Effect Of Microcystin-RR On Water Metabolism And Neurobehavior In Mice And Related Mechanism Exploration

Part 1:Effect of microcystin-RR on water metabolism in mice and its mechanism studyObjective:To investigate the effect of microcystin-RR(MC-RR)on water metabolism in mice with a prolonged exposure and to further explore the inner mechanism.Methods:In the first stage,mice were divided into four groups at random,with 7 mice per group.MC-RR was diluted with 0.9%physiological saline and administered daily to the mice at doses of 140 ?g/kg(Group A),70 ?g/kg(Group B),35 ?g/kg(Group C),and 0 ?g/kg(Group D)BW via intraperitoneal injection for 30 days.During the administration period,the body weight,urine volume and water intake were measured daily.On the 30th day,the mice were euthanized to collect blood samples and main organs for biochemical and toxicological analyses.In the second stage,the same experiments applied in the first stage were performed on a new batch of 28 animals(in 4 groups,n=7)but with an additional 12 days of observation after the cessation of MC-RR exposure on day 31 to investigate the water metabolism recovery process in mice.Another batch of 28 animals(in 4 groups,n=7)was administered with MC-RR for 60 days in order to observe the water metabolism dysfunction induced by prolonged exposure.In the third stage,the same experiments applied in the first stage were performed on a new batch of animals(total 168 mice divided into 4 dose groups)todetermine the dynamic process of injury and recovery.In this experiment,the toxin was administered to the animals for one month,and then the animals were kept feeding for another 12 days.During the experiment 7 animals in each group were taken out and euthanized on days 7,14,21,28.35 and 42 for examination of the blood and urine indices.In the fourth stage,aliskiren was chosen as the RAS blocker,and three groups of animals were treated daily for 30 days with dosages of MC-RR 140 ?g/kg BW + aliskiren 100 mg/kg BW,MC-RR 140 ?g/kg BW+aliskiren 50 mg/kg BW,and MC-RR 140 ?g/kg BW +physiological saline.Results:(1)On the 9th day of treatment,an increase in water intake and urine output was observed in the high-dose group compared with the control,and the symptoms worsened with the repeated administration of the toxin until day 30.After the toxin treatment was completed,the symptoms of polyuria and polydipsia disappeared within 1week.Besides,more severe water dysfunction occurred in animals in the 60 days of administration with high dose MC-RR.Two plateaus of increased water intake or urine output were observed during day 30 to day 42(urine volume was 0.21-0.25 ml/g/day and water intake volume was 0.51-0.60 ml/g/day)and day 43 to day 60(urine volume was 0.25-0.32 ml/g/day and water intake volume was 0.60-0.68 ml/g/day).(2)A relatively less increase of body weight and serious liver injury,such as the obvious swelling and inflammatory hepatocytes and the increase of serum enzymes like ALT.AST.ALP,were observed in the mice treated with 140?g/kg MC-RR compared with the control(P<0.05).However,signs of renal injury were not observed throughout the experiment,such as BUN,CREA,UA,mALB and urinary electrolytes being in normal ranges.(3)The mRNA level of angiotensinogen(AGT)in hepatocytes was upregulated to approximately 150%of the control(P<0.05).and the serum renin-angiotensin system(RAS)and AVP was activated in the high-dose group.(4)After the toxin treatment was completed,the high levels of the RAS and vasopressin in group A returned to normal levels within 1 week.As expected,the symptoms of polyuria and polydipsia also disappeared.In addition,the observed polyuria and polydipsia and the high level of the RAS induced by MC-RR could be alleviated by RAS blocker in a dose-dependent mannerConclusion:The evidence obtained in this study strongly supports that water metabolism dysfunction caused by MC-RR in mice is via RAS activation route resulted from liver damage.It is the first time that the symptoms of water disorder in mice were discovered and its mechanisms were clearly investigated by us.Part 2:Effect of microcystin-RR on neurobehavior in mice and its mechanism explorationObjective:To investigate the effect of repeated exposure to MC-RR on the neurobehaviour in mice and the underlying mechanisms.Methods:KM male mice were divided into four groups at random,with 7 mice per group.MC-RR was diluted with 0.9%physiological saline and administered daily to the mice at doses of 140 ?g/kg(Group A),70 ?g/kg(Group B).35 ?g/kg(Group C),and 0 ?g/kg(Group D)BW via intraperitoneal injection for 7 days.During the administration period,the water maze test training was done from the 3rd day to the 6th day.Then the open field test and water maze test were done on 7th day.Clinical chemistry and histopathology were performed after the tests,including observation of the ultrastructurer alteration of blood brain barrier in mice using transmission electron microscope,detection of the accumulation of MC-RR in the brain using Immunohistochemical and LC/MS method and the concentration of four amino acid neurotransmitters in the brain by HPLC.The expression of Oatps in the brain and liver of the mice were detected by fluorescence quantitative PCR method.Results:Compared with the control,140 ?g/kg MC-RR could enhace the spontaneous activity of mice when administered for 7 days.However,the effect of MC-RR on learning and memory in mice has not been found.In capillary epithelial cells of the cerebral cortex of the mice,the expansion of endoplasmic reticulum and ruption of mitochondria were observed in high-dose group.The concentration of MC-RR in the brain was much higher than the control when they were detected using immunohistochemical method,while the result was negative when MMPB-LC/MS method was used,which did not found the accumulation of MC-RR in the brains of any group.The concentration of Glu,Asp,Gly and GABA in mouse brain increased to 7.79 ± 2.40 mg/kg,4.30 ± 0.67 mg/kg,2.30 ± 0.67 mg/kg,1.30 ± 0.19 mg/kg respectively in 140 ?g/kg group,which were much higher than the control(P<0.05).The expression levels of Oatplcl,Oatp3al and Oatp1a4 were higher in mouse brain than those in the mouse liver.The other Oatps such as Oatp1a1,Oatp1b2,Oatp2b1,Oatp2a1 and Oatp1a5 expressing highly in mouse liver were also detected in mouse brains.Conclusions:The present suggests that MC-RR can be transferred into mouse brains via Oatps inducing the structural failure of endoplasmic reticulum and mitochondria of the brains in high dose administration,accompanied by the increased release of neurotransmitters,ultimately resulting in the behavioral alteration of the mice.