Effects Of Dietary Pattern And Genetic Variation On The Internal Exposure Of POCs In Non-occupational Population

BackgroundPersistent organochlorine compounds(POCs),represented by organochlorine pesticides(OCPs)and polychlorinated biphenyls(PCBs),are a class of persistant organic pollutants(POPs)that have been banned for many years,they are environmentally persistent,lipophilicity,biomagnification,and accumulate in human adipose tissue.The POCs have carcinogenicity,toxicity to neuroendocrine system,and reproductive system,may resulting in diabetes and hypertension.Compared with external environmental exposure,internal exposure assess accurately the total exposure levels of pollutans which have been entered into human body via all kinds of routes and help to evaluate effects of environmental pollutans on human health.Age,gender,lifestyle and diet make more contribution to internal exposure doses.Owing to low volatility of POCs,the major routes of exposure of for non-occupation crowd to POCs is via food,especially fish and meat.The POCs intake via food were affected by food contamination levels and food consumption.Diet patterns are a comprehensive method of assessing the content and proportion of various foods ingested by the population,which might influence the health of human body through altering the exposure of pollutants.In addition,genetic factors may also be related to human exposure of pollutants,particularly metabolic enzyme gene variation can change the metabolic activity of the body of the prototype and the amount of metabolites,affecting the health hazard outcome.Genome-wide association research(GWAS)is an effective pool for genetic varation study of complex traits.However,a few research on the relationship between genetic variation and internal exposure were reported.Based on the above strategies,we can clarify the role of the genes and the environment in influencing exposure concentrations and characteristics of human to pollutants,and the different adverse effects on human health,find risk factors and susceptible populations.It is of great significance to comprehensively elaborate the relationship between environment and health and implement precise prevention.ObjectiveWe conducted environmental assessment and epidemiology research among non-occupation population in two typical regions of Huaihe River of Jiangsu Provience,to explore effects of environmental exposure,diet pattern and genetic variation on human exposure to POCs and disclose intrinsic link among them.The study might provide theoretical and practical basis for clarifying the health hazards,risk assessment and precision control strategies of POCs.MethodsWith preliminary study of our research group,two typical towns named A,B in were selected as the study districts on the basis of information surveys and historical resources observation.Area A is a typical agricultural town,B area is a typical heavy industry town.Accordancing with China's relevant sample criterions,collected different types of water samples,sediment samples and main foods(staple food,vegetables,fruits,chicken,fish,pork).According to the principle of multi-level cluster sampling,a total of 2353 resident polulations were enrolled,face-to-face questionnaires(including dietary frequency surveys)and physical examinations were carried out after the signing of informed consent and blood samples were collected.Then use solid phase extraction,the disc membrane extraction,and soxhlet extraction to process the samples,and gas chromatography-mass spectrometry to analyse the target pollutants including organochlorine pesticides(OCPs)and polychlorophenyls(PCBs).The dietary patterns were analyzed using principal component method,the determinants of exposure concentrations of human in two areas were observed using multiple linear regression model.We initially performed a GWAS for POCs markers in 511 subjects of A area,and further replicated using Taqman genotyping in two-inependant stage,finally SNPs were identified using multiple linear regression additive model.Based on the results of genetic variations(located in CYP2B6 gene),we conducted a series of experiments.Plasimd carrying C/T allele of rs3181842 were transfected and measure luciferase activity using a Dual-Luciferase Reporter Assay System.The enzymatic activity and expression of CYP2B6 were measured using fluorescence assay kit and western bloting in 7901 cells with low-expression of CYP2B6.The cubation of p,p'-DDT and rCYP2B6 with 8-MOP in vivo were conducted to observe changes of p,p' DDT and its metabolites at different time intervals.The activity of CYP2B1(homologous to human CYP2B6)and changes of p,p'-DDT and its metabolites in serum at different time intervals were observed in control group and in treatment with the specific inhibitor KR-II group after the injection of p,p'-DDT by tail vein injection.Finally,combination of dietary patterns and genetic variations were analyzed using multiple linear regression model.All P values were bilateral test(P<0.05).Results1.Exposure status of representive POCs in environment,food and human serumThe main pollutants in water and soil were HCHs and DDTs.The concentrations of HCHs,DDTs and PCBs in water of B area was significantly higher than that of A area.In the B area,the concentrations of HCHs in surface water(74.14 ng/g)were significantly higher than that in peripheral water(41.19 ng/g).HCHs were the main exposed POCs in water,of which the isomer of ?-HCH ranked the top,whose concentrations exceed that of DDTs.The average concentrations of HCHs,DDTs,PCBs of A area(2.94,81.18,17.17ng/g,respectively)were significantly lower than that of B area(3.13,131.46,17.68 ng/g,respectively).In the B area,the concentrations of HCHs below to that of DDTs,of which sort by the concentration for p,p'-DDT,p,p-DDE,o,p'-DDT,p,p'-DDD,o,p'-DDD,o,p'-DDE.The range of PCBs were ND?19.74 ng/g,which far below to that of HCHs and DDTs.The source analysis show that area A was a typical agricultural town,and source of area B was industrial,which is in line with previously survey.Consistent with characteristics of exposure in environmental medium,concentrations of food of B area were significantly higher than that of A area.The dose of HCHs,DDTs,PCBs in vegetables of B area were 27.74,76.59,9.29 ng/g,which exceed that of A area presented with 2.77,5.57,6.45,as well as concentrations in another foods,the exposure dose in fish were 7.32,105.48,50.23 ng/g of B area and 76.59,156.37,72.25 ng/g of A area.The concentrations of animal foods far beyond that of plant foods in same area,the exposure dose of DDTs of B area increased gradually(2.77 ng/g,76.59,2.24,26.24,110.67,196.78,156.37 ng/g)in seven kinds of foods namely fruits,vegetables,rice,wheat,pork,chicken,and fish.The(3-HCH?p,p'-DDE and p,p'-DDT were main pollutants.The average serum concentration of ?-HCH?p,p'-DDE and PCBs of A area,were 0.76,4.62 and 10.85 ?g/g lipid,respectively,which were significantly higher that of A area(0.48,3.87 and 15.52 ?g/g lipid,respectively).Age,gender and BMI were major determinants using multiplel linear regression model in both two areas.2.Effects of dietary patterns on the levels of the representative POCs in human serum.The average estimated dietary intake(EDI)of HCHs,DDTs and PCBs of A area were eatimated to be 26.66,24.09 and 5.32 ?g/kg.bw.d,which beyond that of B area(1.93,2.94 and 3.48 ?g/kg.bw.d).The EDIp,p'-DDE of A and B area was 0.33 and 0.22?g/kg.bw.d?There was a significant positive correlation between EDI p,p'-DDE and serum p,p-DDE concentrations in the whole population and high exposure population(rA=0.053,rB=0.081,P<0.05;rA= 0.330,rB=0.129,P<0.05),indicating that EDIp,p'-DDE were more representative,similar dietary sources.Three dietary patterns was modeled for the three representative POCs,including staple patterns,fish patterns and poultry meat patterns.In area A,serum PCB153 levels increased with the enhancement of fish pattern score,compared with the low fish pattern group,the exposure level in the moderate and high group increased by 22.9%and 27.2%,respectively.In area B,serum p,p'-DDE increased with the enhancement of fish pattern score,compared with the low fish pattern group,the exposure level in the moderate and high group increased by 1.2%and 4.9%,respectively.3.Effects of genetic varation on the levels of the representative POCs in human serum.In the present study,we initially performed a GWAS for serum POC marker,in 511 subjects of A area,and coincidentally identified two common genetic variants of cytochrome P450 2B6(CYP2B6),rs2279345 and rs3181842,associated with low level of serum p,p'-DDE(Paddiive=1.78×10-6 for rs2279345,Padditive=9.86×10-7 for rs3181842),which was validated in two regions(A:Padditive=1.86×10-25,B:Padditive=1.86×10-25 for rs2279345;A:Padditive=1.86×10-25,B:Padditive=2.93×10-17for rs3181842),and association of combined studies appeared stronger.The rs3181842 presented a mutant in 3'-UTR C/T of CYP2B6 and the high linkage equilibrium with rs2279345(r2>0.99),thus it was introduced to the functional study for these variants.T allele of rs3181842 led to low activity and expression of CYP2B6 in 7901 cells.CYP2B6 was reported to metabolize p,p'-DDT to its metabolites p,p'-DDE and others.The present study showed that inhibition of rCYP2B6 activity by 8-MOP generated a low formation ofp,p,p'-DDE from p,p'-DDT metabolism in vitro,and KR-?(a specific inhibitor of rat CYP2B1)decreased the levels of p,p'-DDE in liver and serm in rats treated with p,p'-DDT.4.Combined effects of dietary pattern and genetic variation on the p,p,-DDE concentration in human serumThere are no significance difference between dietary patterns and genotypes of rs2279345 and rs318182.In the same dietary patterns,the concentrations ofp,p p DDE were sinificantly redued in different genotypes of SNP rs2279345 and rs3181842(TT/CT/CC for rs2279345;CC/CT/CC for rs3181842).In the area B,the levels of serum p,p'-DDE in rs3181842 CT and TT genotype were significantly increased among the same dietary patterns,presented with 3.36,3.50,4.02 ng/g lipid and 3.07,3.34,5.48 ng/g lipid.There was no synergistic effect or interaction between rs2279345 and dietary patterns.In area B,the model including the rs3181842,fish patterns and "fish patterns*rs3181842" could explain varibility of serum p,p'-DDE exposure for 25.4%?37.0%· For serump,p,p'-DDE concentrations,the rs3181842 could significantly reduce the content by 19.95%,while the high fish patterns group significantly increased the content of 7.68%.The combined effect between rs3181842 and fish patterns appeared a significant reduction of 15.8%.ConclusionThe concentration of POCs in area B was higher than that in area A,and latter is agricultural sources of pollution,the former is for industrial sources of pollution.The major exposure POCs in water and soil were HCHs and DDTs,respectively,in which,the ?-HCH,a-HCH,p,p'-DDT and p,p'-DDE are primary isomer.Similarily,the exposure levels of POCs of B area in seven food types far beyond that of A area,the animal food make more contribution,especially fish and chicken.Owing to high food consumption of A area,serum POCs concentration of B area below to that of A area.Three dietary patterns were modeled in two regions,namely staple patterns,fish patterns and poultry meat patterns.High fish pattern group appeared heavy concentrations ofp,p-DDE in B area.The new genetic variants rs2279345 and rs3181842 of CYP2B6 associated with human exposure level of p,p'-DDE,could greatly reduce the content.In the B region,The SNP rs3181842 combined with the fish patterns had a negative effect on serum p,p'-DDE,and the effect of the genetic loci was greater.In summary,the study suggests that the exposure level of POCs in the environmental medium of industrial pollution is higher,but the consumption of food and dietary patterns can significantly affect the internal exposure level.The SNP rs3181842 plays an important role in the human exposure level of p,p'-DDE.The fish patterns can increase the accumulation of compounds,but the effect of intensity is less than that of genetic variation.