| BackgroundWith the rapid development of human activities and production industries such as chemicals,drugs and pesticides,domestic and industrial water consumption has increased rapidly.The most direct manifestation of water bloom phenomenon is the excessive growth of blue-green algae and the production of MCs(Microcystins).When these water bodies carrying various pollutants enter the surface water,they cause various serious water environmental pollution problems,and ultimately lead to complex water pollution characteristics.There are many kinds of pollutants co-existing in the complex water environment.At present,heavy metals,persistent organic pollutants and various new pollutants have attracted more and more attention.Due to the small volume and large surface area of micro plastics,MCLR(Microcystin LR)can be adsorbed on the surface of microplastics to increase its bioavailability.Previous studies on MCLR have focused on liver toxicity.As the second largest target organ of MCLR,the reproductive toxicity mechanism of gonad is not clear,and the study on reproductive toxicity when co exposed with microplastics is even less.ObjectiveThis study explored the effects of MCLR and microplastics on the reproductive function of the parent zebrafish through animal experiments and studied the mechanism of mitochondrial damage in reproductive toxicity caused by MCLR and microplastics through in vitro experiments,providing support for clarifying the mechanism of male reproductive toxicity caused by MCLR and microplastics,and providing theoretical basis for providing prevention and control strategies.Methods:Part 1 In vivo1.630 healthy adult males aged 5 months were randomly divided into control group,5 μg/L MCLR group,25 μg/L MCLR group,5 μg/L MCLR+PSMPs group,5 μg/L MCLR+PSNPs group,25 μg/L MC LR+PSMPs group,25 μg/L MCLR+PSNPs group.They were raised in water with corresponding concentration and exposed for 45 days.After exposure,20 of them were taken for mating and spawning experiment.The male fish exposed to the poison and the female fish not exposed to the poison copulated and spawned.These eggs are collected every day,and the hatchability and survival rate of young fish were calculated.2.Eighteen samples were randomly selected from each dose group,and their liver,brain,and testicular tissues were dissected and weighed to calculate the HSI(Hepatic Steatosis Index),BSI(Brain physical index),and GSI(Gonadophysical index)indices.3.Four adult fish were randomly selected from each group for fixation,and observed the ultrastructure of testicular tissue with transmission electron microscope in the later stage,and freeze the rest with liquid nitrogen and transfer them to-80 ℃refrigerator for preservation.4.Use a reagent kit to detect the levels of SOD and CAT in testicular tissue,as well as the level of oxidative stress in the testes.5.Immunohistochemical observation γ-H2AX(histone H2 AX phosphorylation)and calculate the positive rate.6.Western blot test the expression of H2 AX,PCNA(proliferating cell nuclear antigen),CDK1(cyclin dependent kinase 1),and Cyclin D1(cyclin 1).Part 2 In vitro7.Selecting the mouse GC-2 cell line as the experimental object,eight dose groups were established for cell experiments: control group,2.5 μ g/m L MCLR group,5 μg/m L MCLR group,10 μg/m L MCLR group,2.5 μg/m L MCLR +PSNPs(10 μg/m L)group,5 μg/m L MCLR + PSNPs(10 μg/m L)group,10 μg/m L MCLR + PSMPs(10 μg/m L)group and 10 μg/m LPSNPs group.8.The oxidative stress response of GC-2 cells was detected by reactive oxygen species detection kit.9.Mito-Tracker Green fluorescence probe was used to detect the morphological changes of mitochondria.10.The level of mitochondrial membrane potential was measured by JC-1 method.11.The activity of mitochondrial complex IV was detected with mitochondrial complex IV detection kit.12.ATP content of cells was detected with ATP detection kit.13.Detect whether GC-2 cell DNA has been damaged through comet assay.14.Western blot expression of,PCNA,CDK1,Cyclin D1,Fis1(Fission 1 Protein),and Drp1(dynamin-related protein 1)and other proteins;The co-localization of γ-H2 AX was detected by immunofluorescence and the co-localization rate was calculated.15.Cell cycle and apoptosis detection kit were used to detect the progress of cell cycle.Results Part 1 Male zebrafish exposed to MCLR and microplastics would affect the reproductive function of the father and the growth and development of the offspring(1)Exposure to MCLR and microplastics affects the development of the gonads of the father and the growth of the offspring :Compared with the control group,the GSI of 5 μg/L MCLR + PSNPs group,5 μg/L MCLR + PSNPs group and 25μg/L MCLR + PSNPs group decreased significantly,but had almost no effect on HSI and BSI;The offspring of the infected group were found to be deformed during the growth process;The survival rate of offspring in the 5 μg/L MCLR + PSMPs、25 μg/L MCLR + PSMPs、25 μg/L MCLR + PSNPs groups decreased.(2)The exposure of MCLR and microplastics affected the ultrastructure of the paternal gonads : Compared with control group and MCLR single exposure group,In the combined exposure group,the mitochondria of testicular tissue were seriously vacuolated and swollen,and the mitochondrial spines were broken or even disappeared.The outer membrane of the mitochondria ruptured.And at 25 μg/L MCLR+ PSMPs,25 μg/L MCLR + PSNPs group observed that the sperm morphology was obviously abnormal,and the sperm cell membrane was seriously damaged.(3)Exposure to MCLR and microplastics lead to oxidative stress in the gonadal tissue of the father: The SOD level in 5 μg/L MCLR+PSMPs,25 μg/L MCLR,25 μg/L MCLR + PSMPs,25 μg/L MCLR + PSNPs group was higher than control group,and5 μg/L MCLR + PSMPs treatment group was significantly higher than MCLR single exposure group.The level of CAT decreased with the increase of MCLR concentration,and 5 μg/L MCLR + PSMPs,5 μg/L MCLR + PSNPs and 25 μg/L MCLR + PSNPs coexposure group was lower than its MCLR single exposure group respectively.(4)Exposure to MCLR and microplastics lead to DNA damage and cycle arrest of spermatogenic cells in paternal gonad tissue: immunohistochemistry and WB results showed that the expression of DNA damage key proteins γ-H2 AX was up-regulated,the results showed that the exposure of MCLR and microplastics resulted in DNA damage of testicular spermatogenic cells;Compared with the control group,and 25μg/L MCLR + PSMPs,25 μg/L MCLR + PSNPs group was significantly higher than25 μ g/L MCLR single exposure group.The expression of Cyclin D1 in 5 μg/L MCLR+ PSNPs,25 μg/L MCLR + PSMPs,25 μg/L MCLR + PSNPs treatment group was significantly down-regulated,5 μg/L MCLR + PSMPs,25 μg/L MCLR + PSNPs group was significantly lower than MCLR single exposure group.Part 2 Exposure of MCLR and microplastics caused excessive ROS production and DNA damage(1)When the concentration of MCLR reached 10 μg/ml,significantly higher levels of reactive oxygen species were observed in the MCLR group,co-exposure group,and PSNPs group than in the control group,and co-exposure group were significantly higher than in the MCLR single exposure group.(2)MCLR and PSNPs could induce obvious granular changes and swelling of mitochondria.The results of JC-1 staining flow cytometry showed that the cell membrane potential decreased significantly in all exposed groups,and the cell membrane potential of PSNPs combined exposure group was significantly lower than that of PSNPs alone exposure group.The activity of mitochondrial complex IV in 10μg/m L MCLR single exposure group and 10 μg/m L MCLR + PSNPs co-exposure group decreased significantly.Moreover,10 μg/m L MCLR + PSNPs group was significantly lower than PSNPs single staining group.The ATP detection results also showed that MCLR caused a dose-dependent decrease in intracellular ATP content,and 10 μg/m L MCLR + PSNPs group was significantly lower than the single exposure group of PSNPs,suggesting that the production of mitochondrial ATP was inhibited under the conditions of MCLR and PSNPs exposure.WB results showed that the expression of mitochondrial membrane division fusion protein was affected.The expression in Fis1,Drp1 at 2.5 μg/m L MCLR,5 μg/m L MCLR and 10 μg/m L MCLR + PSNPs groups exhibited a significantly higher expression of MCLR than the control group,and when the concentration of MCLR was at 10 μg/m L,the combined exposure group was higher than that of single exposure group.Compared with the control group,the expression of OPA1(Optic Atrophy 1)and Mfn1(mitochondrial fusion 1)were 2.5 μg/m L MCLR,5 μg/m L MCLR,10 μg/m L MCLR,all MCLR+PSNPs co-exposure group and PSNPs single exposure group were down-regulated,and the expression in each co-exposure group was lower than that in MCLR single exposure group.The expression of NRF1(Nuclear respiratory factor-1)and mt TFA(mitochondrial transcription factor A)was not significantly impacted by MCLR and PSNPs;however,the combined exposure group’s expression was greater than that of the single exposure group.The expression of PGC1(Peroxisome proliferator-activated receptor-gamma coactivator)in each dose group was significantly diminished when compared to the control group.And when MCLR concentration at 2.5 μg/m L,5μg/m L,the combined exposure group was significantly lower than that of the single exposure group.Compared with PSNPs group,the difference was not statistically significant.(3)Compared with the control group,the expression of γ-H2 AX in all exposure groups was significantly increased.When MCLR concentration was 5 μg/m L,the expression of MCLR+PSNPs combined group was higher than that of MCLR alone group.Compared with the control group,all dose groups could up-regulate the expression of proliferating cell nuclear antigen.When MCLR concentration was 2.5 μg/m L,5 μg/m L,the expression of PCNA in MCLR + PSNPs group was significantly higher than that in MCLR single exposure group.The expression of γ-H2 AX and PCNA was significantly greater in the co-exposure group than in the PSNPs simple exposure group.The comet experiment results showed that compared with the control group,the tail length,tail distance,and Olive tail distance of the treatment group were significantly increased,and the tail distance and Olive tail distance of the co exposure group were higher than those of the MCLR single exposure group and PSNPs single exposure group,and the tail dragging phenomenon was more significant.(4)Compared with the control group,the expression of PARP(poly(ADP-ribose)polymerase)and DNA PKcs(DNA-dependent protein kinase catalytic subunit)in each dose group increased significantly.The expression of PARP in all co-exposure groups was significantly higher than that in the control group;The expression of DNA PKcs at5 μg/m L MCLR + PSNPs,10 μg/m L MCLR + PSNPs treatment group was higher than that in MCLR single exposure group.Compared with the control group,the expression of ATM(ataxia telangiectasia-mutated gene)at 2.5 μg/m L MCLR,10 μg/m L MCLR,10 μg/m L MCLR + PSNPs group were increased significantly.The expression of ATM in the co-exposure group at 10μg/m L MCLR + PSNPs was greater than that of the single exposure group.It can be seen that when the MCLR concentration was 10 μg/m L,the expression of PARP and DNA PKcs in the combined exposure group was higher than that in the PSNPs exposure group alone.Flow cytometry’s quantitative analysis revealed that,in comparison to the control group,the number of G0/G1 cells in 2.5μg/m L MCLR + PSNPs,5 μg/m L MCLR + PSNPs,10 μg/m L MCLR,10 μg/m L MCLR+ PSNPs treatment group increased significantly.And 2.5 μg/m L MCLR + PSNPs,5μg/m L MCLR + PSNPs group was significantly higher than MCLR single exposure group.the proportion of G2/M cells at 2.5 μg/m L MCLR + PSNPs,5 μg/m L MCLR +PSNPs,10 μg/m L,10 μg/m L MCLR + PSNPs and PSNPs treatment group decreased significantly.The proportion of cells in S phase had no significant change.The above results indicate that the exposure of microcystins and microplastics activates the DNA repair pathway mediated by NHEJ and BER,and the cell cycle is blocked in G1 phase,providing more time for DNA repair.conclusionAdult male zebrafish exposed to microcystins and microplastics for 45 days would affect the reproductive system of male zebrafish.We found that the exposure of microcystins and microplastics caused excessive ROS production and induced mitochondrial and DNA damage.Mitochondrial damage activates the DNA damage repair pathway mediated by NHEJ and BER,and the cell cycle is blocked in G1 phase,which leads to reproductive dysfunction and further affects the development of offspring. |
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