Vol. 58, Issue 3, 542-551, September 2000

Human Microsomal Epoxide Hydrolase Is the Target of Germander-Induced Autoantibodies on the Surface of Human Hepatocytes

Veronique De Berardinis, Claude Moulis, Michele Maurice, Philippe Beaune, Dominique Pessayre, Denis Pompon, and Jacqueline Loeper

Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Laboratoire propre associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette (V.D.B., D.Po., J.L.); Laboratoire de Pharmacognosie, Université Toulouse III (C.M.); de l'Institut National de la Santé et de la Recherche Médicale:Unité 538, Université Saint-Antoine, Paris (M.M.); Unité 490, Université Paris V (P.B.); and Unité 481, Hôpital Beaujon, Clichy, France (D.Pe.)

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b₅ in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkykated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.