title: The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress.
abstract: Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.
authors.affiliation: Faculty of Pharmacy, University of Toronto, 19 Russell Street, Room 522, Toronto, ON M5S 2S2, Canada.
authors.forename: Tom S
authors.initials: TS
authors.lastname: Chan
authors.forename: Nandita
authors.initials: N
authors.lastname: Shangari
authors.forename: John X
authors.initials: JX
authors.lastname: Wilson
authors.forename: Helen
authors.initials: H
authors.lastname: Chan
authors.forename: Roger F
authors.initials: RF
authors.lastname: Butterworth
authors.forename: Peter J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001205:Ascorbic Acid / Q000096:biosynthesis*; D001555:Benzene Derivatives / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006168:Guinea Pigs; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D048289:Imidazolidines / Q000494:pharmacology; D007659:Ketones / Q000494:pharmacology; D015227:Lipid Peroxidation; D018384:Oxidative Stress; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000975:Antioxidants; D001555:Benzene Derivatives; D048289:Imidazolidines; D007659:Ketones; D017382:Reactive Oxygen Species; C018637:phorone; C026411:sorbinil; D005978:Glutathione; C007164:cumene hydroperoxide; D001205:Ascorbic Acid