Potent tumorigenicity of 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene in the neonatal B6C3F (1) male mouse.

The tumorigenicity of 7-chlorobenz[a]anthracene (7-Cl-BA, and environmental contaminant, and 7 bromobenz[a]anthracene (7-Br-BA) was determines in the male B6C3F(1) newborn mouse. Mice receiving 7-Cl-BA and 7-Br-BA by i.p. injections at a dose of 1600 nmol per mouse on 1, 8, and 15 days after birth developed 92 and 96% hepatocellular adenomas, and 100 and 83% hepatocellular carcinoma, respectively. Metabolism by liver microsomes of 15-day-old mice each produced the corresponding trans-3,4-dihydrodiol. Analysis by (32)P-postlabeling/HPLC indicated the presence of DNA adducts derived from 7-Cl-BA trans-3,4-dihydrodiol and 7-Br-BA trans-3,4-dihydrodiol. Our results indicate that both 7-Cl-BA and 7-Br-BA are potent carcinogens and that bay-region diol epoxides are the ultimate metabolites that lead to DNA adduct formation and tumor initiation.

Liver tumors induced in B6C3F1 mice by 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene contain K-ras protooncogene mutations.

We previously examined the tumorigenicity of 7-chlorobenz[a]anthracene (7-Cl-BA) and 7-bromobenz[a]anthracene (7-Br-BA) in the neonatal mouse bioassay and found that 7-Cl-BA and 7-Br-BA induced hepatocellular adenoma in 92 and 96% of the mice and hepatocellular carcinoma in 100 and 83% of the mice, respectively. In the present study, mRNA was isolated from each of the liver tumors induced by the two compounds and reverse-transcribed to cDNA. Portions of the K- and H-ras oncogene coding sequences were then amplified and analyzed for DNA sequence alterations. Eighty-three percent (20/24) of 7-Cl-BA-induced and 91% (20/22) of 7-Br-BA-induced liver tumors had activated ras protooncogenes. In contrast to the general finding of H-ras mutations in B6C3F1 mouse liver tumors, both compounds had 95% (19/20) of the mutations located at the first base of K-ras codon 13, resulting in a pattern of GGC --> CGC. Thus, our results demonstrate that 7-Cl-BA and 7-Br-BA induce a unique type of ras (K-ras) oncogene activation in liver tumors of B6C3F1 mice.

Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzo[a]pyrene trans-7,8- diol-anti-9,10-epoxide.

Both 1- and 3-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites,trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-epoxy -7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1-nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]- pyrene (3-nitro-BaPDE), together with trans-7,8-dihydroxy-anti-9, 10-ep- oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10(6) clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: 1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G-->T transversion; 2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and 3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. The mutagens induced few frameshifts or complex mutations. Among the differences in mutational specificity for the three diolepoxides, the proportion of substituted dGs with 3' purines was significant (P < 0.05) for BaP-DE (16/19, 84%) and 3-nitro-BaP-DE (17/20, 85%), but not significant for 1-nitro-BaP-DE-induced mutants (11/17, 65%, P > 0.05). Also, high proportions of BaP-DE and 3-nitro-BaP-DE base pair substitutions at G:C occurred in DNA sequence contexts of 5'-GG-3', 5'-GGA-3', and 5'-TGGA-3', while the proportions of 1-nitro-BaP-DE mutants in these contexts were often lower. The results indicate that nitro substitution at C1 or C3 of BaP-DE reduces mutational potency in CHO cells and appears to have only subtle effects upon the mutational pattern in the hprt gene.

Molecular characterization of hprt mutations from Chinese hamster ovary cells treated with 1-, 3-, and 6-nitrosobenzo[a]pyrene.

1-, 3-, and 6-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants that can be metabolized to genotoxic derivatives by either nitroreduction or ring-oxidation. In this study, we examined the types of mutations produced by the primary nitroreduced metabolites, 1-, 3-, and 6-nitroso-BaP (NO-BaP) in the hprt gene of Chinese hamster ovary cells. RNA from 6-thioguanine-resistant mutants was reverse-transcribed to cDNA and the hprt coding sequence was amplified and sequenced. The mutational patterns produced by the three compounds exhibited extensive similarities: 1) base pair substitutions accounted for 67% (28/42) of 1-NO-BaP, 51% (26/51) of 3-NO-BaP, and 50% (11/22) of 6-NO-BaP mutations; 19-36% of the mutations were exon deletions and 14-18% were frameshifts; 2) most (64-84%) of the simple base pair substitutions occurred at G:C, mainly G:C-->T:A and G:C-->C:G transversions; 3) 98% (46/47) of the simple base pair substitutions at G:C had the mutated dG on the non-transcribed strand and 81% (38/47) were located with the mutated dG flanked 3' by at least one purine; and 4) most simple base pair substitutions (48/62, 77%) occurred in exons 2, 3, and 8 of the hprt gene. Although there were no significant differences among the mutation profiles of the NO-BaPs, a significant difference did exist between the mutation pattern produced by 3-NO-BaP and the mutation pattern previously determined for the ring-oxidized product of 3-nitro-BaP metabolism, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydro-3-nitrobenzo[a]pyrene. This observation indicates that differences in the structures of closely related adducts can be important enough to have an effect on mutation profiles.

Metabolism of isomeric nitrobenzo[a]pyrenes leading to DNA adducts and mutagenesis.

We have been interested in determining the structural and electronic features that may be useful in predicting the mutagenic activity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs). We have previously found that a correlation between structural and electronic features and direct-acting mutagenicity in Salmonella typhimurium cannot be made using nitro-PAHs with different molecular size. In this study, a series of structurally related nitro-PAHs, the environmental contaminants 1-, 3-, and 6-nitrobenzo[alpha]pyrene (NBaP) and their derivatives, was used to determine structure-activity relationships. It was found that isomeric NBaPs are activated to DNA damaging and mutagenic derivatives by nitroreduction, ring-oxidation, or by a combination of these two pathways. A general finding was that NBaPs and derivatives with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in S. typhimurium strains TA98 and TA100. In this paper, we also discuss the effect of the location of the nitro group on the metabolism and the mutagenicity of NBaPs and the effect of oxygen-containing functional groups on the mutagenicity of NBaP derivatives. These findings provide a useful molecular basis for interpreting and predicting the direct-acting mutagenicity of nitro-PAHs.

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.

Structure, tumorigenicity, microsomal metabolism, and DNA binding of 7-Nitrodibenz[a,h]anthracene.

It has been previously proposed that a nitropolycyclic aromatic hydrocarbon (nitro-PAH) with its nitro functional group perpendicular or nearly perpendicular to the aromatic moiety exhibits lower tumorigenicity than the corresponding parent aromatic hydrocarbon. We also hypothesized that reduction of the nitro group is not involved, or contributed less significantly in the metabolic activation of this class of nitro-PAHs. To verify this hypothesis, we selected 7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A) for study. The X-ray crystallographic structure of 7-NDB[a,h]A was determined and indicated that the dihedral angle between the nitro functional group and the aromatic dibenz[a,h]anthracenyl moiety was 80.6 degrees, indicating the nitro group preferentially adopts a nearly perpendicular orientation. The tumorigenicity of 7-NDB[a,h]A and dibenz[a,h]anthracene (DB[a,h]A) was determined in the male B6C3F1 neonatal mouse. Mice were administered ip injections of 1/7, 2/7, and 4/7 of the total dose of 7-NDB[a,h]A (400 nmol in 35 microL of DMSO per mouse) within 24 h of birth and at 8 and 15 days of age, respectively, and sacrificed at 12 months of age. DB[a,h]A induced 78 and 96% hepatocellular adenomas and carcinomas, respectively. However, 7-NDB[a,h]A induced only 50 and 8% hepatocellular adenomas and carcinomas compared with the 8 and 4% hepatocellular adenomas and carcinomas induced by the solvent vehicle, DMSO. Aerobic metabolism of 7-NDB[a,h]A by liver microsomes of 15-day old male B6C3F1 neonatal mice resulted in trans-3,4-dihydroxy-3, 4-dihydro-7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A trans-3, 4-dihydrodiol) and trans-10,11-dihydroxy-10, 11-dihydro-7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A trans-10, 11-dihydrodiol) as predominant metabolites. Under anaerobic conditions, 7-NDB[a,h]A was not metabolized (nitroreduced). The DNA adduct levels in liver and lung tissues of male B6C3F1 mice treated with 7-NDB[a,h]A and sacrificed 24 h and 6 days after final dosing were determined by 32P-postlabeling/TLC. In all cases, the DNA adducts derived from 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a, h]A trans-10,11-dihydrodiol were formed. These results suggest that both of the metabolites, 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol, are involved in the metabolic activation of 7-NDB[a,h]A, leading to tumor induction in the neonatal mouse. Thus, our results described in this paper support our hypotheses that a nitro-PAH with a perpendicular nitro orientation exhibits lower tumorigenicity than the corresponding parent PAH and that nitroreduction contributes less significantly in the metabolic activation.