Acetaminophen-glutathione conjugate formation in a coupled cytochrome P-450-glutathione S-transferase assay system mediated by subcellular preparations from adult and weanling rat tissues

Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP-GSH, conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP-GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver > BHA-treated weanling liver > control adult liver > control weanling liver > BHA-adult kidney > control adult kidney > BHA weanling kidney > control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug. (C) 2002 Elsevier Science Ltd. All rights reserved.