Study of N-nitrosomorpholine-induced DNA strand breaks in Caco-2 cells by the classical and modified comet assay: influence of vitamins E and C.

We have studied the genotoxic effects of the well-known heterocyclic liver carcinogen N-nitrosomorpholine (NMOR), an N-nitroso compound, which was prepared in our laboratory by nitrosation of the secondary amine morpholine with NaNO2. NMOR induced DNA strand breaks in human colon carcinoma Caco-2 cells. The concentration-dependent DNA-damaging effects of NMOR were proved by the comet assay. We further characterized DNA strand breaks induced by NMOR as follows: 1) We pretreated cells with vitamins E and C, which are able to eliminate oxidative DNA damage. 2) We varied the pH of the comet assay (12.1 and 13). In general, alkali-labile sites are stable until pH is raised to 12.5. 3) We used the site-specific repair enzymes exonuclease III and formamidopyrimidine-DNA glycosylase in the modified comet assay. Results showed that NMOR-induced DNA strand breaks have their origin exclusively in alkali-labile sites. Nevertheless, vitamins E and C decreased the level of DNA strand breaks. These results showed that antioxidants may have biological activities other than free radical scavenging that relate to their cancer-prevention properties. Our conceptions about reduction of NMOR-induced DNA lesions in Caco-2 cells by vitamins E and C are presented in this work.