敌百虫对异育银鲫抗氧化应激系统的影响及抗坏血酸对鱼体的保护作用
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摘要
有机磷杀虫剂——敌百虫是水产养殖过程中大量应用的水体消毒剂和鱼体杀虫剂,但是敌百虫在杀灭寄生虫的同时也会对非标靶生物如鱼类产生毒副作用,危害鱼体健康。基于上述问题,本研究以异育银鲫为模式动物,探讨了敌百虫对异育银鲫抗氧化应激系统的影响及抗坏血酸对肝脏的保护作用。试验分别研究了敌百虫对异育银鲫仔鱼(1.0±0.3g)和幼鱼(43.3±3.9g)96h的LC50和安全浓度及对鱼体的抗乙酰胆碱酯酶作用；敌百虫胁迫对异育银鲫的氧化应激,肝脏细胞凋亡及肝脏脂肪代谢的作用；并结合体外试验,应用体外培养的原代肝脏细胞进一步研究了敌百虫诱导肝脏细胞凋亡的途径；探讨了抗坏血酸对敌百虫胁迫下异育银鲫的保护作用。
1敌百虫对异育银鲫急性中毒试验及其抗AChE活性的作用
本研究探讨了敌百虫对异育银鲫仔鱼和幼鱼的96h半致死浓度(LC50)及敌百虫对鱼体血浆中乙酰胆碱酯酶(AChE)活性的变化。试验采用静水法生物测试,根据试验期间死亡鱼数及死亡时间,用Bliss-Finney去计算了96h的LC50。并测定了当敌百虫浓度为1和45mg·L-1时,幼鱼在3、5、8、10、24、48、72、96h血浆AchE的活性。结果表明,敌百虫对异育银鲫仔鱼96h的LC50为47.82mg·L-1,安全浓度为4.78mg·L-1；对异育银鲫幼鱼96h的LC50分别为39.06mg·L-1,安全浓度为3.91mg·L-1。与对照组相比,当幼鱼在敌百虫浓度为1和45mg·L-1时,血浆AChE的活性显著降低,并随着时间的延长其活性不可恢复。
2敌百虫对异育银鲫肝脏抗氧化应激系统和细胞凋亡的影响
试验采用静水式补水换药法,将异育银鲫分别在浓度为0、0.5、1、2和4mg·L-1的敌百虫水体中连续饲养30d后,取血清和肝脏组织进行抗氧化应激系统相关指标以及肝脏细胞凋亡的测定,探讨了敌百虫对异育银鲫抗氧化应激系统和肝脏细胞凋亡的影响。结果表明：与对照组相比,在敌百虫胁迫下,肝脏中总一氧化氮合成酶(T-NOS)和黄嘌呤氧化酶(XOD)活性显著升高；肝脏脂质过氧化物丙二醛(MDA)含量除在4mgg·L-1处理组显著性增加(p<0.05)。血浆超氧化物歧化酶(SOD)活性在0.5和1mg·L-1组增加,在2和4mg·L-1组降低；过氧化氢酶(CAT)活性在1mg·L-1组增高,其余各处理组其活性降低；血浆维生素E(VE)含量增加。对肝脏TUNEL细胞凋亡原位检测表明,在处理组1、2和4mg·L-1中鱼体肝细胞凋亡数量呈增加趋势。试验各组的细胞凋亡率分别为0.24%,0.66%,3.72%,11.51%and28.33%。结果表明敌百虫对鱼体可以产生长期慢性的毒性,促使机体发生氧化应激,过量的自由基破坏抗氧化应激系统,引发生物膜脂质过氧化反应产生脂质过氧化物,造成细胞损伤,引起细胞凋亡。
3敌百虫诱导异育银鲫体外原代肝脏细胞凋亡的研究
试验将体外培养的异育银鲫原代肝脏细胞暴露在含敌百虫浓度为0,0.01,0.1和1.0mg·L-1的培养液24h后,分别测定细胞活性、细胞氧化应激和细胞凋亡率并通过透射电镜观察肝脏细胞的细胞器结构。结果表明,随着敌百虫浓度的增加,细胞内活性氧(ROS)和丙二醛(MDA)含量显著升高；细胞活性随敌百虫浓度的增加而降低,细胞凋亡率升高；线粒体中细胞色素C (cyt C)含量降低,胞质中cyt C水平增高；细胞凋亡蛋白酶半胱天冬酶-3(caspase-3)的活性升高。通过AO/EB荧光染色显示肝脏细胞核DNA断裂,出现凋亡小体；透射电镜观察结果表明细胞器结构出现损伤,线粒体空泡化,并且在细胞中脂肪大量堆积。敌百虫可以引起肝脏细胞脂质过氧化反应,过量的自由基可以对细胞器结构和功能造成损伤使线粒体内的cyt C大量释放进入细胞质中,激活了胞质中半胱天冬酶系的级联活化,最终由caspase-3启动凋亡。
4敌百虫对异育银鲫肝脏组织脂肪代谢的影响
本试验采用静水式补水换药法,将异育银鲫分别在浓度为0、0.5、1、2和4mg·L-1的敌百虫水体中连续饲养30d后,取血清和肝脏组织测定脂肪代谢系统相关指标,探讨了敌百虫对鱼体肝脏组织脂肪代谢的影响。结果表明,敌百虫诱发血浆中胰岛素(Insulin)的含量上升、肝脏中环一磷酸腺苷(cAMP)水平降低、激素敏感脂肪酶(HSL)活性下降、肝脏中甘油三酯(TG)含量增高、载脂蛋白B (apo B)和极低密度脂蛋白(VLDL)含量降低。综上所述,敌百虫阻止肝脏中脂肪分解和转运。
5抗坏血酸对异育银鲫保护作用的研究
本试验分两个部分：试验一,用含不同浓度的抗坏血酸(50、100、200、400和800μM)的培养液培养异育银鲫原代肝脏细胞,测定细胞活性、细胞内抗坏血酸含量和乳酸脱氢酶(LDH)活性；再将经抗坏血酸培养后的细胞用0.01mg·L-1浓度的敌百虫胁迫24h,测定细胞内总抗氧化能力(T-AOC)、谷胱甘肽-S-转移酶(GST)活性、丁酰胆碱酯酶(B-CHE)活性和细胞色素P450(cyt P450)的含量。结果表明,当体外添加的抗坏血酸的含量为50-200μM剂量时,肝脏细胞增殖能力增强,细胞抗应激能力提高。试验二,给鱼口服剂量为4.5和9mg·kg BW-1的抗坏血酸及联合剂量为1.5/4.5mg·kg BW-1的生育酚和抗坏血酸后,测定血浆中一氧化氮(NO)、免疫球蛋白M(IgM)和溶菌酶的含量。结果表明,给鱼口服灌注剂量为9mg·kg BW-1的抗坏血酸可以提高鱼血浆中的NO、IgM和溶菌酶的含量,提高鱼体的免疫能力。
综上所述,有机磷杀虫剂——敌百虫可引起非标靶生物异育银鲫神经中毒,鱼体活力下降。在敌百虫长时间胁迫下,鱼体产生过量的自由基,发生氧化应激；过量的自由基导致脂质过氧化反应生成氧化脂质,并在肝脏中堆积；此外脂质过氧化反应损伤线粒体膜结构,使线粒体膜的通透性发生改变,线粒体内的凋亡因子cyt C外泄到胞质中激活胞质中caspase-3的活性最终启动细胞凋亡。敌百虫胁迫下,肝脏中apo B数量减少降低了肝脏中VLDL的组装水平,使由肝脏细胞合成的脂肪转运受阻,引起细胞中脂肪堆积。敌百虫可诱发血浆中胰岛素含量升高,导致肝脏中cAMP含量减少,降低了HSL磷酸化水平致使酶活性下降,脂肪分解作用减弱。抗坏血酸可以提高肝脏细胞的活力,增强肝脏细胞的抗氧化应激能力,提高鱼体的免疫力。
Trichlorfon, an organophosphorus pesticide, is widely used as antiseptic or insecticide in aquaculture; meanwhile, trichlorfon causes adverous health effects in non-targeted species such as fish. The aim of this study is to reveal the effect of trichlorfon on anti-oxidative systerm and protection of ascorbic acid on hepar of Carassais.auratus gibebio. The lethal toxicity of trichlorfon based on96h-LCso (median lethal concentration) bioassays and effect of trichlorfon on acetylcholinesterase (AChE) activity were determined. The effect of trichlorfon on anti-oxidation, hepatocyte apoptosis and hepatic lipometabolism were investigated. Then C.auratus gibebio primary hepatocytes culture was established, and based on the optimized culture of hepatocytes, underlying apoptotic mechanism was investigated. Based on the toxic results, the protection of ascorbic acid on hepar was studied both in vitro and vivo.
1Actute toxicity of trichlorfon to Carassius auratus gibelio and its anti-acetylcholinesterase effects
This study evaluates the toxic effects of the organophosphate pesticide (OP) trichlorfon to C.auratus gibebio. The lethal toxicity of trichlorfon based on96h-LC50bioassays was determined in static water, and effects of trichlorfon on acetylcholinesterase (AChE) activity were investigated. The result showed that the96h-LC50values of larvae (1.0±0.3g BW) and fingerling development (23.2±3.6g BW) of C. auratus gibebio were47.82and39.06mg·L-1detennined by Bliss-Finney analysis. And the safty concentration were4.78and3.91mg·L-1, respectively. AChE activity in plasma was significantly inhibited in all concentrations tested (1and45 mg·L-1) after in vivo trichlorfon exposure3,5,8,10,24,48,72,96h. And the enzyme activity did not return to control levels.
2. Effect of trichlorfon on oxidative stress and hepatocyte apoptosis of Carassius auratus gibelio
To investigate the effect of trichlorfon on oxidative stress and hepatocyte apoptosis of C. auratus gibelio in vivo, the fish were exposed to0,0.5,1.0,2.0and4.0mg·L-1trichlorfon concentrations for30d. We determined the changes in the level of hepatic oxidative stress status and antioxidants in the serum. In addition, hepatocytes apoptosis were measured via TUNEL assay and hepatic ultrastructure was observed by transmission electron microscope. Compared with the0mg L-1treatment, hepatic total nitric oxide synthase (T-NOS) activities in1,2and4mg L-1treatments were increased significantly. The activities of hepatic xanthine oxidase (XOD) showed an increasing trend in all trichlorfon treatments, and increased significantly in4mg L-1treatment. Hepatic malondialdehyde (MDA) contents did not show any significant alterations in0.5,1and2mg·L-1treatments, but it was significantly increased in4mg L-1treatment. An apparent increase in superoxide dismutase (SOD) activity in serum was found at1mg L-1treatment, whereas no significant alteration in the0.5,2and4mg L-1treatments. Activities of catalase (CAT) in serum were increased in1mg L-1treatment and decreased in t0.5,2and4mg L-1treatments. Vitamin E (VE) levels in serum increased significantly after exposure to2and4mg L-1trichlofon. We also observed that hepatocyte apoptic ratios were0.24%,0.66%,3.72%,11.51%and28.33%after exposure to0,0.5,1,2and4mg L-trichlorfon by TUNEL assay, respectively. And transmission electron microscope examination revealed ultrastructure change in0.5,1,2and4mg L-1trichlorfon treatments. In conclusion, the result of the current study revealed that trichlorfon activated oxidative stress, induced lipid peroxidation and hepatocyte apoptosis.
3. Trichlorfon-induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio
In the present study, the effect of trichlorfon on apoptosis and the underlying apoptotic mechanism were investigated in primary cultures of C. auratus gibelio hepatocytes. Analyses of cultures exposed to0,0.01.0.1. and1.0mg·L-1trichlorfon concentrations for24h indicated that trichlorfon induced apoptosis and caused nuclear shrinkage, cell membrane rupture, cytoskeletal collapse, loss of cytoplasm, mitochondria vacuolization, and apoptotic body formation, as well as lipid droplet accumulation. Trichlorfon increased intracellular reactive oxygen species and malondialdehyde concentrations, decreased the cell vialility and caused cytochrome c (cyt C) release from mitochondria into the cytoplasm, leading to caspase-3activation. These findings contributed to a better understanding of the mechanisms underlying trichlorfon-induced apoptosis via activation of mitochondrial pathways while clearly indicating that trichlorfon-induced cell death was via apoptosis accompanied by mitochondrial cyt C release and consequent caspase-3activation.
4The effect of trichlorfon on hepatic lipometabolism of Carassius auratus gibelio
Trichlorfon can disrupt metabolism, reproduction and immune functions of some aquatic animals. In the present study, the effect of trichlorfon on hepatic lipometabolism of C. auratus gibelio was investigated. The fish were exposed to0,0.5,1.0,2.0and4.0mg·L-1trichlorfon concentrations for30d. We determined the changes in plasma insulin and triglyceride (TG) concentration, hepatic TG, hormone-sensitive lipase (HSL) concentration and activity, hepatic cyclic adenosine3',5'-monophosphate (cAMP), apoprotein B (apo B) and very low-density lipoprotein (VLDL) concentrations. The result showed that plasma TG concentrations were decreased but insulin concentrations were increased significantly with trichlorfon concentration increase compared with control. The hepatic HSL activities were decreased significantly and in1,2and4mg L-1treatments the activities can not be detected. But there was no change in the hepatic HSL concentrations in all test groups. Hepatic cAMP, apo B and VLDL concentrations were decreased with trichlorfon concentration increase. In conclusion, the result of the current study revealed that trichlorfon increased insulin concentration which resulted in hepatic cAMP concentration and HSL activity declined, leading to the function of hepatic lipoclasis disturbance. In addition, the hepatic lipid transport function was decreased.
5The protection of ascorbic acid on Carassius auratus gibelio in vitro and vivo
To investigate the anti-oxidative function of ascorbic acid on primary cultured hepatocytes in vitro and plasma immunity of C. auratus gibelio in vivo. The hepatocytes were cultured with media contained0,50,100,200,400and800μM concentration ascorbic acid. Cell viability, the changes in hepatocytes (lactate dehydrogenase) LDH activity, ascorbic acid concentration and antioxidative status with trichlorfon stress (0.01mg·L-1) were assayed. In addition, with oral administration of ascorbic acid (0,4.5and9mg·kg BW-1) in vivo, plasma immune NO,(immunoglobulin M) IgM and lysozyme were determined.The results showed that compared with free-ascorbic acid group, ascorbic acid could increase hepatocytes viability, enhance intracellular total antioxidant capacity (T-AOC), glutathione-S-epoxide transferase (GST) and butyrylcholinesterase (B-CHE) activities and cytochrome P450(cyt P450) concentration, when cell were cultured with50to200μM concentrations ascorbic acid in vitro. And immunologic function of fish was increased when oral administration of ascorbic acid was9mg·kg BW-1. In conclusion, ascorbic acid facilitated hepatocytes function of anti-oxidative stress and plasma immunity in vivo immunity, which which can protect hepar.
Collectively, the result of the current study revealed that the fish exposed to trichlorfon exhibited behavioral changes in the form of neurotoxin toxicity:less general activity than control group. Under long term trichlorfon-stress, trichlorfon can cause oxidation stress, lead to lipid peroxidation (LPO), resulting in hepatocytes lipid accumulation. Cellular ROS led to altering mitochondrial permeability, subsequent cytochrome c release from mitochondria, and formation of the apoptosome, a catalytic multiprotein platform that activates caspase-9which then cleaves caspase-3, apoptosis occurs. Trichlorfon can increase plasma insulin concentration, and lead to HSL activity decreased, so the lipolysis declined. And ascorbic acid could enhance hapetocytes anti-oxidantive function in vitro and plasma immunity in vivo, which facilitated protection of hepar.
1敌百虫对异育银鲫急性中毒试验及其抗AChE活性的作用
本研究探讨了敌百虫对异育银鲫仔鱼和幼鱼的96h半致死浓度(LC50)及敌百虫对鱼体血浆中乙酰胆碱酯酶(AChE)活性的变化。试验采用静水法生物测试,根据试验期间死亡鱼数及死亡时间,用Bliss-Finney去计算了96h的LC50。并测定了当敌百虫浓度为1和45mg·L-1时,幼鱼在3、5、8、10、24、48、72、96h血浆AchE的活性。结果表明,敌百虫对异育银鲫仔鱼96h的LC50为47.82mg·L-1,安全浓度为4.78mg·L-1；对异育银鲫幼鱼96h的LC50分别为39.06mg·L-1,安全浓度为3.91mg·L-1。与对照组相比,当幼鱼在敌百虫浓度为1和45mg·L-1时,血浆AChE的活性显著降低,并随着时间的延长其活性不可恢复。
2敌百虫对异育银鲫肝脏抗氧化应激系统和细胞凋亡的影响
试验采用静水式补水换药法,将异育银鲫分别在浓度为0、0.5、1、2和4mg·L-1的敌百虫水体中连续饲养30d后,取血清和肝脏组织进行抗氧化应激系统相关指标以及肝脏细胞凋亡的测定,探讨了敌百虫对异育银鲫抗氧化应激系统和肝脏细胞凋亡的影响。结果表明：与对照组相比,在敌百虫胁迫下,肝脏中总一氧化氮合成酶(T-NOS)和黄嘌呤氧化酶(XOD)活性显著升高；肝脏脂质过氧化物丙二醛(MDA)含量除在4mgg·L-1处理组显著性增加(p<0.05)。血浆超氧化物歧化酶(SOD)活性在0.5和1mg·L-1组增加,在2和4mg·L-1组降低；过氧化氢酶(CAT)活性在1mg·L-1组增高,其余各处理组其活性降低；血浆维生素E(VE)含量增加。对肝脏TUNEL细胞凋亡原位检测表明,在处理组1、2和4mg·L-1中鱼体肝细胞凋亡数量呈增加趋势。试验各组的细胞凋亡率分别为0.24%,0.66%,3.72%,11.51%and28.33%。结果表明敌百虫对鱼体可以产生长期慢性的毒性,促使机体发生氧化应激,过量的自由基破坏抗氧化应激系统,引发生物膜脂质过氧化反应产生脂质过氧化物,造成细胞损伤,引起细胞凋亡。
3敌百虫诱导异育银鲫体外原代肝脏细胞凋亡的研究
试验将体外培养的异育银鲫原代肝脏细胞暴露在含敌百虫浓度为0,0.01,0.1和1.0mg·L-1的培养液24h后,分别测定细胞活性、细胞氧化应激和细胞凋亡率并通过透射电镜观察肝脏细胞的细胞器结构。结果表明,随着敌百虫浓度的增加,细胞内活性氧(ROS)和丙二醛(MDA)含量显著升高；细胞活性随敌百虫浓度的增加而降低,细胞凋亡率升高；线粒体中细胞色素C (cyt C)含量降低,胞质中cyt C水平增高；细胞凋亡蛋白酶半胱天冬酶-3(caspase-3)的活性升高。通过AO/EB荧光染色显示肝脏细胞核DNA断裂,出现凋亡小体；透射电镜观察结果表明细胞器结构出现损伤,线粒体空泡化,并且在细胞中脂肪大量堆积。敌百虫可以引起肝脏细胞脂质过氧化反应,过量的自由基可以对细胞器结构和功能造成损伤使线粒体内的cyt C大量释放进入细胞质中,激活了胞质中半胱天冬酶系的级联活化,最终由caspase-3启动凋亡。
4敌百虫对异育银鲫肝脏组织脂肪代谢的影响
本试验采用静水式补水换药法,将异育银鲫分别在浓度为0、0.5、1、2和4mg·L-1的敌百虫水体中连续饲养30d后,取血清和肝脏组织测定脂肪代谢系统相关指标,探讨了敌百虫对鱼体肝脏组织脂肪代谢的影响。结果表明,敌百虫诱发血浆中胰岛素(Insulin)的含量上升、肝脏中环一磷酸腺苷(cAMP)水平降低、激素敏感脂肪酶(HSL)活性下降、肝脏中甘油三酯(TG)含量增高、载脂蛋白B (apo B)和极低密度脂蛋白(VLDL)含量降低。综上所述,敌百虫阻止肝脏中脂肪分解和转运。
5抗坏血酸对异育银鲫保护作用的研究
本试验分两个部分：试验一,用含不同浓度的抗坏血酸(50、100、200、400和800μM)的培养液培养异育银鲫原代肝脏细胞,测定细胞活性、细胞内抗坏血酸含量和乳酸脱氢酶(LDH)活性；再将经抗坏血酸培养后的细胞用0.01mg·L-1浓度的敌百虫胁迫24h,测定细胞内总抗氧化能力(T-AOC)、谷胱甘肽-S-转移酶(GST)活性、丁酰胆碱酯酶(B-CHE)活性和细胞色素P450(cyt P450)的含量。结果表明,当体外添加的抗坏血酸的含量为50-200μM剂量时,肝脏细胞增殖能力增强,细胞抗应激能力提高。试验二,给鱼口服剂量为4.5和9mg·kg BW-1的抗坏血酸及联合剂量为1.5/4.5mg·kg BW-1的生育酚和抗坏血酸后,测定血浆中一氧化氮(NO)、免疫球蛋白M(IgM)和溶菌酶的含量。结果表明,给鱼口服灌注剂量为9mg·kg BW-1的抗坏血酸可以提高鱼血浆中的NO、IgM和溶菌酶的含量,提高鱼体的免疫能力。
综上所述,有机磷杀虫剂——敌百虫可引起非标靶生物异育银鲫神经中毒,鱼体活力下降。在敌百虫长时间胁迫下,鱼体产生过量的自由基,发生氧化应激；过量的自由基导致脂质过氧化反应生成氧化脂质,并在肝脏中堆积；此外脂质过氧化反应损伤线粒体膜结构,使线粒体膜的通透性发生改变,线粒体内的凋亡因子cyt C外泄到胞质中激活胞质中caspase-3的活性最终启动细胞凋亡。敌百虫胁迫下,肝脏中apo B数量减少降低了肝脏中VLDL的组装水平,使由肝脏细胞合成的脂肪转运受阻,引起细胞中脂肪堆积。敌百虫可诱发血浆中胰岛素含量升高,导致肝脏中cAMP含量减少,降低了HSL磷酸化水平致使酶活性下降,脂肪分解作用减弱。抗坏血酸可以提高肝脏细胞的活力,增强肝脏细胞的抗氧化应激能力,提高鱼体的免疫力。
Trichlorfon, an organophosphorus pesticide, is widely used as antiseptic or insecticide in aquaculture; meanwhile, trichlorfon causes adverous health effects in non-targeted species such as fish. The aim of this study is to reveal the effect of trichlorfon on anti-oxidative systerm and protection of ascorbic acid on hepar of Carassais.auratus gibebio. The lethal toxicity of trichlorfon based on96h-LCso (median lethal concentration) bioassays and effect of trichlorfon on acetylcholinesterase (AChE) activity were determined. The effect of trichlorfon on anti-oxidation, hepatocyte apoptosis and hepatic lipometabolism were investigated. Then C.auratus gibebio primary hepatocytes culture was established, and based on the optimized culture of hepatocytes, underlying apoptotic mechanism was investigated. Based on the toxic results, the protection of ascorbic acid on hepar was studied both in vitro and vivo.
1Actute toxicity of trichlorfon to Carassius auratus gibelio and its anti-acetylcholinesterase effects
This study evaluates the toxic effects of the organophosphate pesticide (OP) trichlorfon to C.auratus gibebio. The lethal toxicity of trichlorfon based on96h-LC50bioassays was determined in static water, and effects of trichlorfon on acetylcholinesterase (AChE) activity were investigated. The result showed that the96h-LC50values of larvae (1.0±0.3g BW) and fingerling development (23.2±3.6g BW) of C. auratus gibebio were47.82and39.06mg·L-1detennined by Bliss-Finney analysis. And the safty concentration were4.78and3.91mg·L-1, respectively. AChE activity in plasma was significantly inhibited in all concentrations tested (1and45 mg·L-1) after in vivo trichlorfon exposure3,5,8,10,24,48,72,96h. And the enzyme activity did not return to control levels.
2. Effect of trichlorfon on oxidative stress and hepatocyte apoptosis of Carassius auratus gibelio
To investigate the effect of trichlorfon on oxidative stress and hepatocyte apoptosis of C. auratus gibelio in vivo, the fish were exposed to0,0.5,1.0,2.0and4.0mg·L-1trichlorfon concentrations for30d. We determined the changes in the level of hepatic oxidative stress status and antioxidants in the serum. In addition, hepatocytes apoptosis were measured via TUNEL assay and hepatic ultrastructure was observed by transmission electron microscope. Compared with the0mg L-1treatment, hepatic total nitric oxide synthase (T-NOS) activities in1,2and4mg L-1treatments were increased significantly. The activities of hepatic xanthine oxidase (XOD) showed an increasing trend in all trichlorfon treatments, and increased significantly in4mg L-1treatment. Hepatic malondialdehyde (MDA) contents did not show any significant alterations in0.5,1and2mg·L-1treatments, but it was significantly increased in4mg L-1treatment. An apparent increase in superoxide dismutase (SOD) activity in serum was found at1mg L-1treatment, whereas no significant alteration in the0.5,2and4mg L-1treatments. Activities of catalase (CAT) in serum were increased in1mg L-1treatment and decreased in t0.5,2and4mg L-1treatments. Vitamin E (VE) levels in serum increased significantly after exposure to2and4mg L-1trichlofon. We also observed that hepatocyte apoptic ratios were0.24%,0.66%,3.72%,11.51%and28.33%after exposure to0,0.5,1,2and4mg L-trichlorfon by TUNEL assay, respectively. And transmission electron microscope examination revealed ultrastructure change in0.5,1,2and4mg L-1trichlorfon treatments. In conclusion, the result of the current study revealed that trichlorfon activated oxidative stress, induced lipid peroxidation and hepatocyte apoptosis.
3. Trichlorfon-induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio
In the present study, the effect of trichlorfon on apoptosis and the underlying apoptotic mechanism were investigated in primary cultures of C. auratus gibelio hepatocytes. Analyses of cultures exposed to0,0.01.0.1. and1.0mg·L-1trichlorfon concentrations for24h indicated that trichlorfon induced apoptosis and caused nuclear shrinkage, cell membrane rupture, cytoskeletal collapse, loss of cytoplasm, mitochondria vacuolization, and apoptotic body formation, as well as lipid droplet accumulation. Trichlorfon increased intracellular reactive oxygen species and malondialdehyde concentrations, decreased the cell vialility and caused cytochrome c (cyt C) release from mitochondria into the cytoplasm, leading to caspase-3activation. These findings contributed to a better understanding of the mechanisms underlying trichlorfon-induced apoptosis via activation of mitochondrial pathways while clearly indicating that trichlorfon-induced cell death was via apoptosis accompanied by mitochondrial cyt C release and consequent caspase-3activation.
4The effect of trichlorfon on hepatic lipometabolism of Carassius auratus gibelio
Trichlorfon can disrupt metabolism, reproduction and immune functions of some aquatic animals. In the present study, the effect of trichlorfon on hepatic lipometabolism of C. auratus gibelio was investigated. The fish were exposed to0,0.5,1.0,2.0and4.0mg·L-1trichlorfon concentrations for30d. We determined the changes in plasma insulin and triglyceride (TG) concentration, hepatic TG, hormone-sensitive lipase (HSL) concentration and activity, hepatic cyclic adenosine3',5'-monophosphate (cAMP), apoprotein B (apo B) and very low-density lipoprotein (VLDL) concentrations. The result showed that plasma TG concentrations were decreased but insulin concentrations were increased significantly with trichlorfon concentration increase compared with control. The hepatic HSL activities were decreased significantly and in1,2and4mg L-1treatments the activities can not be detected. But there was no change in the hepatic HSL concentrations in all test groups. Hepatic cAMP, apo B and VLDL concentrations were decreased with trichlorfon concentration increase. In conclusion, the result of the current study revealed that trichlorfon increased insulin concentration which resulted in hepatic cAMP concentration and HSL activity declined, leading to the function of hepatic lipoclasis disturbance. In addition, the hepatic lipid transport function was decreased.
5The protection of ascorbic acid on Carassius auratus gibelio in vitro and vivo
To investigate the anti-oxidative function of ascorbic acid on primary cultured hepatocytes in vitro and plasma immunity of C. auratus gibelio in vivo. The hepatocytes were cultured with media contained0,50,100,200,400and800μM concentration ascorbic acid. Cell viability, the changes in hepatocytes (lactate dehydrogenase) LDH activity, ascorbic acid concentration and antioxidative status with trichlorfon stress (0.01mg·L-1) were assayed. In addition, with oral administration of ascorbic acid (0,4.5and9mg·kg BW-1) in vivo, plasma immune NO,(immunoglobulin M) IgM and lysozyme were determined.The results showed that compared with free-ascorbic acid group, ascorbic acid could increase hepatocytes viability, enhance intracellular total antioxidant capacity (T-AOC), glutathione-S-epoxide transferase (GST) and butyrylcholinesterase (B-CHE) activities and cytochrome P450(cyt P450) concentration, when cell were cultured with50to200μM concentrations ascorbic acid in vitro. And immunologic function of fish was increased when oral administration of ascorbic acid was9mg·kg BW-1. In conclusion, ascorbic acid facilitated hepatocytes function of anti-oxidative stress and plasma immunity in vivo immunity, which which can protect hepar.
Collectively, the result of the current study revealed that the fish exposed to trichlorfon exhibited behavioral changes in the form of neurotoxin toxicity:less general activity than control group. Under long term trichlorfon-stress, trichlorfon can cause oxidation stress, lead to lipid peroxidation (LPO), resulting in hepatocytes lipid accumulation. Cellular ROS led to altering mitochondrial permeability, subsequent cytochrome c release from mitochondria, and formation of the apoptosome, a catalytic multiprotein platform that activates caspase-9which then cleaves caspase-3, apoptosis occurs. Trichlorfon can increase plasma insulin concentration, and lead to HSL activity decreased, so the lipolysis declined. And ascorbic acid could enhance hapetocytes anti-oxidantive function in vitro and plasma immunity in vivo, which facilitated protection of hepar.
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