Comparative Studies On The Toxicity Of Microcystins To Fish And Mammals

With the progress of eutrophication in aquatic environments,massive freshwater cyanobacterial blooms and the associated cyanotoxins contamination have been documented worldwide.Among cyanotoxins,the hepatotoxic microcystins(MCs) are the most common group.A lot of studies have demonstrated that mammals are more sensitive to exposure of mlcrocystins than fish.There are a few deductions on the difference of the toxic effects between fish and mammals.First,in fish,MCs result in the malfunction of liver,while hypotensive shock and circulation system obstacle is responsible for mammal death after MCs exposure.Second the velocity of blood perfusion in liver of fish is only 1/4 of mammals,which slows down velocity of the MCs transportation.Third,fish has underdeveloped circulation systems with low blood pressure(about 1/4 of mammals).These traits enable fish to be less sensitive to MC. However,few studies have been conducted to test the above hypothesis.Therefore,our studies aim to reveal the toxic effects of MCs on fish and mammals.Crucian carp and rabbit are selected for representations of fish and mammals.We focused on liver injury and damage of blood circulation system of animals.Additionally,in order to get a better understanding of toxic effects of MC on the mammal blood circulation,we investigated the hemopoiesis changes of rabbit.The main results are as follows:1.Toxin exposure and anatomical traitsLD₅₀ of crucian carp injected i.p.with MC(48h) was 250μg MC-LR/kg BW,while LD₅₀ of rabbit was 33μg MC-LR/kg BW.Exposure doses for crucian carp and rabbit were 200,50μg MC-LR/kg BW and 50,12.5μg MC-LR/kg BW,respectively,and samples were taken at 1,3,12,24 and 48h post injection.A great amount of ascites was observed in crucian carp in the high dose group at 12h post injection.Ascites contained some erythrocytes(5.46±1.51)×10⁴ cells/μL and other blood ingredients.Red blood cells in the ascites exhibited prominent pathological changes:swoln cells,cracked cell membrane,and dissociative nucleolus.While no ascites was observed in the rabbit treated with MC.2.Liver damageWe evaluated alterations in activities of some serum liver-correlated enzymes(ALT, AST,ALP and LDH).Serum enzyme activities showed marked increases with increase of MC doses.There were some differences in response mechanism against ROS caused by MC between crucian carp and rabbit.Activities of SOD,CAT,GPx and GR in crucian carp increased in the early period of the experiment(1-3h),SOD and CAT activities at 3h post injection increased 33.46±8.97%and 68.38±12.47%,espectively.Antioxidant enzymes activities continuously decreased during 12-48h.LPO levels significantly increased during 12-48h post injection.Antioxidant enzymatic activities significantly decreased from 1h,with prominent decreases of 40.47±8.15%,35.33±6.75%,41.67±10.23%and 76.58±13.46%in SOD,CAT,GPx and GR,respectively.The LPO levels of rabbit significantly increased from 1h post injection.3.Blood circulation system dysfunctionCrucian carp of both dose groups and the rabbit of the low dose group all presented evident increases of liver microvascular permeability and prominent decreases of circulation blood volume.Tissue blood vessel leakage and decrease of blood circulation volume were responsible for anemia of MC-treated animal.Significant increase(with 118.18±12.39%) of the liver microvascular permeability in crucian carp of thehigh dose group were observed at 1h post injection.Marked decreases(55.45±4.50%) of blood volume were recorded in crucian carp of the high dose group at 48h post injection. Significant increases of liver microvascular permeability(with152.63±8.53%) were also observed in rabbit of the high dose group at 3h post injection.However,blood volume had no prominent changes in rabbit of the high dose group.On the other hand,circulation blood volume decreased(with 19.20±2.21%) at 48h in rabbit of the low dose group. Crucian carp and rabbit all presented evident decline of blood pressure after MC exposure.Crucian carp exhibited compensative increase of heart rates,while rabbit lacked such compensative mechanism.Blood pressure of crucian carp significantly decreased from lh post injection,with prominent decrease(60.99±4.38%) in the high dose group.Heart rates of both dose groups all increased with an increase of 120.78±11.25%in the high dose group at lh post injection.Evident deceases of blood pressure were also observed in rabbit of both dose groups.Blood pressure of rabbit in high dose group was only 30.02±5.78%of the control at 2 h 30 min.There were no prominent changes in heart rates of rabbit in the low dose group,while significant decrease(with 36.68±6.73%) of heart rates were observed in rabbit of the high dose group at 3h post injection,with amort depression.4.Anemia and damage of hematopoietic organsAnimals treated with MC exhibited anemia symptom during 24-48h post injection, and main hematopoietic organs were damnified.Hematological indices in crucian carp, including RBC,Hb and Ht,significantly decreased 58.48±8.78%,5579±7.54%and 56.86±8.32%,respectively,at 48h post injection,presenting a severe anemia.Erythrocyte parameters(MCV,MCH and MCHC) showed no significant changes,indicating normocytic anemia in crucian carp.The significantly decreased Hb content indicates that no hemolysis occurred in crucian carp.The main hematopoietic organs(kidney and liver) of crucian carp also showed evident pathological changes,especially in the high dose group.The rabbits of the low dose group also suffered from severe normocytic anemia with RBC,Hb and Ht levels significantly decreased 62.71±8.31%,81.03±11.76%and 72.22±9.46%respectively,at 48h post injection.The results of ultramicro observation with TEM indicated that marrow,the main hematopoietic organ,exhibited pathological changes with abnormally agglomerated chromatin and erose nucleus.5.Toxic effects of microcystins on the hematopoietic function in mammalsWe conducted our tentative experiments to evaluate the toxic effects of MCs on the hematopoietic function in rabbit.After a 14d durative i.p.injection of MCs,rabbits exhibited anemia with significant decreases in hematological indices(RBC,Hb,and Ht). MCs exposure(14d) also resulted in decreases of hematopoietic growth factors, including EPO,IL-3 and IL-6,with 57.59±5.50%,51.63±6.38%and 68.59±7.91%, respectively.Using MTT method to determine the multiplication ability of bone marrow cells,and the results indicate that the multiplication ability significantly decreased with 69.23±8.56%.Rabbit bone marrow micronucleus frequency significantly increased at 14d,and the micronucleus frequency of rabbit treated with MC was 3.9-fold of the control.TEM observations indicate that pathological changes occurred in bone marrow cells with crescent or hemicycle nucleus,and swoln ER and mitochondrial.These indicate that MCs exposure might result in bone marrow damage in mammals,leading to hematopoietic hypofunction.