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

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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Effect of styrene oxide and diethyl maleate on expression of cytochrome P450 family 1 and glutathione store in mouse liver

Purpose: To determine the effect of the glutathione (GSH) suppressors styrene oxide (SO) and diethyl maleate (DEM) on the hepatic expression of cytochrome P450 family 1 (Cyp1) isoforms that are related to carcinogenesis including Cyp1a1, Cyp1a2, and Cyp1b1. Methods: Seven-week-old ICR mice were intraperitoneally injected with SO (150 and 300 mg/kg/day), DEM (175 and 350 mg/kg/day), or N-acetylcysteine (NAC; 300 and 600 mg/kg/day) for 7, 14, or 28 days. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, hepatic Cyp1 expression, total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were determined. Results: ALT and AST levels were markedly increased by SO and DEM while GSH/GSSG ratio was decreased by SO in all treatments (p < 0.05), while high dose (350 mg/kg/day) DEM significantly suppressed GSH/GSSG ratio at 28 days (p < 0.05). The expressions of Cyp1a1, Cyp1a2, and Cyp1b1 were induced by SO and DEM, corresponding with induction of ethoxy/methoxy-resorufin O- dealkylase activities. Conclusion: The Cyp1 family metabolizes procarcinogens to carcinogenic DNA adducts; exposure to the industrial solvents, SO and DEM, raises the risk of carcinogenesis via GSH depletion coupled with Cyp1 induction

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 3-HSD and 17-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). Maternalfetal glucocorticoid transport has to be tightly regulated in order to ensure healthy fetal growth and development. For that purpose, the placenta expresses the enzymes 11-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.(VLID)473887

A High-Fat, High-Fructose Diet Induces Antioxidant Imbalance and Increases the Risk and Progression of Nonalcoholic Fatty Liver Disease in Mice

Excessive fat liver is an important manifestation of nonalcoholic fatty liver disease (NAFLD), associated with obesity, insulin resistance, and oxidative stress. In the present study, the effects of a high-fat, high-fructose diet (HFFD) on mRNA levels and activities of the antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), were determined in mouse livers and brains. The histomorphology of the livers was examined and the state of nonenzymatic reducing system was evaluated by measuring the glutathione system and the lipid peroxidation. Histopathology of the liver showed that fat accumulation and inflammation depended on the period of the HFFD-consumption. The levels of mRNA and enzymatic activities of SOD, CAT, and GPx were raised, followed by the increases in malondialdehyde levels in livers and brains of the HFFD mice. The oxidized GSSG content was increased while the total GSH and the reduced GSH were decreased, resulting in the increase in the GSH/GSSG ratio in both livers and brains of the HFFD mice. These observations suggested that liver damage and oxidative stress in the significant organs were generated by continuous HFFD-consumption. Imbalance of antioxidant condition induced by long-term HFFD-consumption might increase the risk and progression of NAFLD