Functional High Level Expression of Cytochrome P450 CYP2D6 Using Baculoviral Expression Systems (original) (raw)
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Expression and Characterization of Canine Cytochrome P450 2D15
Archives of Biochemistry and Biophysics, 1998
CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 in Spodoptera frugiperda (Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1-hydroxylase and dextromethorphan O-demethylase activities when coexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1-hydroxylase, apparent K m values were 4.9, 3.7, and 2.5 M and the V max values were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphan O-demethylase, apparent K m values were 0.6, 0.6, and 2.0 M and the V max values were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.
Archives of Biochemistry and Biophysics, 1996
this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant sug-The purpose of the present studies was to define the gests that leucine homozygotes would eliminate (S)role of the I359L allelic variant of CYP2C9 in the mewarfarin, and probably many other CYP2C9 subtabolism of the low therapeutic index anticoagulant strates, at much slower rates in vivo than individuals warfarin, by performing in vitro kinetic studies with expressing the wild-type enzyme. ᭧ 1996 Academic Press, Inc. the two enantiomers of the drug. To obtain sufficient Key Words: cytochrome P450; expression; baculoviquantities of these variants to perform kinetic studies rus; warfarin; stereoselectivity; CYP2C9. at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to
Drug Metabolism and Disposition, 2003
Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), -naphthoflavone (33 M), phenobarbital (100 or 250 M), isoniazid (100 M) and/or rifampin (20 or 50 M), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by -naphthoflavone (on average 13-fold, n ؍ 28 preparations), and weakly induced by phenobarbital (1.9-fold, n ؍ 25) and rifampin (2.3-fold, n ؍ 22); CYP2A6 activity tended to be increased with phenobarbital (n ؍ 7) and rifampin (n ؍ 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n ؍ 13) and rifampin (13-fold, n ؍ 14); CYP2C8 was induced by phenobarbital (4.0-fold, n ؍ 4) and rifampin (5.2-fold, n ؍ 4); CYP2C9 was induced by phenobarbital (1.8-fold, n ؍ 14) and rifampin (3.5-fold, n ؍ 10); CYP2C19 was markedly induced by rifampin (37-fold, n ؍ 10), but relatively modestly by phenobarbital (7-fold, n ؍ 9); CYP2D6 was not significantly induced by phenobarbital (n ؍ 5) or rifampin (n ؍ 5); CYP2E1 was induced by phenobarbital (1.7-fold, n ؍ 5), rifampin (2.2-fold, n ؍ 5), and isoniazid (2.3-fold, n ؍ 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n ؍ 42) and rifampin (10-fold, n ؍ 61), but not by -naphthoflavone. Based on these observations, we generalize that -naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.
Mutagenesis, 2006
This corrigendum report describes the study of the comparison of human cytochrome b(5) (b(5)) with rat b(5) when coupled with human cytochrome P450 CYP1A2, 2A6 or 2E1. Results indicate a role of the N-terminal part of b(5) in the coupling with CYP. Indeed, the plasmid pLCM-b(5)-RED used in our former study on b(5) [Duarte et al. (2005) Mutagenesis, 20(2), 193-100] erroneously contained rat b(5). Plasmid pLCM-b(5)-RED was corrected with human b(5) and subsequently all experimental work was repeated as was described for the rat b(5) plasmid. Although absolute values of contents and activities were lower, all key-findings as found for rat b(5) could be confirmed using human b(5). The physiological relevant co-expression of the members of the cytochrome P450 complex, CYP, NADPH-cytochrome P450 oxidoreductase (RED) and human b(5) could be demonstrated in the different BTC strains, as was found before. The stimulatory effect of human b(5) on the activity of CYP1A2, CYP2A6 and CYP2E1 was i...
Archives of Biochemistry and Biophysics, 1997
of endogenous substrates such as steroids , bile acids Quantitative reverse-transcriptase polymerase chain (7), and retinoic acid (8), but can also metabolize a large reaction was used to determine the content of mRNA number of foreign substances including procarcinogens derived from four CYP3A genes (CYP3A2, CYP3A9, (9) and pharmaceutical agents, e.g., immunosuppres-CYP3A18, and CYP3A23) in rat liver. CYP3A2 and sive and psychotropic drugs, antibiotics, and anticancer CYP3A9 gene expression was age-and sex-dependent, drugs (6, 10-12).
Archives of Biochemistry and Biophysics, 1996
chrome P450 reductase; metabolism; testosterone; nifedipine; midazolam; V79 Chinese hamster cells. V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs Cytochrome P450 3A4 (CYP3A4) 3 may constitute as was shown by Northern and Western analyses. Activity much as 60% of all cytochrome P450 enzymes exwas tested by 6b-hydroxylation of testosterone for cypressed in human liver (1). CYP3A4 is also expressed tochrome P450 3A4 and by cytochrome c reduction for in jejunum, colon, and pancreas (2). Numerous drugs, NADPH-cytochrome P450 reductase. Five V79 cell environmental toxins, and endogenous substrates are lines were obtained expressing cytochrome P450 3A4, metabolized by CYP3A4 (3-8). Detailed studies on human NADPH-cytochrome P450 oxidoreductase, and CYP3A4-dependent metabolic activation of these both. Cytochrome P450 3A4 activity depended highly chemicals are needed to understand the pharmacologion cytochrome P450 reductase activity, with lowest accal potency of drugs and the mechanisms causing toxictivity when only the parental Chinese hamster cytochrome P450 reductase was present, 5-and 10-fold ity. In recent years, several genetically engineered in higher when coexpressed with the human NADPH-cy-vitro tools for cytochrome P450 have been developed to tochrome P450 reductase. Correspondingly, cytotoxic facilitate these studies. Those studies have proven that and genotoxic potency of aflatoxin B1 was increased cDNA-directed expression of cytochrome P450 is a powby orders of magnitudes when human cytochrome erful means for studying xenobiotic metabolism (9). Ge-P450 3A4 was coexpressed with the human NADPHnetically engineered in vitro tools are particularly indicytochrome P450 reductase. The effect of NADPH-cytocated for human cytochrome P450, because availability chrome P450 reductase coexpression on cytochrome of human tissue is extremely limited for practical and P450 3A4 activity was also tested by nifedipine oxidaethical reasons. tion and midazolam hydroxylation. Nifedipine oxida-As part of our efforts to establish a metabolically tion was increased about 10-fold, 1-hydroxylation of competent cell battery based on V79 Chinese hamster midazolam and 4-hydroxylation of midazolam were incells (10), additional V79 cell lines were constructed for creased 15-fold. ᭧ 1996 Academic Press, Inc.
Physiological Research, 2008
The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of...
Biochemical and Biophysical Research Communications, 2000
Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistanceassociated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and -naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.
European journal of pharmacology, 1995
An NIH/3T3 cell line, stably expressing human cytochrome P450-3A4 (CYP3A4) cDNA has been developed. This cell line was used in combination with a shuttle vector, containing the bacterial lacZ' gene as reporter gene, to study mutagenicity. Ethylmethanesulphonate and aflatoxin B1 were used as model agents to test this system. The mutation frequency of ethylmethanesulphonate increased concentration dependently and was the same in CYP3A4-expressing cells as in parental NIH/3T3 cells, demonstrating that CYP3A4 activity has no influence on the mutagenicity of ethylmethanesulphonate. The mutation frequency of aflatoxin B1 increased concentration dependently only in the CYP3A4-expressing cells and not in parental nor in vector-transfected cells. This increase in mutation frequency could be completely inhibited by ketoconazole, an inhibitor of cytochrome P450 activity, demonstrating the role of CYP3A4 in the activation of aflatoxin B1. The system described in this paper opens the possibi...