甲氧氯是一種有機氯殺蟲劑。研究顯示，甲氧氯是一種內分泌干擾劑，在許多不同細胞中會影響細胞的鈣離子恆定及細胞活性，然而它對人類肝癌的效應並不清楚。本研究在探討甲氧氯在人類肝癌細胞株HA59T細胞中對於細胞質內游離鈣離子移動與對於細胞凋亡的影響。研究方法是利用鈣離子敏感的螢光劑fura-2測量細胞內鈣離子濃度，WST-1測量細胞存活率，細胞凋亡之螢光染劑Alexa Fluor® 488 Annexin V-FITC和propidium iodide偵測細胞凋亡。結果顯示，甲氧氯在濃度範圍0.1-1 μM 之間會引起細胞內鈣離子濃度的上升且有濃度依賴的效應。移除細胞外的鈣離子會去除甲氧氯的作用。甲氧氯誘發之鈣離子流入可由錳離子對fura-2螢光減弱的效應來確認。甲氧氯誘發之鈣離子流入會被nifedipine、econazole、 SKF96365及蛋白質激酶C調節劑所抑制。甲氧氯在濃度範圍10-130 μM 之間會以濃度依賴的方式引起細胞死亡。使用BAPTA/AM 螯合細胞質中的鈣離子並不能避免甲氧氯引起的細胞毒性。此外，甲氧氯在濃度10 μM 及50 μM時會引起濃度依賴之細胞凋亡。總結來說，在HA59T人類肝癌細胞株中，甲氧氯會經由打開蛋白質激酶C敏感之鈣離子可通透通道來引發鈣離子流入細胞且不會引起鈣池的鈣離子釋出，甲氧氯也會引發與鈣離子濃度上升無關之細胞凋亡。

Methoxychlor is an organochlorine pesticide. It has been shown that methoxychlor is thought to be an endocrine disrupter that affects Ca2+ homeostasis and cell viability in different cell models. However, the effect of methoxychlor on human hepatoma cell is unclear. This study explored the action of methoxychlor on cytosolic free Ca2+ concentrations ([Ca2+]i) and apoptosis in HA59T human hepatoma cells. Fura-2, a Ca2+-sensitive fluorescent dye, was applied to measure [Ca2+]i. Viability was measured by using the probe WST-1. Apoptosis was measured by Alexa Fluor® 488 Annexin V/propidium iodide (PI) staining. Methoxychlor at concentrations of 0.1-1 µM caused a [Ca2+]i rise in a concentration-dependent manner. Removal of external Ca2+ abolished methoxychlor’s effect. Methoxychlor-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Methoxychlor-induced Ca2+ entry was inhibited by nifedipine, econazole, SKF96365, and protein kinase C modulators. Methoxychlor killed cells at concentrations of 10-130 µM in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid/AM (BAPTA/AM) did not prevent methoxychlor’s cytotoxicity. Furthermore, methoxychlor (10 and 50 µM) induced apoptosis concentration- dependently. Together, in HA59T cells, methoxychlor induced a [Ca2+]i rise by inducing Ca2+ entry via protein kinase C-sensitive Ca2+-permeable channels, without causing Ca2+ release from stores. Methoxychlor also induced apoptosis that was independent of [Ca2+]i rises.

1 Introduction+1
2 Materials and methods+5
2.1 Chemicals+5
2.2 Cell culture+ 5
2.3 Solutions used in [Ca2+]i measurements+5
2.4 [Ca2+]i measurements+6
2.5 Cell viability assays+7
2.6 Alexa Fluor® 488 Annexin V/PI staining for detection of apoptosis+8
2.7 Statistics+8
3 Results+9
3.1 Concentration-dependent effect of methoxychlor on [Ca2+]i+9
3.2 Methoxychlor-induced Mn2+ influx+9
3.3 Modulations of methoxychlor-induced [Ca2+]i rise+10
3.4 Effect of methoxychlor on cell viability+10
3.5 Lack of a relationship between methoxychlor-induced [Ca2+]i rise and cell death+11
3.6 A possible involvement of apoptosis in methoxychlor-induced cell death+11
4 Discussion+12
5 Figure legends+15
5.1 Effect of methoxychlor on [Ca2+]i in fura-2 loaded cells+15
5.2 Effect of methoxychlor on Ca2+ influx by measuring Mn2+ quenching of fura-2 fluorescence+17
5.3 Effect of Ca2+ channel modulators on methoxychlor-induced [Ca2+]i rise+18
5.4 Effect of methoxychlor on viability of cells+20
5.5 Apoptosis induced by methoxychlor as measured by Annexin V/PI staining+21
6 References+23

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