Microcystins(MC-LR)Exposure Induce Prostate Toxicity In Male Mice

In recent years,with the accelerated industrialization and urbanization in the world,a large number of industrial and domestic wastewater discharged into the natural water body,serious eutrophication caused cyanobacterial blooms,the cyanotoxins has posed a serious threat to survival environment and human health.Microcystins(MCs)is a class of monocyclic heptapeptide compounds produced by freshwater algae,more than 100 kinds of its congeners have been identified so far,and microcystin-LR(MC-LR)is most widely distributed and has the strongest toxicity.Previous studies have shown that MC-LR possesses hepatotoxicity,nephrotoxicity,gastrointestinal toxicity,neurotoxicity and so on.Our study is the first to find the male reproductive toxicity of MC-LR,it can lower testosterone level in rats,impact the quality of sperm and damage the testicular structure.In order to fully illustrate the male reproductive toxicity of MC-LR and its underlying mechanisms,to study the distribution features in male reproductive system after MC-LR exposure is extremely necessary.Therefore,the prostate is a male-specific sexual gland,which plays an important role in maintaining male normal reproductive function.Therefore,the study of the prostate as the object of study,we make a thorough detection of distribution in tissue and cells of MC-LR.Based on above,in this study,we investigated the changes of ACP and PSA expression in prostate epithelial cells after MC-LR exposure,and further explored the mechanism of MC-LR male reproductive toxicity by in vivo and in vitro experiments.The full text is divided into two parts:Part I Effects of acute and chronic exposure to MC-LR on the structure and function of prostate in miceObjectiveThrough the acute and chronic experiments,we explored the distribution of MC-LR in the prostate and the impact of the prostate structure and function.Methods1.Male BALB/c mice aged 6-weeks(specific pathogen free,SPF)(n=96)weighing from 18 g to 22 g were purchased from Experimental Animal Center of the Academy of Military Medical Science Institute,China.They were randomly divided into 3 time-groups:1 day,4 days and 7 days group,each group was divided into 4 groups:1 control group and 3 experiment groups,each group of 8 mice,the experimental groups,mice intraperitoneal injection containing MC-LR concentrations were 7.5 ?g kg bw-1 day-1,15 ?g kg bw-1 day-1 and 30 ?g kg bw-1 day-1 of saline.In the control group,mice were injected intraperitoneally with saline.Respectively,mice were injected 1 day,4 days and 7 days,the eye to take blood,and then cut off the neck,take the prostate and other organs spare.2.Male BALB/c mice aged 6-weeks(specific pathogen free,SPF)(n=100)weighing from 18 g to 22 g were randomly divided into two groups:3 months and 6 months,each group was divided into 5 groups:1 control group and 4 experimental groups.In the experimental group,the mice were fed with drinking water containing 1?g/L,10 ?g/L,20 ?g/L and 30 ?g/L,respectively.In the control group,contains normal drinking water without MC-LR.Respectively,mice were feeding 3 months and 6 months later,the eye to take blood,and then cut off the neck,take the prostate and other organs spare.3.The conventional method was weighed the mice and prostate.Western Blot and immunofluorescence were used to detect whether MC-LR was able to enter the mouse prostate.4.The paraffin sections of the prostate were made,and the sections were stained continuously and stained with H&E.The changes of the prostate structure were observed under the optical microscope.TUNEL was used to detect the apoptosis of the prostate tissue.5.The levels of acid phosphatase(ACP)and prostate specific antigen(PSA)in serum and prostate lysate were measured by enzyme-linked immunosorbent assay.Results1.MC-LR intraperitoneal injection for 1 day,the experimental group compared with the control group,weight loss,prostate index(prostate and body weight ratio)and prostate tissue apoptosis did not change significantly;MC-LR intraperitoneal injection for 4 days and 7 days,weight decreased significantly,the prostate index did not change significantly.MC-LR exposure for 3 months and 6 months later,the experimental group compared with the control group,weight gain was significantly reduced;prostate index did not change significantly.2.Western Blot and immunofluorescence results show that MC-LR can enter the prostate.3.We used paraffin sections,H&E staining revealed 15 ?g kg b.w.-1 day-1 and 30 ?g kg b.w.-1 day-1 structural damage significantly,after MC-LR exposure 7 days the integrity of prostate acinar were destructed,epithelial cells serious fall off,alveolar fibrous tissue around the slight proliferation.After 3 months and 6 months of exposure to MC-LR,the structure of the prostate acinar remained intact at 1 ?g/L,10 ?g/L,20 pg/L and 30 ?g/L,respectively.The epithelial cells were disordered,local hyperplasia.TUNEL detection showed that the apoptosis of the cells in the prostate tissue was significantly increased in the acute experiment,and showed time and dose-dependent.Compared with the acute experiment,the apoptotic rate was low in the chronic experiment.4.ACP and PSA were up-regulated in serum and prostate tissue lysate by enzyme-linked immunosorbent assay.Conclusion1.Intraperitoneal injection of MC-LR makes the weight loss of male mice,long-term low-dose natural drinking water exposure MC-LR male mice net weight loss decreased;no significant increase in prostate index.2.MC-LR can enter the male mouse prostate.3.MC-LR can damage the structure of the prostate tissue,leading to increased cell death,and showing time and dose-dependent.4.MC-LR can up-regulated the levels of ACP and PSA in the serum and prostate tissue lysate.Part II Study on the Toxicity of MC-LR to Prostate Epithelial CellsObjectiveThe effect of MC-LR on the toxicity to male reproductive system was investigated by human prostate epithelial RWPE-1 cell line in vitro after MC-LR exposure.Methods1.Cells were exposed to 0,5,50,250,500 and 5000 nM MC-LR for 48h.Immunofluorescence was used to detect whether MC-LR could enter into RWPE-1 cells.2.Human prostate epithelial cell line RWPE-1 was cultured in vitro,exposed to MC-LR for 48 h at different concentrations(0,5,50,250,500,5000 nM),then detected the cell viability using CCK-8 assay.3.Based on results of cell viability assay,prostate epithelial cell line RWPE-1 was treated with MC-LR for 48 h at different concentrations(0,5,50,250,500,5000 nM),then observed the cell survival rates by FDA/PI detection.4.Cells were exposed to 0,5,50,250,500 and 5000 nM MC-LR for 48h.LDH methods was used to determine the membrane leakage rate of RWPE-1 cells.5.Western Blot was used to detect ACP,PSA,AR,ERK1/2 and MEK expression changes in RWPE-1 cells after exposure to MC-LR.Results1.Prostate epithelial RWPE-1 cells were treated with different concentrations of MC-LR for 48h.Then CCK-8 assay were employed to detect cell viability.Results showed that treating with 250 nM,500 nM and 5000 nM MC-LR for 48 h can result in significantly declined cell viability.2.Prostate epithelial RWPE-1 cells were treated with different concentrations of MC-LR for 48 h.Then FDA/PI assay were explored to detect cell survival rate.Results showed that treating with 5 nM,50 nM,250 nM MC-LR for 48 h can result in relatively better survival rate.But treating with 500 nM and 5000 nM MC-LR result in survival rate decreased significantly.3.In LDH tests,we found that the membrane leakage rate of RWPE-1 cells decreased significantly as MC-LR concentration increased.4.Immunofluorescence assay showed that MC-LR could enter human prostate epithelium RWPE-1 cells after exposure to MC-LR.5.Western Blot detection found that prostate epithelial cells ACP,PSA,AR,pERK1/2 and pMEK expression increased as MC-LR concentration increased.Conclusion1.MC-LR can enter the human prostate epithelial RWPE-1 cells,destructing cell membrane integrity,and ultimately lead to cell death.2.MC-LR can cause cell viability and cell viability decreased,and showed time and dose-dependent effect in prostate epithelial RWPE-1 cells.3.MC-LR may increase the expression of pERK1/2,pMEK,and activate MAPK pathway,which makes AR,PSA,and ACP expression increased in prostate epithelial RWPE-1 cells.Features and innovations in this study1.We are the first to perform the study on toxicity of MC-LR in male prostate and find that MC-LR can enter into the mouse prostate and human prostate epithelial RWPE-1 cells.2.We are the first find the MC-LR can enter the mouse prostate and human prostate epithelial RWPE-1 cells.3.We are the first study of intraperitoneal injection and long-term low-dose exposure to MC-LR,male mammalian specific gonadal prostatic toxicity.