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.     

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.     

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.     

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.     

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.     

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.     

Induction of cytochrome P-450 by glucocorticoids in rat liver. II. Evidence that glucocorticoids regulate induction of cytochrome P-450 by a nonclassical receptor mechanism

In the companion report we used primary cultures of adult rat hepatocytes to demonstrate that glucocorticoids comprise a "class" of compounds that stimulate de novo synthesis of a form of cytochrome P-450 (P450PCN) indistinguishable from that induced by the nonhormonal steroid pregnenolone 16 alpha-carbonitrile (PCN). Because induction of P450PCN is stereospecific for glucocorticoids and is dependent on the concentration of and the length of exposure to steroids it seemed possible that P450PCN represented another of the many genes whose expression is coordinately regulated by glucocorticoids bound to their specific cytoplasmic receptor and translocated into the nucleus. However, in cultured hepatocytes treated with glucocorticoids, synthesis of P450PCN failed to parallel synthesis of a typical glucocorticoid-responsive liver function, tyrosine aminotransferase, in the time course of induction, in the concentrations of glucocorticoids required for half-maximal induction, and in the order of effective steroids ranked by potency. Indeed, two moderately potent inducers of P450PCN either failed to induce tyrosine aminotransferase (spironolactone) or actually antagonized induction of tyrosine aminotransferase synthesis by glucocorticoids (PCN). Moreover, in the same cultures in which glucocorticoid induction of tyrosine aminotransferase was blocked by the presence of PCN or other previously identified antiglucocorticoids, synthesis of P450PCN was actually enhanced. We conclude that synthesis of P450PCN is a specific glucocorticoid-responsive liver function evoked by a novel mechanism readily distinguishable from the classic glucocorticoid receptor pathway.     

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.     

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.     

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 mutant rat strain deficient in induction of a phenobarbital-inducible form of cytochrome P-450 in liver microsomes

Two phenobarbital-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(PB-4), were purified from the liver microsomes of phenobarbital-treated rats and identified with P-450b and P-450e, respectively. It was found, however, that the content of P-450(PB-4) in the liver microsomes of a strain of SD rat, Qdj:SD, was very low even after phenobarbital-induction. The levels of the mRNAs for P-450(PB-1) and P-450(PB-4) were separately determined using Northern blot hybridization with specific oligonucleotide probes. It was found that the level of P-450(PB-4) mRNA in the livers of phenobarbital-treated Qdj:SD rats was much lower than that of phenobarbital-treated Slc:SD rats. Slc:SD rats are widely used in Japanese laboratories. Genetic analysis using the crossbred animals between Qdj:SD and Slc:SD rats showed that the low expression of P-450(PB-4) in Qdj:SD rats is a recessive trait and is caused by a single gene mutation. However, no difference in the 5' flanking region in P-450(PB-4) gene was found between Qdj:SD rats and Slc:SD rats.     

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.     

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.     

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.     

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.