Intestinal Toxicity And Biodetoxification Mechanism Of MC-LR

[Objective]Cyanobacterial blooms frequently occur in eutrophic water bodies,which seriously affect the environment and endanger human health.MC-LR is the most widely distributed and highly toxic cyanobacterial toxin.However,the intestinal toxicity and mechanism of long-term low-dose MC-LR exposure are unknown,and the mechanism of microbial biodegradation and removal of MC-LR is unclear.The purpose of this study is as follows:1.Assess the chronic effects of MC-LR on microstructure and expression levels of inflammatory-related factors in jejunum and ileum in mice.2.Investigate the biodegrading characteristics and mechanisms of bacterial community YFMG and Sphingopyxis sp.YF1 against MC-LR.[Method]（1）Effects of MC-LR on the microstructure and inflammation-related factors of jejunum and ileum in miceMice were treated with 0,1,30,60,90 and 120μg/L MC-LR for six months and microstructure and m RNA expression levels of the inflammation-related factors in jejunum and ileum were analyzed.（2）Isolation and characterization of cyanobacteria and extraction of crude MC-LR from eutrophic water in Hunan for biodegradation.Cyanobacteria were isolated from eutrophic water in Hunan Province by serial dilution and identified using PCR.The generated cyanotoxins were detected by HPLC-ESI-MS.Besides MC-LR were extracted from the cyanobacteria cells via 70%aqueous methanol,freezing and thawing and ultrasonication.The extract was centrifuged and supernatant subjected to SPE and rotary evaporation.（3）Biodegrading characteristics and mechanisms of bacterial community YFMG and Sphingopyxis sp.YF1 against MC-LRA novel MC-biodegrading bacterial community YFMG and Sphingopyxis sp.YF1 were isolated respectively from the eutrophic water and Lake Taihu,and their biodegrading characteristics against MC-LR were assessed under diverse environmental factors including temperature,p H and MC-LR concentration.Response surface methodology（RSM）was used to optimize the environmental factors on MC-LR biodegradation efficiency using bacterial community YFMG and strain YF1.The biodegrading mechanisms of the bacterial community and strain were also investigated.[Results]（1）Effects of MC-LR on the microstructure and inflammation-related factors of jejunum and ileum in miceThe microstructure of jejunum and ileum were destroyed and expression levels of inflammation-related factors IL-1β,IL-8,TNF-α,TGF-β1 and IL-10 were altered at different MC-LR concentrations.（2）Isolation and characterization of cyanobacteria and extraction of crude MC-LR from eutrophic water in Hunan for biodegradationOne species of cyanobacterium was isolated and identified as Microcystis sp.YFM1.Morphologically the cyanobacterium was round and green in color.YFM1 contained mcy B gene and produced MC-LR,MC-RR and MC-YR.The HPLC analysis indicated successful extraction of crude MC-LR and could be used for the subsequent biodegradation experiments.（3）Biodegrading characteristics and mechanisms of bacterial community YFMG and Sphingopyxis sp.YF1 against MC-LRBacterial community YFMG and strain YF1 completely biodegraded MC-LR influenced by the environmental factors and the highest biodegradation rate was 0.313 and 5μg/m L/h respectively.The main intermediate（s）biodegradation product（s）were also biodegraded.RSM analysis disclosed 30°C,p H 7 and 3μg/m L initial MC-LR as the optimal biodegradation conditions with efficiency of 100%after 16 h and 60 min by bacterial community YFMG and strain YF1 respectively.Using high-throughput pyrosequencing bacterial community YFMG was found to contain genus including Sphingobacterium,Acinetobacter and Stenotrophomonas.The 16S r DNA sequences uncovered strain YF1 was affiliated with the genus Sphingopyxis.Via PCR,mlr ABD genes were discovered in bacterial community YFMG while mlr ABCD and paa AGZ genes were demonstrated in stran YF1.[Conclusion]（1）The study found that a long-term chronic low-dose oral exposure to MC-LR could cause intestinal toxicity by destroying the microstructure of the jejunum and ileum,and induce inflammatory reaction.（2）A toxigenic Microcystis sp.YFM1 found to produce MC-LR was successfully isolated from eutrophic water in Hunan,and crude MC-LR was extracted from YFM1 for biodegradation experiments.（3）Bacterial community YFMG and Sphingopyxis sp.YF1 which have the ability to efficiently biodegrade MC-LR were isolated.In addition,the optimal conditions for MC-LR biodegradation by the bacterial community and strain were obtained using RSM at 30^oC,p H 7and 3μg/m L initial MC-LR,respectively.Bacterial community YFMG biodegraded MC-LR into Adda through the MC-biodegrading mlr ABD enzyme.Strain YF1 through the MC-biodegrading mlr ABCD genes and paa AGZ gene biodegraded MC-LR into linear MC-LR,tetrapeptide,Adda,PAA and CO₂,thus achieving the detoxification effect.18 Tables,42 Pictures,251 References...