Evaluation of a Bacillus subtilis rec-assay for the detection of mutagens which may occur in water environments

Abstract

A recombination-deficient mutant of Bacillus subtilis, NIG 45 and a wild-type strain NIG 17, were used as a mutagenicity test microorganism. The strain NIG 45 has a very high sensitivity to gamma rays, u.v. and chemical drugs. Kada et al. ( Mutation Res. 31, 347–364, 1972), Tazima, et al. ( Mutation Res. 32, 55–80, 1975) and Sadaie & Kada ( J. Bacteriol. 125, 489–500, 1976) developed the rec-assay method of Bacillus subtilis, which showed more sensitive to mutagens than the Ames reversion tests of Salmonella. In this research, the liquid-cultivation method of Bacillus subtilis was applied rather than the originally developed plate-cultivation method, resulting in detection of mutagens in very low concentration. Among test heavy metals, chromium sexivalent indicated very strong mutagenicity and manganese heptavalent indicated weak mutagenicity. NIG 45 showed an adverse growth effect at the Cr(+6) concentration of 0.5 mg l −1, while NIG 17 showed the adverse effect at 1.0 mg l −1, indicating the rec-assay of Bacillus subtilis as a good method of monitoring mutagens in water environment. A test of finding the combination effect of two mutagenic substances Cr(+6) and Mitomycin showed an additive effect rather than multiple. The combination of Cr(+6) and a non-mutagenic substance Hg, showed no enhancing effect of Hg to a mutagenic effect of Cr(+6). Among test dyes, acridine orange, acridine yellow and methyl violet indicated strong mutagenicity. Application of the target and hit theory to experimental results showed that mutagenicity of Mitomycin-C and Cr(+6) fitted to the one-target-one-hit theory, and mutagenicity of acridine orange and acridine yellow fitted to the one-target-two-hits theory. The term, mean lethal concentration of chemical mutagens was introduced to evaluate the experimental results of one-target-one-hit and one-target-two-hits cases.