phosphorus-radioisotopes and 4-biphenylamine

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-ho

Topics: 4-Nitroquinoline-1-oxide; Aminobiphenyl Compounds; Animals; Animals, Newborn; Benzo(a)pyrene; Carcinogens; Diethylstilbestrol; DNA; Female; Maternal-Fetal Exchange; Nicotiana; Phosphorus Radioisotopes; Plants, Toxic; Pregnancy; Safrole; Smoke

DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.

Topics: Aminobiphenyl Compounds; Azo Compounds; Biphenyl Compounds; Carcinogens, Environmental; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Deoxyribonucleotides; DNA; DNA Adducts; Humans; Magnetic Resonance Spectroscopy; Phosphorus Radioisotopes; Urinary Bladder Neoplasms; Urothelium

32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P-postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N-Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 +/- 0.8 adducts/10(8) nucleotides (mean +/- SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 +/- 1.7 dG-C8-4-ABP/10(8) nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG-C8-4-ABP/10(8) nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 +/- 26 and 63 +/- 20 dG-C8-4-ABP/10(8) nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.

Topics: Aminobiphenyl Compounds; Animals; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA; DNA Adducts; Hydrolysis; Liver; Male; Mass Spectrometry; Mice; Mice, Inbred Strains; Phosphorus Radioisotopes

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the human urothelium. Since preliminary studies indicated that even higher levels of ABP-DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by 32P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N-(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP-DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N-oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the p

Topics: Acyltransferases; Aminobiphenyl Compounds; Benzidines; Benzo(a)pyrene; Biotransformation; Biphenyl Compounds; Carcinogens; Cytosol; DNA Adducts; Enzyme-Linked Immunosorbent Assay; Gas Chromatography-Mass Spectrometry; Guanosine; Humans; Liver; Lung; Microsomes; Oxidation-Reduction; Peroxidase; Peroxidases; Phosphorus Radioisotopes; Smoking; Sulfotransferases

To characterize the DNA adducts in human uroepithelial cells (HUC) exposed to 4-aminobiphenyl and its proximate N-hydroxy metabolites, we used 32P-postlabeling analyses following butanol extraction of the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3',5'-bisphospho-8-yl)-4-aminobiphenyl (pdGp-ABP) as a major adduct and N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-aminobiphenyl (pdAp-ABP) as a minor adduct in an immortalized non-tumorigenic cell line of HUC following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeled the synthetic N-(deoxyadenosin-3'-phospho-8-yl)-4-aminobiphenyl (dAp-ABP) adduct to generate pdAp-ABP and determined its chromatographic (TLC and HPLC) properties and sensitivity to nuclease P1 digestion. In contrast to pdGp-ABP, which was cleaved to the corresponding 5'-monophosphate by nuclease P1, the pdAp-ABP adduct was unaffected when incubated with nuclease P1 under similar conditions. To test whether nuclease P1 digestion could be adopted for enrichment of the dAp-ABP adduct in HUC samples, postlabeling analyses were carried out after butanol extraction following nuclease P1 digestion of the DNA hydrolysate. Under these conditions, the pdAp-ABP adduct was detected in DNA from HUC E7 cells treated with N-OH-ABP and in calf thymus DNA reacted with N-OH-ABP under acidic (pH 5.0) conditions. These data indicate that pdGp-ABP and pdAp-ABP adducts are generated in HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichment may provide a method for qualitative and quantitative analyses of the pdAp-ABP adduct in DNA.

Topics: Aminobiphenyl Compounds; Carcinogens; Cells, Cultured; Chromatography, Thin Layer; Deoxyadenosines; DNA; DNA Adducts; Epithelial Cells; Epithelium; Humans; Isotope Labeling; Phosphorus Radioisotopes; Sensitivity and Specificity; Single-Strand Specific DNA and RNA Endonucleases; Urinary Tract

Recent studies have demonstrated the presence of DNA adducts from 4-aminobiphenyl (4-ABP) in the bladder cells of humans; however, the correlation between the concentration of these adducts and the tumorigenic response is not clear. To help elucidate this relationship, we have investigated DNA adduct formation in experimental animals continuously administered 4-ABP. Male and female BALB/c mice were treated for 28 days with 4-ABP. hydrochloride in their drinking water. DNA adducts in target tissues (liver of females and bladder of males) were identified and quantified by 32P-postlabeling analyses and radioimmunoassays. These results were compared to previously reported tumor incidences obtained from the lifetime administration of 4-ABP hydrochloride. The major 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 saturation was observed at high doses. In the livers of males, the adduct levels were linearly correlated with dose throughout the entire dose range. A comparison between DNA adducts and tumorigenesis indicated a linear correlation between adduct levels and the incidence of liver tumors in female mice. In the bladders of male mice, however, the relationship was markedly nonlinear. These data suggest that adduct formation alone is insufficient for tumorigenesis in the bladder and that other factors such as cell proliferation are necessary for tumor production.

Topics: Aminobiphenyl Compounds; Animals; DNA; DNA Adducts; Female; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred BALB C; Phosphorus Radioisotopes; Radioimmunoassay; Urinary Bladder Neoplasms

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.

Topics: Acetylation; Aminobiphenyl Compounds; Animals; Autoradiography; Deoxyguanosine; DNA; DNA Damage; Female; Genotype; Isotope Labeling; Liver; Male; Mice; Mice, Inbred C57BL; Phenotype; Phosphorus Radioisotopes; Time Factors; Urinary Bladder; Urinary Bladder Neoplasms

Transplacental exposure of fetuses to carcinogens is known to induce tumors in the offspring, often with a high incidence and short latency. While covalent adduction of DNA appears to be essential for tumor initiation, little is known about the binding of carcinogens to the DNA of fetal tissues. A sensitive 32P-postlabeling method enabled us to study the binding of the environmental carcinogens safrole (600 mumol/kg p.o.), 4-aminobiphenyl (800 mumol/kg), and benzo(a)pyrene (200 mumol/kg) to the DNA of various maternal and fetal tissues after administration of test carcinogens to pregnant ICR mice on day 18 of gestation. The results show that these carcinogens bound to the DNA of maternal and fetal liver, lung, kidney, heart, brain, intestine, skin, maternal uterus, and placenta, with organ-specific quantitative and qualitative differences. It was possible for the first time to analyze DNA adduct patterns in minute amounts of tissue, for example those available from fetal heart. The covalent binding index (mumol adducted nucleotides per mol of DNA nucleotides/mumol carcinogen administered per g body weight) 24 h after safrole treatment was estimated for the different organs and ranged from 0.1 to 247 and 0.1 to 5.8 for maternal and fetal DNA, respectively. Covalent binding index values of 0.2 to 13 and 0.1 to 0.3 for maternal and fetal DNA, respectively, were found for 4-aminobiphenyl. Benzo(a)pyrene treatment yielded covalent binding index values of 0.6 to 6.5 and 0.3 to 0.7 for maternal and fetal DNA, respectively. In both maternal and fetal tissues, safrole exhibited preferential binding to liver DNA. 4-Aminobiphenyl bound preferentially to DNA of maternal liver and kidney but showed no preference among fetal tissues. Benzo(a)pyrene exhibited weak tissue preference in both maternal and fetal organs. For all of the compounds studied, the fetal adduct levels were generally lower than the corresponding maternal adduct levels, especially when the level of maternal adduction was high. The major finding was that several carcinogens of diverse structure or their metabolites readily crossed the placenta and gave rise to DNA adducts in fetal organs. The resulting DNA damage in rapidly proliferating tissues may play a critical role in transplacental carcinogenesis.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Aminobiphenyl Compounds; Animals; Benzo(a)pyrene; Benzopyrenes; Biotransformation; Carcinogens, Environmental; Dioxoles; DNA; DNA Adducts; Female; Fetus; Kidney; Liver; Male; Maternal-Fetal Exchange; Mice; Mice, Inbred ICR; Phosphorus Radioisotopes; Placenta; Pregnancy; Safrole