CYTOTOXIC/GENOTOXIC EFFECTS OF NO IN THE IMMATURE BRAIN

Nitric oxide (NO) has recently been identified as a key intracellular messenger substance in the central nervous system. Nitric oxide has recently been proposed as a key mediator of glutamatergic neurotoxicity; glutamate, in turn, has been established or proposed as a key mediator of neurotoxicity in numerous pathophysiologic mechanisms including hypoxic- ischemic injury, prolonged convulsive injury and a variety of neurometabolic diseases. A deeper understanding of the role and cellular injury mechanisms of nitric oxide in the mediation of neurotoxicity will be important step in developing a deeper understanding of a wide variety of neurological disease, in both the infant and adult. The focus in these studies will be the immature brain. Several lines of preliminary evidence support the notion that nitric oxide is a key mediator of injury to the immature brain: 1) Focal cerebral hypoxic-ischemic injury and intrastriatal injection of the NMDA-agonist quinolinic acid (QA) both lead to brain nitric oxide production in the rat ; 2) Pharmacologic inhibition of de novo nitric oxide synthesis exerts a marked neuroprotective effect against both focal cerebral ischemic injury and intrastriatal QA injection in the 7-day old rat and 3) The toxicity of the xenobiotic neurotoxin 3- nitropropionic acid (3-NPA) , which has been implicated as the toxin in an epidemic of dystonia in Chinese children, is mediated by NO, directly generated from 3-NPA. Focal cerebral hypoxic-ischemic injury, intrastriatal QA injection and exposure to 3-NPA provide rat model systems in which to study mechanisms of nitric oxide neurotoxicity in the immature brain. We propose to perform studies which extend these preliminary findings, and so more firmly establish the role of NO in these models. Nitric oxide, a highly reactive free radical, is very likely toxic when produced in excess. Evidence from non-neural systems suggests that mitochondrial enzymes enriched in iron-sulfur centers, such as Complex l of the respiratory chain , are prime targets for NO. We propose to study the activity of these key mitochondrial targets in the model systems discussed above. Preliminary evidence obtained in the sponsor's laboratory suggest that mitochondrial DNA (mtDNA) injury appears in ischemic cortex several weeks after exposure of 7-day old rats to focal cerebral hypoxic- ischemic injury. In light of recent data suggesting a genotoxic role for nitric oxide, and our preliminary data noted above, we speculate that NO may mediate the mitochondrial genotoxicity. We will explore this possibility in each of the three models discussed above by assessing the presence of mtDNA injury, utilizing a PCR strategy which will selectively amplify mtDNA with a pre-determined deletion. We will then examine animals in whom de novo nitric oxide synthesis has been pharmacologically blocked; if these fail to show mtDNA deletions, then one may conclude that NO plays some mediatory role. Further experiments, designed to search for cytosine to thymine transitions in mtDNA (which appear to be characteristic of NO) will help establish a direct genotoxic role for NO.