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Hormonal context in the regulation of cytochrome P450 (CYP) during liver insufficiency with serotonergic system dysfunction
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/b-catenin signaling via Sp1 induction. P40 CYP1B1 INHIBITS CANCER CELL APOPTOSIS THROUGH SP1-MEDIATED SUPPRESSION OF TRAIL EXPRESSION Yeo-Jung Kwon, Hyoung-Seok Baek, Dong-Jin Ye, Sangyun Shin, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and shares the feature as an enzyme for drug metabolisms. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate cancer cells. To explore the role of CYP1B1 on cancer cell progression, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of anti-apoptotic factors such as Bcl-2, BclxL, XIAP, survivin and Mcl-1 were examined and showed to be promoted followed by CYP1B1 overexpression or treatment with CYP1B1 inducer, 7, 12-dimethylbenz[a]anthracene (DMBA). These promoting results were reversed when cells were transfected with siRNA for CYP1B1 or treated with a CYP1B1 inhibitor, tetramethoxystilbene (TMS). Moreover, AIF, a widely known pro-apoptotic factor, showed to be translocated to cytosol from mitochondria after treatment with TMS. The expression of TRAIL, a member of tumor necrosis factor superfamily, was also examined followed by alteration of CYP1B1 expression and it showed similar changes in expression level to pro-apoptotic factors. Sp1, a transcription factor, has been reported to suppress TRAIL expression by binding TRAIL promoter. To identify whether Sp1 mediates the CYP1B1-induced suppression of TRAIL, we examined Sp1 expression after changes of CYP1B1 expression level and it showed that Sp1 is promoted by CYP1B1. Moreover, Sp1 knockdown with specific siRNA showed promotion of TRAIL-related apoptotic factors and these results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a well-known Sp1 binding inhibitor, resulted in recovery of TRAIL expression formerly suppressed by treatment with DMBA. Taken together, these data suggest that CYP1B1 blocks apoptosis via TRAIL suppression and this is mediated by Sp1 induction. P41 DIFFERENT SUSCEPTIBILITY OF CYTOCHROME P450 2C ENZYMES TO DOWN-REGULATION BY NITRIC OXIDE Ji Won Park, Choon-myung Lee, Edward T. Morgan. Department of Pharmacology, Emory University, Atlanta, GA, USA Cytochromes P450 (P450) are key enzymes in metabolizing both endogenous and xenobiotic compounds and can be induced or inhibited by numerous drugs, environmental chemicals, diet and diseases. Such changes in P450 expression and activity can cause drug toxicity and drugdrug interactions. High levels of nitric oxide (NO) are produced during activation of the immune system and previously our lab showed that nitric oxide (NO) mediates the down-regulation of several P450 enzymes including CYP2C22 and CYP2B proteins by inflammatory cytokines. The purpose of this study was to determine if human CYP2C proteins are down-regulated by NO. HeLa and Huh7 cells were transduced with lentiviral vectors containing either human CYP2C enzymes or rat CYP2C11 tagged with v5, and the transduced cells were incubated with the nitric oxide donor, DPTA-NONOate (DPTA). Preliminary studies indicated that CYP2C8 was the most sensitive of the CYP2C enzymes. With 4 hr of DPTA treatment, CYP2C8 enzymes were significantly down-regulated to 50-55% of control. However, the level of CYP2C11 was not affected by NO. For longer exposure of cells to NO, NO synthase was induced in the cells using a doxycycline inducible pLIX304-hNOS2 vector. At 24hr after doxycycline treatment, media were collected to determine NO production and cells were harvested. The treatment resulted in a ~50% downregulation of CYP2C8 protein. This downregulation was attenuated by the NOS inhibitor, L-NG-nitroarginine methyl ester (L-NAME). At both 4 hr and 24 hr, no changes of CYP2C8 mRNA levels were observed. Remarkably, the downregulation of CYP2C8 by either DPTA or doxycycline was insensitive to the
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proteasome inhibitors bortezomib or MG132. Pretreatment with calpain inhibitors ALLN and Calpain inhibitor III did not block CYP2C8 downregulation by DPTA, but partially inhibited CYP2C8 degradation after 24hr of doxycycline treatment. Taken together, these data show different effects of NO on the downregulation of CYP2C enzymes and non-lysosomal protease are partially involved in CYP2C8 downregulation by NO. Supported by grant 2R01GM69971 from the National Institutes of Health. P42 EFFECT OF CYP2C9 GENOTYPE FOR PHARMACOKINETICS OF EFAVIRENZ IN VIVO IN CYNOMOLGUS MONKEYS Kazuhide Iwasaki 1, Yusuke Kitsugi 1, Kanami Ikeda 1, Takahiro Yoshikawa 1, Shotaro Uehara 2, Yasuhiro Uno 1, Masahiro Utoh 1, Hiroshi Yamazaki 2. 1 Shin Nippon Biomedical Laboratories (SNBL), Ltd., Kainan, Japan; 2 Showa Pharmaceutical University, Machida, Japan Cynomolgus monkeys are frequently used in nonclinical studies of new drug development due to their evolutionary closeness to humans. Efavirenz (EFV) is metabolized by CYP3A4 and CYP2B6 in humans, but is mainly metabolized by CYP2C9 in cynomolgus monkeys. Because cynomolgus CYP2C9 is polymorphic, in this study, the effect of CYP2C9 polymorphisms for the pharmacokinetics of EFV and its metabolites was assessed in the genotyped cynomolgus monkeys. EFV was administered at 1 mg/kg intravenous (IV) administration and 2 mg/kg oral (PO) administration to six cynomolgus monkeys including homozygotes (homo, n¼2), heterozygote (hetero, n¼1), and wild type animals (wild, n¼3). Plasma concentrations of EFV and its metabolites were determined by high performance liquid chromatography with tandem mass spectrometry. After IV administration, EFV exhibited biphasic elimination from the plasma. Terminal half-life values of EFV were about 3 to 6 hours in wild and hetero, and about 9 hours in homo. The area under the plasma concentration-time curve (AUC) of EFV after IV administration was similar between wild and hetero, but was 1.5-fold higher in homo than in wild. After PO administration, the AUC of EFV was similar between wild and hetero, but was 2fold higher in homo than in wild. These results indicated that CYP2C9 genotype was associated with EFV metabolism. Therefore, CYP2C9 polymorphisms might account for interindividual variations of EFV clearance in cynomolgus monkeys. P43 HORMONAL CONTEXT IN THE REGULATION OF CYTOCHROME P450 (CYP) DURING LIVER INSUFFICIENCY WITH SEROTONERGIC SYSTEM DYSFUNCTION Marta Kot. Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland Pioneering studies in the rat have demonstrated that the serotonergic system is an unknown earlier factor that influences the activity and protein levels of CYP isoforms in the liver [1,2,3]. However, the expression of CYP isoforms depends on hepatic function and can be altered by different exogenous or endogenous factors. Because liver insufficiency is associated with predisposition to e.g., hepatocellular carcinoma, the aim of this study was to show the functional consequences of serotonergic system dysfunction in the regulation of cytochrome CYP2B and CYP3A isoforms during liver insufficiency. A rat model of liver insufficiency with dysfunction of the serotonergic system was developed. To induce abnormal liver function, animals were treated with nitrosodiethylamine (DEN) at 50 mg/ kg of body weight twice a week for 7 weeks. To induce dysfunction of the serotonergic system, the removal of tryptophan in the diet was employed. The rate of serotonin synthesis is controlled by the availability of tryptophan. The serum levels of AST (aspartate transaminase), ALT (alanine transaminase) and AFP (alpha-fetoprotein) were significantly increased during DEN-evoked liver insufficiency (determined by ELISA kits). The concentration of serum serotonin was significantly increased during liver insufficiency alone and decreased after serotonergic system dysfunction during liver insufficiency (estimated using HPLC). Dysfunction of the serotonergic system during liver insufficiency generated up-regulation of CYP2B and down-regulation CYP3A gene expression (real time RT PCR analysis) with a concomitant change in protein levels (evaluated by
S40
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
western blot analysis) and activity (examined by measuring the rate of CYP specific reactions using HPLC) in the same direction. In addition, liver insufficiency led to reduction in T4 level and increased the TSH level. Serotonergic system dysfunction during liver insufficiency evoked a stronger drop in the level T3 and T4 without changing TSH levels. Either liver insufficiency or dysfunction of the serotonergic system did not influence the level of glucocorticoids. All hormones were determined with ELISA kits. In conclusion, the serotonergic system is engaged in the regulation of CYP2B and CYP3A isoforms during liver insufficiency. Moreover, the interplay between the serotonergic system and the regulation of CYP2B or CYP3A is dependent on hepatic function and can be observed without influence from thyroid hormones and glucocorticoids. This work was supported by the Polish National Science Center grant SONATA 2012/07/ D/NZ4/00814 to M.K. 1. Kot M., Daniel W.A. The brain noradrenergic and serotonergic systems as unknown factors regulating the activity of cytochrome P450. Eur. Neuropsychopharmacol. 2009 March;19(1):17-18. 2. Kot M., Daniel W.A. Cytochrome P450 is regulated by noradrenergic and serotonergic systems. Pharmacological Research. 2011 Oct;64(4):371-80. 3. Kot M., Pilc A., Daniel W.A. Simultaneous alterations of brain and plasma serotonin concentrations and liver cytochrome P450 in rats fed on a tryptophan-free diet. Pharmacological Research. 2012 Oct;66(4):292-9. P44 ABSTRACT WITHDRAWN P45 IDENTIFICATION AND CHARACTERIZATION OF CYTOCHROME P450 4F ENZYMES IN MARMOSETS Shotaro Uehara 1, Yasuhiro Uno 2, Yukako Yuki 1, Takashi Inoue 3, Erika Sasaki 3, Hiroshi Yamazaki 1. 1 Showa Pharmaceutical University, Machida, Japan; 2 Shin Nippon Biomedical Laboratories (SNBL), Ltd., Kainan, Japan; 3 Central Institute for Experimental Animals, Kawasaki, Japan Common marmosets (Callithrix jacchus), belonging to New World monkeys, are attractive as non-human primate models in drug metabolism study because of small body size, ease handling, high fertility associated with early sexual maturity. However, the drug metabolizing properties has not been fully understood due to insufficiency of the information for cytochrome P450 (P450), major drug metabolizing enzymes. We identified four novel marmoset P450 4F genes by sequence homology searching using marmoset genome data. By RT-PCR using gene-specific primers, marmoset P450 4F2, 4F3B, 4F11, and 4F12 cDNAs were successfully isolated from the liver, and its deduced amino acid sequences exhibited high identities (87-93%) to the homologous human and cynomolgus monkey P450 4F enzymes. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Next, the catalytic activities by marmoset and human P450 4F proteins heterologously expressed in Escherichia coli were investigated using anti-allergic drug ebastine, a human P450 2J/4F probe substrate. Enzyme assays indicated that marmoset P450 4F12 effectively catalyzed ebastine 4-hydroxylation among four marmoset P450 4F enzymes. Ebastine 4-hydroxylation activities by small intestine and liver microsomes from marmosets showed greatly higher values than those of humans, and ebastine 4-hydroxylation activity by marmoset small intestine microsomes was inhibited (~60%) by anti-P450 4F antibodies, unlike the cases in humans, suggesting that P450 4F enzymes might involve in the species differences of ebastine metabolism among marmosets and humans. P46 ABSTRACT WITHDRAWN P47 INFLUENCES OF CYTOCHROME B5 EXPRESSION AND ITS GENETIC POLYMORPHISM ON THE ACTIVITY OF CYP2C9, CYP2C19 AND CYP3A4 Sung-Eun Yoo 1, Myeongjin Yi 1, Sun-Ah Cho 1, Woo-Young Kim 1, DongHyun Kim 1, Jae-Gook Shin 1, 2, Su-Jun Lee 1. 1 Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of
Medicine, Busan, South Korea; 2 Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan, South Korea Cytochrome b5 (Cytb5) is a membrane-associated protein involved in various biological processes, such as transferring electron to P450s. The objective of present study was to investigate the effects of Cytb5 expression and its genetic mutation on the drug metabolism catalyzed by CYP2C9, CYP2C19 and CYP3A4. Expression of Cytb5 protein and mRNA in human liver tissues was examined using Western blot and Quantitative real-time PCR, respectively. Distribution of genetic polymorphisms in Cytb5 gene was extensively analyzed in HapMap and 1000 genome data. The putative splicing variant, c.288G>A (rs7238987) was selected as a possible functional variant and it was screened in 36 liver tissues by direct DNA sequencing. Activities of CYP2C9, CYP2C19, and CYP3A4 in liver microsomes were determined by the metabolism of Tolbutamide, Omeprazole and Midazolam, respectively. Cytb5 protein and mRNA contents showed inter-individual variations in expressions with 11- and 6-fold, respectively. Cytb5 expression was correlated with the activity of CYP2C9 and CYP3A4. A splicing variant, c.288G>A, was identified at 5.6% in human liver tissues (N¼36) by direct DNA sequencing. Liver tissues having a splicing variant exhibited a decreased expression of Cytb5 protein content compared to the wild-type. The CYP2C19 dependent Omeprazole metabolism was significantly decreased in the liver tissues carrying the splicing variant when compared to the liver tissues having Cytb5 wild-type. The present results propose that different expression of Cytb5 and its genetic polymorphism would cause variations in CYP mediated drug metabolism, which may explain, at least in part, inter-individual drug responses in humans in addition to CYP polymorphisms. P48 NITRIC OXIDE EVOKED HUMAN CYP2B6 DOWN REGULATION Choon-Myung Lee, Edward T. Morgan. Department of Pharmacology, Emory University, Atlanta, GA, USA We showed previously that rat CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 downregulation, we made several cell lines (HeLa, HuH7, and HepG2 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag are expressed from a lentiviral vector with a CMV promoter. Native CYP2B6 protein was rapidly down regulated within 3 h following NO donor DETA NONOate treatment, while its mRNA level was not down regulated, suggesting posttranslational down regulation. A cycloheximide chase experiment showed that half-life of CYP2B6 protein was 1h 20 min with DPTA NONOate treatment compared to 2h 40 min with mock treatment. DPTA NONOate treatment resulted in rapid down regulation of CYP2B6 activity measured as the formation of 7-hydroxy-4-trifluoromethylcoumarin as well as 2B6 protein in the CYP2B6 HeLa cell line. In addition, the concentration of DPTA NONOate required to down-regulate CYP2B6 was significantly lower than that for rat CYP2B1. We generated a tet-on hNOS2 HeLa cell line to study the effect of endogenous NO on the down regulation. The endogenous NO induced by doxycycline treatment also effectively down regulated CYP2B6 protein, which was blocked by the co-treatment of NOS2 competitive inhibitor L-NG-nitroarginine methyl ester (L-NAME). We investigated the proteolytic enzymes responsible for NO-dependent CYP2B6 degradation. Calpain inhibitors (MDL 28170, ALLN, Cal III, and E64d) and lysosomal protease inhibitors (NH4Cl, 3-methyladenine, and chloroquine) did not inhibit the NO dependent CYP2B6 down-regulation. Although the proteasome inhibitors MG132 and bortezomib tended to partially suppress the NO-induced down regulation in the HuH7 cell line, the suppression was not statistically significant, in contrast to previous observations with rat CYP2B1 protein. When cells were co-treated with NO donor and proteasome inhibitors, a smear of high molecular mass could be detected on CYP2B6 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by cotreatment with DPTA NONOate and bortezomib. Taken together, our data suggest that CYP2B6 is down regulated in an NO-dependent manner via the proteasome. We also found that the tight binding inhibitor of CYP2B6, 4-(4-chlorophenyl)imidazole (4-CPI) completely abolished NO-induced
CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/b-catenin signaling via Sp1 induction. P40 CYP1B1 INHIBITS CANCER CELL APOPTOSIS THROUGH SP1-MEDIATED SUPPRESSION OF TRAIL EXPRESSION Yeo-Jung Kwon, Hyoung-Seok Baek, Dong-Jin Ye, Sangyun Shin, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and shares the feature as an enzyme for drug metabolisms. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate cancer cells. To explore the role of CYP1B1 on cancer cell progression, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of anti-apoptotic factors such as Bcl-2, BclxL, XIAP, survivin and Mcl-1 were examined and showed to be promoted followed by CYP1B1 overexpression or treatment with CYP1B1 inducer, 7, 12-dimethylbenz[a]anthracene (DMBA). These promoting results were reversed when cells were transfected with siRNA for CYP1B1 or treated with a CYP1B1 inhibitor, tetramethoxystilbene (TMS). Moreover, AIF, a widely known pro-apoptotic factor, showed to be translocated to cytosol from mitochondria after treatment with TMS. The expression of TRAIL, a member of tumor necrosis factor superfamily, was also examined followed by alteration of CYP1B1 expression and it showed similar changes in expression level to pro-apoptotic factors. Sp1, a transcription factor, has been reported to suppress TRAIL expression by binding TRAIL promoter. To identify whether Sp1 mediates the CYP1B1-induced suppression of TRAIL, we examined Sp1 expression after changes of CYP1B1 expression level and it showed that Sp1 is promoted by CYP1B1. Moreover, Sp1 knockdown with specific siRNA showed promotion of TRAIL-related apoptotic factors and these results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a well-known Sp1 binding inhibitor, resulted in recovery of TRAIL expression formerly suppressed by treatment with DMBA. Taken together, these data suggest that CYP1B1 blocks apoptosis via TRAIL suppression and this is mediated by Sp1 induction. P41 DIFFERENT SUSCEPTIBILITY OF CYTOCHROME P450 2C ENZYMES TO DOWN-REGULATION BY NITRIC OXIDE Ji Won Park, Choon-myung Lee, Edward T. Morgan. Department of Pharmacology, Emory University, Atlanta, GA, USA Cytochromes P450 (P450) are key enzymes in metabolizing both endogenous and xenobiotic compounds and can be induced or inhibited by numerous drugs, environmental chemicals, diet and diseases. Such changes in P450 expression and activity can cause drug toxicity and drugdrug interactions. High levels of nitric oxide (NO) are produced during activation of the immune system and previously our lab showed that nitric oxide (NO) mediates the down-regulation of several P450 enzymes including CYP2C22 and CYP2B proteins by inflammatory cytokines. The purpose of this study was to determine if human CYP2C proteins are down-regulated by NO. HeLa and Huh7 cells were transduced with lentiviral vectors containing either human CYP2C enzymes or rat CYP2C11 tagged with v5, and the transduced cells were incubated with the nitric oxide donor, DPTA-NONOate (DPTA). Preliminary studies indicated that CYP2C8 was the most sensitive of the CYP2C enzymes. With 4 hr of DPTA treatment, CYP2C8 enzymes were significantly down-regulated to 50-55% of control. However, the level of CYP2C11 was not affected by NO. For longer exposure of cells to NO, NO synthase was induced in the cells using a doxycycline inducible pLIX304-hNOS2 vector. At 24hr after doxycycline treatment, media were collected to determine NO production and cells were harvested. The treatment resulted in a ~50% downregulation of CYP2C8 protein. This downregulation was attenuated by the NOS inhibitor, L-NG-nitroarginine methyl ester (L-NAME). At both 4 hr and 24 hr, no changes of CYP2C8 mRNA levels were observed. Remarkably, the downregulation of CYP2C8 by either DPTA or doxycycline was insensitive to the
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proteasome inhibitors bortezomib or MG132. Pretreatment with calpain inhibitors ALLN and Calpain inhibitor III did not block CYP2C8 downregulation by DPTA, but partially inhibited CYP2C8 degradation after 24hr of doxycycline treatment. Taken together, these data show different effects of NO on the downregulation of CYP2C enzymes and non-lysosomal protease are partially involved in CYP2C8 downregulation by NO. Supported by grant 2R01GM69971 from the National Institutes of Health. P42 EFFECT OF CYP2C9 GENOTYPE FOR PHARMACOKINETICS OF EFAVIRENZ IN VIVO IN CYNOMOLGUS MONKEYS Kazuhide Iwasaki 1, Yusuke Kitsugi 1, Kanami Ikeda 1, Takahiro Yoshikawa 1, Shotaro Uehara 2, Yasuhiro Uno 1, Masahiro Utoh 1, Hiroshi Yamazaki 2. 1 Shin Nippon Biomedical Laboratories (SNBL), Ltd., Kainan, Japan; 2 Showa Pharmaceutical University, Machida, Japan Cynomolgus monkeys are frequently used in nonclinical studies of new drug development due to their evolutionary closeness to humans. Efavirenz (EFV) is metabolized by CYP3A4 and CYP2B6 in humans, but is mainly metabolized by CYP2C9 in cynomolgus monkeys. Because cynomolgus CYP2C9 is polymorphic, in this study, the effect of CYP2C9 polymorphisms for the pharmacokinetics of EFV and its metabolites was assessed in the genotyped cynomolgus monkeys. EFV was administered at 1 mg/kg intravenous (IV) administration and 2 mg/kg oral (PO) administration to six cynomolgus monkeys including homozygotes (homo, n¼2), heterozygote (hetero, n¼1), and wild type animals (wild, n¼3). Plasma concentrations of EFV and its metabolites were determined by high performance liquid chromatography with tandem mass spectrometry. After IV administration, EFV exhibited biphasic elimination from the plasma. Terminal half-life values of EFV were about 3 to 6 hours in wild and hetero, and about 9 hours in homo. The area under the plasma concentration-time curve (AUC) of EFV after IV administration was similar between wild and hetero, but was 1.5-fold higher in homo than in wild. After PO administration, the AUC of EFV was similar between wild and hetero, but was 2fold higher in homo than in wild. These results indicated that CYP2C9 genotype was associated with EFV metabolism. Therefore, CYP2C9 polymorphisms might account for interindividual variations of EFV clearance in cynomolgus monkeys. P43 HORMONAL CONTEXT IN THE REGULATION OF CYTOCHROME P450 (CYP) DURING LIVER INSUFFICIENCY WITH SEROTONERGIC SYSTEM DYSFUNCTION Marta Kot. Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland Pioneering studies in the rat have demonstrated that the serotonergic system is an unknown earlier factor that influences the activity and protein levels of CYP isoforms in the liver [1,2,3]. However, the expression of CYP isoforms depends on hepatic function and can be altered by different exogenous or endogenous factors. Because liver insufficiency is associated with predisposition to e.g., hepatocellular carcinoma, the aim of this study was to show the functional consequences of serotonergic system dysfunction in the regulation of cytochrome CYP2B and CYP3A isoforms during liver insufficiency. A rat model of liver insufficiency with dysfunction of the serotonergic system was developed. To induce abnormal liver function, animals were treated with nitrosodiethylamine (DEN) at 50 mg/ kg of body weight twice a week for 7 weeks. To induce dysfunction of the serotonergic system, the removal of tryptophan in the diet was employed. The rate of serotonin synthesis is controlled by the availability of tryptophan. The serum levels of AST (aspartate transaminase), ALT (alanine transaminase) and AFP (alpha-fetoprotein) were significantly increased during DEN-evoked liver insufficiency (determined by ELISA kits). The concentration of serum serotonin was significantly increased during liver insufficiency alone and decreased after serotonergic system dysfunction during liver insufficiency (estimated using HPLC). Dysfunction of the serotonergic system during liver insufficiency generated up-regulation of CYP2B and down-regulation CYP3A gene expression (real time RT PCR analysis) with a concomitant change in protein levels (evaluated by
S40
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
western blot analysis) and activity (examined by measuring the rate of CYP specific reactions using HPLC) in the same direction. In addition, liver insufficiency led to reduction in T4 level and increased the TSH level. Serotonergic system dysfunction during liver insufficiency evoked a stronger drop in the level T3 and T4 without changing TSH levels. Either liver insufficiency or dysfunction of the serotonergic system did not influence the level of glucocorticoids. All hormones were determined with ELISA kits. In conclusion, the serotonergic system is engaged in the regulation of CYP2B and CYP3A isoforms during liver insufficiency. Moreover, the interplay between the serotonergic system and the regulation of CYP2B or CYP3A is dependent on hepatic function and can be observed without influence from thyroid hormones and glucocorticoids. This work was supported by the Polish National Science Center grant SONATA 2012/07/ D/NZ4/00814 to M.K. 1. Kot M., Daniel W.A. The brain noradrenergic and serotonergic systems as unknown factors regulating the activity of cytochrome P450. Eur. Neuropsychopharmacol. 2009 March;19(1):17-18. 2. Kot M., Daniel W.A. Cytochrome P450 is regulated by noradrenergic and serotonergic systems. Pharmacological Research. 2011 Oct;64(4):371-80. 3. Kot M., Pilc A., Daniel W.A. Simultaneous alterations of brain and plasma serotonin concentrations and liver cytochrome P450 in rats fed on a tryptophan-free diet. Pharmacological Research. 2012 Oct;66(4):292-9. P44 ABSTRACT WITHDRAWN P45 IDENTIFICATION AND CHARACTERIZATION OF CYTOCHROME P450 4F ENZYMES IN MARMOSETS Shotaro Uehara 1, Yasuhiro Uno 2, Yukako Yuki 1, Takashi Inoue 3, Erika Sasaki 3, Hiroshi Yamazaki 1. 1 Showa Pharmaceutical University, Machida, Japan; 2 Shin Nippon Biomedical Laboratories (SNBL), Ltd., Kainan, Japan; 3 Central Institute for Experimental Animals, Kawasaki, Japan Common marmosets (Callithrix jacchus), belonging to New World monkeys, are attractive as non-human primate models in drug metabolism study because of small body size, ease handling, high fertility associated with early sexual maturity. However, the drug metabolizing properties has not been fully understood due to insufficiency of the information for cytochrome P450 (P450), major drug metabolizing enzymes. We identified four novel marmoset P450 4F genes by sequence homology searching using marmoset genome data. By RT-PCR using gene-specific primers, marmoset P450 4F2, 4F3B, 4F11, and 4F12 cDNAs were successfully isolated from the liver, and its deduced amino acid sequences exhibited high identities (87-93%) to the homologous human and cynomolgus monkey P450 4F enzymes. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Next, the catalytic activities by marmoset and human P450 4F proteins heterologously expressed in Escherichia coli were investigated using anti-allergic drug ebastine, a human P450 2J/4F probe substrate. Enzyme assays indicated that marmoset P450 4F12 effectively catalyzed ebastine 4-hydroxylation among four marmoset P450 4F enzymes. Ebastine 4-hydroxylation activities by small intestine and liver microsomes from marmosets showed greatly higher values than those of humans, and ebastine 4-hydroxylation activity by marmoset small intestine microsomes was inhibited (~60%) by anti-P450 4F antibodies, unlike the cases in humans, suggesting that P450 4F enzymes might involve in the species differences of ebastine metabolism among marmosets and humans. P46 ABSTRACT WITHDRAWN P47 INFLUENCES OF CYTOCHROME B5 EXPRESSION AND ITS GENETIC POLYMORPHISM ON THE ACTIVITY OF CYP2C9, CYP2C19 AND CYP3A4 Sung-Eun Yoo 1, Myeongjin Yi 1, Sun-Ah Cho 1, Woo-Young Kim 1, DongHyun Kim 1, Jae-Gook Shin 1, 2, Su-Jun Lee 1. 1 Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of
Medicine, Busan, South Korea; 2 Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan, South Korea Cytochrome b5 (Cytb5) is a membrane-associated protein involved in various biological processes, such as transferring electron to P450s. The objective of present study was to investigate the effects of Cytb5 expression and its genetic mutation on the drug metabolism catalyzed by CYP2C9, CYP2C19 and CYP3A4. Expression of Cytb5 protein and mRNA in human liver tissues was examined using Western blot and Quantitative real-time PCR, respectively. Distribution of genetic polymorphisms in Cytb5 gene was extensively analyzed in HapMap and 1000 genome data. The putative splicing variant, c.288G>A (rs7238987) was selected as a possible functional variant and it was screened in 36 liver tissues by direct DNA sequencing. Activities of CYP2C9, CYP2C19, and CYP3A4 in liver microsomes were determined by the metabolism of Tolbutamide, Omeprazole and Midazolam, respectively. Cytb5 protein and mRNA contents showed inter-individual variations in expressions with 11- and 6-fold, respectively. Cytb5 expression was correlated with the activity of CYP2C9 and CYP3A4. A splicing variant, c.288G>A, was identified at 5.6% in human liver tissues (N¼36) by direct DNA sequencing. Liver tissues having a splicing variant exhibited a decreased expression of Cytb5 protein content compared to the wild-type. The CYP2C19 dependent Omeprazole metabolism was significantly decreased in the liver tissues carrying the splicing variant when compared to the liver tissues having Cytb5 wild-type. The present results propose that different expression of Cytb5 and its genetic polymorphism would cause variations in CYP mediated drug metabolism, which may explain, at least in part, inter-individual drug responses in humans in addition to CYP polymorphisms. P48 NITRIC OXIDE EVOKED HUMAN CYP2B6 DOWN REGULATION Choon-Myung Lee, Edward T. Morgan. Department of Pharmacology, Emory University, Atlanta, GA, USA We showed previously that rat CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 downregulation, we made several cell lines (HeLa, HuH7, and HepG2 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag are expressed from a lentiviral vector with a CMV promoter. Native CYP2B6 protein was rapidly down regulated within 3 h following NO donor DETA NONOate treatment, while its mRNA level was not down regulated, suggesting posttranslational down regulation. A cycloheximide chase experiment showed that half-life of CYP2B6 protein was 1h 20 min with DPTA NONOate treatment compared to 2h 40 min with mock treatment. DPTA NONOate treatment resulted in rapid down regulation of CYP2B6 activity measured as the formation of 7-hydroxy-4-trifluoromethylcoumarin as well as 2B6 protein in the CYP2B6 HeLa cell line. In addition, the concentration of DPTA NONOate required to down-regulate CYP2B6 was significantly lower than that for rat CYP2B1. We generated a tet-on hNOS2 HeLa cell line to study the effect of endogenous NO on the down regulation. The endogenous NO induced by doxycycline treatment also effectively down regulated CYP2B6 protein, which was blocked by the co-treatment of NOS2 competitive inhibitor L-NG-nitroarginine methyl ester (L-NAME). We investigated the proteolytic enzymes responsible for NO-dependent CYP2B6 degradation. Calpain inhibitors (MDL 28170, ALLN, Cal III, and E64d) and lysosomal protease inhibitors (NH4Cl, 3-methyladenine, and chloroquine) did not inhibit the NO dependent CYP2B6 down-regulation. Although the proteasome inhibitors MG132 and bortezomib tended to partially suppress the NO-induced down regulation in the HuH7 cell line, the suppression was not statistically significant, in contrast to previous observations with rat CYP2B1 protein. When cells were co-treated with NO donor and proteasome inhibitors, a smear of high molecular mass could be detected on CYP2B6 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by cotreatment with DPTA NONOate and bortezomib. Taken together, our data suggest that CYP2B6 is down regulated in an NO-dependent manner via the proteasome. We also found that the tight binding inhibitor of CYP2B6, 4-(4-chlorophenyl)imidazole (4-CPI) completely abolished NO-induced