Multistage transformation of cultured rat urothelium: the effects of N-methyl-N-nitrosourea, sodium saccharin, sodium cyclamate and 12-O-tetradecanoylphorbol-13-acetate.

Rat bladder epithelial cell transformation was found to involve at least three distinct phenotypic stages in vitro. The effects on this process of N-methyl-N-nitrosourea (MNU), sodium saccharin, sodium cyclamate and 12-O-tetradecanoylphorbol-13-acetate (TPA) alone or in combination were investigated. MNU was a potent inducer of proliferating preneoplastic epithelial foci. Sodium saccharin alone induced a transient hyperplastic response of the cultured urothelium but did not induce significantly more foci than in controls, though a late toxic effect of saccharin may have masked focus induction. In combination with a single dose of 250 micrograms/ml MNU, sodium saccharin did not increase focus incidence compared with MNU treatment alone but following a single treatment with 25 micrograms/ml MNU which alone induced no foci, saccharin induced a significant number of foci indicating a promoting effect under these conditions. Sodium cyclamate alone induced a marked and prolonged epithelial hyperplasia and a significant increase in focus incidence above controls and above MNU-treated cultures. Following a single treatment with 250 micrograms/ml MNU, cyclamate increased focus incidence still further. TPA, the potent skin tumour promoting agent, alone induced significantly more foci than in controls. Rapidly-proliferating cell lines were established from nine foci induced by treatment with MNU alone or MNU + saccharin. Four of these were tumorigenic. These results indicate that MNU is a potent inducer of preneoplastic foci in rat urothelial cultures. Sodium saccharin and sodium cyclamate can act as promoting agents in this in vitro system as in the in vivo induction of tumours and sodium cyclamate appears to be active as a complete inducer under these conditions.