DNA METHYLATION ADDUCT FORMATION AND H-RAS GENE-MUTATIONS IN PROGRESSION OF N-BUTYL-N-(4-HYDROXYBUTYL)NITROSAMINE-INDUCED BLADDER-TUMORS CAUSED BY A SINGLE EXPOSURE TO N-METHYL-N-NITROSOUREA

After receiving 500 p.p.m. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN ) in their drinking water for an initial 10 weeks, rats were given a s ingle i.p. injection of N-methyl-N-nitrosourea (MNU) at a dose of 50 m g/kg body wt at week 20 (at a stage when bladder tumor development had already occurred), and then maintained until they were killed at week 40. Three and six hours after the MNU injection, the DNA methylation adducts, O-6-methyldeoxyguanine (O-6-medG) and 7-methyldeodeoxyguanine (7-medG), were immunohistochemically revealed to be markedly more fre quent in urothelial preneoplasias or neoplasias than in normal cells. These adducts were rapidly repaired, and although 7-dmeG in tumor cell s still persisted after 72 h, they appeared essentially to have return ed to normal levels. At the termination, conversion of transitional ce ll carcinomas (TCC) to squamous cell carcinomas (SCC) of the urinary b ladder was significantly increased in the BBN + MNU group. The extent of invasion was also significantly greater with the additional MNU tre atment. Expression of p21 protein, detected by immunohistochemistry, w as comparable between the groups. Mutations in the H-ras gene were obs erved in one case each of the BBN and BBN + MNU groups, and both cases showed a G:C to A:T transition at codon 12. The present study thus su ggested that while an additional single treatment with MNU of rats bea ring BBN-induced bladder neoplasias is associated with significant, po ssibly mutation-dependent tumor progression, H-ras mutations are not n ecessary events.