Interaction and distinction of genotoxic and non-genotoxic events in carcinogenesis.

Multistage carcinogenesis involves genotoxic as well as non-genotoxic mechanisms. The importance of genotoxic events in human carcinogenesis is apparent from the analysis of tumours: for example, five to six genetic alterations can be found in most malignant colorectal tumours. While such measurable "footprints" (e.g. ras, p53 mutations) can be left in tumours by genotoxic events, non-genotoxic events cannot directly generate them. Thus, the lack of specific indicators of non-genotoxic events in carcinogenesis makes the identification of non-genotoxic carcinogens difficult. It is also important to emphasize that apparent "genotoxic" endpoints (mutations, chromosome aberrations) could be induced by "non-genotoxic" agents through indirect mechanisms (e.g. induced cell proliferation and/or genomic instability, oxidative damage, deamination of 5-methyl cytosine). This emphasizes the need for differentiating "events" from the actual "activities" of chemicals and the difficulty of classification of carcinogens into genotoxic and non-genotoxic. One of the best models for the study of interaction of genotoxic and non-genotoxic mechanisms during carcinogenesis is a two-stage carcinogenesis system using mouse skin, rat liver or cultured cells. Molecular analysis of tumours produced on mouse skin by the classical initiation-promotion protocol indicates that the mutation spectra of oncogenes, e.g. Ha-ras, are determined by initiating (genotoxic) and not by promoting (non-genotoxic) agents. However, since usually no tumours appear without the application of tumour-promoting agents, the manifestation of genotoxic events (Ha-ras mutation) is dependent on the action of non-genotoxic agents. Using a BALB c 3T3 two-stage cell transformation system, we have now succeeded in confirming this and have quantitated the initiation and promotion events. These studies may help us not only in understanding mechanisms of carcinogenesis but also in developing molecular quantitative risk assessment in terms of multistage carcinogenesis.