William J. Bement

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.

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-mediated increase in cytochrome P-450 in cultured hepatocytes.

Abstract Cellular levels of cytochrome P-450 were increased on exposure of cultured chick embryo hepatocytes to ethanol. This increase correlated with increases in the enzymatic activities of both benzphetamine demethylase [associated with the species of P-450 induced by propylisoproplyacetamide (PIA) or phenobarbital] and ethoxyresorufin deethylase [associated with the P-448 species induced by β-naphthoflavone (β-NF)]. The increased enzymatic activity relative to P-450 more closely resembled that caused by PIA than by β-NF. Glucuronidation of phenol red was also increased by ethanol; the dose response resembled that for increases in cytochrome P-450. PIA induced the glucuronidation of phenol red, whereas β-NF did not. Therefore, ethanol exposure was found to increase cytochrome P-450 and glucuronyltransferase in these cells in a pattern similar to increases observed with PIA.

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.

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.

Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix☆

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.

Increases in cytochrome P-450 in cultured hepatocytes mediated by 3- and 4-carbon alcohols

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 the 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.

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.

Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells

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.

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.