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Toxicology 293 (2012) 78– 88

Contents lists available at SciVerse ScienceDirect

Toxicology

j ourna l ho me pag e: www.elsev ier .com/ locate / tox ico l

opper-induced immunotoxicity involves cell cycle arrest and cell death in thepleen and thymus

oham Mitra1, Tarun Keswani1, Manali Dey, Shaswati Bhattacharya, Samrat Sarkar,uranjana Goswami, Nabanita Ghosh, Anuradha Dutta, Arindam Bhattacharyya ∗

mmunology Lab, Department of Zoology, University of Calcutta, Kolkata 700019, West Bengal, India

r t i c l e i n f o

rticle history:eceived 30 October 2011eceived in revised form3 December 2011ccepted 29 December 2011vailable online 8 January 2012

eywords:opper

mmunotoxicitypleenhymusndoGaxbiquitin

a b s t r a c t

Copper is an essential trace element for human physiological processes. To evaluate the potential adversehealth impact/immunotoxicological effects of this metal in situ due to over exposure, Swiss albino micewere treated (via intraperitoneal injections) with copper (II) chloride (copper chloride) at doses of 0,5, or 7.5 mg copper chloride/kg body weight (b.w.) twice a week for 4 wk; these values were derivedfrom LD50 studies using copper chloride doses that ranged from 0 to 40 mg/kg BW (2×/wk, for 4 wk).Copper treated mice evidenced immunotoxicity as indicated by dose-related decreases and increases,respectively, in thymic and splenic weights. Histomorphological changes evidenced in these organs werethymic atrophy, white pulp shrinkage in the spleen, and apoptosis of splenocytes and thymocytes; theseobservations were confirmed by microscopic analyses. Cell count analyses indicated that the proliferativefunctions of the splenocytes and thymocytes were also altered because of the copper exposures. Amongboth cell types from the copper treated hosts, flow cytometric analyses revealed a dose related increasein the percentages of cells in the Sub-G0/G1 state, indicative of apoptosis which was further confirmed byAnnexin V binding assay. In addition, the copper treatments altered the expression of selected cell deathrelated genes such as EndoG and Bax in a dose related manner. Immunohistochemical analyses revealedthat there was also increased ubiquitin expression in both the cell types. In conclusion, these studies

show that sublethal exposure to copper (as copper chloride) induces toxicity in the thymus and spleen,and increased Sub G0/G1 population among splenocytes and thymocytes that is mediated, in part, by theEndoG–Bax–ubiquitin pathway. This latter damage to these cells that reside in critical immune systemorgans are likely to be important contributing factors underlying the immunosuppression that has beendocumented by other investigators following acute high dose/chronic low-medium dose exposures tocopper agents.

. Introduction

Copper is present in all plant and animal tissues (May andilliams, 1981) at their threshold level and it exert toxicity due

o over exposure. Copper is one of the members of the U.S. Envi-onmental Protection Agency’s “Priority List of Chemicals,” (CASo. 7440-50-8) has been classified by the International Agency foresearch on Cancer as unclassifiable to carcinogenicity to humansGroup 3; 2002).

The widespread potential for human exposure to copper occurs

ia consumption of food and water contaminated with coppernd also by inhalation of industrial or cosmetics contained dust,ist or fumes with copper has raised questions about the health

∗ Corresponding author. Tel.: +91 3324615445; fax: +91 3324614849.E-mail address: arindam19@yahoo.com (A. Bhattacharyya).

1 Equal contribution.

300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2011.12.013

© 2012 Elsevier Ireland Ltd. All rights reserved.

hazards of these compounds. Copper, as an important trace ele-ment, performs various biological functions (Camakaris et al., 1999)in the form of metalloproteins. In addition, copper ions serve aselectron acceptors and help in incorporating iron into transferrinfor its ultimate utilization in heme synthesis (in the bone mar-row). Copper–zinc protein, like cytoplasmic superoxide dismutasepresent in the liver, erythrocytes, neurons, and phagocytic leuko-cytes acts in the conversion of superoxide anion (•O2

−) free radicalsinto hydrogen peroxide (H2O2) (May and Williams, 1981). Coppercan also enhance the carcinogenic potential of other metal or non-metal agents when it is present in elevated concentrations; thisoccurs in part, by copper acting as a catalytic agent through whichredox reactions occur (DNA damaging reactive oxygen species likeH2O2 and •O2

− are produced) during the metabolism of these other

agents (Theophanides and Anastassopoulou, 2002).

However, copper containing compounds have been found to betoxic in repeat dose toxicity studies where accumulation of copperultimately surpasses cell detoxification/metabolizing capabilities

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Aburto et al., 2001). Diseases like Wilson disease, Menke’s disease,nd idiopathic copper toxicosis are diseases associated with hep-tic and extra-hepatic copper accumulation. Animal studies havehown that various forms of copper are carcinogenic, mutagenic,nd/or teratogenic; other studies have shown that various typesf copper agents can also cause reproductive, developmental, andeurologic toxicities (Bodensteiner et al., 2004; Gerber et al., 2002;olmes et al., 2001; Klinefelter et al., 2004; Moser et al., 2004).

The immune system is one of the main adaptation mechanismshrough which the body defends itself against harmful agents andathogens. The potential adverse effects of copper contaminants onhe immune system are a raising concern for regulatory authorities.lsabbagh and El-Tawil (2001) demonstrated the immunomod-latory effects of selective copper compounds on both B- and-lymphocytes. Specifically, in their studies, dietary exposure ofemale mice either to cupravit (an inorganic copper agent) or pre-icur (a carbamate compound) for 8 wk reduced their humoralmmune responses to both T-cell-dependent (sheep red blood cells)nd independent (Escherichia coli lipopolysaccharide [LPS]) anti-ens. Cellular immunity was also suppressed in these hosts.

In general, apoptosis plays an important role in vertebrateevelopment and morphologic homeostasis (Steller, 1995). Many

ymphocytes undergo apoptosis at the termination of the acutehase of an immune response and apoptosis is involved in theeletion of the majority of T-lymphocytes in the thymus (Palmer,003). Apoptosis, as a vital regulator of the immune system, coulde a potential target for immunotoxicants that are known to dam-ge splenic and thymic tissues. Therefore, apart from any overtffects on these two key organs involved in the immune system,he study here also investigated whether copper triggered apopto-is among thymocytes and/or splenocytes. In addition, to provideome mechanistic explanation for any observed changes in levels ofpoptosis among these cell types, this study also evaluated whetherhese induced outcomes occurring via copper-mediated changes inxpression of selective apoptosis regulating molecules (i.e., EndoGnd/or Bax).

. Materials and methods

.1. Materials

Antibodies against Bax, EndoG, and ubiquitin were procured from Santaruz Biotechnology (Santa Cruz, CA). Alkaline phosphatase (AP)-conjugated antiouse and anti-rabbit secondary antibodies were obtained from Cell Signaling

echnology, Inc. (Danvers, MA). Pre-stained protein molecular weight marker,,3′-diaminobenzidine tetrahydrochloride (DAB) system, as well as horseradish per-xidase (HRP)-conjugated secondary anti mouse and anti rabbit antibodies wereought from Bangalore Genei (Bangalore, India). Hematoxylin and eosin stains wereurchased from Merck Chemicals (Mumbai, India). An Annexin V-propidium iodidePI) kit was procured from Becton Dickinson Immunocytometry system, San Jose,A. All remaining chemicals cited in this paper were procured from local firms in

ndia and were of the highest purity grade.

.2. Animal handling

Swiss albino mice (∼25 g each; five mice in each group) were obtained from theational Institute of Nutrition (Hyderabad, India). Each was housed in an animal

acility (maintained at 25–28 ◦C; with 55 [±5]% relative humidity, and a 12 h/12 hight/dark cycle) located at the Animal Housing Unit in the Department of Zoology,niversity of Calcutta. All animals were provided rodent chow (National Institutef Nutrition) and filtered water ad libitum. All animal experiments were performedollowing the “Principles of Laboratory Animal Care” (NIH publication No. 85-23,evised 1985), as well as by following specific Indian law on “Protection of Animals”nder the supervision of authorized investigators.

For the experiments, the mice were randomly divided into different groups com-rising (A) control and (B) copper (II) chloride (copper chloride)-treated sets. In an

nitial study, the mice were given copper chloride as intraperitoneal (IP) injections

t different concentrations (0, 2.5, 5, 7.5, 10, 12.5, 15, 20, or 40 mg/kg b.w.) twice

week for four consecutive weeks (a total of eight dosages) to determine the LD50

ose. Thereafter, based on this information, new sets of mice were provided sub-ethal doses (5 or 7.5 mg copper chloride/kg b.w.) twice a week for 4 wk to permit thexperiments below to be performed. In each case, control mice received injections

293 (2012) 78– 88 79

of normal saline (0.9% NaCl) as vehicle. At 72 h after the final dosing, the mice in eachgroup were euthanized by overdose with sodium thiopentone (Mancure Drugs Pri-vate Ltd., Mumbai, India) and spleen and thymus were then harvested for analysesas described in the various assays below.

As the studies here were designed to investigate potential mechanisms thatunderlie the immune suppression that has been documented following chronic low-medium dose exposures to copper agents, and given the: time constraints for thesestudies (i.e., exposures for no longer than 4 wk); earlier evidence by Hebert et al.(1993) that showed that a 2-wk exposure of rodents (rats and mice) to water con-taining < 300 ppm copper (III) sulfate (note: this exposure was to a cupric form ofthe metal that would correspond to a level of 45 mg/kg b.w./day here) yielded noremarkable untoward effects; and that toxicities by either ingestion or by injection(IP, intra-venous [IV], or subcutaneous [SC]) can often bring about similar patholo-gies in a host (although inherent values (like LDX) can vary wildly as a function ofroute of exposure (i.e., see representative route comparison studies by Ali-Ali et al.(2008) and Garber (2008) for thymoquinone and ricin, respectively), the use of theIP exposure route here for such a low dose as 5 or 7.5 mg Copper chloride/kg b.w.was deemed just and appropriate.

2.3. Histological analysis

At sacrifice, from subsets of mice in each group, the spleen, thymus, and liverwere removed from each host and immediately washed in phosphate buffered saline(PBS, pH 7.4). The tissues were then fixed for 24 h in buffered formaldehyde solution(10% in PBS) at room temperature, dehydrated by graded ethanol, and embeddedin paraffin (MERCK, solidification point 60–62 ◦C). Tissue sections (5-�m thickness)were then deparaffinized with xylene, re-hydrated with graded alcohols (100–50%ethanol), stained with eosin and hematoxylin, and then mounted in DPX resin(Merck, Mumbai, India). Digital images were captured in Olympus BX51 microscopefitted with an Olympus DP70 camera (U-TVO 63XC; Olympus Corp., Tokyo, Japan)having both a 40× and 100× (wide-zoom) lens.

2.4. Isolation of the thymic and splenic cells and cell viability assay

Spleen and thymus tissues recovered from other subsets of mice in each treat-ment group were aseptically removed at sacrifice. Single cell suspensions were thenprepared in RPMI 1640 medium (Hyclone Laboratories, Inc., Logan, UT) by pass-ing the cell population through a 50-�m pore size nylon mesh (Becton Dickinson,Franklin Lakes, NJ). Cells were then centrifuged at 1500 rpm for 5 min at 4 ◦C. Thepelleted cells were resuspended in RBC lysis buffer (0.83% [w/v] ammonium chlo-ride, 0.1% [w/v] potassium bicarbonate, 0.004% [w/v] EDTA) and incubated at roomtemperature for 5 min. The reaction was stopped by addition of an equal volume ofPBS. The samples were then centrifuged at 1500 rpm at 4 ◦C, and the generated pelletwas re-suspended in PBS. Total viable (non-RBC) cells were then counted in haemo-cytometer by trypan blue exclusion test and used for further analysis. Briefly, cellsundergoing either apoptotic or necrotic death can be distinguished from viable cellsby enhanced uptake of trypan blue. Cell viability was assessed using a hemocytome-ter (American Optical Corporation, NY) and the trypan blue assay by adding 100 �l ofcell suspensions to 400 �l of trypan blue stain (1% w/v) (Sigma–Aldrich) and 500 �lPBS. Approximately 10 �l of each suspension were added to a haemocytometer. Liveand dead cells were enumerated under a light phase contrast microscope. Cell countin the form of percent viability was assessed for the thymus and the spleen at thetime of isolation.

2.5. Isolation of the thymocyte and splenocytes for cell cycle analyses

Aliquots (each containing 5 × 106 cells) of isolated splenocytes and thymocyteswere then removed, recentrifuged, and suspended in 1 ml PBS. Phosphate-citratebuffer (200 �l, pH 7.8) was then added and the cells incubated for 60 min at roomtemperature. After centrifugation, the cells were resuspended in 0.5 ml of propid-ium iodide stain (10 mg PI, 0.1 ml Triton-X 100, and 3.7 mg EDTA in 100 ml PBS) andthen received 0.5 ml of RNase A (50 �g/ml) solution before being further incubatedfor 30 min in the dark. The PI fluorescence was then measured using a FL-2 filter(585 nm) in a BD FACS Calibur flow cytometer (Becton Dickinson); a minimum of10,000 events was acquired for each sample. The flow cytometric data was ulti-mately analyzed using WinMDI 2.9 and histogram display of DNA content (X-axis,PI-fluorescence) vs. counts (Y-axis) has been displayed.

2.6. Determination of apoptotic cells by flow cytometry

From other subsets of mice in each treatment group, thymocytes and spleno-cytes were isolated as described previously. In this study, apoptotic and necroticcell distributions were analyzed by Annexin V binding and PI uptake, respectively.Briefly, cells were adjusted to 5 × 105/ml in binding buffer (10 mM HEPES [pH 7.4],140 mM NaCl, 2.5 mM CaCl2, provided in an Annexin V – PI kit; Becton Dickinson)

and 10 �l of FITC-Annexin V was combined with 190 �l of the suspension. The mix-ture was then incubated for 10 min at room temperature. After centrifugation, thecells were re-suspended in 190 �l binding buffer and 10 �l PI (50 �g PI/ml) solu-tion was added. Cells were then analyzed in the FACS Calibur flow cytometer; foreach sample, a minimum of 10,000 events was acquired. Using WinMDI 2.9 software,

8 cology 293 (2012) 78– 88

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Fig. 1. Effect of copper on survival of mice. Each value in Y axis represents thepercentage (%) of survival of the copper chloride treated populations comparedto vehicle treated control mice. The treatment groups represent mice injectedintraperitoneally with copper chloride solution at concentrations of 2.5, 5.0, 7.5,10, 12.5, 15, 20 and 40 mg/kg body weight (b.w.). Results are presented as arith-

0 S. Mitra et al. / Toxi

istogram displays of DNA content (X-axis, PI-fluorescence) vs. counts (Y-axis) wereenerated. Cells that were positive for both PI and Annexin V were considered asecrotic cells and thus excluded from analysis. All results were expressed as theercentage of apoptotic cells (±SD)/sample.

.7. Preparation of cell lysates

Spleen and thymus tissues recovered from other subsets of mice in each treat-ent group were each placed in RIPA Lysis buffer (150 mM sodium chloride, 1.0%

riton-X-100, 50 mM Tris [pH 8.0], 0.01% SDS, and 0.5% sodium deoxycholate) con-aining 1 mM PMSF (phenyl-methanesulfonylfluoride), 1 �g approtinin/ml, and 1 �geupeptin/ml (all Sigma, St. Louis, MO) and homogenized. Supernatants were thenollected following centrifugation of each mixture at 14,000 rpm for 15 min at 4 ◦C.stimations of protein content in each supernatant were then performed using theradford reagent (Sigma) and subsequent measures of absorbance at 595 nm in a UV-700 PharmaSpec spectrophotometer (Shimadzu Scientific Instruments, Columbia,D). Samples were then normalized to a fixed concentration (i.e., 5 �g/ml) to permit

nbiased Western blot analyses using equal protein loadings into gel wells.

.8. Western blot analysis

To perform Western blot analysis of Endo-G expression, cell lysates (50 �gliquots) from each spleen and thymus were loaded into dedicated wells in a 10–12%olyacrylamide gel. After resolution of the sample contents, the gel proteins wereransferred to a nitrocellulose membrane and the latter blocked for 30 min at 4 ◦Cith non-fat dry milk in TBS containing 0.1% Tween-20. Each primary antibody wasiluted to 1:1000 in 5% BSA and then applied to the membrane. After overnight incu-ation at 4 ◦C, the membrane was rinsed free of unbound primary antibody and againlocked with non-fat dry milk in TBS/Tween-20. Secondary antibodies were theniluted to 1:1000 ratios in 5% BSA and applied to the membrane. After a 2 h incuba-ion at 4 ◦C, unbound antibody was rinsed away and the membrane developed usingBT/BCIP (nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate;i-Media, Mumbai, India) chromagens. �-Actin was also analyzed on each mem-rane for confirmation of gel sample loading (i.e., based on constitutive expression).

.9. Immunohistochemistry

Tissue sections (5 �m thickness) were cut from paraffin embedded tissuesnd mounted on positively charged Super frost slides (Export Mengel CF, Menzel,raunschweig, Germany). Tissues were deparaffinized, dehydrate through gradedlcohols, and then blocked for endogenous peroxidase in 3% hydrogen peroxide inethanol. All tissues were preblocked in Tris-buffer saline (TBS) containing 0.3%

riton-X 100 and 0.5% blocking agent (bovine serum albumin [BSA], Sisco Researchaboratories Pvt. Ltd. [SRL], Mumbai, India) and incubated with anti-Bax and anti-biquitin primary antibody overnight at 4 ◦C as a positive control. Anti-sera specificor those antigens were diluted 1:30 in Tris-buffered saline containing 0.3% Triton-

and 0.5% blocking agent. Immunoreactive complexes were then detected using aAB system. Slides were counter-stained briefly in hematoxylin and then mounted

n DPX resin. Slides that received no primary antibody served as negative controls.

.10. Statistical analysis

Each experiment was performed three times; to illustrate results from the tissueistology or Western blot analyses, the best representative data from among eachxperimental set are presented. Data were analyzed and values between groupsere analyzed using single way ANOVA followed by post hoc LSD test. All values are

hown as mean ± SEM, except where otherwise indicated. Results were consideredignificant at p < 0.05.

. Results

.1. Effect of copper on survival of mice

To explore the appropriate doses of copper for use in themmunotoxicological studies LD50 for the copper chloride wasetermined by administrating two doses/week during 4 wk expo-ure regimen using 0, 2.5, 5, 7.5, 10, 12.5, 15, 20 or 40 mg/kg b.w.f copper chloride. The mice that died in all the higher dose groupsi.e., >10 mg copper chloride/kg b.w.) displayed severe losses of

ovement, respiratory distress, and alopecia pre-mortem. In the0 mg/kg b.w. group, ≈50% of the population died over the 28-dayeriod; in the 12.5 and 15 mg/kg b.w. groups’ ≈70% and 80% mortal-

ties were noted, respectively. In the 20 and 40 mg/kg b.w. groups allosts died after the very first treatment. With the lower concentra-ions tested like 7.5 mg/kg b.w. mortalities ≤10% were consistentlyoted. No death was observed within 5 mg/kg b.w. group of mice

metic mean (±SE) of ten mice per group. Error bar represent that animals were alivefor different days after treatment of copper chloride with dose of 7.5, 10, 12.5 and15 mg/kg b.w.

(Fig. 1). As a result of the LD50 study, 5 and 7.5 mg copper chlo-ride/kg b.w. were chosen as the treatment doses for use in theimmunotoxicology studies (at 20 mice/experimental group [with 5mice each sub-group used for various endpoint analyses]). Over thecourse of these 28-day exposures, there were no behavioral abnor-malities or aberrant body weight changes observed in the coppertreated (5 mg and 7.5 mg copper chloride/kg b.w. group) mice.

3.2. Effect of copper on weight changes of spleen and thymus

Firstly we evaluated whether copper can induce any changesin physical appearances of spleen and thymus in treated mice ornot. At sacrifice (i.e., Day 31, 72 h after the final treatment), thespleen and thymus of mice in each of the copper and vehicle treatedgroups were recovered to evaluate any physical changes to eachorgan. It was seen that the (dry) weights of spleens were increasedsignificantly in mice of the 5 mg as well as 7.5 mg copper chlo-ride/kg b.w. group as compared to those of control mice (Fig. 2A).With the thymus, dry weights of the organ were found to be sig-nificantly decreased in mice from both dose groups (5 mg/kg and7.5 mg/kg b.w.) when compared to those of vehicle treated con-trol mice (Fig. 2B). The morphological appearances of spleen andthymus (Fig. 2C and D, respectively) of control, 5 mg/kg b.w. and7.5 mg/kg b.w. also signify its dry weight changes due to coppertreatment.

3.3. Effect of copper on the status of splenocyte and thymocytecounts

Increment and reduction of size and dry weight of spleen andthymus, respectively, might relate to splenocyte and thymocytenumber alteration in copper treated mice. To test the hypothesis weperformed counting of cells from spleen and thymus. It was inter-estingly noted that there was a significant reduction in the numberof splenic and thymic lymphocytes in either set of copper treatedmice as compared to their control counterparts. From Fig. 3A it canbe observed that treatments with 5 or 7.5 mg copper chloride/kgb.w. regimens resulted in ∼16 ± 3% and ∼40 ± 3% reductions in the

numbers of live splenocytes, respectively. In the thymus, there wasalso significant decrease in live thymocyte numbers in each coppertreatment group; the numbers of live cells dropped by ∼25 ± 3% and∼35 ± 3% as compared to those in the control mice thymus (Fig. 3B).

S. Mitra et al. / Toxicology 293 (2012) 78– 88 81

Fig. 2. Differential weight changes in the spleen and thymus induced by copper. Graphical representation indicates weight changes (with respect to control) in (A) spleenand (B) thymus of host mice that received 5 or 7.5 mg copper chloride/kg b.w. over the 28 day regimen. Changes of morphological appearances in (C) spleen and (D) thymusof copper chloride treated mice (both 5 mg/kg and 7.5 mg/kg b.w. groups) compared to vemice per group. Asterisks (*) indicate significant differences (p < 0.05, ANOVA followed byletters indicate significant differences (p < 0.05) between groups.

Fig. 3. Effect of copper on splenic and thymic cell counts. Graphical representationindicates effect of 5 or 7.5 mg copper chloride/kg b.w. on (A) splenic (cells × 108)and (B) thymic cell counts (cells × 106) compare to the respective vehicle treatedcontrol. Results shown are presented as arithmetic mean (±SE) of five mice pergroup. Asterisks (*) indicate significant differences (p < 0.05, ANOVA followed bypost hoc LSD test) in values for different doses compared to controls. The differentletters indicate significant differences (p < 0.05) between groups.

hicle treated control. Results shown are presented as arithmetic mean (±SE) of five post hoc LSD test) in values for different doses compared to controls. The different

3.4. Histological changes induced by copper in spleen and thymus

To confirm the adverse effect of copper on splenocytes andthymocytes in tissue architecture, H/E staining of the splenic andthymic tissues was done. After 28 days of treatment, distinct his-tological changes had occurred in spleens and thymus of mice thatreceived 5 or 7.5 mg copper chloride/kg b.w. Spleens of the controlmice presented distinct T- and B-cell zones in the white pulp, sur-rounded by well defined marginal zones, trabeculae, and red pulp(Fig. 4A and B). In the spleens of copper chloride treated hosts, cellsin the white pulp had proliferated considerably and enlarged tothe limits wherein the margin between white and red pulp beganto disappear and hollow spaces without cells appeared (Fig. 4C–F).Significant differences between control and copper exposed ani-mals were also noted in the thymus. The thymus of control micedisplayed thymocytes of variable size (small, medium, large) inthe cortical region, distributed in a typical cell cluster organiza-tion (Fig. 5A and B). In contrast, organs of the 5 mg/kg treated miceevidenced thymocyte hypertrophy and a decrease in the numberof thymocyte nuclei/field (Fig. 5C and D). Furthermore, the tissuearchitecture in these organs was completely damaged, as evidencedby cellular margin isolation/rupture (Fig. 5E and F). This form of tis-sue disruption was even more evident in the thymuses of mice thatreceived 7.5 mg copper chloride/kg b.w. (Fig. 5F).

3.5. Flow cytometric analysis of copper induced splenic andthymic cell cycle phase distribution

From histological point of view and cell count analysis it wasrevealed that disrupted cellular distribution pattern and decreased

cells number are present in spleen and thymus of copper treatedmice compared to control one. Cell cycle analyses of cells from bothspleen and thymus were performed to investigate one potentialmechanism underlying the reduction in splenic and thymic cell

82 S. Mitra et al. / Toxicology 293 (2012) 78– 88

Fig. 4. Histopathological changes in splenic tissues in response to copper treatment. Hematoxylin and eosin stains were used to prepare sections of spleen from mice treatedwith (A and B) vehicle, (C and D) 5 mg copper chloride/kg b.w. and (E and F) 7.5 mg copper chloride/kg b.w. White arrows represent no change in marginal zone of whitepulp. In control tissue section, distinct and defined separate cluster of white pulp marked by yellow arrows. In treatment tissue sections, black arrows represent areas whereappearances of white pulp and red pulp are not distinct. Appearances of hollow spaces without cells observed in discrete and disorganized white pulp area compared to thatof control. Magnification indicated 40× (A, C, and E) and 100× (B, D, and F). (For interpretation of the references to color in this figure caption, the reader is referred to theweb version of the article.)

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ounts (as well as any significant changes in splenic and thymicorphology) in the mice that had been treated with vehicle, 5 and

.5 mg copper chloride/kg b.w. The flow cytometry data revealedhat compare to the percentage of control host splenocytes in var-ous stations of the cell cycle, among the copper treated animalshere was a dose related substantive increase (i.e., 2–3 fold) inhe percentages of cells at Sub-G0/G1 (might be apoptotic cells;

1) and decreases in those at G0/G1 (M2)(i.e., ≈25 - 30%) spleno-ytes (identified in Fig. 6A and B). In contrast, among thymocytes,here was also 2–3 fold increase in the percentages of cells at the

ub-G0/G1 phase (might be apoptotic cells; M1) for the copper chlo-ide treated mice as compared to cells from the control hosts, aotable decrease in percentages of thymocyte cells at G0/G1 (M2)i.e., ∼10–12% decrement) (Fig. 7A and B).

3.6. Copper induces apoptosis in splenocytes and thymocytes

Annexin V binding assay was performed with the isolatedsplenocytes and thymocytes (as earlier) of mice treated with 0, 5,or 7.5 mg copper chloride/kg b.w. to confirm our findings in cellcycle analysis whether the populations in Sub-G0/G1 are apoptoticor not. Decreased cell count in copper treated spleen and thymusmight correlate the percentages of apoptotic cells in the spleen andthymus. In both sets of copper treated mice, the number of apop-totic spleen cells significantly increased compared to that among

splenocytes from the control hosts (Fig. 8A–C). With respect to thethymocytes, there was also significant increase in apoptotic events(i.e., apoptotic cells) with respect to levels seen in thymus from thecontrol animals (Fig. 8B–D).

S. Mitra et al. / Toxicology 293 (2012) 78– 88 83

Fig. 5. Histopathological changes in thymic tissues in response to copper treatment. Hematoxylin and eosin stains were used to prepare sections of thymus from micetreated with (A and B) vehicle, (C and D) 5 mg copper chloride/kg and (E and F) 7.5 mg copper chloride/kg b.w. White arrows represent no changes and distinct cell clusterlike organizations in control tissue. Green, blue and yellow arrows indicate representative large, medium and small sized cells, respectively, in control section. Black arrowsrepresent decrease in the number of thymocyte nuclei/field and ruptured margin of cell clusters with hollow spaces without cells in treated tissue sections compared toc retatiov

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ontrol. Magnification indicated 40× (A, C and E) and 100× (B, D and F). (For interpersion of the article.)

.7. Expression of EndoG and Bax in the spleen and thymus ofopper treated mice

The Bcl-2-associated X protein, or Bax is a protein of the Bcl-2ene family. It promotes apoptosis. To investigate whether theread been an induction of apoptosis in the spleen/thymus of theopper treated mice, Bax expression patterns were also analyzed.ctopic expression of Bax induces apoptosis (with an early releasef cytochrome c preceding many apoptosis associated morpho-ogical alterations as well as caspase activation and subsequentubstrate proteolysis). The immunohistochemistry results revealedhat Bax immunoreactivity in both the spleen (Fig. 9A–C) and in the

hymus (Fig. 9D–F) increased with the increasing copper chlorideose.

To confirm apoptotic event in spleen and thymus of copperreated mice, we evaluated the expression of another apoptotic

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marker proteins EndoG compared to that of control. The resultsillustrate that EndoG expression in the spleen increased signifi-cantly in mice that received 5 mg copper chloride/kg b.w. as well aswith 7.5 mg copper chloride/kg b.w. dose compared to that of con-trol (Fig. 10A and B). In the thymus, the EndoG expression patternwas also significantly altered due to the 5 mg copper chloride/kgb.w. regimen; however, as in the spleen, a 7.5 mg copper chlo-ride/kg b.w. dose resulted to a much increased expression in EndoGas compared to that in control mice tissues (Fig. 10C and D).

3.8. Ubiquitin expression in spleen and thymus of copper treatedmice

Apoptotic event due to copper treatment might be associatedwith ubiquitination of apoptosis inhibiting proteins. Thereforethe study also assessed whether copper exposure promote

84 S. Mitra et al. / Toxicology 293 (2012) 78– 88

Fig. 6. Flow cytometric analysis of splenocyte cell cycle phase distribution. Cells from copper chloride treated and control mice were fixed and nuclear DNA was labeled withPI. Cell cycle phase distribution of splenic lymphocyte nuclear DNA was determined by single label flow cytometry. Histogram display of DNA content (X-axis, PI-fluorescence)vs. counts (Y-axis) is shown in control, 5 mg copper chloride/kg b.w., and 7.5 mg copper chloride/kg b.w. treatments, respectively (A). Bar graphs represent the % of splenocyteDNA population in different stages of cell cycle for control, 5 mg copper chloride/kg b.w., and 7.5 mg copper chloride/kg b.w. treatments, respectively (B). The results showna nt diffc betwet

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re representative of three independent experiments. Asterisks (*) indicate significaompared to controls. The different letters indicate significant differences (p < 0.05)he respective upper level of the bar graph).

biquitin aggregation in the spleen and thymus of treated miceompared to control mice or not. The immunohistochemistryesults illustrated that in the spleens of copper treated miceboth in 5 mg/kg b.w. and 7.5 mg/kg b.w.), ubiquitin expres-ion levels increased in tandem with the copper chloride doseFig. 11A–C). Immunopositivity of ubiquitin in copper treated thy-

us were similarly affected as in the spleen of copper treated miceFig. 11D–F).

. Discussion

In recent years, many toxicologic endpoints, such as sperma-otoxicity, hepatotoxicity, neuromuscular toxicity, carcinogenicity,eproductive, developmental, and immune system have been iden-ified in animals treated with any one of a variety of chemical agentsKlinefelter et al., 2004; Linder et al., 1994; Holmes et al., 2001;hristian et al., 2002; Bodensteiner et al., 2004; Moser et al., 2004). Aumber of these induced immunotoxic effects are characterized as

mmunosuppressive (Tryphonas et al., 1991); these are often man-fested in marked decreases in immune function. In animals, theseutcomes may be expressed in many ways, including as thymictrophy, splenomegaly, inhibition of immune cell function, or evenpoptosis of splenocytes or thymocytes.

Copper is a crucial trace metal required for normal growth andevelopment of living organisms. However, there is limited infor-ation regarding the immunotoxicity of this metal. Thus, the main

im of this study was to examine the immunotoxicity of copper (inhe form of copper (II) chloride [copper chloride]) and, in particular,

ny occurrence of apoptosis among immunocytes in Swiss albinoice treated with this agent twice a week for 4 wk.Copper induced changes in organ (spleen and thymus) weights

nd physical appearances in treated mice could be associated with

erences (p < 0.05, ANOVA followed by post hoc LSD test) in values for different dosesen groups within their respective similar cell cycle phases accordingly (marked by

a decrease in cell population levels, even in spite of the appear-ance of the splenic enlargement. Specifically, this splenomegalycould be associated with the observed disarray in the white pulpand the expansion of the red pulp (erythrocyte-rich) areas ofthe organ and dead cell accumulation. In contrast, the decreasesin thymocyte cell count also decreased with the advancementof thymus atrophy, suggesting a more straightforward linkagebetween these outcomes in the copper treated hosts. Thesechanges in the immune system organs (sizes and cellularity)are similar in many ways to those noted with many othertypes of agents across a wide spectrum of chemical types, i.e.,3,4-dichloropropionanilide, tetrachlorodibenzo-p-dioxin (TCDD),ethanol, and cadmium (Blaylock et al., 1992; Han et al., 1993; Cuffet al., 1996; Pathak and Khandelwal, 2007). One of the most plau-sible explanations for these (both in the treated hosts here and inthese other cited studies) is that there was an inhibition of lym-phocyte proliferation alone or in conjunction with an inductionof apoptosis among the cells (i.e., lymphocytes) in the thymus orspleen (Kamath et al., 1997; Vandebriel et al., 1999).

The function of the spleen is dependent on the systemic circu-lation; as such, it lacks afferent lymphatic vessels. When copperis present in excess amounts, it will automatically be circulatedand affect all the body’s organs, including lymphoid organs. Anexamination of histopathological features here revealed some mor-phological changes in the thymus and spleen of the copper exposedmice. The changes in immune system organ size/morphology werealso analyzed in the context of flow cytometry data to ascer-tain if copper was causing the changes, in part, by induction

of necrosis/apoptosis or cell cycle arrest among lymphocytes (orother cell types) in these organs. The cytometric analyses revealeda dose related increase in splenocytes and thymocytes locatedat the Sub-G0/G1 phase of the cell cycle; this outcome could

S. Mitra et al. / Toxicology 293 (2012) 78– 88 85

Fig. 7. Flow cytometric analysis of thymocyte cell cycle phase distribution. Cells from copper treated as well as vehicle treated control mice were fixed and nuclear DNAwas labeled with PI. Cell cycle phase distribution of thymocyte nuclear DNA was determined by single label flow cytometry. Histogram display of DNA content (X-axis,PI-fluorescence) vs. counts (Y-axis) is shown for control, 5 mg copper chloride/kg b.w. and 7.5 mg copper chloride/kg b.w. treatments, respectively (A). Bar graphs representthe % of thymocyte DNA population in different stages of cell cycle for control, 5 mg copper chloride/kg b.w., and 7.5 mg copper chloride/kg b.w. treatments, respectively(B). The results shown are representative of three independent experiments. Asterisks (*) indicate significant differences (p < 0.05, ANOVA followed by post hoc LSD test)in values for different doses compared to controls. The different letters indicate significant differences (p < 0.05) between groups within their respective similar cell cyclephases accordingly (marked by the respective upper level of the bar graph).

Fig. 8. Level of apoptosis in splenocyte and thymocyte in response to copper treatments. (A and B) Spleen and (C and D) thymus cells recovered from mice that receivedvehicle, 5 mg copper chloride/kg b.w. and 7.5 mg copper chloride/kg b.w. during the 28 day regimen. For each cell population sample, a minimum of 10,000 events wereacquired during flow cytometric analyses of Annexin V-FITC and PI fluorescence levels. Apoptotic cells (Annexin V+/PI−) were analyzed flow cytometrically and dot plotdisplay Annexin V fluorescence (X-axis, logarithmic scale) vs. PI fluorescence (Y-axis, logarithmic scale). Results are one representative of five mice per group (A and C). Thebar graph represents the % of apoptotic population at different treatment conditions (B and D). Asterisks (*) indicate significant differences (p < 0.05, ANOVA followed by posthoc LSD test) in values for different doses compared to controls. The different letters indicate significant differences (p < 0.05) between groups.

86 S. Mitra et al. / Toxicology 293 (2012) 78– 88

Fig. 9. Immunoreactivity of Bax in spleen and thymus response to copper treatments. Differential expression of Bax in spleens recovered from mice that received (A) vehicle,( ely, dui per chi

bAeiwpgbstcsalh

Fcpdb

B) 5 mg copper chloride/kg b.w. and (C) 7.5 mg copper chloride/kg b.w., respectivn the thymic tissues recovered from mice that received (D) vehicle, (E) 5 mg copndicated 40×.

e indicative of ongoing cell cycle arrest or, based upon PI andnnexin V staining patterns here, an increase in apoptotic/necroticvents among the cells (i.e., compared to controls, nearly 50%ncrease in apoptotic splenocytes and thymocytes, respectively,

ere noted). It is therefore quite plausible that the increases in apo-tosis/cell cycle arrest among splenocytes and thymocytes (and ofreater importance, potentially lymphocytes in particular) coulde a key factor underlying the thymic atrophy and decreasedplenic white pulp size/content that evolved from the 28 dayreatments with copper. Regardless of whether it was the lympho-ytes that in fact underwent the shift to apoptotic and/or necrotic

tates, the overall changes to spleen and thymus size/morphologyre suggestive of multiple pathologies in each organ that wouldikely/eventually apparent as immunosuppression in the exposedost.

ig. 10. Effect of copper chloride treatments on EndoG expression in spleen and thymus ofopper chloride/kg b.w.) mice underwent Western blot analyses using anti-Endo G antibrotein in samples shown in A and C. �-Actin used as loading control. Data shown is rifferences (p < 0.05, ANOVA followed by post hoc LSD test) in values for different doses cetween groups.

ring the 28 day regimen. Differential immunoreactivity of Bax was also observedloride/kg b.w. and (F) 7.5 mg copper chloride/kg b.w., respectively. Magnification

In trying to explain the observations regarding effects of the cop-per on the cell cycle, it is important to examine the potential role ofEndoG, a mitochondrion-specific nuclease that translocates to thenucleus during cell cycle arrest and apoptosis. Once released frommitochondria, EndoG cleaves chromatin DNA into nucleosomalfragments (independently of caspases); thus, EndoG represents anovel caspase independent apoptotic pathway. Huang et al. (2006)illustrated the link between this EndoG cleaving function and apo-ptosis/cycle arrest in a study that showed that elevation of EndoGexpression produced a change in the cell cycle that resembled G2arrest (i.e., one caused primarily by accumulation of damaged DNA).

In an attempt to relate changes in EndoG expression to the copperexposures here, we note previous reports that have shown thatan increased presence of copper can lead to a decrease in lev-els of most mitochondrial membrane fatty acids and a break in

mice. Lysates of (A) spleen and (C) thymus from control and treated (0, 5 and 7.5 mgody. (B and D) Bars represent quantitative densitometric values of the expressedepresentative of three comparable experiments. Asterisks (*) indicate significantompared to controls. The different letters indicate significant differences (p < 0.05)

S. Mitra et al. / Toxicology 293 (2012) 78– 88 87

Fig. 11. Immunohistochemical analysis of ubiquitin in response to copper in spleen and thymus of treated mice. Differential immunoreactivity of ubiquitin in spleens recoveredfrom mice that received (A) vehicle, (B) 5 mg copper chloride/kg b.w. and (C) 7.5 mg copper chloride/kg b.w., respectively, during the 28 day regimen. Immunoreactivity ofu ehicleM

a(psacactemct2piemw

ipetibeihpdeaXidttici

biquitin was also seen in the thymic tissues recovered from mice that received (D) vagnification indicated 40×.

cardiolipin like molecule that aids in organelle pore formationSokol et al., 1990; Arciello et al., 2005). Thus, should the cop-er be inducing increasing mitochondrial pore formation in theplenocytes/thymocytes, this would provide a means for increasedmounts of EndoG in each cell’s nucleus. The Western blot data herelearly suggests that the amount of EndoG protein in the spleennd thymus was increasing in tandem with the dose of copperhloride employed. To further verify whether mitochondrial pro-eins were potentially involved in the pathologies noted here, baxxpression was also assessed. In keeping with the above-suggestedechanism(s) of effect, bax levels in spleen and thymus of copper

hloride-treated mice were seen to be up-regulated. These results,aken together, allow us to surmise that over the course of the8 day exposure regimen, copper was promoting increased EndoGrotein expression in the spleen and thymus and that this led to

ncreased EndoG-mediated mitochondrial-dependent damage inach organ’s cells (including, potentially, local lymphocytes) ulti-ately giving rise to the observed increases in levels of cells thatere apoptotic/in cell cycle arrest.

On the other hand, it was reported that copper metabolisms tightly regulated through ubiquitination. Normally, cop-er metabolism related genes like COMMD1 are ubiquitouslyxpressed and basal levels are controlled by an ubiquitin ligasehat belongs to a family of inhibitor of apoptosis, XIAP (X-linkednhibitor of apoptosis protein). Interestingly, XIAP protects cellsy both caspase dependent and independent mechanisms (Mainet al., 2009; Arnesano et al., 2009). Further, XIAP was recentlydentified as a copper-binding protein and regulator of copperomeostasis. Although the mechanism by which XIAP binds cop-er in the cytosol is unclear, the copper chaperone for superoxideismutase (CCS) is thought to serve as a mediator of copper deliv-ry to XIAP in cells. CCS is a target of the E3 ubiquitin ligasectivity of XIAP, although interestingly, ubiquitination of CCS byIAP was found to lead to enhancement of its chaperone activ-

ty toward its physiologic target, SOD1, rather than proteasomalegradation (Brady et al., 2010). Thus, we hypothesize that if copperoxicity is increased in a cell (i.e., here, a splenocytes/thymocytes),

hen XIAP/ubiquitin up regulation occurs and this, in turn, leads tonitiation of cell death. It is evident from the immunohistochemi-al analyses performed here that ubiquitin expression was in factncreased in both the spleen and thymus of the copper treated mice.

, (E) 5 mg copper chloride/kg b.w. or (F) 7.5 mg copper chloride/kg b.w., respectively.

Several studies have implicated dynamic post-translational reg-ulation (including ubiquitination and phosphorylation) of copperchaperones and transporters in the maintenance of cellular copperhomeostasis (Liu et al., 2007; Caruano-Yzermans et al., 2006). A bet-ter understanding of how copper trafficking (i.e., from transportersto chaperones to copper dependent proteins) is dynamically regu-lated in response to changing cellular requirements will be essentialfor developing improved therapies for disorders of dysregulatedcopper metabolism. Whether copper aggregation stimulates ubiq-uitination in the spleen (or liver) is even now still not clear. Takentogether, our data and other previous reports of copper induceddegradation are consistent with a model in which interaction ofcopper free CCS results in non degradative ubiquitination of CCS(whereas copper bound CCS would transfer copper and be ubiqui-tinated and targeted for proteasomal degradation). We have seenthat the increased ubiquitination in the spleen and thymus of thecopper treated mice; our hypothesis is that this ubiquitinationmight have been stimulated by copper aggregation and failure oflocal proteasomal systems to remove these aggregated and ubiq-uitinated protein (these events, in turn, may initiate local celldeath).

In summary, these studies represent an important step toincreasing our understanding of the mechanism(s) underlyingcopper-induced toxicity in situ. The present study clearly showedthat sub lethal exposure to copper led to an induction of bothsplenomegaly and thymic atrophy. These studies also showed thatthese copper induced outcomes in the spleen and thymus werevery likely related to an induction of apoptosis and cell cycle arrestamong their resident cells, including lymphocytes. Importantly,this study also established that the apoptosis induced by copperin the thymus and spleen appeared to be mediated, in part, by theEndoG-Bax pathway and up regulation of ubiquitin expression.

While our inclination is to conclude that the potential effects ofcopper on the immune system would seem to be derived primarilyfrom effects on the above-noted pathways within splenic/thymicpopulations. It is clear that splenomegaly is commonly observedevents arising from exposure(s) to a wide variety of agents and in

many instances, thymic atrophy accompanies these pathologies.Experimental hepatitis induces immunomorphological changes inthe spleen and thymus (Obernikhin et al., 2006) quite similar tothose described here.

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8 S. Mitra et al. / Toxi

Ultimately, whether through direct effects on resident cellsr indirectly via damage to non-immune system organs, thesehanges in/damage to cells of the two critical immune systemrgans - the spleen and thymus are likely to be major factors thatould underlie the immune suppression that has been documented

ollowing acute high dose/chronic low medium dose exposures toopper agents.

onflict of interest

The authors report no conflicts of interest. The authors are aloneesponsible for the content and writing of the paper.

cknowledgements

The Authors want to thank Department of Biotechnology andenetic Engineering, University of Calcutta, for their Flow cytome-

ry instrument facility. This work was supported by grants fromepartment of Science and Technology, Govt. of India FIST Pro-ram in Department of Zoology, University of Calcutta. Also weike to thank Indian Council of Medical Research, Govt. of IndiaICMR 5/8/4-4(Env)/2008/NCD-I] for equipment and fellowshipupport.

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