氨氮对鱼类毒性的影响因子及气呼吸型鱼类耐氨策略

张云龙张海龙王凌宇顾贝易樊启学

(1. 安徽农业大学动物科技学院, 合肥 230036; 2. 华中农业大学水产学院, 农业部淡水生物繁育重点实验室, 武汉 430072)

氨氮对鱼类毒性的影响因子及气呼吸型鱼类耐氨策略

张云龙1张海龙2王凌宇2顾贝易2樊启学2

(1. 安徽农业大学动物科技学院, 合肥 230036; 2. 华中农业大学水产学院, 农业部淡水生物繁育重点实验室, 武汉 430072)

氨氮广泛存在于养殖水体中, 在氨氮过高的养殖环境下可能会导致鱼类的大量死亡。从生态、环境及养殖效益角度来看, 研究氨氮对鱼类的毒性以及鱼类应对环境或体内高氨浓度的策略均具有重要意义。某些鱼类具有其特殊的策略来降低氨毒性, 使得这些种类能适应极高的环境或体内氨浓度。这些耐氨策略主要为(1)合成谷氨酰胺、(2)合成尿素排出、(3)增强机体排泄、(4)Rh蛋白促进氨解毒、(5)降低周围环境pH、(6)NH3挥发和体表碱化、(7)降低体内氨生成、(8)特定氨基酸代谢生成丙氨酸、(9)组织高氨耐受性。鱼类的氨耐受策略较多而且是可变的, 主要受特定种类的生活习性和栖息环境影响。文章综述了氨氮对鱼类的毒性机理以及鱼类的应对策略, 为相关的研究提供基础资料。

气呼吸型鱼类; 氨氮; 氨毒性; 耐氨策略

人工饲料的过量投喂, 是导致养殖水体中氨氮含量偏高的重要原因之一。氨氮对水生动物具有较强的毒性, 可引起其抽搐、昏迷和死亡, 这可能是由于过高浓度的取代了K+以及神经元去极化, 随后会造成中枢神经系统细胞的死亡[1]。大量研究也报道了氨氮对鱼类的毒性作用, 如草鱼(Ctenopharyngodon idella)[2]、黄颡鱼(Pelteobagrus fulvidraco)[3]以及舌齿鲈(Dicentrarchus labrax)[4]等。环境中氨氮由农业径流以及生物废弃物分解产生, 然而鱼类机体中的氨主要是由氨基酸代谢产生。鱼类消化食物中的蛋白质会产生氨基酸, 但鱼类却不能贮存多余的氨基酸, 因此除去用于生长所需, 鱼类摄入食物中过量的氨基酸和蛋白质会转化成脂肪和碳水化合物储存于肝脏中[5], 而转化过程需要去氮, 氨则在这一过程中产生。由于氨对水生动物的高毒性, 在通常情况下鱼组织或器官中氨含量应该保持在较低的水平[5]。当鱼体内氨含量达到了一定量的时候, 其多数鱼类会表现出致死作用。但是, 某些特殊种类却具有其独特的氨耐受机制,以应对环境或者体内过高的氨浓度。本文依据国内外的研究资料, 系统地总结了氨对鱼类毒性的影响因子及气呼吸型鱼类的耐氨策略, 为今后相关的研究提供基础资料。

1 氨氮对鱼类的毒性

氨氮广泛存在于水环境中, 由动植物排放或者微生物分解有机质产生[1]。而水生动物体内的氨则主要是由于氨基酸代谢产生的, 动物肠道分解食物中的蛋白质是氨基酸的主要来源[5,6]。研究表明鱼类摄入食物中40%—60%的氮会在24h内排泄出[7,8]。除此之外, 鱼类在饥饿状态下也会将肌肉蛋白质代谢为氨基酸, 以提供ATP源或者碳水化合物[5,9]。而当暴露于较高的环境氨氮中时, 鱼类会降低自身的氨基酸代谢速度以减少体内氨的生成,以保护机体免于氨氮毒性[7,10]。已有大量的研究报道了氨氮对鱼类的毒性, 如高环境氨氮会导致虹鳟(Oncorhynchus mykiss)、鲤(Cyprinus carpio)和鲫(Carssius auratus)鳃结构异化[11], 也会导致牙鲆(Paralichthys olivaceus)鳃结构变化[12]、造成薄氏大弹涂鱼(Boleophthalmus boddaerti)大脑氧化应激反应[13]、引起军曹鱼(Rachycentron canadum)鳃、食管和大脑的组织损伤[14]等, 类似的研究还可见亚马逊沼虾(Macrobrachium amazonicum)[15]、凡纳滨对虾(Litopenaeus vannamei)[16]、高体雅罗鱼(Leuciscus idus)[17]、细鳞肥脂鲤(Piaractus mesopotamicus)[18]、小锯盖鱼(Centropomus parallelus)[19]等。

在液态环境下, 氨可以非离子态的NH3和离子态的两种形态存在, 其转化方程式为NH3+H3O–=此反应的解离常数(pKa)为9.0—9.5左右。鱼类血液的pH较氨反应的pKa低, 因此氨在鱼类血液中主要(＞95%)以的形式存在[5]。血氨可通过血液循环累积至不同的组织中, 但氨具有较强的细胞毒性[1,9,20], 机体必须排泄多余的氨。氨可激活细胞液中的磷酸果糖激酶I以促进机体糖酵解作用, 也可通过影响核糖体三羧酸循环来干扰机体能量代谢[5]。也有研究表明高浓度氨氮暴露可通过增加谷氨酸盐的释放量或者(以及)降低谷氨酸盐的重吸收致使细胞外谷氨酸盐含量的升高[21]。此外, N-甲基-D-天门冬氨酸型谷氨酸(N-methyl-D-aspartate-type glutamate, NMDA)受体也被大量的激活, NMDA的大量增加导致机体Ca2+及Na+的大量增加[22]。高浓度氨氮暴露引起的过量NMDA具有神经毒性, 可引起氧化应激反应, 造成神经元变形和死亡[23]以及线粒体渗透性的改变。在体内高氨条件下, 线粒体渗透性的改变会导致谷氨酸盐通过线粒体膜的渗透以及通过星形胶质细胞线粒体基质中的谷氨酰胺酶生成氨[5,9,24,25]。但与哺乳动物相比, 鱼类通常具有较强的氨氮耐受能力, 这可能是由于其中枢神经系统没有其他高等脊椎动物的中枢神经系统发达[26]。

NH3可渗透过大多数生物细胞膜, 但的渗透性则相对小的多[1], 因此氨氮毒性很大程度上取决于这两种氨形态的存在比例。而两种氨形态的转化在正常情况下是趋于平衡, 而外界环境(如pH、温度、盐度)以及动物自身状况均可影响两种氨形态的转化平衡, 因此氨氮对鱼类的毒性也受到许多因素的影响, 主要有以下几类:

1.1 主要环境因子影响氨氮对鱼类的毒性

pH在水生动物内稳态中也起到重要作用, 研究表明pH的变化可影响动物体内的酸碱平衡、离子调节以及氨排泄[27]。已有大量研究阐述了pH对氨氮毒性的影响, 介质的pH越高则NH3的存在比例越高, 相对来说毒性也就越大, 可见斑点叉尾(Ictalurus punctaus)[28]、克林雷氏鲶(Rhamdia quelen)[29]、金体美鳊(Notemigonus crysoleucas)[30]等。

与pH相比, 温度对离子态氨向非离子态转化的影响作用要小的多, 但也有研究表明温度升高会增强氨氮对水生动物的毒性[31]。Kır等[32]研究发现了26℃时总氨氮(Total ammonia-nitrogen, TAN)对短沟对虾(Penaeus semisulcatus)的安全浓度是14℃的4倍, 非离子氨的安全浓度则为2倍。将温度从15℃升高至25℃后, 氨氮暴露24、48、72以及96h后, 细鳞肥脂鲤对氨氮的敏感性分别上升21.80%、9.55%、31.92%和30.87%[18]。相似的, 随着温度的升高, 大西洋白姑鱼(Argyrosomus regius)对氨氮的耐受性明显地降低[33]。

盐度也被证实对氨氮毒性具有一定的影响, 在多数鱼类的研究中均发现降低盐度会提高铵态氮和亚硝态氮对鱼类的毒性作用, 如南方滨对虾(Litopenaeus schmitti)[34]、锯齿长臂虾(Palaemon serratus)[35]等。盐度对氨氮毒性的影响机制可能是由于盐度升高会增加钠和钙的含量, 这两种物质已被证明可通过增加鳃细胞和Na+的交换来促进机体氨的排泄, 从而阻止氨氮通过渗透作用流入细胞内[36]。

1.2 运动对氨氮毒性的影响

运动是鱼类的重要生理活动之一, 低密度下鱼类会进行常规游动, 高密度下会进行力竭游动, 鱼类依靠游泳来适应水流速度、捕食、逃跑以及洄游等[37]。力竭游动时鱼类的能量源主要来自于白肌的无氧糖酵解[38]。此外, 力竭游动也被证明会加速机体氨的生成、提高机体氨含量[1], 也就是会放大氨氮的毒性。在虹鳟和银大马哈鱼(Oncorhynchus kisutch)的研究中已经发现剧烈游动会诱导其血液氨浓度的上升[39]。对鲤[37]和鲫[40]的研究中也发现氨氮暴露明显降低了鱼类的游动能力。这可能是因为氨氮环境下的增加改变了鱼类机体的代谢方式[41],还会替代K+造成肌肉细胞膜去极化, 对肌肉造成损伤[42]。因此, 游动和氨氮暴露对鱼类是两个相互作用的因素, 游动放大了氨氮毒性, 氨氮暴露降低了鱼类的游动能力。然而这种相互作用的机制以及其生理生化水平上的表现等均缺乏深入的研究报道, 这也是以后值得研究的一个方向。

1.3 摄食对氨氮毒性的影响

在鱼类摄食之后, 大量的蛋白质被分解以维持机体的正常生理活动, 而机体内氨的生成和排泄明显增加[1,43]。大量的氨生成会导致细胞内碱中毒,引起一系列的鱼类病理反应[44]。就此来说, 当鱼类暴露于氨氮环境下, 相比于投喂的鱼类, 饥饿似乎更有利于鱼类应对氨氮毒性。但是在对鲫的研究中却得出了相反的结论[40,45], 他们发现投喂的鲫比饥饿的鲫对氨氮的耐受性更高。鱼类具有大量、可调节的生理生化活动, 其对营养状况具有较强的适应调节性, 如离子平衡、代谢、内分泌等[46]。因此, 食物也会对鱼类应对氨氮毒性起到一定的作用。Wicks和Randall[43]发现虹鳟在摄食之后, 谷氨酰胺合成酶活力明显上升, 其可将血液中过量的氨转化为无毒的谷氨酰胺, 这一现象在肌肉中表现尤为明显。总体来说, 谷氨酰胺合成酶活力的上升可能就是某些鱼类摄食之后具有较强氨氮耐受力的原因。

2 鱼类的氨耐受策略

多数鱼类都是以排氨为主要的氨代谢途径, 但也有一些鱼类可将氨氮代谢为谷氨酸盐或者尿素以降低氨氮毒性。某些鱼类的大脑具有很高的氨氮耐受性, 也可通过加快的转运来加速氨排泄、调控周围环境pH以及降低鳃上皮和皮肤的氨渗透性以降低氨氮毒性。此外, 也有大量报道认为氨氮的排泄与鱼类鳃上皮和皮肤上的Rh糖蛋白(Rhesus glycoproteins, Rh)相关。本文综述了几种鱼类常用的生理策略, 以应对氨氮环境暴露。

2.1 合成谷氨酰胺

在鱼体内, 谷氨酰胺合成酶(Glutamine synthetase, GS)可催化谷氨酸和合成谷氨酰胺, 而谷氨酸则是由谷氨酸脱氢酶(Glutamate dehydrogenase, GDH)催化α-酮戊二酸和合成的。而谷氨酰胺的合成来源于谷氨酸还是α-酮戊二酸主要是由细胞内GS活性决定的[5]。谷氨酰胺在鱼类大脑降解氨氮毒性中起到了重要的作用, 如海湾豹蟾鱼(Opsanus beta)、尖齿胡鲶(Clarias gariepinus)、虹鳟及黄鳝(Monopterus albus)等[25,47—50], 谷氨酰胺合成后会从各组织中作为一种无毒的氨代谢物释放到血液中, 最后进入肝脏中。通常来说, 鱼类肌肉中GS活性是很低的[51], 但在一些气呼吸型鱼类在空气暴露或氨氮暴露条件下却可在大脑和肝脏及肌肉中将氨代谢为谷氨酰胺。这些气呼吸型鱼类的肝脏、肌肉及肠道组织中均可检出较高的GS活性[52]。以谷氨酰胺的形式应对较高的体内氨氮含量具有其特定的优势, 谷氨酰胺可储存于机体中, 当环境条件改善后, 这些谷氨酰胺可用于合成嘌呤、嘧啶及黏多糖等机体生理活动[5]。

由于GDH涉及联合脱氨作用, 因此鱼类机体中谷氨酰胺含量的增加不太可能是因为GDH胺化反应导致谷氨酸含量增加。云斑尖塘鳢(Oxyeleotris marnorata)在摄食之后12h之后肠道GDH含量显著增加[6], 谷氨酸含量的增加可能是鱼类应对摄食后体内氨氮含量显著升高的主要措施之一[9]。鱼类肠道中过量的谷氨酸可能会被转运至肝脏和肌肉中,促进氨基酸合成用以细胞容积调节[53,54]。肝脏是谷氨酸代谢的主要场所, 因此肠道通过GDH胺化作用合成谷氨酰胺转运至肝脏中是非常必要的[55]。此外, 过量的游离氨基酸并不会用于合成蛋白且会在一些必要的生理活动中代谢产生氨氮。因此, 肠道和肝脏在鱼类以谷氨酰胺形式进行氨氮解毒的过程中是互相协作的关系。

在氨氮暴露下, 鱼体内谷氨酰胺累积的报道已见于多种鱼类。云斑尖塘鳢暴露于空气中72h后其肌肉中谷氨酰胺含量增加了3倍, 而肝脏中谷氨酰胺含量却在暴露24h后到达峰值, 说明了肝脏中谷氨酰胺可能随后转移至肌肉中存贮[56]。空气暴露48h后泥鳅(Misgurnus anguillicaudatus)大脑、肌肉以及肝脏中谷氨酰胺含量显著增加, 而GDH活性则明显降低[57]。空气暴露黄鳝72h后, 其体内谷氨酰胺含量达到最大峰值, 其肝脏GS活性在空气暴露144h后明显高于对照组[58]。虹鳟暴露于670和1000 μmol/ L NH4Cl溶液24h和96h之后, 相比于空白对照组, 其大脑谷氨酰胺含量升高而谷氨酸含量降低[50]。中华乌塘鳢(Bostrychus sinensis)暴露于含有15 mmol/L的海水中6d之后, 其肠道GS和GDH活性均显著提高[59]。此外, 空气暴露中华乌塘鳢24h内其肌肉中累积谷氨酰胺, 而在48h后则又恢复至正常水平, 说明鱼体内累积的谷氨酰胺可能通过某些同化作用转化为其他的含氮化合物[60]。类似的研究报道还可见于许氏齿弹涂鱼(Periophthalmodon schlosseri)、薄氏大弹涂鱼、海湾豹蟾鱼、尖齿胡鲶及仿刺参(Apostichopus japonicus)等[24,25,47,61]。

2.2 合成尿素排出

在脲生成和排尿素的动物中, 其保持体内较低氨含量最主要的措施即将氨转化为尿素, 再通过尿液排出体外[9]。尿素合成主要在动物肝脏内进行,这一过程被称为鸟氨酸-尿素循环(Ornithine-urea cycle, OUC)。就鱼类来说, 并非所有的鱼类都可生成脲, 但也有部分鱼类具有功能性的OUC, 具有脲生成功能, 如软骨鱼类中的板鳃亚纲[62]。一些脲生成型鱼类的OUC可凭借肝脏细胞中的氨甲酰磷酸合成酶III将谷氨酰胺转化为低毒的尿素, 尿素分子更小, 易于排出。投喂许氏齿弹涂鱼[7]和细鳞非洲肺鱼(Protopterus dolloi)[8]24h之后, 其尿素合成速度和排泄速率均明显增加, 也有研究发现板鳃类摄食之后尿素合成速度提高, 但其尿素主要用于调控机体的渗透压而非排出体外[20]。

相比于脲生成型鱼类, 仅有少量鱼类具有排泄尿素的功能。海湾豹蟾鱼在拥挤胁迫下可以尿素形式排泄出50%的氮代谢废物[63], 而阿部鲻鳅虎鱼(Mugilogobius abei)在氨氮暴露条件下只能以很少量尿素的形式排泄体内氨[64]。然而, 对鱼类来说,合成尿素对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, 鱼类在水中都是排氨的, 而且一些气呼吸型鱼类在空气暴露条件下也有许多策略来应对体内较高的氨浓度。对一些气呼吸型鱼类的研究中发现, 当其处于较高的体内氨浓度(空气暴露)或者较高的环境氨浓度(氨氮暴露)时, 多数鱼类并不以合成尿素作为主要的氨耐受策略, 如云斑尖塘鳢[56]、泥鳅[57,65]、黄鳝[58]、大鳞副泥鳅(Paramisgurnus dabryanus)[66]及龟壳攀鲈(Anabas testudineus)[67]等。在已有的报道中, 通过OUC以尿素作为主要氨排泄的鱼类只有格氏雀丽鱼(Alcolapia grahami)一种, 其生活环境pH高达10左右, 在这种情况下氨排泄受到严重的阻碍。因此, 其通过OUC合成尿素的能力很强, 以保证机体免于氨氮毒性[9]。尽管合成尿素并不是多数鱼类应对高浓度氨氮的主要策略, 但其在氨氮环境下仍会增加尿素的合成和累积, 如细鳞非洲肺鱼[68]、石花肺鱼(Protopterus aethiopicus)和非洲肺鱼(Protopterus annectens)[69]等。由于尿素合成是非常耗能的, 而且肺鱼合成尿素也并非为了降解氨氮毒性,由此可推断其合成尿素是其某些生理活动所需要,如维持夏眠[9]。

理论上来说, 鱼类在氨氮或者空气暴露条件下,最有效的应对措施是活跃的排泄, 这样可以确保机体内较低的氨水平, 以保护大脑免于氨氮毒性[5,9]。一些气呼吸型鱼类会特化其鳃或者辅助呼吸器官以适应快速的排泄, 这在龟壳攀鲈[67]、尖齿胡鲶[70]及许氏齿弹涂鱼[71,72]上已得到证实。大量的研究表明鱼类的鳃具有一定的可塑性[73], 环境条件的变化以及个体自身的发育均可能导致鱼类鳃表面积的变化, 如水体中溶解氧状况[73]、离子强度[74]及稚鱼向成鱼转换[75]等。鳃功能异化在气呼吸型鱼类中可能表现的更为明显, 因为其可能会暴露于空气中, 无法通过鳃与水体进行物质交换。泥鳅和花溪鳉在空气暴露状态下可通过肠道或者皮肤挥发NH3以排泄体内一部分的氨[65,76], 许氏齿弹涂鱼在这种情况下会在其鳃腔中保留少量的水以保证的快速排泄[77], 而龟壳攀鲈暴露于空气中仍可通过鳃和皮肤快速的排泄体内过量的氨, 排泄速度甚至比正常状态下更快[67]。

仔稚鱼阶段鳃结构的形态发育对其氨排泄具有重要的意义。在胚胎以及鳃功能不完善的仔鱼阶段, 其主要以皮肤作为主要的氨排泄位点, 这在卵黄囊膜上尤为明显[83]。在仔稚鱼鳃功能成熟之后, 其氨排泄也就与成鱼相似, Na+和的交换与NHE、氢ATP酶及Rh糖蛋白密切相关[83,84]。仔稚鱼氨排泄位点从皮肤向鳃转换的标志在于鳃小片结构的分化、成熟[85]。

2.4 Rh蛋白的作用

Rhesus糖蛋白(Rhgp, Rhesus glycoproteins)是溶质转运家族SLC42中的一员, 在氨跨膜转运中起重要的作用[86]。Rh基因在鱼类中最先报道于红鳍东方鲀(Takifugu rubripes), 包括Rhag (Rh-associated glycoprotein)、Rhcg1、Rhcg2以及Rhbg[87]。Rh蛋白促进氨排泄的可能机制有以下3个: (1) 促进NH3的扩散, (2) 电中性的交换, (3) 产电的转运[88]。后续的研究发现, 其他硬骨鱼类中也发现一种或者多种与氨转运相关Rh蛋白的表达, 如虹鳟[89]、斑马鱼(Danio rerio)[83]、海湾豹蟾鱼[90]、鲤[91]、大西洋盲鳗(Myxine glutinosa)[92]等, 这些结果说明了Rh蛋白在硬骨鱼类中普遍存在, 且与其氨排泄具有密切关系。

Wright和Wood[88]认为淡水鱼类存在一种交换综合体”, 包含一些膜转运蛋白, 这些转运蛋白共同协作促进鳃的氨排泄, 而且其与Na+的摄入及酸排泄均有着动态的联系。在这一模式下, 血浆和红细胞均可以将氨带入鳃组织中。由于pH得差异及膜渗透性, 氨在这一状态下主要是以离子态的形式存在, 因此红细胞内部的总氨浓度要明显的高于血浆中的浓度(约3—4倍)。然而,红细胞与血浆之间存在一个明显的氨浓度差, 这种非稳态的条件会促进血液流经鳃组织[88]。这一现象会导致红细胞中的Rhag促进NH3由红细胞流向血浆中。如果鳃上皮和鳃上血管之间柱状细胞中含有Rhag, 其还会进一步促进NH3流向鳃上皮细胞。之后, NH3可以通过Rhbg (Rhbg 1 and/or 2)从鳃细胞膜基底以及通过Rhcg (Rhcg 1 and/or 2)从鳃细胞膜顶层排泄出体外, 以降低红细胞与血浆之间的氨浓度差。然而, 在正常的生理状态下, 氨的排泄可能更加依赖于代谢产生的CO2在碳酸酐酶催化下的水合作用, 以此来提供额外的H+用于酸化[88]。在斑马鱼H+质子泵细胞中发现碳酸酐酶-4同工型mRNA的细胞外表达[93], 这为上述观点提供了证据。CO2的水合作用速度远比的脱水作用要快, 因此以CO2水合来提供H+是有可能的, 而且可能在鳃上皮表明的酸化中也起到了重要的作用。

2.5 降低环境pH

在静水条件下, 鱼类排泄出的H+和CO2对周围环境酸化具有很大的影响。在酸性条件下, 以NH3形式存在的氨比例降低, 使得水生动物免于环境氨毒性。许氏齿弹涂鱼可在滩涂地上建造巢穴用于繁殖, 有研究表明其巢穴中pH约在7.0左右, 而滩涂地其他位置pH则高达7.84[72], 而且在实验室的研究也证实许氏齿弹涂鱼可降低周围环境pH[71]。当许氏齿弹涂鱼在洞穴中的时候, 由于缺乏充足的水, 其排泄速率会明显增加, 因此其周围环境氨浓度显著提高。在这种情况下, 许氏齿弹涂鱼排泄并不是为了排出体内的NH3和H+, 因为外界环境与其体内的NH3浓度差增加, NH3会流回体内。因为酸的排泄是受外界环境氨浓度影响的, 因此即使在中性环境中, 酸的排泄也会持续进行[9]。这样一来, 许氏齿弹涂鱼鳃上皮的离水界面的pH会呈酸性(较高的H+浓度), 以保证不会分解为NH3, 避免NH3回流入鱼体内[72]。因此,和酸排泄的同时进行也是某些鱼类有效的耐氨策略。

2.6 NH3挥发和体表碱化

NH3挥发是气呼吸型鱼类应对氨氮毒性的重要策略之一。由于氨可以NH3的形式存在, 因此理论上鱼类是有可能直接将NH3排放到空气中。鱼类可以直接挥发NH3最初见于大头鳚中, 但NH3挥发只占总氨排泄量的8%左右[94]。但后续的一些研究发现一些鱼类如柯克氏跳弹鳚(Alticus kirki)[95]、花溪鳉[76]以及泥鳅[65]等均可在空气暴露条件下挥发相当大比例的NH3。而且, 温度以及湿度均与NH3挥发量具有正相关关系。泥鳅暴露于空气中会挥发相当大比例的NH3[65], 可能是因为泥鳅后肠壁非常薄, 血管分布密集[96], 这种器官特化非常有利于气体的流动。此外, 空气暴露会导致泥鳅细胞膜流动性的显著增加, 这可能会增加NH3在其鳃细胞膜中的渗透性[97]。这一发现可能意味着鳃与泥鳅的氨气挥发无关。NH3应该是由泥鳅直接挥发至空气中, 而且是通过肠道挥发再经肛门排出[65]。花溪鳉也具有挥发NH3的能力[76], 而且其在空气暴露条件下皮肤表面pH为增加0.4—0.5个单位, 这增加了其皮肤表面NH3的累积[98]。此外, 有研究表明,花溪鳉在氨氮和空气暴露条件下, Rhcg在其鳃和皮肤的表达量都是非常显著的[99]。因此, Hung等[99]认为Rh蛋白提高了NH3从血液中向皮肤的流动性, 而且可能促进NH3的挥发。

由于NH3的存在量极大程度上受介质pH影响,因此鱼类体表碱化应该有利于氨以NH3的形式排泄。泥鳅在空气暴露条件下, 前肠明显碱化, 是其NH3挥发位点之一[65], 在一些陆生蟹类中也发现其可以通过碱化的尿液排出NH3[100]。相比于海水鱼类, 淡水鱼类肠腔碱化的机制并不清楚[101], 海水鱼类肠腔碱化主要是与肠道阴离子交换相关[102,103]。淡水鱼类肠腔内壁碱化的机制也鲜有报道, 但其很可能也是通过分泌替代Cl–完成的, 这一现象在泥鳅中已得到证实[104]。

2.7 降低体内氨的生成

鱼体内的氨主要由氨基酸代谢产生, 因此鱼类可通过降低氨基酸代谢以减少氨的生成来防止机体内氨浓度过高。鱼类蛋白质水解和合成的平衡会保持其体内游离氨基酸(Free amino acids, FAA)含量的稳态。如果非必需FAA含量在机体内累积可认为是GDH及一些转氨酶催化氨和α-酮酸合成氨基酸的增加, 而必需FAA含量在饥饿鱼类体内累积则可能是由于氨基酸合成的减少[105]。降低体内氨基酸代谢可能是鱼类应对氨氮毒性的有效策略之一, 其可降低鱼类机体内氨含量, 而且并不需要外源能量的参与[5]。但是, 这种情况只能在体内氨浓度已达到一定程度才会出现, 此时鱼类体内氨的累积速度要明显大于排泄速率。

这一耐氨策略已在一些气呼吸型鱼类中得到证实, 如空气暴露泥鳅一段时间后, 泥鳅体组织中氨累积量会明显超过氨排泄量[57,65], 因此作者认为泥鳅可通过降低蛋白质水解和氨基酸代谢来适应空气暴露。黄鳝在高环境氨氮中暴露较长一段时间后, 其肝脏和肌肉中总FAA含量会显著增加[106],而且总FAA的增加主要体现在谷氨酰胺以及几种必需氨基酸含量的增加, 这些结果表明了黄鳝在长时间高氨氮暴露下会降低氨基酸代谢[58]。相似的,空气暴露中华乌塘鳢24h后, 其氨基酸代谢并未受到抑制, 而在暴露72h后则发现其体内N保留量达到595 μmol, 因此氨基酸代谢降低也是发生在长时间空气暴露后[60]。细鳞非洲肺鱼进入夏眠的前6d及后续的34d, 其机体氨生成速度相较于对照组(0)分别降低了26%和28%[68]。石花肺鱼在夏眠的前12d内氨生成速度仅降低20%, 而在夏眠的第34—46d其体内氨生成速度则降低96%[107]。非洲肺鱼经历12d的夏眠后, 其组织中尿素含量却明显上升(增加约2.7倍)而非氨浓度升高, 但其肝脏OUC相关酶活性却没有明显的变化。当其在空气中夏眠46d后, 其组织中尿素含量较对照组升高至1.4倍、氨生成速度降低56%, 说明了非洲肺鱼主要以合成尿素和降低氨生成来应对空气暴露[108]。类似的研究还可见于许氏齿弹涂鱼和薄氏大弹涂鱼等[10]。

2.8 特定氨基酸代谢生成丙氨酸

抑制体内蛋白水解和氨基酸代谢可能是鱼类应对氨氮毒性的有效策略之一, 其可降低鱼类机体内氨含量。然而, 在这种情况下也同样抑制了利用氨基酸作为能量源, 这对某些特定鱼类来说并不合适, 如许氏齿弹涂鱼需要在滩涂地上运动[109]。因此, 某些气呼吸型鱼类在抑制体内蛋白水解和氨基酸代谢的同时会部分代谢氨基酸以保证能量供应。谷氨酸和丙酮酸在转氨基作用下会产生α-酮戊二酸, 其在三羧酸循环和电转移链的作用下可被完全氧化为CO2和H2O, 此反应可提供ATP。α-酮戊二酸通过三羧酸循环可被转化为苹果酸, 而苹果酸在苹果酸酶的作用下又可变为丙酮酸, 丙酮酸加上谷氨酸在丙氨酸转氨酶的转氨基作用下可生成丙氨酸。从本质上来说, 这一系列转氨反应生成丙氨酸并未涉及到氨的释放。因此, 部分氨基酸代谢生成丙氨酸虽然不能降低氨的毒性却很好的抑制了体内氨的生成。从这一点来说, 氨基酸部分代谢生成丙氨酸也是鱼类应对氨氮毒性的有效策略之一, 而且这一过程还可提供机体必要的ATP。有许多氨基酸可被部分代谢为丙氨酸且不产生氨, 如1 mol谷氨酸转化为丙氨酸可产生10 mol ATP, 而精氨酸和脯氨酸转化为丙氨酸生成的ATP量则更大。

泥鳅在空气中暴露12h之后, 其肝脏中丙氨酸含量升高两倍, 说明其在空气中可部分代谢氨基酸生成丙氨酸以抑制体内氨浓度的升高[57]。许氏齿弹涂鱼在滩涂地中排泄氨是很困难的, 氨基酸部分代谢生成丙氨酸对其来说是较为理想的耐氨策略, 且可为其在滩涂地中活动提供必要的能量。Ip等[110]发现许氏齿弹涂鱼在陆地上运动3h后, 其体内糖原含量并没有发生变化, 尽管其肌肉中乳酸含量明显升高, 而且肌肉中氨和丙氨酸含量也明显升高, 这些结果说明氨基酸部分代谢的出现与暴露时间及机体能耗相关。氨基酸部分代谢使得一些鱼类降低了对碳水化合物的依赖性, 节约了贮存在体内的糖原, 使得它们在离水条件下依然能够保持较高的代谢速率。鳢科的月鳢(Channa asiatica)也是一种典型的气呼吸型鱼类, 月鳢在干旱季节会经常面临空气暴露。月鳢在离水条件下无法进行运动、摄食等生活习性, 待其重新回到水中也会经历较长时间的恢复期。月鳢在空气中暴露48h后, 其肌肉中丙氨酸含量从3.7升高至12.6 μmol/g, 这补偿了其体内氨累积与氨排泄差值的70%[111]。这说明月鳢可利用一些氨基酸作为能量源, 与此同时最大程度上降低了其体内的氨浓度。

2.9 组织高氨耐受性

一些气呼吸型鱼类的组织和细胞具有很高的氨耐受性, 其组织或者器官中可累积较高浓度的氨,如泥鳅及黄鳝等[57,65,106]。但是氨在其机体内并不是均匀分布的, 一些组织和器官中的氨浓度显著高于其他组织和器官。泥鳅在正常情况下血浆中氨含量为0.81 μmol/L, 而在空气中暴露6h后血浆氨浓度升高至2.46 μmol/L, 空气暴露48h后血浆、肌肉和肝脏中氨含量均显著升高, 分别为5.09、14.8和15.2 μmol/g[57]。然而, 在通常情况下多数鱼类组织和器官中氨含量均＜1 μmol/g。空气暴露黄鳝72h后,其肝脏、大脑和血浆中氨浓度分别升高为对照组的3倍、3.5倍和5倍, 肌肉和肠道氨含量在144h后达到峰值, 分别为6.9和4.5 μmol/g[58]。黄鳝在75 mmol/ L的NH4Cl溶液中暴露72h后, 其肌肉、肝脏、肠道、大脑和血浆中也发现有明显的氨累积现象[106]。Tsui等[65]认为泥鳅组织中氨累积有助于其进行NH3挥发。但黄鳝并不能进行NH3挥发, 其组织中氨的累积可能是由于其对环境氨氮极高的耐受性, NH4Cl溶液(pH 7.0, 28℃)对黄鳝48, 72和96h的半致死浓度分别为209.9、198.7和193.2 mmol/L[106]。

与其他组织不同, 鱼类大脑对氨的耐受性可能较低, 当血液中氨浓度升高, 通过血液循环, 氨就可影响到脑组织。增加谷氨酰胺的合成是鱼类常用的应对脑组织氨浓度过高的策略, 但脑组织中谷氨酰胺的累积也会导致一些其他的问题, 因此这一策略也是暂时性的[5]。然而, 鱼类的神经中枢神经系统较高等脊椎动物并不是非常发达, 因此鱼类大脑通常具有较高的氨耐受能力[26]。在对一些鱼类的研究中发现其脑组织高氨耐受性机制与高等脊椎动物是不同, 如海湾豹蟾鱼、许氏齿弹涂鱼、薄氏大弹涂鱼、尖齿胡鲶及黄鳝[24,25,47,49]。Ip等[24]给许氏齿弹涂鱼和薄氏大弹涂鱼注射致死剂量的CH3COONH4和100 μg/g的蛋氨酸亚砜酰亚胺(Methionine sulfoximine, MSO), 而MSO是一种GS抑制剂, 以此来抑制其大脑中谷氨酰胺的累积。结果表明MSO并不能降低许氏齿弹涂鱼和薄氏大弹涂鱼的死亡率。同样的方法和剂量, MSO (100 μg/g)可在27—48 min内将注射了致死剂量CH3COONH4的尖齿胡鲶的死亡率降低20%[25], 他们认为MSO抑制尖齿胡鲶脑组织氨累积可能是通过抑制了脑组织中GDH和丙氨酸转氨酶的活性。类似地, MSO抑制黄鳝脑组织氨累积也不是抑制其GS活性, 而是通过影响GDH活性来实现的[49]。前文已经叙述了NMDA受体对脑组织的神经毒性作用。因此, NMDA受体的拮抗剂可能具有保护脑组织免于氨氮毒性的作用。已有研究的NMDA受体拮抗剂为(5R,10S)-(+)-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminehydrogenmaleate (MK801)。然而, 2 μg/ g的MK801对注射致死剂量的CH3COONH4许氏齿弹涂鱼和薄氏大弹涂鱼并未起到保护作用[24], 说明了NMDA受体的激活并不是氨氮急性暴露时致死的主要原因。综合这些结果来看, 鱼类大脑具有间接降低脑组织氨含量的功能。

3 展望

本文综述了氨氮对鱼类的毒性机理以及鱼类的应对策略, 为相关方向的研究提供了理论资料。鉴于国内外对氨氮毒理的研究仍停留在基础的生理学范畴, 后续的相关研究应该从分子和细胞的角度分析氨氮对水生动物的毒理作用, 这样可更直接、更迅速地反应氨氮对水生动物的毒性及其作用机制。同样地, 对鱼类氨解毒策略的研究也应加强深度和广度, 如探讨每一种耐氨机制的分子调控机制、常规非气呼吸型鱼类是否也具有独特的氨解毒策略及其调控机制等等。这些研究资料的累积将丰富氨氮对水生动物的毒理机制及水生动物的氨解毒机制, 具有重要的科研价值, 也可为水生动物健康养殖提供理论依据。

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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

安徽省高等学校自然科学研究项目(KJ2017A130)；国家科技支撑计划(2012BAD25B08和2012BAD25B00)资助 [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)]

张云龙(1989—), 男, 安徽合肥人; 讲师, 博士; 主要从事鱼类增养殖研究。E-mail: zhangyunlong@ahau.edu.cn

樊启学(1962—), 男, 湖北潜江人; 教授; 主要从事鱼类增养殖及水域生态学研究。E-mail: fanqixue@mail.hzau.edu.cn