Genotoxicity of N-nitrosochlordiazepoxide in cultured mammalian cells

Chlordiazepoxide, a benzodiazepine derivative commonly used for the treatment of anxiety, was found to react with sodium nitrite in HCl aqueous solution yielding, at pH ranging from 0.5 to 5,N-nitrosochlordiazepoxide (NO-CDE). In the absence of a metabolic activation system, a dose-dependent frequency of DNA single-strand breaks was revealed by the alkaline elution technique in V79 cells exposed to subtoxic NO-CDE concentrations ranging from 33 to 330 microM. DNA lesions were only partially repaired within 48 hr, and their promutagenic character was demonstrated by the induction of 6-thioguanine resistance in the same cells. The genotoxicity of NO-CDE was confirmed by results obtained in metabolically competent primary cultures of both rat and human hepatocytes, which displayed similar dose-related amounts of DNA fragmentation and of DNA repair synthesis after treatment with concentrations ranging from 33 to 1000 microM. In conclusions similar to those which might occur in the stomach of a patient taking chlordiazepoxide the concentration of NO-CDE in the reaction mixture (50 microM) was of the same order as the concentrations found to induce a genotoxic effect in cultured mammalian cells.