147-Pharmacology-Chen-BB

E-Mail karger@ O riginal Paper P harmacology 2015;95:145–153D O I:10.1159/000380883 Effects of Methoxychlor and 2,2-bis ( p -Hydroxyphenyl)-1,1,1-Trichloroethane on Cytochrome P450 Enzyme Activities in Human and Rat LiversB ingbing Chen a Peipei Pan c Li Wang a Menchun Chen a Yaoyao Dong a Ren-Shan Ge b Guo-Xin Hu aaS chool of Pharmacy, and b T he 2nd Affiliated Hospital, Wenzhou Medical University, W enzhou , and cT aizhou Central Hospital, T aizhou , ChinaBoth chemicals had no effect on human CYP3A4 and ratC YP3A1 activity. In summary, MXC and HPTE are potent in-hibitors of some human and rat CYPs. © 2015 S. Karger AG, BaselI ntroduction Cytochrome P450 (CYP) enzymes are a superfamily of hemoproteins. They are involved in the metabolism of endogenous and exogenous compounds, including ste-roids, fatty acids, bile acids, lipid-soluble vitamins, drugs,and environmental pollutants[1–3] . Most CYPs are ex-pressed in the liver, but some are localized in extrahepat-ic tissues. In humans, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are the major drug-metabolizing CYPs in the liver, which are homologous with CYP2C11, CYP2C6,CYP2D2, and CYP3A1, respectively, in rats[4–6].These CYPs are responsible for the metabolism of many drugs. CYP induction or inhibition can alter the biotransforma-tion and bioavailability of many drugs. However, directK ey WordsC ytochrome P450 enzyme · Enzyme inhibition · Methoxychlor · 2,2-bis( p -Hydroxyphenyl)-1,1,1-trichloroethane · HPTEA b stract C ytochrome P450 (CYP) enzymes are involved in the metab-olism of endogenous and exogenous compounds. Human and rat liver microsomes were used to investigate the inhib-itory effects of methoxychlor (MXC) and its metabolite 2,2-bis( p -hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on the activities of corresponding human and rat CYPs. Probe drugs were used to test the inhibitory effects of MXC and HPTE on human and rat CYPs. The results showed that MXC and HPTE inhibited both human CYP2C9 and rat liver CYP2C11 activity, with half-maximal inhibitory concentra-tion (IC 50 ) values of 15.47 ± 0.36 (MXC) and 8.87 ± 0.53 μmol/l (HPTE) for human CYP2C9, and of 22.45 ± 1.48 (MXC) and 24.63 ± 1.35 μmol/l (HPTE) for rat CYP2C11. MXC and HPTE had no effects on human CYP2C19 activity but inhibited rat CYP2C6 activity with IC 50 values of 14.84 ± 0.04 (MXC) and 8.72 ± 0.25 μmol/l (HPTE). With regard to human CYP2D6 and rat CYP2D2 activity, only HPTE potently inhibited human C YP2D6 activity, with an IC 50 value of 16.56 ± 0.69 μmol/l.Received: December 12, 2014A ccepted after revision: February 1, 2015P ublished online: March 31, 2015 G uo-Xin HuS chool of PharmacyW enzhou Medical UniversityC hashan District, Wenzhou, Zhejiang 325035 (China) E -Mail hgx @ w w /phaR .-S. Ge and G.-X. Hu contributed equally to this work. w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A MC hen/Pan/Wang/Chen/Dong/Ge/HuP harmacology 2015;95:145–153DOI: 10.1159/000380883146inhibition of these enzymes by environmental pollutantshas been investigated far less.O ne of the environmental pollutants is methoxychlor (MXC), an organochlorine pesticide. MXC replaced di-chlorodiphenyltrichloroethane (DDT) for wide use in ag-riculture, since DDT has a half-life of elimination (T 1/2)about 8 years and is easily absorbed in animal bodies[7].MXC is considered the preferred alternative to DDT for reasons of its much lower toxicity and shorter eliminationrate (T 1/2 is about 6 months)[8] . After ingestion, MXC is predominantly metabolized into 2,2-bis( p -hydro x y p he-nyl)-1,1,1-trichloroethane (HPTE)[9] . Both MXC and HPTE were characterized to be endocrine disruptors. A previous observation indicates that MXC is a reproduc-tive toxicant [10] . Interestingly, HPTE was found to exert a greater reproductive toxicity than the parent compoundMXC in both sexes[8, 9] . Further study indicated that both MXC and HPTE bind to human estrogen receptor-α, with the latter being more potent than the parent com-pound [11] . In addition, MXC and HPTE may have other toxicities, such as interfering with the metabolism of en-dogenous compounds or exogenous chemicals.O ur previous study has shown that MXC and HPTE suppressed the activities of some steroidogenic enzymes of both human and rat testes in vitro, including 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD3,and 11β-HSD[8, 12] . Recently, we have demonstrated that they are also potent inhibitors of 17α-hydrox-ylase/17,20-lyase (CYP17A1)[13] . However, sinceC YP17A1 belongs to the family of CYP enzymes, it is presently unclear whether both MXC and its metabolite HPTE effect direct inhibition of other CYP enzymes. In this study, we compared the potencies of MXC and HPTE in inhibiting the activities of four major types of CYP en-zyme in the human or rat liver.M aterials and Methods M ateri alsC ytochrome enzyme substrates and their metabolites includ-ing diclofenac and 4 ′ -hydroxydiclofenac for homologous CYP2C9 and CYP2C11, omeprazole and 5 ′ -hydroxy omeprazole for ho-mologous CYP2C19 and CYP2C6, dextromethorphan and dex-trorphan for homologous CYP2D6 and CYP2D2, midazolam and 1 ′ -hydroxy midazolam for homologous CYP3A4 and CYP3A1, as well as internal control diazepam, bisoprolol, and carbamazepine were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). MXC and HPTE were purchased from Sigma (St. Louis, Mo., USA). All the other chemicals and solvents used were of analytical grade. MXC and HPTE were prepared using ethanol as a solvent.M ale Sprague-Dawley rats (250–300 g) were purchased from the Animal Center of Wenzhou Medical University. All animal procedures were carried out in strict accordance with the recom-mendations of the Guide for the Care and Use of Laboratory Ani-mals of Wenzhou Medical University. The protocol was approved by the Committee on the Ethics of Animal Experimentation of Wenzhou Medical University (permit No. 2014-0055). All surger-ies were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Human liver microsomes were purchased from Gentest (Woburn, Mass., USA).P reparation of Microsomal ProteinM icrosomal preparations of rat livers were made as described previously [8, 14] . Briefly, liver samples from rats were homoge-nized in cold 0.01 mmol/l PBS buffer containing 0.25 mmol/l of sucrose and centrifuged at 700 g for 30 min. The supernatants were transferred to new tubes and centrifuged at 10,000 g for an addi-tional 30 min. Then, the they were centrifuged at 105,000 g for 1 h twice. The pellets were resuspended with cold 0.01 mmol/l PBS, and protein concentrations were measured using the Bio-Rad Pro-tein Assay Kit according to the manufacturer’s protocol. The mi-crosomes were used for the measurement of CYP activity.E nzyme Inhibition Assays for CYPs in Human and Rat LiverT he inhibition assays for the respective human and rat enzyme pairs CYP2C9 and CYP2C11, CYP2C19 and CYP2C6, CYP2D6 and CYP2D2, and CYP3A4 and CYP3A1 were performed as de-scribed previously [15] . Preliminary experiments were performed to determine whether the metabolite formation rate was linear with the time of incubation and microsomal protein concentration, and the result showed that the metabolite formation rate was linear within 30 min. 285-μl incubation mixtures were prepared, contain-ing microsomes (0.25 mg/ml of liver microsomes), 100 mmol/l Tris-HCl buffer (pH 7.4), 10 μmol/l substrate, and different con-centrations of MXC or HPTE. After 5 min of preincubation at 37 °C , the abovementioned incubation mixture was initiated by ad-dition of 15 μl of 1 mmol/l NADPH. The substrate concentration was selected based on the K m of the substrate for human CYPs [16] .T o determine the effects of MXC or HPTE on CYP2C9 and CYP2C11, the inhibition assays were performed as follows. Diclo-fenac (10 μmol/l) was used as the common probe substrate for de-tecting human CYP2C9 or rat CYP2C11 activity. Varying concen-trations (0.1, 1.0, 5.0, 10, 25, 50, and 100 μmol/l) of MXC or HPTE were added to an incubation mixture to determine the half-maxi-mal inhibitory concentration (IC 50 ). The conversion rate of diclo-fenac (10 μmol/l) to 4 ′ -hydroxydiclofenac was measured to evalu-ate the inhibition in the presence of 100 μmol/l inhibitor (MXC or HPTE) in human and rat liver microsomes. When CYP2C9 and CYP2C11 were obviously inhibited by MXC or HPTE, the inhibi-tory modes of MXC and HPTE were further investigated.I n a separate study, different final concentrations of NADPH (20, 50, 100, and 200 μmol/l) were incubated together with the reac-tion mixture containing 0.25 mg/ml liver microsomes and 0–50μmol/l MXC or HPTE to determine whether MXC or HPTE com-petes with the cofactor NADPH. All reactions were terminated by adding 30 μl cold HCl (0.1 mol/l). Then, 50 μl diazepam (10 μg/ml) internal standard and 1 ml ethyl acetate were added. After mixing thoroughly for 2 min, the samples were centrifuged at 13,000 rpm for 5 min, and the organic layer was transferred to a clean glass tubeand dried under nitrogen. The organic phase was evaporated under w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A ME ffects of MXC and HPTE on Series CYPs P harmacology 2015;95:145–153DOI: 10.1159/000380883147a nitrogen stream at 40 °C . The residue was dissolved in 100 μl of mobile phase for measuring the conversion of diclofenac into 4 ′-hy-droxydiclofenac by HPLC, and 20 μl was injected into the HPLC system. The percent inhibitory activity of CYP2C9 or CYP2C11 was calculated depending on the percent conversion between the en-zyme substrate and its metabolite. The inhibition assays for ther espective human and rat enzyme pairs CYP2C19 and CYP2C6, CYP2D6 and CYP2D2, and CYP3A4 and CYP3A1 were performed as mentioned above with the corresponding common substrates omeprazole, dextromethorphan, and midazolam, respectively; the concentration of the corresponding substrate selected was based on the K m [17, 18] . A different internal standard was added to the assay mixture for determination of the activity of the above enzymes.D etermination of Substrates and Products of CYPs by HPLCT he HPLC system (Agilent 1100) was equipped with a quater-nary pump, on-line vacuum degasser, autosampler, thermostat-controlled column compartment, fluorescence detector, diodearray detector, and Agilent ChemStation Rev A.10.02 (Agilent, Santa Clara, Calif., USA). The Agilent ZORBAX SB-C18 column (4.6 × 150 mm, ID 5 μm) was used for the separation of substrates and their metabolites; the methods have been described previous-ly[15, 19–21] . Diazepam, midazolam, bisoprolol, and carbamaze-pine were used as an internal control for determination of diclofe-nac, omeprazole, dextromethorphan, and midazolam, respective-ly. The percent conversion of the substrate into its metabolite was used to calculate CYP activity.S tati sti csT he percent conversion of the probe substrate into its metabo-lite was calculated to render a value for CYP activity by HPLC. The positive control was chosen as 100% activity of the enzyme. The inhibition was repeated 5 times. The IC 50 value was calculatedu sing GraphPad (version 5; GraphPad Software Inc., San Diego, Calif., USA), and nonlinear regression of curve fit with one-site competition was performed. Lineweaver-Burk analysis was used to determine the mode of inhibition. The data were subjected to analysis by one-way ANOVA followed by Duncan’s multiple com-parisons to identify significant differences between groups when 3 and more groups were analyzed. All data are expressed as means ± SEM. Differences were regarded as significant at p < 0.05.R esults Effects of MXC and HPTE on CYP2C9 Activity in Human and on CYP2C11 Activity in Rat Liver Microsomes A s shown in f igure 1 , when the maximum concentra-tion of MXC or HPTE (100 μmol/l) was used, MXC and HPTE inhibited human CYP2C9 by 72.24 and 100%, re-spectively, while they inhibited rat CYP2C11 by 81.91 and 85.76%, respectively (p < 0.05). F urthermore, the IC 50 values for the inhibition of human CYP2C9 activity by MXC and HPTE were 15.47 ± 0.36 and 8.87 ± 0.53 μmol/l, while those for inhibition of rat CYP2C11 activ-ity by MXC and HPTE were 22.45 ± 1.48 and 24.63 ± 1.35 μmol/l, respectively ( t able 1 ). These data indicate that MXC and HPTE are inhibitors of CYP2C9 or CYP2C11, and that they inhibit human CYP2C9 more potently than rat CYP2C11.F ig. 1. Effects of MXC and HPTE on human CYP2C9 ( a ) and rat CYP2C11 activity ( b ) in liver microsomes. The activity was mea-sured by the conversion rate of diclofenac (10 μmol/l) to 4 ′-hy-droxydiclofenac in the presence of 100 μmol/l inhibitor (MXC or HPTE). The conversion rate was 21.87 ± 1.19% (%CON, MXC) and 0% (%CON, HPTE) for human liver microsomes and 20.99 ± 1.82% (%CON, MXC) and 14.70 ± 0.76% (%CON, HPTE) for rat liver microsomes. Means ± SEM (n = 5). * * *Significant difference compared with the control at p < 0.05. CON = Control.T ab le 1. I C 50 values (μmol/l) for human and rat CYPsMXC 15.47 ± 0.3622.45 ± 1.48ND 14.84 ± 0.04NDND HPTE8.87±0.5324.63±1.35ND8.72±0.2516.56±0.69NDV alues are means ± SEM (n = 5). ND = Not determined.w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A MC hen/Pan/Wang/Chen/Dong/Ge/HuP harmacology 2015;95:145–153DOI: 10.1159/000380883148M echanism Explaining the Effect of MXC and HPTE on Human CYP2C9 ActivityT he mechanisms of the inhibitory effect of MXC and HPTE on CYP2C9 activity were investigated. Lineweav-er-Burk plot analysis showed that both MXC and HPTEwere competitive inhibitors of human CYP2C9 activityagainst the substrate diclofenac ( f ig. 2 a , b). This indicatesthat they inhibit human CYP2C9 enzyme activity by competing with the substrate diclofenac at its enzyme-binding site. However, when NADPH was investigated, Lineweaver-Burk analysis showed that MXC and HPTE inhibited human CYP2C9 activity in an uncompetitive mode against the cofactor NADPH ( f ig. 2 c , d), indicating that both MXC and HPTE bind to the CYP2C9-NADPH-P450-reductase complex.E ffects of MXC and HPTE on CYP2C19 Activity in Human and on CYP2C6 Activity in Rat Liver MicrosomesO meprazole (10 μmol/l) was used as the common probe substrate for detecting human CYP2C19 or ratC YP2C6 activity. The conversion rate of omeprazole (10 μmol/l) to 5 ′ -hydroxy omeprazole was measured to eval-F ig. 2. Lineweaver-Burk plots of human CYP2C9 kinetics in the presence of MXC or HPTE. CYP2C9 activity was measured by the conversion rate of diclofenac (10 μmol/l) to 4 ′ -hydroxydiclofenac in human liver microsomes for 30 min in the pres-ence of varying concentrations of the sub-strate diclofenac or the cofactor NADPH. The experiments were repeated 5 times. a ,b Effects of MXC ( a ) and HPTE ( b )on human CYP2C9 activity against diclofe-nac. c ,d Effects of MXC ( c ) and HPTE ( d )on human CYP2C9 activity against NADPH. pro = Protein.F ig. 3. Effects of MXC and HPTE on human CYP2C19 ( a ) and ratCYP2C6 activity ( b ) in liver microsomes. The activity was mea-sured by the conversion rate of omeprazole (10 μmol/l) to 5 ′-hy-droxy omeprazole in the presence of 100 μmol/l inhibitor (MXC orHPTE). The conversion rate was 70.67 ± 0.06% (%CON, MXC) and 18.57 ± 0.07% (%CON, HPTE) for human liver microsomes and 4.09 ± 0.24% (%CON, MXC) and 1.16 ± 0.11% (%CON, HPTE) for rat liver microsomes. Means ± SEM (n = 5). * * *Significant differ-ence compared with the control at p < 0.05. CON = Control.w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A ME ffects of MXC and HPTE on Series CYPs P harmacology 2015;95:145–153DOI: 10.1159/000380883149uate the inhibition in the presence of 100 μmol/l inhibitor (MXC or HPTE) in human and rat liver microsomes. Both MXC and HPTE inhibited human CYP2C19 and rat CYP2C6 activity in the liver microsomes when compared to controls (p < 0.05). MXC showed 29.33 ± 0.06% inhibi-tion of the conversion of omeprazole to 5 ′-hydroxy omeprazole by CYP2C19 in human liver microsomes, while HPTE showed 81.43 ± 0.07% inhibition of the h uman enzyme activity ( f ig. 3 a ). MXC and HPTE also showed 95.91 ± 0.24 and 98.84 ± 0.11% inhibition of rat CYP2C6 activity compared to the control activity, respec-tively ( f ig. 3 b ). The IC 50 values for the inhibition of rat CYP2C6 activity were 14.84 ± 0.041 μmol/l for MXC and 8.72 ± 0.25 μmol/l for HPTE, respectively ( t able 1 ). These data indicate that MXC and HPTE induce a more potent inhibition of rat CYP2C6 than of human CYP2C19 activ-ity, and that HPTE is a more potent inhibitor of both hu-man CYP2C19 and rat CYP2C6 activity than its parent compound MXC.M echanism Explaining the Effect of MXC and HPTE on Rat CYP2C6 ActivityT he mechanisms of the inhibitory effect of MXC and HPTE on rat CYP2C6 activity were investigated. Line-weaver-Burk plot analysis showed that both MXC and HPTE were noncompetitive inhibitors of rat CYP2C6 a ctivity ( f ig. 4 a , b). However, when NADPH was used for the measurement of CYP2C6 activity in the rat liver mi-crosomes, Lineweaver-Burk analysis showed that MXC and HPTE were in a competitive mode ( f ig. 4 c , d). These results indicate that MXC and HPTE inhibited the C YP2C6 enzyme activity by competing with the substrate NADPH at its enzyme-binding site of P450 reductase.E ffects of MXC and HPTE on CYP2D6 Activity in Human and on CYP2D2 Activity in Rat Liver MicrosomesD extromethorphan (10 μmol/l) was used as the com-mon probe substrate for detecting human CYP2D6 or ratF ig. 4. Lineweaver-Burk plots of ratC YP2C6 kinetics in the presence of MXC or HPTE. CYP2C6 activity was measured by the conversion rate of omeprazole to 5 ′ -hydroxy omeprazole in rat liver micro-somes for 30 min in the presence of varying concentrations of the substrate omeprazole or the cofactor NADPH. The experiments were repeated 5 times. a ,b Effects of MXC ( a ) and HPTE ( b ) on rat CYP2C6 activity against omeprazole. c ,d Effects of MXC ( c )and HPTE ( d ) on human CYP2C6 activity against NADPH.pro =Protein.w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A MC hen/Pan/Wang/Chen/Dong/Ge/HuP harmacology 2015;95:145–153DOI: 10.1159/000380883150CYP2D2 activity. The conversion rate of dextrometho-rphan (10 μmol/l) to dextrorphan was measured to evalu-ate the inhibition in the presence of 100 μmol/l inhibitor (MXC or HPTE) in human and rat liver microsomes. MXC inhibited human CYP2D6 activity by 19.70 ± 8.29%, while HPTE inhibited the activity of this enzyme by 66.31 ± 1.05% compared to the control ( f ig. 5 a ). MXC inhibited rat CYP2D2 activity by 21.34 ± 2.45%, whereas HPTE in-hibited the enzyme activity by 57.18 ± 6.90% ( f ig. 5 b ). The IC 50 value for the inhibition of human CYP2D6 activity by HPTE was 16.56 ± 0.69 μmol/l ( t able 1 ). These data indicate that MXC induces weak inhibition of both hu-man CYP2D6 and rat CYP2D2 activity, and that HPTE is a more potent inhibitor of these enzymes than its parent compound MXC. Meanwhile, HPTE exhibited a more potent inhibition of human CYP2D6 than of rat CYP2D2 activity.M echanism Explaining the Effect of HPTE on Human CYP2D6 ActivityT he mechanisms of the inhibitory effect of HPTE on human CYP2D6 activity were investigated. Lineweaver-Burk plot analysis showed that HPTE was a mixed-type inhibitor of human CYP2D6 activity ( f ig. 6 a ). However, when NADPH was used for the measurement of human CYP2D6 activity, Lineweaver-Burk analysis showed thatHPTE was in an uncompetitive mode ( f ig. 6 b ). The resultsindicate that HPTE inhibits human CYP2D6 enzyme ac-tivity by competing with the substrate dextromethorphan at its enzyme-binding site.E ffects of MXC and HPTE on CYP3A4 Activity in Human and on CYP3A1 Activity in Rat Liver MicrosomesM idazolam (10 μmol/l) was used as the common probe substrate for detecting human CYP3A4 or rat CYP3A1 activity. The conversion rate of midazolam (10 μmol/l) to 1 ′ -hydroxy midazolam was measured to evaluate the in-hibition in the presence of 100 μmol/l inhibitor (MXC or HPTE) in human and rat liver microsomes. MXC and HPTE induced almost no inhibition of human CYP3A4 and rat CYP3A1 activity ( f ig. 7 ).F ig. 5. Effects of MXC and HPTE on human CYP2D6 ( a ) and ratCYP2D2 activity (b ) in liver microsomes. The activity was mea-sured by the conversion rate of dextromethorphan (10 μmol/l) to N-dextromethorphan in the presence of 100 μmol/l inhibitor (MXC or HPTE). The conversion rate was 81.3 ± 8.29% (%CON, MXC) and 33.69 ± 1.05% (%CON, HPTE) for human liver micro-somes and 78.66 ± 2.45% (%CON, MXC) and 42.82 ± 6.9% (%CON, HPTE) for rat liver microsomes. Means ± SEM (n = 5). * * * Significant difference compared with the control at p < 0.05. CON =Control.F ig. 6. Lineweaver-Burk plots of human CYP2D6 kinetics in the presence of HPTE. CYP2D6 activity was measured by the conver-sion rate of dextromethorphan (10 μmol/l) to N-dextromethor-phan in human liver microsomes for 30 min in the presence of varying concentrations of the inhibitor or the cofactor NADPH. The experiments were repeated 5 times. Effects of HPTE on humanCYP2D6 activity against dextromethorphan (a ) and NADPH (b ). w n l o a d e d b y : n z h o u M e d ic a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A ME ffects of MXC and HPTE on Series CYPs P harmacology 2015;95:145–153DOI: 10.1159/000380883151D iscussion MXC is an organochlorine pesticide which is widely used in agriculture[22] . Thus, it may pose a health risk not only to agricultural workers but also to the general long-term-exposed population. In the present study, we investigated the dose-dependent inhibition of human liv-er CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activity as well as of the homologous rat liver CYP2C11, CYP2C6, CYP2D2, and CYP3A1 activity by MXC and its metabo-lite HPTE.C YP2C9, CYP2C19, CYP2D6, and CYP3A4 are the main members of the CYP group of enzymes; they are localized to human liver microsomes and are homolo-gous with CYP2C11, CYP2C6, CYP2D2, and CYP3A1 in the rat liver, respectively [16, 23–25] . These enzymes play a critical role in the metabolism of a variety of endoge-nous and exogenous compounds, including steroids,drugs, and environmental pollutants[3, 25].Diclofenac,omeprazole, dextromethorphan, and midazolam were used as the common substrates of human CYP2C9 and rat CYP2C11, human CYP2C19 and rat CYP2C6, hu-man CYP2D6 and CYP2D2, and human CYP3A4 and CYP3A1, respectively. These substrates were used as the probe drugs for measuring the activity of the above CYPs by calculating the percent conversion of the substrates totheir metabolites[16, 23–25] .O ur results indicate that of these homologous CYPs, MXC and HPTE can completely inhibit human CYP2C9 and rat CYP2C11 activity, and that the inhibition of hu-man CYP2C9 activity was more potent than that of rat CYP2C11 activity. The same concentration of inhibitor (MXC and HPTE) also had an obvious inhibitory effect on both human CYP2C19 and rat CYP2C6 activity, but the inhibition was more sensitive to the rat than theh uman enzyme. However, for human CYP2D6 and rat CYP2D2 activity, MXC showed weak inhibition, but HPTE had a more obvious inhibitory effect on these en-zymes than the parent compound MXC. Different from the abovementioned CYPs, MXC and HPTE induced no inhibition of human CYP3A4 and rat CYP3A1 activity.O ur study also showed that MXC and HPTE were competitive inhibitors of human CYP2C9 and ratC YP2C11 activity, noncompetitive inhibitors of human CYP2C19 and rat CYP2C6 activity, and mixed inhibitors of human CYP2D6 and rat CYP2D2 activity against their respective substrates. We investigated whether MXC or HPTE competed with the cofactor NADPH. The present results indicate that MXC and HPTE were noncompeti-tive inhibitors of human CYP2C9 and rat CYP2C11 ac-tivity, competitive inhibitors of human CYP2C19 and rat CYP2C6 activity, and noncompetitive inhibitors of hu-man CYP2D6 and rat CYP2D2 activity against NADPH. Thus, it is reasonable to infer that MXC and HPTE show different modes of inhibition of these enzymes. MXC and HPTE also possibly interfere with the CYP2C9 (CYP2C11)-NADPH-P450 reductase complex. It sug-gests that the inhibition caused by MXC and HPTE may affect CYP catalysis of endogenous and exogenous com-pounds. Among CYPs, CYP2C9, CYP2C19, and CYP2D6 are responsible for the metabolism of 10, 5, and 30%, re-spectively, of the drugs metabolized by the cytochromeenzyme family in humans[26] . MXC and HPTE expo-sure can decrease the rate of hepatic biotransformation of the drugs, causing increased serum concentrations andtoxicity by inhibiting these CYP enzymes[27] . If there is a narrow therapeutic index for the drugs, the elevated se-rum levels may elicit unwanted toxicities. For example, CYP2C9 is responsible for the metabolism of ibuprofen and tolbutamide, among other drugs. Inhibition ofC YP2C9 by MXC and HPTE can cause an increasing hy-poglycemia risk and other adverse reactions. Drugs me-tabolized by CYP2C19 include omeprazole, diazepam, and lansoprazole. More than 80 drugs in the clinical set-ting are metabolized by CYP2D6, including several anti-psychotics and antidepressants; thus, long-time MXC or HPTE exposure is a risk factor for postural hypotensionF ig. 7. Effects of MXC and HPTE on human CYP3A4 ( a ) and rat CYP3A1 activity ( b ) in liver microsomes. The activity was mea-sured by the conversion rate of midazolam (10μmol/l)to 1′-hy-droxy midazolam in the presence of 100 μmol/l inhibitor (MXC or HPTE). The conversion rate was 96.17 ± 3.00% (%CON, MXC) and 97.82 ± 4.31% (%CON, HPTE) for human liver microsomes and 92.97 ± 5.22% (%CON, MXC) and 80.99 ± 1.65% (%CON, HPTE) for rat liver microsomes. Means ± SEM (n = 5). CON = Control.w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A MC hen/Pan/Wang/Chen/Dong/Ge/HuP harmacology 2015;95:145–153DOI: 10.1159/000380883152and extrapyramidal side effects [26] . MXC and HPTE in-hibit hepatic CYPs, reducing presystemic drug metabo-lism, resulting in an increase in oral bioavailability of the drug absorbed.B ecause CYP2C9 is mainly involved in the metabolism of estrogen [28] , the inhibition of CYP2C9 by MXC or its metabolite HPTE may be a factor contributing to the es-trogen-like effect of MXC and HPTE. When estrogen me-tabolism is reduced, the estrogen level is increased. This can cause many male reproductive diseases such as hypo-spadias, cryptorchidism, adult testiculoma, breast carci-noma, and carcinoma of the prostate[29] . In this study, we demonstrated that concentrations as low as 5 μmol/l of MXC or HPTE suppressed human CYP2C9 activity. This indicates that this inhibition may be involved in the reduced metabolism of estrogen. The level of MXC instream water can reach levels as high as 128 μg/l[30].Thus, these observations are relevant to public health, and such concentrations of MXC or HPTE have the po-tential to affect CYP2C9 activity. A heightened estrogen level in the human body may increase estrogen activity via binding to estrogen receptor activity. It is perhaps rea-sonable to infer that exposure to low concentrations of MXC and HPTE will increase the estrogen level. The re-sults presented herein identify additional mechanisms of action of MXC and HPTE, i.e. causing estrogen-like ac-tion, affecting normal endocrine function. An identifica-tion of the mechanisms of action of hormonally active agents present in the environment is required to facilitate the process of risk assessment in a population.C onclusion The present study demonstrates that MXC and HPTE inhibited human CYP2C9 and rat CYP2C11 as well as human CYP2C19 and rat CYP2C6 activity but had no ef-fects on human CYP3A4 and rat CYP3A1 activity. For CYP2D6 and CYP2D2, only HPTE potently inhibited the activity of this enzyme. MXC and HPTE showed com-petitive inhibition of human CYP2C9 and rat CYP2C11 activity against their substrate, but they displayed non-competitive inhibition of human CYP2C19 and ratC YP2C6 activity against their substrate. HPTE showed noncompetitive inhibition of human CYP2D6 and rat CYP2D2 activity against its substrate. MXC and HPTE are potent disruptors of endogenous and exogenous c ompound metabolism.D isclosure Statement T he authors report no conflicts of interest.R eferences 1 Gonzalez FJ, Lee YH: Constitutive expressionof hepatic cytochrome p450 genes. FASEB J 1996; 10:1112–1117. 2 Girennavar B, Jayaprakasha GK, Patil BS: Po-tent inhibition of human cytochrome P450 3A4, 2D6, and 2C9 isoenzymes by grapefruit juice and its furocoumarins. J Food Sci 2007; 72:C417–C421. 3 Belic A, Temesvari M, Kohalmy K, Vrzal R,Dvorak Z, Rozman D, Monostory K: Investi-gation of the CYP2C9 induction profile in hu-man hepatocytes by combining experimental and modelling approaches. Curr Drug Metab 2009; 10:1066–1074. 4 Ohkura T, Ohta K, Nagao T, Kusumoto K,Koeda A, Ueda T, Jomura T, Ikeya T, Ozeki E, Wada K, Naitoh K, Inoue Y, Takahashi N, Iwai H, Arakawa H, Ogihara T: Evaluation of human hepatocytes cultured by three-dimen-sional spheroid systems for drug metabolism. Drug Metab Pharmacokinet 2014; 29:373–378.5 Wang Z, Sun W, Huang CK, Wang L, Ia MM,Cui X, Hu GX, Wang ZS: Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver mi c rosomes. Drug Dev Ind Pharm 2014, Epub ahead of print.6 Videau O, Pitarque S, Troncale S, Hery P,Thevenot E, Delaforge M, Benech H: Can a cocktail designed for phenotyping pharmaco-kinetics and metabolism enzymes in human be used efficiently in rat? Xenobiotica 2012; 42:349–354. 7 Kapoor IP, Metcalf RL, Nyström RF, SanghaGK: Comparative metabolism of methoxy-chlor, methiochlor, and DDT in mouse, in-sects, and in a model ecosystem. J Agric Food Chem 1970; 18:1145–1152. 8 Hu GX, Zhao B, Chu Y, Li XH, AkingbemiBT, Zheng ZQ, Ge RS: Effects of methoxy-chlor and 2,2-bis( p -hydroxyphenyl)-1,1,1-trichloroethane on 3β-hydroxysteroid dehy-drogenase and 17β-hydroxysteroid dehydro-genase-3 activities in human and rat testes. Int J Androl 2011; 34:138–144. 9 Akingbemi BT, Ge RS, Klinefelter GR, Gun-salus GL, Hardy MP: A metabolite of me-thoxychlor, 2,2-bis( p -hydroxyphenyl)-1,1,1-trichloroethane, reduces testosterone biosyn-thesis in rat Leydig cells through suppression of steady-state messenger ribonucleic acid levels of the cholesterol side-chain cleavage enzyme. Biol Reprod 2000; 62:571–578.10 Lafuente A, Marquez N, Pousada Y, Pazo D, Esquifino AI: Possible estrogenic and/or anti-androgenic effects of methoxychlor on pro-lactin release in male rats. Arch Toxicol 2000; 74:270–275.11 Paulose T, Hernandez-Ochoa I, Basavarajap-pa MS, Peretz J, Flaws JA: Increased sensitiv-ity of estrogen receptor alpha overexpressing antral follicles to methoxychlor and its me-tabolites. Toxicol Sci 2012; 120:447–459.12 Guo J, Deng H, Li H, Zhu Q, Zhao B, Chen B, Chu Y, Ge RS: Effects of methoxychlor and its metabolite 2,2-bis( p -hydroxyphenyl)-1,1,1-trichloroethane on 11β-hydroxysteroid dehy-drogenase activities in vitro. Toxicol Lett 2013; 218:18–23.w n l o a d e d b y : n z h o u M e d i c a l C o l l e g e .107.222.187 - 5/27/2015 8:27:47 A M。