Oncogene (2002) 21, 7376 – 7391 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

DNA adduct burden and tobacco carcinogenesis

John K Wiencke*,1

1Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California, CA 94143-0560, USA

Background DNA adducts associated with tobacco smoking could provide a marker of biologically effective dose of tobacco Introduction carcinogens and improve individual cancer risk predic- tion. A significant number of clinical and epidemiologic DNA adducts are physical complexes formed between studies have reported associations of increased DNA reactive chemical species and sites within the DNA adduct levels with the occurrence of the prevalent molecule. DNA adducts have been proposed as tobacco related cancers including cancer of the lung, potential markers of ‘biologically effective dose’ from head and neck, and bladder. The inducibility of DNA exposure to tobacco carcinogens that may help to adducts following in vitro treatments using blood provide an integrated measure of carcinogen exposure lymphocytes also appears to be a risk factor in the relevant to individual cancer risk assessment. The development of lung and head and neck cancer. emphasis of this review is on human in vivo studies Corroborative evidence pointing to the importance of except where model in vitro or animal studies are DNA adducts in tobacco carcinogenesis include numer- relevant to a specific issue. This review emphasizes ous studies showing associations of tobacco smoke literature appearing since 1998; below are cited reviews exposure with the induction of DNA adducts in humans of earlier literature. The field of investigation that in vivo. Further effort is necessary, however, to more addresses DNA modifications in human cancer is fully characterize the dose – response relationship expansive. This review is restricted to epidemiologic between smoking and DNA adducts in exposed target and clinical studies that address five areas bearing on and surrogate tissues. The relationship between gene the etiologic role of DNA adducts in tobacco polymorphisms thought to modify tobacco-related cancer carcinogenesis: risk and DNA adduct levels is complex. Results of some DNA adduct studies (both in vitro and in vivo) appear (1) The evidence that genetic polymorphisms impli- inconsistent with the epidemiologic findings. This is cated in tobacco related cancers modify DNA evident for polymorphisms involving both carcinogen adduct concentrations in human tissues; metabolism (e.g. GSTP1) and DNA repair (e.g. (2) The evidence that DNA adducts are risk factors in XRCC1). Molecular studies of human tumors suggest the development of tobacco related cancers; associations of p53 mutation with DNA adducts and (3) The association of DNA adducts with molecular have revealed correlations of DNA adduct levels with alterations involving oncogenes and tumor sup- somatic alterations (e.g. 3p21 LOH) that are thought to pressor genes in human tumors; occur at the very earliest stages of tobacco carcinogen- (4) The potential importance of endogenous and esis. More research is needed to assess the relationship chemically unstable DNA adducts in tobacco between endogenous sources of DNA adducts and carcinogenesis; and tobacco smoke exposure and the relative oncogenic (5) Methodologic issues that affect the validity of effects of chemically stable versus unstable DNA investigations into the role of DNA adducts in adducts. Many potentially fruitful new avenues of cancer tobacco carcinogenesis. research are emerging that integrate DNA adduct analyses with assessments of smoking, genetics, diet Recent reviews and ambient air quality. These investigations aim to understand the multifactorial nature of interindividual Several recent reviews have addressed the use of DNA variability in response to tobacco carcinogens. As these adducts in risk assessment and toxicology studies (van trends continue a variety of innovative study designs and Delft et al., 1998a; Vainio, 1998; Timbrell, 1998; Kriek approaches will become important in human populations. et al., 1998; Garner, 1998; Carmichael, 1998; Hemmin- Oncogene (2002) 21, 7376 – 7391. doi:10.1038/sj.onc. ki et al., 2001). Other reviews emphasize the chemistry 1205799 of adduct formation and laboratory methodology (Phillips et al., 2000; Reddy, 2000; Szeliga and Dipple, Keywords: DNA adducts; tobacco; risk assessment; 1998). Although most recent studies have employed molecular epidemiology only a few analytical approaches (i.e. 32P-postlabeling or immunologically based assays), new technologies and improved applications have also been reviewed *Correspondence: JK Wiencke; E-mail: [email protected] (Turteltaub and Dingley, 1998, accelerated mass DNA adduct burden and tobacco carcinogenesis JK Wiencke 7377 spectrometry); (Marzilli et al., 2001, capillary-electro- been associated with GSTP1 genotypes presumed to phoresis-mass spectrometry); (Shinozaki et al., 1998, result in greater not less PAH detoxification (Ryberg et flow cytometry); (Ni et al., 1998, electrospray tandem al., 1997; Kihara and Noda, 1999; Harries et al., 1997; mass spectrometry); (Petruzzelli et al., 1998, serum van Lieshout et al., 1999). BPDE-DNA antibodies by ELISA); (Bucci et al., 1998, DNA adduct studies thus far have not fully resolved competitive ELISA); (Xing et al., 2001, capillary this apparent anomaly. In studies of newborns, PAH – electrophoresis); (Tan et al., 2001, immunoelectrophor- DNA adducts were marginally higher among subjects esis with laser induced fluorescence). 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 Genetic susceptibility observed among non-smoking women carrying the GSTP1 Val/Val genotype (Grzybowska et al., 2000) Previous reviews of genetic polymorphisms have compared to others, but in occupationally exposed assessed the potential for adducts to provide an populations no effect of the GSTP1 polymorphism by intermediary tool for genotyping studies (Hemminki itself has been reported on variations in PAH – DNA et al., 2000). The relationship of genotype to adduct adduct levels (Zhang et al., 2000; Viezzer et al., 1999; formation in determining risk for lung, esophagus and Grzybowska et al., 2000; Schoket et al., 2001). The oral cancer have been evaluated (Bartsch et al., 1998). GSTP1 polymorphism was also not found to be not An overall weight of evidence approach concluded that predictive of PAH – DNA adduct levels in bronchial under conditions giving rise to detectable PAH – DNA tissues of lung cancer patients (Ozawa et al., 1999). adducts levels in tissues, most studies reviewed reported There have been suggestions of interactions between a significant role for GSTM1 deletion either alone or in GSTP1 and other polymorphisms, but again the combination with CYP1A1 variants on variations in picture is far from clear. Whereas PAH – DNA adducts adduct levels and urinary metabolites of genotoxic in occupationally exposed workers were elevated in substances (Pavanello and Clonfero, 2000). GSTM1 null subjects and in persons carrying a combined GSTM1 null and GSTP1 variants (Schoket et al., 2001), another study of cigarette smokers found Metabolic polymorphisms evidence of the GSTM1 non-null genotype interacting Glutathione S-transferases and cytochrome with GSTP1 variants and being associated with P4501A1 Although early studies suggested a role of elevated DNA adducts (Butkiewicz et al., 2000). A GSTM1 deficiency in aromatic-hydrophobic DNA suggested interaction of the MspI variant of CYP1A1 adduct levels (Ryberg et al., 1994; Grinberg-Funes et with the GSTP1 Val/Val genotype on adduct levels in al., 1994; Shields et al., 1993; Kato et al., 1995) these newborns was reported in one study (Whyatt et al., studies were criticized (Cuzick, 1995) for not defining 2001). It has been proposed (Coles et al., 2000b) that cigarette smoking exposure adequately and for not GSTP1 like GSTM1 is coordinately regulated with the having sufficient size to adjust for potential statistical GSTM3 locus. However, GSTM3 genotypes were not artifacts. Continued work on the GSTs has appeared found to affect the relationship of GSTP1 with lung (Table 1) and more recent studies have included cancer in a recent study of 389 lung cancer cases and polymorphism of the GSTT1 and GSTP1 genes. The 353 controls in Germany (Risch et al., 2001). GSTP1 isoforms could be particularly relevant in lung A re-examination of GSTP1 genotype – phenotype carcinogenesis as GSTP1 is highly expressed in lung relationships (Sundberg et al., 2002) has suggested that and shows high activity in detoxifying diol epoxides of the GSTP1 enzyme containing valine at position 105 PAHs. Human GSTP1 expression in cell culture has may actually have less than, or at most equal activity been shown to be very effective in preventing the in reducing, PAH – DNA adduct formation compared formation of BPDE – DNA adducts produced in vitro with the wildtype GSTP1 Ile105/Ala114 isoform. The (Fields et al., 1998). Two common polymorphisms in authors caution against extrapolating catalytic infor- GSTP1 give rise to four genotypic variants in human mation from pure enzymes to the complex situation populations. Although allelic variants (i.e. A313G; within the intact cell. Notably, they found only about Ile105Val) show reduced enzyme activity toward the 1 – 2% of the expected rate of BPDE conjugation to be model substrate 1-chloro-2,4-dinitrobenzene (Watson observed in vivo in cells, which the authors postulate et al., 1998) when transfected into human HepG2 cells may be a result of the reduced concentrations of these variants were shown to provide greater protection lipophilic substrates available for conjugation by the against the formation of (+)-anti-BPDE – DNA soluble GSTs (Sundberg et al., 2002). adducts (Hu et al., 1999) compared with the wildtype Among coke oven workers the GSTM1 null GSTP1 isoform. The wildtype GSTP1 contains genotype was associated with adducts among highly isoleucine at position 105. Catalytic properties of both PAH exposed groups but GSTT1 null subjects, who are recombinant and native variants of GSTP1 enzymes deficient in GSTT1 mediated GSH conjugation, were compared with wildtype forms and the variants demonstrated lower not higher adduct burdens (Viezzer were shown to display greater catalytic efficiency et al., 1999). Negative studies have also appeared in towards carcinogenic PAHs (Coles et al., 2000a). Lung, which adduct levels in non-tumorous lung or blood bladder and esophageal cancer risk, however, have MNCs were not modified by GSTM1 (Wiencke et al.,

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7378 Table 1 Genetic polymorphisms and DNA adducts levels Endpoint/Methods Tissue Gene Loci Study design/population Results Reference 1 Bulky-DNA adducts Bronchial tissues GSTM1 Lung cancer case series Current smokers and short term Schoket et al., CYP1A1 (n=124) exsmokers had higher adduct 1998 32P-postlabeling Referent non-malignant lung levels than former smokers (1 yr conditions (n=26) quit); no association with genotype, cig/day 2 BPDE-DNA adducts Non-tumorous lung GSTM1 Lung cancer case series Non detectable (low) adduct levels Rojas et al., tissue CYP1A1 (n=30), occupational PAH associated with GSTM1 non-null 1998 HPLC/fluorometry exposed coke oven workers genotypes; gene interactions WBCs (n=20) suggested 3 Aromatic-DNA adducts Total WBC CYP1A1 Occupational case referent No association of cumulative Pan et al., 32P-postlabeling (China) exposure and adducts; among 1998 25 coke oven male workers, CYP1A1 variants adducts 25 referent male subjects correlated with 1-hydroxyprene Urinary 1-OHPY levels 4 Bulky aromatic-DNA Total WBC and GSTM1 Occupational case referent NAT2 slow acetylators had higher Binkova, adducts lymphocytes NAT2 adduct levels no effect of GSTM1 et al., 1998 68 male PAH exposed coke Urinary cotinine associated with 32P-postlabeling oven workers adduct levels Urinary continine 56 non exposed machine Plasma vitamin levels workers 5 PAH-DNA adducts Oral mucosa GSTM1 Exposure case referent No association of GSTM1 or Motykiewicz CYP1A1 Polish non-cmoking women CYP1A1 with adducts, adducts et al., 1998 Immunohistochemical 67 PAH-exposed 72 referent higher in PAH exposed group, subjects evidence of seasonal variation 6 BPDE-DNA adducts (in T47D cells GSTP1 In vitro Cell Culture 70 – 90% reduction in DNA adducts Fields et al., vitro MCF-7 cells expression in T47D cells highly expressing 1998 only BPDE (30 – 600 nM) GSTP1 [3H]BPDE binding 7 PAH-DNA adducts Placental tissue CYP1A1 PAH exposure case referent Neither CYP1A1 expression nor Whyatt et al., Msp1 smoking associated with adduct 1998 Immunochemical (Genotype) 70 exposed newborns levels, higher placental adducts in 90 referent newborns CYP1A1 variant infants Infant plasma cotinine CYP1A1 mRNA (expression) 8 PAH-DNA adducts Maternal and GSTM1 Cross-sectional Maternal smoking associated with Whyatt et al., umbilical white blood CYP1A1 70 mothers and newborns mothers adduct levels; higher 1998 Immunochemical cells adducts among newborns with CYP1A1 variants 9 PAH-DNA adducts Blood lymphocytes GSTM1 Cross-sectional Smokers had higher adduct levels Wang et al., GSTT1 compared with nonsmokers; 1998b 32P-postlabeling CYP1A1 158 subjects CYP1A1 variants and GSTM1 null NAT2 subjects had higher adduct levels; (plasma beta-carotene, ALDH2 interactions with plasma alpha-tocopherol) antioxidants 10 PAH-DNA adducts Blood lymphocytes GSTM1 Cross-sectional DNA adduct levels positively Soni et al., correlated with daily cigarette 1998 32P-postlabeling 21 smokers consumption and inversely with GSTM1 status 11 Bulky hydrophobic – Blood MNCs GSTM1 Lung cancer case series Among former smokers Wiencke et al., DNA adducts Non tumorous lung CYP1A1 polymorphisms were not 1999 tissue 143 lung associated with DNA adduct 32P-postlabeling 54 blood levels; early age of smoking initiation associated with increased adduct levels 12 Aromatic/hydrophobic – Nontumorous Expression Case series Female cases had higher smoking Mollerup, DNA adducts Lung tissues only Lung cancer patients adjusted adduct levels; adducts 1999 CYP1A1 29 females were positively correlated with 32P-postlabeling 93 males CYP1A1 expression 13 PAH-DNA adducts, Blood MNCs GSTM1 Occupational cross-sectional Positive interaction of high PAH Viezzer et al., GSTT1 70 male coke oven workers, exposure and GSTM1 null 1999 32P-postlabeling GSTP1 Italian steel workers (mean genotype, GSTT1 null had lower mEPHX age 41.3 yrs) adduct levels urinary 1-pyrenol PAH exposure subgroups by urinary 1-pyrenol 14 BPDE-DNA adducts Total WBC CYP1A1 Occupational Increased adduct levels in GSTM1 Rojas et al., GSTM1 case-referent null but not GSTT1 null subjects 2000 HPLC/fluorometry GSTT1 89 coke oven Significant effects of CYP1A1 (35 smokers, 36 exsmokers, genotypes on adduct levels Continued

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7379 Table 1 (Continued ) Endpoint/Methods Tissue Gene Loci Study design/population Results Reference 18 nonsmokers) 44 power plant workers (all smokers) 15 PAH-DNA adducts Maternal and CYP1A1 Cross Sectional Among mothers no association of Whyatt et al., Newborn WBCs GSTP1 160 Polish mothers and their adduct levels and gene 2000 Immunoassay newborns polymorphisms; Among newborns CYP1A1 MSPI variants had higher adducts, positive interaction with GSTP1 variants 16 PAH-DNA adducts Total WBC CYP1A1 Occupational Case referent Marginal association of adducts Zhang et al., GSTM1 65 coke oven workers (China) with high versus low PAH 2000 32P-postlabeling GSTP1 exposure, significant associations NAT2 of adducts with urinary cotinine, Urinary cotinine P53 CYP1A1 and p53 among subgroups 17 Aromatic-DNA adducts Total WBC CYP1A1 Occupational Nonsignificant differences in Grzybowska 32P-postlabeling CYP2D6 133 nonsmoking Polish combined subgroups of et al., 2000 GSTM1 women (ages 35 – 45 years) genotypes; no clear seasonal GSTP1 associations 18 PAH-DNA adducts Bronchoaleveolar Expression Smoking Case-referent threefold higher adduct levels in Piipari et al., Macrophages only smokers; no detectable CYP1A1 2000 32P-postlabeling CYP1A1 31 smokers protein or mRNA CYP1B1 16 non smokers CYP3A 19 PAH-DNA adducts Blood MNCs GSTP1 Cross sectional Smokers with combine GSTP1 Butkiewicz GSTM1 GSTM1 positive had higher adduct et al., 2000 32P-postlabeling 170 healthy volunteers levels; seasonal variation 20 Hydrophobic – DNA WBC GSTM1 Cross-sectional GSTT1 null associated with higher Palli et al., adducts Buffy coat GSTT1 309 subjects adduct levels and suggested 2000 32P-postlabeling CYP1A1 interaction of GSTT1 and GSTM1 NAT2 21 PAH-DNA adducts Peripheral blood GSTM1 Occupational case referent No effects of polymorphisms on Van Delft, 32P-postlabeling Lymphocytes GSTP1 adducts. Smoking associated with et al., 2001 (w/ butanol) 32 Dutch coke oven workers Increased DNA adducts and 33 nonexposed subjects urinary 1-OHPY excretion Urinary 1-OHPY 22 Aromatic-DNA adducts Isolated blood MNC GSTM1 Case Control Among current smokers, NAT2 Hou et al., NAT2 170 cases slow acetyltors show higher 2001 32P-postlabeling 146 controls adducts; postive interaction with (Stockholm Sweden) GSTM1 null 23 4-ABP DNA adducts MNCs GSTM1 Cross Sectional No overall effect of genotypes on Godschalk GSTT1 67 Smokers Adduct levels et al., 2001 GC-MS NAT1 NAT2 23 4-ABP-DNA adducts Bladder biopsy GSTM1 Bladder cancer 4ABP-DNA adducts higher in Martone et al., 32P-postlabeling NAT2 Case series smokers of air cured tabacco; no 1998 association with NAT2 p53 mutation 23 PAH-DNA adducts Peripheral blood CYP1A1 Occupationally DNA adducts correlated with Schocket et al., Lymphocytes, CYP1B1 Exposed aluminum plant urinary 1-OHPY in GSTM1 null 2001 32P-postlabeling CYP2C9 workers, lung cancer patients workers; interaction of Bronchial tissue CYP2C GSTM1 and P1 suggested Urinary 1-OHPY NQO1 GSTM1 GSTP1 24 4-ABP-DNA adducts Bladder cancer GSTM1 Case series No association of GSTM1 or NAT2 Martone biopsies NAT2 45 bladder cancers and adducts; adducts higher in et al., 1998 GC/MS current smokers of air cured tobacco 25 Aromatic-DNA adducts WBC NAT2 Badder cancer NAT2 genotype associated with Peluso et al., 32P-postlabeling Case (n=114) control (n=46) adducts; adducts associated with 1998; See also case status Peluso et al., 2000 26 Aromatic-DNA adducts Pancreas tissue GSTM1 Cross sectional Cyp1A1 var/var (n=3) showed Li et al., 32P-postlabeling CYP1A1 30 pancreas cancer higher adduct levels compared 2002 CYP2E1 24 organ donors with hets and wildtype (n=61) 8-oxo-dG: HPLC-EC NAT1 6 non-pancreas cancer NAT2 5 chronic pancreatitis MNSOD hOGG1 Continued

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7380 Table 1 (Continued ) Endpoint/Methods Tissue Gene Loci Study design/population Results Reference 27 Oxidative Stress related Pancreas tissue GSTM1 Smoking case referent Neither GSTM1 nor NQO1 Kadlubar DNA adducts GSTT1 polymorphisms associated with et al., 1998 NQO1 15 smokers adduct levels 8-oxo-dG: HPLC-electro- 15 non-smokers chemical detection m1G:GC-MS edA, edC:immunoaffinity/ 32P-postlabeling 28 Polyphenol-related Isolated Blood MNCs XRCC1 Cross sectional analysis Positive gene dosage effect for Duell et al., DNA adducts ERCC2 Controls is lung cancer case – detectable DNA adducts with the 2000 control study XRCC1 399GIn allele; positive 32P-postlabeling n=61 (median age 65 yrs) interaction with age 29 Bulky DNA adducts WBC XRCC1 Cross sectional Higher adduct levels in non- Matullo et al., XRCC3 n=302 Italian subjects smoking XRCC1 399GIn 2001b 32P-postlabeling XPD EPIC Study homozygotes; no effect on smoking adducts 30 Bulky-DNA adducts WBC XRCC1 Case control Higher adduct levels in XRCC3 Matullo et al., XRCC3 124 bladder ca cases, 85 non smokers, Slow NAT2 2001a 32P-postlabeling XPD hospital controls acetlyators showed higher adduct NAT2 levels 31 Bulky-DNA adducts Peripheral blood XPD Occupational XPDLys751GIn Palli et al., Leukocytes Case-referent Associated with higher adduct 2001 32P-postlabeling levels; twofold increase with traffic Traffic exposed workers exposure; (n=114) Significant seasonal variation (n=100) general population

1999). Although several analytic factors have been 1998), nor in lung cancer patients (Schoket et al., 1998; discussed in regards to discordant results (Phillips and Wiencke et al., 1999). One small study of adducts in Castegnaro, 1999), some studies point to factors in the lung cancer patients, however, did suggest an interac- diet that may influence these types of comparisons. For tion of the GSTM1 null genotype with homozygosity example, GSTM1 null smokers were reported to have of the CYP1A1 MspI variant (Rojas et al., 1998). higher adduct levels compared with non-null smokers, Several studies have reported CYP1A1 variants to be and these associations were found to be modified by associated with higher adduct levels in newborn blood plasma antioxidants (Wang et al., 1998b). DNA and placental tissues (Whyatt et al., 1998, 2001). adducts were found higher in GSTM1 null subjects in Despite years of study, the role of CYP1A1 gene the EPIC – Italy study, and were inversely associated polymorphisms in PAH activation remains unclear. with the consumption of fresh fruit, vegetables, olive The notion that DNA adduct studies in vivo among oil and antioxidants (Palli et al., 2000). Earlier studies smokers and occupationally exposed populations or suggested an important role for dietary antioxidants on patients with smoking related cancers could help to DNA adduct results in smokers (Grinberg-Funes et al., elucidate the toxicologic functionality of CYP poly- 1994; Mooney et al., 1997). Given the significant role morphisms has not yet been proven valid. The dietary constituents are now thought to play in lung unexpected and paradoxical finding of increased cancer (Williams and Sandler, 2001) further work in hepatic DNA adducts of BP in CYP1A1 knockout this area is needed. Such information may help to mice also suggests that further study is warranted on improve preventive strategies involving anti-oxidants the kinetics of metabolic clearance of PAHs and its and dietary interventions (Saha et al., 2001; Jacobson impact on DNA adduct formation (Uno et al., 2001). et al., 2000). Expression of CYP1A1 as assessed by mRNA levels N-acetylation and aromatic amines in lung tissue was shown to be positively correlated with variations in hydrophobic-DNA adduct levels in After cancer of the lung and bronchi, bladder cancer is currently smoking lung cancer patients (Mollerup et the most prevalent tobacco related malignancy. A al., 1999). Earlier work showed a strong correlation of series of earlier studies suggested that differences in microsomal aryl hydrocarbon hydroxylase activity in carcinogen-adduct levels can correlate closely to lung with BPDE – DNA adduct levels (Alexandrov et differences in bladder cancer risks that are determined al., 1992). The importance of CYP1A1 gene poly- both by carcinogen exposure intensity and metabolic morphisms and other CYP related gene polymorphisms genotype (Vineis and Martone, 1996; Vineis and in adduct formation is more ambiguous. Polymorph- Ronco, 1992). Generally these studies support the idea isms at the CYP1A1 locus were not associated with that adduct levels correlate with smoking related risk, variations in adduct levels in occupationally exposed particularly those differences in risk associated with workers (Schoket et al., 2001; Grzybowska et al., 2000; smoking black (air cured) versus blonde (flue cured) Zhang et al., 2000; Pan et al., 1998; Motykiewicz et al., tobacco. Protein – carcinogen adduction has been the

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7381 focus of most of the research. Some potential DNA adducts, however, the evidence supporting inconsistencies were raised, however, in studies of genetic modification of DNA adducts from cigarette DNA adducts. For example, no evidence of case smoking in vivo in man must be considered equivocal. control differences in urothelial cell DNA adduct levels Strong associations of CYP1A1 expression and enzyme were recorded in bladder biopsies (Talaska et al., activity with PAH – DNA adducts in vivo and negative 1994), nor were associations observed between 4- or inconsistent findings with CYP1A1 genetic variants aminobiphenyl-DNA adduct levels and p53 mutations argues against the functional importance of the known in bladder tumors (Martone et al., 1998). In urine, the CYP1A1 polymorphisms. Indeed, the relationships major adduct forming species associated with smoking between the common genetic variations and enzyme black tobacco was suggested to be PhIP (Peluso et al., inducibility is still unclear. Extensive characterization 1991). More relevant to the more widely consumed flu of the catalytic properties of GSTP1 variants and the cured tobacco is the identification of the putative N- weak or inconsistent relationships in vivo require (deoxyguanosin-8-yl)-4-aminobiphenyl DNA adduct in further evaluation. Hemoglobin carcinogen adducts exfoliated bladder cells, the concentration of which was appear a better marker of biological effective dose for found to be related to the number of cigarettes smoked bladder cancer compared with DNA adducts. per day (Talaska et al., 1991). Recently, in a case control study of bladder cancer, DNA repair polymorphisms NAT2 slow acetylator genotypes were associated with increased aromatic DNA adducts in white blood cells XRCC1, XCRCC3, XPD, ERCC2, hOGG1, p53 from buffy coats obtained from cancer cases, but no such association was evident among the controls (Peluso et While several epidemiological studies have implicated al., 2000). Researchers also reported that dietary fruits polymorphisms in DNA repair enzymes in cancers of and vegetables were inversely related to adduct levels. the lung (Ratnasinghe et al., 2001; Park et al., 2002; Curiously, smoking was not found to be associated with Divine et al., 2001; David-Beabes and London, 2001; the DNA adducts levels measured (Peluso et al., 2000). Butkiewicz et al., 2001; Cheng et al., 2000a; Sugimura In the aforementioned case series of bladder cancer et al., 1999), head and neck (Olshan et al., 2002; biopsies no association of NAT2 genotypes was observed Sturgis et al., 1999) and bladder (Stern et al., 2001), with 4-ABP DNA adduct measurements although several of these reports have indicated protective effects current smoking and smoking air cured tobacco were of alleles that are presumed to compromise DNA positively associated with increased adduct levels repair activities (David-Beabes and London, 2001; (Martone et al., 1998). NAT2 genotypes associated with Olshan et al., 2002; Stern et al., 2001). One possible slow acetylator phenotypes were associated with higher explanation for this apparent inconsistency, drawn aromatic-DNA adduct levels (Hou et al., 2001; Matullo from recent investigations of ultraviolet radiation et al., 2001a). No effect of either NAT1 or NAT2 related basal and squamous skin cancer, is that there polymorphisms was found in an analysis of aromatic are different effects of DNA damage in cells with intact amine-DNA adducts in another study (Godschalk et al., apoptotic mechanisms versus those that have already 2001). These mixed results relating adduct measurements lost the apoptotic response to DNA damage stimuli. and NAT2 genotypes should be considered in light of the Lower repair capacity could thus be protective for overall modest increase in risk for bladder cancer cancer, in that deficient cells would be targeted for cell associated with polymorphisms at this locus indicated death rather than survive and harbor deleterious in a recent meta-analysis of epidemiologic studies mutations (Nelson et al., 2002). Such a model predicts (Marcus et al., 2000). interactions of DNA repair polymorphisms with tobacco carcinogen dose and/or dose intensity. Further study is obviously required, as both positive (Park et Quinone reductases and oxidative DNA damage al., 2002) and negative interactions (David-Beabes and Only a few studies have examined DNA adducts in London, 2001) of heavy smoking and the XRCC1 Gln pancreatic tissues of smokers and non-smokers. One of allele have already been recorded for lung cancer risk. these concluded that neither GSTM1 nor NQO1 Although these epidemiologic observations make it modified DNA adducts related to oxidative stress problematic to formulate a priori hypotheses regarding (Kadlubar et al., 1998). This study must be considered DNA adducts and repair polymorphisms, several preliminary with regard to the less prevalent genotypes reports indicate increased adduct burdens in indivi- (e.g. NQO1 and GSTT1 homozygous variant indivi- duals who carry DNA repair variants. In a study of duals), and more studies could be valuable. Cigarette blood MNC DNA adducts with chromatographic smoking is one of only a few identified environmental properties similar to adducts detected in cells treated risk factors for pancreatic cancer. A recent study with simple phenols and quinones, researchers (Duell et al., 2002) indicated an increased risk of observed a significant association of the XRCC1 Gln GSTT1 null subjects among heavy smokers and allele with detectable DNA adduct levels. Adduct levels perhaps a higher risk among women who were GSTT1 also showed a positive interaction with blood donor null and who smoked heavily. age but appeared unrelated to cigarette smoking (Duell A strong mechanistic rationale implicates metabolic et al., 2000). No association was observed for polymorphisms in the formation of smoking related polymorphisms in the ERCC2 gene and phenol related

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7382 adducts. Higher levels of bulky hydrophobic – DNA DNA adducts, cancer risk associated with the induction adducts were observed in white blood cells of non- of DNA adducts in biopsied cells, and correlative smokers who were homozygous for the XRCC1 399Gln studies of adduct levels with exposures to known allele (Matullo et al., 2001b). In another study, XRCC3 smoking carcinogens in target or surrogate tissues. variant alleles were associated with higher adduct levels The latter indirectly support a causal relationship of in non-smokers (Matullo et al., 2001a). Among traffic DNA adduct formation with cancer risk, Table 2. exposed workers the XPD 751Gln allele was associated with higher adduct levels (Palli et al., 2001). DNA adducts associated with lung cancer A polymorphism involving the endonuclease 8-oxo- guanine DNA glycosylase I (hOGG1;[C1245G;Exon7,- Studies of DNA adducts in lung cancer patients have Ser326Cys]) was not found to affect aromatic DNA indicated higher adduct levels in lung tissue of cancer adducts in pancreatic tissues (Li et al., 2002). No cases (Cheng et al., 2000b, 2001) and in their peripheral association of 8-oxo-dG levels and hOGG1 were found white blood cells (Vulimiri et al., 2000; Perera et al., in lung tumor and normal lung tissue (Hardie et al., 1989) compared with controls. Higher adduct levels 2000). Recent work has questioned the functional were reported in lung tissue from women compared importance of the hOGG1 Ser326Cys variant in repair with men (Cheng et al., 2001). In a prospective study, of 8-oxo-dG lesions (Janssen, 2001, #399) and in lung researchers reported that increased adduct levels in cancer pathogenesis itself (Wikman et al., 2000). white blood cells were associated with lung cancer risk Finally, PAH – DNA adduct levels in white blood cells among those who were current smokers at the time of were suggested to be associated with the occurrence of blood sampling (Tang et al., 2001). A small cohort a P53 polymorphism (Zhang et al., 2000). study of heavy smokers reported that high adduct These early studies suggest that DNA repair levels in bronchoalveolar cells were associated with polymorphisms may help us to understand endogenous higher cancer mortality (Bonassi et al., 2001), though or age related DNA adduct formation and removal. not specifically lung cancer. DNA adducts induced in More comprehensive studies of smokers are required to vitro have been associated with lung cancer risk in address the role of these genetic variants in smoking- case – control studies (Li et al., 2001b; Wei et al., 2000). induced DNA damage. Studying interactions among gene loci and carcinogen dose will require more DNA adducts and cancers of the bladder, head and neck, sophisticated models and statistical analyses. At this pancreas and uterine cervix time it is difficult to predict whether high risk genotypes will be associated with increased or DNA adduct levels in white blood cells were signifi- decreased adduct level. The relationship may be cantly associated with bladder cancer risk (Peluso et al., different at early phases of a chronic exposure 1998, 2000). A series of studies have shown that compared with later times and the establishment of smoking is associated with smoking related adducts in genetically altered tissues. 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 New candidate gene polymorphisms to be associated with risk of head and neck cancer (Li et Additional polymorphic candidate genes with potential al., 2001a). Particularly striking was the finding of to modify tobacco related cancer risk via modulation specific adducts in gingival tissue derived from of DNA adduct levels, include the phenol sulfotrans- unsaturated aldehydes in cigarette smoke (Nath et al., ferases such as SULT1A1 (Nowell et al., 2000), 1998). A small study of pancreatic cancer patients myeloperoxidase (Petruska et al., 1992; London et al., reported higher levels of several DNA adduct species in 1997; Le Marchand et al., 2000; Schabath et al., 2000; nontumorous pancreatic tissues from cases compared Misra et al., 2001; Rojas et al., 2001; Kantarci et al., with control tissues (Wang et al., 1998a). Smoking is a 2002), dihydronicotinamide riboside (NRH)-quinone risk factor in cervical cancer and DNA adducts related oxidoreductase 2 (NQQ2) (Jaiswal et al., 1999; Long to smoking have been observed in cervical cells of and Jaiswal, 2000), UDP-glucuronosyl transferases smokers (Melikian et al., 1999a). Interestingly, HPV (Ren et al., 2000; Grant and Bell, 2000; Yueh et al., infection appeared to enhance induction of DNA 2001), and the transcription factor Nrf2, which is adducts by BPDE in cervical epithelial cells (Melikian essential for inducible and constitutive expression of a et al., 1999b) suggesting a mechanism for the interaction group of detoxification and antioxidant enzymes (Aoki of HPV and smoking in cervical cancer risk. et al., 2001). DNA-adducts and exposure to lung carcinogens DNA adducts and cancer risk Studies of highly occupationally exposed populations have generally reported DNA adducts to be related to Three lines of epidemiologic evidence implicate DNA airborne PAH exposure but sorting out the indepen- adducts in the etiology of smoking related cancers. dent effects of occupational and smoking related These include: case – control and cohort studies showing contributions to total PAH burdens has been proble- associations of cancer occurrence with smoking related matic. Some exposures postulated to pose an increased

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7383

Table 2 Studies of DNA adducts in tabacco related cancer risk Hypothesis/question Adduct Tissue Study design/Population Results Reference

1Isin vivo adduct level PAH-DNA WBC Hospital based lung cancer No overall association of Tang, a risk factor for adducts case cotnrol study adducts with lung cancer; suggested et al., 1998 lung cancer? Case (n=136) interaction of adducts and GSTM1 Control (n=115) genotype on case-control status 2Isin vivo adduct level Aromatic-DNA WBC Prospective lung cancer study Among current smokers mean Tang et al., a risk factor for adducts Nested case (n=89) control adduct levels were greater among 2001 lung cancer? (n=173) study (all males) cases compared with controls 3Isin vivo adduct Aromatic/ Non-tumorous Hospital based Lung cancer patients had greater Cheng level in lung a risk factor hydrophopic – DNA lung tissue Case (n=73) DNA adduct levels compared with et al., 2000b for lung cancer adducts Control (n=33) controls 4Isin vivo adduct level in PAH-DNA Non-tumorous Lung cancer case-referent Lung cancer patient had higher Cheng lung associated with lung adducts lung (Taiwan) adduct levels measured by two et al., 2001 cancer, are there gender 62 non-smoking lung cancer techniques, women had higher differences? patients, 20 non-cancer adducts compared with men controls 5 Is adduct level in Aromatic-DNA Blood Hospital based Higher adduct levels in cases Vulimiri blood a risk factor for adducts lymphocytes Case (n=43) compared with controls; includes et al., 2000 lung cancer? Control (n=47) minority populations 8-oxo-dG 6 Is cancer mortality PAH-DNA Broncho- Cohort Smokers dying from cancer had Bonassi associated with adduct adducts alveolar 40 heavy smokers twofold higher levels than smokers et al., 2001 level? Cells still alive at follow-up 7 Is induction of DNA BPDE-DNA In vitro Hospital based case control Cases were 2.15-fold more likely Li et al., adducts a risk factor for adducts treated blood Case n=221) to have induced adduct levels 2001 lung cancer? lymphocytes Control above the median control level; (n=229) positive dose response of induced adduct level and risk 8 Is the detection of DNA Total aromatic White blood cells Bladder cancer case control Cases were 3.7-fold more likely to Peluso adducts a risk factor for DNA adducts Case (n=162) have detectable DNA adducts et al., 2000 bladder cancer? Controls (n=104) compared with controls; suggested See also interaction with low fruit/vegetable Peluso consumption; no association of et al., 1998 adducts with tobacco smoking 9 Is induced DNA adduct BPDE-DNA In vitro treated Hospital based head and Cases were 2.2-fold more likely to Li et al., level a risk factor in head adducts blood neck case control study have adduct levels above the 2001a and neck cancer? lymphocytes median control level; highest Case (n=91) induced adduct levels observed in Control (n=115) current smokers 10 Are adducts in oral PAH-DNA Mouth floor and Smoking case referent Adduct levels higher among Besarati Nia, tissues related to smoking adducts buccal mucosal Smokers (n=26) current smokers; adduct levels et al., 2000 and oral cancer risk? cells greater in mouth floor cells Non-smokers (n=22) compared with buccal mucosal cells 11 Are adducts correlated PAH-DNA Laryngeal tissues, Case series Adduct levels positively correlated Banaszewski with cigarette smoking in Adducts Squamous cell 33 primary laryngeal cancer with cigarettes smoked per day, et al., 2000 laryngeal tissues? tumors patients patient age, male gender and TNM See also (5 women) stage. Adduct levels higher in Szyfter (28 men) normal versus tumor tissues. et al., 1999b Adducts higher in low risk anatomic area 12 Does smoking induce 1,N2-propanodeoxy- Gingical tissue Smoking case referent Total levels of cyclic adducts (AdG Nath et al., &, &-unsaturated guanosine derived 11 smokers and CdG) 4.4-fold greater in smokers 1998 aldehyde related DNA from acrolein (AdG) 12 non-smokers compared with non-smokers. One adducts in gingival crotonaldehyde (CdG) CdG adduct increased 8.8-fold in tissue? smokers compared with non-smokers 13 Does smoking induce PAH-DNA adducts Oral epithelial Smoking case referent PAH-DNA adducts associated with Ramano, DNA adducts in oral cells 33 smokers smoking and increased with daily et al., 1999 mucosal cells? 64 non-smokers cigarette consumption; results not affected by diet, infection, alsohol 14 Are in vitro DNA Aromatic and lipid Pancreatic tumors Pancrease cancer case series, Smoking associated adducts detected Wang adducts associated with peroxidation-related and normal case-referent in non-tumorous tissues; higher adduct et al., 1998 the occurence of DNA adducts pancreas tissues Cases (13 normal, 20 tumor) levels detected in nontumor tissues pancreas cancer? Non-cancer (n=19 organ from cases compared with noncancer donors) tissues 15 Is working a carbon BP – DNA adducts WBC Cross-sectional study of 17 No correlation between adduct level Arnould electrode manufacturing workers in a plant producing and PAH air concentrations was et al., 1999 plant a risk factor for carbon electrodes observed increased DNA adduct levels? 16 Is working in a carbon PAH-DNA adducts Blood Cross sectional study of High PAH exposure increased urinary Van Delft electrode manufacturing lymphocytes workers exposed to 5200 mg 1-OHPY excretion but not DNA et al., 1998b plant a risk factor for PAH/m3 in air adducts; tobacco smoking was increased DNA adduct associated with increases in DNA levels? adducts 17 Do cooking oil fumes BPDE-DNA In vitro treatment In vitro study Cooking oil fumes implicated in lung Yang induce DNA adducts? adducts of lung cancer among nonsmokers induce et al., 2000 adenocarcinoma BPDE-DNA adducts cell lines Continued

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7384 Table 2 (Continued ) Hypothesis/question Adduct Tissue Study design/Population Results Reference 18 Does smoking lead to Lipophilic-DNA Inhaled saline Smoking case referent 20 Adduct levels greater in sputum from Nia, detectable DNA adducts in adducts inhaled sputum smokers 24 healthy smokers compared with non-smokers; et al., 2000 sputum? non-smokers enrichment by the P1 nuclease method more sensitive thn butanol to smoking 19 Does occupational PAH BPDE-DNA White blood cells Cross-sectional study of coke BPDE-DNA adducts in blood Arnould, exposure correlated with adducts oven workers positively correlated urinary 1-OHPY et al., 2000 adduct levels in blood? 20 Do smoking related adducts Lipophilic-DNA Inhaled saline Smoking case referent Current smoking was associated with Nia, in sputum correlated levels adducts induced sputum increased adduct levels in both tissues; 2000 in peripheral lymphocytes? 9 smokers higher levels in sputum; stable adduct Blood 9 non-smokers levels over time lymphocytes 21 Do PAH levels correlate BPDE-DNA levels Lung tissues Cross-sectional autopsy BPDE-DNA adduct levels correlated Lodovici with PAH-DNA adduct obtained at study with BP concentration in tissues and et al., 1998 levels in lung tissues? autopsy were higher in smokers compared with non-smokers 22 Are adducts associated BPDE-DNA adducts Peripheral blood Cross-sectional study of Smokers with more than 20 pack- Shinozaki, with age? lymphocytes smokers and non-smokers years had increased adduct levels; et al., 1999 adducts positively correlated with age but not daily cigarette consumption 23 Are DNA adduct levels PAH-DNA adducts Isolated peripheral Population-based comparison Despite higher urban air PAH Kyrtopoulos associated with moderate to blood of 194 young adult subjects exposure, adduct levels in subjects et al., 2001 low levels of PAH exposure lymphocytes living in two Greek cities from Athens were not lower than subjects from urban air pollution? (Halkida, Athens) from Halkida; adduct levels greater in men compared with women 24 Are blood MNCs a good Aromatic DNA Isolated MNCs, Cross-sectional study of 78 Adduct levels in MNCs or BAL were Godschalk surrogate for detection of adducts broncho-alveolar smokers correlated with daily cigarette tar et al., 1998 smoking related DNA lavage cells, white exposure using the P1 nuclease adducts? cell subtypes method but not butanol extraction 25 Are blood MNCs a good Bulky hydrophobic- Isolated MNCs Cross-sectional study of 147 Blood MNC adduct levels highly Wiencke surrogate for detection of DNA adducts lung tissue lung cancer patients correlate with lung adduct levels; et al., 1999 smoking related DNA cigarettes per day a predictor of adducts? adduct levels in current smokers, early age of smoking predicts adduct level in former smokers 27 Does high PAH exposure BPDE-DNA adducts Isolated MNCs Occupational PAH case Occupational (coke oven) but not Pavenello, increase DNA adduct level referent medicinal (coal tar) exposure et al., 1999 In blood cells? 26 psoriasis patients associated with increased adduct (coal tar), levels; neither charcol grilled meat 15 coke oven workers, nor smoking associated with adduct 19 chimney sweeps, 36 levels non-PAH exposed 28 Does smoking induce DNA PAH-DNA adducts Cervical cells Smoking case referent Mean adduct level higher in epithelial Vineis adducts in cells of the 16 smokers cervical cells of smokers compared et al., 2000 uterine cervix? 16 non-smokers with non-smokers 29 Is smoking associated with BPDE-DNA adducts Cervical epithelial Case series of tissues from 17 Mean BPDE-adduct levels in Melikian increased levels of DNA and stromal tissue women undergoinf surgery epithelial but not stromal tissues et al., 1999a adducts in cervical tissue? samples for non-malignant disease higher in smokers compared with non-smokers 30 Does HPV-16 infection BPDE-DNA In vitro treated In vitro cell culture Greater levels of BPDE adducts Melikian potentiate adduct formation adducts epethelial cervical experiment induced in HPV-16 immortalized cells et al., 1999b from smoking carcinogens? cells compared with normal cells

risk for lung cancer have failed to lead to detectable the DNA from the short-lived granulocyte fraction of increases in adduct levels. For example, among workers WBC (Savela and Hemminki, 1991). In a subsequent exposed to PAHs in carbon electrode manufacturing study, hydrophobic-DNA adducts in blood MNCs no correlations were found between DNA adducts and were found to be a reliable surrogate of lung DNA occupational PAH exposures (Arnould et al., 1999; van adduct levels (Wiencke et al., 1995). Another researcher Delft et al., 1998b). In another study, and one of the used induced sputum to assess smoking related DNA only population based investigations on DNA adducts, adducts (Nia et al., 2000). White blood cell adduct researchers did not find that PAH – DNA adducts in levels were shown to provide a suitable surrogate for blood lymphocytes correlated with differences in PAH DNA adducts in bronchoalveolar cells (Godschalk et exposure related to urban air pollution (Kyrtopoulos et al., 1998). The lung retains PAHs, and higher al., 2001). concentrations can be found in men compared with As indicated above, adduct levels in white blood cells women, and perhaps among high risk ethnic minority have been associated with tobacco related cancer risk. groups (Seto et al., 1993; Goldman et al., 2001). Direct correlations among surrogate tissue and target Interestingly, concentrations of PAHs measured tissues, however, are few in number. An early directly in lung tissue at autopsy were found to important observation was that smoking effects on correlate well with PAH – DNA adduct levels measured adducts were stronger when measurements were made in the same tissues (Lodovici et al., 1998). This using DNA isolated from blood MNC compared with observation suggests that exposed lung may constitute

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7385 a tissue reservoir of unmetabolized PAHs. Immune important predictor of interindividual variations in cells from the peripheral blood or bronchoalveolar cells lung DNA adduct levels after adjusting for other that migrate and circulate through lung tissue may be measures of cigarette consumption (Wiencke et al., exposed to such accumulated PAHs. This may help 1999). Aromatic-DNA adducts were also found to be explain the correlation of peripheral blood or bronch- increased in smokers who started smoking at an early oalveolar cell DNA adduct levels with those measured age in another study (Vulimiri et al., 2000). Time in lung parenchyma and airways. elapsed since quitting smoking would seem a logical inverse predictor of DNA adducts in former smokers but this has not been reported. Although animal Dose response for DNA adducts and cigarette smoking models have been used to address these questions The nature of the dose – response for cigarette smoking (Helleberg et al., 2001; Izzotti et al., 1999) they do not and DNA adduct formation has significant implica- take into account the protracted exposures typical in tions in assessing cancer risks associated with tobacco humans, nor the cellular and physiologic changes use (Vineis et al., 2000). It is significant, therefore, that induced in tissues undergoing chronic exposure, which it is still somewhat unclear which measures of tobacco may modify adduct induction and repair. As indicated smoke exposure are most highly correlated with below, widespread accumulations of genetically altered smoking related DNA adduct levels. Several studies clones of cells is known to occur in smokers now support the idea that current smokers have higher chronically exposed to tobacco carcinogens (Franklin adduct levels compared with former smokers, and that et al., 1997). daily cigarette consumption (cigarettes per day) is the most predictive measure of variations in adduct levels DNA adducts and somatic mutation in tumors in target (Wiencke et al., 1999; Szyfter et al., 1999a; Banaszewski et al., 2000; Romano et al., 1999) and DNA adducts and p53 surrogate tissues of current smokers (Nia et al., 2000; Hou et al., 2001). In one study the daily cigarette tar A review several years ago cited the need for human consumed, but not the number of cigarettes, was found clinical studies of DNA adducts and the mutational to be predictive of adduct levels in blood MNCs spectra of cancer-relevant genes, and noted the paucity (Godschalk et al., 1998). Daily cigarette consumption of studies in which mutations as a consequence of in current smokers also predicted PAH – DNA adduct DNA damage at specific genes had been investigated in levels in cardiac tissue from patients undergoing open vivo (Kriek et al., 1998). This conclusion is still relevant heart surgery (van Schooten et al., 1998). Nonetheless today. The important mechanistic hypotheses on p53 some investigations have reported higher levels of mutational spectrum and smoking (see Hussain in this aromatic-DNA adducts in former smokers (Vulimiri volume) raise significant challenges for clinical and et al., 2000). Earlier suggestions (Phillips et al., 1988) epidemiological investigators to translate these insights that DNA adducts may reflect lifetime cumulative into human populations. Early relatively small studies tobacco smoke exposure as estimated by the pack – suggested that aromatic-DNA adduct levels were year variable do not appear to have been substantiated. increased in tissues from patients with p53 mutant Few studies have examined predictors of adduct lung cancer compared with cases bearing non-p53 levels among former smokers specifically. One of the mutant tumors (Kure et al., 1996; Ryberg et al., 1997). earliest studies of lung PAH – DNA adducts in former Another study reported only a weak association smokers reported that adduct levels were similar to between PAH – DNA adduct levels in lung tissue and those observed among never smokers after about 5 p53 mutations (Andreassen et al., 1996). Our group years of smoking cessation (Phillips et al., 1988). found that lung cancer patients with high hydro- Later, a biphasic loss of PAH – DNA adducts was phobic – DNA adduct levels in non-tumorous lung proposed that included a rapid initial phase followed tissue (above the median adduct level) were threefold by a second slower phase of adduct loss (Schoket et more likely to have a tumor containing a p53 mutation al., 1993). Studies of subjects in a smoking cessation (Wiencke et al., 1998). This association was significant, program indicated an estimated half-life of PAH – even after adjusting for smoking status. In contrast, in DNA adducts of about 9 – 13 weeks in blood a study of bladder cancer, 4-aminbiphenyl – DNA leukocytes (Mooney et al., 1995). The half-life of adducts were not found to be associated with the p53 PAH – DNA adducts in the lung is probably much mutational status of tumors (Martone et al., 1998). more protracted. One study of lung cancer patients Larger studies with carefully collected smoking and indicated a half-life of approximately 1.7 years lifestyle histories are necessary to define the relation- (Schoket et al., 1998) among short-term quitters. In ship of DNA adduct burden in vivo and the p53 a study of lung tissues from former smokers that mutational status and spectra of human lung cancer. included long-term quitters (410 years), several statistical models were applied that suggested an DNA adducts and LOH at 3p21 approximate 10% decline in adducts per year for about 9 years and a much slower residual decline after An event more common than p53 mutation in lung 9 years (Thurston et al., 2000). Among these former cancer is the LOH and deletion of regions of the short- smokers early age of smoking initiation was the most arm of chromosome 3 (e.g. 3p14, 3p12). LOH in these

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7386 regions is strongly associated with smoking history not imply, however, that endogenous agents do not (Nelson et al., 1998; Hirao et al., 2001; Zienolddiny et play a role in tobacco carcinogenesis, because tobacco al., 2001) and is thought to be among the earliest exposures can directly and indirectly increase oxidative events in lung carcinogenesis (Wistuba et al., 1999, stress. In animals, cigarette smoke exposures were 2000; Park et al., 1999; Endo et al., 2000; Mitsudomi et shown to enhance endogenous DNA adducts by al., 1996). These early somatic events may be the several fold in different tissues (Gupta et al., 1999; molecular basis for what Slaughter first termed ‘field Zhou et al., 2000). Significant progress has been made cancerization’. According to this idea, grossly normal in characterizing carbonyl-containing products of lipid epithelium can be ‘preconditioned’ by carcinogen peroxidation (e.g. malondialdehyde, 4-hydroxy-2-alke- exposure. Such preconditioning may result in early nals) and the adducts they form with DNA (Leuratti et tobacco-induced somatic mutations that are propa- al., 1998; Chen et al., 1998; Moller and Wallin, 1998; gated by enhanced cell replication into a clonal ‘field’ Yi et al., 1998) as well as the contributions of of genetically altered progenitor cells. physiologic mediators such as nitric oxide in these Consistent with these observations, we found a reactions (Nair et al., 1998). The DNA adducts formed strong correlation of early age of smoking initiation in reactions with carcinogenic aldehydes may produce with LOH at a loci near the hMLH1 gene on structurally unique adducts (Hecht et al., 2001); these chromosome 3p21 in surgically resected lung cancer hold promise as sensitive markers of oral cancer risk patients (Hirao et al., 2001). Interestingly, our associated with tobacco use (Nath et al., 1998). Some investigations also revealed a link between these events endogenously derived DNA adducts are thought to and increased hydrophobic – DNA adduct levels in increase with age (Josyula et al., 2000; Yang et al., lung tissue from the same patients (Hirao et al., 2001). 1998). Several reviews have been presented on Another group, using microsatellite markers close to endogenous DNA adducts and cancer risk (Burcham, hMLH1, reported a similar increase in PAH – DNA 1998; Bartsch and Nair, 2001; Blair, 2001). adduct levels in lung tissues of lung cancer patients Future studies of genetic susceptibility studies should whose tumors contained LOH at 3p21 (Zienolddiny et consider the potential overlap between endogenous and al., 2001), although the question of age at smoking exogenous pathways and sources of DNA adducts. For initiation was not addressed in the latter study. In a example, ethylene oxide, which is an endogenous study of lymphocytes from lung cancer patients and metabolite of ethylene, is also present in cigarette controls, the highest aromatic DNA adduct levels were smoke. Studies of blood hemoglobin adducts formed observed among cases who started smoking before 15 from ethylene oxide indicate that genetic deficiency in years of age (Vulimiri et al., 2000). GSTT1 leads to higher adduct levels in smokers These links between DNA adducts and somatic (Fennell et al., 2000) and non-smokers (Thier et al., alterations in the lung and early smoking initiation 2001). DNA adducts in blood leukocytes were also should be considered in light of the epidemiologic reported to be higher in GSTT1 null subjects in the observations that early smoking initiation is an EPIC – Italy study (Palli et al., 2000). independent risk factor for lung cancer (Hegmann et al., 1993). Taken together these associations lead to a Chemically unstable DNA adducts novel ‘critical period’ hypothesis for lung cancer susceptibility (Wiencke and Kelsey, 2002): growth and The majority of studies of tobacco-related cancer and development of the lungs during adolescence may set DNA adducts have focused on chemically stable up a critical period of susceptibility to tobacco-related modifications of DNA, like those produced by the DNA damage. This idea is consistent with the burst of reaction of BPDE with the exocyclic amino group of lung growth that is known to occur in the early teen guanine. PAHs present in tobacco smoke, including years. Exposure to tobacco carcinogens during this BP, can also be metabolized via radical cation period may lead to the propagation of fields of altered intermediates to electrophiles that bind to DNA bases epithelial cells, particularly in the airway, that later and destabilize the N-glycosyl bond, causing rapid evolve into frank malignancy. Exposure after the depurination or depyrimidination of the adducted critical growth period may have a much lower bases (Bodell et al., 1989; Chen et al., 1996; Devanesan probability of inducing large, clonal outgrowths with et al., 1996). In some experimental systems (e.g. mouse the same malignant potential. skin treated with BP) these unstable DNA adducts are much more prevalent than the stable DNA adducts. Understanding the relative importance of unstable Emerging issues in tobacco carcinogenesis and DNA versus stable adducts in tobacco carcinogenesis in adducts humans and the role of DNA repair mechanisms that act on these different lesions will require further Endogenous adducts, oxidative DNA damage and indirect investigation (Melendez-Colon et al., 1997, 1999; action Chakravarti et al., 2001). Future epidemiologic studies It has been suggested that damage to DNA from may be the only way to accurately assess the endogenous exposures may be orders of magnitude importance of depurinating adducts in tobacco carci- greater than those from exogenous sources including nogenesis. This will require improvements in the exposure to tobacco smoke (Povey, 2000). This does methods for detecting such compounds in biological

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7387 specimens. One sensitive method for detecting unstable Alternatives to case – control or cross-sectional DNA adducts in urine (i.e. using capillary electrophor- studies include those employing prospective and esis – fluorescence line narrowing spectroscopy) has interventional designs. For example, in the SELECT been developed (Roberts et al., 2000). Initial studies study (Selenium and Vitamin E Cancer Prevention using this approach found BP adducted bases (e.g. BP- Trial) the goal is to enroll 32 400 men aged 55 years 6 N7Gua) in the urine of three of seven cigarette and older and follow them for a 12 year study period smokers and three of seven women exposed to coal (Hoque et al., 2001). Blood samples collected at smoke, but none were detected in urine from 13 control baseline could be used for DNA adduct and candidate subjects (Casale et al., 2001). gene studies; an expected 800 lung cancer cases are predicted to occur within the study period. Factors affecting the validity of DNA adduct studies Bias caused by seasonal variation in DNA adducts Exposure misclassification, statistical analysis, study Finally, a question still unresolved is whether studies of design DNA adducts can be significantly biased by seasonal Others have reviewed some of the statistical and variations in adduct levels. Several studies have reported analytical issues that can compromise the validity of statistically significant variations in DNA adduct levels DNA adduct studies (Cuzick, 1995). The literature according to the season of study (Butkiewicz et al., 2000; reviewed here alludes to a divergence in adduct results Grzybowska et al., 1993; Moller et al., 1996; Motykie- in current versus former smokers. This has important wicz et al., 1998; Palli et al., 2001; Perera et al., 1992; implications for the design of future investigations into Tang et al., 1995; Topinka et al., 2000). Many of these the role of DNA adducts in tobacco-related cancer. At reported higher ‘background’ adduct levels in winter any single point in time the levels of tobacco-related compared with summer, and these variations appear to DNA adducts in a human tissue represent a dynamic be related to the airborne concentrations of PAHs and process that is dependent on a number of factors; these nitro-PAHs, particularly for studies carried out in include the intensity and recency of exposure to industrialized and polluted areas of Poland and the tobacco smoke, the metabolic balance of activation Czech Republic (Moller et al., 1996; Topinka et al., and detoxification mechanisms and the repair or 2000). These observations suggest that seasonal variation removal of adducts by DNA repair and/or cell in DNA adducts may not be a generalized phenomenon turnover. Therefore, to observe relationships between but is rather an issue that is regionally specific to those putative modifiers of DNA adduct concentrations, it areas where ambient air quality varies widely with the may be important to stratify on smoking status season of the year. (current, former, never) in all statistical analyses or However, recent research also indicates the complex- to employ multivariate statistical techniques that ities of these ecological associations. For example, carefully address potential interactions of modifiers concentrations of PAH – DNA adducts levels in WBCs and smoking status. It may be expected, for example, were found to peak in Northern Italy (Turin, Varese) that genetic modification of adduct levels arising in winter and in summer in Central and Southern Italy through a metabolic mechanism (adduct formation) (Florence, Naples, Ragusa), which suggested opposite may be more evident among current smokers. Genetic seasonal trends according to latitude (Palli et al., 2000). factors affecting repair and tissue renewal (adduct The authors speculate that in summer photochemical removal) may be more evident in comparisons pollution may be higher at lower latitudes and play a involving former smokers. relatively greater role in the induction of adducts In addition, many of the case – control studies cited compared to the winter months. In another study, above can be criticized, on the basis that DNA adducts blood PAH – DNA adducts in non-smokers were found may be linked to cancer occurrence through their to be higher in the small Greek city of Halkida association with tobacco smoke exposure. As DNA compared with adduct levels in non-smokers living in adducts are thought to be part of the causal pathway Athens, despite the far greater air pollution burden in tobacco-related cancer they are not formally that exists in Athens (Kyrtopoulos et al., 2001). considered to be confounders. Nonetheless, these Consequently, it would appear prudent to consider considerations raise concerns about the clinical value seasonal information and ambient air quality in of DNA adducts as independent predictors of tobacco- planning future studies to avoid potential biases that related cancer risk. To mitigate against these concerns, may arise from regional or seasonal variations in DNA the question of adducts as risk factors could be adducts in human populations. addressed in studies that carefully match cases and controls on cigarette smoking exposure. Case – control studies matching on smoking may also provide a more Abbreviations robust way to examine the role of candidate genes as GSTM1, GSTT1, GSTP1, Glutathione S-transferase M1, T1, modifiers of DNA adduct formation or repair. Because P1; NAT1, NAT2, N-acetyltransferase type I and type II; of the difficulty in recruiting suitable controls, however, NQQ1 NAD(P)H:quinone oxidoreductase type I; BP, such studies are more difficult to perform than benzo(a)pyrene; BPDE, 7,8-dihydro-7,8-dihydroxybenzo(a)- traditional case – control studies. pyrene 9, 10-oxide; MNC, mononuclear cells; PAH,

Oncogene DNA adduct burden and tobacco carcinogenesis JK Wiencke 7388 polynuclear aromatic hydrocarbons; GSH, glutathione; yguanosine; PhIP, 2-amino-1-methyl-6-phenylimidazao[4,5- HPLC, high performance liquid chromatography; 1-OHPY, b]pyridine; HPV, human papilloma virus; EPIC, European 1-hydroxypyrene; WBC, white blood cells; GS/MS, gas Prospective Investigation into Cancer and Nutrition chromatography-mass spectrometry; eA, 1,N6-ethenodeoxya- denosine; eC, 3,N4-etheneodeoxycytidine; M1G, 3-(2-deoxy- b-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one; BP-6 N7Gua, 7-(benzo[a]pyren-6-yl)guanine; ELISA, enzyme Acknowledgements linked immunosorbent assay; 8-oxo-dG, 8-hydroxy-2’-deox- This work was supported by NIH ES06717.

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