The effects of sodium saccharin with and without N-methyl-N-nitrosourea co-treatment on anchorage-independent growth of human diploid fibroblasts.
In order to examine the effects of sodium saccharin on in vitro cell transformation, neonatal human diploid fibroblasts were treated with various levels of the compound during specific phases of a synchronized cell cycle, then subcultured for a suitable expression period, and assayed for anchorage-independent growth in soft agar. The dose-relatedness and cell-cycle-dependency of the effects were determined both in cells treated with sodium saccharin and in cells co-treated with saccharin and the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Cell treatment with saccharin at ng/ml levels for 10 hr following release from a metabolic block (G(1) phase), with or without MNU treatment (70 mug/ml) at 10 hr, was an effective transforming regimen. Continuous saccharin treatment for 12 days (from the end of the synchronized cell cycle until seeding into soft agar) was often as effective as G(1) exposure, but treatment during the metabolic block or during S-phase and mitosis was always less effective. The dose-relatedness of the effects was similar both for saccharin-induced transformation at ng/ml treatment levels, and for co-transformation with MNU. Maximum colony growth (>100/50,000 seeded cells) was observed at a concentration of 50 ng/ml, and a variable, but decreased, response was seen at higher and lower exposures. MNU co-treatment served to maintain the transformation response at higher (mug/ml) saccharin levels at which saccharin alone was not effective. Thus, sodium saccharin demonstrated significant transforming activity over a wide dose range, both when used alone, and in combination with MNU.
West
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Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.
Kadlubar
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