The Associations Between Aflatoxins/Microcystins Exposure And Hepatocellular Carcinoma Risk By A Case-control Study

Background and ObjectivesHepatocellular carcinoma(HCC)is one of the most common malignant tumor in the world.New HCC cases in our country account for about 54% of the world new cases.Hepatitis B virus(HBV)infection and aflatoxins(AFs)exposure are the significant risk factors of hepatocellular carcinoma in the world.Obviously,they are insufficient to explain all the reasons.Other biotoxin,such as microcystins(MCs)may play an important role in HCC development.Chongqing is located in the upper and middle areas of Yangtze River.It has the warm and wet climate,which is especially suitable for the growth of aspergillus flavus and blue-green algae.And people have the risk of aflatoxin and microcystin exposure.In fact,the incidence of hepatocellular carcinoma is high in Chongqing.But there is no standardized case-control study to estimate the associations between biotoxins exposure and hepatocellular carcinoma risk.So,we aim to investigate the associations between aflatoxins/microcystins exposure and HCC by a case-control study.The interaction effects of aflatoxins/microcystins coexposure with other risk factors are estimated,respectively.We hope to provide a new strategy for HCC prevention.MethodsWe conducted a 1:1 case-control study based on three hospitals of ChongqingSouthwest Hospital,Daping Hospital affiliated to Third Military Medical University,and The Second Affiliated Hospital of Chongqing Medical University.HCC cases and controls were matched by age(±3 yesrs)and sex.214 Cases were confirmed with HCC diagnosis standard.The cases caused by clear reasons such as drug-induced liver injury,alcoholic liver damage were excluded.214 controls were included with no cancer and no digestive system diseases,and recruited simultaneously with cases.The questionnaire included personal basic information,life habits(smoking,alcohol drinking,tea drinking,eating),the history of diseases,and the family history of diseases.The levels of serum MC-LR,serum AFB1-ALB adduct and AFB1-N7-GUA adduct were anlysed by enzyme-linked immunosorbent assay.The associations between biotoxins exposure and HCC risk were anlysed by binary logistic regression,and interaction effects of biotoxins co-exposure with other risk factors were estimated in HCC development.Results1.The average ages were 50.74 ± 9.67 years for HCC patients,51.15 ± 9.90 years for controls.The proportion of men and women was 183:31 in both two groups.There were no significant differences in age,gender,nationality,education,and occupation distribution in HCC cases and controls by statistical analysis.2.After the adjustment of age,sex,smoking,alcohol drinking,family histroy of cancer and diabetes et.al,the HBV infection is still the most dangerous factor for HCC in Chongqing region by binary logistic regression analysis(OR=58.462,95%CI: 28.388~ 120.397).3.There was a significant difference on the median of serum AFB1-ALB adduct between HCC cases and controls(cases vs controls: 146.23 vs 72.42 ng/g albumin,P<0.001),but no significant difference on the median of urinary AFB1-N7-GUA adduct in two groups(cases vs controls: 0.17 vs 0.14 ng/mg creatinine,P=0.210).The adjusted(HBV,smoking,alcohol drinking,family histroy of cancer and diabetes et.al)ORs of HCC risk were 1.9(95%CI: 1.1~3.4)for serum AFB1-ALB adduct,and 2.1(95%CI: 1.03~4.2)for urinary AFB1-N7-GUA adduct.4.We observed positive interactions of aflatoxins co-exposure with HBV,alcohol drinking,and diabetes.The S was 4.7(95%CI: 2.8~7.9),3.5(95%CI: 1.0~12.0),and 12.4(95%CI: 1.8~84.2),respectively for serum AFB1-ALB adduct with each of the three factors mentioned,and was 1.9(95%CI:1.1~3.1),2.0(95%CI: 1.1~3.6),and 2.0(95%CI: 1.1~3.6),respectively for urinary AFB1-N7-GUA adduct with each of the three factors mentioned.5.The serum MC-LR high exposure is an independent risk factor(OR=2.9;95% CI: 1.5~5.5)for HCC after adjustment(HBV,smoking,alcohol drinking,family histroy of cancer,diabetes and aflatoxin et.al).If we group the population by the quartiles of serum MC-LR level,HCC risk also increased gradually as serum MC-LR levels(Q2,Q3 and Q4).The OR values were 1.5(95% CI: 0.6~3.8),3.0(95% CI: 1.2~7.4)and 4.4(95% CI: 1.7~10.9)for MC-LR Q2,Q3 and Q4 exposure,respectively.6.We also observed positive interactions of MC-LR co-exposure with HBV,alcohol drinking.The S was 3.4(95%CI: 2.3~5.0)and 7.9(95%CI: 1.4~43.8),respectively for serum MC-LR with each of the two factors mentioned.But the S was 0.4(95%CI: 0.3~0.6)for MC-LR co-exposure with aflatoxin,which suggested a negative interaction.ConlusionsHBV infection(OR=58.462)was still the main risk factor of HCC in Chongqing.AFs(OR=1.9~2.1)and MCs(OR=2.9)exposure were also independent risk factors for HCC.MCs exposure was associated with higher HCC risk than AFs in human.There were positive interactions of AFs co-exposure with HBV(S=1.9~4.7),alcohol drinking(S=2.0~3.5),diabetes(S=2.0~12.4).And there were also positive interactions of MCs co-exposure with HBV(S=3.4),alcohol drinking(S=7.9),but was a negative interaction of MCs co-exposure with AFs(S=0.4).Innovation1.The same population was used simultaneously to estimate the relationshaip between two kinds of biotoxins exposure in the environment(aflatoxins and microcystins)and HCC risk.2.We used two aflatoxin biomarkers to estimate the association between aflatoxin exposure and HCC risk in southwest China,and to find that aflatoxin exposure was an independent risk factor for HCC.The positive interactions of aflatoxin co-exposure with HBV,alcohol drinking,and diabetes were observed in HCC pathogenesis.3.The serum MC-LR levels were tested in HCC cases for the first time.We observed that MC-LR exposure was an independent risk factor for HCC.There were positive interactions of MC-LR co-exposure with HBV,alcohol drinking.4.For the first time,we found that there was an obvious negative interaction of MC-LR co-exposure with aflatoxin in the HCC pathogenesis,which was the opposite of expected results and previous experimental reports.At present,we speculated that microcystins may competively consume metabolic enzymes with aflatoxin,reduce aflatoxin activation,which may decrease theses carcinogenic aflatoxin adducts.LimitationsFirst of all,this study is based on the hospital case-control study,and there may be some selection biases.Secondly,we investigated some HCC cases after surgery with complex content of the epidemiological questionnaire,and there may be some memory biases and information biases.Again,we did not detect all subtypes of microcystins in the serum,which may underestimate the effects of microcystins.Finally,we observed the significant negative interaction of MC-LR co-exposure with aflatoxin,but we didn’t explore and explain the possible mechanism in next step.Future planIn future,we will further expand the sample size to estimate relationship between microcystins exposure and HCC risk.The phenomena and mechanism of negative interaction effect of two toxins co-exposure should be further validation,analysis,and interpretation.In addition,we will track postoperative HCC caeses health to analyze relationship between biotoxins exposure and HCC prognosis.