Efficacy of crude extract of Emblica officinalis (amla) in arsenic-induced oxidative damage and apoptosis in splenocytes of mice

Introduction: Arsenic, an environmental contaminant naturally occurred in groundwater and has been found to be associated with immune-related health problems in humans. Objective: In view of increasing risk of arsenic exposure due to occupational and non-occupational settings, the present study has been focused to investigate the protective efficacy of amla against arsenic-induced spleenomegaly in mice. Results: Arsenic exposures (3 mg/kg body weight p.o for 30 days) in mice caused an increase production of ROS (76%), lipid peroxidation (84%) and decrease in the levels of superoxide dismutase (53%) and catalase (54%) in spleen as compared to controls. Arsenic exposure to mice also caused a significant increase in caspases-3 activity (2.8 fold) and decreases cell viability (44%), mitochondrial membrane potential (47%) linked with apoptosis assessed by the cell cycle analysis (subG1-28.72%) and annexin V/PI binding in spleen as compared to controls. Simultaneous treatment of arsenic and amla (500 mg/kg body weight p.o for 30 days) in mice decreased the levels of lipid peroxidation (33%), ROS production (24%), activity of caspase-3 (1.4 fold), apoptosis (subG1 12.72%) and increased cell viability (63%), levels superoxide dismutase (80%), catalase (77%) and mitochondrial membrane potential (66%) as compared to mice treated with arsenic alone. Conclusions: Results of the present study indicate that the effect of arsenic is mainly due to the depletion of glutathione in liver associated with enhanced oxidative stress that has been found to be protected following simultaneous treatment of arsenic and amla.

from As poisoning. One of the most common sources of As contamination is drinking water, where levels ranges from 0.01 to 4.0 mg arsenic/L. [1] Dietary intake of As through consumption of contaminated food, as well as exposures through medicines and occupational settings, is also reported. [2] Comparing all three oxidation states of As, the trivalent form is considered to be more toxic due to its ability to bind with sulfhydryl groups of proteins and disrupt enzyme activity. [3] Alterations in the expression of genes involved in immune function have been reported in mouse embryonic cells following exposure to arsenic. [4] Studies have INTRODUCTION High levels of arsenic (As) in groundwater have caused a great concern, especially in the Indo-Bangladesh region where over a million of people are reported to be suffering also reported that exposure to As significantly inhibits mitogen-induced lymphocyte proliferation in cells from exposed humans and mice. [5,6] It has been shown that As induces immunosuppression via apoptosis in thymocytes and splenocytes in a dose-dependent manner in epidemiological and experimental studies. [7] Exposure to As during pregnancy was found to be associated with decreased thymus size in children and increased infectious diseases in both mothers and children, indicating an As-related immunosuppression. [8,9] Arsenic exposure has also been found to give rise to reductions in CD4 + cell counts, CD4 + :CD8 + cell ratios, and T-regulatory cell levels in adults and children. [10][11][12][13][14] Chronic exposure to As has been shown to be involved in the activation of inflammatory signaling pathways in humans and rodents. [15] Apart from mammals, low levels of As exposure were also seen to cause reduced immune defense responses in birds and fish. [16] An involvement of oxidative stress in a large number of pathological states (including cancer) has been proven clinically. [17] As toxicity is known to be mediated in great part through the generation of reactive oxygen species (ROS), possibly in combination with changes in host anti-oxidative defenses. [3,18,19] A prime target of As intoxication is mitochondria affected either by ROS generation or through a condensing of the mitochondrial matrix and an opening of permeability transition pores. [20] Irregular apoptosis of immune cells due to As exposure may lead to immune-related disorders that in turn may result in immunodeficiency, autoimmune diseases, or malignancies. [21,22] Recently, we reported that As induced oxidative damage and apoptosis in thymocytes of mice and that these changes were associated with increased oxidative stress. [23] We also found that As accumulated in the liver and impaired its anti-oxidant status and induced alterations in the levels of serum hepatic enzymes associated with hepatic toxicity. [24] Natural products, mainly those obtained from dietary sources, contain a wide variety of anti-oxidants. Phytoconstituents, an important source of anti-oxidants, are capable of terminat-ing free radical chain reactions. [25] Recently, a number of studies have been carried out to assess the protective potential of these herbal and natural agents against As-induced toxicity and related conditions. [3,20,23] The fruit extract of amla and its individual constituents have shown to impart anti-oxidant, inflammatory, cancer, and microbial effects as well as to appear to induce immuno-modulation in situ. [26] Amla is a rich source of Vitamin C, a water-soluble anti-oxidant that acts as a scavenger of free radicals and plays an important role to protect against lipid damage, protein oxidation, and DNA oxidation. [27] Amla also contains a variety of active constituents like polyphenols, flavones, tannins, anthocyanins, flavonols, ellagic acid, and derivatives that protect against ROS and exhibit a wide range of biological effects. [28] Studies have shown that the aqueous extract of amla induced apoptosis and inhibited lung metastasis by injected murine colon 26-L5 carcinoma cells [29] and B16-BL6 melanoma cells in mice. We recently reported on the protective efficacy of amla against As-induced enhanced oxidative stress and apoptosis in thymocytes and hepatotoxicity in mice. [23,24] Although the anti-oxidant potential of amla and its various constituents has already been reported, [30] not much is known about the immunomodulatory effects of the crude extract of amla and associated outcomes. As whole amla fruit is taken as a dietary supplement therefore, the present study was undertaken to investigate the immunomodulatory role of crude extract of amla during As-induced toxicities in the spleens of mice.

Plant material
The ethyl acetate extract of amla contains many phenolics and flavonoids including gallic acid, ellagic acid, isocorilagin, chebulanin and chebulagic acid. [31,32] Therefore, this extract was been selected for use in the present study. Briefly, the fresh fruits of Emblica officinalis were collected from an authentic source; the materials were verified by the Herbarium of Birbal Sahni Institute of Palaeobotany, Lucknow, India (plant identification no-13394). The samples were then allowed to dry at 40°C and then the materials were pulverized and screened through a 60-mesh sieve. Thereafter, the fruit powder (100 g) was extracted three times using 95% ethanol. The combined extracts were then filtered through 0.45 µm Whatman filter paper and the filtrate then evaporated to dryness with a rotary evaporator under reduced pressure. The residue was then suspended in water and extracted successively with diethyl ether and ethyl acetate. The extract was evaporated under reduced pressure to obtain the powdered form of the ethyl acetate fraction. Yields of 5.5% (w/v; in ethyl acetate fraction) were routinely obtained and used for the present study.

Animals and treatment
The study was approved by the institutional animal ethics committee of King George Medical University, Lucknow (No. 121 IAH/Pharma-11), India, and all experiments were carried out in accordance with guidelines set by the committee for the purpose of control and super-vision of experiments on animals (CPCSEA), Ministry of Environment and Forests (Government of India), New Delhi, India. Male Balb/c mice (15 ± 2 g) were obtained from the animal breeding colony of CSIR-Indian Institute of Toxicology Research (Lucknow). Mice were housed in an air-conditioned room at 25 ± 2ºC with a 12 h light/dark cycle under standard hygiene conditions and had ad libitum access to a pellet diet and filtered water. The dose of fruit extract of Embilica officinalis and As is based on our previous studies [23,24] and other literature. [33][34][35][36] The study is a part of ongoing research work in our laboratory where we have previously carried out the studies on the doses of As (3 mg/kg BW) and Emblica officinalis (500 mg/kg BW) and further to avoid variations in the study we have selected these doses for the present study.
For the study, the mice were randomly divided into four groups with 10 animals/group: Group I Mice treated with vehicle (2% gum acacia) for duration of treatment (control) Group II Mice treated with sodium arsenite (dissolved in distilled water at 3 mg arsenic/kg BW, per os daily for 30 days) Group III Mice treated with fruit extract of Emblica officinalis (500 mg/kg BW, suspended in 2% gum acacia, per os daily for 30 days) Group IV M i c e c o -t re a t e d d a i l y w i t h A s a n d Emblica officinalis extract as in Groups II and III. 24 h after the final dosing, all mice were euthanized by cervical dislocation and each spleen isolated. The spleens of three mice/group were cleaned and placed in phosphate-buffered saline (PBS, pH 7.4) and subsequently processed for measures of apoptotic parameters. The remaining spleens in each set were placed in a ice-cold saline solution (0.15 M), blot-dried, weighed, and then immediately processed for use in enzyme/non-enzyme anti-oxidant assays.

Preparation of splenocytes
Each spleen removed at necropsy was made into a single-cell suspension using a 400 µm pore stainless steel mesh. [37] Erythrocytes in the suspension were then lysed with a hypotonic buffered solution and the cells washed with PBS. The remaining cells were suspended in PBS, counted in a hemocytometer and then diluted with PBS to appropriate concentrations for use in the various assays outlined below.

Cell viability
Cell viability was measured by the MTT reduction method Mosman. [38] In brief, cells were seeded at 10 4 cells/well in a 96-well plate and then a 10 µl MTT (5 mg/ml PBS) solution was added to each well. After the plate was incubated 4 he at 37°C in a CO 2 incubator, it was centrifuged (1200 × g, 10 min) and then the supernatant in each well was removed. Thereafter, 100 µl DMSO was added to each well to dissolve the formazan that had formed in the wells and, after 5 min, the absorbance in each well was read and then measured at 530 nm using a Biotek Multiwell plate reader (Biotek Synergy HT, Winooski, VT).
For several of the splenocyte-based assays below, tissue homogenates were required and prepared. Briefly, cells were pelleted by centrifugation (200 × g, 10 min, 4°C) and then re-suspended in PBS. The final lysate was obtained by re-centrifugation (200 × g, 10 min, 4°C) to remove debris. The supernatant was collected and then total protein content measured using the method of Lowry et al., [39] and bovine serum albumin as a standard.

ROS generation
The generation of ROS was measured using DCFH-DA by flow cytometry as described previously by Wang et al. [40] Single cell suspensions (10 6 /ml) of splenocytes from control and treated mice were prepared in PBS and incubated with DCFH-DA (100 µM final conc.) at 37°C for 1 hour. Hydrolysis of DCFH-DA leads to formation of fluorescent DCFH that was measured by fluorescence intensity (FL-1, 530 nm) in a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA). All data were analyzed using the 'Cell Quest'3.3 software (Becton Dickinson). A minimum of 10,000 events per sample were acquired for each analysis.

Lipid peroxidation
To assess intensity of oxidative stress, lipid peroxidation (as reflected in levels of malondialdehyde (MDA) present) was measured using the procedure of Ohkawa et al. [41] The absorbance of each reaction mixture was read at 532 nm using a Cary 300 Bio UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Total peroxidation levels were then expressed in µmole MDA/mg protein.

Superoxide dismutase and catalase activity
To assess superoxide dismutase activity in splenocyte homogenates, the assay of Marklund and Marklund [42] was utilized. A unit of activity was defined as the amount of enzyme required for 50% inhibition of pyrogallol auto-oxidation. All results were expressed as U/min/mg protein. Catalase activity in the homogenates was assayed using spectrophoto-metrically by the protocol of Aebi (1984), and using hydrogen peroxide (H 2 O 2 ) as substrate. All activities were expressed in µmole/min/mg protein.

Caspase-3 activity
Splenocyte caspase-3 activity was measured using kit (Chemi-Con USA). In brief, cells (3.0 × 10 6 /ml) were placed in lysis buffer and incubated on ice for 10 min. Reaction buffer (10 mM Tris•HCl, 1 mM EDTA, 10 mM dithiothreitol, 5% glycerol) and DEVD-AFC substrate (50 µM) were then added and the mixture then incubated for 2 h at 37°C in the dark. AFC was used as a standard; fluorescence was measured at excitation and emission wavelengths of, respectively, 400 and 505 nm on a microplate reader. Enzyme activity was expressed as nmoles AFC/60 min.

Assay of mitochondrial membrane potential
Mitochondrial membrane potential was assessed by flow cytometry using the procedure of Bai et al. [43] Single cell suspensions (10 6 /ml) of splenocytes from each mouse were incubated with Rh123 (at 5 µg/ml final concentration) at 37°C for 60 min in the dark. Membrane potential was then measured using an FL-1 filter (at fluorescence intensity of 530 nm) in the LSR II flow cytometer. A minimum of 10,000 events per sample were acquired for each analysis.

Analysis of apoptotic DNA
Assays of apoptotic DNA levels were performed using the procedure of Darzynkiewicz et al. [44] Briefly, sets of 10 6 splenocytes from individual control and treated mice were prepared cell cycle analysis. The cells were washed with PBS and fixed with 70% ethanol. The fixed cells were then again washed with PBS, suspended in phosphate citrate buffer (200 µl, pH 7.8), and incubated for 60 min at room temperature. After centrifugation, the cells were re-suspended in 0.5 ml propidium iodide (100 µg/ml) and 0.5 ml RNAse (50 µg/ml) and incubated a further 30 min in the dark. PI fluorescence was measured using an FL-2 filter (585 nm) in the LSR II flow cytometer. A minimum of 10,000 events per sample were acquired for each analysis.

Assessment of apoptotic and necrotic cell
Apoptotic and necrotic cell distribution among the splenocytes was analyzed via Annexin V binding and PI uptake, following the procedure of Vermes et al. [45] Briefly, sets of 10 6 splenocytes from individual control and treated mice were each suspended in 1 ml binding buffer (1X), and then a 100 µl aliquot was incubated with 5 µl Annexin V-FITC and 10 µl PI (As provided with kit obtained from Biovision, San Francisco, CA) at room temperature for 15 min in the dark. Thereafter, 400 µl binding buffer (1X) was added to each sample and the associated FITC and PI fluorescence then measured through, respectively, FL-1 (530 nm) and FL-2 (585 nm) filters in the LSR II flow cytometer. A minimum of 10,000 events/sample were acquired each time.

Statistical analysis
Data were analyzed using one-way analysis of variance (ANOVA) followed by a Newman-Keuls test for multiple pair-wise comparisons among the groups. All values were expressed as mean (±SEM). P < 0.05 were considered significant.

Effect on cell viability in splenocytes of mice
The effect of As and co-treatment of As and amla on cell viability in spleen has been presented in Figure 1. Mice exposed to As exhibited a significant decrease in cell viability (44%, P < 0.01) as compared to controls. Co-treatment with As and amla increased the cell viability (63%, P < 0.05) in spleen as compared to those treated with As alone. No significant effect on cell viability was observed in mice treated with amla alone as compared to controls [ Figure 1].

Effect on splenocytes ROS generation
Exposure to As caused an increased generation of ROS (76%) in splenocytes as compared to in cells from control mice [ Figure 2]. Co-treatment with amla decreased ROS generation (24%) compared to values in cells from mice treated with As alone, suggesting an anti-oxidative free-radical scavenging activity by the extract. No significant effect on ROS production was seen in splenocytes from mice treated with amla alone as compared to controls.

Effect on the lipid peroxidation in splenocytes
The effects of As and co-treatment with amla on lipid peroxidation in splenocytes are shown in Figure 3. Exposure to As caused a significant increase in lipid peroxidation (84%) levels in splenocyte as compared to values in cells from control mice. Co-treatment with amla significantly decreased the level of peroxidation (33%) compared to values in cells of mice treated with As alone. No significant effect on lipid peroxidation levels were seen in cells from mice treated with amla alone as compared to controls.

Effect on the activity of superoxide dismutase in splenocytes
Exposure to As caused a significant decrease (53%) in splenocyte SOD activity compared to values in cells from control mice. Co-treatment with amla increased the activity (80%) compared to values in cells of the mice treated with As alone. No significant effect on activity was noted in splenocytes of mice treated with amla alone as compared to controls [ Figure 4].

Effect on the activity of catalase in splenocytes
Arsenic exposure caused significant decreases (54%) in catalase activity in splenocytes compared to values in cells from control hosts [ Figure 5]. Amla co-treatment increased the activity (77%) as compared to values in cells from mice treated with As alone, suggesting a protective effect against oxidative insult. No significant effect on activity was seen in splenocytes of mice treated with amla alone as compared to controls.

Effect on the mitochondrial membrane depolarization in splenocytes
Mice exposed to As displayed decreased mitochondrial membrane depolarization (47%) in their splenocytes as compared to those in cells from controls [ Figure 6]. Co-treatment with amla increased the membrane depolarization (66%) compared to values in cells from mice treated with As alone. No significant effect on depolarization was observed in splenocytes from mice treated with amla alone as compared to controls.

Effect on caspase-3 activity in splenocytes
The effects of As and co-treatment with amla on caspase-3 activity in splenocytes from treated mice are presented in [ Figure 7]. Exposure to As caused a significant increase in the caspase activity (2.8 fold) in splenocytes as compared to levels noted in cells from control hosts. Amla co-treatment decreased this activity (1.4 fold) in splenocytes as compared to values in cells from mice treated with As alone. No significant effect on caspase activity was noted in splenocytes from mice treated with amla alone as compared to controls.

Effect on cell cycle in splenocytes
Exposure of mice to As caused an increased number of cells in the sub-G 1 portion of the cell cycle, indicating DNA damage (28%) in these hosts' splenocytes as compared to in the cells from control hosts [ Figure 8]. Co-treatment with amla reduced the number of cells in sub-G 1 (12%) relative to the numbers seen in mice treated with As alone. No significant effect on cell cycle was noted splenocytes from mice treated with amla alone as compared to controls.

Effect on annexin V/PI binding by splenocytes
Annexin binding was used to reflect numbers of apoptotic and necrotic cells in a given test population. Exposure of As caused an increased in the numbers of necrotic (22%) and apoptotic cells (15%) among all splenocytes as compared to values seen in cells recovered in control mice spleens [ Figure 9]. Amla co-treatment decreased the number of necrotic (1.37%) and apoptotic cells (6.02%) relative to levels seen in mice treated with As alone. No significant effect on apoptotic/necrotic cell levels in splenocyte populations were documented as a result of the treatment with amla alone.

DISCUSSION
Exposure to As impairs lymphocyte, monocyte and macrophage activity and affects the cellular immune responses, resulting in immunosuppression in mammals. [46,47] Arsenic is a potent immunotoxicant that modulates non-specific immune responses and alters expression of cytokines often in dose and time-dependent manners. [48] Studies have demonstrated that As exposure decreases body weight and impairs anti-oxidant defenses leading to enhanced oxidative damage to biological membranes in part via increased levels of lipid peroxidation, protein carbonyl contents, and reduced anti-oxidant levels in situ. [3,7,23,36] The increased generation of free radicals/ROS associated with enhanced oxidative stress has been implicated in the induction of apoptosis in both patho and physiologic states. [7,36] Such radicals react with biomolecules and so modify protein structure and function and increase the extent of oxidative damage in DNA. Chronic As exposure has been found associated with apoptosis in lymphocytes; this may be an additional under pinning to the documented immunomodulation arising from As exposure.
Arsenic-induced depletion of anti-oxidant defense enzymes, e.g., superoxide dismutase, catalase, and glutathione peroxidase, has also been shown to occur in dose and time-dependent manners. [49] It has been shown that glutathione, the most abundant non-protein thiol-bearing agent in cells, could play an important role in As-induced oxidative stress. [50] Recently, we showed that As treatment resulted in increased generation of ROS and enhanced oxidative stress, outcomes linked with apoptosis in hepatcytes and thymocytes of mice. [23,24] The mechanisms of As toxicity in case of hepatocytes and thymocytes are linked with the enhanced oxidative  stress associated with the free radicals generations. In the present study, the decreased viability of splenocytes following exposure to As suggested to us there was likely increased production of ROS and lipid peroxidation, in conjunction with decreased levels of reduced glutathione, activity of superoxide dismutase and catalase, was in line with observations in earlier studies [7,36] and indicative of enhanced oxidative stress in these hosts following exposure to arsenic. The effect on cell viability and on lipid peroxidation following exposure to As is quite high in splenocytes as compare to thymocytes as observed in the earlier study. [23] Mitochondria are prime targets for As toxicity, either indirectly via ROS accumulation or directly through a condensing of mitochondrial matrices and opening of permeability transition pores (by virtue of the thiol-oxidizing property of some As agents). [20] In either case, the As-induced mitochondrial insult initiates apoptosis. [51] Because decreased MMP results in further generation of ROS, it may be difficult to conclude whether As-induced ROS are the cause or consequence of mitochondrial damage. Studies have reported that As-induced mitochondrial damage in guinea pig liver leads to leakage of super-oxide ions into the cytosol; this suggested to us that here, the NaAsO 2 may in fact have been inducing apoptosis by activating intrinsic mitochondrial pathways. [52] Apoptosis is linked to increased mitochondrial oxidative stress that causes cytochrome c release and consequent activation of caspases and cell death. [53] Arsenic exposure has not only been found to be strongly associated with production of ROS, decreased mitochondrial membrane potential, caspase activation and increased DNA fragmentation, but also with decreased expression of anti (and increased expression of pro) apoptotic proteins linked to apoptosis in normal and transformed cells. [54][55][56][57] Exposure to As is known to result in increased ROS production and also altered expression of genes associated with immune responses [58] in particular those related to T-cell activity. [6] As such, the findings here of decreases in splenocyte cytochrome c oxidase activity and mitochondrial membrane potential, along with the cellular increases in activity of mitochondrial caspase-3 and both the number of cells in sub-G 1 portion of cell cycle and those that are apoptotic/necrotic due to exposure to As are consistent with many previous findings.
A number of studies have been carried to investigate the protective efficacy of herbal and/or plant extracts with strong anti-oxidant potential against As-induced oxidative damage and apoptosis. [59,36] Amla is widely accepted as an "immune booster" and possesses multiple pharmacological and immunomodulatory properties [24,36,59] due in part to its content of a wide variety of phenolics and its derivatives. Use of amla to mitigate oxidative stress has been widely accepted. [28] In addition, the metal-binding properties of amla due by many of its constituents have been reported. [36] Amla has been scientifically studied for its protective role against toxicities associated with exposure to ethanol, [60] carbon tetrachloride, or hexachlorocyclohexane. [61] Studies showed that amla extract could modulate levels of certain of kidney and liver anti-oxidants and helped to restore activities of anti-oxidant enzymes in cyclo-phosphamide-treated animals. [62] The extract of amla has also been reported to enhance cyto-protection and to reduce levels of apoptosis and DNA fragmentation in cells exposed to toxicants. [63] The extract has also been found to impart protection against metal-induced clastogenicity, including that by chromium (Cr VI ). [59] Further, Cr VI -induced apoptosis, DNA fragmentation and immunosuppression were ameliorated by treatment with amla. [59] We recently noted immunomodulatory and anti-oxidative potentials of amla against As-induced oxidative stress and apoptosis in the thymocytes of exposed mice. [23] In the present study, changes in levels of lipid peroxidation and reduced glutathione, and in superoxide dismutase and catalase activities following exposure to As were found to be mitigated/inhibited by co-treatment with amla, suggesting a protective effect. This effect was also reflected by the impact of the extract against As-induced changes in mitochondrial membrane potential, caspase-3 activity, and numbers of sub-G 1 , apoptotic, and/or necrotic splenocytes.

CONCLUSIONS
The results of this study clearly indicated that while arsenic-induced free radical generation and enhanced oxidative stress, leading to apoptosis/necrosis among splenocytes of mice, co-treatment with amla mitigated/prevented many of these effects. Taken together, the findings here suggest to us that amla could provide a new approach for combating arsenic-induced immunotoxicity. Further studies are required to understand the detailed mechanisms of any potential immunoprotection being afforded by this particular extract.