Comparison of CYP3A4 and CYP3A5: The Effects of Cytochrome b5 and NADPH-cytochrome P450 Reductase on Testosterone Hydroxylation Activities

Lee, S.J.,Goldstein, J.A. 日本藥物動態學會 2012 Drug metabolism and pharmacokinetics Vol.27 No.6

CYP3A4 and CYP3A5 require cytochrome b5 (b5) and NADPH-cytochrome P450 oxidoreductase (CPR) for optimum metabolism, but little is known about the specific requirements for b5 and CPR to produce optimal activities for these enzymes. The metabolism of testosterone (TT) by CYP3A4 and CYP3A5 was analyzed by various combinations of b5 and CPR using a fixed amount of recombinant P450 which had been purified from an Escherichia coli expression system. CYP3A4 and CYP3A5 required 4- and 8-fold more of CPR than of the P450s, respectively, for optimal activity. The requirement of b5 for optimal activity showed the same pattern for both CYP3A4 and CYP3A5, exhibiting a gradual stimulation of the activity reaching a maximum at 16 fold more b5 than P450. Although CYP3A4 exhibited higher activities than CYP3A5 in all combinations, both enzymes exhibited the same dependency profile for b5 and CPR. Therefore, the stronger activity of CYP3A4 compared to CYP3A5 appears to be intrinsic to the CYP3A4 protein itself and not to different requirements for b5 and CPR. Since the relative amounts of b5 and CPR are important in the maintenance of CYP3A4 and CYP3A5 activities, different levels of these proteins in vitro and in vivo may cause altered metabolism of their substrates or misinterpretation of enzyme properties.

Ultraviolet B radiation induces impaired lifecycle traits and modulates expression of cytochrome P450 (<i>CYP</i>) genes in the copepod <i>Tigriopus japonicus</i>

Puthumana, Jayesh,Lee, Min-Chul,Park, Jun Chul,Kim, Hui-Su,Hwang, Dae-Sik,Han, Jeonghoon,Lee, Jae-Seong Elsevier 2017 Aquatic toxicology Vol.184 No.-

<P><B>Abstract</B></P> <P>To evaluate the effects of ultraviolet B (UV-B) radiation at the developmental, reproductive, and molecular levels in aquatic invertebrates, we measured UV-B-induced acute toxicity, impairments in developmental and reproductive traits, and UV-B interaction with the entire family of cytochrome P450 (<I>CYP</I>) genes in the intertidal benthic copepod <I>Tigriopus japonicus</I>. We found a significant, dose-dependent reduction (<I>P<</I> 0.05) in the survival of <I>T. japonicus</I> that began as a developmental delay and decreased fecundity. The 48h LD10 and LD50 were 1.35 and 1.84kJ/m<SUP>2</SUP>, and the CYP inhibitor (PBO) elevated mortality, confirming the involvement of <I>CYP</I> genes in UV-B induced toxicity. Low-dose UV-B (1.5kJ/m<SUP>2</SUP>) induced developmental delays, and higher doses (6–18kJ/m<SUP>2</SUP>) caused reproductive impairments in ovigerous females. The significant up-regulation of <I>CYP</I> genes belonging to clans 2/3/MT/4/20 in <I>T. japonicus</I> exposed to UV-B (12kJ/m<SUP>2</SUP>) confirmed molecular interaction between UV-B and <I>CYP</I> genes. Moreover, orphan CYPs, such as <I>CYP20A1</I>, provide good insight on the deorphanization of invertebrate <I>CYPs</I>. Overall, these results demonstrate the involvement of UV-B radiation in the expression of all the <I>CYP</I> genes in <I>T. japonicus</I> and their susceptibility to UV-B radiation. This will provide a better understanding of the mechanistic effects of UV-B in copepods through the predicted AhR-mediated up-regulation of <I>CYP</I> genes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Impaired effects of UV-B on the copepod <I>Tigriopus japonicus</I> were examined. </LI> <LI> Modulation of entire CYP genes were analyzed in response to UV-B. </LI> <LI> CYP inhibitor (PBO) confirmed the role of CYP in UV-B induced mortality. </LI> <LI> Low-dose UV-B found induce developmental delays, and higher doses cause reproductive impairments. </LI> <LI> Study predicted the mechanistic effects of UV-B in copepods through the AhR-mediated up-regulation of <I>CYP</I> genes. </LI> </UL> </P>

파밤나방 중장으로부터 cytochrome P450 4계열의 유전자 단편들의 클로닝

문재유 서울대학교 농업개발연구소 1999 농업생명과학연구 Vol.3 No.-

Cytochrome P450 plays a most important role in metabolizing the exogous materials. In insect, P450 protein is related to metabolism of hormones and pheromes. But have been studied in most detail for their roles in insecticide resistance. In addition to this roles, P450-dependent metabolism has been associated with the adaptation of insect herbibores to host plant chemicals. Recently It is revealed that cytochrome P450 4 family gene is related to metabolism of host plant material. In this research, cloning of cytochrome P450 4 family gene fragments is carried out from monophagous insect (Bombyx mori), oligophagous insect(Helicoverpa assulta), and polyphagous insect (Spodoptera exigua), two conserved regions in the alignment of cytochrome P450 4 family proteins served as guide to the synthesis of degenerate oligonucleotide primers. The primers were used in RT-PCR from midgut and fat body total RNA. Five new genes of 440-449bp were cloned and sequence from spodoptera exigua midgut. Deduced amino acids had conserved regions which is contained in all cytochrome P450 (ΦΦKE-LR Φ-P : Φ, hadyrophobic amino acid) and another conserved region (PERF) and similar sequence Amino acid sequences of sample 1, 3 and 4 were shown to have 82%, 61%, and 73% identity with that of CYP 4M4, So, Sample 1, 3 and 4 were thought to belong to CYP 4M family, Protein sequence of sample 2 and 5 were shown to have 75% and 82% identity with that of 4S1 and 4S2 So, Sample 2 and 5 were thought to belong to CYP 4S family

Effect of <i>CYP3A5*3</i> genotype on serum carbamazepine concentrations at steady-state in Korean epileptic patients

Park, P.-W.,Seo, Y. H.,Ahn, J. Y.,Kim, K.-A.,Park, J.-Y. Blackwell Publishing Ltd 2009 Journal of clinical pharmacy and therapeutics Vol.34 No.5

<P>Abstract</P><P>Background and Objective: </P><P>Carbamazepine (CBZ) is metabolized mainly by the CYP3A family of enzymes, which includes CYP3A4 and CYP3A5. Several studies have suggested that the <I>CYP3A5*3</I> genotype influences the pharmacokinetics of CYP3A substrates. The present study aimed to assess the effect of the <I>CYP3A5*3</I> genotype on serum concentration of CBZ at the steady-state in Korean epileptic patients.</P><P>Method: </P><P>The serum concentrations of CBZ in 35 Korean epileptic patients were measured and their <I>CYP3A5</I> genotype was determined. Fourteen patients were <I>CYP3A5</I> expressors (two for <I>CYP3A5*1/*1</I> and 12 for <I>CYP3A5*1/*3</I>) and 21 patients were <I>CYP3A5</I> non-expressors (<I>CYP3A5*3/*3</I>). Dose-normalized concentrations (mean ± SD) of CBZ were 9·9 ± 3·4 ng/mL/mg for <I>CYP3A5</I> expressors and 13·1 ± 4·5 ng/mL/mg for <I>CYP3A5</I> non-expressors (<I>P</I> = 0·032). The oral clearance of CBZ was significantly higher in <I>CYP3A5</I> non-expressors than that of <I>CYP3A5</I> expressors (0·056 ±0·017 L/h/kg vs. 0·040 ± 0·014 L/h/kg, <I>P</I> = 0·004). The <I>CYP3A5</I> genotype affected the CBZ concentrations in Korean epileptic patients and is a factor that may contribute to inter-individual variability in CBZ disposition in epileptic patients.</P>

Effects of Triflusal and Clopidogrel on the Secondary Prevention of Stroke Based on Cytochrome P450 2C19 Genotyping

Han, Sang Won,Kim, Yong-Jae,Ahn, Seong Hwan,Seo, Woo-Keun,Yu, Sungwook,Oh, Seung-Hun,Nam, Hyo Suk,Choi, Hye-Yeon,Yoon, Sung Sang,Kim, Seo Hyun,Lee, Jong Yun,Lee, Jun Hong,Hwang, Yang-Ha,Lee, Kee Ook,J Korean Stroke Society 2017 Journal of stroke Vol.19 No.3

<P><B>Background and Purpose</B></P><P> To compare the efficacy and safety of antiplatelet agents for the secondary prevention of ischemic stroke based on cytochrome P450 2C19 (CYP2C19) polymorphisms. </P><P><B>Methods</B></P><P> This study was a prospective, multicenter, randomized, parallel-group, open-label, blind genotype trial. First time non-cardiogenic ischemic stroke patients were enrolled and screened within 30 days. Participants were randomized to receive either triflusal or clopidogrel for secondary stroke prevention. The primary outcome was the time from randomization to first recurrent ischemic stroke or hemorrhagic stroke. </P><P><B>Results</B></P><P> The required sample size was 1,080 but only 784 (73%) participants were recruited. In patients with a poor CYP2C19 genotype for clopidogrel metabolism (n=484), the risk of recurrent stroke among those who received triflusal treatment was 2.9% per year, which was not significantly different from those who received clopidogrel treatment (2.2% per year; hazard ratio [HR], 1.23; 95% confidence interval [CI], 0.60–2.53). In the clopidogrel treatment group (n=393), 38% had good genotypes and 62% poor genotypes for clopidogrel metabolism. The risk of recurrent stroke in patients with a good CYP2C19 genotype was 1.6% per year, which was not significantly different from those with a poor genotype (2.2% per year; HR, 0.69; 95% CI, 0.26–1.79). </P><P><B>Conclusions</B></P><P> Whilst there were no significant differences between the treatment groups in the rates of stroke recurrence, major vascular events, or coronary revascularization, the efficacy of antiplatelet agents for the secondary prevention of stroke according to CYP2C19 genotype status remains unclear.</P>

Cytochrome P450 2C8 and CYP3A4/5 are Involved in Chloroquine Metabolism in Human Liver Microsomes

Kyoung-AhKim,Ji-YoungPark,Ji-SukLee,SabinaLim 대한약학회 2003 Archives of Pharmacal Research Vol.26 No.8

Chloroquine has been used for many decades in the prophylaxis and treatment of malaria. It is metabolized in humans through the N-dealkylation pathway, to desethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ), by cytochrome P450 (CYP). However, until recently, no data are available on the metabolic pathway of chloroquine. Therefore, the metabolic pathway of chloroquine was evaluated using human liver microsomes and cDNA-expressed CYPs. Chloroquine is mainly metabolized to DCQ, and its Eadie-Hofstee plots were biphasic, indicating the involvement of multiple enzymes, with apparent Km and Vmax values of 0.21 mM and 1.02 nmol/min/mg protein 3.43 mM and 10.47 nmol/min/mg protein for high and low affinity components, respectively. Of the cDNA-expressing CYPs examined, CYP1A2, 2C8, 2C19, 2D6 and 3A4/5 exhibited significant DCQ formation. A study using chemical inhibitors showed only quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4/5 inhibitor) inhibited the DCQ formation. In addition, the DCQ formation significantly correlated with the CYP3A4/5-catalyzed midazolam 1-hydroxylation (r=0.868) and CYP2C8-catalyzed paclitaxel 6a-hydroxylation (r = 0.900). In conclusion, the results of the present study demonstrated that CYP2C8 and CYP3A4/5 are the major enzymes responsible for the chloroquine N-deethylation to DCQ in human liver microsomes.

Cytochrome P450 1A1(CYP1A1)과 Glutathione S-Transferase P1(GSTP1) 유전자 다형성이 후두암 발생에 미치는 영향

이국행,심윤상,이용식,이병철,성명식,홍영준,김현주,김헌,김용정 대한이비인후과학회 2002 대한이비인후과학회지 두경부외과학 Vol.45 No.1

Background and Objectives:Smoking has been reported as an important risk factor of laryngeal cancer. Cytochrome P450 1A1 (CYP1A1) and glutathione S-transferase P1 ( ) are genes that encode enzymes which are involved in the metab-olism of carcinogens in cigarette smoke. In this study, we statistically tested the significances of smoking and genotypes of CYP1A1 and GSTP1 as risk factors of laryngeal cancer. Materials and Method:In this case-control study, 84 pathologically proven laryngeal cancer patients and 168 age- and sex-matched controls were included as the study subjects. Information on smoking habit was collected using a self-administered questionnaire, and CYP1A1 and GSTP1 genotypes were analyzed using . Results:Smoking was turned out to be a significant risk factor of laryngeal cancer both in univariate and multivariate ana-lyses. The CYP1A1 Ile/Ile genotype was significant in the univariate test, but the statistical significance disappeared in the multivariate conditional logistic model including smoking. The odds ratio (95% confidence interval) of GSTP1 A/A geno-type for laryngeal cancer was 0.71 (0.38, 1.3), which was not statistically significant. Conclusion:Smoking is the most potent risk factor among the thre factors, and the genotypes of CYP1A1 and GSTP1 would not be major risk factors for laryngeal cancer in Koreans. (Korean J Otolaryngol 2002;45:56-61)

인체간 microsome에서의 제 2세대 sulfonylurea계 약물들의 cytochrome P450 약물 대사효소에 대한 억제작용

최지이,김수영,김경아,박지영 침례병원 2003 浸禮病院學術誌 Vol.19 No.-

Modification of N-Terminal Amino Acids of Fungal Benzoate Hydroxylase (CYP53A15) for the Production of p-Hydroxybenzoate and Optimization of Bioproduction Conditions in Escherichia coli

( Shun Tamaki ),( Mitsuhiko Yagi ),( Yuki Nishihata ),( Hideki Yamaji ),( Yasushi Shigeri ),( Tomohide Uno ),( Hiromasa Imaishi ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.3

The aromatic compound p-hydroxybenzoate (PHBA) is an important material with multiple applications, including as a building block of liquid crystal polymers in chemical industries. The cytochrome P450 (CYP) enzymes are beneficial monooxygenases for the synthesis of chemicals, and CYP53A15 from fungus Cochliobolus lunatus is capable of executing the hydroxylation from benzoate to PHBA. Here, we constructed a system for the bioconversion of benzoate to PHBA in Escherichia coli cells coexpressing CYP53A15 and human NADPH-P450 oxidoreductase (CPR) genes as a redox partner. For suitable coexpression of CYP53A15 and CPR, we originally constructed five plasmids in which we replaced the N-terminal transmembrane region of CYP53A15 with a portion of the N-terminus of various mammalian P450s. PHBA productivity was the greatest when CYP53A15 expression was induced at 20°C in 2×YT medium in host E. coli strain ΔgcvR transformed with an N-terminal transmembrane region of rabbit CYP2C3. By optimizing each reaction condition (reaction temperature, substrate concentration, reaction time, and E. coli cell concentration), we achieved 90% wholecell conversion of benzoate. Our data demonstrate that the described novel E. coli bioconversion system is a more efficient tool for PHBA production from benzoate than the previously described yeast system.

Constitutive Expression and Changes of Cytochrome P450 Isozymes mRNAs by Vehicles (Petrolatum, DMSO, Ethanol) in Rat Skin Using Semi-quantitative RT-PCR

Lee, Ai-Young,Lee, Kyung-Hoon,Ko, Duck-Sung,Chey, Won-Young The Korean Society of Pharmacology 2001 The Korean Journal of Physiology & Pharmacology Vol.5 No.5

Many drugs are primarily metabolized by the cytochrome P450s (CYPs). Drug metabolites would be important allergens for adverse drug reactions such as drug eruptions. Skin tests with a suspected drug have conducted to identify causative drugs of drug eruptions, with vehicles such as white petrolatum, DMSO, ethanol. This study will compare the expression of rat CYP isozyme mRNAs between the skin and the liver, with examining an effect of the vehicles on the cutaneous CYPs using semi-quantitative RT-PCR. Thirty-two Sprague-Dawley rats between the ages of six and eight weeks were divided as four groups. One group was used to compare the constitutive mRNA expression between skin and liver, while the others were to examine the effects of three vehicles. The ratios of expression of CYP1A2, CYP2B1/2, CYP2E1, CYP3A1, and CYP4A1 were significantly higher in the liver than the skin. However, CYP1A1 and CYP2C11 were higher in the skin than liver. The effects of vehicles were quite different; white petrolatum significantly induced CYP1A1 (p=0.012) and CYP2C11 mRNAs, while ethanol inhibited CY P1A1 and CYP2B1/2. DMSO did not make any changes. The results suggest that rat skin can participate in drug metabolism with their own CYP isozymes. The effects of vehicles on the cutaneous CYP expression should not be ignored and may be applied for determination of an appropriate vehicle for certain drug(s).