Phthalic acid esters are used as plasticizers and are widely dispersed in the environment. These chemicals induce peroxisomes and have been implicated in hepatocellular carcinoma. Mechanisms for the biological response to phthalates are not clear; however, recent preliminary work from our laboratory suggests that important interactions between phthalate metabolites and lipid metabolism occur. 2-Ethylhexanol, a primary metabolite of the common plasticizer diethylexyl phthalate, inhibits hepatic ketogenesis resulting from Beta-oxidation of fatty acids in the perfused liver. It causes circulating ketone bodies to decline concomitantly with a rapid accumulation of microvascular lipid exclusively in periportal regions of the liver lobule in vivo. The purpose of the experiments is to evaluate the hypothesis that phthalates and a variety of hypolipidemic drugs which induce peroxisomes act via a common mechanism. We propose that peroxisomal inducers initially inhibit ketogenesis and cause long-chain acyl CoA compounds and triglycerides to accumulate in the liver. We suspect that increases in acyl CoA is a common link leading to the biological response to phthalates. To test this hypothesis, we will study the mechanism of inhibition of ketogenesis from endogenous fatty acids by 2-ethylhexanol and monoethylhexyl phthalate in isolated mitochondria and peroxisomes. Temporal changes in critical components of fatty acid metabolism (e.g., acyl CoA, carnitine and malonyl CoA levels and carnitine and acyl transferase activity) following infusion of ethylhexanol and related phthalates into perfused livers will be evaluated. Using micro-light guides, changes in fatty acid metabolism in periportal and pericentral regions of the liver lobule will be studied following ethylhexanol addition based on changes in NADH fluorescence. New methods to study lipid accumulation in specific zones of the liver lobule based on the fluorescence properties of the lipophilic dye Nile Red, and to study sublobular H2O2 production, a product of peroxisomal Beta-oxidation, based on changes in reflected light due to changes in catalase-H2O2 will be developed. The hypothesis that decreases in ketogenesis lead to accumulation of hepatic lipid and increases in peroxisomal marker enzymes will be tested by comparing the effects of a series of alkanols, a variety of hypolipidemic drugs and tetradecylglycidic acid, an inhibitor of acyl CoA transport into the mitochondria. Species with different responses to phthalates in vivo (mice, rats, hamsters, guinea pigs) will be compared. Since phthalates are ubiquitous environmental pollutants it is imperative that we understand their mechanism(s) of action. Information from these studies will lead ultimately to a rational means of prevention of phthalate-induced toxicity in man.
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