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Chain oxygen disorder in deoxygenated YBa2Cu3O7−δ thin films induced by light ion irradiation
Science of the Total Environment 409 (2010) 278–283
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Interaction between chronic arsenic exposure via drinking water and plasma lactate dehydrogenase activity Md. Rezaul Karim a,b,1, Kazi Abdus Salam b,1, Ekhtear Hossain b, Khairul Islam b, Nurshad Ali b, Abedul Haque b, Zahangir Alam Saud b, Tanzima Yeasmin b, Mostaque Hossain c, Hideki Miyataka d, Seiichiro Himeno d, Khaled Hossain b,⁎ a
Department of Applied Nutrition and Food Technology, Islamic University, Kushtia-7003, Bangladesh Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladesh c Department of Medicine, Rajshahi Medical College Hospital, Rajshahi-6000, Bangladesh d 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 29 July 2010 Received in revised form 29 September 2010 Accepted 1 October 2010 Available online 29 October 2010 Keywords: Arsenic exposure Lactate dehydrogenase Contaminated water
a b s t r a c t Arsenic is a potent environmental pollutant that has caused one of the largest public health poisonings in the history of human civilization, affecting tens of millions of people worldwide especially in Bangladesh. Lactate dehydrogenase (LDH) in blood plays an important role in predicting cell or organ damage and as an important clue to the diagnosis of a variety of cancers. However, effect of chronic arsenic exposure on the LDH level in blood has not yet been documented. Since the chronic arsenic exposure is associated with organ damages and multi-site cancers, this research aimed at assaying the plasma level of LDH activity in the population who were exposed to arsenic chronically in Bangladesh. A total of 185 individuals living in arsenic-exposed areas and 121 individuals living in non-exposed area in Bangladesh were recruited as study subjects. Arsenic content in drinking water, hair and nails were estimated by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and LDH activity was assayed by a spectrophotometer. Significant increase in LDH activity was observed with increasing concentrations of arsenic in water, hair and nails. Further, the study subjects were split into four groups based on the three ways of each exposure metrics (water, hair and nail arsenic concentrations) where the study subjects in the non-exposed area were used as a reference (lowest exposure) group. LDH activity was found to be increased in the higher exposure groups of water and hair arsenic concentrations. LDH activity was also increased at low to medium exposure groups of nail arsenic concentrations.Thus, the elevated plasma LDH activity might be helpful for the early prognosis of organ or tissue damage in the individuals who were exposed to arsenic chronically. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Exposure to arsenic contaminated drinking water represents one of the largest public health poisonings in the history of human civilization, affecting tens of millions of people worldwide (Chowdhury, 2004). The general population is exposed to inorganic and organic arsenic through water, food, occupation and other environmental sources. A significant number of toxicity cases have been already reported in the north-west region in Bangladesh and millions more are at risk for arsenic toxicity in the country (WHO, 1997; Chowdhury et al., 2000; BGS and DPHE, 2001; Khan et al., 2003). The situation is deteriorating as new cases of arsenic toxicity are still being reported in different parts of the country. Arsenic
⁎ Corresponding author. Tel.: + 880 721 750041 x4109 (office); fax: + 880 721 750064 (office). E-mail address: [email protected] (K. Hossain). 1 These authors contributed equally to this work. 0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2010.10.001
in drinking water is typically inorganic, and can be present either as As+ 3 (arsenite) or As+ 5 (arsenate). In Bangladesh, arsenic in ground water is primarily in the As+ 3 form (Zheng et al., 2004). As+ 3 forms which have a higher affinity for thiol groups (Vahter and Concha, 2001), are more cytotoxic and genotoxic than As+ 5. Individuals who accumulate the trivalent intermediates are thought to be of greater risk of arsenicinduced diseases (Rossman et al., 2004; Thomas et al., 2004). The major metabolic pathway of inorganic arsenic in humans is its methylation in liver. This methylation of arsenic is proved by the presence of monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in urine and bile (Li et al., 2008; Cui et al., 2004). Individuals who excrete relatively lower proportions of urinary DMA are at increased risk for many types of diseases (Chen et al., 2003a,b; Hsueh et al., 1997; Tseng et al., 2005; Yu et al., 2000). Arsenic toxicity induces dermatitis, multisite cancers, cardiovascular diseases, diabetes mellitus, immune disorders, peripheral neuropathy, liver damage, renal failure, and other ailments (Mazumder et al., 1998; Chen et al., 2007a; Meliker et al., 2007; Mumford et al., 2007; Tapio and Grosche, 2006; Vahidnia et
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al., 2008; Wang et al., 2002; Mazumder, 2005; Ali et al., 2010). In fact, arsenic affects almost all organs including lung, liver, heart and kidney. LDH is an enzyme (Stryer, 1982) found in the cells of many tissues, including the heart, liver, kidneys, skeletal muscle, brain, red blood cells and lungs. The total LDH can be further separated into five components (isozymes) labeled by number: LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5 (Calbreath, 1992). Each of the isotype is specifically present in specific tissue. LDH-1 is found mainly in the heart. LDH-2 is primarily associated with the system in the body that defends against infection. LDH-3 is found in the lungs and other tissues, LDH-4 in the kidney, placenta, and pancreas, and LDH-5 in liver and skeletal muscle. Certain diseases have classic patterns of elevated LDH isoenzyme levels. For example, an LDH-1 level higher than that of LDH-2 is indicative of a heart attack or injury; elevations of LDH-2 and LDH-3 indicate lung injury or disease; elevations of LDH-4 and LDH-5 indicate liver or muscle disease or both. LDH elevations in the blood can be measured from serum or plasma as total LDH or as LDH isoenzymes. Total LDH is the overall measurement of all five LDH isoenzymes found in the body. LDH in blood can be used for the prognosis of tissue damage and a variety of cancers. Furthermore, in monitoring the progress of diseases, LDH has been found to be relevant in establishing the survival duration and rate in Hodgkin's diseases and non-Hodgkin's lymphoma, and in the follow-up of ovarian dysgerminoma (Pressley et al., 1992). Several epidemiological studies showed the relationship between the alteration of several circulating parameters and arsenic exposures elucidating the mechanism and risk of arsenicosis (Ali et al., 2010; Chen et al., 2007a,b,c; Li et al., 2007). However, effect of chronic arsenic exposure on plasma LDH levels has not yet been documented. Since the chronic arsenic exposure is associated with organ damages and multi-site cancers, this research aimed at assaying the plasma level of LDH activity in the population exposed to arsenic chronically in Bangladesh. 2. Materials and methods 2.1. Study areas and study subjects Arsenic-exposed study areas and study subjects were selected as described in our previous study (Ali et al., 2010). Briefly, arsenicexposed study areas were chosen according to the arsenic affected area map (http://www.dchtrust.org/arsenic_map.htm) presented by the School of Environmental Studies (SOES), Jadavpur University, India and Dhaka Community Hospital, Bangladesh. The study areas included Marua in Jessore, Dutpatila, Jajri, Vultie and Kestopur in Chuadanga, and Bheramara in Kushtia district (north-west region) of Bangladesh. Prevalence of typical skin symptoms of arsenicosis such as melanosis on the skin, hyperkeratosis and hard patches on the palms and soles was very high in the local residents of these areas. Local residents 15– 60 years of ages in the arsenic-exposed areas were requested to attend a convenient place nearby locality. We selected the study subjects irrespective of symptoms, however, subjects with skin symptoms were identified as we described previously (Ali et al., 2010). Non-exposed study subjects (as a reference group) with no history of arsenic contamination in drinking water and age group between 15 and 60 years were from Naogaon (northern region) district in Bangladesh. We randomly selected some tube wells (drinking water sources) in the non-exposed area for the measurement of water arsenic level and we found that water of the 95.56% tube wells contained very low levels of arsenic (b10 μg/L) and the remaining 4.44% tube well water contained a little bit higher level of arsenic (b15 μg/L) but still the levels were much lower than the maximum permissive limit of water arsenic concentration (50 μg/L) for Bangladesh. Pregnant and lactating mother and the individuals who had previous and recent history of drug addiction, hepatotoxic drugs, malaria, kalazar, chronic alcoholism, previous and present history of hepatic, renal and severe cardiac diseases have been excluded from
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this study. Of the 196 individuals who were approached, 8 were excluded and 188 were primarily recruited (95.92% participation rate) in the arsenic-exposed areas. Finally, we further excluded 3 individuals after the collection of blood samples as they were found to be hepatitis B positive in our laboratory test. In non-exposed area, 4 from 125 individuals were excluded. The response rate of the individuals from the non-exposed area was 96.8%. We did not find any hepatitis B positive individuals in our laboratory test in nonexposed area. The subjects participated in this study gave their written consent. 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 household 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). 2.2. Ethical permission Ethical permission for this study was approved by the Bangladesh Medical Research Council, Mohakhali, Dhaka-1212. All sorts of confidentialities and rights of the study subjects were strictly maintained as per the guideline of the Bangladesh Medical Research Council. 2.3. Water collection and arsenic analysis Drinking water samples were collected as described previously by Ali et al. (2010). We selected only those tube wells that were being used by the local residents for their drinking water. Water samples from these tube wells were collected in acid-washed containers after the well was pumped for 5 min as previously described (Van green et al., 2008). Total arsenic concentration in water samples was determined by Inductively Coupled Plasma Mass Spectroscopy (ICPMS, HP-4500, Agilent Technologies, Kanagawa, Japan) 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 arsenic 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 As75 was 0.03 μg/L. River water (NMIJ CRM 7202-a No.347 National Institute of Advanced Industrial Science and Technology, Japan) was used as a certified reference material to check the accuracy of the arsenic level in the drinking water for this study. The average value (mean ± SD) of arsenic in the “river water” determined in triplicate by ICP-MS analysis was 1.06 ± 0.04 μg/L (reference value, 1.18 μg/L). 2.4. Collection of nail and hair samples, and analysis of arsenic poisoning Arsenic levels in nail and hair samples have been reported to provide the integrated measures for arsenic exposure (Agahian et al., 1990; Karagas et al., 1996). Nail samples 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 (Paakkanen et al., 1998). Nail and hair samples were cleaned by the method described by Chen et al. (1999). Briefly, 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
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were determined by ICP-MS. Determination of the accurate arsenic concentrations in these biological materials was verified by using a CRM “cod fish powder” (NMIJ CRM 7402-a, National Institute of Advanced Industrial Science and Technology, Japan). The average value (mean ± SD) of arsenic in “cod fish powder” determined 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). 2.5. Collection of blood plasma All study subjects were requested to gather in a convenient place nearby locality. Fasting blood samples were collected from the study subjects. Blood samples (5–7 mL) were collected from each individual by venipuncture in the EDTA-containing blood collection tubes. 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. 2.6. Examination of hepatitis B Hepatitis B was tested in all the collected plasma samples by using 3rd generation HBsAg ELISA test kit (Medivent Diagnostic & Co. LTD. Ireland) according to the manufacturer's protocol. 2.7. Measurement of plasma LDH activity Plasma LDH activity was measured using LDH kit according to the manufacturer's protocol (dds Diagnostic systems). The principle of colorimetric method is briefly described below. LDH catalyzes the following reaction: Pyruvate + NADH + H
þ LDH
⇄ L lactate + NADþ :
Decrease of the absorbance value at 340 nm, due to the NADH oxidation in NAD+, is directly proportional to the enzyme activity, which can be measured spectrophotometrically (Microlab 200, Vital Scientific, Dieren, Netherlands). All human samples were analyzed in triplicate, and then mean values were taken. 2.8. Statistical analysis Statistical analysis for this study was performed by using software of Statistical Packages for Social Sciences (SPSS). Differences of the characteristics between arsenic-exposed and non-exposed (reference) study subjects were analyzed by Independent Sample T-test. Correlations between LDH activity and arsenic concentrations in drinking water or hair or nails were analyzed by Spearman correlation coefficient test after log transformation. All study subjects were split into four (lowest, low, medium and high) groups based on the four concentrations of each exposure metric (drinking water, hair and nail arsenic concentrations). Non-exposed study subjects were in the lowest (reference) group, whereas, exposed population were in the low, medium and high exposure groups with equal proportions determined by frequency test. LDH activity in lowest, low, medium and high exposure groups was evaluated by one way ANOVA (Dunnett's T3 test) after log transformation of LDH values. Effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity were analyzed by linear regression model. Log-transformed water arsenic concentrations and LDH values were used in linear regression model. Best fit model was determined by AIC (Akaike Information Criteria) which was computed by the following formula: AIC = n ln ðRSS = nÞ + 2K; where n = sample size; RSS = residual sum of squares and K = number of parameters.
3. Results 3.1. Descriptive characteristics of the study subjects Descriptive characteristics of the arsenic-exposed and non-exposed (reference) study subjects are summarized in Table 1. After excluding 3 individuals who showed hepatitis B positive in our laboratory test, the total number of study subjects was 306. The numbers of study subjects in arsenic-exposed and non-exposed areas were 185 and 121, respectively. The mean (Mean ± SE) arsenic concentrations in drinking water, hair and nails were significantly higher (p b 0.001) in the exposed individuals than in the non-exposed individuals. The average ages of the study subjects in the arsenic-exposed and non-exposed areas were 37.89 ± 12.08 and 35.26 ± 11.72 years, respectively. In arsenic-exposed areas, there were 112 male and 73 female study subjects, whereas in non-exposed area, there were 55 and 66, respectively. The majority of the male study participants in the arsenic-exposed and non-exposed areas were occupationally farmers (81.25% and 81.82%, respectively) and the majority of the female participants in the arsenic-exposed and non-exposed areas were housewives (87.67% and 89.39%, respectively). The percentage of smokers in the exposed and non-exposed study
Table 1 Descriptive characteristics of the study subjects in arsenic-exposed and non-exposed study subjects. Parameters
All subjects
Non-exposed (reference) subjects
Exposed subjects
Total subjects (n) As concentration in drinking water (mean ± SE; μg/L) Median (IQR) As concentration in hair (mean ± SE; μg/g) Median (IQR) As concentration in nail (mean ± SE; μg/g) Median (IQR) Mean age ± SD (range; years) Median (IQR) Sex (n) Male Female Occupation [n, (%)] Male Farmers Business Students Tailors Rickshaw pullers Others Female Housewives Farm workers Students Others Smoking (%) Yes No Years of drinking water consumption from tube wells (mean ± SD) Skin symptoms (%) (+) symptoms (−) symptoms BMI (kg/m2) ± SD Median (IQR)
306 85.13 ± 5.97
121 3.01 ± 0.25
185 167.25 ± 11.69⁎
8.95 (165.46) 2.80 ± 0.24
2.31 (3.23) 0.44 ± 0.03
117 (238.55) 5.15 ± 0.44⁎
1.24 (3.27) 4.96 ± 0.31
0.35 (0.47) 1.36 ± 0.11
2.95 (4.04) 8.55 ± 0.51⁎
3.14 (7.01) 36.85 ± 11.99 (15–60) 35 (18)
1.01 (1.25) 35.26 ± 11.72 (15–60) 34 (18)
6.54 (8.01) 37.89 ± 12.08 (15–60) 35 (17)
167 139
55 66
112 73
136 (81.44) 3 (1.80) 10 (5.99) 2 (1.20) 2 (1.20) 14 (8.38)
45 (81.82) 0 6 (10.91) 0 0 4 (7.27)
91 (81.25) 3 (2.68) 4 (3.57) 2 (1.79) 2 (1.79) 10 (8.93)
123 (88.49) 13 (9.35) 2 (1.44) 1 (0.72)
59 (89.39) 6 (9.09) 1 (1.52) 0
64 (87.67) 7 (9.59) 1 (1.37) 1 (1.37)
26.17 73.83 16.29 ± 7.78
21.57 78.43 16.59 ± 6.72
30.77 69.23 15.99 ± 8.83
48.04 51.96 20.52 ± 3.19 20.23 (4.05)
– – 21.26 ± 2.98 20.80 (3.85)
79.46 20.54 19.94 ± 3.25⁎ 19.53 (3.71)
Data were presented as Mean ± SD or Mean ± SE (95% CI). BMI was calculated as body weight (kg) divided by height square (m2). Differences were analyzed by Independent Sample T-test. ⁎ p b 0.001.
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Fig. 1. Correlations between the plasma LDH activity and water or hair or nail arsenic concentrations. Effects of drinking water (A), hair (B) and nail (C) arsenic concentrations on plasma LDH activity. Arsenic concentrations and plasma LDH activity were used after log transformation. rs and p-values were from Spearman correlation coefficient test.
subjects was 30.77 and 21.57. In this study, we did not find any female smoker in the study participants. This is very reasonable because generally females in Bangladesh do not smoke cigarette. The mean BMI of the study subjects in arsenic-exposed and non-exposed areas was 19.94± 3.25 and 21.26± 2.98, respectively. We did not find any individual who was alcoholic. Moreover, 79.46% study subjects showed typical skin symptoms of arsenicosis in the arsenic-exposed areas. 3.2. Arsenic exposure and plasma LDH activity Fig. 1 shows the effect of arsenic exposure on LDH activity. A significant increase in plasma LDH activity was observed with the increasing concentrations of arsenic in drinking water (Fig. 1A) with a significant positive correlation (rs = 0.50, p b 0.001). A similar relationship was observed between the LDH activity and hair arsenic concentrations (rs = 0.45, p b 0.001, Fig. 1B), and between LDH activity and nail arsenic concentrations (rs = 0.37, p b 0.001, Fig. 1C). Further, as shown in Table 2 study subjects were split into four groups based on the four concentrations of each exposure metric (drinking water, hair and nail arsenic concentrations). We then compared the plasma LDH activity in between each group. Intriguingly, we found that plasma LDH activity was increased in the higher exposure groups compared to the lower groups of water arsenic
concentrations. An increasing pattern of LDH activity was also observed in the higher groups of hair arsenic concentrations. In the case of nail arsenic concentrations, similar pattern of the elevation of LDH activity was also observed with an exception between medium and high exposure groups. 3.3. Effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity Table 3 summarizes the results from linear regression analyses used to determine the effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity. Because water arsenic concentrations and plasma LDH had skewed distribution, these variables were log-transformed. Age and sex were excluded from the best fit model which was determined by Akaike Information Criteria (AIC). Water arsenic concentrations, BMI and smoking showed significant effects (p b 0.001 for BMI, p b 0.01 for smoking and p b 0.001 for arsenic) on plasma LDH activity. 4. Discussion LDH in blood is often used as a marker for tissue damage. Although it has been well established that prolonged exposure of arsenic causes
Table 2 Relationship between LDH activity and arsenic exposure. Variables
Arsenic concentrations [Mean ± SD] No. of study subject LDH activity (U/L) [Mean ± SD] Log LDH activity (U/L) [Mean ± SD]
Water arsenic concentrations Lowest (reference group): 0.03–13.36 μg/L 3.01 ± 2.74 Low: 0.03–62.81 μg/L 14.49 ± 16.38 Medium: 68.2–198 μg/L 121.85 ± 40.03 High: 214–546 μg/L 353.72 ± 112.08
121 60 61 64
175.84 ± 61.16 209.68 ± 87.68 222.31 ± 66.60 299.91 ± 117.14
2.22 ± 0.14 2.29 ± 0.16a⁎⁎⁎ 2.33 ± 0.12a⁎ 2.45 ± 0.16a⁎,b⁎,c⁎
Hair arsenic concentrations Lowest (reference group): 0.03–1.87 μg/g Low: 0.13–2.09 μg/g Medium: 2.13–4.66 μg/g High: 4.68–34.5 μg/g
0.44 ± 0.33 1.22 ± 0.54 3.11 ± 0.75 11.07 ± 7.30
121 61 62 62
175.84 ± 61.16 213.08 ± 65.40 249.29 ± 111.57 272.28 ± 111.50
2.22 ± 0.14 2.31 ± 0.13a⁎ 2.36 ± 0.18a⁎ 2.41 ± 0.15a⁎,b⁎⁎
Nail arsenic concentrations Lowest (reference group): 0.12–7.54 μg/g Low: 0.11–4.54 μg/g Medium: 4.59–10.34 μg/g High: 10.52–37.42 μg/g
1.36 ± 1.22 2.47 ± 1.20 6.80 ± 1.63 16.33 ± 6.11
121 62 61 62
175.84 ± 61.16 225.65 ± 82.60 258.81 ± 103.29 250.93 ± 113.81
2.22 ± 0.14 2.33 ± 0.15a⁎ 2.38 ± 0.17a⁎ 2.37 ± 0.16a⁎
All p-values were from one way ANOVA after log transformation of LDH activity. *p b 0.001, **p b 0.01, ***p b 0.05. a Significantly different from lowest group. b Significantly different from low group. c Significantly different from medium group.
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Table 3 Evaluation of the effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity through linear regression analyses. Parameters
Coefficient (B)
BMI Water Smoking Constant
0.010 0.080 0.065 1.884
R-square 0.283
t-value
p-value
3.876 10.050 2.837 27.478
0.000 0.000 0.005 0.000
BMI, water arsenic, sex, age and smoking were adjusted in linear regression analysis. Best fit model was presented according to the Akaike Information Criteria (AIC) from which age and sex were finally excluded.
multiple organ dysfunctions and tissue breakdown, however, association between chronic arsenic exposure and plasma LDH activity has not yet been documented clearly. In this study, we evaluated the plasma LDH activity in the population exposed to arsenic in Bangladesh which was found to be correlated with the levels of arsenic in drinking water, hair and nails (Fig. 1). To our knowledge, this is the first systemic analysis of the association between the chronic arsenic exposure and the plasma LDH activity in humans. Further, the study subjects were split into four groups based on the arsenic concentrations in water, hair and nails, and we found that plasma LDH activity was increased in higher groups (Table 2) of water and hair arsenic concentrations. LDH activity was also higher in all the higher (from low to medium) groups of nail arsenic concentrations except the high versus medium exposure groups. LDH activity was almost unchanged or a little bit decreased in high group compared to the medium group. This discrepancy was probably due to the close distribution pattern of nail arsenic concentrations between medium and high exposure groups. In our previous study, we demonstrated that nail and hair arsenic concentrations were strongly correlated with the drinking water arsenic levels in the population exposed to arsenic in Bangladesh (Ali et al., 2010). Therefore, consistent results across these three different exposure metrics explicitly stated the potential role of chronic arsenic exposure in increasing LDH activity in humans (Table 2). Reference value of the LDH kit used for this study was 240 U/L for both male and female. The mean level of LDH activity in high and some medium exposure groups was above the reference level (Table 2). Further, a clear and significant elevation of LDH activity in all the higher (low, medium and high) groups compared to the lowest (reference) group observed in this study (Table 2) suggested that arsenic-exposed populations were more susceptible in increasing plasma LDH activity than the non-exposed (reference) populations. Since several previous studies reported that BMI, age, sex, tobacco smoking and socio economic status might modify the health effects of arsenic toxicity (Watanabe et al., 2001; Maharjan et al., 2007; Mumford et al., 2007; Ahsan et al., 2006; Chen et al., 2007b), we, therefore, checked the effects of BMI, sex, age and smoking along with water arsenic concentrations on the LDH activity in linear regression analyses. BMI and smoking showed significant effects on LDH activity (Table 3). Effects of BMI and smoking on LDH activity observed in this study were consistent with the previous study (Jong, 2003; Anbarasi et al., 2005). Although BMI and smoking could influence the LDH activity, however, still water arsenic concentration showed the biggest contribution in the alteration of LDH activity. From the strong dose–response relationship and the association between plasma LDH activity and the three different ways of exposure metrics (water, hair and nail arsenic concentrations), we conclude that arsenic exposure is a major risk factor for the elevation of plasma LDH activity in humans. Although this study explicitly demonstrated the association between arsenic exposures and plasma LDH activity, there are a few potential limitations, which warrant further discussion. First, we selected the study subjects excluding those who had past and present history of myocardial infarction, severe renal problems, haemolytic
anaemia, hepatitis, hepatotoxic drugs and jaundice. We excluded 3 study subjects after performing laboratory examination for hepatitis B. Clinical doctor involved in this study examined carefully whether study subjects were suffering from jaundice, severe heart ailments and renal problem, however, we did not perform any laboratory examination except hepatitis B for further confirmation of ailments that could interfere plasma LDH activity. Second, LDH has five isoforms. Each of the isotype is present in specific tissue. Plasma LDH represents the total LDH. Although an elevated total LDH level in blood indicates cell and tissue damage, it does not indicate what particular part of the body is damaged. Further study is needed to detect the isotype(s) that are found to be increased in the plasma of the arsenic-exposed population. LDH is a true intracellular enzyme because of its high degree of tissue specificity where overall tissue concentrations of LDH are some 500 fold greater than serum level under normal circumstances (Sullivan and Alpers, 1971; Podlasek and McPherson, 1989). Generally high concentrations of LDH are found in liver, heart, kidney, erythrocyte and skeletal muscle (Calbreath, 1992). Consequently, diseases affecting those organs such as renal dysfunction, hepatic disorders, myocardial infarction and haemolysis, have been reported to be associated with significant elevation in total serum LDH activity. Additionally, elevated LDH activity has also been reported in a variety of cancers, e.g. small cell carcinoma of the lung, neuroblastoma and osteosarcoma. Such elevations have widely applied as diagnostic indices for kidney, liver, heart and red blood cell dysfunction and a variety of cancers (Wills, 1971; Timmis, 1993; González-Billalabeitia et al., 2009). Usually, if tissue damage occurs, LDH is leaked from the damaged tissue to blood where it is measured. Therefore, increased plasma LDH activity with the increasing exposure of arsenic demonstrated in this study might be useful for the early prognosis of lung, muscle, liver, kidney, heart or other tissue damage, and also be an important clue to the early diagnosis of many types of cancers. Either plasma or serum can be used to measure LDH in blood. Sample type (serum or plasma) to measure LDH may vary from one research group to another (Kornberg and Polliack, 1980; Enomoto et al., 2003) depending on the study design. In this study, we measured plasma specimens because plasma is available for analysis sooner than serum, which needs around 30 min of clotting and hemolysis is less extensive in plasma than in serum. As far as we are aware, this is the first systematic population based comprehensive study demonstrating the association between chronic arsenic exposure and the plasma LDH activity published to date. Given the findings reported here, we conclude that high level of plasma LDH activity in the arsenic-exposed population may be a good indicative for early prognosis of the organ or tissue damages and other ailments caused by arsenic. Strength of the study includes the large variation in arsenic exposure levels in the study population, uses of three different ways of arsenic exposure metrics to prove the association between arsenic exposure and plasma LDH activity, and increase level of LDH in higher exposure gradient groups. 5. Conclusions This research demonstrated a novel dose–response relationship between LDH activity and arsenic concentrations in water or hair or nails. Splitting the study subjects into four groups based on the water, hair and nail arsenic concentrations, we found that LDH activity was increased in higher exposure groups. Thus, elevated plasma LDH activity might be helpful for the early prognosis of organ or tissue damage in the individuals who were exposed to arsenic chronically. Acknowledgements This work was funded by the Ministry of Science and Information & Communication Technology, Government of the People's Republic
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of Bangladesh [Grant No. 2007–2008/BS-135/176/1(5)]. This work was also partially supported by The Academy of Sciences for the Developing World (TWAS) [Grant No. 07-177RG/BIO/AS; UNESCO FR: 3240184288]. References Agahian B, Lee JS, Nelson JH, Johns RE. Arsenic levels in fingernails as a biological indicator of exposure to arsenic. Am Ind Hyg Assoc J 1990;51:646–51. Ahsan H, Chen Y, Parvez F, Zablotska L, Argos M, Hussain I, et al. Arsenic exposure from drinking water and risk of premalignant skin lesions in Bangladesh: baseline results from the Health Effects of Arsenic Longitudinal Study. Am J Epidemiol 2006;163: 1138–48. Ali N, Hoque MA, Haque A, Salam KA, Karim MR, Rahman A, et al. Association between arsenic exposure and plasma cholinesterase activity: a population based study in Bangladesh. Environ Health 2010;9:36. Anbarasi K, Sabitha KE, Devi CSS. Lactate dehydrogenase isoenzyme patterns upon chronic exposure to cigarette smoke: protective effect of bacoside A. Environ Toxicol Pharmacol 2005;20:345–50. BGS (British Geological Survey) and DPHE (Department of Public Health Engineering, Bangladesh). Arsenic contamination of groundwater in Bangladesh. Vol 2: final report. In: Kinniburg DG, Smedley PL, editors. BGS technical report WC/00/19. Keyworth, UK: British Geological Survey; 2001. Available:http://www.bgs.ac.uk/ arsenic/Bangladesh/repots.htm [accessed 22 September 2010]. Calbreath DF. Clinical chemistry. Philadelphia: WB Saunders; 1992. Chen KL, Amarasiriwardena CJ, Christiani DC. Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry. Biol Trace Elem Res 1999;67:109–25. Chen YC, Guo YL, Su HJ, Hsueh YM, Smith TJ, Ryan LM, et al. Arsenic methylation and skin cancer risk in southwestern Taiwan. J Occup Environ Med 2003a;45:241–8. Chen YC, Su HJ, Guo YL, Hsueh YM, Smith TJ, Ryan LM, et al. Arsenic methylation and bladder cancer risk in Taiwan. Cancer Causes Control 2003b;14:303–10. Chen CJ, Wang SL, Chiou JM, Tseng CH, Chiou HY, Hsueh YM, et al. Arsenic and diabetes and hypertension in human populations. Toxicol Appl Pharmacol 2007a;222: 298–304. Chen Y, Santella RM, Kibriya MG, Wang Q, Kappil M, Verret WJ, et al. Association between arsenic exposure from drinking water and plasma levels of soluble cell adhesion molecules. Environ Health Perspect 2007b;115:1415–20. Chen Y, Hall M, Graziano JH, Slavkovich V, van Geen A, Parvez F, et al. A prospective study of blood selenium levels and the risk of arsenic-related premalignant skin lesions. Cancer Epidemiol Biomark Prev 2007c;16:207–13. Chowdhury AM. Arsenic crisis in Bangladesh. Sci Am 2004;291:86–91. Chowdhury UK, Biswas BK, Chowdhury TR, Samanta G, Mandal BK, Basu GC, et al. Groundwater arsenic contamination in Bangladesh and West Bengal, India. Environ Health Perspect 2000;108:393–7. Cui X, Kobayashi Y, Hayakawa T, Hirano S. Arsenic speciation in bile and urine following oral and intravenous exposure to inorganic and organic arsenics in rats. Toxicol Sci 2004;82:478–87. Enomoto T, Jiro U, Arata A, Takashi N, Shoji K. Diabetes mellitus may increase risk for idiopathic pulmonary fibrosis. Chest 2003;123:2007–11. González-Billalabeitia E, Hitt R, Fernández J, Conde E, Martínez-Tello F, Enríquez de Salamanca R, et al. Pre-treatment serum lactate dehydrogenase level is an important prognostic factor in high-grade extremity osteosarcoma. Clin Transl Oncol 2009;11:479–83. Hsueh YM, Chiou HY, Huang YL, Wu WL, Huang CC, Yang MH, et al. Serum betacarotene level, arsenic methylation capability and incidence of skin cancer. Cancer Epidemiol Biomark Prev 1997;6:589–96. Jong WC. Association between elevated serum hepatic enzyme activity and total body fat in obese humans. Ann Clin Lab Sci 2003;33:257–64. Karagas MR, Morris JS, Weiss JE, Spate V, Baskett C, Greenberg ER. Toenail samples as an indicator of drinking water arsenic exposure. Cancer Epidemiol Biomark Prev 1996;5:849–52. Khan MM, Sakauchi F, Sonoda T, Washio M, Mori M. Magnitude of arsenic toxicity in tube-well drinking water in Bangladesh and its adverse effects on human health including cancer: evidence from a review of the literature. Asian Pac J Cancer Prev 2003;4:7-14. Kornberg A, Polliack A. Serum lactic dehydrogenase (LDH) levels in acute leukemia: marked elevations in lymphoblastic leukemia. Blood 1980;56:351–5.
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Contents lists available at ScienceDirect
Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Interaction between chronic arsenic exposure via drinking water and plasma lactate dehydrogenase activity Md. Rezaul Karim a,b,1, Kazi Abdus Salam b,1, Ekhtear Hossain b, Khairul Islam b, Nurshad Ali b, Abedul Haque b, Zahangir Alam Saud b, Tanzima Yeasmin b, Mostaque Hossain c, Hideki Miyataka d, Seiichiro Himeno d, Khaled Hossain b,⁎ a
Department of Applied Nutrition and Food Technology, Islamic University, Kushtia-7003, Bangladesh Department of Biochemistry and Molecular Biology, Rajshahi University, Rajshahi-6205, Bangladesh c Department of Medicine, Rajshahi Medical College Hospital, Rajshahi-6000, Bangladesh d 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 29 July 2010 Received in revised form 29 September 2010 Accepted 1 October 2010 Available online 29 October 2010 Keywords: Arsenic exposure Lactate dehydrogenase Contaminated water
a b s t r a c t Arsenic is a potent environmental pollutant that has caused one of the largest public health poisonings in the history of human civilization, affecting tens of millions of people worldwide especially in Bangladesh. Lactate dehydrogenase (LDH) in blood plays an important role in predicting cell or organ damage and as an important clue to the diagnosis of a variety of cancers. However, effect of chronic arsenic exposure on the LDH level in blood has not yet been documented. Since the chronic arsenic exposure is associated with organ damages and multi-site cancers, this research aimed at assaying the plasma level of LDH activity in the population who were exposed to arsenic chronically in Bangladesh. A total of 185 individuals living in arsenic-exposed areas and 121 individuals living in non-exposed area in Bangladesh were recruited as study subjects. Arsenic content in drinking water, hair and nails were estimated by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and LDH activity was assayed by a spectrophotometer. Significant increase in LDH activity was observed with increasing concentrations of arsenic in water, hair and nails. Further, the study subjects were split into four groups based on the three ways of each exposure metrics (water, hair and nail arsenic concentrations) where the study subjects in the non-exposed area were used as a reference (lowest exposure) group. LDH activity was found to be increased in the higher exposure groups of water and hair arsenic concentrations. LDH activity was also increased at low to medium exposure groups of nail arsenic concentrations.Thus, the elevated plasma LDH activity might be helpful for the early prognosis of organ or tissue damage in the individuals who were exposed to arsenic chronically. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Exposure to arsenic contaminated drinking water represents one of the largest public health poisonings in the history of human civilization, affecting tens of millions of people worldwide (Chowdhury, 2004). The general population is exposed to inorganic and organic arsenic through water, food, occupation and other environmental sources. A significant number of toxicity cases have been already reported in the north-west region in Bangladesh and millions more are at risk for arsenic toxicity in the country (WHO, 1997; Chowdhury et al., 2000; BGS and DPHE, 2001; Khan et al., 2003). The situation is deteriorating as new cases of arsenic toxicity are still being reported in different parts of the country. Arsenic
⁎ Corresponding author. Tel.: + 880 721 750041 x4109 (office); fax: + 880 721 750064 (office). E-mail address: [email protected] (K. Hossain). 1 These authors contributed equally to this work. 0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2010.10.001
in drinking water is typically inorganic, and can be present either as As+ 3 (arsenite) or As+ 5 (arsenate). In Bangladesh, arsenic in ground water is primarily in the As+ 3 form (Zheng et al., 2004). As+ 3 forms which have a higher affinity for thiol groups (Vahter and Concha, 2001), are more cytotoxic and genotoxic than As+ 5. Individuals who accumulate the trivalent intermediates are thought to be of greater risk of arsenicinduced diseases (Rossman et al., 2004; Thomas et al., 2004). The major metabolic pathway of inorganic arsenic in humans is its methylation in liver. This methylation of arsenic is proved by the presence of monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in urine and bile (Li et al., 2008; Cui et al., 2004). Individuals who excrete relatively lower proportions of urinary DMA are at increased risk for many types of diseases (Chen et al., 2003a,b; Hsueh et al., 1997; Tseng et al., 2005; Yu et al., 2000). Arsenic toxicity induces dermatitis, multisite cancers, cardiovascular diseases, diabetes mellitus, immune disorders, peripheral neuropathy, liver damage, renal failure, and other ailments (Mazumder et al., 1998; Chen et al., 2007a; Meliker et al., 2007; Mumford et al., 2007; Tapio and Grosche, 2006; Vahidnia et
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al., 2008; Wang et al., 2002; Mazumder, 2005; Ali et al., 2010). In fact, arsenic affects almost all organs including lung, liver, heart and kidney. LDH is an enzyme (Stryer, 1982) found in the cells of many tissues, including the heart, liver, kidneys, skeletal muscle, brain, red blood cells and lungs. The total LDH can be further separated into five components (isozymes) labeled by number: LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5 (Calbreath, 1992). Each of the isotype is specifically present in specific tissue. LDH-1 is found mainly in the heart. LDH-2 is primarily associated with the system in the body that defends against infection. LDH-3 is found in the lungs and other tissues, LDH-4 in the kidney, placenta, and pancreas, and LDH-5 in liver and skeletal muscle. Certain diseases have classic patterns of elevated LDH isoenzyme levels. For example, an LDH-1 level higher than that of LDH-2 is indicative of a heart attack or injury; elevations of LDH-2 and LDH-3 indicate lung injury or disease; elevations of LDH-4 and LDH-5 indicate liver or muscle disease or both. LDH elevations in the blood can be measured from serum or plasma as total LDH or as LDH isoenzymes. Total LDH is the overall measurement of all five LDH isoenzymes found in the body. LDH in blood can be used for the prognosis of tissue damage and a variety of cancers. Furthermore, in monitoring the progress of diseases, LDH has been found to be relevant in establishing the survival duration and rate in Hodgkin's diseases and non-Hodgkin's lymphoma, and in the follow-up of ovarian dysgerminoma (Pressley et al., 1992). Several epidemiological studies showed the relationship between the alteration of several circulating parameters and arsenic exposures elucidating the mechanism and risk of arsenicosis (Ali et al., 2010; Chen et al., 2007a,b,c; Li et al., 2007). However, effect of chronic arsenic exposure on plasma LDH levels has not yet been documented. Since the chronic arsenic exposure is associated with organ damages and multi-site cancers, this research aimed at assaying the plasma level of LDH activity in the population exposed to arsenic chronically in Bangladesh. 2. Materials and methods 2.1. Study areas and study subjects Arsenic-exposed study areas and study subjects were selected as described in our previous study (Ali et al., 2010). Briefly, arsenicexposed study areas were chosen according to the arsenic affected area map (http://www.dchtrust.org/arsenic_map.htm) presented by the School of Environmental Studies (SOES), Jadavpur University, India and Dhaka Community Hospital, Bangladesh. The study areas included Marua in Jessore, Dutpatila, Jajri, Vultie and Kestopur in Chuadanga, and Bheramara in Kushtia district (north-west region) of Bangladesh. Prevalence of typical skin symptoms of arsenicosis such as melanosis on the skin, hyperkeratosis and hard patches on the palms and soles was very high in the local residents of these areas. Local residents 15– 60 years of ages in the arsenic-exposed areas were requested to attend a convenient place nearby locality. We selected the study subjects irrespective of symptoms, however, subjects with skin symptoms were identified as we described previously (Ali et al., 2010). Non-exposed study subjects (as a reference group) with no history of arsenic contamination in drinking water and age group between 15 and 60 years were from Naogaon (northern region) district in Bangladesh. We randomly selected some tube wells (drinking water sources) in the non-exposed area for the measurement of water arsenic level and we found that water of the 95.56% tube wells contained very low levels of arsenic (b10 μg/L) and the remaining 4.44% tube well water contained a little bit higher level of arsenic (b15 μg/L) but still the levels were much lower than the maximum permissive limit of water arsenic concentration (50 μg/L) for Bangladesh. Pregnant and lactating mother and the individuals who had previous and recent history of drug addiction, hepatotoxic drugs, malaria, kalazar, chronic alcoholism, previous and present history of hepatic, renal and severe cardiac diseases have been excluded from
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this study. Of the 196 individuals who were approached, 8 were excluded and 188 were primarily recruited (95.92% participation rate) in the arsenic-exposed areas. Finally, we further excluded 3 individuals after the collection of blood samples as they were found to be hepatitis B positive in our laboratory test. In non-exposed area, 4 from 125 individuals were excluded. The response rate of the individuals from the non-exposed area was 96.8%. We did not find any hepatitis B positive individuals in our laboratory test in nonexposed area. The subjects participated in this study gave their written consent. 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 household 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). 2.2. Ethical permission Ethical permission for this study was approved by the Bangladesh Medical Research Council, Mohakhali, Dhaka-1212. All sorts of confidentialities and rights of the study subjects were strictly maintained as per the guideline of the Bangladesh Medical Research Council. 2.3. Water collection and arsenic analysis Drinking water samples were collected as described previously by Ali et al. (2010). We selected only those tube wells that were being used by the local residents for their drinking water. Water samples from these tube wells were collected in acid-washed containers after the well was pumped for 5 min as previously described (Van green et al., 2008). Total arsenic concentration in water samples was determined by Inductively Coupled Plasma Mass Spectroscopy (ICPMS, HP-4500, Agilent Technologies, Kanagawa, Japan) 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 arsenic 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 As75 was 0.03 μg/L. River water (NMIJ CRM 7202-a No.347 National Institute of Advanced Industrial Science and Technology, Japan) was used as a certified reference material to check the accuracy of the arsenic level in the drinking water for this study. The average value (mean ± SD) of arsenic in the “river water” determined in triplicate by ICP-MS analysis was 1.06 ± 0.04 μg/L (reference value, 1.18 μg/L). 2.4. Collection of nail and hair samples, and analysis of arsenic poisoning Arsenic levels in nail and hair samples have been reported to provide the integrated measures for arsenic exposure (Agahian et al., 1990; Karagas et al., 1996). Nail samples 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 (Paakkanen et al., 1998). Nail and hair samples were cleaned by the method described by Chen et al. (1999). Briefly, 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
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were determined by ICP-MS. Determination of the accurate arsenic concentrations in these biological materials was verified by using a CRM “cod fish powder” (NMIJ CRM 7402-a, National Institute of Advanced Industrial Science and Technology, Japan). The average value (mean ± SD) of arsenic in “cod fish powder” determined 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). 2.5. Collection of blood plasma All study subjects were requested to gather in a convenient place nearby locality. Fasting blood samples were collected from the study subjects. Blood samples (5–7 mL) were collected from each individual by venipuncture in the EDTA-containing blood collection tubes. 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. 2.6. Examination of hepatitis B Hepatitis B was tested in all the collected plasma samples by using 3rd generation HBsAg ELISA test kit (Medivent Diagnostic & Co. LTD. Ireland) according to the manufacturer's protocol. 2.7. Measurement of plasma LDH activity Plasma LDH activity was measured using LDH kit according to the manufacturer's protocol (dds Diagnostic systems). The principle of colorimetric method is briefly described below. LDH catalyzes the following reaction: Pyruvate + NADH + H
þ LDH
⇄ L lactate + NADþ :
Decrease of the absorbance value at 340 nm, due to the NADH oxidation in NAD+, is directly proportional to the enzyme activity, which can be measured spectrophotometrically (Microlab 200, Vital Scientific, Dieren, Netherlands). All human samples were analyzed in triplicate, and then mean values were taken. 2.8. Statistical analysis Statistical analysis for this study was performed by using software of Statistical Packages for Social Sciences (SPSS). Differences of the characteristics between arsenic-exposed and non-exposed (reference) study subjects were analyzed by Independent Sample T-test. Correlations between LDH activity and arsenic concentrations in drinking water or hair or nails were analyzed by Spearman correlation coefficient test after log transformation. All study subjects were split into four (lowest, low, medium and high) groups based on the four concentrations of each exposure metric (drinking water, hair and nail arsenic concentrations). Non-exposed study subjects were in the lowest (reference) group, whereas, exposed population were in the low, medium and high exposure groups with equal proportions determined by frequency test. LDH activity in lowest, low, medium and high exposure groups was evaluated by one way ANOVA (Dunnett's T3 test) after log transformation of LDH values. Effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity were analyzed by linear regression model. Log-transformed water arsenic concentrations and LDH values were used in linear regression model. Best fit model was determined by AIC (Akaike Information Criteria) which was computed by the following formula: AIC = n ln ðRSS = nÞ + 2K; where n = sample size; RSS = residual sum of squares and K = number of parameters.
3. Results 3.1. Descriptive characteristics of the study subjects Descriptive characteristics of the arsenic-exposed and non-exposed (reference) study subjects are summarized in Table 1. After excluding 3 individuals who showed hepatitis B positive in our laboratory test, the total number of study subjects was 306. The numbers of study subjects in arsenic-exposed and non-exposed areas were 185 and 121, respectively. The mean (Mean ± SE) arsenic concentrations in drinking water, hair and nails were significantly higher (p b 0.001) in the exposed individuals than in the non-exposed individuals. The average ages of the study subjects in the arsenic-exposed and non-exposed areas were 37.89 ± 12.08 and 35.26 ± 11.72 years, respectively. In arsenic-exposed areas, there were 112 male and 73 female study subjects, whereas in non-exposed area, there were 55 and 66, respectively. The majority of the male study participants in the arsenic-exposed and non-exposed areas were occupationally farmers (81.25% and 81.82%, respectively) and the majority of the female participants in the arsenic-exposed and non-exposed areas were housewives (87.67% and 89.39%, respectively). The percentage of smokers in the exposed and non-exposed study
Table 1 Descriptive characteristics of the study subjects in arsenic-exposed and non-exposed study subjects. Parameters
All subjects
Non-exposed (reference) subjects
Exposed subjects
Total subjects (n) As concentration in drinking water (mean ± SE; μg/L) Median (IQR) As concentration in hair (mean ± SE; μg/g) Median (IQR) As concentration in nail (mean ± SE; μg/g) Median (IQR) Mean age ± SD (range; years) Median (IQR) Sex (n) Male Female Occupation [n, (%)] Male Farmers Business Students Tailors Rickshaw pullers Others Female Housewives Farm workers Students Others Smoking (%) Yes No Years of drinking water consumption from tube wells (mean ± SD) Skin symptoms (%) (+) symptoms (−) symptoms BMI (kg/m2) ± SD Median (IQR)
306 85.13 ± 5.97
121 3.01 ± 0.25
185 167.25 ± 11.69⁎
8.95 (165.46) 2.80 ± 0.24
2.31 (3.23) 0.44 ± 0.03
117 (238.55) 5.15 ± 0.44⁎
1.24 (3.27) 4.96 ± 0.31
0.35 (0.47) 1.36 ± 0.11
2.95 (4.04) 8.55 ± 0.51⁎
3.14 (7.01) 36.85 ± 11.99 (15–60) 35 (18)
1.01 (1.25) 35.26 ± 11.72 (15–60) 34 (18)
6.54 (8.01) 37.89 ± 12.08 (15–60) 35 (17)
167 139
55 66
112 73
136 (81.44) 3 (1.80) 10 (5.99) 2 (1.20) 2 (1.20) 14 (8.38)
45 (81.82) 0 6 (10.91) 0 0 4 (7.27)
91 (81.25) 3 (2.68) 4 (3.57) 2 (1.79) 2 (1.79) 10 (8.93)
123 (88.49) 13 (9.35) 2 (1.44) 1 (0.72)
59 (89.39) 6 (9.09) 1 (1.52) 0
64 (87.67) 7 (9.59) 1 (1.37) 1 (1.37)
26.17 73.83 16.29 ± 7.78
21.57 78.43 16.59 ± 6.72
30.77 69.23 15.99 ± 8.83
48.04 51.96 20.52 ± 3.19 20.23 (4.05)
– – 21.26 ± 2.98 20.80 (3.85)
79.46 20.54 19.94 ± 3.25⁎ 19.53 (3.71)
Data were presented as Mean ± SD or Mean ± SE (95% CI). BMI was calculated as body weight (kg) divided by height square (m2). Differences were analyzed by Independent Sample T-test. ⁎ p b 0.001.
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Fig. 1. Correlations between the plasma LDH activity and water or hair or nail arsenic concentrations. Effects of drinking water (A), hair (B) and nail (C) arsenic concentrations on plasma LDH activity. Arsenic concentrations and plasma LDH activity were used after log transformation. rs and p-values were from Spearman correlation coefficient test.
subjects was 30.77 and 21.57. In this study, we did not find any female smoker in the study participants. This is very reasonable because generally females in Bangladesh do not smoke cigarette. The mean BMI of the study subjects in arsenic-exposed and non-exposed areas was 19.94± 3.25 and 21.26± 2.98, respectively. We did not find any individual who was alcoholic. Moreover, 79.46% study subjects showed typical skin symptoms of arsenicosis in the arsenic-exposed areas. 3.2. Arsenic exposure and plasma LDH activity Fig. 1 shows the effect of arsenic exposure on LDH activity. A significant increase in plasma LDH activity was observed with the increasing concentrations of arsenic in drinking water (Fig. 1A) with a significant positive correlation (rs = 0.50, p b 0.001). A similar relationship was observed between the LDH activity and hair arsenic concentrations (rs = 0.45, p b 0.001, Fig. 1B), and between LDH activity and nail arsenic concentrations (rs = 0.37, p b 0.001, Fig. 1C). Further, as shown in Table 2 study subjects were split into four groups based on the four concentrations of each exposure metric (drinking water, hair and nail arsenic concentrations). We then compared the plasma LDH activity in between each group. Intriguingly, we found that plasma LDH activity was increased in the higher exposure groups compared to the lower groups of water arsenic
concentrations. An increasing pattern of LDH activity was also observed in the higher groups of hair arsenic concentrations. In the case of nail arsenic concentrations, similar pattern of the elevation of LDH activity was also observed with an exception between medium and high exposure groups. 3.3. Effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity Table 3 summarizes the results from linear regression analyses used to determine the effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity. Because water arsenic concentrations and plasma LDH had skewed distribution, these variables were log-transformed. Age and sex were excluded from the best fit model which was determined by Akaike Information Criteria (AIC). Water arsenic concentrations, BMI and smoking showed significant effects (p b 0.001 for BMI, p b 0.01 for smoking and p b 0.001 for arsenic) on plasma LDH activity. 4. Discussion LDH in blood is often used as a marker for tissue damage. Although it has been well established that prolonged exposure of arsenic causes
Table 2 Relationship between LDH activity and arsenic exposure. Variables
Arsenic concentrations [Mean ± SD] No. of study subject LDH activity (U/L) [Mean ± SD] Log LDH activity (U/L) [Mean ± SD]
Water arsenic concentrations Lowest (reference group): 0.03–13.36 μg/L 3.01 ± 2.74 Low: 0.03–62.81 μg/L 14.49 ± 16.38 Medium: 68.2–198 μg/L 121.85 ± 40.03 High: 214–546 μg/L 353.72 ± 112.08
121 60 61 64
175.84 ± 61.16 209.68 ± 87.68 222.31 ± 66.60 299.91 ± 117.14
2.22 ± 0.14 2.29 ± 0.16a⁎⁎⁎ 2.33 ± 0.12a⁎ 2.45 ± 0.16a⁎,b⁎,c⁎
Hair arsenic concentrations Lowest (reference group): 0.03–1.87 μg/g Low: 0.13–2.09 μg/g Medium: 2.13–4.66 μg/g High: 4.68–34.5 μg/g
0.44 ± 0.33 1.22 ± 0.54 3.11 ± 0.75 11.07 ± 7.30
121 61 62 62
175.84 ± 61.16 213.08 ± 65.40 249.29 ± 111.57 272.28 ± 111.50
2.22 ± 0.14 2.31 ± 0.13a⁎ 2.36 ± 0.18a⁎ 2.41 ± 0.15a⁎,b⁎⁎
Nail arsenic concentrations Lowest (reference group): 0.12–7.54 μg/g Low: 0.11–4.54 μg/g Medium: 4.59–10.34 μg/g High: 10.52–37.42 μg/g
1.36 ± 1.22 2.47 ± 1.20 6.80 ± 1.63 16.33 ± 6.11
121 62 61 62
175.84 ± 61.16 225.65 ± 82.60 258.81 ± 103.29 250.93 ± 113.81
2.22 ± 0.14 2.33 ± 0.15a⁎ 2.38 ± 0.17a⁎ 2.37 ± 0.16a⁎
All p-values were from one way ANOVA after log transformation of LDH activity. *p b 0.001, **p b 0.01, ***p b 0.05. a Significantly different from lowest group. b Significantly different from low group. c Significantly different from medium group.
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Table 3 Evaluation of the effects of BMI, water arsenic concentrations, sex, age and smoking on LDH activity through linear regression analyses. Parameters
Coefficient (B)
BMI Water Smoking Constant
0.010 0.080 0.065 1.884
R-square 0.283
t-value
p-value
3.876 10.050 2.837 27.478
0.000 0.000 0.005 0.000
BMI, water arsenic, sex, age and smoking were adjusted in linear regression analysis. Best fit model was presented according to the Akaike Information Criteria (AIC) from which age and sex were finally excluded.
multiple organ dysfunctions and tissue breakdown, however, association between chronic arsenic exposure and plasma LDH activity has not yet been documented clearly. In this study, we evaluated the plasma LDH activity in the population exposed to arsenic in Bangladesh which was found to be correlated with the levels of arsenic in drinking water, hair and nails (Fig. 1). To our knowledge, this is the first systemic analysis of the association between the chronic arsenic exposure and the plasma LDH activity in humans. Further, the study subjects were split into four groups based on the arsenic concentrations in water, hair and nails, and we found that plasma LDH activity was increased in higher groups (Table 2) of water and hair arsenic concentrations. LDH activity was also higher in all the higher (from low to medium) groups of nail arsenic concentrations except the high versus medium exposure groups. LDH activity was almost unchanged or a little bit decreased in high group compared to the medium group. This discrepancy was probably due to the close distribution pattern of nail arsenic concentrations between medium and high exposure groups. In our previous study, we demonstrated that nail and hair arsenic concentrations were strongly correlated with the drinking water arsenic levels in the population exposed to arsenic in Bangladesh (Ali et al., 2010). Therefore, consistent results across these three different exposure metrics explicitly stated the potential role of chronic arsenic exposure in increasing LDH activity in humans (Table 2). Reference value of the LDH kit used for this study was 240 U/L for both male and female. The mean level of LDH activity in high and some medium exposure groups was above the reference level (Table 2). Further, a clear and significant elevation of LDH activity in all the higher (low, medium and high) groups compared to the lowest (reference) group observed in this study (Table 2) suggested that arsenic-exposed populations were more susceptible in increasing plasma LDH activity than the non-exposed (reference) populations. Since several previous studies reported that BMI, age, sex, tobacco smoking and socio economic status might modify the health effects of arsenic toxicity (Watanabe et al., 2001; Maharjan et al., 2007; Mumford et al., 2007; Ahsan et al., 2006; Chen et al., 2007b), we, therefore, checked the effects of BMI, sex, age and smoking along with water arsenic concentrations on the LDH activity in linear regression analyses. BMI and smoking showed significant effects on LDH activity (Table 3). Effects of BMI and smoking on LDH activity observed in this study were consistent with the previous study (Jong, 2003; Anbarasi et al., 2005). Although BMI and smoking could influence the LDH activity, however, still water arsenic concentration showed the biggest contribution in the alteration of LDH activity. From the strong dose–response relationship and the association between plasma LDH activity and the three different ways of exposure metrics (water, hair and nail arsenic concentrations), we conclude that arsenic exposure is a major risk factor for the elevation of plasma LDH activity in humans. Although this study explicitly demonstrated the association between arsenic exposures and plasma LDH activity, there are a few potential limitations, which warrant further discussion. First, we selected the study subjects excluding those who had past and present history of myocardial infarction, severe renal problems, haemolytic
anaemia, hepatitis, hepatotoxic drugs and jaundice. We excluded 3 study subjects after performing laboratory examination for hepatitis B. Clinical doctor involved in this study examined carefully whether study subjects were suffering from jaundice, severe heart ailments and renal problem, however, we did not perform any laboratory examination except hepatitis B for further confirmation of ailments that could interfere plasma LDH activity. Second, LDH has five isoforms. Each of the isotype is present in specific tissue. Plasma LDH represents the total LDH. Although an elevated total LDH level in blood indicates cell and tissue damage, it does not indicate what particular part of the body is damaged. Further study is needed to detect the isotype(s) that are found to be increased in the plasma of the arsenic-exposed population. LDH is a true intracellular enzyme because of its high degree of tissue specificity where overall tissue concentrations of LDH are some 500 fold greater than serum level under normal circumstances (Sullivan and Alpers, 1971; Podlasek and McPherson, 1989). Generally high concentrations of LDH are found in liver, heart, kidney, erythrocyte and skeletal muscle (Calbreath, 1992). Consequently, diseases affecting those organs such as renal dysfunction, hepatic disorders, myocardial infarction and haemolysis, have been reported to be associated with significant elevation in total serum LDH activity. Additionally, elevated LDH activity has also been reported in a variety of cancers, e.g. small cell carcinoma of the lung, neuroblastoma and osteosarcoma. Such elevations have widely applied as diagnostic indices for kidney, liver, heart and red blood cell dysfunction and a variety of cancers (Wills, 1971; Timmis, 1993; González-Billalabeitia et al., 2009). Usually, if tissue damage occurs, LDH is leaked from the damaged tissue to blood where it is measured. Therefore, increased plasma LDH activity with the increasing exposure of arsenic demonstrated in this study might be useful for the early prognosis of lung, muscle, liver, kidney, heart or other tissue damage, and also be an important clue to the early diagnosis of many types of cancers. Either plasma or serum can be used to measure LDH in blood. Sample type (serum or plasma) to measure LDH may vary from one research group to another (Kornberg and Polliack, 1980; Enomoto et al., 2003) depending on the study design. In this study, we measured plasma specimens because plasma is available for analysis sooner than serum, which needs around 30 min of clotting and hemolysis is less extensive in plasma than in serum. As far as we are aware, this is the first systematic population based comprehensive study demonstrating the association between chronic arsenic exposure and the plasma LDH activity published to date. Given the findings reported here, we conclude that high level of plasma LDH activity in the arsenic-exposed population may be a good indicative for early prognosis of the organ or tissue damages and other ailments caused by arsenic. Strength of the study includes the large variation in arsenic exposure levels in the study population, uses of three different ways of arsenic exposure metrics to prove the association between arsenic exposure and plasma LDH activity, and increase level of LDH in higher exposure gradient groups. 5. Conclusions This research demonstrated a novel dose–response relationship between LDH activity and arsenic concentrations in water or hair or nails. Splitting the study subjects into four groups based on the water, hair and nail arsenic concentrations, we found that LDH activity was increased in higher exposure groups. Thus, elevated plasma LDH activity might be helpful for the early prognosis of organ or tissue damage in the individuals who were exposed to arsenic chronically. Acknowledgements This work was funded by the Ministry of Science and Information & Communication Technology, Government of the People's Republic
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