Oxygen radicals and oxidant stress toxicity have been causally linked to convulsions (grand mal seizures) that are life-threatening and limit the oxygen pressure that can be used in HBO therapy, the treatment of choice for gas gangrene and CO poisoning. Neurodegenerative diseases including Huntington's and amyotrophic lateral sclerosis and brain trauma and stroke have also been linked to oxygen radical toxicity mechanisms. Convulsions can be caused by excessive stimulation of excitatory neuronal receptors which also can kill neuronal cells. Quinolinic acid (QA), a metabolite of the brain kynurenine pathway from tryptophan, is the most potent endogenous excitatory and convulsive agent known, 100-X as potent as aspartate or glutamate. QA is produced in glial cells by the kynurenine pathway which includes 3-hydroxyanthranilate oxidase (HAO), the immediate producer of QA. Oxygen and superoxide are substrates, and stimulatory, for 3 pathway enzymes including HAO which requires Fe 2 + and which we have found is also inactivated by HBO. QA has been measured and found to be elevated in brain and cerebrospinal fluid in the above named neurodegenerative disorders.
The Specific Aims are focused on the thesis that HBO convulsions are caused by imbalances in production and accumulation of QA and other kynurenine pathway intermediates via effects on oxygen and iron requiring enzymes. We will convulse rats in 5 atm HBO and also study their tissues and enzymes in vitro to elucidate biochemical and toxicologic mechanisms of oxygen on the kynurenine pathway. For the in vivo work, brain microdialysis probes and high performance liquid chromatography will be used to collect and quantify fluxes of kynurenine pathway intermediates prior to, during, and after convulsions (while in the HBO chamber) under a variety of test conditions. We also will use brain slices, homogenates and glial cell primary cultures to determine the effects of oxygen (including hyperbaric) tensions, iron, and ascorbate on Km and Vmax of pathway enzymes and fluxes of intermediates through the pathway. Correlated with the latter, we also will pursue the thesis that control of Fe2+ is critical to prevent oxidative inactivation of pathway enzymes. Better understanding of oxygen- and iron-regulation of the production of QA and other receptor agonists and antagonists pathway intermediates may allow development of specific strategies for their control in hyperbaric oxygen therapy and in neurodegenerative brain disorders.
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