Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

Biotransformation of Chrysin to Baicalein: Selective C6-Hydroxylation of 5,7-Dihydroxyflavone Using Whole Yeast Cells Stably Expressing Human CYP1A1 Enzyme.

Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer, and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multistep chemical approaches. Here, we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7-trihydroxyflavone (baicalein). Molecular modeling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake-flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds.

Immunochemical detection of cytochrome P450 enzymes in small intestine microsomes of male and female untreated juvenile cynomolgus monkeys.

The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies. CYP2D, CYP2J2, CYP3A4 and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76 and CYP2E1 were not detected in any of the 35 animals examined. On analysis of pooled microsomes (35 animals), CYP3A (3A4+3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%) and CYP2D (0.4%). On the analysis of individual microsome samples, each P450 content varied 2-to-6-fold between animals, and no sex differences were observed in any P450 content. These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines.

Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Isolation of two cytochrome P450 forms, CYP2A19 and CYP1A, from pig liver microsomes.

Cytochromes P450 comprise a superfamily of proteins that play a crucial role in the biotransformation of numerous chemicals. Purified CYPs can be used e.g. in studies on structure or determining the drug metabolism pathways. In this work, purification of the porcine CYP1A and CYP2A19 to electrophoretic homogeneity from the pig hepatic microsomes using octylamino Sepharose and hydroxylapatite column chromatography is reported. The proteins have been clearly recognized by commercial antibodies against rat and human CYP1A2 (porcine CYP1A) and human CYP2A6 (CYP2A19) respectively, using Western blot. Activities of both enzymes were determined by specific substrates, 7-ethoxyresorufin, 7-methoxyresorufin (CYP1A) and coumarin (CYP2A19). The isolated enzymes show kinetic parameters similar to human counterparts. Taken together, pig cytochromes can be used for the testing of veterinary drug metabolism, useful for the determination of drug residues in meat of pigs. The results obtained show that the pigs may be a suitable model for biotransformation of xenobiotics in humans.

Human cytochromes P450 1A1 and 1A2 participate in detoxication of carcinogenic aristolochic acid.

OBJECTIVES: A carcinogenic and nephrotoxic plant alkaloid, aristolochic acid (AA), causes the development of aristolochic acid nephropathy, which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA may also cause a similar type of kidney fibrosis with malignant transformation of the urothelium, the Balkan endemic nephropathy. The aim of the study was to resolve which cytochromes P450 (CYP) detoxicate the major component of AA, aristolochic acid I (AAI), to its O-demethylated metabolite, aristolochic acid Ia (AAIa). METHODS: High performance liquid chromatography (HPLC) was employed for separation and characterization of AAI metabolites generated by CYPs. RESULTS: Human, rat and mouse hepatic CYPs oxidize AAI into its detoxication metabolite AAIa. Most of the detoxication of AAI in human hepatic microsomes is mediated by CYP1A2 and 1A1, while other CYPs play a minor role. CONCLUSIONS: The data are the first report on identification of human CYP enzymes detoxicating AAI.

Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid.

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K(m) ( approximately 0.4 microM) for all variants but slightly different V(max) values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V(max) of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K(m) values of 111, 83, and 250 microM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.

Isolation of two cytochrome P450 cDNAs, CYP1A1 and CYP1A2, from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus).

Two cytochrome P450 (CYP), CYP1A1 and CYP1A2, cDNA sequences have been isolated and cloned from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus). EROD, a model substrate for CYP1A, and heterologous antibodies have been employed as a biomarker in marine mammals, however the CYP1A sequences have not been characterised in these two seal species. mRNA was used as the template in RT-PCR, rather than DNA as this indicates transcription of the CYP1A gene in these seal species exposed to environmental contaminants. Harp and grey seal CYP1A1 amino acid sequences exhibited >99% identity and the CYP1A2 sequences were >98% identical. Phylogenetic analyses of the two seal species with other mammalian, and avian CYP1A sequences, showed the CYP1A1 and CYP1A2 sequences clustered with corresponding sequences in other mammalian species. The closest sequences to the seal CYP1As was dog CYP1A. The CYP1A sequence information presented in this study has provided the necessary data for the future production of species-specific probes for the use as biomarkers of environmental contaminant exposure.

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT.

Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.

A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.).

The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56 degrees C for 5') which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS-PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity activity relationship. The molecular weight of the native form of the purified protein was determined to be 175 +/- 35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25 degrees C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 microM)-inhibited menadione oxidoreductase activity of up to 46.7 +/- 0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis-Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein).

Purified fusion enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 oxidoreductase.

A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.

Preparation of highly purified cytochrome P4501A1 from leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular and immunochemical properties.

Cytochrome P4501A1 was purified to electrophoretic homogeneity from the liver microsomes of feral fish leaping mullet (Liza saliens) collected in Izmir Bay, Aegean coast of Turkey. Purification of cytochrome P4501A1 involved anion exchange chromatography of Emulgen 913-cholate solubilized microsomes on first- and second-DEAE-cellulose columns, hydrophobic interaction chromatographies of the partially purified cytochrome P4501A1 on Porapak Q and phenyl-Sepharose CL-4B and further purification on adsorption chromatography on the hydroxylapatite column. Finally, it is further concentrated and purified on the third DEAE-cellulose column. The purified cytochrome P4501A1 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Cytochrome P4501A1, purified 32-fold with a specific content of 15-17 nmoles P450 (mg protein)-1, produced a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 58,000 +/- 500. Absolute absorption spectrum of the purified cytochrome P4501A1 fractions showed maximal absorption at 417.5 nm and CO-difference spectrum of dithionite-reduced cytochrome P4501A1 gave a peak at 448 nm. Purified P4501A1 was found to be active in the O-deethylation of 7-ethoxyresorufin in the reconstituted system containing purified fish liver cytochrome P450 reductase and synthetic lipid. However, it was unable to catalyze the oxidation of the other monooxygenase substrates such as benzphetamine and aniline known to be specific for the other isozymes. Purified L. saliens liver microsomal cytochrome P4501A1 showed strong cross-reactivity with the antibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout and scup. Spectral, electrophoretic, immunochemical and biocatalytic properties of the purified cytochrome P4501A1 strongly suggested that it is the CYP1A1 in the L. saliens liver.

Beta-naphthoflavone induced CYP1A1 and 1A3 proteins in the liver of rainbow trout (Oncorhynchus mykiss).

1. Two CYP1A proteins, designated HAP 1 and HAP 2, were isolated from the liver of the beta-naphthoflavone (BNF)-treated rainbow trout. The proteins were initially resolved by chromatography on a DEAE sepharose column and were further purified by hydroxyl-apatite chromatography. 2. Both HAP 1 and HAP 2 proteins exhibited high 7-ethoxyresorufin, methoxy-resorufin and phenacetin O-dealkylase activities and were good catalysts for the oxidation of 7,12-dimethylbenz[a]anthracene (DMBA). No qualitative difference was observed between the two proteins in their ability to catalyse the formation of the individual metabolites of DMBA. 3. The two purified proteins showed identical amino acid sequence for the first 13 amino acids. However, the 14th amino acid was valine for HAP 1 protein and alanine for HAP 2 protein. 4. Alignment of the amino acid sequences showed that HAP 1 protein was identical to the deduced protein of the previously reported trout CYP1A2 (renamed CYP1A1) gene for the first 24 amino acids at the N-terminal region. HAP 2 protein corresponded to the deduced protein sequence of CYP1A3 gene for the first 14 amino acids. However, unlike the deduced sequences of CYP1A1 and 1A3 the N-terminal methionine was absent in the purified proteins. 5. We conclude that HAP 1 and HAP 2 are the products of the CYP1A1 and CYP1A3 genes respectively, and are found in the liver of the BNF-treated rainbow trout.

Separation of three P450 isozymes from liver microsomes of gilthead seabream treated with beta-NF and partial purification of cytochrome P4501A1.

Three distinct cytochrome P450 isozymes, P4501A1, P4502B and an unidentified P450 isozyme were separated and isolated from beta-NF-treated gilthead seabream liver microsomes. Cytochrome P4501A1 was partially purified from beta-NF-treated gilthead seabream liver microsomes in the presence of detergents Emulgen 913 and cholate and protease inhibitors using two DEAE-cellulose, Porapak Q and two hydroxylapatite column chromatographies. The overall yield of purified P4501A1 was 1.2% with respect to microsomal total P450 with a specific content of 3 nmol/mg protein. The purified P4501A1 was characterized with respect to spectral, electrophoretic, biocatalytic and immunochemical properties, which are found to be similar to P4501A1s purified from other teleost species such as trout, scup, cod and perch suggesting that the P450 we have purified belongs to CYP1A1.