Release of Cr(III) from Cr(III) picolinate upon metabolic activation.

Hexavalent and trivalent chromium are released into the environment from a number of different industrial activities. It is known that Cr(VI) can be reduced and subsequently complexed by humic acids to produce Cr(III) humic acid complexes in the soil and aquatic environments. The metabolic fate of Cr(III) humic acid complexes and other Cr(III) organic complexes in mammalian systems is unknown. Therefore, Cr(III) picolinate was chosen as a model complex for Cr(III) humic acid complexes and other environmentally relevant Cr(III) complexes. Both human hepatocyte microsomes and primary cultures of chick hepatocytes were used to generate metabolites of Cr(III) picolinate. The results from both of these treatments show that a significant amount of Cr(III) is released (66 and 100%, respectively) and that N-1-methylpicotinamide is the primary organic metabolite from this compound. These data suggest that the populations of humans who are exposed Cr(III) picolinate or other environmentally relevant organic Cr(III) complexes, such as Cr(III) humic acid complexes, are potentially accumulating high levels of Cr(III) intracellularly. This intracellular accumulation of Cr(III) can result in the formation of covalent bonds between Cr(III) and DNA and/or other macromolecules, causing genotoxic effects. These data should be considered when assessing the risk of an area contaminated with chromium.

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.

Short-term treatment with alcohols causes hepatic steatosis and enhances acetaminophen hepatotoxicity in Cyp2e1(-/-) mice.

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.

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.

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.

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.

Effect of sodium arsenite on heme metabolism in cultured chick embryo hepatocytes.

We had previously reported that low concentrations of sodium arsenite (1-5 microM) decreased the induction of cytochrome P450 CYP1A and CYP2H in cultured chick embryo hepatocytes in parallel with increases in heme oxygenase. However, in those studies exogenous heme did not prevent the decrease in CYPs. In this study, we investigated the effect of arsenite on the synthesis and degradation of heme. Arsenite had no effect on induction of 5-aminolevulinic acid synthase mRNA or activity. Arsenite, at concentrations from 1 to 25 microM, had no effect on protoporphyrin synthesis from 5-aminolevulinic acid and did not increase the accumulation of other porphyrins, indicating that the enzymes in the pathway between 5-aminolevulinic acid synthase and ferrochelatase were unaffected by arsenite. Synthesis of heme from radioactive 5-aminolevulinic acid was slightly decreased (less than 20%) by 2.5 microM arsenite, a concentration that decreased induction of CYP1A and CYP2H by greater than 50%. Rates of biliverdin formation and degradation of exogenous heme were not different in cultures treated simultaneously with arsenite and heme or with heme alone. However, arsenite treatment increased biliverdin formation from heme synthesized from added 5-aminolevulinic acid by 60% and decreased the endogenous heme content of the cells by 30%. Our results suggest that although 2.5 microM arsenite induced heme oxygenase four- to sixfold, this had no effect on degradation of exogenous heme. Degradation of heme synthesized from 5-aminolevulinic acid was increased but this did not affect the regulatory heme pool.

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.

Mechanism of the synergistic induction of CYP2H by isopentanol plus ethanol: comparison to glutethimide and relation to induction of 5-aminolevulinate synthase.

We had previously found that combined treatment with isopentanol and ethanol synergistically induced CYP2H protein and activity in cultured chick nepatoytes. Here we investigated the mechanism of induction of CYP2H by the alcohols and whether they caused a coordinate induction of 5-aminolevulinate synthase (ALAS) mRNA. Treatment with isopentanol alone or in combination with ethanol resulted in coordinate increases in CYP2H1 and ALAS mRNAs. With isopentanol alone, the amounts of CYP2H1 and ALAS mRNAs at 4 to 6 h were similar to those observed after treatment with the alcohol combination, but declined by 11 h. Readdition of isopentanol at 11 h again increased the expression of both mRNAs, indicating that the decreases at 11 h were due to limiting amounts of inducer. Similar results were observed in cells exposed to low concentrations of glutethimide. In the combined alcohol treatment, increases in CYP2H1 and ALAS mRNAs were sustained from 4 h to 11 h after addition of the alcohols, but decreased to control levels by 24 h. Using pulse labeling to measure de novo synthesis of CYP2H1/2 protein, we found that the increases in CYP2H1/2 protein reflected the increases in CYP2H1 mRNA. The half-life of CYP2H1/2 protein, measured from pulse-chase experiments, was approximately twofold greater than the half-life of CYP2H1 mRNA. Our results indicate that the alcohols and glutethimide coordinately increase ALAS and CYP2H1 mRNA, and that increases in CYP2H1/2 protein arise from increases in its mRNA.

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.

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.

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.

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.

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.

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.

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)

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.

Cocaine toxicity in cultured chicken hepatocytes: role of cytochrome P450.

Cocaine (COC) causes liver damage in several species, including man. Chicken embryo hepatocyte cultures were evaluated as a model system to investigate the mechanism of cocaine-mediated hepatotoxicity. Parameters used to assess toxicity were: (1) release of lactate dehydrogenase (LDH); (2) decreased induction of 5-aminolevulinic acid synthase (ALAS), measured as porphyrin accumulation; and (3) decreased protein synthesis. Exposure of untreated cultures to COC or norcocaine (NOR) caused dose-dependent increases in LDH release, decreased protein synthesis, and eventual cell death. Pretreatment with 2-propyl-2-isopropylacetamide (PIA), a phenobarbital-like inducer of cytochrome P450, accelerated toxicity and lowered the threshold dose at which toxicity occurred. PIA pretreatment also increased rates of elimination of both COC and NOR and increased rates of formation of NOR from COC. The toxicity of COC and NOR could also be detected as decreased porphyrin accumulation. Addition of the P450 inhibitor SKF-525A concurrently with COC or NOR decreased their rates of elimination. SKF-525A also prevented the increase in LDH release as well as the decrease in protein synthesis caused by treatment with COC or N-hydroxynorcocaine (N-OH). Addition of SKF-525A up to 3 hr after COC resulted in partial prevention of the LDH increase. Exposure of the cultures to COC induced cytochrome P450 2H protein. We conclude that this hepatocyte culture system is highly sensitive to COC toxicity and that constitutive as well as induced cytochrome P450 isoforms are involved in the production of liver damage from COC.

Ascorbic acid inhibition of cytochrome P450-catalyzed uroporphyrin accumulation.

Previous studies on the mechanism of the uroporphyria caused by polyhalogenated aromatic hydrocarbons have indicated a key role of cytochrome P450 of the 1A subfamily in catalyzing uroporphyrinogen (UROgen) oxidation. Here we report that ascorbic acid (ASC) inhibits UROgen oxidation in primary cultures of chick embryo hepatocytes and hepatic microsomes from chickens and mice. In hepatocyte cultures, 0.15 mM ASC totally prevented the accumulation of uroporphyrin (URO) induced by treatment of cells with the combination of 3,4,3',4'-tetrachlorobiphenyl (TCB) and 2-propyl-2-isopropylacetamide (PIA), but had no effect on the induction of protoporphyrin accumulation by PIA and desferrioxamine. However, addition of 5-aminolevulinic acid (ALA) to cultures treated with PIA plus TCB decreased the ability of ASC to prevent URO accumulation, suggesting that the effectiveness of ASC was dependent on the intracellular concentration of ALA or its metabolites. Similarly, when chick hepatocyte cultures were treated with TCB plus exogenous ALA to produce URO accumulation, the effectiveness of ASC was also less than when ALA was produced endogenously. Under this condition, addition of piperonyl butoxide, a P450 inhibitor, increased ASC inhibition of URO accumulation. ASC competitively inhibited the oxidation of UROgen by hepatic microsomes from chicks or mice treated with 3-methylcholanthrene (MC) with Ki for ASC being about 0.1 mM. ASC prevented formation of a 500-nm absorbing compound, probably tetrahydrouroporphyrin, the first intermediate in UROgen oxidation. These results are consistent with ASC preventing URO accumulation in hepatocytes by competitive inhibition of the first step of UROgen oxidation and suggest a new physiological role of ASC, that of maintaining UROgen in the reduced state.