Mutagenesis vol. 2 no. 5 pp. 375-381, 1987
© 1987 UK Environmental Mutagen Society/Oxford University Press
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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
Section of Chemical Carcinogenesis, Institute of Cancer Research, Royal Cancer Hospital Clifton Avenue, Sutton, Surrey SM2 5PX, UK
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.
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