The DNA repair host-mediated assay as a rapid and sensitive in vivo procedure for the determination of genotoxic factors present in various organs of mice

Abstract

The DNA repair host-mediated assay, in which repairable DNA damage is determined in E. coli cells present in various organs of mice exposed to genotoxic agents, was further developed to broaden the range of organs under study and to simplify the procedure of assessing differential bacterial cell survival. A pair of derivatives of E. coli K-12 strain 343/113 was constructed which differed vastly in DNA repair capacity (uvr ⁺/rec ⁺ vs uvrB/recA), as a means of assessing DNA damaging effects; furthermore, the strains differed in their ability to ferment lactose (Δ Lac vs Lac ⁺), so that the individual survival of both strains could be determined on a single agar medium (containing neutral red as pH indicator), on which the strains had different colony colour morphology (red, Lac ⁺ vs white, Lac ⁻ colonies). Finally, the strains were made streptomycin-dependent, to prevent uncontrolled growth of the bacterial cells within the various organs and also to inhibit contamination of the survival agar medium by representatives of the normal intestinal microflora. The experimental procedure consisted of injecting mixtures of stationary cells of the two strains (ca. 3–5×10⁸ viable cells per mouse) both intravenously and orally into mice, either pretreated or subsequently treated with test chemicals. Ninety minutes after injection of the bacteria, the liver, spleen, lungs, kidneys, stomach, intestine, colon, and ca. 50 μl blood, were removed, suspended in buffer, homogenized, and the survival of the two strains determined on neutral red agar supplemented with streptomycin. In preliminary experiments in which the mice were treated with intraperitoneal injections of mitomycin C (0–2.0 mg per kg body weight), a dose-dependent increase in DNA damaging activity was induced in bacterial cells present in all organs tested, the lowest effects being observed in kidneys and lungs, and the highest in liver and blood. These results need further confirmation in more extensive tests, but they do nevertheless clearly indicate the possible usefulness of the DNA repair host-mediated assay as a rapid biological dose monitor for obtaining information on the genotoxic activity in vivo of compounds for which long-term mutagenicity and carcinogenicity data are not yet available.