Effect of arsenic trioxide in drinking water on adult white rats
A significant surge in heavy metal contamination has been observed in drinking water due to various anthropogenic activities in the modern era. Heavy metal toxicity is known to cause various kinds of health issues but the mechanism is little understood. Arsenic is a Group I human carcinogen, and chronic arsenic exposure through drinking water is a major threat to human population. It is reported that the Indo-Bangladesh region drinks arsenic contaminant water above the permissible limits showing serious detrimental effects on the health of public. Arsenic is metabolised from lethal inorganic form to less lethal organic form as part of the detoxification mechanism. The highest concentration of arsenic accumulation is found in the liver and kidneys due to their role in the detoxification and elimination of arsenic. Arsenic induced toxicities have led to chronic illnesses like hypertension, cardiovascular disease, diabetes; organ dysfunctions; pregnancy related issues like impaired fetal growth, fetal loss during pregnancy, increased post-birth infant mortality; various types of malignancies and solid tumours, including lung, prostate, bladder, cancers, melanoma skin cancer; long-term memory loss and modification of hormonal regulation. Our study focusses on determining the effects of arsenic trioxide in drinking water on adult white rats by oral administration of sub lethal dose (1/10th of LD50) in rats for 48 h, 72 h and 96 h respectively. Arsenic mediated effects were studied by analyzing oxidative stress, liver functionality and histological morphology of hepatic tissue. Arsenic treated samples revealed abnormal levels of MDA, Catalase activity, GSH, alkaline phosphatase, SGOT/AST and SGPT/ALT when compared against untreated samples, suggesting the presence of arsenic induced oxidative stress in liver. In addition, alterations in histological morphology of arsenic treated liver confirmed the detrimental effects of this toxic element.
Keywords: arsenic trioxide, drinking water, oxidative stress, liver damage
|ATSDR||Agency for Toxic Substances and Disease Registry|
|TBARS||Thiobarbituric Acid Reactive Substances|
|SGOT||Serum Glutamate Oxaloacetate Transaminase|
|SGPT||Serum Glutamate Pyruvate Transaminase|
Chronic exposure to arsenic through drinking water is one of the major global issues. Countries like Taiwan, Bangladesh, Chile, India, and Argentina (Naujokas et al., 2013) have been consuming arsenic above the normal limits due to lack of awareness and improper regulation of public health safety. Reportedly, arsenic is ranked as one of the most hazardous chemicals by the U.S. Agency for Toxic Substances and Disease Registry (ATSDR) (2011) concerning public health safety (Naujokas et al., 2013). And yet many states in the U.S.A consume 50 ppm (Tchounwou et al., 2003), parts of India and Bengal consume 3400 ppm (Mazumdar & Dasgupta, 2011) of arsenic via drinking water with the revised permissible limit being 10ppb by the World Health Organization (WHO 1993). Consumption of arsenic on a daily basis exposes our biological system to a toxicant which has led to multiple diseases over time. Arsenic-induced toxicities have led to chronic illnesses like hypertension, cardiovascular disease, diabetes; organ dysfunctions; pregnancy-related issues like impaired foetal growth, foetal loss during pregnancy, increased post-birth infant mortality; various types of malignancies and solid tumours including, lung, prostate, bladder cancers, melanoma skin cancer; long-term memory loss and modification of hormonal regulation (Ghosh & Sil, 2015). Natural arsenic is present scarcely in the environment between 1.5-2 ppm (National Research Council, 1977) but the potential lethality has increased over time due to phenomena such as bioaccumulation in the food chain. Arsenate is mainly categorised as organic and inorganic arsenate. Inorganic arsenate salts are dissociated in all four arsenic acid (As5+) species, H3AsO4, H2AsO4 1–, HAsO42– and AsO43–. Arsenic acid (As5+) is the least toxic of the inorganic forms and arsenous acid (As3+) is more toxic in vivo than arsenic acid and also more inhibitory in vitro (Roy and Saha, 2002). However, inorganic forms of arsenic i.e. arsine, arsenite and arsenate, are more toxic than the organic acidic forms (Benramdane et al., 1999).
Inorganic arsenic is introduced into the biosphere via volcanic eruptions, withering of minerals, mainly arsenopyrites (5%), and groundwater. Organic arsenic is usually bound with hydrocarbons and comparatively less lethal. Common organic arsenic species are monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA). Plants may contain from less than 0.01 to about 5 ppm depending on arsenic-containing treatments used during cultivation and marine fish may contain up to 10 ppm (National Academy of Sciences, 1977). Sea food and rice are a rich source of arsenic. Though 40% (Flora, 2015) of arsenic exposure is consumed through diet it is unlikely that consumption of organic arsenic compounds leads to arsenic poisoning (Ferguson & Gavis, 1972).
Increasing anthropogenic activities have majorly contributed to the rising arsenic contamination in drinking water. Exposure through drinking water is increasing due to contamination from industrial operation and over withdrawal of groundwater for irrigation purposes. Arsenic, an integral part of various metal ores and coal, is released during the smelting process or in coal-burning, which produces dust and gas to contaminate the surrounding soil and water. As a result arsenic pollution in mining places and in smelting or coal-burning in thermal power plants is referred to as “occupational hazard”, threatening the lives of workers (Roy & Saha, 2002). Arsenic is mainly produced in the U.S. with 80% demand for agricultural cultivation. Arsenic-based industries are involved in the manufacturing of insecticides, herbicides, cotton desiccant, wood preservatives, drugs, poisons, drugs in human and veterinary medicine, lead-based battery and semiconductor technology (Flora, 2015). Apart from these sources environmental pollution in air, smoking of cigarettes, consumption of arsenic based drugs and usage of cosmetics exposes humans to arsenic directly.
Arsenic accumulation has been found to have genotoxic effects, mutagenic effect, induced oxidative stress, induced immune imbalance and impaired apoptotic regulation that impends the cellular metabolic process and culminates to organ dysfunction (Florea et al., 2005).
The present study focusses on understanding the induction of oxidative stress by arsenic trioxide upon oral consumption by assessing the levels of reduced glutathione, malondialdehyde and catalase activity. The effect of arsenic on liver was determined by performing regular histological studies and estimating levels of serum glutamic-oxalacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT) and alkaline phosphatase.
Arsenic, a very toxic chemical, is consumed chronically at non-permissible levels by humans around the world resulting in lowering of the health standards and increasing the incidence of death. This study primarily helps in understanding the detrimental effects of arsenic in drinking water with the intention to curb arsenic pollution. By demonstrating the arsenic induced effects on human system, measures to regulate the heavy metal leaching into the natural environment could be exercised seriously by the government bodies globally. The contamination of limited fresh water also leads to ecological imbalance that results in grave issues like species extinction. It is vital to understand the impact of advancing anthropogenic activities and how it affects the biosphere at a basic level, such as consumption of contaminated drinking water especially during an environmental crisis period such as now. This study is limited to understanding the effect of arsenic trioxide at a specific sub lethal concentration i.e.1/10th of LD50 for a short duration of treatment (48 h, 72 h and 96 h). However, it does provide an insight on the mechanism of the action of arsenic trioxide. This study is also limited to understanding the effect of inorganic arsenic in the form of arsenic trioxide that is consumed orally.
Objectives of the Research
· Compare the functionality of liver between arsenic treated and untreated groups.
· Study the histopathological effects of arsenic trioxide on hepatic tissue.
· Study the arsenic induced effect on cellular metabolism.
The methylation pathway involves the formation of a monomethylated metabolite of As (III), which is either rapidly methylated again into a dimethylated derivative or is spontaneously oxidized into the As (V) form MMA. The first methylation reaction is catalyzed by arsenite methyltransferase. The transfer of the second methyl group is catalysed by another enzyme, MMA methyltransferase. The latter enzyme is sensitive to inhibition by As (III). This characteristic explains the increased concentrations of MMA in biological media after acute intoxication. As (V) is reduced to As (III) by arsenate reductase before its methylation. In addition, a small percentage of total arsenic is found in blood and urine as the As(V) form and was shown to originate from in vivo oxidation of absorbed As(III). The methylated metabolites (MMA and DMA) show a very weak affinity for tissues; therefore, their elimination in bile, blood, and urine is rapid. (Benramdane et al., 1999).
Arsenic accumulation has been found to have genotoxic effects, mutagenic effect, induced oxidative stress, induced immune imbalance and impair the apoptotic regulation that impends the cellular metabolic process and culminates to organ dysfunction (Florea et al., 2005). Chronic exposure to different inorganic arsenic compounds (arsenite, arsenic trioxide, or arsenate) produces characteristic pathology in the liver, including fatty infiltration, liver degeneration, inflammatory cell infiltration and focal necrosis (Srivastava et al. 2013).
Oxygen reacts with the organic form of DMA that forms DMA radical and superoxide anion (O2•−). The O2•− which is a primary unstable reactive oxygen species (ROS) initiates a dynamic chain of reactions forming secondary ROS such as dimethylarsinicperoxyl radical. (Ghosh & Sil, 2015) Other arsenic influenced reactive oxygen species are hydroxyl radical (•OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), and peroxyl radicals (Bhattacharya, A. & Bhattacharya S., 2007). These free unstable radicals inhibit essential enzyme functionality such as acetylcholine esterase(AChE), glutathione peroxidase (GPx), glutathione reductase (GR), and GSH-independent antioxidants such as superoxide dismutase (SOD) and catalase (Roy & Bhattacharya, 2007). It is reported that arsenic-induced ROS reduces the mitochondrial membrane potential and impairs oxidative phosphorylation that leads to lower ATP production (Iwama et al., 2001).
Nuclear factor (erythroid-2 related) factor 2 (Nrf2)/Keap1 (Kelch-like ECH-associated protein1)/ ARE (antioxidant response element)—driven target gene system is a major cytoprotective machinery against oxidative stress and helps to maintain cellular oxidative homeostasis. Arsenic induced oxidative stress activates and increases the levels of Nrf2 factor. Keap1 plays an important role in the localization of Nrf2 protein within the cell. Keap1 acts as a negative regulator of Nrf2, it forms an E3 ubiquitin complex with Cullin 3 (Cul 3) and Ring-box 1 (Rbx 1) (Keap1-Cul3-Rbx1) and enables Nrf2 degradation. Therefore under normal conditions Nrf2 levels are low (Lau et al., 2013). p62 plays its role by docking the Keap1 protein through a motif called Keap1 interacting region (KIR) thereby blocking and binding between Keap1 and Nrf2. Nrf2 protein is separated from Keap1 following its migration to the nucleus. Interestingly, induction of p62 results from oxidative stress and is mediated by Nrf2, which binds to the ARE containing cis-element of p62. Therefore p62/SQSTM1 (sequestosome 1) is a target gene for Nrf2, which creates a positive feedback loop by inducing ARE driven gene transcription (Srivastava et al. 2013). ARE driven genes aid in cytoprotection, detoxification and elimination of harmful substance such as arsenic. These genes include intracellular redox balancing proteins (e.g. heme oxygenases) and thioredoxin reductase-1 (TrxR1), phase I and II detoxication enzymes (e.g., NAD(P)H quinone oxidoreductase-1 (NQO1)), glutathione S-transferase (GST), glutamate cysteine ligase catalytic subunit, and regulatory subunit (GCLM)), xenobiotic transporters (multidrug resistance-associated proteins (MRPs)), and other stress response proteins (Lau et al., 2013). Arsenic exposure leads to higher expression of HSP proteins that protect the cell from intracellular damages. An upregulation of transcription of heat shock proteins (HSP) and other antioxidant stress proteins belonging to 27, 32, 60, 70, 90 families were observed as a result of arsenic exposure. To counter the oxidative stress within the cell upregulation of some free radical scavenging proteins such as metallothionein (Albores et al. 1992) and heme oxygenase (Keyse and Tyrrell, 1989) has been observed that helps detoxify heavy metal accumulation.
In addition, arsenic-induced ROS reduces the ratio of GSH/GSSG. GSH is a non protein thiol that acts as a reducing equivalent in the cell and takes part in antioxidant defence, nutrient metabolism and regulation of cellular events (including gene expression, DNA and protein synthesis, cell proliferation and apoptosis, signal transduction, cytokine production and immune response, and protein glutathionylation) intracellular mechanisms (Wu et al., 2004). The decrease in GSH creates an imbalance in the redox potential within the cell that can lead to early senescence of cells.
Arsenic when consumed above the permissible limits can have adverse effects on the human body. The immediate toxicity is high in liver and kidneys due to their role in methylating, detoxifying and eliminating lethally potent arsenic from the system. Arsenic establishes toxicitiy by disrupting the redox potential of the cell. The imbalance in redox potential does not favour numerous biochemical processes and leads to cellular dysfunction culminating in various abnormalities. Therefore it is important to regulate arsenic content in drinking water.
- Lowry’s reagent
- Bovine’s Serum Albumin (BSA)
- Folin’s phenol
- Phosphate Buffer Saline (PBS solution)
- 20% Tricloroacetic acid (TCA)
- 0.2 mg/mL 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) in PBS
- 0.5% Thiobarbituric acid (TBA)
- 2.5N Hydrochloric acid (HCl)
- Assay buffer (50 mM Tris HCL,9 mM H2O2, 0.25 mM EDTA, pH 8.0)
- 10% tissue homogenate
- Paraffin (M.P.: 65-70oC)
- Absolute alcohol
- 90% alcohol
- 70% alcohol
- 50% alcohol
- Eosin stain
- BE70 fixative
- Delafield Haematoxylin stain (haematoxylin-2g, Ammonium alum-3g, Glycerin-100mL, Distilled water-100mL, Alcohol 95% -100mL)
- Glutamate oxaloacetate transaminase GOT (AST), glutamate pyruvate transaminase GPT (ALT) and alkaline phosphatase test kits purchased from Span diagnostics Ltd., Surat, India.
Selection of test organism
In this study, the chosen model organism is the Sprague-Dawley rat. Rats are mammalian vertebrates that mimic the human biological system. In addition, rats possess a larger size which is helpful in monitoring physical symptoms of abnormality.
The classification of the Rat (Rattus norvegicus)
Maintenance of model organism
9 adult healthy male Sprague-Dawley Rats of a single strain were purchased from a supplier. The rats weighed 90 g-120 g and were kept in different stainless steel cages according to their body weight for their respective arsenic trioxide administration. The rats were maintained in clean, dry, hygienic standard conditions (room temperature 20°C±2°C, and 14 hour light/dark cycle). Water and food were made available throughout the experiments. The health of rats was monitored and checked for any signs of abnormality.
In the treatment group, each rat was orally fed with 1/10th of 50% lethal dosage (LD50) of arsenic trioxide for 48 hours, 72 hours and 96 hours. The rats were euthanized using chloroform as required. The experiments were conducted following the guidelines and approval of the Institutional Animal Ethics Committee of Visva-Bharati University.
Preparation of arsenic trioxide solution: 1/10th of LD50 of Arsenic trioxide was administered orally. The value of LD50 reported was 6.6 mg/kg body weight (Vinaykumar Siragam, 2000, Influence of Mercuric Chloride, Cadmium Chloride and Arsenic Trioxide in Induction of Alterations in Rat Platelet Functions, Degree of Doctor of Philosophy (Science), Visva-Bharati University, Shantiniketan, India) .
Based on this 1/10th of LD50 i.e. 0.66 mg/mL of stock solution was prepared. 13.2 mg of arsenic trioxide was weighed and dissolved in 10 mL of 1N HCl solution. The mixture was heated until arsenic trioxide completely dissolved and the final volume was made to 20 mL with double distilled water. The pH was adjusted to 7.4 using 1N NaOH. The solution was diluted according to the body weight of the rats before administration.
After 48 h both treated and untreated rats were dissected and the liver was harvested. A section of liver tissue was excised and stored in a vial containing buffered ethanol (BE70) fixative for histological studies. Another small section was cut and further processed for biochemical analysis. The same was repeated for 72 h and 96 h.
Sample preparation for analysis of biochemical reactions:
Standard 10% tissue homogenate was prepared for all the assays.
A. 700µl of 10% tissue in PBS was homogenized using a hand-held homogenizer followed by centrifugation at 1000 rpm at 4oC for 10 min. The supernatant was collected and used to perform protein, catalase and thiobarbituric acid reactive substances (TBARS) assays.
B. 350 µl of PBS was added to 70 mg of tissue and homogenized using a handheld homogenizer. 350 µl of trichloroacetic acid was added. The mixture was centrifuged 1000 rpm at 4oC for 10 min. The supernatant was collected and used to perform the glutathione (GSH) assay.
C. Blood was drawn using sterile syringe by puncturing the heart of the rat directly. The blood serum was later used to estimate levels of SGOT/AST, SGPT/ALT and alkaline phosphatase.
Biochemical analysis of liver tissue
A) Protein Assay: Protein concentration was estimated using Lowry’s method (1951). Briefly, 120 µl of distilled water was added to 5 µl of the sample. 1.25 mL of Lowry’s reagent was added and then incubated at room temperature for 30 min. The absorbance was measured spectrophotometrically at 660 nm. The standardisation of protein was performed using Bovine’s Serum Albumin (BSA). 5.0, 2.5, 1.0, 0.5, 0.25, 0.01 mg/mL protein standards were used and their OD at 660 nm was found using a spectrometer after performing Lowry’s assay.
B) GSH Assay: The concentration of reduced glutathione (GSH) was estimated using Ellman et al method (1961). Briefly, 625 µl DTNB was added to 125 µl of sample. The reaction mixtures were incubated at room temperature in dark for 5 min. Absorbance was measured spectrometrically at 405 nm. The GSH concentration was expressed as relative GSH concentration.
C) TBARS/ MDA Assay: The concentration of MDA was estimated using Buege’s & August’s method (1978). Briefly, 1 mL of sample was mixed with 2 mL of TBA-TCA-HCl mixture thoroughly and heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1,000 g for 10 min. The absorbance of the supernatant was determined at 535 nm and expressed in terms of relative MDA concentration.
D) Catalase Assay: Rate of catalase activity was determined using Aebi’s method (1974) modified by Kawamura (1994). A 5% homogenate was prepared in 50 mM phosphate buffer (pH 7.0) and centrifuged at 12,500 ×g for 30 min at 4∘C. The supernatant (20 𝜇L) was added to 980 𝜇L of an assay buffer containing 50 mM Tris-HCl (pH 8.0), 9 mM H2O2, and 0.25 mM EDTA to constitute the assay volume of 1 mL. The decrease in ΔOD/min of that assay mixture was recorded at 240 nm for 1 min. The results were expressed as relative catalase activity.
Liver function test
B) Estimation of serum glutamate pyruvate transaminase (SGPT)/ alanine transaminase (ALT): Estimation of SGPT/AST was done using kit. The protocol was followed as instructed in the kit. The concentration was expressed as unit enzyme per litre (U/L).
C) Estimation of Alkaline Phosphatase: Estimation of alkaline phosphatase was done using kit. The protocol was followed as instructed in the kit. The concentration was expressed as unit enzyme per litre (U/L).
Portions of liver tissue of all animals were fixed in BE70 fixative, dehydrated through graded alcohol, and embedded in paraffin, and routine microtomy was carried out to obtain 5 𝜇m thick tissue sections. Sections were stained by routine hematoxylin-eosin (H&E) technique and viewed under light microscope after the slides were mounted using DPX and mild heat was given to even the layer and remove any trapped bubbles.
A) ESTIMATION OF GSH
The concentration of reduced glutathione (GSH) decreased as the duration of arsenic oxide treatment increased. The highest concentration of GSH was observed at 48h whereas 96 h of arsenic treatment showed the least concentration of GSH. Abnormal levels of GSH concentration can be considered as a significant biomarker for presence of oxidative stress. Arsenic being an electrophile binds with nucleophilic sulfhydryl (-SH) group in GSH, which leads to imbalance in the redox potential of the cell. Abnormal levels of GSH suggest it to be a method of adaptive measures by the cell (Zhu et al., 2011).
|48 h||72 h||96 h|
B) ESTIMATION OF MDA
The concentration of MDA increased over the duration of arsenic treatment increased. The peak value (1.16) was observed at 96 h whereas the lowest value (1.05) was observed at 48 h of arsenic treatment. Arsenic is lipophilic in nature and therefore binds with fatty acid in the cell which increases the rate of lipid peroxidation (Bhattacharya, A. & Bhattacharya S., 2007).
|48 h||72 h||96 h|
C) ESTIMATION OF CATALASE ACTIVITY
The rate of catalase activity decreased as the duration of arsenic treatment increased. Catalase is an enzyme that degrades hydrogen peroxide to water and molecular oxygen. A decrease in the rate of catalase activity could suggest unavailability of the substrate molecule i.e. hydrogen peroxide, inhibition of catalase enzyme or maybe lower expression of catalase.
|48 h||72 h||96 h|
Liver functionality test
A) Estimation of SGPT/ALT
SGPT/ALT showed a consistent decrease in concentration as the duration of arsenic treatment increased. ALT acts as a molecular marker for testing liver functionality. The results when compared against control showed elevated SGPT concentrations suggesting signs of stress and damage in the liver. This is also supported by the abnormal levels of other biomarkers that indicate liver functionality such as alkaline phosphatase and SGOT/AST.
|48 h||72 h||96 h|
B) Estimation of SGOT/AST
Overall concentration of SGOT was observed to increase in a time dependent manner. At 72 h the levels of SGOT for treated and untreated samples were similar. The normalising of SGOT levels of the treated group might suggest that though arsenic treatment initially (48 h) elicited hepatic damage, the hepato-protective machinery is trying to cope with the damages and reduce the toxicity.
C) Estimation of Alkaline Phosphatase
The levels of alkaline phosphatase decreased steadily over the period of arsenic treatment. Abnormal levels of alkaline phosphatase indicate arsenic induced stress in liver. The combined results of SGOT, SGPT and ALP can be conclusive for presence of arsenic induced hepatotoxicity. The results of biochemical assays to check the functionality of liver can be evidently supported by the results of histological studies.
|48 h||72 h||96 h|
Images of histological sections of arsenic treated liver tissues revealed alterations in the histo-architecture when compared against the morphology of untreated liver tissues.
Normal hepatic tissue in the control rats appeared with innumerable lobules. Each lobule consisted of hexagonally shaped hepatocytes with a central lobule. The hepatic cells appeared with a distinct outlined cell membrane and a centrally placed conspicuous nucleus. There appears to be no abnormal patches of cellular disintegration.
Treatment of 1/10th of LD50 of arsenic trioxide over 48 h, 72 h and 96 h showed various morphological stress signals. The degree of alteration varies depending on the duration of treatment. Maximum distortion of liver tissue can be observed at 96 h where the intercellular integrity is lost and white patches are clearly visible due to cellular degradation. At 48 h treatment initial symptoms of hepatoxicity is revealed via the formation of pycnotic nuclei where the large conspicuous nuclei shrink. At 72 h the cellular shape is distorted, and the cell’s volume evidently decreases.
CONCLUSION AND RECOMMENDATIONS
Arsenic activates different signalling pathways that control important functions such as proliferation, differentiation, and apoptosis. The present study is conclusive for establishment of arsenic induced oxidative stress and hepatoxicity upon oral administration of 1/10th of LD50 of arsenic trioxide on adult white rats for 48 h, 72 h and 96 h. This is suggested by presence of abnormal levels of biomarkers indicating: oxidative stress - MDA/TBARS, catalytic activity and GSH; abnormal liver functionality- alkaline phosphatase, SGOT/AST and SGPT/ALT and histological images of altered tissue architecture of the liver. However further studies must be performed inorder to understand the mode of action of arsenic toxicity in human system.
Industrial effluents, air pollutants, agricultural drains must be constantly monitored and ground water must be purified before consumption. Traces of arsenic from drinking water can be eliminated via precipation (using coagulants such as alum, iron, lime), adsorption (using adsorbents ferric oxide, goethite, ferrihydrite, Fe oxide-coated sand, alumina, gibbsite, kaolinite, illite, etc.) and phytoremediation (using hyperaccumulators such as Pityrogramma calomelanos , Pteris cretica, Pteris longifolia, and Pteris umbrosa) (Flora, 2015). Exposure to arsenic in the natural environment must be regulated inorder to prevent arsenic related disorders on long term basis.
The author of this study would like to thank Proffessor Shelley Bhattacharya for her guidance and support.
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