Elevated levels of plasma Big endothelin-1 and its relation to hypertension and skin lesions in individuals exposed to arsenic

Toxicology and Applied Pharmacology 259 (2012) 187–194

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Elevated levels of plasma Big endothelin-1 and its relation to hypertension and skinlesions in individuals exposed to arsenic

Ekhtear Hossain a,1, Khairul Islam a,1, Fouzia Yeasmin a, Md. Rezaul Karim b, Mashiur Rahman a,Smita Agarwal a, Shakhawoat Hossain a, Abdul Aziz a, Abdullah Al Mamun a, Afzal Sheikh a, Abedul Haque a,M. Tofazzal Hossain a, Mostaque Hossain c, Parvez I. Haris d, Noriaki Ikemura e, Kiyoshi Inoue e,Hideki Miyataka e, Seiichiro Himeno e, Khaled Hossain a,⁎a Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladeshb Department of Applied Nutrition and Food Technology, Islamic University, Kushtia-7003, Bangladeshc Department of Medicine, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka, Bangladeshd Faculty of Health & Life Sciences, De Montfort University, Leicester, LE1 9BH, UKe Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770–8514, Japan

Abbreviations: As, Arsenic; ICP-MS, Inductively CoupBig ET-1, Big endothelin-1.⁎ Corresponding author at: Department of Biochem

Rajshahi University, Rajshahi-6205. Fax: +880 721 750E-mail address: [email protected] (K. Hossain

1 These authors contributed equally to this work.

0041-008X/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.taap.2011.12.023

a b s t r a c t

Article history:Received 31 October 2011Revised 20 December 2011Accepted 26 December 2011Available online 5 January 2012

Keywords:ArsenicBig endothelinHypertensionSkin lesionsBangladesh

Chronic arsenic (As) exposure affects the endothelial system causing several diseases. Big endothelin-1 (BigET-1), the biological precursor of endothelin-1 (ET-1) is a more accurate indicator of the degree of activationof the endothelial system. Effect of As exposure on the plasma Big ET-1 levels and its physiological implica-tions have not yet been documented. We evaluated plasma Big ET-1 levels and their relation to hypertensionand skin lesions in As exposed individuals in Bangladesh. A total of 304 study subjects from the As-endemicand non-endemic areas in Bangladesh were recruited for this study. As concentrations in water, hair and nailswere measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The plasma Big ET-1 levels weremeasured using a one-step sandwich enzyme immunoassay kit. Significant increase in Big ET-1 levels wereobserved with the increasing concentrations of As in drinking water, hair and nails. Further, before andafter adjusting with different covariates, plasma Big ET-1 levels were found to be significantly associatedwith the water, hair and nail As concentrations of the study subjects. Big ET-1 levels were also higher inthe higher exposure groups compared to the lowest (reference) group. Interestingly, we observed that BigET-1 levels were significantly higher in the hypertensive and skin lesion groups compared to the normoten-sive and without skin lesion counterpart, respectively of the study subjects in As-endemic areas. Thus, thisstudy demonstrated a novel dose–response relationship between As exposure and plasma Big ET-1 levels in-dicating the possible involvement of plasma Big ET-1 levels in As-induced hypertension and skin lesions.

© 2012 Elsevier Inc. All rights reserved.

Introduction

In the arseniasis-endemic areas of the world, the main source ofexposure to As is through drinking water and As toxicity throughdrinking water represents one of the biggest catastrophes in history,affecting millions of people in the world (British Geological Surveyand Department of Public Health and Engineering, 2001; WorldHealth Organization, 2001). Bangladesh is one of the most severelyaffected regions in that approximately 80 million people consumewater containing As levels greater than the 10 μg/L standard set by

led Plasma Mass Spectroscopy;

istry and Molecular Biology,064.).

rights reserved.

the World Health Organization (Caldwell et al., 2003; Chowdhury,2004). There is great temporal and spatial variation in groundwaterAs levels in different regions of Bangladesh. The precise reasonfor the high levels of As in groundwater is not fully understood butvarious theories have been proposed including role of microbial mo-bilization, anthropogenic activities, etc. (Harvey et al., 2002; Hossainet al., 2011; Islam et al., 2004; Polizzotto et al., 2006; Sutton et al.,2009). As is widely present in natural waters, in the form of inorganicarsenite (AsIII) and arsenate (AsV). After consumption, inorganicAs is converted to methylated derivatives. Although methylation ofAs has been commonly considered a mechanism for detoxification,recent studies have shown that methylated trivalent arsenicals aremore toxic than inorganic As (Kligerman et al., 2003). Still thereare no appropriate animal models available for investigating healtheffects of As. Therefore, significant uncertainties remain regardingmechanisms by which As exerts its deleterious health effects on thehuman population.

188 E. Hossain et al. / Toxicology and Applied Pharmacology 259 (2012) 187–194

Several epidemiological studies suggest exposure to As correlateswith endothelial dysfunction (Chen et al., 2007; Lee et al., 2003). En-dothelial dysfunction, defined as an imbalance of endothelium-derived vasoconstrictor and vasodilator substances, is a common de-nominator in the pathogenesis and progression of both macro andmicro vascular complications. Previously it has been reported thatAs increases vasoconstrictor activity in rat blood vessels throughthe endothelium-dependent vasoconstrictor activity by compromis-ing basal endothelium nitric acid function (Bilszta et al., 2006).Endothelin receptor A (ETA) and B (ETB) are two distinct receptorsfor endothelins. The endothelins are a family of peptides (ET-1, ET-2, ET-3 and ET-4) consisting of 21 amino acids. They are producedby the endothelial cells, the smooth muscle cells of the blood vessels,and the cardiac myocytes (Levin, 1995; Yanagisawa et al., 1988).Among the four, ET-1 is a principal isoform found in human plasma(Yamaji et al., 1990). ET-1 is a potent vasoconstrictor (MacCumberet al., 1990) and the overall function of ET-1 is to increase blood pres-sure and vascular tone (Rubanyi and Polokoff, 1994; Wagner et al.,1992). ET-1 is formed from its biological precursor Big ET-1, a 38amino acid-long peptide that, after synthesis in the cytoplasm, iscleaved by endothelin conversion enzyme to yield active ET-1(amino acids 1–21) and a C terminal fragment (amino acids 22–38)(Yanagisawa et al., 1988). Big ET-1 has a circulating half-life of23 min (Hemsen et al., 1995) compared with only 3.5 min for ET-1.Circulating ET-1 may grossly underestimate local tissue concentra-tions (de Nucci et al., 1988), while Big ET-1 with its longer half-lifehas been implicated as a more sensitive indicator of endothelialsystem activation (Ishibashi et al., 1994; Jordan et al., 2005; Nelsonet al., 1998; Teng et al., 2006). Previous studies have reported thatAs induces hypertension (Chen et al., 1995; Rahman et al., 1999).Hypertension is implicated with endothelial dysfunction (Lermanet al., 1995). However, association between As-induced hyperten-sion and endothelial dysfunction has not yet been established.Therefore, in this study, we for the first time evaluated plasma BigET-1 levels and their association with hypertension in human sub-jects who were exposed to As through drinking water in Bangladesh.Furthermore, prolonged exposure to As induces typical skin symp-toms of arsenicosis such as melanosis and hyperkeratosis (Ahsanet al., 2000; Guha Mazumder et al., 1998). ET-1 has been reportedto be involved in the hyperpigmentation of the skin (Hachiya et al.,2004; Murase et al., 2009; Vural et al., 2001) but the involvementof this molecule in As-induced skin lesions has not yet been investi-gated. To obtain further information in this area, we explored therelationship between Big ET-1 levels and skin lesions in As exposedpopulation in our study group.

Methods

Study areas and study subjects. Ethical permission was obtainedfrom the Bangladesh Medical Research Council, Mohakhali, Dhaka-1212. As-endemic study areas for this study were chosen as describedpreviously (Ali et al., 2010; Karim et al., 2010). The study areas in-cluded Marua in Jessore, Dutpatila, Jajri, Vultie and Kestopur inChuadanga, and Bheramara in Kushtia district (north-west region)of Bangladesh. The prevalence of typical skin symptoms of arsenicosissuch as melanosis on the skin, hyperkeratosis and hard patches on thepalms of the hands and soles of the feet were very high among thelocal residents of the areas selected for sampling. Local residents15–60 years of age were invited to participate in the study. Thosewho responded spontaneously were asked to convene at a specificlocation in their village for initial screening purposes in light ofthe exclusion criteria. The study subjects were selected from thisconvened group, irrespective of the presence or absence of skinsymptoms or hypertension. Subsequently, individuals who exhibitedsymptoms were first identified by a general physician and then diag-nosis was confirmed by a dermatologist. The physician involved in

this study carefully examined various parts of the body to confirmthe presence of melanosis and hyperkeratosis. Adults who had livedfor at least last 5 years in As-endemic areas of Bangladesh were rec-ruited for this study.

Attempt was made to match, as much as possible the following:age (individual matching), sex and socioeconomic parameters of As-endemic population and the non-endemic study subjects (as a refer-ence group). The non-endemic study subjects with no history ofAs contamination in the drinking water were selected from Naogaondistrict (northern region) in Bangladesh. Socioeconomic parametersincluded occupation, education, monthly income and house types ofthe study subjects. We randomly selected some tube wells (drinkingwater sources) in the non-endemic area for the measurement ofwater As levels and we found that water in 96.6% of the tube wellscontained very low levels of As (b10 μg/L) and water in the remain-ing 3.4% of the tube wells contained a little bit higher levels ofAs (b15 μg/L) but still the levels were lower than the maximum per-missive limit of water As concentration (≤50 μg/L) for Bangladesh.As in the endemic areas, adults (15–60 years of ages) who hadlived for at least last 5 years in non-endemic area were recruitedfor this study.

Pregnant and lactating mothers and individuals who had a previ-ous and recent history of drug addiction, hepatotoxic and anti-hypertensive drugs, malaria, kalazar, chronic alcoholism, previousand present history of hepatic, renal or severe cardiac diseases havebeen excluded from this study. Of the 219 individuals who wereapproached, 9 were excluded according to the exclusion criteria[i.e., study candidates (n=4) who had resided in the As-endemicareas for less than 5 years, pregnant and lactating mothers (n=3),and had hematological diseases (n=2)]; thus, a total of 210 werefinally recruited (95.89% participation rate) in the As-endemic areas.In non-endemic area, 3 [i.e., study candidates (n=2) who had resid-ed in the non-endemic area for less than 5 years, pregnant and lactat-ing mother (n=1)] from 97 individuals were excluded. The responserate of the individuals from the non-endemic area was 96.91%.Household visits were carried out to interview residents. Personal in-terview of the study subjects was carried out by the trained membersof our research team using a standard questionnaire. Informationobtained from the interview included the sources of water for drink-ing and daily house hold uses, water consumption history, socio-economic status, occupation, food habit, cigarette smoking, alcoholintake, personal and family medical history, history of diseases, phys-iological complications, major diseases, previous physician's reportsand Body Mass Index (BMI). During the sample collection process,we were blinded to As levels in the drinking water, and to those inthe hair and nails of the study participants. We collected all bloodand other specimens (including water samples) on the same day foreach site.

Collection of nail and hair samples, and analysis of As. As levels infinger nails and hair have been reported to provide the integrated mea-sures for As exposure (Agahian et al., 1990;Gault et al., 2008). Nailswerecollected from each study subject as described previously (Schmitt et al.,2005). Hair samples with the length of about 1 cm were collected fromthe region of the head close to the scalp behind the ear by using a ceram-ic blade cutter andkept in polypropylene bottles (Mäki-Paakkanen et al.,1998). Nail and hair samples were cleaned by the method described byChen et al. (1999). Samples were immersed in 1% Triton X-100, sonicat-ed for 20 min, and then washed five times with milli-Q water. Thewashed samples were allowed to dry at 60°C overnight in a dryingoven. Nail and hair samples were digested with concentrated nitricacid using a hot plate at 70°C for 15 min and 115°C for 15 min. Aftercooling, the samples were diluted with 1.0% nitric acid containing yttri-um (10 ppb), and concentrations of As75 and Y79 in these samples weredetermined by ICP-MS (HP-4500, Agilent Technologies, Kanagawa,Japan). Accuracy of As measurement was verified by using a certified

189E. Hossain et al. / Toxicology and Applied Pharmacology 259 (2012) 187–194

reference material (CRM) “cod fish powder” (NMIJ CRM 7402-a,National Institute of Advanced Industrial Science and Technology,Japan). The average value (mean±SD) of As in “cod fish powder” deter-mined in triplicate by the above-mentioned digestion followed by ICP-MS analysis was 34.9±2.35 μg/g (reference value, 36.7 μg/g).

Water collection and As analysis. Study subjects identified the tubewells they used as their primary sources of drinking water. Watersamples were collected for this study as described by Ali et al.(2010). Water samples from these tube wells were collected inacid-washed containers after the well was pumped for 5 min as pre-viously described (Van Geen et al., 2008). Total As concentration inwater samples was determined by ICP-MS after the addition of a so-lution of yttrium (10 ppb in 1.0% nitric acid) to all water samples asan internal standard for ICP-MS analysis. The ion signals for As andyttrium were monitored at m/z of 75 and 79, respectively. All sam-ples were determined in triplicate and the average values wereused for data analysis. The detection limit of As75 was 30 ppt. Riverwater (NMIJ CRM 7202-a No.347 National Institute of Advanced In-dustrial Science and Technology, Japan) was used as a CRM. The av-erage value (mean±SD) of As in the “river water” determined intriplicate by ICP-MS analysis was 1.06±0.04 μg/L (reference value,1.18 μg/L).

Blood pressure measurement. The standard protocol for measuringblood pressure recommended by the World Health Organizationwas used in this study. After study subjects had rested for 20 minor longer, both systolic and diastolic blood pressures (SBP and DBP)were measured three times with a mercury sphygmomanometerwith subjects sitting. SBP and DBP were defined at the first andfifth phase Korotkoff sounds, respectively. The average of threemeasurements was used for the analysis. Hypertension was definedas a SBP of≥140 mm Hg and a DBP of≥90 mm Hg on three repeatedmeasurements.

Collection of plasma. Fasting blood samples were collectedfrom the study subjects. Blood samples (5–7 ml) were collected inEDTA-containing blood collection tubes from each individual byvenipuncture. Whole blood was then placed immediately on iceand subsequently centrifuged at 1600×g for 15 min at 4 °C. Plasmasupernatant was then taken and stored at −80 °C.

Measurement of plasma Big ET-1. The plasma levels of Big ET-1were measured using one-step sandwich enzyme immunoassay kit(Biomedica, Divischgasse, Austria). All standards and samples wereanalyzed in duplicate and the mean value was taken. On completionof the assay, the observed color change was read on a standardplate reader (Mikura Ltd. UK) and plasma values were calculated byextrapolation from a standard curve. A separate standard curve wasconstructed for each immunoassay batch.

Statistical analysis. Statistical analysis for this study was performedby using the Statistical Packages for Social Sciences (SPSS) software.Characteristics of the study subjects from As-endemic and non-endemic areas were analyzed by Independent Samples T-test andChi-square test. Because of skewed distributions, log transformationwas performed for drinking water, hair and nail As concentrations.Log-transformed values were reconverted to antilogarithm forms inthe table. The nature of associations between As exposure metrics(water, hair and nails) and plasma Big ET-1 levels were evaluatedthrough analysis of scatter plots. Subsequently, bivariate associationsbetween different exposure metrics and plasma Big ET-1 levels wereexamined using Pearson correlation coefficient test. Before and afteradjusting for covariates (age, sex, BMI, smoking, and hypertension),univariate linear regression analysis was performed to examine theassociations between plasma Big ET-1 levels and As exposure metrics.

Study subjects in the As-endemic area were split into tertile groups(low, medium and high) based on the three concentrations of eachexposure metric with equal proportion through frequency test andstudy subjects in the non-endemic area were used as a referencegroup (lowest exposure group). Plasma Big ET-1 levels in lowest,low, medium and high exposure groups were analyzed by linear re-gression. Univariate linear regression analysis were performed forthe comparison of Big ET-1 levels between hypertensive and normo-tensive, and skin lesion and without skin lesion groups of the studysubjects in As-endemic areas.

Results

Descriptive characteristics of the study subjects

Table 1 shows the characteristics of the study subjects in theAs-endemic and non-endemic areas. Of the 304 participants 210were from As-endemic areas and 94 from non-endemic area. Non-endemic individuals were selected for this study as a referencegroup. As concentrations in the drinking water, hair and nails of thestudy subjects in the As-endemic areas were approximately 60, 19and 8 times higher, respectively than those of non-endemic controlarea. The average age of the study subjects in the As-endemic andnon-endemic areas were 38.42±12.15 and 35.77±10.22 years, re-spectively. The SBP and DBP of the study subjects in the As-endemicareas were 122.67±20.45 and 80.05±11.84 mm Hg, respectively,whereas thesewere 111.49±14.20 and 71.12±9.65 mm Hg, respec-tively for the non-endemic population. In the As-endemic areas, therewere 106 male and 104 female study subjects, whereas in the non-endemic area, these were 53 and 41, respectively. Most of the malestudy subjects in both As-endemic and non-endemic areas werefarmers, whereas most of the female study subjects were housewives. Socioeconomic characteristics (occupation, monthly incomeand housing) of the study subjects from non-endemic and endemicareas were almost similar. The percentage of tobacco smokers in theAs-endemic and non-endemic areas were 19.5 and 33, respectively.We did not find any female who admitted to be a smoker in the As-endemic and non-endemic areas as generally Bangladeshi women donot smoke cigarette. The mean BMI of the study subjects in the As-endemic and non-endemic areas were 20.84±3.37 and 21.37±2.71,respectively. The average (mean±SD) levels of plasma Big ET-1in As-endemic and non-endemic population were 0.83±0.23 and0.57±0.21 fmol/mL, respectively. The differences in Big ET-1 levelsbetween As-endemic and non-endemic population were statisticallysignificant (pb0.001). Big ET-1 levels were 45.61% higher in As-endemic population than the non-endemic population.

Correlation between As exposure and plasma Big ET-1 levels

Fig. 1 shows the effect of As exposure on plasma Big ET-1 levels. Asignificant increase in plasma Big ET-1 levels was observed with theincreasing concentrations of As in the drinking water (r=0.428,pb0.001, Fig. 1A). A similar relationship was also observed betweenBig ET-1 and hair As concentrations (r=0.441, pb0.001, Fig. 1B), andbetween Big ET-1 and nail As concentrations (r=0.406, pb0.001,Fig. 1C).

Table 2 shows the association between As exposure metrics withplasma Big ET-1 levels through linear regression analysis. Beforeand after adjusting for covariates, we found that water As concentra-tions were significantly associated with plasma Big ET-1 levels. Simi-larly, hair and nail As concentrations also displayed significantpositive association with plasma Big ET-1 levels. To investigate theexposure–response relationship between water As concentrationsand plasma Big ET-1 levels, we evaluated plasma Big ET-1 levels inthe tertile groups (low, medium and high) of the study subjects inthe As-endemic areas compared with the non-endemic population

Table 1Descriptive characteristics of the study subjects in As-endemic and non-endemic areas.

Parameters All subjects Non-endemic (reference subjects) As-endemic

Total subjects (n) 304 94 210Sex (n)

Male 159 53 106Female 145 41 104

Age (mean±SD) 37.60±11.63 35.77±10.22 38.42±12.15As concentration in drinking water (mean±SD; μg/L) 133.65±160.14 3.19±2.99 192.05±161.54⁎

As concentration in hair (mean±SD; μg/g) 4.11±5.83 0.31±0.21 5.81±6.31⁎

As concentration in nail (mean±SD; μg/g) 6.77±7.06 1.20±0.85 9.27±7.20⁎

SBP (mean±SD; mm Hg) 119.21±19.42 111.49±14.20 122.67±20.45⁎

DBP (mean±SD; mm Hg) 77.29±11.93 71.12±9.65 80.05±11.84⁎

Occupation [n, (%)]MaleFarmers 134 (84.3) 41 (77.4) 93 (87.7)Business 2 (1.3) 0 2 (1.9)Students 14 (8.8) 9 (17) 5 (4.7)Tailors 4 (2.5) 0 4 (3.8)Others 5 (3) 3 (5.7) 2 (1.9)FemaleHousewives 137 (94.5) 41 (100) 96 (92.3)Farm workers 5 (3.4) 0 5 (4.8)Students 2 (1.4) 0 2 (2)Others 1 (0.7) 0 1 (1)

Education [n, (%)]No formal education 127 (41.8) 38 (40.4) 89 (42.4)Primary 109 (35.9) 33 (35.1) 76 (36.2)Secondary 51 (16.8) 17 (18.1) 34 (16.2)Higher 17 (5.6) 6 (6.4) 11 (5.2)

Income/month (US$) 23.83±8.12 23.12±7.34 24.15±8.44House [n, (%)]

Brick house with concrete roof (pakka) 41 (13.5) 12 (12.8) 29 (13.8)Brick house with corrugated tin roof 111 (36.5) 36 (38.3) 75 (35.7)Mud house with corrugated tin roof 98 (32.2) 29 (30.9) 69 (32.9)Straw house with corrugated tin roof 43 (14.1) 14 (14.9) 29 (13.8)Others 11 (3.6) 3 (3.3) 8 (3.8)

Smoking [n, (%)]Yes 72 (23.7) 31 (33) 41 (19.5)No 232 (76.3) 63 (67) 169 (80.5)

BMI (mean±SD; kg/m2) 21.01±3.18 21.37±2.71 20.84±3.37Big ET-1 (mean±SD; fmol/ml) 0.75±0.26 0.57±0.21 0.83±0.23⁎

Data were presented as mean±SD (95% CI).BMI (Body Mass Index) was calculated as body weight (Kg) divided by height squared (m2).DBP, diastolic blood pressure; SBP, systolic blood pressure. Differences were analyzed by independent samples T-test and Chi-square test.⁎ pb0.001.

190 E. Hossain et al. / Toxicology and Applied Pharmacology 259 (2012) 187–194

(lowest or reference group). Intriguingly, we found that plasma BigET-1 (Table 3) levels were significantly increased in the higher expo-sure groups before and after adjusting for covariates compared tothe lowest exposure group. Further, we explored the dose–responserelationship between the internal exposure metrics (hair and nail Asconcentrations) and plasma Big ET-1 levels. Levels of plasma Big ET-1were significantly higher in the higher exposure groups compared tothe lowest exposure group (Table 3).

As exposure, plasma Big ET-1 levels and hypertension

Since ET-1 is a potent vasoconstrictor and As exposure is a risk fac-tor for hypertension (Chen et al., 1995; Rahman et al., 1999), we nextevaluated whether increased Big ET-1 levels, observed in the studysubjects, were associated with their blood pressure or not. Study sub-jects in the As-endemic areas were split into two groups based on thestatus of hypertension. After adjusting for covariates (age, sex, BMIand smoking), the plasma Big ET-1 levels in the hypertensive groupwere significantly higher compared to the normotensive counterpart(Table 4).

As exposure, plasma Big ET-1 levels and skin lesions

Relationship between exposure to As from drinking water and de-velopment of skin lesions (hyperkeratosis and hyperpigmentation)

has been well established (Ahsan et al., 2000; Guha Mazumder et al.,1998). However, the mechanism underlying the development ofsuch skin lesions remain unknown. Recent studies have suggestedthat hyperkeratosis and hyperpigmentation in skin can result fromelevated levels of plasma ET-1 (Sacar et al., 2005; Vural et al., 2001).Therefore, for the first time, we investigated the relationship betweenplasma Big ET-1 levels and As-induced skin lesions in the study sub-jects of As-endemic areas. We found that the study subjects withskin lesions had a significantly higher level of plasma Big ET-1(Table 4) than those without skin lesions. This result led us to makea hypothesis that increased plasma levels of Big ET-1 (by implicationits final form — ET-1) might be one of the factors responsible for thedevelopment of skin lesions in As-endemic population.

Discussion

Significant gaps remain in the mechanistic understanding ofAs-induced disorders in humans including endothelial dysfunction.Increased plasma level of ET-1 is associated with endothelial activa-tion or dysfunction. Previous studies have suggested that Big ET-1,the immediate biological precursor of ET-1, may be a more accurateindicator of the degree of activation of the endothelial system(Leveson et al., 1985; Nelson et al., 1998; Teng et al., 2006) comparedto ET-1. ET-1 is a vasoconstrictor peptide derived mainly from vascu-lar endothelial cells (Yanagisawa et al., 1988). Several studies have

A B C

Pla

sma

Big

ET

-1 (

fmol

/ml)

r = 0.428p < 0.001

r = 0.441p < 0.001

r = 0.406p < 0.001

0

0.5

1

1.5

2

-2 0 2 4 -2 -1 0 1 2 -1 0 1 2Log nail arsenic

(µg/g)Log water arsenic

(µg/L)Log hair arsenic

(µg/g)

Fig. 1. Correlations between the plasma Big ET-1 levels and water, hair or nail As concentrations. Effects of drinking water (A), hair (B) and nail (C) arsenic concentrations on plasmaBig ET-1 levels. Arsenic concentrations were used after log transformation. r- and p-values were from Pearson correlation coefficient test.

191E. Hossain et al. / Toxicology and Applied Pharmacology 259 (2012) 187–194

indicated that ET-1 has a wide range of physiological effects in the de-velopment of diseases including cardiovascular disease, differenttypes of cancer and pigmentation of the skin (Best et al., 1999; Jiaoet al., 2008; Lerman et al., 1995; Sacar et al., 2005; Simpson et al.,2000; Vural et al., 2001). Although As exposure targets endothelialsignals in the development of pathogenesis, the effect of As exposureon the plasma Big ET-1 levels and its physiological implications havenot yet been documented. In this study, we evaluated the plasma BigET-1 levels in a population of As-endemic areas in Bangladesh andmonitored their relationship with hypertension and skin lesions.

We found that Big ET-1 levels of the study subjects in As-endemicareas were significantly higher than those (reference group) of thenon-endemic area. Previous study suggested that As contents of hairand nail samples might be used as effective biomarkers for As expo-sure (Gault et al., 2008). In our previous study, we also showedthat drinking water As levels were strongly correlated with hair andnail As levels (Ali et al., 2010). Similar and consistent results wereobserved in this study in the correlation between water and hair ornail As concentrations (data not shown). We found that plasma BigET-1 levels were strongly associated with drinking water, hair andnail As concentrations (Fig. 1). The study subjects were separatedinto four groups based on the four concentrations of As in the drink-ing water where non-endemic study subjects (reference group)were considered as the lowest exposure group. Significantly higherlevels of plasma Big ET-1 were observed in the higher exposuregroups (Table 3) compared to the lowest exposure group (referencegroup). Since the relationship between water As and plasma Big ET-1 levels suggested only an external exposure–response relationship,we next examined, the dose–response relationship using candidatebiomarkers (hair and nails) of As exposure. Similar patterns as ob-served in the exposure-response relationship were also found in the

Table 2Association between As exposure and plasma Big ET-1 levels by linear regression analysis.

Independent variables Before adjusting covariates After adju

Coefficient (95% CI) p-value (t-test) Coefficien

Water As (μg/L) 0.097 (0.074–0.120) b0.001 0.095 (0.Hair As (μg/g) 0.165 (0.127–0.203) b0.001 0.164 (0.Nail As (μg/g) 0.194 (0.144–0.243) b0.001 0.191 (0.

Before data analysis, log transformed values of As exposure metrics were used.Degree of freedom (df) before and after adjustment for covariates were (1, 302), (5, 298) a

a Adjusted for age, sex, BMI and smoking.b Adjusted for age, sex, BMI, smoking and blood pressure.

case of dose–response relationship. Even after adjusting for differentcovariates, water, hair and nail As showed significant effects in in-creasing plasma Big ET-1 levels which suggested that As exposurewas an independent risk factor for the elevation of plasma Big ET-1.In this study, we found that SBP and DBP were significantly higherin the population exposed to As compared to the non-endemic refer-ence group (Table 1). These results were in agreement with previousstudies in which As exposure was associated with increased levels ofblood pressure (Kwok et al., 2007; Yang et al., 2007).

Raised concentrations of ET-1 and its precursor Big ET-1, providean important indicator of heart failure, congestive heart disease(Pacher et al., 1993) and are related to pulmonary hypertension inpatients with this problem (Cody et al., 1992; Pacher et al., 1993),the severity of their overall condition, and their prognosis. Further,moderate-to-severe hypertensive patients presented enhanced ex-pression of prepro ET-1 in the endothelium of subcutaneous resis-tance arteries (Schiffrin et al., 1992). Interestingly, in this study, wealso found that plasma Big ET-1 levels were significantly higher inthe hypertensive group compared to the normotensive study subjectsin the As-endemic areas (Table 4). After excluding the hypertensivestudy subjects (n=47), all the different associations between Asexposure and plasma Big ET-1 were significant (data not shown).Therefore, these findings were consistent with the notion that Asexposure was responsible for inducing the elevation of plasma BigET-1 which in turn might be responsible for hypertension. In spiteof the more potent biological action of ET-1 than Big ET-1, we mea-sured the plasma Big ET-1 level in this study, since active peptide,ET-1 is cleared more rapidly from the organism and its paracrineactivity is not reflected by its blood concentrations (Wei et al.,1994). It has been shown that Big ET-1 has a longer half-life andslower clearance than ET-1. Synthesis of ET-1 is closely related to

sting covariatesa After adjusting covariatesb

t (95% CI) p-value (t-test) Coefficient (95% CI) p-value (t-test)

072–0.118) b0.001 0.080 (0.056–0.103) b0.001125–0.203) b0.001 0.143 (0.105–0.181) b0.001140–0.241) b0.001 0.162 (0.113–0.211) b0.001

nd (6, 297), respectively.

Table 3Dose–response relationship of plasma Big ET-1 levels in the lowest, low, medium and high exposure groups by univariate linear regression analysis.

Independent variables Before adjusting covariates After adjusting covariatesa After adjusting covariatesb

Coefficient (95% CI) p-value (t-test) Coefficient (95% CI) p-value (t-test) Coefficient (95% CI) p-value (t-test)

Water As (μg/L)Lowest (0.03–13.37) – – – – – –

Low (0.08–87.8) 0.202 (0.133–0.271) b0.001 0.206 (0.136–0.276) b0.001 0.188 (0.119–0.256) b0.001Medium (88.6–242) 0.334 (0.264–0.403) b0.001 0.337 (0.266–0.407) b0.001 0.296 (0.226–0.367) b0.001High (243–546) 0.256 (0.188–0.324) b0.001 0.259 (0.190–0.328) b0.001 0.219 (0.150–0.288) b0.001

Hair As (μg/g)Lowest (0.03–1.18) – – – – – –

Low (0.13–2.37) 0.231 (0.161–0.301) b0.001 0.228 (0.156–0.300) b0.001 0.201 (0.131–0.271) b0.001Medium (2.40–5.59) 0.251 (0.181–0.321) b0.001 0.260 (0.189–0.330) b0.001 0.216 (0.146–0.286) b0.001High (5.66–37.24) 0.306 (0.237–0.375) b0.001 0.309 (0.238–0.379) b0.001 0.275 (0.205–0.344) b0.001

Nail As (μg/g)Lowest (0.16–4.55) – – – – – –

Low (0.65–4.77) 0.230 (0.160–0.300) b0.001 0.236 (0.165–0.307) b0.001 0.214 (0.144–0.283) b0.001Medium (4.87–10.86) 0.258 (0.188–0.327) b0.001 0.258 (0.187–0.329) b0.001 0.214 (0.144–0.285) b0.001High (10.93–37.42) 0.302 (0.233–0.372) b0.001 0.306 (0.235–0.376) b0.001 0.267 (0.197–0.337) b0.001

Study subjects in the non-endemic area were used as lowest exposure group.Before data analysis, log transformed values of As exposure metrics were used.Degree of freedom (df) before and after adjustment for covariates were (3, 300), (7, 296) and (8, 295), respectively.

a Adjusted for age, sex, BMI and smoking.b Adjusted for age, sex, BMI, smoking and blood pressure.

192 E. Hossain et al. / Toxicology and Applied Pharmacology 259 (2012) 187–194

Big ET-1 (Levin, 1995; Rubanyi and Polokoff, 1994). Measurement ofBig ET-1 substantially assists interpretation of plasma endothelinlevels (Plumpton et al., 1995, 1996). Therefore, increased plasmalevels of Big ET-1 in As-endemic study participants observed in thisstudy ultimately reflected an increase in plasma ET-1 levels. In thisstudy, we did not explore the mechanisms by which As increasesBig ET-1 levels. One possible mechanism is that As exposure couldgenerate free radicals that might increase the expression of themRNA of Big ET-1 since oxidative stress-mediated expression ofET-1 was reported previously (Michael et al., 1997).

Physiological importance of ET-1 and its implication with diseaseprogression have been well studied previously. Intravenous infusionof ET-1 into conscious rats causes an initial decrease in blood pres-sure that is followed by intense and prolonged hypertension(Rubanyi and Polokoff, 1994; Wagner et al., 1992). In the heart,ET-1 affects the coronary circulation through the vasoconstrictiveresponse in coronary circulation and may play a role in the etiologyof coronary vasospasm (Best et al., 1999; Lerman et al., 1995). Plasmaconcentration of Big ET-1 has been implicated with heart failure(Rivera et al., 2005). Elevated plasma Big ET-1 levels were also ob-served in diabetes (Ergul, 2011) and cancers (Jiao et al., 2008;Simpson et al., 2000). Increased Big ET-1 levels observed in thisstudy may provide new insights into As-induced development ofcardiovascular diseases and cancers. Additionally, the findings ofthis study have identified Big ET-1 level as a potential biomarkerand a therapeutic drug target for reducing the risk of cardiovasculardiseases in As-endemic population. Further, we have demonstrated asimple link between Big ET-1 levels and skin lesion. In light of thisfinding, we hypothesize that the increased Big ET-1 is responsiblefor the skin lesions observed in As exposed populations. Since this

Table 4Association between plasma Big ET-1 levels with hypertension and skin lesions.

Parameters Categories No. of study subjects

Blood pressure Normotensive 163Hypertensive 47

Skin symptom (−) Symptom 33(+) Symptom 177

p-values were from univariate linear regression.a Data were adjusted for age, sex, BMI and smoking status.b Data were adjusted for age, sex, BMI, smoking status and hypertension.

hypothesis is based on a very simple link between Big ET-1 levelsand skin lesions, it needs to be tested through a more detailed andcomprehensive study including large number of study subjectsfrom high and low As exposed population with and without skinlesions. However, our hypothesis is consistent with previous studieson UVB-induced hyperpigmentation through ET-1 and other mole-cules (Hachiya et al., 2004; Murase et al., 2009) although these stud-ies were not in relation to As exposure. Vural et al. (2001) havesuggested that increased ET-1, amongst other factors, may be re-sponsible for increase in hyperpigmentation, hyperkeratinisationand keratinocyte proliferation in actinic keratosis and basal cell car-cinoma patients compared to a control group. They have suggestedthat the increased levels of ETs and nitric oxide may be responsiblefor the cytotoxic and mitogenic properties that may further causesuch types of skin tumors to proliferate. More recently, it has beenreported by Lan et al. (2005) that in basal cell carcinoma, an in-creased expression of ET-1 is responsible for the hyperpigmentationof this skin tumor.

Themajor strengths of this study were 1) to show for the first timethe effects of As exposure on plasma Big ET-1 levels, through moni-toring three different exposure metrics (water, hair and nail Aslevels), in a study population who showed large variations in theirAs exposure levels and 2) to demonstrate a relationship of the in-creased plasma Big ET-1 levels in the As exposed population withhypertension and skin lesions. Although this study presents extensiveepidemiological research demonstrating the effects of As exposure onplasma Big ET-1 and its association with hypertension and skin le-sions, there are some limitations warranting further discussion.First, we showed the association between the As exposure and plas-ma Big ET-1 levels after adjusting for BMI, age, sex and smoking

Plasma Big ET-1 (fmol/mL) (mean±SD) p-value

0.80±0.23 pb0.001a

0.96±0.190.75±0.22 pb0.05b

0.84±0.23

193E. Hossain et al. / Toxicology and Applied Pharmacology 259 (2012) 187–194

habits. However, there may be some other factors such as co-exposure to other metals, insecticides or pesticides or other toxic sub-stances or individual variations that could influence the Big ET-1levels. If any accompanying metals (or other contaminants) could in-fluence the association between As exposure and plasma Big ET-1,then they would also be expected to follow the same concentra-tion gradients as As in the drinking water, hair and nails. This isunlikely, but more detailed and extensive study of the other metalsand their association with Big ET-1 are required in future. Further-more, the results of the study are consistent with the previousanimal studies (Soucy et al., 2005; Yamaguchi et al., 2007) whichhave demonstrated that As exposure increases endothelin levels.We also found a dose–response relationship between As exposureand plasma Big ET-1 levels. When this is taken into considerationwith the findings of the previous animal studies, a cause-effect rela-tionship between As exposure and plasma Big ET-1 levels is highlyplausible. Second, most of our study population was lean with regardto BMI. Third, the study subjects without skin lesions are much fewerthan those with symptoms. So the results in relation to plasma Big ET-1 and skin lesions would require further confirmation by increasingthe sample size. Fourth, this study was designed to be cross-sectional, but not prospective. Further verification of the cause-effect relationship between plasma Big ET-1 levels and hypertension,and skin lesions would require a cohort based study. Thus, the find-ings of the current study may not be generalizable to other studypopulations, given the possible different distributions of risk factorsfor endothelial dysfunction, hypertension and skin lesions that mayinfluence the effect of As exposure. Nevertheless, increased plasmaBig ET-1 levels with increasing levels of As and their correlationwith hypertension and skin lesions may be significant for obtainingnovel mechanistic insights into the endothelial dysfunction, hyper-tension and skin lesions induced by As.

As far as we are aware, this research for the first time has demon-strated the interaction between As exposure and plasma Big ET-1levels in human subjects who are chronically exposed to As. Further-more, this study has demonstrated a dose–response relationship be-tween As exposure and plasma Big ET-1. There are two importantfindings of this study that are noteworthy. Firstly, we have demon-strated that As exposed study subjects, who were hypertensive, hadsignificantly higher levels of plasma Big ET-1, raising the possibilitythat As-induced hypertension is due to vasoconstrictor activity ofplasma Big ET-1 or ET-1. Secondly, we have for the first time showna relationship between the Big ET-1 and skin lesions suggesting arole for Big ET-1 in the development of skin lesions.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the Grants of Ministry of Science andInformation & Communication Technology, Government of the People'sRepublic of Bangladesh [Grant No. 2007-2008/BS-135/176/1(5)], andalso partially supported by a grant of TWAS (Grant No. Ref-09–153RG/BIO/AS_I; UNESCO FR: 3240230321) and Heiwa Nakajima Founda-tion, Japan.

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