The chlorinated dioxin TCDD Is prototypical of many halogenated, polycyclic hydrocarbons that exist as environmental pollutants. A lethal dose of TCDD in animals results in a progressive weight loss and general debiliation, with death occurring in a few weeks. However, the mechanism of toxicity of TCDD and related compounds is not known. Initial studies in our laboratories have demonstrated that administration of TCDD to rats induces hepatic lipid peroxidation as measured by increase in: NAHPH-dependent hepatic membrane production of malondialdehyde (MDA), a by- product of lipid peroxidation; whole liver MDA content; microsomal content of conjugated dienes; and hepatic DNA damage (single strand breaks). Other investigators have shown that iron deficiency provides partial protection against the toxic effects of TCDD, while TCDD added in vitro to microsomes in the presence of complexed ferric iron enhances NAHPH-dependent lipid peroxidation. These results suggest that iron and free radicals are involved in TCDD induced lipid peroxidation, although direct evidence is lacking. The role of iron in TCDD induced hepatic lipid peroxidatin can be assessed by varying dietary levels of iron, adding varius forms of iron to microsomes, treating animals with the potent iron chelator desferrixamine, adding desferrioxamine to hepatic microsomes, determining effect of TCDD on hepatic content and distribution of iron and analyzing the various forms in which iron exists in microsomes before and after TCDD administration. Lipid peroxidation will be measured colorimetrically and by HPLC. We shall demonstrate the production of free radicals after TCDD administration to rats both in vivo and in vitro using the spin trapping agents, alpha- phenyl N-tert-butyl nitrone (PBN) and trimethoxyphenyl-tert- butyl nitrone (MO3PBN) in conjunction with an electron spin resonance (ESR) spectrometer. Rats will be treated with TCDD and given one of the spin traps 2-4 hours prior to sacrifice. Hepatic lipids will be isolated and analyzed by ESR. For the in vitro studies, hepatic microsomes will be isolated from TCDD treated animals and incubated in the presence of NADPH, with or without added iron, and a spin trap. The resulting products will be analyzed by ESR. The above studies will enable us to verify our hypothesis concerning the role of iron and free radicals in TCDD induced lipid peroxidation, and provide the basis for advanced investigations of the molecular basis of action of TCDD.
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