The mechanism of action of the novel tetrazepinone 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3H)-one (NIME), structurally related to the antitumour drug temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. NIME preferentially inhibited DNA synthesis over protein and RNA syntheses at 3 and 24 hr post-treatment. A Maxam-Gilbert sequencing assay showed that NIME induced barely detectable levels of guanine N7 alkylation in an isolated DNA strand, in contrast to temozolomide, a strong alkylating agent containing, like NIME, a cyclic 3-methyl-1,2,3-triazene moiety. Alkaline sucrose density-gradient sedimentation, at concentrations 2- to 10-fold lower than the ones used in the DNA sequencing assay, showed significant DNA damage in OVCAR-3 cells 24 hr after treatment with NIME. This was accompanied by a significant accumulation of cells in late S and G2M. Cell cycle arrest was transient and was reversed after 2-3 days following drug treatment. This was in agreement with bivariate bromodeoxyuridine/propidium iodide analysis, which showed that at 100 microM, a concentration at which the majority of the cells arrested in late S and G2M, a significant fraction of bromodeoxyuridine positive (S-phase) cells escaped the block. In an attempt to elucidate the mechanism underlying these effects, the degradation of NIME in cell culture medium was analyzed by GC-MS (gas chromatography coupled with mass spectrometry). The results showed that, in contrast to temozolomide, NIME did not convert to an open-chain alkyltriazene in cell culture medium, but to a major benzimidazole product, which exerted a minor effect on the cell cycle. This suggests that NIME, despite containing a 3-(alkyl)-1,2,3-triazene moiety, does not act by DNA alkylation but probably by generating a short-lived genotoxic species during its degradation to 6,5-benzofused derivatives.