title: Metabolic activation of 4-hydroxyanisole by isolated rat hepatocytes.
abstract: A tyrosinase-directed therapeutic approach for treating malignant melanoma uses depigmenting phenolic prodrugs such as 4-hydroxyanisole (4-HA) for oxidation by melanoma tyrosinase to form cytotoxic o-quinones. However, in a recent clinical trial, both renal and hepatic toxicity were reported as side effects of 4-HA therapy. In the following, 4-HA (200 mg/kg i.p.) administered to mice caused a 7-fold increase in plasma transaminase toxicity, an indication of liver toxicity. Furthermore, 4-HA induced-cytotoxicity toward isolated hepatocytes was preceded by glutathione (GSH) depletion, which was prevented by cytochrome p450 inhibitors that also partly prevented cytotoxicity. The 4-HA metabolite formed by NADPH/microsomes and GSH was identified as a hydroquinone mono-glutathione conjugate. GSH-depleted hepatocytes were much more prone to cytotoxicity induced by 4-HA or its reactive metabolite hydroquinone (HQ). Dicumarol (an NAD(P)H/quinone oxidoreductase inhibitor) also potentiated 4-HA- or HQ-induced toxicity whereas sorbitol, an NADH-generating nutrient, prevented the cytotoxicity. Ethylenediamine (an o-quinone trap) did not prevent 4-HA-induced cytotoxicity, which suggests that the cytotoxicity was not caused by o-quinone as a result of 4-HA ring hydroxylation. Deferoxamine and the antioxidant pyrogallol/4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL) did not prevent 4-HA-induced cytotoxicity, therefore excluding oxidative stress as a cytotoxic mechanism for 4-HA. A negligible amount of formaldehyde was formed when 4-HA was incubated with rat microsomal/NADPH. These results suggest that the 4-HA cytotoxic mechanism involves alkylation of cellular proteins by 4-HA epoxide or p-quinone rather than involving oxidative stress.
authors.affiliation: Department of Pediatric Laboratory Medicine, Hospital for Sick Children and Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
authors.forename: M Y
authors.initials: MY
authors.lastname: Moridani
authors.forename: S S
authors.initials: SS
authors.lastname: Cheon
authors.forename: S
authors.initials: S
authors.lastname: Khan
authors.forename: P J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D000840:Anisoles / Q000737:chemistry / Q000493:pharmacokinetics* / Q000633:toxicity; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D051381:Rats
chemical_list: D000840:Anisoles; C009760:mequinol