CYTOTOXICITY AND ACTIVATION OF CB1954 IN A HUMAN TUMOR-CELL LINE

CB1954 (5-aziridin-1-yl-2,4-dinitrobenzamide) is a monofunctional alkylating agent, to which Walker 256 cells are very sensitive. These cells express a nitroreductase which reduces CB1954 to a bifunctional crosslinking agent 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. In vitro testing on the human colon line LS174T showed that the differential cytotoxicities between the monofunctional agent (CB1954), and the active species (generated in situ by the addition of NADH and the Walker rat nitroreductase) were smaller than anticipated due to the unexpected toxicity of CB1954 (IC50 value for CB1954 on LS174T cells of 78-mu-M). The toxicity of the chemically synthesised active form was less than if it had been generated in situ (on LS174T cells). Further experiments showed that NADH was toxic at the levels used to generate the active species (500-mu-M). Gel filtration and electrophoresis experiments demonstrated that the human colon carcinoma and choriocarcinoma cell lines MAWI and JAR, as well as LS174T express an enzyme of similar molecular weight to that of the 33 kD Walker cell nitroreductase, which is capable of reducing CB1954 to its toxic metabolite, and reducing MTT to its insoluble formazan salt. The expression of this enzyme presumably accounts for the unexpected toxicity of CB1954.