白鲢罗非鱼对微囊藻毒素急性、亚急性毒性响应

沈 强,刘永定,李敦海,李嗣新



白鲢罗非鱼对微囊藻毒素急性、亚急性毒性响应

沈 强1*,刘永定2,李敦海2,李嗣新1

(1.水利部中国科学院水工程生态研究所,水利部水工程生态效应与生态修复重点实验室,湖北 武汉 430079；2.中国科学院水生生物研究所,湖北 武汉 430072)

为了探究不同控藻鱼类对产毒微囊藻的适应机制,为生物操纵的鱼种选择提供依据,研究了白鲢和罗非鱼对微囊藻毒素(MC)的生物富集、降解,及两种鱼对毒素的抗性、解毒机制的差异性.结果发现:在喂食微囊藻实验中,白鲢、罗非鱼对MC日摄入量达到10mg/kg,2种鱼均对MC有较强抗性.微囊藻经鱼摄入后,MC总含量在白鲢、罗非鱼粪便中分别下降到71.5%、6.0%,罗非鱼对MC降解能力远高于白鲢.白鲢和罗非鱼的肝系数分别从(1.19±0.21)%、(2.24±0.19)%下降到(0.79±0.06)%、(1.72±0.07)%,均表现出显著差异性下降(<0.05).微囊藻毒素在白鲢、罗非鱼肌肉中积累量分别为(1.57±0.31)μg/kg、(10.81±6.52)μg/kg(鲜重)、肝脏中积累量分别为(4.28±1.64)mg/kg、(2.48±0.15)mg/kg(鲜重).MC在白鲢、罗非鱼肌肉、肝脏中的积累量均存在显著差异性(<0.05).罗非鱼肌肉中毒素含量是白鲢的6.9倍.在微囊藻毒素LR(MC-LR)对白鲢和罗非鱼的急性毒性效应实验中,MC-LR对白鲢、罗非鱼的LD50为270和790µg/kg,罗非鱼对毒素有更强耐受性.喂食毒藻和i.p.注射MC均导致白鲢和罗非鱼肝细胞内脂滴大量出现.2种鱼在MC-LR注射后,谷胱甘肽(GSH)含量均表现出6h内明显下降.6h后两种鱼GSH含量均逐步回升,二者差异显著(<0.05).实验结果表明,罗非鱼对MC降解能力远高于白鲢.白鲢主要摄食群体微囊藻的群体胶鞘和附着细菌,胞内微囊藻毒素释放量小,白鲢这种摄食机制导致它能以产毒微囊藻为食而受到较轻危害.罗非鱼体内消化酶对微囊藻和MC具较强的消化降解能力;GSH含量及相关酶活性水平高,对体内毒素清除效率高.从食用安全性角度出发,与罗非鱼相比,白鲢是更适合用于控制蓝藻水华的鱼种,可广泛应用于蓝藻水华控制中.

有害藻类水华；微囊藻毒素；白鲢；罗非鱼；抗性机制

湖泊水体富营养化引起的藻类水华已成为世界范围重大环境污染问题之一.在我国,不少大型浅水湖泊在每年夏秋季节均有大量蓝藻水华形成,如太湖[1-2],巢湖[3-4]和滇池[5-6].其中大多数水华以微囊藻水华为主.微囊藻细胞通常呈球形,天然情况下多为群体形态,在藻细胞破裂时释放出大量毒素,即微囊藻毒素(MC).目前已发现100种以上微囊藻毒素变体[7],微囊藻毒素能够作用于蛋白磷酸酶,使蛋白磷酸化失调,增加活性氧(ROS)形成而具有肝脏毒性[8].微囊藻毒素给多种动植物带来危害,包括农作物[9-11],水生植物[12],浮游动物[13],鱼类[14-15],野生动物,家畜,鸟类[16-17]和人类[18]等,甚至导致其死亡.藻类水华及其所产生的毒素成为人们关注的热点问 题.

研究表明,与哺乳动物相比,鱼类对微囊藻毒素的耐受性较强,某些鱼类如白鲢,花鲢和罗非鱼等对微囊藻毒素有一定抗性[19-20].这些鱼类常被广泛应用于国内外湖泊水库中控制蓝藻水华[21-23].控藻鱼类以产毒微囊藻为食物,并能与之长期共存,但其生理适应机制尚不完全清楚.因此,本文选取国内外2种常见的控藻鱼类白鲢、罗非鱼为研究对象,研究微藻藻毒素对2种鱼类的亚急性和急性毒性效应,探讨不同控藻鱼类对产毒微囊藻在生理生化上的解毒机制,为今后生物操纵的鱼种优选及水华污染控制方面提供科学依据.

1 材料和方法

1.1 实验材料

微囊藻水华采集:在武汉市郊某一水华频发的鱼塘,使用孔径60μm浮游生物网采集藻类水华样品.经显微镜检测,水华中铜绿微囊藻()为主要种类,优势度>98%.采集的水华除去杂物、蒸馏水清洗后,再次用孔径60μm浮游生物网浓缩成浓藻浆,测定藻浆含水量,4℃下备用.初步纯化的浓藻浆用于亚急性实验.

微囊藻毒素制备:以采集的产毒微囊藻水华为原料制备.毒素提取方法参照文献[24-25],毒素提取物用制备型HPLC纯化,收集毒素出峰中段,反复制备2次,得到MC-LR纯品(色谱纯度>93%).纯化后的MC-LR用于急性毒性实验.

实验鱼:白鲢() (27.0±2.0)g和罗非鱼() (15.2±2.6)g分别购自武汉关桥鱼种基地和赤壁鱼种养殖基地.实验前鱼种先在室内水池(1m×1m×0.4m)内暂养7d,使之适应实验环境.

1.2 亚急性毒性实验

亚急性毒性实验分组参照文献[26-27],分成2组.2个为处理组,分别投放30条白鲢和30条罗非鱼,每天定时投喂经过上述处理后的微囊藻藻浆,投喂的微囊藻生物量参照文献方法[28],控制为0.5g干重藻/L.显微镜计数表明该投喂量相当于水中微囊藻细胞密度为6×109cells/L.2个为对照组,分别投放30条白鲢和30条罗非鱼,用商业鱼饲料喂食.所有水族箱中的水均用增氧泵并每天定时用曝气脱氯后的水更换,使用增氧泵一是为了保持水中的溶解氧,二是使水体中微囊藻细胞充分混合并被鱼类所摄食.水温用加热棒维持在(23±2)℃.分别在实验前后测量鱼体重、肝脏重量,实验持续28d.

1.2.1 对微囊藻及毒素摄食、消化和降解情况的观测 两种鱼对毒藻和微囊藻毒素的日摄入量:每7d对处理组水族箱中的水和投喂前的微囊藻取样.鱼对微囊藻的日摄入量()的计算公式为:

＝0.5g/L×200L×藻细胞密度日变化(%)/

水族箱内鱼重(kg) (1)

藻细胞密度日变化测定方法:水样取样2次,第1次取样在投喂微囊藻后;第2次在第2d换水喂藻前进行.藻细胞密度日变化根据2次取样的藻细胞密度计算.鱼对微囊藻毒素的日摄入量根据鱼对微囊藻的日摄入量和微囊藻中的毒素含量计算.

显微镜观察:收集白鲢和罗非鱼粪便和投喂前微囊藻,Nikon E600型显微镜下观察(´200,´400)并拍照.

白鲢和罗非鱼粪便、投喂前微囊藻胞内毒素含量测定:每7d对白鲢和罗非鱼粪便和投喂前的微囊藻取样,真空干燥器(Yamato, NEOCOOL)干燥后,参照文献方法[24-25]提取测定微囊藻毒素含量.

1.2.2 鱼类组织器官中毒素含量测定 喂藻实验结束后,取处理组白鲢、罗非鱼各15条,取肝脏组织,用0.85％生理盐水漂洗,除去血液等杂物,滤纸拭干水分,分析天平上称重,真空干燥器(Yamato, NEOCOOL)干燥脱水.用解剖刀取鱼约1g肌肉,剔除鱼刺,蒸馏水清洗后用滤纸吸干水分,分析天平上称重,用手术剪将鱼肉剪碎,真空干燥器干燥脱水.

鱼肌肉和肝脏组织的毒素提取:采用纯甲醇提取方法[29-30],取3批鱼作为平行样.每批取5条鱼肝脏组织样品、肌肉样品分别混合, 放入瓷研钵中,加入液氮,将样品冰冻并研磨成粉末状,加入100%色谱纯甲醇20~30mL,磁力搅拌器上搅拌提取,1h后,10000r/min离心,沉淀再加甲醇提取2次,离心合并上清液.用等体积正已烷萃取上清液2次,弃去正已烷层.上清液在旋转蒸发器中浓缩干燥后,加入20mL去离子水充分振荡,过预先已活化的Sep-pak C18固相萃取柱(10mL 100%甲醇活化,10mL 去离子水调整).20mL 20%甲醇淋洗小柱后,用20~30mL 100% 纯甲醇洗脱.洗脱液在旋转蒸发器中浓缩干燥,加入10mL去离子水,充分振荡,用ELISA试剂盒(Envirologix microcystin plate kit, Portland, ME)测定毒素浓度,根据所提取样品重量计算出微囊藻毒素在肌肉和肝脏中含量.

1.2.3 病理学检测 肝系数(HSI)测定:分别在实验前后取处理组的白鲢、罗非鱼各5条,测定鱼体重、肝脏重量,根据Al-Ghais方法计算肝系数[31].计算公式:

HSI＝肝重量(g)/鱼体重(g)´100% (2)

鱼肝脏超微结构观察:喂藻实验结束后,分别取处理组、对照组白鲢、罗非鱼肝脏组织,切成1mm3小块,先后用2.5%戊二醛溶液和1%OsO4固定,酒精梯度脱水后环氧树脂包埋,LKB型超薄切片机切片,乙酸双氧铀和柠檬酸铅染色[32].日立H-600型透射电镜下观察.

1.3 急性毒性实验

1.3.1 半致死剂量(LD50)测定 纯化后的MC-LR用无菌0.85%生理盐水溶解并稀释成不同浓度,腹腔注射入白鲢和罗非鱼体内,注射部位为鱼的腹鳍下端.每个不同浓度下分别采用5条鱼,记录鱼死亡率,根据死亡率－剂量关系计算出半致死剂量LD50[33].

1.3.2 鱼类组织器官中GSH及相关酶测定 腹腔注射MC-LR:纯化后的MC-LR用无菌0.85%生理盐水溶解稀释后,腹腔注射入白鲢和罗非鱼体内(各20条).注射剂量均为100μg/kg.分别在注射MC-LR后0,6,12,24,48,72h取样,每次各取5条白鲢和罗非鱼.对照组也按上述方法设置:取等体积0.85%无菌生理盐水腹腔注射白鲢和罗非鱼(各20条),分别在注射MC-LR后0,6,12,24,48,72h取样,每次各取3条白鲢和罗非鱼.取出鱼肝脏制备10%肝组织匀浆液,测定GSH、GST和GSH-Px活性.

GSH及相关抗氧化酶活性测定:取1g左右上述鱼肝脏组织块除去杂质,拭干称重.加9倍预冷的0.86%生理盐水,冰水浴下剪碎匀浆.4℃下3000r/min离心15min,取上清液,即得到10%肝脏组织匀浆[34]. GSH、GSH-ST、GSH-Px活性的测定参照文献[34-35],分别采用商业试剂盒(GSH assay kit, A006-4; GSH- ST assay kit, A004; GSH-PX assay kit, A005; 南京建成生物工程研究所)进行测定.在分光光度计(Pharmacia Biotech,Ultrospec 3000) 412nm处测定光吸收值.

1.3.3 病理学检测 取注射100μg/kg MC-LR后48h的两种鱼肝脏组织制备电镜切片.为观察外源性GSH对MC-LR的解毒作用,另取3条白鲢,预先注射剂量为1mg/kg 的GSH,2h后再腹腔注射(i.p.) 100μg/kg MC-LR.注射毒素48h后取鱼肝脏组织制备电镜切片,透射电镜下观察.

1.4 数据处理

数据统计分析使用SPSS V19.0,用One-Way ANOVA法进行相关性分析和差异显著性分析,绘图在OriginPro 2018软件中进行.数据表示方式为均值±标准差.

2 结果

2.1 微囊藻及毒素对白鲢和罗非鱼的亚急性毒性效应

在28d喂藻实验中没有鱼死亡,表明白鲢、罗非鱼在以产毒微囊藻为单一食物条件下均能存活, 2种鱼都对微囊藻毒素表现出较强抗性.

2.1.1 鱼类对产毒微囊藻和微囊藻毒素摄食、消化和降解 鱼类对毒藻和微囊藻毒素的日摄入量:白鲢、罗非鱼对产毒微囊藻的日摄入量依次为(6.02± 1.59),(7.94±2.73) g干重藻/kg. 2种鱼对产毒微囊藻和微囊藻毒素的日摄入量大致相同,罗非鱼摄食能力略高于白鲢(图1).白鲢、罗非鱼对微囊藻毒素MC-RR的日摄入量依次为(7.34±1.95),(9.69± 3.34)mg/kg;对微囊藻毒素MC-LR日摄入量依次为(1.81±0.48)、(2.38±0.82)mg/kg.2种鱼对微囊藻毒素的总摄入量(MC-RR＋MC-LR)达到10mg/kg左右.由于采集的铜绿微囊藻所含的毒素中 MC-RR占大部分比例,且MC-RR的毒性较低,约为MC-LR的1/10[19],这也可能是白鲢、罗非鱼在该条件下能够生存的原因之一.

白鲢对毒藻、MC日摄入量数据引自文献[28]

2种鱼对微囊藻的消化能力:白鲢粪便中基本以完整群体形态的微囊藻为主,粪便丝状,颜色翠绿色,表明白鲢对微囊藻消化能力差;罗非鱼粪便中未观察到群体形态微囊藻,呈咖啡色,表明藻类色素被完全分解,罗非鱼对微囊藻具较强消化能力.显微鉴定进一步表明,白鲢粪便中微囊藻占大部分比例(图2B),且微囊藻群体形态相对于喂食前微囊藻(图2A)无明显变化;而在罗非鱼粪便中观察不到有完整藻细胞存在,微囊藻基本上被消化吸收(图2C).显微鉴定表明,白鲢对摄入的微囊藻消化能力差,而罗非鱼对微囊藻的消化吸收能力远比白鲢强.这与2种鱼消化道内消化酶对微囊藻毒素的降解能力差异一致.

图2 投喂前微囊藻和白鲢及罗非鱼粪便的显微观察

A.铜绿微囊藻水华, ×200; B.白鲢粪便, ×200; C.罗非鱼粪便, ×400

鱼类对摄入的微囊藻毒素的降解:采集的铜绿微囊藻主要含MC-RR和MC-LR,毒素以MC-RR为主(图3).微囊藻毒素被2种鱼摄入后,由于鱼体内消化酶等作用,相对于喂食前的微囊藻,白鲢、罗非鱼粪便中MC总含量分别下降到71.5%、6.0%,罗非鱼消化降解MC的能力远高于白鲢(图4).该数据一定程度上解释了罗非鱼对毒藻具较强耐受性.此外对比鱼粪便和喂食前的毒藻,MC-RR与MC-LR比值的变化显著,依次为3.66:1(喂食前微囊藻)、2.77:1(白鲢粪便)和1.97:1(罗非鱼粪便).该数据表明:微囊藻毒素在被白鲢、罗非鱼摄入,到经粪便排出的过程中,部分毒素被鱼消化道内的消化酶降解.由于MC-RR、MC-LR的稳定性存在差异,导致鱼粪便中MC-RR和MC-LR的比值发生改变.罗非鱼粪便中,MC含量急剧下降,且MC-RR和MC-LR比值变动更为显著,表明了罗非鱼对蓝藻细胞及毒素具有很强的裂解、消化能力.

图3 产毒微囊藻及2种鱼粪便中毒素分析HPLC图谱

Fig.3 The HPLC chromatogram of microcystins in Microcystis, sliver carp feces and tilapia feces

白鲢粪便MC含量数据引自文献[28]

2.1.2 微囊藻毒素在鱼肌肉和肝脏中的累积 喂食产毒微囊藻28d后,微囊藻毒素在白鲢、罗非鱼肌肉中积累量依次为(1.57±0.31)μg/kg、(10.81±6.52) μg/kg(鲜重);在白鲢、罗非鱼肝脏中积累量依次为(4.28±1.64)mg/kg、(2.48±0.15)mg/kg (鲜重).微囊藻毒素在白鲢、罗非鱼肌肉、肝脏中的积累量均存在显著差异性(<0.05).罗非鱼肌肉中毒素含量是白鲢的6.9倍;而肝脏中毒素含量上,罗非鱼是白鲢的58%,显著低于白鲢(图5).

图5 微囊藻毒素在白鲢、罗非鱼肌肉和肝脏中的积累

其中白鲢体内MC积累数据引自文献[28],*<0.05

总体分析,微囊藻毒素在2种鱼体内积累量均很低,在鱼肉和肝脏中积累量仅为´10-9和´10-6级.相对于摄入的微囊藻毒素量[10mg/(kg·d)],微囊藻毒素在2种鱼体内积累量微乎其微.

2.1.3 病理学反应 肝系数变化:通过28d喂藻实验,白鲢肝脏重量显著下降(<0.01),罗非鱼肝脏重量变化不明显;白鲢和罗非鱼的肝系数(HIS)均表现出显著差异性下降(<0.05)(图6).喂食毒藻实验中,白鲢肝脏重量、两种鱼的肝系数均显著下降的原因,可能是两种鱼在逆境条件下能量摄取不足,肝脏中糖元等储能物质被大量消耗利用导致(见图7电镜照片,2种鱼肝细胞糖元消耗明显).

鱼肝脏超微结构变化:对照组正常白鲢肝细胞的粗面内质网排列整齐、线粒体结构正常、富含糖原颗粒且基本无脂肪滴(图7A,B);正常罗非鱼肝细胞内线粒体正常、有少量脂滴、富含大量雪花状糖原颗粒、粗面内质网排列整齐、核结构正常(图7C,D);喂食产毒微囊藻28d后,白鲢肝细胞内出现大量巨大的脂滴,线粒体嵴较清晰,但内质网和线粒体均有肿大的现象(图7E,F,G);喂食产毒微囊藻28d后的罗非鱼肝细胞内同样有大量脂滴出现,但线粒体嵴清晰可见,细胞超微结构无明显改变(图7H,I,J).脂滴病变较明显.2种鱼在喂食产毒微囊藻28d后,肝组织细胞均表现出较明显脂滴病变,白鲢肝细胞病变程度高于罗非鱼.

*<0.05,**<0.01

A.正常白鲢肝细胞,×8000;B.正常白鲢肝细胞,×15000;C.正常罗非鱼肝细胞,×8000;D.正常罗非鱼肝细胞,×17000;E.喂食毒藻28d后白鲢肝细胞,×6000;F.喂食毒藻28d后白鲢肝细胞,×12000;G.喂食毒藻28d后白鲢肝细胞,×12000;H.喂食毒藻28d后罗非鱼肝细胞,×8000;I.喂食毒藻28d后罗非鱼肝细胞,×8000;J.喂食毒藻28d后罗非鱼肝细胞,×30000

2.2 微囊藻毒素对白鲢和罗非鱼的性毒性效应

2.2.1 半致死剂量(LD50)比较 依据表1做出致死率-对数剂量曲线,求出MC-LR对白鲢和罗非的LD50.计算结果得出MC-LR对白鲢、罗非鱼的LD50依次为270,790µg/kg.而MC-LR对小鼠的LD50为50µg/kg[19],因此在对MC的耐受性上,罗非鱼>白鲢>小鼠,罗非鱼对毒素耐受性比白鲢更强.

表1 i.p.注射不同剂量MC-LR对白鲢和罗非的致死率

Table 1 Mortality of sliver carp and tilapia after injected with different doses of MC-LR

2.2.2 GSH含量、GST、GSH-Px活性变化 与对照组相比,白鲢注射100µg/kg MC-LR 6h后,肝脏组织中GSH急剧下降至74%,与对照差异显著(< 0.05).此后GSH含量缓慢恢复,在72h后恢复到MC-LR注射前水平(图8).罗非鱼注射100µg/kg MC-LR后,其肝脏组织中GSH含量同样在短时间内显著下降,在6~12h期间GSH含量迅速恢复并上升至较高浓度, 与对照差异显著(<0.05),12h后GSH含量高达MC-LR注射前的1.4倍.该结果显示出罗非鱼在毒素急性暴露后,肝脏组织内GSH快速参与了对MC的解毒作用,并表现出极强的GSH补偿性再生合成能力.

图8 i.p.注射100µg/kg MC-LR后白鲢、罗非鱼肝脏组织GSH含量的变化

*<0.05

对比100µg/kg MC-LR后两种鱼肝组织内GSH含量变化结果(图8),两种鱼在MC-LR注射后,GSH含量均在6h内明显下降,表明GSH在注射MC-LR 6h内被大量消耗.6h后两种鱼GSH含量均逐步回升,其中罗非鱼肝脏GSH含量在6,12,24,72h分别是白鲢的78%、128%、127%和107%,二者差异显著(<0.05).罗非鱼显示出更强的GSH再生能力和解毒潜力.

GSH-Px、GST都能以GSH为底物,清除机体的过氧化氢和有机氢过氧化物,在消除微囊藻毒素进入机体后带来的氧化胁迫中起到重要作用.对比两种鱼注射MC-LR前后肝脏组织GST活性(图9),罗非鱼肝脏组织GST在MC-LR注射前后无明显变化,而白鲢GST活性在MC-LR注射6h后显著下降至注射前的47%,并在随后6~72h期间持续维持在较低水平.

*<0.05

在MC-LR注射后,罗非鱼肝脏组织GSH-Px活性持续上升,72h后上升至注射前184%.而白鲢GSH-Px活性在MC-LR注射后6h下降明显,为注射前的72%;随后活性略有恢复,但72h仍无法恢复至注射前水平.

对比2种鱼的GST、GSH-Px活性,罗非鱼肝脏GST活性远显著高于白鲢(<0.05),且GSH-Px活性在毒素暴露后得到激活并持续上升.该结果也表明了罗非鱼体内GSH相关酶活性在毒素暴露下能迅速激活,从而为解毒提供了有利环境.

图10 急性毒性实验白鲢罗非鱼肝细胞超微结构病变

A.注射MC-LR 48h白鲢肝细胞,×20000;B.注射MC-LR 48h白鲢肝细胞,×15000; C.注射MC-LR 48h白鲢肝细胞,×5000;D.注射MC-LR 48h罗非鱼肝细胞,×5000;E.注射MC-LR 48h罗非鱼肝细胞,×10000; F.注射MC-LR 48h罗非鱼肝细胞,×20000; G.预先注射1mg/kg GSH 2h后再注射MC-LR,48h白鲢肝细胞,×20000;H.预先注射1mg/kg GSH 2h后再注射MC-LR,48h白鲢肝细胞,×10000

2.2.3 鱼肝细胞超微结构变化情况 注射100µg/ kg MC-LR 48h后的白鲢肝细胞发生了明显病变.表现为:糖原颗粒减少,脂滴大量出现(图10A,B);线粒体肿大,部分发生髓鞘样结构病变(图10A,B的箭头处);大量出现死亡的肝细胞(图10C).注射100µg/kg MC-LR 48h后的罗非鱼肝细胞病变同注射毒素后的白鲢相似,表现为:数目众多巨大脂肪滴出现,糖原颗粒很少(图10D,E);部分核膜消失(图10F);线粒体肿大,基质密度降低(图10E),有髓鞘样结构病变发生(图10F箭头处).预先注射1mg/kg GSH 2h后再注射100µg/kg MC-LR,48h后白鲢肝细胞表现为:细胞内基本无脂滴出现、糖原颗粒较丰富、部分线粒体肿大.和直接注射100µg/kg MC-LR 48h后的白鲢肝细胞相比,死亡细胞数明显下降(图10G,H).

急性毒性暴露实验中,白鲢和罗非鱼肝细胞内再次出现大量脂滴,而预先注射1mg/kg GSH的肝细胞无此现象发生.急性暴露实验中肝细胞脂滴大量出现的现象与亚急性暴露实验中2种鱼肝细胞病变情况相同.

3 讨论

3.1 微囊藻毒素对白鲢和罗非鱼的急性、亚急性毒性效应

在28d喂食毒藻的亚急性实验中,罗非鱼肌肉中毒素含量是白鲢的6.9倍;而肝脏中毒素含量罗非鱼是白鲢的58%,显著低于白鲢.可能由于罗非鱼体内消化酶活性较高,导致摄入的微囊藻细胞大量裂解,从而释放出较多的微囊藻毒素进入体内导致.罗非鱼肝脏中毒素积累量相对较低,表明罗非鱼肝脏对微囊藻毒素的降解功能强于白鲢.

对比两种不同毒素暴露方式下,MC对2种鱼的毒性效应、结合2种鱼肝脏超微结构病变的结果,注射MC-LR方式的急性毒性实验对白鲢和罗非鱼肝细胞的损伤远大于喂食产毒微囊藻的方式.在同等毒素暴露的条件下,肝脏细胞的罗非鱼肝细胞受损程度略小于白鲢,揭示了罗非鱼比白鲢对微囊藻毒素具更强的抗性,与2种鱼肝脏中毒素积累量的测定结果一致.

罗非鱼对外源微囊藻毒素比白鲢有更强的解毒能力.在鱼体内,GSH可防御MC带来的氧化性损伤,并与MC相结合形成GSH-MC结合物是解毒的第一步[36].因此,两种控藻鱼体内GSH水平的应激性修复能力、GSH相关酶系统的活性恢复能力是表征两种鱼解毒能力的重要标志.本研究证明了两种鱼对MC-LR的解毒作用与GSH及其合成代谢相关的酶系统相关联.罗非鱼在外源毒物进入后,体内的GSH能在较短时间内迅速上升并维持在较高水平.电镜观察结果表明外源性GSH对白鲢肝脏细胞也起到了很好保护作用,证明了GSH在2种控藻鱼类解毒过程中的重要作用.

3.2 微囊藻毒素暴露下2种鱼肝细胞脂滴病变

综合两种MC暴露途径,均导致了白鲢和罗非鱼肝细胞内脂滴大量出现,而两种鱼的对照组、预先注射GSH保护后再注射MC-LR的白鲢肝细胞内脂滴大小和数量基本正常.这种MC暴露下白鲢和罗非鱼肝细胞内脂滴大量出现的现象尚少有文献报道.肝细胞脂滴病变是一种可逆性病变,鱼类暴露在铜、镉等重金属污染条件下,肝细胞内有形成大量脂肪滴的类似报道[37-38].结合GSH及相关酶的实验推测,该脂滴病变现象表明微囊藻毒素可能参与了干扰鱼肝脏细胞的脂肪代谢途径.其原因有待进一步研究.

3.3 白鲢和罗非鱼对产毒微囊藻和微囊藻毒素的适应机制

本实验中喂食微囊藻的细胞密度达到6× 109cell/L,超过绝大多数有微囊藻水华爆发的天然水体的藻细胞密度.白鲢和罗非鱼均对产毒微囊藻和微囊藻毒素有较强的抗性.其中罗非鱼抗性又略高于白鲢.

白鲢对产毒微囊藻的适应机制:在喂食产毒微囊藻的实验中,白鲢摄入的微囊藻毒素大部分经粪便直接被排出体外.由于白鲢无真正的胃[39],该滤食性鱼类对摄入微囊藻营养的获取主要取自微囊藻的群体胶鞘和附着的细菌,而非微囊藻细胞本身[40],且微囊藻毒素是胞内毒素,只有当藻细胞死亡裂解后才能释放出来[41-42],因此白鲢摄食产毒微囊藻后,微囊藻毒素能较完整地保存在藻细胞内,从而大大减轻了对其危害.另一方面,白鲢肝细胞中GSH也具有良好的补偿性再生能力,保证了对毒素具一定的抗性.白鲢的这种摄食机制保证了它能以产毒微囊藻为食而受到较轻的危害.

罗非鱼对产毒微囊藻的适应机制:罗非鱼胃中消化液呈强酸性,pH值最低仅为1.4[43],这种强酸环境可有效地裂解蓝藻细胞,同时对蓝藻细胞具很强的裂解、消化和利用能力[43-44],也保证了对微囊藻毒素有较强的降解能力.另一方面,相对于白鲢,罗非鱼体内GSH含量在外源性毒素进入体内后能迅速地应激性上升,同时GSH相关酶活性维持在较高水平,保证了罗非鱼对进入体内的微囊藻毒素具较高的清除效率.在上述综合作用下,保证了罗非鱼对进入体内的微囊藻毒素有更强耐受能力.

3.4 白鲢和罗非鱼用于控制蓝藻水华风险评估

目前有多种鱼类如白鲢、鳙鱼和罗非鱼被用于控制水体中蓝藻水华.由于微囊藻毒素能在鱼体内积累从而对人类健康带来潜在的危害,WHO推荐人体每日可允许摄入的微囊藻毒素量TDI不大于0.04μg/kg人体重[19].根据WHO推荐的标准,按成人平均体重60kg计算,则每人每天可摄入的微囊藻毒素量不超过2.4μg.假设人均每天吃鱼300g,则鱼肉中微囊藻毒素含量不能超过8.0µg/kg鱼湿重.

根据喂藻28d的实验结果,微囊藻毒素在白鲢、罗非鱼肌肉中的积累量分别为(1.57±0.31),(10.8± 2.1)μg/kg鱼湿重.从食用安全性角度评价,毒素在罗非鱼肌肉中积累量超过8.0µg/kg鱼重的标准,蓝藻水华爆发水体中的罗非鱼不适于食用,而白鲢肌肉中微囊藻毒素积累量则在WHO推荐的标准范围内.

其他文献也支持了本文的研究.长期暴露在产毒蓝藻水华的情况下,微囊藻在罗非鱼肌肉中的积累量为0~337.3μg/kg 鱼重[30],远超WHO推荐范围;通过对北美10多个湖泊中鱼体微囊藻毒素含量的监测表明,在所有存在罗非鱼的5个湖泊中,罗非鱼体内微囊藻毒素含量依次为2.7~6.2,2.0,2.1~19.3, 7.1~33.8,3.4~14.1μg/kg鱼湿重[45],半数湖泊中罗非鱼体内毒素数据均超过了WHO推荐的标准.而在水华严重爆发的太湖中,白鲢体内MC含量最高为0.094μg/kg鱼干重(以常规鱼的肌肉含水量80%计,相当于MC最高含量0.024μg/kg鱼湿重)[46].Zhang等[47]对国内8个富营养化的湖泊中白鲢体内MC进行了监测,白鲢肌肉中MC含量范围为0.014~ 0.036μg/g DW(相当于2.8~7.2μg/kg鱼湿重,以鱼的肌肉含水量80%计),均在WHO推荐的食用安全范围之内.

由此可见,从食用安全性角度出发,与罗非鱼相比,白鲢是更适合用于控制蓝藻水华的鱼种,可以广泛应用于蓝藻水华控制的实际操作中.

4 结论

4.1 在亚急性毒性实验中,白鲢、罗非鱼对MC日摄入量达10mg/kg,2种鱼均对MC有较强抗性.微囊藻经鱼摄入后,MC含量在白鲢、罗非鱼粪便中分别下降到71.5%、6.0%,罗非鱼对MC降解能力远高于白鲢.白鲢和罗非鱼肝系数分别从(1.19±0.21)%、(2.24±0.19)%下降到(0.79±0.06)%、(1.72±0.07)%,均显著下降(<0.05).微囊藻毒素在白鲢、罗非鱼肌肉中积累量分别为(1.57±0.31)μg/kg、(10.81±6.52)μg/ kg(鲜重)、肝脏中积累量分别为(4.28±1.64)mg/kg、(2.48±0.15)mg /kg (鲜重).MC在白鲢、罗非鱼的肌肉、肝脏中积累量均存在显著差异性(<0.05).

4.2 在急性毒性实验中,MC-LR对白鲢、罗非鱼的LD50为270,790µg/kg,罗非鱼对MC有更强耐受性.注射MC-LR后2种鱼肝组织谷胱甘肽(GSH)含量和相关GST、GSH-Px酶均发生显著变动.罗非鱼GSH、GSH-Px酶恢复能力和GST酶活性水平显著高于白鲢.

4.3 毒性实验中微囊藻毒素急性、亚急性暴露均导致白鲢和罗非鱼肝细胞内脂滴大量出现,但白鲢、罗非鱼对微囊藻及毒素具有不同生理适应机制,白鲢主要摄食群体微囊藻的群体胶鞘和附着细菌,胞内微囊藻毒素释放量小,导致其能以毒藻为食而受危害较轻.而罗非鱼体内消化酶对微囊藻和MC具较强消化降解能力;且GSH含量及相关酶活性水平高,对体内毒素清除效率高.

4.4 从食用安全性角度出发,白鲢是更适合用于控制蓝藻水华的鱼种,可广泛应用于蓝藻水华控制中.

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The acute and subacute response of sliver carp and tilapia to microcystin.

SHEN Qiang1*, LIU Yong-ding2, LI Dun-hai2, LI Si-xin1

(1.Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem of Ministry of Water Resources, Institute of Hydroecology, Ministry of Water Resources and Chinese Academy of Sciences, Wuhan 430079, China；2.Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan 430072, China)., 2019,39(6)：2633~2643

In order to study adaptative mechanisms of sliver carp and tilapia on harmful algal blooms and provide scientific methods of fish species selection in biological manipulation, systematic research was conducted on bioaccumulation, degradation of microcystin and the differences in resistance and detoxification mechanisms on sliver carp () and tilapia (). In toxicfeeding experiment, the daily intake of microcystin by sliver carp and tilapia was up to 10mg/kg body weight. Both fishes show strong resistance to MC. Microcystin concentrations in feces of sliver carp and tilapia afterintake were significantly decreased to 71.5% and 6.0% respectively (<0.05). The degradation ability of tilapia to toxicand microcystin is much higher than silver carp. The hepatosomatic index of sliver carp and tilapia was significantly decreased from (1.19±0.21)% and (2.24±0.19)% to (0.79±0.06)% and (1.72±0.07)% respectively (<0.05). Bioaccumulations of MC of the two species were (1.57±0.31) and (10.81±6.52)μg/kg (fresh weight) in the muscle, (4.28±1.64) and (2.48±0.15)mg/kg (fresh weight) in the liver. There were significant differences between MC accumulation in the muscle and liver of each species (<0.05). Microcystin concentration in the muscle of tilapia was 6.9 times higher than that of silver carp. During the toxic experiment, LD50of microcystin-LR was 270µg/kg on sliver carp and 790µg/kg on tilapia, which suggested microcystin tolerance of tilapia is stronger than that of sliver carp. Enormous lipid droplets were observed in the liver cell of the two species whether fed withor intraperitoneally injected with microcystin. After intraperitoneal injection with microcystin-LR, the content of GSH in the two species showed a significant decrease in 6h and then increased gradually. Significant difference of GSH content was found between the two species (<0.05). The results showed that the degradation ability of tilapia to microcystin is much higher than silver carp. Silver carp mainly feeds on the mucilage sheath and adhesion bacteria of colonialwith small amount of intracellular microcystin released. This mechanism can effectively protect silver carp fed withto less damage. The digestive enzymes in tilapia have strong digestion and degradation ability toand microcystin, and the high level of GSH content and related enzyme activity ensure efficient detoxification of toxins. From the view of food safety, compared with tilapia, silver carp is the species which is more suitable and to be widely used for cyanobacteria bloom control.

harmful algal blooms (HABs)；microcystin；sliver carp；tilapia；resistance mechanism

X174

A

1000-6923(2019)06-2633-11

沈 强(1975-),男,河南潢川人,高级工程师,博士,主要研究方向为藻类学和水生态监测新技术研究.发表论文20余篇.

2018-11-12

湖北省自然科学基金资助项目(2017CFB678);948计划项目(201509)

*责任作者, 博士, shenqiang2005@gmail.com