title: Assessment of the role of the glutathione and pentose phosphate pathways in the protection of primary cerebrocortical cultures from oxidative stress.
abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H2O2). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-(13)C2, 6,6-(2)H2] glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H2O2, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H2O2 exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H2O2-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.
authors.affiliation: Department of Radiology, University of Michigan, Ann Arbor 48109-0648, USA.
authors.forename: O
authors.initials: O
authors.lastname: Ben-Yoseph
authors.forename: P A
authors.initials: PA
authors.lastname: Boxer
authors.forename: B D
authors.initials: BD
authors.lastname: Ross
mesh_terms: D000640:Amitrole / Q000494:pharmacology; D000818:Animals; D019328:Buthionine Sulfoximine; D002273:Carcinogens / Q000494:pharmacology; D002374:Catalase / Q000037:antagonists & inhibitors; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000502:physiology; D002540:Cerebral Cortex / Q000166:cytology; D002614:Chelating Agents / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors; D005978:Glutathione / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000633:toxicity; D007501:Iron / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents; D018384:Oxidative Stress / Q000502:physiology*; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D051381:Rats; D017262:Siderophores / Q000494:pharmacology; D004113:Succimer / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D002273:Carcinogens; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007773:Lactates; D018696:Neuroprotective Agents; D017262:Siderophores; C060658:dimethyl mercaptosuccinate; D008717:Methionine Sulfoximine; D019344:Lactic Acid; D019328:Buthionine Sulfoximine; D006861:Hydrogen Peroxide; D004113:Succimer; D007501:Iron; D002374:Catalase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D005947:Glucose; D003676:Deferoxamine; D000640:Amitrole