BACKGROUND:Among the pathogenic factors of cervical spondylosis,herniation of the intervertebral disc,dislocation of the facet joint and the stenosis of the intervertebral foramen are important factors leading to symptoms in patients.Moreover,inappropriate manipulation may aggravate the possibility of cervical disc rupture,leading to exacerbation of symptoms in patients. OBJECTIVE:To compare the effect between sagittal cervical manipulation and traditional cervical rotation manipulation on the area of the intervertebral disc,facet joint and intervertebral foramen at the operative segment by the finite element analysis. METHODS:The neck CT data of a male volunteer with a normal neck were selected and imported into Mimics 17.0 three-dimensional reconstruction software.Geo-magic Studio 12.0,Solidworks 2017 and Ansys Workbench 17.0 software were used for the construction of the finite element model of cervical vertebrae(C3-6)including intervertebral disc and articular cartilage.The lower end plate of the C5 vertebral body was fixed.A uniformly distributed vertical downward 50 N load was applied on the upper surface of the upper vertebral body(C3).The stress,deformation and deformation direction of the C4-5 intervertebral disc,joint capsule stress,the displacement of facet joints and the area of bilateral intervertebral foramen were compared between sagittal cervical manipulation and traditional rotation reduction. RESULTS AND CONCLUSION:(1)When using the rotation technique,the maximum normal equivalent stress(von Mises stress)of the C4-5 disc was 8.06 MPa;the total deformation was 1.05 mm,and the fiber ring expanded to the left and outside.When using the sagittal tip lifting technique,the maximum normal equivalent stress(von Mises stress)of the C4-5 disc was 2.60 MPa;the total deformation was 0.90 mm,and the fiber ring expanded to the left and back.Compared with the rotation technique,the pressure of the cervical manipulation technique on the disc was less(about 32.3%of the rotation technique),and the deformation degree of the disc was also light(about 85.7%of the rotation technique).(2)When the rotation technique was used,the maximum stresses of the left and right articular capsule ligaments were 0.37 MPa and 1.69 MPa,respectively.The overall displacement of the facet joint was 2.21 mm.The area of the right intervertebral foramen decreased by about 3.8%and the area of the left intervertebral foramen increased by about 0.9%.When the sagittal end lifting manipulation was performed,the maximum stresses of the left and right articular capsule ligaments were 0.27 MPa and 1.70 MPa,respectively;the overall displacement of the facet joint was 1.63 mm;the area of the right intervertebral foramen increased by about 2.6%,and the area of the left intervertebral foramen decreased by about 0.9%.Compared with rotation manipulation,sagittal end lifting manipulation had fewer changes in the displacement of facet joint,joint capsule stress and intervertebral foramen area,so it was safer to operate.(3)In conclusion,compared with cervical rotation manipulation,sagittal end lifting manipulation has fewer changes in facet joint displacement,intervertebral disc stress/deformation degree,joint capsule stress,and foraminal area.In clinical practice,more appropriate manipulation should be selected based on biomechanical results after an accurate assessment of patients'conditions.

Objective:To analyze the relationship between dietary composition of residents in endemic fluorosis areas and skeletal fluorosis.Methods:A case-control study was used to analyze the difference of dietary composition between patients with skeletal fluorosis (case group) and residents without skeletal fluorosis (control group). In August 2019, taking the drinking water-borne endemic fluorosis area in Wenshui County, Lvliang City, Shanxi Province as the survey site, a cluster sampling method was adopted to select local residents aged over 18 years old, and a questionnaire survey was conducted by face-to-face interview. The survey contents included gender, age and consumption frequency of various foods. Binary logistic regression was used to analyze the relationship between food consumption frequency and skeletal fluorosis. The diagnosis of skeletal fluorosis was made by using portable digital radiography (DR) to take X-ray films of forearm and lower leg, combining with clinical signs, and according to the Diagnostic Standard for Endemic Skeletal Fluorosis (WS/T 192-2008) to determine.Results:A total of 1 061 subjects were included in this study, including 376 in the case group and 685 in the control group. The age composition of patients in the case group (≤60, > 60 years old: 162, 214 cases) was significantly different from that in the control group (≤60, > 60 years old: 423, 261 cases, χ 2 = 34.52, P < 0.001). There was no statistically significant difference in gender ratio (χ 2 = 1.37, P = 0.251). The proportion of patients in the case group who ate meat and eggs > 1 time/week was lower than that in the control group (χ 2 = 8.06, 5.46, P < 0.05), the proportion of patients who ate milk > 1 time/week was higher than that in the control group (χ 2 = 4.01, P = 0.046), and the proportion of patients who ate seafood ≥1 time/week was lower than that in the control group (χ 2 = 4.16, P = 0.046). The results of binary logistic regression analysis showed that after adjusting for age, sex, and urinary fluoride, the frequency of eating meat, eggs or milk > 1 time/week and the frequency of eating seafood ≥1 time/week were not related to the risk of skeletal fluorosis ( P > 0.05); however, in the group ≤60 years old, the frequency of eating eggs > 1 time/week was associated with the risk of skeletal fluorosis [odds ratio ( OR) = 0.59, 95% confidence interval ( CI): 0.39, 0.88]. Conclusions:The consumption frequency of meat, milk, eggs and seafood is significantly different between the skeletal fluorosis patients and the control people. In the population ≤60 years old, consumption frequency of eggs > 1 time/week may reduce the risk of skeletal fluorosis.

Objective:Through determination of urinary arsenic metabolites in high water arsenic exposed areas of Jilin and Shanxi provinces, to explore the mode and possible influencing factors of arsenic metabolism in different populations.Methods:From October 2018 to August 2019, a cluster sampling was carried out in villages (arsenic in drinking water ≥0.05 mg/L) of some townships (towns) in Lyuliang City, Shanxi Province and Baicheng City, Jilin Province for epidemiological investigation and general health examination. The residents over 35 years old drinking water from local centralized water supply and small well water sources were selected as arsenic exposure group, and people (nearby low-arsenic water source areas) with the same diet and living habits and similar economic conditions were selected as control group. Urine samples were collected. Liquid chromatography-atomic fluorescence spectrometry(LC-AFS) technology was used to separate and detect 4 species of arsenic compounds, including trivalent inorganic arsenic (iAs Ⅲ), pentavalent inorganic arsenic (iAs Ⅴ), methylated arsine (MMA), and dimethylated arsine (DMA). Total arsenic (tAs), inorganic arsenic percentage (iAs%), MMA percentage (MMA%), DMA percentage (DMA%), primary methylation index (PMI) and the secondary methylation index (SMI) were calculated. The influencing factors of arsenic metabolism were analyzed by multiple linear regression. Results:A total of 1 415 villagers were investigated, including 1 256 in arsenic exposure group and 159 in control group. Compared with the control group, there were no significant differences in age, gender ratio and occupation distribution between arsenic exposure group and control group ( P > 0.05), but there were significant differences in smoking, drinking, body mass index (BMI) and education level distribution ( P < 0.05). The median of urinary tAs, iAs%, MMA%, DMA%, PMI and SMI in control group and arsenic exposure group were 12.86 μg/L, 15.03, 5.23, 76.35, 84.97, 93.68 and 69.68 μg/L, 10.24, 8.37, 79.31, 89.76, 90.65, respectively, the levels of urinary tAs, DMA% and PMI in arsenic exposed group were higher than those in control group, while iAs% and SMI were lower than those in control group, the differences were statistically significant ( U=- 13.87, - 4.30, - 6.64, - 6.64, - 1.99, P < 0.05). After analysis of the factors influencing urinary arsenic metabolism in the population, we found that age and BMI had an impact on iAs% ( β=- 0.08, - 0.08, P < 0.05); gender, drinking, BMI and education level were influencing factors of MMA% ( β =- 0.11, - 0.09, - 0.07, 0.08, P < 0.05); DMA% was mainly affected by age, gender, BMI and education level ( β = 0.06, 0.09, 0.10, - 0.09, P < 0.05); PMI was mainly affected by age and BMI ( β = 0.08, 0.08, P < 0.05); while SMI was affected by gender, drinking, BMI and education level ( β=0.09, 0.08, 0.08, - 0.09, P < 0.05). Conclusions:The urinary arsenic metabolism models of different arsenic exposed groups are different. Age, gender, smoking, drinking, BMI and education level may be influencing factors of different arsenic metabolism models.

Objective:To investigate the association of single nucleotide polymorphism at the estrogen receptor 1(ESR1) gene rs1801132 with the risk of brick-tea type skeletal fluorosis.Methods:The typical brick-tea type fluorosis areas in Qinghai, Xinjiang, and Inner Mongolia were selected as the survey sites for a cross-sectional study. An epidemiological questionnaire was conducted by the staffs on the sites for participants older than 16 years, and physical examination and X-ray diagnosis were performed. Brick tea, blood, and urine samples were collected at the same time. The diagnosis of skeletal fluorosis through X-ray was based on the "Diagnostic Criteria for Endemic Skeletal Fluorosis" (WS/T 192-2008); The determination of tea's fluoride and urinary fluoride was performed by fluoride ion-selective electrode method; gene sequencing analysis of rs1801132 locus of ESR1 gene was done by Sequenom MassARRAY flight mass spectrometry system.Results:A total of 994 patients were included in this study. The total prevalence of skeletal fluorosis was 23.9% (238/994). The prevalence of skeletal fluorosis in Tibetans(39.9%, 123/308) was higher than those of Mongolian and Han nationality [22.2% (58/261), 13.4% (57/425), χ 2=20.435, 67.811, P < 0.05]. Based on binary logistic analysis, the daily tea fluoride intake ≤ 3.5 mg, urinary fluoride content ≤1.6 mg/L, and age ≤45 years were used as the reference groups, and then, when the daily tea fluoride intake > 7.0 mg ( OR=2.865, 95% CI: 1.923-4.268), urinary fluoride content > 1.6-3.2 mg/L ( OR=2.368, 95% CI: 1.686-3.326) and > 3.2 mg/L ( OR=3.559, 95% CI: 2.401-5.276), the age > 45-65 years old ( OR=2.361, 95% CI: 1.603-3.477) and > 65 years old ( OR=4.556, 95% CI: 2.845-7.296), the risk of fluorosis was higher than that of the reference group, respectively. When the daily tea fluoride intake was > 3.5-7.0 mg and the level of urinary fluoride was > 1.6-3.2 mg/L, G allele had a protective effect on skeletal fluorosis in Mongolian population (adjusted OR=0.207, 95% CI: 0.044-0.974); when the daily tea fluoride intake was > 3.5-7.0 mg, gender was male group, G allele had a protective effect on skeletal fluorosis in Han population (adjusted OR=0.315, 95% CI: 0.112-0.887). Conclusion:The single nucleotide polymorphism of the rs1801132 locus at the ESR1 gene may be associated with the risk of susceptibility to brick-tea type skeletal fluorosis in Mongolian and Han nationality.

Objective:To study and establish a high-throughput and rapid detection method for fluorine ion content in water based on 96-well plate and microplate reader, and to evaluate its performance.Methods:According to the principle of blue ternary complex formed by reaction of fluorine ion with fluorine reagent and lanthanum nitrate, the absorbance of the complex at the wavelength of 650 nm was proportional to the concentration of fluoride ion, and the content of fluoride ion in water samples was quantitatively determined by microplate reader. The accuracy, precision and detection limit of the method were evaluated by the standard recovery rate method and the reference material method.Results:The linear correlation coefficient of the standard curve of this method was > 0.999; the standard recovery rates of different concentrations were 99.00% - 103.33% in the accuracy experiment of this method, and the average recovery rate was 101.08%. In the standard material method, the measured values are all within the calibration range of the standard solution. The relative standard deviation of water samples with different concentrations was ≤10% in the precision experiment and the detection limit was 0.06 mg/L.Conclusion:The method has good accuracy and precision, low detection limit and small sample consumption, and this method can greatly improve the detection efficiency.

Objective To investigate the effects of arsenic trioxide (As2O3) on oxidative stress and apoptosis of neuroblastoma cells (SH-SY5Y) and the protective effect of folic acid (FA).Methods SH-SY5Y cells were cultured in vitro,and were divided into six groups:control group,low arsenic group (2.5 μmol/L As2O3),medium arsenic group (5.0 μmol/L As2O3),high arsenic group (10.0 μmol/L As2O3),FA intervention group (10.0 μmol/L As2O3,0.3 mmol/L FA),and FA control group (0.3 mmol/L FA).Each group of cells was cultured for 24 h or 5 h.Cell chromatin agglutination was observed by fluorescence staining.The ultrastructure of cells was observed by transmission electron microscope.The changes of oxidative stress related indicators of glutathione (GSH),malondialdehyde (MDA),superoxide dismutase (SOD),and reactive oxygen species (ROS) were detected;caspase 3 activity was also detected.Results Under fluorescence microscope,as the dose of arsenic increased,the nucleus became increasingly highlighted and a small number of cells in the medium and high arsenic groups showed chromatin agglutination,and FA intervention reduced chromatin agglutination.Under transmission electron microscope,the mitochondria of low and medium arsenic groups were slightly swollen and the endoplasmic reticulum was expanded;while the mitochondria of high arsenic group were significantly swollen and the nuclear membrane was ruptured,and the apoptotic bodies were observed.Mitochondria were slightly swollen after FA intervention.There were statistically significant differences in GSH content,SOD activity,ROS level and caspase 3 activity between groups (F =14.905,6.120,12.714,36.657,P ＜ 0.05).GSH content and SOD activity in high arsenic group [0.104 ± 0.074,(12.673 ± 5.106) U/mg prot] were lower than those in control group [1.000 ± 0.000,(34.699 ±3.998) U/mg prot,P ＜ 0.05].GSH content in FA intervention group (0.411 ± 0.344) was higher than that in high arsenic group (P ＜ 0.05).The ROS level and caspase 3 activity in high arsenic group were higher than those in control group (P ＜ 0.05),and the ROS level and caspase 3 activity in FA intervention group were lower than those in high arsenic group (P ＜ 0.05).There was no significant difference in MDA content between groups (F =8.207,P ＜ 0.05).Conclusions Arsenic exposure can inhibit the activity of antioxidants,cause oxidative stress injury,and increase the activity of caspase 3,leading to cell apoptosis.FA plays an antagonistic role in arsenicinduced oxidative damage and apoptosis of nerve cells.

Objective To study the effect of fluoride exposure on bone growth in zebrafish.Methods The zebrafish larvaes at 3 days post fertilization (3 dpf) were exposed to the conventional fish water and 25,50,100 mg/L of NaF for 5 days until the skeletal bone was formed (8 dpf) and the temperature was kept at 28 ℃.The fluoride content of zebrafish embryos was detected by F-ion selective electrode.The fluoride exposure model was re-established as the control group (0.0 mg/L),the low doses group (0.5,1.0,4.0 mg/L) and the high doses group (50.0,100.0 mg/L).The survival rates of the zebrafish embryos were calculated and the morphology of zebrafish embryos was observed under 40 times microscope.The zebrafish skeleton was stained with alizarin red.The staining areas and the integrated optical density (IOD) of the bone staining were quantitatively analyzed by digital microscope to analyze the sclerotic and osteoporosis of the skull.Results The fluoride contents of the control group and 25,50,100 mg/L NaF groups were (0.32 ± 0.01),(0.63 ± 0.01),(0.86 ± 0.02) and (1.21 ± 0.01) μg/150 embryos.Compared with the control group,the fluoride contents of zebrafish embryos in fluoride exposed groups were increased (P ＜ 0.05),and the dose-response relationship was obvious.The survival rates of zebrafish embryos in control group and fluoride exposed groups were 96.67％,96.67％,96.67％,98.33％,98.33％ and 98.33％.There was no significant difference among different groups (x2 =7.309,P ＞ 0.05);under a 40 times microscope,there were no obvious deformities of the spin in different groups;the areas of the alizarin red staining of the skull were 84 380.51 ± 11 711.41 in the control group,92 592.16 ± 7 143.81,92 164.85 ± 10 136.18 and 95 112.26 ± 13 721.91 in the low doses exposure groups (0.5,1.0,4.0 mg/L NaF),67 778.92 ± 8 597.11 and 64 272.93 ± 9 302.57 in the high doses groups (50.0,100.0 mg/L NaF).The areas of the alizarin red staining of the skull in the low doses exposure groups were significantly higher than that of the control group (P ＜ 0.05),while the high doses exposure groups were lower (P ＜ 0.05);the IOD of the alizarin red staining of the skull was 25 094.13 ± 6 571.86 in the control group,29 754.95 ± 3 836.45,28 747.36 ± 4 677.86 and 30 776.49 ± 5 589.63 in the low doses exposure groups (0.5,1.0,4.0 mg/L NaF),19 263.10 ± 4 754.72 and 18 202.58 ± 4 897.15 in the high doses groups (50.0,100.0 mg/L NaF).The IOD of the alizarin red staining of the skull in the low doses exposure groups was significandy higher than that of the control group (P ＜ 0.05),while the high doses exposure groups was lower (P ＜ 0.05).Conclusion Low doses of fluoride exposure may cause bone sclerosis in zebrafish embryos,while the high dose of fluoride exposure may cause osteoporosis.

Objective To investigate the relationship between single nucleotide polymorphism(SNP)of the peroxisome proliferator activated receptor γ (PPARγ) gene Rs1801282 and brick-tea type fluorosis. Methods From 2012 to 2013, this cross-sectional study was performed in 16 endemic fluorosis areas of brick-tea type in Inner Mongolia Autonomous Region,Qinghai and Xinjiang Uygur Autonomous Region of China,to select adults>18 years old as subjects, who were diagnosed as skeletal fluorosis by X-ray. All of the subjects filled in demography survey questionnaire; the survey contents included general characteristic s, and average daily brick tea intake. Drinking tea samples and urine samples of each subject were collected, and fluoride content of urine and brick-tea was determined via the ion selective electrode method (WS/T 89-2006). X-ray scintigraphy was used to diagnose skeletal fluorosis, according to the "Diagnostic Criteria of Endemic Skeletal Fluorosis" (WS/T 192-2007); the subjects were divided into skeletal fluorosis group (case group) and non-skeletal fluorosis group (control group). To collect venous blood 5 ml, whole blood DNA was extracted, and polymorphism at Rs1801282 of PPARγ was detected by MassARRAY time-of-flight mass spectrometry, to calculate odds ratio (OR) and 95% confidence interval (CI). Results There were 1 414 people included in this study,including 347 in case group and 1 067 in control group. By the Hardy-Weinberg balance test, the PPARγ gene Rs1801282 genotype was representative in case group, control group and each nationality (P > 0.05). The difference of PPARγ gene Rs1801282 genotype in case group and control group was not statistically significant (OR was 0.991, 95%CI: 0.704 - 1.395, the adjusted OR was 1.026, 95%CI: 0.707-1.489).The difference of PPARγ gene Rs1801282 genotype(CC,CG+GG)in case group and control group in different nationality was not statistically significant (Tibetan: OR was 1.400, 95%CI: 0.576 - 3.404, the adjusted OR was 1.258, 95%CI: 0.474 - 3.340; Kazak: OR was 0.898, 95%CI:0.516 -1.562,the adjusted OR was 0.936,95%CI:0.532 -1.648;Mongolia: OR was 1.148,95%CI:0.508-2.594, the adjusted OR was 1.644, 95%CI: 0.683 - 3.956; Han: OR was 1.058, 95%CI: 0.451 - 2.482, the adjusted OR was 0.959, 95%CI: 0.388 - 2.371; Russian: OR was 0.000, 95%CI: 0.000 - 0.000, the adjusted OR was 0.000, 95% CI: 0.000 - 0.000) with binary Logistic regression analysis. Conclusion We have found no association between SNP of PPARγ gene Rs1801282 and skeletal fluorosis of brick-tea type fluorosis in China.

Objective To observe the effect of fluoride on fibroblast growth factor 23 (FGF23) in bone tissue of mice,and to explore the role of FGF23 in fluoride-induced bone injury.Methods Sixty-four Balb/c mice,half male and female,were divided into 4 groups based on body weight via the random number table method and 16 mice were in each group.The mice in control group,low fluoride group,middle fluoride group and high fluoride group were treated with 0,25,50,and 100 mg/L F-distilled water,respectively.After three months,the mice were put to death and the prevalence of dental fluorosis was calculated.The fluoride contents in spine were detected via the fluoride-ion selective electrode method,serum content of calcium and phosphorus were detected by micro enzyme labeled method.The levels of FGF23,parathyroid hormone (PTH) and 1,25 dihydroxy vitamin D3 [1,25 (OH)2D3] in serum were measured by enzyme-linked immunosorbent assay.The FGF23 protein expression levels in bone tissue were determined by immunohistochemistry and Western blotting.Results The rates of dental fluorosis in low,medium and high fluoride groups were 75％ (4/16),100％ (16/16) and 100％ (16/16),respectively.Compared with control group [0 (0/16)] the differences were statistically significant (P ＜ 0.05).The levels of fluoride in the fluoride group [low,medium,high fluoride groups:(1 730.86 ± 165.90),(2 400.58 ± 286.65),(3 980.88 ± 511.65) mg/kg] were higher than that of control group [(854.30 ± 89.05) mg/kg,P ＜ 0.05].There was no difference in serum calcium content among groups (F =0.05,P ＞ 0.05).The contents of phosphorus in the serum of the medium and the high fluoride groups [(2.46 ± 0.32),(2.48 ± 0.73) mmol/L] were lower than those in the control and the low fluoride groups [(2.89 ± 0.45),(3.25 ± 0.69) mmol/L,P ＜ 0.05].The serum PTH and 1,25 (OH)2D3 content increased first and then decreased.The expression of FGF23 in middle and high fluoride groups [(660.84 ± 64.18),(638.74 ± 121.23) ng/L] was up-regulated compared with that of control group [(613.53 ± 98.18) ng/L].The expression of FGF23 protein in cortical bone increased gradually with the dose of fluoride.Western blotting results showed that the content of FGF23 protein in the bone tissue of mice was significantly increased in the low fluoride group (1.58 ± 0.46) and the middle fluoride group (1.40 ± 0.41) compared with that of control group (1.00 ± 0.41),the differences were statistically significant (P ＜ 0.05).Conclusions The phosphorus,FGF23,PTH,and 1,25 (OH)2D3 levels in the serum and FGF23 protein levels in the bone tissue of fluorosis mice have changed.It may be suggested that FGF23 interacts with PTH and 1,25 (OH)2D3 to influence the level of calcium and phosphorus metabolism in the body and participate in the formation of skeletal fluorosis.

Objective To study the urinary arsenic safety guideline value of a population for evaluating the arsenic exposure level in a certain population and providing evidence for the implementation of prevention and control measures in endemic arsenicosis area.Methods According to the data from the national high-arsenic drinking water sources screening in endemic arsenicosis area of drinking water type and quality supervision and inspection for water-improving project to decrease arsenic from 2005 to 2014,census data on arsenic poisoning in endemic arsenicosis area,data on surveillance of endemic arsenicosis,10 722 people with detailed personal information,complete water arsenic exposure data and accurate urinary arsenic detection data were selected to be the research objects.The relationship between urinary arsenic and water arsenic was analyzed based on the surveillance data of 4 501 people from 2013 to 2014.The safety guidance value of urinary arsenic was determined based on the geometric mean value of urinary arsenic in people exposed to water arsenic in the range of (0.050 ± 0.005) mg/L,and verified using the data of 6 221 people from 2005 to 2012.Every time,a random sample of 2 000 people was taken as the verification sample,the sensitivity and specificity of the index for determining whether water arsenic exposure exceeded the standard were determined by area under the ROC curve (AUC),and a total of 10 sample tests was performed.Results When the water arsenic concentration was less than 0.01 mg/L,the correlation coefficient of water arsenic concentration with urinary arsenic concentration was 0.097 (P ＜ 0.01);when the water arsenic concentration was more than 0.01 mg/L and less than 0.05 mg/L,the correlation coefficient of arsenic concentration with water arsenic concentration was 0.456 (P ＜ 0.01);when the water arsenic concentration was more than 0.05 mg/L,the correlation coefficient of water arsenic concentration with urinary arsenic concentration was 0.630 (P ＜ 0.01).With increase of water arsenic concentration,the concentration of urinary arsenic increased significantly,and the difference was statistically significant (x2 =2 337.956,P ＜ 0.01).When water arsenic concentration was in the range of (0.050 ± 0.005) mg/L,the urinary arsenic geometric mean was 0.032 mg/L.AUC analysis of 10 random samples of 2 000 people showed that the geometric mean of urinary arsenic was 0.032 mg/L in the population,which can accurately distinguish whether the water arsenic level exceeded 0.05 mg/L,and the AUC value was higher than 0.94.And the sensitivity and specificity were achieved 0.898 and 0.844.Conclusions The geometric mean of urinary arsenic is 0.032 mg/L,which can be used as a safety guideline value for urinary arsenic in the population.When the geometric mean of urinary arsenic exceeds this value,the population may be exposed to high arsenic.