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.

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 percent 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 percent 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

Studies on the catalytic activity of human hepatic porphobilinogen deaminase.

Porphobilinogen deaminase was purified from human hepatocytes. A variety of group specific reagents have been used to achieve site-specific modifications to evaluate the potential role of such groups in the whole catalytic cycle. Treatment with dicarbonyl reagents caused a rapid loss in activity that was time and concentration, dependent. Protection experiments revealed that arginine residues are involved in the binding of the substrate. Treatment with Woodward's reagent K showed the fastest inactivation of deaminase (85% in 30 sec at 30mM) which was pH dependent and could be prevented by the presence of substrate, suggesting that deprotonated carboxylated groups from Asp/Glu are essential for catalytic activity. Kinetic analysis gave values of 0.3 sec-1 for the k3 rate constant and 8x10-2 M for the KI of the non covalent complex between deaminase-Woodward's Reagent K.

Kinetic and molecular parameters of human hepatic porphobilinogen deaminase.

A basal level of human liver porphobilinogen deaminase of 3.66 units/g wet weight was found in adult tissue. Activity in neonatal liver was at least three fold higher. Physico-chemical studies revealed that the enzyme has the approximate form of a symmetrical molecule and exhibits hyperbolic kinetics with a Km value of 3.6 microM at pH 7.6. Two ionizable groups with pK values of 7.35 and 8.90 are prominent for catalysis. A set of pI (5.8-4.9) were observed under different conditions. Results demonstrate the existence of a single protein differentially charged with multiple molecular forms in adult liver.

Characterization of porphobilinogen deaminase from rat liver.

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.

Mechanism of hexachlorobenzene-induced porphyria in rats. Effect of phenobarbitone pretreatment.

The effect of a pretreatment with phenobarbitone (PB) on the porphyrinogenic action exerted by hexachlorobenzene (HCB) was examined in female rats. Kinetic studies of enzyme function after HCB poisoning showed that porphyrinogen carboxy-lyase was the only enzyme of haem biosynthesis that markedly lowered its activity. Both stages of uroporphyrinogen (UPG) III decarboxylation were decreased. This enzyme, together with UPG I synthase (increased levels) were the first enzymes altered. Subsequently, an increase in delta-aminolaevulinate (AmLev) synthase and ferrochelatase was detected; AmLev dehydratase was the last to increase. On long-term exposure, PB alone did not modify the basal values of haem intermediates; only the content of cytochrome P-450 increased. All the enzyme activities studied showed no significant changes, except ferrochelatase, which increased. With both drugs the metabolic impairment promoted by HCB was accelerated and enhanced by prior PB treatment leading to the onset of an earlier and stronger porphyria. A more noticeable accumulation and excretion of higher carboxylated porphyrins and precursors was more promptly observed as a consequence of the early porphyrinogen carboxy-lyase blockade and the concomitant induction of AmLev synthase. Although the enzymic activities of both AmLev dehydratase and ferrochelatase were enhanced, this response differed in time. For UPG I synthase this pretreatment elicited lower values than those found in the HCB group. Cytochrome P-450 contents were immediately and slightly enhanced by all the drugs, but the values for the combined treatment were the lowest. Of the several hypotheses that could explain the action of HCB on the haem pathway, our results would suggest that the porphyrinogenic action of HCB is mediated by some of its metabolic products.

Porphyrins and porphyrinogen carboxy-lase in hexachlorobenzene-induced porphyria.

1. Qualitative and quantitative studies of the porphyrins and the porphyrinogen carboxylyase of the liver, spleen, kidney, harderian gland and erythrocytes from normal rats and from those hexachlorobenzene-induced porphyria were carried out. 2. Hexachlorobenzene has no effect on erythrocyte porphyrin content, but produces a decrease in that of Harderian gland and an increase in the porphyrin content of the kidney and spleen, and a marked increase in the liver (1 mumol/g of tissue). Octacarboxylic (isomer III) and heptacarboxylic porphyrins accumulated in kidney, spleen and liver, the former porphyrin being predominant. 3. Hexachlorobenzene has no effect on the activity of porphyrinogen carboxy-lase in erythrocytes; there is a slight decrease in enzyme activity in the Harderian gland, and a marked decrease in the liver and kidney enzyme activities. In the liver the removal of each carboxyl group from uroporphyrinogen III appears to be affected by this treatment. 4. The liver is the principal site of action of hexachlorobenzene, with the kidney next in decreasing order of effect, and erythropoietic tissue is unaffected. The marked decrease in porphyrinogen carboxy-lyase activities observed in liver and kidney could explain the high accumulation of octacarboxylic and heptacarboxylic porphyrins found in these tissues. 5. The results are discussed in relation to changes promoted by hexachlorobenzene in other enzymes of the haem pathway.

Cobalt inhibition of synthesis and induction of delta-aminolevulinate synthase in liver.

Cobalt has complex actions on the metabolism of heme in the liver. In this organ the metal potently induces heme oxygenase (EC 1.14.99.3), and decreases cellular heme and hemoprotein content. The metal also displays biphasic effects on hepatic heme synthesis. These effects are reflected in the ability of cobalt to initially inhibit synthesis of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37], the rate limiting enzyme of the heme pathway, following which a later enhanced rate of formation of this enzyme occurs. In this study, cobalt was shown to block almost entirely the ability of the barbiturate analogue allylisopropylacetamide to induce delta-aminolevulinate synthase in liver. The blocking effect of cobalt on the otherwise potent enzyme inducing action of this drug was time-dependent; if the metal was injected 30 min prior to allylisopropylacetamide, inhibition of enzyme induction was complete. When the metal was administered 1.5 or more hours after allylisopropylacetamide, inhibition of enzyme induction was incomplete. Cobalt did not block the ability of the drug to directly degrade heme to "green pigment" thus the enzyme inducing action of allylisopropylacetamide and its degradative action on heme are separately mediated.

Tetrapyrroles as substrates and inhibitors of porphyrinogen carboxy - lyase from rat liver.

Porphyrinogen carboxy-lyase is an enzyme of the haem pathway which catalyses the stepwise decarboxylation of porphyrinogens with different number of carboxyl groups. This enzyme has a low substrate specificity since at least eighteen porphyrinogens were proved to be decarboxylated by the enzyme. In order to clarify this complex process of decarboxylation, studies were carried out using a purified enzyme preparation from rat liver. We studied the behavior of the enzyme in the presence of uroporphyrinogens I, II, III, and IV. The effect of different porphyrins, porphyrinogens and haemin on uroprophyrinogen decarboxylation was also studied to see the influence of nature and position of the side chains of pyrroles as well as the oxidation state in the tetrapyrrolic ring. The liver enzyme decarboxylates the four isomers or uroporphyrinogen. The relative accumulation of intermediates porphyrinogens formed was different from that of isomer III. Uroporphyrinogen IV is an efficient substrate for the porphyrinogen carboxyl-lyase since it was decarboxylated at higher rate than the normal uroporphyrinogen III. The elimination of a carboxyl group of an acetic acid residue located between an acetic and a propionic acid side chain appears to be easier than the one corresponding to an acetic between two propionics or between a methyl and a propionic acid residue. The presence of vicinal propionic side chains in the position 6 and 7 of the reduced porphyrin ring is an important, but not essential requirement for the binding of the enzyme to porphyrinogen. It was found that coproporphyrinogen III inhibits markedly uroporphyrinogen decarboxylation and that haemin also has inhibitory effect on this reaction. The results of the inhibitory studies suggest one or both of the propionic acid residues located in positions 2 and 4 as important factors in the tetrapyrrole enzyme binding. Some other evidence would indicate that possibly the propionic acid side chain at the position 4 may be particularly important. The reduced state of the tetrapyrrolic ring is essential for the decarboxylation process: thus would allow the side chains to adopt a steric disposition facilitating its binding to the enzyme.

Tetrapyrroles as substrates and inhibitors of porphyrinogen carboxy - lyase from rat liver.

Porphyrinogen carboxy-lyase is an enzyme of the haem pathway which catalyses the stepwise decarboxylation of porphyrinogens with different number of carboxyl groups. This enzyme has a low substrate specificity since at least eighteen porphyrinogens were proved to be decarboxylated by the enzyme. In order to clarify this complex process of decarboxylation, studies were carried out using a purified enzyme preparation from rat liver. We studied the behavior of the enzyme in the presence of uroporphyrinogens I, II, III, and IV. The effect of different porphyrins, porphyrinogens and haemin on uroprophyrinogen decarboxylation was also studied to see the influence of nature and position of the side chains of pyrroles as well as the oxidation state in the tetrapyrrolic ring. The liver enzyme decarboxylates the four isomers or uroporphyrinogen. The relative accumulation of intermediates porphyrinogens formed was different from that of isomer III. Uroporphyrinogen IV is an efficient substrate for the porphyrinogen carboxyl-lyase since it was decarboxylated at higher rate than the normal uroporphyrinogen III. The elimination of a carboxyl group of an acetic acid residue located between an acetic and a propionic acid side chain appears to be easier than the one corresponding to an acetic between two propionics or between a methyl and a propionic acid residue. The presence of vicinal propionic side chains in the position 6 and 7 of the reduced porphyrin ring is an important, but not essential requirement for the binding of the enzyme to porphyrinogen. It was found that coproporphyrinogen III inhibits markedly uroporphyrinogen decarboxylation and that haemin also has inhibitory effect on this reaction. The results of the inhibitory studies suggest one or both of the propionic acid residues located in positions 2 and 4 as important factors in the tetrapyrrole enzyme binding. Some other evidence would indicate that possibly the propionic acid side chain at the position 4 may be particularly important. The reduced state of the tetrapyrrolic ring is essential for the decarboxylation process: thus would allow the side chains to adopt a steric disposition facilitating its binding to the enzyme.

Tetrapyrroles as substrates and inhibitors of porphyrinogen carboxy - lyase from rat liver

Porphyrinogen carboxy-lyase is an enzyme of the haem pathway which catalyses the stepwise decarboxylation of porphyrinogens with different number of carboxyl groups. This enzyme has a low substrate specificity since at least eighteen porphyrinogens were proved to be decarboxylated by the enzyme. In order to clarify this complex process of decarboxylation, studies were carried out using a purified enzyme preparation from rat liver. We studied the behavior of the enzyme in the presence of uroporphyrinogens I, II, III, and IV. The effect of different porphyrins, porphyrinogens and haemin on uroprophyrinogen decarboxylation was also studied to see the influence of nature and position of the side chains of pyrroles as well as the oxidation state in the tetrapyrrolic ring. The liver enzyme decarboxylates the four isomers or uroporphyrinogen. The relative accumulation of intermediates porphyrinogens formed was different from that of isomer III. Uroporphyrinogen IV is an efficient substrate for the porphyrinogen carboxyl-lyase since it was decarboxylated at higher rate than the normal uroporphyrinogen III. The elimination of a carboxyl group of an acetic acid residue located between an acetic and a propionic acid side chain appears to be easier than the one corresponding to an acetic between two propionics or between a methyl and a propionic acid residue. The presence of vicinal propionic side chains in the position 6 and 7 of the reduced porphyrin ring is an important, but not essential requirement for the binding of the enzyme to porphyrinogen. It was found that coproporphyrinogen III inhibits markedly uroporphyrinogen decarboxylation and that haemin also has inhibitory effect on this reaction. The results of the inhibitory studies suggest one or both of the propionic acid residues located in positions 2 and 4 as important factors in the tetrapyrrole enzyme binding. Some other evidence would indicate that possibly the propionic acid side chain at the position 4 may be particularly important. The reduced state of the tetrapyrrolic ring is essential for the decarboxylation process: thus would allow the side chains to adopt a steric disposition facilitating its binding to the enzyme.