Date of Award

1-1-1982

Embargo Period

1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Rosalie Crouch

Abstract

Superoxide dismutase (SOD) is a scavenger of the toxic superoxide radical and thereby protects the integrity of cell components from oxidative damage. This enzyme is an essential part of the biological defense system of aerobic cells, both directly by removing the superoxide anion and indirectly by preventing the formation of hydroxyl radical when superoxide reacts with hydrogen peroxide. The chemical diabetogenic agent streptozotocin, alloxan and Vacor are reported to directly inhibit the activity of this enzyme. The role of SOD in protection of pancreatic beta cells from the toxicity of diabetogenic drugs and the interaction of the drug with enzyme was thus investigated. By immunochemical techniques, immunoreactive SOD was localized to the beta cells of human islets of Langerhans. Relatively little immunoreactive SOD was associated with either pancreatic acinar cells or with the glucagon-secreting alpha cells. By immunoelectron microscopy, the enzyme was associated with the cytosol and hormone secretory granules of beta cells while relatively little was associated with the plasma membrane, mitochondria, rough endoplasmic reticulum or nucleus. These data were confirmed by biochemical assay of SOD enzymatic activity/mg soluble protein in the extracts of isolated islets as compared with similar assays of soluble protein from whole pancreas. SOD was purified by chloroform-ethanol extraction, ion exchange chromatography and gel filtration of soluble extracts from erythrocytes and islets of the same dog to test an hypothesis of the presence of an islet-specific isoenzyme of SODs. By the criteria of pl, electrophoretic mobility and uv spectroscopy, these proteins are identical. The islet SOD was confirmed to be of the copper-zinc type by cyanide sensitivity and chloroform-ethanol stability. Both erythrocyte and islet SODs were inhibited by 40-50% upon direct exposure of the extracts to either streptozotocin, alloxan or Vacor. Neither enzyme could be protected from the drugs by inclusion of high concentrations of D-glucose or 3-0-methylglucose which are known to protect intact islets from these drugs. Therefore, if inhibition of endogenous SOD is part of the mechanism of alloxan and/or streptozotocin toxicity, and if this inhibition can be prevented by D-glucose or 3-0-methylglucose, respectively, protection by these carbohydrates must not be at the level of drug/enzyme interaction but rather must involved denial of access of the drugs to the intracellular enzyme either by surface receptor blockade or by induction of intracellular processes which rapidly dispose of the drug preventing accumulation of a concentration sufficient to inhibit the SOD. The mechanism of the interaction was further characterized by using 12C-ailoxan and bovine erythrocyte SOD. 14C-alloxan was seen to associate with the protein over the first 5 min, remain associated for 20 min and then slowly dissociate. The initial association phase was dependent upon accessibility to the active site copper. Visible spectroscopy of the interaction of bovine erythrocyte SOD with alloxan revealed changes in the visible spectrum of bovine erythrocyte SOD and cyanide-SOD which were comparable to these seen after treatment of either SOD with hydrogen peroxide. These changes were also dependent upon the accessibility of active site copper. Alloxan was observed to interact with either bovine erythrocyte SOD or copper to result in prevention of the rapid changes in the UV spectrum of alloxan seen over 20 min in basic solution. Zinc could not prevent these changes. The relevance of these interactions to viable cellular systems was examined by exposure of islets to alloxan in tissue culture. Extracts of these islets showed 40-60% inhibition of endogenous SOD enzymatic activity with no effect of SOD immunoreactivity. This inhibition was blocked by the presence of high concentrations of ambient glucose in the medium. Prior to extraction of soluble protein, islets were challenged with low or high glucose to stimulate insulin secretion and to confirm the inhibitory effects of alloxan in presence of low glucose and protection by high glucose. In these studies, maintenance of SOD specific activity was seen to correlate with maintenance of beta cell secretory response to glucose challenge. Since these data implied a critical role for SOD in the maintenance of beta cell integrity, the use of SOD and SOD-like copper salicylates were explored as a potential prophylaxis against chemical toxins. Native bovine erythrocyte SOD protected against streptozotocin toxicity in isolated islets in tissue culture. Cyanide-inactivated bovine erythrocyte SOD was ineffective. In vivo, native bovine erythrocyte SOD could protect against streptozotocin given 10 sec or 50 min after SOD but protection was less effective when the two agents were mixed and injected simultaneously, suggesting that protection likely occurred at the beta cell level, possible involving associated and/or internalization. Cyanide-inactivated bovine erythrocyte SOD was ineffective as a prophylactic as was a polyethylene glycol-derivatized bovine erythrocyte SOD with prolonged circulation time. Copper-(II)-(3,5-diisopropylsalicylate)2, a lipophilic SOD-like compound with a rate constant for dismutation in the same order of magnitude as that for native SOD, was also effective in attenuation of streptozotocin diabetes when injected 15 min prior to the drug. Copper-free 3,5-diisopropylsalicylote was ineffective; salicylate-induced hyperglycemia, renal glycosuria and copper hepatotoxicity were eliminated as possible interferences. Although significant morbidity resulted from certain of these copper complexes with SOD activity, the ease and economy with which they may be employed recommends them as possible model prophylactic agents in experimental diabetic syndromes.

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