Acetaminophen alters microsomal ryanodine Ca2+ channel in HepG2 cells overexpressing CYP2E1

Acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding of drug metabolites to liver proteins. Acetaminophen metabolites have been shown to affect rat liver microsomal Ca2+ stores, but the mechanism is not well understood. The aim of the current work was to find out if the metabolism of acetaminophen by CYP2E1 affects ryanodine-sensitive Ca2+ stores in the endoplasmic reticulum of transduced HepG2 cells. Five millimoles acetaminophen decreased proliferation of CYP2E1-overexpressing HepG2 cells, increased cytosolic Ca2+ levels and produced significant cytotoxicity, while only little, mostly anti-proliferative effects were found in HepG2 cells lacking CYP2E1. CYP2E1 inhibitor-4-methylpyrazole decreased drug cytotoxicity in transduced cells and normalized elevated Ca2+ levels. Acetaminophen cytotoxicity was significantly higher in CYP2E1 expressing cells with depleted glutathione. In the cells engineered to overexpress CYP2E1, an increased [3H]ryanodine affinity (by 45%) and increased ligand maximal binding to ryanodine receptors (by 64%) was observed, most probably due to increased association rate of [3H]ryanodine. Ca2+ loading was decreased by about 53% in microsomal fractions isolated from transduced cells treated with acetaminophen and by 92% in glutathione depleted transfected cells treated with the drug. Ca2+/Mg2+-ATPase activity was unchanged in all microsomal fractions. Such effects were not observed in cells lacking CYP2E1. Our results confirm significant role of CYP2E1 in metabolic activation of acetaminophen and indicate that ryanodine receptors located in the liver endoplasmic reticulum are sensitive targets for acetaminophen metabolites.