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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES006263-03
Application #
2155121
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1993-07-01
Project End
1998-05-31
Budget Start
1994-06-01
Budget End
1995-05-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Gorman, Nadia; Trask, Heidi S; Robinson, Susan W et al. (2007) Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2. Toxicol Appl Pharmacol 221:235-42
Gorman, Nadia; Zaharia, Adrian; Trask, Heidi S et al. (2007) Effect of an oral iron chelator or iron-deficient diets on uroporphyria in a murine model of porphyria cutanea tarda. Hepatology 46:1927-834
Gorman, Nadia; Zaharia, Adrian; Trask, Heidi S et al. (2007) Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda. Hepatology 45:187-94
Trask, Heidi W; Gorman, Nadia; Dwyer, Barney E et al. (2005) Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures. Arch Biochem Biophys 439:1-11
Uno, Shigeyuki; Dalton, Timothy P; Sinclair, Peter R et al. (2004) Cyp1a1(-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria. Toxicol Appl Pharmacol 196:410-21
Grundy, Martin A; Gorman, Nadia; Sinclair, Peter R et al. (2004) High-throughput non-heme iron assay for animal tissues. J Biochem Biophys Methods 59:195-200
Gorman, Nadia; Trask, Heidi W; Bement, William J et al. (2004) Genetic factors influence ethanol-induced uroporphyria in Hfe(-/-) mice. Hepatology 40:942-50
Hon, Thomas; Dodd, Athena; Dirmeier, Reinhard et al. (2003) A mechanism of oxygen sensing in yeast. Multiple oxygen-responsive steps in the heme biosynthetic pathway affect Hap1 activity. J Biol Chem 278:50771-80
Sinclair, Peter R; Gorman, Nadia; Trask, Heidi W et al. (2003) Uroporphyria caused by ethanol in Hfe(-/-) mice as a model for porphyria cutanea tarda. Hepatology 37:351-8
Gorman, Nadia; Ross, Kerry L; Walton, Heidi S et al. (2002) Uroporphyria in mice: thresholds for hepatic CYP1A2 and iron. Hepatology 35:912-21

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