De-Jin Zhan

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.

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.

Potent tumorigenicity of 7-chlorobenz[α]anthracene and 7-bromobenz[α]anthracene in the neonatal B6C3F1 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.

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‐8aP) 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‐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‐Bap‐DE), together with trans‐7,8‐dihydroxy‐anti‐9,10‐epoxy‐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/106 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.

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.

Effect of Caloric Restriction on Metabolism of Polycyclic Aromatic Hydrocarbons by Male B6C3F1 Mouse Liver Microsomes

Abstract Effect of caloric restriction (CR) on metabolism of 7-fluorobenz-[a]anthracene (7-F-BA), 7-chlorobenz[a]anthracene (7-Cl-BA), 7-bromo-benz[a]anthracene (7-Br-BA), and 7-nitrobenz[a]anthracene (7-nitro-BA) by mouse liver microsomes was studied. The results indicate that CR can affect metabolism rate, modulate the regioselectivity and stereoselectivity of the cytochrome P-450 metabolizing enzymes, and lower the yield of the proximate mutagenic metabolites. These findings indicate that CR may in part modulate chemical carcinogenesis by altering the metabolic activation.

Effect of Nitro Orientation on Ras -Protooncogene Mutation in Liver Tumors from 7-Nitrodibenz[ a,h ]anthracene-Treated Mice

Dibenz[ a,h ]anthracene (DB[ a,h ]A) and 7-nitrodibenz[ a,h ]anthracene (7-NDB[ a,h ]A) induced liver tumors when administered to neonatal B6C3F 1 mice. For protooncogene analysis, RNA was isolated from each of the liver tumors from treated mice and reverse-transcribed into cDNA. Portions of the K- and H- ras protein coding sequences were then amplified and analyzed for DNA sequence alterations. DB[ a,h ]A-induced liver tumors had a 100% (23/23) frequency of ras -protooncogene mutation, with 83% (19/23) occurring at the first base of K- ras codon 13 and resulting in G GC M C GC transversion; the remaining 17% (4/23) of the mutations were located at the second base of H- ras codon 61. In contrast, only four of nine (44%) of 7-NDB[ a,h ]A-induced liver tumors had ras -protooncogene mutations, with two each at K- ras codon 13 and H- ras codon 61. Combined with previous observations, the results indicate that the nitro substituent perpendicular to the aromatic moiety alters the chemical-induced protooncogene activation frequency and mutational pattern in liver tumors of B6C3F 1 mice.

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 tranversions; 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 hae an effect on mutation profiles. Environ. Mol. Mutagen. 31:60–69, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.