EGF Receptors of Hepatocytes from Rats Treated with Phenobarbital Are Sensitized to Down-Regulation by Phenobarbital in Culture

Abstract

Hepatocytes from Fisher 344 rats treated with the liver tumor promoter phenobarbital (PhB; 0.1% in the drinking water, 2–3 months) exhibit reduced epidermal growth factor (EGF) binding and EGF-induced mitogenesis in culture. Similar responses are induced by >1 mM PhB added to the culture medium of hepatocytes from untreated rats. In this study, we demonstrated that hepatocyte EGFr protein, as determined by immunoblotting, was unchanged by treatment of rats with PhB. However, hepatocytes from PhB-treated rats are more sensitive to PhB in culture in that decreased EGF binding occurred with 0.05 mM PhB, a concentration also attained in plasma of rats exposed to PhB. Sensitization was reversible, as is tumor promotion, since hepatocytes from rats withdrawn from PhB for 1 month were unresponsive to <3 mM PhB. EGFr down-regulation by a series of barbiturates correlated well with their known activities as tumor promoters and CYP2B1/2 inducers, with pentobarbital and PhB yielding high activities, while barbital was intermediate and barbituric acid, 5-phenylbarbituric acid, and 5-ethylbarbituric acid were ineffective. Differentiated hepatocyte function was required for PhB-induced EGFr down-regulation since HepG2 and rat liver epithelial cells were unresponsive, but involvement of CYP2B1/2 activity was discounted by the failure of metyrapone to inhibit the response in PhB-induced hepatocytes. These studies support a role for impaired EGFr function in PhB liver tumor promotion due to effects on existing EGFr protein and suggest that EGFr down-regulation by PhB in culture is independent of CYP2B1/2 activity but shares mechanistic components involved in its transcriptional activation by PhB.