The Chihuahua cohort. All procedures involving human subjects were approved by institutional review boards at the University of North Carolina at Chapel Hill (UNC) and Cinvestav-IPN (Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico). Individuals participating in the present study were among 1,163 men and women recruited for the Chihuahua cohort (Mexico); all study participants provided informed consent. This cohort was established between 2008 and 2013 to study chronic diseases associated with iAs exposure in drinking water. Only adults (≥ 18 years of age) with ≥ 5 years of uninterrupted residency in the study area were recruited. Pregnant women and participants who reported kidney or urinary tract infection were excluded because these conditions could affect the urinary pattern of iAs metabolites. Individuals with a potential for occupational exposure to As were also excluded. Data on residency, occupation, drinking-water sources and consumption, smoking, use of alcohol, drugs, or medication, and medical history were gathered at the time of enrollment using a questionnaire. Specific questions were asked about previous diagnosis of diabetes and the use of antidiabetic drugs. Samples of household tap water were collected for As analysis. The participants were then transported to Universidad Autónoma de Chihuahua to undergo a medical examination. Body weight and height were recorded and used to calculate BMI. A single spot urine sample was collected in sterile plastic cups and placed immediately on ice. Aliquots of urine samples were frozen and stored at –80°C for speciation analysis of As; the rest was used for EUC isolation. A single sample of fasting venous blood was drawn, followed by a standard oral glucose tolerance test (OGTT) in which a sample of venous blood was drawn 2 hr after an oral load of 75 g glucose. All blood samples were placed on ice immediately after collection. Plasma was isolated from the fasting and 2-hr blood by centrifugation at 4°C and frozen at –80°C.
Isolation of EUC. EUC were isolated from individuals recruited between March 2011 and August 2012. A total of 466 individuals underwent medical examination during this period; 428 provided urine for EUC isolation. EUCs were isolated from freshly collected urine (100 mL/participant) by centrifugation at 4°C. The cell pellet was washed with ice-cold phosphate-buffered saline (PBS) and again centrifuged. Cells were then resuspended in PBS, counted, and checked for the presence of bacteria, yeast, and red or white blood cells using a microscope. Only EUC free of microbial contamination and with < 5% of the total cell count represented by red or white blood cells (from a total of 374 participants) were used in the present study. All cells other than bacteria, yeast, and red or white blood cells were assumed, but not confirmed, to be EUC. EUC were stored at –80°C and shipped along with the urine samples on dry ice to UNC once per month for As speciation analysis.
Diagnosis of diabetes. Glucose levels in fasting and 2-hr plasma samples were measured using a Prestige 24i Chemistry Analyzer (Tokyo Boeki). The analyzer was calibrated before analysis, and reference human sera with normal and elevated glucose levels (Serodos and Serodos PLUS; Human Diagnostics Worldwide) were used for quality control. Study participants with fasting plasma glucose (FPG) ≥ 126 mg/dL or 2-hr plasma glucose (2HPG) ≥ 200 mg/dL, or with a self-reported doctor’s diagnosis or self-reported use of antidiabetic medication were classified as diabetic.
Analyses of As in household water and urine. Concentrations of As in acid-digested water samples were determined at Cinvestav-IPN using HG-CT-AAS (Del Razo et al. 2011 (link)). Urine samples were analyzed at UNC after storage at –80°C for up to approximately 1 month, which is known to result in oxidation of MAsIII and DMAsIII (Del Razo et al. 2011 (link)). Thus, only analysis of total iAs (iAsIII + V), MAs (MAsIII + V), and DMAs (DMAsIII + V) was performed using HG-CT-AAS (Hernández-Zavala et al. 2008a (link)). A certified standard reference material (SRM), Arsenic Species in Frozen Human Urine (SRM 2669; National Institute of Standards and Technology) was used with every shipment to assure accuracy. The concentrations of As species measured by HG-CT-AAS in SRM 2669 ranged from 86.7% to 106.4% of the certified values: 90.3–106.4% for iAs, 86.7–96.4% for MAs, and 88.2–99.0% for DMAs. The limits of detection (LODs) using 200 μL urine per sample were 0.05 ng As/mL for MAs or DMAs and 0.1 ng As/mL for iAs. The creatinine concentration in urine was determined by a colorimetric assay (Cayman Chemical Company); specific gravity was measured using a digital Atago PAL refractometer (Atago USA). It should be noted that the HG-CT-AAS cannot detect organic As species commonly found in seafood (e.g., arsenobetaine), and thus accounts for As species associated mainly with iAs exposure.
Analyses of As species in EUCs. AsIII and AsV species in EUCs were analyzed at UNC using HG-CT-ICP-MS (Matoušek et al. 2013 (link)). Briefly, cell pellets were lysed in ice-cold deionized water. The trivalent species (AsIII, MAsIII, and DMAsIII) were measured in an aliquot of cell lysate directly, without pretreatment. Another aliquot was treated with 2% cysteine and analyzed for total iAs (iAsIII + V), MAs (MAsIII + V), and DMAs (DMAsIII + V). The concentrations of iAsV, MAsV, and DMAsV were determined as a difference between AsIII + V values obtained for cysteine-treated aliquots and AsIII values from untreated sample aliquots. For AsIII species concentrations below LOD, the values of LOD divided by the square root of 2 were used when calculating the corresponding AsV values. Calibration curves were generated using cysteine-treated pentavalent As standards (at least 98% pure) as previously described (Hernández-Zavala et al. 2008a (link)). The instrumental LODs for As species analyzed by HG-CT-ICP-MS ranged from 0.04 to 2.0 pg As. The analyses of As species in EUC were performed by a researcher who was unaware of the diabetes status of the individual study participant or of the As concentrations in the corresponding urine and water samples.
Statistical analysis. Continuous variables were described using means and SDs or medians and interquartile ranges (IQRs; for nonnormally distributed variables). Categorical variables were described using frequencies. For As species concentrations below LOD, the values of LOD divided by the square root of 2 were used for statistical analysis, including regression and descriptive analyses and to determine IQRs. The statistical significance of differences in characteristics of study participants with versus without diabetes was assessed using Student’s t-tests or one-way analyses of variance (ANOVA). Associations between As species in EUC and urine were estimated using linear regression models with log-transformed (log10) variables as well as with Spearman correlations. Associations of diabetes with concentrations of As species in EUC and urine were estimated using logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). To control for potential confounding, sex (as a categorical variable) and age and BMI (as continuous variables) were included a priori as covariates. We also used linear regression models adjusted for age, sex, and BMI to estimate associations of log10-transformed FPG and 2HPG concentrations with concentrations of iAs metabolites and the sum of speciated As in urine. Age, sex, and BMI were included as covariates in these models. The linearity of the associations between As species and FPG/2HPG was assessed graphically and by linear regression using log10-transformed values. Slopes were significantly nonzero for all As species except pentavalent species in EUC. Unless otherwise specified, ORs, regression coefficients, and CIs are reported for a 1-IQR increment of exposure to facilitate comparison because of the different concentration ranges of As in EUCs and urine. Analyses of urinary metabolites of iAs were conducted both with and without urinary creatinine concentration or specific gravity adjustment. All statistical analyses were performed in Epi Info 7, version 1.0.6 (Centers for Disease Control and Prevention) and graphical representations were generated using GraphPad Instat software package (GraphPad Software Inc.). Statistical significance was considered at the level of p < 0.05.
Isolation of EUC. EUC were isolated from individuals recruited between March 2011 and August 2012. A total of 466 individuals underwent medical examination during this period; 428 provided urine for EUC isolation. EUCs were isolated from freshly collected urine (100 mL/participant) by centrifugation at 4°C. The cell pellet was washed with ice-cold phosphate-buffered saline (PBS) and again centrifuged. Cells were then resuspended in PBS, counted, and checked for the presence of bacteria, yeast, and red or white blood cells using a microscope. Only EUC free of microbial contamination and with < 5% of the total cell count represented by red or white blood cells (from a total of 374 participants) were used in the present study. All cells other than bacteria, yeast, and red or white blood cells were assumed, but not confirmed, to be EUC. EUC were stored at –80°C and shipped along with the urine samples on dry ice to UNC once per month for As speciation analysis.
Diagnosis of diabetes. Glucose levels in fasting and 2-hr plasma samples were measured using a Prestige 24i Chemistry Analyzer (Tokyo Boeki). The analyzer was calibrated before analysis, and reference human sera with normal and elevated glucose levels (Serodos and Serodos PLUS; Human Diagnostics Worldwide) were used for quality control. Study participants with fasting plasma glucose (FPG) ≥ 126 mg/dL or 2-hr plasma glucose (2HPG) ≥ 200 mg/dL, or with a self-reported doctor’s diagnosis or self-reported use of antidiabetic medication were classified as diabetic.
Analyses of As in household water and urine. Concentrations of As in acid-digested water samples were determined at Cinvestav-IPN using HG-CT-AAS (Del Razo et al. 2011 (link)). Urine samples were analyzed at UNC after storage at –80°C for up to approximately 1 month, which is known to result in oxidation of MAsIII and DMAsIII (Del Razo et al. 2011 (link)). Thus, only analysis of total iAs (iAsIII + V), MAs (MAsIII + V), and DMAs (DMAsIII + V) was performed using HG-CT-AAS (Hernández-Zavala et al. 2008a (link)). A certified standard reference material (SRM), Arsenic Species in Frozen Human Urine (SRM 2669; National Institute of Standards and Technology) was used with every shipment to assure accuracy. The concentrations of As species measured by HG-CT-AAS in SRM 2669 ranged from 86.7% to 106.4% of the certified values: 90.3–106.4% for iAs, 86.7–96.4% for MAs, and 88.2–99.0% for DMAs. The limits of detection (LODs) using 200 μL urine per sample were 0.05 ng As/mL for MAs or DMAs and 0.1 ng As/mL for iAs. The creatinine concentration in urine was determined by a colorimetric assay (Cayman Chemical Company); specific gravity was measured using a digital Atago PAL refractometer (Atago USA). It should be noted that the HG-CT-AAS cannot detect organic As species commonly found in seafood (e.g., arsenobetaine), and thus accounts for As species associated mainly with iAs exposure.
Analyses of As species in EUCs. AsIII and AsV species in EUCs were analyzed at UNC using HG-CT-ICP-MS (Matoušek et al. 2013 (link)). Briefly, cell pellets were lysed in ice-cold deionized water. The trivalent species (AsIII, MAsIII, and DMAsIII) were measured in an aliquot of cell lysate directly, without pretreatment. Another aliquot was treated with 2% cysteine and analyzed for total iAs (iAsIII + V), MAs (MAsIII + V), and DMAs (DMAsIII + V). The concentrations of iAsV, MAsV, and DMAsV were determined as a difference between AsIII + V values obtained for cysteine-treated aliquots and AsIII values from untreated sample aliquots. For AsIII species concentrations below LOD, the values of LOD divided by the square root of 2 were used when calculating the corresponding AsV values. Calibration curves were generated using cysteine-treated pentavalent As standards (at least 98% pure) as previously described (Hernández-Zavala et al. 2008a (link)). The instrumental LODs for As species analyzed by HG-CT-ICP-MS ranged from 0.04 to 2.0 pg As. The analyses of As species in EUC were performed by a researcher who was unaware of the diabetes status of the individual study participant or of the As concentrations in the corresponding urine and water samples.
Statistical analysis. Continuous variables were described using means and SDs or medians and interquartile ranges (IQRs; for nonnormally distributed variables). Categorical variables were described using frequencies. For As species concentrations below LOD, the values of LOD divided by the square root of 2 were used for statistical analysis, including regression and descriptive analyses and to determine IQRs. The statistical significance of differences in characteristics of study participants with versus without diabetes was assessed using Student’s t-tests or one-way analyses of variance (ANOVA). Associations between As species in EUC and urine were estimated using linear regression models with log-transformed (log10) variables as well as with Spearman correlations. Associations of diabetes with concentrations of As species in EUC and urine were estimated using logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). To control for potential confounding, sex (as a categorical variable) and age and BMI (as continuous variables) were included a priori as covariates. We also used linear regression models adjusted for age, sex, and BMI to estimate associations of log10-transformed FPG and 2HPG concentrations with concentrations of iAs metabolites and the sum of speciated As in urine. Age, sex, and BMI were included as covariates in these models. The linearity of the associations between As species and FPG/2HPG was assessed graphically and by linear regression using log10-transformed values. Slopes were significantly nonzero for all As species except pentavalent species in EUC. Unless otherwise specified, ORs, regression coefficients, and CIs are reported for a 1-IQR increment of exposure to facilitate comparison because of the different concentration ranges of As in EUCs and urine. Analyses of urinary metabolites of iAs were conducted both with and without urinary creatinine concentration or specific gravity adjustment. All statistical analyses were performed in Epi Info 7, version 1.0.6 (Centers for Disease Control and Prevention) and graphical representations were generated using GraphPad Instat software package (GraphPad Software Inc.). Statistical significance was considered at the level of p < 0.05.
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