DNA cross-linking and cytotoxicity in normal and transformed human cells treated in vitro with 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d] -1,2,3,5-tetrazin-4(3H)-one

Abstract

Normal (IMR-90) and SV40-transformed (VA-13) human embryo cells were treated with 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d] -1,2,3,5-tetrazin-4(3H)-one (M&B 39565), and the effects of the drug on cell viability and cellular DNA integrity were studied. The effects of M&B 39565 were compared with one of its potential decomposition products 5-[3-(2-chloroethyl)triazen-1 -yl]imidazole-4-carboxamide (MCTIC). M&B 39565 and MCTIC were 5- to 6-fold more toxic to VA-13 cells than to IMR-90 cells for drug concentrations which produced a 2-log cell kill, as measured by colony-forming assays. Using alkaline elution analysis, VA-13 cells exhibited concentration-dependent DNA interstrand cross-link formation. In IMR-90 cells, little or no interstrand cross-link formation was detected. The DNA interstrand cross-link formation in VA-13 cells was found to peak 12 hr after drug removal. A linear correlation between DNA interstrand cross-link formation and log cell kill was observed in VA-13 cells but not in IMR-90 cells. DNA-protein cross-link formation was found to be comparable in both cell lines for each drug, suggesting that drug penetration and intracellular drug reactivity were similar. Initial chemical decomposition studies suggest that both M&B 39565 and MCTIC may produce a chloroethyldiazo species. This species has been implicated in the formation of chloroethyl-DNA adducts which convert to DNA interstrand cross-links in mammalian cells treated with chloroethylnitrosoureas [Erickson et al., Nature (Lond.), 288: 727, 1980]. These data suggest that DNA interstrand cross-link formation may be a common mechanism for the in vitro cytotoxicity of M&B 39565 and MCTIC.