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

Breast cancer is one of the major cancers around the world but its etiology is still not well understood. Only approximately 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.     

Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells

Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.     

White blood cell DNA adducts and fruit and vegetable consumption in bladder cancer

The 'Mediterranean diet', a diet rich in cereals, fruit and vegetables, has been associated with lowering the risk of a variety of cancers of the digestive tract and the bladder. In a previous study, we showed that the high phenolic content these dietary components produce in the urine could be associated with higher antimutagenic properties of the urine and lower arylamine-DNA adducts in exfoliated bladder cells. We have conducted a case-control study on 162 bladder cancer patients and 104 hospital controls. Total aromatic DNA adducts were measured in white blood cells (WBC) of all subjects by (32)P-post-labelling. Genetically based metabolic polymorphisms were analysed by PCR-RFLP (NAT2, GSTM1, GSTT1, GSTP1, COMT and NQO1). All subjects were interviewed about their tobacco use, dietary habits and other risk factors. The odds ratio (OR) for the risk of bladder cancer according to the presence/absence of WBC DNA adducts (detection limit 0.1 RALx10(8)) was 3.7 [95% confidence interval (CI) 2.2-6.3] and a dose-response relationship with levels of adducts was apparent. The association between case/control status and the presence of WBC DNA adducts was significantly stronger in the subjects who consumed fewer portions of fruit or vegetables per day (OR 7.80, 95% CI 3.0-20.30 for 0-1 portions of vegetables) than in the heavy consumers (OR 4.98 for consumers of 2 portions daily, OR 1.97 for consumers of > or =3 portions; similar but lower estimates were found for the intake of fruit). No association was noticed between tobacco smoking and WBC DNA adducts. Only NAT-2, among the several genotypes considered, was associated in a statistically significant way with the risk of bladder cancer (OR 1.72, 95% CI 1.03-2.87) and with the levels of WBC DNA adducts. Our report suggests that fruit and vegetables could protect against bladder cancer by inhibiting the formation of DNA adducts.     

4-aminobiphenyl is a major etiological agent of human bladder cancer: evidence from its DNA binding spectrum in human p53 gene

4-aminobiphenyl (4-ABP) is a major etiological agent of human bladder cancer, and its metabolites are able to form DNA adducts that may induce mutation and initiate bladder carcinogenesis. Thirty to sixty percent of human bladder cancer has a mutation in the p53 gene, and the mutational spectrum bears two characteristics: compared with other cancers, the pattern of mutations is more evenly distributed along the p53 gene, and the mutational hotspots occur at both CpG sites, such as codons 175, 248 and 273, and non-CpG sites, such as codons 280 and 285, the latter two being unique mutational hotspots for bladder and other urinary tract cancers. These findings raise the possibility that the special p53 mutational features in human bladder cancer are due to the unique binding spectrum of metabolically activated 4-ABP in bladder cells. To address this question, here we have mapped the 4-ABP-DNA adduct distribution in the p53 gene at the nucleotide sequence level in human bladder cells. We found that, unlike benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide-DNA adduction, which preferentially occurs at CpG sites, 4-ABP-DNA adduction is not biased for CpG sites, and the adducts are more evenly distributed along the p53 gene; nonetheless, the p53 mutational hotspots in bladder cancer at codons 175, 248, 280 and 285 are also the preferential sites for 4-ABP adduct formation. These results strongly suggest that the unique binding spectrum of 4-ABP contributes greatly to the unique mutational spectrum in the p53 gene of human bladder cancer, and provide further molecular evidence to directly link 4-ABP to bladder cancer.     

Organ specificity of the bladder carcinogen 4-aminobiphenyl in inducing DNA damage and mutation in mice

Aromatic amines are a widespread class of environmental contaminants present in various occupational settings and tobacco smoke. Exposure to aromatic amines is a major risk factor for bladder cancer development. The etiologic involvement of aromatic amines in the genesis of bladder cancer is attributable to their ability to form DNA adducts, which upon eluding repair and causing mispairing during replication, may initiate mutagenesis. We have investigated the induction of DNA adducts in relation to mutagenesis in bladder and various nontarget organs of transgenic Big Blue mice treated weekly (i.p.) with a representative aromatic amine compound, 4-aminobiphenyl (4-ABP), for six weeks, followed by a six-week recovery period. We show an organ-specificity of 4-ABP in inducing repair-resistant DNA adducts in bladder, kidney, and liver of carcinogen-treated animals, which accords with the bioactivation pathway of this chemical in the respective organs. In confirmation, we show a predominant and sustained mutagenic effect of 4-ABP in bladder, and much weaker but significant mutagenicity of 4-ABP in the kidney and liver of carcinogen-treated mice, as reflected by the elevation of background cII mutant frequency in the respective organs. The spectrum of mutations produced in bladder of 4-ABP-treated mice matches the known mutagenic properties of 4-ABP-DNA adducts, as verified by the preponderance of induced mutations occurring at G:C base pairs (82.9%), with the vast majority being G:C→T:A transversions (47.1%). Our data support a possible etiologic role of 4-ABP in bladder carcinogenesis and provide a mechanistic view on how DNA adduct-driven mutagenesis, specifically targeted to bladder urothelium, may account for organ-specific tumorigenicity of this chemical.     

DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.     

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.     

Immunohistochemical quantitation of 4-aminobiphenyl-DNA adducts and p53 nuclear overexpression in T1 bladder cancer of smokers and nonsmokers

An immunoperoxidase method, using a monoclonal antibody whichrecognizes 4-aminobiphenyl (4-ABP)-DNA adducts, was developedfor the detection and quantitation of DNA damage in bladdertissue and applied to stored paraffin blocks of transurethralresection specimens of 46 patients with T1 bladder cancer. Meanrelative staining intensity for 4-ABP-DNA adducts was significantlyhigher in current smokers (275 ± 81, n = 24) comparedto nonsmokers (113 ± 71, n = 22) (P < 0.0001). Therewas a linear relationship between mean levels of relative stainingand number of cigarettes smoked with lower levels in the 1–19cigiday group (205 ± 30, n = 5), compared to the 20–40(289 ± 40, n = 7) and the >40 cig/day group (351 ±57, n = 3)(P < 0.001). Nuclear overexpression of p53, analyzedby immunoperoxidase staining, was observed in 27 (59%) of the45 stage T1 tumors analyzed. There was a significant correlationbetween p53 overexpression and recurrence of disease (odds ratio= 12.3, P < 0.01). Nuclear staining of p53 was also correlatedwith smoking status, cig/day and 4-ABP-DNA adducts. This workdemonstrates that the immunohistochemical method has sufficientsensitivity for detection of 4-ABP-DNA adducts in human bladdersamples. The method has several advantages including small samplesize, the possibility of retrospective analysis of stored paraffinblocks, the ability to analyze binding in specific cell types,and a relatively low cost.     

Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling.

Tamoxifen (TAM), a nonsteroidal antiestrogen used as a chemotherapeutic and chemopreventive agent for breast cancer, induces liver tumors in rodents and covalent DNA adduct formation in hepatic DNA. Here, we report the development and validation of highly sensitive and specific immunoassays for the determination of TAM-DNA adducts. Rabbits were immunized with calf thymus DNA, chemically modified with alpha-acetoxytamoxifen to 2.4 adducts per 100 nucleotides, and the resulting antisera were characterized by competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemiluminescence immunoassay (CIA). Compared with DELFIA, the CIA has a much lower background and a 20-fold increase in sensitivity. For the immunogen TAM-DNA, 50% inhibition was at 2.0 +/- 0.11 (mean +/- SE, n = 18) fmol of (E)-alpha-(N2-deoxyguanosinyl)tamoxifen (TAM-dG) adduct in TAM-DNA by DELFIA. For TAM-DNA modified to 4.8 adducts in 10(6) nucleotides, 50% inhibition was at 20.6 +/- 6.6 (mean +/- SE, n = 8) fmol of TAM-dG in TAM-DNA by DELFIA and at 0.92 +/- 0.11 (mean +/- SE, n = 10) fmol of TAM-dG in TAM-DNA by CIA. No inhibition was observed in either assay with up to 20 microg (62.5 nmol of nucleotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-alpha-(N2-deoxyguanosinyl)tamoxifen and the individual compounds TAM and 4-OH-TAM gave DELFIA 50% inhibitions at 828, 2229, 5440, and 8250 fmol, respectively. For assay validation, TAM-dG levels were determined by DELFIA, CIA, and 32P-postlabeling in TAM-DNA samples modified in vitro to different levels, and comparable values were obtained in all three assays. Further validation was obtained in vivo in rat liver. DNA adducts of TAM were measurable in rat liver 24 h after a single i.p. dose of 45 mg TAM/kg body weight and after daily p.o. dosing for 7 days with 5.0, 10.0, and 20.0 mg TAM/kg body weight. In addition, TAM-DNA adducts disappeared slowly over 21 days in rats on a control diet that were first given p.o. TAM at 45 mg/kg/day for 4 days. In the rat experiments, TAM-DNA adduct levels determined by CIA compared well with those determined by 32P-postlabeling, although the CIA gave an underestimation at the highest doses. For rat liver samples, the detection limit by CIA was 3 adducts per 10(9) nucleotides (0.2 fmol of adducts per 20 microg of DNA).     

DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 4-aminobiphenyl are infrequently detected in human mammary tissue by liquid chromatography/tandem mass spectrometry

Some epidemiological investigations have revealed that frequent consumption of well-done cooked meats and tobacco smoking are risk factors for breast cancer in women. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed in well-done cooked meat, and 4-aminobiphenyl (4-ABP) is an aromatic amine that arises in tobacco smoke and occurs as a contaminant in the atmosphere. Both compounds are rodent mammary carcinogens, and putative DNA adducts of PhIP and 4-ABP have been frequently detected, by immunohistochemistry (IHC) or (32)P-post-labeling methods, in mammary tissue of USA women. Because of these findings, PhIP and 4-ABP have been implicated as causal agents of human breast cancer. However, the biomarker data are controversial: both IHC and (32)P-post-labeling are non-selective screening methods and fail to provide confirmatory spectral data. Consequently, the identities of the lesions are equivocal. We employed a specific and sensitive liquid chromatography/mass spectrometry (MS) method, to screen tumor-adjacent normal mammary tissue for DNA adducts of PhIP and 4-ABP. Only 1 of 70 biopsy samples obtained from Minneapolis, Minnesota breast cancer patients contained a PhIP-DNA adduct. The level was three adducts per 10(9) nucleotides, a level that is 100-fold lower than the mean level of PhIP adducts reported by IHC or (32)P-post-labeling methods. The occurrence of 4-ABP-DNA adducts was nil in those same breast tissues. Our findings, derived from a specific mass spectrometry method, signify that PhIP and 4-ABP are not major DNA-damaging agents in mammary tissue of USA women and raise questions about the roles of these chemicals in breast cancer.     

DNA adduct formation and tumorigenesis in mice during the chronic administration of 4-aminobiphenyl at multiple dose levels

Recent studies have demonstrated the presence of DNA adductsfrom 4-aminobiphenyl (4-ABP) in the bladder cells of humans;however, the correlation between the concentration of theseadducts and the tumorigenic response is not clear. To help elucidatethis relationship, we have investigated DNA adduct formationin experimental animals continuously administered 4-ABP. Maleand female BALB/c mice were treated for 28 days with 4-ABP hydro-chloridein their drinking water. DNA adducts in target tissues (liverof females and bladder of males) were identified and quantifiedby ³²P-postlabeling analyses and radio-immunoassays. These resultswere compared to previously reported tumor incidences obtainedfrom the lifetime administration of 4-ABP hydrochloride. Themajor adduct observed in both tissues was N-(deoxyguanosin-8-yl)-4-ABP.In the bladders of both sexes and the livers of female mice,adduct levels increased with dose at low doses, but saturationwas observed at high doses. In the livers of males, the adductlevels were linearly correlated with dose throughout the entiredose range. A comparison between DNA adducts and tumorigenesisindicated a linear correlation between adduct levels and theincidence of liver tumors in female mice. In the bladders ofmale mice, however, the relationship was markedly nonlinear.These data suggest that adduct formation alone is insufficientfor tumorigenesis in the bladder and that other factors suchas cell proliferation are necessary for tumor production.     

Environmental tobacco smoke and bladder cancer risk in never smokers of Los Angeles County

Cigarette smoking is a major risk factor for bladder cancer and a prominent point source of 4-aminobiphenyl (4-ABP), a recognized human bladder carcinogen. 4-ABP-hemoglobin (Hb) adducts are established biomarkers of 4-ABP exposure in humans. The role of environmental tobacco smoke (ETS) in the etiology of bladder cancer is largely unknown. As part of a large population-based bladder cancer study in Los Angeles County, California, lifetime exposure to ETS was ascertained for 148 cases and 292 control subjects who had never used any tobacco products over their lifetime. 4-ABP-Hb adducts were quantitatively measured on 230 control subjects. Female lifelong nonsmokers living with two or more smokers during childhood were significantly related to risk of bladder cancer [odds ratio (OR), 3.08; 95% confidence interval (95% CI), 1.16-8.22]. During adulthood, approximately 2-fold risks were seen among women living with a spouse/domestic partner who smoked for > or =10 years or having a coworker who smoked in an indoor environment for > or =10 years. When all sources of ETS exposure were combined, a statistically significant, dose-dependent association (P for trend = 0.03) was noted in women, with the OR for the highest category of ETS exposure being 5.48 (95% CI, 1.06-28.36). Levels of 4-ABP-Hb adducts varied by ETS exposure status among female control subjects. Mean level was lowest in women never exposed to ETS (16.4 pg/g Hb) and highest in those with current ETS exposure (23.6 pg/g Hb). ETS exposure was associated with neither bladder cancer risk nor 4-ABP-Hb adduct levels in male lifelong nonsmokers. In conclusion, ETS is a risk factor for bladder cancer in women who were lifelong nonusers of any tobacco products.     

4-aminobiphenyl-DNA adducts and p53 mutations in bladder cancer

Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms. Int. J. Cancer 75:512–516, 1998.© 1998 Wiley-Liss, Inc.     

4-Aminobiphenyl induces liver DNA adducts in both neonatal and adult mice but induces liver mutations only in neonatal mice

The mechanisms underlying the susceptibility of neonatal mice to genotoxic carcinogens were investigated by analyzing the DNA adducts and mutations induced in the livers of neonatal and adult Big Blue transgenic mice by 4-aminobiphenyl (4-ABP), a potent human and rodent carcinogen. Neonatal and adult mice were treated with a regimen of 4-ABP known to induce tumors in neonatal mice. Animals were sacrificed 1 day after the last treatment for DNA adduct analysis and 8 weeks after the last treatment for analysis of lacI and cII mutant frequency (MF). N-(Deoxyguanosin-8-yl)-4-ABP was the major DNA adduct identified in the livers of the 4-ABP-treated mice and levels of this adduct were significantly higher in treated animals than in the controls for both the neonates and adults. Adduct levels for adult females (44.0 +/- 4.8 adducts/10(6) nucleotides) were higher than in neonatal females (25.9 +/- 2.2 adducts/10(6) nucleotides), while adduct levels in adult males (13.5 +/- 2.0 adducts/10(6) nucleotides) were lower than in neonatal males (33.8 +/- 4.1 adducts/10(6) nucleotides). 4-ABP treatment significantly increased the liver cII MFs in both sexes of neonatal mice but not in adult mice. Sequence analysis of cII mutant DNA revealed that 4-ABP induced a unique spectrum of mutations in neonatal mice, characterized by a high frequency of G:C-->T:A transversion, while the mutation spectrum in 4-ABP-treated adults was similar to that of control mice. Our results indicate that DNA adduct formation by 4-ABP depends as much on sex as it does on age, whereas the conversion of DNA adducts into mutations differed with animal age. These observations suggest that neonates are more sensitive than adults to genotoxic carcinogens because the relatively high levels of cell division in the developing animal facilitate the conversion of DNA damage into mutation. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html     

Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl

To assess target-tissue exposure to the human urinary bladder carcinogen 4-aminobiphenyl (ABP), we have developed a sensitive immunochemical method for measuring the major arylamine-DNA adduct formed, N-(guan-8-yl)-ABP (Gua-C8-ABP). High-affinity polyclonal antisera from rabbits immunized with N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin were characterized and shown to have high specificity for antigenic determinants on the purine and biphenyl rings of Gua-C8-ABP and minimal cross-reactivity with ABP, deoxyguanosine, or hydrolyzed DNA. Assay standards containing ABP-modified DNA were prepared by reacting [3H]N-hydroxy-ABP with calf thymus DNA. DNA samples were hydrolyzed with trifluoroacetic acid and dried under vacuum, and the residues were dissolved in dimethyl sulfoxide under argon. Using a streptavidin-biotin amplified enzyme-linked immunosorbent assay, DNA hydrolysates competed at 25 micrograms DNA/microtiter well for a limiting amount of anti-keyhole limpet hemocyanin-(Gua-C8-ABP) in the presence of excess solid-phase bovine serum albumin-(Gua-C8-ABP) coating antigen. The limit of sensitivity for this assay using 25 micrograms DNA was 2 adducts/10(8) nucleotides. Gua-C8-ABP adducts in liver and bladder epithelial DNAs were readily quantified after p.o. administration of 5 mg/kg ABP to dogs. This methodology is capable of detecting adducts at levels of biological significance and should be applicable to human target-tissue dosimetry.     

Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS.

Electrospray mass spectrometry (ES-MS) is a powerful tool for analysis of carcinogen-adducted DNA. In this study, we developed a quantitative isotope dilution method for analysis of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP), the principal nucleoside adduct derived from enzymatic hydrolysis of 4-aminobiphenyl (4-ABP)-modified DNA. The method used column switching valves to perform on-line sample concentration and cleanup, which permitted direct analysis of enzymatic DNA hydrolysates using narrow-bore liquid chromatography (LC). ES-MS detection was performed using a single quadrupole instrument by monitoring M+H+ and two fragment ions characteristic for dG-C8-4-ABP, along with M+H+ and a fragment ion for the deuterated internal standard. The detection limit for dG-C8-4-ABP in DNA hydrolysates was approximately 10 pg on-column, equivalent to 0.7 dG-C8-4-ABP adducts in 10(7) normal nucleotides for a sample containing 100 microg DNA. The method was applied to the analysis of calf thymus DNA modified in vitro through reaction with N-hydroxy-4-ABP and of hepatic DNA isolated from mice treated in vivo with two dose levels of 4-ABP.     

4-Aminobiphenyl-DNA adducts in laryngeal tissue and smoking habits: an immunohistochemical study

4-Aminobiphenyl (4-ABP)-DNA adducts and p53 overexpression were evaluated in laryngeal biopsies from 38 patients by immunohistochemical methods. Samples were categorized as tumors (n = 9), polyps (n = 28) or normal tissue (n = 1). 4-ABP-DNA adducts were evaluated with a quantitative immunoperoxidase method using monoclonal antibody 3C8 in both the lesion and adjacent tissue. Relative staining intensity data showed a log-normal distribution and values found in adjacent tissue from smokers were significantly higher (median: 173.5, geometric mean: 159.9) than those measured in adjacent tissue from non-smokers (median: 75.5, geometric mean: 7.40). Statistical significance was assessed both by non-parametric testing on raw data (P = 0.0007 on rank sum test) and by parametric testing on log-transformed data (P = 0.0002 on an unpaired t-test). Furthermore, relative staining intensity in the lesional tissue showed the same significant difference between smokers and non-smokers in patients affected by polyps, whereas no significant difference was detected in patients with laryngeal tumors. Overexpression of p53, also measured with an immunoperoxidase method, was observed in 44% of the malignant tumors and in 3.5% of the polyps. This work demonstrates that 4-ABP-DNA adducts can be evaluated in laryngeal tissue and are related to smoking exposure.      

Tamoxifen DNA damage detected in human endometrium using accelerator mass spectrometry

This study was aimed to establish whether tamoxifen binds irreversibly to uterine DNA when given to women. Patients were given a single therapeutic dose of [(14)C]tamoxifen citrate orally (20 mg, 0.37 or 1.85 MBq) approximately 18 h prior to hysterectomy or breast surgery. Nonmalignant uterine tissue was separated into myometrium and endometrium. DNA and protein were isolated and bound radiolabel determined by the sensitive technique of accelerator mass spectrometry. Levels of irreversible DNA binding of tamoxifen in the endometrium of treated patients were 237 +/- 77 adducts/10(12) nucleotides (mean +/- SE, n = 10). In myometrial tissues, a similar extent of DNA binding was detected (492 +/- 112 adducts/10(12) nucleotides). Binding of tamoxifen to endometrial and myometrial proteins was 10 +/- 3 and 20 +/- 4 fmol/mg, respectively. In breast tissue, sufficient DNA could not be extracted but protein binding was an order of magnitude higher than that seen with endometrial proteins (358 +/- 81 fmol/mg). These results demonstrate that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers relative to those previously reported in women after long-term tamoxifen treatment where levels, when detected, ranged from 15000 to 130000 adducts/10(12) nucleotides. Our findings support the hypothesis that the low level of DNA adducts in human uterus is unlikely to be involved with endometrial cancer development.     

Smoking is associated with a decrease of O6-alkylguanine-DNA alkyltransferase activity in bronchial epithelial cells

O6-alkylguanine-DNA alkyltransferase (MGMT) represents the first line of defense against the toxic, mutagenic and carcinogenic effects of O6-alkylguanine adducts in DNA. These adducts mediate the biological activity from a series of alkylating agents, such as the tobacco-specific nitrosamines, believed to contribute to the carcinogenicity of tobacco smoke. There have been conflicting reports on the effects of smoking on MGMT activity in lung and other tissues. Here, we investigate MGMT activity in peripheral blood mononuclear cells (PBMC) and lung bronchial epithelial cells (BEC), extracted by lung brushings, from smokers and nonsmokers attending a bronchoscopy clinic. MGMT activity was significantly lower in BECs (geometric mean; 95% confidence interval 1.02; 0.86-1.20 fmol/microg DNA) than in PBMCs (7.86; 6.70-9.59 fmol/microg DNA; p < 0.001), suggesting that bronchial epithelia may be particularly sensitive to alkylation damage. More importantly our results indicate that activity in BECs is significantly decreased in samples from current smokers (0.71; 0.54-0.93 fmol/microg DNA) compared to nonsmokers (1.25; 1.03-1.51 fmol/microg DNA; p = 0.002). This could represent an important contribution to the carcinogenicity of tobacco smoke.     

Levels of the adducts of 4-aminobiphenyl to hemoglobin in control subjects and bladder carcinoma patients

We determined the hemoglobin adduct levels of one aromatic amine of cigarette smoke, 4-aminobiphenyl (4-ABP), in smoking controls and in patients with transitional cell bladder carcinoma. Covalently bound 4-ABP was measured by capillary gas-chromatography and negative-ion chemical ionization mass-spectrometry, using deuterated 4-ABP as internal standard. Smoking was quantified measuringthe urinary excretion of cotinine. Thirteen cases and controls were paired for urinary continine levels. Bladder carcinoma patients had slightly higher levels of 4-ABP hemoglobin adducts than controls (means ± S.D. were 103 ± 47 and 65 ± 44, respectively). This difference was significant using a t-test for paired samples (P = 0.04) and nonparametric Kruskal-Wallis rank analysis (P = 0.033).