Factors Influencing Regulation of CYP2B Expression

ABSTRACTCytochrome P450 (P450) enzymes participate in a wide array of metabolic reactions. Of these P450s, CYP2B subfamily plays animportant role in the metabolism of endogenous compounds and xenobiotics. CYP2B1 and CYP2B2 in rats and CYP2B9 and CYP2B10in mice are major CYP2B isoenzymes constitutively and inducible expressed. Their constitutive expression is sexually dimorphic,specifically more expression in male than female rats, and more in female than male mice. Recent studies have shown that regulation ofCYP2B expression is markedly influenced by not only various endogenous and exogenous compounds, but also age, sex, strain, andnutritional status. Regarding regulation of P450 expression in mouse liver, a C57BL/6 strain is one of the most suitable mouse modelsbecause of its marked response to CYP2B induction. The regulation of sexual dimorphism of CYP2B highly depends on numerousendogenous hormones including glucocorticoids, sex hormones and growth hormones. Adrenalectomy suggested that glucocorticoidsinduced CYP2B10 but simultaneously suppressed CYP2B9 expression in both sexes. β-estradiol (ES) up-regulated the expression ofCYP2B9, while testosterone showed reverse activity of ES. Hypophysectomy and the age-expression profile revealed that growthhormone (GH) exerts suppressive effect on regulation of CYP2B9 and CYP2B10 expression in the males, but only on CYP2B10 in thefemales. Xenobiotics, i.e., phenobarbital, dexamethasone, DDT (1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane), are exogenous factorsinfluencing the CYP2B expression. For example, phenobarbital and DDT induce both CYP2B9 and CYP2B10 while dexamethasonepredominantly induces CYP2B10, but simultaneously suppresses CYP2B9. Therefore, the factors that affect regulation of CYP2Bexpression should be thoroughly considered to eliminate their confounding effects, leading to accurate and precise outcome measures.Keywords: CYP2B, C57BL/6, sexual dimorphism, endogenous hormones, xenobioticsThai Pharm Health Sci J 2009;4(4):524-531

Cytochrome P450 expression-associated multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD) in HepG2 cells

Purpose: To establish a free fatty acid (FFA)-induced non-alcoholic fatty liver disease (NAFLD) model in HepG2 cells.Methods: HepG2 cells were incubated with 0.1, 1, or 5 mM oleic acid (OA) or palmitic acid (PA) for 24 h. Histological features were examined by oil-red-O staining. Expression levels of metabolic genes (peroxisome proliferator activated receptors α/γ, sterol regulatory element binding proteins 1a/1c, acetyl-CoA carboxylase, acyl-CoA oxidase, and fatty acid synthase), antioxidative genes (catalase and superoxide dismutases 1/2), and  cytochrome P450 genes (CYP1A2, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11) were determined by reverse transcription-real time polymerase chain reaction (RT-qPCR).Results: Intracellular lipid storage was observed in cells treated with 1 mM OA or PA while cell shrinkage was present at 5 mM concentrations of both. Expression of all metabolic genes were elevated by 1 mM PA and 5 mM OA and PA. Expression of all antioxidative genes were diminished by 5 mM OA whereas 5 mM PA only reduced superoxide dismutase-2 expression. Expression of CYP1A2, CYP2D6, and CYP3A4 genes were down-regulated by both FFAs, CYP2C19 was induced by PA, while CYP2E1 and CYP4A11 were up-regulated in a concentration-dependent manner.Conclusion: PA was the more potent steatogenic agent in an OA- or PA- induced NAFLD model in HepG2 cells. Increase in intracellular hepatic lipid and expression of metabolic genes, suppression of antioxidative genes, suppression of CYP1A2, CYP2D6, and CYP3A4, and induction of CYP2E1 andCYP4A11 correlated with the multiple-hit pathogenesis model of NAFLD. These findings suggest that PA-induced NAFLD model in HepG2 cells is a suitable in vitro model for studying novel therapeutic approaches to NAFLD treatment. Keywords: NAFLD, Multiple-hit pathogenesis, Free fatty acid, Oleic acid, Palmitic aci

Optimized models of xenobiotic-induced oxidative stress in HepG2 cells

Purpose: To evaluate the molecular impact of ethanol, sodium selenite, and tert-butyl hydroperoxide (TBHP) on oxidant-antioxidant balance in HepG2 cells to establish an optimized oxidative stress model of HepG2 cells. Methods: HepG2 cells were treated with ethanol (10 - 500 mM) and sodium selenite (1 - 10 µM) for 24 and 48 h and with TBHP (50 - 200 µM) for 3 and 24 h, respectively. Biomarkers for cellular injury, ie, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA), and for antioxidant system, i.e., superoxide dismutase (SOD), catalase (CAT), and total glutathione content, were determined. Results: All treatments increased the levels of LDH, AST, ALT, and MDA but decreased SOD and CAT activities and the total glutathione content in HepG2 cells. Oxidative stress was induced by these oxidative stressors in HepG2 cells via oxidant-antioxidant imbalance, with TBHP (100 µM, 3 h) acting as a powerful oxidant based on the minimal time to induce oxidative stress. The antioxidants, ascorbic acid and gallic acid, improved oxidant-antioxidant imbalance against xenobiotic-induced oxidative stress in HepG2 cells. Conclusion: These oxidative stress models are suitable for investigating the antioxidant and/or hepatoprotective potential of chemicals, including natural compounds

CYP1A1 Gene: A Cancer Risk Modifier

AbstractCYP1A1gene is an important target in cancer, due to its role in metabolic bioactivation of polycyclic aromatic hydrocarbons (PAHs),to electrophilic intermediates referred to bay region epoxides, to ultimate carcinogens and capable of causing oncogenic mutations in DNA. Expression of CYP1A1 mRNA is elevated by activation of the arylhydrocarbon receptor (AhR) through binding of exogenous ligands such as PAHs, of which the halogenated derivative 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a prototype, then translocates from cytoplasm to nucleus upon the activation. Hence, disruption of these regulatory pathways is implicated in tumor progression. In addition, up-regulation of protein kinase C (PKC) and tyrosine kinase, as well as activation of mitogen-activated protein kinases (MAPKs) result in increase of AhR signal transduction. Glucocorticoid receptor (GR) and estrogen receptor (ER) also affect AhR-mediated pathway, but undergoing via different aspects Namely, GR decreases AhR-mediated expression by interacting with xenobiotic responsive element-binded AhR, while ER has a direct interaction with CYP1A1 promotor by acting as a co-regulator of AhR-mediated transcriptional activation. In normal condition, AhR plays a promoting role in cell cycle progression. In the existence of exogenous ligands, AhR shows inhibitory effect vice vesa . CYP1A1 is expressed constitutively in several extrahepatic tissues such as intestine, lung, placenta, and kidney, but not in liver. However, CYP1A1 expression has been demonstrated in the liver after inducer treatment. Therefore, cancer progression regarding overexpression of CYP1A1 possibly occurred in several organs related to exogenous ligands in every day exposures, e.g. smoking, diet, and the environment. Apart from these, genetic polymorphism of CYP1A1 gene has recently been noted to involve in difference types of cancer.Key words:CYP1A1 gene, aryl hydrocarbon receptor, cancer, AhR บทคัดย่อยีนไซโตโครม พี 450 1 เอ 1 (CYP1A1) เป็นยีนเป้าหมายสำคัญหนึ่งของมะเร็ง เนื่องจากมีหน้าที่หลักในการเปลี่ยนแปลงทางชีวภาพของสารกลุ่มpolycyclic aromatic hydrocarbon (PAHs) ให้อยู่ในรูปสารกึ่งกลางจำพวกepoxide หรือสารก่อมะเร็งที่มีฤทธิส์ ูงสุดซึ่งสามารถกระตุ้นกระบวนการกลายพันธุ์ในสายดีเอ็นเอที่เกี่ยวข้องกับการเกิดมะเร็งได้ mRNA ของCYP1A1 จะถูกเหนี่ยวนำให้เพิ่มขึ้นด้วยการกระตุ้นการจับระหว่างตัวรับaryl hydrocarbon (AhR) กับ ligand)ได้แก่ สาร PAH ที่มีอนุพันธ์ของ2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) เป็นสารต้นแบบ จากนั้นสารประกอบของตัวรับ AhR และ ligand จะผ่านเข้าสู่นิวเคลียสอันเป็นจุดเริ่มการกระตุ้นยีน CYP1A1 ดังนั้นการรบกวนกลไกการควบคุมใด ๆ จะส่งผลต่อกระบวนการเจริญเติบโตของเซลล์มะเร็ง ยิ่งไปกว่านี้การเพิ่มการทำงานของ protein kinase C (PKC), tyrosine kinase และการกระตุ้นmitogen-activated protein kinases (MAPKs) จะส่งผลให้เพิ่มการส่งสัญญาณของตัวรับ AhR นอกจากนี้ glucocorticoid receptor (GR) และestrogen receptor (ER) ยังแสดงผลร่วมต่อกระบวนการส่งสัญญาณของตัวรับ AhR ได้ อาทิ GR สามารถลดการแสดงออกของยีน CYP1A1 ที่แสดงผลผ่านกลไกของตัวรับ AhR โดยจับกับองค์ประกอบของตัวรับ AhRหรือองค์ประกอบภายในยีนที่ตอบสนองต่อสารแปลกปลอม (xenobioticresponsive element) ในขณะที่ ER สามารถส่งผลโดยตรงต่อส่วนโปรโมเตอร์ภายในยีน CYP1A1 โดยเป็นเสมือนตัวควบคุมร่วมในการเหนี่ยวนำผ่านตัวรับ AhR ในสภาวะปกติตัวรับ AhR มีบทบาทส่งเสริมการเจริญเติบโตของเซลล์แต่ตัวรับ AhR สามารถแสดงผลตรงกันข้ามในสภาวะที่มีลิแกนด์ เนื่องจากยีน CYP1A1 สามารถพบได้ในเนื้อเยื่อหลายชนิดเช่น ปอด รก ตับและไต แม้ว่าจะพบยีนนี้ในตับเมื่อถูกเหนี่ยวนำด้วยสารกระตุ้นต่าง ดังนั้นกระบวนการเจริญของมะเร็งที่เกี่ยวข้องกับการแสดงออกที่มากเกินไปของยีน CYP1A1 จึงมีโอกาสเกิดขึ้นในอวัยวะหลายชนิดจากการได้รับลิแกนด์ภายนอกที่ปนเปื้อนอยู่ทั่วไปในสภาวะแวดล้อมนอกจากนี้ความหลากหลายทางพันธุกรรมของยีน CYP1A1 พบว่าสัมพันธ์กับโอกาสเกิดมะเร็งหลายประเภทด้วยคำสำคัญ: ยีนไซโตโครม พี 450 1 เอ 1, ตัวรับแอริลไฮโดรคาร์บอน,มะเร็ง, AhR

Physiology and Pathophysiology of Steroid Biosynthesis, Transport and Metabolism in the Human Placenta

The steroid hormones progestagens, estrogens, androgens, and glucocorticoids as well as their precursor cholesterol are required for successful establishment and maintenance of pregnancy and proper development of the fetus. The human placenta forms at the interface of maternal and fetal circulation. It participates in biosynthesis and metabolism of steroids as well as their regulated exchange between maternal and fetal compartment. This review outlines the mechanisms of human placental handling of steroid compounds. Cholesterol is transported from mother to offspring involving lipoprotein receptors such as low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SRB1) as well as ATP-binding cassette (ABC)-transporters, ABCA1 and ABCG1. Additionally, cholesterol is also a precursor for placental progesterone and estrogen synthesis. Hormone synthesis is predominantly performed by members of the cytochrome P-450 (CYP) enzyme family including CYP11A1 or CYP19A1 and hydroxysteroid dehydrogenases (HSDs) such as 3β-HSD and 17β-HSD. Placental estrogen synthesis requires delivery of sulfate-conjugated precursor molecules from fetal and maternal serum. Placental uptake of these precursors is mediated by members of the solute carrier (SLC) family including sodium-dependent organic anion transporter (SOAT), organic anion transporter 4 (OAT4), and organic anion transporting polypeptide 2B1 (OATP2B1). Maternal–fetal glucocorticoid transport has to be tightly regulated in order to ensure healthy fetal growth and development. For that purpose, the placenta expresses the enzymes 11β-HSD 1 and 2 as well as the transporter ABCB1. This article also summarizes the impact of diverse compounds and diseases on the expression level and activity of the involved transporters, receptors, and metabolizing enzymes and concludes that the regulatory mechanisms changing the physiological to a pathophysiological state are barely explored. The structure and the cellular composition of the human placental barrier are introduced. While steroid production, metabolism and transport in the placental syncytiotrophoblast have been explored for decades, few information is available for the role of placental-fetal endothelial cells in these processes. With regard to placental structure and function, significant differences exist between species. To further decipher physiologic pathways and their pathologic alterations in placental steroid handling, proper model systems are mandatory

Deep ocean minerals minimize eccentric exercise-induced inflammatory response of rat skeletal muscle.

Background: We have previously shown an accelerated recovery from muscle fatigue in men challenged by prolonged exercise after oral deep ocean minerals (DOM) supplementation. Here, we hypothesized a decrease in eccentric exercise-induced muscle inflammation in rats regularly consuming DOM-containing drinks (hardness 600 mg/L and fructose 11%). Methods: Forty-seven male Sprague Dawley rats were randomized into 4 groups: Control (C, N = 12), Fructose (F, N = 12), Fructose+Exercise (FE, N = 12), and Fructose+Exercise+DOM (FED, N = 11). Since fructose is a commonly used ingredient in beverages, 11% of fructose was added as a vehicle of the study. Soleus muscles of rats were analyzed 24 h after an acute bout of downhill running following 9 weeks of DOM supplementation. Results: Leukocyte infiltration and TNF-a mRNA of muscle in the FE group were 5 times and 4 times greater the F group, respectively, (P eight fold greater than the C group (P < 0.05). The reduced glutathione (GSH) of muscle in the F group was 34% lower than that in the C group (P < 0.05). However, GSH levels were similar for the C and FED groups. Conclusion: Prolonged fructose supplementation modulates inflammatory balance of rat skeletal muscle. The results of the study suggest that DOM can minimize eccentric exercise-induced inflammatory cytokine responses in rat skeletal muscle.This work was supported by grants from Taiwan Yes Corporation, Ministry of Science and Technology (Grant No. 102-2410-H-845-018-MY3), and University of Taipei. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results

Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis

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In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

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Differential gene expression between wild-type and Gulo-deficient mice supplied with vitamin C

The aim of this study was to test the hypothesis that hepatic vitamin C (VC) levels in VC deficient mice rescued with high doses of VC supplements still do not reach the optimal levels present in wild-type mice. For this, we used a mouse scurvy model (sfx) in which the L-gulonolactone oxidase gene (Gulo) is deleted. Six age- (6 weeks old) and gender- (female) matched wild-type (WT) and sfx mice (rescued by administering 500 mg of VC/L) were used as the control (WT) and treatment (MT) groups (n = 3 for each group), respectively. Total hepatic RNA was used in triplicate microarray assays for each group. EDGE software was used to identify differentially expressed genes and transcriptomic analysis was used to assess the potential genetic regulation of Gulo gene expression. Hepatic VC concentrations in MT mice were significantly lower than in WT mice, even though there were no morphological differences between the two groups. In MT mice, 269 differentially expressed transcripts were detected (≥ twice the difference between MT and WT mice), including 107 up-regulated and 162 down-regulated genes. These differentially expressed genes included stress-related and exclusively/predominantly hepatocyte genes. Transcriptomic analysis identified a major locus on chromosome 18 that regulates Gulo expression. Since three relevant oxidative genes are located within the critical region of this locus we suspect that they are involved in the down-regulation of oxidative activity in sfx mice

Sexually dimorphic gene expression in the heart of mice and men

The prevalence and clinical manifestation of several cardiovascular diseases vary considerably with sex and age. Thus, a better understanding of the molecular basis of these differences may represent a starting point for an improved gender-specific medicine. Despite the fact that sex-specific differences have been observed in the cardiovascular system of humans and animal models, systematic analyses of sexual dimorphisms at the transcriptional level in the healthy heart are missing. Therefore we performed gene expression profiling on mouse and human cardiac samples of both sexes and young as well as aged individuals and verified our results for a subset of genes using real-time polymerase chain reaction in independent left ventricular samples. To tackle the question whether sex differences are evolutionarily conserved, we also compared sexually dimorphic genes between both species. We found that genes located on sex chromosomes were the most abundant ones among the sexually dimorphic genes. Male-specific expression of Y-linked genes was observed in mouse hearts as well as in the human myocardium (e.g. Ddx3y, Eif2s3y and Jarid1d). Higher expression levels of X-linked genes were detected in female mice for Xist, Timp1 and Car5b and XIST, EIF2S3X and GPM6B in women. Furthermore, genes on autosomal chromosomes encoding cytochromes of the monoxygenase family (e.g. Cyp2b10), carbonic anhydrases (e.g. Car2 and Car3) and natriuretic peptides (e.g. Nppb) were identified with sex- and/or age-specific expression levels. This study underlines the relevance of sex and age as modifiers of cardiac gene expression