Development of Caco-2 cells expressing high levels of cDNA-derived cytochrome P4503A4

PURPOSE: To develop Caco-2 cell derivatives expressing high levels of human cytochrome P450 drug metabolizing enzymes. METHODS: The cDNAs for two cytochrome P450 forms, CYP2A6 and CYP3A4, were introduced into an extrachromosomal vector under control of the cytomegalovirus early intermediate promoter. Vector-bearing cells were selected via resistance to hygromycin B. RESULTS: Transfected cells exhibited high levels of cDNA-derived protein as measured by Western blot, spectrophotometric P450 determination and/or cytochrome P450 form-selective enzyme assay. CYP3A4 and CYP2A6 catalytic activities were about 100 fold higher than in control cells. cDNA-expressing cells were found to form tight monolayers and were suitable for study of xenobiotic transport and metabolism. The permeabilities of cephalexin, phenylalanine, mannitol and propranolol across transfected monolayers were found to be similar to those across untransfected monolayers. The appropriate transfected monolayers metabolized the CYP2A6 substrate coumarin and the CYP3A4 substrates testosterone and nifedipine. CONCLUSIONS: A Caco-2 cell system to simultaneously study drug transport and metabolism has been developed.     

Modeling of a three-dimensional structure of cytochrome P-450 1A2 and search for its new ligands

The substances inhibiting cytochrome P450 1A2 (CYP1A2) represent a perspective class of new drugs, which application in clinical practice can become the important part in preventive maintenance in oncology. The present work is devoted to computer modelling of 3-D structure of CYP1A2 and searching of new inhibitors by database mining. The modelling of CYP1A2 was done based on homology with 4 bacterial cytochromes P450 with known 3-D structure. For optimization of CYP1A2 active site structure the models of its complexes with characteristic substrates (caffeine and 7-ethoxyresorufin) were designed. These complexes were optimized by molecular dynamics simulation in water. The models of 24 complexes of CYP1A2 with known ligands with known Kd were designed by means of DockSearch and LeapFrog programs. 3D-QSAR model with good predictive force was created based on these complexes. On a final stage the search of knew CYP1A2 ligands in testing database (more than 23.000 substances from database Maybridge and 112 known CYP1A2 ligands from database Metabolite, MDL) was executed. 680 potential ligands of CYP1A2 with Kd values, comparable with known ones were obtained. This number has included 73 compounds from 112 known ligands, introduced in tested database as the internal control.     

The substrate binding site of human liver cytochrome P450 2C9: an NMR study

Purified recombinant human liver cytochrome P450 2C9 was produced, from expression of the corresponding cDNA in yeast, in quantities large enough for UV-visible and 1H NMR experiments. Its interaction with several substrates (tienilic acid and two derivatives, lauric acid and diclofenac) and with a specific inhibitor, sulfaphenazole, was studied by UV-visible and 1H NMR spectroscopy. At 27 degrees C, all those substrates led to an almost complete conversion of CYP 2C9 to high-spin (S = 5/2) CYP 2C9-substrate complexes characterized by a Soret peak at 390 nm; their KD values varied between 1 and 42 microM. On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon binding of its NH2 group to CYP 2C9 iron. Interactions of the five substrates with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the 1H NMR spectrum of each substrate. Distances between the heme iron atom and substrate protons were calculated from the NMR data, and the orientation of the substrate relative to iron was determined from those distances. Finally, a model for substrate positioning in the CYP 2C9 active site was constructed by molecular modeling studies under the constraint of the iron-proton distances. It points out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to establish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hydroxylation site and the anionic site. Sulfaphenazole was easily included in that model; its very high affinity for CYP 2C9 is due to a third structural feature, the presence of its NH2 function which binds to CYP 2C9 iron.     

Fluvoxamine inhibits the CYP2C19-catalysed metabolism of proguanil in vitro

OBJECTIVE: The potent CYP1A2 inhibitor fluvoxamine has recently been shown also to be an effective inhibitor of the CYP2C19-mediated metabolism of the antimalarial drug proguanil in vivo. The purpose of the present study was to confirm this interaction in vitro. METHODS: A high-performance liquid chromatography (HPLC) method was developed to assay 4-chlorophenylbiguanide (4-CPBG) and cycloguanil formed from proguanil by microsomes prepared from human liver. The limit of detection was 0.08 nmol mg-'. h-I. RESULTS: The formation of 4-CPBG and cycloguanil could be described by one-enzyme kinetics, indicating that the formation of the two metabolites is almost exclusively catalysed by a single enzyme, i.e. CYP2C19 within the concentration range used, or that the contribution of an alternative low-affinity enzyme, probably CYP3A4, is very low. This notion was confirmed by the lack of potent inhibition by four CYP3A4 inhibitors: ketoconazole, bromocriptine, midazolam and dihydroergotamine. Fluvoxamine was a very effective inhibitor of the oxidation of proguanil, displaying Ki values of 0.69 micromol x l(-1) for the inhibition of cycloguanil formation and 4.7 micromol x l(-1) for the inhibition of 4-CPBG formation. As expected, the CYP2C19 substrate omeprazole inhibited the formation of both metabolites with an IC50 of 10 micromol x l(-1). Norfluoxetine and sulfaphenazole inhibited proguanil oxidation with Ki values of 7.3-16 micromol x l(-1), suggesting that the two compounds are moderate inhibitors of CYP2C19. CONCLUSIONS: Fluvoxamine is a fairly potent inhibitor of CYP2C19 and it has the potential for causing drug-drug interactions with substrates for CYP2C19 such as imipramine, clomipramine, amitriptyline and diazepam. The combination of fluvoxamine and proguanil can not be recommended.     

Genes and regulatory sites of the "host-takeover module" in the terminal redundancy of Bacillus subtilis bacteriophage SPO1

Members of the CYP2F gene subfamily are selectively expressed in lung tissues and have been implicated as important catalysts in the formation of reactive intermediates from several pneumotoxic chemicals. Human CYP2F1 bioactivates 3-methylindole (3MI), while mouse CYP2F2 bioactivates naphthalene. Although 3MI is a potent pneumotoxin in ruminants and rodents, the participation of cytochrome P450s from the 2F subfamily in 3MI bioactivation has not been fully defined. To test the hypothesis that a goat lung 2F homologue uniquely catalyzes the dehydrogenation of 3MI to the putative electrophile 3-methylene-indolenine, the CYP2F3 cDNA was cloned from a goat lung cDNA library and expressed in Escherichia coli. The predicted amino acid sequence of CYP2F3 possessed 82% identity to both human CYP2F1 and mouse CYP2F2. CYP2F3 was mutated at the 5' end, expressed in E. coli, and shown to have a molecular mass of 50 kDa. The reconstituted enzyme uniquely catalyzed only the dehydrogenation of 3MI to form 3-methylene-indolenine, an electrophilic intermediate, without detectable formation of other products, thus demonstrating highly unusual selectivity for dehydrogenation rather than hydroxylation of a substrate. Immunoinhibition studies demonstrated that about 20% of the production of the intermediate in goat lung microsomal samples was produced by CYP2F3. The CYP2F3 enzyme had a specific activity that was similar to that of human cDNA-expressed CYP2F1. CYP2F3 also stereoselectively catalyzed the formation of the 1R,2S-oxide from naphthalene; this stereoisomer is the putative pneumotoxin. The enzyme, however, lacked catalytic activity with other common P450 substrates including 7-ethoxycoumarin, a substrate for CYP2F1, indicating that the substrate selectivity of CYP2F3 appears to be high.     

Cytochromes P450 in crustacea

Since the last review of this topic, further insight has been gained into the presence and functions of cytochrome P450 proteins in the hepatopancreas and other organs of aquatic crustacean species, although progress has been slow relative to the advances in other species. Recent studies with several lobster, shrimp, crab and crayfish species suggest that cytochromes P450 in the 2 and 3 families are the most abundant forms in hepatopancreas microsomes. Substrates normally metabolized by CYP2 and CYP3 family members are monooxygenated more rapidly by crustacea than substrates normally metabolized by CYP1 family enzymes, e.g. erythromycin, testosterone and aminopyrine are much more rapidly monooxygenated than ethoxyresorufin. Some progress has been made in cloning and sequencing crustacean P450 forms. CYP2L1 and CYP2L2 cDNA sequences have been cloned from spiny lobster hepatopancreas libraries, and there was evidence for at least two more cytochromes P450 in spiny lobster hepatopancreas. An area of continued interest, but of no consensus or general findings, relates to the presence and inducibility of CYP1 family members in crustacea. Some studies indicate weak induction of total cytochrome P450 and increased turnover of substrates normally associated with CYP1, while others show no effect of the classic inducers that act at the Ah receptor in vertebrates. A few studies of the roles of cytochromes P450 in the biosynthesis and degradation of steroids, including ecdysteroids, have been published. Further studies are needed to understand the regulation and normal function of the crustacean cytochromes P450.     

Molecular modelling of mammalian CYP2B isoforms and their interaction with substrates, inhibitors and redox partners

1. The construction of three-dimensional models of CYP2B isozymes from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6), based on a multiple sequence alignment with CYP102, a unique eukaryotic-like bacterial P450 (in terms of possessing an NADPH-dependent FAD- and FMN-containing oxidoreductase redox partner) of known crystal structure, is reported. 2. The enzyme models described are shown to be consistent with experimental evidence from site-directed mutagenesis studies, antibody recognition sites and amino acid residues identified as being associated with redox partner interactions, together with the location of a key serine residue (Ser-128) likely to be involved in protein kinaseA-mediated phosphorylation. 3. A substantial number of known substrates and inhibitors of CYP2B isozymes are shown to fit the putative active sites of the enzyme models in agreement with their reported position of metabolism or mode of inhibition respectively. In particular, there is complementarity between the characteristic non-planar geometries of CYP2B substrates and key groups in the enzymes' active sites. 4. Molecular modelling of CYP2B isozymes appears to rationalize a number of the reported findings from quantitative structure-activity relationship investigations on series of CYP2B substrates and inhibitors.     

Metabolism of epinastine, a histamine H1 receptor antagonist, in human liver microsomes in comparison with that of terfenadine

Epinastine is a non-sedative second-generation antiallergic drug, like terfenadine. In the present study, the metabolism of epinastine in human liver microsomes was investigated and compared with that of terfenadine. Terfenadine was extensively metabolized to terfenadine acid with a Km value of 1.78 microM, a Vmax value of 173.8 pmol/min/mg and a metabolic clearance (Vmax/Km) of 103.9. Epinastine, in contrast, was poorly metabolized by microsomes from the same source with a high Km value of 232 microM. Metabolic clearance of epinastine was only 0.832, which was lower by three orders of magnitude than that of terfenadine. Studies with microsomes expressing recombinant cytochrome P450 (CYP) species revealed that the CYP isoforms responsible for epinastine metabolism are CYP3A4, 2D6 and (to a minor extent) 2B6. Epinastine and terfenadine had no effect on CYP1A2 (theophylline 1-demethylation), 2C8/9 (tolbutamide hydroxylation) or 2E1 (chlorzoxazone 6-hydroxylation) activity, but weakly inhibited CYP2D6 (debrisoquine 4-hydroxylation) activity. CYP3A4 (testosterone 6 beta-hydroxylation) activity was strongly inhibited by terfenadine with a Ki value of 25 microM, whereas epinastine had no effect at up to 100 microM. Thus, epinastine is very poorly metabolized compared to terfenadine in human liver microsomes and does not inhibit CYP3A4 activity in vitro, unlike terfenadine.     

Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fimi

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Br?nsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.     

Increasing compliance with medical procedures: application of the high-probability request procedure to a toddler

The microsome of insect cells expressing CYP Ge-5 (CYP93B1), a cytochrome P450 cDNA of licorice (Glycyrrhiza echinata L.), catalyzed the formation of [14C]licodione and [14C]2-hydroxynaringenin from (2S)-[14C]liquiritigenin and (2S)-[14C]naringenin, respectively. On acid treatment, the products were converted to 14C-labeled 7,4'-dihydroxyflavone and apigenin. Eriodictyol was also converted to luteolin by the reaction with the microsome of yeast expressing CYP93B1 and subsequent acid treatment. CYP93B1 was thus shown to encode (2S)-flavanone 2-hydroxylase, which has previously been designated to licodione synthase and flavone synthase II depending on the substrates employed.     

In-vitro metabolism of YM17E, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, by liver microsomes in man

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl) ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [14C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6 beta-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and alpha-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.     

Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.     

Inhibition and induction of cytochrome P450 and the clinical implications

The cytochrome P450s (CYPs) constitute a superfamily of isoforms that play an important role in the oxidative metabolism of drugs. Each CYP isoform possesses a characteristic broad spectrum of catalytic activities of substrates. Whenever 2 or more drugs are administered concurrently, the possibility of drug interactions exists. The ability of a single CYP to metabolise multiple substrates is responsible for a large number of documented drug interactions associated with CYP inhibition. In addition, drug interactions can also occur as a result of the induction of several human CYPs following long term drug treatment. The mechanisms of CYP inhibition can be divided into 3 categories: (a) reversible inhibition; (b) quasi-irreversible inhibition; and (c) irreversible inhibition. In mechanistic terms, reversible interactions arise as a result of competition at the CYP active site and probably involve only the first step of the CYP catalytic cycle. On the other hand, drugs that act during and subsequent to the oxygen transfer step are generally irreversible or quasi-irreversible inhibitors. Irreversible and quasi-irreversible inhibition require at least one cycle of the CYP catalytic process. Because human liver samples and recombinant human CYPs are now readily available, in vitro systems have been used as screening tools to predict the potential for in vivo drug interaction. Although it is easy to determine in vitro metabolic drug interactions, the proper interpretation and extrapolation of in vitro interaction data to in vivo situations require a good understanding of pharmacokinetic principles. From the viewpoint of drug therapy, to avoid potential drug-drug interactions, it is desirable to develop a new drug candidate that is not a potent CYP inhibitor or inducer and the metabolism of which is not readily inhibited by other drugs. In reality, drug interaction by mutual inhibition between drugs is almost inevitable, because CYP-mediated metabolism represents a major route of elimination of many drugs, which can compete for the same CYP enzyme. The clinical significance of a metabolic drug interaction depends on the magnitude of the change in the concentration of active species (parent drug and/or active metabolites) at the site of pharmacological action and the therapeutic index of the drug. The smaller the difference between toxic and effective concentration, the greater the likelihood that a drug interaction will have serious clinical consequences. Thus, careful evaluation of potential drug interactions of a new drug candidate during the early stage of drug development is essential.     

The catalytic site of cytochrome P4504A11 (CYP4A11) and its L131F mutant

CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.     

Peroxisome proliferator-activated receptor alpha controls the hepatic CYP4A induction adaptive response to starvation and diabetes

The hepatic CYP4A enzymes are important fatty acid and prostaglandin omega-hydroxylases that are highly inducible by fibric acid hypolipidemic agents and other peroxisome proliferators. Induction of the CYP4A enzymes by peroxisome proliferators is mediated through the nuclear peroxisome proliferator-activated receptor alpha (PPARalpha). Fatty acids have recently been identified as endogenous ligands of PPARalpha, and this receptor has been implicated in the regulation of lipid homeostasis. In the present report we characterized the induction of the hepatic CYP4A genes in rats during the altered lipid metabolism associated with starvation and diabetes. The mRNA levels of CYP4A1, CYP4A2, and CYP4A3 were induced 7-17-fold in the livers of fasted animals and 3-8-fold in the livers of diabetic animals. This was accompanied by corresponding changes in CYP4A protein levels and arachidonic and lauric acid omega-hydroxylase activity. Interestingly, feeding animals after the fasting period caused as much as an 80% suppression of CYP4A mRNA levels, whereas CYP4A protein levels and functional activity returned to control values. A second PPARalpha-responsive gene, acyl-CoA oxidase, was also induced in rat liver by diabetes and fasting. By using PPARalpha-deficient mice, we unambiguously demonstrated that PPARalpha is strictly required for hepatic CYP4A induction by starvation and diabetes. Similarly, induction of hepatic thiolase and bifunctional enzyme also required expression of PPARalpha. This represents the first evidence for the pathophysiologically induced activation of a nuclear receptor.     

Activities of recombinant human cytochrome P450c27 (CYP27) which produce intermediates of alternative bile acid biosynthetic pathways

The primary physiological significance of cytochrome P450c27 (CYP27) has been associated with its role in the degradation of the side chain of C27 steroids in the hepatic bile acid biosynthesis pathway, which begins with 7alpha-hydroxylation of cholesterol in liver. However, recognition that in humans P450c27 is a widely or ubiquitously expressed mitochondrial P450, and that there are alternative pathways of bile acid synthesis which begin with 27-hydroxylation of cholesterol catalyzed by P450c27, suggests the need to reevaluate the role of this enzyme and its catalytic properties. 27-Hydroxycholesterol was thought to be the only product formed upon reaction of P450c27 with cholesterol. However, the present study demonstrates that recombinant human P450c27 is also able to further oxidize 27-hydroxycholesterol giving first an aldehyde and then 3beta-hydroxy-5-cholestenoic acid. Kinetic data indicate that in a reconstituted system, after 27-hydroxycholesterol is formed from cholesterol, it is released from the P450 and then competes with cholesterol for reentry the enzyme active site for further oxidation. Under subsaturating substrate concentrations, the efficiencies of oxidation of 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenal to the acid by human P450c27 are greater than the efficiency of hydroxylation of cholesterol to 27-hydroxycholesterol indicating that the first hydroxylation step in the overall conversion of cholesterol into 3beta-hydroxy-5-cholestenoic acid is rate-limiting. Interestingly, 3beta-hydroxy-5-cholestenoic acid was found to be further metabolized by the recombinant human P450c27, giving two monohydroxylated products with the hydroxyl group introduced at different positions on the steroid nucleus.     

Prostaglandin-E2 9-reductase from corpus luteum of pseudopregnant rabbit is a member of the aldo-keto reductase superfamily featuring 20 alpha-hydroxysteroid dehydrogenase activity

The prostaglandin-E2 9-reductase (PGE2 9-reductase) activity in the corpus luteum of rabbits corresponds to a cytosolic, NADPH-dependent enzyme with a molecular mass of 36 kDa. This enzyme was purified from corpora lutea on day 12 of pseudopregnancy with a 266-fold enrichment. The main purification step was affinity chromatography using Red Sepharose CL-6B. The efficiency of this column was improved by elution with 1 mM NADH prior to elution of the active fractions with 1 mM NADPH. Amino acid sequence data demonstrate that the rabbit luteal PGE2 9-reductase has to be classified as a member of the aldo-keto reductase superfamily. The enzyme revealed a wide substrate specificity comprising the reduction of aldehydes, ketones, and quinones. Apparent kinetic constants were determined using methylglyoxal, DL-glyceraldehyde, and 9,10-phenanthrenquinone as substrates. The fully purified enzyme showed two catalytic activities of particular interest: PGE2 9-reductase and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities. The competitive inhibition of 20 alpha-HSD activity by PGE2 indicates that progesterone and PGE2 are substrates for the same enzyme. From these results, we conclude that prostaglandin and steroid metabolism are tightly linked to each other. For this reason the aldo-keto reductase could be a key enzyme in the cascade of events leading to the regression of the corpus luteum in the rabbit.