Aluminum neurotoxicity to earthworms (Eisenia andrei)

鋁是一種普遍存在且使用廣泛的金屬，然而對生物而言是非必需元素。鋁在中性的土壤中通常溶解度很低，但一些人類的活動，特別是會使土壤酸化的動作如酸雨或施肥，會釋出鋁離子。這些釋出的鋁離子會影響很多生物。本研究中用蚯蚓(Eisenia andrei)為對象來了解鋁離子對蚯蚓的神經毒性。蚯蚓腦及腹神經索樣本以石蠟包埋後，組織切片以Walton stain或H&E stain進行染色，以了解鋁在神經系統內的累積情形及病理變化。Walton stain的結果顯示鋁會大量累積在腦的細胞中，而控制組中則僅有核仁和部份的胞外基質中出現微量鋁的累積。而在腹神經索中，也有類似的現象，但未若腦部顯著。H&E stain的結果顯示：與控制組比較，在鋁實驗組的腦部和腹神經索中，均出現明顯嗜伊紅(eosinophilic)區域。進一步實驗顯示，鋁累積區域和嗜伊紅區域有重疊現象。另外，前述切片經退染後再以剛果紅進行染色，結果顯示剛果紅染到的區域和鋁累積的區域，以及嗜伊紅區域間均有重疊現象。這樣的結果表示鋁會在蚯蚓神經系統，特別是腦造成近似類澱粉蛋白沉積的現象。為了進一步了解鋁的毒性機制，在本實驗中使用鈣離子、鐵離子，或四種抗氧化劑(EUK134、迷迭香酸、兒茶酚和薑黃素)和鋁同時處理蚯蚓，結果顯示僅有鈣離子能顯著延長蚯蚓的存活時間。然而，隨後的鑭離子（鈣離子通道抑制劑）對蚯蚓進行前處理，並未顯著延長蚯蚓之存活時間。依這些結果推測鋁進入蚯蚓體內與鈣離子有關，但不是透過鈣離子通道，而可能透過細胞間隙（paracellular route）進入蚯蚓體內，再經過運送進入神經細胞。進入神經細胞的鋁可能會造成近似類澱粉蛋白沉積的現象，產生毒性，而非經氧化壓力導致細胞死亡。Aluminum (Al) , a widely spread metal, is a non-essential element for most animals in the earth. Normally, aluminum solubility is low in neutral soil. But human activities, like the use of fertilizer, or acidic rain cause aluminum ion being release from the soil, which affect soil organisms such as earthworms due to its neurotoxicity. In this study, earthworms (Eisenia andrei) were exposed to aluminum ion (Al3+) to investigate its neurotoxicity. The results showed the cytoplasms in aluminum-exposed earthworms were heavily stained by Walton staining but not in the control group. In the brain, the proportion of stained area in aluminum-exposed group was significantly increased. In the ventral nerve cord, however, the increasing was not significant. The results of H&E staining significantly showed that large eosinophilic regions appeared both brain and the ventral nerve cord of the aluminum-exposed earthworms, but not in control earthworms. Furthermore, the aluminum accumulation region and the eosinophilic region showed similar patterns. Also, the Congo red staining result showed similar pattern to the aluminum accumulation region and the eosinophilic region. These results imply that the accumulation of aluminum may result in amyloidosis-like symptom. To realize the possible mechanism of aluminum intoxication, calcium ion (Ca2+), ferric ion (Fe3+) or four antioxidants (EUK134, rosmarinic acid, catechin and curcumin) were simultaneously used with aluminum to treat the earthworms. Calcium ion, but not ferric ion, EUK134, rosmarinic, catechin or curcumin could increase the survival time of the aluminum treated earthworms. The presence of lanthanum ion (La3+), a calcium channel blocker, did not significantly increase the survival time. These results inferred that aluminum entered earthworm body was not associated with calcium channel, but might be absorbed by the paracellular route