A CYP2B6-humanized mouse model and its potential applications

CYP2B6 is a human microsomal cytochrome P450 enzyme with broad substrate selectivity. CYP2B6 is the only functional member of the human CYP2B gene subfamily, which differs from the situation in rodents, such as mouse, where multiple functional Cyp2b genes are expressed. Recent studies with Cyp2b knockout or knockdown mouse models have yielded insights into the in vivo roles of mouse CYP2B enzymes in drug disposition and xenobiotic toxicity. A CYP2B6-humanized mouse model (CYP2A13/2B6/2F1-transgenic/Cyp2abfgs-null), which expresses human CYP2B6 in the liver, and human CYP2A13 and CYP2F1 in the respiratory tract, but not any of the mouse Cyp2b genes, has also been established. In the CYP2B6-humanized mouse, the CYP2B6 transgene is expressed primarily in the liver, where it was found to be active toward prototype CYP2B6 substrate drugs. The regulatory elements of the CYP2B6 transgene appear to be compatible with mouse nuclear receptors that mediate CYP2B induction. Therefore, the CYP2B6-humanized mouse is a valuable animal model for studying the impact of CYP2B6 expression or induction on drug metabolism, drug efficacy, drug-drug interaction, and drug/xenobiotic toxicity. In this mini-review, we provide a brief background on CYP2B6 and the Cyp2b-knockout and CYP2B6-humanized mice, and discuss the potential applications and limitations of the current models.     

C. elegans DAF-12, Nuclear Hormone Receptors and human longevity and disease at old age

In Caenorhabditis elegans, DAF-12 appears to be a decisive checkpoint for many life history traits including longevity. The daf-12 gene encodes a Nuclear Hormone Receptor (NHR) and is member of a superfamily that is abundantly represented throughout the animal kingdom, including humans. It is, however, unclear which of the human receptor representatives are most similar to DAF-12, and what their role is in determining human longevity and disease at old age. Using a sequence similarity search, we identified human NHRs similar to C. elegans DAF-12 and found that, based on sequence similarity, Liver X Receptor A and B are most similar to C. elegans DAF-12, followed by the Pregnane X Receptor, Vitamin D Receptor, Constitutive Andosteron Receptor and the Farnesoid X Receptor. Their biological functions include, amongst others, detoxification and immunomodulation. Both are processes that are involved in protecting the body from harmful environmental influences. Furthermore, the DAF-12 signalling systems seem to be functionally conserved and all six human NHRs have cholesterol derived compounds as their ligands. We conclude that the DAF-12 signalling system seems to be evolutionary conserved and that NHRs in man are critical for body homeostasis and survival. Genomic variations in these NHRs or their target genes are prime candidates for the regulation of human lifespan and disease at old age.     

Lipopolysaccharide down-regulates carbolesterases 1 and 2 and reduces hydrolysis activity in vitro and in vivo via p38MAPK-NF-κB pathway

Carboxylesterases constitute a class of enzymes that hydrolyze drugs containing such functional groups as carboxylic acid ester, amide, and thioester. Hydrolysis of many drugs is reduced in liver diseases such as hepatitis and cirrhosis. In this study, we have demonstrated, in vitro and in vivo, treatment with LPS decreased the expression of HCE1 and HCE2 and the capacity of hydrolytic activity. In HepG2 cells, the decreased expression by LPS occurred at both mRNA and protein levels. Both HCE1 and HCE2 promoters were significantly repressed by LPS, and the repression was comparable with the decrease in HCE1 and HCE2 mRNA, suggesting the transrepression is responsible for suppressed expression. Further study showed that both PDTC, a NF-κB inhibitor, and SB203580, a p38MAPK inhibitor, could abolish the repression of HCE1 and HCE2 mediated by LPS, but U0126, a selective ERK1/2 inhibitor, could not do so, suggesting the repression of HCE1 and HCE2 by LPS through the p38MAPK-NF-κB pathway. In addition, being pretreated with LPS, HepG2 cells altered the cellular responsiveness to ester therapeutic agents, including clopidogrel (hydrolyzed by HCE1) and irinotecan (hydrolyzed by HCE2). The altered cellular responsiveness occurred at low micromolar concentrations, suggesting that suppressed expression of carboxylesterases by LPS has profound pharmacological and toxicological consequences, particularly with those that are hydrolyzed in an isoform-specific manner. This study provides new insight into the understanding of the pharmacological and toxicological effects and the mechanisms for repressing drug metabolism enzymes in inflammation.     

Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines

BACKGROUND: Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors. METHODS: Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining. RESULTS: Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole. CONCLUSION: The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance.     

Natural substance rutin versus standard drug atorvastatin in a treatment of metabolic syndrome-like condition

BackgroundMetabolic syndrome is a cluster of metabolic risk factors. The clear causes of its development are not known yet and there is no comprehensive treatment of this disease. There is a trend to use natural substances in the treatment of various diseases, but their effects need to be well explored. We decided to test effect of rutin compared to the effect of the standard drug atorvastatin.MethodsAs a model of metabolic syndrome we used males of hypertriacylglycerolemic rats in combination with high-fat-high-fructose diet. Rutin (100 mg/kg) and atorvastatin (50 mg/kg) were administered orally daily for 5 weeks.ResultsWe determined biochemical parameters from blood: HDL-cholesterol, LDL-cholesterol, total cholesterol, triacylglycerols. Relaxation and contraction response of aorta was measured to determine vessel dysfunctions and possible predisposition to cardiovascular disease. The negative influence on cognitive functions could be associated with the development of metabolic cognitive syndrome. Therefore we aimed to monitor spatial memory by Morris water maze test. Both rutin and atorvastatin had a tendency to decrease levels of serum triacylglycerols, but only atorvastatin significantly reduced levels od LDL-cholesterol and increased HDL-cholesterol levels. Both compounds significantly reduced the phenylephrine-induced contractile response of the aorta and improved the relaxation response. Further, treated animals learned better compared to untreated rats in the Morris water maze.ConclusionBased on our results we can assume that atorvastatin and rutin had positive effect on spatial memory and vessel reactivity. Atorvastatin optimized lipid profile of blood serum.     

Improvac does not modify the expression and activities of the major drug metabolizing enzymes cytochrome P450 3A and 2C in pigs

In the present study, we investigated hepatic mRNA expression and activities of CYP3A and 2C in entire, surgically castrated and pigs vaccinated with Improvac. Additionally, we examined the mRNA expression of the two nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), known to regulate CYP3A and 2C mRNA expression, respectively. Activities of CYP3A and 2C were estimated as a rate of 7-benzyloxy-4-trifluoromethylcoumarin and 7-benzyloxyquinoline metabolism (CYP3A) and tolbutamide metabolism (CYP2C). We found no effect of Improvac treatment or surgical castration on either CYP3A or 2C activities. Similarly, the mRNA expressions of CYP3A29, 2C33 and PXR were not changed. CAR mRNA expression differed only between entire and surgically castrated male pigs (p=0.005), being greater in surgically castrated pigs. Our results indicated that neither CYP3A nor 2C are affected by Improvac.     

芒果苷上调HepG2细胞膜蛋白MRP3和核受体PXR、CPF表达

目的 体外HepG2细胞培养观察芒果苷(mangiferin)对多耐药相关蛋白3(multidrug resistance-associate protein 3, MRP3)和核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响.方法 用芒果苷刺激HepG2细胞72 h后,分别抽提细胞RNA、膜蛋白及核蛋白,采用半定量RT-PCR和蛋白免疫印迹检测膜转运蛋白MRP3和核受体PXR、CPF在转录与蛋白水平的表达变化.熊脱氧胆酸(ursodeoxycholic acid,UDCA)处理的HepG2细胞作为阳性对照、DMSO处理细胞为阴性对照.结果 芒果苷可显著刺激HepG2细胞膜转运蛋白MRP3的mRNA(比阴性对照组高3.0倍,P＜0.05)和蛋白(比阴性对照组高3.3倍,P＜0.05)表达,其作用强于UDCA.芒果苷也可明显上调核受体PXR [mRNA水平增高1.7倍(P＜0.05),蛋白水平增高3.7倍(P＜0.01)]、CPF[mRNA水平增高2.1倍(P＜0.05),蛋白水平增高4.9倍(P＜0.05)]的表达.结论 芒果苷刺激肝癌细胞HepG2细胞膜转运蛋白MRP3的表达上调可能与核受体PXR、CPF途径相关.     

CYP3A4和CYP286药物诱导剂体外筛选体系的建立

目的构建CYP酶药物诱导剂的体外筛选技术平台。方法利用双荧光素酶报告基因系统，将包含hPXR蛋白识别和结合调控元件的CYP3A4和286启动子序列插入报告基因上游，将表达载体和报告载体共转染HepG2细胞，用含有利福平或DMSO培养基培养48h后裂解进行双荧光素酶活性检测。结果经利福平处理的细胞裂解物荧光素酶比活性值与阴性对照组和溶剂组差异显著。结论本研究构建的CYP3A4和286体外筛选平台，可以有效地用于体外诱导剂的筛选。     

Therapeutic regulation of autophagy in hepatic metabolismOA

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ thatgoverns body energy metabolism. Autophagy’s role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. T...