五氯酚(PCP, pentachlorophenol)為一種常被使用於木材保存劑及殺草劑備製的化學物質，已被證實是一種環境污染物，其代謝產物四氯對苯二酚TCHQ (tetrachlorohydroquinone)一直被認為是五氯酚的主要毒性來源。在過去的研究中發現TCHQ在小鼠的致癌模式中扮演promoter的角色，但是對於其中的機制仍不清楚，因此本篇的研究目的在於探討Bcl-2蛋白在TCHQ誘發細胞凋亡中所扮演的角色，同時探討其他可能導致細胞凋亡之訊號傳遞路徑。使用正常及Bcl-2過度表現的兩株NIH3T3細胞進行實驗，在研究中發現，經TCHQ處理後可引發Bcl-2表現量的增加，而Bcl-2的過度表現又可抑制細胞凋亡，因此推測TCHQ的promotion作用可能與促進Bcl-2表現量的增加有直接的關係。在給予TCHQ的NIH3T3細胞中可以明顯觀察到活性氧物種(ROS)的增加，DNA損傷及細胞凋亡的發生，而在Bcl-2過度表現的NIH3T3細胞中則發現DNA損傷及細胞凋亡的程度降低，但卻不能有效降低ROS的量，因此推測Bcl-2的作用是在ROS生成的下游位置。另一方面，在訊號傳遞路徑的研究中主要針對與DNA損傷有直接相關的兩條路徑作探討，即ATM (ataxia telangiectasia mutated)-dependent pathway和MAPK (mitogen-activated protein kinases) pathway。TCHQ可引發NIH3T3細胞ATM蛋白的表現，但卻沒有觀察到下游p53及Bax等蛋白的累積，且加入wortmannin (ATM-inhibitor)後細胞凋亡的現象並沒有明顯的下降，但是卻可以觀察到與MAPK有關的phospho-p38和phospho-JNK表現量增加。根據以上結果推測，四氯對苯二酚引起細胞凋亡的訊號傳遞路徑可能與MAPK pathway有較直接的關係。

Tetrachlororhydroquinone (TCHQ) is the major toxic metabolite of widely used wood preservative pentachlorophenol (PCP). Recently, we found that TCHQ was a promoter in a mouse skin carcinogenesis model. However, the mechanism is still not clear. In this study, we intended to discover the role of Bcl-2 in TCHQ-induced apoptosis and its related possible signaling pathways. Over-expression of Bcl-2 effectively suppresses TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphological changes and DNA fragmentation. Although production of reactive oxygen species (ROS) contributes to TCHQ-induced apoptosis, Bcl-2 fails to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, over-expressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. These data suggest that Bcl-2 prevents TCHQ-induced apoptosis at the downstream of oxidative damage events. Interestingly, TCHQ induced a significant up-regulation of Bcl-2 expression, and over-expressed Bcl-2 can inhibit TCHQ induced apoptosis. Thus, our results suggest TCHQ-induced tumor promotion may be through a mechanism of up-regulation of Bcl-2 protein and subsequent apoptosis inhibition. On the other hand, we found that TCHQ increased ATM (ataxia telangiectasia mutated) protein expression, but did not induce downstream p53 accumulation and Bax up-regulation. We also found that TCHQ induced both phospho-p38 and phospho-JNK expression, which belongs to MAPK (mitogen-activated protein kinases) cascade pathway. According to these results, the possible signaling pathway of TCHQ-induced apoptosis may relate to MAPK pathway instead of ATM-dependent pathway.

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