| 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 effectsThis 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 gibelioTo 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 gibelioIn 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 gibelioTrichlorfon 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 vivoTo 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. |
PDF Full Text Request