Identification of Novel Electrophilic Metabolites of Piper methysticum Forst. (Kava) Benjamin M. Johnson, †,‡ Sheng-Xiang Qiu, § Shide Zhang, § Fagen Zhang, † Joanna E. Burdette, †,‡ Linning Yu, † Judy L. Bolton, †,‡ and Richard B. van Breemen* ,†,‡ Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, and Herbstandard, Inc., 1743 Canyon View Court, Chesterfield, Missouri 63017 Received December 16, 2002 Dietary supplements containing Piper methysticum Forst. (kava) have been implicated in multiple cases of liver injury in humans, including 10 recently reviewed cases in which patients required liver transplantation following the usage of kava-containing products (Centers for Disease Control and Prevention, reprinted. (2003) J. Am. Med. Assoc. 289, 36-37). To investigate a possible mechanism(s) of kava-induced hepatotoxicity, an extract of kava was incubated in vitro with hepatic microsomes, NADPH, and GSH. Electrophilic intermediates that were generated via metabolic activation were trapped as GSH conjugates and removed from the protein mixture using ultrafiltration. Positive ion electrospray LC-MS/MS with precursor ion scanning was used for the selective detection of GSH conjugates, and LC-MS n product ion scanning was used to elucidate their structures. Using this in vitro MS-based screening assay, two novel electrophilic metabolites of kava, 11,12-dihydroxy-7,8-dihydrokavain- o-quinone and 11,12-dihydroxykavain-o-quinone, were identified. Mercapturic acids of these quinoid species were not detected in the urine of a human volunteer following ingestion of a dietary supplement that contained kava; instead, the corresponding catechols were metabolized extensively to glucuronic acid and sulfate conjugates. These observations indicate that quinoid metabolites, under most circumstances, are probably not formed in substantial quantities following the ingestion of moderate doses of kava. However, the formation of electrophilic quinoid metabolites by hepatic microsomes in vitro suggests that such metabolites might contribute to hepatotoxicity in humans when metabolic pathways are altered (e.g., because of a drug interaction, genetic difference in enzyme expression, etc.) or if conjugation pathways become saturated. Introduction In March 2002, a consumer advisory was issued by the United States Food and Drug Administration warning that severe liver injury might be caused by the consump- tion of dietary supplements containing kava (1). Accord- ing to the advisory, more than 25 cases of liver injury associated with kava including hepatitis, cirrhosis, and liver failure have been reported in other countries. The Centers for Disease Control and Prevention recently reviewed 10 such case reports (two in the United States, six in Germany, and two in Switzerland) in which liver transplants were necessary following hepatic failure that was associated with the use of kava-containing supple- ments (2). Adverse neurological and dermatological reac- tions to kava have also been reported (3-8). These cases have prompted several European regulatory agencies to issue warnings about the safety of supplements contain- ing kava or remove them from the marketplace alto- gether. Regulatory action by the Food and Drug Admin- istration concerning the legal sale of such supplements in the United States might be necessary as more details about kava toxicity become available (1). Mechanisms that have been proposed to explain ad- verse reactions to kava include an immunoallergic re- sponse (9) and the inhibition of cytochrome P450 isoforms by various kava constituents (10). Elevated levels of γ-glutamyltransferase have also been reported among heavy kava users (11). The chemical compounds that are unique to kava and appear to be responsible for its anxiolytic activity (12) include the 5,6-dihydro-R-pyrones KV, 1 MT, and their 7,8-dihydro derivatives (Figure 1). Several stable metabolites of these compounds were identified in urine and feces of rats following administra- tion via gavage (13); however, reactive metabolites resulting from their bioactivation have not been reported previously. * To whom correspondence should be addressed. Tel: (312)996-9353. Fax: (312)996-7107. E-mail: breemen@uic.edu. † Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago. ‡ UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago. § Herbstandard, Inc.. 1 Abbreviations: CID, collision-induced dissociation; DDKV, 11,12- dihydroxy-7,8-dihydrokavain; DHKV, 11,12-dihydroxykavain; DKV, 7,8-dihydrokavain; DMT, 7,8-dihydromethysticin; KV, kavain; MT, methysticin. 733 Chem. Res. Toxicol. 2003, 16, 733-740 10.1021/tx020113r CCC: $25.00 © 2003 American Chemical Society Published on Web 05/15/2003