P-450 PB-4 Research Articles

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.

Hepatic and renal cytochrome P-450s in spontaneously hypertensive rats

The differences in the levels of cytochrome P-450s in hepatic and renal microsomes between spontaneously hypertensive rats (SHR) and normotensive control rats (Wistar Kyoto rats, WKY) were investigated by Western blotting with a specific antibody. Differences in the metabolic activity of the microsomes were also studied. In hepatic microsomes, the content of P450 PB-1 (IIIA2) was 140% higher in SHR than in WKY and the content of P450 IF-3 (IIA1) in SHR was one-seventh that in WKY. The differences reflected the increase in testosterone 6 beta-hydroxylation activity and decrease in testosterone 7 alpha-hydroxylation activity in hepatic microsomes of SHR. The level of P450 K-5 (IVA2) in hepatic microsomes of SHR was 4-times that in microsomes of WKY. The levels of other cytochrome P-450s in SHR were not very different from those in WKY. In renal microsomes, the levels of three renal cytochrome P-450s, P450 K-2, K-4, and K-5, were measured. The level of P450 K-5 (fatty acid omega-hydroxylase) in SHR was 50% higher than that in WKY and the difference reflected the increase in lauric acid omega- and (omega-1)-hydroxylation activities of the renal microsomes of SHR. The levels of P450 K-2 and K-4 did not differ in both rats.

Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes

1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days. 2. Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures. 4. These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

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.

ω- and (ω − 1) -hydroxylation of arachidonic acid, lauric acid and prostaglandin A 1 by multiple forms of cytochrome P-450 purified from rat hepatic microsomes

The metabolism of arachidonic acid, lauric acid and prostaglandin A 1 by rat hepatic microsomes and multiple forms of cytochrome P-450 purified from rat hepatic microsomes was studied. Arachidonic acid was hydroxylated by hepatic microsomes of male rats by ω- and (ω − 1)-hydroxylation. Phenobarbital treatment of rats decreased the hydroxylation activity slightly, but 3-methylcholanthrene treatment increased the hydroxylation activity 2-fold. However, lauric acid and prostaglandin A 1 ω- and ω − 1)-hydroxylation activities decreased after treatment with phenobarbital and 3-methylcholanthrene. Arachidonic acid and lauric acid were metabolized with similar ratios of ω- and (ω − 1)-hydroxylation, but prostaglandin A 1 was efficiently metabolized at the ω-position by hepatic microsomes of untreated male rats. In a reconstituted system with purified cytochromes P-450, P-450 UT-1, UT-2 ( P-450 h), MC-1 ( P-450 d) and MC-5 ( P-450 c) effectively hydroxylated arachidonic acid at both the ω- and (ω − 1)-position. P-450 UT-8 hydroxylated arachidonic acid only at the ω-position. P-450 DM ( P-450 J) hydroxylated arachidonic acid at the (ω−1)-position efficiently. Lauric acid was also hydroxylated by P-450 UT-1, UT-2, PB-1, PB-2, MC-1, IF-3 ( P-450 a) and DM, at the (ω−1)-position only. Only P-450 UT-8 could hydroxylate lauric acid at the ω-position. Prostaglandin A 1 was efficiently and specifically metabolized by P-450 UT-8 with ω-hydroxylation. P-450 UT-2 and PB-1 could hydroxylate prostaglandin A 1 by (ω − 1)-hydroxylation, but with low activity.

Changes in the amount of cytochrome P450s in rat hepatic microsomes with starvation

The effects of starvation on the composition of 12 different cytochrome P450s in rat hepatic microsomes were studied with a specific antibody. Changes in the metabolic activity of the microsomes were studied at the same time. P450 DM (P450j) was induced 2.5-fold by a 48-h starvation and its increase reflected the increase of metabolic activity of hepatic microsomes toward aniline, 7-ethoxycoumarin, and N-nitrosodimethylamine. P450 K-5, the major renal cytochrome P450 in untreated male rat, was also induced 2.5-fold by a 48-h starvation. P450 UT-2 (P450h) and P450 UT-5 (P450g), typical male-specific forms, decreased with starvation. P450 UT-2 had high testosterone 2α-and 16α-hydroxylation activities. These activities of hepatic microsomes were reduced with the decrease in P450 UT-2. P450 PB-1, testosterone 6β-hydroxylase, was increased time-dependently by starvation. P450 UT-4 (RLM2), a minor male-specific form, was not changed by starvation. P450 PB-2 (P450k), present in both sexes, was changed little by starvation. P450 PB4 (P450b) and P450 PB-5 (P450e) are strongly induced in rat liver by phenobarbital in coordinate fashion. Starvation increased P45O PB-4 12-fold but reduced P450 PB-5 to 22% of the control level. P450 MC-1 (P450d) was decreased by starvation. P450 MC-5 (P450c) was barely detected in control rats and was not changed by starvation. P450 IF-3 (P450a), rich in immature rats, was increased by starvation, accompanied by an increase in testosterone 7α-hydroxylation activity in the hepatic microsomes. We further investigated whether new cytochrome P450s appeared upon starvation by comparison of Chromatographic profiles of cytochrome P450 from starved rats with those of cytochrome P450 from control rats using HPLC. Three new cytochrome P450s were detected in the starved rats. These cytochrome P450s were purified to homogeneity. One of them was P450 DM, judging from spectral properties, catalytic activity, and the NH 2-terminal sequence. The two other forms were designated P450 3b and 4b. The minimum molecular weights of P450 3b and 4b were 53,000 and 52,000, respectively, and their CO-reduced absorption maxima were at 449 and 452 nm, respectively. P450 3b metabolized aminopyrine, N-nitrosodimethylamine, 7-ethoxycoumarin, and lauric acid, but with low activity. P450 4b was efficient in lauric acid ω- and (ω-1)-hydroxylation only. The spectral properties, catalytic activity, peptide map, and NH 2-terminal sequence of P450 4b agreed with those of P450 K-5. P450 3b was a new cytochrome P450, judged by these criteria.

Metabolism of lidocaine by purified rat liver microsomal cytochrome P-450 isozymes

The metabolism of lidocaine was studied using rat liver microsomes or a reconstituted lidocaine monooxygenase system with one of eight forms of cytochrome P-450 purified from liver microsomes from untreated- (P450 UT-2 and UT-5), phenobarbital- (P450 PB-1, PB-2, PB-4, and PB5) or 3-methylcholanthrene- (P450 MC-1 and MC-5) treated rats. A reverse phase high-performance liquid chromatography system capable of simultaneously assaying four major lidocaine metabolites, namely, monoethylglycinexylidide (MEGX), 3-hydroxylidocaine (3-OH LID), methylhydroxylidocaine (Me-OH LID) and glycinexylidide (GX), was employed to determine the rate of formation of each metabolite. Untreated microsomes generated MEGX, Me-OH LID, and 3-OH LID, but the formation of GX was not detected. In male rat liver microsomes, MEGX was the major metabolite of lidocaine when a concentration of 1 mM was employed. The formation of MEGX and Me-OH LID was increased significantly (P < 0.01) by microsomes from phenobarbital-treated rats, and the formation of 3-OH LID was increased with 3-methylcholanthrene. The study with the reconstituted system with purified cytochrome P-450 isozymes revealed that all eight forms of cytochrome P-450 used have an ability to N-deethylate lidocaine to form MEGX. Among these isozymes, cytochrome P450 PB-4 and P450 UT2 showed a higher turnover number for the formation of MEGX. Me-OH LID was formed exclusively by P450 PB-5, and 3-OH LID exclusively by P450 MC-1. Selectivity of cytochrome P450 PB-5 for aromatic methyl hydroxylation of lidocaine was confirmed by an inhibition study; formation of Me-OH LID by microsomes of rats treated with phenobarbital was inhibited completely by antibody against P450 PB-5. It was concluded that different cytochrome P-450 isozymes metabolize lidocaine with a different rate and different position selectivities. Since a specific substrate of cytochrome P450 PB-5 (P450e) is not known, lidocaine may be a useful substrate for the identification of P450 PB-5.

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats.

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.

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)

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.

Constitutive testosterone 6 beta-hydroxylase in rat liver.

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: Simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography

Four aminopyrine metabolites generated by hepatic microsomes were simultaneously assayed by high-performance liquid chromatography. The metabolites were 4-monomethylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 3-hydroxymethyl-2-methyl-4-dimethylamino-l-phenyl-3-pyrazoline-5-one (AM-OH), and one unidentified metabolite. MAA was the major metabolite generated by the microsomes; its formation was induced by phenobarbital but not by 3-methylcholanthrene. Female rats had lower N-demethylation activity of aminopyrine than male rats. The production of AA by microsomes was low. The formation of AM-OH was strongly induced by phenobarbital, but treatment with 3-methylcholanthrene reduced its formation. These differences in the microsomal aminopyrine monooxygenase activity are dependent on the relative amounts of the individual cytochrome P-450 isozymes. Therefore, we examined aminopyrine metabolism in a reconstituted system with purified cytochrome P-450s. P-450 UT-2 ( P-450h) had high aminopyrine N-demethylation and hydroxylation activities, but P-450 F-2 ( P-450i) had low N-demethylation activity and no hydroxylation activity. P-450 PB-4 ( P-450b) and P-450 PB-5 ( P-450e) had high aminopyrine hydroxylation activity and their N-demethylation activity also was high. The 3-methylcholanthrene-inducible forms P-450 MC-1 ( P-450d) and MC-5 ( P-450c) had aminopyrine N-demethylation activity but no hydroxylation activity. P-450 UT-4 (RLM2) is a unique form that produced a large amount of the unknown metabolite. P-450 UT-7 had the highest N-demethylation activity. Addition of cytochrome b 5 to the reconstituted system enhanced the aminopyrine hydroxylation activities of P-450s UT-1, UT-2, PB-2, and PB-5. Also, the N-demethylation activities of P-450s UT-1, PB-1, PB-2, and MC-1 were increased by cytochrome b 5. Metyrapone inhibited the catalytic activities of P-450s PB-4, PB-5, MC-1, and MC-5, and especially those of P-450s UT-4, and UT-7. The kinetics of the four major cytochrome P-450s ( P-450 UT-2, UT-4, PB-4, and MC-5) for aminopyrine N-demethylation and hydroxylation activities were studied. P-450s PB-4 and UT-2 had similar K m values (0.50 and 0.62 m m, respectively) in aminopyrine N-demethylation activity. These K m values were similar to the K m of aminopyrine N-demethylase in microsomes from phenobarbital-treated rats. P-450 UT-4 had a higher K m (3.23 m m) than P-450 UT-2 by an order of magnitude. P-450 MC-5 gave the highest K m value for aminopyrine N-demethylation (8.55 m m).

Sequence of the 5' end of the developmentally regulated rat P450 PB1 (P450IIC6) gene.

Sequence of the 5' end of the developmentally regulated rat P450 PB1 (P450IIC6) gene.

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16α-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes ( r = 0.72). In addition, anti- P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450 s ( r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.

Phenobarbital, dexamethasone and benzanthracene induce several cytochrome P450 mRNAs in rat hepatoma cells

Hepatoma cells derived from the Reuber H35 rat hepatoma express cytochrome P450 enzymes of two major families: polycyclic aromatic hydrocarbon-inducible forms are found in both differentiated and dedifferentiated cells while phenobarbital (PB)-inducible forms are found only in differentiated cells. We report here that (i) benzanthracene and PB induce P450 c mRNA in differentiated and dedifferentiated cells and (ii) dexamethasone and PB induce P450 b/e and/or P450 PB1 mRNAs in differentiated cells but not in dedifferentiated cells.

The effect of terpenoid compounds on cytochrome P-450 levels in rat liver

We have investigated the ability of camphor, menthol, pinene, limonene and myrcene to induce in rats members of a cytochrome P-450 sub-family termed PB P-450. These proteins have recently been designated as members of the P450IIB sub-family. None of these naturally occurring terpenoids significantly changed the total content of cytochromes P-450 or cytochrome b 5. Radioimmunoassay results showed that PB P-450 was induced 6-fold by camphor and to a lesser extent by menthol and pinene. The induction was confirmed by Western blotting. It was shown by nucleic acid hybridization that induction of PB P-450 by terpenoids was mediated by an increase in the amount of the corresponding mRNA. Analysis of the denaturation of mRNA-cDNA hybrids demonstrated that the mRNA induced by the terpenoids was encoded by a member of the P450IIB sub-family. None of the terpenoids had an effect on the amount of mRNA coding for P450IA2 (a cytochrome P-450 inducible by β-naphthoflavone and isosafrole). The results indicate that cytochromes P-450 induced by a synthetic compound, phenobarbital, may have originally evolved in response to terpenoid compounds normally present in the environment.

CDNA cloning, sequence, and regulation of a major female-specific and growth hormone-inducible rat liver cytochrome P-450 active in 15 beta-hydroxylation of steroid sulfates.

cDNA clones encompassing the complete coding sequence for the female-specific rat liver microsomal 15 beta-hydroxylase have been isolated and sequenced. This cytochrome P-450 species (P-450 15 beta) has a total of 490 amino acid residues, and its deduced amino acid sequence as well as direct sequencing of the amino-terminal portion of the purified protein revealed its identity with P-450 i. P-450 15 beta was found to be 66% similar to the male-specific rat liver microsomal 16 alpha-hydroxylase, P-450 16 alpha. Furthermore, P-450 15 beta is 47% similar to the major phenobarbital-inducible cytochrome P-450 in rat liver, P-450 b, while its structural similarity to P-450 c, P-450 pregnenolone-16a-carbonitrile, and P-450 lauric acid omega-hydroxylase is less than 30%. However, the developmentally regulated cytochromes P-450 f and P-450 PB-1 both have 68% similarity to P-450 15 beta. It is therefore concluded that P-450 15 beta belongs to the same gene subfamily (P450IIC) that also includes cytochromes P-450 f, PB-1, and 16 alpha. The plasma pattern of growth hormone (GH) is known to be a major determinant for the sex-specific expression of P-450 15 beta and P-450 16 alpha. Continuous infusion of GH to hypophysectomized male rats causes an increase in P-450 15 beta mRNA after 6 days of treatment, while intermittent injections have no effect. On the other hand, no effect on P-450 16 alpha mRNA is observed after continuous administration of GH, while intermittent injections cause an increase after 2 days of treatment. This relatively long period before P-450 15 beta and P-450 16 alpha mRNA induction is seen might indicate that protein factors mediating GH action are involved.

Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2, 6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca 2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450 h, P-450 PB-1/k, and P-450 p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16α-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2β- and 6β-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450 p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450 p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based “suicide” inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450 p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450 p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.

Expression and inducibility of drug-metabolizing enzymes in preneoplastic and neoplastic lesions of rat liver during nitrosamine-induced hepatocarcinogenesis.

The expression, inducibility, and regulation of four different cytochrome (cyt.) P-450 isoenzymes (PB1, PB2, MC1, and MC2) NADPH-cytochrome P-450 reductase, the glutathione transferases (GSTs) B and C and microsomal epoxide hydrolase (mEHb) have been studied during nitrosamine-induced hepatocarcinogenesis using immunohistochemical techniques. The investigations revealed basic differences in the expression of the individual drug metabolizing enzymes in the course of neoplastic development. While the two GSTs and mEHb were increased in all preneoplastic and benign neoplastic lesions, the levels of the distinct cyt. P-450 isoenzymes were characteristically different from each other. Following initial changes in the expression of these enzymes in early preneoplastic lesions (i.e., increase of cyt. P-450 PB1 versus slight decrease of the other cyt. P-450 isoenzymes), a continuous reduction of all cyt. P-450 isoenzymes was observed during the further course of hepatocarcinogenesis. In progressed neoplastic nodules, all cyt. P-450-isoenzymes and NADPH cyt. P-450 reductase were decreased to varying extents. Treatment of animals with inducers of the monooxygenase system, such as phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls, led to a rather heterogenous pattern of enzyme alterations in preneoplastic and neoplastic lesions. Following administration of phenobarbital, some islets responded to the same degree as the surrounding tissue, others were less or not at all inducible and a few of the lesions showed a prominent increase in cyt. P-450 PB2 and NADPH-cyt. P-450 reductase levels. The interesting finding that these two enzymes always showed concurrent changes may be indicative of a common regulation.(ABSTRACT TRUNCATED AT 250 WORDS)