Among the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other planar polyhalogenated hydrocarbons, such as hexachlorobenzene and PCBs, is an hepatic porphyria indistinguishable in its symptoms from clinical Porphyria Cutanea Tarda. This porphyria is characterized by massive hepatic accumulation of uroporphyrin (URO), produced by oxidation of uroporphyrinogen (UROgen), the first cyclic tetrapyrrole of the heme biosynthetic pathway. Hepatic UROgen decarboxylase (URO-D) is highly decreased in this condition. Previous years of this project have established (using mice, rats and chick in vivo and in hepatocytes in culture) that this porphyria is due to several factors which include the induction of liver specific cytochrome P4501A2 and the amount of UROgen made in the liver. We were the first to show that P4501A2 oxidizes UROgen to URO, and that ALA feeding of mice induced for p4501A2, decreases the time to onset of massive uroporphyria from 5 to less than 2 weeks, indicating the importance of UROgen availability. An important preliminary finding for this proposal is that ascorbic acid prevents URO accumulation in chick hepatocyte cultures and inhibits UROgen oxidation by isolated microsomes from animals induced for P4501A2.
Specific Aims : [1] We will continue investigations of the mechanism of the UROgen oxidation in culture and in cell free systems and examine how this oxidation leads to the inhibition of URO-D. This experiments are made feasible in cell-free systems using combinations of microsomal or reconstituted P450s with 100,000g supernatants or purified enzyme as source of URO-D. We will determine the role and mechanism of ascorbic acid in preventing the UROgen oxidation. [2] We will investigate the possible physiological role of ascorbic acid in preventing URO accumulation in hepatocyte cultures and in intact animals (mice and guinea pigs). [3] We will determine the relationship to human disease of these previous findings with experimental systems, in particular to show that human P4501A2 catalyzes UROgen oxidation and that human hepatocytes accumulate URO when treated with inducers of P4501A2 and ALA. [4] Patients with PCT will be evaluated to see if their plasma ascorbic levels are below normal and whether hepatic P4501A2 is elevated. We will also determine if there is a correlation between P4501A2 phenotype (as determined by caffeine metabolism in humans) dietary AscA deficiency and urinary excretion of URO.
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