Distribution and induction of cytochrome P-450 in rat liver nuclear envelope

Induction of cytochrome P-450s by 3-methylcholanthrene (MC) and phenobarbital (PB) and distribution of P-450s in the rat liver nuclear envelope were investigated by biochemical analyses and ferritin immunoelectron microscopy using specific antibodies against the major molecular species of MC- and PB-induced cytochrome P-450. It was found, in agreement with Kasper (J. Biol. Chem., 1971, 246: 577-581), that the total amount of cytochrome P-450s determined by biochemical analysis was markedly increased by MC, but not by PB, treatment. Immunoelectron microscopic analysis, however, showed marked and slight increases in ferritin labeling by MC and PB treatment, respectively. The latter finding was interpreted as resulting from the induction of a particular molecular species of PB-induced cytochrome P-450s. Ferritin immunoelectron microscopic analysis of intact isolated nuclei, naked nuclei from which the outer membrane of the nuclear envelope was partially detached (mechanically), and isolated nuclear envelopes have shown that the ferritin particles are found exclusively on the cytoplasmic face of the outer nuclear envelopes. Neither the nucleoplasmic face of the inner membrane of the nuclear envelope nor the cisternal face of both membranes of the nuclear envelope showed any labeling with ferritin. This indicates that cytochrome P-450 is located only on the outer membrane of the nuclear envelope and does not diffuse laterally into the domain of the inner membrane of the nuclear envelope across the nuclear pores. Our results suggest that a marked heterogeneity exists in the enzyme distribution between the outer and inner membrane of the nuclear envelope and that microsomal marker enzymes such as cytochrome P-450 exist exclusively in the outer membrane. In addition, it appears that cytochrome P-450 is probably not a transmembrane protein but an intrinsic protein located on the cytoplasmic face of the outer membrane of the nuclear envelope.     

Gamma-glutamyltransferase induction by dexamethasone in cytochrome P-450-depleted rat liver

The effects of the cytochrome P-450 depletion by cobaltic protoporphyrin IX on the postnatal glucocorticoid-inducibility of the membrane-bound enzyme gamma-glutamyltransferase have been assessed in the rat liver. Dexamethasone-induced gamma-glutamyltransferase activity in 14-, 28- and 77-day-old rats was high, weak and absent, respectively, and inversely correlated with the physiological cytochrome P-450 activity. In the liver acinus, the enzyme was reexpressed by the zone 1 and zone 2 hepatocytes in suckling rats, substantially only by the zone 1-hepatocytes in just weaned rats. Following cytochrome P-450 depletion, gamma-glutamyltransferase induction by dexamethasone was more rapid, more intense and more extended in the liver, acinus, occurring also in the zone 3 hepatocytes in suckling rats, in the zone 2 and a few zone 3 hepatocytes in just weaned rats. Further, the enzyme induction occurred also in adult rats in the zone 1 and in some zone 2 cells. This shows that cytochrome P-450 modulates the extent of hepatic gamma-glutamyltransferase induction by dexamethasone in postnatal rat-hepatocytes. The phenomenon may be consequent on hormone biotransformation changes caused by the cytochrome P-450 depletion.     

Activity of cytoplasmic NADP-dependent dehydrogenase in rat liver during induction of cytochrome P-450 by phenobarbital

Activity of oxidation enzymes of the pentosephosphate way (glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), cytoplasmic malate dehydrogenase (decarboxylating oxaloacetate) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) as well as the content of microsomal cytochromes b5 and P-450 in the rat liver have been studied 24 hours after 1, 2, 3, 4 and 5 intraperitoneal administrations of phenobarbital (4 mg per 100 g of the body weight). It is shown that the cytochrome P-450 content increases after a single administration of phenobarbital and then it gradually grows reaching its maximum after 4 administrations and falls after 5 administrations (though it remains high as compared to the control animals). The content of cytochrome b5 increases only after 4 administrations of phenobarbital and after 5th one it returns to the initial level. The content of microsomal gangliosides calculated per 1 mg of microsomal protein decreases after a single administration of phenobarbital and 5 days later it returns to the initial level. Activity of glucose-6-phosphate dehydrogenase increases after a single administration of phenobarbital, that of malate dehydrogenase--after 3 administrations, 6--phosphogluconate-dehydrogenase--after 4 administrations of the preparation. The 5 administrations of phenobarbital makes activity of all the mentioned dehydrogenases return to the initial level. Activity of isocitrate dehydrogenase under given conditions of the experiment does not change.     

Effects of emotional-pain stress on induction of P-450 cytochrome in the rat liver

Stress effect on cytochrome P-450 content in rat liver has been studied. There is evidence to the effect that stress factors of different origin cause the decline in cytochrome P-450 content both in the control group and in the conditions of induction. The data obtained enable us to suppose that stress causes intensification of cytochrome P-540 degradation process, the induction efficiency being steady.     

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.     

A role for xanthine oxidase in the loss of cytochrome P-450 evoked by interferon.

It has been suggested that the loss of cytochrome P-450, which is mediated by interferon and its inducers, can result from the generation of free radical species by the enzyme xanthine oxidase. Cytochrome P-450, aminopyrine N-demethylase, and ethoxyresorufin deethylase were depressed by 35, 36, 38%, respectively, in the livers of hamsters 24 h following the administration of a synthetic interferon (IFN-alpha-Con1) which contains the most frequent amino acid sequences of the human subtypes. Interferon increased the activities of the D and O forms of xanthine oxidase by 65 and 74%, respectively, in the same animals. The induction of the D form of xanthine oxidase, which is the precursor of the O form, preceded the loss in cytochrome P-450. The protein synthesis inhibitor, actinomycin D, prevented the interferon-induced loss of drug biotransformation and the increase in xanthine oxidase. The free radical scavenger, alpha-tocopherol, and the xanthine oxidase inhibitor, allopurinol, also prevented the loss of cytochrome P-450 mediated by the interferon inducer poly rI.rC. In chickens in which xanthine oxidase cannot be formed, poly rI.rC had no effect on cytochrome P-450 levels. These results suggest that xanthine oxidase induction may play some role in the interferon-mediated loss of cytochrome P-450.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Multi-functional property of rat liver mitochondrial cytochrome P-450

To solve the problem of whether a common enzyme catalyzes both 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1 alpha-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol competitively inhibited 25-hydroxylation of 1 alpha-hydroxy-vitamin D3 and vice versa. From these results it was concluded that 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 1 alpha-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Age-dependent expression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

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.     

Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p

We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of liver microsomal cytochrome P-450 (P-450) examined to date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the major form of P-450 induced in liver microsomes of rats treated with pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies to P-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN, the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and the ethylmorphine and erythromycin demethylase activities. Antibodies to P-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p.     

Segmental homologies in the coding and 3' non-coding sequences of rat liver cytochrome P-450e and P-450b cDNAs and cytochrome P-450e-like genes

The nucleotide sequence of a cloned cDNA insert carried by pHDQ14 was determined and found to code for the 107 C-terminal amino acids of rat liver cytochrome P-450e. Comparison of the pHQ14 cDNA sequence with those of cloned cDNAs for cytochrome P-450b and of 2 P-450e-like genes revealed segmental homologies that may have resulted from gene conversion. These results suggest that gene conversion may generate sequence variants of genes for rat liver cytochrome P-450s.     

Functional reconstitution of rat liver cytochrome P-450 with mesohemin

After allylisopropylacetamide-mediated "suicide" destruction of their prosthetic heme moieties, certain rat liver cytochrome P-450 isozymes can be effectively reconstituted by addition of exogenous hemin in vitro. We now report that two of these isozymes will equally accept mesohemin , a 2,4-diethyl heme-analog and result in a "meso-hemoprotein" with altered spectral but not functional characteristics.     

In vitro effects of corticosteroids, DDT, and 4, 9-dichlorodibenzodioxin on rat liver xanthine oxidase activity. Interactions between xanthine oxidase and cytochrome P450 in rat liver in vivo

It was established that deoxycorticosterone, cortisol, dexamethasone, DDT, and 4,9-dichlorodibenzodioxin inhibit in vitro binding of xanthine to highly purified rat liver xanthine oxidase. They are suggested to be allosteric inhibitors. The corresponding inhibition and binding constants were estimated. Also established was that cortisol and DDT, in dose 20 mg per kg of weight, inhibit xanthine oxidase and increase microsomal cytochrome P450 level in rat liver. There is an inverse relationship between xanthine oxidase activity and cytochrome P450 level in rat liver.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.