Inhibition of protein synthesis: a basis for tunicamycin-induced decrease in rat liver cytochrome P-450

Tunicamycin caused a dose and time dependent decrease in cytochrome P-450 in rat liver. A dose of 50 micrograms/kg caused a decrease of about 50% in 72 hours. A similar decrease in the activities of rat liver microsomal aniline hydroxylase, aminopyrine N-demethylase and ethoxycoumarin O-deethylase were also seen after the tunicamycin treatment. Tunicamycin also suppressed food and water intake but the decrease in cytochrome P-450 was not related to these effects. NADPH cytochrome c reductase was not markedly decreased by tunicamycin. A decrease in cytochrome P-450 was also observed in cultured rat hepatocytes treated with tunicamycin. It decreased incorporation of [35S]-methionine into total proteins as well as into various cytochrome P-450 isozymes of rat hepatocytes. This indicates that a decrease in protein synthesis may be responsible for the tunicamycin-induced decrease in cytochrome P-450 and drug metabolism.     

Antioxidants as stabilizers of cytochrome P-450 in hepatocytes

Spontaneous destruction of cytochrome P-450 arising from activation of lipid peroxidation (LPO) occurs during incubation of hepatocytes. LPO activation in hepatocyte suspension by a catalytic system containing Fe2+--ADP plus NADP X H makes the destruction of cytochrome P-450 more rapid. Supplementation of the incubation medium with the antioxidant, 2-ethyl-6-methyl-3-hydroxypyridine (HP-6), inhibits LPO, on the one hand, and stabilizes cytochrome P-450, on the other one. Ionol appeared to be a more effective LPO inhibitor in hepatocytes and, accordingly, a more effective stabilizer of cytochrome P-450 than water-soluble HP-6.     

A mechanism for the loss of cytochrome P-450 in primary mouse hepatocytes

This study examined various biochemical parameters such as mitochondria and mitochondrial DNA (mtDNA), total heme and cyto P450 content in fresh hepatocytes and dedifferentiated hepatocytes. These parameters were chosen in order to understand the dramatic decrease in drug metabolism in cultured hepatocytes. The data in this study shows a temporal decrease in cytochrome P450, total heme and also a decrease in mitochondria. Also, the ratio of mtDNA content to mitochondrial density was found to increase as hepatocytes underwent dedifferentiation. Stereological analysis of cell preparations provided a measure of mitochondrial density per cell area and mtDNA content was assessed by the use of a specific radiolabelled probe. This study demonstrates that a loss of the organelle which is partially responsible for synthesis of heme correlates with a decrease in cytochrome P450.     

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.     

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.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Effect of thioacetamide on cytochrome P-450 synthesis in rat liver.

Both calcitonin and prostaglandin E2 (PGE2) stimulate adenylate cyclase activity in the human breast cancer cell line (T 47D). The maximum cyclic AMP response to calcitonin exceeds that of PGE2. When maximal concentrations of the two hormones were added simultaneously to the cells, the amount of cyclic AMP generated was less than that seen with calcitonin alone. When cells were treated with the protein toxin of Bordetella pertussis (islet-activating protein; IAP) which inactivates the inhibitory regulatory component (Ni) of adenylate cyclase, there was no change in basal or calcitonin-responsive adenylate cyclase in intact cells. However, the PGE2 response was augmented at all dose levels, and this effect was dependent on the concentration of IAP. Moreover, in cells pretreated with IAP, simultaneous addition of PGE2 and calcitonin resulted in additivity rather than in inhibition of cyclic AMP production. The additivity of the response to calcitonin and PGE2 after IAP treatment implies activation of separate pools of adenylate cyclase catalytic subunit by the two hormones. These data are consistent with a model in which calcitonin acts on adenylate cyclase in T 47D cells through stimulatory regulatory components alone, whereas PGE2 acts on the same cells through both stimulatory and inhibitory components. The Ni input can limit the maximum effect of PGE2 and is capable of limiting calcitonin effects when the two agonists are used simultaneously.     

Hormonal requirements for the induction of cytochrome P-450 in hepatocytes cultured in a serum-free medium

Drug mediated induction of cytochrome P450 was studied in cultures of hepatocytes that had never been cultured in the presence of serum. Propylisopropylacetamide induced a five-fold increase in cytochrome P450, approximating $\text{in ovo}$ induced levels, when triiodothyronine and/or dexamethasone were included in the culture medium. Insulin was apparently not required for this induction. Cytochrome P450, free of cytochrome oxidase, could be fully recovered from cell homogenates in a 8700g supernatant, by use of a buffer containing 0.2% Emulgen.     

The properties of cytochrome P-450 induced in the rat small intestine by estradiol

The properties of cytochrome P-450 induced in the rat small intestine by estradiol were investigated. The interaction of substrates with intestinal microsomal cytochrome P-450 was compared to that of the enzyme induced in the rat liver by phenobarbital. The results obtained indicate that in the rat small intestine estradiol increases the concentration of the enzyme which differs from the liver type cytochrome P-450 but resembles the liver type cytochrome P-448. The difference spectroscopy data were supported by a parallel study on the action of specific inhibitors of the hydroxylation reaction.     

Hydroxylation products of hydrophobic xenobiotics--stabilizers of cytochrome P-450 in the hepatocytes

The effects of the hydroxylation product 3,4-benzo(a)pyrene and the free radical scavenger 1,2,3-trioxybenzene on cytochrome P-450 degradation in isolated rat hepatocytes induced by the Fe2+-ADP + NADPH system activating lipid peroxidation (LPO) were investigated. During incubation of hepatocytes, cytochrome P-450 is destroyed due to accumulation of LPO products. Addition of the free radical scavenger 1,2,3-trioxybenzene and the monoxygenase substrate 3,4-benzo(a)pyrene to the incubation medium induces inhibition of LPO and simultaneous stabilization of cytochrome P-450. Deceleration of malonic dialdehyde production by the free radical scavenger of the monoxygenase substrate suggests that both the compounds stabilize cytochrome P-450. It is assumed that in liver hepatocytes, exogenous free radical scavengers of the phenolic type and the products of their decarboxylation protect cytochrome P-450 against the LPO-induced destruction via oxidative metabolism of hydrophobic substrates.     

Detergents as probes of reconstituted rat liver cytochrome P-450 function

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.     

Ascorbic acid deficiency and cytochrome P-450 in adult rat hepatocytes in primary monolayer culture.

Adult rat hepatocytes in primary monolayer culture exhibit selective alteration of microsomal constituents and functions during the first hours of incubation ex vivo, including a striking decrease in the concentration of cytochrome P-450. The present studies document that these alterations are due in part to deficiency of l-ascorbate in the cultured cells. The deficiency appears to develop both by loss of the vitamin from the cells during their preparation and by a diminished synthetic capacity for ascorbate. Supplementation of the culture medium with l-ascorbate, at a concentration sufficient to restore intracellular levels of vitamin C to normal, results in maintenance of significantly increased concentrations of cytochromes P-450 and b₅. The activity of NADPH cytochrome c reductase similarly is ascorbate-dependent, suggesting that the vitamin plays a role in the formation and/or stabilization of membrane protein or lipid. Microsomal heme metabolism appeared to be unaffected by the presence or absence of ascorbate.     

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.     

Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.     

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.