Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis: direct measurement of communication in vivo.

We have developed a simple method to measure gap-junctional intercellular communication (GJIC), by means of microinjection/dye transfer assay, in liver slices freshly removed from the rat. Using this method and immunostaining of connexin 32 (cx32), the major liver gap junction protein, we studied sequential changes of GJIC during chemical hepatocarcinogenesis in male Fischer-344 rats under a modified Solt-Farber protocol (3 weeks 4 day exposure regimen). Four weeks after commencement of the protocol, there was a substantial decrease in GJIC in the liver parenchyma, which was free from focal lesions. The decrease in GJIC persisted up to at least the 15th week of treatment, while a decrease in the number of immunoreactive cx32 spots was evident only at 4 weeks of post-protocol commencement. Most enzyme-altered (GST-P-positive) focal lesions showed markedly lower GJIC and a significantly lower number of cx32-positive spots than surrounding hepatocytes. Most GST-P-positive foci showed a selective lack of GJIC with surrounding heptocytes. Hepatocellular carcinomas arising 1 year after the carcinogenic regimen had significantly reduced communicational capacity accompanied by a large decrease in cx32 expression. These results suggest that a progressive decrease in homologous as well as heterologous GJIC in preneoplastic lesions occurs during rat hepatocarcinogenesis, and that preneoplastic lesions with the most prominent disorders in GJIC may be more likely to develop into carcinomas.