A Population And Mechanism Study On Association Of Co-exposure To Aflatoxin And Microcystin With Hepatic And Renal Injury

Aflatoxin(AFT)and microcystin(MC)are ubiquitously exist in humid and warm regions,and people living there are exposed to the toxins mainly through the contaminated food and water.In some developing countries(including China),contamination with AFT and MC is very common.Presently,a number of lab experiments have confirmed that AFT and MC can cause damage to liver and kidney of organism;however,epidemiological studies and mechanism researches of exposure to each toxin separately or to two toxins in combination on liver and kidney injuries are scarce.AFB1 and MC-LR are the most toxic subtypes(highly contaminating,most toxic and mostly studied)of AFT and MC,respectively.Those toxins share the very important mechanism of toxicity,which is reactive oxygen.Plenty of reactive oxygen species(ROS)can be generated in body after exposure to the toxins,leading to the oxidative damage to cells.Furthermore,ROS can cause genetic damage to cells through DNA oxidative damage.Our previous study found that AFT and MC polluted the here Gorges Reion commonly.In the first part of this program,we established a cross-sectional study in Three Gorges Region and made a survey of the detailed information on basic living and dietary habits,disease history of families and health status of the participants based on our previous studies.In the second part,we discussed the effects of exposure to the two toxins separately or combinatively on liver damage in population affected with or without HBV,using the baseline information and physical examination data.In the third part,we discussed the effects of exposure to the two toxins separately or combinatively on renal damage in population,using the baseline information and physical examination data.The fourth part is at the cellular level,in which,we discussed the influences of exposure to the two toxins separately or combinatively on active oxygen of cells and the DNA oxidative damage resulted from it.Part ?.Establishment of AFB1 and MC-LR exposed population in Three Gorges RegionMethods:1.Study sitesLidu and Yihe are two towns in the rural area of the Three Gorges Region,lying between 28°28?N to 31°44?N and 105°49?E to 110°12?E.Residents living in the towns have the similar living habits but different water and food resources for geographic and economic reasons.2.Study populationAfter cluster sampling,we interviewed 6467 participants living in the two towns(Lidu and Yihe)in Fuling,Chongqing,in a questionnaire in which we collected the data of basic information,living habit,medication history,drinking and dietary habit,disease history of their families,etc.3.Detecting AFB1 and MC-LR levels in environment of the two townsWe detected the concentrations of AFB1 in the daily food(13 kinds)of residents in the two towns with ELISA,and concentrations of MC-LR in their daily water(three types of drinking water and five living water resources for basic life)and aquatic products(fish and duck)for food as well.4.Testing viral hepatitisWe tested HBsAg and HBcAg in serum of the participants with automatic biochemical analyzer,and the levels of HBV DNA in serum of participants who were positive for HBsAg.5.Detecting indicators of liver and kidney in serumWe segregated serum samples from each 5 ml venous blood collected from participants in June 2013,for the detection of alkaline phosphatase(ALP),aspartate aminotransferase(AST),alanine aminotransferase(ALT),?-glutamyltransferase(GGT)triglyceride(Tg),total cholesterol(Tch),creatinine(Cr),usea nitrogen(UN),and glucose(Glu),using automatic biochemical analyzer.6.Statistical analysis of dataData were analyzed with Microsoft Excel 2007 and SPSS 20.0.According to the data distribution,methods including frequency distribution,Chi-square test(?2 test),Kruskal-Wallis test and were adopted for statistical analysis.Results:1.AFB1 and MC-LR in the environment in the study sitesOf all the water and aquatic food samples,68.70% were detected positive for MC-LR,with concentrations ranging from 0.02 to 1.74 ?g/kg.Mean levels of MC-LR in well water and finished water,as well as that in the total mean of water were significantly higher in Yihe than in Lidu.So were the mean levels of MC-LR in muscle of fish and duck,as well as that in the total mean of muscle.Of all the food samples,42.24% were detected positive for AFB1,with concentrations ranging from 0.04 to 4.87 ?g/kg.There were no significant differences between the two towns.2.Participants characteristicsOf all the 6467 participants,5493(Lidu : Yihe =1857 : 3636)finished information and blood collection,with mean age of 58.76±12.56 years old.62.37% of the participants were females and 99.58% of them were Han ethnicity.There were significant differences in age,sex,education levels,incomes,smoking,passive smoking and medication history between the two towns.There were no significant differences in BMI,nationality,born place and alcoholic consumption between the two towns.3.Health parameters of the participantsIn the study participants,abnormal rates of ALP,AST,ALT and GGT were 3.44%,10.16%,4.81% and 9.92%,respectively;abnormal rates of Cr,BUN and e GFR were 4.50%,6.97% and 2.35%,respectively;and abnormal rates of Tch,Tg and Glu were 16.15%,24.84 and 9.40%,respectively.Conclusion:In this study,we found that residents living in Fuling district were exposed to AFB1 and MC-LR,and that concentrations of AFB1 and MC-LR,and levels of renal function in the two towns are different between the two towns.This result indicated that AFB1 and MC-LR might be the principal reason resulting in the differences of renal function levels in the two towns.Part ?.Association of exposure to HBV,AFB1 and MC-LR in population of Three Gorges Region with liver damage in adultsMethods:1.Calculating exposure levels of study populationWe calculated the estimated daily intake(EDI)of AFB1 and MC-LR based on the concentrations of the toxins in food and water recorded in food frequency table of every resident.2.Comparing liver enzymes,HBV infection,and AFB1 and MC-LR exposure levels between two towns.3.Comparative and combinative effects of the three hazards with liver enzmes in all participants.4.Statistical analysis of dataAccording to the data distribution,methods including frequency distribution,Chi-square test(?2 test),Kruskal-Wallis test and multivariate logistic analysis were adopted for statistical analysis.Results:1.Relationship of HBV DNA,EDIAFB1 and EDIMC-LR with liver enzymesAST and ALT were significantly positively correlated with HBV titers,EDIAFB1 and EDIMC-LR,with correlation coefficients as HBV > MC-LR > AFB1.ALT and GGT were only positively correlated with EDIMC-LR,but not with HBV titers and EDIAFB1.2.Comparative effects of the three hazards.The participants with positive HBsAg,especially those with detectable HBV DNA,had higher AST and ALT levels than those with negative HBs Ag.Compared with none-HBV-infections,those with HBV infection had significantly higher risks of abnormal AST(OR=2.413,95%CI=1.701-3.424)and ALT(OR=2.974,95%CI=2.009-4.403),and those with detectable HBV DNA had much significantly higher risks of abnormal AST(OR=5.322,95%CI=3.535-8.014)and ALT(OR=5.633,95%CI=3.512-9.033).Participants with higher AFB1 exposure or higher MC-LR exposure had higher mean levels of AST and ALT,but had no significant differences in abnormal rates of the liver enzymes.3.Co-effects of the three hazardsIn non-HBV-infections,those with both high EDIs of AFB1 and MC-LR had higher mean levels of AST and ALT than those with both low EDIs of AFB1 and MC-LR However,there were no significantly different ORs of abnormal AST and ALT in these participants with different EDIs of toxins.There was a significant increase in mean levels and ORs of abnormal AST and ALT in those with HBV-infection at different MC-LR/AFB1 exposure levels,especially in those with detectable HBV DNA.Adjusted ORs of abnormal AST and ALT in participants with detectable HBV,compared with none-HBV-infections,were 16.402(95%CI = 4.862-55.340)and 18.052(95%CI = 4.968-65.597)when both of their EDIs of AFB1 and MC-LR were high,and 4.647(95%CI = 2.253-9.585)and 5.504(95%CI = 2.457-12.328)when both of their EDIs of AFB1 and MC-LR were low.Conclusion:Among the three liver hazards(HBV,AFB1 and MC-LR),HBV is the most serious risk and can strongly increase the risk of liver injury induced by AFB1 and MC-LR.Control of HBV infection should be the key measure of preventing population from suffering the hazards of environmental toxins.Part ?.Association of exposure to AFB1 and MC-LR in population of Three Gorges Region with renal impairment in adultsMethods:1.Association between toxin exposure and renal function indicators2.Comparative and combinative effects of the two toxins with renal function indicators3.Statistical analysis of dataData were analyzed with Microsoft Excel 2007 and SPSS 20.0.According to the data distribution,methods including frequency distribution,Chi-square test(?2 test),Kruskal-Wallis test and multivariate logistic analysis were adopted for statistical analysis.Results:1.Comparative effects of the three hazards.Participants with higher MC-LR exposure had higher levels of Cr and BUN but lower levels of eGFR.ORs for abnormal Cr,BUN and e GFR were 4.58(95%CI=2.92~7.21),1.80(95%CI =1.34~2.42)and 4.41(95%CI=2.55~7.63),respectively.However,there were no significant differences in abnormal rates of renal function parameters in participants with AFB1 exposure alone.2.Co-effects of AFB1 and MC-LRWhen exposed to AFB1 and MC-LR in combination,participants with higher MC-LR exposure,no matter AFB1 exposure was low or high,had significantly increased OR values in Cr,BUN and e GFR.Results indicated that MC-LR might be the major risk to the renal function in population.Compared with those with both low EDIs of AFB1 and MC-LR,ORs for abnormal Cr,BUN and eGFR were 1.46(95%CI=1.10-1.94),2.92(95%CI=1.96-4.36)and 2.92(95%CI=1.66-5.16),respectively in those with both high EDIs of AFB1 and MC-LR.Conclusion:MC-LR,rather than AFB1,might be one important risk of renal function impairment.Control of MCs pollution should be the key measure of preventing population from kidney damage.Part IV.DNA oxidative damage and oxidative stress change induced by combined exposure to AFB1 and MC-LR in L02 cellMethods:1.Association of exposure to AFB1 and MC-LR with DNA oxidative damageWe confirmed the effects of AFB1 and MC-LR on DNA oxidative damage in L02 cell with micronucleus assay and comet assay.2.Association of exposure to AFB1 and MC-LR with oxidative stressWe monitored the change of ROS in L02 cells treated with AFB1 and MC-LR,to determine the oxidative response of the two toxins to cells.3.Statistical analysis of dataIn experiments conducted in cells,we compared ROS,micronucleus percentage and DNA percentage in tails among different exposed groups using mean test.Accordingly,P<0.05(two-sided)was considered to be statistically different.Results:1.Effects of AFB1 and MC-LR on DNA damage in L02 cellsThe higher exposure concentrations of AFB1,the higher percentage rates of DNA in the tails and micronucleus in L02 cells.Percentage of MN and DNA in the tails were 41.33 ± 7.78% and 5.00±2.00%,respectively,in the group with both high doses of AFB1(10mg/L)and MC-LR(10mg/L),and 18.19±15.06% and 1.03±0.59%,respectively,in the control group(DMSO group).2.Effects of AFB1 and MC-LR on ROS expression in L02 cellsIn a relatively lower exposure concentration,as the co-exposure levels of AFB1 and MC-LR increased,ROS expression significantly increased;in a relatively higher exposure concentration,as the co-exposure levels of AFB1 and MC-LR increased,ROS expression significantly decreased.Conclusion:When exposed to AFB1 and MC-LR separately or combinatively,cells would suffer oxidative stress changes and DNA damage.Overall Conclusion:Our results showed that residents living in the Three Gorges Region commonly exposed to aflatoxin and microcystin,two important factors for liver and kidney damage.HBV is the most risk for liver injury and can increase liver injury induced by AFB1 and MC-LR.MCs,rather than AFT,might be one important risk of renal function impairment.Control of MCs pollution should be the key measure of preventing population from kidney damage.When exposed to AFB1 and MC-LR separately or combinatively,cells would suffer oxidative stress changes and DNA damage.Innovations:1.In this study,we compared effects of HBV,AFB1 and MC-LR,three major factors inducing liver injury,analyzed the combined effects of those three factors in a same population,and found that HBV is the most serious risk for liver injury and can increase the liver injury induced by AFB1 and MC-LR;thus this study might provide a new theoretical basis for confirming the hazards of the two toxins and their susceptible populations.2.This was the first population study to provide epidemiological evidence of positive association of environmental exposure to MC and AFT with renal function impairment,whose results indicated that MC,rather than AFT,is the major risk for renal function impairment.This study provides a new perspective to causes of chronic kidney disease.3.This study was the first attempt to explore the effects of the two biotoxins in cells.We found that AFT and MC can cause DNA oxidative injury in cells,and the effect of the combined exposure was significantly stronger than that of one factor,which revealed the synergic genotoxicity of the botoxins.Consequently,this study would provide an experimental basis for the mechanism of the two botoxins on their synergic genotoxicity.There were no similar reports found at home and abroad.Limitations:However,there are several limitations in this study.Firstly,we did not test all the liver and kidney biomarkers due to financial matter.Secondly,we were unable to test all kinds of MCs and AFT,which may underestimate the overall exposure to AFT and MC.Thirdly,although we have adjusted for some potential environmental factors,there are some other risk factors that may have effects on liver and kidney damage.Lastly,although we found ROS was one of the important pathway through which liver damage was induced by AFT and MC,other important pathways involved in the co-effects need to be explored.The next step of our work:In the future,we will analyze the relationship between toxins and metabolic disease.We hope to establish a cohort study in order to raise long-term concern about the health problems of the population.What's more,underling mechanism of the two toxins needs more exploration.