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Ethanol and isopentanol are the predominant alcohols in alcoholic beverages. We have reported previously that pretreatment of rats with a liquid diet containing 6.3% ethanol plus 0.5% isopentanol for 7 days results in a synergistic increase in acetaminophen hepatotoxicity, compared with rats treated with either alcohol alone. Here, we investigated the role of CYP3A in acetaminophen hepatotoxicity associated with the combined alcohol treatment. Triacetyloleandomycin, a specific inhibitor of CYP3A, protected rats pretreated with ethanol along with isopentanol from acetaminophen hepatotoxicity. At both 0.25 and 0.5 g acetaminophen/kg, triacetyloleandomycin partially prevented elevations in serum levels of alanine aminotransferase. At 0.25 g acetaminophen/kg, triacetyloleandomycin completely protected 6 of 8 rats from histologically observed liver damage, and partially protected the remaining 2 rats. At 0.5 g acetaminophen/kg, triacetyloleandomycin decreased histologically observed liver damage in 7 of 15 rats. In rats pretreated with ethanol plus isopentanol, CYP3A, measured immunohistochemically, was decreased by acetaminophen treatment. This effect was prevented by triacetyloleandomycin. These results suggest that CYP3A has a major role in acetaminophen hepatotoxicity in animals administered the combined alcohol treatment. We also found that exposure to ethanol along with 0.1% isopentanol for only 3 days resulted in maximal increases in acetaminophen hepatotoxicity by the combined alcohol treatment, suggesting that short-term consumption of alcoholic beverages rich in isopentanol may be a risk for developing liver damage from acetaminophen.

CYP2E is considered the only form of cytochrome P450 responsible for ethanol-mediated increases in acetaminophen hepatotoxicity. However, in experimental systems used for investigating ethanol-mediated increases in acetaminophen hepatotoxicity, animals are withdrawn from ethanol for 16 to 24 hr before the administration of acetaminophen to ensure the clearance of ethanol from the circulation. In rats, CYP2E has been shown to decrease to control levels after this time period of withdrawal from ethanol. We have previously shown in cultured human and rat hepatocytes, and in intact rats, that ethanol induces CYP3A in addition to CYP2E. To determine if there might be a role for CYP3A in ethanol-mediated APAP hepatotoxicity in addition to the recognized role for CYP2E, we investigated the effect of triacetyloleandomycin (TAO) on acetaminophen hepatotoxicity in ethanol-pretreated rats, as well as the effect of 11 hr withdrawal from ethanol on hepatic levels of CYP3A and CYP2E. TAO was dissolved in saline instead of dimethylsulfoxide, the solvent most usually employed, since dimethylsulfoxide inhibits CYP2E. Rats were administered 6.3% ethanol as part of the Lieber-DeCarli diet for 7 days, followed by replacement of the liquid diet with water for 11 hr. This 11-hr withdrawal from ethanol resulted in a decrease in hepatic levels of ethanol-induced CYP2E; however, considerable induction was still evident. There was no significant decrease in CYP3A. TAO completely prevented the histologically observed liver damage from acetaminophen in ethanol-pretreated rats, but did not prevent the increase in serum levels of AST. In ethanol-pretreated rats, exposure to APAP in the absence of TAO was associated with a 75% decrease in CYP3A, compared to animals exposed to APAP in the presence of TAO. These results suggest that CYP3A may have been suicidally inactivated by acetaminophen in the absence of TAO. Our findings suggest that CYP3A has a major role in ethanol-mediated increases in acetaminophen hepatotoxicity.

Acetaminophen (APAP) hepatotoxicity was investigated in rats fed ethanol and isopentanol alone or in combination in a liquid diet for 7 days. Serum levels of aspartate aminotransferase (AST) and histological examination of liver slices were used to assess hepatotoxicity. At 7 hr after intragastric administration of 0.5 or 1.0 g APAP/kg, there was no significant increase in serum levels of AST in rats treated with APAP alone, or in rats pretreated with ethanol or isopentanol alone followed by APAP. There was mild central lobular congestion in the livers of rats pretreated with ethanol alone followed by APAP. In contrast, in rats pretreated with the combination of ethanol and isopentanol, administration of APAP caused a dramatic increase in serum levels of AST, along with marked central lobular necrosis, including steatosis and ischemic changes. Hepatic glutathione levels were decreased to 40-50% of control values in APAP-treated rats that had been pretreated with ethanol either alone or in combination with isopentanol. The serum concentrations of APAP were significantly lower in rats pretreated with the combination of ethanol and isopentanol followed by 1 g APAP/kg than in rats treated with APAP alone, suggesting a greater rate of APAP metabolism. We had reported previously that combined treatment of rats with ethanol and isopentanol resulted in additive to synergistic increases in CYP3A, with no further increases in CYP2E than that caused by ethanol alone. CYP3A may, therefore, be responsible for the increased APAP hepatotoxicity caused by the combined alcohol treatment.

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.

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.

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 wild-type mice. However, in Cyp1a2 (−/−) 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.

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.

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.

The mechanism by which phenobarbital and similar compounds regulate gene expression has remained elusive for many years despite intense investigation. We had previously reported that the mRNA expression for the phenobarbital-inducible CYP2H1 gene was increased by cycloheximide treatment as rapidly and to a similar extent as by the phenobarbital-type drugs glutethimide and 2-propyl-2-isopropylacetamide (PIA), in primary cultures of chick embryo hepatocytes or in chick embryo liver in vivo (J. W. Hamilton, W. J. Bement, P. R. Sinclair, J. F. Sinclair, and K. E. Wetterhahn, 1988, Biochem. J. 255, 267-275). To examine the mechanism of this induction further, we determined the effects of various structurally related and unrelated inhibitors of protein synthesis on CYP2H1 expression in cultured chick embryo hepatocytes. Cycloheximide increased the transcription rate of the CYP2H1/2 genes to a similar extent as did PIA, and had little or no effect on CYP2H1 mRNA half-life. A number of other protein synthesis inhibitors, including streptovitacin, acetoxycycloheximide, pactamycin, and ricin, all increased CYP2H1 mRNA expression to a similar extent. The dose responses for induction of CYP2H1 mRNA and inhibition of protein synthesis by these agents were closely correlated. There was no relationship between the effectiveness of these agents to induce CYP2H1 mRNA expression and their structures or lipophilicity. Cycloheximide acetate required deesterification to cycloheximide for both inhibition of protein synthesis and induction of CYP2H1 mRNA. These results suggest that a labile negative regulatory protein is involved in CYP2H1/2 gene expression. It is also possible that this factor is involved in regulating the phenobarbital response of CYP2H1/2.

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. Light-induced 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 protoporphyrinogen 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.

Hepatic 5-aminolevulinate (ALA) synthase, the first and rate-limiting enzyme in the heme biosynthetic pathway, is known to be feedback repressed by the end product of the pathway, heme. We investigated whether heme regulates ALA synthase mRNA expression transcriptionally or post-transcriptionally in primary cultures of chick embryo hepatocytes. 2-Propyl-2-isopropylacetamide increased the rate of transcription of the ALA synthase gene, whereas heme or an inhibitor of heme biosynthesis, desferrioximine, had no effect on the drug-induced transcription rate. Heme decreased the half-life of ALA synthase mRNA from approximately 3.5 h to 1.2 as recently reported by Drew and Ades (1989, Biochem. Biophys. Res. Commun. 162, 102–107). We also found that the heme-mediated decrease in mRNA stability was prevented by cycloheximide treatment, suggesting that the heme effect was mediated by a labile protein. These results support a model for hepatic ALA synthase regulation in which inducing drugs directly stimulate ALA synthase gene transcription, whereas heme regulates ALA synthase expression post-transcriptionally by modulating mRNA stability as well as by blocking translocation of ALA synthase enzyme into the mitochondrion.

When hepatocytes are cultured on matrigel, a reconstituted basement membrane matrix, mRNAs for cytochrome P450 class IIB1/2 and class III genes can be induced by treatment with phenobarbital. We took advantage of this new system to critically evaluate the role of heme as a regulator of these cytochromes P450 and of 5-aminolevulinate synthase (ALA-S), the rate-limiting enzyme in heme biosynthesis. Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450, activities catalyzed by IIB1/2 (benzyloxy- and pentoxyresorufin O-dealkylases) and ALA-S activity, and ALA-S mRNA. Treatments with phenobarbital combined with succinyl acetone, an inhibitor of heme biosynthesis at the step of 5-aminolevulinate dehydrase, blocked the induction of the proteins for cytochrome P450IIB1/2 and cytochrome P450IIIAI, as indicated by spectral, immunological, and enzymatic assays. However, at the same time, succinyl acetone cotreatment failed to inhibit the induction of the mRNAs for cytochrome P450IIB1/2 and cytochrome P450IIIA. Lack of effect on the cytochrome P450 mRNAs was selective inasmuch as treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures. In contrast to earlier concepts, we conclude that in the intact hepatocyte, phenobarbital-induced cytochrome P450 induction is independent of changes in heme synthesis.

Radioactivity-labelled heme has usually been isolated from liver to which unlabelled carrier has been added by long, laborious techniques involving organis solvent extraction followed by crystallization. We have devised a simpler, rapid method for obtaining radiochemically-pure heme synthesized in vivo in rat liver from δ-amino[4- 14 C]levulinate, by modifying our previous procedure (Bonkowsky et al. (1975) J. Clin. Invest. 56, 1139–1148). This method, in which the heme is extracted into ethyl acetate/glacial acetic acid and in which porphyrins are removed from the heme-containing organic phase with HCl washes, does not require addition of carrier heme. The new method gives better heme recoveries than and heme specific activities identical to, those obtained using the crystallization method. In this new method heme must by synthesized from δ-amino[4- 14 C]levulinate; it not satisfactory to use [2- 14 C]-glycine substrate because non-heme counts are isolated in the heme fraction.

Chick embryo liver cells, when cultured for 41 h in the presence of [2-14C]mevalonic acid, took up label and incorporated radioactivity into heme a, but not into protoheme. Incubation of cells with delta-[4-14C]aminolevulinic acid (ALA) resulted in uptake of label and incorporation of radioactivity into both protoheme and heme a. These results show that both protoheme and heme a are synthesized during the incubation period, and that mevalonic acid is a specific precursor of the farnesyl moiety of heme a. Incubation of cells with [1,2-14C]acetate plus N-methyl mesoporphyrin IX, an inhibitor of heme synthesis, resulted in negligible incorporation of label into protoheme and heme a, although cellular lipids were highly labeled. This result indicates that the heme purification methods employed were capable of separating hemes from lipids, and that the measured incorporation of label into hemes from [14C]mevalonic acid and [14C]ALA was not due to lipid contamination.

Cultured chick embryo hepatocytes were iron-loaded with ferric nitrilotriacetate. Iron-loading was confirmed by both quantitative cellular iron determinations and ultrastructural studies. With iron-loading, lipid peroxidation, as detected by malonaldehyde released into the medium, occurred at a linear rate for 12h, after which time the rate of malonaldehyde production decreased. No cell toxicity, as detected by lactate dehydrogenase release, was noted. The amount of malonaldehyde recovered in the medium after 18h of exposure to iron represented 24-33% of the total malonaldehyde that could be produced by incubating lysed cells with iron and ascorbate. Cellular glutathione was not affected by iron-stimulated lipid peroxidation, but was increased by allylisopropylacetamide. Although iron-loading by itself had no effect on activity of 5-aminolaevulinate synthase, the first and rate-limiting step in haem synthesis, iron-loading in the presence of the porphyrogenic drug allylisopropylacetamide increased levels of 5-aminolaevulinate synthase 6-fold over levels induced by the drug alone. The antioxidant, butylated hydroxytoluene, totally inhibited iron-stimulated lipid peroxidation, but did not interfere with the effect of iron-loading to potentiate an increase in 5-aminolaevulinate synthase. After 18h of exposure to iron, followed by a change to fresh medium, the iron remaining within the cells did not stimulate further lipid peroxidation over the following 18h, but did potentiate an increase in 5-aminolaevulinate synthase on exposure to allylisopropylacetamide. It therefore appears that lipid peroxidation is not the mechanism by which iron potentiates induction of hepatic 5-aminolaevulinate synthase.

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

Uroporphyrinogen decarboxylase activity was investigated in cultures of chick embryo liver by two different methods: (1) analysis of porphyrin composition following incubation of intact cells with δ-aminolevulinic acid; and (2) a more conventional direct enzymic assay of cell homogenates. Activity was detectibly decreased following exposure of cells to 100 ng/ml 2,4,5,3′,4′-pentabromobiphenyl using the first method, but not the second. This decrease in activity was reversed by homogenizing the cells treated with 100 ng/ml pentabromobiphenyl. It is concluded that the direct homogenate assay of the enzyme may miss or underestimate decreases in its in vivo activity.

A role of haemopexin in transporting haem to hepatocytes for degradation has been inferred from the high affinity of haemopexin for haem. We have examined this question in primary cultures of chick-embryo and adult rat liver cells. We present here the results of four sets of experiments which indicate that haemopexin retarded haem uptake by hepatocytes in culture. (1) Haem bound to bovine serum albumin is known to repress the activity of delta-aminolaevulinate synthase in chick cultures as indicated by decreased porphyrin accumulation. When haem-albumin was added in the presence of excess purified or freshly secreted chicken haemopexin, no haem-mediated repression of porphyrin production was observed. The haem-mediated repression of porphyrin accumulation was partially prevented when human, but not chicken, albumin was added to cultures. This finding reflected the higher affinity of human albumin for haem compared with that of chicken albumin. (2) Haemopexin inhibited the ability of haem to be incorporated into cytochrome P-450 induced in the chick cultures in the presence of the iron chelator desferrioxamine. (3) The rate of association of [55Fe]haem with cultured rat hepatocytes when [55Fe]haem-haemopexin was added was one-eighth of the rate observed when [55Fe]haem-bovine serum albumin was used as the haem donor. (4) The presence of haemopexin also diminished the catabolism of haem by both rat and chick-embryo liver cell cultures. It is concluded that the uptake and subsequent metabolic effects of haem are inhibited in cultured hepatocytes by proteins such as haemopexin which have a high affinity for haem.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.

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.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was assayed in cultures of chick-embryo hepatocytes by the changes in composition of porphyrins accumulated after addition of excess 5-aminolaevulinate. Control cells accumulated mainly protoporphyrin, whereas cells treated with 3,4,3′,4′-tetrachlorobiphenyl or 2,4,5,3′,4′-pentabromobiphenyl accumulated mainly uroporphyrin, indicating decreased activity of the decarboxylase. 3-Methylcholanthrene and other polycyclic-hydrocarbon inducers of the P-448 isoenzyme of cytochrome P-450, did not affect the decarboxylase in the absence of the biphenyls. Induction of P-448 was detected as an increase in ethoxyresorufin de-ethylase activity. Pretreatment of cells with methylcholanthrene decreased the time required for the halogenated biphenyls to inhibit the decarboxylase. The dose response of methylcholanthrene showed that less than 40% of the maximal induction of cytochrome P-448 was needed to produce the maximum biphenyl-mediated inhibition of the decarboxylase. In contrast, induction of the cytochrome P-450 isoenzyme by propylisopropylacetamide had no effect on the biphenyl-mediated decrease in decarboxylase activity. Use of inhibitors of the P-450 and P-448 isoenzymes (SKF-525A, piperonyl butoxide and ellipticine) supported the concept that only the P-448 isoenzyme is involved in the inhibition of the decarboxylase by the halogenated biphenyls. The effect of preinduction with methylcholanthrene to enhance inhibition of the decarboxylase was also shown by the increased rate at which porphyrin accumulated from endogenously synthesized 5-aminolaevulinate after treatment of cells with the combination of propylisopropylacetamide and the biphenyls. Antioxidants, chelators of iron, and chromate affected the decrease in decarboxylase activity only if they prevented the induced increase in cytochrome P-448. We conclude that the P-448 and not the P-450 isoenzyme of cytochrome P-450 plays an obligatory role in the inhibition of uroporphyrinogen decarboxylase caused by halogenated biphenyls.

The amount of cytochrome P-450 was increased to different extents after treatment of cultured chick embryo hepatocytes with n -propanol, isopropanol, n -butanol, or isobutanol, These increases were associated with increases in benzphetamine demethylase activity, a cytochrome P-450-catalyzed oxidation, and glucuronidation of phenol red, catalyzed by UDP-glucuronyl transferase. The responses were similar to those obtained with ethanol or propylisopropylacetamide, which are phenobarbital-like inducers. Pretreatment of cells with cycloheximide prevented the increases in both cytochrome P-450 and glucuronidation of phenol red, indicating that protein synthesis was required for these responses.

Iron-loaded male C57BL/6 mice allowed free access to an aqueous solution of 5-aminolaevulinic acid (ALA) (2 mg/ml) as their only drink, develop severe uroporphyria within 9 days of a single intraperitoneal dose of 20-methylcholanthrene (MC) (125 mg/kg). At 21 days, uroporphyrinogen decarboxylase (EC 4.1.1.37) activities are less than 10% of control activities. The porphyria is not dependent on pretreatment with iron and persists for at least 21 days after withdrawal of ALA. The same intraperitoneal dose of MC does not produce porphyria within 21 days when given without ALA. Continuous administration of ALA markedly accelerates the onset of porphyria in iron-loaded male C57BL/6 mice after a single intraperitoneal dose of hexachlorobenzene (200 mg/kg); mice given phenobarbitone and ALA do not become porphyric. MC with ALA does not produce porphyria in iron-loaded male DBA/2 mice. At least two separate events are needed to produce uroporphyria in mammals: induction of a specific form of cytochrome P-450 and stimulation of the formation of intermediates of haem biosynthesis in the liver. These results show that severe, persistent porphyria can be produced in mammals by compounds other than polyhalogenated aromatic hydrocarbons and suggest that a similar mechanism underlies the porphyrogenic action of halogenated and non-halogenated compounds.

Uroporphyrin (URO) accumulation caused by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) and 3,4,3′,4′-tetrachlorobiphenyl (TCB) in cultured chick embryo hepatocytes was found to depend on the concentration of the added polyhalogenated aromatic compound, and on either the addition of 5-aminolevulinic acid or the induction of 5-aminolevulinic acid synthase. TCDD alone did not cause more than a slight increase in uroporphyrin, whereas TCB alone caused considerable uroporphyrin accumulation associated with increased 5-aminolevulinic acid synthase activity. However, in the presence of exogenous 5-aminolevulinic acid, TCDD was more potent than TCB in causing uroporphyrin accumulation. The concentrations of TCDD or TCB which maximally induced ethoxyresorufin deethylase activity, an indicator of induced cytochrome P450 activity, were lower than those required for maximal uroporphyrin accumulation. Furthermore, ethoxyresorufin deethylase activity was found to decline at concentrations of TCDD or TCB which caused maximum uroporphyrin accumulation. Pretreatment with 3-methylcholanthrene enhanced uroporphyrin accumulation, whereas addition of inhibitors of cytochrome P450 decreased uroporphyrin accumulation. Uroporphyrin accumulation occurred without a decrease in uroporphyrinogen decarboxylase activity, and was unrelated to the degree of conversion of the polyhalogenated aromatic compounds to water-soluble metabolites. Our results indicate that URO accumulation caused by TCDD and TCB requires two separate actions; (1) induction of cytochrome P450 which occurs at low concentrations of the halogenated chemicals, and (2) increased uroporphyrinogen oxidation which is catalyzed by the induced cytochrome P450 and which occurs at higher concentrations of the halogenated chemicals.

Cytochrome P450 and conjugation of phenol red are inducible in cultures of chick embryo hepatocytes. Cobaltous and manganous ions cause decreases in P450, which is partially restored in the intact cells by hemin.

To examine current models for the co-ordinate regulation of 5-aminolaevulinate (ALA) synthase and cytochrome P-450 we have determined the effect of drugs, inhibitors of haem biosynthesis, haem and cycloheximide on the steady-state expression of mRNAs for ALA synthase and a phenobarbital-inducible cytochrome P-450 (PB1 P-450), in chick embryo hepatocytes in vivo and in primary culture. We found that the mRNAs for ALA synthase and PB1 P-450 were rapidly and simultaneously induced by the porphyrinogenic drugs glutethimide and 2-propyl-2-isopropylacetamide. Inhibitors of haem biosynthesis when administered alone had a small effect on ALA synthase mRNA induction, but in combination with the drugs synergistically increased induction of both ALA synthase mRNA and enzyme activity. However, there were concentrations of inhibitors that increased induction of enzyme activity without increasing mRNA induction. Haem suppressed ALA synthase mRNA induction by drugs by only 50%, whereas induction of ALA synthase enzyme activity was completely suppressed. This suppression of ALA synthase mRNA by haem was blocked by cycloheximide treatment which did not block the induction of ALA synthase mRNA by drugs. In fact, cycloheximide synergistically increased the drug induction of ALA synthase mRNA, suggesting the presence of a labile protein factor which may interact with a haem-responsive element of the ALA synthase gene. Cycloheximide treatment alone did not significantly affect ALA synthase mRNA expression, but induced PB1 P-450 mRNA to a similar extent to that caused by porphyrinogenic drugs, suggesting the presence of a labile repressor which modulates PB1 P-450 gene expression. Basal and drug-inducible PB1 P-450 mRNA levels were unaffected by haem or by inhibitors of haem biosynthesis, indicating that the PB1 P-450 gene is not regulated by haem in chick embryo hepatocytes. Our results indicate that drugs simultaneously induce ALA synthase and PB1 P-450 mRNA expression, and that ALA synthase activity is regulated by haem principally at a post-transcriptional site rather than at the transcriptional level.