| Abstract: | 棒曲毒素（patulin, 簡稱PAT）是一種由青黴菌（Penicillium）和麴菌（Aspergillus）菌屬所分泌的黴菌毒素（mycotoxin），通常在發霉的水果和其再製品中被偵測出來。當以PAT處理人類胚胎腎臟細胞株（human embryonic kidney cell line，簡稱HEK293）後，發現隨著PAT的劑量增加及處理時間的延長會增加ERK1/2、p38 和 JNK的磷酸化蛋白表達量，同樣地在人類周邊血液單核球細胞（peripheral blood mononuclear cells，簡稱PBMCs）和犬類腎臟細胞株（Madin-Darby canine kidney cell line，簡稱MDCK）中，ERK1/2磷酸化蛋白的量也會增加。使用ERK1/2途徑的專一性抑制劑U0126處理HEK293細胞，會令PAT所誘發的ERK1/2蛋白磷酸化的現象被抑制，而且PAT所引起的DNA損傷的情形也會顯著降低；但是U0126並不會改變由PAT所導致的細胞死亡、乳酸去氫的釋放和DNA合成等現象。微陣列晶片的結果顯示，以PAT處理HEK293細胞後，可令早期生長基因（early growth response gene-1, egr-1）mRNA的表達量上升。從p38和JNK訊息傳導途徑的角度來看，使用p38途徑專一性的抑制劑SB203580會降低p38的磷酸化蛋白表達量，並且增加HEK293細胞的存活率，同時也會延緩PAT所引起的細胞形態的傷害，但是JNK的抑制劑SP600125卻不會產生相同的效應。在JNK途徑中，PAT除了可以引起JNK磷酸化蛋白的表達量上升，另外位於JNK上游的MKK4蛋白和下游的ATF-2及c-JUN蛋白也都有磷酸化的現象。PAT會抑制細胞中蛋白質的生合成，使用double-stranded RNA (dsRNA)-dependent protein kinase R（簡稱PKR）的抑制劑adenine會抑制MAPKs訊息傳導途徑的磷酸化現象，由此推測PAT是藉由ribotoxic stress response來誘發MAPK訊息傳導途徑。以PAT處理人類白血病細胞株（HL-60）時發現超氧陰離子（superoxide anion）在細胞內的含量增高，然而SB203580的前處理會使得超氧陰離子的含量比只處理PAT時來的高，這個結果顯示p38訊息傳導途徑與PAT所刺激產生的氧化壓力相關。綜合以上實驗數據可知，PAT在HEK293細胞株中會造成MAPKs訊息傳導途徑的快速活化，其中ERK1/2途徑的活化與PAT所造成的DNA損傷及egr-1基因mRNA表達量上升有密切關聯，而細胞形態的改變與自由基的產生則和p38訊息傳導途徑有高度的相關性。JNK訊息途徑的活化則並未參與上述所觀察到的現象。PAT可能是透過ribotoxic stress response來活化MAPKs訊息傳導途徑。
Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration- and time-dependent increase in phosphorylation of ERK1/2, p38 and JNK in human embryonic kidney (HEK293) cells. Phosporylated ERK1/2 was also observed in PAT-treated PBMC and MDCK cells. In HEK293 U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage. Conversely, U0126 did not affect cell death, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated culture. In cDNA microarrays assays, exposure of HEK293 cells to PAT elevated the levels of early growth response gene-1 (egr-1) mRNA. On the other hand, the p38 inhibitor, SB203580, markedly suppressed p38 activation and significantly reduced the cell death caused by PAT. Nevertheless, inhibition of the JNK signal by SP600125 did not exhibit such effects. Neither p38 nor JNK played a role in PAT-elicited DNA damage. In PAT-treated cells, inactivation of double-stranded RNA-activated protein kinase R (PKR) with an inhibitor adenine strongly prevented the phosphorylation of JNK and ERK. On the other hand, administration of HEK293 cells with PAT-cysteine adducts, a chemical derivative of PAT, did not modulate MAPK signaling pathways, cell viability and DNA integrity. Exposure of HL-60 cells with PAT elevated the cellular superoxide anion amounts, and the pre-treatment of SB203580 further increased the superoxide anion levels, suggesting that p38 pathways is involved in PAT-elicited oxidative stress. In conclusion, PAT causes a rapid and persistent activation of MAPKs in human cells. ERK1/2 pathway plays an important role in mediating PAT-induced DNA damage and the p38 signaling pathway contributes to the PAT-modulated cell death and ROS production. PKR also plays a role in PAT-mediated MAPK activation. |
