Presence of a threshold for promoting effects of phenobarbital on diethylnitrosamine-induced hepatic foci in the rat.

The dose dependence of the hepatopromoting effects of phenobarbital (PB) was investigated in a rat liver medium-term bioassay (Ito test) to elucidate a practical threshold level. F344 rats were given a single i.p. injection of diethylnitrosamine (200 mg/kg body wt) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0, 1, 2, 4, 7.5, 15 or 500 p.p.m. in experiment 1 and 0, 0.01, 0.1 or 0.5 p.p.m. in experiment 2 were fed to the rats for 6 weeks. Experiment 3 was conducted to confirm previous data using the same medium-term bioassay, with PB at doses of 0, 1, 2, 4, 7.5, 15, 30, 60, 125, 250 or 500 p.p.m. fed to the rats. All surviving animals were killed at week 8 in these experiments and their livers were immunohistochemically examined for expression of glutathione S-transferase placental form (GST-P). Quantitative values for GST-P-positive foci in the liver were increased dose dependently in rats given 60-500 p.p.m. PB. However, those for doses in the range 1-7.5 p.p.m. demonstrated a decrease as compared with the control group (0 p.p.m.), with significant differences observed for 1 and 2 p.p.m.. The results for 15-30 and 0.01-0.5 p.p.m. were comparable with the control values. Examination of transforming growth factor-alpha (TGF-alpha)-positive foci also produced similar results to those for GST-P in experiment 1. Immunohistochemical staining of TGF-alpha and GST-P using serial liver sections demonstrated that the TGF-alpha-positive foci comprised a sub-population of the GST-P-positive lesions, being approximately 1/8-1/10th as common in livers of animals treated with PB. TGF-alpha-positive foci were almost always negative on immunostaining for TGF-beta. Western blotting for proteins CYP2B1, 2C6 and 3A2 revealed a good correlation between changes in GST-P-positive foci and CYP3A2 protein expression. The finding of inhibition effects at low doses of PB confirms the presence of a threshold level for promoting effects by PB on liver carcinogenesis in rats.