Date of Award


Degree Type


Degree Name

Doctor of Philosophy


This research was concerned with the role of metallothionein (MT) in chemical toxicity mediated by reactive intermediates generated from xenobiotic and oxygen metabolism. Metallothionein(s) are ubiquitous, low molecular weight, cysteine-rich (30% of total amino acids), metal binding proteins. These proteins have been shown to be important in the regulation of essential metal metabolism and in the detoxication of toxic metals. In the present research it was hypothesized that (i) because of its high cysteinyl thiolate concentration, MT is reactive towards intermediates generated from xenobiotic metabolism; and (ii) the metal (Zn, Cu) released from MT concomitant with the inactivation of metal binding sites would influence the modulating role of MT in chemical toxicity.;To determine the role of MT thiolate groups in scavenging free radicals generated from xenobiotic metabolism, experiments were carried out to characterize the reaction between CCl{dollar}\sb4{dollar} and purified Cd,Zn-MT, focusing on MT thiols as potential sites of interaction. Incubation of MT with CCl{dollar}\sb4{dollar} resulted in time-dependent depletion of MT thiols with concurrent reduction in the metal binding sites of the protein; this was due to CCl{dollar}\sb4{dollar}-linked oxidation of MT, rather than the covalent binding of {dollar}\sp{lcub}14{rcub}{dollar}CCl{dollar}\sb4{dollar} metabolites, indicating an antioxidative property of MT thiol groups.;Because Zn and Cu ions by themselves have antioxidant or prooxidant properties, respectively, the influence of Zn-MT or Cu-MT in oxidative stress was examined to determine the role of metal complement of MT in chemical toxicity. For this, Ehrlich cells with different concentrations of Zn-MT or Cu-MT were exposed to H{dollar}\sb2{dollar}O{dollar}\sb2{dollar}. In vitro toxicity testing revealed that the H{dollar}\sb2{dollar}O{dollar}\sb2{dollar} toxicity was negatively correlated with cellular Zn-MT concentrations but directly related to cellular Cu-MT concentrations. H{dollar}\sb2{dollar}O{dollar}\sb2{dollar} treatment resulted in oxidation of MT thiolate groups, loss of its metal-binding capacity, and translocation of MT-bound Zn or Cu to other cellular sites. Study with Cu and Fe chelating agents as well as antioxidant showed that Cu-MT enhanced sensitivity to H{dollar}\sb2{dollar}O{dollar}\sb2{dollar} by a Cu-dependent Fenton chemistry. The direct effect of Zn or Cu in H{dollar}\sb2{dollar}O{dollar}\sb2{dollar} was also examined in control cells. Zn and Cu produced inhibition and enhancement of H{dollar}\sb2{dollar}O{dollar}\sb2{dollar} toxicity, respectively, indicating the inherent antioxidative and prooxidative properties of Zn and Cu, respectively.;To investigate the toxicological significance of the prooxidative property of Cu-MT, the influence of Cu-MT in vivo was also investigated. Neonatal guinea pigs were used as the biological model because copper and Cu-MT are known to exist in high concentrations in the liver of 3-day-old guinea pigs but declined to low adult levels by day 7 of life. Comparison of the hepatotoxic responses to iron nitrilotriacetate (FeNTA) in the three age groups revealed heightened sensitivity in the 3-day-old guinea pigs but not in 7-day-old and adult animals. FeNTA treatment resulted in oxidation of MT thiolate groups, loss of its metal binding capacity and translocation of MT-bound Cu to other cellular sites. Results of in vitro studies confirmed the prooxidative function of Cu-MT and indicated involvement of Cu released from MT.;In conclusion, the data of these studies showed that MT thiolate groups possess antioxidative property and that MT can act either as an antioxidant or a prooxidant, a property related to the metal complement of the metalloprotein.



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