title: Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells.
abstract: The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA.
authors.forename: Nikhil M
authors.initials: NM
authors.lastname: Vad
authors.forename: Imam H
authors.initials: IH
authors.lastname: Shaik
authors.forename: Reza
authors.initials: R
authors.lastname: Mehvar
authors.forename: Majid Y
authors.initials: MY
authors.lastname: Moridani
mesh_terms: D000818:Animals; D001711:Biotransformation; D045744:Cell Line, Tumor; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008297:Male; D008545:Melanoma / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000502:physiology; D010226:Parabens / Q000493:pharmacokinetics* / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D012995:Solubility
chemical_list: D010226:Parabens; D017382:Reactive Oxygen Species; C012313:ethyl-p-hydroxybenzoate; D014442:Monophenol Monooxygenase; D005978:Glutathione