Xenobiotic Metabolism

Environmental Health Sciences

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Abstract

Pulegone is a monoterpene found in the essential oil of several mints, including Pennyroyal. These mints are used in flavoring food and beverages. Pennyroyal and Pennyroyal oil have also been used as an abortifacient. The use of Pennyroyal oil, which contains about 85% pulegone, as an abortifacient has resulted in serious toxicity and death. Pulegone has been nominated to the NTP for toxicity and carcinogenicity studies. Recent studies on pulegone have involved characterization of its metabolism in mice. Based on urinary metabolites pulegone is metabolized by rats by three major pathways: 1) hydroxylation followed by glucuronidation; 2) reduction to menthone/isomenthone followed by hydroxylation; 3) Michael addition of glutathione. Metabolism of pulegone in mice differs from rats in that several mercapturic acid and aromatic metabolites identified in rat urine are not present in mouse urine. This difference in metabolite profile could be due to the much more rapid biotransformation in mice via pathway 1. The presence of mercapturic acids implies the formation of alkylating agents. The lack of mercapturic acid metabolites correlates the observation that pulegone is less toxic to mice in the NTP studies. However, there are literature reports that high doses of pulegone deplete GSH in mice. Bile duct cannulation studies in mice revealed the presence of metabolites formed from direct addition of GSH to pulegone. Probably due to differences in transport, GSH adducts in rats are converted in liver and kidney to mercapturic acids, while in mice the adducts are excreted in bile. In both rats and mice the GSH adducts and /or the corresponding mercapturic acids are derived from direct conjugation via Michael addition with pulegone. The accepted mechanism for pulegone toxicity is metabolism first to menthofuran followed by further oxidation of the furan ring to an enonal. Interestingly none of the nearly 20 pulegone metabolites we have identified are unambiguously required to be formed via menthofuran. These observations led us to conduct metabolism studies of menthofuran to determine if there are metabolites in common and to test the hypothesis that a reactive intermediate is derived from menthofuran. In male rats 4 urinary metabolites were common to both chemicals supporting the intermediacy of menthofuran in pulegone metabolism. Other menthofuran metabolites were clearly derived from oxidation of the furan ring followed by reaction with nucleophiles such as GSH, water, sulfite, and taurine, verifying the postulated reactive intermediate. Juglone is an hydroxynaphthoquinone that has been isolated from walnut hulls. Interest in juglone arose from proposed NTP studies of black walnut extract derived from walnut hulls and sold as a dietary supplement. As a naphthoquinone juglone was expected to be reactive. In fact the reaction of juglone with GSH was almost instantaneous. Addition of GSH to the quinone gives a hydroquinone which is easily oxidized back to a quinone which in turn reacts with an additional GSH. Double reaction of quinones/hydroquinones and the related nephrotoxicity has been well explored. Thus it would expected that juglone may have a deleterious effect on the kidney. Tissue distribution studies substantiate the reactivity of juglone; there was notably more radioactivity at the site of application following dermal, iv and gavage exposures. The tissue to blood ratio was persistently high in kidney (not from contained urine), consistent with the expected nephrotoxicity. Many local anesthetics, such as lidocaine and prilocaine, contain aromatic amines (2,6-xylidene and o-toluidene in the examples cited) as part of their structure, as amides. Amides are cleaved by esterases/amidases freeing the amine. o-Toluidene and 2,6-xylidene are both positive in carcinognicity studies and are the amine component in several anesthetics. This was the basis for the nomination of this class of chemicals for NTP study. Because of the pharmacological activity of the anesthetics, a 2-year bioassay was not recommended. Instead a metabolism/mechanism study was suggested. To explore the possibility that potentially mutagenic metabolites result from biotransformation of anesthetics, the Ames mutagenicity of urine from rats treated with either 2,6-xylidine, o-toluidene, prilocaine or lidocaine was determined. The intention was to ultimately isolate and identify the mutagen/premutagen. Male rats were dosed by gavage with each of the chemicals (all doses at 100 mg/kg; n=4 rats/treatment group). Urine samples were collected for 8 hr after dosing, filter-sterilized, incubated with or without glucuronidase/sulfatase and Aroclor 1254-induced rat S9, and tested with Salmonella typhimurium strains TA98 and TA100. Results demonstrated that urine of rats receiving either of the four test chemicals (lidocaine, prilocaine, 2,6-xylidine, or o-toluidine), were neither toxic nor mutagenic to either strain TA98 or TA100. Urine from benzpyrene-treated animals (positive control) was mutagenic. No potent anesthetic-derived mutagens were detected in this study; therefore, the mutagenic potential of either lidocaine, prilocaine, 2,6-xylidine, or o-toluidine will not be further investigated. Studies of the polybrominated diphenyl ethers have only recently started. Initial results from the tetrabromodiphenyl ether indicates that the chemical is rapidly absorbed from an oral exposure. Absorption appears to be the same whether the vehicle is aqueous or corn oil. Ten daily doses of 0.1 umol/kg results in tissue concentrations almost exactly 10 times a single 0.1 umol/kg dose and approximately the same as a 1.0 umol/kg dose. This result implies nearly 100% absorption, a long half life and therefore bioaccumulation. Another interesting observation out of these preliminary studies is that thymus and thyroid have high tissue concentrations of radioactivity.