氨暴露導致斑馬魚胚胎離子調節損傷及成魚行為改變

Ammonia exposure impairs ion regulation in zebrafish embryos and changes behaviors in adult zebrafish

鄭倢安(Cheng Chieh-An)

指導教授 ： 林豊益

國立臺灣師範大學/理學院/生命科學系/碩士(2021年)

https://doi.org/10.6345/NTNU202100082

摘要

氨（包含氣態的NH3以及離子態的NH4+）為魚類代謝胺基酸後產生的主要含氮廢物，也是常見的環境汙染物。當魚體內氨濃度提高，將會導致魚隻中樞神經受損，抽搐、昏迷甚至死亡。然而，目前研究中多著重在高氨處理後魚類的適應機制，關於氨對魚隻離子調節功能及行為的毒性作用尚不清楚。本研究分為兩個部分，首先利用斑馬魚胚胎作為模式動物，探討氨如何對胚胎離子調節功能造成損傷，接著利用斑馬魚成魚作為模式動物，評估氨處理後斑馬魚的行為改變。在胚胎毒性研究中，浸泡於不同濃度（0、10、15、20 mM）的氯化銨溶液中96小時（4-100 hpf）後，觀察胚胎卵黃囊上離子細胞及表皮角質細胞。結果指出，20 mM氨處理後離子細胞內氧化壓力上升（CellROX螢光亮度顯著上升）且由Rhodamine 123標定的具粒線體活性離子細胞數目顯著下降，顯示粒線體活性降低。此外，以細胞免疫螢光染色標定20 mM氨處理後凋亡細胞數目顯著上升，並觀察到表皮角質細胞結構損傷。綜合以上結果發現，在高氨處理下，斑馬魚胚胎離子細胞及表皮角質細胞損傷，導致斑馬魚胚胎失去體表屏障，體內離子大量流失。而在行為實驗中，將斑馬魚浸泡於不同濃度（0、1、5、10 mM）的氯化銨溶液中4小時後，對游泳行為、社交行為、學習與記憶能力等面向進行不同實驗。結果顯示1 mM氨處理時可以促進學習記憶能力；5 mM時焦慮及恐懼程度提升且群游下降；10 mM氨處理時活動力、社交行為及焦慮程度下降，但恐懼程度上升。綜上所述，在不同濃度氨暴露以及不同的環境刺激下，斑馬魚的游泳、社交、學習等行為改變，而這些改變可能使斑馬魚存活率下降，進一步使個體適存度降低。

關鍵字

氨 ； 斑馬魚 ； 離子細胞 ； 表皮角質細胞 ； 細胞凋亡 ； 氧化壓力 ； 粒線體損傷 ； 行為改變

並列摘要

Ammonia (including NH3 and NH4+) is a toxic nitrogenous product of fish, and also a common environmental pollutant. Accumulation of ammonia is toxic to fishes causing convulsions, coma and death. However, the toxic effects of ammonia on fish ion regulation and behaviors are not fully understood. The 1st purpose of this study was to investigate how ammonia impairs ion regulation in fish embryos, and 2nd purpose was to investigate how ammonia influences behaviors in adult fish. Zebrafish were used in this study because it is a popular animal model for toxicological studies. In embryonic experiments, zebrafish embryos were exposed to NH4Cl (0, 10, 15, or 20 mM) for 96 h (4-100 hpf) and the ionocytes and keratinocytes in the skin were examined. Results showed that 20 mM NH4Cl exposure increased the oxidative stress (indicated by CellROX staining) and decreased mitochondria activity (indicated by rhodamine-123 staining) in the skin ionocytes. Also, 20 mM NH4Cl increased apoptosis (indicated by caspase immunostaining) and impaired the apical structure of keratinocytes. Taken together, high concentration ammonia impaired the skin ionocytes and keratinocytes, causing severe ion losses in zebrafish embryos. In behavior experiments, adult zebrafish were exposed to NH4Cl (0, 1, 5 or 10 mM) for 4 h, and several behaviors including swimming behavior, social behavior, learning and memory were analyzed. The results showed that 1 mM NH4Cl promoted learning behavior; 5 mM NH4Cl increased the anxiety level and fear response, but decreased the shoaling behavior; 10 mM NH4Cl decreased swimming activity, social behaviors, and anxiety level but increased fear response. Taken together, ammonia exposure altered the social behaviors, learning and memory in adult zebrafish.

並列關鍵字

ammonia ； apoptosis ； ionocyte ； keratinocyte ； mitochondria damage ； oxidative stress ； zebrafish ； behavioral alteration

參考文獻

Agetsuma, M., Aoki, T., Aoki, R., Okamoto, H., 2012. Cued fear conditioning in zebrafish (Danio rerio), Zebrafish protocols for neurobehavioral research. Springer, pp. 257-264.

Albrecht, J., Norenberg, M.D., 2006. Glutamine: a Trojan horse in ammonia neurotoxicity. Hepatology 44, 788-794.

Amo, R., Fredes, F., Kinoshita, M., Aoki, R., Aizawa, H., Agetsuma, M., Aoki, T., Shiraki, T., Kakinuma, H., Matsuda, M., 2014. The habenulo-raphe serotonergic circuit encodes an aversive expectation value essential for adaptive active avoidance of danger. Neuron 84, 1034-1048.

Arillo, A., Margiocco, C., Melodia, F., Mensi, P., Schenone, G., 1981. Ammonia toxicity mechanism in fish: studies on rainbow trout (Salmo gairdneri Rich.). Ecotoxicology and environmental safety 5, 316-328.

Baldisserotto, B., Martos-Sitcha, J.A., Menezes, C.C., Toni, C., Prati, R.L., Garcia, L.d.O., Salbego, J., Mancera, J.M., Martínez-Rodríguez, G., 2014. The effects of ammonia and water hardness on the hormonal, osmoregulatory and metabolic responses of the freshwater silver catfish Rhamdia quelen. Aquatic Toxicology 152, 341-352.