Hepatic Metabolism of Carcinogenic β‑Asarone

β-Asarone (<b>1</b>) belongs to the group of naturally occurring phenylpropenes like eugenol or anethole. Compound <b>1</b> is found in several plants, e.g., <i>Acorus calamus</i> or <i>Asarum europaeum</i>. Compound <b>1</b>-containing plant materials and essential oils thereof are used to flavor foods and alcoholic beverages and as ingredients of many drugs in traditional phytomedicines. Although <b>1</b> has been claimed to have several positive pharmacological effects, it was found to be genotoxic and carcinogenic in rodents (liver and small intestine). The mechanism of action of carcinogenic <i><b>allylic</b></i> phenylpropenes consists of the metabolic activation via cytochrome P450 enzymes and sulfotransferases. <i>In vivo</i> experiments suggested that this pathway does not play a major role in the carcinogenicity of the <i><b>propenylic</b></i> compound <b>1</b> as is the case for other propenylic compounds, e.g., anethole. Since the metabolic pathways of <b>1</b> have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of <b>1</b> in liver microsomes of rats, bovines, porcines, and humans using ¹H NMR, HPLC-DAD, and LC-ESI-MS/MS techniques. We synthesized the majority of identified metabolites which were used as reference compounds for the quantification and final verification of metabolites. Microsomal epoxidation of the side chain of <b>1</b> presumably yielded (<i>Z</i>)-asarone-1′,2′-epoxide (<b>8a</b>) which instantly was hydrolyzed to the corresponding <i>erythro</i>- and <i>threo</i>-configurated diols (<b>9b</b>, <b>9a</b>) and the ketone 2,4,5-trimethoxyphenylacetone (<b>13</b>). This was the main metabolic pathway in the metabolism of <b>1</b> in all investigated liver microsomes. Hydroxylation of the side chain of <b>1</b> led to the formation of three alcohols at total yields of less than 30%: 1′-hydroxyasarone (<b>2</b>), (<i>E</i>)- and (<i>Z</i>)-3′-hydroxyasarone (<b>4</b> and <b>6</b>), with <b>6</b> being the mainly formed alcohol and <b>2</b> being detectable only in liver microsomes of Aroclor 1254-pretreated rats. Small amounts of <b>4</b> and <b>6</b> were further oxidized to the corresponding carbonyl compounds (<i>E</i>)- and (<i>Z</i>)-3′-oxoasarone (<b>5</b>, <b>7</b>). 1′-Oxoasarone (<b>3</b>) was probably also formed in incubations with <b>1</b> but was not detectable, possibly due to its rapid reaction with nucleophiles. Eventually, three mono-<i>O</i>-demethylated metabolites of <b>1</b> were detected in minor concentrations. The time course of metabolite formation and determined kinetic parameters show little species-specific differences in the microsomal metabolism of <b>1</b>. Furthermore, the kinetic parameters imply a very low dependence of the pattern of metabolite formation from substrate concentration. In human liver microsomes, 71–75% of <b>1</b> will be metabolized via epoxidation, 21–15% via hydroxylation (and further oxidation), and 8–10% via demethylation at lower as well as higher concentrations of <b>1</b>, respectively (relative values). On the basis of our results, we hypothesize that the genotoxic epoxides of <b>1</b> are the ultimate carcinogens formed from <b>1</b>