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

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

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

Diabetic Induction in Experimental Mouse Model - การชักนำภาวะเบาหวานในหนูทดลอง

AbstractDiabetes Mellitus (DM) is a chronic disease negatively influencing quality of life and economic status of a patient and society According to rapid increase in numbers of DM patients worldwide; there is an extensive effort to develop a new anti-diabetic drug. One important step in a new drug development is to study pharmacological and toxicological activities in the experimental animal prior subsequently performing risk-benefit assessment in human volunteers. Hence, a diabetic mouse model which possessed a corresponded DM condition to the objective of drug development is the first important necessity. Several pevious studies normally employed chemical agents such as streptozotocin, alloxan, or bromocriptine with different dose ranged from 10-200 mg/kg depending on route of administration and experimental animal species to induce DM type I. Likewise, induction of DM type II related to high fat-diet feeding and/or co-administration of the chemical agent to predispose obesity and insulin tolerance condition in animals. Besides DM induction using the chemical agent or high fat-diet feeding, a genetic modifying and viral infected techniques have been introduced to produce a specific DM animal species. Therefore, a DM experimental animal model optimally supporting a drug development objective leads to reliability and valuable knowledge of its outcome.Keywords: diabetes mellitus, diabetic induction, streptozotocin บทคัดย่อโรคเบาหวาน (Diabetes Mellitus) เป็นโรคที่ส่งผลกระทบด้านลบต่อคุณภาพชีวิต เศรษฐกิจและสังคม ปจั จุบันจำนวนผู้ป่วยทัว่ โลกมีแนวโน้มเพิ่มขึ้นอย่างรวดเร็ว จึงมีการมุ่งพัฒนายาใหม่เพื่อรักษาเบาหวานอย่างต่อเนื่อง ขัน้ ตอนที่สำคัญในการพัฒนายาคือ การศึกษากลไกการออกฤทธิ ์และความเป็นพิษของยาในสัตว์ทดลอง เพื่อการประเมินความปลอดภัยก่อนการทดสอบต่อไปในมนุษย์ ดังนั้นการชักนำหนูทดลองให้เกิดภาวะเบาหวานที่สอดคล้องกับวัตถุประสงค์ของการพัฒนายา จึงเป็นขัน้ ตอนแรกที่สำคัญ การศึกษาที่ผ่านมานิยมใช้สารเคมี อาทิ streptozotocin, alloxanหรืออาจพบสารเคมีอื่นบ้าง เช่น bromocriptine เป็นต้น ในขนาดและจำนวนครัง้ ที่แตกต่างกันตั้งแต่ 10-200 mg/kg ขึ้นกับวิธีการบริหารยาและสายพันธุ์หนูทดลอง เพื่อชักนำภาวะเบาหวานชนิดที่ 1 ในขณะที่การชักนำภาวะเบาหวานชนิดที่ 2 อาจใช้เฉพาะการให้อาหารที่มีส่วนประกอบของไขมันในปริมาณสูง เพื่อชักนำให้หนูทดลองเกิดภาวะอ้วน และภาวะดื้ออินซูลิน หรือในบางกรณีอาจใช้อาหารไขมันสูงร่วมกับการให้สารเคมีดังกล่าวด้วย นอกจากวิธีการที่กล่าวมาข้างต้นแล้ว ยังมีการพัฒนาวิธีต่างๆเพื่อชักนำภาวะเบาหวานให้ใกล้เคียงกับที่เกิดขึ้นในมนุษย์ ได้แก่ การพัฒนาพันธุกรรมหนูทดลองเป็นสายพันธุ์ที่มีภาวะเบาหวาน การชักนำด้วยไวรัสเพื่อกระตุ้นให้เกิดภาวะต้านภูมิตัวเอง เป็นต้น ดังนั้นจะเห็นได้ว่า การชักนำภาวะเบาหวานในหนูทดลองที่สอดคล้องกับวัตถุประสงค์ของการพัฒนายาหรือสมุนไพรที่ศึกษาย่อมนำมาซึ่งผลการทดลองที่เชื่อถือได้ และองค์ความรู้ต่อการพัฒนายารักษาโรคเบาหวานต่อไปคำสำคัญ: โรคเบาหวาน, การชักนำภาวะเบาหวาน, streptozotoci