Effects of using a low-copy plasmid and controlling membrane permeability in SOS-based genotoxic bioassay

Abstract

A whole-cell assay based on bacterial SOS response has been used for genotoxicity evaluation of chemical substances. In this study, for improvement of induction ratio and detection limit in the genotoxic assay, three modifications to an original tester strain, Escherichia coli harboring a multi-copy plasmid-borne fusion of firefly luciferase and the umuD promoter, were examined singly or in combinations. First, the effect of replacement of the multi-copy plasmid with a low-copy one was examined. Bioluminescence (BL) emission from the E. coli strain having the low-copy plasmid induced by mitomycin C (MMC) as a genotoxic substance was decreased compared to that from the original strain, but the BL emission ratio of the induced strain to the non-induced one was increased by two-fold at maximum due to reduced blank BL emission from the non-induced strain. Next, two modifications were made in view of control of the membrane permeability for substances by using toluene or a tolC mutant. Toluene promoted the membrane permeation of substances, while tolC mutation inhibited the efflux of them via cell membranes. The addition of toluene to the strain resulted in an increase in the BL ratio by increasing BL emission from the induced strain. On the other hand, by the use of tolC mutation, the dose-dependency curve of the BL ratio shifted to a lower concentration range of MMC, and the detection limit of MMC was therefore greatly improved. Compared to the original tester strain, in combination with the use of the low-copy plasmid, the maximum induction ratio was increased about four-fold to above 400 by the addition of toluene, whereas the detection limit was improved 1000-fold to below 10 pg ml −1 by the use of a tolC mutant. The effects of each modification and their combination on the induction ratio and detection limit were examined with various substances.