Uroporphyrinogen III cosynthase-deficient mutant of Salmonella typhimurium LT2.

A new type of heme-deficient mutant of Salmonella typhimurium LT2 was isolated using neomycin. The mutant, designated as strain SASY74, accumulated uroporphyrin I and coproporphyrin I. Extracts of the mutant converted 5-aminolevulinic acid to uroporphyrin I. Extracts of the mutant SASY74 and of the uroporphyrinogen synthase-deficient mutant SASY32 complemented each other and converted, when incubated together, 5-aminolevulinic acid to protoporphyrin. This finding excludes the possibility that uroporphyrinogen I synthase in strain SASY74 is deficient in its cosynthase-binding ability. Hence, the most probable explanation for the accumulation of uroporphyrin I and coproporphyrin I by the mutant is the lack of the uroporphyrinogen III cosynthase activity. This mutant is the first isolated in bacteria with such deficiency, and the mutation is analogous, as far as porphyrin synthesis is concerned, to human congenital porphyria. Mapping of the corresponding gene (hemD) by conjugation and P22-mediated transduction suggests the following gene order on the chromosome: ilv....hemC, hemD, cya....metE. The hemC and hemD genes are probably adjacent; this is the first case in which two hem genes of Enterobacteriaceae are contiguous on the chromosomal map.     

A molecular study of congenital erythropoietic porphyria in cattle

Previous studies have shown that congenital erythropoietic porphyria (CEP) in cattle is caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS) encoded by the UROS gene. In this study, we have established the pedigree of an extended Holstein family in which the disease is segregating in a manner consistent with autosomal recessive inheritance. Biochemical analyses demonstrated accumulation of uroporphyrin, thus confirming that it is indeed insufficient activity of UROS which is the cause of the disease. We have therefore sequenced all nine exons of UROS in affected and non-affected individuals without detecting any potential causative mutations. However, a single nucleotide polymorphism (SNP) located within the spliceosome attachment region in intron 8 of UROS is shown to segregate with the disease allele. Our study supports the hypothesis that CEP in cattle is caused by a mutation affecting UROS; however, additional functional studies are needed to identify the causative mutation.     

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.     

Study of the origin of urinary porphyrins in experimental hexachlorobenzene-induced porphyria

The activity of the enzyme uroporphyrinogen decarboxylase was determined in the liver and the kidneys of C57BL/6 mice and Wistar albino rats with chronic hexachlorobenzene intoxication and the amount of the deposited uroporphyrin was measured in the both organs. In the control animals the activity of hepatic uroporphyrinogen decarboxylase was several times higher than the renal one. The administration of hexachlorobenzene led to an inhibition of the enzyme activity, which was equally expressed (about 2.5 times) in the liver and kidneys of the both species. The accumulation of uroporphyrin was more pronounced in the hepatic tissue than in the kidneys (about 9 times in mice and 5 times in rats on average). Taking into consideration the much higher uroporphyrin accumulation in the liver, the more active haem biosynthesis in this organ, as well as its larger size, one could accept that the predominant part of the urinary porphyrins in hexachlorobenzene porphyria has a hepatic and not a renal origin.     

Leishmania spp.: delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.     

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.     

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.     

Survivors of Myocardial Infarction due to “Essential” Atherosclerosis

Currently, the porphyrias are classified in four main groups: congenital porphyria, acute intermittent porphyria, porphyria cutanea tarda hereditaria, and porphyria cutanea tarda symptomatica. The acquired form of porphyria (porphyria cutanea tarda symptomatica) occurs in older males and is nearly always associated with chronic alcoholism and hepatic cirrhosis. The main clinical changes are dermatological, with excessive skin fragility and photosensitivity resulting in erosions and bullae. Biochemically, high levels of uroporphyrin are found in the urine and stools. Treatment to date has been symptomatic and usually unsuccessful.A case of porphyria cutanea tarda symptomatica is presented showing dramatic improvement of both the skin lesions and porphyrin levels in urine and blood following repeated phlebotomy.Possible mechanisms of action of phlebotomy on porphyria cutanea tarda symptomatica are discussed.     

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.     

The one-electron reduction of uroporphyrin I by rat hepatic microsomes

Uroporphyrin I, which accumulates in body tissues of congenital erythropoietic porphyria patients, can undergo an enzymatic one-electron reduction to the porphyrin anion radical when a suitable reducing cofactor is present. We have demonstrated, in the absence of light, that anaerobic microsomal incubations containing NADPH and uroporphyrin I give an electron spin resonance spectrum consistent with the enzymatic formation of a porphyrin anion free radical. This radical undergoes a second-order decay (k2 approximately 10(5) M-1 s-1) due to nonenzymatic disproportionation of the radical. Aerobic microsomal incubations were also investigated for the reduction of oxygen to superoxide by monitoring oxygen consumption and the spin-trapping of superoxide. These experiments demonstrate that electron transfer from the porphyrin radical to molecular oxygen does occur, but due to the slow formation of the radical anion, no oxygen consumption above the basal level could be detected in the microsomal incubations. The photoreduction of uroporphyrin I in aerobic and anaerobic incubations was also investigated.     

Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide.

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3',4'-tetrachlorobiphenyl and 2,4,5,3',4'-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3',4'-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3',4',5'-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3',4'-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.     

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.     

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.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Export of a heterologous cytochrome P450 (CYP105D1) in Escherichia coli is associated with periplasmic accumulation of uroporphyrin

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases.     

Relative roles of CYP2E1 and CYP1A2 in mouse uroporphyria caused by acetone

Porphyria cutanea tarda is a liver disease characterized by excess production of uroporphyrin. We previously reported that acetone, an inducer of CYP2E1, enhances hepatic uroporphyrin accumulation in mice treated with iron dextran (Fe) and 5-aminolevulinic acid (ALA). Cyp2e1(-/-) mice treated with Fe and ALA were used to investigate whether CYP2E1 is required for the acetone effect. Hepatic uroporphyrin accumulation was stimulated by acetone in Cyp2e1(-/-) mice to the same extent as in wild-type mice. In the absence of acetone, uroporphyrin accumulated in Cyp2e1(-/-) mice treated with Fe and ALA, but less than in wildtype mice. However, in Cypla2(-/-) mice, uroporphyrin accumulation caused by Fe and ALA, with or without acetone, was completely prevented. Acetone was not an inducer of hepatic CYP1A2 in the wild-type mice. Although acetone is an inducer of CYP2E1, CYP1A2 appears to have the essential role in acetone-enhancement of uroporphyria.     

Identification of meso-hydroxyuroporphyrin I in the urine of a patient with congenital erythropoietic porphyria.

A new porphyrin, meso-hydroxyuroporphyrin I, was isolated from the urine of a patient with congenital erythropoietic porphyria. The structure was characterized as the methyl ester, ethyl ester and acetoxy derivatives by fast-atom-bombardment m.s., by conversion into uroporphyrin I and by chemical synthesis.     

Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda.

The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins. Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation. Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type. The excreted porphyrins comprised mainly dehydroisocoproporphyrin. Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme. A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant. The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda.     

Acute porphyrias in the Argentinean population: a review.

The porphyrias are a group of inherited metabolic disorders of heme biosynthesis which result from a partial deficiency in one of its seven specific enzymes, after its first and rate limiting enzyme, delta-aminolevulinic acid synthetase. They can be classified on the basis of their clinical manifestations into cutaneous, acute and mixed disorders. Acute intermittent porphyria (AIP) is the most common type of hepatic acute porphyrias, inherited as an autosomal dominant trait, caused by a defect in the gene which codifies for the heme enzyme porphobilinogen deaminase. Its prevalence in the Argentinean population is about 1:125,000. A partial deficiency in another enzyme, protoporphyrinogen oxidase, produces variegate porphyria (VP), the second acute porphyria most frequent in the Argentinean population (1:600,000). Here, we review all the mutations we have found in 46 AIP and 9 VP unrelated Argentinean patients. To screen for mutations in symptomatic patients, we have proposed a geneticresearch strategy.     

A new opaque variant of maize by a single dominant RNA-interference-inducing transgene

In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.