Dose-related effects of phenobarbital on hepatic glutathione-S-transferase activity and ligandin levels in the rat.

To determine which individual parameters contribute to the increased bilirubin clearance which follows phenobarbital administration in the rat, dose response studies are being conducted relating changes in various aspects of bilirubin transport to the dose of phenobarbital administered. The relationships between phenobarbital dose, immunoreactive ligandin concentrations, and cytosolic glutathione-S-transferase (GSHT) enzymatic activities were determined in the 100,000g liver cytosol obtained from non-fasted male Sprague-Dawley rats, treated for 6 days (ip) with either phenobarbital at various doses ranging from 1 to 125 mg/kg/day or distilled water. Ligandin levels were measured by radioimmunoassay employing an antiserum which reacts with both GSHT-1 (Ya) and -2 (Yc) subunits. Ligandin concentration increased in a dose-dependent fashion, achieving a maximal observed value of 278% of control at the highest administered phenobarbital dose. Values were significantly elevated compared to controls at doses as low as 3 mg/kg/day. GSH-dependent delta 5-3-ketosteroid isomerase (KSI) activity, which reflects predominantly GSH transferase subunit 1, and GSHT activity against 1-chloro-2,4-dinitrobenzene (CDNB) also increased over the entire range of phenobarbital doses administered. Both of these enzymatic activities were highly correlated with immunoreactive ligandin levels (KSI: r = 0.89, p less than 0.005; GSHT (CDNB): r = 0.92, p less than 0.001). By contrast, GSHT activity against 1,2-dichloro-4-nitrobenzene (DCNB), which resides principally on GSHT subunits not present in ligandin, did not correlate significantly with measured ligandin concentrations. These studies indicate that phenobarbital is capable of inducing immunoreactive ligandin concentrations and related enzymatic activities at doses as small as 5% of those commonly employed to demonstrate this effect.