鄰苯二甲酸二(2-乙基已基)酯及代謝物對哺乳類中國倉鼠卵巢細胞之細胞毒性及基因毒性

鄰苯二甲酸二(2-乙基己基)酯(DEHP)為一種添加於生產PVC的塑化劑，能被代謝成具高生物活性的代謝物鄰苯二甲酸單乙基己酯(MEHP)。流行病學研究指出囓齒動物暴露DEHP及代謝物MEHP後具有誘導肝癌和生殖毒性的能力。然而，MEHP引發基因毒性和致癌性的機制迄今仍尚未明朗。因此，本研究目的為闡明MEHP基因毒性和致突變性，使用hprt缺陷型CHO細胞進行試驗，正常修復型AA8細胞系以及其衍生的核苷酸修復缺陷型UV5和鹼基修復缺陷型EM9亞克隆型細胞，帶有細菌型鳥嘌呤次黃嘌呤磷酸核糖基轉移酶基因(gpt)AS52細胞及其衍生的AS52-XPD/PARP-1敲落型細胞評估DEHP及MEHP的劑量反應關係。突變篩選模型AS52細胞暴露MEHP後導致細胞內產生活性氧物質和DNA單股斷裂且具有劑量依賴性關係。突變篩選模型結果顯示，gpt突變型AS25克隆體(ASMC)之主要突變位點為G：C至A：T和A：T至G：C。接著以惡性細胞轉化及體內異種移植物模型分析收集到的AS52突變克隆體，顯示ASMC具有惡性轉化性質，並且20個ASMC中有4個具有腫瘤生長能力。此外在微核糖核酸微陣列分析(microRNA array)結果顯示ASMC的microRNA let-7家族成員和miR-125b為下調表現，可能提高致癌基因MYC表現並活化ErbB訊號途徑，在qPCR結果顯示ASMC的c-myc表現約提高7.5倍。綜合以上結果，代謝物MEHP的細胞癌化作用機制可能是通過產生ROS，導致鹼基損傷，增加細胞突變發生率，同時造成許多抑癌功能microRNA不正常表現，進而使致癌基因表現量提高，促使細胞轉化為前癌細胞。

關鍵字

鄰苯二甲酸單乙基己酯；活性氧化物質；遺傳毒性；致突變性；致癌性；中國倉鼠卵巢細胞； AS52細胞株； AS52突變細胞

並列摘要

DEHP, di-(2-ethylhexyl) phthalate, a kind of chemical materials is serve as plasticizer in the production of PVC, can be converted to the more potent metabolite mono-ethylhexyl phthalate (MEHP). Epidemiological studies have shown an association with elevated induction of rat hepatic cancer and reproductive toxicity in response to MEHP exposure. However, the mechanism of genotoxicity and carcinogenicity induced by MEHP treatment remains unclear. This study therefore aimed to elucidate the mechanisms of action, genotoxicity and mutagenicity in the hypoxanthine-guanine phosphoribosyltransferase (hprt＋/－) gene induced in several CHO cell types by MEHP were assessed. Dose response relationships were determined in the parental AA8 cell line, its nucleotide repair-deficient UV5 and base repair-deficient EM9 subclones, and also in AS52 cells harboring the bacterial guaninehypoxanthine phosphoribosyltransferase (gpt) gene and its derived AS52-XPD-knockdown and AS52-PARP-1-knockdown cells. Treatment of AS52 with MEHP led to intracellular production of reactive oxygen species (ROS) and DNA strand breaks in a dose-dependent manner. Separately, mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Independent AS52-mutant cell (ASMC) clones were collected for the sequential in vivo xenograft tumorigenic studies, 4 of total 20 clones had aggressive tumor growth. Moreover, microarray analysis indicated down-regulated miRNA let-7 family and miR-125b in ASMC, which might raise oncogenic c-myc level and activate ErbB signaling pathway. The qPCR results show that level of c-myc mRNA is 7 times change in ASMC compare to AS52. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action of MEHP is likely to be through generation of ROS, causing base excision damage resulting in carcinogenicity. In addition, the dysregulated miRNA expressions may also play an important role in tumor promotion in ASMC.

並列關鍵字

Mono-ethylhexyl phthalate ； Reactive Oxygen Species ； genotoxicity ； mutagenicity ； carcinogenicity ； Chinese Hamster Ovary ； AS52 ； AS52-mutant cells

參考文獻

ADIBI, J. J., PERERA, F. P., JEDRYCHOWSKI, W., CAMANN, D. E., BARR, D., JACEK, R. & WHYATT, R. M. 2003. Prenatal exposures to phthalates among women in New York City and Krakow, Poland. Environmental health perspectives, 111, 1719.

AL-KURAYA, K., SCHRAML, P., TORHORST, J., TAPIA, C., ZAHARIEVA, B., NOVOTNY, H., SPICHTIN, H., MAURER, R., MIRLACHER, M. & K CHLI, O. 2004. Prognostic relevance of gene amplifications and coamplifications in breast cancer. Cancer Research, 64, 8534-8540.

ALBRO, P. W., CHAPIN, R. E., CORBETT, J. T., SCHROEDER, J. & PHELPS, J. L. 1989. Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats. Toxicology and applied pharmacology, 100, 193-200.

ALBRO, P. W. & LAVENHAR, S. R. 1989. Metabolism of DI (2= Ethylhexyl) Phthalate. Drug metabolism reviews, 21, 13-34.

ANDERSON, D., YU, T. W. & HINCAL, F. 1999. Effect of some phthalate esters in human cells in the comet assay. Teratogenesis, carcinogenesis, and mutagenesis, 19, 275-280.

國際替代計量

鄰苯二甲酸二(2-乙基已基)酯及代謝物對哺乳類中國倉鼠卵巢細胞之細胞毒性及基因毒性

全文下載

主題瀏覽