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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 skin lesions 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, Bangladesh Department of Applied Nutrition and Food Technology, Islamic University, Kushtia-7003, Bangladesh c Department of Medicine, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka, Bangladesh d Faculty of Health & Life Sciences, De Montfort University, Leicester, LE1 9BH, UK e Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770–8514, Japan b
a r t i c l e
i n f o
Article history: Received 31 October 2011 Revised 20 December 2011 Accepted 26 December 2011 Available online 5 January 2012 Keywords: Arsenic Big endothelin Hypertension Skin lesions Bangladesh
a b s t r a c t Chronic arsenic (As) exposure affects the endothelial system causing several diseases. Big endothelin-1 (Big ET-1), the biological precursor of endothelin-1 (ET-1) is a more accurate indicator of the degree of activation of the endothelial system. Effect of As exposure on the plasma Big ET-1 levels and its physiological implications have not yet been documented. We evaluated plasma Big ET-1 levels and their relation to hypertension and skin lesions in As exposed individuals in Bangladesh. A total of 304 study subjects from the As-endemic and non-endemic areas in Bangladesh were recruited for this study. As concentrations in water, hair and nails were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The plasma Big ET-1 levels were measured using a one-step sandwich enzyme immunoassay kit. Significant increase in Big ET-1 levels were observed with the increasing concentrations of As in drinking water, hair and nails. Further, before and after adjusting with different covariates, plasma Big ET-1 levels were found to be significantly associated with the water, hair and nail As concentrations of the study subjects. Big ET-1 levels were also higher in the higher exposure groups compared to the lowest (reference) group. Interestingly, we observed that Big ET-1 levels were significantly higher in the hypertensive and skin lesion groups compared to the normotensive and without skin lesion counterpart, respectively of the study subjects in As-endemic areas. Thus, this study demonstrated a novel dose–response relationship between As exposure and plasma Big ET-1 levels indicating 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 of exposure to As is through drinking water and As toxicity through drinking water represents one of the biggest catastrophes in history, affecting millions of people in the world (British Geological Survey and Department of Public Health and Engineering, 2001; World Health Organization, 2001). Bangladesh is one of the most severely affected regions in that approximately 80 million people consume water containing As levels greater than the 10 μg/L standard set by
Abbreviations: As, Arsenic; ICP-MS, Inductively Coupled Plasma Mass Spectroscopy; Big ET-1, Big endothelin-1. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205. Fax: + 880 721 750064. E-mail address: [email protected] (K. Hossain). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.12.023
the World Health Organization (Caldwell et al., 2003; Chowdhury, 2004). There is great temporal and spatial variation in groundwater As levels in different regions of Bangladesh. The precise reason for the high levels of As in groundwater is not fully understood but various theories have been proposed including role of microbial mobilization, anthropogenic activities, etc. (Harvey et al., 2002; Hossain et 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 inorganic arsenite (As III) and arsenate (As V). After consumption, inorganic As is converted to methylated derivatives. Although methylation of As has been commonly considered a mechanism for detoxification, recent studies have shown that methylated trivalent arsenicals are more toxic than inorganic As (Kligerman et al., 2003). Still there are no appropriate animal models available for investigating health effects of As. Therefore, significant uncertainties remain regarding mechanisms by which As exerts its deleterious health effects on the human population.
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Several epidemiological studies suggest exposure to As correlates with endothelial dysfunction (Chen et al., 2007; Lee et al., 2003). Endothelial dysfunction, defined as an imbalance of endotheliumderived vasoconstrictor and vasodilator substances, is a common denominator in the pathogenesis and progression of both macro and micro vascular complications. Previously it has been reported that As increases vasoconstrictor activity in rat blood vessels through the endothelium-dependent vasoconstrictor activity by compromising basal endothelium nitric acid function (Bilszta et al., 2006). Endothelin receptor A (ETA) and B (ETB) are two distinct receptors for endothelins. The endothelins are a family of peptides (ET-1, ET2, ET-3 and ET-4) consisting of 21 amino acids. They are produced by 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 (MacCumber et al., 1990) and the overall function of ET-1 is to increase blood pressure and vascular tone (Rubanyi and Polokoff, 1994; Wagner et al., 1992). ET-1 is formed from its biological precursor Big ET-1, a 38 amino acid-long peptide that, after synthesis in the cytoplasm, is cleaved 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 of 23 min (Hemsen et al., 1995) compared with only 3.5 min for ET-1. Circulating ET-1 may grossly underestimate local tissue concentrations (de Nucci et al., 1988), while Big ET-1 with its longer half-life has been implicated as a more sensitive indicator of endothelial system activation (Ishibashi et al., 1994; Jordan et al., 2005; Nelson et al., 1998; Teng et al., 2006). Previous studies have reported that As induces hypertension (Chen et al., 1995; Rahman et al., 1999). Hypertension is implicated with endothelial dysfunction (Lerman et al., 1995). However, association between As-induced hypertension and endothelial dysfunction has not yet been established. Therefore, in this study, we for the first time evaluated plasma Big ET-1 levels and their association with hypertension in human subjects who were exposed to As through drinking water in Bangladesh. Furthermore, prolonged exposure to As induces typical skin symptoms of arsenicosis such as melanosis and hyperkeratosis (Ahsan et al., 2000; Guha Mazumder et al., 1998). ET-1 has been reported to be involved in the hyperpigmentation of the skin (Hachiya et al., 2004; Murase et al., 2009; Vural et al., 2001) but the involvement of this molecule in As-induced skin lesions has not yet been investigated. To obtain further information in this area, we explored the relationship between Big ET-1 levels and skin lesions in As exposed population in our study group. Methods Study areas and study subjects. Ethical permission was obtained from the Bangladesh Medical Research Council, Mohakhali, Dhaka1212. As-endemic study areas for this study were chosen as described previously (Ali et al., 2010; Karim et al., 2010). The study areas included Marua in Jessore, Dutpatila, Jajri, Vultie and Kestopur in Chuadanga, and Bheramara in Kushtia district (north-west region) of Bangladesh. The prevalence of typical skin symptoms of arsenicosis such as melanosis on the skin, hyperkeratosis and hard patches on the palms of the hands and soles of the feet were very high among the local residents of the areas selected for sampling. Local residents 15–60 years of age were invited to participate in the study. Those who responded spontaneously were asked to convene at a specific location in their village for initial screening purposes in light of the exclusion criteria. The study subjects were selected from this convened group, irrespective of the presence or absence of skin symptoms or hypertension. Subsequently, individuals who exhibited symptoms were first identified by a general physician and then diagnosis was confirmed by a dermatologist. The physician involved in
this study carefully examined various parts of the body to confirm the presence of melanosis and hyperkeratosis. Adults who had lived for at least last 5 years in As-endemic areas of Bangladesh were recruited for this study. Attempt was made to match, as much as possible the following: age (individual matching), sex and socioeconomic parameters of Asendemic population and the non-endemic study subjects (as a reference group). The non-endemic study subjects with no history of As contamination in the drinking water were selected from Naogaon district (northern region) in Bangladesh. Socioeconomic parameters included occupation, education, monthly income and house types of the study subjects. We randomly selected some tube wells (drinking water sources) in the non-endemic area for the measurement of water As levels and we found that water in 96.6% of the tube wells contained very low levels of As (b10 μg/L) and water in the remaining 3.4% of the tube wells contained a little bit higher levels of As (b15 μg/L) but still the levels were lower than the maximum permissive limit of water As concentration (≤ 50 μg/L) for Bangladesh. As in the endemic areas, adults (15–60 years of ages) who had lived for at least last 5 years in non-endemic area were recruited for this study. Pregnant and lactating mothers and individuals who had a previous and recent history of drug addiction, hepatotoxic and antihypertensive drugs, malaria, kalazar, chronic alcoholism, previous and present history of hepatic, renal or severe cardiac diseases have been excluded from this study. Of the 219 individuals who were approached, 9 were excluded according to the exclusion criteria [i.e., study candidates (n = 4) who had resided in the As-endemic areas for less than 5 years, pregnant and lactating mothers (n = 3), and had hematological diseases (n = 2)]; thus, a total of 210 were finally recruited (95.89% participation rate) in the As-endemic areas. In non-endemic area, 3 [i.e., study candidates (n = 2) who had resided in the non-endemic area for less than 5 years, pregnant and lactating mother (n = 1)] from 97 individuals were excluded. The response rate of the individuals from the non-endemic area was 96.91%. Household visits were carried out to interview residents. Personal interview of the study subjects was carried out by the trained members of our research team using a standard questionnaire. Information obtained from the interview included the sources of water for drinking and daily house hold uses, water consumption history, socioeconomic status, occupation, food habit, cigarette smoking, alcohol intake, personal and family medical history, history of diseases, physiological complications, major diseases, previous physician's reports and Body Mass Index (BMI). During the sample collection process, we were blinded to As levels in the drinking water, and to those in the hair and nails of the study participants. We collected all blood and other specimens (including water samples) on the same day for each site. Collection of nail and hair samples, and analysis of As. As levels in finger nails and hair have been reported to provide the integrated measures for As exposure (Agahian et al., 1990; Gault et al., 2008). Nails were collected from each study subject as described previously (Schmitt et al., 2005). Hair samples with the length of about 1 cm were collected from the region of the head close to the scalp behind the ear by using a ceramic blade cutter and kept in polypropylene bottles (Mäki-Paakkanen et al., 1998). Nail and hair samples were cleaned by the method described by Chen et al. (1999). Samples were immersed in 1% Triton X-100, sonicated for 20 min, and then washed five times with milli-Q water. The washed samples were allowed to dry at 60° C overnight in a drying oven. Nail and hair samples were digested with concentrated nitric acid using a hot plate at 70° C for 15 min and 115° C for 15 min. After cooling, the samples were diluted with 1.0% nitric acid containing yttrium (10 ppb), and concentrations of As75 and Y79 in these samples were determined by ICP-MS (HP-4500, Agilent Technologies, Kanagawa, Japan). Accuracy of As measurement was verified by using a certified
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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” determined in triplicate by the above-mentioned digestion followed by ICPMS analysis was 34.9 ±2.35 μg/g (reference value, 36.7 μg/g). Water collection and As analysis. Study subjects identified the tube wells they used as their primary sources of drinking water. Water samples were collected for this study as described by Ali et al. (2010). Water samples from these tube wells were collected in acid-washed containers after the well was pumped for 5 min as previously described (Van Geen et al., 2008). Total As concentration in water samples was determined by ICP-MS after the addition of a solution of yttrium (10 ppb in 1.0% nitric acid) to all water samples as an internal standard for ICP-MS analysis. The ion signals for As and yttrium were monitored at m/z of 75 and 79, respectively. All samples were determined in triplicate and the average values were used for data analysis. The detection limit of As 75 was 30 ppt. River water (NMIJ CRM 7202-a No.347 National Institute of Advanced Industrial Science and Technology, Japan) was used as a CRM. The average value (mean ± SD) of As in the “river water” determined in triplicate by ICP-MS analysis was 1.06 ± 0.04 μg/L (reference value, 1.18 μg/L). Blood pressure measurement. The standard protocol for measuring blood pressure recommended by the World Health Organization was used in this study. After study subjects had rested for 20 min or longer, both systolic and diastolic blood pressures (SBP and DBP) were measured three times with a mercury sphygmomanometer with subjects sitting. SBP and DBP were defined at the first and fifth phase Korotkoff sounds, respectively. The average of three measurements was used for the analysis. Hypertension was defined as a SBP of ≥ 140 mm Hg and a DBP of ≥ 90 mm Hg on three repeated measurements. Collection of plasma. Fasting blood samples were collected from the study subjects. Blood samples (5–7 ml) were collected in EDTA-containing blood collection tubes from each individual by venipuncture. Whole blood was then placed immediately on ice and subsequently centrifuged at 1600 × g for 15 min at 4 °C. Plasma supernatant was then taken and stored at − 80 °C. Measurement of plasma Big ET-1. The plasma levels of Big ET-1 were measured using one-step sandwich enzyme immunoassay kit (Biomedica, Divischgasse, Austria). All standards and samples were analyzed in duplicate and the mean value was taken. On completion of the assay, the observed color change was read on a standard plate reader (Mikura Ltd. UK) and plasma values were calculated by extrapolation from a standard curve. A separate standard curve was constructed for each immunoassay batch. Statistical analysis. Statistical analysis for this study was performed by using the Statistical Packages for Social Sciences (SPSS) software. Characteristics of the study subjects from As-endemic and nonendemic areas were analyzed by Independent Samples T-test and Chi-square test. Because of skewed distributions, log transformation was performed for drinking water, hair and nail As concentrations. Log-transformed values were reconverted to antilogarithm forms in the table. The nature of associations between As exposure metrics (water, hair and nails) and plasma Big ET-1 levels were evaluated through analysis of scatter plots. Subsequently, bivariate associations between different exposure metrics and plasma Big ET-1 levels were examined using Pearson correlation coefficient test. Before and after adjusting for covariates (age, sex, BMI, smoking, and hypertension), univariate linear regression analysis was performed to examine the associations between plasma Big ET-1 levels and As exposure metrics.
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Study subjects in the As-endemic area were split into tertile groups (low, medium and high) based on the three concentrations of each exposure metric with equal proportion through frequency test and study subjects in the non-endemic area were used as a reference group (lowest exposure group). Plasma Big ET-1 levels in lowest, low, medium and high exposure groups were analyzed by linear regression. Univariate linear regression analysis were performed for the comparison of Big ET-1 levels between hypertensive and normotensive, and skin lesion and without skin lesion groups of the study subjects in As-endemic areas. Results Descriptive characteristics of the study subjects Table 1 shows the characteristics of the study subjects in the As-endemic and non-endemic areas. Of the 304 participants 210 were from As-endemic areas and 94 from non-endemic area. Nonendemic individuals were selected for this study as a reference group. As concentrations in the drinking water, hair and nails of the study subjects in the As-endemic areas were approximately 60, 19 and 8 times higher, respectively than those of non-endemic control area. The average age of the study subjects in the As-endemic and non-endemic areas were 38.42 ± 12.15 and 35.77 ± 10.22 years, respectively. The SBP and DBP of the study subjects in the As-endemic areas were 122.67 ± 20.45 and 80.05 ± 11.84 mm Hg, respectively, whereas these were 111.49 ± 14.20 and 71.12 ± 9.65 mm Hg, respectively for the non-endemic population. In the As-endemic areas, there were 106 male and 104 female study subjects, whereas in the nonendemic area, these were 53 and 41, respectively. Most of the male study subjects in both As-endemic and non-endemic areas were farmers, whereas most of the female study subjects were house wives. Socioeconomic characteristics (occupation, monthly income and housing) of the study subjects from non-endemic and endemic areas were almost similar. The percentage of tobacco smokers in the As-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 Asendemic and non-endemic areas as generally Bangladeshi women do not smoke cigarette. The mean BMI of the study subjects in the Asendemic and non-endemic areas were 20.84 ± 3.37 and 21.37 ± 2.71, respectively. The average (mean ± SD) levels of plasma Big ET-1 in As-endemic and non-endemic population were 0.83 ± 0.23 and 0.57 ± 0.21 fmol/mL, respectively. The differences in Big ET-1 levels between As-endemic and non-endemic population were statistically significant (p b 0.001). Big ET-1 levels were 45.61% higher in Asendemic 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. A significant increase in plasma Big ET-1 levels was observed with the increasing concentrations of As in the drinking water (r = 0.428, p b 0.001, Fig. 1A). A similar relationship was also observed between Big ET-1 and hair As concentrations (r = 0.441, p b 0.001, Fig. 1B), and between Big ET-1 and nail As concentrations (r = 0.406, p b 0.001, Fig. 1C). Table 2 shows the association between As exposure metrics with plasma Big ET-1 levels through linear regression analysis. Before and after adjusting for covariates, we found that water As concentrations were significantly associated with plasma Big ET-1 levels. Similarly, hair and nail As concentrations also displayed significant positive association with plasma Big ET-1 levels. To investigate the exposure–response relationship between water As concentrations and plasma Big ET-1 levels, we evaluated plasma Big ET-1 levels in the tertile groups (low, medium and high) of the study subjects in the As-endemic areas compared with the non-endemic population
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Table 1 Descriptive characteristics of the study subjects in As-endemic and non-endemic areas. Parameters
All subjects
Non-endemic (reference subjects)
As-endemic
Total subjects (n) Sex (n) Male Female Age (mean ± SD) As concentration in drinking water (mean ± SD; μg/L) As concentration in hair (mean ± SD; μg/g) As concentration in nail (mean ± SD; μg/g) SBP (mean ± SD; mm Hg) DBP (mean ± SD; mm Hg) Occupation [n, (%)] Male Farmers Business Students Tailors Others Female Housewives Farm workers Students Others Education [n, (%)] No formal education Primary Secondary Higher Income/month (US$) House [n, (%)] Brick house with concrete roof (pakka) Brick house with corrugated tin roof Mud house with corrugated tin roof Straw house with corrugated tin roof Others Smoking [n, (%)] Yes No BMI (mean ± SD; kg/m2) Big ET-1 (mean ± SD; fmol/ml)
304
94
210
159 145 37.60 ± 11.63 133.65 ± 160.14 4.11 ± 5.83 6.77 ± 7.06 119.21 ± 19.42 77.29 ± 11.93
53 41 35.77 ± 10.22 3.19 ± 2.99 0.31 ± 0.21 1.20 ± 0.85 111.49 ± 14.20 71.12 ± 9.65
106 104 38.42 ± 12.15 192.05 ± 161.54⁎ 5.81 ± 6.31⁎ 9.27 ± 7.20⁎ 122.67 ± 20.45⁎ 80.05 ± 11.84⁎
134 (84.3) 2 (1.3) 14 (8.8) 4 (2.5) 5 (3)
41 (77.4) 0 9 (17) 0 3 (5.7)
93 (87.7) 2 (1.9) 5 (4.7) 4 (3.8) 2 (1.9)
137 (94.5) 5 (3.4) 2 (1.4) 1 (0.7)
41 (100) 0 0 0
96 (92.3) 5 (4.8) 2 (2) 1 (1)
127 (41.8) 109 (35.9) 51 (16.8) 17 (5.6) 23.83 ± 8.12
38 (40.4) 33 (35.1) 17 (18.1) 6 (6.4) 23.12 ± 7.34
89 (42.4) 76 (36.2) 34 (16.2) 11 (5.2) 24.15 ± 8.44
41 (13.5) 111 (36.5) 98 (32.2) 43 (14.1) 11 (3.6)
12 (12.8) 36 (38.3) 29 (30.9) 14 (14.9) 3 (3.3)
29 (13.8) 75 (35.7) 69 (32.9) 29 (13.8) 8 (3.8)
72 (23.7) 232 (76.3) 21.01 ± 3.18 0.75 ± 0.26
31 (33) 63 (67) 21.37 ± 2.71 0.57 ± 0.21
41 (19.5) 169 (80.5) 20.84 ± 3.37 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. ⁎ p b 0.001.
(lowest or reference group). Intriguingly, we found that plasma Big ET-1 (Table 3) levels were significantly increased in the higher exposure groups before and after adjusting for covariates compared to the lowest exposure group. Further, we explored the dose–response relationship between the internal exposure metrics (hair and nail As concentrations) and plasma Big ET-1 levels. Levels of plasma Big ET-1 were significantly higher in the higher exposure groups compared to the 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 factor for hypertension (Chen et al., 1995; Rahman et al., 1999), we next evaluated whether increased Big ET-1 levels, observed in the study subjects, were associated with their blood pressure or not. Study subjects in the As-endemic areas were split into two groups based on the status of hypertension. After adjusting for covariates (age, sex, BMI and smoking), the plasma Big ET-1 levels in the hypertensive group were 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 development 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 of such skin lesions remain unknown. Recent studies have suggested that hyperkeratosis and hyperpigmentation in skin can result from elevated levels of plasma ET-1 (Sacar et al., 2005; Vural et al., 2001). Therefore, for the first time, we investigated the relationship between plasma Big ET-1 levels and As-induced skin lesions in the study subjects of As-endemic areas. We found that the study subjects with skin lesions had a significantly higher level of plasma Big ET-1 (Table 4) than those without skin lesions. This result led us to make a hypothesis that increased plasma levels of Big ET-1 (by implication its final form — ET-1) might be one of the factors responsible for the development of skin lesions in As-endemic population. Discussion Significant gaps remain in the mechanistic understanding of As-induced disorders in humans including endothelial dysfunction. Increased plasma level of ET-1 is associated with endothelial activation or dysfunction. Previous studies have suggested that Big ET-1, the immediate biological precursor of ET-1, may be a more accurate indicator of the degree of activation of the endothelial system (Leveson et al., 1985; Nelson et al., 1998; Teng et al., 2006) compared to ET-1. ET-1 is a vasoconstrictor peptide derived mainly from vascular endothelial cells (Yanagisawa et al., 1988). Several studies have
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A
B
191
C
Plasma Big ET-1 (fmol/ml)
2
r = 0.406 p < 0.001
r = 0.441 p < 0.001
r = 0.428 p < 0.001 1.5
1
0.5
0-2
0
2
4 -2
Log water arsenic (µg/L)
-1
0
1
2 -1
0
1
2
Log nail arsenic (µg/g)
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 plasma Big ET-1 levels. Arsenic concentrations were used after log transformation. r- and p-values were from Pearson correlation coefficient test.
indicated that ET-1 has a wide range of physiological effects in the development of diseases including cardiovascular disease, different types of cancer and pigmentation of the skin (Best et al., 1999; Jiao et al., 2008; Lerman et al., 1995; Sacar et al., 2005; Simpson et al., 2000; Vural et al., 2001). Although As exposure targets endothelial signals in the development of pathogenesis, the effect of As exposure on the plasma Big ET-1 levels and its physiological implications have not yet been documented. In this study, we evaluated the plasma Big ET-1 levels in a population of As-endemic areas in Bangladesh and monitored their relationship with hypertension and skin lesions. We found that Big ET-1 levels of the study subjects in As-endemic areas were significantly higher than those (reference group) of the non-endemic area. Previous study suggested that As contents of hair and nail samples might be used as effective biomarkers for As exposure (Gault et al., 2008). In our previous study, we also showed that drinking water As levels were strongly correlated with hair and nail As levels (Ali et al., 2010). Similar and consistent results were observed in this study in the correlation between water and hair or nail As concentrations (data not shown). We found that plasma Big ET-1 levels were strongly associated with drinking water, hair and nail As concentrations (Fig. 1). The study subjects were separated into four groups based on the four concentrations of As in the drinking water where non-endemic study subjects (reference group) were considered as the lowest exposure group. Significantly higher levels of plasma Big ET-1 were observed in the higher exposure groups (Table 3) compared to the lowest exposure group (reference group). Since the relationship between water As and plasma Big ET1 levels suggested only an external exposure–response relationship, we next examined, the dose–response relationship using candidate biomarkers (hair and nails) of As exposure. Similar patterns as observed in the exposure-response relationship were also found in the
case of dose–response relationship. Even after adjusting for different covariates, water, hair and nail As showed significant effects in increasing plasma Big ET-1 levels which suggested that As exposure was an independent risk factor for the elevation of plasma Big ET-1. In this study, we found that SBP and DBP were significantly higher in the population exposed to As compared to the non-endemic reference group (Table 1). These results were in agreement with previous studies in which As exposure was associated with increased levels of blood pressure (Kwok et al., 2007; Yang et al., 2007). Raised concentrations of ET-1 and its precursor Big ET-1, provide an important indicator of heart failure, congestive heart disease (Pacher et al., 1993) and are related to pulmonary hypertension in patients 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 expression of prepro ET-1 in the endothelium of subcutaneous resistance arteries (Schiffrin et al., 1992). Interestingly, in this study, we also found that plasma Big ET-1 levels were significantly higher in the hypertensive group compared to the normotensive study subjects in the As-endemic areas (Table 4). After excluding the hypertensive study subjects (n = 47), all the different associations between As exposure and plasma Big ET-1 were significant (data not shown). Therefore, these findings were consistent with the notion that As exposure was responsible for inducing the elevation of plasma Big ET-1 which in turn might be responsible for hypertension. In spite of the more potent biological action of ET-1 than Big ET-1, we measured the plasma Big ET-1 level in this study, since active peptide, ET-1 is cleared more rapidly from the organism and its paracrine activity is not reflected by its blood concentrations (Wei et al., 1994). It has been shown that Big ET-1 has a longer half-life and slower clearance than ET-1. Synthesis of ET-1 is closely related to
Table 2 Association between As exposure and plasma Big ET-1 levels by linear regression analysis. After adjusting covariatesa
After adjusting covariatesb
Independent variables
Before adjusting covariates 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) Hair As (μg/g) Nail As (μg/g)
0.097 (0.074–0.120) 0.165 (0.127–0.203) 0.194 (0.144–0.243)
b0.001 b0.001 b0.001
0.095 (0.072–0.118) 0.164 (0.125–0.203) 0.191 (0.140–0.241)
b 0.001 b 0.001 b 0.001
0.080 (0.056–0.103) 0.143 (0.105–0.181) 0.162 (0.113–0.211)
b0.001 b0.001 b0.001
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) and (6, 297), respectively. a Adjusted for age, sex, BMI and smoking. b Adjusted for age, sex, BMI, smoking and blood pressure.
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Table 3 Dose–response relationship of plasma Big ET-1 levels in the lowest, low, medium and high exposure groups by univariate linear regression analysis. Independent variables
After adjusting covariatesa
Before adjusting covariates
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) Medium (88.6–242) High (243–546)
– 0.202 (0.133–0.271) 0.334 (0.264–0.403) 0.256 (0.188–0.324)
– b0.001 b0.001 b0.001
– 0.206 (0.136–0.276) 0.337 (0.266–0.407) 0.259 (0.190–0.328)
– b 0.001 b 0.001 b 0.001
– 0.188 (0.119–0.256) 0.296 (0.226–0.367) 0.219 (0.150–0.288)
– b0.001 b0.001 b0.001
Hair As (μg/g) Lowest (0.03–1.18) Low (0.13–2.37) Medium (2.40–5.59) High (5.66–37.24)
– 0.231 (0.161–0.301) 0.251 (0.181–0.321) 0.306 (0.237–0.375)
– b0.001 b0.001 b0.001
– 0.228 (0.156–0.300) 0.260 (0.189–0.330) 0.309 (0.238–0.379)
– b 0.001 b 0.001 b 0.001
– 0.201 (0.131–0.271) 0.216 (0.146–0.286) 0.275 (0.205–0.344)
– b0.001 b0.001 b0.001
Nail As (μg/g) Lowest (0.16–4.55) Low (0.65–4.77) Medium (4.87–10.86) High (10.93–37.42)
– 0.230 (0.160–0.300) 0.258 (0.188–0.327) 0.302 (0.233–0.372)
– b0.001 b0.001 b0.001
– 0.236 (0.165–0.307) 0.258 (0.187–0.329) 0.306 (0.235–0.376)
– b 0.001 b 0.001 b 0.001
– 0.214 (0.144–0.283) 0.214 (0.144–0.285) 0.267 (0.197–0.337)
– b0.001 b0.001 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.
Big ET-1 (Levin, 1995; Rubanyi and Polokoff, 1994). Measurement of Big ET-1 substantially assists interpretation of plasma endothelin levels (Plumpton et al., 1995, 1996). Therefore, increased plasma levels of Big ET-1 in As-endemic study participants observed in this study ultimately reflected an increase in plasma ET-1 levels. In this study, we did not explore the mechanisms by which As increases Big ET-1 levels. One possible mechanism is that As exposure could generate free radicals that might increase the expression of the mRNA of Big ET-1 since oxidative stress-mediated expression of ET-1 was reported previously (Michael et al., 1997). Physiological importance of ET-1 and its implication with disease progression have been well studied previously. Intravenous infusion of ET-1 into conscious rats causes an initial decrease in blood pressure 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 vasoconstrictive response in coronary circulation and may play a role in the etiology of coronary vasospasm (Best et al., 1999; Lerman et al., 1995). Plasma concentration of Big ET-1 has been implicated with heart failure (Rivera et al., 2005). Elevated plasma Big ET-1 levels were also observed in diabetes (Ergul, 2011) and cancers (Jiao et al., 2008; Simpson et al., 2000). Increased Big ET-1 levels observed in this study may provide new insights into As-induced development of cardiovascular diseases and cancers. Additionally, the findings of this study have identified Big ET-1 level as a potential biomarker and a therapeutic drug target for reducing the risk of cardiovascular diseases in As-endemic population. Further, we have demonstrated a simple link between Big ET-1 levels and skin lesion. In light of this finding, we hypothesize that the increased Big ET-1 is responsible for the skin lesions observed in As exposed populations. Since this
hypothesis is based on a very simple link between Big ET-1 levels and skin lesions, it needs to be tested through a more detailed and comprehensive study including large number of study subjects from high and low As exposed population with and without skin lesions. However, our hypothesis is consistent with previous studies on UVB-induced hyperpigmentation through ET-1 and other molecules (Hachiya et al., 2004; Murase et al., 2009) although these studies were not in relation to As exposure. Vural et al. (2001) have suggested that increased ET-1, amongst other factors, may be responsible for increase in hyperpigmentation, hyperkeratinisation and keratinocyte proliferation in actinic keratosis and basal cell carcinoma patients compared to a control group. They have suggested that the increased levels of ETs and nitric oxide may be responsible for the cytotoxic and mitogenic properties that may further cause such types of skin tumors to proliferate. More recently, it has been reported by Lan et al. (2005) that in basal cell carcinoma, an increased expression of ET-1 is responsible for the hyperpigmentation of this skin tumor. The major strengths of this study were 1) to show for the first time the effects of As exposure on plasma Big ET-1 levels, through monitoring three different exposure metrics (water, hair and nail As levels), in a study population who showed large variations in their As exposure levels and 2) to demonstrate a relationship of the increased plasma Big ET-1 levels in the As exposed population with hypertension and skin lesions. Although this study presents extensive epidemiological research demonstrating the effects of As exposure on plasma Big ET-1 and its association with hypertension and skin lesions, there are some limitations warranting further discussion. First, we showed the association between the As exposure and plasma Big ET-1 levels after adjusting for BMI, age, sex and smoking
Table 4 Association between plasma Big ET-1 levels with hypertension and skin lesions. Parameters
Categories
No. of study subjects
Plasma Big ET-1 (fmol/mL) (mean ± SD)
p-value
Blood pressure
Normotensive Hypertensive (−) Symptom (+) Symptom
163 47 33 177
0.80 ± 0.23 0.96 ± 0.19 0.75 ± 0.22 0.84 ± 0.23
p b 0.001a
Skin symptom
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.
p b 0.05b
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habits. However, there may be some other factors such as coexposure to other metals, insecticides or pesticides or other toxic substances or individual variations that could influence the Big ET-1 levels. If any accompanying metals (or other contaminants) could influence the association between As exposure and plasma Big ET-1, then they would also be expected to follow the same concentration gradients as As in the drinking water, hair and nails. This is unlikely, but more detailed and extensive study of the other metals and their association with Big ET-1 are required in future. Furthermore, the results of the study are consistent with the previous animal studies (Soucy et al., 2005; Yamaguchi et al., 2007) which have demonstrated that As exposure increases endothelin levels. We also found a dose–response relationship between As exposure and plasma Big ET-1 levels. When this is taken into consideration with the findings of the previous animal studies, a cause-effect relationship between As exposure and plasma Big ET-1 levels is highly plausible. Second, most of our study population was lean with regard to BMI. Third, the study subjects without skin lesions are much fewer than those with symptoms. So the results in relation to plasma Big ET1 and skin lesions would require further confirmation by increasing the sample size. Fourth, this study was designed to be crosssectional, but not prospective. Further verification of the causeeffect relationship between plasma Big ET-1 levels and hypertension, and skin lesions would require a cohort based study. Thus, the findings of the current study may not be generalizable to other study populations, given the possible different distributions of risk factors for endothelial dysfunction, hypertension and skin lesions that may influence the effect of As exposure. Nevertheless, increased plasma Big ET-1 levels with increasing levels of As and their correlation with hypertension and skin lesions may be significant for obtaining novel mechanistic insights into the endothelial dysfunction, hypertension and skin lesions induced by As. As far as we are aware, this research for the first time has demonstrated the interaction between As exposure and plasma Big ET-1 levels in human subjects who are chronically exposed to As. Furthermore, this study has demonstrated a dose–response relationship between As exposure and plasma Big ET-1. There are two important findings of this study that are noteworthy. Firstly, we have demonstrated that As exposed study subjects, who were hypertensive, had significantly higher levels of plasma Big ET-1, raising the possibility that As-induced hypertension is due to vasoconstrictor activity of plasma Big ET-1 or ET-1. Secondly, we have for the first time shown a relationship between the Big ET-1 and skin lesions suggesting a role 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 and Information & Communication Technology, Government of the People's Republic of Bangladesh [Grant No. 2007-2008/BS-135/176/1(5)], and also partially supported by a grant of TWAS (Grant No. Ref-09–153 RG/BIO/AS_I; UNESCO FR: 3240230321) and Heiwa Nakajima Foundation, Japan. References Agahian, B., Lee, J.S., Nelson, J.H., Johns, R.E., 1990. Arsenic levels in fingernails as a biological indicator of exposure to arsenic. Am. Ind. Hyg. Assoc. J. 51, 646–651. Ahsan, H., Perrin, M., Rahman, A., Parvez, F., Stute, M., Zheng, Y., Milton, A.H., BrandtRauf, P., van Geen, A., Graziano, J., 2000. Associations between drinking water and urinary arsenic levels and skin lesions in Bangladesh. J. Occup. Environ. Med. 42, 1195–1201.
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Elevated levels of plasma Big endothelin-1 and its relation to hypertension and skin lesions 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, Bangladesh Department of Applied Nutrition and Food Technology, Islamic University, Kushtia-7003, Bangladesh c Department of Medicine, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka, Bangladesh d Faculty of Health & Life Sciences, De Montfort University, Leicester, LE1 9BH, UK e Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770–8514, Japan b
a r t i c l e
i n f o
Article history: Received 31 October 2011 Revised 20 December 2011 Accepted 26 December 2011 Available online 5 January 2012 Keywords: Arsenic Big endothelin Hypertension Skin lesions Bangladesh
a b s t r a c t Chronic arsenic (As) exposure affects the endothelial system causing several diseases. Big endothelin-1 (Big ET-1), the biological precursor of endothelin-1 (ET-1) is a more accurate indicator of the degree of activation of the endothelial system. Effect of As exposure on the plasma Big ET-1 levels and its physiological implications have not yet been documented. We evaluated plasma Big ET-1 levels and their relation to hypertension and skin lesions in As exposed individuals in Bangladesh. A total of 304 study subjects from the As-endemic and non-endemic areas in Bangladesh were recruited for this study. As concentrations in water, hair and nails were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The plasma Big ET-1 levels were measured using a one-step sandwich enzyme immunoassay kit. Significant increase in Big ET-1 levels were observed with the increasing concentrations of As in drinking water, hair and nails. Further, before and after adjusting with different covariates, plasma Big ET-1 levels were found to be significantly associated with the water, hair and nail As concentrations of the study subjects. Big ET-1 levels were also higher in the higher exposure groups compared to the lowest (reference) group. Interestingly, we observed that Big ET-1 levels were significantly higher in the hypertensive and skin lesion groups compared to the normotensive and without skin lesion counterpart, respectively of the study subjects in As-endemic areas. Thus, this study demonstrated a novel dose–response relationship between As exposure and plasma Big ET-1 levels indicating 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 of exposure to As is through drinking water and As toxicity through drinking water represents one of the biggest catastrophes in history, affecting millions of people in the world (British Geological Survey and Department of Public Health and Engineering, 2001; World Health Organization, 2001). Bangladesh is one of the most severely affected regions in that approximately 80 million people consume water containing As levels greater than the 10 μg/L standard set by
Abbreviations: As, Arsenic; ICP-MS, Inductively Coupled Plasma Mass Spectroscopy; Big ET-1, Big endothelin-1. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205. Fax: + 880 721 750064. E-mail address: [email protected] (K. Hossain). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.12.023
the World Health Organization (Caldwell et al., 2003; Chowdhury, 2004). There is great temporal and spatial variation in groundwater As levels in different regions of Bangladesh. The precise reason for the high levels of As in groundwater is not fully understood but various theories have been proposed including role of microbial mobilization, anthropogenic activities, etc. (Harvey et al., 2002; Hossain et 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 inorganic arsenite (As III) and arsenate (As V). After consumption, inorganic As is converted to methylated derivatives. Although methylation of As has been commonly considered a mechanism for detoxification, recent studies have shown that methylated trivalent arsenicals are more toxic than inorganic As (Kligerman et al., 2003). Still there are no appropriate animal models available for investigating health effects of As. Therefore, significant uncertainties remain regarding mechanisms by which As exerts its deleterious health effects on the human population.
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Several epidemiological studies suggest exposure to As correlates with endothelial dysfunction (Chen et al., 2007; Lee et al., 2003). Endothelial dysfunction, defined as an imbalance of endotheliumderived vasoconstrictor and vasodilator substances, is a common denominator in the pathogenesis and progression of both macro and micro vascular complications. Previously it has been reported that As increases vasoconstrictor activity in rat blood vessels through the endothelium-dependent vasoconstrictor activity by compromising basal endothelium nitric acid function (Bilszta et al., 2006). Endothelin receptor A (ETA) and B (ETB) are two distinct receptors for endothelins. The endothelins are a family of peptides (ET-1, ET2, ET-3 and ET-4) consisting of 21 amino acids. They are produced by 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 (MacCumber et al., 1990) and the overall function of ET-1 is to increase blood pressure and vascular tone (Rubanyi and Polokoff, 1994; Wagner et al., 1992). ET-1 is formed from its biological precursor Big ET-1, a 38 amino acid-long peptide that, after synthesis in the cytoplasm, is cleaved 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 of 23 min (Hemsen et al., 1995) compared with only 3.5 min for ET-1. Circulating ET-1 may grossly underestimate local tissue concentrations (de Nucci et al., 1988), while Big ET-1 with its longer half-life has been implicated as a more sensitive indicator of endothelial system activation (Ishibashi et al., 1994; Jordan et al., 2005; Nelson et al., 1998; Teng et al., 2006). Previous studies have reported that As induces hypertension (Chen et al., 1995; Rahman et al., 1999). Hypertension is implicated with endothelial dysfunction (Lerman et al., 1995). However, association between As-induced hypertension and endothelial dysfunction has not yet been established. Therefore, in this study, we for the first time evaluated plasma Big ET-1 levels and their association with hypertension in human subjects who were exposed to As through drinking water in Bangladesh. Furthermore, prolonged exposure to As induces typical skin symptoms of arsenicosis such as melanosis and hyperkeratosis (Ahsan et al., 2000; Guha Mazumder et al., 1998). ET-1 has been reported to be involved in the hyperpigmentation of the skin (Hachiya et al., 2004; Murase et al., 2009; Vural et al., 2001) but the involvement of this molecule in As-induced skin lesions has not yet been investigated. To obtain further information in this area, we explored the relationship between Big ET-1 levels and skin lesions in As exposed population in our study group. Methods Study areas and study subjects. Ethical permission was obtained from the Bangladesh Medical Research Council, Mohakhali, Dhaka1212. As-endemic study areas for this study were chosen as described previously (Ali et al., 2010; Karim et al., 2010). The study areas included Marua in Jessore, Dutpatila, Jajri, Vultie and Kestopur in Chuadanga, and Bheramara in Kushtia district (north-west region) of Bangladesh. The prevalence of typical skin symptoms of arsenicosis such as melanosis on the skin, hyperkeratosis and hard patches on the palms of the hands and soles of the feet were very high among the local residents of the areas selected for sampling. Local residents 15–60 years of age were invited to participate in the study. Those who responded spontaneously were asked to convene at a specific location in their village for initial screening purposes in light of the exclusion criteria. The study subjects were selected from this convened group, irrespective of the presence or absence of skin symptoms or hypertension. Subsequently, individuals who exhibited symptoms were first identified by a general physician and then diagnosis was confirmed by a dermatologist. The physician involved in
this study carefully examined various parts of the body to confirm the presence of melanosis and hyperkeratosis. Adults who had lived for at least last 5 years in As-endemic areas of Bangladesh were recruited for this study. Attempt was made to match, as much as possible the following: age (individual matching), sex and socioeconomic parameters of Asendemic population and the non-endemic study subjects (as a reference group). The non-endemic study subjects with no history of As contamination in the drinking water were selected from Naogaon district (northern region) in Bangladesh. Socioeconomic parameters included occupation, education, monthly income and house types of the study subjects. We randomly selected some tube wells (drinking water sources) in the non-endemic area for the measurement of water As levels and we found that water in 96.6% of the tube wells contained very low levels of As (b10 μg/L) and water in the remaining 3.4% of the tube wells contained a little bit higher levels of As (b15 μg/L) but still the levels were lower than the maximum permissive limit of water As concentration (≤ 50 μg/L) for Bangladesh. As in the endemic areas, adults (15–60 years of ages) who had lived for at least last 5 years in non-endemic area were recruited for this study. Pregnant and lactating mothers and individuals who had a previous and recent history of drug addiction, hepatotoxic and antihypertensive drugs, malaria, kalazar, chronic alcoholism, previous and present history of hepatic, renal or severe cardiac diseases have been excluded from this study. Of the 219 individuals who were approached, 9 were excluded according to the exclusion criteria [i.e., study candidates (n = 4) who had resided in the As-endemic areas for less than 5 years, pregnant and lactating mothers (n = 3), and had hematological diseases (n = 2)]; thus, a total of 210 were finally recruited (95.89% participation rate) in the As-endemic areas. In non-endemic area, 3 [i.e., study candidates (n = 2) who had resided in the non-endemic area for less than 5 years, pregnant and lactating mother (n = 1)] from 97 individuals were excluded. The response rate of the individuals from the non-endemic area was 96.91%. Household visits were carried out to interview residents. Personal interview of the study subjects was carried out by the trained members of our research team using a standard questionnaire. Information obtained from the interview included the sources of water for drinking and daily house hold uses, water consumption history, socioeconomic status, occupation, food habit, cigarette smoking, alcohol intake, personal and family medical history, history of diseases, physiological complications, major diseases, previous physician's reports and Body Mass Index (BMI). During the sample collection process, we were blinded to As levels in the drinking water, and to those in the hair and nails of the study participants. We collected all blood and other specimens (including water samples) on the same day for each site. Collection of nail and hair samples, and analysis of As. As levels in finger nails and hair have been reported to provide the integrated measures for As exposure (Agahian et al., 1990; Gault et al., 2008). Nails were collected from each study subject as described previously (Schmitt et al., 2005). Hair samples with the length of about 1 cm were collected from the region of the head close to the scalp behind the ear by using a ceramic blade cutter and kept in polypropylene bottles (Mäki-Paakkanen et al., 1998). Nail and hair samples were cleaned by the method described by Chen et al. (1999). Samples were immersed in 1% Triton X-100, sonicated for 20 min, and then washed five times with milli-Q water. The washed samples were allowed to dry at 60° C overnight in a drying oven. Nail and hair samples were digested with concentrated nitric acid using a hot plate at 70° C for 15 min and 115° C for 15 min. After cooling, the samples were diluted with 1.0% nitric acid containing yttrium (10 ppb), and concentrations of As75 and Y79 in these samples were determined by ICP-MS (HP-4500, Agilent Technologies, Kanagawa, Japan). Accuracy of As measurement was verified by using a certified
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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” determined in triplicate by the above-mentioned digestion followed by ICPMS analysis was 34.9 ±2.35 μg/g (reference value, 36.7 μg/g). Water collection and As analysis. Study subjects identified the tube wells they used as their primary sources of drinking water. Water samples were collected for this study as described by Ali et al. (2010). Water samples from these tube wells were collected in acid-washed containers after the well was pumped for 5 min as previously described (Van Geen et al., 2008). Total As concentration in water samples was determined by ICP-MS after the addition of a solution of yttrium (10 ppb in 1.0% nitric acid) to all water samples as an internal standard for ICP-MS analysis. The ion signals for As and yttrium were monitored at m/z of 75 and 79, respectively. All samples were determined in triplicate and the average values were used for data analysis. The detection limit of As 75 was 30 ppt. River water (NMIJ CRM 7202-a No.347 National Institute of Advanced Industrial Science and Technology, Japan) was used as a CRM. The average value (mean ± SD) of As in the “river water” determined in triplicate by ICP-MS analysis was 1.06 ± 0.04 μg/L (reference value, 1.18 μg/L). Blood pressure measurement. The standard protocol for measuring blood pressure recommended by the World Health Organization was used in this study. After study subjects had rested for 20 min or longer, both systolic and diastolic blood pressures (SBP and DBP) were measured three times with a mercury sphygmomanometer with subjects sitting. SBP and DBP were defined at the first and fifth phase Korotkoff sounds, respectively. The average of three measurements was used for the analysis. Hypertension was defined as a SBP of ≥ 140 mm Hg and a DBP of ≥ 90 mm Hg on three repeated measurements. Collection of plasma. Fasting blood samples were collected from the study subjects. Blood samples (5–7 ml) were collected in EDTA-containing blood collection tubes from each individual by venipuncture. Whole blood was then placed immediately on ice and subsequently centrifuged at 1600 × g for 15 min at 4 °C. Plasma supernatant was then taken and stored at − 80 °C. Measurement of plasma Big ET-1. The plasma levels of Big ET-1 were measured using one-step sandwich enzyme immunoassay kit (Biomedica, Divischgasse, Austria). All standards and samples were analyzed in duplicate and the mean value was taken. On completion of the assay, the observed color change was read on a standard plate reader (Mikura Ltd. UK) and plasma values were calculated by extrapolation from a standard curve. A separate standard curve was constructed for each immunoassay batch. Statistical analysis. Statistical analysis for this study was performed by using the Statistical Packages for Social Sciences (SPSS) software. Characteristics of the study subjects from As-endemic and nonendemic areas were analyzed by Independent Samples T-test and Chi-square test. Because of skewed distributions, log transformation was performed for drinking water, hair and nail As concentrations. Log-transformed values were reconverted to antilogarithm forms in the table. The nature of associations between As exposure metrics (water, hair and nails) and plasma Big ET-1 levels were evaluated through analysis of scatter plots. Subsequently, bivariate associations between different exposure metrics and plasma Big ET-1 levels were examined using Pearson correlation coefficient test. Before and after adjusting for covariates (age, sex, BMI, smoking, and hypertension), univariate linear regression analysis was performed to examine the associations between plasma Big ET-1 levels and As exposure metrics.
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Study subjects in the As-endemic area were split into tertile groups (low, medium and high) based on the three concentrations of each exposure metric with equal proportion through frequency test and study subjects in the non-endemic area were used as a reference group (lowest exposure group). Plasma Big ET-1 levels in lowest, low, medium and high exposure groups were analyzed by linear regression. Univariate linear regression analysis were performed for the comparison of Big ET-1 levels between hypertensive and normotensive, and skin lesion and without skin lesion groups of the study subjects in As-endemic areas. Results Descriptive characteristics of the study subjects Table 1 shows the characteristics of the study subjects in the As-endemic and non-endemic areas. Of the 304 participants 210 were from As-endemic areas and 94 from non-endemic area. Nonendemic individuals were selected for this study as a reference group. As concentrations in the drinking water, hair and nails of the study subjects in the As-endemic areas were approximately 60, 19 and 8 times higher, respectively than those of non-endemic control area. The average age of the study subjects in the As-endemic and non-endemic areas were 38.42 ± 12.15 and 35.77 ± 10.22 years, respectively. The SBP and DBP of the study subjects in the As-endemic areas were 122.67 ± 20.45 and 80.05 ± 11.84 mm Hg, respectively, whereas these were 111.49 ± 14.20 and 71.12 ± 9.65 mm Hg, respectively for the non-endemic population. In the As-endemic areas, there were 106 male and 104 female study subjects, whereas in the nonendemic area, these were 53 and 41, respectively. Most of the male study subjects in both As-endemic and non-endemic areas were farmers, whereas most of the female study subjects were house wives. Socioeconomic characteristics (occupation, monthly income and housing) of the study subjects from non-endemic and endemic areas were almost similar. The percentage of tobacco smokers in the As-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 Asendemic and non-endemic areas as generally Bangladeshi women do not smoke cigarette. The mean BMI of the study subjects in the Asendemic and non-endemic areas were 20.84 ± 3.37 and 21.37 ± 2.71, respectively. The average (mean ± SD) levels of plasma Big ET-1 in As-endemic and non-endemic population were 0.83 ± 0.23 and 0.57 ± 0.21 fmol/mL, respectively. The differences in Big ET-1 levels between As-endemic and non-endemic population were statistically significant (p b 0.001). Big ET-1 levels were 45.61% higher in Asendemic 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. A significant increase in plasma Big ET-1 levels was observed with the increasing concentrations of As in the drinking water (r = 0.428, p b 0.001, Fig. 1A). A similar relationship was also observed between Big ET-1 and hair As concentrations (r = 0.441, p b 0.001, Fig. 1B), and between Big ET-1 and nail As concentrations (r = 0.406, p b 0.001, Fig. 1C). Table 2 shows the association between As exposure metrics with plasma Big ET-1 levels through linear regression analysis. Before and after adjusting for covariates, we found that water As concentrations were significantly associated with plasma Big ET-1 levels. Similarly, hair and nail As concentrations also displayed significant positive association with plasma Big ET-1 levels. To investigate the exposure–response relationship between water As concentrations and plasma Big ET-1 levels, we evaluated plasma Big ET-1 levels in the tertile groups (low, medium and high) of the study subjects in the As-endemic areas compared with the non-endemic population
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Table 1 Descriptive characteristics of the study subjects in As-endemic and non-endemic areas. Parameters
All subjects
Non-endemic (reference subjects)
As-endemic
Total subjects (n) Sex (n) Male Female Age (mean ± SD) As concentration in drinking water (mean ± SD; μg/L) As concentration in hair (mean ± SD; μg/g) As concentration in nail (mean ± SD; μg/g) SBP (mean ± SD; mm Hg) DBP (mean ± SD; mm Hg) Occupation [n, (%)] Male Farmers Business Students Tailors Others Female Housewives Farm workers Students Others Education [n, (%)] No formal education Primary Secondary Higher Income/month (US$) House [n, (%)] Brick house with concrete roof (pakka) Brick house with corrugated tin roof Mud house with corrugated tin roof Straw house with corrugated tin roof Others Smoking [n, (%)] Yes No BMI (mean ± SD; kg/m2) Big ET-1 (mean ± SD; fmol/ml)
304
94
210
159 145 37.60 ± 11.63 133.65 ± 160.14 4.11 ± 5.83 6.77 ± 7.06 119.21 ± 19.42 77.29 ± 11.93
53 41 35.77 ± 10.22 3.19 ± 2.99 0.31 ± 0.21 1.20 ± 0.85 111.49 ± 14.20 71.12 ± 9.65
106 104 38.42 ± 12.15 192.05 ± 161.54⁎ 5.81 ± 6.31⁎ 9.27 ± 7.20⁎ 122.67 ± 20.45⁎ 80.05 ± 11.84⁎
134 (84.3) 2 (1.3) 14 (8.8) 4 (2.5) 5 (3)
41 (77.4) 0 9 (17) 0 3 (5.7)
93 (87.7) 2 (1.9) 5 (4.7) 4 (3.8) 2 (1.9)
137 (94.5) 5 (3.4) 2 (1.4) 1 (0.7)
41 (100) 0 0 0
96 (92.3) 5 (4.8) 2 (2) 1 (1)
127 (41.8) 109 (35.9) 51 (16.8) 17 (5.6) 23.83 ± 8.12
38 (40.4) 33 (35.1) 17 (18.1) 6 (6.4) 23.12 ± 7.34
89 (42.4) 76 (36.2) 34 (16.2) 11 (5.2) 24.15 ± 8.44
41 (13.5) 111 (36.5) 98 (32.2) 43 (14.1) 11 (3.6)
12 (12.8) 36 (38.3) 29 (30.9) 14 (14.9) 3 (3.3)
29 (13.8) 75 (35.7) 69 (32.9) 29 (13.8) 8 (3.8)
72 (23.7) 232 (76.3) 21.01 ± 3.18 0.75 ± 0.26
31 (33) 63 (67) 21.37 ± 2.71 0.57 ± 0.21
41 (19.5) 169 (80.5) 20.84 ± 3.37 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. ⁎ p b 0.001.
(lowest or reference group). Intriguingly, we found that plasma Big ET-1 (Table 3) levels were significantly increased in the higher exposure groups before and after adjusting for covariates compared to the lowest exposure group. Further, we explored the dose–response relationship between the internal exposure metrics (hair and nail As concentrations) and plasma Big ET-1 levels. Levels of plasma Big ET-1 were significantly higher in the higher exposure groups compared to the 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 factor for hypertension (Chen et al., 1995; Rahman et al., 1999), we next evaluated whether increased Big ET-1 levels, observed in the study subjects, were associated with their blood pressure or not. Study subjects in the As-endemic areas were split into two groups based on the status of hypertension. After adjusting for covariates (age, sex, BMI and smoking), the plasma Big ET-1 levels in the hypertensive group were 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 development 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 of such skin lesions remain unknown. Recent studies have suggested that hyperkeratosis and hyperpigmentation in skin can result from elevated levels of plasma ET-1 (Sacar et al., 2005; Vural et al., 2001). Therefore, for the first time, we investigated the relationship between plasma Big ET-1 levels and As-induced skin lesions in the study subjects of As-endemic areas. We found that the study subjects with skin lesions had a significantly higher level of plasma Big ET-1 (Table 4) than those without skin lesions. This result led us to make a hypothesis that increased plasma levels of Big ET-1 (by implication its final form — ET-1) might be one of the factors responsible for the development of skin lesions in As-endemic population. Discussion Significant gaps remain in the mechanistic understanding of As-induced disorders in humans including endothelial dysfunction. Increased plasma level of ET-1 is associated with endothelial activation or dysfunction. Previous studies have suggested that Big ET-1, the immediate biological precursor of ET-1, may be a more accurate indicator of the degree of activation of the endothelial system (Leveson et al., 1985; Nelson et al., 1998; Teng et al., 2006) compared to ET-1. ET-1 is a vasoconstrictor peptide derived mainly from vascular endothelial cells (Yanagisawa et al., 1988). Several studies have
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A
B
191
C
Plasma Big ET-1 (fmol/ml)
2
r = 0.406 p < 0.001
r = 0.441 p < 0.001
r = 0.428 p < 0.001 1.5
1
0.5
0-2
0
2
4 -2
Log water arsenic (µg/L)
-1
0
1
2 -1
0
1
2
Log nail arsenic (µg/g)
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 plasma Big ET-1 levels. Arsenic concentrations were used after log transformation. r- and p-values were from Pearson correlation coefficient test.
indicated that ET-1 has a wide range of physiological effects in the development of diseases including cardiovascular disease, different types of cancer and pigmentation of the skin (Best et al., 1999; Jiao et al., 2008; Lerman et al., 1995; Sacar et al., 2005; Simpson et al., 2000; Vural et al., 2001). Although As exposure targets endothelial signals in the development of pathogenesis, the effect of As exposure on the plasma Big ET-1 levels and its physiological implications have not yet been documented. In this study, we evaluated the plasma Big ET-1 levels in a population of As-endemic areas in Bangladesh and monitored their relationship with hypertension and skin lesions. We found that Big ET-1 levels of the study subjects in As-endemic areas were significantly higher than those (reference group) of the non-endemic area. Previous study suggested that As contents of hair and nail samples might be used as effective biomarkers for As exposure (Gault et al., 2008). In our previous study, we also showed that drinking water As levels were strongly correlated with hair and nail As levels (Ali et al., 2010). Similar and consistent results were observed in this study in the correlation between water and hair or nail As concentrations (data not shown). We found that plasma Big ET-1 levels were strongly associated with drinking water, hair and nail As concentrations (Fig. 1). The study subjects were separated into four groups based on the four concentrations of As in the drinking water where non-endemic study subjects (reference group) were considered as the lowest exposure group. Significantly higher levels of plasma Big ET-1 were observed in the higher exposure groups (Table 3) compared to the lowest exposure group (reference group). Since the relationship between water As and plasma Big ET1 levels suggested only an external exposure–response relationship, we next examined, the dose–response relationship using candidate biomarkers (hair and nails) of As exposure. Similar patterns as observed in the exposure-response relationship were also found in the
case of dose–response relationship. Even after adjusting for different covariates, water, hair and nail As showed significant effects in increasing plasma Big ET-1 levels which suggested that As exposure was an independent risk factor for the elevation of plasma Big ET-1. In this study, we found that SBP and DBP were significantly higher in the population exposed to As compared to the non-endemic reference group (Table 1). These results were in agreement with previous studies in which As exposure was associated with increased levels of blood pressure (Kwok et al., 2007; Yang et al., 2007). Raised concentrations of ET-1 and its precursor Big ET-1, provide an important indicator of heart failure, congestive heart disease (Pacher et al., 1993) and are related to pulmonary hypertension in patients 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 expression of prepro ET-1 in the endothelium of subcutaneous resistance arteries (Schiffrin et al., 1992). Interestingly, in this study, we also found that plasma Big ET-1 levels were significantly higher in the hypertensive group compared to the normotensive study subjects in the As-endemic areas (Table 4). After excluding the hypertensive study subjects (n = 47), all the different associations between As exposure and plasma Big ET-1 were significant (data not shown). Therefore, these findings were consistent with the notion that As exposure was responsible for inducing the elevation of plasma Big ET-1 which in turn might be responsible for hypertension. In spite of the more potent biological action of ET-1 than Big ET-1, we measured the plasma Big ET-1 level in this study, since active peptide, ET-1 is cleared more rapidly from the organism and its paracrine activity is not reflected by its blood concentrations (Wei et al., 1994). It has been shown that Big ET-1 has a longer half-life and slower clearance than ET-1. Synthesis of ET-1 is closely related to
Table 2 Association between As exposure and plasma Big ET-1 levels by linear regression analysis. After adjusting covariatesa
After adjusting covariatesb
Independent variables
Before adjusting covariates 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) Hair As (μg/g) Nail As (μg/g)
0.097 (0.074–0.120) 0.165 (0.127–0.203) 0.194 (0.144–0.243)
b0.001 b0.001 b0.001
0.095 (0.072–0.118) 0.164 (0.125–0.203) 0.191 (0.140–0.241)
b 0.001 b 0.001 b 0.001
0.080 (0.056–0.103) 0.143 (0.105–0.181) 0.162 (0.113–0.211)
b0.001 b0.001 b0.001
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) and (6, 297), respectively. a Adjusted for age, sex, BMI and smoking. b Adjusted for age, sex, BMI, smoking and blood pressure.
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Table 3 Dose–response relationship of plasma Big ET-1 levels in the lowest, low, medium and high exposure groups by univariate linear regression analysis. Independent variables
After adjusting covariatesa
Before adjusting covariates
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) Medium (88.6–242) High (243–546)
– 0.202 (0.133–0.271) 0.334 (0.264–0.403) 0.256 (0.188–0.324)
– b0.001 b0.001 b0.001
– 0.206 (0.136–0.276) 0.337 (0.266–0.407) 0.259 (0.190–0.328)
– b 0.001 b 0.001 b 0.001
– 0.188 (0.119–0.256) 0.296 (0.226–0.367) 0.219 (0.150–0.288)
– b0.001 b0.001 b0.001
Hair As (μg/g) Lowest (0.03–1.18) Low (0.13–2.37) Medium (2.40–5.59) High (5.66–37.24)
– 0.231 (0.161–0.301) 0.251 (0.181–0.321) 0.306 (0.237–0.375)
– b0.001 b0.001 b0.001
– 0.228 (0.156–0.300) 0.260 (0.189–0.330) 0.309 (0.238–0.379)
– b 0.001 b 0.001 b 0.001
– 0.201 (0.131–0.271) 0.216 (0.146–0.286) 0.275 (0.205–0.344)
– b0.001 b0.001 b0.001
Nail As (μg/g) Lowest (0.16–4.55) Low (0.65–4.77) Medium (4.87–10.86) High (10.93–37.42)
– 0.230 (0.160–0.300) 0.258 (0.188–0.327) 0.302 (0.233–0.372)
– b0.001 b0.001 b0.001
– 0.236 (0.165–0.307) 0.258 (0.187–0.329) 0.306 (0.235–0.376)
– b 0.001 b 0.001 b 0.001
– 0.214 (0.144–0.283) 0.214 (0.144–0.285) 0.267 (0.197–0.337)
– b0.001 b0.001 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.
Big ET-1 (Levin, 1995; Rubanyi and Polokoff, 1994). Measurement of Big ET-1 substantially assists interpretation of plasma endothelin levels (Plumpton et al., 1995, 1996). Therefore, increased plasma levels of Big ET-1 in As-endemic study participants observed in this study ultimately reflected an increase in plasma ET-1 levels. In this study, we did not explore the mechanisms by which As increases Big ET-1 levels. One possible mechanism is that As exposure could generate free radicals that might increase the expression of the mRNA of Big ET-1 since oxidative stress-mediated expression of ET-1 was reported previously (Michael et al., 1997). Physiological importance of ET-1 and its implication with disease progression have been well studied previously. Intravenous infusion of ET-1 into conscious rats causes an initial decrease in blood pressure 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 vasoconstrictive response in coronary circulation and may play a role in the etiology of coronary vasospasm (Best et al., 1999; Lerman et al., 1995). Plasma concentration of Big ET-1 has been implicated with heart failure (Rivera et al., 2005). Elevated plasma Big ET-1 levels were also observed in diabetes (Ergul, 2011) and cancers (Jiao et al., 2008; Simpson et al., 2000). Increased Big ET-1 levels observed in this study may provide new insights into As-induced development of cardiovascular diseases and cancers. Additionally, the findings of this study have identified Big ET-1 level as a potential biomarker and a therapeutic drug target for reducing the risk of cardiovascular diseases in As-endemic population. Further, we have demonstrated a simple link between Big ET-1 levels and skin lesion. In light of this finding, we hypothesize that the increased Big ET-1 is responsible for the skin lesions observed in As exposed populations. Since this
hypothesis is based on a very simple link between Big ET-1 levels and skin lesions, it needs to be tested through a more detailed and comprehensive study including large number of study subjects from high and low As exposed population with and without skin lesions. However, our hypothesis is consistent with previous studies on UVB-induced hyperpigmentation through ET-1 and other molecules (Hachiya et al., 2004; Murase et al., 2009) although these studies were not in relation to As exposure. Vural et al. (2001) have suggested that increased ET-1, amongst other factors, may be responsible for increase in hyperpigmentation, hyperkeratinisation and keratinocyte proliferation in actinic keratosis and basal cell carcinoma patients compared to a control group. They have suggested that the increased levels of ETs and nitric oxide may be responsible for the cytotoxic and mitogenic properties that may further cause such types of skin tumors to proliferate. More recently, it has been reported by Lan et al. (2005) that in basal cell carcinoma, an increased expression of ET-1 is responsible for the hyperpigmentation of this skin tumor. The major strengths of this study were 1) to show for the first time the effects of As exposure on plasma Big ET-1 levels, through monitoring three different exposure metrics (water, hair and nail As levels), in a study population who showed large variations in their As exposure levels and 2) to demonstrate a relationship of the increased plasma Big ET-1 levels in the As exposed population with hypertension and skin lesions. Although this study presents extensive epidemiological research demonstrating the effects of As exposure on plasma Big ET-1 and its association with hypertension and skin lesions, there are some limitations warranting further discussion. First, we showed the association between the As exposure and plasma Big ET-1 levels after adjusting for BMI, age, sex and smoking
Table 4 Association between plasma Big ET-1 levels with hypertension and skin lesions. Parameters
Categories
No. of study subjects
Plasma Big ET-1 (fmol/mL) (mean ± SD)
p-value
Blood pressure
Normotensive Hypertensive (−) Symptom (+) Symptom
163 47 33 177
0.80 ± 0.23 0.96 ± 0.19 0.75 ± 0.22 0.84 ± 0.23
p b 0.001a
Skin symptom
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.
p b 0.05b
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habits. However, there may be some other factors such as coexposure to other metals, insecticides or pesticides or other toxic substances or individual variations that could influence the Big ET-1 levels. If any accompanying metals (or other contaminants) could influence the association between As exposure and plasma Big ET-1, then they would also be expected to follow the same concentration gradients as As in the drinking water, hair and nails. This is unlikely, but more detailed and extensive study of the other metals and their association with Big ET-1 are required in future. Furthermore, the results of the study are consistent with the previous animal studies (Soucy et al., 2005; Yamaguchi et al., 2007) which have demonstrated that As exposure increases endothelin levels. We also found a dose–response relationship between As exposure and plasma Big ET-1 levels. When this is taken into consideration with the findings of the previous animal studies, a cause-effect relationship between As exposure and plasma Big ET-1 levels is highly plausible. Second, most of our study population was lean with regard to BMI. Third, the study subjects without skin lesions are much fewer than those with symptoms. So the results in relation to plasma Big ET1 and skin lesions would require further confirmation by increasing the sample size. Fourth, this study was designed to be crosssectional, but not prospective. Further verification of the causeeffect relationship between plasma Big ET-1 levels and hypertension, and skin lesions would require a cohort based study. Thus, the findings of the current study may not be generalizable to other study populations, given the possible different distributions of risk factors for endothelial dysfunction, hypertension and skin lesions that may influence the effect of As exposure. Nevertheless, increased plasma Big ET-1 levels with increasing levels of As and their correlation with hypertension and skin lesions may be significant for obtaining novel mechanistic insights into the endothelial dysfunction, hypertension and skin lesions induced by As. As far as we are aware, this research for the first time has demonstrated the interaction between As exposure and plasma Big ET-1 levels in human subjects who are chronically exposed to As. Furthermore, this study has demonstrated a dose–response relationship between As exposure and plasma Big ET-1. There are two important findings of this study that are noteworthy. Firstly, we have demonstrated that As exposed study subjects, who were hypertensive, had significantly higher levels of plasma Big ET-1, raising the possibility that As-induced hypertension is due to vasoconstrictor activity of plasma Big ET-1 or ET-1. Secondly, we have for the first time shown a relationship between the Big ET-1 and skin lesions suggesting a role 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 and Information & Communication Technology, Government of the People's Republic of Bangladesh [Grant No. 2007-2008/BS-135/176/1(5)], and also partially supported by a grant of TWAS (Grant No. Ref-09–153 RG/BIO/AS_I; UNESCO FR: 3240230321) and Heiwa Nakajima Foundation, Japan. References Agahian, B., Lee, J.S., Nelson, J.H., Johns, R.E., 1990. Arsenic levels in fingernails as a biological indicator of exposure to arsenic. Am. Ind. Hyg. Assoc. J. 51, 646–651. Ahsan, H., Perrin, M., Rahman, A., Parvez, F., Stute, M., Zheng, Y., Milton, A.H., BrandtRauf, P., van Geen, A., Graziano, J., 2000. Associations between drinking water and urinary arsenic levels and skin lesions in Bangladesh. J. Occup. Environ. Med. 42, 1195–1201.
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