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, ratswere given a single i.p. injection of N-methyl-N-nitrosourea(MNU) at a dose of 50 mg/kg body wt at week 20 (at a stage whenbladder tumor development had already occurred), and then maintaineduntil they were killed at week 40. Three and six hours afterthe MNU injection, the DNA methylation adducts, O⁶-methyldeoxyguanine(O⁶-medG) and 7-methyldeoxyguanine (7-medG), were immunohistochemicallyrevealed to be markedly more frequent in urothelial preneoplasiasor neoplasias than in normal cells. These adducts were rapidlyrepaired, and although 7-dmeG in tumor cells still persistedafter 72 h, they appeared essentially to have returned to normallevels. At the termination, conversion of transitional cellcarcinomas (TCC) to squamous cell carcinomas (SCC) of the urinarybladder was significantly increased in the BBN + MNU group.The extent of invasion was also significantly greater with theadditional MNU treatment Expression of p21 protein, detectedby immunohistochemistry, was comparable between the groups.Mutations in the H-ras gene were observed in one case each ofthe BBN and BBN + MNU groups, and both cases showed a G:C toA:T transition at codon 12. The present study thus suggestedthat while an additional single treatment with MNU of rats bearingBBN-induced bladder neoplasias is associated with significant,possibly mutation-dependent tumor progression, H-ras mutationsare not necessary events.