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-g enotoxic rodent hepatocarcinogens when administered b vivo. In the present study the ability of phenoharbitone (PB) to induce S phase DNA synthesis in primary cultures of rat hepatocytes was investigated, In the absence of se rum or growth factors PB was not a mitogen per sa, However, stimulation of S phase DNA synthesis by epidermal growth factor (EGF) was enhanced by co-c ulture with PB, This effect was both time and concentration dependent, The lowest concentration of PB that significantly enhanced the effect of EGF wa s 10 muM 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 cultur es pretreated with PB (0.1 mM) for 2 days were more responsive to the induc tion 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 d imethylthiourea, reduced glutathione was not depleted by PB treatment nor w ere oxidized glutathione or lipid peroxides elevated. Western blotting anal ysis showed that PB had no effect on epidermal growth factor receptor (EGFR ) autophosphorylation per se after 1 and 48 h culture, enhanced sensitizati on 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 fo r 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 r edistribution of enzyme activity from a soluble to a particulate compartmen t of hepatocytes. Therefore, PB-mediated changes in protein kinase activity may contribute to the potentiation this compound affords.