Department of Zoology, Panjab University, Chandigarh,160014,India
Received: 16 May 2017 Revised and Accepted: 31 Aug 2017
Objective:Cadmium (Cd) exposure develops various serious pathological conditions because of its long-term retention in tissues. Withania somnifera(WS) is a known antioxidant due to its pharmacological properties, whereas vitamin E (VE) is a dietary antioxidant. The present study tries to study the combinational effect of WS and VE against Cd-induced toxicity.
Methods: Amelioration of Cd-induced toxicity by WS andVE was evaluated in mice liver by studying biochemical, morphological and genotoxic parameters. For this study mice (n=6) were given short-term Cdexposure of 5 mg/kg b. wt.intraperitoneally (ip) for 5 d. WSleaf extract was given orally (500 mg/kg b. wt.)and in combination with VE (100 mg/kg b. wt.) to study its preventive effects against Cd-induced oxidative stress-mediated histopathological and genotoxic alterations.
Results: Cd significantly enhanced lipid peroxidation (LPO (p<0.0001) and declined the levels of antioxidant viz. reduced glutathione (GSH (p ≤ 0.0001), glutathione-S-Transferase (GST (p ≤ 0.0001), superoxide dismutase (SOD (p ≤ 0.0001) and catalase (CAT) (p ≤ 0.0001) activity. Histopathological examination of Cd-treated mice liver exhibited marked structural damage. Metal generated oxidative stress and imbalance of pro-and antioxidants forms the basis for marked structural changes and genotoxicity.Individual treatments of WS and VE reduced the Cd-induced toxicity but when WSwas given in combination with VE for the same period,levels of antioxidants were brought to near normal levels.
Conclusion: WS in combination with VE reduced Cd-induced oxidative burden and restored the levels of anti-oxidant enzymes resulting into improved histoarchitecture and reduced genotoxic insult.
Keywords: Cadmium, Withania Somnifera, Vitamin E, Liver, Oxidative stress
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Essential heavy metals are required in very small amount for various metabolic processes in thecell, but at high concentration, they create physiological stress leading to thegeneration of free radicals.Cadmium (Cd) has been ranked 7thin the top 20 Hazardous Substances Priority List . Acute Cd poisoning causes pulmonary edema, fulminate hepatitis, haemorrhage, testicular injury and lethality whereas prolonged exposure to Cd causes bio-accumulative damage to various organ systems [2,3]. Liver is the main target organ forshort-term Cd toxicity. Tolerance to acute Cd hepatotoxicity depends on the pre-synthesized metallothioneins (MT) in the liver, which functions to sequester Cd in the cytosol, with areduction in the amount of available Cd for other critical organelles . Soluble salts of Cd accumulate in the body, leading to toxicity in the liver, kidney, lungs, brain, testes, heart and central nervous system.
Withania somnifera(WS) is popularly known as ashwagandha is a palntin the solanaceaefamily.It has active constituents like withaferin A and withanolide D which are responsible for its bio-preventive properties . These constituents are known to elevate the levels of endogenous antioxidants such as superoxide dismutase (SOD), catalase (CAT), ascorbic acid and decrease lipid peroxidation (LPO) [6,7]. Khare (2007) has demonstrated thehepato-protective efficacy of withaferin A . Vitamin E (α-tocopherol) (VE) is an important component of thehuman diet and is the most effective lipid-soluble antioxidant in the biological system. It is one of the most efficient chain-breaking antioxidant  and allows free radicals to extract a hydrogen atom from its antioxidant molecules by rendering them inactive and breaks the chain of free radical reactions .
Thus the present study was designed to evaluate the preventive efficacy of thecombination of WS and VE against Cd-induced hepatic cytotoxicity and genotoxicity.
Selection of animals
Adult male BALB/c mice (n=6/group) weighing 25-30 gm were used throughout the studies. The mice were obtained from animal house of Panjab University after approval from theethics committee of the institute (IAEC/282). All the animals were housed in polypropylene cages, fed standard rat pellet diet (Ashirwad Industries, Punjab, Hindustan Lever, India) and were given water and maintained on a 12 hour day and night regime.They were acclimatised for 10 d prior to experimental use.All animals used in the study were housed, cared and used experimentally in accordance with the ‘Guide for the Care and Use of Experimental Animals’.
Plant used in the study
Leaves of WS Plant were collected from Chandigarh and were identified and voucher numbers were obtained from Botany department of Panjab University, Chandigarh (voucher no. 21250). The leaf of the plant was air dried and the extraction was done using Soxhlet extraction method and ethanol was used as a solvent.
Chemicals used in the study
CdSO4 was obtained from Sigma Chem. co., St. Louis, Missouri, USA. VE was obtained from MERCK India Limited. All chemicals of analytical grade specifications were obtained from HIMEDIA Ltd., India.
Preparation of homogenate and post-mitochondrial supernatant from liver tissue
10% homogenates of liver tissue were prepared in 50 mmol Tris-HCl buffer (pH-7.4) using a homogenizer at 0-4 °C. The homogenates were used for biochemical estimation of LPO, reduced glutathione (GSH) and protein content. Post mitochondrial supernatants (PMS) were prepared by centrifuging these homogenates at 10,000 rpm for 10 min at 4 °C and supernatants were used for the estimation of anti-oxidant enzymes (using Jenway 6305 UV/vis spectro-photometer).
|S. No.||Groups||Treatment (for 5d)|
|1||Control group||normal water and food pellet|
|2||Cd group||5 mg/kg b. wt.(ip)|
|3||WSgroup||500 mg/kg b. wt.(orally)|
|4||Cd+WS||5 mg/kg b. wt.(ip)+500 mg/kgb. wt.(orally)|
|5||VE group||100 mg/kg b. wt. (orally)|
|6||Cd+VE group||5 mg/kg b. wt.(ip)+100 mg/kgb. wt.(orally)|
|7||Cd+WS+VE||5 mg/kg b. wt.(ip)+500 mg/kgb. wt.(orally))+100 mg/kg b. wt.(orally)|
Homogenate and PMS of liver were prepared and various biochemical tests were performed by standard methods.
LPO was estimated by following thestandard method of Beuge and Aust .Homogenate (10%) was incubated in 150 mmol Tris-HCl (pH 7.4), 1.5 mmolascorbic acids, 1.0 mmol ferrous sulphate and incubated for 15 min. Added 10% trichloroacetic acid and 0.375% thiobutyric acid (TBA) and kept in boiling water bath for 15 min. The absorbance of theclear supernatant was measured at 532 nm.
GSH was measured in the homogenate by thestandardmethodofbeutleret al. .Homogenate and 0.2M di-sodium hydrogen phosphate buffer were mixed thoroughly. Then added 1.0 mmol Dithio-bisnitro benzoic acid (DTNB) prepared in 1% potassium citrate. The absorbance of theclear supernatant was taken at 412 nm.
The activity of GST was measured in the PMS by the method of Habiget al. . PMS was incubated in 0.2 M phosphate buffer, 20 mmol1-chloro-2,4-dinitrobenzene (cDNB). Kept this mixture at 37 °C for 5 min. and increase in absorbance/30 seconds was measured at 340 nm for 3 min.
CAT activity was measured in PMS by using thestandardmethod ofLuck . H2O2 phosphate buffer is formed by mixing 0.067M phosphate buffer in 12.5 mmol H2O2 (pH 7). An assay mixture consisted of 2.9 ml of H2O2 phosphate buffer and 0.01 ml PMS. The decrease in absorbance at 240 nm was noted after 30 seconds for 3 min.
SOD was measured in PMS according to the method of Kono . Solution A was prepared by 50 mmol Sodium carbonate in 0.1 mmol EDTA (pH 10.8), Solution B contained 96 µM Nitroblue tetrazolium (NBT) in solution A, Solution C was prepared by mixing 0.6% (v/v) Triton X-100 (w/v) in solution A and Solution D contained 20 mmol Hydroxylamine hydrochloride (pH 6.0). The rate of NBT reduction by SOD enzyme was recorded at 560 nm for 30 min.
Protein content was determined by the method of Lowry .
The structural changes in the liver were evaluated by Hematoxylin and Eosin staining by themethodofpearse .Tissues were fixed into Bouin’s fixative and cut at 5 microns in amicrotome and stained with haematoxylin and eosin and observed under light microscope (Leica DC 100, PCI Interface Digital Camera).
Bone marrow chromosomal analysis
Bone marrow chromosomal analysis was done by the method of Das . Prior to sacrifice, mice were injected with 4 mg/kg b. wt.of colchicineintraperitoneally(ip). Mice were sacrificed and bone marrow was flushed out of femur with hypotonic KCl (0.075M) solution, converted into afine suspension of cells by repeated aspiration and pipetting. Then cell suspension was incubated at 37 °C and centrifuged to get a pellet.Cells were then fixed in Carnoy’s fixative, slides were prepared, air dried and stained with 4% giemsaand observed under light microscope.
Allvalues were expressed as mean±standard deviation (SD) of 6 animals per group. Statistical significance in all groupswasanalysed by applying ONE WAY ANOVA at 5% significant level using SPSS software.
Amelioration of oxidative stress by WS and VE
The oxidative stress was assessed by analysing LPO, levels of cellular non-enzymatic (GSH) and enzymatic anti-oxidants (SOD, CAT, GST). These parameters were evaluated spectrophotometrically in the liver tissues of mice exposed to Cd, as well asWS (500 mg/kg b. w.) and VE (100 mg/kg b. w.). Short term Cd exposure (5 mg/kg b. w.) caused statistically significant elevation (p<0.0001) in the levels of LPO (97%) in liver tissue as compared to the control mice. Individualtreatments ofWSand VEto Cd-treatedmicesignificantly decreased the elevated levels of LPO (~24%, p ≤ 0.001) but theconcurrent treatment of WSand VE (6.96%)normalized the levels of LPO to control values (fig. 1 [A]).
A statistically significant decrease (p ≤ 0.0001) was observed in the level of GSH (57%) and GST (80%) in liver tissues of Cd intoxicated mice as compared to control mice.WS and VE individual treatments restored the levels of GSH and GST in the hepatic tissue (p ≤ 0.001). However, Concurrent treatment of WS with VE in theCd-treated group more effectively preserved the anti-oxidant status of GSH (10.40%, p ≤ 0.05) (fig. 1 [B]) and GST (17.79%, p ≤ 0.05) (table 1).
Five days Cd exposure significantly decreased the levels of CAT (63.82%, p ≤ 0.0001) and SOD (70.26%, p ≤ 0.0001) in liver tissue as compared to the control. Individual treatments of WSand VE significantly (p ≤ 0.001) increased the levels of these antioxidants as compared to the Cd-treated group but thecombinational treatment of WS and VE to Cd intoxicated mice, worked in combination in elevating the levels of CAT and SOD (p ≤ 0.05) (table 1).
Modulatory potential of WS and VE on Cd-induced histopathological alterations
Light microscopic examination of liver from control mice depicted the presence of cords of hepatocytes arranged around the central vein, occurrence of hepatic sinusoids in between the hepatic cords with theoccasional distribution of Kupffer cell. (fig. 2 [A]). Similar histoarchitecture of theliver was also observed in WS (fig. 2 [B]) and VE treated mice (fig. 2 [C]). Cd intoxication induced marked structural alterations in liver including hepatocytic degeneration, multiple foci of Kupffer cells infiltration, Kupffer cells hyperplasia, sinusoidal widening and lobular inflammation. Multiple foci of thenecroticlesions and damaged portal triad were also observed (fig. 2 [D-F]).
Fig.1: Graphs showing levels of lipid peroxidation (LPO) and reduced glutathione (GSH) in liver tissue, (A) levels of LPO (n moles/mg protein) in livers of various treatment groups; (B) levels of GSH (µ moles/mg protein) in livers of various treatment groups.▀p ≤ 0.0001 (extremely statistically significant), ▲p ≤ 0.001 (very statistically significant), ●p<0.05 (statistically significant)
Table 1: Table showing protective effect of WS and VE on level of GST, CAT and SOD in mice with Cd-induced toxicity in liver
Values of glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) represented as mean±SD. The level of significance was considered as●p<0.05 (statistically significant), ▲p ≤ 0.001 (very statistically significant), ▀p ≤ 0.0001 (extremely statistically significant). Units: CAT-µmoles of H2O2 decomposed/min./mg protein; SOD–units/min./mg protein (1unit of theenzyme is defined as the amount of enzyme-inhibiting 50% nitroblue tetrazolium reduction);GST-µmoles GSH adduct formed/min./mg protein.
Fig.2: Light micrographs of hepatic tissues from control and Cd-treated mice, (A) control mice showing normal histoarchitecture of liver with hepatocytes arranged in cords around central vein and well-defined sinusoids (X200); (B) WSand (C) VE treated mice liver showing normal hepatocytes arranged in cords around central vein. Well organized sinusoids separating the hepatocytes and few Kupffer cells present in the sinusoids (400X); (D-F) Cd-treated mice liver: (D) Large Kupffer cells infiltration, lobular inflammation, damaged hepatocytes (X100), (E) Widening of sinusoids and massive infiltration around portal triad (X400), (F) Appearance ofbinucleated and trinucleated cells in hepatocytes, damaged endothelial lining of central vein (X400)
Fig.3: Light micrographs of hepatic tissues from different groups getting various treatments in combination with Cd, (A) Cd+WStreated mice liver revealednormal hepatocytes and sinusoids with increased Kupffer cells infiltration (X100); (B) Cd+VE treated mice liver revealed normal hepatocytes and sinusoids, enhanced Kupffer cells infiltration and slight lobular inflammation (X100); (C-D) Cd+WS+VE treated mice liver revealed normal hepatocytes with well-organized sinusoids and very few foci of Kupffer cell infiltration (X100, X200)
Treatment with WS effectively prevented Cd mediated structural alterations in the liver (fig. 3 [A]). VE also modulated the structural alteration induced by Cd (fig. 3 [B]) but WS was found to be more effective than VE in individual treatments. However, the concurrent treatment of WS and VE prevented the structural alterations produced by Cd more promptly as compared to their individual treatments indicating their combinational efficacy (fig. 3 [C-D]).
Abbreviations for fig. 2 and 3: BD–bile duct, CV–Central vein, KC–Kupffer cells, HA–hepatic artery, HP–hepatocytes, HS–Hepatic sinusoids, HV–hepatic vein, PT–portal triad.
Genoprotective efficacy of WS and VE against Cd-induced chromosomal alterations
Metaphasic plates (100) were analysed from bone marrow chromosomes of all the groups. Metaphasic spread of control group revealed 40 chromosomes with normal contour and well-structured chromatids (fig. 4(A)andtable2).WS (fig. 4 [B] and table 2) and VE treatments also (fig. 4 [C]andtable 2) revealed 40 chromosomes with normal contour and well-structured chromatids in light micrographs.
Five days Cd exposure induced genotoxic damage at the cellular and subcellular level. Bone marrow chromosomal analysis of Cd-treated mice revealed various chromosomal aberrations, which were then further classified into structural, physiological and numerical aberrations. Structural aberrations included centromeric fusion (18%), chromosomal breaks (2%), elongated chromosomes (4%), ring formation (10%) and Y chromatid separation (16%). Whereas physiological aberrations included the wooly (4%), sticky (4%) and condensed chromosomes (8%). Aneuploidy observed in 10% of the cells that signifies the numerical aberrations. (fig. 4 [D-I])and table 2).
Fig.4: Metaphasic spreads from bone marrow of various control groups (Saline, WS, VE) and Cd-treated mice, (A) control Group: normal metaphasic spreads with 40 acrocentric chromosomes; (B) WSGroup: normal metaphasic plates with 40 chromosomes; (C) VE Group: Normal metaphasic spreads of 40 chromosomes; (D-I) Cd-treated mice showing chromosomal aberrations; (D) centromeric fusion; (E)ring formation and centromeric fusion; (F)Y chromatid break; (G)shortened and wooly chromosomes; (H) aneuploidy; (I)chromosome break
WStreatment ameliorated Cd-induced chromosomal defects like ring formation (2%), centromeric fusion (2%), Y chromatid breaks (2%) and aneuploidy (4%) (fig. 5 [A-C]and table 2). Although VE was also found to be effective in reducing Cd mediated genotoxicity by decreasing abnormalities like ring formation (2%), condensed chromosomes (2%), Y chromatid breaks (2%), chromosomal breaks (2%), chromatid elongation (2%),but WSwas found more effective in case of individual treatments (fig. 5 [D-F]and table 2).
Whereas, concurrent treatment of WS and VE was found to be most effective in attenuating Cd mediated chromosomal aberrations. A large number of normal metaphasic plates were seen in this group (90%) with very few cells revealing centromeric fusion (2%), Y chromatid breaks (2%), chromatid elongation (2%), sticky chromosomes (2%) and aneuploidy (2%) (fig. 5 [G-I]and table 2).
Fig.5: Metaphasic spreads from bone marrow of different groups getting various treatments in combination with Cd (A-C) Cd+WS treated mice; (A)-normal extended 40 acrocentric chromosomes, (B)-ring formation, (C)-sticky and elongated chromosomes; (D-F) Cd+Vitamin E treated mice. (D)-Y chromatid break, (E)-centromeric fusion, (F)-aneuploidy; (G-I) Cd+WS+VE treated mice G-normal extended 40 acrocentric chromosomes, H-chromosomal break, I-Y chromatid breaks
Abbreviations for fig. 4 and 5: CB-chromosomal break, CF-centromeric fusion, R-Ring formation, Y-Y chromatid break.
Table 2: Effect of WS and VE on Cd-induced chromosomal aberrations in bone marrow cells of mice (5 d treatment) (%age of cells)
|Multiple chromatid breaks||4||2|
|Normal metaphase plate||98||14||96||96||84||80||90|
Cd exposure generates oxidative stress and contributes to the development of serious pathological conditions because of its long-termretention in tissues . The exact mechanism of toxicity is not known but a number of mechanisms have been proposed including enhanced oxidative stress, theinvolvement of genotoxic mediators and failure of preventive mechanisms . Thus present study tries to correlate the major prospective parameters to deduce the basis of Cd toxicity. Short term Cd exposure significantly increased the levels of hepatic LPO plausibly by both direct contribution and indirectly by activation of free radicals. The increased lipid peroxide levels might have contributed towards altered membrane functions . Furthermore, enhanced LPOalso contributes towards the piling up of the oxidative stressby adding more free radicals to the stock and by inhibiting the antioxidant enzymes activities.SOD is the first line of defence against free radicals which catalyzes the dismutation of superoxide anion radical (O2•ˉ) into hydrogen peroxide (H2O2) by reduction, whereas CAT plays an important role in theelimination of H2O2 by initiating its hydrolysis into thewater and prevents its accumulation in the body . Thus increasedMDA levels and decreased activities of SOD and CAT enzymes formed the basis of observed Cd-inducedhepatic toxicity. These observations strongly correlate LPO mediated depression in hepatic levels of catalase and SOD. Casalino also suggested Cd-induced displacement of Zn, Mn from the active sites of SOD, resulting in its decreased activity [23, 24].
Cd exposure caused more than 50% decline in the level of GSH and GST in liver. The decreased activity of GSH could be possibly due to competition between Cd-MT complex and GSH for sulphur containing amino acids. As Cd-MT complex has more affinity for sulphur containing amino acids thus masking the activity of GSH . Furthermore, decreased level of GSH caused the decline in the level of GST and GR making the cell more susceptible to attack by free radicals .
In the present study, the histoarchitecture of Cd-exposed mice liver revealed many histopathological alterations like sinusoidal dilation, hepatocytic damage, kupffer cells infiltrations and multiple foci of lobular inflammation due to mononuclear cell and kupffer cell hyperplasia around the portal triad. This could appear due to inflammatory mediators released by kuppfer cells and generation of ROS which caused asubsequent elevation in LPO. In the present study, marked elevation in hepatic LPO and reduced antioxidants must have caused the observed histopathological alterations in the liver. These results are in accordance with the previous observations [25-26]who have also indicated that the imbalance of pro and antioxidant mediated LPO formed the basis of cellular and subcellular damage.
The genotoxicity of Cd is well established as Cd is regarded as a human carcinogen . The observed Cd-inducedstructural, physiological and numerical chromosomal aberrations seemingly have developed due to theunderlying stockpile of free radicals and by direct interaction of Cd with the DNA.Elevated levels of LPOenhanced the probability of interaction between Cd and DNA due to the leaky membranes . Furthermore, theimbalance between oxidants and antioxidants adds fuel to the building up cellular oxidative stress and enhances the clastogenic potential of the metal. Most plausibly the duo of metal and oxidative stress could have played the significant role in themanifestation of Cd-induced genotoxicity.
In the present study, the anti-oxidant property of WS, VE andtheir combination has been exploited against Cd-induced toxicity. Treatment with WS markedly decreased the LPO and increased the levels of endogenous enzymes like CAT, GST, GSH and SOD. The observed antioxidant activity of WScould be attributed to its biologically active constituent’swithaferin A and withanolides provide free phenolic hydrogen atom . These phenolic hydrogen atoms are capable of scavengingCd initiated free radical generation. Furthermore, WS instigated elevation in the levels of antioxidants and lowered metal induced biochemical and structural alterations. These observations are in conformity with Bhattacharya and his co-authors who have also documented WSmediated elevation in antioxidant enzymes [29-31]. As WSreduced the observed oxidative stress, so the genotoxic alterations were also seen to be reduced. Biologically active constituents of WSlike steroidal lactones, alkaloids and flavonoids by reducing LPO might have prevented the free radicals to interact directly and indirectly with DNA and established the genoprotective potential of WS [32, 33].
VE treatment also reduced the Cd-induced oxidative stress by reducing lipid peroxidation and oxidative stress due to its high lipid solubility . VE rapidly transfers its phenolic H-atom to a lipid peroxyl radical, converting it into a lipid hydroperoxide and a VE radical . VE is the most potent lipid peroxyl radical scavenger that significantly lowers LPO and oxidative stress-mediated chromosomal damage by stabilizing leaky membranes . This protective efficacy of VE could have inhibited Cd-induced free radical formation and its stockpiling and furthermore prevented histopathological and chromosomal alterations.
WSand VE both being strong free radicals scavengers interfered well with the Cd instigated toxic reactions. Both these antioxidants reduced the Cd-induced hepatotoxicity but the combinational treatment of WSand VE most effectively attenuated Cd mediated alterations. Their reactive constituents acted in a duo that inhibited ROS and prevented biochemical, structural and genotoxic changes. VE by preventingthe formation of peroxyl radical reduced LPO and inhibited all the chain reactions instigated by this radical and WSbalanced the pro-and antioxidant status in the system by providing free phenolic hydrogen atoms to the free radicals and enhancing the levels of endogenous antioxidants. Thus this duo synergistically acted on the Cd generated ROS and prevented the building up oxidative stress and resulted in abrogation of Cd mediated toxicity. Thus the present study proposes WS and VE as potential therapeutic drug supplements for ameliorating metal-induced toxicity.
Seema Rani, Vijay Lakshmi Sharma and Mani Chopra designed the research work; Seema Rani and Mani Chopra performed the experimental research work; Seema Rani, Vijay Lakshmi Sharma and Mani Chopra analysed the data; Seema Rani and Mani Chopra wrote the manuscript.
We acknowledge UGC-CAS II and DST-PURSE for financial assistance.
The authors declare no conflict of interest
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How to cite this article
Seema Rani, Vijay Lakshmi Sharma, Mani Chopra. Evaluating the protective efficacy ofcombination Withania somnifera and vitamin E against cadmium-induced oxidative stress-mediated hepatic histopathology and genotoxicity in murine model.Int J Pharm Pharm Sci 2017;9(10):230-235.