壬基苯酚誘導人類胎盤滋養層細胞氧化傷害及粒線體功能缺損

Induction of Oxidative Damage and Mitochondrial Dysfunction of Human Trophoblast Cells by Nonylphenol

林子瑜(Tzu-Yu Lin)

指導教授 ： 高淑慧

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2005年)

摘要

壬基苯酚（nonylphenol, NP）是常用的非離子型清潔劑，壬基苯酚聚乙氧基醇類非離子型界面活性劑（nonylphenol ethoxylate, NPEO）的最終產物。由於目前此類清潔劑使用數量龐大，NP在水環境中不易被分解。由於其構造和雌性激素類似，會產生類似雌性激素的作用。 在許多環境研究中也指出，壬基苯酚會造成雄性魚類生殖能力下降，甚至有中性魚的出現。 本實驗將探討壬基苯酚對人類胎盤滋養層細胞（trophoblast）粒線體功能的影響，並進一步探討壬基苯酚造成傷害的機制。在壬基苯酚刺激下，人類胎盤滋養層細胞的存活率有下降的趨勢，以1.5uM 壬基苯酚經24小時培養，其細胞存活率降低為對照組的51.54±16.65%。此外，經壬基苯酚刺激後，隨處理時間延長，細胞內活性氧族（reactive oxygen species, ROS）亦呈漸增的趨勢，並且對細胞造成氧化性傷害，包括脂質過氧化物（lipid peroxides）的產生。以1uM 壬基苯酚處理48小時後，經HPLC測得細胞內脂質過氧化物明顯增加55.92±14.54%。在粒線體產生ATP方面，經壬基苯酚刺激後，細胞內ATP產量隨著壬基苯酚濃度增加而有明顯的下降，經1.5uM 壬基苯酚，24小時處理後，則細胞內ATP的含量減少為對照組的64.80±17.05%。顯示壬基苯酚降低粒線體氧化磷酸化的能力。此外，我們又以real-time PCR探討粒線體DNA的拷貝數。結果發現，粒線體DNA拷貝數隨著壬基苯酚濃度的增加有稍減少的趨勢。我們更進一步發現，壬基苯酚可經由ROS的產生來使缺氧誘發因子-1a(hypoxia inducible factor-1a, HIF-1a）的蛋白表現穩定，在加入3 mM的L-NAC（ROS清除劑）後，HIF-1a蛋白表現明顯減少32%。以及壬基苯酚誘發誘導型一氧化氮合成?（inducible nitric oxide sythase, iNOS）表現增加以及一氧化氮的產生。細胞經1uM 壬基苯酚處理8小時後，可觀察到iNOS蛋白表現增加58.00±7.94%；在24小時的時候，一氧化氮增加了23.27±6.91%。我們以Annexin V-FITC/Propidium iodide（PI）染色偵測細胞死亡情形，發現在2uM壬基苯酚處理細胞24小時候，有46.47±0.26%的細胞呈現向壞死（necrosis）現象，而有22.05±1.61%的細胞則是走向細胞凋亡（apoptosis）。我們還繼續探討壬基苯酚對ICR老鼠胚胎發育的影響。經1uM 壬基苯酚培養的受精卵，其孵化率（hatching rate）為87.50%；但是其胚胎外觀與對照組相比，則是有形狀不規則，且較破碎的情形發生。 綜合以上實驗結果顯示，壬基苯酚確實對人類胎盤滋養層細胞產生氧化傷害，並造成粒線體功能缺損，由此推測壬基苯酚可能造成胎盤的氧化傷害，進而影響到胚胎的存活及發育能力。

關鍵字

壬基苯酚 ； 粒線體 ； 活性氧族

並列摘要

Nonylphenol (NP) is a degradation product of a widely used nonionic surfactant nonylphenol ethoxylate (NPEO). NP has been identified as an environmental hormone with estrogenic activity, and suggested to disrupt normal reproductive function of mammals. In this study, we investigated the causal roles of NP in inducing trophoblast cell death and impaired ICR mouse embryo development. We conducted several experiments to address the NP-induced effects on cellular viability, mitochondrial function, and identified the molecular mechanism of cytotoxicity in NP-treated human trophoblast cells (3A-Sub-E). The results showed that the cell viability was declined to 51.54±16.65% in 1.5 uM NP-treated cells by dye exclusion assay. Reactive oxygen species (ROS) generation and oxidative damages were increased in a dose- and time-dependent manner. The dose-dependent decrease of ATP production and mtDNA copy number in NP-treated cells were detected by luminescence assay and real-time quantitative PCR. Furthermore, we found the hypoxia inducible factor-1a (HIF-1a) was increased by NP via ROS generation. The increased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) generation were also detected in NP-treated cells. The iNOS protein was increased 58.00±7.94% and NO generation was measured 23.27±6.91% in the 1 uM NP-treated cells. After 2 uM NP treatment, approximate 46.47±0.26% cells underwent necrosis and 22.05±1.61% cells were apoptotic when detected by Annexin V and PI staining. In addition, we also demonstrated the detrimental effects of NP on embryonic development capacity. The increased hatching rate were found in 4-cell embryos with 1 uM NP treatment. However, the increased abnormal morphology was found in the hatched embryos with NP treatment. In conclusion, that NP contributes to the induction of oxidative damages in placenta, and may affect embryo viability and developmental competence.

並列關鍵字

Nonylphenol ； Mitochondria ； Reactive oxygen species

參考文獻

Adeoya-Osiguwa, S. A., Markoulaki, S., Pocock, V., Milligan, S. R., and Fraser, L. R. (2003). 17beta-Estradiol and environmental estrogens significantly affect mammalian sperm function. Hum Reprod 18, 100-107.

Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 90, 7915-7922.

An, B. S., Kang, S. K., Shin, J. H., and Jeung, E. B. (2002). Stimulation of calbindin-D(9k) mRNA expression in the rat uterus by octyl-phenol, nonylphenol and bisphenol. Mol Cell Endocrinol 191, 177-186.

Beckman, J. S., and Koppenol, W. H. (1996). Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271, C1424-1437.

Bicknell, R. J., Herbison, A. E., and Sumpter, J. P. (1995). Oestrogenic activity of an environmentally persistent alkylphenol in the reproductive tract but not the brain of rodents. J Steroid Biochem Mol Biol 54, 7-9.