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tumor promotion, since hepatocytes from rats withdrawn from PhB for 1 month were unresponsive to 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.