Study On Arsenic Methylation Pattern And Its Association With Oxidative Stress Status In Populations Exposed To Arsenic In Drinking Water

ObjectiveArsenic has been identified as a human carcinogen by the International Agency for Research on Cancer(IARC).The long exposure to arsenic in drinking water can cause the classical dermal stigmata and even skin cancers,and also is related to the development of cancers of several organs,cardiovascular diseases,diabetes and the impairment of intelligence in children.It is estimated that 200 million people are being under the threat of high arsenic in drinking water in the world.However,the susceptibility to arsenicosis is different in subjects exposed to high arsenic.Arsenic is a xenobiotic,and the metabolism of arsenic in the body may be closely related to the development of its toxicity.The prime biotransformation of inorganic arsenic(iAs) in human bodies is methylation,which results in arsenic metabolites different in toxicity and target organs.Epidemiological studies have suggested that subjects with higher secondary arsenic methylation capacity or lower relative content of monomethylated arsenic(MMA) in the urine have lower risk of arsenic-related diseases.The susceptibility to arsenicosis is at least partly related to the pattern of arsenic methylation.Thus,the study on factors influencing arsenic methylation is essential to the further study on the mechanism and the prevention of arsenicosis.Oxidative stress is one of the most important theories in the mechanism of arsenicosis.Could arsenic methylation pattern affect the status of oxidative stress? Is it possible that arsenic methylation pattern related to the susceptibility to arsenicosis through oxidative stress? These answers remain unclear.The present study was conducted in subjects exposed to arsenic through drinking water subacutely and chronically in China.Arsenic methylation pattern among different kinds of populations,as well as effects of polymorphisms of glutathione S-transferases omega 1(GSTO1) and glutathione S-transferases omega 2(GSTO2) genes on arsenic methylation pattern,were studied. In addition,the status of oxidative stress status for study subjects were assessed on the bases of antioxidants in blood and oxidants DNA lesions in urine.Associations of oxidative stress with arsenic methylation pattern and polymorphisms of GSTO1 and GSTO2 were also analyzed.Methods1.Study subjects(1)Subacute arsenic-exposed subjects:76 subjects were from patients admitted to the Centre Hospital of Fuxin in accidental subacute arsenic poisoning of Fuxin in December,2004.In the accident,pollution of drinking water was caused by the leakage of arsenic-containing waste from the drain pipe in a copper-smelting factory.The concentration of arsenic in the polluted well water was(48.5±4.3) mg/L.(2)Chronic high arsenic-exposed subjects:The subjects were from 3 high arsenic-exposed villages(Naimoban,Koukenban and Shiligetu) near hohhot in Inner Mongolia,China.In these villages,centralized tap-water systems were established and supplied water to all villagers for daily life for at least 6 years.Unfortunately,the tap-water contained high concentrations of arsenic,as much as 0.09 mg/L,0.16 mg/L and 0.24 mg/L,respectively.100 subjects in 0.09 mg/L arsenic-exposed group,108 subjects in 0.16 mg/L arsenic-exposed group,and 72 subjects in 0.24 mg/L arsenic-exposed group were recruited in this study.(3)Chronic low arsenic-exposed(control) subjects:The subjects were from Tianjiaying village around Hohhot in Inner Mongolia,China.Centralized tap-water with 0.02 mg/L arsenic,less than the maximum allowable concentration of arsenic in drinking water(0.05 mg/L) in rural of China,was provided in this village.2.Epidemiological investigationStudy on the subacute and chronic arsenic-exposed subjects:data on subacute arsenic-exposed subjects were obtained from the Central Hospital of Fuxin.Cross sectional studies were conducted in the chronic arsenic-exposed subjects compared with the subacute exposed,in which data on drinking method,daily ingestion of water, disease history were collected.Study on arsenic methylation and oxidative stress of subjects chronically exposed to high arsenic in drinking water:Data on age,sex,smoking,drinking,dietary habits, daily water ingestion,medical history and others were obtained by questionnaire. Trained doctors conducted detailed physical examinations and arsenicosis identification according to the Diagnosis Standards on Arsenicosis of China(WS/T211-2001).3.Sample collection(1)Urine samples:Urine samples of subacute arsenic-exposed subjects were collected prior to any therapeutic intervention after they were admitted to the hospital. Spot urine samples of chronic arsenic-exposed subjects were collected.All urine samples were shipped to lab in 0-4℃ice box and kept in-80℃before analysis.(2)Blood samples:15 ml of fasting venous blood was taken from the study subjects,inoculated into anticoagulated tubes(containing heparin) and aliquoted.Then the samples were shipped to lab in liquid nitrogen and kept in-80℃before analysis.4.Determination of arsenic metabolitesUrine samples were mix with 2 mol/L NaOH(1/1,v/v) and digested at 100℃for 3 h.Then cold trap hydride generation-atomic absorption spectrometry were applied to determine the content of iAs,MMA,dimethylated arsenic(DMA) and trimethylated arsenic(TMA).The total arsenic(TAs) content was calculated by summing up the content of all above arsenicals.The levels of urinary creatinine(Cr) determined with jaffe assay were used to correct the concentrations of arsenic in the urine.Proportions of urinary arsenicals(iAs%,MMA%and DMA%) and the 2 arsenic methylation ratios [first methylation ratio(FMR) and secondary methylation ration(SMR)]were used to assess arsenic methylation capacity of the body.5.Genotype analysisPolymerase Chain Reaction/Restriction fragment length polymorphism (PCR/RFLP) was used to detect polymorphisms of GSTO1 and GSTO2 genotypes.6.Determination of redued glutashione(GSH) content and superoxide dismutase(SOD) activity in blood5,5'-dithiobis-2-nitrobenzoic acid(DTNB) method and nitrite-generating method were used to determine GSH content and SOD activity in blood,respectively.levels of haemoglobin were assayed to correct GSH content and SOD activity。7.Determination of urinary 8-hydroxy-2'-deoxyguanosine(8-OHdG) levelsEnzyme linked immunobsorbentassy(ELISA) Kit was applied for the determination of urinary 8-OHdG levels.Urinary Cr levels determined with jaffe assay were used to correct urinary 8-OHdG levels.8.Statistical analysisStatistical analysis was conducted by using the SPSS software(version 11.0). Nonparametric tests were applied to data which were not fit or approximately fit normal distribution even after transformation.Mann-Whitney U test and Kruskal-Wallis H test were used to determine the statistical significance for the differences between 2 and 3 groups,respectively.Parametric tests were applied to data which were fit or approximately fit normal distribution after transformation.One-way ANOVA and LSD test were performed to determine the statistical significance for the differences among 3 groups.T-test was used to determine the statistical significance for the differences between 2 groups.Multiple linear regression analyses were applied to assess the associations of oxidative stress status with arsenic methylation pattern.Results1.Comparison of arsenic methylation pattern among subacute, chronic high and low arsenic-exposed subjectsIndividuals in subacute and chronic arsenic-exposed groups were all different in concentrations and proportions of urinary arsenic metabolites.Concentrations of urinary arsenicals and TAs were highest for subacute arsenic-exposed subjects and lowest for chronic low arsenic-exposed subjects,and the differences between groups were statistically significant(P＜0.05);The order of urinary iAs%and MMA%values, from highest to lowest,was as follows:subacute arsenic-exposed subjects＞chronic high arsenic exposed subjects＞chronic low arsenic-exposed subjects;The order of DMA%,FMR and SMR values,from highest to lowest,was as follows:chronic low arsenic exposed subjects＞chronic high arsenic exposed subjects＞subacute arsenic-exposed subjects.All above differences between groups were statistically significant(P＜0.05).2.Comparison of urinary 8-OHdG levels among subacute,chronic high and low arsenic-exposed subjectsThe levels of urinary 8-OHdG were highest for subacute arsenic-exposed subjects and lowest for chronic low arsenic-exposed subjects,and the differences were statistically significant(P＜0.05).3.Urinary arsenic concentrations and arsenic methylation pattern of subjects chronically exposed to different levels of arsenic in drinking waterThe order of urinary arsenic concentrations of for the 3 groups was as follows: 0.16 mg/L-arsenic-exposed subjects＞0.09 mg/L-arsenic-exposed subjects＞0.02 mg/L-arsenic-exposed subjects,and the differences were statistically significant(P＜0.05).The values of urinary iAs%and MMA%were significantly higher,whereas the values of DMA%,FMR and SMR were significantly lower(P＜0.05) in 0.16 mg/L and 0.09 mg/L high arsenic-exposed subjects,compared with 0.02 mg/L low arsenic-exposed subjects.Only the values of urinary DMA%and SMR were significantly different between 0.16 mg/L and 0.09 mg/L arsenic exposed groups,both of which were higher for the 0.16 mg/L-exposed(P＜0.05).4.Urinary arsenic concentrations and arsenic methylation pattern of children and adultsIn 0.16 mg/L and 0.09 mg/L high arsenic-exposed groups,children and adults were not significantly different in urinary TAs concentration(P＞0.05);however, children were significantly lower in the values of urinary MMA%(P＜0.05),but significantly higher in the values of DMA%and SMR(P＜0.05) than adults of the same group.In 0.02 mg/L low arsenic-exposed group,children were significantly higher in urinary TAs concentration than adults(P＜0.05);no significant difference in proportions of urinary arsenicals or arsenic methylation ratios was observed between children and adults(P＞0.05).5.Gender differences in urinary arsenic concentrations and arsenic methylation patternNo statistical difference was absorbed in urinary arsenic concentrations and arsenic methylation pattern between males and females in the same arsenic-exposed group(P＞0.05).6.Variations of urinary arsenic concentrations and arsenic methylation capacity in high arsenic-exposed population over 2 yearsFor both children and adults,there was no significant variation in the average concentration of urinary TAs over the 2 years.On the whole,iAs%and MMA%were significantly elevated,but DMA%,FMR and SMR were significantly decreased for 9-year exposed subjects,compared with that of 7-year exposed subjects(P＜0.05).The significant difference in urinary iAs%(P＜0.05) of 3 subgroups after 7-year exposure to arsenic disappeared after 9-years exposure to arsenic.Arsenic methylation pattern of individuals,relative to the entire population,altered over the 2 years.7.Family correlation of arsenic methylation patternThe proportions of urinary arsenicals and the 2 arsenic methylation ratios were significantly correlated between siblings,as well as between parents and children(P＜0.05).Correlation of arsenic methylation capacity was not observed between couples.8.The relationship between arsenic methylation pattern and the genotypes of GSTO1 and GSTO2There were no significant differences in urinary arsenic profile among individuals who carried with different genotypes in A140D site of GSTO1 gene or different genotypes in N142D site of GSTO2 gene(P＞0.05).9.Effect of arsenic exposure on blood GSH content and SOD activityBlood GSH content were significantly lower for 0.16 mg/L-arsenic-exposed children and adults compared with the 009 mg/L-and 0.02 mg/L-arsenic-exposed(P＜0.05).Significant difference between children and adults of the same group was only found in 0.09 mg/L-arsenic-exposed group,in which children had significantly higher blood GSH content than adults(P＜0.05).Blood SOD activity was significantly lower for 0.16 mg/L-arsenic-exposed Children and adults compared with the 0.09 mg/L arsenic exposed(P＜0.05).The increase of blood SOD activity was observed in 0.09 mg/L arsenic exposed children and adults compared with the 0.02 mg/L arsenic exposed,however the difference was only significant between children.No significant difference in blood SOD activity was found between children and adults if the same group.10.Effect of arsenic exposure on urinary 8-OHdG levels Among the 3 studied groups,urinary 8-OHdG levels were highest for the 0.16 mg/L-arsenic-exposed adults and lowest for the 0.02 mg/L-arsenic-exposed adults.All above differences between groups were statistically significant(P＜0.05).Children in 0.16 mg/L and 0.09 mg/L were not significantly different in urinary 8-OHdG levels,but both had significantly higher urinary 8-OHdG levels than the 0.02 mg/L-arsenic-exposed(P＜0.05).Urinary 8-OHdG levels were significantly increased for children and adults with 9-year exposure to arsenic,compared with that with 7-year exposure to arsenic(P＜0.05).11.Association of arsenic methlyation capacity with GSH,SOD and 8-OHdGThe levels of urinary 8-OHdG were significantly positively correlated with the values of urinary iAs%,MMA%,but negatively correlated with the values of urinary DMA%,FMR and SMR for both children and adults.The content of blood GSH was significantly negatively correlated with the value of urinary iAs%and MMA%for both children and adults.In children,significant positive correlation of blood GSH with FMR was observed,and in adults significant positive correlation of blood GSH with urinary DMA%,FMR and SMR.Significant correlation of blood SOD activity with proportions of urinary arsenic metabolites and the 2 methylaton ratios was only observed in Adults.12.The relationship between oxidative stress and the genotypes of GSTO1 and GSTO2No significant differences were found in blood GSH content,SOD activity or urinary 8-OHdG among individuals who carried with different genotypes in A140D site of GSTO1 gene or different genotypes in N142D site of GSTO2 gene.Conclusion1.Arsenic methylation capacity decreases with the increase in the dose rate or the time of arsenic exposure.Male and female are not different in arsenic methylation pattern,while children have higher arsenic methylation capacity than adults,when exposed to the same level of arsenic in drinking water.In addition,the variability of arsenic methylation pattern is not affected by polymorphism of GSTO1 A140D or GSTO2 N142D.The effects of exogenous factors on arsenic methylation capacity could not be ignored besides genetic factors. 2.The exposure to high levels of arsenic in drinking water results in the decrease of anti-oxidant capacity,but the aggravation of oxidative DNA lesions in human bodies. Oxidative DNA lesions elevate with the increase in the dose rate or the time of arsenic exposure.3.The oxidative stress status of arsenic-exposed population is closely related to arsenic methylation capacity,which underlies the possible mechanism of relationship between variability of arsenic methylation pattern and susceptibility to arsenicosis.