In vitro and in vivo response of hepatocytes from hepatic nodules to the mitoinhibitory effects of phenobarbital.

Abstract

One of the proposed mechanisms by which phenobarbital (PB) promotes hepatocarcinogenesis in the rat is by differential mitoinhibition. However, our earlier studies indicated that PB inhibited DNA synthesis in vitro in hepatocytes isolated from both surrounding non-nodular liver and hepatic nodules promoted by orotic acid (OA). Since nodules generated by one promoter need not necessarily be resistant to another promoter, the present study was undertaken to determine whether foci/nodules promoted by PB itself are resistant to the mitoinhibitory effects of PB. Accordingly, rats were initiated with diethylnitrosamine (DENA, 200 mg/kg i.p) and promoted with PB (0.07% of PB as its sodium salt) in their drinking water for 16 or 33 weeks. In vitro studies indicated that PB (3-5 mM) inhibited DNA synthesis induced by epidermal growth factor (EGF) in hepatocytes from surrounding non-nodular liver as well as from nodules promoted by PB for 33 weeks. In another experiment, initiated rats exposed to PB for 33 weeks were subjected to either two-thirds partial hepatectomy (PH) or sham hepatectomy. Hepatocytes were labelled with tritiated thymidine in vivo for 48 h. Autoradiographic analysis indicated that in the presence of PB, the hepatocytes from both foci/nodules and the surrounding non-nodular liver responded to PH to the same extent. In addition, they both responded to PH less efficiently as compared to the corresponding controls. Further, initiated rats exposed to PB for 16 weeks when subjected to PH and killed 4 weeks thereafter, the percentage area occupied by gamma-glutamyltranspeptidase-positive foci/nodules in the PB group increased, but to the same extent as in initiated control rats not exposed to PB. The above results raised an interesting possibility that the lack of resistance of the PB-promoted nodules to the mitoinhibitory effects of PB may be because the PB-promoted nodule does not express a resistant phenotype. To examine this aspect, the response of hepatocytes from 33 week PB-promoted nodules to the mitoinhibitory effects of OA was examined. The results indicated that OA (60-120 microM) inhibited EGF-induced DNA synthesis in hepatocytes isolated from both nodules as well as from surrounding non-nodular liver. These results suggest that PB is a mitoinhibitor but may not provide a strong differential growth advantage to foci/nodules in response to a proliferative stimulus. Further, the nodules promoted by PB do not appear to express the resistant phenotype, defined as being resistant to the mitoinhibitory effects of OA and PB.