Molecular dosimetry of DNA damage caused by alkylation: I. Single-strand breaks induced by ethylating agents in cultured mammalian cells in relation to survival

Abstract

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (ssb) or alkali-labile sites were measured by centrifugation in alkaline sucrose gradients after lysis in alkali. 4 agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl- N′-nitro- N-nitrosoguanidine (ENNG), N-ethyl- N-nitrosourea (ENU), ethyl methanesulphonate (EMS) and diethyl sulphonate (DES). The compounds differed greatly in their potency to induce the lesions measured when compared on a molar basis, but comparison at equicytotoxic doses showed relatively small differences. Upon prolonged incubation of the DNA in alkali, the number of ssb increased considerably. DNA from untreated cells showed biphasic kinetics: slow ssb formation for about 10 h, then the rate increased and remained constant for up to 40h. Treated cells showed an accelerated, dose-dependent linear generation of ssb for 10 h, followed by a short plateau; then ssb were formed again at a constant rate, somewhat higher than that in controls. Ssb formed in the initial phase are ascribed to phosphotriester hydrolysis, those after the plateau to unidentified causes. Zero intercepts appeared to be a measure of apurinic sites generated intracellularly.