Evaluation of 4-aminobiphenyl-DNA adducts in human breast cancer: the influence of tobacco smoke

Beatrice Faraglia1,7, Shu Yuan Chen1, Marilie D. Gammon2, Yujing Zhang1, Susan L. Teitelbaum3, Alfred I. Neugut4, Habibul Ahsan4, Gail C. Garbowski1, Hanina Hibshoosh5, Dongxin Lin6,8, Fred F. Kadlubar6 and Regina M. Santella1,9

1 Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 701 West 168th Street, 10032, New York, NY 10032
2 Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina
3 Department of Environmental Health Sciences, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029
4 Department of Epidemiology, Mailman School of Public Health, Columbia University, 701 West 168th Street, New York, NY 10032
5 Department of Pathology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032
6 National Center for Toxicological Research, Jefferson, AR 72079, USA

9 To whom correspondence should be addressed Email: rps1{at}columbia.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is one of the major cancers around the world but its etiology is still not well understood. Only ~50% of the disease is associated with known risk factors including highly penetrant genes and lifestyle factors. Thus, environmental carcinogens may play an important role in the etiology of breast cancer. The arylamine 4-aminobiphenyl (4-ABP) is a tobacco smoke constituent, an environmental contaminant, and a well-established bladder carcinogen in rodents and humans. In this study, we investigated the role of 4-ABP in the etiology of human breast cancer by measuring 4-ABP–DNA adducts using a monoclonal antibody based immunoperoxidase method that had been validated by comparison with gas chromatography/mass spectroscopy analysis of liver tissues from 4-ABP-treated mice. Adducts were analyzed in 150 paraffin-embedded breast tumors and in 55 adjacent normal tissues collected from cases in the Long Island Breast Cancer Study Project. The role of polymorphisms in genes involved in the metabolism of 4-ABP including N-acetyl transferase 2 (NAT2), cytochrome P4501A2 (CYP1A2) and glutathione S-transferase M1 (GSTM1) and the nucleotide excision repair gene XPD was also explored in the same patients. The mean log-transformed relative staining intensity for 4-ABP–DNA adducts was higher in normal (5.93 ± 0.54) than in the corresponding tumor (5.44 ± 0.62, P < 0.0001) tissues. However, a highly significant positive correlation was observed between the levels of 4-ABP–DNA in both tissues (r = 0.72, P < 0.0001). Smoking status was correlated with the levels of 4-ABP–DNA in tumor adjacent normal tissues with a significant linear trend (P = 0.04) for current, former and never smokers; adducts were not related to smoking status in tumor tissues. No correlation was observed between the levels of 4-ABP–DNA and polymorphisms in the genes analyzed even when subjects were stratified by smoking status. These results demonstrate that smoking is associated with increased levels of 4-ABP–DNA adducts in human mammary tissue. In this study, genetic polymorphisms did not significantly affect the formation of 4-ABP–DNA adducts in breast cancer cases, perhaps due to the small number of samples.

Abbreviations: 4-ABP, 4-aminobiphenyl; CYP, cytochrome P450; GST, glutathione S-transferase; NAT, N-acetyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is one of the major cancers in the United States and its incidence is still increasing (1). Established risk factors for breast cancer include reproductive and menstrual characteristics, family history, postmenopausal obesity, exogenous estrogen use and alcohol consumption (2). These factors, however, account for only about half of breast cancer cases (3). The inheritance of mutations in high penetrance genes (e.g. BRCA1 and BRCA2, ATM, p53) appears to be important in no more than 5–10% of cases (4). Lifestyle and environmental factors are believed to play an important role although no clear epidemiological links have been established with any specific environmental factors except alcohol and ionizing radiation (2). Considerable evidence suggests that chemical carcinogens may be involved in the development of human breast cancers. Various chemicals that have been shown to induce tumors in experimental animals are present in the environment (5) and the spectra of gene mutations observed in human breast tumors suggest the involvement of exogenous agents in inducing these mutations (6,7). A recent analysis of p53 mutations in breast tumors suggested that cigarette smoking modifies the prevalence and spectrum of mutations (8). To investigate the role of chemical carcinogens in breast cancer, DNA adducts have been measured in breast tissues in several studies (915). However, most of these studies have concentrated on those of polycyclic aromatic hydrocarbons.

4-Aminobiphenyl (4-ABP) is a well-studied aromatic amine and a known bladder carcinogen in both experimental animals and humans and a possible breast carcinogen. It is metabolized by hepatic cytochrome P4501A2 (CYP1A2) to yield N-hydroxy-aminobiphenyl, a direct-acting mutagen (16). However, the accumulation of biologically active metabolites of 4-ABP is dependent on the balance between activation and detoxification by N-acetylation, mainly by N-acetyltransferase 2. Studies of human 4-ABP exposure have demonstrated higher levels of DNA adducts in smokers than in non-smokers in directly exposed tissues such as oral cells, sputum and laryngeal tissues (1719) but also in bladder tissues (20,21). Elevated levels of 4-ABP–DNA have also been detected in liver tissues of hepatocellular cancer cases compared with controls (22). A more recent study has detected 4-ABP–DNA adducts in exfoliated ductal epithelial cells in human breast milk using 32P post-labeling (23). Using an appropriate standard, 18 of 64 samples were found to have 4-ABP–DNA with levels ranging from 1 to 300/108 nucleotides.

Previous reports on breast cancer risk associated with exposure to environmental carcinogens, including tobacco smoke, are extremely variable [(8) and references therein]. Therefore, it has been hypothesized that genetic factors might also play a role in modulating this risk (24,25). Individual variability in enzymes responsible for activating (phase I) and detoxifying (phase II) carcinogens and for repairing DNA damage may explain varying susceptibilitiy to breast cancer development and individual variation in DNA adducts with similar exposures [reviewed in (24,25)]. Several studies on CYP1A2 have suggested a relationship between polymorphisms in this gene and cancer susceptibility (26). Glutathione S-transferase (GST) family members are among the most studied phase II detoxifying enzymes regarding cancer susceptibility and well defined genetic polymorphisms in GSTM1 and GSTT1 have been associated with individual cancer risk (27). Polymorphisms in NAT2 have also been associated with individual susceptibility to bladder and other cancers, including breast cancer although the findings have been inconsistent for breast cancer (28). DNA adduct levels in tissues and cancer risk may also be related to individual differences in DNA repair capacity (29). The XPD gene encodes a helicase involved in the excision repair pathway and in transcription and its variants have also been investigated in relation to cancer risk (30).

Despite the strong biological plausibility for the role of metabolizing enzymes in breast tumorigenesis, there have been relatively few epidemiological studies that have evaluated the role of metabolizing gene polymorphisms in breast DNA adduct levels (9,10,13,31). Moreover, the role of 4-ABP–DNA adducts and their relationship with tobacco exposure and the presence of an at-risk genotype in breast tumorigenesis have also not been extensively evaluated. We report the results of a study in which the levels of 4-ABP–DNA adducts were evaluated in a series of breast cancer cases from the Long Island Breast Cancer Study Project (32). In those cases in which adjacent normal tissue was also present, adducts in the two tissue types were compared. The relationship between 4-ABP–DNA adduct levels and risk factors and gene polymorphisms (CYP1A2, NAT2, GSTM1 and XPD) were also examined.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
The present study used tissue and blood samples from participants in the Long Island Breast Cancer Study Project undertaken to determine whether environmental factors are associated with breast cancer (32). This population-based, case-control study was conducted in the counties of Nassau and Suffolk in New York State. Eligible women were residents of these two counties, spoke English and were newly diagnosed between August 1, 1996 and July 21, 1997. After informed consent was obtained, a questionnaire was administered eliciting information on major cancer risk factors, such as current and former smoking status, menopausal status, age or race and grilled food intake. The complete questionnaire is available on the National Cancer Institute web site http://epi.grants.cancer.gov/LIBCSP/Questionnaire.html. A total of 1508 (82.1%) eligible subjects completed the questionnaire of which 93.8% were white and 4.6% black. A total of 150 cases were selected from those cases assayed previously for polycyclic aromatic hydrocarbon–DNA adducts in blood (33) based on the order in which the paraffin blocks were retrieved. These samples were submitted by ~15 of the 33 hospitals providing patients. Sections from two subjects were lost from the slides during the staining process and thus the final analysis consisted of 148 subjects.

Immunohistochemistry
The immunoperoxidase method was first validated using stored liver tissues of Balb/c female mice treated with single i.p. doses of 4-ABP from 0 to 80 mg/kg in corn oil plus 0.1% ethanol for 24 h (17). Adducts had been determined previously on DNA isolated from liver tissues at the National Center for Toxicologic Research using gas chromatography with negative ion chemical ionization mass spectrometry that involved alkaline hydrolysis to release the 4-ABP followed by derivatization and analysis. For the immunoperoxidase method, formalin-fixed, paraffin-embedded tissues were stained using monoclonal antibody 3C8, which specifically recognizes 4-ABP–DNA adducts (17) essentially as described previously (34). Briefly, after being dewaxed and rehydrated, 5-µm sections were treated sequentially with RNase A (100 g/ml for 1 h at 37°C) and proteinase K (10 g/ml at room temperature for 10 min) to enhance the sensitivity of the staining and eliminate cross-reactivity with RNA. Sections were then treated with 4 N HCl for 5 min, and neutralized with 50 mM Tris base before being incubated in 0.3% H2O2 in methyl alcohol at room temperature for 30 min. Non-specific binding was then blocked by 10% horse serum and the slides were incubated with antibody 3C8 overnight at 4°C. After washing with PBS, peroxidase-labeled anti-mouse ABC and diaminobenzidine kits were used (Vector Laboratories, Burlingame CA). Staining was quantified with a Cell Analysis System 200 microscope (Becton Dickinson, San Jose, CA) using the Cell Measurement Program software package to measure nuclear average optical density. A total of 50 randomly selected cells of each section (five fields, 10 cells/field) were quantified. The fields were arbitrarily selected based on large numbers of cells being present and that tissue edges were not included.

The levels of 4-ABP–DNA adducts were evaluated in the human samples using formalin-fixed, paraffin-embedded tissues as for the animal tissues. Sections had been cut and stored at -20°C for 1–6 months before staining. In 55 cases, sections included normal adjacent mammary gland tissue that was also analyzed for 4-ABP–DNA adducts levels.

Genotype analysis
DNA isolated previously from blood mononuclear cells was genotyped for NAT2, CYP1A2 and GSTM1 using polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism with visualizaion of gel bands on a Molecular Devices fluoroimager. For NAT2 genotypes, the four most common functional (NAT2*4) and low activity alleles (NAT2*5, NAT2*6, NAT2*7) were determined. Patients were classified as low, intermediate and rapid acetylators when they expressed any two, only one or no low activity NAT2 alleles, respectively (35). The CYP1A2*1F allele (www.imm.ki.se/CYPparallels/cyp1a2.htm), a C to A at -164 polymorphism was determined as reported in Chida et al. (36). Deletion of GSTM1 was determined as described in (37).

The exon 23, codon 751 A to C polymorphism in XPD, was determined using template-directed primer extension with detection of incorporated nucleotides by fluorescence polarization in a 96 microwell-based format essentially as described (38). Master DNA 96 well plates containing 10 ng/µl were used to make replica plates containing 25 ng DNA/well. For PCR amplification, the primers (forward 3'-CCC TCT CCC TTT CCT CTG TT-5', reverse 3'-GGC AAG ACT CAG GAG TCA CC-5') gave a 171 bp product. Conditions for amplification were 0.2 µl (8 pmol/µl) forward and reverse primers, 0.4 µl 25 mM MgCl2, 1 µl 10x PCR buffer, 0.1 µl (5 U/ml) Taq polymerase (Roche Molecular Biochemicals, Indianapolis, IN), 0.25 µl (10 mM) dNTPs (Roche) and 5.35 µl water. Denaturation at 94°C for 5 min 30 s was followed by 34 cycles of 94°C for 30 s, 60°C for 45 s and 72°C for 1 min, followed by 4 min at 72°C. Primers and dNTPs were digested with 1 U of shrimp alkaline phosphatase (1 U/µl, Roche) after addition of 1 µl of 10x buffer and 1 U Escherichia coli exonuclease I (10 U/µl, United States Biochemical, Cleveland, OH) and 7.9 µl of water for 45 min at 37°C followed by heating at 95°C for 15 min. The reverse extension primer was 3'-CTG AGC AAT CTG CTC TAT CCT CT-5'. Acycloprime FP SNP Detection kit G/T contained the ddNTPs labeled either with R110 or TAMRA (Perkin Elmer Life Sciences, Boston, MA). To 7 µl of reaction mixture was added 0.05 µl acycloprimer enzyme, 1 µl G/T Terminatior mix, 2 µl 10x reaction buffer, 0.5 µl extension primer (10 pmol/µl) and 9.45 µl water. Extension was carried out by heating at 95°C for 2 min followed by 30 cycles of 95°C for 15 s and 55°C for 30 s. Plates were read on a Perkin Elmer Victor instrument.

Statistical analysis
Definitions for exposure to environmental tobacco smoke and for cigarette smoking are based on self-reported data from the main questionnaire. A current active cigarette smoker was defined as a regular smoker within the last 12 months prior to the date of diagnosis; a former active smoker was defined as a regular smoker who reported quitting >12 months prior to the reference date; a passive smoker was defined as either a current or former smoker or non-smoker who reported ever living with an active smoker; and a never smoker is a non-smoker who also did not report living with an active smoker. Trend tests were used to examine the relationship between adduct levels with increasing smoke exposure, assuming that exposure of current smokers is higher than that of former or never smokers. For analyses examining active smoking or passive smoking, variables were coded as 1 = never, 2 = former; 3 = current and for combined active/passive smoking variables were coded as 1 = never either, 2 = ever passive only, 3 = ever active only, 4 = ever both.

A subject was defined as postmenopausal if her last menstrual period was more than 6 months before the reference date or if she had both ovaries removed before the date of diagnosis. If a subject was taking hormone replacement therapy or had a hysterectomy without removal of both ovaries, her menopausal status was initially classified as unknown. Any smoker with unknown status was categorized as postmenopausal if her age at reference was >=54.8 years (90th percentile for natural menopause among smoking controls) and any non-smoker with unknown menopausal status was categorized as postmenopausal if her age at reference was >=55.4 years (90th percentile for natural menopause among non-smoking controls).

Data on relative staining intensity for 4-ABP–DNA adducts were log-transformed to normalize the distribution. Paired t-test was used to test for differences in adduct levels between normal and tumor tissues (n = 55). The Pearson correlation between normal and tumor tissues was also determined. Differences in means of 4-ABP–DNA adduct intensity (log-transformed) by different groups (age, smoking, genotype) were tested by t-test or ANOVA. There were no differences between subjects with both tumor and non-tumor tissue data (n = 55) compared with those with data only in tumor tissue (n = 93) for all variables examined except menopausal status with 55% of subjects with both tissues being postmenopausal compared with 75% (P = 0.01) of subjects with only tumor tissue. Comparison of all cases to subjects analyzed for 4-ABP–DNA adducts in this study indicated significant differences in age (mean 59.5 versus 56.9 years, P = 0.02) and passive smoking status (19.0, 66.3 and 14.7 versus 10.6, 73.2 and 16.2% for categories 1–3, P = 0.05), respectively. SAS version 8.2 was used for all analyses.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The immunoperoxidase method for detection of 4-ABP–DNA was validated by analysis of liver tissues of mice treated with 0–80 mg/kg 4-ABP for 24 h. Relative nuclear staining intensity values in the nuclei ranged from 189 ± 42 to 656 ± 76. DNA isolated from these tissues had been analyzed previously for 4-ABP–DNA adducts by gas chromatography/mass spectroscopy. Adduct levels ranged from 0 to 629/108 nucleotides. The comparison of 4-ABP dose to 4-ABP–DNA adducts measured by the two methods is given in Figure 1. For the immunoperoxidase method the relative staining intensity for the control tissue (189 arbitrary units) has been subtracted from all values for tissues of animals treated with 4-ABP. There is a good correlation between adducts determined by the two methods (r = 0.94, P < 0.005).



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Fig. 1. Comparison of immunoperoxidase ({diamondsuit}) and gas chromatography/mass spectroscopy ({blacksquare}) analysis of 4-ABP-DNA adducts in liver tissues of mice treated with various doses of 4-ABP. Immunoperoxidase data are expressed as relative nuclear staining intensity minus the value (189 arbitrary units) for the untreated control animal. GC/MS data are expressed as adducts per 108 nucleotides.

 
The method was then applied to the tissue samples of breast cancer cases. Representative immunoperoxidase stainings of nuclei in breast tumor and adjacent non-tumor tissues with high and low 4-ABP–DNA adduct levels are illustrated in Figure 2. Staining intensity values ranged from 19 to 955. The means and standard deviations of relative staining intensity values and log-transformed values according to the patients' age at diagnosis and smoking status are given in Table I. There was a positive correlation between log-transformed relative staining intensity in tumor and adjacent non-tumor tissues in the same individual (r = 0.72, P < 0.0001, n = 55, Figure 3). However, mean log relative staining intensity was significantly higher in adjacent normal tissues (5.93 ± 0.54) than in tumor tissues (5.44 ± 0.62) with P < 0.0001 for paired t-test among the 55 subjects.



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Fig. 2. Representative immunoperoxidase staining for 4-ABP–DNA adducts using monoclonal antibody 3C8 of breast tumor tissues with high (A) and low (B) adduct levels and adjacent non-tumor tissue with high (C) and low (D) adduct levels.

 

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Table I. Distribution of mean original and log-transformed relative staining intensity of 4-ABP–DNA adducts in normal adjacent tissue and tumor tissue of breast cancer cases

 


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Fig. 3. Correlation between log-transformed relative staining intensity for 4-ABP–DNA adducts in tumor tissue and adjacent nontumor tissues of the same subject (r = 0.72, P < 0.0001, n = 55).

 
4-ABP–DNA adducts levels in tumor or adjacent non-tumor tissues were not related to patients' age (Table I). Nor was there a relationship with menopausal status (data not shown). When the data were stratified according to smoking status, a significant increasing trend (P = 0.04) was observed between the levels of 4-ABP–DNA adducts in normal tissues and active smoking habits with values of 5.77 ± 0.60 in never smokers, 5.95 ± 0.50 for former smokers and 6.20 ± 0.43 for current smokers. There was also a significant difference (P = 0.04) between current and never smokers. The increasing trend relationship was clear when subjects with both active and passive smoke exposure and/or ever active smoke exposure only were compared with those with ever passive smoke exposure or neither (trend test, P = 0.03). There were significantly higher adducts for those with only passive smoke exposure (5.90 ± 0.48) compared with those with never either active or passive exposure (4.63 ± 0.14) (multiple comparisons t-test before Bonferroni rule adjustment, P = 0.001) though there were only two subjects in the later category. There was no difference in adduct levels between those with active smoke exposure only and ever both active and passive smoke exposure. In contrast, for tumor tissues, levels of 4-ABP–DNA were not related to either active or passive smoking status. PAH–DNA adduct levels in blood mononuclear cells determined by ELISA were available for all subjects (33). There was no relationship with 4-ABP–DNA in breast tissue.

The relationships between 4-ABP–DNA and genotypes for CYP1A2, NAT2, GSTM1 and XPD was also investigated. No significant relationships between polymorphisms in these genes and log-transformed staining intensity for 4-ABP–DNA were observed in all cases (Table II) or when cases were stratified according to smoking status (data not shown).


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Table II. Distribution of mean of log-transformed relative staining intensity of 4-ABP–DNA adducts in normal adjacent and tumor tissues of breast cancer cases according to genotype

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study provides evidence for a link between tobacco smoke exposure and the levels of 4-ABP–DNA adducts in normal breast tissue adjacent to tumors from breast cancer cases. We found that relative staining intensity for 4-ABP–DNA adducts in tumor adjacent mammary tissues obtained from active current smokers was significantly higher than that in tissues obtained from non-smokers. 4-ABP is a main component of tobacco smoke and several studies have confirmed a higher exposure in smokers than in non-smokers (1721). Our results of an increased level of 4-ABP–DNA adducts in normal breast tissues of smokers is consistent with the data. Passive smoking did not significantly affect the levels of 4-ABP–DNA adducts in adjacent tissues. A significant correlation was observed in the levels of 4-ABP–DNA adducts in tumor and normal adjacent breast tissues. However, it is of interest that a significant relationship between tobacco exposure and 4-ABP–DNA adducts was only observed for normal but not for tumor tissues. The reasons for this are not clear but may be due to the small sample size.

While this is the first report of 4-ABP–DNA adducts in breast tissue, several other studies have reported on the presence of PAH–DNA adducts in breast tissue by immunohistochemistry (11,12,39) or of aromatic DNA adducts by 32P post-labeling (9,10,14,15). PAH–DNA adducts were higher in breast tissue of cases compared with tissue from women undergoing reduction mammoplasty (14) or from women with benign breast disease (39). Elevated adducts in smokers compared with non-smokers were found in some studies (10,15) but not others (39). The parent Long Island Breast Cancer Study Project examined the relationship between PAH–DNA adducts in blood mononuclear cells, measured by ELISA, in cases and controls. Among women in the highest quintile of PAH–DNA adducts, the odds ratio for breast cancer was elevated (OR 1.49, 95% CI 1.00–2.21) but there was no increased risk with increasing adduct levels (33). No relationship was found between blood PAH–DNA and 4-ABP–DNA in breast tissue. This was not surprising given the different sources of exposure to these compounds and the lifespan of the different cell types.

We also found that the levels of 4-ABP–DNA adducts were significantly lower in tumor tissues (mean = 5.44 ± 0.62, n = 55) compared with adjacent normal mammary tissues (mean = 5.93 ± 0.54) in those subjects with paired tissues. These differences may be due to alterations in metabolic enzymes in tumor tissue. The production of biologically active metabolites of 4-ABP is dependent on the balance between the activity of CYP1A2 (activating) and NAT2 and GSTM1 (inactivating) enzymes. Accordingly, lower adduct levels in tumor tissues compared with adjacent normal tissues may be due, excluding variation in 4-ABP exposure and/or in the DNA repair mechanisms, to decreased levels of CYP1A2 and/or increased levels of NAT2 and GSTs. However, as NAT but not CYP1A2 mRNA, protein or enzyme activity have been measured in breast mammary epithelial cells [reviewed in (25)], our results suggest that neoplastic transformation of mammary epithelial cells is probably associated with a change in the activity of NAT or GST that would affect the accumulation of active metabolites of 4-ABP and, in turn, the formation of 4-ABP–DNA adducts. This may also explain the lack of an association between adducts and smoking in tumor tissues. However, other mechanisms cannot be excluded and further studies will be needed to confirm these results and to elucidate the underlying molecular mechanisms.

We also evaluated the genotype of several genes which have been reported to be associated with cancer risk or DNA adduct levels (CYP1A2, NAT2, GSTM1 and XPD) in the same cases but found no associations between the level of 4-ABP–DNA adducts and genotype.

As with any study such as this, however, these results should be interpreted in light of study limitations. A limitation of the immunoperoxidase method is that absolute adduct levels cannot be determined. The animal studies indicated that samples with staining intensities in the range of 189–656 contain 4-ABP–DNA adducts in the range of 0–629/108 nucleotides. However, animals were treated with a single carcinogen and contain primarily 4-ABP–DNA adducts. In contrast, humans exposed to 4-ABP are also exposed to many other chemicals that may also form adducts. While antibody 3C8 appears to be highly specific based on cross-reactivity testing with other carcinogen–DNA adducts, the number of other adducts tested was small (17). Thus, absolute levels should be interpreted with caution. However, a prior study using post-labeling reported adducts in the range of 1–300/108 in exfoliated ductal epithelial cells (23).

A potentially important consideration is the relatively small number of tissues studied (n = 148). With two groups of 75, assuming a within group variance of 0.57 (calculated from Table II) and alpha = 0.05, there was 80% power to detect a difference of 0.275 and 70% power to detect a difference of 0.245. It is of interest, however, that several smaller studies have observed significant relationships between genotype and adducts. GSTM1 genotype and PAH–DNA adducts were correlated in breast tumor tissues (n = 95) but not in control tissues from women with benign breast disease (n = 87) (13). With a sample size of 42 breast tissues from women undergoing surgery for breast cancer or reduction mammoplasty, aromatic DNA adducts by post-labeling were elevated in tissues of women with slow NAT2 acetylator status (9). In another study, among ever-smokers, higher levels of aromatic adducts were observed in adjacent normal tissue of breast cancer cases with CYP1A1*1/*2 or *2/*2 genotypes (n = 10) compared with those with the CYP1A1*1/*1 genotype (n = 33) (10). This study also found that individuals having slow NAT2 alleles had a significantly higher frequency of the typical smoking-related adduct pattern (15/45, 25%) in tumor adjacent tissue than those with rapid acetylator status (4/53, 7%). Only one other study investigated the role of CYP1A2 genotype on adducts by post-labeling and also found no association (31). However, only 35 subjects, 26 reduction mammoplasty samples and nine tumor adjacent tissues were analyzed. Thus, our study had larger numbers of subjects than other studies but was not able to detect a relationship between adducts and genotype suggesting the potential for erroneous results in the smaller studies (40). Clearly, studies with even larger numbers of subjects will be required to definitively address this issue.

In conclusion, regardless of the underlying molecular mechanisms, we believe that our results suggest the need to use breast normal, rather than cancer, tissues to evaluate individual exposure to specific carcinogens and its role in breast cancer development. This study also provides further clues into the mechanisms underlying the possible association of smoking with breast cancer risk suggesting that 4-ABP present in tobacco smoke might play an important role in this process. However, evidence for causality cannot be drawn on the basis of this study alone and many questions remain. It is unclear whether 4-ABP–DNA adducts directly affect the process of neoplastic transformation (i.e. by leading to DNA mutation) or they are simply biomarkers of a high exposure (i.e. tobacco smoke). Further research on the relation between 4-ABP–DNA adducts and breast tumorigenesis will provide clues into the mechanisms underlying the effects of tobacco smoke and other environmental carcinogens on breast cancer development and will help to elucidate more effective breast cancer preventive measures.


    Notes
 
7 Present address: Institute of General Pathology and 'Giovanni XXIII' Cancer Research Center, Catholic University School of Medicine, Largo Francesco Vito 1, 00168 Rome, Italy Back

8 Present address: Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China 100021 Back


    Acknowledgments
 
The efforts of Drs M.Terry, M.S.Wolff, S.D.Stellman, G.Kabat, B.Levin, L.H.Bradlow, M.Hatch, J.Beyea, D.Camann, M.Trent, R.Senie, C.Maffeo, P.Montalvan, G.Berkowitz, M.Kemeny, M.Citron, F.Schnabel, A.Schuss, S.Hajdu, V.Vinceguerra and J.Britton in the parent study are gratefully acknowledged. This work was supported by NIH grants CA/ES66572 and CA77185, DAMD17-98-1-8052, ES09089, ES10126 and an award from the AVON Products Foundation Breast Cancer Research and Care Program at the Herbert Irving Comprehensive Cancer Center.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 11, 2002; revised January 10, 2003; accepted January 27, 2003.