Biomedical subjects

N Gorman

Publications and source records attributed to N Gorman.

At least 19 recordsLinked to original sources

PubMed2001

Role of cytochrome P450 1A2 in bilirubin degradation Studies in Cyp1a2 (-/-) mutant mice.

In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.

Animals↗

PubMed2001

Uroporphyria in Hfe mutant mice given 5-aminolevulinate: a new model of Fe-mediated porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

Aminolevulinic Acid↗

PubMed2000

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.

Acetone↗

PubMed2000

Effect of arsenite on the induction of CYP1A4 and CYP1A5 in cultured chick embryo hepatocytes.

We had reported previously that 2.5-5 microM sodium arsenite decreased the phenobarbital-mediated induction of CYP2H activity and protein but not CYP2H1 mRNA in chick-embryo hepatocyte cultures. Induction of a CYP1A activity and protein by 3-methylcholanthrene was also decreased by low arsenite concentrations; however, CYP1A mRNAs were not measured in those studies. We report here that low concentrations of arsenite decreased induction of activities and mRNAs of two chicken cytochromes P450, CYP1A (1A4 and 1A5), by 3-methylcholanthrene in chick-embryo hepatocyte cultures. Arsenite treatment did not affect the turnover of either mRNA, nor did it decrease the superinduction of each mRNA caused by treatment with cycloheximide in addition to 3-methylcholanthrene. Glutathione depletion enhanced the effect of arsenite to decrease induction of CYP1A4. These results indicate the induction of CYP1A4 and 1A5 is inhibited by sodium arsenite at the level of transcription, suggesting that the Ah receptor complex may be involved.

Animals↗

PubMed2000

CYP1A2 is essential in murine uroporphyria caused by hexachlorobenzene and iron.

Using Cyp1a2(-/-) mice we previously showed that CYP1A2 is absolutely required for hepatic uroporphyrin accumulation caused by iron and 5-aminolevulinate (ALA) treatment, both in the presence and absence of an inducer of CYP1A2. In this study we have used these mice to investigate whether CYP1A2 has an obligatory role in hepatic uroporphyria caused by hexachlorobenzene (HCBZ), an inducer of CYP2B and CYP3A, as well as CYP1A2. Here we treated mice with HCBZ and iron, with and without the porphyrin precursor, ALA, in the drinking water. In iron-loaded wild-type mice given a single dose of HCBZ and ALA, hepatic uroporphyrin (URO) accumulated to 300 nmol/g liver after 37 days, whereas in Cyp1a2(-/-) mice, there was no hepatic URO, even after an additional dose of HCBZ, and a further 29 days of ALA treatment. A similar requirement for CYP1A2 was found in uroporphyria produced in HCBZ and iron-treated mice in the absence of ALA. As detected by Western immunoblotting, HCBZ induced small increases in CYP2B and CYP3A in the livers of all animals. In the wild-type animals, HCBZ also induced CYP1A2 and associated enzyme activities, including uroporphyrinogen oxidation, by about 2-3-fold. In the Cyp1a2(-/-) mice, HCBZ did not increase hepatic microsomal uroporphyrinogen oxidation. These results indicate that, in mice, CYP1A2 is essential in the process leading to HCBZ-induced uroporphyria. Contributions by other CYP forms induced by HCBZ appear to be minimal.

Aminolevulinic Acid↗

PubMed1999

Role of small differences in CYP1A2 in the development of uroporphyria produced by iron and 5-aminolevulinate in C57BL/6 and SWR strains of mice.

Previous work has implicated CYP1A2 in experimental uroporphyria caused by polyhalogenated aromatic compounds, and in uroporphyria caused by iron and 5-aminolevulinate (ALA) in the absence of inducers of CYP1A2. Here we examined whether the different susceptibilities of SWR and C57BL/6 strains of mice to uroporphyria in the absence of inducers of CYP1A2 are related to different levels of CYP1A2. Enzymological assays (ethoxy- and methoxyresorufin dealkylases, and uroporphyrinogen oxidation) and immunoblots indicated that there was about twice the amount of hepatic CYP1A2 in SWR mice compared with C57BL/6 mice. Immunohistochemistry revealed that CYP1A2 was located centrilobularly in the liver, and the staining was more intense in SWR mice than in C57BL/6 mice. Hepatic non-heme iron was about double in SWR compared with C57BL/6 mice. In SWR mice given iron dextran, hepatic iron was 1.7-fold that of C57BL/6 mice given iron dextran. SWR mice administered ALA in the drinking water accumulated much less hepatic protoporphyrin than did C57BL/6 mice. To confirm the importance of small increases in CYP1A2, C57BL/6 mice were given a low dose of 3-methylcholanthrene (MC) (15 mg/kg), as well as iron and ALA. There was about a 5- to 6-fold increase in hepatic uroporphyrin accumulation after 32 days on ALA compared with animals not given MC. In these animals, CYP1A2 was increased by 10-fold at 2 days, but returned to basal levels by 14 days. We conclude that small and transient differences in CYP1A2 may be important in the development of uroporphyria.

Aminolevulinic Acid↗

PubMed1998

Uroporphyria produced in mice by iron and 5-aminolaevulinic acid does not occur in Cyp1a2(-/-) null mutant mice.

In the present study we have investigated the putative requirement for the cytochrome P-450 isoform CYP1A2 in murine uroporphyria, by comparing Cyp1a2(-/-) knockout mice with Cyp1a2(+/+) wild-type mice. Uroporphyria was produced by injecting animals with iron-dextran and giving the porphyrin precursor 5-aminolaevulinic acid in the drinking water. Some animals also received 3-methylcholanthrene (MC) to induce hepatic CYP1A2. In both protocols, uroporphyria was elicited by these treatments in the Cyp1a2(+/+) wild-type mice, but not in the null mutant mice. Uroporphyrinogen oxidation activity in hepatic microsomes from untreated Cyp1a2(+/+) mice was 2.5-fold higher than in Cyp1a2(-/-) mice. Treatment with MC increased hepatic CYP1A1 in both mouse lines and hepatic CYP1A2 only in the Cyp1a2(+/+) line, as determined by Western immunoblotting. MC increased hepatic ethoxy- and methoxy-resorufin O-dealkylase activities in both mouse lines, but increased uroporphyrinogen oxidation activity in the Cyp1a2(+/+) wild-type mice only. These results indicate the absolute requirement for hepatic CYP1A2 in causing experimental uroporphyria under the conditions used.

Aminolevulinic Acid↗

PubMed1998

Formation of zinc protoporphyrin in cultured hepatocytes: effects of ferrochelatase inhibition, iron chelation or lead.

The formation of zinc protoporphyrin in response to lead or iron depletion has previously been investigated in erythroid systems. Because of its possible metabolic role in non-erythroid tissue, we investigated the formation of zinc protoporphyrin in cultured hepatocytes. The effects of lead and inhibitors of ferrochelatase, the iron insertion step of heme synthesis, on the conversion of 5-aminolevulinic acid to zinc protoporphyrin, protoporphyrin and heme were compared in rat and chick embryo hepatocyte cultures. In rat cultures, zinc protoporphyrin was synthesized enzymatically by ferrochelatase, since N-methylmesoporphyrin, an inhibitor of ferrochelatase. caused 40% or greater decreases in both heme and zinc protoporphyrin accumulation and markedly stimulated protoporphyrin accumulation. In addition, chelation of ferrous iron with 2,2'-dipyridyl decreased heme accumulation by 50%, but increased ZPP accumulation by 200%. Zinc protoporphyrin formation in chick embryo hepatocytes required the addition of zinc as well as 5-aminolevulinic acid and apparently was non-enzymatic, since it was not inhibited by N-methylmesoporphyrin nor increased by iron chelation. In the presence of 5-aminolevulinic acid, lead had no effect on zinc protoporphyrin, protoporphyrin or heme accumulation in chick hepatocytes, but decreased all three in rat hepatocytes, with the decrease in protoporphyrin being far greater than that of zinc protoporphyrin or heme. These findings indicate that, in contrast to the effect of lead in erythroid tissue, it did not specifically increase zinc protoporphyrin accumulation or alter iron availability in cultured hepatocytes.

Aminolevulinic Acid↗

PubMed1998

CYP1A-catalyzed uroporphyrinogen oxidation in hepatic microsomes from non-mammalian vertebrates (chick and duck embryos, scup and alligator).

Uroporphyrin (URO) accumulation in the liver of animals treated with polyhalogenated aromatic hydrocarbons (PHAH) is associated with increased microsomal oxidation of uroporphyrinogen catalyzed by rodent CYP1A2 and by a similar form in chicken, CYP1A5. The planar biphenyl, 3,3',4,4'-tetrachlorobiphenyl (TCB) stimulates uroporphyrinogen oxidation (UROX) in chick hepatic microsomes, but inhibits UROX activity in hepatic microsomes from mice and rats pre-induced by CYP1A2. Here we investigated whether TCB would stimulate or inhibit UROX in other non-mammalian species. UROX was stimulated 1.5-3-fold by TCB and 2-4-fold by 3,3',4,4',5,5'-hexachlorobiphenyl in hepatic microsomes from duck, alligator and scup treated with inducers of CYP1A. Hexachlorobenzene stimulated chick UROX, but was ineffective with microsomes from the other species. The stimulation of UROX by TCB was also observed in chick hepatocyte cultures. Pretreatment with up to 5 nM TCB induced CYP1A, but did not result in accumulation of URO. However, URO did accumulate if additional (post-induction) TCB was added along with 5-aminolevulinic acid. In this post-inductional TCB treatment, cycloheximide was included to prevent further induction of CYP1A. In duck hepatocytes, pretreatment with 25 nM TCB resulted in URO accumulation from 5-aminolevulinic acid. Post-induction TCB was not required and caused no further increase in URO accumulation. The differences in PHAH stimulation of UROX among the non-mammalian species have implications in the evolutionary changes in CYP1A, as well as the mechanism of development of PHAH-stimulated uroporphyria in different species.

Animals↗

PubMed1998

Uroporphyrinogen oxidation catalyzed by human cytochromes P450.

Porphyria cutanea tarda is associated with excess hepatic production of uroporphyrin. Oxidation of uroporphyrinogen to uroporphyrin was previously demonstrated to be specifically catalyzed by cytochrome P450 (CYP) 1A2. Here, we investigated the ability of human CYP1A2 to catalyze uroporphyrinogen oxidation (UROX). UROX activity in human liver microsomes was maximally only 10% of the activity in microsomes from livers of untreated mice. There was a poor correlation of UROX activity with methoxyresorufin demethylation, an activity catalyzed predominantly by CYP1A2 and strongly correlated with immunodetectable CYP1A2. With CYP forms expressed in HepG2 cells, the methoxyresorufin demethylation and (ethoxyresorufin deethylation) activities of murine and human CYP1A2 forms were similar, but UROX activity catalyzed by human CYP1A2 was only 15-20% of the activity catalyzed by murine CYP1A2. Human CYP1A1, CYP1A2, and CYP3A4 expressed in lymphoblastoid cells all catalyzed UROX. In insect cells, CYP1A2 was more active in catalyzing UROX than was CYP1A1, CYP2E, CYP3A4, or CYP3A5. Human CYP1A2 expressed in Escherichia coli as a fusion protein with rat CYP oxidoreductase also catalyzed UROX. Reconstituted human CYP1A2 and CYP3A4 were active in catalyzing UROX, with reconstituted CYP1A2 having the highest specific activity obtained in this study. From inhibitor studies, it was concluded that some of the UROX activity in the insect cell microsomes was attributable to expressed CYP and some to an unidentified source. These results indicate that human CYP1A2 is active in catalyzing UROX but has lower activity than the murine orthologue. The results also indicate that most of the UROX activity found in human liver microsomes is not due to CYP1A2.

Animals↗

PubMed1997

Multiple roles of polyhalogenated biphenyls in causing increases in cytochrome P450 and uroporphyrin accumulation in cultured hepatocytes.

Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes treated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) that are known inducers of cytochrome P4501As (CYP1A). Previous dose response studies had shown that URO accumulation does not begin until CYP1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is maximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD had not been explained. PHAHs, such as 3,3',4,4'-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) oxidation by microsomes from 3-methylcholanthrene (MC)-treated chick embryos. Here we used a new protocol to investigate whether the requirement for more TCB to stimulate in vitro microsomal UROGEN oxidation extended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other PHAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further increases in CYP1A as determined by immunoblots. 5-Aminolevulinic acid and a high concentration of TCB ("postinduction TCB") were then added to stimulate intracellular UROGEN oxidation. Using the protocol with postinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postinduction TCB. Increases in CYP1A proteins, measured immunochemically, were detected at about the same inducing TCB concentrations that began to increase URO accumulation. The new protocol, with postinduction TCB, using URO accumulation as the end point, greatly increased the sensitivity of the culture system for detection of PHAHs with EC50s (nM) for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), TCB, 3,3',4,4',5,5'-hexachlorobiphenyl, MC, and hexachlorobenzene being about 0.003, 0.11, 0.75, 3.5, and 30, respectively. As little as 2-4 fmol TCDD per culture dish caused detectible increases in URO accumulation. We conclude that URO accumulation in chick hepatocyte cultures is limited not only by the induction of CYP1A, but also by the stimulation of intracellular UROGEN oxidation.

Animals↗

PubMed1997

Ascorbic acid deficiency in porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), the most common form of porphyria, is manifested as skin photosensitivity caused by excess hepatic production of uroporphyrin and heptacarboxylporphyrin. In experimental animal models, ascorbic acid modulates chemically induced uroporphyrin accumulation. The purpose of this study was to determine whether ascorbic acid is decreased in the plasma of patients with PCT. Plasma was obtained after an overnight fast from 21 PCT patients, 16 of whom were infected with hepatitis C virus (HCV), and from a separate group of 9 patients with HCV infection but not PCT. Thirteen PCT patients were studied when they had active disease and 8 after treatment-induced remission. Plasma ascorbic acid was low (<23 micromol/L) in 11 (85%) of the 13 untreated PCT patients and deficient (<11 micromol/L) in 8 (62%). Two patients with normal ascorbic acid levels (45 and 62 micrommol/L) had consumed multivitamins. In 2 patients with deficient ascorbic acid, plasma levels returned to normal after phlebotomy treatment. Of the 8 patients studied during remission, 4 had normal ascorbic acid values and 4 were deficient (5 to 8 micromol/L). Plasma ascorbic acid values were normal for all patients who had HCV but no PCT. These data suggest that plasma ascorbic acid concentrations are commonly low in PCT, but this decrease is unrelated to HCV infection. Ascorbic acid deficiency may be one of the factors that contributes to the pathogenesis of PCT.

Adult↗

PubMed1997

Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by AH receptor ligands in chicken liver and kidney.

Uroporphyrinogen is an intermediate of the heme biosynthetic pathway. The oxidation of uroporphyrinogen to uroporphyrin (UROX) has been demonstrated to be catalyzed by mammalian CYP1A2. This reaction has an important role in uroporphyria caused by halogenated aromatic compounds. Two CYP enzymes induced by Ah receptor ligands were purified recently from chick embryo liver. One, designated CYP1A5, was preferentially active in arachidonic acid epoxygenation and the other, designated CYP1A4, in 7-ethoxyresorufin deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH), reactions mainly catalyzed by CYP1A1 in rodents. The amino acid sequences of both CYP1A5 and CYP1A4 are more similar to CYP1A1 than to 1A2, and neither can be classified as an ortholog of mammalian CYP1A1 or 1A2. Here we report that reconstituted purified CYP1A5 was eight times more active than CYP1A4 in catalyzing UROX. The stimulation of UROX by 3,4,3',4'-tetrachlorobiphenyl that has been observed in microsomes was also observed with the reconstituted enzymes. Similar dose response relationships were found for induction of UROX and EROD in both chick embryo liver microsomes and in cultured chick hepatocytes, indicating coinduction of CYP1A5 and CYP1A4. UROX was induced by the Ah receptor ligand, 3-methylcholanthrene, in chicken kidney as well as liver. The findings reported here and other evidence that CYP1A4 and CYP1A5 tend to exhibit CYP1A1 and 1A2-like enzyme activites, respectively, indicate that the division of some enzyme activities among CYP1A enzymes applies to different vertebrate classes.

Animals↗

PubMed1996

Oxidation of porphyrinogens by horseradish peroxidase and formation of a green pyrrole pigment.

When humans or plants are exposed to certain chemicals which interfere with heme biosynthetic enzymes, porphyrinogen intermediates accumulate and are oxidized to cytotoxic porphyrins. Here we have investigated the role of peroxidases in porphyrinogen oxidation. Horseradish peroxidase (HRP) rapidly oxidizes uroporphyrinogen to uroporphyrin and this is inhibited by ascorbic acid. HRP also oxidizes deuteroporphyrinogen (a synthetic porphyrin similar to protoporphyrinogen), but the yield of porphyrin is lower than with uroporphyrinogen as substrate. This low yield is in part due to a rapid, HRP-dependent conversion of deuteroporphyrin (but not uroporphyrin) to a green compound with spectral characteristics of a chlorin with a large peak at 638 nm. This reaction requires addition of a sulfhydryl reductant such as glutathione and is inhibited by ascorbic acid. These findings suggest that cellular peroxidases and ascorbic acid levels may play a role in modifying the phototoxic tetrapyrroles which accumulate in plants and humans after certain environmental exposures.

Horseradish Peroxidase↗

PubMed1996

Cloning and identification of HEM14, the yeast gene for mitochondrial protoporphyrinogen oxidase.

A respiratory-defective mutant (C54) of Saccharomyces cerevisiae was found to have a phenotype consistent with a mutation in either mitochondrial protoporphyrinogen oxidase or ferrochelatase. The mutant is grossly deficient in hemes, accumulates protoporphyrin and is rescued by exogenous heme. The increased levels of protoporphyrin at the expense of heme is indicative of a block in one of the two last steps of the heme biosynthetic pathway. Complementation of C54 by a known ferrochelatase mutant suggested that the defect was most likely in HEM14 encoding protoporphyrinogen oxidase. A plasmid capable of complementing C54 was obtained by transformation with a yeast genomic plasmid library. A partial sequence of the insert identified the gene as reading frame YER014 of yeast chromosome V (GenBank Accession Number U18778). This reading frame codes for a protein homologous to human protoporphyrinogen oxidase. Disruption of this gene elicits a respiratory defect and accumulation of protoporphyrin. The phenotype of the null mutant together with the homology of YER014p to human protoporphyrinogen oxidase provide compelling evidence that YER014 is HEM14.

Cloning, Molecular↗

PubMed1995

Ascorbic acid inhibits chemically induced uroporphyria in ascorbate-requiring rats.

Ascorbate was previously shown to suppress accumulation of uroporphyrin (URO) in cultured chick embryo hepatocytes and to competitively inhibit microsomal oxidation of uroporphyrinogen catalyzed by cytochrome P4501A2. Here we used the Osteogenic Disorder Shionogi (ODS) mutant rat, which cannot synthesize ascorbic acid, to examine the in vivo effect of ascorbic acid on hepatic URO accumulation caused by treatment with 3-methylcholanthrene (MC) and 5-aminolevulinate (ALA). Female mutant rats maintained on three levels of dietary ascorbate (15,200, and 800 ppm) were treated for a total of 24 days. On the 11th and 16th days, rats were administered 3-methylcholanthrene, and 5-aminolevulinate was present continuously in the drinking water from day 14. Hepatic URO accumulated at the two lowest ascorbate levels, but not at 800 ppm ascorbate. The latter dose produced normal hepatic ascorbate levels. Plasma ascorbate levels were proportional to the hepatic values. Male rats also accumulated URO at the low dietary dose of ascorbic acid. The methylcholanthrene-induced increase in microsomal levels of CYP1A1 and CYP1A2, total cytochrome P450, and activities of uroporphyrinogen oxidation and ethoxyresorufin deethylase were not affected by the dietary level of ascorbate. Neither male nor female Fischer 344 rats accumulated URO when treated with the MC/ALA regime. Hepatic ascorbate concentrations in these rats were five-fold to seven-fold higher than they were in mutant rats that developed uroporphyria on 150 ppm dietary ascorbate. In ODS rats fed ascorbate at 90 but not 900 ppm in the diet, hexachlorobenzene caused hepatic URO accumulation, indicating that the effect of ascorbic acid is not unique to the regimen using methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminolevulinic Acid↗

PubMed1994

Protoporphyrinogen accumulation in cultured hepatocytes treated with the diphenyl ether herbicide, acifluorfen.

Diphenyl ether (DPE) herbicides such as acifluorfen inhibit both the plant and mammalian forms of protoporphyrinogen oxidase, a heme biosynthetic enzyme. Only small amounts of protoporphyrin accumulated in primary cultures of chick embryo and rat hepatocytes treated with acifluorfen and the porphyrin precursor, 5-aminolevulinic acid. However, there was a large accumulation of the porphyrin precursor, protoporphyrinogen, which was detected after oxidation to protoporphyrin by an E. coli membrane enzyme. In contrast, conventional methods of porphyrin analysis which depend on quantitative autoxidation of protoporphyrinogen failed to detect this accumulation of protoporphyrinogen. This is the first demonstration that protoporphyrinogen can accumulate to high levels and remain stable in liver cells. In addition, we found that the effect of a protoporphyrinogen oxidase inhibitor such as acifluorfen on the regulation of heme synthesis in hepatocyte cultures differed from that of an iron chelator.

Aminolevulinic Acid↗

PubMed1994

Effects of hemopexin on heme-mediated repression of 5-aminolevulinate synthase and induction of heme oxygenase in cultured hepatocytes.

The serum protein hemopexin is considered to have a major role in the mechanism of the uptake of heme by hepatocytes by means of a heme-hemopexin receptor. Therefore, we examined in primary cultures of adult rat and embryonic chick hepatocytes whether the presence of hemopexin would affect the heme-mediated repression of 5-aminolevulinate synthase activity (the rate-limiting enzyme of heme biosynthesis) and the heme-induced increase of heme oxygenase activity (the rate-limiting step of heme degradation). Both of these heme-mediated effects were partly or entirely prevented by the presence of hemopexin. We conclude that homologous hemopexin, at molar concentrations exceeding that of heme, inhibited the uptake of heme into hepatocytes. These results suggest that heme, in amounts sufficient to affect the rate-limiting steps of heme synthesis and degradation, can only enter hepatocytes in primary culture when the binding capacity of hemopexin for heme has been exceeded or altered.

5-Aminolevulinate Synthetase↗