張云龍張海龍王凌宇顧貝易樊啟學(xué)

(1. 安徽農(nóng)業(yè)大學(xué)動物科技學(xué)院, 合肥 230036; 2. 華中農(nóng)業(yè)大學(xué)水產(chǎn)學(xué)院, 農(nóng)業(yè)部淡水生物繁育重點實驗室, 武漢 430072)

氨氮對魚類毒性的影響因子及氣呼吸型魚類耐氨策略

張云龍1張海龍2王凌宇2顧貝易2樊啟學(xué)2

(1. 安徽農(nóng)業(yè)大學(xué)動物科技學(xué)院, 合肥 230036; 2. 華中農(nóng)業(yè)大學(xué)水產(chǎn)學(xué)院, 農(nóng)業(yè)部淡水生物繁育重點實驗室, 武漢 430072)

氨氮廣泛存在于養(yǎng)殖水體中, 在氨氮過高的養(yǎng)殖環(huán)境下可能會導(dǎo)致魚類的大量死亡。從生態(tài)、環(huán)境及養(yǎng)殖效益角度來看, 研究氨氮對魚類的毒性以及魚類應(yīng)對環(huán)境或體內(nèi)高氨濃度的策略均具有重要意義。某些魚類具有其特殊的策略來降低氨毒性, 使得這些種類能適應(yīng)極高的環(huán)境或體內(nèi)氨濃度。這些耐氨策略主要為(1)合成谷氨酰胺、(2)合成尿素排出、(3)增強機體排泄、(4)Rh蛋白促進氨解毒、(5)降低周圍環(huán)境pH、(6)NH3揮發(fā)和體表堿化、(7)降低體內(nèi)氨生成、(8)特定氨基酸代謝生成丙氨酸、(9)組織高氨耐受性。魚類的氨耐受策略較多而且是可變的, 主要受特定種類的生活習(xí)性和棲息環(huán)境影響。文章綜述了氨氮對魚類的毒性機理以及魚類的應(yīng)對策略, 為相關(guān)的研究提供基礎(chǔ)資料。

氣呼吸型魚類; 氨氮; 氨毒性; 耐氨策略

人工飼料的過量投喂, 是導(dǎo)致養(yǎng)殖水體中氨氮含量偏高的重要原因之一。氨氮對水生動物具有較強的毒性, 可引起其抽搐、昏迷和死亡, 這可能是由于過高濃度的取代了K+以及神經(jīng)元去極化, 隨后會造成中樞神經(jīng)系統(tǒng)細胞的死亡[1]。大量研究也報道了氨氮對魚類的毒性作用, 如草魚(Ctenopharyngodon idella)[2]、黃顙魚(Pelteobagrus fulvidraco)[3]以及舌齒鱸(Dicentrarchus labrax)[4]等。環(huán)境中氨氮由農(nóng)業(yè)徑流以及生物廢棄物分解產(chǎn)生, 然而魚類機體中的氨主要是由氨基酸代謝產(chǎn)生。魚類消化食物中的蛋白質(zhì)會產(chǎn)生氨基酸, 但魚類卻不能貯存多余的氨基酸, 因此除去用于生長所需, 魚類攝入食物中過量的氨基酸和蛋白質(zhì)會轉(zhuǎn)化成脂肪和碳水化合物儲存于肝臟中[5], 而轉(zhuǎn)化過程需要去氮, 氨則在這一過程中產(chǎn)生。由于氨對水生動物的高毒性, 在通常情況下魚組織或器官中氨含量應(yīng)該保持在較低的水平[5]。當(dāng)魚體內(nèi)氨含量達到了一定量的時候, 其多數(shù)魚類會表現(xiàn)出致死作用。但是, 某些特殊種類卻具有其獨特的氨耐受機制,以應(yīng)對環(huán)境或者體內(nèi)過高的氨濃度。本文依據(jù)國內(nèi)外的研究資料, 系統(tǒng)地總結(jié)了氨對魚類毒性的影響因子及氣呼吸型魚類的耐氨策略, 為今后相關(guān)的研究提供基礎(chǔ)資料。

1 氨氮對魚類的毒性

氨氮廣泛存在于水環(huán)境中, 由動植物排放或者微生物分解有機質(zhì)產(chǎn)生[1]。而水生動物體內(nèi)的氨則主要是由于氨基酸代謝產(chǎn)生的, 動物腸道分解食物中的蛋白質(zhì)是氨基酸的主要來源[5,6]。研究表明魚類攝入食物中40%—60%的氮會在24h內(nèi)排泄出[7,8]。除此之外, 魚類在饑餓狀態(tài)下也會將肌肉蛋白質(zhì)代謝為氨基酸, 以提供ATP源或者碳水化合物[5,9]。而當(dāng)暴露于較高的環(huán)境氨氮中時, 魚類會降低自身的氨基酸代謝速度以減少體內(nèi)氨的生成,以保護機體免于氨氮毒性[7,10]。已有大量的研究報道了氨氮對魚類的毒性, 如高環(huán)境氨氮會導(dǎo)致虹鱒(Oncorhynchus mykiss)、鯉(Cyprinus carpio)和鯽(Carssius auratus)鰓結(jié)構(gòu)異化[11], 也會導(dǎo)致牙鲆(Paralichthys olivaceus)鰓結(jié)構(gòu)變化[12]、造成薄氏大彈涂魚(Boleophthalmus boddaerti)大腦氧化應(yīng)激反應(yīng)[13]、引起軍曹魚(Rachycentron canadum)鰓、食管和大腦的組織損傷[14]等, 類似的研究還可見亞馬遜沼蝦(Macrobrachium amazonicum)[15]、凡納濱對蝦(Litopenaeus vannamei)[16]、高體雅羅魚(Leuciscus idus)[17]、細鱗肥脂鯉(Piaractus mesopotamicus)[18]、小鋸蓋魚(Centropomus parallelus)[19]等。

在液態(tài)環(huán)境下, 氨可以非離子態(tài)的NH3和離子態(tài)的兩種形態(tài)存在, 其轉(zhuǎn)化方程式為NH3+H3O–=此反應(yīng)的解離常數(shù)(pKa)為9.0—9.5左右。魚類血液的pH較氨反應(yīng)的pKa低, 因此氨在魚類血液中主要(＞95%)以的形式存在[5]。血氨可通過血液循環(huán)累積至不同的組織中, 但氨具有較強的細胞毒性[1,9,20], 機體必須排泄多余的氨。氨可激活細胞液中的磷酸果糖激酶I以促進機體糖酵解作用, 也可通過影響核糖體三羧酸循環(huán)來干擾機體能量代謝[5]。也有研究表明高濃度氨氮暴露可通過增加谷氨酸鹽的釋放量或者(以及)降低谷氨酸鹽的重吸收致使細胞外谷氨酸鹽含量的升高[21]。此外, N-甲基-D-天門冬氨酸型谷氨酸(N-methyl-D-aspartate-type glutamate, NMDA)受體也被大量的激活, NMDA的大量增加導(dǎo)致機體Ca2+及Na+的大量增加[22]。高濃度氨氮暴露引起的過量NMDA具有神經(jīng)毒性, 可引起氧化應(yīng)激反應(yīng), 造成神經(jīng)元變形和死亡[23]以及線粒體滲透性的改變。在體內(nèi)高氨條件下, 線粒體滲透性的改變會導(dǎo)致谷氨酸鹽通過線粒體膜的滲透以及通過星形膠質(zhì)細胞線粒體基質(zhì)中的谷氨酰胺酶生成氨[5,9,24,25]。但與哺乳動物相比, 魚類通常具有較強的氨氮耐受能力, 這可能是由于其中樞神經(jīng)系統(tǒng)沒有其他高等脊椎動物的中樞神經(jīng)系統(tǒng)發(fā)達[26]。

NH3可滲透過大多數(shù)生物細胞膜, 但的滲透性則相對小的多[1], 因此氨氮毒性很大程度上取決于這兩種氨形態(tài)的存在比例。而兩種氨形態(tài)的轉(zhuǎn)化在正常情況下是趨于平衡, 而外界環(huán)境(如pH、溫度、鹽度)以及動物自身狀況均可影響兩種氨形態(tài)的轉(zhuǎn)化平衡, 因此氨氮對魚類的毒性也受到許多因素的影響, 主要有以下幾類:

1.1 主要環(huán)境因子影響氨氮對魚類的毒性

pH在水生動物內(nèi)穩(wěn)態(tài)中也起到重要作用, 研究表明pH的變化可影響動物體內(nèi)的酸堿平衡、離子調(diào)節(jié)以及氨排泄[27]。已有大量研究闡述了pH對氨氮毒性的影響, 介質(zhì)的pH越高則NH3的存在比例越高, 相對來說毒性也就越大, 可見斑點叉尾(Ictalurus punctaus)[28]、克林雷氏鯰(Rhamdia quelen)[29]、金體美鳊(Notemigonus crysoleucas)[30]等。

與pH相比, 溫度對離子態(tài)氨向非離子態(tài)轉(zhuǎn)化的影響作用要小的多, 但也有研究表明溫度升高會增強氨氮對水生動物的毒性[31]。K?r等[32]研究發(fā)現(xiàn)了26℃時總氨氮(Total ammonia-nitrogen, TAN)對短溝對蝦(Penaeus semisulcatus)的安全濃度是14℃的4倍, 非離子氨的安全濃度則為2倍。將溫度從15℃升高至25℃后, 氨氮暴露24、48、72以及96h后, 細鱗肥脂鯉對氨氮的敏感性分別上升21.80%、9.55%、31.92%和30.87%[18]。相似的, 隨著溫度的升高, 大西洋白姑魚(Argyrosomus regius)對氨氮的耐受性明顯地降低[33]。

鹽度也被證實對氨氮毒性具有一定的影響, 在多數(shù)魚類的研究中均發(fā)現(xiàn)降低鹽度會提高銨態(tài)氮和亞硝態(tài)氮對魚類的毒性作用, 如南方濱對蝦(Litopenaeus schmitti)[34]、鋸齒長臂蝦(Palaemon serratus)[35]等。鹽度對氨氮毒性的影響機制可能是由于鹽度升高會增加鈉和鈣的含量, 這兩種物質(zhì)已被證明可通過增加鰓細胞和Na+的交換來促進機體氨的排泄, 從而阻止氨氮通過滲透作用流入細胞內(nèi)[36]。

1.2 運動對氨氮毒性的影響

運動是魚類的重要生理活動之一, 低密度下魚類會進行常規(guī)游動, 高密度下會進行力竭游動, 魚類依靠游泳來適應(yīng)水流速度、捕食、逃跑以及洄游等[37]。力竭游動時魚類的能量源主要來自于白肌的無氧糖酵解[38]。此外, 力竭游動也被證明會加速機體氨的生成、提高機體氨含量[1], 也就是會放大氨氮的毒性。在虹鱒和銀大馬哈魚(Oncorhynchus kisutch)的研究中已經(jīng)發(fā)現(xiàn)劇烈游動會誘導(dǎo)其血液氨濃度的上升[39]。對鯉[37]和鯽[40]的研究中也發(fā)現(xiàn)氨氮暴露明顯降低了魚類的游動能力。這可能是因為氨氮環(huán)境下的增加改變了魚類機體的代謝方式[41],還會替代K+造成肌肉細胞膜去極化, 對肌肉造成損傷[42]。因此, 游動和氨氮暴露對魚類是兩個相互作用的因素, 游動放大了氨氮毒性, 氨氮暴露降低了魚類的游動能力。然而這種相互作用的機制以及其生理生化水平上的表現(xiàn)等均缺乏深入的研究報道, 這也是以后值得研究的一個方向。

1.3 攝食對氨氮毒性的影響

在魚類攝食之后, 大量的蛋白質(zhì)被分解以維持機體的正常生理活動, 而機體內(nèi)氨的生成和排泄明顯增加[1,43]。大量的氨生成會導(dǎo)致細胞內(nèi)堿中毒,引起一系列的魚類病理反應(yīng)[44]。就此來說, 當(dāng)魚類暴露于氨氮環(huán)境下, 相比于投喂的魚類, 饑餓似乎更有利于魚類應(yīng)對氨氮毒性。但是在對鯽的研究中卻得出了相反的結(jié)論[40,45], 他們發(fā)現(xiàn)投喂的鯽比饑餓的鯽對氨氮的耐受性更高。魚類具有大量、可調(diào)節(jié)的生理生化活動, 其對營養(yǎng)狀況具有較強的適應(yīng)調(diào)節(jié)性, 如離子平衡、代謝、內(nèi)分泌等[46]。因此, 食物也會對魚類應(yīng)對氨氮毒性起到一定的作用。Wicks和Randall[43]發(fā)現(xiàn)虹鱒在攝食之后, 谷氨酰胺合成酶活力明顯上升, 其可將血液中過量的氨轉(zhuǎn)化為無毒的谷氨酰胺, 這一現(xiàn)象在肌肉中表現(xiàn)尤為明顯?？傮w來說, 谷氨酰胺合成酶活力的上升可能就是某些魚類攝食之后具有較強氨氮耐受力的原因。

2 魚類的氨耐受策略

多數(shù)魚類都是以排氨為主要的氨代謝途徑, 但也有一些魚類可將氨氮代謝為谷氨酸鹽或者尿素以降低氨氮毒性。某些魚類的大腦具有很高的氨氮耐受性, 也可通過加快的轉(zhuǎn)運來加速氨排泄、調(diào)控周圍環(huán)境pH以及降低鰓上皮和皮膚的氨滲透性以降低氨氮毒性。此外, 也有大量報道認為氨氮的排泄與魚類鰓上皮和皮膚上的Rh糖蛋白(Rhesus glycoproteins, Rh)相關(guān)。本文綜述了幾種魚類常用的生理策略, 以應(yīng)對氨氮環(huán)境暴露。

2.1 合成谷氨酰胺

在魚體內(nèi), 谷氨酰胺合成酶(Glutamine synthetase, GS)可催化谷氨酸和合成谷氨酰胺, 而谷氨酸則是由谷氨酸脫氫酶(Glutamate dehydrogenase, GDH)催化α-酮戊二酸和合成的。而谷氨酰胺的合成來源于谷氨酸還是α-酮戊二酸主要是由細胞內(nèi)GS活性決定的[5]。谷氨酰胺在魚類大腦降解氨氮毒性中起到了重要的作用, 如海灣豹蟾魚(Opsanus beta)、尖齒胡鯰(Clarias gariepinus)、虹鱒及黃鱔(Monopterus albus)等[25,47—50], 谷氨酰胺合成后會從各組織中作為一種無毒的氨代謝物釋放到血液中, 最后進入肝臟中。通常來說, 魚類肌肉中GS活性是很低的[51], 但在一些氣呼吸型魚類在空氣暴露或氨氮暴露條件下卻可在大腦和肝臟及肌肉中將氨代謝為谷氨酰胺。這些氣呼吸型魚類的肝臟、肌肉及腸道組織中均可檢出較高的GS活性[52]。以谷氨酰胺的形式應(yīng)對較高的體內(nèi)氨氮含量具有其特定的優(yōu)勢, 谷氨酰胺可儲存于機體中, 當(dāng)環(huán)境條件改善后, 這些谷氨酰胺可用于合成嘌呤、嘧啶及黏多糖等機體生理活動[5]。

由于GDH涉及聯(lián)合脫氨作用, 因此魚類機體中谷氨酰胺含量的增加不太可能是因為GDH胺化反應(yīng)導(dǎo)致谷氨酸含量增加。云斑尖塘鱧(Oxyeleotris marnorata)在攝食之后12h之后腸道GDH含量顯著增加[6], 谷氨酸含量的增加可能是魚類應(yīng)對攝食后體內(nèi)氨氮含量顯著升高的主要措施之一[9]。魚類腸道中過量的谷氨酸可能會被轉(zhuǎn)運至肝臟和肌肉中,促進氨基酸合成用以細胞容積調(diào)節(jié)[53,54]。肝臟是谷氨酸代謝的主要場所, 因此腸道通過GDH胺化作用合成谷氨酰胺轉(zhuǎn)運至肝臟中是非常必要的[55]。此外, 過量的游離氨基酸并不會用于合成蛋白且會在一些必要的生理活動中代謝產(chǎn)生氨氮。因此, 腸道和肝臟在魚類以谷氨酰胺形式進行氨氮解毒的過程中是互相協(xié)作的關(guān)系。

在氨氮暴露下, 魚體內(nèi)谷氨酰胺累積的報道已見于多種魚類。云斑尖塘鱧暴露于空氣中72h后其肌肉中谷氨酰胺含量增加了3倍, 而肝臟中谷氨酰胺含量卻在暴露24h后到達峰值, 說明了肝臟中谷氨酰胺可能隨后轉(zhuǎn)移至肌肉中存貯[56]?？諝獗┞?8h后泥鰍(Misgurnus anguillicaudatus)大腦、肌肉以及肝臟中谷氨酰胺含量顯著增加, 而GDH活性則明顯降低[57]?？諝獗┞饵S鱔72h后, 其體內(nèi)谷氨酰胺含量達到最大峰值, 其肝臟GS活性在空氣暴露144h后明顯高于對照組[58]。虹鱒暴露于670和1000 μmol/ L NH4Cl溶液24h和96h之后, 相比于空白對照組, 其大腦谷氨酰胺含量升高而谷氨酸含量降低[50]。中華烏塘鱧(Bostrychus sinensis)暴露于含有15 mmol/L的海水中6d之后, 其腸道GS和GDH活性均顯著提高[59]。此外, 空氣暴露中華烏塘鱧24h內(nèi)其肌肉中累積谷氨酰胺, 而在48h后則又恢復(fù)至正常水平, 說明魚體內(nèi)累積的谷氨酰胺可能通過某些同化作用轉(zhuǎn)化為其他的含氮化合物[60]。類似的研究報道還可見于許氏齒彈涂魚(Periophthalmodon schlosseri)、薄氏大彈涂魚、海灣豹蟾魚、尖齒胡鯰及仿刺參(Apostichopus japonicus)等[24,25,47,61]。

2.2 合成尿素排出

在脲生成和排尿素的動物中, 其保持體內(nèi)較低氨含量最主要的措施即將氨轉(zhuǎn)化為尿素, 再通過尿液排出體外[9]。尿素合成主要在動物肝臟內(nèi)進行,這一過程被稱為鳥氨酸-尿素循環(huán)(Ornithine-urea cycle, OUC)。就魚類來說, 并非所有的魚類都可生成脲, 但也有部分魚類具有功能性的OUC, 具有脲生成功能, 如軟骨魚類中的板鰓亞綱[62]。一些脲生成型魚類的OUC可憑借肝臟細胞中的氨甲酰磷酸合成酶III將谷氨酰胺轉(zhuǎn)化為低毒的尿素, 尿素分子更小, 易于排出。投喂許氏齒彈涂魚[7]和細鱗非洲肺魚(Protopterus dolloi)[8]24h之后, 其尿素合成速度和排泄速率均明顯增加, 也有研究發(fā)現(xiàn)板鰓類攝食之后尿素合成速度提高, 但其尿素主要用于調(diào)控機體的滲透壓而非排出體外[20]。

相比于脲生成型魚類, 僅有少量魚類具有排泄尿素的功能。海灣豹蟾魚在擁擠脅迫下可以尿素形式排泄出50%的氮代謝廢物[63], 而阿部鯔鰍虎魚(Mugilogobius abei)在氨氮暴露條件下只能以很少量尿素的形式排泄體內(nèi)氨[64]。然而, 對魚類來說,合成尿素對能量的消耗是巨大的, 研究表明每合成1 mol尿素需要消耗5 mol ATP[9]。此外, 魚類在水中都是排氨的, 而且一些氣呼吸型魚類在空氣暴露條件下也有許多策略來應(yīng)對體內(nèi)較高的氨濃度。對一些氣呼吸型魚類的研究中發(fā)現(xiàn), 當(dāng)其處于較高的體內(nèi)氨濃度(空氣暴露)或者較高的環(huán)境氨濃度(氨氮暴露)時, 多數(shù)魚類并不以合成尿素作為主要的氨耐受策略, 如云斑尖塘鱧[56]、泥鰍[57,65]、黃鱔[58]、大鱗副泥鰍(Paramisgurnus dabryanus)[66]及龜殼攀鱸(Anabas testudineus)[67]等。在已有的報道中, 通過OUC以尿素作為主要氨排泄的魚類只有格氏雀麗魚(Alcolapia grahami)一種, 其生活環(huán)境pH高達10左右, 在這種情況下氨排泄受到嚴(yán)重的阻礙。因此, 其通過OUC合成尿素的能力很強, 以保證機體免于氨氮毒性[9]。盡管合成尿素并不是多數(shù)魚類應(yīng)對高濃度氨氮的主要策略, 但其在氨氮環(huán)境下仍會增加尿素的合成和累積, 如細鱗非洲肺魚[68]、石花肺魚(Protopterus aethiopicus)和非洲肺魚(Protopterus annectens)[69]等。由于尿素合成是非常耗能的, 而且肺魚合成尿素也并非為了降解氨氮毒性,由此可推斷其合成尿素是其某些生理活動所需要,如維持夏眠[9]。

理論上來說, 魚類在氨氮或者空氣暴露條件下,最有效的應(yīng)對措施是活躍的排泄, 這樣可以確保機體內(nèi)較低的氨水平, 以保護大腦免于氨氮毒性[5,9]。一些氣呼吸型魚類會特化其鰓或者輔助呼吸器官以適應(yīng)快速的排泄, 這在龜殼攀鱸[67]、尖齒胡鯰[70]及許氏齒彈涂魚[71,72]上已得到證實。大量的研究表明魚類的鰓具有一定的可塑性[73], 環(huán)境條件的變化以及個體自身的發(fā)育均可能導(dǎo)致魚類鰓表面積的變化, 如水體中溶解氧狀況[73]、離子強度[74]及稚魚向成魚轉(zhuǎn)換[75]等。鰓功能異化在氣呼吸型魚類中可能表現(xiàn)的更為明顯, 因為其可能會暴露于空氣中, 無法通過鰓與水體進行物質(zhì)交換。泥鰍和花溪鳉在空氣暴露狀態(tài)下可通過腸道或者皮膚揮發(fā)NH3以排泄體內(nèi)一部分的氨[65,76], 許氏齒彈涂魚在這種情況下會在其鰓腔中保留少量的水以保證的快速排泄[77], 而龜殼攀鱸暴露于空氣中仍可通過鰓和皮膚快速的排泄體內(nèi)過量的氨, 排泄速度甚至比正常狀態(tài)下更快[67]。

仔稚魚階段鰓結(jié)構(gòu)的形態(tài)發(fā)育對其氨排泄具有重要的意義。在胚胎以及鰓功能不完善的仔魚階段, 其主要以皮膚作為主要的氨排泄位點, 這在卵黃囊膜上尤為明顯[83]。在仔稚魚鰓功能成熟之后, 其氨排泄也就與成魚相似, Na+和的交換與NHE、氫ATP酶及Rh糖蛋白密切相關(guān)[83,84]。仔稚魚氨排泄位點從皮膚向鰓轉(zhuǎn)換的標(biāo)志在于鰓小片結(jié)構(gòu)的分化、成熟[85]。

2.4 Rh蛋白的作用

Rhesus糖蛋白(Rhgp, Rhesus glycoproteins)是溶質(zhì)轉(zhuǎn)運家族SLC42中的一員, 在氨跨膜轉(zhuǎn)運中起重要的作用[86]。Rh基因在魚類中最先報道于紅鰭東方鲀(Takifugu rubripes), 包括Rhag (Rh-associated glycoprotein)、Rhcg1、Rhcg2以及Rhbg[87]。Rh蛋白促進氨排泄的可能機制有以下3個: (1) 促進NH3的擴散, (2) 電中性的交換, (3) 產(chǎn)電的轉(zhuǎn)運[88]。后續(xù)的研究發(fā)現(xiàn), 其他硬骨魚類中也發(fā)現(xiàn)一種或者多種與氨轉(zhuǎn)運相關(guān)Rh蛋白的表達, 如虹鱒[89]、斑馬魚(Danio rerio)[83]、海灣豹蟾魚[90]、鯉[91]、大西洋盲鰻(Myxine glutinosa)[92]等, 這些結(jié)果說明了Rh蛋白在硬骨魚類中普遍存在, 且與其氨排泄具有密切關(guān)系。

Wright和Wood[88]認為淡水魚類存在一種交換綜合體”, 包含一些膜轉(zhuǎn)運蛋白, 這些轉(zhuǎn)運蛋白共同協(xié)作促進鰓的氨排泄, 而且其與Na+的攝入及酸排泄均有著動態(tài)的聯(lián)系。在這一模式下, 血漿和紅細胞均可以將氨帶入鰓組織中。由于pH得差異及膜滲透性, 氨在這一狀態(tài)下主要是以離子態(tài)的形式存在, 因此紅細胞內(nèi)部的總氨濃度要明顯的高于血漿中的濃度(約3—4倍)。然而,紅細胞與血漿之間存在一個明顯的氨濃度差, 這種非穩(wěn)態(tài)的條件會促進血液流經(jīng)鰓組織[88]。這一現(xiàn)象會導(dǎo)致紅細胞中的Rhag促進NH3由紅細胞流向血漿中。如果鰓上皮和鰓上血管之間柱狀細胞中含有Rhag, 其還會進一步促進NH3流向鰓上皮細胞。之后, NH3可以通過Rhbg (Rhbg 1 and/or 2)從鰓細胞膜基底以及通過Rhcg (Rhcg 1 and/or 2)從鰓細胞膜頂層排泄出體外, 以降低紅細胞與血漿之間的氨濃度差。然而, 在正常的生理狀態(tài)下, 氨的排泄可能更加依賴于代謝產(chǎn)生的CO2在碳酸酐酶催化下的水合作用, 以此來提供額外的H+用于酸化[88]。在斑馬魚H+質(zhì)子泵細胞中發(fā)現(xiàn)碳酸酐酶-4同工型mRNA的細胞外表達[93], 這為上述觀點提供了證據(jù)。CO2的水合作用速度遠比的脫水作用要快, 因此以CO2水合來提供H+是有可能的, 而且可能在鰓上皮表明的酸化中也起到了重要的作用。

2.5 降低環(huán)境pH

在靜水條件下, 魚類排泄出的H+和CO2對周圍環(huán)境酸化具有很大的影響。在酸性條件下, 以NH3形式存在的氨比例降低, 使得水生動物免于環(huán)境氨毒性。許氏齒彈涂魚可在灘涂地上建造巢穴用于繁殖, 有研究表明其巢穴中pH約在7.0左右, 而灘涂地其他位置pH則高達7.84[72], 而且在實驗室的研究也證實許氏齒彈涂魚可降低周圍環(huán)境pH[71]。當(dāng)許氏齒彈涂魚在洞穴中的時候, 由于缺乏充足的水, 其排泄速率會明顯增加, 因此其周圍環(huán)境氨濃度顯著提高。在這種情況下, 許氏齒彈涂魚排泄并不是為了排出體內(nèi)的NH3和H+, 因為外界環(huán)境與其體內(nèi)的NH3濃度差增加, NH3會流回體內(nèi)。因為酸的排泄是受外界環(huán)境氨濃度影響的, 因此即使在中性環(huán)境中, 酸的排泄也會持續(xù)進行[9]。這樣一來, 許氏齒彈涂魚鰓上皮的離水界面的pH會呈酸性(較高的H+濃度), 以保證不會分解為NH3, 避免NH3回流入魚體內(nèi)[72]。因此,和酸排泄的同時進行也是某些魚類有效的耐氨策略。

2.6 NH3揮發(fā)和體表堿化

NH3揮發(fā)是氣呼吸型魚類應(yīng)對氨氮毒性的重要策略之一。由于氨可以NH3的形式存在, 因此理論上魚類是有可能直接將NH3排放到空氣中。魚類可以直接揮發(fā)NH3最初見于大頭鳚中, 但NH3揮發(fā)只占總氨排泄量的8%左右[94]。但后續(xù)的一些研究發(fā)現(xiàn)一些魚類如柯克氏跳彈鳚(Alticus kirki)[95]、花溪鳉[76]以及泥鰍[65]等均可在空氣暴露條件下?lián)]發(fā)相當(dāng)大比例的NH3。而且, 溫度以及濕度均與NH3揮發(fā)量具有正相關(guān)關(guān)系。泥鰍暴露于空氣中會揮發(fā)相當(dāng)大比例的NH3[65], 可能是因為泥鰍后腸壁非常薄, 血管分布密集[96], 這種器官特化非常有利于氣體的流動。此外, 空氣暴露會導(dǎo)致泥鰍細胞膜流動性的顯著增加, 這可能會增加NH3在其鰓細胞膜中的滲透性[97]。這一發(fā)現(xiàn)可能意味著鰓與泥鰍的氨氣揮發(fā)無關(guān)。NH3應(yīng)該是由泥鰍直接揮發(fā)至空氣中, 而且是通過腸道揮發(fā)再經(jīng)肛門排出[65]?；ㄏ氁簿哂袚]發(fā)NH3的能力[76], 而且其在空氣暴露條件下皮膚表面pH為增加0.4—0.5個單位, 這增加了其皮膚表面NH3的累積[98]。此外, 有研究表明,花溪鳉在氨氮和空氣暴露條件下, Rhcg在其鰓和皮膚的表達量都是非常顯著的[99]。因此, Hung等[99]認為Rh蛋白提高了NH3從血液中向皮膚的流動性, 而且可能促進NH3的揮發(fā)。

由于NH3的存在量極大程度上受介質(zhì)pH影響,因此魚類體表堿化應(yīng)該有利于氨以NH3的形式排泄。泥鰍在空氣暴露條件下, 前腸明顯堿化, 是其NH3揮發(fā)位點之一[65], 在一些陸生蟹類中也發(fā)現(xiàn)其可以通過堿化的尿液排出NH3[100]。相比于海水魚類, 淡水魚類腸腔堿化的機制并不清楚[101], 海水魚類腸腔堿化主要是與腸道陰離子交換相關(guān)[102,103]。淡水魚類腸腔內(nèi)壁堿化的機制也鮮有報道, 但其很可能也是通過分泌替代Cl–完成的, 這一現(xiàn)象在泥鰍中已得到證實[104]。

2.7 降低體內(nèi)氨的生成

魚體內(nèi)的氨主要由氨基酸代謝產(chǎn)生, 因此魚類可通過降低氨基酸代謝以減少氨的生成來防止機體內(nèi)氨濃度過高。魚類蛋白質(zhì)水解和合成的平衡會保持其體內(nèi)游離氨基酸(Free amino acids, FAA)含量的穩(wěn)態(tài)。如果非必需FAA含量在機體內(nèi)累積可認為是GDH及一些轉(zhuǎn)氨酶催化氨和α-酮酸合成氨基酸的增加, 而必需FAA含量在饑餓魚類體內(nèi)累積則可能是由于氨基酸合成的減少[105]。降低體內(nèi)氨基酸代謝可能是魚類應(yīng)對氨氮毒性的有效策略之一, 其可降低魚類機體內(nèi)氨含量, 而且并不需要外源能量的參與[5]。但是, 這種情況只能在體內(nèi)氨濃度已達到一定程度才會出現(xiàn), 此時魚類體內(nèi)氨的累積速度要明顯大于排泄速率。

這一耐氨策略已在一些氣呼吸型魚類中得到證實, 如空氣暴露泥鰍一段時間后, 泥鰍體組織中氨累積量會明顯超過氨排泄量[57,65], 因此作者認為泥鰍可通過降低蛋白質(zhì)水解和氨基酸代謝來適應(yīng)空氣暴露。黃鱔在高環(huán)境氨氮中暴露較長一段時間后, 其肝臟和肌肉中總FAA含量會顯著增加[106],而且總FAA的增加主要體現(xiàn)在谷氨酰胺以及幾種必需氨基酸含量的增加, 這些結(jié)果表明了黃鱔在長時間高氨氮暴露下會降低氨基酸代謝[58]。相似的,空氣暴露中華烏塘鱧24h后, 其氨基酸代謝并未受到抑制, 而在暴露72h后則發(fā)現(xiàn)其體內(nèi)N保留量達到595 μmol, 因此氨基酸代謝降低也是發(fā)生在長時間空氣暴露后[60]。細鱗非洲肺魚進入夏眠的前6d及后續(xù)的34d, 其機體氨生成速度相較于對照組(0)分別降低了26%和28%[68]。石花肺魚在夏眠的前12d內(nèi)氨生成速度僅降低20%, 而在夏眠的第34—46d其體內(nèi)氨生成速度則降低96%[107]。非洲肺魚經(jīng)歷12d的夏眠后, 其組織中尿素含量卻明顯上升(增加約2.7倍)而非氨濃度升高, 但其肝臟OUC相關(guān)酶活性卻沒有明顯的變化。當(dāng)其在空氣中夏眠46d后, 其組織中尿素含量較對照組升高至1.4倍、氨生成速度降低56%, 說明了非洲肺魚主要以合成尿素和降低氨生成來應(yīng)對空氣暴露[108]。類似的研究還可見于許氏齒彈涂魚和薄氏大彈涂魚等[10]。

2.8 特定氨基酸代謝生成丙氨酸

抑制體內(nèi)蛋白水解和氨基酸代謝可能是魚類應(yīng)對氨氮毒性的有效策略之一, 其可降低魚類機體內(nèi)氨含量。然而, 在這種情況下也同樣抑制了利用氨基酸作為能量源, 這對某些特定魚類來說并不合適, 如許氏齒彈涂魚需要在灘涂地上運動[109]。因此, 某些氣呼吸型魚類在抑制體內(nèi)蛋白水解和氨基酸代謝的同時會部分代謝氨基酸以保證能量供應(yīng)。谷氨酸和丙酮酸在轉(zhuǎn)氨基作用下會產(chǎn)生α-酮戊二酸, 其在三羧酸循環(huán)和電轉(zhuǎn)移鏈的作用下可被完全氧化為CO2和H2O, 此反應(yīng)可提供ATP。α-酮戊二酸通過三羧酸循環(huán)可被轉(zhuǎn)化為蘋果酸, 而蘋果酸在蘋果酸酶的作用下又可變?yōu)楸? 丙酮酸加上谷氨酸在丙氨酸轉(zhuǎn)氨酶的轉(zhuǎn)氨基作用下可生成丙氨酸。從本質(zhì)上來說, 這一系列轉(zhuǎn)氨反應(yīng)生成丙氨酸并未涉及到氨的釋放。因此, 部分氨基酸代謝生成丙氨酸雖然不能降低氨的毒性卻很好的抑制了體內(nèi)氨的生成。從這一點來說, 氨基酸部分代謝生成丙氨酸也是魚類應(yīng)對氨氮毒性的有效策略之一, 而且這一過程還可提供機體必要的ATP。有許多氨基酸可被部分代謝為丙氨酸且不產(chǎn)生氨, 如1 mol谷氨酸轉(zhuǎn)化為丙氨酸可產(chǎn)生10 mol ATP, 而精氨酸和脯氨酸轉(zhuǎn)化為丙氨酸生成的ATP量則更大。

泥鰍在空氣中暴露12h之后, 其肝臟中丙氨酸含量升高兩倍, 說明其在空氣中可部分代謝氨基酸生成丙氨酸以抑制體內(nèi)氨濃度的升高[57]。許氏齒彈涂魚在灘涂地中排泄氨是很困難的, 氨基酸部分代謝生成丙氨酸對其來說是較為理想的耐氨策略, 且可為其在灘涂地中活動提供必要的能量。Ip等[110]發(fā)現(xiàn)許氏齒彈涂魚在陸地上運動3h后, 其體內(nèi)糖原含量并沒有發(fā)生變化, 盡管其肌肉中乳酸含量明顯升高, 而且肌肉中氨和丙氨酸含量也明顯升高, 這些結(jié)果說明氨基酸部分代謝的出現(xiàn)與暴露時間及機體能耗相關(guān)。氨基酸部分代謝使得一些魚類降低了對碳水化合物的依賴性, 節(jié)約了貯存在體內(nèi)的糖原, 使得它們在離水條件下依然能夠保持較高的代謝速率。鱧科的月鱧(Channa asiatica)也是一種典型的氣呼吸型魚類, 月鱧在干旱季節(jié)會經(jīng)常面臨空氣暴露。月鱧在離水條件下無法進行運動、攝食等生活習(xí)性, 待其重新回到水中也會經(jīng)歷較長時間的恢復(fù)期。月鱧在空氣中暴露48h后, 其肌肉中丙氨酸含量從3.7升高至12.6 μmol/g, 這補償了其體內(nèi)氨累積與氨排泄差值的70%[111]。這說明月鱧可利用一些氨基酸作為能量源, 與此同時最大程度上降低了其體內(nèi)的氨濃度。

2.9 組織高氨耐受性

一些氣呼吸型魚類的組織和細胞具有很高的氨耐受性, 其組織或者器官中可累積較高濃度的氨,如泥鰍及黃鱔等[57,65,106]。但是氨在其機體內(nèi)并不是均勻分布的, 一些組織和器官中的氨濃度顯著高于其他組織和器官。泥鰍在正常情況下血漿中氨含量為0.81 μmol/L, 而在空氣中暴露6h后血漿氨濃度升高至2.46 μmol/L, 空氣暴露48h后血漿、肌肉和肝臟中氨含量均顯著升高, 分別為5.09、14.8和15.2 μmol/g[57]。然而, 在通常情況下多數(shù)魚類組織和器官中氨含量均＜1 μmol/g。空氣暴露黃鱔72h后,其肝臟、大腦和血漿中氨濃度分別升高為對照組的3倍、3.5倍和5倍, 肌肉和腸道氨含量在144h后達到峰值, 分別為6.9和4.5 μmol/g[58]。黃鱔在75 mmol/ L的NH4Cl溶液中暴露72h后, 其肌肉、肝臟、腸道、大腦和血漿中也發(fā)現(xiàn)有明顯的氨累積現(xiàn)象[106]。Tsui等[65]認為泥鰍組織中氨累積有助于其進行NH3揮發(fā)。但黃鱔并不能進行NH3揮發(fā), 其組織中氨的累積可能是由于其對環(huán)境氨氮極高的耐受性, NH4Cl溶液(pH 7.0, 28℃)對黃鱔48, 72和96h的半致死濃度分別為209.9、198.7和193.2 mmol/L[106]。

與其他組織不同, 魚類大腦對氨的耐受性可能較低, 當(dāng)血液中氨濃度升高, 通過血液循環(huán), 氨就可影響到腦組織。增加谷氨酰胺的合成是魚類常用的應(yīng)對腦組織氨濃度過高的策略, 但腦組織中谷氨酰胺的累積也會導(dǎo)致一些其他的問題, 因此這一策略也是暫時性的[5]。然而, 魚類的神經(jīng)中樞神經(jīng)系統(tǒng)較高等脊椎動物并不是非常發(fā)達, 因此魚類大腦通常具有較高的氨耐受能力[26]。在對一些魚類的研究中發(fā)現(xiàn)其腦組織高氨耐受性機制與高等脊椎動物是不同, 如海灣豹蟾魚、許氏齒彈涂魚、薄氏大彈涂魚、尖齒胡鯰及黃鱔[24,25,47,49]。Ip等[24]給許氏齒彈涂魚和薄氏大彈涂魚注射致死劑量的CH3COONH4和100 μg/g的蛋氨酸亞砜酰亞胺(Methionine sulfoximine, MSO), 而MSO是一種GS抑制劑, 以此來抑制其大腦中谷氨酰胺的累積。結(jié)果表明MSO并不能降低許氏齒彈涂魚和薄氏大彈涂魚的死亡率。同樣的方法和劑量, MSO (100 μg/g)可在27—48 min內(nèi)將注射了致死劑量CH3COONH4的尖齒胡鯰的死亡率降低20%[25], 他們認為MSO抑制尖齒胡鯰腦組織氨累積可能是通過抑制了腦組織中GDH和丙氨酸轉(zhuǎn)氨酶的活性。類似地, MSO抑制黃鱔腦組織氨累積也不是抑制其GS活性, 而是通過影響GDH活性來實現(xiàn)的[49]。前文已經(jīng)敘述了NMDA受體對腦組織的神經(jīng)毒性作用。因此, NMDA受體的拮抗劑可能具有保護腦組織免于氨氮毒性的作用。已有研究的NMDA受體拮抗劑為(5R,10S)-(+)-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminehydrogenmaleate (MK801)。然而, 2 μg/ g的MK801對注射致死劑量的CH3COONH4許氏齒彈涂魚和薄氏大彈涂魚并未起到保護作用[24], 說明了NMDA受體的激活并不是氨氮急性暴露時致死的主要原因。綜合這些結(jié)果來看, 魚類大腦具有間接降低腦組織氨含量的功能。

3 展望

本文綜述了氨氮對魚類的毒性機理以及魚類的應(yīng)對策略, 為相關(guān)方向的研究提供了理論資料。鑒于國內(nèi)外對氨氮毒理的研究仍停留在基礎(chǔ)的生理學(xué)范疇, 后續(xù)的相關(guān)研究應(yīng)該從分子和細胞的角度分析氨氮對水生動物的毒理作用, 這樣可更直接、更迅速地反應(yīng)氨氮對水生動物的毒性及其作用機制。同樣地, 對魚類氨解毒策略的研究也應(yīng)加強深度和廣度, 如探討每一種耐氨機制的分子調(diào)控機制、常規(guī)非氣呼吸型魚類是否也具有獨特的氨解毒策略及其調(diào)控機制等等。這些研究資料的累積將豐富氨氮對水生動物的毒理機制及水生動物的氨解毒機制, 具有重要的科研價值, 也可為水生動物健康養(yǎng)殖提供理論依據(jù)。

[1]Randall D J, Tsui T K N. Ammonia toxicity in fish [J]. Marine Pollution Bulletin, 2002, 45(1-12): 17—23

[2]Zhou X, Dong Y W, Wang F, et al. The effect of high ammonia concentration on gill structure alternation and expression of sod and hsp90 genes in grass carp, Ctenopharyngodon idella [J]. Acta Hydrobiologica Sinica, 2013, 37(2): 321—328 [周鑫, 董云偉, 王芳, 等. 急性氨氮脅迫對于草魚sod和hsp90基因表達及鰓部結(jié)構(gòu)的影響. 水生生物學(xué)報, 2013, 37(2): 321—328]

[3]Zhang L, Xiong D M, Li B, et al. Toxicity of ammonia and nitrite to yellow catfish (Pelteobagrus fulvidraco) [J]. Journal of Applied Ichthyology, 2012, 28(1): 82—86

[4]Sinha A K, Rasoloniriana R, Dasan A F, et al. Interactive effect of high environmental ammonia and nutritional status on ecophysiological performance of European sea bass (Dicentrarchus labrax) acclimated to reduced seawater salinities [J]. Aquatic Toxicology, 2015, 160: 39—56

[5]Chew S F, Ip Y K. Excretory nitrogen metabolism and defence against ammonia toxicity in air-breathing fishes [J]. Journal of Fish Biology, 2014, 84(3): 603—638

[6]Tng Y Y M, Wee N L J, Ip Y K, et al. Postprandial nitrogen metabolism and excretion in juvenile marble goby, Oxyeleotris marmorata (Bleeker, 1852) [J]. Aquaculture, 2008, 284(1—4): 260—267

[7]Ip Y K, Lim C K, Lee S M L, et al. Postprandial increases in nitrogenous excretion and urea synthesis in the giant mudskipper Periophthalmodon schlosseri [J]. Journal of Experimental Biology, 2004, 207(17): 3015—3023

[8]Lim C K, Wong W P, Lee S M L, et al. The ammonotelic African lungfish Protopterus dolloi increases the rate of urea synthesis and becomes ureotelic after feeding [J]. Journal of Comparative Physiology B, 2004, 174(7): 555—564

[9]Ip Y K, Chew S F. Ammonia production, excretion, toxicity, and defense in fish: a review [J]. Frontiers in Physiolgoy, 2010, 1: 134, doi: 10.3389/fphy.2010. 00134

[10]Lim C B, Anderson P M, Chew S F, et al. Reduction in the rates of protein and amino acid catabolism to slow down the accumulation of endogenous ammonia: a strategy potentially adopted by mud skipper (Periophthalmodon schlosseri and Boleophthalmus boddaerti) during aerial exposure in constant darkness [J]. Journal of Experimental Biology, 2001, 204(9): 1605—1614

[11]Sinha A K, Matey V, Giblen T, et al. Gill remodeling in three freshwater teleosts in response to high environmental ammonia [J]. Aquatic Toxicology, 2014, 155: 166—180

[12]Dong X, Zhang X, Qin J, et al. Acute ammonia toxicity and gill morphological changes of Japanese flounder Paralichthys olivaceus in normal versus su-persaturated oxygen [J]. Aquacuture Research, 2013, 44(11): 1752—1759

[13]Ching B, Chew S F, Wong W P, et al. Environmental ammonia exposure induces oxidative stress in gills and brain of Boleophthalmus boddaerti (mudskipper) [J]. Aquatic Toxicology, 2009, 95(3): 203—212

[14]Rodrigues R V, Schwarz M H, Delbos B, et al. Acute exposure of juvenile cobia Rachycentron canadum to nitrate induces gill, esophageal and brain damage [J]. Aquaculture, 2011, 322/323: 223—226

[15]Pinto M R, Lucena M N, Faleiros R O, et al. Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae) [J]. Aquatic Toxicology, 2016, 170: 13—23

[16]de Lourdes Cobo M, Sonnenholzner S, Wille M, et al. Ammonia tolerance of Litopenaeus vannamei (Boone) larvae [J]. Aquaculture Research, 2014, 45(3): 470—475

[17]Gomu?ka P, ?arski D, Kupren K, et al. Acute ammonia toxicity during early ontogeny of ide Leuciscus idus (Cyprinidae) [J]. Aquaculture International, 2014, 22(1): 225—233

[18]Barbieri E, Bondioli A C V. Acute toxicity of ammonia in Pacu fish (Piaractus mesopotamicus, Holmberg, 1887) at different temperatures levels [J]. Aquaculture Research, 2015, 46(3): 565—571

[19]Medeiros L S, Pavione P M, Baroni V D, et al. Ammonia excretion in fat snook (Centropomus parallelus Poey, 1860) at different salinities [J]. Aquaculture Research, 2015, 46(12): 3084—3087

[20]Chew S F, Poothodiyil N K, Wong W P, et al. Exposure to brackish water leads to increases in conservation of nitrogen and retention of urea in the Asian freshwater stingray, Himantura signifer, upon feeding [J]. Journal of Experimental Biology, 2006, 209(3): 484—492

[21]Schimdt W, Wolf G, Grungreiff K, et al. Adenosine influences the high-affinity uptake of transmitter glutamate and aspartate under conditions of hepatic encephalophthy [J]. Metabolic Brain Disease, 1993, 8(2): 73—80

[22]Tsui TKN. Mechanisms of ammonia tolerance in the oriental weatherloach, Misgurnus anguillicaudatus [D]. City University of Hongkong, Hongkong China. 2005 [徐權(quán)能. 泥鰍的耐氨機制. 博士學(xué)位論文. 香港城市大學(xué), 中國香港. 2005]

[23]Mi?ana M D, Hermenegildo C, Llansola M, et al. Carnitine and choline derivatives containing a trimethylamine group prevent ammonia toxicity in mice and glutamate toxicity in primary cultures of neurons [J]. The Journal of Pharmacology and Experimental Therapeutics, 1996, 279(1): 194—199

[24]Ip Y K, Leong M W F, Sim M Y, et al. Chronic and acute ammonia toxicity in mudskipper, Periophthalmodon schlosseri and Boleophthalmus boddaerti: brain ammonia and glutamine contents, and effects of methionine sulfoximine and MK801 [J]. Journal of Experimental Biology, 2005, 208(7): 1993—2004

[25]Wee N L J, Tng Y Y M, Cheng H T, et al. Ammonia toxicity and tolerance in the brain of the African sharptooth catfish, Clarias gariepinus [J]. Aquatic Toxicology, 2007, 82(3): 204—213

[26]Evans D H, Piermarini P M, Choe K P. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste [J]. Physiological Reviews, 2005, 85(1): 97—177

[27]Wood C M. Toxic response of the gill. In: Schlenk D, Benson W H (eds), Target Organ Toxicity in Marine and Freshwater Teleosts [M]. Volume 1, Organs. Taylor & Francis, London, 2001, 1—89

[28]Sheehan R J, Lewis W M. Influence of pH and ammonia salts on ammonia toxicity and water balance in young channel catfish [J]. Transactions of American Fisheries Society, 1986, 115(6): 891—899

[29]Miron Ddoss, Moraes B, Becker A G, et al. Ammonia and pH effects on some metabolic parameters and gill histology of silver catfish, Rhamdia quelen (Heptapteridae) [J]. Aquaculture, 2008, 277(3-4): 192—196

[30]Sink T D. Influence of pH, salinity, calcium, and ammonia source on acute ammonia toxicity to golden shiners, Notemigonus crysoleucas [J]. Journal of the World Aquaculture Society, 2010, 41(3): 411—420

[31]Romano N, Zeng C. Toxic effects of ammonia, nitrite, and nitrate to crustaceans: a review on factors influencing their toxicity, physiological consequences, and coping mechanisms [J]. Reviews in Fisheries Science, 2013, 21(1): 1—21

[32]K?r M, Kumlu M, Eroldo?an O T. Effects of temperature on acute toxicity of ammonia to Penaeus semisulcatus juveniles [J]. Aquaculture, 2004, 241(1-4): 479—489

[33]K?r M, Topuz M, Sunar M C, et al. Acute toxicity of ammonia in Meagre (Argyrosomus regius Asso, 1801) at different temperatures [J]. Aquaculture Research, 2015, doi: 10.1111/are.12811

[34]Barbieri E. Acute toxicity of ammonia in white shrimp (Litopenaeus schmitti) (Burkenroad, 1936, Crustacea) at different salinity levels [J]. Aquaculture, 2010, 306(1—4): 329—333

[35]K?r M, ?z O. Effects of salinity on acute toxicity of ammonia and oxygen consumption rates in common prawn, Palaemon serratus (Pennat, 1777) [J]. Journal of the World Aquaculture Society, 2015, 46(1): 76—82

[36]Soderberg R W, Meade J W. Effects of sodium and Calcium on acute toxicity of un-ionized ammonia toAtlantic salmon and lake trout [J]. Journal of Applied Aquaculture, 1992, 1(4): 83—92

[37]Diricx M, Sinha A K, Liew H J, et al. Compensatory responses in common carp (Cyprinus carpio) under ammonia exposure: Additional effects to feeding and exercise [J]. Aquatic Toxicology, 2013, 142/143: 123—137

[38]Kieffer J D, Wakefield A M, Litvak M K. Juvenile sturgeon exhibit reduced physiological responses to exercise [J]. Journal of Experimental Biology, 2001, 204(24): 4281—4289

[39]Wicks B J, Joensen R, Tang Q, et al. Swimming and ammonia toxicity in salmonids: the effect of sub lethal ammonia exposure on the swimming performance of coho salmon and the acute toxicity of ammonia in swimming and resting rainbow trout [J]. Aquatic Toxicology, 2002, 59(1-2): 55—69

[40]Sinha A K, Liew H J, Diricx M, et al. The interactive effects of ammonia exposure, nutritional status and exercise on metabolic and physiological responses in gold fish (Carssius auratus L.) [J]. Aquatic Toxicology, 2012, 109: 33—46

[41]Beaumont M W, Butler P J, Taylor E W. Exposure of brown trout, Salmo trutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential [J]. Aquatic Toxicology, 2000, 51(2): 259—272

[42]Beaumont M W, Taylor E W, Butler P J. The resting membrane potential of white muscle from brown trout (Salmo trutta) exposed to copper in soft, acidic water [J]. Journal of Experimental Biology, 2000, 230(14): 2229—2236

[43]Wicks B J, Randall D J. The effect of feeding and fasting on ammonia toxicity in juvenile rainbow trout, Oncorhynchus mykiss [J]. Aquatic Toxicology, 2002, 59(1-2): 71—82

[44]Lemarie G, Dosdat A, Coves D, et al. Effect of chronic ammonia exposure on growth of European seabass (Dicentrarchus labrax) juveniles [J]. Aquaculture, 2004, 229(1—4): 471—491

[45]Sinha A K, Liew H J, Diricx M, et al. Combined effects of high environmental ammonia, starvation and exercise on hormonal and ion-regulatory response in goldfish (Carssius auratus L.) [J]. Aquatic Toxicology, 2012, 114—115: 153—164

[46]Bucking C, Wood C M. Gastrointestinal processing of Na+, Cl–and K+during digestion: implication for homeostatic balance in freshwater rainbow trout [J]. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2006, 291(6): R1764—R1772

[47]Veauvy C M, McDonald M D, van Audekerke J, et al. Ammonia affects brain nitrogen metabolism but not hydration status in the Gulf toadfish (Opsanus beta) [J]. Aquatic Toxicology, 2005, 74(1): 32—46

[48]Wright P A, Steele S L, Hvitema A, et al. Introduction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia [J]. Journal of Experimental Biology, 2007, 210(16): 2905—2911

[49]Tng Y Y M, Chew S F, Wee N L J, et al. Acute ammonia toxicity and the protective effects of methionine sulfoximine on the swamp eel, Monopterus albus [J]. Journal of Experimental Zoology Part A Ecological Genetics and Physiology, 2009, 311(9): 676—688

[50]Sanderson L A, Wright P A, Robinson J W, et al. Inhibition of glutamine synthetase during ammonia exposure in rainbow trout indicates a high reserve capacity to prevent brain ammonia toxicity [J]. Journal of Experimental Biology, 2010, 213(13): 2343—2353

[51]Mommsen T P, Walsh P J. Biochemical and environmental perspectives on nitrogen metabolism in fishes [J]. Experientia, 1992, 48(6): 583—593

[52]Anderson P M, Broderius M A, Fong K C, et al. Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichyths sinensis in response to exposure to a high exogenous ammonia concentration [J]. Journal of Experimental Biology, 2002, 205(14): 2053—2065

[53]Chew S F, Tng Y Y M, Wee N L J, et al. Nitrogen metabolism and branchial osmoregulatory acclimation in the juvenile marble goby, Oxyeleotris marnorata, exposed to seawater [J]. Comparative Biochemistry and Physiology Part A Molecular & Intergrative Physiology, 2009, 154(3): 360—369

[54]Chew S F, Tng Y Y M, Wee N L J, et al. Intestinal osmoregulatory acclimation and nitrogen metabolism in juveniles of the freshwater marble goby exposed to seawater [J]. Journal of Comparative Physiology Biochemical, Systems, and Environmental Physiology, 2010, 180(4): 511—520

[55]Campbell J W. “Excretory nitrogen metabolism” In: Prosser C L (Eds.), Environmental and Metabolic Animal Physiology. Comparative Animal Physiology [M]. 4th edn. New York: Wiley-Interscience. 1991, 277—324

[56]Jow L Y, Chew S F, Lim C B, et al. The marble goby Oxyeleotris marnorata activates hepatic glutamine synthetase and detoxifies ammonia to glutamine during air exposure [J]. Journal of Experimental Biology, 1999, 202(3): 237—245

[57]Chew S F, Jin Y, Ip Y K. The loach Misgurnus anguillicaudatus reduces amino acid catabolism and accumulates alanine and glutamine during aerial exposure [J]. Physiological and Biochemical Zoology, 2001, 74(2): 226—237

[58]Tay A S L, Chew S F, Ip Y K. The swamp eel Monopterus albus reduces endogenous ammonia production and detoxifies ammonia to glutamine during 144 h of aerial exposure [J]. Journal of Experimental Biology, 2003, 206(14): 2473—2486

[59]Peh W Y X, Chew S F, Ching B Y, et al. Roles of intestinal glutamate dehydrogenase and glutamine synthetase in environmental ammonia detoxification in the euryhaline four-eyed sleeper, Bostrychus sinensis [J]. Aquatic Toxicology, 2010, 98(1): 91—98

[60]Ip Y K, Chew S F, Leong I W A, et al. The sleeper Bostrichyths sinensis (Family Eleotridae) stores glutamine and reduces ammonia production during aerial exposure [J]. Journal of Comparative Pysiology Biochemical, Systems, and Environmental Physiology, 2001, 171(5): 357—367

[61]Wang G, Pan L, Ding Y. Defensive strategies in response to environmental ammonia exposure of the sea cucumber Apostichopus japonicus: Glutamine and urea formation [J]. Aquaculture, 2014, 432: 278—285

[62]Steele S L, Yancey P H, Wright P A. The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge [J]. Physiological, and Biochemical Zoology, 2005, 78(2): 216—226

[63]Walsh P J, Danulat E, Mommsen T P. Variation in urea excretion in the gulf toadfish (Opsanus beta) [J]. Marine Biology, 1990, 106(3): 323—328

[64]Iwata K, Kajimura M, Sakamoto T. Functional ureogenesis in the gobiid fish Mugilogobius abei [J]. Journal of Experimental Biology, 2000, 203(24): 3703—3715

[65]Tsui T K N, Randall D J, Chew S F, et al. Accumulation of ammonia in the body and NH3volatilization from alkaline regions of the body surface during ammonia loading and exposure to air in the weather loach Misgurnus anguillicaudatus [J]. Journal of Experimental Biology, 2002, 205(5): 651—659

[66]Zhang Y L, Zhang H L, Wang L Y, et al. Changes of ammonia, urea contents and transaminase activity in the body during aerial exposure and ammonia loading in Chinese loach Paramisgurnus dabryanus [J]. Fish Physiology and Biochemisty, 2017, 43(2): 631—640

[67]Tay A S L, Loong A M, Hiong K C, et al. Active ammonia transport and excretory nitrogen metabolism in the climbing perch, Anabas testudineus, during 4 days of emersion or 10 minutes of forced exercise on land [J]. Journal of Experimental Biology, 2006, 209(22): 4475—4489

[68]Chew S F, Chan N K Y, Tam W L, et al. The African lungfish, Protopterus dolloi, increases the rate of urea synthesis despite a reduction in ammonia production during 40 days of aestivation in a mucus cocoon [J]. Journal of Experimental Biology, 2004, 207(5): 777—786

[69]Loong A M, Hiong K C, Lee S L M, et al. Ornithineurea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for 6 days [J]. Journal of Experimental Zoology Part A Ecological Genetics and Physiology, 2005, 303(5): 354—365

[70]Ip Y K, Subaidah R M, Liew P C, et al. The African catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids during ammonia loading but is capable of excreting ammonia against an inwardly driven ammonia concentration gradient [J]. Physiological and Biochemical Zoology, 2004, 77(2): 255—266

[71]Chew S F, Hong L N, Wilson J M, et al. Alkaline environmental pH has no effect on the excretion of ammonia in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti [J]. Physiological and Biochemical Zoology, 2003, 76(2): 204—214

[72]Ip Y K, Randall D J, Kok T K T, et al. The giant mudskipper Periophthalmodon schlosseri facilitates activeNH+4excretion by increasing acid excretion and decreasing NH3permeability in the skin [J]. Journal of Experimental Biology, 2004, 207(5): 787—801

[73]Matey V, Richards J G, Wang Y, et al. The effect of hypoxia on gill morphology and ionoregulatory status in the Lake Qinghai scaleless carp, Grmnocypris przewalskii [J]. Journal of Experimental Biotogy, 2008, 211(7): 1063—1074

[74]LeBlanc D M, Wood C M, Fudge D S, et al. A fish out of water: gill and skin remodeling promotes osmo-and ionoregulation in the mangrove killifish Kryptolebias marmoratus [J]. Physiological and Biochemical Zoology, 2010, 83(6): 939—949

[75]Brauner C J, Matey V, Wilson J M, et al. Transition in organ function during the evolution of air-breathing; insights from Arapaima gigas, an obligate air-breathing teleost from the Amazon [J]. Journal of Experimental Biology, 2004, 207(9): 1433—1438

[76]Frick N T, Wright P A. Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus II. Significant ammonia volatilization in a teleost during air-exposure [J]. Journal of Experimental Biology, 2002, 205(1): 91—100

[77]Chew S F, Sim M Y, Phua Z C, et al. Active ammonia excretion in the giant mudskipper, Periophthalmodon schlosseri (Pallas), during emersion [J]. Journal of Experimental Zoology Part A Ecological Genetics and Physiology, 2007, 307(6): 357—369

[78]Randall D J, Ip Y K, Chew S F, et al. Air breathing and ammonia excretion in the giant mudskipper, Periophthalmodon schlosseri [J]. Physiological and Bioche-mical Zoology, 2004, 77(5): 783—788

[79]Loong A M, Chew S F, Wong W P, et al. Both seawater acclimation and environmental ammonia exposure lead to increases in mRNA expression and protein abundance of Na+: K+: 2Cl–cotransporter in the gills of the freshwater climbing perch, Anabas testudineus [J]. Journal of Comparative Physiology Biochemical, Systems, and Environmental Physiology, 2012, 182(4): 491—506

[80]Zidi-Yahiaoui N, Mouro-Chanteloup I, D’Ambrosio A M, et al. Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells [J]. Biochemical Journal, 2005, 391(1): 33—40

[81]Mak D O, Dang B, Weiner I D, et al. Characterization of transport by the kidney Rh glycoproteins, RhBG and RhCG [J]. American Journal of Physiology - Renal Physiology, 2006, 290(2): F297—F305

[82]Mouro-Chanteloup I, Cochet S, Chami M, et al. Functional reconstitution into lipsomes of purified human RhCG ammonia channel [J]. PLoS One, 2010, 5: e8921

[83]Shih T H, Horng J L, Hwang P P, et al. Ammonia excretion by the skin of zebrafish (Danio rerio) larvae [J]. American Journal of Physiology - Cell Physiology, 2008, 295(6): C1625—C1632

[84]Kumai Y, Perry S F. Ammonia excretion via Rhcg 1 facilitates Na+uptake in larval zebrafish, Danio rerio, in acidic water [J]. American Journal of Physiology -Regulatory, Integrative and Comparative Physiology, 2011, 301(5): R1517—R1528

[85]Fu C, Wilson J M, Rombough P J, et al. Ions first: Na+uptake shift from the skin to the gills before O2uptake in developing rainbow trout, Oncorhynchus mykiss [J]. Proceedings of the Royal Society B Biological Science, 2010, 277(1687): 1553—1560

[86]Nakhoul N L, Hamm L L. The challenge of determining the role of Rh glycoproteins in transport of NH3and[J]. Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 2014, 3(3): 53—61

[87]Nakada T, Westhoff C M, Kato A, et al. Ammonia secretion from fish gill depends on a set of Rh glycoprotein [J]. FASEB Journal, 2007, 21(4): 1067—1074

[88]Wright P A, Wood C M. A new paradigm for ammonia excretion in aquatic animals: role of Rhesus (Rh) glycoproteins [J]. Journal of Experimental Biology, 2009, 212(15): 2303—2312

[89]Tsui T K N, Hung C Y C, Nawata C M, et al. Ammonia transport in cultured gill epithelium of freshwater rainbow trout: the importance of Rhesus glycoproteins and the presence of an apicalexchange complex [J]. Journal of Experimental Biology, 2009, 212(6): 878—892

[90]Rodela T M, Esbaugh A J, Weihrauch D, et al. Revisiting the effects of crowding and feeding in the gulf toadfish, Opsanus beta: the role of Rhesus glycoproteins in nitrogen metabolism and excretion [J]. Journal of Experimental Biology, 2012, 215(2): 301—313

[91]Wright P A, Wood C M, Wilson J M. Rh versus pH: the role of Rhesus glycoproteins in renal ammonia excretion during metabolic acidosis in a freshwater teleost fish [J]. Journal of Experimental Biology, 2014, 217(16): 2855—2865

[92]Edwards S L, Arnold J M, Blair S D, et al. Ammonia excretion in the Atlantic hagfish (Myxine glutinosa) and responses of an Rhc glycoprotein [J]. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2015, 308(9): R769—R778

[93]Lin L Y, Liao B K, Horng J L, et al. Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+uptake in zebrafish H+-ATPase-rich cells [J]. American Journal of Physiology - Cell Physiology, 2008, 294(5): C1250—C1260

[94]Davenport J, Sayer, M D J. Ammonia and urea excretion in the amphibious teleost Blennius pholis (L.) in sea-water and in air [J]. Comparative Biochemistry and Physiology Part A Physiology, 1986, 84(1): 189—194

[95]Rozemeijer M J C, Plaut I. Regulation of nitrogen excretion of the amphibious blenniidae Alticus kirki (Guenther, 1868) during emersion and immersion [J]. Comparative Biochemistry and Physiology Part A Physiology, 1993, 104(1): 57—62

[96]Gon?alves A F, Castro L F C, Pereira-Wilson C, et al. Is there a compromise between nutrient uptake and gas exchange in the gut of Misgurunus anguillicaudatus, an intestinal air-breathing fish? [J]. Comparative Biochemistry and Physiology Part D Genomics and Proteomics, 2007, 2(4): 345—355

[97]Moreira-Silva J, Tsui T K N, Coimbra J, et al. Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus [J]. Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology, 2010, 151(1): 40—50

[98]Litwiller S L, O’Donnell M J O, Wright P A. Rapid increase in the partial pressure of NH3on the cutaneous surface of air-exposed mangrove killifish, Rivulus marmortus [J]. Journal of Experimental Biology, 2006, 209(9): 1737—1745

[99]Hung C Y C, Tsui K N T, Wilson J M, et al. Rhesus glycoprotein gene expression in the mangrove killifish Kryptolebias marmoratus exposed to elevated environmental ammonia levels and air [J]. Journal of Experimental Biology, 2007, 210(14): 2419—2429

[100]Varley D G, Greenaway P. Nitrogenous excretion in the terrestrial carnivorous crab Geograpsus grayi: site and mechanism of excretion [J]. Journal of Experimental Biology, 1994, 190(1): 179—193

[101]Grosell M. Intestinal anion exchange in marine fish osmoregulation [J]. Journal of Experimental Biology, 2006, 209(15): 2813—2827

[102]Kurita Y, Nakada T, Kato A, et al. Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish [J]. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2008, 294(4): R1402—R1412

[103]Grosell M, Manger E M, Williams C, et al. High rates ofsecretion and Cl–absorption against adverse gradients in the marine teleost intestine: the involvement of an electrogenic anion exchanger and H+-pump metabolon [J]? Journal of Experimental Biology, 2009, 212(11): 1684—1696

[104]Wilson J M, Moreira-Silva J, Delgado I L S, et al. Mechanisms of transepithelial ammonia excretion and luminal alkalinization in the gut of intestinal air-breathing fish Misgurnus anguillicaudatus [J]. Journal of Experimental Biology, 2013, 216(4): 623—632

[105]Ip Y K, Chew S F, Wilson J M, et al. Defences against ammonia toxicity in tropical air-breathing fishes exposed to high concentrations of environmental ammonia: a review [J]. Journal of Comparative Physiology Biochemical, Systems, and Environmental physiology, 2004, 174(7): 565—575

[106]Ip Y K, Tay A S L, Lee K H, et al. Strategies for surviving high concentrations of environmental ammonia in the swamp eel Monopterus albus [J]. Physiological and Biochemical Zoology, 2004, 77(3): 390—405

[107]Ip Y K, Yeo P J, Loong A M, et al. The interplay of increased urea synthesis and reduced ammonia production in the African lungfish Protopterus aethiopicus during 46 days of aestivation in a mucus cocoon on land [J]. Journal of Experimental Zoology Part A Ecological Genetics and Physiology, 2005, 303(12): 1054—1065

[108]Loong A M, Chng Y R, Chew S F, et al. Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia [J]. Journal of Comparative Physiology Biochemical, Systems, and Environmental Physiology, 2012, 182(3): 367—379

[109]Kok T W T, Lim C B, Lam T J, et al. The mudskipper Periophthalmodon schlosseri respires more efficiently on land than in water and vice versa for Boleophthalmus boddaerti [J]. Journal of Experimental Zoology Part A Ecological and Integrative Physiology, 1998, 280(1): 86—90

[110]Ip Y K, Lim C B, Chew S F, et al. Partial amino acid catabolism leading to the formation of alanine in Periophthalmodon schlosseri (mudskipper): a strategy that facilitates the use of amino acids as the energy source during locomotory activity on land [J]. Journal of Experimental Biology, 2001, 204(9): 1615—1624

[111]Chew S F, Wong M Y, Tam W L, et al. The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism [J]. Journal of Experimental Biology, 2003, 206(4): 693—704

IMPACT FACTORS OF AMMONIA TOXICITY AND STRATEGIES FOR AMMONIA TOLERANCE IN AIR-BREATHING FISH： A REVIEW

ZHANG Yun-Long1, ZHANG Hai-Long2, WANG Ling-Yu2, GU Bei-Yi2and FAN Qi-Xue2
(1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China; 2. Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, College of Fishery, Wuhan 430072, China)

Ammonia distributes widely in aquaculture water, and is a major issue in the massive mortality rate of fish species with a high ammonia aquaculture environment. Studies on ammonia toxicity and defense in fish are important because of ecological, environmental, and economical relevance. Some fish species have specific strategies to deal with ammonia loading, so that they can tolerate high levels of environmental or internal ammonia. These strategies can be categorized into: (1) glutamine synthesis; (2) urea synthesis and excretion; (3) activeexcretion; (4) ammonia detoxification, improved by Rh glycoproteins; (5) lowering of ambient pH; (6) NH3volatilization and alkalization of the body surface; (7) reduction in body ammonia production; (8) amino acid catabolism leading to the alanine form; and (9) high tissue and organ ammonia tolerance. The response of fish species that are able to ameliorate ammonia toxicity are many and varied, depending on the behaviour of the species and its habitat environment. This paper summarizes ammonia toxicity, as it is hoped that this review can provide basic information on ammonia detoxification mechanisms in airbreathing fish species.

Air-breathing fish; Ammonia; Toxicity; Strategies of ammonia tolerance

Q178.1

A

1000-3207(2017)05-1157-12

10.7541/2017.144

2016-07-14;

2017-01-25

安徽省高等學(xué)校自然科學(xué)研究項目(KJ2017A130)；國家科技支撐計劃(2012BAD25B08和2012BAD25B00)資助 [Supported by the Provincial Natural Science Research Project of Anhui Provincial Higher University Education (KJ2017A130); the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BAD25B08, 2012BAD25B00)]

張云龍(1989—), 男, 安徽合肥人; 講師, 博士; 主要從事魚類增養(yǎng)殖研究。E-mail: zhangyunlong@ahau.edu.cn

樊啟學(xué)(1962—), 男, 湖北潛江人; 教授; 主要從事魚類增養(yǎng)殖及水域生態(tài)學(xué)研究。E-mail: fanqixue@mail.hzau.edu.cn