Role of cyclic AMP in the inhibition of mouse hepatocyte intercellular communication by liver tumor promoters

Abstract

The liver tumor promoters phenobarbital (PB) (20–500 μg/ml) and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (1–10 μg/ml) inhibited intercellular communication between primary cultured B6C3F1 mouse hepatocytes after 8 hr of treatment. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabeled donor hepatocytes to recipient hepatocytes. The addition of either dibutyryl cyclic AMP (N6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) (0.001–0.1 mm) or caffeine (0.01–1 mm) decreased or completely abolished the inhibitory effects of PB and DDT on intercellular communication. Cyclic AMP (adenosine 3′:5′-cyclic monophosphate; cAMP) in primary cultured mouse hepatocytes was measured by radioimmunoassay. Cyclic AMP in nontreated, freshly plated cultures declined from 4.2 ± 0.7 pmol/mg protein after 1 hr in culture to 2.4 ± 0.5 pmol/mg protein after 8 hr in culture. Phenobarbital at 250 and 500 μg/ml significantly decreased cyclic AMP below control values after 1 hr of treatment. However, no difference in the amount of cyclic AMP was detected between control and PB-treated cultures after 2, 4, and 8 hr in culture or with lower PB concentrations. DDT at 10 μg/ml decreased cAMP levels in the hepatocytes after 1, 2, 4, and 8 hr of treatment. No effects were seen after 8 hr of treatment or with lower DDT concentrations. DDT (10 μg/ml) also decreased cAMP levels in 24-hr-old cultures while PB (500 μg/ml) had no effect. Addition of dibutyryl cAMP (0.1 mm) or caffeine (1.0 mm) to freshly plated cultures elevated cAMP levels 50-fold and twofold, respectively. These data suggest that the inhibition of mouse hepatocyte intercellular communication by PB and DDT at the highest concentrations tested may be mediated by transient decreases in intercellular cAMP levels.