Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Increased oxidation of p-nitrophenol and aniline by intact hepatocytes isolated from pyrazole-treated rats

Induction of cytochrome P-450 IIE1 by pyrazole has been shown in a variety of studies with isolated microsomes or reconstituted systems containing the purified P-450 isozyme. Experiments were conducted to document induction by pyrazole in intact hepatocytes by studying the oxidation of p-nitrophenol to 4-nitrocatechol or of aniline to p-aminophenol. Hepatocytes prepared from rats treated with pyrazole for 2 days oxidized p-nitrophenol or aniline at rates which were 3- to 4-fold higher than saline controls. To observe maximal induction in hepatocytes, it was necessary to add metabolic substrates such as pyruvate, sorbitol or xylitol, which suggests that availability of the NADPH cofactor may be rate-limiting in the hepatocytes from the pyrazole-treated rats. Carbon monoxide inhibited the oxidation of p-nitrophenol and aniline by hepatocytes from the pyrazole-treated rats and controls, demonstrating the requirement for cytochrome P-450. The oxidation of both substrates by the hepatocyte preparations was inhibited by a variety of agents that interact with and are effective substrates for oxidation by P-450 IIE1 such as ethanol, dimethylnitrosamine, pyrazole and 4-methylpyrazole. Microsomes isolated from pyrazole-treated rats oxidized aniline and p-nitrophenol at elevated rats compared to saline controls. These results indicate that induction by pyrazole of the oxidation of drugs which are effective substrates for P-450 IIE1 can be observed in intact hepatocytes. The extent of induction and many of the characteristics of aniline or p-nitrophenol oxidation observed with isolated microsomes from pyrazole-treated rats can also be found in the intact hepatocytes.     

Role of cytochrome P-450IIE1 in N-nitroso-N-methylaniline induced hepatocyte cytotoxicity.

1. The cytotoxicity of N-nitrosomethylaniline (NMA) towards hepatocytes isolated from rats was prevented by acetone or ethanol (inhibitors for cytochrome P-450IIE1) but not by metyrapone or SKF525A (inhibitors for cytochrome P-450IIB1/2). Various alcohols, secondary ketones and isothiocyanates that induced cytochrome P-450IIE1 were also found to be protective. Various aromatic and chlorinated hydrocarbon solvents that are substrates or inducers of cytochrome P-450IIE1 also prevented NMA cytotoxicity. Nitrogen-containing heterocycles that induced cytochrome P-450IIE1 were less effective. Further evidence that cytochrome P-450IIE1 was responsible for the activation of NMA was the marked increase in hepatocyte susceptibility if hepatocytes from pyrazole-induced rats were used. 2. NMA was more cytotoxic to hepatocytes isolated from phenobarbital-pretreated rats than uninduced rats. However, metyrapone now prevented and SKF525A delayed the cytotoxicity whereas ethanol, acetone, allyl isocyanate, isoniazid or trichloroethylene had no effect on the susceptibility of phenobarbital-induced hepatocytes. Furthermore, microsomes isolated from phenobarbital-pretreated rats had higher NMA-N-demethylase activity which was more inhibited by metyrapone and SKF525A than that of uninduced microsomal activity. By contrast the N-demethylase activity of phenobarbital induced microsomes was more resistant to acetone, ethanol, hexanal, trichloroethylene and toluene than uninduced microsome. 3. The above results suggest that cytochrome P-450IIE1 catalyses the cytotoxic activation of NMA in normal or pyrazole-induced hepatocytes whereas cytochrome P-450IIB1/2 is responsible for cytotoxicity in phenobarbital-induced hepatocytes.     

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.     

Expression of rabbit cytochrome P-450IIE2 in yeast and stabilization of the enzyme by 4-methylpyrazole

A rabbit cytochrome P-450IIE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP1) and the iso-1-cytochrome c gene terminator (CYC1), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450IIE2 per mg of protein. The P-450IIE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450IIE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized.     

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.     

Induction of cytochrome P-450 and b5 in hepatocyte subpopulations with phenobarbital

Hepatocytes isolated from intact and phenobarbital-treated rats were separated by Ficoll discontinuous density gradient into five subpopulations. About 85% of the total cell number sedimented within the range of 1.044 to 1.126 g X cm-3. In intact rats, heavy hepatocytes contained about twice as much cytochrome P-450 and about 3 times as much cytochrome b5 as light cell population. Phenobarbital induced cytochrome P-450 in all subpopulations, with a more pronounced increase observed in light hepatocytes. On the contrary, maximum induction of cytochrome b5 was noted in heavy hepatocytes. The results demonstrate the absence of uniform distribution of cytochrome P-450 and b5 in hepatocyte subpopulations. The highest concentrations of both cytochromes are observed in heavy (centrolobular) hepatocytes.     

Intralobular localization of different cytochrome P-450 form dependent monooxygenase activities in the liver of normal and inducer-treated rats.

1. Isolated periportal (PP) and perivenous (PV) hepatocytes from normal and inducer-treated rat livers were used to examine the following: intralobular localization of cytochrome P-450IA, P-450IIB, P-450IIE and P-450IIIA dependent monooxygenase activities and effects of phenobarbital (PB), beta-naphthoflavone (BNF) and pregnenolone-16 alpha-carbonitrile (PCN) on the zonal induction of these monooxygenases. 2. 7-Ethoxyresorufin O-deethylase (7EROD), 7-pentoxyresorufin O-dealkylase (7PROD) and N-nitrosodimethylamine N-demethylase (NAND) activities of PP hepatocytes were not significantly different from those of PV hepatocytes. 3. Ethylmorphine N-demethylase (EMND) activity was significantly higher in PV hepatocytes than in PP hepatocytes of normal rats. 4. EMND activity was induced by PCN and PB treatments. The response of EMND activity to PCN treatment was higher in PP hepatocytes than that in PV hepatocytes, and as a result the PV dominance disappeared following PCN treatment. 5. Extents of the response of this activity to PB treatment were similar in PP and PV hepatocytes, and PV dominance remained unchanged even after induction.     

Inhibition of CCl4 metabolism by oxygen varies between isoenzymes of cytochrome P-450

Oxygen inhibition of CCl4 metabolism by different isoenzymes of cytochrome P-450 was assessed by studying liver microsomes isolated from control rats and rats treated with phenobarbital or isoniazid. Rates of CCl4 metabolism were similar for all microsomes under a nitrogen atmosphere. An air atmosphere inhibited metabolism by microsomes from control rats to 12% of the value under nitrogen and metabolism by microsomes from rats treated with phenobarbital to 5%. It inhibited metabolism by microsomes from rats treated with isoniazid only to 32%. Rats treated with phenobarbital, which increases hepatic cytochrome P-450 content, or isoniazid, which does not increase hepatic cytochrome P-450 content, both metabolized more CCl4 than control rats as indicated by exhalation of greater quantities of CCl4 metabolites and by an increase in CCl4 toxicity. These results indicate that some isoenzymes of cytochrome P-450 are more effective than others in metabolizing CCl4 when oxygen is present.     

Primary cultures of hepatocytes on human fibroblasts.

Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Centrilobular expression of ethanol-inducible cytochrome P-450 (IIE1) in rat liver

Western blot analysis of digitonin eluates as well as immunohistochemical analysis revealed a 30-fold higher concentration of cytochrome P-450IIE1 in the centrilobular than in the periportal regions of the rat liver. Ethanol treatment caused a selective centrilobular induction of P-450IIE1, whereas phenobarbital induced P-450IIB1/2 in both liver lobule regions. The heterogeneous distribution pattern of P-450IIE1 was also observed in cells isolated from either region and correlated to the relative content of P-450IIE1 mRNA in the two cell types. The regiospecific expression and induction of P-450IIE1 may explain why several hepatotoxins, known to be metabolized by this isozyme, primarily damage the centrilobular region in the liver.     

Primary cultures of hepatocytes on human fibroblasts

Summary Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cytochrome P-450 and chromosome damage by cyclophosphamide in LEC strain rats predisposed to hereditary hepatitis and liver cancer

LEC strain rats predisposed to hereditary hepatitis and liver cancer were examined for hepatic drug-metabolizing ability and the inducibility of chromosome damage by cyclophosphamide (CP) in somatic cells. Whereas the hepatic cytochrome P-450 contents and the activities of cytochrome P-450-catalyzed monooxygenases were lower in females than in males of both LEC and control LEA strains, male LEC rats exhibited significantly reduced cytochrome P-450 contents and monooxygenase activities compared with male LEA rats. When exposed to CP, a promutagen/procarcinogen requiring P-450-dependent metabolic activation, the frequencies of chromosome aberrations and sister-chromatid exchanges (SCEs) in bone marrow cells tended to be lower in females than in males of each strain and lower in LEC than in LEA rats of the same sex. In particular, the CP-induced SCEs were substantially lower in LEC rats. However, no such sex and strain differences were found in the SCE frequencies in regenerating hepatocytes of partially hepatectomized rats exposed to CP.     

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

Treatment of rats with ethanol or rabbits with either imidazole or pyrazole, agents known to induce the ethanol-inducible form of liver microsomal cytochrome P-450 (P-450 LMeb), caused, compared to controls, 3-25-fold enhanced rates of CCl4-dependent lipid peroxidation or chloroform production in isolated liver microsomes. No significant differences were seen when the rate of CCl4-dependent lipid peroxidation was expressed relative to the amount of P-450 LMeb in the various types of microsomal preparations. In reconstituted membranous systems, this type of P-450 was a 100-fold more effective catalyst of CCl4 metabolism than either of the cytochromes P-450 LM2 or P-450 LM4. It is proposed that the induction of this isozyme provides the explanation on a molecular level for the synergism seen of ethanol on CCl4-dependent hepatotoxicity.     

Post-translational reduction of cytochrome P450IIE by CCl4, its substrate

The molecular mechanism of cytochrome P450IIE reduction by CCl4 was reexamined by measuring its enzyme activity, immunoreactive protein contents, and mRNA levels. Aniline hydroxylase and the amounts of immunoreactive P450IIE were rapidly decreased in a time-dependent manner after a single dose of CCl4. No changes were observed in the amounts of immunoreactive P450IIC and P450IA despite significant decreases decrease in their catalytic activities. However, the decreases in P450IIE enzyme activity and immunoreactive protein by CCl4 were not accompanied by a decline in its mRNA level. The data thus suggested a post-translational reduction of P450IIE by CCl4, probably due to specific destruction of the P450IIE protein by its own substrate rather than heme moiety.     

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unimpaired induction of drug-metabolizing enzymes in hepatocytes isolated from rats with micronodular cirrhosis

To test further the competence of the cirrhotic liver to metabolize xenobiotics, hepatocytes were isolated from control and CCl4-induced cirrhotic male or female rats. Histologically micronodular cirrhosis was present in all CCl4-treated rats, while control rats had normal livers. Portal perfusion pressure and intrahepatic collagen content were also significantly increased by CCl4 administration. In male rats, no significant differences in levels of circulating transaminases nor in alkaline phosphatase was observed between cirrhotic and control rats, while CCl4-treated females had slightly higher than normal serum transaminase levels at the time of the studies. Hepatocytic cytochrome P-450 and basal xenobiotic biotransformation were unaffected by micronodular cirrhosis in both genders; calculation of the aminopyrine and 7-ethoxycoumarin intrinsic clearances (Cli) revealed, however, a slightly decreased transformation potential in hepatocytes obtained from cirrhotic females, a phenomenon not observed in cirrhotic male rats. It is speculated that the observed reduction in Cli may have been independent of cirrhosis per se, owing to the perduring cytotoxic effect of CCl4 as evidenced by the higher than normal level of transaminases in female rats. Finally, male rats were subjected to in vivo administration of phenobarbital or 3-methylcholanthrene; both compounds led to significant induction of the mixed-function oxidase system, which was similar in magnitude and in selectivity in control and cirrhotic rats as illustrated by calculation of the Michaelis-Menten kinetic parameters for aniline p-hydroxylation, aminopyrine-N-demethylation, 7-ethoxycoumarin-O-deethylation, and p-nitrophenol UDP-glucuronyl transferase. We conclude that in well-established but compensated and hepatolysis-free micronodular cirrhosis, hepatocytes are fully able to transform xenobiotics and to respond normally and selectively to inducers of drug metabolism.