Detection of in vitro genotoxicity of pro-mutagens using the comet assay under human and rat liver S9 fractions

Abstract

The metabolic activating ability of human liver S9, human lung S9 (HLuSP-E), and rat liver S9 were compared. One of the human liver S9 (HLSP-E) was prepared from a pool from 15 donors and the other (HLS-014E) from a liver sample that had high levels of P450 activities. Rat liver S9 was prepared from male Sprague-Dawley rats either untreated (RLSP-E) or pretreated with phenobarbital/5,6-benzoflavone (RL-PB/BF). Human lymphoma WTK1 cells were treated with 37 pro-mutagens that required cytochrome P450 or NADPH-P450 reductase for metabolic activation and their genotoxicity was determined by the comet assay. HLS-014E and RL-PB/BF had a higher P450 enzyme activity and activated a greater variety of pro-mutagens than others. The results of the human liver S9 and RL-PB/BF were discrepant for only 2 out of 37 pro-mutagens; phenacetin was positive with human liver S9 but not with RL-PB/BF, and benz[a]anthrathene was positive with RL-PB/BF but not with human liver S9. Both RL-PB/BF and human liver S9 activated pyrene, IQ, MeIQ, 1-nitropyrene, and N-nitrosopiperidine while RLSP-E did not. In spite of the existence of species differences in CYP isoforms and differences of CYP activity, therefore, there were no appreciable qualitative differences in pro-mutagen activating ability of the human liver S9 and RL-PB/BF, suggesting the possibility that the genotoxicity of some pro-mutagens that are genotoxic with human liver S9 are missed by the use of liver S9 from rats that were not pretreated to induce CYP. Therefore, conventionally used CYP-induced rat liver S9 fraction is useful for the evaluation of the genotoxic risk for humans in spite of the species difference in CYP isoforms.Keywords: rat liver S9, human liver S9, metabolic activation, comet assay MOJ Toxicology Research ArticleOpen Access Detection of in vitro genotoxicity of pro-mutagens using the comet assay under human and rat liver S9 fractions 256

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“…Buccal pouch and blood samples were collected for analysis. S9 fraction was prepared according to the method by Ames et al 10 .…”

Differential Genotoxicity of Polygoni Multiflori in Rat and Human: Insights from Ames Test and S9 Metabolic Activation System

“…In rat liver S9, PMR elicited reverse mutations in the TA100 and TA1537 strains but not in human liver S9. Although differences in the toxicity patterns between metabolic enzymes in rats and humans have been reported in several substances 10,16,17 , the underlying mechanism is unknown. Therefore, we aimed to explore the key factors underlying the differences in the genotoxicity exhibited by PMR in rat and human liver S9.…”