A study of dose response and organ susceptibility of copper toxicity in a rat model

Journal of Trace Elements in Medicine and Biology 29 (2015) 269–274

Toxicology

A study of dose response and organ susceptibility of copper toxicity in arat model

Vijay Kumar a, Jayantee Kalita a,*, U.K. Misra a, H.K. Bora b

aDepartment of Neurology, Sanjay Gandhi Post Graduate Medical Sciences, Raebareily Road, Lucknow 226014, Indiab Laboratory Animal Division, Central Drug Research Institute, Lucknow, India

A R T I C L E I N F O

Article history:Received 4 April 2014Received in revised form 3 June 2014Accepted 5 June 2014

Keywords:CopperLiverKidneyBrainToxicity

A B S T R A C T

Copper (Cu) in higher concentration is toxic and results in various organ dysfunction. We report Cuconcentration in liver, brain and kidney in the rat model following chronic exposure of oral coppersulphate at different subtoxic doses and correlate the tissue Cu concentrations with respective organdysfunction. Fifty-four male wistar rats divided in 3 groups, the control group received saline water andthe experimental group (Group-IIA and IIB) received oral copper sulphate in dose of 100 and 200 mg/kgBody Weight. At the end of 30 days, 60 days and 90 days of exposure, six rats were sacrificed from eachgroup. The maximum peak force in grip strength, latency to fall in rotarod and percentage attention scorein Y-maze were significantly reduced in the copper sulphate exposed rats compared to the controls at alltime points and these were more marked in Group-IIB compared to Group-IIA. Cu concentration wassignificantly higher in liver, kidney and brain in the Group-II compared to the Group-I. The Cuconcentration was highest in the liver (29 folds) followed by kidney (3 folds) and brain (1.5 folds). SerumALT, AST and bilirubin correlated with liver Cu, BUN with kidney Cu, and grip strength, rotarod and Y-maze findings correlated with brain Cu level. In rats, chronic oral copper sulphate exposure at subtoxiclevel results in neurobehavioral abnormality and liver and kidney dysfunctions due to increased Cuconcentration in the respective organs. Liver is the most vulnerable organ and copper toxicity increaseswith increasing dose and duration of exposure.

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1. Introduction

Copper (Cu) is an essential trace metal that plays animportant role in many biological functions and serves as acofactor for several enzymes. Cu uptake, distribution within thecells, detoxification and removal are maintained by an elegantsystem. Mutations in genes involved in Cu homeostasis areresponsible for disorders of Cu metabolism in humans. The bestdescribed human Cu toxicosis disorder is Wilson disease (WD)which is an autosomal recessive disease due to ATP7B genemutation which leads to defective Cu transportation andexcretion into the bile [1]. The main pathogenesis of cellularinjury in WD is due to the presence of excess free Cu which

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;BUN, blood urea nitrogen; CNS, central nervous system; Cu, Copper; kgBWt, kg BodyWeight; LEC, Long Evans Cinnamon; MRI, magnetic resonance imaging; WD, Wilsondisease.* Corresponding author. Tel.: +91 522 2494169; fax: +91 0522 2668811.E-mail addresses: [email protected], [email protected] (J. Kalita).

http://dx.doi.org/10.1016/j.jtemb.2014.06.0040946-672X/ã 2014 Elsevier GmbH. All rights reserved.

results in cirrhosis of the liver, Kayser Fleischer ring, renaltubular dysfunction and brain damage [2]. Clinically, the WDpatients have liver dysfunction in the first decade andneurological dysfunction in the second decade [3]. Many WDpatients with neurological manifestation may not have a historyof hepatic dysfunction. Central nervous system (CNS) isprivileged as it is protected from most of the toxic agentsand infections because of blood brain and blood cerebrospinalbarriers. However, free Cu is capable of crossing blood brainbarrier resulting in oxidative stress mediated cell damage [4,5].On cranial MRI, certain areas of the brain such as corpusstriatum, thalamus and substantia nigra are more frequentlyinvolved in WD suggesting vulnerability of these areas to Cutoxicity [6–8].

Recently Long Evans Cinnamon (LEC) rat and toxic milk micehave been developed to replicate the WD and to evaluate thevarious aspect of Cu induced toxicity [9–11]. The well-establishedmodel for WD, LEC rat has a deletion in the ATP7B gene leading to anon-functioning protein. The animals steadily accumulate Cu inthe liver and develop hepatic manifestation at the age of about 3months [11]. LEC rat has symptoms similar to hepatic manifesta-tion but does not exhibit symptoms similar to neurological

Fig. 1. Error bar diagram shows behavioral changes in the control and experimentalgroup. (A) Grip strength: Maximum peak force declines in the Group-II compared tothe Group-I. The grip strength was not significantly different in the Group-IIA and Bat 30 and 60 days but at 90 days; grip strength was significantly reduced in Group-IIB compared to Group-IIA. (B) Rotarod: Latency to fall time was significantlydecreased in Group-II compared to the Group I. The latency to fall time wassignificantly reduced in Group-IIB at 30 days compared to Group-IIB. (C) Y-maze:The percentage of attention was reduced in Group-II at all time points compared toGroup-I; however this reduction was not significantly different between Group-IIAand B. The differences in the variables between the groups were analyzed by two-way ANOVA with Bonferroni post-hoc multiple comparison tests. Values are in

270 V. Kumar et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 269–274

manifestation of human disease. An animal model of Cu toxicity forlonger duration may simulate the pathological changes similar toWD patients [12,13]. Intraperitonial injection of copper lactateleads to impaired spatial memory and neuromuscular coordina-tion, swelling of astrocytes, decreased serum AChE activity, copperdeposition in the choroid plexus, neuronal degeneration, andincreased levels of Cu in the hippocampus of rats [14]. Cu inducedcellular injury in both humans and animals may be similar and maybe mediated by free radical and oxidative stress [15,16]. Previousexperimental studies had reported Cu induced toxicity in the liver,kidney and brain in isolation [14,17–20]. Only few studiesevaluated liver, brain and kidney dysfunction comprehensivelyin Cu induced toxicity [14,21–23]. It is important to learn whetherthere are simultaneous CNS and hepatic injury in chronicCu toxicity or there is a sequential involvement of differentorgans. None of the experimental study comprehensively evaluat-ed the vulnerability of different organs as well as different part ofthe CNS to chronic Cu toxicity. The experimental study of chronicCu toxicity may help in understanding the vulnerability andsequential changes of different organ dysfunction. The presentstudy therefore aimed to evaluate the Cu concentration in liver,kidney and brain at different time points following different dosesof copper sulphate exposure in the rat. Cu level in different organswould also be correlated with respective biochemical and neuro-behavioral changes.

2. Materials and methods

Male Wistar rats were acclimatized for 7 days to oral gavagefeeding in the laboratory. The animals maintained in an air-conditioned room (25 � 2 �C) with 12 h light and dark cycle. Allanimalshad freeaccess towaterandfood.Thestudywasapprovedbythe Animal Ethics Committee, Central Drug Research Institute, India.

2.1. Study design

Fifty Four male Wistar rats (body weight = 205 �10 g, 2.5months old) were included in this study. Animals were divided intothree groups, 18 animals in each group. Group-I (n = 18, controlgroup), rats were fed saline water daily through oral gavage. Inexperimental group, Group-IIA (n = 18) and Group-IIB (n = 18) werefed copper sulphate (CuSO4�5H2O, Sigma–Aldrich, St. Louis, MO) ina dose of 100 mg/kg and 200 mg/kg Body Weight respectively, dailyupto 90 days through oral gavage. The animals were examineddaily and their body weight and behavioral changes were recordedat baseline, 30 days, 60 days and 90 days. Blood was collected atbaseline, 30 days, 60 days and 90 days in heparinized and plain vialfrom the orbital vein.

Six rats from each group were sacrificed by decapitation atthe end of 30 days, 60 days and 90 days of exposure. Just beforethe sacrifice, blood was also collected in heparinized and plainvial by cardiac puncture under anesthesia after an overnightfast. Thereafter, the rats were perfused with saline from the leftventricle through thoracotomy to remove whole blood from theorgans. The liver, kidney, and brain were removed and werefrozen in liquid nitrogen and then stored at �80 �C untilanalysis.

2.2. Hematological and biochemical studies

Heparinized blood was used for measurement of hemoglobinand white cell count. Measurement of blood urea nitrogen (BUN)and serum bilirubin, transaminases, and creatinine, was doneusing clinical chemistry analyzer (Randox Imbola, Ireland, UnitedKingdom).

2.3. Behavioral studies

All the behavioral tests were performed during the light phase.The following behavioral studies were done at different time points.

2.4. Grip-strength test

The forelimb-grip strength of the rat was measured using acomputerized grip strength meter (TSE-Systems, Bad Homburg,Germany). Each rat was gently placed on the mesh and pulled byholdingthetailintheoppositedirectionuntilitreleaseditsgripfromthe mesh. The peak force developed before the release of grip wasrecorded in Newton (N). We calculated the mean of five measure-ments, allowing 30 s of recovery time in between the test.

2.5. Y-maze test

Y-maze was used for assessing learning and memory. Y-mazeconsists of three arms with an angle of 120� between each arm. Themaze was placed in a separate room with minimal light, and the

Mean � SEM.

Table 1Haematological and biochemical changes in the control (Group-I) and experimental group (Group-IIA and IIB) at 30 days, 60 days and 90 days.

Control (Group-I) Experimental Group (II) P value

100 mg/kg BWt (IIA) 200 mg/kg BWt (IIB) (I) vs. (IIA) (I) vs. (IIB) (IIA) vs. (IIB)

ALT (SGPT) (U/L)30 days 20.28 � 0.92 30.95 � 5.81 44.37 � 4.35 0.10 0.21 0.8760 days 20.51 � 1.48 55.62 � 5.33 69.40 � 10.47 0.01 <0.01 0.8590 days 21.93 � 1.39 63.55 � 8.59 77.37 � 10.53 <0.01 <0.01 0.85AST (SGOT) (U/L)30 days 47.31 � 3.21 226.90 � 6.52 227.73 � 5.59 <0.01 <0.01 1.0060 days 48.83 � 3.10 256.08 � 12.38 257.52 � 18.75 <0.01 <0.01 1.0090 days 50.79 � 3.45 265.47 � 14.34 272.37 � 11.15 <0.01 <0.01 1.00Bilirubin (mg/dL)30 days 0.55 � 0.05 2.45 � 0.15 2.67 � 0.22 <0.01 <0.01 1.0060 days 0.56 � 0.04 3.26 � 0.36 3.25 � 0.47 <0.01 <0.01 1.0090 days 0.57 � 0.05 3.35 � 0.44 4.16 � 0.55 <0.01 <0.01 0.70Hemoglobin (g/dL)30 days 12.78 � 0.49 11.62 � 0.31 11.17 � 0.26 0.25 0.03 0.9960 days 13.07 � 0.40 11.18 � 0.25 10.88 � 0.19 0.01 <0.01 0.9990 days 13.73 � 0.41 10.87 � 0.25 10.23 � 0.27 <0.01 <0.01 0.90BUN (mg/dL)30 days 16.94 � 0.62 21.59 � 2.09 21.67 � 1.88 0.29 0.27 1.0060 days 17.86 � 0.90 24.13 � 1.69 25.97 � 0.28 0.05 <0.01 0.9990 days 17.27 � 0.81 25.74 � 1.39 28.99 � 1.32 <0.01 <0.01 0.74Creatinine (mg/dL)30 days 0.64 � 0.04 0.61 � 0.04 0.53 � 0.01 1.00 0.34 0.8060 days 0.67 � 0.03 0.62 � 0.05 0.62 � 0.02 0.99 0.98 1.0090 days 0.72 � 0.04 0.65 � 0.04 0.68 � 0.02 0.90 1.00 1.00BUN/creatinine ratio30 days 26.61 � 0.95 35.16 � 2.25 40.97 � 3.89 0.24 <0.01 0.7360 days 26.93 � 1.54 38.04 � 3.43 42.27 � 1.78 0.05 <0.01 0.9490 days 24.35 � 0.94 40.58 � 3.21 43.19 � 2.96 <0.01 <0.01 1.00

BUN: blood urea nitrogen, ALT: alanine aminotransferase (SGPT), AST: aspartate aminotransferase (SGOT), kgBWt: kg Body Weight. Value in Mean � SEM.

V. Kumar et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 269–274 271

floor of the maze was dusted with sawdust after each trial toeliminate olfactory stimuli. Two trials were done and the inter-trialinterval was one hour. The first trial (training) was for 10 min, andthe rat was allowed to explore only two arms (starting arm and theother arm). For the second trial (retention), the rat was placed atthe same starting arm and allowed to explore for 5 min with freeaccess to all three arms. All trials were analyzed for the number ofentries that the rat made into each arm. The results were expressedas the percentage of novel arm entries made during the 5 minretention trial.

2.6. Rotarod test

Rotarod was used for the assessment of motor coordination andbalance using an accelerating rotarod apparatus (TSE-Systems, BadHomburg, Germany). The rats were placed on the revolving rodaccelerating at 5–50 rpm. Prior to testing, the rat was trained for atleast three times and then allowed to rest for 15 min in betweenthe trial. The apparatus was wiped with 70% ethanol and dried

Table 2Copper concentrations in different organs at different time point in the control (Group

Organ Control (Group-I) Experimental Group (II)

100 mg/kgBWt (IIA) 200 mg

Liver-Cu (mg/g wet weight)30 days 5.32 � 0.27 141.05 � 06.14 175.1060 days 5.78 � 0.41 186.08 � 03.94 207.9190 days 7.42 � 0.57 224.38 � 05.08 246.55Kidney-Cu (mg/g wet weight)30 days 7.30 � 0.26 14.09 � 0.61 17.4760 days 7.48 � 0.21 18.55 � 0.39 21.0890 days 8.70 � 0.32 21.62 � 0.56 23.93Brain-Cu (mg/g wet weight)30 days 2.0 � 0.02 2.51 � 0.03 2.7760 days 2.04 � 0.02 2.94 � 0.05 3.1090 days 2.13 � 0.04 3.47 � 0.06 3.53

KgBWt: kg Body Weight. Value in Mean � SEM.

before each trial. The latency to fall from the rod was recorded, andany rat remaining on the rod for more than 300 s was removed andreturned to the cage.

2.7. Copper measurement

Copper concentration was measured in liver, kidney and brainby atomic absorption spectrophotometry (GBC Avanta Sigma; GBCScientific Equipment PTY Ltd., Dandenong, Victoria, Australia).0.5 g of each organ was digested with a mixture of nitric acid andperchloric acid (6:1, v/v) and the digest was brought to constantvolume with double distilled deionized water.

2.8. Statistical analysis

SPSS-16 (SPSS, Chicago, IL, USA) and GraphPad Prism-5(GraphPad Software, La Jolla, CA) were used for statistical analysis.The differences in the variables between the groups were analyzed

-I) and experimental group (Group-IIA and IIB).

P value

/kgBWt (IIB) (I) vs. (IIA) (I) vs. (IIB) (IIA) vs. (IIB)

� 03.5 <0.01 <0.01 <0.01 � 07.04 <0.01 <0.01 0.02 � 04.99 <0.01 <0.01 0.02

� 0.36 <0.01 <0.01 <0.01 � 0.60 <0.01 <0.01 <0.01 � 0.52 <0.01 <0.01 0.01

� 0.04 <0.01 <0.01 0.02 � 0.05 <0.01 <0.01 0.33 � 0.04 <0.01 <0.01 0.99

Fig. 2. (A–C) Regression curve shows correlation of Cu concentration with liverdysfunction. The liver Cu concentration was positively correlated with SGPT (A),SGOT (B) and serum bilirubin (C). Values are in Mean � SEM.

Fig. 3. (A, B) Regression curve shows correlation of Cu concentration with kidneydysfunction. The kidney Cu concentration positively correlated with blood ureanitrogen (BUN) (A) and BUN/Creatinine ratio (B). Values are in Mean � SEM.

272 V. Kumar et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 269–274

by two-way ANOVA with Bonferroni post-hoc multiple compari-son test. The correlation of Cu and functional parameters of thedifferent organs were done by Karl Pearson/Spearman correlationtest. The variables having a two tailed P values of <0.05 wereconsidered significant.

3. Results

3.1. Body weight

The body weight increased in all the groups but those animalsexposed to copper sulphate had significantly lower body weightcompared to the controls. Group-IIB had significantly lower bodyweight compared to controls at 30 days (217.87 �4.34 vs.229.03 � 4.60 g; P = 0.001), 60 days (209.42 � 5.74 vs.251.00 � 3.80 g; P < 0.0001) and 90 days (197.70 � 3.98 vs.

268.30 � 4.76 g; P < 0.0001). Group-IIA however had significantlylow body weight at 60 days (219.20 � 4.70 vs. 251.00 � 3.80 g;P < 0.0001) and 90 days (210.63 � 6.51 vs. 268.30 � 4.76 g;P < 0.0001 but not at 30 days (222.93 � 4.97 vs. 229.03 � 4.60 g;P = 0.05) compared to the controls.

4. Behavioral study

4.1. Grip-strength test

The grip strength at baseline in the control (Group-I) andexperimental groups (IIA and IIB) was almost similar. In the controlgroup, it remained same or slightly increased at 30 days, 60 daysand 90 days. The grip strength in the Group-IIA and IIB at 30 days,60 days and 90 days was significantly reduced compared to Group-I. The grip strength was more affected in the Group-IIB comparedto Group-IIA. The changes in the grip-strength in different groupsare shown in Fig. 1A.

4.2. Rotarod test

In the control group (Group-I), the latency to fall time wasstable until 90 days. The latency to fall time was significantlyreduced in Group-IIA and IIB compared to Group-I at different timepoints. The reduction in latency to fall time in Group-IIB comparedto Group-IIA was significant at 30 days (P = 0.03) but not at 60(P = 0.26) and 90 days (P = 0.84). The details are shown in Fig. 1B.

4.3. Y-maze test

The cognitive functions evaluated by Y-maze were similar atbaseline between the groups but was significantly reduced at

Fig. 4. (A–C) Regression curve shows correlation of brain Cu concentration withmotor and cognitive impairment. The brain Cu concentration inversely correlatedwith maximum peak force (A), latency to fall on rotarod (B) and percentage ofattention on Y-maze (C). Values are in Mean � SEM.

V. Kumar et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 269–274 273

60 and 90 days in the rats exposed the copper sulphate comparedto the controls. The percentage of attention was significantlyreduced in the Group-IIB at all time points compared to thecontrols. There was however no significant difference in thepercentage of attention between the Group-IIA and IIB. The detailsshown in Fig. 1C.

4.4. Blood analysis

The hemoglobin level was reduced and serum bilirubin andtransaminases were higher in the Group-IIA and IIB compared to theGroup-I. The change in serum creatininewas however not significantbetween the groups. The details are summarized in Table 1.

4.5. Cu level in liver, kidney and brain

The Cu concentrations in the liver, kidney and brain weresignificantly higher in the Group-IIA and IIB compared to theGroup-I at 30, 60 and 90 days. The increase in Cu concentration wasproportional to the dose of exposure. The details have been shownin Table 2. In the Group-IIB rats, the deposition of Cu was highest inthe liver (29 folds) followed by kidney (3 folds) and brain (1.5 folds)compared to the controls.

The liver Cu level significantly correlated with serum ALT(SGPT)(r = 0.78; P < 0.001; Fig. 2A), AST(SGOT) (r = 0.96; P < 0.001; Fig. 2B)and bilirubin (r = 0.87; P < 0.001; Fig. 2C). The Cu level in kidneytissue correlated with BUN level (r = 0.76; P < 0.001; Fig. 3A) andBUN/creatinine ratio (r = 0.77; P < 0.001; Fig. 3B). The brain Cu levelinversely correlated with grip strength (r = �0.92; P < 0.001;Fig. 4A), retention time in rotarod (r = �0.93; P < 0.001; Fig. 4B)and attention in Y-maze (r = �0.77; P < 0.001; Fig. 4C).

5. Discussion

This study revealed higher Cu concentration in the liver, kidneyand brain in the rats exposed to oral copper sulphate in a dose of100 and 200 mg/kg Body Weight compared to the controls. Thetissue Cu concentration and its toxicity were increased withincreasing dose and duration of exposure. In the Group-IIB rats, thedisposition of Cu in the liver was 29 folds, whereas in the kidneyand brain it was 3 and 1.5 folds as compared to the controls.Cu toxicity of the brain was evident by reduced grip strength,shorter latency to fall time in rotarod and reduced percentageattention score in Y-maze. The hepatic toxicity was evident byraised serum bilirubin and transaminases. Serum creatininealthough was normal, but BUN was significantly elevated in therats exposed to copper sulphate suggesting kidney dysfunction.This study for the first time demonstrated the sequentialconcentrations of Cu in different organs at different doses alongwith their biochemical and behavioral abnormalities followingchronic subtoxic oral copper sulphate administration.

In an earlier study, rats were fed with diet having additionalCu in 2.5, 5, 10 and 20 ppm up to 10–60 days revealed no change inbody weight and Cu concentration in liver, heart and brain [24]. Onthe other hand, rats exposed to intraperitoneal injection of copperlactate (0.15 mg/100 g body weight) daily for 90 days revealedsignificant reduction in serum acetylcholinesterase (AChE) activityand impaired neuromuscular coordination and spatial memory incomparison to the control rats [14]. Cu toxicity of brain and livermay be mediated by the formation of hydroxyl radical formationwith subsequent oxidative damage [15,25].

The earlier studies on copper toxicity have also reportedbehavioral changes, which were associated with higherCu concentration in the brain [14,26–28]. Leiva et al. reportedhigher Cu concentration in the rat hippocampus exposed to 1 mg/kg of copper sulphate intraperitoneally for 30 days, but this was notassociated with learning abnormality assessed by Morris-watermaze [26]. Another rat experimental study reported rotationalbehavioral abnormality following a single dose of 0.25 nmol CuSO4

injection into the right substantia nigra [28]. Hosseini et al.reported that excess of deposited Cu increases lipid peroxidation,protein oxidation, mitochondrial membrane potential decline, andcytochrome c expulsion that start cell death signaling [29].

In our study, we have done both motor (rotarod time and gripstrength) and cognitive functions (Y-maze) and observed abnor-malities in the rats exposed to copper sulphate. These findings aresomewhat similar to the neurological manifestations in WD.Patients with WD neurologically manifest with cognitive, extra-pyramidal and pyramidal dysfunctions. Slow learning, decliningmotor activity and various neuropsychiatric manifestations are

274 V. Kumar et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 269–274

quite characteristic of WD. Extrapyramidal dysfunction in the formof rigidity, dystonia, tremor and chorea are frequently associated inWD. Motor weakness although is not severe in WD but presence ofhyperreflexia suggests the pyramidal dysfunction [7,30,31].Reduced grip strength may suggest pyramidal tract dysfunctionand decreased stability in rotarod may suggest both extra-pyramidal and pyramidal dysfunction.

Cu level was increased 29 folds in liver, 3 folds in kidney and 1.5folds in the brain in Group-IIB compared to the controls. Thisreflects the more frequent and early liver damage in WD patientscompared to brain. The Cu is absorbed in the small intestine and inthe liver its homeostasis is maintained with the help of coppertransporting ATP7B protein. In a normal situation, up to a certainlimit excess Cu is excreted in the bile by the Cu transportingsystem. The circulating Cu is bound to ceruloplasmin, whichmaintains free Cu below the toxic level. In the toxic milk mice andLEC rat, this homeostasis is affected and these animals developCu toxicity due to impaired excretion similar to human WD[27,32,33]. The excess Cu is first accumulated in the liver and onceit is saturated, free Cu is released which subsequently getsaccumulated in the other organs. Neurobehavioral changes in ourstudy were observed at much lower Cu concentration in the braincompared to liver. This may be due to lesser regenerative capabilityof brain tissue. Neuronal regeneration although recently have beendemonstrated [20,28,34] but the proportion is minimal incomparison to the liver, kidney and other tissues.

We have administrated higher dose of copper sulphate than thereported literature to make the Cu toxicity more apparent.Moreover, we have not used knocked-out rat. Further studiesare needed to evaluate the mechanism of Cu induced tissue injuryand their histopathological changes. This Cu toxicity rat model iseasy to produce and may be used for understanding thepathophysiology and therapeutic intervention of Cu toxicity.

Conflict of interest

There is no conflict of Interest to declare by all authors.

Acknowledgements

Funding: Indian Council of Medical Research, New Delhi, India,financially supports Mr. Vijay Kumar in this work.

Ethics Approval: The project was approved by the InstitutionalEthics Committee, Central Drug Research Institute, Lucknow, India.

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