Cohort Study Of Microcystins,hepatitis B Virus And Liver Injury Of Township Population In Three Gorges Region

Background and objectivesMicrocystins（MCs）contamination is very common in China.MCs are the most probable class of toxins from cyanobacterial blooms that endanger human health in the world.Among them,microcystin-LR（MC-LR）is the most common,most toxic,and most studied subtype.Previous studies have shown that MC-LR can cause significant liver damage to the body.Hepatitis B virus（HBV）is a highly hepatotropic DNA virus that is closely related to liver injury.At present,China is still the country with the largest number of HBV infections in the world.Studying the effects of these two factors on liver injury is of great significance to protect the health of Chinese residents.In our previous cross-sectional study in Three Gorges Region,MC-LR exposure levels were closely related to liver damage,and combined exposure of MC-LR and HBV significantly increased liver damage.However,there have been no reports of association between MCs exposure and HBV infection,and cohort studies of MCs exposure and HBV infection on liver injury.Therefore,this study intends to conduct follow-up work on the basis of our previous research to study the above aspects,and to explore the harm of MCs pollution to the health of Chinese residents.Study contents and methodsPart Ⅰ Establishment of follow-up cohort of township population in Three Gorges RegionIn July 2013,our team randomly selected some townships（villages）,streets and communities in two townships in Three Gorges Region as survey points,and conducted questionnaire surveys on volunteers over 30 years old who lived there for a long time and had no major diseases.After MC-LR external exposure levels were assessed and liver function and other health indicators were tested,a cross-sectional survey of 5493 volunteers was completed finally.The results showed that MC-LR external exposure levels were significantly positively correlated with liver and kidney injury,and HBV was an important risk factor for liver injury.To further investigate the association between MCs exposure and HBV infection and the effects of MCs exposure and HBV infection on liver injury in cohort,we conducted a part follow-up of these populations in May 2016.The planned follow-up of 1,000 to 1,200volunteers included（1）418 volunteers of all hepatitis B virus antigen（HBsAg）（+）in the above population;（2）500 volunteers of HBsAg（-）who were paired by the same sex and±3years old in the two towns in the above population,respectively.After completing the questionnaire survey or the detection of health indicators such as liver function of 1055people,and screening out the volunteers with missing blood samples,wrong or incomplete questionnaire information,a follow-up cohort consisting of 900 subjects was finally established.During the follow-up,we found that the living environment and drinking water type of the study subjects have changed greatly compared with the previous ones due to urban and rural construction and drinking water reconstruction.According to the previous external exposure evaluation method,the individual exposure level cannot be accurately evaluated,so we focus on the association of the changes of MC-LR internal exposure level（serum MC-LR）,HBV infection and liver injury of subjects in the follow-up cohort in this study.Part Ⅱ Study of association between MC-LR internal exposure levels and HBV infectionFirst,the serum MC-LR levels in the follow-up cohort were detected by enzyme-linked immunosorbent assay;the HBsAg was detected by electrochemiluminescence method;the HBV-DNA levels of HBsAg（+）patients were detected by PCR-fluorescence probe method.Secondly,we performed a cross-sectional study on the association between serum MC-LR levels and HBV infection of subjects in 2013.Binary logistic regression was then used to analyze the association between the HBV infection and high serum MC-LR exposure levels.Finally,we used the Mann-Whitney test to compare the levels of the differences of serum MC-LR levels（△MC-LR）between the new HBV-infected patients and the subjects without HBV in the follow-up cohort.Differences of levels of△MC-LR were analyzed for the effect of HBV infection on serum MC-LR levels.Part Ⅲ Study of association between MC-LR internal exposure levels,HBV infection and liver injuryFirst,the follow-up cohort was divided into 4 groups:group I was composed of people who were HBsAg（-）in 2013 but HBsAg（+）in 2016;group II was composed of people who were HBsAg（-）both in 2013 and 2016;group III was composed of people who were HBsAg（+）both in 2013 and 2016;group IV was composed of people who were HBsAg（+）in 2013 but HBsAg（-）in 2016.Mann-Whitney test was used to compare the distributions of liver function indicators between groups.Secondly,Spearman test and Mann-Whitney test were used to analyze the association between△MC-LR and changes in liver function indexes in subjects with MC-LR exposure alone in the follow-up cohort（group II）.Finally,Spearman test and Mann-Whitney test were used to analyze the association between△MC-LR,HBV-DNA changes（△HBV-DNA）and changes in liver function indexes in the MC-LR and HBV combined exposure subjects in the follow-up cohort（group III）.Part Ⅳ Evaluation and comparison of non-cancer health risks of MC-LR pollution in ChinaAfter published MC-LR data of river water,lake water,well water,finished water of waterwoks,peripheral water and bottled water in China were collected through searching in databases,we used the model recommended by US Environmental Protection Agency（USEPA）to evaluate and compare the non-carcinogenic health risks of MC-LR contamination in the waters of China,and analyzed the health hazards caused by MC-LR pollution in China.Results1.The mean age of follow-up cohort was 58.76±10.96 years old in 2013,and females accounted for 60.89%.In this cohort,the abnormal rate of aspartate aminotransferase（AST）in 2016（4.8%）was significantly lower than that in 2013（7.6%）（P=0.015）,which indicated liver damage was alleviated.At the time of follow-up,7.4%of subjects the living environment and 18.8%of subjects drinking water source were changed.The exposure environment of MCs changed greatly.2.The serum MC-LR level in July 2016（0.324±0.142μg/L）was significantly greater than that in May 2013（0.257±0.098μg/L）in the follow-up cohort（P=0.000）;there was no significant difference between serum HBV-DNA level of the population in July 2016 and that in May 2013（P=0.313）.In the cohort,there were 41 subjects whose results of HBsAg changed from negative to positive,and 38 subjects whose results of HBsAg changed from positive to negative,672 subjects whose results of HBsAg were always negative,149subjects whose results of HBsAg were always positive.And the median and mean values of△HBV-DNA of subjects whose results of HBsAg were always positive were-73（-1.8×10³²）IU/mL and-2.6×10⁶±2.5×10⁷ IU/mL,respectively.3.Analysis of variance showed that dampness,non-tap water type,daily average fish intake（≥60 g/d）and body mass index（BMI）≥27 kg/m² were risk factors for high serum MC-LR exposure in 2013,while HBsAg（+）was significantly negatively correlated with high serum MC-LR.After adjustment of type of drinking water and other factors,binary logistic regression analysis showed that HBsAg（+）was still significantly negatively correlated with high serum MC-LR level（OR=0.415,95%CI:0.276⁰.625）.4.Cohort analysis showed that the mean value of△MC-LR was 0.015±0.127μg/L in the newly HBV-infected population,and the mean value of△MC-LR in the normal population without HBV was 0.072±0.164μg/L.The former△MC-LR level was significantly lower than the latter（P=0.044）.After adjustment of the effects of living environment,drinking water type and BMI,the△MC-LR levels between the two groups were still significantly different（P=0.040）.5.In the case of MC-LR exposure alone,after adjustment of the effects of alcohol and medication,we found that levels of serum ALT and AST changes（△ALT,△AST）（-3.19±25.68 IU/L and-3.19±17.47 IU/L）in the△MC-LR low-exposure group in the follow-up cohort were significantly smaller than those in the△MC-LR high-exposure group（0.31±13.05 IU/L and 2.16±14.91 IU/L）（P=0.039 and P=0.043）.The△MC-LR in the study subjects were positively correlated with changes of liver function indexes.6.The△ALT and△AST were significantly positively correlated with△HBV-DNA in the follow-up cohort,respectively（r=0.240,P=0.004;r=0.188,P=0.022）.7.In the case of MC-LR and HBV combined exposure,there were significant negative correlations between△MC-LR and△ALT,△AST in the follow-up cohort,respectively（r=-0.257,P=0.002;r=-0.259,P=0.001）.The reasons of these associations were that on the one hand,elevated levels of HBV-DNA in the body lead to increasments in liver function enzyme levels,and on the other hand,elevated levels of HBV-DNA may reduce serum MC-LR levels.It indicated that the HBV-DNA level of hepatitis B patients exposed to MCs and HBV is the most critical factor affecting liver injury in general.8.From April 1998 to June 2016,the non-carcinogenic health risk ranges of river water,lake water,well water,finished water of waterwoks,peripheral water and bottled water in China were 0¹.35×10^-6,0⁵4.63×10^-6,0⁰.69×10^-6,0¹.27×10^-6,0⁰.29×10^-6,0⁰.67×10^-6,respectively.From July 2004 to September 2014,the non-carcinogenic health risk ranges of MC-LR in above water in the area of the follow-up cohort were 0.99×10^-6,0⁰.59×10^-6,0⁰.36×10^-6,0.04×10^-60.14×10^-6,0.02×10^-60.13×10^-6 and 0.03×10^-60.06×10^-6,respectively.The non-carcinogenic health risk ranges of MC-LR in the waters in the area of the follow-up cohort were much lower than many other parts of China.ConclusionsSerum MC-LR levels were significantly negatively associated with HBV infection,and HBV infection may reduce serum MC-LR levels.In the case of MC-LR exposure alone,changes in serum liver function were positively correlated with changes in MC-LR levels;the level of HBV-DNA was the most critical factor for liver injury in the case of combined MC-LR and HBV exposure.Changes in HBV-DNA levels were significantly positively correlated with changes in liver function indicators and may be inversely correlated with changes in serum MC-LR levels.And we recommend strengthening monitoring of pollution of MCs in China.InnovationsFirst,a cohort study of internal exposure of MC-LR in the population was conducted for the first time.Second,it was found that the changes of serum MC-LR level were positively correlated with the changes of liver function indexs;in the case of combined exposure of MC-LR and HBV,HBV-DNA level was the most important factor affecting liver injury.LimitationsWe did not conduct a follow-up of all people in the previous survey,which may affect the promotion of the research results.Secondly,we only detected the serum MC-LR level in the cohort,and did not detect other subtypes of MCs,which would underestimate the impact of MCs on liver damage.Future planWe will study the mechanism of that HBV infection may reduce serum MC-LR levels in the body,and investigate the association between exposure levels of MCs and other health injuries in this cohort.