Copper-induced immunotoxicity involves cell cycle arrest and cell death in the liver

Copper-Induced Immunotoxicity Involves CellCycle Arrest and Cell Death in the Liver

Tarun Keswani, Soham Mitra, Arindam Bhattacharyya

Immunology Laboratory, Department of Zoology, University of Calcutta, 35, BallygungeCircular Road, Kolkata 700019, West Bengal, India

Received 6 May 2013; accepted 9 October 2013

ABSTRACT: Inorganic copper, such as that in drinking water and copper supplements, largely bypassesthe liver and enters the free copper pool of the blood directly and that promote immunosuppression.According to our previous in vivo report, we evaluate the details of the apoptotic mechanism in liver, wehave investigated how copper regulates apoptotic pathways in liver. We have analyzed different proteinexpression by Western blotting and immunohistochemistry expression. We have also have measuredmitochondrial trans-membrane potential, Annexin V assay, ROS, and CD41 and CD81 population inhepatocyte cells by flow cytometry. Copper-treated mice evidenced immunotoxicity as indicated bydose-related, distinct histomorphological changes in liver. Flow cytometric analyses revealed a dose-related increase in the percentages of hepatocyte cells in the Sub-G0/G1 state, further confirmed byAnnexin V binding assay. In addition, the copper treatments altered the expression of apoptotic markers,further ROS generation and mitochondrial trans-membrane potential changes promote intrinsic pathwayof apoptosis that was p53 independent. Apart from the role of inflammation, our findings also have identi-fied the role of other partially responsible apoptotic molecules p73 that differentially changed due to cop-per treatment. Our study demonstrates how apoptotic pathways regulate copper-inducedimmunosuppression in liver. VC 2013 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2013.

Keywords: copper; immunotoxicity; liver; apoptosis; P53; ROS

INTRODUCTION

Wide variety of industrial and agricultural applications uses

copper and its salts, causing continuous contamination of

water supplies (Hebert et al., 1993). Copper-gluconate is

widely used in the United States as a nutritional enhancer of

supplements, candies, and beverages. Widespread consump-

tion of food and water contaminated with copper has raised

questions about the health hazards of these compounds. Dis-

eases like Wilson disease, Menke’s disease, and idiopathic

copper toxicosis are diseases associated with hepatic and

extra-hepatic copper accumulation. Hence, many investiga-

tions have been performed to investigate the potential

adverse effects of copper. Chronic exposure of cells and tis-

sues to excessive amount of copper can result in pro-

grammed cell death or apoptosis. Copper-induced apoptosis

has been demonstrated in splenocytes, thymocytes, hepato-

cytes, and so forth (Linder, 2001; Mitra et al., 2013). The

immune system is one of the main adaptation mechanisms

through which the body defends itself against harmful agents

and pathogens. It was reported previously that excessive

copper intake results in impairment of both cellular and

humoral immune responses (Pocino et al., 1991). The poten-

tial adverse effects of copper contaminants on the immune

system are a concern for regulatory authorities.

In our previous study (Mitra et al., 2012); we reported

that excessive copper exposure in Swiss albino mice pro-

mote cell cycle arrest and cell death in immune organs like

Correspondence to: A. Bhattacharyya; e-mail: [email protected]

Contract grant sponsors: Indian Council of Medical Research, and

Govt. of India [ICMR 5/8/4-4/(Env)/2008/NCD-I, dated 01/02/2010]

Department of Science and Technology and Govt. of India FIST Program

in Department of Zoology, University of Calcutta.

Published online 00 Month 2013 in Wiley Online Library

(wileyonlinelibrary.com). DOI: 10.1002/tox.21916

VC 2013 Wiley Periodicals, Inc.

1

spleen and thymus that ultimately leads to immunosuppres-

sion. Apoptosis, a vital regulator of the immune system and

being a potential target for immunotoxicants that are known

to damage splenic and thymic tissues. Copper has also been

shown to activate p53-dependent and independent pathways

of apoptosis either of reversible growth arrest (Linder, 2001;

Marchetti et al., 2004) or of apoptosis (Hershko et al., 2005;

Yu et al., 2007). p53 is also known to cause cell death by

directly inducing mitochondrial permeability and apoptosis,

independent of the transcriptional up-regulation of pro-

apoptotic genes (Johnstone et al., 2002). More recently,

researchers have shown that p53 can interact at the mitochon-

drial level, with Bax, Bcl-2, and Bcl-xl (Linder, 2001; Mihara

et al., 2003). In our previous study we have identified that

copper-induced immunotoxicity facilitates cell cycle arrest

and apoptosis via differential apoptotic pathways in spleen

and thymus. Therefore, our current study further investigated

whether copper induced apoptosis among liver. In addition, to

observe mechanistic explanation for any observed changes in

levels of apoptosis among hepatocytes, this study also eval-

uated expression of selective apoptosis-regulating molecules.

MATERIALS AND METHODS

Reagents

Copper (II) chloride dihydrate crystal was purchased from

Merck, Mumbai, India. Primary antibodies against Bax, Bcl2,

BclxL, Caspase 3, Caspase 8, Caspase 9, cytochrome c, p27,

p53, p62, p73, TNF-a, and Cox 2 and alkaline phosphatase

(AP)-conjugated anti mouse and anti-rabbit secondary anti-

bodies were obtained from Cell Signaling technology (Dan-

vers, MA). Pre-stained protein molecular weight marker,

3,30-diaminobenzidine tetrahydrochloride (DAB) system as

well as horseradish peroxidase (HRP)-conjugated secondary

anti-mouse and anti-rabbit antibodies were bought from Ban-

galore Genei (Bangalore, India). FITC CD4 and PE CD8b

antibodies were purchased from BD Bioscience (CA).

Dihexyl-oxacarbocyanine (DiOC6) was procured from Bec-

ton Dickinson Immunocytometry system, San Jose, CA. All

remaining chemicals cited in this article were procured from

local firms in India and were of the highest purity grade.

Animals and Treatment

Male Swiss albino mice (�25 g each; five mice in each

group) were obtained from the National Institute of Nutrition

(Hyderabad, India). Each was housed in an animal facility

(maintained at 25–28 [62]�C; with 55 [65]% relative

humidity, and a 12 h/12 h light/dark cycle) located at the

Animal Housing Unit in the Department of Zoology, Univer-

sity of Calcutta. All animals were provided rodent chow

(National Institute of Nutrition) and filtered water ad libitum.

All animal experiments were performed following the

“Principles of Laboratory Animal Care” (NIH publication

No. 85-23, revised 1985) as well as by following specific

Indian law on “Protection of Animals” under the supervision

of authorized investigators.

For the experiments, mice were treated as mentioned by

Mitra et al. (2012). Briefly, the mice were randomly divided

into two groups comprising (A) normal/control and (B) cop-

per (II) chloride (CuCl2)-treated sets. In an initial study, the

mice were given CuCl2 as intraperitoneal (IP) injections at

sub-lethal dose (5 mg CuCl2/kg BW) twice a week for

4 weeks to permit the experiments below to be performed.

Histological Analysis

At sacrifice, from subsets of mice in each group, the liver

was 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 embedded in paraffin (MERCK, solidification point

60–62�C). Tissue sections (thickness 5-lm) were then depar-

affinized with xylene, rehydrated 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 microscope

fitted with an Olympus DP70 camera (U-TVO 63XC;

Olympus, Tokyo, Japan) having both a 403 and 1003

(wide-zoom) lens (Keswani and Bhattacharyya, in press).

Isolation of the Hepatocytes for Cell Cycleand Annexin V Analysis

Briefly, animals were sacrificed and the portal vein was can-

nulated with a 1/2-gauge needle (BD Biosciences, Franklin

Lakes, NJ), and injected with 2 mL 1% (wt/vol) collagenase

IV (Sigma, St. Louis, MO) in PBS. Liver tissues recovered

from other subsets of mice were mechanically disrupted

before incubation in 10 mL 1% collagenase at 37�C for 20

min. Single-cell suspensions, cell cycle analysis, and

Annexin V assay were then performed as previously men-

tioned. The Propidium Iodide (PI) fluorescence was then

measured through a FL-2 filter (585 nm) in a BD FACS Cal-

ibur flow cytometer (Becton Dickinson); a minimum of

10,000 events was acquired for each sample. The flow cyto-

metric data were ultimately analyzed using Cell Quest soft-

ware (Becton Dickinson) and histogram displays of DNA

content (x-axis, PI-fluorescence) versus counts (y-axis) were

generated for analyses (Keswani and Bhattacharyya, 2013).

Analysis of Mitochondrial Trans-MembranePotential by Flow Cytometry

Loss of mitochondrial trans-membrane potential was verified

by flow cytometry at the single-cell level. Aliquots (each

containing 5 3 106 cells) of isolated hepatocytes from

treated and control were removed, recentrifuged, and sus-

pended in 1 mL PBS. Hepatic cells were stained with the

2 KESWANI, MITRA, AND BHATTACHARYYA

Environmental Toxicology DOI 10.1002/tox

potentially sensitive dye DiOC6 (40 nM, 15 min at 37�C in

the dark). Loss of DiOC6 fluorescence indicates disruption

of the mitochondrial inner trans-membrane potential (Pen-

nington, 1961). The probe was excited at 488 nm and emis-

sion was measured through a 530-nm band pass filter.

Flow Cytometric Analysis of ROS

The experimental procedure was followed as mentioned by

Chatterjee et al. (2009). Briefly, the generation of Reactive

Oxygen Species (ROS) was detected by 20,70-dichlorofluores-

cein diacetate (DCFH-DA). The cells were incubated with

(100 lM final concentration) for 60 min in dark at 37�C. The

cells were harvested and suspended in PBS and ROS genera-

tion of cells (104) was measured by the fluorescence intensity

(FL-1, 530 nm). Logarithmic amplification was used to detect

the fluorescence of the probe.

Flow Cytometric Analyses of CD41 andCD81 T Lymphocyte Population inCopper-Treated Liver

The T-lymphocyte phenotyping was conducted to analyze

the effect of copper based on CD4 and CD8 surface mole-

cules. Aliquots (each containing 5 3 106 cells/100 lL) of

isolated hepatocytes were then removed, re-centrifuged, and

suspended in 1 mL PBS. An aliquot of 100 lL was incu-

bated with 5 lL FITC-conjugated anti-CD4 monoclonal

antibody and 5 lL PE-conjugated anti-CD8 monoclonal

antibody for 30 min in dark at room temperature, after which

100 lL PBS was added to each sample. The FITC and PE

fluorescence were measured through FL-1 filter (530 nm)

and FL-2 filter (585 nm), respectively, and 10,000 events

were acquired (Mitra et al., 2013).

Preparation of Cell Lysates

Liver tissues recovered from other subsets of mice in each

treatment group were each placed in RIPA Lysis buffer (150

mM sodium chloride, 1.0% Triton-X-100, 50 mM Tris [pH

8.0], 0.01% SDS, and 0.5% sodium deoxycholate) contain-

ing 1 mM PMSF (phenyl-methanesulfonyl fluoride), 1 lg

aprotinin/mL, and 1 lg leupeptin/mL (all Sigma, St. Louis,

MO) and homogenized. Supernatants were then collected

following centrifugation of each mixture at 14,000 rpm for

15 min at 4�C. Estimations of protein content in each super-

natant were then performed using the Bradford reagent

(Sigma) and subsequent measures of absorbance at 595 nm

in a UV-1700 PharmaSpec spectrophotometer (Shimadzu

Scientific Instruments, Columbia, MD). Samples were then

normalized to a fixed concentration (i.e., 5 lg/mL) to permit

unbiased Western blot analyses using equal protein loadings

into gel wells (Mitra et al., 2013).

For cytoplasmic extracts, cells were washed twice with

ice cold PBS and collected by centrifugation at 1500 r.p.m.

for 10 min at 40�C. Cell pellet was then resuspended in

buffer A (10 mM Hepes pH 7.9; 10 mM KCl; 0.1 mM

EDTA; 0.1 mM EGTA; 1 mM DTT; proteinase inhibitors)

for 15 min and subsequently centrifuged for 2 min at 6800

r.p.m. Supernatant was transferred to new cups and subse-

quently centrifuged at 14,000 r.p.m. for additional 20 min.

Pellet was resuspended in 30–100 mL buffer C (20 mM

Hepes pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1

mM DTT; proteinase inhibitors) and incubated on ice. A

final centrifugation step at 14,000 r.p.m. for 20 min was per-

formed to separate nuclear proteins from cellular debris.

Western Blot Analysis

For Western blot analysis of Bax, Bcl-2, Bcl-xL, Caspase 3,

Caspase 8, Caspase 9, cytochrome c, p27, p53, p62, p73,

TNF-a, and Cox 2, cell lysate was loaded into a 10–15%

SDS–polyacrylamide gel. After resolution of the sample

contents, the gel proteins were transferred to a nitrocellulose

membrane and the latter blocked for 30 min at 4�C with

non-fat dry milk in TBS containing 0.1% Tween-20. Each

primary antibody was diluted to 1:1000 in 5% BSA and then

applied to the membrane. After overnight incubation at 4�C,

the membrane was rinsed free of unbound primary antibody

and again blocked with non-fat dry milk in TBS/Tween-20.

Secondary antibodies were then diluted to 1:1000 ratios in

5% BSA and applied to the membrane. After a 2 h incuba-

tion at 4�C, unbound antibody was rinsed away and the

membrane developed using NBT/BCIP (nitroblue tetrazo-

lium chloride/5-bromo-4-chloro-3-indolyl-phosphate; Hi-

Media, Mumbai, India) chromagens. b-Actin was also ana-

lyzed on each membrane for confirmation of gel sample

loading (i.e., based on constitutive expression).

Immunohistochemistry

Immunohistochemistry was done as previously mentioned

by Mitra et al. (2012). Briefly, tissue sections (5 lm thick-

ness) were cut from paraffin-embedded tissues and mounted

on positively charged Super frost slides (Export Mengel CF,

Menzel, Braunschweig, Germany). Tissues were incubated

with anti-caspase 3, iNOS, TNF-a, COX-2, and p53 primary

antibody overnight at 4�C as a positive control. The immu-

noreactive complexes were then detected using a DAB sys-

tem. Slides were counter-stained briefly in hematoxylin and

then mounted in DPX resin. Slides that received no primary

antibody served as negative controls (data not shown).

Statistical Analysis

All values are shown as mean (6SEM), except where other-

wise indicated. Each experiment was performed three times;

to illustrate results from the tissue immunohistochemistry or

Western blot analyses, the best representative data from

among each experimental set are presented. All data were

analyzed and, when appropriate, significance of differences

COPPER-INDUCED IMMUNOTOXICITY 3


between mean values was determined by a Student’s t-test.

Results were considered significant at p < 0.05.

RESULTS

Histological Changes Induced by Copperin Liver

To confirm the adverse effect of copper on hepatocytes, stain-

ing of the hepatic tissues was done. After 28 days of treat-

ment, distinct histological changes had occurred in mice that

received 5 mg CuCl2/kg. Liver of the control mice presented

no enlargement between the hepatic, typical oval and round

shapes, but a swollen and clear cytoplasm was noted. Nuclei

of hepatocytes exhibited very strong staining, but normal mor-

phological features were observed [Fig. 1(A,B)]. After copper

treatment, liver sections showed extensive histopathological

changes, presented an enlarged liver laden showing showed

areas of coagulative bridging necrosis. Many vacuoles were

observed in most of the hepatocytes. Also, the nucleus–cyto-

plasmic ratio was changed and the hepatic sinusoids narrowed

in some areas compared to control mice [Fig. 1(B,C)].

Flow Cytometric Analysis of Copper-InducedHepatic Cell Cycle Phase Distribution andAnnexin V Apoptosis Assay

In our previous study (Mitra et al., 2012) it was found that

apoptosis and cell cycle arrest appeared in spleen and thymus

due to copper treatment in mice. To investigate one potential

mechanism underlying any reduction in hepatic cell counts,

we observed that in comparison to cells from control hosts,

there was increase in the Sub G0/G1 and decrease in G0/G1

population of hepatocytes [Fig. 2(A,B)]. Further Annexin V

binding assay confirmed, the number of apoptotic liver cells

was nearly fourfold greater in treated mice relative to that

among hepatocytes from the control hosts [Fig. 2(C,D)].

Fig. 1. Histopathological changes in hepatic tissues in response to CuCl2. Hematoxylin and eosin stains were used to preparesections of liver from mice treated with (A and B) vehicle or (C and D) 5 mg CuCl2/kg. White arrows represent no change andblack arrows represent changes in tissues compared with control. Magnification 5 40 3 (A and C) and 5 100 3 (B and D).[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



wileyonlinelibrary.com

Expression Level of Bax, Cytochrome c, Bcl-2, and Bcl-xl in Liver of Copper-Treated Mice

In this study, we have critically evaluated if there were any

complementary changes in the expression of apoptotic

marker protein Bax and anti apoptotic proteins Bcl-2 and

Bcl-xl in the CuCl2-treated mice or not. Western blot analy-

ses revealed that in mice which received 5 mg CuCl2/kg

BW, Bax expression level in the liver increased significantly

[Fig. 3(A)]; whereas Bcl-2 [Fig. 3(B)] and Bcl-xl [Fig. 3(C)]

decreased significantly compared to that in control mice tis-

sues. Bax induces apoptosis with an early release of cyto-

chrome c in spleen and thymus was confirmed in our earlier

study (Mitra et al., 2013). The Western blot experiments

revealed that in mice which received 5 mg CuCl2/kg b.w.,

cytochrome c expression in liver increased significantly

compared to that in control mice tissues [Fig. 3(D)].

Differential Changes in Expression Pattern ofCaspase 9, Caspase 8, Caspase 3, and MMPDue to Copper Treatment in Liver of Mice

We have evaluated caspase 9 expression in both organs of

copper-treated mice. Western blot analysis revealed that

expression level of caspase 9 has increased significantly in

liver of copper-treated mice compared to vehicle-treated

control tissues [Fig. 4(A)]. Therefore, an increased caspase 9

expression may validate the increased release of cytochrome

c in liver cytosol and promotion of intrinsic pathway of apo-

ptosis. Conversely, we also have chased the extrinsic path-

way of apoptosis and found caspase 8 did not alter in liver of

copper-treated animals compared to their respective control

tissue [Fig. 4(B)]. Further Western blot and immuno-

histochemistry analysis revealed that caspase 3 expression

level increased significantly in liver compared to their

respective control [Fig. 4(C,D)]. The activation of caspase-3

and depolarization of the mitochondrial inner membrane are

very rapid and occur in parallel (Liu et al., 1996). Decrease

in DiOC6 fluorescence is a measure of the less integrity of

the mitochondrial membrane, therefore the results from this

study indicated decrease in integrity of the mitochondrial

membrane in liver of copper-treated mice [Fig. 4(E,F)] com-

pared to the vehicle-treated control.

Immunoreactivity of iNOS, COX-2, and TNF-aDue to Copper Treatment in Liver of Mice

Excessive nitric oxide (NO) and especially peroxynitrite

may cause tissue damage (Ba and Garg, 2011). From

Fig. 2. Flow cytometric analysis of hepatic lymphocyte cell cycle phase distribution. (A) Cells from CuCl2-treated as well normalmice were fixed and nuclear DNA was labeled with PI. Cell cycle phase distribution of hepatic lymphocyte nuclear DNA wasdetermined by single label flow cytometry. Histogram display of DNA content (x-axis, PI-fluorescence) versus counts (y-axis) isshown. (B) Bars represent the percentage (%) of cell population in different phases of cell cycle for control and 5.0 CuCl2 mg/kgBW treatments, respectively. Levels of hepatic apoptotic cells in response to CuCl2 treatments. (C and D) Hepatic cells recov-ered from mice that received vehicle and 5 mg CuCl2/kg during the 28-d regimen. For each cell population sample, a minimumof 10,000 events was acquired during flow cytometric analyses of Annexin V-FITC and PI fluorescence levels. Results are pre-sented as arithmetic mean (6SE) of three mice per group. *Value significantly different from control at p < 0.05.



immunohistochemistry it is revealed that immunoreactivity

of iNOS is much higher in copper-treated liver [Fig. 5(A,B)]

compared to that of vehicle-treated control. The product of

iNOS, nitric oxide (NO), has been demonstrated to modulate

and induce COX-2 expression to promote inflammation.

Therefore, apoptosis in the treated immune organs (liver)

may promote inflammation where nitric oxide production is

high. There may be chronic inflammatory condition

appeared in immune organs due to copper treatment and that

may modulate or accelerate apoptotic events in treated liver.

We have evaluated proinflammatory markers COX-2 and

TNF-a in liver. Immunohistochemical analysis revealed that

expression level and immunoreactivity of TNF-a and COX-

2 were significantly higher in liver of copper-treated animal

compared to the vehicle-treated control [Fig. 5(C–F)].

Expression Pattern of p53, p73, and ROS inLiver of Copper-Treated Mice

p53 mediates apoptosis through a linear pathway involving

bax trans-activation, bax translocation from the cytosol to

membranes, cytochrome c release from mitochondria, and

caspase-9 activation (Shen and White, 2001). Western blot

and Immunohistochemical analysis revealed that expression

level of p53 decreased in copper-treated liver compared to

that of vehicle-treated control, respectively [Fig. 6(A,B)].

Therefore, these results demonstrate that in case of copper-

treated liver the apoptotic event was possibly p53 independ-

ent. Conversely, Western blot analysis revealed that expres-

sion level of p73 increased significantly in compared to that

of control [Fig. 6(C)]. Therefore, the expression level of p73

might alter with reference to p53 or it may function inde-

pendent of p53. Each cellular concentration and distribution

of p53 has a distinct cellular function and that ROS act as

both an up-stream signal that triggers p53 activation and as a

downstream factor that mediates apoptosis. To check the

hypothesis, we measured the ROS in liver of copper-treated

mice. Flow-cytometric data revealed that ROS level

increased in the liver of copper-treated liver compared to the

control [Fig. 6(D,E)]. Therefore, results indicated that ROS

generation in treated liver was not p53 dependent.

Changes in Population of CD41and CD81 CellsDue to Copper Treatment in Liver of Mice

Heavy metal toxicity has deleterious effect on helper CD41

and cytotoxic CD81 T cell populations (Pathak and Khan-

delwal, 2009). It was also found that prolonged exposure to

Fig. 3. Effect of copper treatments on expression pattern of Bax, Bcl-2, Bcl-xL, and cytochrome c in liver. Respective lysatesof hepatocytes from control and treated (5 mg CuCl2/kg b.w.) mice underwent Western blot analyses using (A) anti-Bax, (B)anti-Bcl-2, (C) anti-Bcl-xL, and (D) anti-cytochrome c antibody. Respective bars represent quantitative densitometric valuesof the expressed protein in samples shown in A, B, and C with arbitrary units. b-Actin used as loading control. Data shownare representative of three comparable experiments. Asterisk (*) value significantly different from the control at p < 0.05.



low copper concentration had been shown a deleterious

effect on both antioxidant defense system enzymes and phe-

notypic properties of immunocompetent cells of mice (Kli-

nefelter et al., 2004). Therefore, finally we evaluated the

population in terms of percentage of CD41 and CD81 cells

in liver of copper-treated mice. Immunosuppression will be

linked with these changes in population dynamics of CD41

and CD81 T cells. Flow cytometric data revealed that in

liver of copper-treated mice, CD41 CD81 cells increased

significantly, CD41 cells increased significantly and CD81

Fig. 4. Effect of copper treatments on caspase 9, caspase 8, caspase 3, and MMP expres-sion pattern in liver. Lysates of hepatocytes from control and treated (5 mg CuCl2/kg b.w.)mice underwent Western blot analyses using (A) anti-caspase 9, (B) anti-caspase 8, and(C) anti-caspase 3 antibodies. b-Actin used as loading control. Data shown are representa-tive of three comparable experiments. Bars represent quantitative densitometric values ofthe respective expressed proteins in samples with arbitrary units. Asterisk (*) values aresignificantly different from the control at p < 0.05. (D) Differential expression and immuno-reactivity of Caspase 3 observed in Liver recovered from mice that received vehicle and 5mg CuCl2/kg b.w., respectively, during the 28-d regimen. Magnification 5 340. Flow-cytometric analysis of mitochondrial transition pore formation and mitochondrial Transmembrane potential in response to Copper treatment (5 mg/kg b.w.). The mitochondrialmembrane permeability was measured by flow cytometry in a single labeling system usingDiOC6 fluorescent probes and a 530-nm band pass filter. (E) The loss of fluorescenceindicates the disintegration of mitochondrial membrane. (F) Histograms and bars, respec-tively, represent qualitative and quantitative values of the mitochondrial membranepotential changes for liver. Asterisk (*) values are significantly different from the controlat p < 0.05. [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]




cells possess no significant change compared to vehicle-

treated control mice organs, respectively [Fig. 6(F–K)].

DISCUSSION

In recent years, many toxicologic endpoints, such as hepato-

toxicity, spermatotoxicity, neuromuscular toxicity, carcino-

genicity, reproductive, developmental, and immune system

toxicity endpoints have been identified in animals treated

with any one of a variety of chemical agents (Linder et al.,

1994; Basu and Haldar, 1998; Holmes et al., 2001; Christian

et al., 2002; Bodensteiner et al., 2004; Moser et al., 2004). In

our previous study, we have showed how copper promotes

apoptosis and cell cycle arrest in spleen and thymus of Swiss

albino mice that ultimately leads to immunosuppression

(Mitra et al., 2012). Therefore, in this study, we have

observed the expression levels of different cell signaling

molecules related to apoptosis and inflammation and how

their expression levels modulate apoptotic events due to cop-

per toxicity in liver of Swiss albino mice.

The function of the liver is dependent on the systemic cir-

culation. As such, it lacks afferent lymphatic vessels. When

copper is present in excess amounts, it will automatically be

circulated and affect all the body’s organs, including lymph-

oid organs. In the liver, distinct morphological changes were

Fig. 5. Effects of copper treatment on iNOS, COX-12, and TNF-a expression in liver. Dense expression and immunoreactivityof (A and B) iNOS, (C and D) TNF-a, and (E and F). COX-2 was observed in liver recovered from mice that received vehicleand 5 mg CuCl2/kg b.w., respectively, during the 28-d regimen. Magnification 5 340. Magnification 5 340. Data shown arerepresentative of three comparable experiments. [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]




Fig. 6. Effect of copper treatments on p53, p73, and ROS generation in liver. (A) Lysates ofhepatocytes from control and treated (5 mg CuCl2/kg) mice underwent Western blot analy-ses using anti-p53 antibodies. Bars represent quantitative densitometric values of theexpressed protein in samples with arbitrary units. Data shown are representative of threecomparable experiments. *Value significantly different from the control at p < 0.05.(B) Decreased expression and immunoreactivity of p53 observed in liver recovered frommice that received vehicle and 5 mg CuCl2/kg b.w., respectively, during the 28-d regimen.Magnification 5 340. (C) Lysates of hepatocytes from control and treated (5 mg CuCl2/kg)mice underwent Western blot analyses using anti-p73 antibodies. Bars represent quantita-tive densitometric values of the expressed protein in samples A, B, and C. Data shown arerepresentative of three comparable experiments. Asterisk (*) value significantly differentfrom the control at p < 0.05. (D and E) After copper treatment, the single-cell suspensionof liver was incubated with DCFH-DA fluorescent probes and analyzed by flow cytometryin a single labeling system with a 530-nm band pass filter using histogram plot. (F–J) Effectof copper treatments on CD41 and CD81 cells. Flow cytometric analysis of population per-centage of CD41 and CD81 cells in liver of copper-treated mice compared to that of con-trol. Bars represent respective population percentage of the organs. Data shown arerepresentative of three comparable experiments. *Value significantly different from thecontrol at p < 0.05. [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]




observed along with hepatomegaly (data not shown) notable

among the 5 mg CuCl2/kg mice. Based on these observations,

it can be concluded that prolonged (i.e., 28 days) exposure to

sublethal doses of CuCl2 can clearly induce extensive mor-

phological changes in the liver. Further flow cytometric anal-

yses revealed a dose-dependent increase in Sub G0/G1 phase

of the cell cycle and cell cycle arrest (i.e., arrest) in hepato-

cytes. It is quite plausible that the observed morphological

change in the liver of hosts treated for 28 days with CuCl2could primarily be a result of apoptosis and the inhibition of

lymphocyte proliferation. We therefore performed Annexin

V assay to observed apoptosis in hepatocytes. It was interest-

ing to observe that there was nearly fourfold increase in

apoptotic cells in hepatocytes with respect to their controls.

The biological pathway controlling cell fate is sequen-

cially organized at the molecular level. An oncogene-

derived protein Bcl-2 confers negative control in the

pathway of cellular suicide machinery. Although Bax-bax

homodimers act as apoptosis inducers, Bcl-2-Bax hetero-

dimer evokes a survival signal for cells (Elmore, 2007).

Therfore, decreased expression of Bcl-2 and increased

expression of Bax in liver of copper-treated mice promoted

cell death; thus clear indication of apoptosis is observed in

our study. It was thought that the main mechanism of action

of the Bcl-2 family of proteins is the regulation of cyto-

chrome c release from the mitochondria via alteration of

mitochondrial membrane permeability (Jiang and Wang,

2004). Increased Bax expression in liver of copper-treated

mice in our study ultimately leads to increased cytochrome c

release. Cytochrome c has to be folded into the mature,

heme-bound form to activate Apaf-1 and subsequent caspase

activation (Haupt et al., 2003).

Among the caspases, one of the initiator (or apical) is cas-

pase 9, because cytochrome c binds and activates Apaf-1 as

well as procaspase-9, forming an “apoptosome” (Jiang and

Wang, 2004). In this study, apoptotic events in liver of

copper-treated mice also follow this initiation event by over

expressing caspase 9 and no change in Caspase-8 expression

compared to vehicle-treated control. Further, disruption in

mitochondrial transmembrane potential, possibly indicate

involvement of mitochondrial dependent pathway in copper-

induced hepatocyte death in our study. Caspase-3 is consid-

ered to be the most important of the executioner caspases

and is activated by any of the initiator caspases like caspase

9 (Jiang and Wang, 2004). Increased expression pattern as

well as dense immunoreactivity of caspase 3 also appeared

in copper-treated liver in our study supporting previous stud-

ies (Mitra et al., 2013). Definite evidence of apoptosis leads

us to investigate the role of inflammation in copper induced

immunotoxicity. Increased expression pattern of COX-2 and

TNF-a was observed in liver in copper-treated groups, there-

fore, apoptotic events due to copper toxicity also promoted

inflammatory condition in liver for immune surveillance.

A multitude of mechanisms are used by p53 to ensure

efficient induction of apoptosis in a stage, tissue, and stress-

signal specific manner (Haupt et al., 2003, Chatterjee et al.,

2009, Mitra et al., 2013). As evidenced by the protein and

immunohistochemistry level of expression of p53 in copper-

treated liver our study revealed that in liver the apoptotic

mechanism was possibly p53 independent. Recent studies

have revealed that p53 dependent apoptotic pathway

involves ROS as their apoptotic trigger (Liu et al., 2008). It

might be that increased iNOS expression and ROS genera-

tion occurring in liver of copper-treated mice may be p53

independent also evidenced during cadmium toxicity in sple-

nic lymphocytes (Chatterjee et al., 2009). Another protein

p73 is a member of the p53 family that shows homology to

p53 in their respective trans-activation, DNA-binding, and

oligomerization domains. Both p73 transactivate p53-

regulated promoters and induce apoptosis (Sheikh and For-

nace, 2000). During this study, to check the role or involve-

ment of p73 in copper-mediated apoptosis of liver of mice, it

was revealed that p73 expression level increased as per

expression level of p53. Although p73 is predicted to medi-

ate apoptosis via mechanisms that are completely distinct

from those engaged by p53 (Sheikh and Fornace, 2000).

Finally, we have checked the population status of CD41 and

CD81 T lymphocytes in liver of copper-treated mice com-

pared to control. Comparatively, CD81 T cell population

increased non-significantly in liver, whereas significant

increase in CD41CD81 doubles positive T cells was

observed. Therefore, we can conclude that copper-induced

ROS-mediated apoptosis occurred mainly via intrinsic path-

way, which might be caspase 9, caspase 3 dependent and

p53 independent. Further study will be done to evaluate the

role of CD41 T cells and CD81 T cells function in copper-

induced immunotoxicity. For our future study, in vitroassays also will be done to delineate the pathway involved in

the apoptosis of immune cells in liver.

The Authors want to thank Department of Biotechnology

and Genetic Engineering, University of Calcutta, for their

Flow cytometry instrument facility. This work was sup-

ported by University of Calcutta for instrument support.

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Copper-induced immunotoxicity involves cell cycle arrest and cell death in the liver