Modulation of cytochrome P450 activity in the kidney of rats following long-term red wine exposure

Cytochrome P450 (CYP)-dependent oxidation of lauric acid, p-nitrophenol and ethanol by microsomal fractions of kidney were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total CYP content and specifically CYP 2E1, as well as p-nitrophenol and ethanol oxidation. The effects of ethanol treatment on the content and activity of CYP 2E1 were attenuated when red wine was administered, while the alcohol-free red wine values were similar to those of the control group. Although lauric acid hydroxylation was decreased by red wine treatment, the content of CYP 4A1 was not influenced by drinking fluids. We conclude that red wine administration attenuates the ethanol-induced enhancement of microsomal activities dependent on CYP 2E1 of rat kidney. Our results suggest that the non-alcoholic constituents of red wine could account for this modulation.     

Modulation of peroxisomal and microsomal fatty acid oxidation by acetone. A comparative study between liver and kidney

The effect of acetone consumption on some microsomal and peroxisomal activities was studied in rat kidney and these results were compared with data from former investigations in liver. Acetone increased the microsomal lauric acid hydroxylation, the aminopyrine N-demethylation catalyzed by cytochrome P450 and the microsomal UDP-glucuronyltransferase activity. Also, acetone increased the peroxisomal β-oxidation of palmitoyl CoA and catalase activities in kidney. These studies suggest that acetone is a common inducer of the microsomal and peroxisomal fatty acid oxidation, as previously shown in both starved and ethanol treated rats. Our results support the hypothesis that microsomal fatty acid ω-hydroxylation results in the generation of substrates being supplied for peroxisomal β-oxidation. We propose that the final purpose of these linked fatty acid oxidations could be the catabolism of fatty acids or the generation of a substrate for the synthesis of glucose from fatty acids. This pathway would be triggered by acetone treatment in a similar way in liver and kidney.     

In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450

The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.     

Oxidative stress, microsomal and peroxisomal fatty acid oxidation in the liver of rats treated with acetone

Parameters of oxidative stress, microsomal cytochrome P450 activity and peroxisomal fatty acid oxidation were studied in liver of rats following acetone (1% v/v) consumption for 7 days. Acetone treatment increased the activity of catalase and decreased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GTPx), but did not significantly modify the liver content of malondialdehyde (MDA) and reduced glutathione. Also, acetone increased the total content of cytochrome P450, the microsomal lauric acid hydroxylation, aminopyrine N-demethylation and the peroxisomal beta-oxidation of palmitoyl CoA. These effects were similar to those found previously in starved and ethanol-treated rats, supporting the hypothesis that ketone bodies would be the common inducer of microsomal and peroxisomal fatty acid oxidation in these metabolic states.     

Effects of gamma-hexachlorocyclohexane and L-3,3',5-triiodothyronine on rat liver cytochrome P4502E1-dependent activity and content in relation to microsomal superoxide radical generation

Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43% increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37% decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135% enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69% by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45% in HCCH- and T3-treated rats over control values, respectively, with a parallel 31% (HCCH) and 41% (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.     

Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450_alk (P450_alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450_alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450_alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450_alk. The coordinate expression of the P450_alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450_alk monooxygenase activity about 2-fold.     

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.     

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ. Dedicated to the memory of Dr. Susana Afonso     

Inhibition of human LDL lipid peroxidation by phenol-rich beverages and their impact on plasma total antioxidant capacity in humans

Mounting evidence shows that phenol-rich beverages exert strong antioxidant activity. However, in vivo evidence has produced conflicting results. In the present study, we studied the impact of the ingestion of 300 mL of black and green tea, alcohol-free red wine, alcohol-free white wine, or water on plasma total antioxidant capacity in five healthy volunteers. Red wine has the highest content of phenolics (3.63 +/- 0.48 g QE/L), followed by green tea (2.82 +/- 0.07 g QE/L), black tea (1.37 +/- 0.15 g QE/L), and white wine (0.31 +/- 0.01 g QE/L). Plasma total antioxidant capacity values of subjects who drank green tea rose at 30 min (P < 0.05). After black tea and red wine ingestion, the peaks were at 50 min (P < 0.05 and P < 0.01, respectively). No changes were observed in the control and white wine groups. Red wine and green tea were the most efficient in protecting low density lipoprotein from oxidation driven by peroxyl and ferril radicals, respectively. Phenol-rich beverages are a natural source of antioxidants; however, the phenolic content alone cannot be considered an index of their in vivo antioxidant activity.     

Molecular regulation of ethanol-inducible cytochrome P450-IIEI in hamsters

Liver polysomal poly(A)+ RNA, isolated from hamsters treated with ethanol or pyrazole, was translated in vitro to determine the effect of these compounds on specific mRNA encoding P450-IIEI, an ethanol-inducible P450 isozyme. As assessed by immunoprecipitation of translation products, ethanol and pyrazole increased hepatic P450-IIEI mRNA levels by 160% and 45%, respectively, when compared to controls. In liver microsomes from the same animals, ethanol and pyrazole caused a two-fold increase in microsomal P450-IIEI protein and a two- to three-fold enhancement of microsomal ethanol oxidation and p-nitrophenol hydroxylation. Our results show that the induction of P450-IIEI protein in hamsters by ethanol and pyrazole, an "ethanol-like" inducer, is accompanied by an increase in translatable P450-IIEI mRNA.     

The function of the microsomal oxidative system of different organs in guinea pigs administered ethanol

The microsomal cytochrome content and enzyme activity has been determined in liver, kidney, lungs and intestinal mucous from guinea pig males which were injected 25% ethanol intraperitoneally at a dose of 2 g per 1 kg of body mass. The changes in cytochrome P-450 and b5 content, amidopyrine-N-demethylase, aniline hydroxylase, p-nitrophenol hydroxylase. NADP.H-cytochrome-c-reductase activities in investigated organs of the animals have been found depending on the ethanol intoxication period (for 3, 6 or 14 days). Changes of the same type in microsomal enzyme activities have been discovered in liver, lungs and intestine, but not in kidney that is accounted for the substrate specificity and inducibility of the cytochrome P-450 some forms in extrahepatic tissues.     

Rat kidney antioxidant response to long-term exposure to flavonol rich red wine

This study evaluated the antioxidant defense system of the rat kidney following chronic exposure to red wine rich in flavonols. Both ethanol and antioxidant non-alcoholic wine components, mainly polyphenols, could contribute to the antioxidant status of kidney. Adult rats were given separately, water, ethanol (12.5%), red wine or alcohol-free red wine. After ten weeks of treatment, blood samples were obtained to determine plasma antioxidant capacity (FRAP, ferric reducing ability of plasma), uric acid and ethanol levels. Kidney tissues (cortex and papilla) were separated to perform measurements of reduced glutathione (GSH), glutathione disulfide (GSSG), lipid peroxidation (malondialdehyde, MDA) and the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activity of (Na + K)-ATPase, a membrane-bound enzyme, was also assessed. Red wine in plasma, elevated the FRAP without changing the concentration of uric acid; in kidney, it diminished the MDA production and elevated the GSH/GSSG ratio and the activity of CAT and GSH-Px. The activity of SOD did not change. Despite the finding that renal (Na + K)-ATPase activity was upregulated by ethanol, it was not altered by either red wine or alcohol-free red wine. The effects on the antioxidant enzymes could be attributed to ethanol, but the increase in the FRAP and GSH/GSSG ratio is attributed to the non-alcoholic components of red wine. These data suggest that there is an enhancement of the antioxidant defense potential in kidney and plasma, after chronic red wine consumption. Both ethanol and the non-alcoholic antioxidant constituents of red wine could be responsible for these effects.     

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells.

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.     

Increased catalytic activity of cytochrome P-450IIE1 in pericentral hepatocytes compared to periportal hepatocytes isolated from pyrazole-treated rats.

Cytochrome P-450IIE1 is induced by a variety of agents, including acetone, ethanol and pyrazole. Recent studies employing immunohistochemical methods have shown that P-450IIE1 was expressed primarily in the pericentral zone of the liver. In order to evaluate whether catalytic activity of P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, the oxidation of aniline and p-nitrophenol, two effective substrates for P-450IIE1, by periportal and pericentral hepatocytes isolated from pyrazole-treated rats was determined. Periportal and pericentral hepatocytes were prepared by a digitonin-collagenase procedure; the marker enzymes glutamine synthetase and gamma-glutamyl transpeptidase indicated reasonable separation of the two cell populations. Viability, yield and total cytochrome P-450 content were similar for the periportal and pericentral hepatocytes. Pericentral hepatocytes oxidized aniline and p-nitrophenol at rates that were 2-4-fold greater than periportal hepatocytes under a variety of conditions. Carbon monoxide inhibited the oxidation of the substrates with both preparations and abolished the increased oxidation found with the pericentral hepatocytes. Pyrazole or 4-methylpyrazole, added in vitro, effectively inhibited the oxidation of aniline and p-nitrophenol and prevented the augmented rate of oxidation by the pericentral hepatocytes. Western blots carried out using isolated microsomes revealed a more than 2-fold increase in immunochemical staining with microsomes isolated from the pericentral hepatocytes, which correlated to the 2-4-fold increase in the rate of oxidation of aniline or p-nitrophenol by the pericentral hepatocytes. These results suggest that functional catalytic activity of cytochrome P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, and that most of the induction by pyrazole of P-450IIE1 appears to occur within the pericentral zone.     

Microsomal ethanol-oxidizing system in Euglena gracilis. Similarities between Euglena and mammalian cell systems.

1. ADH activity of Euglena grown with 50 mM ethanol decreased, but MEOS activity increased with a corresponding increase in the total amount of cytochrome P-450. 2. Phenobarbital treatment increased the total amount of cytochrome P-450. 3. CO and KCN, cytochrome P-450 ligands, diminished acetaldehyde formed from ethanol oxidation by MEOS. 4. The amounts of NAD(P)H cytochrome c reductases and cytochrome b5 type, components of microsomal monooxygenase reaction, have been spectrophotometrically measured. 5. NAD(P)H cytochrome c reductases activities were induced by phenobarbital. 6. DMSO, an inhibitor of rabbit MEOS, inhibited O2 consumption (11-20%) by Euglena grown with an ethanol, but not a lactate medium. 7. These studies indicate the presence of cytochrome P-450-dependent MEOS in Euglena similar to that in the mammalian hepatic cell.     

10-Undecynoic acid, an inhibitor of cytochrome P450 4A1, inhibits ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

1,12-Dodecanedioic acid, the end-product of omega-hydroxylation of lauric acid, stimulates in a concentration dependent manner, phosphatidylethanolamine synthesis via ethanolamine-specific phospholipid base exchange reaction in rat liver endoplasmic reticulum. On the other hand, administration to rats of 10-undecynoic acid, a specific inhibitor of omega-hydroxylation reaction catalyzed by cytochrome P450 4A1, inhibits the ethanolamine-specific phospholipid base exchange activity by 30%. This is accompanied by a small but significant decrease in phosphatidylethanolamine content in the endoplasmic reticulum and inhibition of cytochrome P450 4A1. On the basis of these results it can be proposed that a functional relationship between cytochrome P450 4A1 and phosphatidylethanolamine synthesis exists in rat liver. Cytochrome P450 4A1 modulates the cellular level of lauric acid, an inhibitor of phospholipid synthesis. In turn, ethanolamine-specific phospholipid base exchange reaction provides molecular species of phospholipids, containing mainly long-chain polyunsaturated fatty acid moieties, required for the optimal activity of cytochrome P450 4A1.     

CYP78A1 Preferentially Expressed in Developing Inflorescences of Zea mays Encoded a Cytochrome P450-Dependent Lauric Acid 12-Monooxygenase

Cytochrome P450 (P450 or CYP) monooxygenases play an important role in the oxidation of a number of lipophilic substrates including secondary metabolites in higher plants. Larkin reported that CYP78A1 was preferentially expressed in developing inflorescences of Zea mays (Larkin, Plant Mol. Biol. 25: 343-353, 1994). However, the enzymatic function of CYP78A1 hasn’t been clarified yet. To characterized the enzymatic activity of CYP78A1, in this study, CYP78A1 cDNA and tobacco or yeast NADPH-cytochrome P450 oxidoreductase (P450 reductase) was expressed in the yeast Saccharomyces cerevisiae AH22 cells under the control of alcohol dehydrogenase promoter I and terminator. The reduced CO-difference spectrum of a microsomal fraction prepared from the transformed yeast cells expressing CYP78A1 and yeast P450 reductase showed a peak at 449 nm. Based on the spectrum, the content of a P450 molecule was estimated to be 45 pmol P450 equivalent/mg of protein in the microsomal fraction. The recombinant yeast microsomes containing CYP78A1 and yeast P450 reductase were found to catalyze 12-monooxygenation of lauric acid. Based on these results, CYP78A1 preferentially expressed in developing inflorescences of Zea mays appeared to have participated in the monooxygenation of fatty acids.     

Degradation of cytochrome P450 2E1: selective loss after labilization of the enzyme.

Mechanism-based inactivation of cytochrome P450 can result in the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. In the present study we took advantage of different modes of inactivation of P450 2E1 by CCl4, 1-aminobenzotriazole, or 3-amino-1,2,4-triazole to investigate parameters which target P450 2E1 for proteolysis from the microsomal membrane. Treatment of mice with CCl4 at the point of maximal induction of P450 2E1 after a single oral dose of acetone resulted in the complete loss of P450 2E1-dependent p-nitrophenol hydroxylation and a 75% loss of immunochemically detectable protein within 1 h of administration. Treatment with 1-aminobenzotriazole at the point of maximal induction caused a complete loss of P450 2E1-dependent p-nitrophenol hydroxylation but only a 12% loss of immunochemically detectable protein 1 h after administration. Treatment of mice with 3-amino-1,2,4-triazole caused a rapid loss of both catalytic activity and microsomal p-nitrophenol hydroxylase activity. However, unlike CCl4 treatment, the activity and enzyme level rebounded 5 and 9 h after treatment. The P450 2E1 ligand, 4-methylpyrazole, administered at the point of maximal induction maintained the acetone-induced catalytic and immunochemical level of P450 2E1. These results suggest that differentially modified forms of P450 2E1 show a characteristic susceptibility to degradation. While there are many potential pathways for protein degradation, the loss of P450 2E1 was associated with increased formation of high molecular weight microsomal ubiquitin conjugates. The formation of ubiquitin-conjugated microsomal protein which correlates with P450 2E1 loss suggests that ubiquitination may represent a proteolytic signal for the rapid and selective proteolysis of certain labilized conformations of P450 2E1 from the endoplasmic reticulum.     

Hepatic and intestinal gamma-glutamyltranspeptidase activity: its activation by chronic ethanol administration.

To study the effect of chronic ethanol administration on the activity of gamma-glutamyltranspeptidase (GGTP) in various tissues, female rats were pair-fed liquid diets with 36% of total calories either as ethanol or isocaloric carbohydrate (controls). Six weeks of ethanol feeding in an increase of cytochrome P450 content by 70%. Hepatic microsomal GGTP activity was more than doubled after ethanol feeding whether expressed per gram of liver or per mg of microsomal protein. Furthermore intestinal GGTP activity was significantly enhanced after ethanol, whereas there was no change in the enzyme activity in either kidney or pancreas. Phenobarbital administration to rats also resulted in an enahancement of GGTP activity in the liver but not in the intestine. These results suggest that enhanced hepatic and intestinal GGTP activities may contribute, at least partly, to increased serum GGTP activity frequently seen in alcoholics.     

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.