Lack of promoting effect of clonazepam on the development of N-nitrosodiethylamine-initiated hepatocellular tumors in mice is correlated with its inability to inhibit cell-to-cell communication in mouse hepatocytes

The tumor-promoting ability of clonazepam (CZP), a widely usedbenzodiazepine anticonvulsant, was investigated in an in vivomouse liver tumor promotion assay and an in vitro mouse hepatocyteintercellular communication assay. The development of preneoplastichepatocellular foci of cellular alteration and hepatocellularneoplasms was studied in male B6C3F1 mice initiated, at 5 weeksof age, with a single i.p. injection of N-nitrosodiethylamine(NDEA; 90 mg/kg body weight) in tricaprylin, followed by administrationof either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%)in diet beginning 2 weeks after carcinogen injection and continuingto 60 weeks of age. Several mice from each group were killedafter 9, 21, 33 or 53 weeks on test diet, and portions of liverand other organs were fixed In formalin and examined histotogically.Unlike PB, CZP did not promote the development of preneoplastichepatocellular foci or neoplasms (adenomas and carcinomas) inNDEA-initiated mice. Following limited (2 weeks) dietary exposureat 0.15%, CZP was a potent inducer of hepatic P450IIBl-mediatedalkoxyresorufin O-dealkylase activities. In contrast, the degreeof induction in hepatic tissue from mice fed 0.136% CZP for53 weeks was markedly lower than that in mice fed 0.05% PB for53 weeks. In the in vitro assay, diazepam, a strong tumor promoterin mouse liver, significantly inhibited mouse hepatocyte gapjunctional intercellular communication, while CZP had no significanteffect on this parameter. Thus, CZP, a drug structurally relatedto diazepam, is inactive as a liver tumor promoter in mice.