sorbinil and phorone

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.

Topics: Animals; Antioxidants; Ascorbic Acid; Benzene Derivatives; Glutathione; Guinea Pigs; Hepatocytes; Imidazolidines; Ketones; Lipid Peroxidation; Oxidative Stress; Rats; Reactive Oxygen Species

Using isolated rat hepatocytes we have shown that glutathione (GSH) depletion by glutathione-S-transferase (GST)-catalyzed conjugation with 1-bromoheptane or phorone was accompanied by a significant elevation in ascorbate synthesis. Glycogenolysis was also stimulated without a significant rise in glucose synthesis. Furthermore, when glycogenolysis was stimulated in control hepatocytes by increasing intracellular cAMP levels (with glucagon or dibutyryl cAMP), cellular glucose levels, but not ascorbate levels, increased. These data suggest that GSH depletion can stimulate ascorbate synthesis independently of glucose synthesis and that hepatocytes can direct glycogenolysis towards ascorbate synthesis during GSH conjugation.

Topics: Animals; Ascorbic Acid; Bucladesine; Cyclic AMP; Diamide; Dithiothreitol; Fructose; Glutathione; Glutathione Disulfide; Glycogen; Hepatocytes; Heptanes; Imidazoles; Imidazolidines; Ketones; Male; Rats; Rats, Sprague-Dawley; Uridine Diphosphate Glucose