Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.     

Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli

Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.     

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l⁻¹ cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.     

Induction of cytochrome P450-dependent monooxygenase in mouse liver and kidney by rutaecarpine, an alkaloid of the herbal drug Evodia rutaecarpa.

Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.     

Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity

Cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of substituted and unsubstituted aromatics. However, since activities were quite low, a combination of site-specific mutagenesis and directed evolution was applied to produce 7800 variants of CYP116B3, which were screened via a newly developed high-throughput screening system based on the dealkylation of 7-ethoxycoumarin catalyzed by recombinant E. coli. The best mutant was found after four rounds of directed evolution and had a 240-fold increased deethylation activity toward 7-ethoxycoumarin (223 nmol product/nmol P450.min) and a 10-fold increased demethylation activity toward 7-methoxycoumarin (9 nmol product/nmol P450.min).     

Cloning,expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Cytochrome P450 monooxygenases of the CYP102A subfamily are single-component natural fusion proteins consisting of a heme domain and a diflavin reductase. The characterised CYP102A enzymes are fatty acid hydroxylases with turnover rates of several thousands per minute. In search of new P450s with similar activities, but with a broader substrate spectrum, we cloned, expressed and characterised CYP102A7 from Bacillus licheniformis. As expected, CYP102A7 was active towards medium-chain fatty acids but showed a strong preference for saturated over unsaturated fatty acids, which could not be observed for either of the CYP102A members so far. Besides fatty acids, CYP102A7 was able to catalyse the oxidation of cyclic and acyclic terpenes with high activity and coupling efficiency. For example, (R)-(+)-limonene was converted with activity of 220 nmol nmol P450(-1) min(-1) and 80% coupling. Unusual for enzymes of the CYP102A subfamily was the deethylation activity of CYP102A7 towards 7-ethoxycoumarin. Furthermore, this monooxygenase, though having a moderate thermal stability, exhibited 50% of its initial activity in the presence of 26% DMSO. Comparison of the homology models of CYP102A7 and other members of the CYP102A subfamily revealed distinct differences in the shape of the substrate access channel and the active site, which might explain differences in catalytic properties of these homologous enzymes.     

Stimulation of mixed-function oxidation of 7-ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol

Rates of O-deethylation of 7-ethoxycoumarin by perfused livers from fasted, phenobarbital-treated rats were 3.7 mumol X g-1 X h-1. Approximately 50% of the product was conjugated. When rates of 7-ethoxycoumarin O-deethylation were varied by infusing different concentrations of substrate, a good correlation (r = 0.91) was found between rates of O-deethylation of 7-ethoxycoumarin and fluorescence of 7-hydroxycoumarin detected from the liver surface. Micro-light guides (tip diameter 170 microns) placed on periportal and pericentral regions on the liver surface were used to monitor the conversion of nonfluorescent 7-ethoxycoumarin to fluorescent 7-hydroxycoumarin. The O-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin increased fluorescence 64% and 28% in pericentral and periportal regions of the liver lobule, respectively. Rates of 7-ethoxycoumarin O-deethylation estimated from these increases in fluorescence were 5.2 mumol X g-1 X h-1 in pericentral and 2.2 mumol X g-1 X h-1 in periportal regions of the liver. During mixed-function oxidation of 7-ethoxycoumarin, the oxidation:reduction state of NADP(H) was similar in both regions of the liver lobule. Xylitol (2 mM) decreased the NADP+/NADPH ratio and stimulated rates of drug metabolism in both regions of the liver lobule. This indicates that conditions exist where the supply of NADPH is an important rate-determining factor for 7-ethoxycoumarin metabolism in both periportal and pericentral regions of the liver lobule.     

Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

A novel ISFET-type biosensor based on P450 monooxygenases

We made a biosensor based on ion-sensitive field effect transistor (ISFET) using P450 monooxygenase. ISFETs are electrical devices and have been used as pH sensors. We used genetically engineered P450 monooxygenase for our research because of its high enzymatic activity. The fusion enzyme between rat CYP1A1P450 monooxygenase and yeast NADPH-cytochrome P450 oxidoreductase was expressed in yeast Saccharomyces cerevisiae strain AH22. Yeast microsomal membranes were immobilized in an agarose layer on the ISFET. o-Deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin was catalyzed by the enzyme in the presence of nicotinamide adenine dinucleotide phosphate reduced form (NADPH). Formation of 7-hydroxycoumarin from 7-ethoxycoumarin was also measured by fluorescence. The difference of the voltage between the ISFET device and control device without enzymes showed a voltage increase along with the enzymatic reaction of P450 monooxygenases, and this voltage increase in the device was inhibited by addition of MnCl(2), an inhibitor of P450 monooxygenase. There was a positive correlation between the voltage increase in the ISFET device and the fluorescence intensity. This is the first electrochemical biosensing using P450 monooxygenases immobilized on the ISFET, and is applicable to the sensing of chlorophenol compounds.     

Inhibition of cytochrome P450 mediated enzyme activity by alkylphosphocholines

The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.     

Cytochrome P450 expression and activities in rat, rabbit and bovine tongue

Xenobiotic metabolism in the tongue has received little attention in the literature. In the present study, we report a comparative analysis of constitutive cytochrome P450 (CYP) expression and activities in the tongue. First we compared catalytic activities of rabbit, rat and bovine tongue samples using the probe substrates 4-nitrophenol, 1-phenylethanol, caffeine and 7-ethoxycoumarin. Rabbit tongue samples showed the highest activities for all substrates. We then compared the activities in rat and rabbit tongue with those in the rabbit liver, along with the effects of P450 inhibitors on specific activities. Combined, the activity studies indicate that CYP1A1 is active in rabbit tongue cells, but CYP1A2, CYP3A6 and CYP2E1 are below limits of detection. RT-PCR was also used to compare mRNA levels of 11 different rabbit and six different rat P450 isoforms in the tongue to those in the liver of these two species. Only CYP2E1, CYP1A1 and CYP4A4 were detected at significant levels in the rabbit tongue. None of the six rat isoforms probed were observed in the tongue. Although 4-nitrophenol activity was observed in the rabbit tongue samples, the kinetic parameter K(m) was inconsistent with the involvement of CYP2E1. We suggest that although CYP2E1 is expressed in the tongue, it is rapidly degraded in this organ, and the nitrophenol hydroxylation and caffeine hydroxylation we observe is the result of activity of CYP1A1.     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.

Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.     

Paradoxical effect of sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5′-diphosphoglucuronic add or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.     

In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC₅₀ values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC₅₀ values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC₅₀ values more than 100 μM. K_i values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations     

Herbicide-resistant tobacco plants expressing the fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase.

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator. The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.