DNA sequence analysis of 1-nitropyrene-4,5-oxide and 1-nitropyrene-9,10-oxide induced mutations in the hprt gene of Chinese hamster ovary cells.
Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were A-->G transition (15/33) and G-->A transition (8/33). The predominant base substitution, A-->G transition (11/27) and G-->A transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.
Kim
Hyun-Jo
HJ
Department of Biochemistry, College of Natural Sciences, Yeungnam University, Gyongsan 712-749, Korea.
Kim
Tae-Ho
TH
Lee
Sun-Young
SY
Lee
Dong-Hoon
DH
Kim
Sang-In
SI
Pfeifer
Gerd P
GP
Kim
Seog K
SK
Lee
Chong-Soon
CS
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017353:Gene Deletion; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000494:pharmacology*; D017354:Point Mutation; D011721:Pyrenes / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D009153:Mutagens; D011721:Pyrenes; C057066:1-nitropyrene-4,5-oxide; C057067:1-nitropyrene-9,10-oxide; D007041:Hypoxanthine Phosphoribosyltransferase