CYP2C9 Expression Research Articles

CYP2C19 gene polymorphisms among Moroccan patients with breast cancer disease: A case-control study

BackgroundCytochrome P450 2C19 (CYP2C19) enzyme is implicated in the metabolism pathway of many exogenous and endogenous substrates, including estrogens. The expression and activity of CYP2C19 are influenced by its genetic variability. Therefore, functional polymorphisms of CYP2C19 gene are expected to affect the catabolism of estrogens and consequently are associated with susceptibility to develop breast cancer. AimsUsing a case-control study, we investigated CYP2C19 polymorphisms: CYP2C19*2(rs4244285), CYP2C19*3(rs4986893) and CYP2C19*17(rs12248560) in Moroccan Breast Cancer patients compared to healthy women controls. We also tested association of these polymorphisms with several characteristics of these patients. MethodsCYP2C19 polymorphisms were genotyped in 440 subjects, including 220 histologically confirmed Breast Cancer patients and 220 healthy women controls. For each polymorphism, association between genotypes and Breast Cancer risk was evaluated as odds ratios with 95% confidence intervals adjusted for age and menopausal status. Furthermore, association of each CYP2C19 polymorphisms with tumor characteristics of Breast Cancer cases was investigated. ResultsNone of the CYP2C19 variants was significantly associated with Breast Cancer risk. However, carriers of CYP2C19*3 allele A showed an association with Triple-Negative Breast Cancer subtypes (P=0.02). ConclusionOur results highlighted a possible implication of CYP2C19*3 polymorphism and triple negative Breast Cancer through an unidentified mechanism. Further studies with more triple negative samples are needed to support this finding.

A preliminary study of pharmacogenetic biomarkers for individuals with autism and gastrointestinal dysfunction

BackgroundAutism Spectrum Disorder (ASD) symptoms are commonly treated with a variety of pharmaceuticals which can have adverse side effects. A study of pharmacogenetic biomarkers for ASD medications and association with gastrointestinal (GI) dysfunction symptoms was conducted in individuals diagnosed with autism and/or GI dysfunction to provide further information on the genetic risk in relation to treatment effectiveness. MethodsA total of sixty participants were recruited, 10 with autism and GI dysfunction, 21 with GI dysfunction (without autism) and 29 without autism or GI dysfunction (typical controls). Buccal cell samples were collected and sequenced. A GI dysfunction questionnaire which included questions regarding prescription of medications associated with treating ASD symptoms was provided to the participants. To calculate Odds Ratios and compare the average of risk allele expression frequency of the SNPS being investigated, the sequencing and questionnaire data were analyzed using the epiR package and Welch Two Sample T-tests, respectively, using the R statistics program. The Bonferroni correction was utilized to correct for multiple comparisions. ResultsPeople in the autism group were more likely to express the risk alleles for the Cytochrome P450 family 2 subfamily C member 9 rs1057910 and Solute Carrier family 6, member 2 rs3785143 SNPs; however, after the Bonferroni correction these findings were not statistically significant (CYP2C9 rs1057910, P = 0.074; SLC6A2, rs3785143, P = 0.4218). ConclusionsFurther research is warranted to reveal the potential use of CYP2C9 and SLC6A2 SNP expression as pharmacogenetic biomarkers to determine the most appropriate medication for individuals with autism and/or GI dysfunction.

Deregulating the CYP2C19/Epoxy-Eicosatrienoic Acid-Associated FABP4/FABP5 Signaling Network as a Therapeutic Approach for Metastatic Triple-Negative Breast Cancer.

Recurrence and metastasis are the main causes of triple-negative breast cancer (TNBC) mortality. On the basis of our clinical cohorts and integrative omics analyses, we hypothesized that understanding the interplay between fatty acid binding protein (FABP) and epoxy-eicosatrienoic acid (EET) driven metastatic progression can uncover a new opportunity for TNBC intervention. In this study, the biological relevance of increased protein expression of CYP2C19, FABP4, and FABP5 in TNBC tumors and in the TNBC cell line (MDA-MB-231), as well as its highly metastatic lung seeking variant (LM6) were delineated from publicly available datasets, shRNA-mediated knockdown, EET supplementation, cancer and stromal cell co-cultures, and an orthotopic and resection xenograft tumor mouse model. We found that the high expression levels of CYP2C19 and FABP4 and FABP5 are critical in TNBC metastatic transformation and stromal cell interactions. Furthermore, EET-associated nuclear translocation of FABP4 and FABP5 and nuclear accumulation of SREBP-2 or PPAR-γ influence TNBC cell proliferation, migratory transformation, and distal metastasis priming. Most notably, we uncovered novel bioefficacy and modes of action of the anticancer drug doxorubicin and a phytogalactolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG), which effectively attenuated TNBC recurrence and lung metastasis through deregulating the FABP/EET dynamics and levels. This study, therefore, introduces a novel approach to combating TNBC by targeting the FABP/EET/CYP-associated metastatic signaling network.

Effects of baicalin on pharmacokinetics of florfenicol and mRNA expression of CYP1A2, CYP2C11, CYP3A1, UGT1A1, MDR1, and ABCC2 in rats

Background: The traditional Chinese medicine Scutellariae radix is often used in combination with antibiotics, such as florfenicol, in Chinese veterinary medicine. Baicalin (5, 6, 7-trihydroxyflavone-7-β-D-glucuronide) is the main active constituent of S. radix. The effects of baicalin on the pharmacokinetics of florfenicol are not known. Thus, we have studied the effects of baicalin on florfenicol pharmacokinetics and the mRNA expression of drug-metabolizing enzymes/efflux transporters in rats. Materials and Methods: Sprague-Dawley rats were given baicalin (50 mg/kg BW or 100 mg/kg BW) or 0.9% sodium chloride solution by intragastric administration for 7 days. They were then fed florfenicol by intragastric administration (25 mg/kg BW) on the 8th day. Blood samples were collected at various times, and the plasma concentrations of florfenicol were estimated with ultra-high performance liquid chromatography. The mRNA expression levels of cytochrome P450 (CYP) CYP1A2, CYP2C11, CYP3A1, UDP-glucuronosyltransferase 1A (UGT1A), multidrug resistance 1, and ATP-binding cassette C2 (ABCC2) in the liver and jejunum were analyzed with a real-time polymerase chain reaction. Results: The area under the concentration-time curve from zero to infinity, mean residence time from zero to infinity, elimination half-life, and peak concentration of florfenicol were significantly increased and the apparent volume of distribution fraction of the dose absorbed and plasma clearance fraction of the dose absorbed were significantly decreased by baicalin; the mRNA expression levels of CYP1A2, CYP2C11, CYP3A1, UGT1A1, and ABCC2 were down-regulated by baicalin. Conclusion: Baicalin affected the pharmacokinetics of florfenicol in rats, increased the plasma concentration and residence time of florfenicol, probably by decreasing the mRNA expression levels of CYP1A2, CYP2C11, CYP3A1, UGT1A1, and ABCC2 in the liver and jejunum.

Copper (I)-Nicotinate Promotes Gene Expression of CYPs that Produce M1 and Q1 in Aflatoxicosed Rats

Aflatoxin-contaminated food poses a serious risk to both human and animal health. Copper (1)-nicotinate (Cu+-nicotinate) complex potentiated the prophylactic effect against chronic aflatoxicosis in the experimental animals through the synthesis of less toxic metabolites M1 and Q1 which are easily excreted in urine. To investigate the action of the safety Cu+-nicotinate complex on gene expression of Cytochrome 450 (CYP450) system, the liver tissue samples of orally administered rats for 3 weeks with aflatoxin B1 (AFB1; 30 μg/kg body weight), with and without association of the copper complex (400 μg/kg body weight) were assayed for their gene expression of CYP450 families including 1A2, 3A2 and 2C11 as well as histopathological examination of the hepatic tissue samples was performed. The obtained data denoted that the Cu+-nicotinate complex significantly reduced gene expression of CYP2C11 and CYP3A2 that enhancing the most toxic epoxide metabolite. On the contrary, this complex enhanced the expression of CYP1A2 that synthesize the less toxic metabolite M1 and Q1. The histopathological examination mostly confirmed such observation as the signs of aflatoxicosis were absent in Cu+-nicotinate-treated group. Consequently, it could be predicted that the Cu+-nicotinate complex may be medically used as an inhibitory food additive agent against exposure of aflatoxicosis in the intact animals since the complex contains the copper and nicotinic acid, the two daily required biochemical elements for sustaining live in healthy conditions.

Effects of Naringin on the Activity and mRNA Expression of CYP Isozymes in Rats

Naringin (NRG) is a common dietary flavonoid in citrus fruits and has been documented to possess multiple pharmacological activities, including anti-oxidant, anti-inflammatory, and neuroprotective effects. Naringin is frequently consumed in combination with common clinical drugs. To date, the effects of NRG on cytochrome P450 enzymes have not been fully investigated yet. In this study, the activities of hepatic CYP1A2, CYP2D2, CYP2C9, CYP2C19, and CYP2E1 in rats after the continuous oral administration of NRG (50 and 500 mg/kg) were evaluated using cocktail probe-drug method. The concentrations of 5 probe drugs (phenacetin, dextromethorphan, diclofenac sodium, omeprazole, and chlorzoxazone) in rat plasma were simultaneously determined with a validated HPLC-MS/MS (high performance liquid chromatography-tandem mass spectrometry) method and then used to calculate corresponding pharmacokinetic parameters. Compared with the control group, the AUC(0- t), AUC(0-∞), t 1/2, and C max of each probe drug in treatment groups showed no significant differences. Meanwhile, fluorescence quantitative polymerase chain reaction (FQ-PCR) analysis revealed that NRG did not significantly affect the mRNA expressions of genes CYP1a2, CYP2d2, CYP2c6, CYP2c11, and CYP2e1 in rat liver. Based on these results, it could be concluded that NRG showed no significant effects on the activities and mRNA expressions of tested CYP450 in rats.

Dual Roles for the TSPYL Family in Mediating Serotonin Transport and the Metabolism of Selective Serotonin Reuptake Inhibitors in Patients with Major Depressive Disorder.

We previously reported that testis‐specific Y‐encoded‐like protein (TSPYLs) are transcription regulators for CYP3A4, CYP2C9, and CYP2C19. Here, we observed dual roles for TSPYLs in mediating serotonin transport and the metabolism of selective serotonin reuptake inhibitors (SSRIs) in patients with major depressive disorder (MDD). The widely prescribed SSRIs, citalopram, and escitalopram are metabolized mainly by CYP2C19. The TSPYL1 rs3828743 single nucleotide polymorphism (SNP), which decreases its suppression of CYP2C19 expression, was associated with rapid escitalopram metabolism and worse treatment response in the Mayo PGRN‐AMPS clinical trial. We also found that TSPYLs can regulate expression of the serotonin transporter protein, SLC6A4, and, in turn, serotonin transport into cells. The SNPs in tight linkage disequilibrium with the TSPYL1 rs10223646 SNP were significantly correlated with baseline severity of depression in patients with MDD in the Sequenced Treatment Alternatives to Relieve Depression and International SSRI Pharmacogenomics Consortium clinical trials. Our findings suggest that genetic variation in TSPYL genes may be novel indicators for baseline severity of depression and SSRI poor response.

Methylation of adenosine at the N6 position post-transcriptionally regulates hepatic P450s expression

The methylation of adenosines at the N6 position (m6A formation) is the most prevalent type of RNA modification in humans. This modification is mediated by methyltransferase like 3 (METTL3)-METTL14 complex, and the methyl group can be removed by RNA demethylases including fat mass and obesity-associated (FTO) and AlkB homolog 5. The formed m6A is recognized by reader proteins such as members of the YT521-B homology (YTH) family, resulting in changes in the splicing, nuclear export, and decay of RNA or translation. In this study, we examined the impact of m6A modification on the expression of drug-metabolizing P450 isoforms. By treatment with 3-deazaadenosine, an inhibitor of RNA methylation, CYP1A2, CYP2B6, and CYP2C8 levels were significantly increased (1.6-fold, 2.2-fold, and 2.7-fold, respectively) in HepaRG cells. In subsequent experiments, we focused on CYP2C8, which showed the largest increase. Consistent with the increase in the mRNA level, CYP2C8 protein level and activity were significantly increased by treatment with 3-deazaadenosine. The CYP2C8 expression levels and activities in HepaRG and Huh-7 cells were increased by knockdown of METTL3/14, whereas they were decreased by knockdown of FTO, suggesting that m6A modification downregulates CYP2C8 expression. With an RNA immunoprecipitation assay using an anti-m6A antibody, it was revealed that the adenosines in the 5′-UTR and the last exon of CYP2C8 are methylated in HepaRG cells and human liver samples. It was demonstrated that YTHDC2, which is known to degrade m6A-containing mRNA, downregulates CYP2C8 expression. In conclusion, we found a novel post-transcriptional regulation mechanism in which the YTHDC2 promotes CYP2C8 mRNA degradation via recognizing the m6A in CYP2C8 mRNA, which is installed by METTL3/14 and removed by FTO.

Down-regulation of SOX17, GATA4 and FoxA2 promotes differentiation potential of hepatocytes from human hematopoietic stem cells

The failure of regeneration of damaged liver in various end-stage liver diseases results in high morbidity and mortality. In this context, we have demonstrated the differentiation ability of human hematopoietic stem cells (HSCs) into hepatocytes. In this study, HSCs were isolated from a donor and cultured which exhibited the presence of CD34 and CD133 and absence of CD90 and CD73 markers. These CD34+ HSCs were induced for 21 days in hepatocyte differentiation medium (HDM). The obtained cells were characterized by immunocytochemical, immunofluorescence, western blot, qRT-PCR and flow cytometry analysis. Further, functional assays were done to accentuate the differentiated cells are hepatocytes. In HDM at day 6 differentiated cells showed the expression of definitive endodermal (DE) markers, SOX17, GATA4 and FoxA2 indicating the beginning of differentiation process. At day 21 the flow cytometry analysis showed 84.2 % positive to ALB-PE, 75.4 % positive to HNF4α-PE, and 77.3% positive to AFP-PE. Further, the qRT-PCR and western blot analysis presented prominent expression of hepatocyte-specific genes AAT, ALB, AFP, CK18, CK19, HNF4α, TFR2, and Hepcidin confirms the generation of hepatocytes in HDM. The ability of albumin secretion, urea production, glycogen storage, uptake of LDL, high ALDH enzyme activity describes the functionality of differentiated hepatocytes. Distinct expression of UGT1A1, CYP2B6, CYP2C9, CYP3A4, and CYP7A1 genes explains the ability to clear toxins and bilirubin as observed in normal hepatocytes. All these results indicate HSCs were differentiated into hepatocytes thus, autologous transplantation of HSCs could be a better option in the regeneration of the damaged liver.

PPTS Inhibits the TGF-β1-Induced Epithelial-Mesenchymal Transition in Human Colorectal Cancer SW480 Cells.

The current study investigates the inhibitory effects of Pulsatilla pentacyclic triterpenoid saponins extract (PPTS) on epithelial-mesenchymal transition (EMT) triggered by the transforming growth factor-β1 (TGF-β1) in human colorectal cancer SW480 cell line, further illustrates the possible mechanism of PPTS inhibition of growth and invasion from the perspective of EMT, and provides new theoretical support for the treatment of tumor by Chinese medicine. The SW480 cells were treated in groups: blank control, TGF-β1 (10 ng/mL), and varying concentrations of PPTS cotreated with TGF-β1-induced (10 ng/mL) groups. CCK8 was used to detect cell viability; transwell was applied to detect invasion ability, cell migration ability was also determined, ELISA and RT-qPCR were utilized for the determination of CYP3A, CYP2C9, CYP2C19, N-cadherin, and MMP-9 expression. Flow cytometry detection was applied to detect cell cycle and apoptosis. The results obtained have shown that PPTS can significantly inhibit the invasion and migration of tumors in SW480 cells and can also block the S phase in the cell cycle but may produce cytotoxicity in higher doses. The present research work provides substantial evidence that PPTS has a significant inhibitory effect on TGF-β1-induced EMT in SW480 cells and it also promotes apoptosis.

Pharmacokinetic interference of doxorubicin with tolbutamide due to reduced metabolic clearance with increased serum unbound fraction in rats.

The study examined the effect of doxorubicin (DOX) on the hepatic expression of CYP2C and its activity for metabolizing tolbutamide (TB), a specific CYP2C substrate, in rats and whether the pharmacokinetics of tolbutamide were altered by doxorubicin exposure. The expression level of hepatic CYP2C11 was depressed 1day after doxorubicin administration (day 1), and this effect on CYP2C11 was augmented on day 4. However, the expression level of hepatic CYP2C6 remained unchanged. The activity of tolbutamide 4-hydroxylation in hepatic microsomes was decreased with time following doxorubicin administration. Regarding the enzyme kinetic parameters for tolbutamide 4-hydroxylation on day 4, the maximum velocity (Vmax ) was significantly lower in the DOX group than that in the control group, while the Michaelis constant (Km ) was unaffected. On pharmacokinetic examination, the total clearance (CLtot ) of tolbutamide on day 4 was increased, despite the decreased metabolic capacity. On the other hand, the serum unbound fraction (fu ) of tolbutamide was elevated with a reduced serum albumin concentration in the DOX group. Contrary to CLtot , CLtot /fu , a parameter approximated to the hepatic intrinsic clearance of unbound tolbutamide, was estimated to be significantly reduced in the DOX group. These findings indicate that the metabolic capacity of CYP2C11 in the liver is depressed time-dependently by down-regulation after doxorubicin exposure in rats, and that the decreased enzyme activity of TB 4-hydroxylation in hepatic microsomes reflects the pharmacokinetic change of unbound tolbutamide, not total tolbutamide, in serum.

Strain‐selective efficacy of sacubitril/valsartan on carotid fibrosis in response to injury in two inbred mouse strains

Sacubitril/valsartan (Sac/val) is more effective than valsartan in lowering BP and mortality in patients with heart failure. Here, we proposed that Sac/val treatment would be more effective in preventing pathological vascular remodelling in 129X1/SvJ (129X1), than in C57BL/6J (B6) inbred mice. Sac/val (60mg·kg-1 ·day-1 ) and valsartan (27mg·kg-1 ·day-1 ) were given as prophylactic or therapeutic treatments, to 129X1 or B6 mice with carotid artery ligation for 14 days. Blood flow was measured by ultrasound. Ex vivo, carotid tissue was analysed with histological and morphometric techniques, together with RNA sequencing and gene ontology. Sac/val was more effective than valsartan in lowering BP in 129X1 compared with B6 mice. Liver expression of CYP2C9 and plasma cGMP levels were similar across treatments. A reduction in carotid thickening after prophylactic treatment with valsartan or Sac/val also resulted in significant arterial shrinkage in B6 mice. In 129X1 mice, Sac/val and prophylactic treatment with valsartan had no effect on carotid thickening but preserved carotid size. BP lowering significantly correlated with a decline in carotid stiffness (R2 =.37, P=.0096) in 129X1 but not in B6 mice. The gene expression signature associated with hyalurononglucosaminidase activity was down-regulated in injured arteries after both regimens of Sac/val only in 129X1 mice. Administration of Sac/val but not valsartan significantly reduced deposition of hyaluronic acid and carotid fibrosis in 129X1 mice. These results underscore the importance of the genetic background in the efficacy of the Sac/val on vascular fibrosis.

Long-term culture and characterization of patient-derived primary hepatocytes using conditional reprogramming.

Cultivation of primary human hepatocytes (PHHs) often faces obstacles including failure of long-term in vitro culture, weak proliferation ability, rapid loss of liver-specific function and morphology, and tendency of fibrosis. Previous research focused on immortalization methods, such as telomerase and viral, to culture immortalized primary human hepatocytes, which may lose some of the normal properties. However, non-immortalized PHHs often fail to maintain long-term viability and functionality. These highlight the urgent need for developing new culture strategy for PHHs. In the present study, we isolated PHHs from fresh human liver tissues representing different liver diseases and age groups. We used conditional reprogramming, without permanent immortalization, for long-term in vitro primary human hepatocytes cultivation and characterization. For functional characterization, we assessed CYP3A4, 1A1 and 2C9 activities and measured the mRNA expression of albumin, s100a4, krt8, krt18, cyp1a1, cyp3a4, cyp2b6, cyp2c8, cyp2c9, and cyp2d6. Additionally, we compared the DNA fingerprint of the cells against their original liver tissues using short tandem repeat (STR) analysis. We found that PHHs-derived from young patients can survive for more than three months, while the lifespan of primary human hepatocytes derived from adult patients ranges from two to three months, which is longer than most commercial primary hepatocytes. Importantly, the cells at early passages retain strong CYP3A4, 1A1 and 2C9 activities and the DNA fingerprints are identical with their original tissues. Through conditional programming, we achieved, for the first time, a high level of success rate in the long-term in vitro cultivation of primary human hepatocytes-derived patients representing diverse liver disease. Moreover, the conditional programming cell culture technology reported in this paper requires neither co-culture with additive cells, nor complex and expensive components, such as collagen sandwich or spheroid culture. We thus believe that the patient-derived PHHs cultivation using conditional programming may provide a viable and valuable cell model to study liver disease-related mechanisms. Impact statement Commercially available primary human hepatocytes rapidly lose their proliferative ability and liver-specific functions over a few cultivation days. The demand for pharmaceutical toxicity screening and liver disease research requires the development of long-term primary hepatocyte culture methods. This manuscript addresses this challenge by introducing for the first time successful long-term in vitro cultivation of primary human hepatocytes from a range of liver transplantation patients using conditional reprogramming technique. The beauty of this technique is that it is not a permanent immortalization and does not require co-culture with additive cells. The primary human hepatocytes retain proliferative capacity, genetic stability, and hepatocyte-specific functions at early passages. In view of these, we believe that scientists and researchers will benefit from using these highly valuable cell models to study diverse liver diseases.

Losartan effects on liver cytochromes CYP3A, CYP2C and CYP2E1 functioning at metabolic syndrome in young and adult rats

CYP450-dependent interactions and toxicological consequences of hypoglycemic and antihypertensive drugs used in treatment of children with metabolic syndrome (MS) remained unclear. Our aim was to carry out a complex estimation of metabolic syndrome and losartan mediated changes in CYP3A, CYP2C, CYP2E1 mRNA expression, corresponding marker enzymes activities, liver antioxidant system and lipid peroxidation parameters of adult and pubertal rats. Wistar albino male rats of two age categories (young animals of 21 days age (50–70 g) and adults (160–180 g) were divided into 6 groups (6 animals in each): 1 – Control 1 (intact young rats); 2 – Control 2 (intact adult rats); 3 –young rats with MS; 4 – adult rats with MS; 5 – young rats with MS+losartan; 6 – adult rats with MS+ losartan. The metabolic syndrome model was induced by full replacement of drinking water with 20% fructose solution (200 g/l). After 60 days of MS modeling, investigation of rat liver CYP3A, CYP2C, CYP2E1 mRNA expression, their marker enzymes activities, lipid peroxidation parameters were carried out. Losartan administration caused increase of CYP3A, CYP2C and CYP2E1 mRNA expression rates in both age groups. Marker enzymes, glutathione transferase and reductase rates were normalized only in adult rats. In group of pubertal animals losartan administration led to CYP3A and CYP2C marker enzymes activities normalization. Liver reduced glutathione contents remained decreased in both age groups. Thus, losartan demonstrates some age-dependent effectiveness towards normalization of CYP450 isoforms expression rates, p-nitrophenol hydroxylase, erythromycin-N-demethylase and diclofenac hydroxylase activities, but not glutathione system and lipid peroxidation rates.

Correction to "RNA Editing Enzymes Modulate the Expression of Hepatic CYP2B6, CYP2C8, and Other Cytochrome P450 Isoforms".

Correction to "RNA Editing Enzymes Modulate the Expression of Hepatic CYP2B6, CYP2C8, and Other Cytochrome P450 Isoforms".

Reversal Effect of Pioglitazone on Multidrug Resistance in K562/ADR Cells and Its Mechanism

To explore the reversal effect of pioglitazone (PIO) on multidrug resistance in K562/ADR cells and its mechanism. The proliferation inhibition rate, half inhibition concentration (IC50) and drug-resistance reversal multipe were detected and the curve of proliferation inhibition rate was drawn by MTT assay, the transcription of PPARγ, CYP2C8 and CYP2J2 genes was detected by RT-PCR; the expression of PPARγ, CYP2C8 and CYP2J2 proteins was detected by Western blot. The IC50 of PIO on K562 and K562/ADR cells for 60 h was 326.7 μmol/L and 349.1 μmol/L respectively. The reversal multiple of 30 μmol/L PIO on ADR-resistance of K562/ADR cells was 6.4. After treatment of K562/ADR cells with PIO, the transcription of CYP2C8 and CYP2J2 and the protein expression of CYP2C8 and CYP2J2 significantly decreased, the transcription of PPARγ gene and the expression of PPARγ protein were not changed. Pioglitazone can reverse the adriamycin-resistance in K562/ADR cells that is closely related to the decrease of protein expression of CYP2C8 and CYP2J2. Pioglitazone is an effective multidrug resistance reversal agent for tumors.

Effect of "Jiaji" (EX-B2)-electroacupuncture preconditioning on expression of myocardial cytochrome P450 enzyme in rats with acute myocardial ischemia-reperfusion injury

To investigate the effect of "Jiaji"(EX-B2)-electroacupuncture(EA) preconditioning on structural changes of myocardium and expression of cytochrome P450 (CYP450) enzymes in acute myocardial ischemia-reperfusion injury (MIRI) rats. Sixty male Wistar rats were randomly divided into sham operation, model, EX-B2, Neiguan (PC6), Yanglingquan(GB34) and Quchi (LI11) groups (n＝10 in each group). The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min, followed by reperfusion for 30 min. EA preconditioning (2 Hz/100 Hz, 1 mA) was respectively applied to EX-B2, PC6, GB34, and LI11 for 30 min, once daily for 7 days. Morphological changes of myocardium were observed by H．E. staining. The ultrastructure of myocardial cells was observed by transmission electron microscope (TEM). The protein expression levels of myocardial CYP450 (CYP1A1, CYP2B2, CYP2C11 and CYP4A2) were detected by Western blot. In MIRI rats, myocardial pathological changes as swollen, disorganized arrangement, and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope, and focal myofilament degeneration, dissolution and necrosis with interstitial edema, vague mitochondria with swelling and vacuolation, ruptured and disorganized arrangement of crests under TEM were found in the ischemic area, which was obviously milder in rats of both EX-B2 and PC6 groups. After modeling, the expression of myocardial CYP1A1 and CYP2B2 protein were significantly up-regulated in the model group relevant to the sham operation group (P<0.05). In EA preconditioning groups, the expression levels of CYP1A1 and CYP4A2 proteins in the EX-B2, PC6 and GB34 groups were obviously down-regulated (P<0.01), and those of CYP2B2 and CYP2C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group (P<0.01). Compared with the EX-B2 group, the expression levels of CYP1A1, CYP4A2 proteins in the LI11 group were significantly up-regulated (P<0.05), and CYP2B2, CYP2C11 proteins were significantly down-regulated (P<0.05). No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2C11 and CYP4A2 proteins, between the LI11 and model groups in the expression of CYP1A1, CYP2B2, CYP2C11 and CYP4A2, between the GB34 and model groups in the expression of CYP2B2, CYP2C11 (all P>0.05)．. EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450 ω-hydroxylase CYP1A1 and CYP4A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2B2 and CYP2C11 proteins in acute MIRI rats, thus playing a role in myocardial protection.

RNA Editing Enzymes Modulate the Expression of Hepatic CYP2B6, CYP2C8, and Other Cytochrome P450 Isoforms.

A-to-I RNA editing, the most frequent type of RNA editing in mammals, is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. Recently, we found that there is a large interindividual variation in the expression of ADAR1 protein in the human livers. In this study, we investigated the possibility that A-to-I RNA editing may modulate the expression of cytochrome P450 (P450), causing interindividual variations in drug metabolism potencies. We found that knockdown of ADAR1 or ADAR2 in HepaRG cells resulted in the decreased expression of CYP2B6 and CYP2C8 mRNA and protein. Knockdown of ADARs significantly decreased the stability of CYP2B6 mRNA but not CYP2C8 mRNA. Luciferase assays revealed that the 3'-untranslated region of CYP2B6 and the promoter region of CYP2C8 would be involved in the decrease in their expression by the knockdown of ADARs. We found that the decreased expression of the hepatocyte nuclear factor 4α (HNF4α) protein by the knockdown of ADARs was one of the reasons for the decreased transactivity of CYP2C8. The mRNA levels of other P450 isoforms, such as CYP2A6, 2C9, 2C19, 2D6, and 2E1, which are known to be regulated by HNF4α, were also decreased by ADAR1 or ADAR2 knockdown. Exceptionally, the CYP3A4 mRNA level was significantly increased by ADAR1 knockdown, suggesting the possibility that the change could be due to the change in the expression or function of other regulatory factors. In conclusion, this study revealed that the RNA editing enzymes ADAR1 and ADAR2 are novel regulatory factors of P450-mediated drug metabolism in the human liver.

The HNF1α-Regulated LncRNA HNF1α-AS1 Is Involved in the Regulation of Cytochrome P450 Expression in Human Liver Tissues and Huh7 Cells.

Expression of cytochrome P450s (P450s) is regulated by epigenetic factors, such as DNA methylation, histone modifications, and noncoding RNAs through different mechanisms. Among these factors, long noncoding RNAs (lncRNAs) have been shown to play important roles in the regulation of gene expression; however, little is known about the effects of lncRNAs on the regulation of P450 expression. The aim of this study was to explore the role of lncRNAs in the regulation of P450 expression by using human liver tissues and hepatoma Huh7 cells. Through lncRNA microarray analysis and quantitative polymerase chain reaction in human liver tissues, we found that the lncRNA hepatocyte nuclear factor 1 alpha antisense 1 (HNF1α-AS1), an antisense RNA of HNF1α, is positively correlated with the mRNA expression of CYP2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 as well as pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Gain- and loss-of-function studies in Huh7 cells transfected with small interfering RNAs or overexpression plasmids showed that HNF1α not only regulated the expression of HNF1α-AS1 and P450s, but also regulated the expression of CAR, PXR, and aryl hydrocarbon receptor (AhR). In turn, HNF1α-AS1 regulated the expression of PXR and most P450s without affecting the expression of HNF1α, AhR, and CAR. Moreover, the rifampicin-induced expression of P450s was also affected by HNF1α and HNF1α-AS1. In summary, the results of this study suggested that HNF1α-AS1 is involved in the HNF1α-mediated regulation of P450s in the liver at both basal and drug-induced levels.

Effect of the metabolic capacity in rat liver S9 on the positive results of in vitro micronucleus tests.

A high incidence of positive results is obtained with in vitro genotoxicity tests, which do not correlate with the in vivo negative results in many cases. To address this issue, the metabolic profile of rat liver 9000 × g supernatant fraction (S9) pretreated with phenobarbital (PB) and 5,6-benzoflavone (BNF) was characterized. Furthermore, the in vitro micronucleus tests of 10 compounds were performed with PB-BNF-induced rat S9. PB-BNF increased cytochrome P450 (CYP) activity and CYP1A1, CYP1A2, CYP2B1/2, CYP2C6, CYP3A1, and CYP3A2 expression in rat S9, whereas it decreased CYP2C11 and CYP2E1 expression. PB-BNF-induced S9 enhanced the micronucleus induction (MI) of benzo[a]pyrene (BaP), cyclophosphamide (CPA), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), which are metabolized by CYP1A1, CYP2C6, and CYP1A2, respectively. In contrast, coumarin and chlorpheniramine showed MI with PB-BNF-induced S9 despite the fact that they show negative results in the in vivo studies. Furthermore, diclofenac, piroxicam, lansoprazole, and caffeine showed MI regardless of the enzyme induction by PB-BNF, whereas phenacetin did not show MI. These results indicate that PB-BNF-induced rat S9 is effective in detecting the genotoxic potential of promutagens, such as BaP, CPA, and PhIP, but not of coumarin and chlorpheniramine, probably due to the differences in the in vitro and in vivo metabolic profile and its exposure levels of the drugs.