The toxicity and mutagenicity of the anti-tumour drug 5-aziridino-2, 4-dinitrobenzamide (CB1954) is greatly reduced in a nitroreductase-deficient strain of E. coli
In the 1970s it was shown that the monofunctional alkylating agent CB1954 (5-aziridino-2, 4-dinitrobenzamide) kills the Walker rat carcinoma 256 in vivo and in vitro, but is inactive against several other tumours. In studies with bacteria, the large differences in survival in DNA-repair-proficient and deficient strains of Escherichia coli B treated with CB1954 were characteristic of a difunctional cross-linking agent. It was concluded that DNA was the only target large enough to receive significantly more than one lethal hit per molecule and that mono-alkylation alone could not acount for the lethal effects of CB1954. In 1986 it was shown that CB1954 induced DNA interstrand cross-links in CB1954-sensitive cultured Walker 256 cells, but not in resistant Chinese hamster V79 cells, suggesting that the sensitivity of Walker cells results mainly from their activation of the drug to a difunctional agent by nitroreduction. To test this, we assayed the toxicity and mutagenicity of CB1954 in nitroreductase-plus and -minus strains of E. coli WP2uvrA. Agar-overlay assays showed that CB1954 was mutagenic to several strains of E. coli WP2, in a dose range 1–100 μg per plate, with slopes (mutants/nmol) of 7.1, 1.05 and 0.16 for WP2uvrA pKM101, WP2uvrA and WP2. Assays with nitrofurazone showed that these strains possessed nitroreductase activity. However, E. coli NFR-343, a nitrofurazone-resistant mutant of WP2uvrA which lacks nitroreductase activity was markedly less sensitive to the mutagenicity of CB1954, giving a mean slope of 0.12 compared with 1.15 for WP2uvrA. Aroclor-induced rat-liver S9 did not change these responses. Treat-&-plate assays with these two strains showed that WP2uvrA was very sensitive to the toxicity of CB1954, with a D37 of 1.1 μg per ml (100 min at 37°C) when tested over a dose range of 0.5 to 5 μg per ml. In contrast, doses up to 50 μg per ml had no significant effect on the viability of NFR-343. CB1954 was mutagenic to both strains, giving slopes of 2.5 (WP2uvrA) and 0.6 (NFR-343) for log-induced mutation frequency as a function of log-dose. Intact cells of WP2uvrA, but not NFR-343, reduced CB1954 with a half-life of 40 min. These data support the idea that CB1954 undergoes nitroreduction to a form which is much more reactive with DNA, resulting in enhanced toxicity and mutagenicity.