Evaluation of low to Moderate Arsenic Exposure, Metabolism and Skin Lesions in a Turkish Rural Population


 Background: There is no human data regarding exposure, metabolism and potential health effects of arsenic (As) contamination in drinking water in the Central Anatolian region, Turkey. Methods: Residents in the 10 villages with drinking water total As (T-As) level >50 µg/L and 10-50 µg/L were selected as an exposed group (n=420) and, <10 µg/L as a control group (n=185). Time-weighted average-As (TWA-As) intakes were calculated from T-As analysis of drinking water samples (DWS). Other metals in DWS, concentrations of T-As in urine and hair samples, urinary As species (S-As) (As (III), As (V), MMA-V and DMA-V), and some micronutrients in serum samples of residents were analyzed. Primary and secondary methylation indexes (PMI, SMI; respectively) were assessed from S-As concentrations. . Skin lesion presence was examined. Results: TWA-As intake was 75.15 ppb in exposed group. Urinary and hair T-As and urinary S-As concentrations were significantly higher in exposed group (p<0.05). PMI and SMI values are revealed that methylation capacities of the residents were efficient and there was no saturation in As metabolism. No significant increase was observed in the frequency of skin lesions (hyperpigmentation, hypopigmentation, keratosis) of exposed group (p>0.05). Only frequency of keratosis either at hand or foot was higher in individuals with hair As concentration >1 µg/g (p<0.05).Conclusion: Current study findings of chronic As exposure through drinking water of individuals living in the study area, could contribute to overall health risk assessment of regulatory agencies’ meta-analysis efforts on low-to moderate As exposure (<100 µg/L).


Introduction
Major factors leading water pollution are known to be microbial factors, chemicals and radionuclides.
Arsenic is one of the major chemical contaminants. Arsenic is a naturally occurring element which has widespread distribution worldwide. It exists in the form of inorganic arsenic species (i As), namely arsenite (As III) and arsenate (As V), in drinking water. The most signi cant environmental exposure results from contamination of drinking water in geographic regions with abundant inorganic arsenic species. The inorganic arsenic species are classi ed within group 1 (human carcinogen) in the International Agency For Research on Cancer classi cation, which is most hazardous contaminant in drinking water regarding longterm health risks (IARC 2004;IARC 2012;Vahter 2002).
It is thought that As contamination in drinking water is global threat affecting 137 million people in more than 70 countries since it is estimated that 57 million people exposes to As levels > 50 µg/L and 137 million to As levels > 5-10 µg/L (Ravenscroft August 2007). In epidemiological studies from Asian and Latin American countries such as Taiwan, Bangladesh, India and Argentina, it was established that there is an association between long-term exposure to water contaminated by high doses of inorganic arsenic species (150-1000 µg/L) and visceral organ cancers (lung, bladder, kidney, liver, colon and prostate cancers), skin lesions (hypo-or hyper-pigmentation and hyperkeratosis) and development of skin cancer other than melanoma. In addition, a dose-response relationship was shown in some of these studies (Ferreccio et al. 2013;IARC 2004;NRC 2001). It can be also suggested that long-term exposure to high doses of inorganic arsenic species has negative in uences on diseases other than cancer including vascular disorders, neuropathy, chronic cough, diabetes mellitus, development and immune system (Naujokas et al. 2013;NRC 2001). Abnormal skin characteristics are known to be early nding seen in arsenic toxicity. Unlike visceral organ cancers related to arsenic, premalignant skin lesion generally presents within a period ranging from 6 months to 10 years after exposure, comprising most important ndings in arsenic toxicity. However, arsenicinduced skin lesions occur in only 15-20% of population (Centeno et al. 2002;Tseng et al. 1968).
In humans, oral route is the most important way of As intake, while exposure via dermal route is minimal. After being ingested, inorganic As is metabolized to methylarsonic acid (MMA) and dimethylarsinic acid (DMA) via methylation. The methylation occurs via alternating reduction of As (V) to As (III) and addition of a methyl group. Because As (III) is more toxic than As (V), this initial step in the biotransformation of arsenate may be regarded as a bioactivation. However, majority of As (III) formed is distributed to the tissues where it is methylated to MMA and DMA (Vahter 2002). As MMA and DMA are readily excreted in urine, evaluation for e ciency of arsenic methylation is mainly based on the relative amounts of the different metabolites in the urine (Vahter 1999). Irrespective of the type and extent of exposure, the average relative distribution of arsenic metabolites in the urine of various population groups seems to be fairly constant, i.e. 10-30% inorganic As, 10-20% MMA, and 60-80% DMA (Vahter 2002). Thus, speciation of As in biological samples from people chronically exposed to As through drinking contaminated water would help elucidating to what extent the methylated metabolites contribute to the observed toxicity (Vega et al. 2001).
There is an individual variation regarding arsenic metabolites in urine, which is interpreted as arsenic methylation activity. Urinary MMA/iAs and DMA/MMA ratios can be used as a marker for activity of primary and secondary methylation. In a few study in humans, it was proposed that there is an association between primary and secondary methylation rates and higher cancer risk (e.g. skin, bladder) and formation of skin lesions (Chen et al. 2003;Steinmaus et al. 2006;Valenzuela et al. 2005;Wei et al. 2017;Yu et al. 2000;Zhang et al. 2014). It has been established that genetic polymorphism is major factor affecting the metabolism of inorganic arsenic species while it is also affected from level of exposure, age and gender by 30% (Lindberg et al. 2007;Nermell et al. 2008).
In several studies, it has been demonstrated that arsenic, a major carcinogenic contaminant, is a natural source for pollution of drinking water and tap water as well as geothermal water in Western and Central Anatolian regions in Turkey (Atabey and Unal 2008;Bundschuh et al. 2013;Dogan and Dogan 2007;Gunduz et al. 2010;Simsek 2013;Yılmaz and Yılmaz 2006). In most recent geological studies from Central Anatolian region, it was found that levels of arsenic varied from 11 to 500 µg/L in drinking water at residential areas of Nevşehir Province (Atabey 2009;Atabey 2013;Atabey and Unal 2008).
In Turkey, the preliminary data regarding adverse health impact in humans due to contamination of ground water with arsenic came from Emet district of Kütahya Province. In this region, the ndings for effects contamination with iAs species on humans were limited and based on ecological data in general (Col et al. 1999;Dağıstanlı 1995;Dogan et al. 2005;Yildiz et al. 2008). In a study on mortality statistics from Simav district of Kütahya Province, it was found that lung, prostate, colon and gastric cancer rates were higher among individuals living in villages with high arsenic levels (> 10 µg/L) when compared to those living in villages with low arsenic levels (< 10 µg/L) in drinking water (Gunduz et al. 2014).
However, there is no data regarding short-and long-term arsenic exposure, arsenic metabolism and potential effects on health resulting from arsenic contamination in drinking water in Nevşehir Province. The main objectives of the present study are: (1) to conduct a study in the population living in several villages of Nevşehir Province, Turkey having different arsenic levels in drinking water (> 50µg/L, 10-50 µg/L and < 10µg/L), (2) to determine recent and long-term iAs exposure by measuring total As levels in urine and hair, (3) to determine As metabolism by measuring As species (As (III), As (V), MMA-V and DMA-V) in urine samples and to evaluate methylation capacity as primary methylation index-PMI; and secondary methylation index; SMI, (4) to determine barium (Ba), cadmium (Cd) chromium (Cr), nickel (Ni), antimony (Sb), selenium (Se) and zinc (Zn) and vanadium (V) concentrations other than total As concentrations in water samples used (drinking and cooking) by villagers, (5) to determine some micronutrient levels (serum folate and vitamin B12) of subjects exposed to iAs, (6) to determine the prevalence of skin lesions.

Study setting
The study was conducted in a region at Nevşehir Province of Turkey. The Nevşehir Province has 8 districts and 153 villages and a total population of 298,389 based on 2018 data retrieved in 19.09.2019 (https://biruni.tuik.gov.tr). Nevşehir Province was build over Western hillside of a wide plateau formed accumulation of ash and lava from Mount Erciyes, Mount Hasan and Mount Melendiz localized within southeastern volcanic region of middle Anatolia. The streambed of river Kızılırmak divides land of Nevşehir Province into 2 regions. There are mostly volcanic rock areas at south to Kızılırmak and those surrounding Nevşehir. In this region, volcanic history and natural hydrogeological conditions can lead elevated arsenic concentrations (up to 500 µg As/L) in groundwater (Atabey 2013).
To select study area, pilot analysis was conducted by measurement of total arsenic levels (µg/L) in drinking water samples from tap water and village fountain water in 19 villages and 2 municipality areas within Nevşehir Province. Based on pilot analysis, overall 10 villages were selected: Dadaği, Küçükayhan, Gülpınar and Emmiler villages with total As level > 50 µg/L and Kızılkaya and Yassıca villages with total As level > 10-50 µg/L as exposed group and Eskiyaylacık, Civelek, Alkan and Altıpınar villages with total As level < 10 µg/L as control group (Fig. 1). Figure 1 shows geographic localizations of study area in geological map of Nevşehir Province. There is no industrial activity in residential areas included to the study or nearby these areas.

Study population and biological sampling
Informed consent was obtained from all individual participants included in the study.
This study was performed in line with the principles of the Declaration of Helsinki. The study was conducted between March, 2011 and April, 2012. The individuals who were residing in the study area for at least one year were included to the study. The individuals previously resided in another region of Nevşehir Province known to have high mesothelioma incidence were excluded. The individuals with cancer or hepatitis were also excluded. All participants gave written informed consent. All participants were asked to complete a survey including items on age, gender, education level, smoking, alcohol consumption (yes/no), nutritional habits, general health status, medication and supplement intake, water sources used for cooking and drinking, residential duration in the area, occupation and degree of relationship. Diabetes mellitus and hypertension were de ned as self-reported use of insulin/oral hypoglycemic agents or antihypertensive medication, respectively. In the selected study areas, tap water, village fountain water and well water are being used for drinking and cooking. The individuals who are smokers for at least one year were considered as current smokers while those quit smoking for more than one year were considered as ex-smokers (Burgaz et al. 2011). In the study region, no pesticide containing arsenic was used within prior 10 years. The subjects were asked not to consume seafood within a week prior to urine sample collection. Thus, the interference between organic As compounds with seafood origin and total As concentrations was minimized. Urine, blood and hair samples were simultaneously drawn from individuals exposed to arsenic (n = 420) and controls (n = 185) early at morning. Skin examination was performed following biological sampling. In each village included to the study, tap water, village fountain water as well as private well water samples (100 mL) were collected in polypropylene containers previously washed with a mixture of nitric acid-water (1:9,v/v) at time of biological sampling.
Total arsenic (AsIII + AsV) concentrations were measured using hydride generation-atomic absorption spectroscopy (HG-AAS) (Unicam 939) (Dedina and Tsalev 1995). All water samples were prepared in three parallel solutions and measured at least three times using HG-AAS. Results were expressed as µg/L ±standard deviation. Limit of detection (LOD) and limit of quanti cation (LOQ) for arsenic determination by HG-AAS was 0.3 µg/L and 1 µg/L, respectively. The accuracy for total arsenic in water samples was tested using Standard Reference Material TMDA61 (National Research Institute, Canada). Observed values (31.8 ± 2.2 µg/L As) were in good agreement with the certi ed values (33.6 ± 5.2 µg/L As) (p > 0.05).

Derivation of time-weighted average (TWA) As intake
Given the different sources of As exposure including tap water, village fountain water and private well water samples in the study areas, we also calculated a time-weighted (TWA) As intake as a function of arsenic concentrations in water sources and of drinking durations of water (TWA-As in µg/L= ∑C i xT i / ∑T i where C i and T i denote water As concentration and drinking duration of i th water sources) ). The duration of water use for each individual is based on data retrieved from questionnaires.
2.3.3 Urinary collection and determination of total arsenic and arsenic species (As III, As V, MMA, DMA) In order to prevent interference from organic arsenicals, individuals were asked to avoid consuming sea food for one week prior to collecting the urine samples. The early morning spot urine samples (50-100 mL) were collected in polypropylene containers previously washed with a mixture of nitric acid-water (1:9, v/v). As soon as the samples were collected, speci c gravity (SG) values were measured using a refractometer and recorded. It has been suggested that urinary creatinine levels are affected by body mass index, age, gender and seasonal factors. In addition, it is also suggested that there is a signi cant correlation between urinary As concentrations and creatinine levels, proposing that one could not de nitely show true elevation in As exposure . Thus, in our study, concentrations of As compounds in urine samples were adjusted according to speci c gravity values.
The speci c gravity-adjusted urinary As concentration was calculated using the formula: Urine As concentration x (1.012-1) / (measured SG-1) (Cone et al. 2009;Nermell et al. 2008). The samples were stored at − 20°C until assays. Arsenic speciation analysis was performed within 6 months of the sample collection.
Total arsenic levels in urine samples were measured with hydride generation-atomic uorescence spectrometry (HG-AFS, PS Analytical Excalibur Millennium System, PS Analytical, UK). The method for total arsenic determination was validated using standard reference material "SRM NIST 2669 arsenic species in frozen human urine" (Level I, Low-Level and Level II, High-Level). The SRM's were digested and analyzed for total arsenic by using HG-AFS. The certi cate values for low and high level SRM were 22.2 ± 4.8 µg/l and 50.7 ± 6.3 µg/l, respectively, while the values found were 22.3 ± 0.4 µg/l and 50.7 ± 0.6 µg/l (n = 5). The results obtained were statistically not different from the certi ed values at 95% con dence level (p > 0.05).
The quanti cation of arsenic speciation [inorganic Arsenic (iAs) including iAs (III) and iAs (V), MMA-V and DMA-V] was carried out by high performance liquid chromatography-hydride generation-atomic uorescence spectrometry (HPLC-HG-AFS) (Gomez-Rubio et al. 2012). In our study, the amounts of MMA (III) and DMA (III) metabolites, excreted by urine and considered as toxic, were not studied since detection of trivalent arsenic species depends on storage conditions of samples and urinary concentration. These species are highly susceptible; thus assays should have to be performed immediately after urine sampling. We were unable to perform such a rapid assay in our study design. Due to such challenges, trivalent arsenic species are not used as a eligible marker for As methylation (Del Razo et al. 2011).
The chromatographic separation of arsenic species was performed using a Hewlett Packard HPLC system, 1050 model (Agilent Technologies, USA) equipped with an isocratic pump, a 100 µL injection loop, a Hamilton PRP-X100 anion-exchange column (250 mm x 4.1 mm, 10 µm) and a Hewlett Packard Hypersil guard column (20 mm x 2.1 mm, 5 µm). The signal output of the atomic uorescence spectrometer (AFS) system was recorded with a computer having a chromatographic software (Agilent ChemStation for LC systems software, Rev. B. 03.01) interfaced to the AFS instrument via an analogue-to-digital converter "Agilent Interface 35900 E". Separations were performed in an isocratic mode. The mobile phase, consisting of 8.0 mM KH 2 PO 4 /K 2 HPO 4 at pH 5.80, was used at a ow rate of 1.0 mL min − 1 and an injection volume of 100 µL. An AFS instrument (PS Analytical Ltd. Millennium Excalibur, UK) was used to detect the separated species. A boosted hollow cathode lamp (Photron Super Lamp) was used as the excitation source at 193.7 nm. Lamp current, argon and nitrogen as the carrier and drying gases, respectively, were used at the ow rates recommended by the manufacturer. The separated arsenic species were derivatized into their volatile hydrides by a post-column hydride generation with 1.5 M HCl and 1.4 % w.w − 1 NaBH 4 (prepared in 0.4 % w.w − 1 NaOH) at a ow rate of 4.0 mL min − 1 .
Results were expressed as µg/L ±standard deviation. LOD values with HPLC-AFS were found to be 0.3µg/L for As (III) and DMA, 0.7µg/L for MMA and As (V). LOQ were found as 1.0 µg/L for As(III) and DMA, 2.3 µg/L for MMA and As(V). Accuracy of HPLC − HG − AFS was assessed by analyzing the standard reference material "SRM NIST 2669 arsenic species in frozen human urine" (level II, High-Level). The certi ed values for As (III), As(V), DMA and MMA were 5.03 ± 0.3, 6.16 ± 0.49, 25.3 ± 0.7 and 7.18 ± 0.56 µg l − 1 while the results obtained were 5.12 ± 0.45, 6.64 ± 0.49, 26.5 ± 1.0 and 7.21 ± 1.04 µg l − 1 , respectively. The results obtained were statistically not different from the certi ed values at 95% con dence level (p > 0.05).

Determination of total As in hair samples
The hairs near to scalp (300-500 mg) were collected from the area behind the ears (post auricular region). The samples were kept in polyethylene cups at 4 °C until analysis. All samples were washed for 15 min with deionized water while magnetically stirred, then washed with a mixture of acetone-deionized water (1:1) (Basu et al. 2002) in order to minimize external As contamination.
For the total arsenic determination, each urine sample was digested in parallel, and two parallel solutions were prepared from each digest and measured as described by Rahman et al. (Rahman et al. 2000).
Results were expressed as µg/g ± standard deviation. LOD and LOQ values for the total arsenic determination in hair sample were 0.01 and 0.03 µg/g, respectively. The accuracy for total arsenic in hair samples was tested using Standard Reference Material NCS DC73347a Human hair, Beijing, China). Observed values (0.28 ± 0.01 µg/g As) were in good agreement with the certi ed values (0.28 ± 0.05 µg/g As) (p > 0.05).

Determination of other metals in drinking water samples
V, Cr, Ni, Zn, Se, Cd, Sb and Ba concentrations in drinking water samples collected from the selected study area were also analyzed with ICP-MS (Inductive Coupled Plasma-Mass Spectrometry-Thermo X Series) equipped with ow injection assembly. 500 µL injection volume was used in the analyses. Interference free isotopes 51 V + , 52 Cr + , 60 Ni + , 66 Zn + , 82 Se + , 111 Cd + and 121 Sb + and 137 Ba + were selected for the determination of these elements with external calibration method. LOD, LOQ (as µg/L) and % RSD values, respectively were

Analysis of serum folate and vitamin B12 levels
Serum total folate and vitamin B12 concentrations were analyzed by using enzyme linked chemiluminescent immunosorbent assay (ECLIA) kit while following the manufacturer's protocol. The manual offered the normal reference value of serum folate and vitamin B12, respectively: 3.1-17.5 ng/ml and 197-866 pg/ml. The inter-assay coe cients of variation (CVs) for folate and Vitamin B12 were 5.82 % and 2.99 %, respectively.

Skin Examination
Skin examination was performed by a dermatologist blinded to arsenic values in tap water at the area where examinations were performed. Skin examination included pigmentation type (hypopigmentationleukomelanosis, hyperpigmentation-melanosis) and presence of keratosis (yes/no) in palm/dorsum of hands, sol/dorsum of feet, trunk, back and upper and lower extremities. In skin examination, clinical conditions mimicking keratosis or melanosis were excluded (Fatmi et al. 2009). In addition, premalignant/malignant lesions (actinic keratosis, Bowen disease etc.) were also assessed. The results of dermatological examination were summarized as hyperpigmentation (yes/no), hypopigmentation (yes/no), keratosis at hand or foot (yes/no), keratosis at both hand and foot (yes/no) and presence of hyperpigmentation plus keratosis (yes/no).

Statistical Analysis
The normal distribution of continuous variables was assessed using Kolmogorov Smirnov test. Levene test was used for the evaluation of homogeneity of variances. The categorical variables are presented as number of cases and percentages while descriptive statistics for continuous variables are presented as mean ± SD or median (min-max), where applicable. The Student's t test was used to compare differences in mean values between groups in case of normal distribution, while Mann Whitney U test was used to compare data with skewed distribution. When the number of independent groups was more than two, the continuous variables were analyzed by Kruskal Wallis test. When the p-values from the Kruskal Wallis test statistics were statistically signi cant, Conover's multiple comparison test was used to determine the group accounting from difference. Categorical data were analyzed by Pearson's Chi-square, Fisher's exact or Likelihood Ratio test, where appropriate. Degrees of association between continuous variables were evaluated by Spearman's Rank Correlation analyses.
Multiple linear regression (MLR) analyses were performed to determine the best predictors which effect on PMI and SMI measurements. Any variable found to have p value < 0.10 in univariate analysis was accepted as a candidate for the multivariable model along with all variables of known clinical importance. Coe cients of regression (B) and 95% con dence intervals (CIs) for each independent variable were also calculated. Logarithmic transformation was used for both PMI and SMI in multiple linear regression analyses due to not normal distribution.
Self-reported data on body weight and height and drinking water consumption (as a glass of water per day) were found to be unreliable, thus, they were not included in the statistical analysis.
We excluded participants with urinary As (III), As (V), MMA and DMA concentrations below detection limit (LOD) for methylation e ciency (Steinmaus et al. 2010). Measured values of urinary As (III), As (V), MMA and DMA concentrations < LOD were replaced with half the LOD for other all statistical analysis (Del Razo et al. 2011;Gomez-Rubio et al. 2012;Gribble et al. 2013).
Data analysis was performed by using IBM SPSS Statistics version 17.0 software (IBM Corporation, Armonk, NY, USA). A p value less than 0.05 was considered statistically signi cant. Table 1 presents major characteristics of individuals living in selected 10 villages as study area based on criteria given in the Sect. 2.1. In our study population, overall 605 individuals were analyzed including 185 controls. In the study, exposed individuals and controls were compatible regarding mean age, age groups of ≤ 50 years and > 50 years, alcohol consumption status, smoking status and smoking frequency. When subgroups were assessed according to age, the number of individuals aged 51-60 years and aged 71-84 years was higher in the exposed group when compared to control data (p = 0.028 and p = 0.011, respectively). When education level was assessed, it was seen that number of individuals with no formal education was signi cantly higher in the exposed group while number of those with secondary school graduation was signi cantly higher in the control group (p < 0.001 and p = 0.044, respectively). The residential duration of individuals at study region was signi cantly longer in exposed group when compared to controls (p = 0.01).

Results
In addition, residential duration of individuals at study region was less than 30 years in 45.4% of controls and 34% of exposed group. On the other hand, the frequency of individuals residing in the study region more than 30 years was signi cantly higher in exposed group than control group (66% vs. 54.6%; p = 0.008). Diabetes Mellitus Type II was more common in the exposed group (p = 0.023) while exposed group and controls were not different regarding history of hypertension. As suggested by residential duration in Table 1, it is possible to propose that mobility of individuals living in the study region is highly restricted and they can represent the study region.

1 Measurement Of As Exposure
As the locations of villages were represented in Fig. 1. Median (min.-max.) time-weighted average As intake (TWA-As-µg/l-year) levels in drinking water were 0.50 (0.40-31.53) for control group and 75.15 (10.42-300.60) for exposed group. The TWA-As level was signi cantly higher for exposed group than that of control group (p < 0.001) which indicated the TWA-As level as appropriate for evaluation of cumulative As exposure for control and exposed groups.
Descriptive statistics regarding levels of total urine As and As species including As (III), As (V), MMA and DMA of the study population were presented in Table 2. As seen in Table 2, total As exposure was signi cantly higher (by 6-folds) in exposed group when compared to controls (p < 0.001). It was found that total urine As level and As (III), As (V), MMA and DMA levels were signi cantly higher in individuals living at areas with As concentration ranged 10-50 µg/L and > 50 in drinking water when compared to those living in areas with As concentration < 10 µg/L in drinking water and that the statistical signi cance was increased with tap water As concentration (p < 0.05, p < 0.001, respectively, data not shown).
Table 2 Descriptive statistics of total As ve As species -As(III), As(V), MMA, DMA-(µg/L) in urine samples a Unsu cient urine sampling from four and ve subjects in control and exposed group, respectively. b Asspecies analysis were not carried out in thirty-one control subjects. c Two missing total As values due to technical problems. and smoking status in the study population. Total hair As levels were signi cantly higher (by 6-folds) in the exposed group when compared to controls (p < 0.001). * Unsu cient hair sampling from ve and one control and exposed subjects,respectively. a,b,c p < 0.001 When assessed according to drinking water As categories, it was found that total hair As level was signi cantly increased by elevating drinking water As level (p < 0.001). Although total hair As level was signi cantly higher in individuals aged > 50 years compared to those aged ≤ 50 years (p < 0.001), no signi cant difference was shown in hair As levels according to gender or smoking status (yes/no, exsmoker) (p > 0.05). A signi cant, positive correlation was detected between total As level in hair samples and TWA-As level (r = 0.752; p < 0.001). When toxic cut-off level was considered as 1 µg/g (Arnold 1990) total As level in hair samples was higher than cut-off level in 8.3% and 91.7% of individuals living in the areas with drinking water As concentration of 10-50 µg/L and > 50 µg/L in drinking water, respectively (p < 0.001).
5.1.1. Measurement of other metals in drinking water samples Table 4 presents concentrations (with min-max values) of metals other than arsenic measured in drinking water samples (tap water, village fountain water and well water) obtained from study areas during biological sampling. It was found that Cr, Ni, Sb, Se and Zn concentrations were at or below the limits proposed by national and international organizations. Ni and Sb values were above limits in only two private wells of two villages with As concentration > 50 µg/L in drinking water (Dadağı and Küçükayhan).
Vanadium concentration in drinking water was lower in villages with As concentration < 10 µg/L while it was higher in villages with As concentration > 10 µg/L in drinking water. There is no national or international limit value for vanadium levels in drinking water.  Fluoride (F) concentrations in drinking water samples obtained from villages with As concentration of 10-50 µg/L and > 50 µg/L were measured by using uoride electrode, resulting the F concentrations between 0.19 and 0.46 mg/L, respectively. The measured concentrations were below the limit value (1.5 mg/L) for F proposed by National Drinking Water Guideline. No uoride measurement was performed in drinking water samples obtained from areas designated as controls.

Arsenic methylation capacity and urinary methylation indices
Tables 5 and 6 present descriptive statistics for methylation capacity and indices of 339 individuals with urine AS (III), As (V), MMA and DMA concentrations > LOD. a It is excluded participants with urinary As (III), As (V), MMA and DMA concentrations < LOD. The percentage of As was signi cantly lower and the percentage of DMA was signi cantly higher in exposed group when compared to controls (p < 0.001; Table 5). No signi cant difference was detected in percentage of MMA. As shown in Table 6, PMI and SMI values were signi cantly increased in exposed group (p = 0.016, p = 0.021, respectively) and in drinking water As categories (p < 0.001). Age, gender and smoking status had no effect on PMI value. However, age > 50 and being in former smoker group were signi cantly increased SMI value (p < 0.001 and p < 0.05, respectively; Table 6). Table 7 presents descriptive statistics for categories (low, normal or high) of serum folate (ng/mL) and

Measurement of serum folate and vitamin B12 levels
vitamin B12 in subjects according to As levels in drinking water. When mean folate levels in low, normal and high categories were assessed, no signi cant difference was found among individuals exposed to As and controls (p > 0.05). In addition, serum folate levels showed no signi cant difference according to drinking water As categories (p > 0.05; Table 7). It was found that mean serum vitamin B12 level was signi cantly lower in individuals exposed to As when compared to controls (p < 0.001). When mean B12 vitamin in low and normal categories were assessed, the frequencies of individuals with normal B12 levels were lower in control group and and those with low B12 levels were signi cantly higher in exposed group (p < 0.001; Table 7). It was observed that serum vitamin B12 levels were decreased according to drinking water As categories (p < 0.001; Table 7).

Skin lesion ndings
It was observed that frequencies(%) of hyperpigmentation, hypopigmentation, keratosis at either hand or foot, keratosis at both hand and foot and keratosis plus hyperpigmentation were similar between exposed group (6.7, 2.9, 11.2, 6.5 and 11.2, respectively) and controls(7.2, 2.2, 8.7, 4.9 and 8.7, respectively) (p > 0.05). The frequency of skin ndings observed did not differ according to As categories of drinking water (p > 0.05). When frequency of skin nding was assessed according to TWA-As levels, the median TWA-As value with no skin nding observed was 34.05µg/L while median TWA-As value where skin ndings were observed ranged from 34.05 to78.07 µg/L. No signi cant difference was detected between TWA-As values in which skin ndings were observed or not (p > 0.05).
Pathological examination was performed by a dermatologist in three individuals living in villages with As concentration > 50 µg/L (Dadağı and Emmiler) and one individual living in village with As concentration of 10-50 µg/L (Kızılkaya) in drinking water, revealing no malignant lesion. The lichenoid dermatitis was found in two individuals and hyper-keratotic skin with non-speci c morphological ndings was detected in remaining two individuals.
When frequency of skin ndings was assessed according to toxic cut-off level for hair samples, it was found that only frequency of keratosis either at hand or foot in individuals with hair As concentration > 1 µg/g (15.8%) was higher than that of As concentration ≤1 µg/g (8.7%) (p < 0.05). Table 8 presents analysis of skin ndings according to methylation indices (PMI and SMI). PMI values were found to be signi cantly lower in individuals with hyperpigmentation (p < 0.05) while SMI values were signi cantly higher in individuals with keratosis at both hand and foot (p < 0.001). It was shown that SMI value was signi cantly higher in individuals with skin ndings compared to those without any skin ndings (p = 0.003; Table 8). signi cantly affected the PMI (p = 0.007, 0.005; respectively). The other factors selected for the analysis (living in villages with As level of > 50 µg As/L in drinking water, current smoking, folic acid, vitamin B12, hyperpigmentation) were not affected the PMI levels (p > 0.05). other factors selected for the analysis (living in villages with As level of 10-50 µg/L in drinking water, current smoking, vitamin B12, diabetes, hypertension, hyperpigmentation + keratosis) were not affected the SMI levels (p > 0.05).

Discussion
The paper presents quantitative information concerning the recent and long-term As exposure, As metabolism and e ciency and skin lesions in the population living in several villages of Nevşehir Province, Turkey having different arsenic levels in drinking water (> 50µg/L, 10-50 µg/L and < 10µg/L).
The major route for As exposure is food and/or drinking water in individuals other than those with occupational As exposure. Although food is a negligible source of pollution in areas with As concentration > 50 µg/L in drinking water, it has become increasingly important to investigate effects of As taken by diet and smoking on human health while As level is decreasing in drinking water (Marchiset-Ferlay et al. 2012).
Recent epidemiological studies aid to identify health risks due to arsenic exposure. In recent years, contradictory results have been reported about cancer risk in lower doses of inorganic arsenic (< 100 µg/L) in many studies (Baastrup et al. 2008;Chen et al. 2010;Dauphine et al. 2013;Snow et al. 2004). Scarcity in validated animal models which may present carcinogenic effect induced by inorganic arsenic has impeditive effect on understanding mechanism of action of inorganic arsenic. However, lack of scienti c data, regarding dose-response curve form (sublinear, linear, supralinear) at low doses of arsenic, adversely in uences legal arrangements about permissible limits of arsenic. Thus, measurements with accurate biomarkers for both exposure and effect in human tissues have become extremely important to stratify health risks, particularly in low exposure (Abernathy et al. 1996;Bates et al. 1995;Kitchin 2001;Mead 2005;Mo et al. 2006).
Furthermore, in the recent guidance report of US Environmental Protection Agency (EPA), it is suggested to carry out meta-analysis studies which take into consideration of health risks (pulmonary disease, bladder cancer, ischemic heart diseases and skin lesions, etc.), individual arsenic exposure (preferably measured by biomarkers) associated with low to moderate arsenic exposure (< 100 µg/L) via drinking water. In addition, it was recommended that 3 or more peer-reviewed manuscripts with 3 or more exposure levels investigated, should be included to meta-analysis on dose-response relationships (NRC 2013). Accordingly, our study ndings would be contributing signi cantly to this guidance report by assessing individuals living in a study region with low-to-moderate As exposure.
In our study, As levels in drinking water sources are measured and at the same time, time-weighted As (TWA-As) levels in drinking water were calculated to determine individual As exposure of residents to eliminate to the variations in water sources and duration of use of drinking waters in the study region. It is known that TWA-As calculation is preferred approach for stratifying exposure groups in epidemiological studies which do not use biological markers for individual As exposure Chen et al. 2011).
In our study, 3 study regions, based on total As level in drinking water as < 10 µg/L (control), 10-50 µg/L and > 50 µg/L, are also con rmed by TWA-As values of individuals living in these regions. Also, it can be concluded that it would be appropriate to use TWA-As values in analyses for cumulative As exposure. Another important suggestion of the study is, the mobility of individuals living in the study region is highly restricted according to the duration of residency data and they can represent the study region.
6.1. Assessment of recent and long-term arsenic exposure Although total urinary As has been used to determine iAs exposure in many studies, it is important to distinguish iAs and its metabolites from organic forms. In particular, some seafood contains organic As in form of arsenobetaine (less toxic), which is rapidly excreted via urine after recent sh consumption; thus, misleadingly showing high iAs. The speciation methods can distinguish iAs, MMA and DMA from remaining As compounds and it is thought that urinary As metabolites are better predictors for As toxicity when compared to total As (ATSDR 2007;Huang et al. 2009). Herewith, to estimate absorbed iAs doses in the individuals living in study region, besides total urinary As measurements As species (AsIII, AsV, DMA and MMA) levels are also used which is allowing better quantitative estimation for recently absorbed As (Huang et al. 2009;WHO 2001).
In our study, total urinary As level was found to be higher than total urinary level of As species. The potential causes of this nding include lack of hydride formation by organic As species other than seafood consumed, inability to detect such species in samples where no degradation could be achieved, and partial analyte loss during ltering of urine samples before chromatographic separation in speciation studies (Chen et al. 2002;Heitland and Koster 2008). Total As and As species data are pointing out individuals living in the Nevşehir Province are still exposing to As in dose-dependent manner.
keratinocyte, broblast and melanocyte while lower doses of Ba (137 µg/L) caused inhibition of As-induced apoptosis in the co-presence of As (224.8 µg/L) (Kato et al. 2013;Yajima et al. 2012). In our study, it can be suggested that Ba concentration in drinking water (97.8-163.1 µg/L; Table 4) may contribute to efforts to understand pathogenesis of diseases caused by As in the future.
On the other hand, uoride is also an important geogenic, inorganic contaminant that may be commonly present in the environment, which can be detected in underground water. However, information about the effects of F exposure on As metabolism and further As toxicity are limited. Although detailed mechanism has not yet been understood, previous experimental data indicate synergistic, antagonist and independent effects of F and As (Mondal and Chattopadhyay 2020). However, a recent epidemiological study by (Jimenez-Cordova et al. 2019) suggested that high F exposure level (≥ 1.5 mg/L in water) is only signi cantly related to an increase in mono-methylated arsenic levels. In our case, F levels in all water samples were below WHO maximum permissible limit in drinking water; 1.5 mg/L. Thus, we have assumed that F levels might not modify As metabolism.

Skin lesions
Skin lesions are the rst symptoms of toxicity in chronic As exposure (Centeno et al. 2002;NRC 2001;Tseng et al. 1968). In several studies, it was shown that the severity and frequency of As-induced skin lesions such as hyperkeratosis at palm and sole and/or hyper-pigmentation and hypopigmentation (typically both may develop concurrently) at areas where sunlight exposure is limited, depend on As concentration in water and duration of use. In regions such as Bangladesh, India, China and Pakistan where arsenic exposure is high, skin lesion incidence varies from 2.08-20.6% (Fatmi et al. 2013;Rahman et al. 2006). In studies on individuals living at regions with arsenic contamination, it was reported that toxic effects other than cancerincluding skin lesions and other effects occur when As level is above 280-350 µg/L (NRC 2001).
On the other hand, (Karagas et al. 2015) published as systematic review including 25 studies on skin lesions (15 studies) and skin cancer (10 studies) in individuals having individual As exposure data with As levels ≤ 100 µg/L in drinking water. In this systematic review, it is reported that majority of studies on skin lesions were from Southern Asia while others from China and As-induced skin lesion development was dose-dependent. On the other hand, it was shown that risk for As-related skin lesions and skin cancer can vary based on several factors including nutritional factors (e.g. folate and selenium status, dietary diversity), lifestyle (e.g. smoking and body mass index) and genetic polymorphism of As metabolism (Karagas et al. 2015).
In individuals living in the villages at study region, TWA-As exposure ranged from 15.0 to 134.35 µg/L. In the residents with higher TWA-As exposure, incidences of hyperpigmentation, hypopigmentation, keratosis at hand or foot, keratosis at hand and foot and keratosis plus hyperpigmentation were similar to those living in the areas without As contamination. The incidence of keratosis at hand and foot was signi cant higher in individuals with hair As level > 1 µg/L (marker of toxicity) when compared to those with As level < 1 µg/L (15.8% vs.8.7%). This may be an incidental nding; however, it will important to evaluate other risk factors modifying skin lesion formation in this risk group.
In Turkey, there is limited number of comprehensive studies on As exposure and related skin lesions in individuals living in the areas with As contamination in drinking water. In this issue, preliminary ndings were noted in individuals living in İğdeköy and Dulkadir villages at Emet district of Kütahya Province, who consumed drinking water with high As levels (Dağıstanlı 1995). Author reported that hyperkeratosis and hyperpigmentation were present in 9 of 30 individuals from İğdeköy village where As concentration was 140-1700 µg/L in drinking water. Chronic As exposure was shown using hair As levels in these individuals. However, there is no data about follow-up of these individuals after con nement of these problematic drinking water sources. (Dogan et al. 2005) found As-related palmo-plantar keratosis frequency as 17.5% and 1.8% in 97 individuals from İğdeköy and 56 individuals from Dulkadir villages at district of Kütahya Province where As concentration was 0.3-0.9 mg/L, respectively. However, there was no data regarding chronic As exposure of individuals. In a study on 303 individuals living in several villages of Kütahya Province, (Arikan et al. 2015) reported that As-related skin lesion frequency was signi cantly higher in individuals living in villages with mean drinking water As level of 10 ppb when compared to those living in villages with drinking water As level of 445 ppb. The chronic As exposure was assessed using hair As level.
Although hair As level was signi cantly higher in individuals living at areas with high As level when compared to those living at areas with low As level in drinking water, it should be noted that hair As levels in both groups were within normal range. Thus, these results were not comparable with hair As levels in the groups (exposed group and controls) in our study. Moreover, it is di cult to compare with results from other studies conducted at Emet district of Kütahya Province since there are differences in study design, sample size and study population.

Methylation e ciency
The percent arsenic metabolites in urine (iAs, MMA and DMA) show e ciency of arsenic metabolism. The metabolite ratios (MMA: iAs and DMA: MMA) are widely used markers for estimation of individual methylation capacity. Similar indices are used in the epidemiological studies from several countries (Del Razo et al. 2011;Huang et al. 2009;McCarty et al. 2007;Normandin et al. 2014;Xu et al. 2008).
For calculation of metabolite proportions, (Steinmaus et al. 2010) suggest that a little mistake in laboratory measurements can derive relatively large errors at low metabolite concentrations; thus, for more appropriate calculations for methylation e ciency, in our study we excluded participants with urinary As (III), As (V), MMA and DMA concentrations below detection limit (LOD).
In our study, urinary iAs% and DMA% were within range seen in other populations (Heck et al. 2007;Hopenhayn-Rich et al. 1996;Hsu et al. 2017;Huang et al. 2008;Lindberg et al. 2008;Yu et al. 2000). However, it was seen that MMA% values in both control and exposed groups were slightly higher (27.71% and 25.40%, respectively) (Table 5) than other studies. Similarly, (Chiou et al. 1997) and (Hsueh et al. 1997) found urine MMA levels as 18.5-31.5% independent from As dose. PMI and SMI values showed that individuals in our study had e cient methylation capacity and that there is no saturation in As metabolism.
The epidemiological studies on individuals with environmental iAs exposure showed associations between skin lesions, skin cancer, bladder cancer, lung cancer, peripheral vascular disease and hypertension incidences and increased PMI (MMA: iAs) or decreased SMI (DMA: MMMA) values Chen et al. 2003;Chung et al. 2013;Hsu et al. 2017;Hsueh et al. 1997;Huang et al. 2008;Lindberg et al. 2008;McCarty et al. 2007;Pu et al. 2007;Tseng et al. 2005). In some studies, it was shown that there was a negative correlation between iAs exposure and DMA: MMA ratio due to saturation or inhibition of iAs methylation pathway at high levels of As exposure (TWA-As: 158 µg/L) , while low DMA: MMA or high %MMA was associated with increased susceptibility to iAs exposure-related diseases including skin lesions (Chung et al. 2013;Hsu et al. 2017;Lindberg et al. 2008).
Low PMI values in individuals with hyperpigmentation and high SMI levels in individuals with keratosis at hand and foot or skin lesions in our study suggest that low-to moderate As levels in drinking water on the risk of pre-malign skin lesions (as PMI and SMI) may modi able by other factors, e.g. nutritional and lifestyle factors and genetic polymorphisms in genes related to arsenic metabolism.
In many studies, it was shown that inorganic arsenic metabolism is affected by several factors including genetics, demographic characteristics (e.g. age, gender), environmental factors (As dose in drinking water, exposure duration and termination of exposure), lifestyle-and diet-related factors, dietary habits and general health status. Although effects of above-mentioned factors could not be shown consistently, they should be considered as factors which must be taken into consideration (Gribble et al. 2013;Lindberg et al. 2008;Lindberg et al. 2007;Tseng 2009). In our study, one limitation is lack of body mass index data (BMI) which has been proposed as a factor that may affect As metabolism.
Reactions involved in arsenic methylation occur via single carbon metabolism. Methylation is mediated by enzymes which use S-adenosyl methionine (SAM) as substrate. Thus, it is proposed that several factors (e.g. folic acid, cysteine, methionine, niacin, choline, S-adenosyl methionin, vitamin B12) affecting single carbon metabolism can alter As metabolism and that it may have in uence on human health (Gamble et al. 2005;Heck et al. 2007;Huang et al. 2009). It is known that folate is an important co-factor involved in transportation and use of methyl groups during single carbon metabolism. In addition, it contributes to DNA biosynthesis process including gene expression, transcription, chromatin structure, genomic repair and stability regulation as single carbon transporter. The role of folate de ciency has become increasingly important in many diseases such as atherosclerotic disorders, cardiovascular diseases, neurological, neuropsychiatric and congenital disorders, and cancer (Choi and Mason 2000;Stanger 2002).
In descriptive epidemiological studies in regions where As-related disorders are seen, it was seen that diet is weak without diversity. In case-control studies from China, Taiwan, Western Bengal and India, diet content is poor from fruits, vegetables and food of animal origin (Heck et al. 2007). It was found that blood folate levels can be more reliable parameter than survey-based folate estimations (Huang et al. 2008). Thus, we measured serum folate levels to estimate dietary folate intake. It can be suggested that dietary folate intake was not different between controls and exposed group ( Table 7). The higher proportion of individuals with low vitamin B12 levels in exposed group may be due to other factors (age, medication). (Chen et al. 2007) suggest that higher dietary selenium intake may reduce the risk of arsenic-related skin lesions. However, blood selenium levels are lacking in our study.

Conclusion
This paper is part of our bigger project aimed at elucidating the effect of low-to moderate As exposure on skin lesions and genotoxicity parameters (e.g. Micronuclei) in target and surrogate tissues as well as individual susceptibilities (e.g. GSTM1, GSTT1, GSTP1, GST01-1 and AS3MT genetic polymorphisms).
In our study, all environmental and biological measurements demonstrated that individuals living in the study area were chronically exposed to low-to-moderate As due to geological contamination in drinking water. No signi cant increase was observed in the frequency of skin lesions in individuals exposed to As in our study area. Given the contradictory results about health risks (e.g. lung, skin and bladder cancer and skin lesions) to low-to-moderate As exposure (Baastrup et al. 2008;Chen et al. 2010;Dauphine et al. 2013;Snow et al. 2004), it is important to initiate long-term follow-up studies about effects on health in this region which would be considered as risky for As contamination.

Ethics approval
This study was performed in line with the principles of the Declaration of Helsinki.This study was approved by Gazi University Faculty of Medicine Local Ethics Committee (15.06.2009, No.340).