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Metabolic denitrosation of diphenylnitrosamine: a possible bioactivation pathway
Abstract:
Nitrosodiphenylamine was tested for induction of DNA single strand breaks in rat hepatocytes and Chinese hamster V 79 cells with the alkaline filter elution assay. While in rat hepatocytes DNA damage was observed, negative results were obtained in V 79 cells. In view of the metabolic capacity of hepatocytes and the chemical structure of nitrosodiphenylamine it seems likely that cytochrome P-450-dependent, reductive denitrosation might be necessary for exerting this effect. Therefore the metabolism of nitrosodiphenylamine was investigated in phenobarbital-induced mouse liver microsomes and some of the metabolites were also tested. One metabolite was identified as diphenylamine whereas the others were identified as a ring-hydroxylated derivative of diphenylamine and its corresponding quinoneimine. Diphenylhydroxylamine which was not detected in the microsomes as a metabolite produced a significant amount of DNA single strand breaks in V 79 cells. When diphenylhydroxylamine was incubated with microsomes electron spin resonance spectrum was observed which indicated the formation of the diphenylnitroxide radical. This radical seems to be mediated by auto-oxidation rather than by enzymatic catalysis. Whether diphenylhydroxylamine might be responsible for the observed genetoxic effects of nitrosodiphenylamine assumed to be produced via active oxygen species is discussed.
Insights
Nitrosodiphenylamine induced DNA damage in rat cells but not hamster cells. This suggests a metabolite, potentially diphenylhydroxylamine, formed via liver enzymes, may be responsible for the genotoxic effects.
Area of Science:
- Toxicology
- Genetics
- Biochemistry
Background:
- Nitrosodiphenylamine (NDPA) is a chemical compound with potential genotoxic properties.
- Cellular responses to genotoxic agents can vary based on cell type and metabolic capacity.
Purpose of the Study:
- To investigate the genotoxicity of NDPA by examining its ability to induce DNA single-strand breaks.
- To explore the metabolic pathways and potential reactive metabolites of NDPA responsible for its genotoxic effects.
Main Methods:
- Alkaline filter elution assay was used to detect DNA single-strand breaks in rat hepatocytes and Chinese hamster V79 cells.
- Metabolism of NDPA was studied using phenobarbital-induced mouse liver microsomes.
- Metabolites were identified, and their genotoxicity was assessed.
- Electron spin resonance (ESR) spectroscopy was employed to detect radical formation.
Main Results:
- NDPA induced DNA damage in rat hepatocytes but not in V79 cells.
- Metabolism of NDPA in liver microsomes yielded diphenylamine, a ring-hydroxylated derivative, and its quinoneimine.
- Diphenylhydroxylamine, though not detected as a direct NDPA metabolite, caused significant DNA breaks in V79 cells.
- Incubation of diphenylhydroxylamine with microsomes led to the formation of a diphenylnitroxide radical, likely via auto-oxidation.
Conclusions:
- The differential genotoxicity of NDPA suggests a requirement for metabolic activation, possibly through cytochrome P-450-dependent reductive denitrosation.
- Diphenylhydroxylamine emerges as a potential genotoxic metabolite of NDPA, possibly acting through reactive oxygen species.
- Further investigation is warranted to elucidate the precise role of diphenylhydroxylamine and radical formation in NDPA-induced genotoxicity.