Journal of Exposure Science and Environmental Epidemiology (2014) 24, 135–144 & 2014 Nature America, Inc. All rights reserved 1559-0631/14

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ORIGINAL ARTICLE

Relationship between drinking water and toenail arsenic concentrations among a cohort of Nova Scotians Zhijie M. Yu1, Trevor J.B. Dummer1, Aimee Adams2, John D. Murimboh2 and Louise Parker1 Consumption of arsenic-contaminated drinking water is associated with increased cancer risk. The relationship between arsenic body burden, such as concentrations in human toenails, and arsenic in drinking water is not fully understood. We evaluated the relationship between arsenic concentrations in drinking water and toenail clippings among a cohort of Nova Scotians. A total of 960 men and women aged 35 to 69 years provided home drinking water and toenail clipping samples. Information on water source and treatment use and covariables was collected through questionnaires. Arsenic concentrations in drinking water and toenail clippings and anthropometric indices were measured. Private drilled water wells had higher arsenic concentrations compared with other dug wells and municipal drinking water sources (Po0.001). Among participants with drinking water arsenic levels Z1 mg/l, there was a significant relationship between drinking water and toenail arsenic concentrations (r ¼ 0.46, Po0.0001). Given similar levels of arsenic exposure from drinking water, obese individuals had significantly lower concentrations of arsenic in toenails compared with those with a normal weight. Private drilled water wells were an important source of arsenic exposure in the study population. Body weight modifies the relationship between drinking water arsenic exposure and toenail arsenic concentrations. Journal of Exposure Science and Environmental Epidemiology (2014) 24, 135–144; doi:10.1038/jes.2013.88; published online 25 December 2013 Keywords: arsenic; toenails; well water; body burden; body weight

INTRODUCTION Chronic arsenic toxicity is a global public health concern.1,2 The most important arsenic exposure pathway is through drinking well water contaminated with naturally occurring arsenic. Substantial evidence suggests that long-term exposure to arsenic from drinking water is associated with increased risk of developing certain cancers, including skin, lung, liver, bladder, and kidney cancers.1,3 A recent report4 shows that chronic arsenic exposure may also have dermatological, developmental, neurological, respiratory, cardiovascular, immunological, and endocrine effects. Precise measurement of arsenic exposure is a critical issue in assessing the association between arsenic exposure and disease risk. In many arsenic endemic areas where arsenic exposure is high, cancer risk has often been evaluated in relation to consumption of arsenic-contaminated drinking water in the study areas.5–7 In some studies, arsenic concentrations in toenail clippings have been used to establish the relationship between arsenic exposure and cancer risk among populations exposed to low-to-moderate levels of arsenic in drinking water.8,9 Human nail clippings are a useful biomarker of long-term environmental exposures in relation to health outcome assessments.10 Compared with fingernails, toenails may reflect a longer exposure time frame given the relatively slower growth rate.11 In North American populations, toenails are also less likely to be exposed to external contaminations than fingernails or hair. Arsenic measurements in toenails reflect long-term chronic exposure. A validation study12 has demonstrated that a single measurement may reliably represent long-term exposure to certain trace elements including arsenic.

Measured arsenic concentrations from toenail clippings are an integrated assessment of arsenic exposure from the environment during the past 7 to 12 months,11–13 which may include water, food, soil, and dust. However, arsenic in drinking water has been reported to have the strongest association with total arsenic concentrations in toenail clippings among all these exposure pathways.14 Thus far, several studies15–18 have reported the relationships between drinking water and toenail arsenic concentrations. Most of the studies15,18,19 have been conducted in arsenic-endemic areas where arsenic doses have been high, and with a small sample size. In some studies carried out in areas with low-to-moderate arsenic levels in drinking water,16,17 participants were recruited based on cancer case–control design, rather than as a population-based sample. Data on the associations between drinking water and toenail arsenic concentrations among general populations with various levels of arsenic in drinking water, ranging from low to moderate to high doses, are scarce. Some studies20–23 have shown that body mass index (BMI) might influence arsenic metabolism in terms of urinary excretion of total arsenic and percentages of two metabolites, that is, monomethylated arsenicals (%MMA) and dimethylated arsenicals (%DMA), although the findings are inconsistent. There is no study, thus far, reporting how body composition (i.e., body fat distribution) may mediate the relationship between arsenic in drinking water and toenail clippings. The primary aim of this study was to evaluate the relationship between arsenic exposure from drinking water and toenail arsenic concentrations (arsenic body burden) among a cohort of Nova

1 Population Cancer Research Program, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada and 2Department of Chemistry, Acadia University, Wolfville, Nova Scotia, Canada. Correspondence to: Dr. Trevor J.B. Dummer, Population Cancer Research Program, Department of Pediatrics, Dalhousie University, 1494 Carlton Street, PO Box 15000, Halifax, NS, B3H 4R2, Canada. Tel.: þ 1902 494 8059. Fax: þ 1902 494 2089. E-mail: [email protected] Received 7 June 2013; accepted 2 November 2013; published online 25 December 2013

Arsenic in well water and toenails Yu et al

136 Scotians recruited from the general population who were exposed to arsenic concentrations in well water ranging from very low to high. We evaluated whether individuals with different body compositions, who were exposed to similar levels of drinking water arsenic, had different levels of arsenic body burden, controlling for a large number of covariables.

MATERIALS AND METHODS Study Population The Atlantic Partnership for Tomorrow’s Health (PATH) cohort study is a part of the Canadian Partnership for Tomorrow Project (CPTP), a national study examining the role of genetic, environmental, behavioral, and lifestyle factors in the development of cancer and chronic disease. Atlantic PATH is a general population-based cohort and the only restriction on recruitment was that participants must be resident of one of the Atlantic Canada Provinces (Nova Scotia, New Brunswick, Prince Edward Island, and Newfoundland and Labrador) and aged between 35 and 69 years. The study subjects of this analysis are individuals who participated in the Atlantic PATH Nova Scotia Arsenic Sub-Study and provided both drinking water samples and toenail clippings at the Sydney and Halifax assessment centres in Nova Scotia from 2009 through 2010. Participants attending the Sydney Assessment Centre lived on Cape Breton Island, whereas participants attending the Halifax Assessment Centre lived throughout mainland Nova Scotia, although the majority were from Halifax County. The distribution of the study population is shown in Figure 1. The present analysis includes 960 participants who have had both drinking water and toenail clipping samples analysed for trace elements, including arsenic. The study protocol was approved by the Capital District Health Authority and Cape Breton District Health Authority Research Ethics Boards.

Sample and Data Collection Participants were instructed on how to collect and store water samples. Before collecting the water sample, participants were asked to run the water tap for 10 min to flush the system. To eliminate air gaps, participants were instructed to fill the sample bottles until they overflowed. Samples were stored in a refrigerator before being returned to the study team. During attendance at an assessment centre, toenail clipping samples were collected by trained staff. A set of standardized questionnaires on sociodemographic, health, and lifestyle factors was administered and physical measurements (including height, weight, and body composition) were measured by research nurses.

Figure 1.

Water samples were collected predominantly from the participants’ principle residence, although some participants provided a sample from a second home if that water supply was from a well. All 960 participants provided water samples and 923 of the water samples were taken from the main home drinking water supply. Water sources were classified as municipal-treated water, private drilled well, private dug well, and other (including natural spring, lake, river, lagoon, dugout, and other unspecified sources). Well water treatment use was either yes, no, or unknown. Very few participants reported the type of treatment system that was in use, or the age or depth of the well, and these data could not be included in the analysis.

Anthropometric Indices and Body Composition Body weight, percentage body fat, fat mass and fat-free mass were measured using the Tanita bioelectrical impedance device (Tanita BC-418, Tanita Corporation of America, Arlington Heights, IL, USA), which is a validated instrument for body composition measurement.24,25 Body height was measured by a Seca stadiometer. BMI was calculated as weight in kg divided by height in m2. We also calculated fat mass index (FMI) and fatfree mass index (FFMI) by dividing fat mass and fat-free mass in kg by height in m2, respectively.26 Waist circumference was measured by using Lufin steel tape. Overweight was defined as 25r BMI o30 kg/m2 and obesity was defined as BMI Z30 kg/m2, respectively. Abdominal obesity was defined as waist circumference Z102 cm for men or Z88 cm for women.27 The top sex-specific tertiles of FMI and FFMI were classified as high levels of FMI and FFMI, respectively.

Measurement of Arsenic in Drinking Water and Toenail Clipping Samples Determination of arsenic was obtained from water samples by adapting US EPA method 200.8. Samples were acidified to 1% (v/v) with ultrapure nitric acid (Fisher Optima grade) and analysed using a PerkinElmer Elan DRC-e inductively coupled plasma–mass spectrometer (ICP-MS) equipped with an SC4 DX autosampler (ESI). Arsenic was measured in dynamic reaction cell mode using oxygen as a reaction gas. Coefficients of variation were typically o0.12 for arsenic concentrations o1 mg/l and o0.05 for concentrations Z1 mg/l. Certified reference materials (CRMs) NIST 1643e (trace elements in drinking water) and SLRS-4 (trace elements in river water) were analysed for each batch to assess the accuracy of the method. Toenail samples were prepared according to the method of Ryabukhin.28 Samples were washed sequentially with 25 ml of acetone (HPLC grade) and sonicated for 10 min, then washed three times with 25 ml of deionized (DI) water and sonicated for 10 min each time, and finally

Distribution of study participants across Nova Scotia.

Journal of Exposure Science and Environmental Epidemiology (2014), 135 – 144

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Arsenic in well water and toenails Yu et al

137 washed with 25 ml of acetone (HPLC grade) and sonicated for 10 min, discarding the wash solution between each step. After washing, toenails were dried overnight at 60 1C on a DigiPrep MS hot block (SCP Science). The toenails were digested by adapting the method of Gault et al.29 First, 1 ml of HNO3 (Fisher Optima grade) was added to the samples and these were heated at 100 1C for 40 min on a DigiPrep MS hot block (SCP Science). After cooling, 1 ml of ultrapure H2O2 (Fisher Optima grade) was added to the samples, and they were heated again to 100 1C for 40 min. After cooling, the samples were diluted to 15 ml with DI water. The digested toenail samples were analysed using a PerkinElmer Elan DRC-e ICP-MS equipped with an SC4 DX autosampler (ESI). Arsenic was measured in dynamic reaction cell mode using oxygen as a reaction gas. Coefficients of variation were typically o0.10 for arsenic concentrations o0.3 mg/g and o0.05 for concentrations Z0.3 mg/g. CRM BCR-397 (trace elements in human hair) was used to assess the accuracy of the method for each batch. Method detection limits (MDLs) were calculated for each batch of the measurements. The average MDLs were 0.066 mg/l for water samples and 0.046 mg/g for toenail samples, respectively. Those arsenic values lower than the corresponding MDLs in each batch were replaced by a half of the average MDLs for water (6.0%) and toenail (29.6%) samples, respectively.15

Assessment of Covariables There was little ethnic diversity in the sample and ethnicity was grouped as white and non-white. For socioeconomic status, levels of educational attainment were categorized as high school or lower, college level, and university level or higher. Occupation was grouped as blue collar, white collar, unemployed home-maker, and retired, and other, including students and professionals who did not belong to the above categories. Household income was classified into three categories according to selfreported household income levels, that is, rCAD $49,999, CAD $50,000– 99,999, and ZCAD $100,000. According to the respondents’ self-reported smoking status, participants were grouped as never smoker, former smoker, and current smoker. We used the long form of the International Physical Activity Questionnaire (IPAQ) to estimate the participants’ levels of physical activity. For each participant, total metabolic equivalent (MET)-min per week were calculated according to the Guidelines for Data Processing and Analysis of the IPAQ (www.ipaq.ki.se). Levels of total physical activity were classified as low, medium, and high by using sex-specific total METmin/week tertiles.

Statistical Analysis We evaluated relationships between arsenic concentrations in water and toenails by using both the Pearson partial correlation analysis and multiple linear regression models with Robust M estimator, adjusted for age, sex, and other covariables. Differences in water and toenail arsenic concentrations across different levels of categorical variables were assessed using multiple general linear models. We utilized multiple logistical regression modelling to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) of higher levels of either water or toenail arsenic concentrations with respect to water sources, geographic locations, and body adipose measurements. We made natural log-transformations for both arsenic concentrations in water and toenail clipping samples to normalize the data. Statistical significance was defined as P-values of o0.05 (two sided). The statistical analyses were performed with SAS statistical package version 9.2 (SAS Institute, Cary, NC, USA).

RESULTS General Characteristics of Study Participants Of the water samples, 69% were taken from non-regulated private well water sources (Table 1). Half of the study participants did not use any well water treatment system, and of those who reported using a treatment system, only a small number provided details regarding the type of system. The prevalence of cigarette smoking was 7.9% and the prevalence of overweight or obesity was 67.1% among study participants. The geometric mean (95% CI) and median (interquartile range (IQR)) of tap water arsenic concentrations were 0.280 (95% CI: 0.243–0.322) mg/l and 0.256 (IQR: 0.105– 0.957) mg/l, respectively. The corresponding values for toenail clipping samples were 0.057 (95% CI: 0.054–0.060) mg/g and 0.056 (IQR: 0.023–0.088) mg/g, respectively. The highest arsenic concentrations were 478 mg/l in drinking water samples and 12.7 mg/g & 2014 Nature America, Inc.

in toenail clippings, respectively. In addition, 4.5% of all water samples and 8% of drilled well samples had an arsenic concentration greater than or equal to the Health Canada maximum acceptable concentration (MAC) of 10 mg/l. Relationships Between Water and Toenail Arsenic Concentrations The overall Pearson correlation coefficient between water and toenail arsenic concentrations was 0.26 (Po0.0001) with adjustment for age and sex. For individuals with a water arsenic concentration of Z1 mg/l, the correlation coefficient increased to 0.46 (Po0.0001). Among those with a water arsenic concentration of o1 mg/l, the correlation became null (r ¼ 0.04, P ¼ 0.27). In the linear regression analysis adjusted for age and sex, a 1% increase in water arsenic concentrations was associated with a 0.10% (95% CI: 0.08–0.12) increase in toenail arsenic (Table 2). The association remained unchanged after further adjustment for year and season of sample collection, ethnicity, socioeconomic status, smoking, physical activity, and body composition (model 3). In the stratified analyses by water arsenic levels, the association was increased substantially by B4 times (b ¼ 0.377, 95% CI: 0.229– 0.463; model 3) among individuals with a water arsenic concentration of Z1 mg/l, but became null among those with o1 mg/l. Determinants of Water and Toenail Arsenic Concentrations In the multivariable regression analysis, drilled wells had significantly higher levels of arsenic concentrations as compared with other types of wells and water sources (Table 3). For arsenic in toenail clippings, private drilled and dug wells were associated with higher toenail arsenic concentrations (Table 4). Participants who were female, obese (as defined by BMI, fat mass, and abdominal obesity), were residents of Cape Breton Island, or had higher levels of household income had significantly lower levels of toenail arsenic. We found that compared with municipal-treated water samples from Cape Breton, water samples from drilled wells across mainland Nova Scotia were associated with significantly increased likelihood of containing arsenic Z1 mg/l (Table 5). However, the significant associations between water samples from the nonregulated (private) water sources and the increased risk of high levels of toenail arsenic concentrations (Z85 percentile) were only observed among individuals living in mainland Nova Scotia and not for those in Cape Breton Island (P for interaction ¼ 0.0002). Further analyses suggested that arsenic concentrations in nonregulated water source were lower in Cape Breton than the rest of Nova Scotia (P for interaction o0.0001, Table 6). The associations between non-regulated water source and increased levels of toenail arsenic were more evident among individuals attending the Halifax assessment centre but not for those living in Cape Breton Island (P for interaction ¼ 0.0008). No significant interactions were observed between water source and household income in relation to either water arsenic Z1 mg/l or toenail arsenic Z85 percentile (data not shown). The association of high levels of water arsenic with increased likelihood of having elevated toenail arsenic concentrations was not apparent among individuals with both high levels of FMI and FFMI (Table 7). The effect of obesity on reduced arsenic retention remained using other anthropometric indices, that is, BMI and waist circumference, as a marker of body adiposity. DISCUSSION Our study is the first comprehensive analysis investigating the relationship between arsenic in drinking water and arsenic in toenails in a general population-based sample of 960 participants, taking into account a large variety of covariables. In this analysis, we found that drinking water arsenic concentrations were strongly associated with arsenic concentrations in toenail clippings. The

Journal of Exposure Science and Environmental Epidemiology (2014), 135 – 144

Arsenic in well water and toenails Yu et al

138 Table 1.

General characteristics of the study participants.a

Characteristics Age, years Age group, n (%) 35–49 years 50–69 years

Table 1.

(n ¼ 960)

c

Abdominal obesity , n (%) Weight, kg Height, cm Waist, cm Hip, cm Waist-to-hip ratio BMI, kg/m2 Fat mass index, kg/m2 Fat-free mass index, kg/m2 Water arsenic, mg/l High water arsenic (Z10 mg/l), n (%) Toenail arsenic, mg/g High toenail arsenicd (Z0.121 mg/g), n (%)

214 (22.3) 746 (77.7) 297 (30.9) 663 (69.1)

Assessment centre location, n (%) Halifax (mainland Nova Scotia participants) Sydney (Cape Breton Island participants)

482 (50.2) 478 (49.8)

Main home drinking water, n (%)

923 (96.9)

Home water source, n (%) Municipal treated water Private drilled well Private dug well Other

297 489 144 29

(31.0) (51.0) (15.0) (3.0)

Water treatment, n (%) Yes No Unknown PNA

422 471 54 7

(44.2) (49.4) (5.7) (0.7)

Year of participant recruitment, n (%) 2009 2010

89 (9.3) 871 (90.7)

Season of participant recruitment, n (%) Winter Spring Summer Autumn

570 280 77 33

Ethnicity, n (%) White Non-white

933 (97.2) 27 (2.8)

Education, n (%) Less than high school College level University level or higher

202 (21.1) 376 (39.2) 381 (39.7)

Occupation, n (%) Blue collar White collar Unemployed, home-maker, and retired Other

106 326 399 121

(11.1) (34.2) (41.9) (12.7)

Household income, n (%) rCAD $49,999 CAD $50,000–99,999 ZCAD $100,000 PNA

247 351 291 71

(25.7) (36.6) (30.3) (7.4)

Smoking status, n (%) Never Former Current PNA

471 409 76 4

(49.1) (42.6) (7.9) (0.4)

Physical activityb, n (%) Low Medium High

310 (33.2) 312 (33.4) 311 (33.3)

Obesity status, n (%) Normal weight (BMI o25 kg/m2) Overweight (25rBMIo30 kg/m2) Obesity (BMI Z30 kg/m2)

644 316 387 257

(59.4) (29.2) (8.0) (3.4)

(67.1) (32.9) (40.3) (26.8)

(n ¼ 960)

Characteristics

56.1 (8.4)

Sex, n (%) Male Female

(Continued ).

417 77.3 166.6 92.1 104.6 0.88 27.8 9.6 18.2 0.256 43 0.056 144

(43.4) (16.4) (8.8) (13.8) (10.4) (0.08) (5.4) (4.2) (2.5) (0.105, 0.957) (4.5) (0.023, 0.088) (15.0)

Abbreviation: PNA, prefer not to answer. a Data are arithmetic mean (SD), number of participants (percentage), or median (interquartile range). Percentages may not total 100 because of rounding. Number of participants may not sum to 960 for specific variables because of missing values that vary slightly among variables: main home drinking water (n ¼ 7), home water source (n ¼ 1), water treatment (n ¼ 6), education (n ¼ 1), occupation (n ¼ 8), and physical activity (n ¼ 27). b Classified according to sex-specific total MET-min/week tertiles. c Waist circumference Z102 cm for men or Z88 cm for women. d Defined as toenail arsenic Z85 percentile.

strength of the association was mainly dependent on the levels of arsenic in drinking water. Private drilled wells were associated with higher levels of arsenic compared with other water sources, including dug wells. Importantly, our study showed that body composition mediates the relationship between arsenic in drinking water and arsenic in toenails; individuals who were obese had significantly lower toenail arsenic levels for any level of arsenic in drinking water than did those with a normal weight. Thus far, several studies have reported the relationships between arsenic concentrations in drinking water and in nail clippings. In Australia, Hinwood et al.15 assessed the important predictors of arsenic concentrations in hair and toenails among 153 residents exposed to high water arsenic, high soil arsenic, high water/soil arsenic, and low arsenic. They found that arsenic concentrations in drinking water had the highest correlation coefficient with toenail arsenic concentrations among age, fish intake, water, dust, and soil arsenic concentrations, numbers of glasses of water, and years lived in the current address. In a US population-based case–control study,16 208 study subjects provided both drinking water and toenail clipping samples. The overall correlation coefficient between arsenic concentrations in drinking water and in toenail clippings was 0.46 (Po0.001). However, it was 0.65 (Po0.001) among those with drinking water arsenic concentrations of Z1 mg/l and was 0.08 (P40.05) among those with concentrations of o1 mg/l, respectively. Slotnick et al.17 analysed the relationships of arsenic from drinking water and food with the arsenic concentrations in toenails. The study subjects were 440 controls of a bladder cancer case–control study recruited from 11 counties of southeastern Michigan. They found that arsenic from drinking water at home explained most of the variations in toenail arsenic concentrations. In Asia, arsenic concentrations were significantly and positively associated with arsenic concentrations in toenails in Taiwan18 and in fingernails in India19 among the selected study participants exposed to drinking water containing high levels of inorganic arsenic. In our analyses, a significant interaction was observed between geographic region (mainland Nova Scotia compared with Cape Breton Island) and water sample source in relation to levels of arsenic concentrations in toenails. This was mainly explained by the differences in water arsenic concentrations from different types of water sources

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& 2014 Nature America, Inc.

Arsenic in well water and toenails Yu et al

139 Table 2. Associations of arsenic concentrations in water samples with those of toenail clippings in Nova Scotians.a

Table 3.

b (95% CI) All (n ¼ 960) Model 1 Model 2 Model 3 By water arsenic levels Model 1 Model 2 Model 3

Z1 mg/l (n ¼ 233)

0.010 (  0.020 to 0.040) 0.007 (  0.024 to 0.038) 0.005 (  0.026 to 0.035)

0.371 (0.287 to 0.455) 0.371 (0.288 to 0.453) 0.377 (0.292 to 0.463)

Assessment centre location Halifax (mainland Reference Nova Scotia participants) Sydney (Cape  0.283 (  0.544 to  0.021) Breton Island participants) Main home drinking water Yes  0.656 (  1.238 to  0.073) No Reference

Model 1, adjusted for age and sex. Model 2, further adjusted for year and season of interview, ethnicity, education, occupation, household income, smoking, and physical activity (MET-min/week tertile) based on model 1. Model 3, further adjusted for fat mass index and fat-free mass index tertiles based on model 2. a Changes in toenail arsenic concentrations are calculated from parameter estimate representing percentage change in the geometric mean.

between the two geographic areas (Tables 5 and 6). Our data are in line with some studies carried out in an American population reporting that water arsenic concentration is the most important determinant of the individual variations in toenail arsenic levels in both cross-sectional analyses15,17 and longitudinally repeated measures.30 Our study is the first attempt to elucidate how body composition, as indicated by BMI, abdominal obesity, and percentage body fat (FMI and FFMI), may influence arsenic in toenails given similar water arsenic exposure. The methylation of ingested inorganic arsenic is the key mechanism for arsenic metabolism, disposition, and retention.31 Ingested inorganic arsenic, via drinking water, is transformed to MMA and DMA in the liver through consecutive reductive methylation and then excreted into urine. Therefore, the relative amount of inorganic arsenic, MMA, and DMA in urine may reflect the arsenic methylation efficiency in vivo.32 It is speculated that high methylation efficiency, as indicated by the relatively decreased proportion of MMA and increased proportion of DMA in urine, might result in lowered arsenic retention.31,32 Human nails are largely constituted of keratin-rich proteins that incorporate arsenic in proportion to the exposures.10,14 Hence, toenail arsenic concentration is considered as a biomarker of both levels of arsenic exposure and retention.14,32 In this study, we found that given similar levels of arsenic in drinking water, obese individuals (defined through BMI and abdominal obesity) and those with both the highest levels of fat mass and fat-free mass indices tended to be associated with the relatively lower levels of arsenic in toenails compared with those with a normal weight. Several studies21–23 have shown that overweight and obesity, as estimated by BMI, might increase arsenic methylation efficiency in terms of urinary excretion of total arsenic, %MMA, and %DMA. This might imply lowered arsenic retention. However, the results to date have been inconsistent. The association of increased BMI with improved arsenic methylation efficiency was observed in some studies20–22 but not all.23 Three studies20–22 reported that BMI was positively associated with arsenic methylation efficiency as indicated by decreased %MMA and increased %DMA in adults. In contrast, another study23 found that urinary total arsenic excretion was significantly reduced among obese adolescents compared with their non-obese counterparts, suggesting a reduction in arsenic methylation capacity among obese adolescents. The discrepancy may be because of the relatively smaller sample size and inadequate assessment of confounders in these studies. Some studies carried out in the arsenic-endemic areas in Bangladesh33 & 2014 Nature America, Inc.

b (95% CI)

Variable

0.098 (0.076 to 0.120) 0.100 (0.078 to 0.122) 0.099 (0.077 to 0.121) o1 mg/l (n ¼ 727)

Determinants of water arsenic concentrations in Nova

Scotia.a P-value

0.0542

0.0273

Home water source Municipal-treated water Private drilled well Private dug well Other

1.424 (1.153 to 1.695)  0.197 (  0.558 to 0.164) 0.629 (  0.096 to 1.355)

o0.0001 0.2846 0.0890

Water treatment Yes No Unknown

 0.121 (  0.356 to 0.113) Reference  0.110 (  0.600 to 0.381)

0.3109

Reference

0.6611

Year of participant recruitment 2009 Reference 2010  0.360 (  0.852 to 0.132)

0.1519

Season of participant recruitment Winter Spring 0.141 Summer 0.349 Autumn  0.040

0.3293 0.1179 0.9175

Reference (  0.143 to 0.425) (  0.089 to 0.787) (  0.790 to 0.711)

a From a multivariable linear regression model with robust M estimator by using naturally log-transformed water arsenic concentrations as the dependent variable and all the variables listed in the table as the independent variables.

and Pakistan34 reported that arsenic-related skin lesions, such as melanosis and keratosis, were more prevalent among individuals with lower levels of BMI (o18.5 kg/m2) compared with their normal BMI counterparts, indicating that there might be greater arsenic retention in these individuals. Our study suggests that increasing obesity influenced arsenic retention, but by using precisely measured fat mass and fat-free mass our findings do not seem to support the assertion that separated body mass per se directly influences arsenic metabolism (Table 7). Increased levels of body mass (both fat mass and fat-free mass) result from interactions between genetic, behavioral, and dietary factors.35 There is evidence suggesting that some nutrients might influence arsenic metabolism.36,37 Interestingly, a recent study carried out in a population from New Hampshire reported that animal protein and total fat intake was inversely associated with arsenic concentrations in toenail clippings, suggesting that dietary factors leading to increased body weight might be related to decreased arsenic body burden.38 Moreover, some in vitro studies39–41 have shown that gastrointestinal microbiota might methylate inorganic arsenic after oral ingestion to methylated metabolites before systemic metabolism. This preabsorptive metabolism may have profound implications on the bioavailability, systemic distribution, and toxicity of inorganic arsenic from the oral ingestion.42 On the other hand, there is some evidence indicating that obese and non-obese people might have different compositions of gastrointestinal microorganisms43 that might further affect energy harvest from diet.44 Different dietary

Journal of Exposure Science and Environmental Epidemiology (2014), 135 – 144

Arsenic in well water and toenails Yu et al

140 Table 4.

Determinants of toenail arsenic concentrations in Nova Scotians.a

Variable

All b (95% CI) 0.085 (0.063 to 0.108)

Men P-value o0.0001

b (95% CI)

Water arsenic Age group 35–49 Years 50–69 Years

Reference  0.026 (  0.144 to 0.093)

0.6729

Reference  0.240 (  0.489 to  0.009)

Sex Male Female

Reference  0.132 (  0.238 to  0.026)

0.0268

— —

Assessment centre location Halifax Reference Sydney  0.426 (  0.537 to  0.314)

o0.0001

Main home drinking water Yes  0.063 (  0.306 to 0.180) No Reference Home water source Municipal-treated water Private drilled well Private dug well Other

0.6135

Reference

0.096 (0.055 to 0.138)

Reference  0.371 (  0.579 to 0.163)  0.162 (  0.567 to 0.243) Reference

Women P-value o0.0001 0.0585

b (95% CI) 0.087 (0.060 to 0.114) Reference 0.053 (  0.083 to 0.190)

P-value o0.0001 0.4419

— —

0.0005 0.4329

Reference

Reference  0.455 (  0.593 to  0.317)  0.024 (  0.335 to 0.288) Reference

o0.0001 0.8815

Reference

0.300 (0.177 to 0.420) 0.280 (0.127 to 0.432) 0.283 (  0.033 to 0.599)

o0.0001 0.0006 0.0793

0.354 (0.126 to 0.582) 0.505 (0.217 to 0.794) 0.547 (  0.069 to 1.164)

0.0023 0.0006 0.0817

0.253 (0.109 to 0.396) 0.198 (0.014 to 0.382) 0.188 (  0.184 to 0.560)

0.0006 0.0351 0.3211

 0.022 (  0.120 to 0.077) Reference 0.015 (  0.192 to 0.222)

0.6658

0.1452 0.8947

0.014 (  0.104 to 0.131) Reference  0.007 (  0.239 to 0.226)

0.8212

0.8852

 0.140 (  0.328 to 0.048) Reference 0.032 (  0.446 to 0.510)

Year of participant recruitment 2009 Reference 2010  0.047 (  0.249 to 0.155)

0.6483

Reference 0.243 (  0.100 to 0.586)

0.1648

Reference  0.219 (  0.475 to 0.037)

0.0934

Season of participant recruitment Winter Reference Spring  0.091 (  0.210 to 0.028) Summer  0.012 (  0.200 to 0.176) Autumn 0.123 (  0.184 to 0.429)

0.1324 0.8998 0.4327

Reference  0.206 (  0.419 to 0.008) 0.457 (  0.031 to 0.945) 0.142 (  0.456 to 0.739)

0.0593 0.0661 0.6426

Reference  0.035 (  0.181 to 0.111)  0.055 (  0.262 to 0.153) 0.027 (  0.341 to 0.394)

0.6384 0.6063 0.8870

Water treatment Yes No Unknown

Ethnicity White Non  white Education Less than high school College level University level or higher

 0.135 (  0.412 to 0.142) Reference

0.3395

Reference

 0.236 (  0.810 to 0.338) Reference

0.4196

Reference

 0.095 (  0.419 to 0.229) Reference

0.9554

0.5648

Reference

 0.032 (  0.161 to 0.097) 0.025 (  0.113 to 0.163)

0.6263 0.5438

 0.064 (  0.314 to 0.186)  0.060 (  0.317 to 0.197)

0.6165 0.6480

0.008 (  0.147 to 0.164) 0.104 (  0.065 to 0.273)

0.9169 0.2281

Reference  0.006 (  0.172 to 0.160)  0.089 (  0.280 to 0.103)

0.9445 0.3641

Reference 0.031 (  0.245 to 0.308)  0.068 (  0.388 to 0.252)

0.8255 0.6761

Reference  0.074 (  0.302 to 0.153)  0.134 (  0.389 to 0.120)

0.5206 0.3004

 0.106 (  0.266 to 0.054)

0.1931

 0.018 (  0.258 to 0.222)

0.8825

 0.198 (  0.424 to 0.028)

0.0865

Reference  0.084 (  0.207 to 0.040)

0.1846

Reference  0.137 (  0.385 to 0.110)

0.2777

Reference  0.087 (  0.231 to 0.058)

0.2394

 0.152 (  0.292 to  0.011)  0.033 (  0.161 to 0.226)

0.0345 0. 7414

 0.248 (  0.526 to 0.029)  0.228 (  0.599 to 0.142)

0.0796 0.2276

 0.139 (  0.307 to 0.028) 0.130 (  0.102 to 0.362)

0.1024 0.2729

Smoking status Never Former Current

Reference  0.049 (  0.147 to 0.049) 0.058 (  0.124 to 0.240)

0.3921 0.5315

Reference  0.016 (  0.206 to 0.174) 0.059 (  0.271 to 0.390)

0.8663 0.7251

Reference  0.071 (  0.188 to 0.046) 0.071 (  0.152 to 0.294)

0.2351 0.5304

Physical activity Low Medium High

Reference 0.049 (  0.065 to 0.162)  0.002 (  0.112 to 0.115)

0.3999 0.7890

Reference 0.064 (  0.152 to 0.280)  0.57 (  0.270 to 0.155)

0.5614 0.5964

Reference 0.031 (  0.106 to 0.167) 0.012 (  0.125 to 0.148)

0.6584 0.8652

Occupation Blue collar White collar Unemployed, home-maker, retired Other Household income rCAD $49,999 CAD $50,000– 99,999 ZCAD $100,000 PNA

Journal of Exposure Science and Environmental Epidemiology (2014), 135 – 144

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Arsenic in well water and toenails Yu et al

141 Table 4. (Continued ). Variable

All

Men

b (95% CI) Obesity statusb Normal weight (BMI o25 kg/m2) Overweight (25rBMI o30 kg/ m2) Obesity (BMI Z30 kg/m2)

Women

b (95% CI)

P-value

Reference

P-value

Reference

 0.094 (  0.205 to 0.016)

b (95% CI)

P-value

Reference

0.0944

0.030 (  0.198 to 0.259)

0.7943

 0.129 (  0.259 to 0.001)

 0.263 (  0.386 to  0.141)

o0.0001

 0.066 (  0.335 to 0.202)

0.6287

 0.335 (  0.475 to  0.195)

o0.0001

Fat mass indexb Low (T1) Medium (T2) High (T3)

Reference  0.086 (  0.207 to 0.036)  0.166 (  0.311 to  0.020)

0.1687 0.0255

Reference 0.173 (  0.065 to 0.411) 0.036 (  0.220 to 0.292)

0.1547 0.7820

Reference  0.177 (  0.321 to  0.034)  0.294 (  0.476 to  0.111)

0.0157 0.0016

Fat-free mass indexb Low (T1) Medium (T2) High (T3)

Reference  0.011 (  0.130 to 0.109)  0.106 (  0.248 to 0.036)

0.8630 0.1419

Reference 0.008 (  0.224 to 0.240)  0.152 (  0.404 to 0.099)

0.9456 0.2351

Reference  0.012 (  0.154 to 0.131)  0.061 (  0.239 to 0.117)

0.8743 0.5031

Abdominal obesityb No Yes

Reference  0.140 (  0.235 to  0.044)

0.0042

Reference 0.003 (  0.190 to 0.196)

0.9728

Reference  0.193 (  0.305 to  0.081)

0.0007

0.1611 0.4223 0.0018

Reference  0.116 (  0.559 to 0.327) 0.051 (  0.377 to 0.479)  0.010 (  0.431 to 0.412)

0.6072 0.8155 0.9639

Reference  0.048 (  0.192 to 0.096)  0.064 (  0.222 to 0.095)  0.308 (  0.467 to  0.149)

0.5094 0.4324 0.0001

0.0525

b

Waist circumference quartiles Q1 Q2  0.095 Q3  0.057 Q4  0.222

Reference (  0.228 to 0.038) (  0.195 to 0.082) (  0.361 to  0.083)

a

From a multivariable linear regression model with robust M estimator by using naturally log-transformed toenail arsenic concentrations as the dependent variable and all the variables listed in the table as the independent variables. b Bodyweight/body composition variables examined in multivariable regression models adjusting for all other variables except other body weight indicators.

Table 6. Differences in water and toenail arsenic concentrations according to water source and investigation site in participants who provided main home drinking water samples (n ¼ 923).

Table 5. Odds ratios of higher water and toenail arsenic according to water source and investigation site in participants who provided main home drinking water samples (n ¼ 923). Water source

Water arsenic Z1 mg/l Municipal-treated water Private dug well or other Private drilled well P for trend P for interaction

Water source

OR (95% Cl) Sydney assessment centre

Halifax assessment centre

1.0 (Reference)

0.31 (0.06 to 1.74)

2.99 (0.91 to 9.89)

2.45 (0.67 to 9.03)

Water arsenic, mg/l Municipal-treated water Private dug well or other Private drilled well P for water source P for investigation site P for interaction

13.10 (4.67 to 36.74) 25.07 (8.92 to 70.41) o0.0001 0.0520

Toenail arsenic Z85 percentilea Municipal-treated 1.0 (Reference) 0.77 (0.30 to 1.98) water Private dug well or 0.39 (0.10 to 1.55) 4.12 (1.54 to 11.00) other Private drilled well 0.82 (0.35 to 1.93) 5.42 (2.36 to 12.48) P for trend o0.0001 P for interaction 0.0002

Sydney assessment centre

Halifax assessment centre

0.175 (0.034)

0.066 (0.010)

0.109 (0.022)

0.155 (0.037)

0.526 (0.065)

o0.0001 0.7363

0.840 (0.113)

o0.0001

a

Toenail arsenic , mg/g Municipal-treated water Private dug well or other Private drilled well P for water source P for investigation site P for interaction

a

Adjusted for age, sex, water treatment use, year and season of interview, ethnicity, education, occupation, household income, smoking, physical activity, fat mass index, and fat-free mass index.

0.051 (0.006)

0.058 (0.007)

0.059 (0.006)

0.106 (0.012)

0.048 (0.006)

o0.0001 o0.0001

0.088 (0.012)

0.0008

a

Adjusted for age, sex, water treatment use, year and season of interview, ethnicity, education, occupation, household income, smoking, physical activity, fat mass index, and fat-free mass index.

patterns that may influence body adiposity might also have impact on changes in the compositions of gut flora.43,45 We, herein, hypothesize that the observed effect modifications of body mass on the relationships between arsenic in water samples and & 2014 Nature America, Inc.

Geometric mean (SE)

toenail clippings in our study participants might result from presystemic metabolism of ingested arsenic with different compositions of gut microbiota among people with different

Journal of Exposure Science and Environmental Epidemiology (2014), 135 – 144

Arsenic in well water and toenails Yu et al

142 Table 7. Interactions between water arsenic and body composition in relation to toenail arsenic levels among participants who provided main home drinking water samples (n ¼ 923).

FMI Water arsenic o1 mg/l High (T3) Low (T1 þ T2) Water arsenic Z1 mg/l High (T3) Low (T1 þ T2) P for trend P for interaction FFMI Water arsenic o1 mg/l High (T3) Low (T1 þ T2) Water arsenic Z1 mg/l High (T3) Low (T1 þ T2) P for trend P for interaction FMI and FFMI combined Water arsenic o1 mg/l FMI high FMI low Water arsenic Z1 mg/l FMI high FMI low P for trend P for interaction BMI Water arsenic o1 mg/l Q4 Q3 Q2 Q1 Water arsenic Z1 mg/l Q4 Q3 Q2 Q1 P for trend P for interaction Waist circumference Water arsenic o1 mg/l Q4 Q3 Q2 Q1 Water arsenic Z1 mg/l Q4 Q3 Q2 Q1 P for trend P for interaction

FFMI high low FFMI high low FFMI high low FFMI high low

Water arsenic

Toenail arsenica

Toenail arsenic Z85 percentilea

GM (SE)

GM (SE)

OR (95% CI)

0.134 (0.014) 0.110 (0.008)

0.054 (0.007) 0.062 (0.008)

1.0 (Reference) 1.12 (0.57 to 2.21)

4.121 (0.790) 4.294 (0.588) o0.0001 0.9835

0.089 (0.014) 0.094 (0.013) o0.0001 0.5585

2.35 (0.99 to 5.61) 3.33 (1.60 to 6.92) o0.0001 0.6406

0.115 (0.012) 0.118 (0.009)

0.052 (0.007) 0.063 (0.008)

1.0 (Reference) 2.08 (1.02 to 4.25)

4.174 (0.790) 4.267 (0.589) o0.0001 0.9835

0.085 (0.013) 0.096 (0.014) o0.0001 0.5468

3.43 (1.41 to 8.34) 5.41 (2.41 to 12.12) o0.0001 0.5838

0.122 0.168 0.101 0.111

0.050 0.057 0.052 0.067

1.0 1.34 0.45 1.88

(0.016) (0.033) (0.021) (0.009)

4.050 (0.983) 4.242 (1.329) 4.373 (1.322) 4.274 (0.657) o0.0001 0.7083

0.129 0.123 0.106 0.117

(0.015) (0.016) (0.013) (0.014)

(0.007) (0.009) (0.008) (0.009)

0.082 (0.014) 0.092 (0.018) 0.085 (0.016) 0.099 (0.014) o0.0001 0.9094

1.96 (0.63 4.23 (1.26 4.46 (1.46 4.75 (2.09 o0.0001 0.4260

to to to to

6.14) 14.18) 13.67) 10.80)

(0.007) (0.008) (0.009) (0.008)

1.0 1.40 1.73 1.56

(Reference) (0.64 to 3.07) (0.81 to 3.69) (0.73 to 3.32)

3.750 (0.829) 4.535 (0.907) 4.238 (0.937) 4.082 (0.978) o0.0001 0.8356

0.081 (0.013) 0.092 (0.014) 0.093 (0.015) 0.097 (0.016) o0.0001 0.8696

2.72 3.64 4.44 3.53

(1.01 to (1.55 to (1.78 to (1.38 to 0.0001 0.9922

0.132 0.105 0.128 0.109

0.050 0.062 0.057 0.067

1.0 1.98 1.26 2.42

(Reference) (0.92 to 4.24) (0.56 to 2.86) (1.12 to 5.24)

(0.016) (0.013) (0.016) (0.013)

4.425 (0.969) 3.970 (0.819) 4.047 (0.894) 4.264 (0.989) o0.0001 0.9276

0.049 0.059 0.065 0.063

(Reference) (0.46 to 3.94) (0.09 to 2.17) (0.93 to 3.83)

(0.007) (0.008) (0.008) (0.009)

0.082 (0.013) 0.100 (0.015) 0.085 (0.014) 0.092 (0.015) o0.0001 0.3746

3.75 (1.50 to 4.97 (2.09 to 3.91 (1.57 to 3.70 (1.35 to o0.0001 0.5206

7.28) 8.52) 11.09) 9.06)

9.41) 11.80) 9.70) 10.11)

Abbreviations: BMI, body mass index; FFMI, fat-free mass index; FMI, fat mass index; GM, geometric mean. a Adjusted for age, sex, water source, water treatment use, year and season of interview, ethnicity, education, occupation, household income, smoking, and physical activity. FMI and FFMI were mutually adjusted in the separated analyses.

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Arsenic in well water and toenails Yu et al

143 levels of body weight. Further investigations are merited to confirm the hypothesis. Our study participants were recruited from different geographic regions of Nova Scotia with various water sources and arsenic contamination levels, ranging from very low to very high. Hence, our findings are more likely to reflect the relationship between water and toenail arsenic concentrations in general populations. However, the proportion of dug wells in our study sample was B15%, which is higher than that of the registered wells (B5% of 115,172 wells) at the Nova Scotia Environment from 1920 through 2011.46 Hence, our study might underestimate the degree of arsenic exposure from the drilled wells. Moreover, most of our study participants were Caucasian, and this may limit the generalizability of our study results to other ethnicities. Our analyses did not include information on daily water consumption. Given the lack of water consumption data, we could not assess whether obesity modifies drinking water consumption rates and therefore potentially arsenic intake. However, this is unlikely as previous studies have shown that daily water consumption is not significantly correlated with toenail arsenic concentrations.15,16 We were unable to include information on consumption of food products, and it is known that some food such as shell fish and rice may contain high levels of (mostly) organic arsenic. However, drinking water is considered to be the most important human source of arsenic exposure.15,17 In our analyses, approximately one third of water samples were taken from municipal-treated tap water. These samples had very low arsenic concentrations compared with the arsenic concentrations in samples from private wells and other water sources (geometric means: 0.11 vs 0.43 mg/l). This might explain that approximately one third of arsenic concentrations of toenail samples were lower than the MDL and this might lead to an underestimation of the relationship between arsenic in well water and toenail clippings. In conclusion, private drilled well water is an important source of arsenic exposure in our study population. The close relationship between water and toenail arsenic concentrations is mediated, to some extent, by body mass. Given that low-to-moderate dose arsenic exposure through drinking water may lead to increased cancer risk and a large variety of adverse health outcomes, public health actions targeting individualized arsenic exposure reduction may significantly improve health status among the populations of interest. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS Production of this study has been made possible through financial support from the Canadian Partnership against Cancer and Health Canada and a grant from the Canadian Cancer Society (grant 19989). We acknowledge the support of all participants in the Atlantic PATH Project. The views expressed herein represent the views of the authors and do not necessarily represent the views of Health Canada. We thank MS Laura Nauta for producing Figure 1.

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