Decreased Uroporphyrinogen Decarboxylase Activity Research Articles

S2631 Porphyria Cutanea Tarda, Type 2, in a Man With a Novel UROD Mutation

Introduction: Porphyria cutanea tarda (PCT) is usually caused by acquired defects in uroporphyrinogen decarboxylase (UROD) activity in the liver. 20% of cases occur in patients who have a genetically determined partial decrease in UROD activity. We describe a case of a man with a novel UROD mutation causing PCT type 2. Case Description/Methods: A 77-year-old man was evaluated for 6 months of painful blistering over his dorsal hands, forearms and head [Fig 1a]. His medical history was notable for hypertension, melanoma, and stroke. He reported heavy alcohol and tobacco use. He had no family history of porphyria. He worked for years at a manufacturing facility, with exposure to various industrial chemicals. More recently, he had exposure to weed killers, carburetor cleaners, and wood varnish. Exam revealed tense bullae with milia and hyperpigmented macules on dorsal hands and forearms. Biopsies revealed a subepidermal blister containing a small number of neutrophils with fibrin and evidence of festooning of the vessels consistent with porphyria. Labs revealed AST 63 U/L, ALT 64 U/L, alkaline phosphatase 54 U/L, and total bilirubin 0.8 mg/dL. Iron profile was unremarkable, and antibodies against HIV and hepatitis A, B and C were negative. Total plasma porphyrins were elevated to 13.4 mcg/dL. Fractionated urine, stool and plasma porphyrin levels were obtained [Table 1]. Urinary uro- and hepta-carboxyl porphyrins were markedly elevated, consistent with PCT. The patient was advised to start hydroxychloroquine 100 mg 3 times weekly and to avoid alcohol, tobacco use and sun exposure. Genetic testing revealed a novel mutation of UROD gene (c.224 G >C; p. Arg 75 Pro) with red blood cell UROD activity decreased by 50%. He returned to clinic 3 and 7 months after the index visit. He had stopped alcohol use, but continued smoking cigarettes. His hands were improved [Fig 1b]. Repeat urinary porphyrins showed improvement [Table 1]. The mutation in UROD is predicted to have a major effect on protein structure and function [Fig 1c]. Discussion: Inherited partial deficiency in UROD is not sufficient to cause clinically manifest PCT. Other factors that increase oxidative stress are also required. This patient has a mutation in the UROD gene not heretofore described in PCT type 2, but that we show markedly affects the structure and activity of the protein product. Nonetheless, the patient did not develop clinically manifest PCT until age 77, emphasizing the importance of acquired factors in disease pathogenesis.Figure 1.: A. Dorsal right hand at presentation B. Dorsal right hand at 7 month follow-up C. Structure of URO-D showing the substrate (yellow) in the active site located at the C-terminal end of the beta-sheets (magenta) that form the core of the TIM-barrel structure. The location of arginine 75 (Arg 75) is shown in green. Arg 75 is the penultimate residue in one of the eight corresponding alpha-helices that surround the central core. Arg 75 forms two hydrogen bonds with glutamic acid 45 (Glu 45) of the preceding helix in the structure. This hydrogen bonding network supports the overall tertiary fold of the protein in this otherwise solvent-exposed region of the protein. The UROD mutation of the patient (Arg 75 Pro) disrupts the helix with premature termination and more importantly eliminates the hydrogen bonding network.Table 1-A.: Results of Selected Laboratory Studies.Table 1-B.: Results of Selected Laboratory Studies.

Comprehensive cytochrome P450 CYP1A2 gene analysis in French caucasian patients with familial and sporadic porphyria cutanea tarda

Porphyria cutanea tarda (PCT), the most frequent type of porphyria, results from decreased uroporphyrinogen decarboxylase (UROD) activity. Two forms of PCT have been described: a familial form (fPCT) characterized by the inherited decrease of UROD activity in all tissues and a sporadic form (sPCT) characterized by decreased UROD activity in the liver. Cytochrome P450 CYP1A2 plays a major role in triggering experimental uroporphyria in rodents. It has been suggested that the highly inducible -163A/A genotype of the CYP1A2 gene could confer a heightened risk of PCT in patients. To examine the impact of CYP1A2 polymorphisms on the clinical course of PCT. We performed an extensive CYP1A2 gene analysis in 96 (48 fPCT and 48 sPCT) unrelated French caucasian patients with PCT and in 99 healthy volunteers of similar ethnic origin. Results We did not observe any difference in CYP1A2 allele distribution, including a novel and rare CYP1A2 c.1063C>T (p.R355W) single nucleotide polymorphism. In addition, we compared the frequency of the -163A highly inducible allele both in patients with symptomatic fPCT (n = 48) and in asymptomatic UROD gene mutations carrier relatives (n=54). This variant was not over-represented in patients with PCT vs. either healthy volunteers or asymptomatic UROD gene mutation carriers. The CYP1A2 genotype does not appear to be a major susceptibility factor in the development of fPCT or sPCT in the French population.

Liver Cirrhosis Induced by Porphyria Cutanea Tarda: A Case Report and Review

Porphyria cutanea tarda (PCT) is a metabolic disorder that results in a decrease in uroporphyrinogen decarboxylase activity. It is characterized by photosensitivity, bullae formation, and skin pigmentation. There are four types of PCT: acquired, familial, toxic, and hepatoerythropoietic. Uroporphyrin levels are elevated in the urine of PCT patients. PCT can be differentiated from other porphyrias by its clinical characteristics and the porphyrin levels in the serum, erythrocytes, urine, and feces. This metabolic disorder can lead to liver dysfunction as well as histological changes such as fatty infiltration or hepatic fibrosis. PCT rarely manifests as liver cirrhosis. We report herein a case of PCT-induced liver cirrhosis that progressed to hepatic failure.

Porphyrie cutanée tardive révélée par le voriconazole

Voriconazole is a systemic antifungal drug that can induce phototoxic reactions suggestive of porphyria cutanea tarda (PCT); however, porphyrin levels in urine, blood and stool remain within the normal range. Superficial cheilitis is frequently associated with this clinical picture; it is believed to be related to drug-induced impairment of endogenous retinoid metabolism. We report a case of true PCT associated with cheilitis, both occurring soon after the introduction of voriconazole and partially disappearing after withdrawal of this drug. A 65-year-old man with a past history of excessive alcohol consumption presented with typical features of PCT associated with a mild superficial desquamating cheilitis. Both symptoms had appeared 12 days after initiation of oral voriconazole for a cavitary aspergillosis. Laboratory tests confirmed a sporadic case of PCT. Withdrawal of voriconazole (replaced by itraconazole) resulted in complete disappearance of the cheilitis but incomplete remission of the PCT. Ultimately, the patient was successfully treated by venous puncture. This patient had both voriconazole-induced superficial cheilitis and a true PCT which seemed related to the same drug. The mechanism by which voriconazole may have revealed PCT remains elusive and could possibly have involved decreased uroporphyrinogen decarboxylase activity in the liver or potentiation of the phototoxic effects of porphyrins by the cutaneous toxicity of voriconazole. On presentation of a clinical picture of PCT-like photosensitivity in a patient on voriconazole, laboratory investigations should be performed routinely to rule out true PCT, even in cases of associated cheilitis.

Toxicity of hexachlorobenzene and its transference from microalgae ( Chlorella kessleri) to crabs ( Chasmagnathus granulatus)

The aim of this work was to study the transference of hexachlorobenzene from a green alga ( Chlorella kessleri) to an estuary crab ( Chasmagnathus granulatus), and to analyze the toxic effects that the xenobiotic has on the latter. The effect of hexachlorobenzene uptake was evaluated measuring oxidative stress, Uroporphyrinogen decarboxylase activity and morphometric parameter alteration, and also performing a histological analysis of crab hepatopancreas. Results demonstrated that hexachlorobenzene enters the alga, is accumulated in it, and then transferred into the crab, causing a decrease in Uroporphyrinogen decarboxylase activity in both organisms. The high malondialdehyde levels detected in crab hepatopancreas after the toxic treatment suggested the existence of hexachlorobenzene-induced lipid peroxidation. Antioxidant defenses such as superoxide dismutase activity and reduced glutathione content fell below normal values on the fourth week of treatment. At the same time, the hepatosomatic index, used as a morphometric parameter, reduced 20% with respect to the control. The histological analysis revealed epithelium disorganization in hepatopancreas tubules, confirming the existence of structural damage caused by hexachlorobenzene.

Genetic factors influence ethanol-induced uroporphyria in Hfe (—/—) mice

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain. (Hepatology 2004;40:942-950).

Genetic factors influence ethanol-induced uroporphyria in Hfe(-/-) mice.

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.

The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage.

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.

Porphyrins, porphyrin metabolism, porphyrias. III. Diagnosis, care and monitoring in porphyria cutanea tarda - suggestions for a handling programme

Deficiency of the fifth enzyme in haem synthesis, uroporphyrinogen decarboxylase (UPGD), may give rise to accumulation and excretion of poly-carboxylated porphyrins, as well as to clinical manifestations in the form of a phototoxic skin reaction and liver engagement leading to cirrhosis and hepatocellular cancer. The cutaneous reaction, presenting as skin fragility and blisters on areas exposed to sun-porphyria cutanea tarda (PCT)-develops only in individuals with a remaining hepatic UPGD activity less than 20% of normal. Experimental results and clinical observation give evidence that PCT is a multifactorial disease. In some individuals a 50% decrease in UPGD activity is a consequence of inheritance of an allele with a mutation in the gene programming for the enzyme, but in these gene carriers, as well as in the other patients with overt PCT, the activity of the hepatic enzyme is reduced below the critical level by the action of specific inhibitors. In the generation of the enzyme inhibitors, iron plays a central role by promoting the formation of reactive oxygen species, a process where a specific class of cytochrome enzymes; cytochrome P450 1A (CYP4501A), participates. The varying individual susceptibility to development of the disease can be discussed in terms of differences in a spectrum of factors that affect the availability of the free form of this element in the liver, or its pathogenic action. In the article the roles of chronic viral infection, alcohol abuse and exposition to polyhalogenated cyclic hydrocarbons are considered in the light of effects on the availability of iron in the liver. Some genetic prerequisites for susceptibility to PCT-inducing agents are included in a tentative model for the disease, i.e. mutations in the UPGD gene and in the HFE gene affected in haemochromatosis, as well as genetically steered inducibilities of the genes programming for CYP4501A and the rate-limiting enzyme in haem synthesis, 5-aminolevulinate synthase. With the pathogenic model as a basis the different therapeutic strategies that can be applied are discussed, and suggestions for a handling programme for the patient presenting with PCT put forward.

A conserved cysteine residue in yeast uroporphyrinogen decarboxylase is not essential for enzymatic activity.

Uroporphyrinogen decarboxylase catalyzes the fifth step of heme biosynthesis in Saccharomyces cerevisiae. Studies utilizing sulfhydryl-specific reagents suggest that the enzyme requires a cysteine residue within the catalytic site. This hypothesis was tested directly by site-directed mutagenesis of highly conserved cysteine-52 to serine or alanine. Plasmids containing these mutations were able to complement a hem6 mutant strain. In addition, properties associated with decreased uroporphyrinogen decarboxylase activity were not detected in the mutant strain transformed with these mutant plasmids. These results suggest that the conserved cysteine-52 by itself is not essential for enzymatic activity.

Studies on the Mechanism of Uroporphyrinogen Decarboxylase Inhibition in Hexachlorobenzene-Induced Porphyria in the Female Rat

Hexachlorobenzene (HCB)-induced porphyria occurs in female, but not male, rats after a delay of 35 days following HCB treatment. Uroporphyrinogen decarboxylase (UROD) inhibition has been proposed as a primary causative event. To determine whether there also exists a delay phase and a sexual dimorphism for UROD inhibition, groups of male and female rats were given HCB (100 mg/kg/day) from Days 1 to 5. Hepatic uroporphyrin III was markedly increased only after Day 33. Liver cytosol UROD activity in HCB-treated female rats with porphyria at Days 33, 40, 47, 54, and 100 was decreased by over 70% compared to concurrent control, whereas treated male rats as well as nonporphyric female rats had UROD activity comparable to control levels at Days 6, 12, 19, 26, 33, 40, 47, and 54. Level of immunoreactive UROD in cytosol of porphyric rats was not modified by HCB. No gender-related differences in liver cytosol radiolabel level ([14C]HCB given as the fifth dose) were found at Days 6 and 30. Chromatography of liver cytosol showed nonspecific binding of radiolabel to proteins for males, porphyric and nonporphyric females, and loss of UROD activity did not correlate with the amount of radiolabel in the UROD-containing fractions. Thus, the gender-specific decrease in UROD activity observed when porphyria develops in female rats (delay of about 4 weeks), as well as the persistence of low activity and porphyria for months, suggests that UROD inhibition was causally related to porphyria.

Time course of hexachlorobenzene-induced alterations of lipid metabolism and their relation to porphyria

A great deal of information concerning the effects of hexachlorobenzene on the haem metabolic pathway has been obtained but little is known about the effects of the drug on lipid metabolism. Consequently, the time course of phospholipid metabolism alteration caused by this xenobiotic was evaluated as related to changes in porphyrin metabolism with the aim to understand better the interregulation of both metabolisms. Female Wistar rats were treated with HCB (1 g/kg) over a 1–8 week period. Individual phospholipid content, [ 32P] incorporation, total lipid content, lipid peroxidation, uroporphyrinogen decarboxylase activity, its inhibitor generation and porphyrin content, were the parameters measured in the liver of treated rats. Phospholipid metabolism—with the exception of sphingomyelin—presents a biphasic behaviour, in both the endogenous contents and de novo synthesis. The turning point between both phases is the time at which levels of porphyrin and conjugated dienes increase, the latter compounds being involved in oxidative processes. On the other hand, sphingomyelin decreases continuously during the 8 weeks of treatment. It was also found that the malondialdehyde content increased during the early stages. The time sequence for haem metabolism parameters showed that the accumulation of porphyrins occurs after the decrease in uroporphyrinogen decarboxylase activity and the enzyme inhibitor formation, which are early events (first and second weeks). Porphyrins could not by themselves exacerbate uroporphyrinogen decarboxylase impairment or inhibitor generation. This study shows that hexachlorobenzene alters simultaneously phospholipid and porphyrin metabolisms from the early stages, and generates an oxidative environment that favours porphyrinogens and lipid oxidation at later stages. So, this oxidative environment links the alterations on both metabolisms.

Decreased Uroporphyrinogen Decarboxylase Activity in Patients with End-Stage Renal Disease Undergoing Hemodialysis

Raised plasma uroporphyrin levels were found in all the 14 patients with end-stage renal disease studied, 7 of whom were on continuous ambulatory peritoneal dialysis (CAPD) and 7 on hemodialysis (HD). The elevation observed in the HD group was higher than that noted in the CAPD group; 18-fold in the former and 13-fold in the latter. The difference in uroporphyrin plasma levels between the two dialysis populations might be explained, at least partially, by the reduced activity of uroporphyrinogen decarboxylase (UROD), the enzyme which converts uroporphyrinogen to coproporphyrinogen. A decrease of 48% was noted in the HD group, whereas no change was observed in the CAPD group. A significant negative correlation (r = -0.37, p < 0.01) was found between the concentration of uroporphyrin in plasma and the activity of UROD. In view of the data shown, it is suggested that erythrocyte UROD activity should be interpreted with caution in HD patients suspected of having porphyria cutanea tarda.

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture.

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10,000g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein.min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3'4,4'-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This results accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.

Development of hexachlorobenzene porphyria in rats: Time sequence and relationship with lipid peroxidation

The relationship between the development of porphyria and free-radical formation induced by hexachlorobenzene was studied in iron-overloaded rats. The first sign of porphyria, an increase in porphyrins in the liver, was detected at day 22. Liver malondialdehyde was also increased at day 22. During the following weeks, liver porphyrins and malondialdehyde increased simultaneously, accompanied by a decrease in uroporphyrinogen decarboxylase activity and glucose-6-phosphate activity in liver, and a high excretion of porphyrins in the urine. In the rats given hexachlorobenzene, changes were detected in the pattern of lipids in the liver microsomes. In comparison with the controls, there were decreases in C20:4 and C22:5 fatty acids, whereas the fatty acid C20:3w6 was increased. In this study of hexachlorobenzene-induced liver damage there was no difference in the time course of the development of porphyria and that of lipid peroxidation.

Uroporphyrin accumulation in cultured chick embryo hepatocytes: Comparison of 2,3,7,8-tetrachlorodibenzo- p-dioxin and 3,4,3′,4′-tetrachlorobiphenyl

Uroporphyrin (URO) accumulation caused by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and 3,4,3′,4′-tetrachlorobiphenyl (TCB) in cultured chick embryo hepatocytes was found to depend on the concentration of the added polyhalogenated aromatic compound, and on either the addition of 5-aminolevulinic acid or the induction of 5-aminolevulinic acid synthase. TCDD alone did not cause more than a slight increase in uroporphyrin, whereas TCB alone caused considerable uroporphyrin accumulation associated with increased 5-aminolevulinic acid synthase activity. However, in the presence of exogenous 5-aminolevulinic acid, TCDD was more potent than TCB in causing uroporphyrin accumulation. The concentrations of TCDD or TCB which maximally induced ethoxyresorufin deethylase activity, an indicator of induced cytochrome P450 activity, were lower than those required for maximal uroporphyrin accumulation. Furthermore, ethoxyresorufin deethylase activity was found to decline at concentrations of TCDD or TCB which caused maximum uroporphyrin accumulation. Pretreatment with 3-methylcholanthrene enhanced uroporphyrin accumulation, whereas addition of inhibitors of cytochrome P450 decreased uroporphyrin accumulation. Uroporphyrin accumulation occurred without a decrease in uroporphyrinogen decarboxylase activity, and was unrelated to the degree of conversion of the polyhalogenated aromatic compounds to water-soluble metabolites. Our results indicate that URO accumulation caused by TCDD and TCB requires two separate actions; (1) induction of cytochrome P450 which occurs at low concentrations of the halogenated chemicals, and (2) increased uroporphyrinogen oxidation which is catalyzed by the induced cytochrome P450 and which occurs at higher concentrations of the halogenated chemicals.

Hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase activity in cultured chick-embryo hepatocytes and in Japanese quail (Coturnix coturnix japonica) and mice treated with polyhalogenated aromatic compounds.

The relationship between hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was investigated in cultured chick-embryo hepatocytes, Japanese quail (Coturnix coturnix japonica) and mice that had been treated with polyhalogenated aromatic compounds. Chick-embryo hepatocytes treated with 3,3',4,4'-tetrachlorobiphenyl accumulated uroporphyrin in a dose-dependent fashion without a detectable decrease in uroporphyrinogen decarboxylase activity when either pentacarboxyporphyrinogen III or uroporphyrinogen III were used as substrates in the assay. Other compounds, such as hexachlorobenzene, parathion, carbamazepine and nifedipine, which have been shown previously to cause uroporphyrin accumulation in these cells, did not decrease uroporphyrinogen decarboxylase activity. Japanese quail treated with hexachlorobenzene for 7-10 days also accumulated hepatic uroporphyrin without any decrease in uroporphyrinogen decarboxylase activity. In contrast, hepatic uroporphyrin accumulation in male C57BL/6 mice treated with iron and hexachlorobenzene was accompanied by a 20-80% decrease in uroporphyrinogen decarboxylase activity, demonstrating that the assay used for uroporphyrinogen decarboxylase, using pentacarboxyporphyrinogen III as substrate, could detect decreased enzyme activity. Our results with chick hepatocytes and quail, showing uroporphyrin accumulation without a decrease in uroporphyrinogen decarboxylase activity, are consistent with a new two-stage model of the uroporphyria: initially uroporphyrinogen is oxidized by a cytochrome P-450-mediated reaction, followed in rodents by a progressive decrease in uroporphyrinogen decarboxylase activity.

Studies of the influence of chloro-substituent sites and conformational energy in polychlorinated biphenyls on uroporphyrin formation in chick-embryo liver cell cultures.

Treatment of cultured chick-embryo liver cells with polychlorinated biphenyls (PCB) results in decreased uroporphyrinogen decarboxylase activity and increased uroporphyrin accumulation. In the present study we examined the effect of the chloro- or bromo-substituent sites in biphenyls (BP) on uroporphyrin accumulation in cultured hepatocytes and the three-dimensional structure of these congeners determined by molecular orbital calculations using a MNDO ('modified neglect of diatomic overlap') method. Among 20 congeners examined, those which were effective in stimulating porphyrin accumulation contained at least two Cl or Br atoms at the lateral adjacent positions in each phenyl ring, e.g. 3,4,3',4'-tetrachloro-, 2,4,3',4'-tetrachloro-, 3,4,5,3',4',5'-hexachloro- and 3,4,5,3',4',5'-hexabromobiphenyl, whereas those which contained less than two halogen atoms or more than three halogen atoms in each phenyl ring or those which contained halogen atoms at 2,2'-positions were not effective. On the basis of the conformational energy (delta E, difference from the most stable conformational energy), which is calculated as a function of the dihedral angle (theta) between the two phenyl rings, biphenyl congeners can be classified into four groups with different conformations. The conformation of active PCB was relatively flexible, whereas inactive species had a rigidly angulated conformation. Furthermore, the calculated probability of the conformation distribution for each congener indicated that the probability of co-planarity was higher for active biphenyls than for inactive congeners. These structural characteristics suggest the significance of both the chloro-substituent sites and the conformational energy reflecting the phenyl-ring twist angles in determining the inhibitory effect of PCB on uroporphyrinogen decarboxylase activity.

Alcohol-induced decrease in uroporphyrinogen decarboxylase activity in rat liver and spleen.

Chronic alcohol consumption in rats leads to a decrease in uroporphyrinogen decarboxylase activity in liver and spleen, associated with a pathologic porphyrinuria. These findings show the toxic effect of alcohol in the biochemical pathogenesis of chronic hepatic porphyria. The results confirm experimentally the transition of symptomatic coproporphyrinuria to chronic hepatic porphyria as observed in man, and the progression of biochemical phases of chronic hepatic prophyria into the clinical phase, i.e., the development from latent to manifest stages under chronic alcohol ingestion.