Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

William A. Pryor

Abstract

When micromolar concentrations of benzoyl peroxide (BPO) are added to rat liver mitochondria, inhibition of mitochondrial NADH-oxidase and succinoxidase is observed. The addition of a classical uncoupler of oxidative phosphorylation results in only partial release of this inhibition, suggesting that BPO inhibits both electron- and energy-transfer in mitochondria. Difference spectroscopy and artificial electron donor results indicate that BPO interacts at coupling site II between cytochromes b and c$\sb1$. BPO only yields radicals in mitochondria via inhibition of electron transport, which results in the formation of superoxide radicals. BPO also induces rapid, large-amplitude swelling of mitochondria; the swelling is dependent on the presence of monovalent cations but is independent of the presence of calcium, oxygen and respiratory substrate. BPO-induced swelling appears to be disassociated from radical production and lipid peroxidation. When isolated rat liver mitochondria are treated with millimolar concentrations of tert-butyl hydroperoxide (TBHP), methyl, tert-butoxyl and tert-butylperoxyl radicals are detected using the ESR spin- trapping technique. The addition of respiratory substrate results in a significant increase in methyl radical production, no change in the concentration of tert-butoxyl radicals, and complete loss of tert-butylperoxyl radicals. One-electron reduction of TBHP by the electron transport chain results in the formation of tert-butoxyl radicals, which subsequently undergo beta-scission to form methyl radicals. The site of interception of electrons by TBHP is shown to be cytochrome c$\sb 1$ by the use of classical chain inhibitors and depletion of mitochondrial cytochrome c. TBHP also appears to interact with a tightly-liganded metal in non-respiring mitochondria, as metal chelators have no effect on TBHP-induced radical production while sodium cyanide inhibits this process. TBHP induces lipid peroxidation in both respiring- and non-respiring mitochondria, as measured by the thiobarbituric acid test, although the degree of peroxidation is less in respiring mitochondria. Radical production is also observed when either cumene hydroperoxide or linoleic acid hydroperoxide is added to mitochondria in the presence of spin trap. The mechanisms of TBHP-induced radical production in both respiring- and non-respiring mitochondria are discussed.

Pages

182

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