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Strategies for optimization of short-term genotoxicity tests: the synergistic effect of NADPH and NADH on P450
Abstract:
The synergistic effect of NADPH and NADH on P450 functions upon pre-mutagens requiring metabolism during the incubation conditions used in the liver microsomal assay (LMA) was studied. The mean specific activity (Asp) during 1 h of pre-incubation (LMA) of some microsomal mono-oxygenases (i.e. ethylmorphine N-demethylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase) examined with S9 fractions from sodium phenobarbital and beta-naphthoflavone pre-treated mice, was doubled when both NADPH and NADH were present. In contrast, when lipid peroxidation was used as the main enzymatic inactivation index, there was no appreciable change. In agreement with biochemical data, in vitro DNA binding of the pre-mutagenic agent [14C]-1,1,1,2-tetrachloroethane ([14C]TTCE), mediated by mouse hepatic enzymes, showed a significant enhancement (4.4-fold) of specific activity in the presence of both pyridine nucleotides. Mutagenesis experiments using TTCE in the diploid D7 strain of Saccharomyces cerevisiae (from stationary growth phase) as a biological test system, showed a significant enhancement of mitotic gene conversion and reverse point mutation frequencies when using NADPH plus NADH in the medium. Conversely, no positive results without NADH were seen. These findings lead us to suggest the routine use of both NADPH and NADH in order to increase the 'sensitivity' of in vitro mutagenicity screens.
Insights
Adding both NADPH and NADH significantly enhances the detection of pre-mutagens in liver microsomal assays. This synergistic effect increases the sensitivity of in vitro mutagenicity testing for chemicals requiring metabolic activation.
Area of Science:
- Biochemistry
- Toxicology
- Genetics
Background:
- Liver microsomal assays (LMA) are crucial for evaluating chemical mutagenicity.
- Metabolic activation of pre-mutagens by cytochrome P450 enzymes is essential for their toxic effects.
- The role of specific cofactors like NADPH and NADH in these assays requires further elucidation.
Purpose of the Study:
- To investigate the synergistic effects of NADPH and NADH on P450-mediated metabolism of pre-mutagens.
- To assess the impact of combined NADPH and NADH on in vitro DNA binding and mutagenesis.
- To determine if including both cofactors enhances the sensitivity of mutagenicity screening.
Main Methods:
- Liver microsomal assay (LMA) with S9 fractions from pre-treated mice.
- Measurement of specific activities for microsomal mono-oxygenases.
- In vitro DNA binding assays using [14C]-1,1,1,2-tetrachloroethane ([14C]TTCE).
- Mutagenesis experiments in Saccharomyces cerevisiae (D7 strain).
Main Results:
- The presence of both NADPH and NADH doubled the specific activity of key microsomal mono-oxygenases.
- In vitro DNA binding of [14C]TTCE showed a 4.4-fold enhancement with both cofactors.
- Significant increases in mitotic gene conversion and reverse point mutation frequencies were observed with NADPH plus NADH.
- No positive mutagenic results were obtained without NADH.
Conclusions:
- NADPH and NADH exhibit a synergistic effect on P450 functions relevant to mutagenicity testing.
- The combined use of NADPH and NADH significantly enhances the detection of mutagenic potential for pre-mutagens.
- Routine inclusion of both NADPH and NADH is recommended to increase the sensitivity of in vitro mutagenicity screens.