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.     

Levels of 4-aminobiphenyl-induced somatic H-ras mutation in mouse liver DNA correlate with potential for liver tumor development

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation.     

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.     

Determinants of 4-aminobiphenyl-DNA adducts in bladder cancer biopsies

Exposure to 4-aminobiphenyl (4-ABP) is an important determinant of urinary bladder cancer in humans. We have analyzed by gas chromatography-mass spectrometry the DNA adducts of 4-ABP in 75 bladder cancer biopsies. The purpose was to understand whether smoking, N-acetyltransferase 2 (NAT2) polymorphism, diet or tumor grade were determinants of 4-ABP-DNA levels. 4-ABP-DNA adducts were above the detection limit of 0.1 fmol/microg DNA for 37/75 patients. Overall the level of adducts was 2.7 +/- 0.7 (mean +/- SE) fmol/microg DNA (86 +/- 22 adducts/10(8) normal nucleotides, mean +/- SE). A strong association with grade was observed. In the group of patients with detectable 4-ABP-DNA adducts the odds ratio for having a tumor grade of 2 or 3 was respectively 4.3 (95% CI 0.8-21.9) and 6 (1.3-27.5), compared with grade 1. A non-statistically significant association was found between adduct levels and the deduced slow acetylator phenotype in grades 2 and 3. The intake of fruit and vegetables produced a lower frequency of detectable adducts, though the association was not statistically significant. Detectable 4-ABP-DNA adducts were clearly associated with current smoking in higher tumor grades (grade 3 versus grades 1 + 2, odds ratios 10.4; 95% CI 1.7-63.1). Overall, our findings indicate that higher levels of DNA adducts characterize more invasive tumors (higher tumor grades). This seems to be facilitated by smoking and contrasted by the intake of fruit and vegetables.     

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.     

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.     

Differences in the covalent binding of benzo[a]pyrene, safrole, 1'-hydroxysafrole, and 4-aminobiphenyl to DNA of pregnant and non-pregnant mice

The effects of pregnancy on the covalent binding of several carcinogens to DNA were investigated in mice. Non-pregnant or timed-pregnant (18th day of gestation) ICR mice of similar age were treated with benzo[a]pyrene (BP, 200 mumol/kg), safrole (600 mumol/kg), 1'-hydroxysafrole (400 mumol/kg), 4-aminobiphenyl (4-ABP, 800 mumol/kg), or trioctanoin (4 ml/kg) per os. Tissue DNA adduct levels at 24 h after carcinogen treatment were analyzed via a 32P-postlabeling assay. Pregnancy lowered the binding of the ultimate carcinogenic metabolite of BP, 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I), to liver and lung DNA by 29-41%, but not the binding of other metabolites. The binding of safrole and its proximate carcinogen, 1'-hydroxysafrole, to liver and kidney DNA was increased 2.3-3.5 fold. Pregnancy decreased the binding of 4-ABP to liver DNA by approximately 18% but increased its binding to kidney DNA by 67%. The results suggest that exposure to some genotoxic compounds, especially those requiring conjugation reactions for metabolic activation, may be more hazardous during pregnancy than in the non-pregnant state.     

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.     

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.     

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.     

Influence of cigarette smoking on the levels of DNA adducts in human bronchial epithelium and white blood cells

The presence of carcinogen-DNA adducts in human tissues is evidence of exposure to carcinogens and may be an indicator of cancer risk. DNA was isolated from non-tumorous bronchial tissue of 37 cigarette smokers, 8 former smokers and 8 non-smokers and analyzed for the presence of aromatic and/or hydrophobic DNA adducts in the 32P-post-labelling assay. Adducts were detected as bands of radioactive material when 5'-32P-labelled deoxyribonucleoside 3',5'-bisphosphates were chromatographed on polyethyleneimine-cellulose tlc plates, and the patterns indicated the formation of adducts by a large number of compounds. Adduct levels detected in DNA from non-smokers, former smokers and current smokers were 3.45 +/- 1.62, 3.93 +/- 1.92 and 5.53 +/- 2.13 adducts/10(8) nucleotides, respectively. The differences in adduct levels between smokers and former and non-smokers were statistically significant (p less than 0.01); and among the smokers, significant correlations were found between adduct levels and both daily cigarette consumption and total cigarette consumption (daily consumption X number of years smoked). DNA was also isolated from the peripheral-blood leukocytes of 31 heavy smokers (greater than 20 cigarettes/day) and 20 non-smokers and analyzed by 32P-post-labelling. Adduct levels in the smokers' samples were not significantly different from levels in the non-smokers' samples (2.53 +/- 1.31 and 2.12 +/- 1.44 adducts/10(8) nucleotides, respectively). Thus, evidence for carcinogen exposure was found in human bronchial epithelium, a target tissue for tobacco-induced tumour formation, but not in peripheral-blood cells, indicating possible limitations in the use of the latter as a surrogate, non-target tissue source of DNA for monitoring human exposure to inhaled carcinogens.     

The genotoxic air pollutant 3-nitrobenzanthrone and its reactive metabolite N-hydroxy-3-aminobenzanthrone lack initiating and complete carcinogenic activity in NMRI mouse skin

3-Nitrobenzanthrone (3-NBA), a genotoxic mutagen found in diesel exhaust and ambient air pollution and its active metabolite N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) were tested for initiating and complete carcinogenic activity in the NMRI mouse skin carcinogenesis model. Both compounds were found to be inactive as either tumour initiators or complete carcinogens in mouse skin over a dose range of 25–400 nmol. Topical application of 3-NBA and N-OH-3-ABA produced DNA adduct patterns in epidermis, detected by ³²P-postlabelling, similar to those found previously in other organs of rats and mice. 24 h after a single treatment of 100 nmol DNA adduct levels produced by 3-NBA (18 ± 4 adducts/10⁸ nucleotides) were 6 times lower than those by 7,12-dimethylbenz[a]anthracene (DMBA; 114 ± 37 adducts/10⁸ nucleotides). In contrast, identical treatment with N-OH-3-ABA resulted in adduct levels in the same range as with DMBA (136 ± 25 adducts/10⁸ nucleotides), indicating that initial DNA adduct levels do not parallel tumour initiating activity. When compounds were tested for tumour initiating activity by a single treatment followed by twice-weekly applications of TPA, DNA adducts formed by DMBA, but not by 3-NBA or N-OH-3-ABA, were still detectable 40 weeks after treatment. When tested for activity as complete carcinogens by twice-weekly topical application, 3-NBA and N-OH-3-ABA produced identical DNA adduct profiles in mouse skin, with adducts still detectable after 40 weeks. Only 3-NBA produced detectable adducts in other organs.     

Applicability of induced sputum for molecular dosimetry of exposure to inhalatory carcinogens: 32P-postlabeling of lipophilic DNA adducts in smokers and nonsmokers.

The lung is a major target organ for smoking-associated cancer. We examined the applicability of induced sputum for molecular dosimetry of exposure to tobacco smoke-related carcinogens. Sputum induction was performed by inhalation of 4.5% saline delivered from an ultrasonic nebulizer for a period of up to 21 min in a group of smoking (n = 20) and nonsmoking (n = 24) healthy individuals. Samples were analyzed for total and differential cell counts and cell viability. Subsequently, DNA contents of the samples were isolated, and measurement of lipophilic DNA adducts was done by the 32P-postlabeling assay using nuclease P1 (NP1) and butanol enrichment methods. All subjects tolerated the induction procedure without experiencing any troublesome symptoms, and 90% of smokers (18 of 20) and 88% of nonsmokers (21 of 24) succeeded in producing sufficient amounts of sputum. Total cell counts and percentages of viable cells in smokers were higher than those in nonsmokers (6.7+/-6.0 versus 4.7+/-6.0 x 10(6), P = 0.40 and 80+/-15 versus 63+/-17, P = 0.01, respectively). In cell differentials, smokers had lower percentages of bronchoalveolar macrophages and higher percentages of neutrophils (69+/-24 versus 92+/-5, P = 0.002 and 26+/-26 versus 4+/-4, P = 0.008, respectively). Using the NP1 digestion method, all smokers and only one nonsmoker showed a diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (3.1+/-1.4 versus 0.6+/-0.8/10(8) nucleotides; P = 0.0007), and also, adduct levels were significantly related to smoking indices. Applying the butanol extraction method, however, only half of the smokers and three nonsmokers showed the diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (4.6+/-3.7 versus 1.0+/-1.9/10(8) nucleotides; P = 0.02), and the levels of adducts were significantly related to the smoking indices. There was a correlation between the levels of adducts determined by the two enrichment methods (r = 0.7; P = 0.02). Paired comparison showed no differences between the levels of adducts measured by the two methods (P = 0.55). We conclude that induced sputum can serve for molecular dosimetry of inhalatory exposure to carcinogens and that the NP1 version of the 32P-postlabeling assay is a choice of preference for studying smoking-induced DNA adducts in the lower respiratory tract.     

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).     

Differential induction of N(2),3-ethenoguanine in rat brain and liver after exposure to vinyl chloride

Although vinyl chloride (VC) clearly induces hepatic angiosarcoma in humans and rodents, a causal association with brain tumors has not been definitively established with the available epidemiological and experimental evidence. Because VC acts by genotoxic mechanisms, DNA adduct formation is thought to be a sensitive biomarker of early events in carcinogenesis. Adult male Sprague Dawley rats were exposed to 0 or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week) by inhalation. Male weanlings were similarly exposed for 5 days. Another group of male adults was exposed to 1100 ppm [(13)C(2)]VC in a nose-only inhalation apparatus for 5 days (6 h/day). A sensitive gas chromatography high-resolution mass spectrometry assay was used to measure the major promutagenic DNA adduct, N(2),3-ethenoguanine (N(2),3-epsilonG), in rat brain and hepatocyte (HEP) DNA. The respective concentrations of N(2),3-epsilonG in control rat brain DNA at 1 and 4 weeks were 5.0 +/- 0.9 and 5.6 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. There was no change in N(2),3-epsilonG in adult rat brain after exposure to 1100 ppm VC for 1 or 4 weeks. In HEPs from the same animals, these adduct concentrations increased from 5.5 +/- 1.4 to 55 +/- 2.0 N(2),3-epsilonG/10(8) unmodified guanine after a 1-week exposure and from 3.0 +/- 0.3 to 110 +/- 20 N(2),3-epsilonG/10(8) unmodified guanine after a 4-week exposure. When weanlings were exposed to 1100 ppm VC for 5 days, there was a statistically significant (P = 0.04) increase in N(2),3-epsilonG in brain from 1.5 +/- 0.2 to 4.4 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. Weanlings exposed to 1100 ppm VC had an even greater increase in N(2),3-epsilonG in HEPs from 1.6 +/- 0.1 to 97 +/- 5.0 N(2),3-epsilonG/10(8) unmodified guanine. [(13)C(2)]N(2),3-epsilonG was not detected in brain DNA from adult rats exposed to 1100 ppm [(13)C(2)]VC for 5 days but was present in HEP DNA at 55 +/- 4.0 [(13)C(2)]N(2),3-epsilonG/10(8) unmodified guanine. The concentrations of the endogenous adduct in both organs were unchanged after this exposure. 7-(Oxoethyl)guanine (OEG), the major DNA adduct formed by VC, was reduced to 7-(2-hydroxyethyl)guanine and measured by liquid chromatography-electrospray ionization-tandom mass spectrometry in brain and HEP DNA from rats exposed to 1100 ppm VC for 1 week. Whereas 4.0 +/- 0.8 OEG/10(6) unmodified guanine were present in HEP DNA from VC-exposed rats, no adducts were detectable in brain DNA (detection limit, 0.3 OEG/10(6) unmodified guanine). These findings indicate that the genotoxic metabolite of VC is not formed in or transported to adult rat brain. Thus, it is unlikely that N(2),3-epsilonG or other VC-induced promutagenic DNA adducts play a significant role in initiating carcinogenesis in adult rat brain after exposure to VC. The data for weanling rats are less clear. Whereas a small increase in N(2),3-epsilonG in the brains of weanlings was found after exposure to 1100 ppm VC, the resulting adduct concentration was similar to that measured in unexposed adults. Future exposures of weanling rats to the stable isotopically labeled compound will be necessary to conclusively determine whether this increase was due to VC.     

Polycyclic aromatic hydrocarbon--DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung.

Smokers of cigarettes are exposed to a number of carcinogens, including polycyclic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE)-DNA and the 32P-post-labeling technique. The ELISA results showed BPDE-DNA antigenicity in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to greater than 150 adducts in 10(8) nucleotides. The autoradiographs of chromatograms of 32P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 10(8) nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in 10(8) nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and 32P-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.     

Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers

A quantitative method has been developed for the analysis of 4-aminobiphenyl (4-ABP) covalently bound as the sulfinic acid amide to the 93 beta cysteine of human hemoglobin. The method uses mild basic hydrolysis of hemoglobin to release the parent amine, derivatization to form the pentafluoropropionamide, and capillary gas chromatography with detection by negative-ion chemical ionization mass spectrometry. The method is precise and gives reproducible results on multiple blood samples taken from individuals over 48 h. Application of this method to blood samples from cigarette smokers and nonsmokers revealed consistently higher adduct levels in smokers. The mean value for smokers was 154 pg 4-ABP per g Hb compared to 28 pg/g Hb for nonsmokers, with no overlap of adduct levels between the two groups. Studies on quitting smokers revealed that adduct levels declined over a period of 6-8 weeks to nonsmoker levels. The finding of 4-ABP adducts in all nonsmokers was not anticipated but is consistent with low-level ubiquitous contamination of air, food, or water. In other animals sampled, rats and dogs had measurable adduct levels, but monkeys and fish did not. The hemoglobin adduct of 4-ABP is the product of a series of reactions between the hemoprotein and N-hydroxy-4-ABP. The formation of hydroxylamines from carcinogenic aromatic amines and their subsequent reactions with DNA are generally thought to be critical events in the initiation of bladder tumors. We suggest that the observed hemoglobin adduct levels formed by this proximate carcinogen will reflect the extent to which these steps have occurred. This is the first report of 4-ABP adducts in human blood.     

Mucosa-specific DNA adducts in human small intestine: a comparison with the colon

The presence of several covalent DNA adducts in the human colonwas demonstrated by ³²p-postlabeling in a previous study. Wedemonstrated that DNA of all the colonlc mucosa tested wereselectively adducted by a single genotoxic agent and this modificationwas completely absent in the DNA of muscular layers. In thisstudy, the DNA adducts of the small intestine are compared withthose of the colon to understand the role of mucosa-specificDNA adduct (MSA) in intestinal carcinogenesis. The mucosal DNAof the small Intestine from 19 adults undergoing surgery dueto gastric carcinoma (seven cases), pancreatic carcinoma (fourcases), colon carcinoma (four cases), small intestinal tumor(two cases), intestinal trauma (one case) and ectopic pancreas(one case) were analyzed. The mucosa adjacent muscular layerDNA of the corresponding samples was examined as a control.Several common DNA adducts were observed in both mucosal andmuscular layers of all the adults. Besides these common backgroundDNA adducts, two MSAs (Si1, Si2) were detected in most of theadults as in colon cases. Si1 was present in all adults examined(19/19 cases) at a level of 0.04–0.22 adducts/10⁸ nucleotides(average 0.09) and Si2 was found in 13/19 patients at a levelof 0.03–0.08 adducts/10 nucleotides (average 0.05). Si2was the same adduct detected in the adult colonic mucosa. However,they were absent in the adjacent muscular layer as well as inthe neonatal intestine tested as a control. The total levelof the mucosa-specific DNA adducts of the small Intestine was     

Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis

The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sulfation-dependent formation of N-acetylated and deacetylated DNA adducts of N-hydroxy-4-acetylaminobiphenyl in male rat liver in vivo and in isolated hepatocytes.

Administration of 3H-labeled N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to male Wistar rats with or without prior partial hepatectomy (PH) resulted in covalent binding of 3H activity to liver macromolecules. Pretreatment with the sulfotransferase inhibitor pentachlorophenol (PCP) 45 min before administration of the arylhydroxamic acid strongly decreased the covalent binding. Analysis of aminobiphenyl adducts after TFA hydrolysis of DNA and RNA showed that PCP decreased the formation of both the N-acetylated adduct N-[deoxy)guanosin-8-yl)-4-acetylaminobiphenyl [(d)G-C8-AABP] and the deacetylated adduct N-[deoxy)-guanosin-8-yl)-4-aminobiphenyl [(d)G-C8-ABP] by 60-80%. In incubations with hepatocytes from male Wistar or Sprague-Dawley rats, omission of inorganic sulfate also strongly decreased the covalent binding of 3H-labeled N-OH-AABP to RNA and protein. Analysis of RNA adducts showed a 70-80% decrease in the formation of G-C8-ABP in the absence of sulfate. Another, as yet unidentified, adduct was only slightly decreased. Similar results were obtained with the structurally related carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP). Pretreatment with PCP decreased the incidence of gamma-glutamyltranspeptidase-positive foci in the liver of male rats when analyzed 30 days after a single injection of N-OH-AABP or N-OH-FAABP by 60 and 80% respectively. Thus, both N-acetylated and deacetylated RNA and DNA adducts of N-OH-AABP in rat liver are formed by sulfation and this metabolic activation pathway is responsible for the formation of genotoxic metabolites involved in the generation of preneoplastic cells.