Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay

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Abstract
We have examined the effects of three buffers–-Vogel-Bonner minimal E (VBM) and 0.15 and 0.015 M sodium phosphate, pH 7.4–-and two concentrations of overnight cells (5× and 10×) on the mutagenicity of two pure compounds and two complex mixtures in strain TA98 using a modification of a microsuspension assay developed by Kado et al. (Mutation Research 121:25–32, 1983). The assay was performed by adding 50 μ1 of cell concentrate of an overnight culture of TA98 resuspended in the appropriate buffer; 50 μ1 of the same buffer or S9 mix; and 2 μ1 of mutagen or dimethyl sulfoxide to a 1-dram vial or 13 × 150-mm test tube. The suspension was incubated for 90 min at 37°C, top agar was added, and the contents poured onto a bottom agar of minimal medium. Cell concentration (5× vs. 10×) had little effect on the mutagenic potencies of the agents tested. The mutagenic potencies of the direct-acting agents (1-nitropyrene and diesel exhaust) were consistently lower with 0.15 M buffer compared to the mutagenic potencies of these agents with the other two buffers. The three buffers gave similar results for the indirect-acting agents (2-aminoanthracene and environmental tobacco smoke). The 0.15 M buffer was considerably cytotoxic in the absence of S9, which may explain why the direct-acting agents were less potent with this buffer compared to the other buffers. VBM was also somewhat cytotoxic, but this did not appear to affect the mutagenic potencies of the agents studied. Thus, other factors in addition to cytotoxicity may account for the effects of the different buffers on the mutagenic potencies of the direct-acting mutagens. Based on these results, we recommend the use of 0.015 M sodium phosphate buffer or VBM and either 5× or 10× cells for use with the microsuspension assay described here.