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Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells
Abstract:
For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.
Insights
Optimizing mutagenesis assays requires careful adjustment of liver postmitochondrial fraction (S15) concentration, as optimal levels vary significantly depending on the specific chemical mutagen tested, influencing cytotoxicity and mutagenicity. Different compounds, like benzo[a]pyrene and nitrosamines, show distinct responses to S15 concentration, highlighting the importance of this factor for accurate mutagenicity testing.
Area of Science:
- Toxicology
- Biochemistry
- Genetics
Background:
- The concentration of liver postmitochondrial fraction (S15) is critical for optimizing mutagenesis assays in V79 Chinese hamster cells.
- Different chemical mutagens exhibit varying optimal S15 concentrations for maximum induction of cytotoxicity and mutagenicity.
Purpose of the Study:
- To investigate the impact of varying liver S15 concentrations on the mutagenicity and cytotoxicity induced by different chemical agents.
- To determine how factors like metabolic activation and lipophilicity influence the optimal S15 concentration for mutagenesis.
Main Methods:
- V79 Chinese hamster cells were exposed to various chemical mutagens (benzo[a]pyrene, aflatoxin B1, N-nitrosodimethylamine) at different concentrations of rat liver S15 fraction.
- Cytotoxicity and mutagenicity were assessed using 8-azaguanine and ouabain resistance assays.
- Metabolism of benzo[a]pyrene was analyzed using high-pressure liquid chromatography.
- The relationship between nitrosamine lipophilicity and mutagenesis at different S15 concentrations was examined.
Main Results:
- Benzo[a]pyrene and aflatoxin B1 showed peak mutagenicity/cytotoxicity at low S15 concentrations (1-5% and 2%, respectively), with higher concentrations leading to reduced responses due to rapid metabolism.
- N-nitrosodimethylamine exhibited a linear increase in mutagenicity/cytotoxicity with increasing S15 concentration.
- Nitrosamine mutagenicity at high versus low S15 concentrations inversely correlated with their lipophilicity, suggesting lipid solubility influences optimal S15 levels.
Conclusions:
- The optimal concentration of liver S15 fraction for mutagenesis assays is chemical-specific.
- Metabolic competition and compound lipophilicity are key factors determining the ideal S15 concentration for accurate mutagenicity assessment.