鄰苯二甲酸酯(Phthalates)是親脂性化合物，製造塑膠製品之增塑劑。Phthalates在環境中無所不在，人體主要暴露途徑由食物攝入，或經生產、食品加工設備及包裝等均有機會汙染食品，成為間接之食品添加物，對人類造成危害。Phthalates會干擾內分泌系統，又稱為內分泌干擾物質（EDCs），會導致肥胖和代謝失調等影響。本實驗研究主旨在於了解Phthalates對於脂肪細胞分化之影響。以三種Phthalates鄰苯二甲酸二（正辛基）酯（DOP）、鄰苯二甲酸二甲酯（DMP）、鄰苯二甲酸二乙酯（DEP）及誘導分化劑處理3T3-L1前脂肪細胞刺激分化。以油紅O (ORO)染色評估三種Phthalates促進脂肪生成之能力，結果顯示DOP脂質累積最為顯著，故以DOP做後續探討。首先以Dual-Luciferase® Reporter Assay System測定PPARγ配體對於過氧化物酶體增殖物反應元件(PPRE)轉錄活性之影響，結果指出DOP對轉錄活性具劑量依賴性，顯示DOP可能與PPAR及類視黃醇X受體(RXR)形成異二聚體，再與啟動子區域之PPRE結合，調節下游基因之轉錄。進一步以即時聚合酶連鎖反應（Real-time polymerase chain reaction）分析DOP對於PPARγ轉錄因子及下游基因之表現，結果顯示在分化期間，DOP會經由活化特定基因誘導細胞分化並生成脂肪。最後以2-NBDG Glucose Uptake Assay及流式細胞儀檢測葡萄糖吸收之能力，以DMEM Low glucose 培養基培養HepG2細胞，模擬正常血糖之條件下並以DOP處理，會抑制細胞之葡萄糖吸收能力；而以DMEM High glucose 培養基誘導細胞，模擬高血糖下胰島素阻抗之狀態，其抑制攝取葡萄糖能力更為顯著，顯示DOP可能使胰島素阻抗之情形更為惡化。人體常經由不同途徑暴露於塑化劑風險，因此需要對其結果做進一步探討。

Phthalates are lipophilic compounds that are used as plasticizers for the manufacture of plastic products. Phthalates are ubiquitous in the environment. The main exposure route of phthalates into human beings is through food intake. Through production, food processing equipment or packaging, there are numerous opportunities to contaminate food and phthalates become indirect food additives, causing harm to humans. Phthalates interfere with the endocrine system, also known as endocrine disrupting substances (EDCs), which can cause effects such as obesity and metabolic disorders. The main purpose of this experimental study was to understand the effect of phthalates on adipocyte differentiation. Three phthalates di-n-octyl phthalate (DOP), dimethyl phthalate (DMP), diethyl phthalate (DEP) and differentiation inducing agents were used to treat 3T3-L1 preadipocytes to stimulate differentiation. Oil red O (ORO) staining was used to evaluate the ability of three phthalates to promote lipogenesis. The results showed that DOP treated cell lipid accumulation was the most significant, so DOP treatment was followed up with further analysis. First, the effect of PPARγ ligand on the transcriptional activity of peroxisome proliferator response element (PPRE) was determined by Dual-Luciferase® Reporter Assay System. This indicates that DOP may have bind to the heterodimer formed by PPAR and RXR that binds to PPRE of the promoter region and thus regulated the transcription of downstream genes. Furthermore, the expression of DOP for PPARγ transcription factor and downstream genes were analyzed by Real-Time polymerase chain reaction. The results showed that during differentiation, DOP induced cell differentiation and lipid droplets accumulation via activation of specific genes. Finally, glucose uptake analysis was performed by 2-NBDG Glucose Uptake Assay and flow cytometer. HepG2 cells were cultured in DMEM medium with low (normal human glucose levels) and high glucose (hyperglycemic) conditions. Results showed that DOP treatment under normal glucose medium inhibited the glucose uptake capacity of the cells. In the high glucose medium, DOP treatment mimics the state of insulin resistance under hyperglycemia and the ability of DOP to inhibit glucose uptake was more significant. This indicates that DOP may worsen insulin resistance. The human body is often exposed to the risk of plasticizers through different routes, so it is necessary to further explore the results.

Chen, M.-L., Chen, J.-S., Tang, C.-L., & Mao, I. F. (2008). The internal exposure of Taiwanese to phthalate—An evidence of intensive use of plastic materials. Environment International, 34(1), 79-85.

Costantini, S., Di Bernardo, G., Cammarota, M., Castello, G., & Colonna, G. (2013). Gene expression signature of human HepG2 cell line. Gene, 518(2), 335-345.

Diradourian, C., Girard, J., & Pégorier, J.-P. (2005). Phosphorylation of PPARs: from molecular characterization to physiological relevance. Biochimie, 87(1), 33-38.

Echeverría, F., Ortiz, M., Valenzuela, R., & Videla, L. A. (2016). Long-chain polyunsaturated fatty acids regulation of PPARs, signaling: Relationship to tissue development and aging. Prostaglandins, Leukotrienes and Essential Fatty Acids, 114, 28-34.

El-Jack, A. K., Kandror, K. V., & Pilch, P. F. (1999). The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation. 10(5), 1581-1594.

Fasano, E., Bono-Blay, F., Cirillo, T., Montuori, P., & Lacorte, S. (2012). Migration of phthalates, alkylphenols, bisphenol A and di(2-ethylhexyl)adipate from food packaging. Food Control, 27(1), 132-138.

Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: Peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions. Progress in Lipid Research, 45(2), 120-159.

Furuhashi, M., Saitoh, S., Shimamoto, K., & Miura, T. (2014). Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases. 8s3, CMC.S17067.

Ghaben, A. L., & Scherer, P. E. (2019). Adipogenesis and metabolic health. Nature Reviews Molecular Cell Biology, 20(4), 242-258.

GREGOIRE, F. M., SMAS, C. M., & SUL, H. S. (1998). Understanding Adipocyte Differentiation. 78(3), 783-809.

Kersten, S. (2014). Physiological regulation of lipoprotein lipase. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1841(7), 919-933.

Kota, B. P., Huang, T. H.-W., & Roufogalis, B. D. (2005). An overview on biological mechanisms of PPARs. Pharmacological Research, 51(2), 85-94.

Larsen, T. M., Toubro, S., & Astrup, A. (2003). PPARgamma agonists in the treatment of type II diabetes: is increased fatness commensurate with long-term efficacy? International Journal of Obesity, 27(2), 147-161.

Liu, X., Shi, J., Bo, T., Zhang, H., Wu, W., Chen, Q., & Zhan, X. (2014). Occurrence of phthalic acid esters in source waters: a nationwide survey in China during the period of 2009–2012. Environmental Pollution, 184, 262-270.

Montagner, A., Rando, G., Degueurce, G., Leuenberger, N., Michalik, L., & Wahli, W. (2011). New insights into the role of PPARs. Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA), 85(5), 235-243.

Moseti, D., Regassa, A., & Kim, W.-K. (2016). Molecular Regulation of Adipogenesis and Potential Anti-Adipogenic Bioactive Molecules. 17(1), 124.

Pomatto, V., Cottone, E., Cocci, P., Mozzicafreddo, M., Mosconi, G., Nelson, E. R., Palermo, F. A., & Bovolin, P. (2018). Plasticizers used in food-contact materials affect adipogenesis in 3T3-L1 cells. The Journal of Steroid Biochemistry and Molecular Biology, 178, 322-332.

Poulsen, L. l. C., Siersbæk, M., & Mandrup, S. (2012). PPARs: Fatty acid sensors controlling metabolism. Seminars in Cell & Developmental Biology, 23(6), 631-639.

Rizzo, G., & Fiorucci, S. (2006). PPARs and other nuclear receptors in inflammation. Current Opinion in Pharmacology, 6(4), 421-427.

Saillenfait, A.-M., Roudot, A.-C., Gallissot, F., & Sabaté, J.-P. (2011). Prenatal developmental toxicity studies on di-n-heptyl and di-n-octyl phthalates in Sprague-Dawley rats. Reproductive Toxicology, 32(3), 268-276.

Serôdio, P., & Nogueira, J. M. F. (2006). Considerations on ultra-trace analysis of phthalates in drinking water. Water Research, 40(13), 2572-2582.

Serrano, S. E., Braun, J., Trasande, L., Dills, R., & Sathyanarayana, S. (2014). Phthalates and diet: a review of the food monitoring and epidemiology data. Environmental Health, 13(1), 43.

Silva, M. J., Kato, K., Gray, E. L., Wolf, C., Needham, L. L., & Calafat, A. M. (2005). Urinary metabolites of di-n-octyl phthalate in rats. Toxicology, 210(2), 123-133.

Sugimoto, R., Ishibashi-Ohgo, N., Atsuji, K., Miwa, Y., Iwata, O., Nakashima, A., & Suzuki, K. (2018). Euglena extract suppresses adipocyte-differentiation in human adipose-derived stem cells. PloS one, 13(2), e0192404-e0192404.

Teil, M. J., Blanchard, M., & Chevreuil, M. (2006). Atmospheric fate of phthalate esters in an urban area (Paris-France). Science of The Total Environment, 354(2), 212-223.

Wang, J., Chen, G., Christie, P., Zhang, M., Luo, Y., & Teng, Y. (2015). Occurrence and risk assessment of phthalate esters (PAEs) in vegetables and soils of suburban plastic film greenhouses. Science of The Total Environment, 523, 129-137.

Wang, X., & Hai, C. (2015). Redox modulation of adipocyte differentiation: hypothesis of “Redox Chain” and novel insights into intervention of adipogenesis and obesity. Free Radical Biology and Medicine, 89, 99-125.

Zeng, F., Cui, K., Xie, Z., Liu, M., Li, Y., Lin, Y., Zeng, Z., & Li, F. (2008). Occurrence of phthalate esters in water and sediment of urban lakes in a subtropical city, Guangzhou, South China. Environment International, 34(3), 372-380.