Inhibition of Mouse Hepatocyte Gap Junctional Intercellular Communication by Phenobarbital Correlates with Strain-Specific Hepatocarcinogenesis

The inhibition of gap junctional intercellular communication (GJIC) is a common effect of nongenotoxic carcinogens and might be a biomarker for these agents. To further test this relationship, we hypothesized that phenobarbital would inhibit mouse hepatocyte GJIC and this would correlate with strain-specific hepatocarcinogenicity. Phenobarbital is a strong nongenotoxic hepatocarcinogen in B6C3F1 mice, but not in C57BL/6 mice. Hepatocytes were isolated from males of both strains, placed in coculture with rat liver epithelial cells, and treated with phenobarbital for up to 14 days. Male mice were also administered PB by single intraperitoneal injection (0.1 mg/kg), then sacrificed 24 h later, or given phenobarbital in the drinking water (500 ppm) for 14 days before sacrifice. GJIC was assayed in cocultures by fluorescent dye microinjection and in isolated liver tissue by fluorescent dye “cut-loading.” Phenobarbital decreased GJIC only in cultured B6C3F1 hepatocytes; this was dose-responsive and temporary, because hepatocyte GJIC returned to control levels within 24 h of phenobarbital exposure. Administration of phenobarbital to mice for 14 days also decreased hepatocyte dye coupling in B6C3F1 liver, but this effect was not seen in C57BL/6 mice or observed after a single administration of the drug. Phenobarbital did not alter connexin32 and connexin26 expression, but increased hepatic Cyp2b1 expression and the liver weight:body weight ratio in both strains. In summary, phenobarbital inhibited mouse hepatocyte GJIC in vivo and in vitro and in correlation with strain-specific hepatocarcinogenicity. These data support the hypothesis that decreased GJIC is a biomarker for nongenotoxic carcinogens and involved in their carcinogenic mechanism.