Study on genotype and phenotype of novel CYP2D6 variants using pharmacokinetic and pharmacodynamic models with metoprolol as a substrate drug.

To investigate the pharmacokinetic and pharmacodynamic profiles of volunteers carrying CYP2D6 genotypes with unknow metabolic phenotypes, a total of 22 volunteers were recruited based on the sequencing results. Peripheral blood and urine samples were collected at specific time points after oral administration of metoprolol. A validated high-performance liquid chromatography (HPLC) method was used to determine the concentrations of metoprolol and α-hydroxymetoprolol. Blood pressure and electrocardiogram were also monitored. The results showed that the main pharmacokinetic parameters of metoprolol in CYP2D6*1/*34 carriers are similar to those in CYP2D6*1/*1 carriers. However, in individuals carrying the CYP2D6*10/*87, CYP2D6*10/*95, and CYP2D6*97/*97 genotypes, the area under the curve (AUC) and half-life (t1/2) of metoprolol increased by 2-3 times compared to wild type. The urinary metabolic ratio of metoprolol in these genotypes is consistent with the trends observed in plasma samples. Therefore, CYP2D6*1/*34 can be considered as normal metabolizers, while CYP2D6*10/*87, CYP2D6*10/*95, and CYP2D6*97/*97 are intermediate metabolizers. Although the blood concentration of metoprolol has been found to correlate with CYP2D6 genotype, its blood pressure-lowering effect reaches maximum effectiveness at a reduction of 25 mmHg. Furthermore, P-Q interval prolongation and heart rate reduction are not positively correlated with metoprolol blood exposure. Based on the pharmacokinetic-pharmacodynamic model, this study clarified the properties of metoprolol in subjects with novel CYP2D6 genotypes and provided important fundamental data for the translational medicine of this substrate drug.

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 µM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 µM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17ß-hydroxysteroid dehydrogenase 3 activity at 10-100 µM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17ß-hydroxysteroid dehydrogenase 3 activity.

Effects of Resveratrol on Hepatitis B Virus Replication: In vitro and in vivo Experiments.

INTRODUCTION: Hepatitis B virus (HBV) infection is a disease with high incidence and lack of effective treatment. In this study, we further explored the mechanism of resveratrol (RVT) in the inhibition of HBV replication. The effects of RVT on HBV replication were verified using in vitro and in vivo experiments. METHODS: HepG2 and HepG2.2.15 cell lines were cultured in vitro, and different concentrations of RVT were used to determine its effect on the proliferation of the two cell lines. Autophagy agonists and inhibitors were given, and whether RVT exerts its effect on the proliferation of HepG2 and HepG2.2.15 cells through autophagy was determined. Reverse transcription-quantitative polymerase chain reaction and Western blot were used to detect changes in autophagy-related factors LC3-II, LC3-I, Beclin 1, and p62. Through transfection of pmiR-155, shmiR-155, and the corresponding control group, the relevant mechanism of RVT in inhibiting the proliferation of HepG2 and HepG2.2.15 cells was analyzed. RVT inhibited the toxicity for HepG2.2.15 cells and reduced HBV replication in vitro (p < 0.05). This effect of RVT was enhanced by rapamycin (RAPA; autophagy activator; p < 0.05) but was partially reversed by 3-MA (autophagy inhibitor; p < 0.05). In addition, our results showed that miR-155 expression was higher in HepG2.2.15 cells than in HepG cells (p < 0.05). miR-155 expression in the RVT treatment group was significantly reduced (p < 0.05). We designed an miR-155 overexpression plasmid, low miR-155 expression plasmid, and the corresponding negative control for transfection and found that transfection of pmiR-155 can partially reverse the effect of RVT (p < 0.05), while transfection with shmiR-155 can enhance the effect of RVT (p < 0.05). DISCUSSION: RVT inhibits miR-155, activates autophagy, inhibits the toxicity for HepG2.2.15 cells, and reduces HBV replication, providing a new research direction for the treatment of HBV infection.

The Clinical Application of Growth Hormone and Its Biological and Molecular Mechanisms in Assisted Reproduction.

Growth hormone (GH) has been used as a co-gonadotrophin in assisted reproduction, particularly in poor ovarian responders. The application of GH has been alleged to activate primordial follicles and improve oocyte quality, embryo quality, and steroidogenesis. However, the effects of GH on the live birth rate among women is controversial. Additionally, although the basic biological mechanisms that lead to the above clinical differences have been investigated, they are not yet well understood. The actions of GH are mediated by GH receptors (GHRs) or insulin-like growth factors (IGFs). GH regulates the vital signal transduction pathways that are involved in primordial follicular activation, steroidogenesis, and oocyte maturation. However, the therapeutic windows and duration of GH administration during assisted reproductive technology require further investigation. The review aimed to clarify the role of GH in human fertility from a molecular and biological point of view to provide evidence for proper GH administration.

Characterization of Recruited Mononuclear Phagocytes following Corneal Chemical Injury.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12-/- mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6Chigh (proinflammatory) cells being most abundant at 1 day post-injury. Ly6Cint cells were highly expressed at 3 days post-injury and Ly6Cneg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c+ dendritic cells were abundant in corneas from Mmp12-/- mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

Intensive care unit dignified care: Development and validation of a questionnaire.

BACKGROUND: Patient dignity is sometimes neglected in intensive care unit (ICU) settings, which may potentially cause psychological harm to critically ill patients. However, no instrument has been specifically developed to evaluate the behaviors of dignified care among critical care nurses. AIM: This study aimed to develop and evaluate ICU Dignified Care Questionnaire (IDCQ) for measurement of self-assessed dignity-conserving behaviors of critical care nurses during care. METHODS: The instrument was developed in 3 phases. Phase 1: item generation; phase 2: a two-round Delphi survey and a readability pilot study; phase 3: cross-sectional survey with model estimation. The questionnaire was evaluated by item analysis, exploratory and confirmatory factor analysis, assessment of internal consistency reliability, and test-retest reliability. The investigation was conducted using a convenience sample of 392 critical care nurses from 6 cities in Zhejiang Province, China, of which 30 participated in the test-retest reliability survey 2 weeks later. ETHICAL CONSIDERATIONS: The study was approved by ethics committee. All participants provided written informed consent before the survey. The questionnaire survey was anonymous. RESULTS: The results showed acceptable reliability and validity of the IDCQ. The 17-item final version questionnaire was divided into 2 dimensions: absolute dignity and relative dignity. These two factors accounted for 62.804% of the total variance, and model fitting results were acceptable. The Cronbach's alpha coefficient of the questionnaire was 0.94, and the test-retest intraclass correlation coefficient (ICC) was 0.88 after 2 weeks. CONCLUSIONS: This study developed a brief and reliable instrument (IDCQ) to assess dignified care in ICU nursing. It can help critical care nurses identify their behaviors in maintaining patient dignity and discover their deficiencies. It may also serve as a clinical nursing management tool to help reduce patient disrespect experience in ICU.

Perfluorotridecanoic Acid Inhibits Leydig Cell Maturation in Male Rats in Late Puberty via Changing Testicular Lipid Component.

Perfluorotridecanoic acid (PFTrDA) is a long-chain (C13) perfluoroalkyl carboxylic acid. Here, we report the influence of PFTrDA exposure on the maturation of rat Leydig cells in late puberty in vivo. Male Sprague-Dawley rats were administered PFTrDA by gavage of 0, 1, 5, and 10 mg/kg/day from 35 days to 56 days postpartum. PFTrDA had no effect on body weight, testis weight, and epididymis weight. It significantly decreased the serum testosterone level after 5 and 10 mg/kg exposure, while it did not alter the serum estradiol level. The serum luteinizing hormone level was markedly reduced after 10 mg/kg PFTrDA exposure, while the follicle-stimulating hormone level was unchanged. Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3 transcript levels in the testis were markedly lowered in the 1-5 mg/kg PFTrDA group and the Lhb transcript level in the pituitary in the 10 mg/kg group. CYP11A1 and HSD11B1-positive Leydig cell numbers were markedly reduced after 10 mg/kg PFTrDA exposure. Testicular triglyceride and free fatty acid (palmitic acid, oleic acid, and linoleic acid) levels were significantly reduced by PFTrDA, while Mgll (up-regulation) and Scarb1 and Elovl5 (down-regulation) expression were altered. AKT1 and AMPK phosphorylation was stimulated after 10 PFTrDA mg/kg exposure. In conclusion, PFTrDA delays the maturation of Leydig cells in late puberty mainly by altering the free fatty acid profile.

Perfluorotridecanoic acid inhibits fetal Leydig cell differentiation after in utero exposure in rats via increasing oxidative stress and autophagy.

Perfluorotridecanoic acid (PFTrDA) is a long-chain perfluoroalkyl substance, and its effect on the differentiation of fetal Leydig cells remains unclear. The objective of this study is to explore the effect of in utero PFTrDA exposure on the differentiation of fetal Leydig cells and investigate its underlying mechanisms. Pregnant Sprague-Dawley female rats were daily administered by gavage of PFTrDA at doses of 0, 1, 5, and 10 mg/kg from gestational day 14 to 21. PFTrDA had no effect on the body weight of dams, but significantly reduced the body weight and anogenital distance of male pups at birth at a dose of 10 mg/kg. PFTrDA significantly decreased serum testosterone levels as low as 1 mg/kg. PFTrDA did not affect fetal Leydig cell number, but promoted abnormal aggregation of fetal Leydig cells at doses of 5 and 10 mg/kg. PFTrDA down-regulated the expression of Insl3, Lhcgr, Scarb1, Star, Hsd3b1, Cyp17a1, Nr5a1, and Dhh as well as their proteins. PFTrDA lowered the levels of antioxidants (SOD1, CAT, and GPX1), induced autophagy as shown by increased levels of LC3II and beclin1, and reduced the phosphorylation of mTOR. In conclusion, PFTrDA inhibits the differentiation of fetal Leydig cells in male pups after in utero exposure mainly through increasing oxidative stress and inducing autophagy.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Proximal Fallopian Tubal Embolization by Interventional Radiology prior to Embryo Transfer in Infertile Patients with Hydrosalpinx: A Prospective Study of an Off-label Treatment.

STUDY OBJECTIVE: To evaluate in vitro fertilization (IVF) outcomes of proximal fallopian tube embolization by interventional radiology compared with laparoscopic salpingectomy before embryo transfer (ET) in patients with hydrosalpinx. DESIGN: A single-center, off-label, nonrandomized prospective study. SETTING: Academic university hospital. PATIENTS: One hundred fifty-five patients with hydrosalpinx were identified on ultrasound or hysterosalpingography desiring IVF between April 2016 and December 2017. INTERVENTIONS: Radiologically guided tubal occlusion with embolization microcoils (RTO-EM) and laparoscopic salpingectomy. MEASUREMENTS AND MAIN RESULTS: Of the 155 analyzed patients, 42 were treated with RTO-EM and 113 with laparoscopic salpingectomy. The subsequent IVF outcomes, including implantation, clinical pregnancy, miscarriage, ectopic pregnancy, and ongoing pregnancy (i.e., a fetal heartbeat on ultrasound beyond 10 weeks) were compared between the 2 groups. Implantation and clinical pregnancy per ET cycle in the RTO-EM group were similar to that of the salpingectomy group (26.7% vs 30.2% [p = .51] and 39.0% vs 45.3% [p = .40], respectively), with a similar miscarriage rate. There was no statistically significant difference in ectopic pregnancies between the 2 groups. Moreover, no difference was detected in ongoing pregnancy per cycle between the 2 groups (33.9% vs 41.2%; p = .32). The ongoing-pregnancy rate per patient following RTO-EM was 47.6% (20 of 42) compared with 61.9% (70 of 113) following salpingectomy (odds ratio, 0.56; 95% confidence interval, 0.27-1.14; p = .11). CONCLUSION: Pregnancy in the RTO-EM group was comparable to the salpingectomy group in patients with hydrosalpinx before ET treatment. RTO-EMs may be an alternative to salpingectomy for patients with hydrosalpinx planning for IVF-ET.

Gestational vinclozolin exposure suppresses fetal testis development in rats.

Vinclozolin is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor and is thought to be related to abnormalities of the reproductive tract. However, its mechanism of inducing abnormalities of the male reproductive tract is still unclear. The purpose of this study was to study the effect of gestational vinclozolin exposure on the development of rat fetal Leydig cells. Female pregnant Sprague-Dawley rats were exposed to vinclozolin (0, 25, 50, and 100 mg/kg body weight/day) by gavage from gestational day 14-21. Vinclozolin dose-dependently reduced serum testosterone levels at doses of 50 and 100 mg/kg and the anogenital distance at 100 mg/kg. RNA-seq, qPCR, and Western blotting showed that vinclozolin down-regulated the expression of Nr5a1, Sox9, Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3, Amh, Pdgfa, and Dhh and their encoded proteins. Vinclozolin reduced the number of NR2F2-positive stem Leydig cells at a dose of 100 mg/kg and enhanced autophagy in the testes. In conclusion, vinclozolin disrupts reproductive tract development and testis development in male fetal rats via several pathways.

Effects of MMP12 on cell motility and inflammation during corneal epithelial repair.

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

In vitro and in vivo characterization of 13 CYP2C9 allelic variants found in Chinese Han population.

Our previous study detected totally 35 CYP2C9 allelic variants in 2127 Chinese subjects, of whom 21 novel alleles were reported for the first time in Chinese populations. The aim of the present study was to characterize the 13 CYP2C9 allelic variants both in vitro and in vivo. Different types of CYP2C9 variants were highly expressed in COS-7 cells, and 50 µM tolbutamide was added as the probing substrate to evaluate their metabolic abilities in vitro. Subsequently, the concentrations of tolbutamide and its metabolite in the plasma and urine within individuals with different types of genotypes were determined by HPLC to evaluate the catalytic activity of the 13 mutant CYP2C9 proteins in vivo. Our results showed that compared with *1/*1 wild-type subjects, subjects with *1/*40 genotype showed increased oral clearance (CL/F), whereas individuals with *1/*3, *1/*13, *3/*3, *3/*13, *1/*16, *1/*19, *1/*34, *1/*42, *1/*45, *1/*46, and *1/*48 genotype exhibited significantly decreased CL/F, and those with *1/*27, *1/*29, *1/*40, and *1/*41 genotype presented similar CL/F value. When expressed in COS-7 cells, the CYP2C9 variants showed similar pattern to the results in clinical study. The study suggests that, besides two typical defective alleles, *3 and *13, seven CYP2C9 allelic variants (*16, *19, *34, *42, *45, *46, and *48) cause defective effects on the enzymatic activities both in vitro and in vivo. In clinic, patients with these defective alleles should be paid close attention to.

Impact of CYP2C9 polymorphism found in the Chinese population on the metabolism of propofol in vitro.

The microsomal CYP2C9 alleles involved in the biotransformation of propofol, a widely used anesthetic agent, were investigated in vitro. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for propofol 4-hydroxylation were determined in recombinant human P450s CYP2C9 microsomes from Sf21 insects cells carrying CYP2C9*1 and other variants. Some of the variants showed decreased enzyme activity compared with the wild type, as previously reported. Two variants (CYP2C9*36 and *56) were found substantially to increase intrinsic clearance relative to the wild type variant. Most variants significantly (p<0.05) decreased intrinsic clearance of propofol compared with the wild type, except *11, *47, and *54. This study is the first to report these rare alleles for propofol metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles for propofol metabolism in vivo.

Effects of methoxychlor and 2,2-bis ( p -hydroxyphenyl)-1,1,1-trichloroethane on cytochrome P450 enzyme activities in human and rat livers.

Cytochrome P450 (CYP) enzymes are involved in the metabolism of endogenous and exogenous compounds. Human and rat liver microsomes were used to investigate the inhibitory 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 concentration (IC50) 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 IC50 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 CYP2D6 activity, with an IC50 value of 16.56 ± 0.69 µmol/l. Both chemicals had no effect on human CYP3A4 and rat CYP3A1 activity. In summary, MXC and HPTE are potent inhibitors of some human and rat CYPs.

In vitro and in vivo drug-drug interaction of losartan and glimepiride in rats and its possible mechanism.

BACKGROUND: Losartan and glimepiride are commonly used drugs to treat chronic diseases of hypertension and diabetes; they are both substrates of CYP2C9. The aim of the present study was to investigate the possible interaction of losartan and glimepiride both in vitro (rat liver microsomes) and in vivo (healthy Sprague-Dawley rats). METHODS: In rat liver microsomes, 1-10 µmol/l losartan and glimepiride were coincubated, and the inhibitory effect was analyzed. In the subsequent pharmacokinetic study, 15 healthy Sprague-Dawley rats received administrations of 5 mg/kg losartan or 1 mg/kg glimepiride or a coadministration. RESULTS: In the rat liver microsome system, glimepiride showed a slight inhibition of losartan at concentrations of 1-10 µmol/l, whereas losartan exhibited no inhibitory effect on glimepiride. In vivo, glimepiride did not modify the plasma concentration of losartan and its metabolite E-3174. The alteration of an increased AUC and Cmax was observed in the pharmacokinetic parameters of glimepiride and hydroxy glimepiride. CONCLUSIONS: Glimepiride did not affect losartan pharmacokinetics in rats, while losartan potently altered glimepiride metabolism; this result was inconsistent with the in vitro outcome. The mechanism requires further investigation. In clinical settings, attention should be paid to the interaction of these two drugs in the human body as well as the possible adverse reactions of glimepiride.

Effect of CYP2C9 genetic polymorphism on the metabolism of flurbiprofen in vitro.

CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4'-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo.

Effects of cytochrome P450 2C9 polymorphism on bosentan metabolism.

Cytochrome P450 (P450) 2C9 is an important member of the P450 enzyme superfamily, with 58 CYP2C9 allelic variants previously reported. Genetic polymorphisms of CYP2C9 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to assess the catalytic activities of 38 human CYP2C9 alleles, including 24 novel alleles (*36-*60) found in the Han Chinese population, toward bosentan (BOS) in vitro. Insect microsomes expressing the 38 CYP2C9 alleles were incubated with 10-625 µM bosentan for 30 minutes at 37°C and terminated by cooling to -80°C immediately. BOS and hydroxyl bosentan, the major metabolite of BOS, were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry system. Thirty-eight defective alleles can be classified into three categories according to the relative clearance value compared with wild type: nine alleles exhibited significantly increased intrinsic clearance values (Vmax/Km) compared with the wild type (1.5-fold-∼4.9-fold relative clearance); nine alleles exhibited significantly reduced intrinsic clearance values compared with the wild type (0.6-28.9% relative clearance). The remaining 20 alleles exhibited no significant difference (1-fold) in enzyme activity compared with the wild type. These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2C9 alleles when administering BOS in the clinic. This is the first report of all these rare alleles for BOS metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles.

A systems-level approach to understanding transcriptional regulation by p53 during mammalian hibernation.

Presumably to conserve energy, many mammals enter into hibernation during the winter. Homeostatic processes such as transcription and translation are virtually arrested. To further elucidate transcriptional regulation during hibernation, we studied the transcription factor p53. Here, we demonstrate that changes in liver mRNA and protein concentrations of known regulators of p53 are consistent with activation. p53 mRNA and protein concentrations are unrelated. Importantly, p53 protein concentration is increased ~2-fold during the interbout arousal that punctuates bouts of torpor. As a result, both the interbout arousal and the torpid state are characterized by high levels of nuclear-localized p53. Chromatin immunoprecipitation assays indicate that p53 binds DNA during the winter. Furthermore, p53 recruits RNA polymerase II, as indicated by nuclear run-on data. However, and consistent with previous data indicating an arrest of transcriptional elongation during torpor, p53 'activity' does not result in expected changes in target gene transcripts. These data demonstrate the importance of using a systems level-approach in understanding a complex phenotype such as mammalian hibernation. Relying on interpretations of data that are based on steady-state regulation in other systems may be misleading in the context of non-steady-state conditions such as torpor.

Effect of 36 CYP2C9 variants found in the Chinese population on losartan metabolism in vitro.

Abstract 1. CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, with 57 CYP2C9 allelic variants being previously reported. Among these variants, we recently identified 21 novel alleles (*36-*56) in the Han Chinese population. The aim of this study was to assess the catalytic activities of 36 CYP2C9 variants found in the Chinese population toward losartan in vitro. 2. Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.5-25 µM losartan for 30 min at 37 °C. Next, the products were extracted, and signal detection was performed using high-performance liquid chromatography. 3. Compared with wild-type CYP2C9.1, the intrinsic clearance (Vmax/Km) values of all variants except for CYP2C9.56 were significantly altered. One variant exhibited markedly increased values (>250%), whereas 33 variants exhibited significantly decreased values (from 20 to 96%) due to increased Km and/or decreased Vmax values. 4. These findings suggest that more attention should be paid to subjects carrying these infrequent CYP2C9 alleles when administering losartan in the clinic.