Whole livers were quickly removed from the body and the gall bladder was excised. After weighing, the livers were divided into two portions. the first of which was frozen in liquid N2 for subsequent lipid extractions. The second portion of liver was washed with ice-cold 0.25 M sucrose-1 mM EDTA-0.1% ethanol, finely minced, and homogenized with 10-12 strokes (3:1, buffer:tissue) with a glass-Teflon tissue homogenizer on ice. A portion of this homogenate was frozen in liquid N2 for later determinations of peroxisomal fatty acyl-CoA oxidase activity and liver protein concentrations. An S-9 fraction was prepared from the remainder of the homogenate by ultracentrifugation for 20 min at 9000g and 4°C. The supernatant was aspirated and frozen in liquid N2 until assayed for ethoxyresorufin O-deethylase (EROD) activity.

Ethoxyresorufin O-deethylase (EROD) The hepatic EROD activity was measured in S-9 homogenates as described by Pohl and Fouts (1980). The presence of 10 uM of dicumarol in the reaction buffer inhibited DT-diaphorase activity (Lubet et al., 1985). Fluorescence of resorufin was measured after incubation of the homogenate mixture at 37°C for 20 min using a Perkin-Elmer (Oak Park, IL) fluorimeter. Under these conditions. the production of resorufin from ethoxyresorufin was in the linear range of the standard curve. The fluorescence of resorufin in the supernatant of the reaction buffer after the addition of methanol was used as a standard. Resorufin was obtained from Aldrich Chemical Co.. Inc. and ethoxyresorufin was from Pierce Chemical Co. (Rockford. IL).

Peroxisomal fatty acyl-CoA oxidase determination Peroxisomal fatty acyl-CoA oxidase activity was determined using the methodology of Walusimbi-Kisitu and Harrison (1983) with palmitoyl CoA as the substrate. In this assay the peroxidase-catalysed decrease in the fluorescence of scopoletin is coupled with palmitoyl CoA dependent H2O2 production. The original method was modified as follows: 50 ul scopoletin ( 1.15 mg/100 ml ethanol), 50 ul Triton X-100 (0.1% Research Products Int. Corp., Elk Grove Village, IL). 50 ul fatty acid-free BSA (15 mg/ml buffer), 30 ul palmitoyl CoA (2.5 mg/ml buffer). 30 ul horseradish peroxidase (4 mg/ml buffer; 1300 U/ml). and 30 ul FAD (4.15 mg/ml buffer) were added to 2.8 ml potassium phosphate buffer (50 mM, pH 7.4). This mixture was warmed for 5 min at 37°C and 10 ul of homogenate (diluted 1:9 or I :99) was added to start the reaction. The fluorescence emission at 467 nm (slit width 5 nm) was recorded at room temperature using a Perkin-Elmer fluorimeter with excitation at 365 nm (slit width 2 nm). The fatty acyl-CoA oxidase activity was estimated by the rate of change in fluorescence compared to controls (no enzyme and/or substrate). Varying concentrations of H2O2 (Fisher Scientific, Fair Lawn, NJ) were used to prepare the standard curve.

Determination of hepatic lipid concentrations Livers were thawed and extracted with chloroform:methanol (2:1) according to the procedure of Folch et al. (1956). After washing with 0.9% NaCl to remove nonlipid contaminants the chloroform layer was aspirated and dried under N2 and the residue was dissolved in 5 ml of fresh chloroform. Total hepatic lipids were determined with the sulpho-phospho-vanillin reaction of Frings and Dunn ( 1970) using olive oil as a standard.

Hepatic protein concentration The protein concentration was determined by the dye-binding method of Bradford (1976) using bovine serum albumin as a standard and is expressed as the percentage of milligrams. defined as milligrams of protein per milligram of liver times 100.

Statistical analysis Each variable was analyzed by random factorial ANOVA. Multiple comparisons were made with Duncan’s multiple range test with p < 0.05 where significance was observed (p < 0.05).

Female C57BL/6J Mice