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.     

Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes

We wished to determine if phenobarbital (PB)-inducible cytochrome P-450 [P-450(PB)] and autolysosomal membrane antigens could be localized immunocytochemically on the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by the post-embedding protein A-gold method. P-450(PB) was maximally induced by PB treatment; then formation of autophagosomes and accumulation of autolysosomes were induced by cessation of PB treatment and by injection of leupeptin, respectively. P-450(PB) was detected neither on the isolation membranes nor on the limiting membranes of autophagosomes and autolysosomes. Autolysosomal membrane antigens, which were localized by the immunogold technique exclusively in post-Golgi compartments such as lysosomes, endosomes, and plasma membrane but were not found in pre-Golgi compartments such as endoplasmic reticulum (ER) and nuclear envelope, were detected in large amounts on the isolation membranes. These results suggest that the isolation membranes originate not from ER membranes but from post-Golgi membranes. We also present direct immunoelectron microscopic evidence that P-450(PB) is indeed degraded in the autolysosomes: when rats were treated with leupeptin, P-450(PB) was detected not only within the autophagosomes but also within the autolysosomes, whereas without leupeptin treatment, P-450(PB) was detectable only within the autophagosomes.     

The effect of separate and consecutive administration of cytochrome P-450 inducers to rats on the localization of various isoforms of the enzyme in liver lobe cells

By means of immunohistochemical methods a study of the liver intralobular localization of cytochromes P-450b(+e) and P-450c(+d) has been carried out after separate and consecutive treatment of rats with phenobarbital (PB) and 3-methylcholanthrene (MC). PB-treatment leads to localization of P-450b in the centrilobular region, whereas homogeneous distribution of P-450c in the lobule is observed after MC-treatment. The consecutive treatment with PB and MC is accompanied by the localization of P-450c only in cells of the periportal region of the lobule. PB-treatment of rats after preliminary MC-injection also results in the periportal localization of P-450c, and a small quantity of P-450b is localized in hepatocytes of the centrilobular region. Hence, the consecutive treatment with inducers of different molecular forms of cytochrome P-450 is accompanied by the redistribution of these isoenzymes in parenchymatous cells of the liver lobule. That is confirmed also by biochemical testing and immunochemical analysis of the microsomal fraction of hepatocytes.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.     

The expression and metabolic activity of cytochrome P-450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes

The expression and activity of the phenobarbital (PB)-inducible P-450 isozymes, P-450b and P-450e, and the major 3-methylcholanthrene (MC)-inducible form, P-450c, were studied in primary cultures of adult rat hepatocytes in T1, Leibovitz L-15 (L-15), and a modification of Waymouth 752/1 (Way) media. P-450 isozymes in initially isolated hepatocytes and control and PB-treated cultures were quantitated by Western blot analysis, and activity was determined with 7,12-dimethylbenz[a]anthracene (DMBA) as substrate. Data from the Western blot analysis correlated well with the metabolic activity toward DMBA. P-450b was consistently induced by PB in hepatocytes in T1 and to a lesser extent in Way. P-450e protein was constitutive in initially isolated cells, expressed in control cultures at a reduced level, and increased or maintained by PB in all three media. DMBA metabolite formation associated with P-450b and P-450e activity was induced by PB in hepatocytes in T1 and Way and was inhibited by antibodies to P-450b. P-450c was only infrequently expressed in freshly prepared hepatocytes, but was detected in all control and PB-treated cultures although at a much higher level in T1. Thus, the amounts of P-450 isozymes, their inducibility by PB, and their activity toward DMBA were found to be dependent on the medium. We have demonstrated enzyme induction and increased activity of the major PB-inducible isozymes in hepatocytes in T1; these are also associated with a change in the control of P-450c expression leading to enhanced constitutive expression and inducibility by phenobarbital.     

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.     

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.     

Macrolide antibiotics inhibit the degradation of the glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in primary monolayer culture

Treatment of rats with macrolide antibiotics such as triacetyloleandomycin (TAO) dramatically increases the hepatic concentration of a cytochrome P-450 indistinguishable from P-450p, the major liver cytochrome induced by glucocorticoids such as dexamethasone (Wrighton, S. A., Maurel, P., Schuetz, E. G., Watkins, P. B., Young, B., and Guzelian, P. S. (1985) Biochemistry 24, 2171-2178). To investigate the mechanism of induction of P-450p, we treated rats for 4 days with these agents and found that dexamethasone and TAO induced the synthesis of P-450p at least 70- and 35-fold over control values, respectively, as estimated from measurements of P-450p mRNA translatable in a cell-free system. However, the accumulation of P-450p holocytochrome (measured as TAO-metabolite spectral complex) or P-450p protein (measured by quantitative immunoblotting) increased at least 150-fold by TAO but only 32-fold by dexamethasone. The possibility that TAO decreases the degradation of P-450p was supported by the observation that administration of TAO to dexamethasone-treated rats labeled with NaH[14C]O3 and [3H]-delta-aminolevulinic acid retarded the decay of radioactive immunoprecipitable P-450p protein (t1/2 = 60 versus 14 h) and heme (t1/2 = 73 versus 10 h). To confirm these results, P-450p protein synthesis was measured as radioactivity incorporated into immunoprecipitable P-450p in primary monolayer cultures of adult rat hepatocytes incubated with [3H]leucine. Dexamethasone treatment of the cultures stimulated P-450p synthesis by at least 30-fold whereas macrolides were without effect. However, macrolide antibiotics but not dexamethasone inhibited the disappearance of radiolabeled P-450p from cultured hepatocytes similar to the results obtained in vivo. We conclude that macrolide antibiotics induce P-450p, the most rapidly turning over cytochrome yet reported, by stimulating its synthesis indirectly and by blocking its degradation, significantly.     

Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture

In the presence of phenobarbital (PB) at 3 mM, hepatocytes isolated from adult rats by a collagenase-perfusion technique survived well on plastic dishes for at least 49 days after initiation of primary culture. PB at concentrations less than 3 mM was ineffective for the maintenance of hepatocytes, and the maintenance of them was attained only in the continuous presence of 3 mM PB. The hepatocytes surviving in the presence of 3 mM PB were morphologically indistinguishable from the hepatocytes after 1-day attachment period, except for the presence of prominent nucleoli in the former. Although both the albumin secretion and tyrosine aminotransferase (TAT) activities of the cells decreased gradually up to day 7 with time in culture, both were thereafter maintained at relatively high levels at least up to day 35 of primary culture. The addition of 10 microM dexamethasone caused a 3-5-fold induction in TAT activity, and the cells were capable of responding to the hormone in this manner at least up to day 28 of primary culture. Furthermore, the cells also had glucose-6-phosphatase activity, even though the level of this enzyme activity was relatively low as compared with that of TAT activity. Survival of hepatocytes in the presence of 3 mM PB was further enhanced by simultaneous addition of dexamethasone (10 microM) and insulin (10 micrograms/ml). The sensitivity of hepatocytes to 3'-methyl-4-dimethylaminoazobenzene (0.24 mM) was remarkably reduced by treatment with PB at 3 mM. PB treatment decreased efficiently the falling rate of total cytochrome P-450 content, but did not induce P-450PB, which is the specific form of cytochrome P-450 induced by PB, in primary cultured hepatocytes. On the other hand, 3-methylcholanthrene (MC, 10 microM) caused an increase of both contents of total cytochrome P-450 and P-450MC, which is the specific form of cytochrome P-450 induced by MC, in primary cultured hepatocytes. However, MC was ineffective for the maintenance of hepatocytes in primary culture. The possible biological actions of PB on primary cultured hepatocytes are discussed on the basis of the experimental data obtained.     

Evidence that 2-allyl-2-isopropylacetamide and phenobarbital induce the same cytochrome P-450 in cultured chick embryo hepatocytes

The induction of cytochrome P-450 in cultured chick embryo hepatocytes was studied using two structurally unrelated compounds, 2-allyl-2-isopropylacetamide and phenobarbital. Pulse-labeling of these cells showed enhanced de novo synthesis of cytochrome P-450. The cytochrome induced by 2-allyl-2-isopropylacetamide, as well as the one induced by phenobarbital, reacted immunologically with antibodies raised against the major hepatic phenobarbital-induced isozyme. Additional form of cytochrome P-450 is induced exclusively by phenobarbital. These results clearly demonstrate that these two drugs induce at least one form of cytochrome P-450 in common.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.     

Synthesis of liver cytochrome P-450b in a cell-free protein synthesizing system

Live ppolysomes isolated from rats that had been treated with phenobarbital (PB) are able to incorporate [³H]leucine into total protein $\text{in}\text{vitro}$ at a rate almost five times that of polysomes prepared from control animals. Specific immunoprecipitation of translational products has shown that polysomes from induced animals synthesize cytochrome P-450b at a rate almost seven times greater than polysomes from control animals. The increased protein and cytochrome P-450b synthesis can be detected as early as 6 h following phenobarbital administration and reaches a maximum at 12–18 h. The results suggest that PB administration effects an increase in mRNA for cytochrome P-450b.     

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.     

Synthesis of sex-specific forms of cytochrome P-450 in rat liver is transiently suppressed by hepatic monooxygenase inducers

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), and alpha-naphthoflavone (alpha-NF) on the synthesis of drug-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), and sex-specific forms of cytochrome P-450, P-450(M-1), and P-450(F-1), in male and female rats were studied. Whereas P-450(PB-1) and P-450(MC-1) in liver microsomes were markedly induced in both sexes by treatment with PB and MC, respectively, the contents of P-450(M-1) and P-450(F-1) were significantly decreased by the treatments. alpha-NF, which is not a P-450 inducer, did not change the contents of sex-specific forms of cytochrome P-450. The translatable mRNAs of the P-450s were also determined by using an in vitro translation system. The mRNAs coding for P-450(PB-1) and P-450(MC-1) were increased by drug administrations. On the other hand, the mRNAs coding for P-450(M-1) and P-450(F-1) were transiently decreased by the drugs, and then returned to the normal levels. The time courses of the induction of the drug-inducible P-450s and the repression of the sex-specific P-450s showed no close correlation. alpha-NF had no effect on the synthesis of P-450(M-1) and P-450(F-1). We also found that the synthesis of P-450(M-1) in the livers of untreated rats showed no diurnal variations.