Tumorigenicity of nitropolycyclic aromatic hydrocarbons in the neonatal B6C3F1 mouse bioassay and characterization of ras mutations in liver tumors from treated mice.

The nitropolycyclic aromatic hydrocarbons (nitro-PAHs) 1-, 2-, and 3-nitrobenzo[a]pyrene, 1- and 3-nitrobenzo[e]pyrene, 2- and 3-nitrofluoranthene, 9-nitrodibenz[a,c]anthracene, and two of the parent PAHs fluoranthene and dibenz[a,c]anthracene were tested for tumorigenicity in the neonatal male B6C3F1 mouse. 6-Nitrochrysene was used as a positive control. Mice were administered three intraperitoneal injections of test agent (400 nmol total) on 1, 8, and 15 days after birth and evaluated for liver and lung tumors at 12 months of age. 2-Nitrobenzo[a]pyrene and 6-nitrochrysene induced a high incidence of liver tumors (91-100%), while the remaining test compounds did not induce tumors at a rate significantly higher than the solvent control. 6-Nitrochrysene was the only test agent to produce a significant increase in the frequency of lung tumors. K- and H-ras mutations were analyzed in liver tumors of treated mice and mainly occurred at the first base of K-ras codon 13, resulting in GGC --> CGC transversion. Since most of the tested nitro-PAHs are mutagens in vitro, the results of this study indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity in the neonatal B6C3F1 mouse bioassay. Also, the results indicate that liver tumors from mice treated with nitro-PAHs possess ras mutations typical of PAHs and their derivatives.

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)

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.

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.

Nitroreduction of 1- and 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene and 1-nitrobenzo[a]pyrene resulting in formation of N2-deoxyguanosinyl adducts through long-range migration.

We recently reported that the reaction of N-hydroxy-3-aminobenzo[a] pyrene with calf thymus DNA produced 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene as the predominant adduct. The deoxyguanosinyl group of this adduct resides at the C6 position, which is remote from the reaction site, the nitrenium ion. It is significant to determine if formation of this type of DNA adduct is general and whether or not adduct formation is due to an increase in the stabilization of the nitrenium ion by increasing aromaticity. Thus, reduction of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene, both chemically and enzymatically, followed by reaction with calf thymus DNA was investigated. DNA was isolated and enzymatically digested, and the resulting modified nucleosides were separated by HPLC. Upon spectral analyses by mass and proton nuclear magnetic resonance spectroscopy, 6-(deoxyguanosin-N2-yl)-1-amino-7,8,9,10-tetrahydrobenzo[a] pyrene, 6-(deoxyguanosin-N2-yl)-3-amino-7,8,9,10-tetrahydrobenzo[a]pyrene, and 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene were identified, respectively. The same DNA adducts were formed from xanthine oxidase-mediated reductive metabolism of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene in the presence of calf thymus DNA. Thee results indicate that formation of N2-deoxyguanosinyl adducts of this type is common and that increasing the aromaticity by increasing the number of aromatic rings is not a decisive factor in directing their formation.

Effects of 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ) and doxorubicin on lens regeneration in the adult newt: a morphological study.

The antitumor drug 3-nitrobenzothiazolo [3,2-a] quinolinium chloride (NBQ) stimulates the in vivo lens regeneration in the adult newt Notophthalmus viridescens and induces a differentiated state in HL-60 leukemia cells. Because the cytotoxic drug doxorubicin (Adriamycin) induces differentiation of HL-60 cells in vitro we decided to compare the effect(s) of doxorubicin with NBQ on lens regeneration in vivo. Both drugs were injected intraperitoneally at six different schedules. Morphological criteria of the different regeneration stages were used in the analysis of the regenerates. NBQ stimulated lens regeneration independently of the time intervals and the stage of regeneration at which the drug was administered. There was an increase in the mean number of mitoses suggesting that NBQ stimulated cell proliferation. Doxorubicin administered for five days did not modify the regenerative process. On the other hand, doxorubicin given for periods of nine or more days after lentectomy, strongly inhibited the formation of a new lens. Thus, the inhibitory effect of doxorubicin is dependent on the continuous long term contact with the tissue. Although NBQ and doxorubicin are both DNA intercalators, they induced the effects on lens regeneration through different mechanisms.

Formation of the adduct 6-(deoxyguanosin-N2-yl)-3-amino-benzo[a]pyrene from the mutagenic environmental contaminant 3-nitrobenzo[a]pyrene.

3-Nitrobenzo[a]pyrene (3-nitro-B[a]P) is a potent bacterial mutagen as a result of nitroreduction. Reaction of N-hydroxy-3-amino-B[a]P, prepared in situ from reduction of 3-nitro-B[a]P with calf thymus DNA, was studied. After enzymatic digestion of the DNA, the resulting modified nucleosides were analyzed by thermospray HPLC-MS and high-resolution proton NMR spectroscopy. The major adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubation of DNA with 3-nitro-B[a]P in the presence of the mammalian nitroreductase xanthine oxidase, and hypoxanthine. These data indicate that a mammalian nitroreductase can metabolize 3-nitro-B[a]P to an activated derivative that reacts with DNA to give a novel adduct distant from the site of N-hydroxylation.

Identification of two N2-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene.

1-Nitropyrene, the most abundant nitro-polycyclic aromatic hydrocarbon in the environment, is a known mammalian and bacterial mutagen and a tumorigen in animals. Early studies on DNA adduct characterization for 1-nitropyrene identified N-(deoxyguanosin-8-yl)-1-aminopyrene as the major product from the modification of calf thymus DNA with N-hydroxy-1-aminopyrene, the activated metabolite from nitroreduction of 1-nitropyrene. In this paper, we report the identification of two N2-deoxyguanosinyl adducts, in addition to N-(deoxyguanosin-8-yl)-1-aminopyrene, formed from the reaction of N-hydroxy-1-aminopyrene, prepared in situ, with calf thymus DNA. These DNA adducts were identified as 6-(deoxyguanosin-N2-yl)-1-aminopyrene and 8-(deoxyguanosin-N2-yl)-1-aminopyrene. The two N2-deoxyguanosinyl adducts were also identified in an ascorbic acid-catalyzed activation of 1-nitrosopyrene and in the mammary gland of female Sprague-Dawley rats administered 1-nitropyrene. The DNA adducts were also formed when 1-nitropyrene was metabolized by xanthine oxidase in the presence of calf thymus DNA, and when 1-nitropyrene was activated by rat liver microsomes and cytosols, as well as from DNA isolated from Salmonella typhimurium suspension cultures incubated with 1-nitropyrene.

In vitro metabolism and DNA adduct formation from the mutagenic environmental contaminant 2-nitrofluoranthene.

The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.