| Abstrakt: |
We are attempting to develop the human lymphocyte culture system for study of rates and mechanisms of chemically-induced mutation. Three loci are being examined: the hpt locus which specifies HGPRT synthesis; the as locus which specifies argininosuccinate synthetase (AS) synthesis; and the o locus which determines ouabain responsiveness. Mutation from hpt+→hpt−, using 6-thioguanine (6TG) selection, is being studied, as is mutation from as−→as+, using high citrulline-low arginine medium, and Os→Or, using 106M ouabain. The tester line UM-21-5, of citrullinemic (as−/as−) origin, has, via selection, produced a line triply mutant (hpt−, as−, and Or), as determined by growth in selective media and by HGPRT and AS specific activities. The Wi-L2 tester line, derived originally from a spherocytosis patient, has produced, by selection, an hpt−and as−derivative. Spontaneous mutation rates in Wi-L2 from 6TtS→6TGr(hpt+→hgt−), via fluctuation analyses, ranged from 0.1-0.3 × 10−7cell/generation. Using sister chromatid exchange (SCE) frequency as an indirect measure of mutagenicity, incubation of Wi-L2 in ethyl methanesulfonate (EMS), 50 μg/ml for 24 hours, resulted in a 3-fold increase in SCE's (mean in controls of 7.7 SCE's/taetaphase vs. 21.9 in EMS-exposed cells). The mutation rates at these 3 loci after EMS mutagenesis are now being determined, as a model for demonstrating the feasibility of monitoring for chemical mutagens using these lymphoid lines. |
