Diogenes Herreno-Saenz

Characterization of DNA adducts in Chinese hamster ovary cells treated with mutagenic doses of 1- and 3-nitrosobenzo[a]pyrene and the trans-7,8-diol-anti-9,10-epoxides of 1- and 3-nitrobenzo[a]pyrene

The environmental contaminants 1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are mutagens in Chinese hamster ovary (CHO) cells with exogenous metabolic activation. Previous studies demonstrated the potent direct-acting mutagenicity of the oxidized metabolites, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzo[a] pyrene (1-NBaPDE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydro-3-nitrobenzo[a]pyrene (3-NBaPDE), and the partially nitroreduced metabolites, 1- and 3-nitrosobenzo[a]pyrene (1- and 3-NO-BaP). In this study, we have identified the major adduct formed by incubation of calf thymus DNA with 1-NBaPDE and used this standard in conjunction with other adduct standards to characterize the 32P-postlabeled DNA adducts produced by 1- and 3-nitro-BaP metabolites in CHO cultures. The major adduct from 1-NBaPDE exposure was 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1- nitrobenzo[a]pyrene; from 3-NBaPDE, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene; from 1-NO-BaP, 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene; and from 3-NO-BaP, 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene. For comparison, the adducts formed by trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the related nitroreduced derivative 6-nitrosobenzo[a]pyrene were also examined. The nitrobenzo[a]pyrene DNA adducts described in this study are proposed to be involved in the mutagenicity of 1- and 3-nitro-BaP upon either oxidative or reductive metabolism.

Comparative Formation of DNA Adducts of Nitro-Polycyclic Aromatic Hydrocarbons in Mouse and Rat Liver Microsomes and Cytosols

Abstract We have characterized the DNA adducts of a series of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) formed under anaerobic conditions in vitro. Although the DNA adducts of nitro-PAHs formed through enzymatic nitroreduction are generally the C8-deoxyguanosyl adducts, nitroreduction of 1-nitrobenzo[a]pyrene (1-nitro-BaP), 3-nitro-BaP, and two derivatives in the presence of calf thymus DNA resulted in the N 2-deoxyguanosyl adducts with the deoxyguanosyl moiety remote from the reaction site. Two DNA adducts of this type were also formed from 1-nitropyrene as minor products. These DNA adducts were formed from anaerobic incubation with rat and mouse liver microsomes and cytosols in the presence of calf thymus DNA. Our results suggest that biological formation of this type of DNA adduct is independent of the enzymatic system, but dependent on the geometric structure and/or electronic features of the nitro-PAH molecules.

Separation of 32P-labeled 3′,5′-bisphosphate nucleotides of polycyclic aromatic hydrocarbon anti-diol-epoxides and derivatives

Abstract 32 P-Postlabeling-HPLC is a highly sensitive analytical method for identification of chemical-modified DNA adducts isolated from samples obtained from experimental animals or humans exposed to carcinogenic chemicals. To determine optimal 32 P-postlabeling-HPLC conditions for efficient separation, we report here the use of ten diol-epoxide-modified 3′,5′-bisphosphate deoxynucleotides derived from benzo[ a ]pyrene (BaP), nitrated BaP, and related compounds. After testing ODS-modified, C 4 -modified, phenyl-modified, diphenyl-modified, and cyclodextrin-bonded reversed-phase HPLC columns, we found that the Vydac diphenyl-modified column can efficiently separate these 3′,5′-bisphosphate deoxynucleotides. The results suggest that 32 P-postlabeling-HPLC is a potentially useful methodology for detecting environmental carcinogens that can be metabolized to diol-epoxides. The relationships between the structures of anti -diol-epoxides and HPLC retention order are also discussed.

DNA Adducts and Carcinogenicity of Nitro-polycyclic Aromatic Hydrocarbons

We have been interested in the structure-activity relationships of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), and have focused on the correlation of structural and electronic features with biological activities, including mutagenicity and tumorigenicity. In our studies, we have emphasized 1-, 2-, 3-, and 6-nitrobenzo[a]pyrenes (nitro-B[a]Ps) and related compounds, all of which are derived from the potent carcinogen benzo[a]pyrene. While 1-, 2-, and 3-nitro-B[a]P are potent mutagens in Salmonella, 6-nitro-B[a]P is a weak mutagen. In vitro metabolism of 1- and 3-nitro-B[a]P has been found to generate multiple pathways for mutagenic activation. The formation of the corresponding trans-7,8-dihydrodiols and 7,8,9,10-tetrahydrotetrols suggests that 1- and 3-nitro-B[a]P trans-7,8-diol-9,10-epoxides are ultimate metabolites of the parent nitro-B[a]Ps. We have isolated a DNA adduct from the reaction between 3-nitro-B[a]P trans-7,8-diol-anti9,10-epoxide and calf thymus DNA, and identified it as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-ni tro-B[a]P . The same adduct was identified from in vitro metabolism of [3H]3-nitro-B[a]P by rat liver microsomes in the presence of calf thymus DNA. A DNA adduct of 3-nitro-B[a]P formed from reaction of N-hydroxy-3-amino-B[a]P, prepared in situ with calf thymus DNA was also isolated. This adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubating DNA with 3-nitro-B[a]P in the presence of the mammalian nitroeductase, xanthine oxidase, and hypoxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Adduct Formation by 2-Nitrofluoranthene in Salmonella typhimurium and Neonatal B6C3F1 Mice

Abstract In vivo studies of the metabolic activation of 2-nitrofluoranthene (2-NFA) were conducted by incubating Salmonella typhimurium TA98 suspension cultures with 2-NFA (10 or 20μM) for 4 hr. The DNA was isolated and the resulting DNA adducts were analyzed by HPLC and 32P-postlabeling. In addition, neonatal mice were administered a total dose of 400 nmol 2-NFA in 35 μ1 dimethylsulfoxide (DMSO) by i.p. injection on 1, 8, and 15 days after birth with 1/7, 2/7, and 4/7, respectively, of the total dose. Livers were removed from the mice 24 hr, 48 hr, and 7 days after the last dose and the DNA isolated for 32P-postlabeling adduct analysis. N−(Deoxyguanosin-8-yl)-2-aminofluoranthene and a second adduct were identified in the DNA from the Salmonella suspensions and one of these adducts was found in the neonatal mice. These results suggest that 2-NFA is metabolically activated to N−hydroxy-2-aminofluoranthene via nitroreduction and that the resulting DNA adduct is responsible for the mutagenic activities of 2-NFA.

Metabolic Activation of the Potent Mutagen and Tumorigen 2-Nitrobenzo[a]pyrene

Abstract 2-Nitrobenzo[a]pyrene (2-nitro-BaP) induced 100% liver tumor incidence in the neonatal mouse tumorigenicity assay and exhibited the highest mutagenicity among the 1-, 2-, 3-, and 6-nitro-BaP when tested in Salmonella typhimurium TA98 and TA100. To determine the metabolic activation pathways leading to tumor initiation and mutation induction, we have studied the aerobic metabolism of 2-nitro-BaP by Sprague-Dawley rat and B6C3F1 mouse liver microsomes. Metabolites were isolated by HPLC and characterized by spectral analyses, including UV-visible absorption, mass and proton NMR spectroscopy. With both rat and mouse liver microsomes, 2-nitro-BaP trans-9,10-dihydrodiol and 2-nitro-BaP trans-7,8-dihydrodiol were formed as the predominant metabolites, and 2-nitro-BaP 7,8,9,10-tetrahydrotetrol was produced in trace quantities. 2-Nitro-BaP and its two dihydrodiol metabolites were assayed in Salmonella typhimurium tester strain TA98. 2-Nitro-BaP trans-7,8-dihydrodiol, in the presence of S9, was the most mu...

Products formed from the in vitro reaction of metabolites of 3-aminochrysene with calf thymus DNA

3-Aminochrysene, a mutagenic geometric isomer of the mutagenic and carcinogenic aromatic amine 6-aminochrysene, has been synthesized and its metabolic activation studied by characterization of the products formed from the reaction of metabolites with calf thymus DNA. DNA adducts produced by 3-aminochrysene via N-oxidation were examined by preparing 3-nitrosochrysene and incubating the nitroso derivative with calf thymus DNA in the presence of ascorbic acid (to generate the N-hydroxy derivative) at pH 5. The major adduct, as determined by 1H-NMR and thermospray-mass spectrometry of the modified nucleoside obtained after enzymatic hydrolysis of the modified DNA, was N-(deoxyguanosin-8-yl)-3-aminochrysene. Thus, the reaction of N-hydroxy-3-aminochrysene with DNA differs from that of N-hydroxy-6-aminochrysene, which had previously been shown to generate N-(deoxyguanosin-8-yl)-6-aminochrysene, 5-(deoxyguanosin-N2-yl)-6-aminochrysene and N-(deoxyinosin-8-yl)-6- aminochrysene as major adducts. 32P-Postlabeling analysis of DNA treated with 3-aminochrysene in the presence of liver microsomes from rats pretreated with phenobarbital indicated an adduct pattern identical to that seen with DNA that had been treated with 3-nitrosochrysene and ascorbic acid. However, DNA treated with 3-aminochrysene (3-AC) in the presence of liver microsomes from rats pretreated with 3-methylcholanthrene contained a major adduct that was chromatographically distinct from N-(deoxyguanosin-8-yl)-3-aminochrysene.