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Bromodeoxyuridine resistance induced in mouse lymphoma cells by microsomal activation of dimethylnitrosamine
Abstract:
Many chemicals are not mutagenic per se, but when metabolized by mammalian tissues yield mutagenic products. Dimethylnitrosamine (DMN) is such a promutagen. It has no effect on cell growth or mutant frequency when incubated alone with L5178Y mouse lymphoma cells, but exerts both mutagenic and toxic effects when incubated in a microsome reaction mixture. Microsomes were prepared from C3H/f We 16-wk-old male mice by the calcium preciptation technique. L5178Y continuously cultured mouse lymphoma cells heterozygous for thymidine kinase (TK+/-) were incubated for 15 min with calcium-precipitated microsomes and various concentrations of DMN in appropriate reaction mixtures. After a 48-hr expression time, treated cells were cloned in soft agar with and without bromodeoxyuridine (BUdR) (50 mug/ml); 10 days later colonies grown to greater than about 200 mum diameter were counted. The frequency of BUdR-resistant (mutant) colonies increased linearly with the DMN concentration. A reconstruction experiment showed that the assay conditions did not significantly alter the relationship between parent and BUdR-resistant cells in growth and cloning efficiency. The smallest dose of DMN used in these experiments was 100mumol/liter, the one-sided (100 mumol greater than control frequency) -p value is 0.036. The locus is extremely sensitive to mutagenesis by DMN compared with other known mutagens at similar levels of cell survival.
Insights
Dimethylnitrosamine (DMN) is a promutagen that becomes mutagenic after metabolism by mammalian tissues. Studies show DMN significantly increases mutations in mouse lymphoma cells when activated by liver microsomes.
Area of Science:
- Toxicology and Mutagenesis
- Mammalian Cell Culture
- Biochemical Assays
Background:
- Many chemicals require metabolic activation to become mutagenic.
- Dimethylnitrosamine (DMN) is a known promutagen.
- L5178Y mouse lymphoma cells (TK+/-) are a standard model for mutagenicity testing.
Purpose of the Study:
- To investigate the mutagenic potential of Dimethylnitrosamine (DMN) after metabolic activation.
- To quantify the mutagenic effects of DMN on L5178Y mouse lymphoma cells.
- To assess the sensitivity of the thymidine kinase (TK) locus to DMN-induced mutagenesis.
Main Methods:
- Preparation of mouse liver microsomes using calcium precipitation.
- Incubation of L5178Y mouse lymphoma cells (TK+/-) with microsomes and varying concentrations of DMN.
- Quantification of mutations using bromodeoxyuridine (BUdR) resistance assay in soft agar.
Main Results:
- DMN alone did not affect cell growth or mutant frequency.
- Metabolic activation by microsomes rendered DMN mutagenic and toxic.
- A linear increase in BUdR-resistant (mutant) colonies was observed with increasing DMN concentration.
Conclusions:
- Dimethylnitrosamine (DMN) requires metabolic activation to exert mutagenic effects.
- The thymidine kinase (TK) locus in L5178Y cells is highly sensitive to DMN-induced mutagenesis.
- The assay system effectively demonstrates DMN's promutagenic activity and the sensitivity of mammalian cells.