Synergism of iron and hexachlorobenzene inhibits hepatic uroporphyrinogen decarboxylase in inbred mice.

The combination of a single subcutaneous dose of iron (12.5 mg/mouse) and subsequent treatment with hexachlorobenzene (0.02% of the diet) caused a progressive inhibition of hepatic uroporphyrinogen decarboxylase in male C57BL/10 mice leading to the accumulation of uroporphyrin in 4-6 weeks. There was only a slight inhibition of the enzyme in the absence of iron and none without hexachlorobenzene. Females were less sensitive than males. In addition, comparisons between the C57BL/10, BALB/c, AKR and DBA/2 strains indicated that the susceptibilities of mice to induction of porphyria did not completely correlate with their classification as Ah-responsive or Ah-non-responsive.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Chemically-induced formation of an inhibitor of hepatic uroporphyrinogen decarboxylase in inbred mice with iron overload.

An inhibitor of hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was demonstrated in heat-treated extracts of livers from C57BL/10ScSn mice with iron overload after a single dose (100 mg/kg; 350 mumol/kg) of hexachlorobenzene (HCB). Inhibition was not due to accumulated uroporphyrin since this could be removed by a SEP-PAK C18 cartridge without affecting inhibitor activity. The presence of the inhibitor could be first demonstrated 2 weeks after mice received HCB and before major elevation of hepatic porphyrin levels. Maximum inhibitory potential was reached at about 8 weeks and was still detected 25 weeks after the chemical, thus paralleling the depression of enzyme activity reported previously [Smith, Francis, Kay, Greig & Stewart (1986) Biochem. J. 238, 871-878]. The inhibitor was not detected following treatment of mice with either iron or HCB alone or after the decarboxylase activity was destroyed in vitro by the combination of uroporphyrin and light. The formation of the inhibitor by inbred mouse strains nominally Ah-responsive (C57BL/6J, C57BL/10ScSn, BALB/c, C3H/HeJ, CBA/J and A/J) and Ah-nonresponsive (SWR, AKR, 129, SJL, LP and DBA/2) did not correlate fully with their reported Ah-phenotype. There was a correlation amongst the Ah-responsive strains only, with hepatic ethoxyphenoxazone de-ethylase activity induced in parallel experiments by treatment with beta-naphthoflavone. De-ethylase activity induced by HCB, however, was considerably less than that with beta-naphthoflavone, which has not been reported as porphyrogenic. Other polyhalogenated chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,2',3',4'-hexachlorobiphenyl and hexabromobenzene, also caused the formation of the inhibitor of uroporphyrinogen decarboxylase.     

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.     

The role of iron in experimental porphyria and porphyria cutanea tarda

Porphyria cutanea tarda (PCT) and experimental porphyria are characterized by a decreased activity of the enzyme uroporphyrinogen decarboxylase, and accumulation of uroporphyrins and heptacarboxylporphyrins in the liver. Iron (Fe) plays an important role in PCT and experimental porphyria. Biochemically and electron microscopically, we examined the relationship between Fe and porphyrins in liver tissue of C57BL/10 mice made porphyric by administration of iron dextran as Imferon^® (IMF), and in liver biopsies of patients with symptomatic PCT. Accumulation of uroporphyrins and heptacarboxylporphyrins, and an increased amount of Fe were observed in livers of mice treated with IMF and in liver biopsies of patients with PCT. In mice treated with IMF, the activity of uroporphyrinogen decarboxylase was decreased. Both in livers of mice treated with IMF and in livers of patients with PCT, needle-like structures, representing uroporphyrin crystals, were observed by electron microscopy. Uroporphyrin crystals and Fe (as ferritin) were observed in the same hepatocyte. Moreover, there was a striking morphological correlation between uroporphyrin crystals and ferritin-Fe, suggesting a role for (ferritin-)Fe in the pathogenesis of porphyria.     

Immunoreactive uroporphyrinogen decarboxylase is unchanged in porphyria caused by TCDD and hexachlorobenzene

A method has been developed for the immuno-titration of rodent liver uroporphyrinogen decarboxylase (porphyrinogen carboxy-lyase, EC 4.1.1.37) and used to show that two porphyrogenic polyhalogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin and hexachlorobenzene, cause porphyria in rodents by decreasing the catalytic activity of uroporphyrinogen decarboxylase without altering the amount of immunoreactive enzyme protein. Investigation of the nature of the inactive form of uroporphyrinogen decarboxylase produced by these compounds should provide new information about the mechanism of their toxicity.     

A difference between two strains of rats in their liver non-haem iron content and in their response to the porphyrogenic effect of hexachlorobenzene.

Female Agus rats developed hepatic porphyria at a much faster rate than female Porton-Wistar rats when fed a diet containing 0.01% of hexachlorobenzene (HCB). They also showed a greater inhibition of liver uroporphyrinogen decarboxylase [EC 4.1.1.37] activity and a marked stimulation of 5-aminolaevulinate synthetase [EC 2.3.1.37]. The difference between the two strains could not be correlated with differences in the liver concentrations of HCB. However, control Agus rats were found to possess significantly higher levels of total non-haem iron in their livers than the Porton animals. This was particularly apparent after 24 h of starvation and is further evidence for the involvement of iron in the pathogenesis of HCB-induced porphyria. The posterior lobes of the livers from the Agus rats given HCB became porphyric more slowly than the remainder with less severe inhibition of uroporphyrinogen decarboxylase. In contrast to their increased susceptibility to HCB, the Agus rats were less susceptible to another prophyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine.     

How does hexachlorobenzene treatment affect liver uroporphyrinogen decarboxylase?

The aims of the present work were: (1) to investigate whether the strong decrease of liver uroporphyrinogen decarboxylase (UroD) activity observed in experimental porphyria cutanea tarda is due to alteration of the enzymatic protein and (2) to improve the knowledge about the normal liver enzyme. With these purposes, several physicochemical studies for enzymatic characterization were carried out comparatively on the 12-fold purified liver enzyme of both normal and hexachlorobenzene porphyric rat. The study shows that the enzyme from porphyric rats has a higher activation energy, lower reactivity index and lower optimum pH than the normal one. In addition, it did not reach the Vmax at any of the substrate concentrations assayed (up to 28 microM uroporphyrinogen III), while the normal enzyme reached the plateau around 14 microM. The porphyric enzyme appears to be more protected than the normal against the inhibitory action of several metals, particularly Cu2+ and Pb2+, and against thermal inactivation. Zn2+ did not affect enzymatic activity, whereas Cu2+, Hg2+, Fe2+, Pb2+, and Cd2+ lowered the activities of both normal and porphyric enzyme in a dose-related way. It was also observed that the larger the atomic radius in its hydrated state, the lower the effect of the metal. Neither glutathione nor dithiothreitol significantly altered enzymatic activity in the range of concentrations assayed. beta-Mercaptoethanol had diverse effects, as regards both the concentration assayed and the enzymatic sample used. Assays with cystine showed a dual behaviour of both normal and porphyric enzymatic activity. Western blots for both preparations revealed a single band (65 kDa) with a similar intensity.This study show that hexachlorobenzene treatment modifies the physicochemical properties of liver UroD leading to a sharp decrease of its activity, without affecting its antigenic reactivity probably as a consequence of changes at the conformational level promoted by the binding of its reported inhibitor.     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.     

Photodynamic inactivation of red cell uroporphyrinogen decarboxylase by porphyrins

The effects of light and porphyrins on the activity of red cell uroporphyrinogen decarboxylase were studied. Photoinactivation of uroporphyrinogen decarboxylase was dependent on uroporphyrin concentration, irradiation time and temperature. Using 40 W/m2 of UV light intensity, 40-45% decreased activity was produced with 200 microM uroporphyrin I, at 37 degrees C and after 2 hr of illumination. It has been demonstrated that porphyrins photoinactivate uroporphyrinogen decarboxylase and a mechanism for this action in relation to skin lesions is proposed.     

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker''s yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.     

Ferritin accumulation and uroporphyrin crystal formation in hepatocytes of C57BL/10 mice: a time-course study

To establish the time-sequence relationship between ferritin accumulation and uroporphyrin crystal formation in livers of C57BL/10 mice, a biochemical, morphological and morphometrical study was performed. Uroporphyria was induced by the intraperitoneal administration of hexachlorobenzene plus iron dextran and of iron dextran alone. Uroporphyrin crystal formation started in hepatocytes of mice treated with hexachlorobenzene plus iron dextran at 2 weeks and in mice treated with iron dextran alone at 9 weeks. In the course of time, uroporphyrin crystals gradually increased in size. Uroporphyrin crystals were initially formed in hepatocytes in the periportal areas of the liver, in which also ferric iron staining was first detected. The amount and the distribution of the main storage form of iron in hepatocytes, ferritin, did not differ between the two treatment groups. Ferritin accumulation preceded the formation of uroporphyrin crystals in hepatocytes in both treatment groups. Moreover, uroporphyrin crystals were nearly always found close to ferritin iron. We conclude that uroporphyrin crystals are only formed in hepatocytes in which also iron (ferritin) accumulates. Hexachlorobenzene accelerates the effects of iron in porphyrin metabolism, but does not influence the accumulation of iron into the liver.     

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.     

Macrocyclic intermediates in the biosynthesis of porphyrins.

The hepta-, hexa- and penta-carboxylic porphyrins found in the faeces of rats poisoned with hexachlorobenzene have been separated by high-pressure liquid chromatography and characterized largely by spectroscopie methods. Their structures were confirmed by total synthesis, as part of a programme in which eleven of the fourteen hepta-, hexa- and penta-carboxylic porphyrins derived from uroporphyrin III have now been synthesized as their methyl esters. The four isomeric heptacarboxylic and three of the pentacarboxylic porphyrinogens have been incubated with haemolysates of chicken erythrocytes, and they are all converted into protoporphyrin IX but at different rates. On the basis of this and other evidence we conclude that the decarboxylation of uroporphyrinogen III to coproporphyrinogen III is a stepwise process taking place by a preferred pathway (both in normal and abnormal metabolism); the acetic acid groups are decarboxylated in a sequential clockwise fashion starting with that on the D ring and followed by those on the A, B and C rings. In the poisoned rats the uroporphyrinogen decarboxylase enzyme (or group of enzymes) is probably partially inhibited and the pentacarboxylic porphyrinogen with an acetic acid group on ring C accumulates. The latter is then transformed by a side pathway into dehydroisocoproporphyrinogen and thence into dehydroisocoproporphyrin and its congeners.     

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.     

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

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers

Uroporphyrinogen decarboxylase (EC 4.1.1.37) which converts uroporphyrinogen I or III into coproporphyrinogen I or III, respectively, was purified about 5,500-fold from chicken erythrocytes. Purification was accomplished by chromatography on DEAE-cellulose, ammonium sulfate fractionation, chromatography on Sephadex G-100, and chromatofocusing. The most purified preparation was homogeneous on polyacrylamide gel electrophoresis and had a specific activity of 1,420 units/mg of protein, the highest value so far reported. The molecular weight, as determined by Sephadex G-150 gel chromatography, is 79,000. The subunit molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 39,700, suggesting that uroporphyrinogen decarboxylase is dimeric in form. The purified enzyme had an isoelectric point of 6.2 and a pH optimum of 6.8. The SH reagents inhibited the enzyme activity, but neither metal ions nor cofactor requirements could be demonstrated. A new and simple method for the separation of free uroporphyrin, hepta-, hexa-, and pentacarboxylic porphyrins and coproporphyrin was developed using a high pressure liquid chromatograph equipped with a spectrofluorometric detector. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified enzyme were performed. 3,4,3',4'-Tetrachlorobiphenyl and 3,4,5,3',4'5'-hexachlorobiphenyl which specifically induce delta-aminolevulinic acid synthetase also strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

In vivo 13C nuclear magnetic resonance spectroscopy using a solenoid transmit/receiver coil

The administration of cadmium (1.25 mg as Cd2+/kg, ip.) to male rats resulted in a significant increase of hepatic and renal ornithine decarboxylase activity. The maximum increase of ornithine decarboxylase activity to about 10-fold of the controls was seen at 4 hr after the administration of cadmium, and the increased enzyme activity was returned to control levels by 12 hr. Cadmium produced somewhat dose-dependently the increase of ornithine decarboxylase activity. The increase of ornithine decarboxylase seen on the administration of cadmium was cancelled by pretreatment of rats with cycloheximide. The treatment of female rats with cadmium also caused the increase of hepatic ornithine decarboxylase activity, but not renal enzyme activity.     

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.