DEHP為最常用於塑料的化合物，與人們的生活息息相關，暴露在醫療器材、食品包裝、建築材料、化妝品、玩具等。DEHP一旦經由各種途徑吸收，就會快速代謝成MEHP。之前的研究指出，塑化劑可能提高胰島素阻抗及第二型糖尿病的風險，而骨骼肌又與代謝症候群有關，因此我們以C2C12為研究題材，觀察C2C12在處理100μM MEHP後對分化、代謝基因表現量及粒線體功能的影響。 在肌肉生成(myogenesis)過程中，細胞增殖狀態下給予MEHP會抑制分化，而分化4天後給予MEHP抑制分化的效果會顯著降低，因此以分化3天後再給予MEHP作為後續實驗，避免代謝作用受分化影響。葡萄糖攝取實驗顯示MEHP對細胞因受insulin刺激而增加的葡萄糖攝取並沒有影響，而在醣類與脂肪酸代謝方面，ERRα、PDK4和CPT1B基因的表現量皆有顯著上升。透過穩定細胞株C2C12-MTS-RFP觀察粒線體細胞形態，在CMB時期給予2天MEHP其粒線體趨向分裂狀態，而透過測定粒線體的DNA含量，發現不論在哪一個時期給予MEHP皆無影響，此外Tfam基因的表現量也沒有差異。為了得知粒線體功能是否受到影響，我們測定一系列在電子傳遞鏈過程中會發生的反應。給予MEHP不會影響到肌管時期的膜電位但是會讓succinate dehydrogenase活性下降，並且使ROS含量與SOD活性些許上升，而HO-1基因表現量也有所上升。接著我們測定電子傳遞鏈上各個具代表性的complex subunit 蛋白質的表現量，發現MEHP會影響到complex I subunit-Ndufb8的含量。為了確認MEHP影響Ndufb8的轉錄或轉譯，以Real time PCR及promoter activity 測定結果皆沒有受到MEHP影響。透過給予轉譯抑制劑cycloheximide 顯示MEHP可能影響Ndufb8的轉譯。綜合以上所述，塑化劑可能造成粒線體功能失調，並減少insulin的敏感度。

Di(2-ethylhexyl) phthalate (DEHP) is widely used in industry to increase the malleability of polyvinyl chloride. It can be found in plastic package, toys, clinic, cosmetic, building, and so on. Once entered the body, it is quickly metabolized to MEHP. Previous studies have shown that the level of phthalate is associated with the development of type-2 diabetes and insulin resistance. In this study, the effects of MEHP on myogenic differentiation, the metabolism of glucose and fatty acid oxidation, and mitochondrial function were observed. We treated C2C12 cells with 100 μM MEHP for two days and cells treated with MEHP in the proliferation stage during myogenesis failed to form myotubes. After three days of differentiation, MEHP treatment did not influence differentiation, we used this stage for measuring the effects of MEHP on metabolism so the intereference from differentiation could be avoided. First, we measured glucose uptake, in the stimulation of insulin situation, MEHP group caused the cells to absorb less glucose compared to the DMSO group. Moreover, we investigated the metabolism of glucose and fatty acid oxidation by using Real-time PCR. The gene expression of ERRα、PDK4、CPT1B is up-regulated. Second, we observed the effect of MEHP on mitochondrial function. C2C12-MTS-RFP was culture for three days in CMB stage, then treated with MEHP for two days and checked the mitochondrial morphology. The cells tended to become fission as compared to the DMSO group. It probably had poor function in mitochondria. So we checked the content of mitochondria. MITO DNA and the gene expression of Tfam was not affected by MEHP. To investigate the function of mitochondria, a series assays on the functions of electron transport system was measured. Membrane potential was not influenced by MEHP, while succinate dehydrogenase was decreased and produced ROS then enhance SOD activity. The expression of ROS-related gene HO-1 was significantly up-regulated. Third, we measured the protein level of 5 complexes in the electron transport system. In the myotube stage, treatment with MEHP decreased the lelvel of complex I subunit-Ndufb8. To ensure whether MEHP influenced transcription or translation stage, mRNA level and promoter activity were measured, and we found MEHP did not influence the transcription of Ndufb8. Through adding translation inhibitor cycloheximide in the medium, we found MEHP might interfere with the translation of Ndufb8. Taken together, MEHP can affect the function of mitochondria and by affecting the functions/levels of key factors in the ETC system.

Agarwal, D.K., Eustis, S., Lamb 4th, J., Reel, J.R., and Kluwe, W.M. (1986). Effects of di (2-ethylhexyl) phthalate on the gonadal pathophysiology, sperm morphology, and reproductive performance of male rats. Environmental Health Perspectives 65, 343-350.
Albro, P.W. (1986). Absorption, metabolism, and excretion of di (2-ethylhexyl) phthalate by rats and mice. Environmental Health Perspectives 65, 293-298.
Alexeyev, M.F. (2009). Is there more to aging than mitochondrial DNA and reactive oxygen species? The FEBS journal 276, 5768-5787.
Chen, S.-S., Hung, H.-T., Chen, T.-J., Hung, H.-S., and Wang, D.-C. (2013). Di-(2-ethylhexyl)-phthalate reduces MyoD and myogenin expression and inhibits myogenic differentiation in C2C12 cells. The Journal of Toxicological Sciences 38, 783-791.
Constantin-Teodosiu, D. (2013). Regulation of muscle pyruvate dehydrogenase complex in insulin resistance: effects of exercise and dichloroacetate. Diabetes & Metabolism Journal 37, 301-314.
Duchen, M.R. (2004). Mitochondria in health and disease: perspectives on a new mitochondrial biology. Molecular Aspects of Medicine 25, 365-451.
El Bacha, T., Luz, M., and Da Poian, A. (2010). Dynamic adaptation of nutrient utilization in humans. Natl Educ 3, 8.
Feige, J.N., Gelman, L., Rossi, D., Zoete, V., Métivier, R., Tudor, C., Anghel, S.I., Grosdidier, A., Lathion, C., and Engelborghs, Y. (2007). The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor γ modulator that promotes adipogenesis. Journal of Biological Chemistry 282, 19152-19166.
Handschin, C., Chin, S., Li, P., Liu, F., Maratos-Flier, E., LeBrasseur, N.K., Yan, Z., and Spiegelman, B.M. (2007). Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1α muscle-specific knock-out animals. Journal of Biological Chemistry 282, 30014-30021.
Huang, X.-F., Li, Y., Gu, Y.-H., Liu, M., Xu, Y., Yuan, Y., Sun, F., Zhang, H.-Q., and Shi, H.-J. (2012). The effects of Di-(2-ethylhexyl)-phthalate exposure on fertilization and embryonic development in vitro and testicular genomic mutation in vivo. PloS one 7, e50465.
Kelly, D.P., and Scarpulla, R.C. (2004). Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes & Development 18, 357-368.
Knutti, D., and Kralli, A. (2001). PGC-1, a versatile coactivator. Trends in Endocrinology & Metabolism 12, 360-365.
Li, X., Fang, E.F., Scheibye-Knudsen, M., Cui, H., Qiu, L., Li, J., He, Y., Huang, J., Bohr, V.A., and Ng, T.B. (2014). Di-(2-ethylhexyl) phthalate inhibits DNA replication leading to hyperPARylation, SIRT1 attenuation, and mitochondrial dysfunction in the testis. Scientific Reports 4, 6434.
Li, X.b., Gu, J.d., and Zhou, Q.h. (2015). Review of aerobic glycolysis and its key enzymes–new targets for lung cancer therapy. Thoracic Cancer 6, 17-24.
Lin, J., Wu, H., Tarr, P.T., Zhang, C.-Y., Wu, Z., Boss, O., Michael, L.F., Puigserver, P., Isotani, E., and Olson, E.N. (2002). Transcriptional co-activator PGC-1α drives the formation of slow-twitch muscle fibres. Nature 418, 797.
Martinelli, M.I., Mocchiutti, N.O., and Bernal, C.A. (2006). Dietary di (2-ethylhexyl) phthalate-impaired glucose metabolism in experimental animals. Human & Experimental Toxicology 25, 531-538.
McBride, H.M., Neuspiel, M., and Wasiak, S. (2006). Mitochondria: more than just a powerhouse. Current Biology 16, R551-R560.
Melnick, R.L., and Schiller, C.M. (1982). Mitochondrial toxicity of phthalate esters. Environmental health perspectives 45, 51-56.
Mootha, V.K., Lindgren, C.M., Eriksson, K.-F., Subramanian, A., Sihag, S., Lehar, J., Puigserver, P., Carlsson, E., Ridderstråle, M., and Laurila, E. (2003). PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genetics 34, 267.
Ngo, H.B., Kaiser, J.T., and Chan, D.C. (2011). The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA. Nature Structural & Molecular Biology 18, 1290.
Posnack, N.G., Swift, L.M., Kay, M.W., Lee, N.H., and Sarvazyan, N. (2012). Phthalate exposure changes the metabolic profile of cardiac muscle cells. Environmental Health Perspectives 120, 1243-1251.
Randle, P., Garland, P., Hales, C., and Newsholme, E. (1963). The glucose fatty-acid cycle its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. The Lancet 281, 785-789.
Ren, Q.-G., Yang, S.-L., Hu, J.-L., Li, P.-D., Chen, Y.-S., and Wang, Q.-S. (2016). Evaluation of HO-1 expression, cellular ROS production, cellular proliferation and cellular apoptosis in human esophageal squamous cell carcinoma tumors and cell lines. Oncology Reports 35, 2270-2276.
Rhodes, C., Orton, T.C., Pratt, I.S., Batten, P.L., Bratt, H., Jackson, S.J., and Elcombe, C.R. (1986). Comparative pharmacokinetics and subacute toxicity of di (2-ethylhexyl) phthalate (DEHP) in rats and marmosets: extrapolation of effects in rodents to man. Environmental Health Perspectives 65, 299-307.
Roden, M. (2004). How free fatty acids inhibit glucose utilization in human skeletal muscle. Physiology 19, 92-96.
Rosado-Berrios, C.A., Vélez, C., and Zayas, B. (2011). Mitochondrial permeability and toxicity of diethylhexyl and monoethylhexyl phthalates on TK6 human lymphoblasts cells. Toxicology in Vitro 25, 2010-2016.
Signes, A., and Fernandez-Vizarra, E. (2018). Assembly of mammalian oxidative phosphorylation complexes I–V and supercomplexes. Essays in biochemistry 62, 255-270.
Srinivasan, C., Khan, A.I., Balaji, V., Selvaraj, J., and Balasubramanian, K. (2011). Diethyl hexyl phthalate-induced changes in insulin signaling molecules and the protective role of antioxidant vitamins in gastrocnemius muscle of adult male rat. Toxicology and Applied Pharmacology 257, 155-164.
Suliman, H.B., Carraway, M.S., Welty-Wolf, K.E., Whorton, A.R., and Piantadosi, C.A. (2003). Lipopolysaccharide stimulates mitochondrial biogenesis via activation of nuclear respiratory factor-1. Journal of Biological Chemistry 278, 41510-41518.
Suárez-Rivero, J., Villanueva-Paz, M., de la Cruz-Ojeda, P., de la Mata, M., Cotán, D., Oropesa-Ávila, M., de Lavera, I., Álvarez-Córdoba, M., Luzón-Hidalgo, R., and Sánchez-Alcázar, J. (2017). Mitochondrial dynamics in mitochondrial diseases. Diseases 5, 1.
Tang, X., Luo, Y.-X., Chen, H.-Z., and Liu, D.-P. (2014). Mitochondria, endothelial cell function, and vascular diseases. Frontiers in Physiology 5, 175.
Terada, S., and Tabata, I. (2004). Effects of acute bouts of running and swimming exercise on PGC-1α protein expression in rat epitrochlearis and soleus muscle. American Journal of Physiology-Endocrinology and Metabolism 286, E208-E216.
Viswanathan, M.P., Mullainadhan, V., Chinnaiyan, M., and Karundevi, B. (2017). Effects of DEHP and its metabolite MEHP on insulin signalling and proteins involved in GLUT4 translocation in cultured L6 myotubes. Toxicology 386, 60-71.
Wu, H., and Ballantyne, C.M. (2017). Skeletal muscle inflammation and insulin resistance in obesity. The Journal of Clinical Investigation 127, 43-54.
Youle, R.J., and Van Der Bliek, A.M. (2012). Mitochondrial fission, fusion, and stress. Science 337, 1062-1065.
Zhang, W., Shen, X.-Y., Zhang, W.-W., Chen, H., Xu, W.-P., and Wei, W. (2017). The effects of di 2-ethyl hexyl phthalate (DEHP) on cellular lipid accumulation in HepG2 cells and its potential mechanisms in the molecular level. Toxicology Mechanisms and Methods 27, 245-252.