in lung tissue from the same patients (Hirao et al., 2001). Another group, using microsatellite markers close to hMLH1, reported a similar increase in PAH–DNA adduct levels in lung tissues of lung cancer patients whose tumors contained LOH at 3p21 (Zienolddiny et al., 2001), although the question of age at smoking initiation was not addressed in the latter study. In a study of lymphocytes from lung cancer patients and controls, the highest aromatic DNA adduct levels were observed among cases who started smoking before 15 years of age (Vulimiri et al., 2000).
These links between DNA adducts and somatic alterations in the lung and early smoking initiation should be considered in light of the epidemiologic observations that early smoking initiation is an independent risk factor for lung cancer (Hegmann et al., 1993). Taken together these associations lead to a novel ‘critical period’ hypothesis for lung cancer susceptibility (Wiencke and Kelsey, 2002): growth and development of the lungs during adolescence may set up a critical period of susceptibility to tobacco-related DNA damage. This idea is consistent with the burst of lung growth that is known to occur in the early teen years. Exposure to tobacco carcinogens during this period may lead to the propagation of fields of altered epithelial cells, particularly in the airway, that later evolve into frank malignancy. Exposure after the critical growth period may have a much lower probability of inducing large, clonal outgrowths with the same malignant potential.
Emerging issues in tobacco carcinogenesis and DNAadducts
Endogenous adducts, oxidative DNA damage and indirect action
It has been suggested that damage to DNA from endogenous exposures may be orders of magnitude greater than those from exogenous sources including exposure to tobacco smoke (Povey, 2000). This does not imply, however, that endogenous agents do not play a role in tobacco carcinogenesis, because tobacco exposures can directly and indirectly increase oxidative stress. In animals, cigarette smoke exposures were shown to enhance endogenous DNA adducts by several fold in different tissues (Gupta et al., 1999; Zhou et al., 2000). Significant progress has been made in characterizing carbonyl-containing products of lipid peroxidation (e.g. malondialdehyde, 4-hydroxy-2-alkenals) and the adducts they form with DNA (Leuratti et al., 1998; Chen et al., 1998; Moller and Wallin, 1998; Yi et al., 1998) as well as the contributions of physiologic mediators such as nitric oxide in these reactions (Nair et al., 1998). The DNA adducts formed in reactions with carcinogenic aldehydes may produce structurally unique adducts (Hecht et al., 2001); these hold promise as sensitive markers of oral cancer risk associated with tobacco use (Nath et al., 1998). Some endogenously derived DNA adducts are thought to increase with age (Josyula et al., 2000; Yang et al., 1998). Several reviews have been presented on endogenous DNA adducts and cancer risk (Burcham, 1998; Bartsch and Nair, 2001; Blair, 2001).
Future studies of genetic susceptibility studies should consider the potential overlap between endogenous and exogenous pathways and sources of DNA adducts. For example, ethylene oxide, which is an endogenous metabolite of ethylene, is also present in cigarette smoke. Studies of blood hemoglobin adducts formed from ethylene oxide indicate that genetic deficiency in GSTT1 leads to higher adduct levels in smokers (Fennell et al., 2000) and non-smokers (Thier et al., 2001). DNA adducts in blood leukocytes were also reported to be higher in GSTT1 null subjects in the EPIC–Italy study (Palli et al., 2000).
Chemically unstable DNA adducts
The majority of studies of tobacco-related cancer and DNA adducts have focused on chemically stable modifications of DNA, like those produced by the reaction of BPDE with the exocyclic amino group of guanine. PAHs present in tobacco smoke, including BP, can also be metabolized via radical cation intermediates to electrophiles that bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination or depyrimidination of the adducted bases (Bodell et al., 1989; Chen et al., 1996; Devanesan et al., 1996). In some experimental systems (e.g. mouse skin treated with BP) these unstable DNA adducts are much more prevalent than the stable DNA adducts. Understanding the relative importance of unstable versus stable adducts in tobacco carcinogenesis in humans and the role of DNA repair mechanisms that act on these different lesions will require further investigation (Melendez-Colon et al., 1997, 1999; Chakravarti et al., 2001). Future epidemiologic studies may be the only way to accurately assess the importance of depurinating adducts in tobacco carcinogenesis. This will require improvements in the methods for detecting such compounds in biological specimens. One sensitive method for detecting unstable DNA adducts in urine (i.e. using capillary electrophoresis–fluorescence line narrowing spectroscopy) has been developed (Roberts et al., 2000). Initial studies using this approach found BP adducted bases (e.g. BP-6 N7Gua) in the urine of three of seven cigarette smokers and three of seven women exposed to coal smoke, but none were detected in urine from 13 control subjects (Casale et al., 2001).
Factors affecting the validity of DNA adduct studies
Exposure misclassification, statistical analysis, study design
Others have reviewed some of the statistical and analytical issues that can compromise the validity of DNA adduct studies (Cuzick, 1995). The literature reviewed here alludes to a divergence in adduct results in current versus former smokers. This has important implications for the design of future investigations into the role of DNA adducts in tobacco-related cancer. At any single point in time the levels of tobacco-related DNA adducts in a human tissue represent a dynamic process that is dependent on a number of factors; these include the intensity and recency of exposure to tobacco smoke, the metabolic balance of activation and detoxification mechanisms and the repair or removal of adducts by DNA repair and/or cell turnover. Therefore, to observe relationships between putative modifiers of DNA adduct concentrations, it may be important to stratify on smoking status (current, former, never) in all statistical analyses or to employ multivariate statistical techniques that carefully address potential interactions of modifiers and smoking status. It may be expected, for example, that genetic modification of adduct levels arising through a metabolic mechanism (adduct formation) may be more evident among current smokers. Genetic factors affecting repair and tissue renewal (adduct removal) may be more evident in comparisons involving former smokers.
In addition, many of the case–control studies cited above can be criticized, on the basis that DNA adducts may be linked to cancer occurrence through their association with tobacco smoke exposure. As DNA adducts are thought to be part of the causal pathway in tobacco-related cancer they are not formally considered to be confounders. Nonetheless, these considerations raise concerns about the clinical value of DNA adducts as independent predictors of tobacco-related cancer risk. To mitigate against these concerns, the question of adducts as risk factors could be addressed in studies that carefully match cases and controls on cigarette smoking exposure. Case–control studies matching on smoking may also provide a more robust way to examine the role of candidate genes as modifiers of DNA adduct formation or repair. Because of the difficulty in recruiting suitable controls, however, such studies are more difficult to perform than traditional case–control studies.
Alternatives to case–control or cross-sectional studies include those employing prospective and interventional designs. For example, in the SELECT study (Selenium and Vitamin E Cancer Prevention Trial) the goal is to enroll 32 400 men aged 55 years and older and follow them for a 12 year study period (Hoque et al., 2001). Blood samples collected at baseline could be used for DNA adduct and candidate gene studies; an expected 800 lung cancer cases are predicted to occur within the study period.
Bias caused by seasonal variation in DNA adducts
Finally, a question still unresolved is whether studies of DNA adducts can be significantly biased by seasonal variations in adduct levels. Several studies have reported statistically significant variations in DNA adduct levels according to the season of study (Butkiewicz et al., 2000; Grzybowska et al., 1993; Moller et al., 1996; Motykiewicz et al., 1998; Palli et al., 2001; Perera et al., 1992; Tang et al., 1995; Topinka et al., 2000). Many of these reported higher ‘background’ adduct levels in winter compared with summer, and these variations appear to be related to the airborne concentrations of PAHs and nitro-PAHs, particularly for studies carried out in industrialized and polluted areas of Poland and the Czech Republic (Moller et al., 1996; Topinka et al., 2000). These observations suggest that seasonal variation in DNA adducts may not be a generalized phenomenon but is rather an issue that is regionally specific to those areas where ambient air quality varies widely with the season of the year.
However, recent research also indicates the complexities of these ecological associations. For example, concentrations of PAH–DNA adducts levels in WBCs were found to peak in Northern Italy (Turin, Varese) in winter and in summer in Central and Southern Italy (Florence, Naples, Ragusa), which suggested opposite seasonal trends according to latitude (Palli et al., 2000). The authors speculate that in summer photochemical pollution may be higher at lower latitudes and play a relatively greater role in the induction of adducts compared to the winter months. In another study, blood PAH–DNA adducts in non-smokers were found to be higher in the small Greek city of Halkida compared with adduct levels in non-smokers living in Athens, despite the far greater air pollution burden that exists in Athens (Kyrtopoulos et al., 2001). Consequently, it would appear prudent to consider seasonal information and ambient air quality in planning future studies to avoid potential biases that may arise from regional or seasonal variations in DNA adducts in human populations.
- GSTM1, GSTT1, GSTP1:
Glutathione S-transferase M1, T1, P1
- NAT1, NAT2:
N-acetyltransferase type I and type II
- NQQ1 :
NAD(P)H:quinone oxidoreductase type I
7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9, 10-oxide
polynuclear aromatic hydrocarbons
high performance liquid chromatography
white blood cells
gas chromatography-mass spectrometry
- BP-6 N7Gua:
enzyme linked immunosorbent assay
human papilloma virus
European Prospective Investigation into Cancer and Nutrition
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This work was supported by NIH ES06717.
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Wiencke, J. DNA adduct burden and tobacco carcinogenesis. Oncogene 21, 7376–7391 (2002). https://doi.org/10.1038/sj.onc.1205799
- DNA adducts
- risk assessment
- molecular epidemiology
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