Reduced inflammatory potential of peritoneal macrophages recruited in mice pretreated with a glycolipid synthesis inhibitor.

Integral components of mammalian cell membranes, glycosphingolipids (GSL) reside in specialized plasma membrane microdomains critical for cell signaling. N-alkylated nojirimycins are compounds developed for GSL substrate deprivation therapy, blocking GSL synthesis by specifically inhibiting an essential enzyme, ceramide glucosyltransferase. Peritoneal macrophages recruited in mice pretreated with an inhibitory N-alkylnojirimycin displayed a reduced capacity to release either TNFalpha or interleukin-6 when re-exposed to whole killed E. coli in vitro. Cell viability and protein content were not affected. A nojirimycin analogue without GSL inhibitory capacity had no effect. The results show inhibition of GSL synthesis in vivo by an N-alkylnojirimycin can reduce the response to an inflammatory stimulus and indicate N-alkylnojirimycins have experimental and potential clinical value for modulating innate immune responses in vivo.

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.

Acetaminophen hepatotoxicity precipitated by short-term treatment of rats with ethanol and isopentanol: protection by triacetyloleandomycin.

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.

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.

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.

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.

Role of CYP3A in ethanol-mediated increases in acetaminophen hepatotoxicity.

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.

Protection of ethanol-mediated acetaminophen hepatotoxicity by triacetyloleandomycin, a specific inhibitor of CYP3A.

Cytochrome P450 2E (CYP2E) is considered responsible for ethanol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, it has been shown in cultured human and rat hepatocytes and intact rats that ethanol induces CYP3A in addition to CYP2E. Therefore, an investigation was made in rats to see whether or not an inhibitor of CYP3A, triacetyloleandomycin (TAO), would protect against ethanol-mediated increases in APAP hepatotoxicity. Rats, treated with 6.3 percent ethanol in the Lieber-DeCarli diet for 7 days, were administered APAP (lg/kg, i.g.) 11 hrs after removal of the diet. Triacetyloleandomycin (500 mg/kg, saline solution) was injected i.p. 2 hrs before the administration of APAP. In rats pretreated with ethanol, treatment with APAP for 7 hrs resulted in focal centrilobular congestion and steatosis. Triacetyloleandomycin completely prevented the histological liver damage in all 8 animals. These results suggest that, in ethanol-treated rats, CYP3A plays a major role in increasing APAP hepatotoxicity.

Acute hepatotoxicity of acetaminophen in rats treated with ethanol plus isopentanol.

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.

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)

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.

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.

Inhibition of protein synthesis increases the transcription of the phenobarbital-inducible CYP2H1 and CYP2H2 genes in chick embryo hepatocytes.

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.

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline induces and inhibits cytochrome P450 from the IA subfamily in chick and rat hepatocytes.

Several heterocyclic amines, found in cooked food, are powerful mutagens in the Ames Salmonella mutagenicity test system. One of these, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is one of the most mutagenic chemicals tested in this assay. In primary cultures of chick and rat hepatocytes, MeIQ, by itself, induced cytochrome P450 from the IA subfamily but was a weak inducer compared to 3-methylcholanthrene. However, in both chick and rat hepatocytes in culture, MeIQ decreased the amount of 3-methylcholanthrene-induced ethoxyresorufin deethylase activity, which is catalyzed by cytochrome P450 IA. The protein moiety of cytochrome P450 IA was decreased at MeIQ concentrations of 2.5 micrograms/ml or greater in chick hepatocytes and 25 micrograms/ml in rat hepatocytes. In hepatic microsomes from methylcholanthrene-treated chicks and rats, MeIQ was a competitive inhibitor of both ethoxyresorufin deethylase activity, a reaction catalyzed mainly by rodent cytochrome P450 IA1, and uroporphyrinogen oxidation, a reaction catalyzed by rodent P450 IA2. In cultured chick hepatocytes, MeIQ also decreased cytochrome P450-mediated oxidation of uroporphyrinogen by intact cells. The ability of MeIQ to inhibit as well as to induce cytochrome P450s of the IA subfamily may be important in assessing the mutagenic and carcinogenic effects of MeIQ in mammals.

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes.

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. 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.

Heme regulates hepatic 5-aminolevulinate synthase mRNA expression by decreasing mRNA half-life and not by altering its rate of transcription.

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.

Effect of interleukin 6 on phenobarbital induction of cytochrome P-450IIB in cultured rat hepatocytes.

Human recombinant interleukin 6 (rhIL-6) caused a dose dependent decrease in the phenobarbital induction of benzyloxyresorufin O-deethylase activity in cultured rat hepatocytes. Decreased enzymatic activity was associated with a decrease in the amount of immunoreactive P-450IIB1/2. rhIL-6 also prevented the PB-induced increase in the steady state level of P-450IIB mRNA. These results suggest that altered P-450 levels observed in vivo during the acute phase reaction may be due to interleukin 6.

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes.

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.