Introduction

Arsenic is a metalloid present in our natural environment. Arsenic toxicity created a great health concern by affecting millions of people in the world including Bangladesh, where people are mainly exposed to arsenic through drinking water (1-3). Long time exposure to arsenic results in its accumulation in hair, nails, muscles and skin (4). This leads to various complications and diseases manifested by cancer (4), diabetes (5), hypertension (6), hepatic damage (7), peripheral neuropathy and multiple vascular diseases, weight loss (1) and miscarriage (8). Arsenic is also known to affect various tissues/organs including liver and heart of human and animal causing increase in the level of various enzymes including LDH, ALT and SGPT (7,9-11). Arsenic toxicity is thought to be caused by the signals generated due to its reaction with sulfhydryl groups of various enzymes and proteins followed by their cross-linking. This cross-linking of various proteins may activate potential intracellular signaling pathways that ultimately lead to arsenic-mediated adverse effects (12,13).
The current available treatments for arsenicosis patients are thiol containing chelating agents such as meso 2,3-dimercaptosuccinic acid (DMSA), 2,3 dimercaptopropane-1-sulfonate (DMPS) or British Anti Lewisite (BAL; 2,3-dimercaprol) (14). However, use of these drugs is still limited due to various accompanying side effects such as hepatotoxicity, renal toxicity, headache, nausea, vomiting, high blood pressure, pain in stomach and chest, anxiety, etc (15). In contrast to chemical drugs, plants are usually less toxic and mostly free from adverse side effects (16). It has been reported that seed powder of M.oleifera reduced uptake of arsenic in kidney, liver and brain (17). Garlic extracts is also reported to revert high level of Reactive Oxygen Species (ROS) in hepatic tissue generated by arsenic (18).
In our experiment we used water Hyacinth (Eichhornia crassipes) Root Powder (HRP) for possible remediation of arsenic-mediated effects in mice. Water hyacinth is a free-floating weed and obtains nutrients from the water by its fibrous roots. The root possesses some mechanisms through which it can chelate various heavy metals (19). It is one of the most studied aquatic plants that can accumulate pollutants especially heavy metals (20-22). A previous report demonstrated the removal of more than 90% of arsenic from a solution (200 μg/L arsenic) within one hour of administrating non-living dried HRP (23). In addition, intraperitoneal injection of ethanolic extract of hyacinth root in arsenic-exposed rat has been shown to reduce arsenic accumulation in various organs (24). The present study has been used to investigate whether HRP could reduce or neutralize arsenic-mediated toxic effects in mice model. Our data have demonstrated that HRP might be a good alternative for remediation of adverse health effects of arsenic.

Materials and Methods

Animal maintenance
Swiss albino male mice (6 weeks of age) of average body weight were purchased from Animal Division of International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). The mice were randomly selected and kept in plastic cages with wood-cobe bedding (5 mice/cage). After five days of acclimation, mice were divided into four groups namely control, hyacinth (Hy), arsenic (As) and arsenic-hyacinth (As+Hy). Control mice were maintained with available supply of distilled water and normal mice feed. Hy was provided with HRP containing feed (8% wt/wt) and distilled water. Arsenic group was given normal feed and sodium arsenite (NaAsO2) containing water (0.2 g/L). The fourth group, As+Hy group was provided with HRP containing feed and arsenic containing water. These different groups of mice were maintained for 8 weeks. All these procedures and experiments using mice were undertaken following the ethical issues set by the Faculty of Biological Sciences, University of Dhaka, Bangladesh.

Preparation of HRP
Water hyacinths were collected from local lakes around Dhaka, Bangladesh and then the root parts were taken. The roots were washed several times with clean water, and finally de-germinated through washing with chlorine containing water. The roots were then cut into smaller pieces and sun dried for a week. HRP were obtained by grinding the dried roots. This powder (8% wt/wt) was mixed with mice feed purchased from ICDDR, B.

Determination of body weight and tail length of mice
Each mouse of a group was weighed every 2 weeks after starting the respective diet and recorded accordingly. A scale in cm was used to measure the tail length.

Blood collection and assay for blood cell morphology, LDH, ALP and SGPT
Surgical blade (size 11) was pinched sharply between ear and eye of mice. Blood came out as drops and collected in test tubes. Blood films were made by placing a drop of blood on one end of a slide, and using a spreader slide blood drop was dispersed over the slide's length. The slide was left to air dry, and fixed immersing it in methanol for 3 to 5 min. After fixation, the slide was stained with Leishman's stain (a mixture based on methylene blue and eosin) to distinguish the cells from each other. Blood cell morphology was then observed under inverted microscope (Kruss Optronic, D22297, Germany). Serum was prepared from collected blood, and LDH, ALP and SGPT were measured using commercially available assay kit (DiaSys Diagnostic Systems, Turkey; Biosystems S.A., Spain; and Human Diagnostic, Germany).

Statistical analysis
Data were statistically analyzed using Student’s t-test with GraphPad Prism 5.

Results

HRP rescued arsenic-mediated loss of body weight and tail wounding
Some previous studies reported that arsenic reduced normal growth and caused loss of body weight and tail wounding in experimental mice (25,26). Here, we investigated whether HRP could rescue the loss of body weight and other physical damage in mice caused by arsenic. At the beginning of the experiment, the average mice weights of control, hyacinth (Hy group), arsenic (As group) and arsenic-hyacinth (As+Hy group) were 30.12±0.58 g, 30.3±0.57 g, 30.82±1.17 g and 30.65±0.73 g, respectively. Weight of the mice in each groups were taken up to 8 weeks at every 2 weeks interval. After 8 weeks, the average body weight of control, Hy, As and As+Hy group were found 43.4±1.03 g, 42±1.84 g, 24.09±1.19 g and 30.7±1.12 g, respectively (Figures 1 and 2). The average body weight of the control mice was increased from 30.12 g to 43.4 g over 8 weeks. This normal increase of mice body weight was retarded after drinking arsenic containing water. A gradual weight loss in As group of mice was observed during those 8 weeks indicating arsenic-mediated growth retardation. Interestingly, HRP partially rescued growth retardation in arsenic exposed mice when supplemented in feed. Body weight of As+Hy mice was significantly higher at each 2 weeks interval (**p<0.05) (Figure 1). The growth pattern of control and Hy groups were found to be similar indicating that HRP alone had no visible effects on the growth of the mice.
Mice of each group were also investigated frequently to observe other physical changes caused by arsenic. Arsenic exposed mice were found relatively restless compared with control mice. After four weeks, As-group mice developed wound at their tail tips. The tails became gradually shorter due to cell death at the tail tip (Figure 3). As-Hy group of mice also showed tail reduction, however, this reduction was significantly less than that of As-group mice. This indicated that HRP might have some effects to neutralize arsenic toxicity that caused tail wounding.

HRP blocked distortion of arsenic-mediated blood cell morphology
The blood cells were then observed under microscope to see the effect of arsenic on cellular morphology. The blood cells of control group were found spherical in shape (Figure 4). However, this normal spherical shape of the cells was distorted in blood collected from arsenic exposed mice. Surprisingly, this distortion of cellular morphology was almost blocked in As-Hy mice. This result suggested that HRP could reduce arsenic-mediated stress causing distortion of blood cell morphology.

Arsenic-induced serum elevation of LDH, ALP and SGPT was reduced by HRP
Some previous reports showed that arsenic could affect heart and liver tissues and increase the level of LDH, ALP and SGPT in serum (7,9-11,27,28). We next investigated whether HRP could reduce the elevation of these serum enzymes in mice caused by arsenic. In arsenic-exposed mice, serum LDH level was found almost double (1268.15±18.98 U/L) when compared with control mice (686.5±17.04 U/L) (Figure 5). This result indicated possibilities of heart and other tissue damage in arsenic-exposed mice that might elevate the level of LDH in the serum. However, this serum LDH elevation was partially blocked (1189.88±22.84 U/L) in As-Hy group mice. The reduction of the enzyme level was found significant (**p<0.05) compared to arsenic-exposed mice (Figure 5). As-mice also showed a significant increase (***p<0.05) in serum ALP (458.5±11.6 U/L) and SGPT (152.02±10.43 U/L) levels compared to control (305.6±11.0 U/L and 92.14±5.11 U/L, respectively). Increased levels of ALP and SGPT refer that arsenic might cause liver damage resulting in release of these enzymes into the serum. Again, in case of As-Hy mice, HRP was found to partially reduce the elevation of serum ALP (409.25±19.15 U/L) and SGPT (126.75±10.14 U/L) values.

Discussion

Epidemiological studies from several arsenic endemic regions have shown that chronic exposure to arsenic can lead to a range of adverse health effects in human (1,2,29, 30). Arsenic can affect different organs including heart, liver, kidney, brain leading to various related diseases (31-33). The effect of arsenic in animal model studies has therefore created immense importance to find out mechanism of toxicity and ways of remediation. In the present study, decrease in body weight was found in arsenic-exposed mice. Arsenic is known to inhibit growth by interfering with various metabolic processes (34). Moreover, arsenic is also shown to induce death of various cells in vitro through activation of apoptotic pathway (13, 35). In our study, we also found that the arsenic-exposed mice developed wound at the tail tips. This tail wounding might be caused by activation of similar apoptotic pathways in mice.
Water hyacinth has been used as sustaining feed source for livestock
especially ruminant animal (36-38). More importantly, it has also been shown that water hyacinth leaf protein concentrate is nontoxic and nutritionally available for applications in food and feed, such as biscuits or seasonings industries (39). Hyacinth root is known to adsorb soluble arsenic in solution (22) and this phenomenon has been used for arsenic removal from contaminated water (20). However, the potentiality of hyacinth for managing arsenic toxicity in an animal model has never been explored before. We, for the first time, found that HRP prevented growth retardation and tail wounding of mice exposed to arsenic. In our experiment, it was also evident that the HRP has itself no toxic or growth retardation effect in mice. Some earlier studies have shown that arsenic can bind to animal and human hemoglobin and other blood cell proteins (40-43). This binding might cause distortion of blood cell shape/ morphology observed in our study (Figure 4). Importantly, HRP supplementation in diet almost restored the normal spherical shape of blood cells in As+Hy group mice. Not only that, HRP also caused partial reduction of arsenic-mediated increased levels of serum LDH, ALP and SGPT.

Conclusion

This study, therefore, indicated the efficacy of HRP in protecting arsenic-induced toxicity in mice. The exact mechanism of HRP action in neutralizing arsenic-induced toxic effects in vivo is still unclear, however, HRP might adsorb ingested arsenic in a way that might mimic arsenic removal from water shown earlier in in vitro studies (22,23,44). Further investigation is needed to explain the mode of HRP action to explore its use as a potential candidate to remediate arsenic effect.

Acknowledgement

This work was supported by a grant from the Ministry of Science and Information & Communication Technology of Bangladesh.