Journal of Trace Elements in Medicine and Biology 30 (2015) 184–193

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TOXICOLOGY

Brain most susceptible to cadmium induced oxidative stress in mice Sandeep K. Agnihotri a , Usha Agrawal b , Ilora Ghosh a,∗ a b

Biochemistry and Environmental Toxicology, Laboratory # 103, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India National Institute of Pathology, Safdarjang Hospital Campus, Post Box No 4909, New Delhi 110029, India

a r t i c l e

i n f o

Article history: Received 12 August 2014 Accepted 28 December 2014 Keywords: Cadmium Brain Lipid peroxidase Oxidative stress

a b s t r a c t Accumulated evidence over the years indicate that cadmium (Cd) may be a possible etiological factor for neurodegenerative diseases. This may possibly be linked to excessive generation of free radicals that damages the organs in the body depending on their defence mechanism. Since Cd is a toxic agent that affect several cell types, the aim of this study was to shed light on the effect of Cd and its consequences on different organs of the mice body. To test the hypothesis of concentration dependent Reactive Oxygen Species (ROS) generation and DNA damage, observations were done in the serum of 4–5 weeks old male Swiss albino mice by treating with cadmium chloride (CdCl2 ) in drinking water for 30 days. The expression of Bcl-2-associated X protein (Bax) an apoptotic marker protein was two times higher in brain compared to liver at an exposure level of 0.5 mg L−1 CdCl2 . Furthermore the correlation and linkage data analysis of antioxidant defence system revealed a rapid alteration in the brain, compared to any other organs considered in this study. We report that even at low dose of Cd, it impaired the brain due to lipid peroxidase sensitivity which favoured the Cd-induced oxidative injury in the brain. © 2015 Elsevier GmbH. All rights reserved.

Introduction Cadmium (Cd), one of the biologically non-essential metals, is currently placed in the list of top 20 hazardous substances [1]. The role of Cd in diseases like Wilson’s disease and Menkes syndrome is well known [2]. An increased production of Reactive Oxygen Species (ROS) which are potentially harmful for the cell components, is a common outcome of Cd exposure. Cd induces the generation of ROS by upregulating the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2) and its associated proteins [3]. It is evident that exposure to Cd may cause adverse health effects through formation of free radicals, that results in DNA damage, lipid peroxidation (LPO), and depletion of protein sulfhydryls [4]. Oxidative stress that results from the state of imbalance between the concentrations of ROS and the antioxidant defence mechanisms, may be connected to various pathological abnormalities [5–7] e.g. neurodegenerative diseases, diabetes, cancer [8]. Prolonged exposure of Cd, a toxic metal, targets the lung, liver, kidney, and testes following acute intoxication, and causing nephrotoxicity, immunotoxicity, osteotoxicity and tumours. Earlier report indicated that Cd stimulates free radical

∗ Corresponding author. Tel.: +91 11 26704306. E-mail address: [email protected] (I. Ghosh). http://dx.doi.org/10.1016/j.jtemb.2014.12.008 0946-672X/© 2015 Elsevier GmbH. All rights reserved.

production, resulting in oxidative damages of lipids, proteins and DNA [9,10]. LPO and protein carbonylation are seen to be the two important parameters, increases with Cd intoxication [11,12]. Its damages were estimated through protein carbonylation and total sulfhydryls degradation [13,14]. The overexpression of Hyaluronan Binding Protein 1 (HABP1), a 34 kDa protein of the hyaladherin family generates ROS in normal murine fibroblasts resulting in induction of apoptosis [15]. Its interaction with various pathogenic proteins under condition of oxidative and pathological stress suggests its generic role in disease conditions [16,17]. In this paper, we report for the first time that Cd toxicity induces differential expression of HABP1 along with Bax, the apoptotic markers in different organs. We also observe that upon oral Cd treatment a significant dose dependent increase in oxidative stress in the brain compared to other organs, signifying the brain to be more vulnerable towards Cd toxicity. Material and methods Material All reagents were obtained from Sigma Aldrich (St. Louis, MO, USA). The anti-HABP1 antibody was raised against purified recombinant human HABP1 in rabbit and characterized in our laboratory [18].

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Animals’ exposure

Catalase assay

Male Swiss Albino mice (4–6 week old and 25 ± 5 g body weight) were obtained from the animal facility of Jawaharlal Nehru University, New Delhi. Animals were categorized in five experimental groups and each group had 5 mice (5 × 5 = 25) of same breed progeny for treatment. To nullify the experimental variation of body weight the consumption of drinking water per day by average weight of a single group, was gauged and the estimated value of CdCl2 concentration in 0.1, 0.5, 1.0, and 2.0 mg L−1 was dissolved in their drinking water and provided for 30 days treatment continuously with reference to earlier reports [19–23]. All the animals were housed in an air conditioned room, where the temperature was maintained at 25–27 ◦ C with constant humidity (40–50%) and kept on 12 h/12 h light/dark cycle throughout the experiment. Animals were fed with standard food pellets (prepared by Brook Bond India Ltd., Backbay Reclamation, Mumbai, India), ad libitum. The spinal dislocation to sacrifice the mice for further experimental protocols for mice described in this study were approved previously by the Institutional Animal Ethical Committee (IAEC) and the committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). All subsequent animal experiments adhered to the ‘Guidelines for Animal Experimentation’ of the University.

Cytosolic fractions were first incubated with 0.3 mol L−1 ethanol and 2% (v/v) Triton X-100 in 0.01 mol L−1 sodium phosphate buffer at pH 7.0 for 10 min in a bath of ice cold water. Catalase activity was then measured at 0 ◦ C in phosphate buffer at pH 7.0 in the presence of 1.5 mmol L−1 peroxide. The reaction was stopped at different times by adding the peroxidase reagent H2 O2 and the disappearance of peroxide was then measured colorimetrically. Results were expressed in terms of the apparent first order reaction rate constant [26].

Preparation of homogenate and cytosol fraction After 30 days treatment mice were sacrificed by spinal dislocation and kidney, brain, liver, spleen and testis were perfused immediately with cold 0.9% saline and washed with chilled 0.15 mol L−1 Tris–potassium chloride (Tris–KCl) buffer (pH 7.4). Tissues were then blotted dry, weighed and homogenized in ice cold 0.15 mol L−1 Tris–KCl buffer (pH 7.4) for 10% homogenate. This homogenate (0.5 mL) was used for assaying glutathione level and remaining homogenate was centrifuged at 1047.19 radian s−1 for 20 min. Then supernatant was transferred into pre-cooled tubes and ultra-centrifuged at 10,500 × g for 60 min. The cytosolic fraction was collected and used for antioxidant enzyme assay, lactate dehydrogenase (LDH) and the pellet was dissolved in homogenizing buffer and used for assaying LPO. Glutathione peroxidase (GSH-Px) assay The activity of GSH-Px was measured in a freshly prepared cytosolic fraction. Glutathione reductase solution (2.4 U mL−1 in 0.1 mol L−1 potassium phosphate buffer, pH 7.0) was added to a 50 mmol L−1 potassium phosphate buffer (pH 7.0), 0.5 mmol L−1 ethylenediaminetetraacetic acid (EDTA), 1 mmol L−1 sodium azide, 0.15 mmol L−1 Nicotinamide adenine dinucleotide phosphate (NADPH) and 0.15 mmol L−1 hydrogen peroxide (H2 O2 ). After the addition of 1 mmol L−1 GSH (reduced glutathione), the NADPHconsumption rate was monitored at 340 nm as described by Flohe and Gunzler [24]. Superoxide dismutase (SOD) assay The cytosolic fraction of tissue homogenate diluted 10 times with water, mixed with 5 ␮L Triton X-100, incubated for 30 min (25◦ C) and thereafter 3 mmol L−1 EDTA was added in assay mixture. The reaction was initiated by the addition of 100 ␮L of freshly prepared 2.6 mmol L−1 pyrogallol solution in 10 mmol L−1 hydrogen chloride (HCl) to attain a final concentration of pyrogallol of 0.13 mmol L−1 in the assay mixture. The assay mixture was transferred to a 1.5 mL cuvette and the rate of increase in the absorbance at 420 nm was recorded for 2 min from 1 min 30 s to 3 min 30 s in a spectrophotometer [25].

Lactate dehydrogenase (LDH) assay Lactate dehydrogenase was assayed by measuring the rate of oxidation of NADH at 340 nm as described by Bergmeyer and Bernt [27]. One unit of enzyme activity was defined as that which causes the oxidation of one ␮mol of NADH per minute. Lipid peroxidation (LPO) estimation LPO in the microsomes was estimated spectrophotometrically by the thiobarbituric acid reactive substances (TBARS) method [28] and is expressed in terms of malondialdehyde (MDA) formed per mg of protein. Reactive Oxygen Species assay N,N-diethyl-para-phenylendiamine (DEPPD) was dissolved in 0.1 mol L−1 sodium acetate buffer (pH 4.8) to a final concentration of 100 ␮g mL−1 (R1 solution), and ferrous sulphate was dissolved in 0.1 mol L−1 sodium acetate buffer (pH 4.8) to a final concentration of 4.37 ␮mol L−1 (R2 solution). Five ␮L of either H2 O2 standard solution (for generating a calibration curve) or serum was added to 140 ␮L of 0.1 mol L−1 sodium acetate buffer (pH 4.8) in 1 well of a 96-well microtiter plate, which reached a temperature of 37 ◦ C after 5 min. One hundred ␮L of the mixed solution, which was prepared from R1 and R2 at a ratio of 1:25 before use, was added to each well as a starter. Then, after pre incubation at 37 ◦ C for 1 min using a multi-mode microplate reader (Spectra Max M5), absorbance at 505 nm was measured for a fixed time (between 60 and 180 s) at intervals of 15 s. A calibration curve was automatically constructed from the slopes, which was calculated based on varying (delta) absorbance at 505 nm each time (min) corresponding to the concentration of hydrogen peroxide. ROS level in serum were calculated by the analyzer of Spectra Max M5 from the calibration curve, and expressed as equivalent to levels of H2 O2 (1 unit = 1.0 mg H2 O2 L−1 ). The serum ROS levels determined by this assay system were compared with those measured by the conventional DROM test. In brief, 20 ␮L serum and 1.2 mL buffered solution (R2 reagent) were mixed in a cuvette, and 20 ␮L chromogen substrate (R1 reagent) was added to the cuvette. After mixing properly, the cuvette was centrifuged for 1 min and incubated for 5 min at 37 ◦ C. Absorbance at 505 nm was monitored for 3 min [29]. In ROS measurement assay all data (10 experiments) were representative of five similar sets of exposure group having at least two mice from each group, in which the relative absorbance intensity was calculated by averaging the values as mean ± S.E. Comet assay Blood was collected (0.6–0.8 mL) from the orbital vessels of each animal post-treatment and the comet assay was performed as described by Singh et al. [30]. Slides were prepared in triplicate per blood cells sample. First the slide was dipped in methanol and burned over a blue flame to remove the machine oil and dust.

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Prepared the 0.5% LMPA (low melting point agarose) and 1% NMA (normal melting agarose) and heated until near boiling point to dissolve the agarose. The slides were dipped one-third into the hot agarose. Wiped the underside of the slide to remove agarose and laid it in a tray on a flat surface to dry. To the coated slide added equal volume of 1% LMPA and phosphate buffered saline (PBS) diluted blood, placed coverslip, and put the slide on a tray resting on ice pack until agarose layer hardened. Gently slide off the coverslip and added a third agarose (80 ␮L LMPA) layer to the slide. Replaced coverslip and kept the slide in the tray until the agarose layer hardens (10–15 min) then slowly lowered slide into cold, freshly made lysing solution (2.5 mol L−1 sodium chloride (NaCl), 100 mmol L−1 Na2 EDTA, 10 mmol L−1 Tris–hydrochloric acid (Tris–HCl), with 1% Triton X-100, and 10% dimethyl sulfoxide (DMSO) added just before use, pH 10), protected from light and refrigerated for minimum two hours. Slides were then placed in alkaline buffer (300 mmol L−1 sodium hydroxide (NaOH) and 1 mmol L−1 EDTA, pH 13) for 20 min to allow unwinding of the DNA to occur. Electrophoresis was conducted for 25 min at 25 V (0.66 V cm−1 ) adjusted to 300 mA by raising or lowering the buffer level in the tank. Slides were then drained, placed on a tray, and washed slowly with three changes of 5 min each of neutralization buffer (0.4 mol L−1 Tris–HCl pH 7.5). DNA was precipitated and the slides were dehydrated in absolute methanol for 10 min and were left at room temperature to dry. The whole procedure was carried out in dim-light to minimize the DNA damage and mortality was observed. Slides were stained with 50 mL of ethidium bromide (20 mg mL−1 ) and viewed under a Nikon fluorescence microscope. Immunoblot analysis Gel electrophoresis under the denaturing condition (in the presence of 0.1% sodium dodecyl sulfate (SDS)) was performed according to the method of Laemmli [31]. Protein (100 ␮g perwell) was loaded in each well and electrophoresed along with the standard molecular weight marker. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The proteins in the gel were transferred onto the nitrocellulose membrane by applying 0.8 mA H−1 current in a wet transfer unit (Bio Rad, Australia) and were immuno-detected with specific antibodies of anti-rHABP1 (1:1000, raised in the laboratory), anti-Bax (0.2 ␮g mL−1 ) and visualized with nitro blue tetrazolium (NBT) 5bromo-4-chloro-3-indolyl phosphate (BCIP) using anti-rabbit or anti-mouse secondary antibody (0.02 ␮g mL−1 ) conjugated to alkaline phosphatase. In immunodetection experiments, western blots were repeated five times using prepared lysate from 5 mice of each exposure group. Immunohistochemical analysis of tissue samples Sections of tissue were deparaffinised, rehydrated and the nonspecific antibody binding was blocked with 3% (w/v) bovine serum albumin (BSA) in 10 mmol L−1 PBS for 1 h at RT. After 5 washes of 5 min each in PBS, the slides were incubated with primary antibodies, 1:100 dilution of anti-rHABP1 (made in the laboratory) [18] or anti-Bax antibody (Sigma), incubated for 1 h in humid chamber. Sections were washed (5 × 5 min in PBS) and incubated with secondary antibody (goat anti-rabbit IgG 0.2 ␮g mL−1 ), conjugated to alkaline phosphatase (AP) (for anti-HABP1 antibody) or conjugated to HRP (for anti-Bax antibody) for 1 h at RT. Fast RedTM (Dako, Denmark) substrate was used for HABP1 detection and 3,3 diaminobenzidine (DAB) for Bax to develop colour. After the desired colour intensity, the sections were washed in PBS, counter-stained in haematoxylin, and mounted in glycerol. At no point during the whole procedure, the sections were allowed to remain dry.

Histopathological analysis For histopathology analysis, tissue sections were fixed in 10% formalin solution for 24 h and then embedded in paraffin wax and 5 ␮m section stained with haematoxylin–eosin. Haematoxylin–eosin stained sections were examined and scored by the pathologist. The degree of necrosis was classified on a scale of 0–III [normal: 0 (0%), mild: I (1–25%), moderate: II (26–49%), severe: III (50–100%)] based on the area of necrosis in each field and expressed as the mean of 10 high power fields (HPFs), chosen at random. The degree of inflammation was evaluated in the same 10 HPFs and classified on a scale of 0–III [no inflammation: 0 (mean of inflammatory cells in 10 HPFs = 0), weak inflammation: I (mean of inflammatory cells in 10 HPFs = 1–10), moderate inflammation: II (mean of inflammatory cells in 10 HPFs = 11–49), and severe inflammation: III (mean of inflammatory cells in 10 HPFs = 50 and over)] [32].

Statistical analysis All enzymatic assays and ROS generation were expressed as means ± standard error of the mean (s.e.m.) with SPSS for windows, version 7.0. Variable of different groups were compared using the Student’s t-test or ANOVA analysis. A level of P < 0.05 was accepted as being statistically significant. Draftsman Plot, a Bray–Curtis similarity is conducted on the data matrix without transformation, and results in a similarity matrix which can then be examined using cluster analysis dendrograms and non-metric, multi-dimensional scaling (MDS) coordination plots. The clustering method used to have high levels of similarity (>65% similarity). Difference in expression level of HABP1 and Bax in immunoblots was determined using Image J 1.42q software.

Results Survivability of mice decreases with increase of CdCl2 concentration To detect in vivo Cd induced toxicity, i.e. LD50 (amount of dose required to kill 50% of test population) in Swiss Albino male mice of age group of 4–6 weeks were treated with different concentrations of CdCl2 (no treatment as control, 0.1 mg L−1 , 0.5 mg L−1 , 1.0 mg L−1 and 2.0 mg L−1 ) for 30 days continuously with drinking water. The mice were randomly divided into 5 groups of 5 mice each and based on their body weight, each was observed against appropriate control and their survivability decreased 90%, 68%, 56%, and 28% corresponding to concentrations 0.1 mg L−1 , 0.5 mg L−1 , 1.0 mg L−1 and 2.0 mg L−1 respectively (Fig. 1A). A survival rate of 56% at a concentration of 1.0 mg L−1 CdCl2 treatment was calculated to be the LD50 and all the subsequent experiments were carried out at this dose of toxicity.

Gradual increase of ROS level in serum due to CdCl2 treatment in a concentration dependent manner Based on previous reports of Cd-induced oxidative stress in different cell lines, the rate of ROS generation in Swiss Albino mice blood serum was checked. Increased level of ROS was observed in the blood stream of CdCl2 treated mice as compared to untreated control mice. Dose dependent gradual increase in ROS of 1.1 fold at 0.1 mg L−1 to more than 2.0 fold at 2.0 mg L−1 was observed, indicating elevated oxidative stress with augmented dose.

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A

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B 300

* Control 0.1

0.5

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* 2.0

Concentration of CdCl2 in mg L-1

ROS (mg/L)

% Survivability of mice

120 100 80 60 40 20 0

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200

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100 0

Control

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Cadmium Treatment (mg

2.0 L-1)

Fig. 1. (A) Rate of survivability of mice with increase in CdCl2 concentration. The histogram presenting the percentage of survivability which showed a decrease with gradual increase of Cd concentration. The data revealed 1.0 mg L−1 CdCl2 triggered LD50 . Asterisk (*) denotes the significance (P < 0.001). All data were representative of mean value of (5 sets of experiments each having 5mice of same breed) experiments with ± S.E. (B) Increase in ROS level upon CdCl2 treatment is concentration dependent. Quantitative estimation of serum ROS level, indicate doubling of oxidant, was reached gradually at 1.0 mg L−1 CdCl2 concentration in mice model, detected by absorbance at 505 nm of N,N-diethyl-para-phenylendiamine (DEPPD) as described in Materials and Methods section, expression is mg L−1 of serum. Asterisk (*) denotes the significance (P < 0.001). All data were representative of five similar sets of experiments having at least three mice from each treatment group, in which the relative absorbance intensity was calculated by averaging the values as mean ± S.E.

Histopathological analysis of Cd toxicity in different organs of swiss albino mice model To analyze the effect of Cd on histopathology of mice organs, haematoxylin–eosin (HE) staining of section of 30 days treated animal was done. Morphological features indicated that increasing concentration of CdCl2 affected the organs (Fig. 2). On the basis of scale (described in Material and Method section), sections of liver tissue showed significant loss of architecture with tissue destruction (scale I at 0.1 mg L−1 , scale II at 0.5 mg L−1 and 1.0 mg L−1 ), presence of microvesicular change and sinusoidal congestion (scale I at 0.1 mg L−1 , scale II at 0.5 mg L−1 and 1.0 mg L−1 and scale III at 2.0 mg L−1 ), increasing inflammation (scale I at 0.1 mg L−1 , scale II at 0.5 mg L−1 and 1.0 mg L−1 and scale III at 2.0 mg L−1 ), and presence of foci of necrosis (scale I at 0.1 mg L−1 ,

scale II at 0.5 mg L−1 and 1.0 mg L−1 and scale III at 2.0 mg L−1 ) with increasing concentration of CdCl2 treatment. Portal triaditis was observed at 2.0 mg L−1 concentration but loss of architecture, which was seen at lower concentrations of CdCl2 was not present. Inflammatory cells including lymphocytes, macrophages and neutrophils were observed in small focal collections at 0.5 mg L−1 , 1.0 mg L−1 and 2.0 mg L−1 concentration of CdCl2 . Inflammation was quantified as the number of foci in one whole section of liver. Inflammatory cells adjacent to the central vein (terminal hepatic venule) causing piecemeal necrosis of hepatocytes were considered more severe. In spleen tissue (extramedullary) haematopoiesis was observed with presence of megakaryocytes which decreased with increasing dosage of CdCl2 (scale I at 0.1 mg L−1 , scale II at 0.5 mg L−1 and 1.0 mg L−1 and 2.0 mg L−1 ). Mild fibrosis was observed in spleens of Cd treated mice. In kidney tissue interstitial

Fig. 2. Effect of Cd on morphology of tissue. Morphology of tissue changed with different CdCl2 concentration and deleterious effect found in kidney, liver, spleen, testis and brain by HE staining as explained in result section in depth, Bars = 50 ␮m.

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haemorrhage (scale I at 0.1 mg L−1 , scale II at 0.5 mg L−1 and 1.0 mg L−1 and 2.0 mg L−1 ) occurred in Cd treated mice and capillary congestion was found in 2.0 mg L−1 CdCl2 treated mice, but there was preservation of corticomedullary differentiation with normal appearing glomerulus, normal thickness of capillary basement membrane and normal cellularity of the glomerulus. Interstitial oedema was not found in renal tissues. In brain, hippocampal disruption occurred and size shrinkage of hippocampus observed with a lesser amount of neuron in dentate gyrus. In testis seminiferous tubules showed normal gametogenesis with maturation of germ cells to spermatids. Occasional sloughing of germ cells into the lumen was present. Leydig cells appeared normal in number.

Confirmation of apoptotic signalling due to CdCl2 treatment with tissue specific expression in mice A pleiotropic protein HABP1 is reported to be associated with stress signalling [15] and in early detection of epidermal carcinoma [33]. Its correlation with ROS generation instigated its in vivo immuno detection in CdCl2 treated mice organs and tissue types. Interestingly, the phenomenon of increased (>2 fold) expression of HABP1 has been observed well before the actual signalling start for apoptosis in the kidney and liver (0.1 mg L−1 ) by immunohistochemical analysis, again establishing its role as an early indicator for programmed cell death (PCD) signalling cascade (Fig. 3A). Immuno-detection (Fig. 3B) in protein samples from tissues showed an insignificant change in the expression pattern of HABP1 in the spleen and testis. The visible induction of HABP1 was observed with 0.5 mg L−1 of CdCl2 treatment in kidney and liver. Whereas in case of brain 0.1 and 0.5 mg L−1 both concentration has clear indication of early expression of HABP1, as compared to control. However, at 1.0 mg L−1 change in the level of expression of HABP1, in all these three (kidney, liver and brain) tissue samples, was insignificant. This immunodetection further corroborated the pathological analysis, confirming that the signalling cascade of HABP1, related to apoptosis was more rapidly activated where LD50 parameters were already switched on. Significance of HABP1 induction in reformatting the pathophysiology of tissues led us to analyze the expression of apoptotic marker Bax in mice system as a confirmatory test. In terms of toxicity and oxidative stress, higher expression level of Bax due to the effect of Cd (1.0 mg L−1 ) was confirmed in different tissues of mice in vivo (Fig. 4A) using immunostaining on tissue sections. In all the tissue samples except the brain, Bax expression was more than 1.5 fold at 1.0 mg L−1 Cd toxicity. In the brain the higher than normal Bax expression from 0.1 mg L−1 and more than 2 fold expression at 0.5 mg L−1 , indicated the acute Cd toxicity on brain tissue compared to other organs at a lower dose.

Confirmation of DNA damage due to oxidative stress upon Cd toxicity In terms of toxicity due to oxidative stress, the DNA damage in blood cells of Cd treated mice was observed by performing the comet assay and visualized the comet in EtBr treated blood cells. The long tail indicated the damage in DNA strands due to ROS generation (Fig. 5A). After analysing the image of comet in Comet Score software, percentage of DNA in tail increased significantly (P < 0.01) from 3.88% to 18.9% (1.0 mg L−1 ) and 21.92% (2.0 mg L−1 ) with increasing concentration of CdCl2 treatments (Fig. 5B) which again correlated with higher expression in ROS level of blood.

Organ specific response due to Cd toxicity in antioxidant enzymatic system Decrease in catalase and superoxide dismutase (SOD) activity under Cd toxicity The formation of ROS is generally prevented by an antioxidant system, e.g. ROS-interacting enzymes such as SOD, peroxidases and catalases. In Swiss Albino mice under Cd induced oxidative stress, activity of the two most important enzymes viz. SOD and catalase (Fig. 6) was studied. In normal mice, SOD activity was highest in the liver tissue followed by kidney, spleen and testis but significantly lower expression of these two enzymes were observed in the brain against all the other four organs. But in case of catalase, brain and spleen showed least activity than other organs at basal level. On increasing the concentration of CdCl2 (1.0 mg L−1 ), enzyme activity of SOD (20%) and catalase (30%) decreased in respective organs, except very little change was seen in case of brain. In both the cases liver is the most affected organ in terms of reduced enzyme activity while the brain enzymes showed minimal change (Fig. 6). Decrease in glutathione peroxidase activity Glutathione (GSH) is a major endogenous antioxidant of the body capable of directly neutralizing free radicals and ROS. Thus, the GSH-Px activity was monitored in the different tissues of the Swiss Albino mice showing continuous decrease in all five organs (Fig. 6). In testis, liver and kidney the change in activity was not significant, yet spleen showed statistically significant drop in the activity level of GSH-Px (P < 0.001). In case of brain the expression level of GSH-Px is very low in normal condition [34] in respect to other organs as change is negligible. Confirmation of tissue damage by lactate dehydrogenase activity with lipid peroxidation The extent of tissue damage due to abnormal ROS generation was assayed by measuring LDH activity, a marker of tissue breakdown and LPO a pathological marker of the cell membrane damage. LDH activity gradually increased in a concentration dependent manner in the liver, kidney, spleen and brain tissue significantly (P < 0.001) (Fig. 6), but in case of testis the increase in activity suddenly stopped at 0.5 mg L−1 concentration, which may be the optimum expression as the activity did not change with higher concentration of treatment. The LPO also changed remarkably with higher expression of MDH in kidney, liver, spleen and testis (Fig. 6). But in case of the brain, the measured MDH was significantly higher in Swiss albino mice for 0.5 mg L−1 to 1.0 mg L−1 of CdCl2 treatment, unlike all other antioxidizing defence system. This confirmed the severity of brain damage due to Cd toxicity. Correlation analysis of antioxidant defence system due to Cd toxicity The organ specific activity of antioxidative defense mechanism in Cd treated groups of Swiss Albino mice was plotted with their corresponding stress induced enzymatic activities. The draftsman correlation plot (Fig. 7A.1) data revealed the hyper-activity of LPO and LDH with correlation to SOD, catalase, GSH peroxidase as well as lower protein content in all the five organs of mice, but activity decreases with higher concentration. To analyze the hierarchy, the mean value for each antioxidant parameters of all those five organs was extracted from each set of data to analyze the main clusters. All of these clusters were significant at the 99% level when analyzed using the correlation test in PRIMER 6 (Fig. 7A.2). The complete linkage of all the five antioxidant machineries provided only two clear clusters with very high (LDH and SOD) and distinctly low groups of

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Fig. 3. (A) Expression of HABP1 is higher with the concentration of CdCl2 . Swiss Albino mice of 4–6 week old were continuously treated with CdCl2 (mg L−1 ) in drinking water ad libitum for 30 day, sacrificed the mice and tissue section processed for Immunohistochemical analysis using anti-HABP1 antibody and eosin stain for detection of HABP1 in tissue structure. Bars = 50 ␮m. (B) Expression pattern of HABP1 in different tissues. After treatment, tissue lysed was processed for immunoblotting with anti-HABP1 first, then same blot was treated with anti-GAPDH for internal control. HABP1 expression is maximum in all the organs at 0.1 mg L−1 which confirmed the initial induction of oxidative stress signals. Whereas, HABP1expression increased with concentration of CdCl2 (mg L−1 ) in brain of Swiss Albino mice (n = 5).

activation status of other enzymes. It is stated that in testis GSH-Px is the best indicator whereas LDH was plotted as significant parameter for all these five organs as their clusters are not significantly distanced. In case of kidney, spleen and testis main clusters broadly correspond with two major groups as LDH showed little identity with all other antioxidant enzymes, separating it as an identifying

characteristic for Cd toxicity analysis in these three organs of Swiss Albino mice (Fig. 7B.1). Both SOD and catalase activation was the indicator for the brain and liver, yet loss of catalase activity was prominent in spleen. LPO activation may be a promising biomarker for brain and liver as shown by linkage clusters mapping performed by Primer 6 software (Fig. 7B.2).

Fig. 4. (A) Expression of Bax is higher with the concentration of Cd. Swiss Albino mice of 4–6 week old were continuously treated with CdCl2 (mg L−1 ) in drinking water ad libitum for 30 day, sacrificed the mice and tissue section processed for immunohistochemical analysis using anti-Bax antibody and eosin stain for detection of expression in tissue structure. (B) Bax protein expression increases with higher Cd concentration (mg L−1 ). Tissue of five different organs lysed to prepared total protein and run on SDS–PAGE for western blotting analysis, using anti-Bax antibody, to check the pro-apoptotic protein expression, showing higher expression level with increased concentration of CdCl2 treatment (n = 5).

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Fig. 5. (A) The comet assay used to measure oxidative damage to CdCl2 treatments. Representation of comet images of blood cells showing increasing level of damage with Comet Score software scored 0 undamaged DNA till 0.1 mg L−1 concentration. With various degrees of damage from minor to severe grade was observed under fluorescence microscope. The degree of damage increased above 0.5 mg L−1 with increasing concentration of treatment. At 1.0 mg L−1 the significant (P < 0.001) level of DNA damage confirming ROS induced toxicity level in mice. (B) DNA damage by comet assay. Graph represented by percentage of DNA in tail degraded which was damage due to Cd toxicity, damage of DNA increases as Cd concentration increases. Score measured by Comet score software.

Discussion Cd exposure due to intake of polluted food or water contributes to a large number of pathological conditions in different organs like liver, kidney, lung, testis and brain, since this heavy metal gets accumulated in the human body [35–40]. It can alter the antioxidant defence system and causes significant change in ROS levels, genotoxicity and cytotoxicity in vivo and in vitro [41,42]. The observed LD50 dose upon CdCl2 exposure in mice was found to be 1.0 mg L−1 . The progressive loss in survivability may be due to Cd exposureinduced ROS generation. It was approximately 2 fold increased in a dose dependant manner, compared to control and in tandem. This study again established Cd induced ROS play a key role in mortality. These toxic effects was studied in Swiss Albino mice liver, kidney, spleen, testis and brain showing change in histopathology of all these organs. ROS generation and their high reactivity caused localized deformation of tissue structure and damage to the organs and can be considered highly toxic at higher concentrations. A range of disorders such as Alzheimer’s disease, Parkinson’s disease, etc. are triggered by excess ROS generation. Toxicity of free radicals contributes to proteins and DNA injury, inflammation, tissue damage and subsequent cellular apoptosis [43] due to increase expression of stress proteins. HABP1 is a protein involved in cellular signalling for apoptosis [44] and it also plays a role in the generation of ROS [15]. Cd induced HABP1 overexpression in different organs in vivo condition possibly illustrated the phenomenon of stress responsiveness of HABP1 protein in all affected organs with respect to stress control and raised the signals. Along with early detection of HABP1, higher expression level of Bax in the different organs examined, confirmed that the apoptosis process was in progress. With the change in oxidative parameters, nuclear signalling cascade was also exhibiting significant DNA damage, 22% DNA tail formation, in blood, indicating Cd induced lethality in the mice. The primary defence of the body against ROS are the antioxidant enzymes. The severity of the effect of ROS is based on the equilibrium between the toxic action and the possible antioxidative

defence mechanism in the living organs. Taking this into account, the analysis of tissues’ antioxidant mechanism like, superoxide dismutase and catalase showed the protective action and free radical scavenger effects [45], as confirmed by decreasing activity of SOD and catalase in liver, spleen, testis, kidney and brain tissue. ROS generation can directly affect the conformation and/or activities of all sulfhydryl-containing molecules, such as proteins or GSH, by oxidation of their thiol moiety. Being an indicator of antioxidant, GSH-Px confirmed the decrease in GSH level as the Cd dose increased. Lipid peroxidation is well defined indicator of cellular injury. Our results showed that LPO increased slightly with increase in CdCl2 concentration. Consistently, MDA level in brain at the same concentration was higher than in other organs, which implied that the brain is more susceptible towards Cd toxicity than other organs. LDH the enzyme responsible for tissue breakdown, expressed with an increased level with the same dose of CdCl2 in brain much more than in kidney and liver, confirming that brain injury was much faster than injury in all other organs in this study. Draftsman plot shows that LDH and LPO were higher in all five organs, but it was significantly higher in the brain tissue, corroborating that the brain tissue got damaged very quickly. The draftsman plot of the organ specific activity of anti-oxidative enzymes of Cd treated swiss albino mice shows that testis GSHPx was the best indicator whereas LDH was plotted as significant parameter for all these five organs as their clusters are not significantly distanced. Both SOD and catalase activation was the indicator of liver and brain, yet loss of catalase activity was prominent in spleen. LPO activation might be confirmed as a promising biomarker for brain and liver using linkage clusters mapping performed by Primer 6 software. Our results indicate that in brain, exposure to Cd (0.5 mg L−1 ) induced an early increase in important cytotoxic markers like LDH and lipid peroxidase than in all other organs. These results seem to suggest the inability of neural cells to induce enzymes against oxidative stress producing ROS. The increase in catalase and SOD activities may be an indication of increased ROS generation due to Cd treatment or could be

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Fig. 6. Effect of CdCl2 on different anti-oxidant enzyme activity. Check the effect of Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidation (GSH-Px), lipid peroxidation (LPO) and lactate dehydrogenase (LDH) enzymes on the kidney, liver, spleen, testis, and brain tissue. Where X-axis represents concentration of CdCl2 and Y-axis represents Unit. (a) Control, (b) 0.1 mg L−1 , (c) 0.5 mg L−1 , (d) 1.0 mg L−1 , (e) 2.0 mg L−1 Cd chloride concentration.

due to an increase in the enzymes expression (catalase and SOD), since it is known that cellular stress response requires the activation of pro-survival pathways. The production of these molecules with anti-oxidant, anti-apoptotic or pro-apoptotic activities and other cellular signalling pathways are conferring protection against oxidative stress [46]. Another possibility is that the catalase and SOD may be activated by the Cd ion directly. The effect of Cd on activities of antioxidant enzymes in different organs and at low or high concentrations for long or short exposure time is worthy of further investigations because after exposure, Cd affected for long time in kidney [47]. Brain seems to be the most affected of the examined organs under stress conditions developed by ROS generation. Cd may increase ROS indirectly by binding to functional protein groups, which disrupts the other protein activity in neural system [48]. The correlation analysis signified the ROS induced Cd toxicity, as LDH activation was shown to be higher confirming morphological deformity in all the organs. Although,

insignificant levels of activation of SOD, catalase and GSH peroxidase in brain upon Cd toxicity was observed, yet significantly higher activation of LPO and LDH was present. This might have resulted in the observed shrinking of the hypothalamus generating instability and brain pathogenesis upon Cd accumulation in the brain. Increased levels of LPO upon treatment with water borne Cd has been implied to cause rupturing of membranes and lipid release from organs. Our observations revealed a significant dose dependent increase in the antioxidant enzymatic activities involved in intermediary catabolism, e.g. LPO, especially in the brain. This might be responsible for considerable histological alteration leading to structural damage and induction of neurodegeneration of the brain tissue. All the above observations indicate a higher susceptibility of the brain towards, Cd induced toxicity than other organs and confirming the existence of organ specific responsiveness in mice model upon exposure to Cd toxicity.

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Fig. 7. (A.1) Correlation of antioxidant enzymatic activity in all the treated organs. Draftsman plots of selected organs e.g. kidney, liver, spleen, testis and brain of variable biochemical parameters as protein, catalase, superoxide dismutase (SOD), glutathione peroxidation (GSH-Px), lipid peroxidation (LPO) and lactate dehydrogenase (LDH) after treatment with different concentration of CdCl2 in drinking water was plotted. The hyper activity of LDH and LPO was clearly visualized against all others which decreased with increasing concentration. (A.2) Complete Linkage plotted with biochemical parameters of Swiss Albino mice exposed to CdCl2 . Its stated that LDH is a promising biomarker than SOD, GSH-Px, LPO and Catalase performed by Primer 6. In all the tissues LDH activity efficiency clustered as a highly significant group confirmed that. (Distance signifies Euclidean Geometrical distance.) (B.1) Draftsman plot for the correlation between all antioxidative enzyme activities due to CdCl2 treatment (1.0 mg L−1 ). Correlation between kidney, liver, spleen, testis and brain of Swiss Albino mice exposed to CdCl2 concentration (1.0 mg L−1 ) and the biochemical parameters confirmed the analogous profiling of all. The expressed activity correlated within the oraganalles when plotted using Primer 6. (B.2) Complete Linkage plot of biochemical parameters showed organ specific Cd toxicity. In Cd treated groups of Swiss Albino mice (1.0 mg L−1 ) was plotted on the basis of their organ specific activity of antioxidative enzymes. It’s stated that in testis GSH-Px is the best indicator whereas LDH was plotted as significant parameter for all these five organs as their clusters using Primer 6 software are not significantly distanced. (Distance signifies Euclidean Geometrical distance.)

Conflict of interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received financial support from University Grant Commission, Jawaharlal Nehru University, UPOE, Department of Science and Technology, and Department of Biotechnology (BT/PR11218/BRB/10/676/2008) funding from Government of India for the research and authorship but no financial support for publication of this article. Acknowledgement We sincerely thank to Professor Kasturi Datta for her scientific inputs.

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