Potentiation of epidermal growth factor-induced DNA synthesis in rat hepatocytes by phenobarbitone: possible involvement of oxidative stress and kinase activation

A transient induction of S phase DNA synthesis is a common feature of non-genotoxic rodent hepatocarcinogens when administered in vivo. In the present study the ability of phenobarbitone (PB) to induce S phase DNA synthesis in primary cultures of rat hepatocytes was investigated. In the absence of serum or growth factors PB was not a mitogen per se. However, stimulation of S phase DNA synthesis by epidermal growth factor (EGF) was enhanced by co-culture with PB. This effect was both time and concentration dependent. The lowest concentration of PB that significantly enhanced the effect of EGF was 10 μM and the effect was maximal at 1.0 mM. At a concentration of 2.0 mM PB no longer enhanced EGF-induced S phase DNA synthesis. Hepatocyte cultures pretreated with PB (0.1 mM) for 2 days were more responsive to the induction of S phase DNA synthesis by EGF for the subsequent 2 days. Despite the inhibition of PB enhancement of S phase DNA synthesis by the antioxidant dimethylthiourea, reduced glutathione was not depleted by PB treatment nor were oxidized glutathione or lipid peroxides elevated. Western blotting analysis showed that PB had no effect on epidermal growth factor receptor (EGFR) autophosphorylation per se after 1 and 48 h culture, enhanced sensitization of EGFR therefore does not appear to contribute to the enhancement of S phase DNA synthesis by PB. In contrast, treatment of hepatocytes with PB for 12 h resulted in a small but statistically significant activation of p42/44 MAP kinase activity and activation of protein kinase C, as measured by redistribution of enzyme activity from a soluble to a particulate compartment of hepatocytes. Therefore, PB-mediated changes in protein kinase activity may contribute to the potentiation this compound affords.