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.
Zhan
D J
DJ
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Heflich
R H
RH
Fu
P P
PP
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000031:analogs & derivatives* / Q000633:toxicity*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects*; D002274:Carcinogens, Environmental / Q000633:toxicity*; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics; D018736:DNA Adducts; D004249:DNA Damage; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004785:Environmental Pollutants / Q000633:toxicity*; D016368:Frameshift Mutation; D005796:Genes / Q000187:drug effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009154:Mutation; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
C057268:7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene-DNA adduct; D002274:Carcinogens, Environmental; D018736:DNA Adducts; D018076:DNA, Complementary; D004785:Environmental Pollutants; D012333:RNA, Messenger; C085532:3-nitrobenzo(a)pyrene-7,8-diol-9,10-epoxide; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C098531:1-nitrobenzo(a)pyrene-7,8-diol-9,10-epoxide; D007041:Hypoxanthine Phosphoribosyltransferase