Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).     

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene

Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.     

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (V_max) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.     

Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P-450b/e IgG established that low levels of "b-like" P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450's b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti-P-450b/e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (greater than 2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (less than 1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (approximately 40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti-P-450b/e. A protein comigrating with P-450e was well above detection (6-7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins)

The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed greatly in activity for the metabolism and binding of the various substrate homologues. The most rapidly metabolised substrates for control microsomes were phenoxazone and its shortest-chain ethers, for PB microsomes phenoxazone and the pentyl ether, and for 3MC microsomes the ethyl and propyl ethers. The variations in activity occurred in Vmax rather than in the apparent K_m-value. All the ethers gave Type I cytochrome P-450-binding spectra. The substrates giving the largest Type I spectra were the same for all microsomes—the ethyl, propyl and butyl ethers—but the magnitudes of the spectra differed in the order 3MC- > PB- > control microsomes. Phenoxazone and resorufin gave Modified Type II cytochrome P-450-binding spectra. PB-induction was most marked for the depentylation reaction (increased 101-fold), whereas 3MC-induction was most marked for depropylation and debutylation (88- and 96-fold).The intermicrosomal differences were interpreted as reflecting the different metabolic specificities of variant forms of cytochrome P-450. Substrate lipophilicity increased with increasing ether chain length and was not a major influence on specificity. The main substrate influence on specificity was steric, due to the presence and length of the ether side chain. The preeminent effect of ether chain length was considered to be on the rate of substrate transformation rather than on substrate interaction with cytochrome P-450.     

Evidence for O-dealkylation of 7-pentoxyresorufin by cytochrome P450 2B1/2B2 isoenzymes in brain

O-dealkylation of 7-pentoxyresorufin (PR) was studied in rat brain to characterise the functional activity specific for cytochrome P450 2B1/2B2 isoenzymes in brain microsomes. Brain microsomes catalyzed the O-dealkylation of PR in the presence of NADPH. Pretreatment with phenobarbital (PB; 80 mg/kg body wt, i.p.× 5 days) resulted in 3-4 fold induction of pentoxyresorufin-O-dealkylase (PROD) activity while 3-methylcholanthrene (MC; 30 mg/kg body wt, i.p. × 5 days) did not produce any significant increase in enzyme activity. Kinetic studies revealed that the rate of velocity (Vmax) for the O-dealkylation of PR was significantly increased to 2.9 times higher in brain microsomes isolated from PB pretreated rats. In vitro studies using metyrapone, an inhibitor of P450 2B1/2B2 catalyzed reactions and antibody for hepatic PB inducible P450s (P450 2B1/2B2) significantly inhibited the activity of PROD in cerebral microsomes prepared from PB pretreated animals. These studies suggest that PB inducible isoenzymes of P450, i.e. P450 2B1/2B2 specifically catalyze the O-dealkylation of PR in brain microsomes.     

Liver microsomal cytochromes P450‐dependent alkoxyphenoxazone O‐dealkylation in vitro by alligator and rat: Activities,inhibition, substrate preference,and discrimination factors

Six substituted alkoxyphenoxazones (resorufins) and four inhibitors of P450‐dependent mixed‐function oxygenases (MFO) were used to probe the breadth and extent of P450 metabolism induced by pretreatment with five xenobiotic chemicals in liver microsomes of the American alligator, Alligator mississippiensis. Phenobarbital (PB), 3‐methylcholanthrene (3MC), and PB–3MC co‐pretreatment elicited major induction of alligator MFO activity measured by alkoxyresorufin O‐dealkylation (AROD). The induced levels of activities observed with appropriate substrate, 7‐ethoxy, 7‐methoxy, 2‐phenylbenzyloxy, 7‐pentoxy, or 7‐benzyloxyresorufin (EROD, MROD, PBROD, PROD and BROD, respectively), were 10 to 100 times lower in alligator as compared to rat. The exception was a higher level of isopropoxyresorufin O‐dealkylation (IPROD) in alligator. The induction regimes used in alligator and rat revealed marked differences in substrate preference, discrimination factors (DF) for various inducible P450 isoforms. EROD, a classic indicator of CYP1A activity in rat, had a low DF in alligator. MROD was the best discriminator in alligator of CYP1A‐type induction. In contrast to rats, pretreatment of alligators with Aroclor 1254, 2,2′,4,4′ tetrachlorobiphenyl, and clofibrate caused minor alterations in AROD relative to untreated controls. The inhibitors, α‐napthaflavone, 1‐ethynylpyrene, SKF 525A, and 9‐ethynylphenanthrene, inhibited AROD activity of the expected P450 isoform. For example, 10 μM α‐napthaflavone inhibited liver microsomal EROD catalyzed by 3MC‐inducible isoforms from alligator by 90% and from rat by 97%. Similarly, 10 μM SKF 525A inhibited PROD catalyzed by PB‐inducible isoforms by 63% and 79% in alligator and rat liver microsomes, respectively. To the best of our knowledge, the present studies are the first to show PB induction of P450 activities typical of the mammalian CYP2 family and their inhibition with classical inhibitors in alligator liver. While our data indicate metabolism of P450 substrates with preferences to certain isoforms, it remains to be established which isoforms exert catalytic function in alligator and whether these are homologues or orthologues of mammalian isoforms. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 17–27, 1999     

Monoclonal antibody-directed phenotyping of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase in mammalian tissues

The distribution of cytochromes P-450 that catalyze aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase were studied with monoclonal antibody (MAb) 1-7-1 which completely inhibits these activities of a purified 3-methylcholanthrene-induced rat liver cytochrome P-450. The degree of inhibition by MAb 1-7-1 quantitatively assesses the contribution of different cytochromes P-450 in the liver, lung, and kidney microsomes from untreated, 3-methylcholanthrene- and phenobarbital (PB)-treated rats, mice, guinea pigs, and hamsters. Enzyme sensitivity to MAb 1-7-1 inhibition defines two types of cytochrome P-450 contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The MAb 1-7-1-sensitive cytochrome P-450 is a major contributor to aryl hydrocarbon hydroxylase in rat liver, lung, and kidney of 3-methylcholanthrene-treated rats, C57BL/6 mice, guinea pigs, and hamsters; this type is also present in lesser amounts in the extrahepatic tissues of the control and PB-treated animals, and in the lungs of the relatively "noninducible" DBA/2 mice treated with 3-methylcholanthrene. This form however makes little or no contribution to liver aryl hydrocarbon hydroxylase of control or PB-treated animals. 7-Ethoxycoumarin O-deethylase is also a function of both the MAb 1-7-1-sensitive and insensitive classes of cytochrome P-450. The ratio of the classes contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase differs in the various tissues and species and after inducer treatment. All of the 7-ethoxycoumarin O-deethylase activity in guinea pigs and hamsters is a function of cytochromes P-450 different than the MAb 1-7-1-sensitive cytochrome P-450 responsible for aryl hydrocarbon hydroxylase activity. Thus, the MAb 1-7-1 antigenically defines the type of cytochromes P-450 contributing to each reaction. Cytochromes P-450 can be viewed as paradigmatic for enzyme systems in which the nature and amount of product is regulated by multiple isoenzymic forms. Analyses using monoclonal antibodies to specific isoenzymes may thus have broad application to a variety of other complex systems which are composed of multiple isoenzymes.     

Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.     

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.     

Immunochemical characterization of some monooxygenase activities in liver microsomes from untreated and phenobarbital-treated rats

Two major forms of liver microsomal cytochrome P 450, one from untreated rats (P 450 A₂NI) and the other from phenobarbital-treated rats (P 450 B₂PB), were partially purified. Reconstitution of monooxygenase activities of purified enzymes and inhibition patterns of these activities by antibodies in microsomes gave the following results: 1) aniline hydroxylase activity is mainly supported by cytochrome P 450 A₂NI. This form is the major one in microsomes from control rats, but is also found at minute amounts in microsomes from phenobarbital-treated rats. It behaves as a constitutive form. 2) 4-nitroanisole-and benzphetamine-demethylase activities are mainly supported by cytochrome P 450 B₂PB which is predominant in phenobarbital-treated rats but is also present in control microsomes at low levels. 3) 4-nitroanisole-O-demethylase activity is less specific than benzphetamine-N-demethylase activity towards cytochrome P 450 B₂PB.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Effect of peroxisomal proliferators on microsomal prostaglandin A omega-hydroxylase

Earlier, we reported the isolation of a cytochrome P-450 highly active in prostaglandin A (PGA) omega-hydroxylation (PGA omega-hydroxylase) from rabbit kidney cortex, small intestine, and colon microsomes. In the present studies, the effects of peroxisomal proliferating agents on the PGA omega-hydroxylase have been examined. Administration of clofibrate or di(2-ethylhexyl)phthalate (DEHP) resulted in a significant increase in the PGA1 omega-hydroxylase activity of kidney cortex, liver, and small intestine microsomes. Similar findings were also obtained for laurate hydroxylase activity in kidney and liver microsomes. Kidney PGA omega-hydroxylase (designated cytochrome P-450ka) was isolated and highly purified from clofibrate- or DEHP-treated rabbits, with a yield 3 times higher than that from untreated, or phenobarbital- or 3-methylcholanthrene-treated rabbits. Cytochrome P-450ka from clofibrate- or DEHP-treated rabbits exhibited the same properties as those from untreated rabbits. Guinea pig antiserum against cytochrome P-450ka strongly inhibited the omega-hydroxylation of PGA1 by kidney cortex microsomes from clofibrate-treated rabbits. The PGA1 omega-hydroxylase activity of clofibrate-treated liver microsomes was also inhibited by this antiserum, suggesting that a PGA omega-hydroxylase immunochemically related to cytochrome P-450ka exists in liver microsomes.     

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.     

CYP3A induction aggravates endotoxemic liver injury via reactive oxygen species in male rats

We carried out this experiment to evaluate the relationship between isoforms of cytochrome P450 (P450) and liver injury in lipopolysaccharide (LPS)-induced endotoxemic rats. Male rats were intraperitoneally administered phenobarbital (PB), a P450 inducer, for 3 days, and 1 day later, they were intravenously given LPS. PB significantly increased P450 levels (200% of control levels) and the activities (300-400% of control) of the specific isoforms (CYP), CYP3A2 and CYP2B1, in male rats. Plasma AST and ALT increased slightly more in PB-treated rats than in PB-nontreated (control) rats with LPS treatment. Furthermore, either troleandomycin or ketoconazole, specific CYP3A inhibitors, significantly inhibited LPS-induced liver injury in control and PB-treated male rats. To evaluate the oxidative stress in LPS-treated rats, in situ superoxide radical detection using dihydroethidium (DHE), hydroxy-2-nonenal (HNE)-modified proteins in liver microsomes and 8-hydroxydeoxyguanosine (8-OHdG) in liver nuclei were measured in control and PB-treated rats. DHE signal intensity, levels of HNE-modified proteins, and 8-OHdG increased significantly in PB-treated rats. LPS further increased DHE intensity, HNE-modified proteins, and 8-OHdG levels in normal and PB-treated groups. CYP3A inhibitors also inhibited the increases in these items. Our results indicate that the induction or preservation of CYP isoforms further promotes LPS-induced liver injury through mechanisms related to oxidative stress. In particular, CYP3A2 of P450 isoforms made an important contribution to this LPS-induced liver injury.     

Photo-induced activation of cytochrome P450/reductase fusion enzyme coupled with spinach chloroplasts

Summary Photoactivation of cytochrome P450 monooxygenase was studied using a combination of spinach chloroplasts and yeast microsomes containing rat P4501A1/yeast reductase fusion enzyme. Under illumination, in the reaction mixture, NADP was reduced, transferring electrons to the P450/reductase fusion enzyme to convert 7-ethoxycoumarin to 7-hydroxycoumarin.     

Hydroxylation of prostaglandin A1 by the microsomes of rabbit intestinal mucosa

Microsomes from rabbit small intestine mucosa were found to catalyze the hydroxylation of PGA1 in the presence of NADPH. The major product was identified as 20-hydroxy PGA1 by using high performance liquid chromatography and gas chromatography-mass spectrometry, and the minor product was assumed to be 19-hydroxy PGA1. The ratio of the former product to the latter was about 24.1. The specific PGA1 omega-hydroxylase activity of small intestine microsomes was comparable to that of liver microsomes, and was significantly higher than those of microsomes from other tissues such as kidney cortex and lung. Microsomes from rabbit colon mucosa also catalyzed the hydroxylation of PGA1 in the presence of NADPH, with the ratio of omega- to (omega-1)-hydroxy PGA1 formed being 33.0. The PGA1 hydroxylase activities of the microsomes from both small intestine and colon were inhibited markedly by carbon monoxide, indicating the participation of cytochrome P-450. A cytochrome P-450 was solubilized from small intestine microsomes, and purified to a specific content of 10.5 nmol of cytochrome P-450/mg of protein. This cytochrome hydroxylated PGA1 at the omega-position with a turnover rate of 38.2 nmol/min/nmol of cytochrome P-450 in the reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and phosphatidylcholine. It is suggested that this cytochrome P-450 is specialized for the omega-hydroxylation of PGA1 in small intestine microsomes.     

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.     

Cloning and characterization of two major 3-methylcholanthrene inducible hamster liver cytochrome P450s

We have studied the immunochemical properties of two major 3-methylcholanthrene inducible hamster liver cytochrome P450 isozymes, P450 MC1 and P450 MC4. Immunoblots using specific antibodies against P450 MC1 and P450 MC4 demonstrated that these two P450s were present in very low levels in control hamster livers and were greatly induced by 3-methylcholanthrene treatment. P450 MC1 was immunochemically different from P450 MC4, rat P450c and P450d, and rabbit LM4. The immunorelated polypeptide to P450 MC1 was not present in the control or the 3-methylcholanthrene-treated rat liver microsomes, whereas it was present in two human liver microsomal preparations. On the other hand, P450 MC4 was immunochemically related to rat P450d and rabbit LM4. The immunorelated polypeptide to P450 MC4 was present in the human and 3-methylcholanthrene-treated rat liver microsomes. We also isolated full-length cDNA clones encoding P450 MC1 and P450 MC4 mRNAs from a 3-methylcholanthrene-induced hamster liver cDNA library. The full-length cDNA clones of P450 MC1 and P450 MC4 contained 1771 and 1868 base pairs, which encoded polypeptides of 494 and 513 amino acids, respectively. RNA blot analysis revealed that the mRNAs for P450 MC1 and P450 MC4 were 2100 and 2600 bases in length, respectively. 3-Methylcholanthrene pretreatment increased the P450 MC1 mRNA level by 16-fold and the P450 MC4 mRNA level by 11-fold in the hamster livers. A comparison of the deduced amino acid sequences with other cytochrome P450s revealed that P450 MC1 was most similar to the mouse P450(15) alpha with 75% sequence identity, whereas P450 MC4 shared 87% identity with the rat P450d or mouse P3(450). These results indicated that P450 MC1 was a unique member (CYP2A8) in the P450IIA subfamily, whereas P450 MC4 was the hamster P450IA2.     

Inhibition of azoreductase activity by antibodies against cytochromes P-450 and P-448

Hepatic microsomal azoreductase activity in mice was induced with phenobarbital (PB) and 3-methylcholanthrene (3-MC). Antibodies against cytochrome P-450 inhibited azoreductase activity of PB-treated animals while antibodies against cytochrome P-448 inhibited liver azoreductase activity of 3-MC-treated animals, each by about 90%. These antibodies also inhibited microsomal 7-ethoxycoumarin-O-deethylase activity to the same extent. It is concluded that hepatic microsomal azoreductase activity is almost totally dependent on cytochromes P-450 and P-448 and the contribution, if any, of other microsomal components is negligible.