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Metabolic activation of 4-hydroxyanisole by isolated rat hepatocytes
M Y Moridani1, S S Cheon, S Khan
1Department of Pediatric Laboratory Medicine, Hospital for Sick Children and Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
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.
Insights
4-hydroxyanisole (4-HA) causes liver toxicity in melanoma treatment by depleting glutathione and forming toxic metabolites. Its cytotoxic mechanism involves protein alkylation, not oxidative stress.
Area of Science:
- Biochemistry
- Toxicology
- Dermatology
Background:
- Melanoma treatment utilizes tyrosinase-directed prodrugs like 4-hydroxyanisole (4-HA).
- Clinical trials reported renal and hepatic toxicity associated with 4-HA therapy.
- Understanding 4-HA's cytotoxic mechanism is crucial for improving melanoma treatment safety.
Purpose of the Study:
- To elucidate the mechanism of 4-hydroxyanisole (4-HA)-induced cytotoxicity.
- To investigate the role of glutathione depletion and reactive metabolites in 4-HA toxicity.
- To differentiate between oxidative stress and other mechanisms in 4-HA-induced cell damage.
Main Methods:
- Administered 4-HA to mice and isolated hepatocytes.
- Assessed liver toxicity via plasma transaminase levels.
- Investigated the role of glutathione (GSH), cytochrome p450, NAD(P)H/quinone oxidoreductase, o-quinone trapping, antioxidants, and formaldehyde formation.
Main Results:
- 4-HA administration significantly increased plasma transaminase levels, indicating liver toxicity.
- Cytotoxicity was preceded by GSH depletion, preventable by cytochrome p450 inhibitors.
- The primary cytotoxic mechanism involves protein alkylation by 4-HA epoxide or p-quinone, not oxidative stress.
Conclusions:
- 4-HA induces hepatotoxicity through a mechanism involving GSH depletion and subsequent protein alkylation.
- The cytotoxic effects are not mediated by o-quinone formation or oxidative stress.
- Findings suggest alternative therapeutic strategies to mitigate 4-HA toxicity in melanoma treatment.