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Differential effects of cadmium administration on peripheral blood granulocytes in rats
Accepted Manuscript Title: DIFFERENTIAL EFFECTS OF CADMIUM ADMINISTRATION ON PERIPHERAL BLOOD GRANULOCYTES IN RATS Author: J. Djokic M. Ninkov I. Mirkov A. Popov Aleksandrov L. Zolotarevski D. Kataranovski M. Kataranovski PII: DOI: Reference:
S1382-6689(13)00267-6 http://dx.doi.org/doi:10.1016/j.etap.2013.11.026 ENVTOX 1887
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
9-9-2013 22-11-2013 28-11-2013
Please cite this article as: Djokic, J., Ninkov, M., Mirkov, I., Aleksandrov, A.P., Zolotarevski, L., Kataranovski, D., Kataranovski, M.,DIFFERENTIAL EFFECTS OF CADMIUM ADMINISTRATION ON PERIPHERAL BLOOD GRANULOCYTES IN RATS, Environmental Toxicology and Pharmacology (2013), http://dx.doi.org/10.1016/j.etap.2013.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Effect of cadmium in vivo on peripheral blood polymorphonuclears (PMN) in rats. Neutrophil infiltration in liver and lungs 48 h following administration observed.
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Inflammation in circulation and rise in neutrophil number noted at that time point. Increase in CD11b+ granular cells and oxidative activity imply their activation.
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Decreased mRNA levels of granulocyte cytokines, but differential production noted.
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DIFFERENTIAL EFFECTS OF CADMIUM ADMINISTRATION ON PERIPHERAL BLOOD GRANULOCYTES IN RATS
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J. Djokica, M. Ninkova, I. Mirkova, A. Popov Aleksandrova, L. Zolotarevskib, D. Kataranovskia,c, M. Kataranovskia,d
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Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia b
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Institute of Pathology, Military Medical Academy, University of Belgrade, Crnotravska 17, 11000 Belgrade, Serbia c
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Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia d
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Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
Corresponding author:
Prof. Milena Kataranovski, PhD
Department of Ecology, Institute for Biological Research “Siniša Stanković” Bulevar Despota Stefana 142, 11000 Belgrade, Serbia Tel: +381 11 2078 375 Fax: +381 11 2761 433 E-mail: [email protected]
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ABSTRACT Infiltration of circulatory inflammatory cells is a common histopathological finding in target organs following cadmium administration, but there is paucity of data concerning their activity. In this study, the effects of sublethal (1 mg/kg) cadmium on peripheral blood polymorphonuclear
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(PMN) cells were examined 48 hours following administration in rats, when tissue (liver and lung) infiltration of these cells was observed. Cadmium administration resulted in systemic
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inflammatory cytokine and acute phase response with an increase in circulatory neutrophil numbers and cells that express CD11b molecules. Rise in basic aspects of oxidative activity
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including intracellular myeloperoxidase (MPO), reactive oxygen (nitroblue tetrazolium/NBT cytochemical assay) and nitrogen (Griess assay) species production was observed in PMNs from
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cadmium-administered rats. A decrease in levels of mRNA for IL-1β, TNF-α and IL-6 was noted, but production of these cytokines was affected differentially. Described effects of cadmium on PMNs add further to the understanding of inflammatory potential of this
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environmental contaminant.
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Keywords
Intraperitoneal cadmium administration; Rat; Systemic inflammatory cytokine and acute phase
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response; Peripheral blood polymorphonuclear leukocyte (PMN); Oxidative activity;
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Inflammatory cytokines (IL-1β, TNF-α, IL-6);
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1. Introduction Cadmium is among the most toxic metals in the environment. It adversely affects the number of organs and tissues including the kidneys, liver, lungs, testes, bone, brain and blood (WHO, 1992; US department of Health and Human Services, 1997). Systemic toxicity of cadmium is
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expressed primarily in liver and kidneys (Kayama et al., 1995a, 1995b) but in other distal organs, including lungs and brain, as well (Manca et al., 1991, 1994). Oxidative stress and inflammation
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are the underlying mechanisms of toxicity to these tissues (Kataranovski et al., 2009b; Kayama et al., 1995b; Manca et al., 1991, 1994; Rikans and Yamano, 2000). A common histopathological
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finding in liver, kidneys and lungs of animals following cadmium administration is the infiltration of leukocytes, primarily neutrophils, during the acute phase (Kataranovski et al.,
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2009a; Kayama et al., 1995a,1995b). Activation of neutrophils in tissue has been suggested as the main mechanism of secondary injury of liver tissue (Rikans and Yamano, 2000). The involvement of neutrophil infiltration in cadmium-induced hepatotoxicity was suggested by data
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that showed the lower intensity of damage of liver tissue following reduction of circulating neutrophil numbers by cyclophosphamide pretreatment of rats (Yamano et al., 1998). Release of
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various inflammatory mediators including reactive oxygen species and hydrolytic enzymes from
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neutrophils is proposed as underlying mechanisms responsible for liver damage (Rikans and Yamano, 2000). Pronounced neutrophil infiltration to liver was observed early (24 hours) after
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administration of single high cadmium doses (3 mg/kg b.m. to 6 mg/kg b.m.) to rats (Yamano et al., 1998), but also after longer (4 consecutive days) time period of administration of lower (2 mg/kg) cadmium doses in mice (Kayama et al., 1995a). Cadmium-induced inflammatory processes in liver progressed with time judging by gradual increase in expression of adhesion molecules (relevant for recruitment of neutrophils from circulation) in sinusoidal endothelial cells after administration of a single (3 mg/kg) cadmium dose (Moussa, 2004). Injury of lung tissue induced by cadmium increased with time as well, judging by an increase in gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) content in lung homogenates as indicators of cadmium toxicity (Manca et al., 1991) though it is not known whether cell-based mechanisms are involved. Despite the evidence of involvement of circulatory neutrophils in hepatotoxicity, the activity of these cells in settings of cadmium administration in vivo (which might be important
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for their behavior in peripheral tissues) is virtually unknown. Cadmium might affect neutrophil functionality, as suggested by investigations in vitro. Using human peripheral blood polymorphonuclear cells, generally stimulatory effects of cadmium in vitro were observed. Increase in oxidative activity (Amoruso et al., 1982; Zhong et al., 1990) and adhesive properties
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of PMNs (Hernandez and Macia, 1996) were demonstrated, though inhibition of some aspects of activity (i.e. phagocytic capacity) of cadmium-exposed human PMNs was shown as well
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(Baginski, 1985).
Our previous investigations showed increase in circulatory levels of bioactive
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inflammatory cytokines TNF-α and IL-6 in rats 24 hours following intraperitoneal administration of sublethal cadmium doses (0.5 mg/kg and 1 mg/kg) and at 2 mg of Cd per kg of body mass,
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when lethal outcome was observed in 25%-33% individuals (Kataranovski et al., 1998). Beside these soluble factors, cellular indicators of inflammation including increase in numbers of
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circulating neutrophils and their activity (activation during in vitro adhesion to noncellular matrix and migration to lungs) were observed at 1 mg/kg and 2 mg/kg (Kataranovski et al., 1998). Our later studies with sublethal cadmium doses confirmed increased activity of peripheral
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blood neutrophils (in vivo aggregability as well as priming for oxidative burst) 24 hours
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following administration of 1 mg of cadmium per kg b.m. (Kataranovski et al., 2009b) and showed activation of granulocytes recovered from lungs of administered animals (Kataranovski
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et al., 2009a; Stosic et al., 2010).
In the view of our above cited data concerning coincidence of effects of acute (24-hour) cadmium administration on circulating granulocytes and granulocyte infiltration and activation in lungs and with the knowledge that systemic cadmium toxicity to tissues progresses with time, we examined changes in peripheral blood granulocytes of rats 48 hours following administration of sublethal dose (1 mg/kg) of cadmium. Beside numerical changes and priming for respiratory burst/reactive oxygen species release (examined in our previous studies), several additional aspects of peripheral blood granulocyte activity were examined and included: expression of CD11b molecules (responsible for leukocyte extravasation and migration to peripheral tissues) and production of molecules involved in effector activity of these cells (intracellular myeloperoxidase activity and reactive nitrogen species release). As granulocytes are also a source of cytokines that might modulate local tissue inflammatory reaction (Cassatella, 1999),
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changes in inflammatory cytokine (IL-1β, TNF-α and IL-6) production were measured as well. Circulating levels of acute phase proteins (haptoglobin and fibrinogen) and inflammatory cytokines (TNF-α and IL-6) were measured to see if granulocyte activity is a part of systemic inflammatory reaction. Data were obtained that showed both stimulatory and suppressive effects
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of cadmium administration on peripheral blood granulocyte activity in rats.
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2. Materials and methods 2.1 Chemicals
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Cadmium chloride (Serva, Feinbiochemica, Germany), lypopolysaccharide (LPS), three-(4,5dimethyl-thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT), N-(1-naphtyl) ethylenediamine
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dihydrochloride, sulfanilamide (p-aminobenzenesulfonoamide), hexadecyltrimethylammonium bromide (HTAB), o-dianisidine dihydrochloride, myeloperoxidase (MPO) and phorbol 12myristate 13-acetate (PMA) (all purchased from Sigma, Sigma Aldrich., St. Louis, MO, USA),
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sodium nitrite (Fluka Chemika, Switzerland) and hydrogen peroxide (H2O2) from Zorka Farma, Sabac (Serbia) were used in experiments. LPS, MTT and PMA were dissolved in culture
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medium and phosphate buffered saline (PBS, pH 7.2), respectively. All solutions for cell culture
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experiments were prepared under sterile conditions and sterile filtered (Minisart, pore size 0.20 µm; Sartorius Stedim Biotech, Goettingen, Germany) before use. Dextran T-500 (Sigma Aldrich,
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St. Louis, MO, USA) 6 % solution was prepared in apyrogenic saline and autoclaved (1100 C). Culture medium RPMI-1640 supplemented with 2 mM glutamine (PAA laboratories, Austria), 20 μg/mL gentamycine (Galenika a.d., Serbia), 5% (v/v) heat inactivated fetal calf serum (PAA laboratories, Austria) was used in cell culture experiments. Monoclonal antibody OX-42 (mouse anti-rat CD11b/CD11c) and FITC-conjugated F(ab')2 goat anti-mouse IgG were purchased from Serotec Ltd, Bicester, UK. Lysis buffer (eBioscience Inc., San Diego, CA, USA) was used for red blood cell lysis. OptiPrep (Nycomed AS, Oslo, Norway) was used for peripheral blood granulocyte isolation. 2.2 Animals and cadmium treatment Animal treatment was carried out in adherence to the guidelines of the Ethical Committee of the Institute for Biological Research "Siniša Stanković" (IBISS), Belgrade, Serbia. Male Dark Agouti (DA) rats 12-14 weeks old, weighing 200 - 240 g were used in experiments. Animals
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were conventionally housed at IBISS, in a controlled environment (24°C with a 60% relative humidity and a 12-hr light:dark cycle) and had access to standard rodent chow and water ad libitum throughout the study. Four to six animals were assigned to each treatment group in at least two independent experiments. Sterile filtered cadmium chloride prepared in pyrogen-free
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saline was administered i.p. in a dosing volume of 0.5 mL at a concentration at which animals received 1 mg of cadmium/kg body mass (b.m.). Pyrogen-free saline was administered as
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control. All measurements were carried out 48 hours following cadmium administration in animals anesthetized by i.p. 40 mg/kg b.m. of thiopental sodium (Rotexmedica, Tritau,
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Germany). Tissue specimens were processed on ice and were subsequently subjected to freezing (for cadmium burden measurements), or to functional studies or preparation for histological
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analysis. 2.3 Cadmium determination
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Cadmium content in tissues and peripheral blood was determined by atomic absorption spectrometry (AAS; SpectrAA-50, Varian, Inc, Palo Alto, Ca, USA) after ashing the lyophilized
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tissue or blood samples at 5500C and dissolving the ash in 1M HCl. The concentrations were
2.4 Clinical biochemistry
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expressed as μmol of Cd per kg of wet tissue or blood weight ± SD.
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Serum aspartate amino transferase (AST) and alanine amino transferase (ALT) were determined with an autoanalyzer (Ciba Corning Express, Oberline, OH, USA) using commercially available reagents. Plasma fibrinogen was measured by Siemens-Dade Behring-BCT analyzer using Multifibren U test for quantitative determination in plasma. Haptoglobin and albumin were measured in serum by BN (Dade Behring) immunochemical system for human blood proteins measured by Siemens BNII (Dade Behring) BCT analyzer. Cross-reactivity with rat blood proteins was checked using serum obtained from turpentine-induced inflammation in rat, known inflammatory model of acute phase reaction in these animals (Giffen et al., 2003). Relative plasma or serum protein levels are expressed as percent changes with respect to the mean value obtained in control animals (considered as 100 %). 2.5 Histology
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Liver and lungs were cut out and immediately fixed in 4% formaldehyde (pH 6.9). After processing, tissue was embedded in paraffin wax for sectioning at 5 m. Hematoxylin and eosin (H&E)-stained histology slides were subsequently analyzed by a certified histopathologist in a
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blinded manner using a Coolscope digital light microscope (Nikon Co, Tokyo, Japan). 2.6 Lung tissue myeloperoxidase (MPO) activity
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The myeloperoxidase (MPO) activity was measured in homogenates of the lung tissue as described (Goldblum et al., 1985). Lungs were removed, cleared of blood and homogenized by
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IKA T18 Basic Homogenizer (IKA Works Inc, Wilmington NC, USA) on the ice in the phosphate buffered saline contaning 1 mM phenylmethanesulfonyl fluoride (PMSF). To one of
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homogenate
two
volumes
of
three
times
concentrated
an
volume
hexadecyltrimethylammonium bromide (HTAB) in potassium phosphate buffer (50 mM, pH 6.5) were added to achieve the final concentration of 0.5 % HTAB. The homogenates were subjected
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to three cycles of freezing and thawing, sonicated for 15-20 sec (Bandelin electronic, UW 2070, Berlin, Germany) at 50 % of maximum intensity amplitude and centrifuged (at 1000 x g at 4°C in
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cooling centrifuge). The MPO was evaluated by the addition of 33 μL of homogenate supernatant to 966 μL of substrate solution (0.167 mg/mL o-dianisidine dihydrochloride and
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0.0005% H2O2 in 50mM potassium phosphate buffer, pH 6.0). The absorbance was read at 450 nm at three-minute intervals up to ten minutes against the standard of myeloperoxidase. The
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values were expressed as MPO units per gram of the lung tissue. 2.7 Peripheral blood and bone marrow leukocyte counts Total and differential peripheral blood leukocyte counts were determined automatically using Siemens ADVIA 120 flow cytometer (Terytown, N.Y., USA). Total bone marrow counts were determined using an improved Neubauer hemocytometer. Differential bone marrow cell counts were determined by differentiating at least 1000 cells from air-dried bone marrow smears stained according to the May Grünwald-Giemsa (MGG) protocol. 2.8 Flow cytometry Adhesion molecule CD11b expression was achieved by flow cytometry analysis of leukocytes from whole-blood following lysis of erythrocytes. Obtained peripheral blood cells were
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incubated on ice with mouse anti-rat CD11b antibody. After 30-min incubation, cells were washed twice and stained with FITC-conjugated F(ab')2 goat anti-mouse IgG for 30 min. After washing, the cells were fixed and assayed for fluorescence intensity on an CyFLOW SPACE (Partec, Munich, Germany). Cells in histogram with position characteristic for granular cells
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were analyzed for percentages of CD11b positive cells and mean fluorescence intensity (MFI).
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2.9 Peripheral blood granulocyte isolation
Cells were isolated from heparinized blood by dextran sedimentation followed by centrifugation
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of leukocyte-rich plasma over OptiPrep (Nycomed AS, Oslo, Norway) density separation medium. Polymorphonuclear cells (granulocytes) were obtained from the pellet fraction following lysis of erythrocytes with lysis bufer. The purity of cells, determined morphologically
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after MGG staining, was ≥ 95 %.
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2.10 MTT assay for granulocyte viability and survival
A quantitative colorimetric assay described for human neutrophils (Oez et al., 1990) in which tetrazolium salt MTT is metabolically reduced by cell’s mitochondrial dehydrogenases
to
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coloured end product, formazan, was used as a measure of metabolical viability of freshly
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isolated peripheral blood leukocytes as well as of survival of cells cultured for 24 hours. Cells were added to wells of a 96-well plate (0.25 × 106 cells/well) and incubated with 500 μg/mL of
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MTT (added immediately or following 24 hours in culture) for 3 hours. Formazan produced by cells was dissolved by overnight incubation in 10% SDS-0.01 N HCl. Absorbance was measured spectrophotometrically at 540 nm/650 nm, by an ELISA 96-well plate reader (GRD, Roma, Italy).
2.11 Peripheral blood granulocyte intracellular myeloperoxidase (MPO) Myeloperoxidase activity was assesssed on the basis of the oxidation of o-dianisidine dihydrochloride by cells (Bozeman et al., 1990). Briefly, 33 μL of PMN cell lysate was added to 966 μL of substrate solution (0.167 mg/mL o-dianisidine dihydrochloride and 0.0005 % H2O2 in 50 mM potassium phosphate buffer, pH 6.0). Absorbance was read at 450 nm (ELISA 96-well plate reader, GRD, Rome, Italy) at three-minute intervals up to ten minutes against the standard of myeloperoxidase.
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2.12 Peripheral blood granulocyte respiratory burst Cytochemical assay for the respiratory burst (that measures intracellular reduction of nitroblue tetrazolium, NBT) (Choi et al., 2006) was employed. Peripheral blood PMNs were cultured (2 ×
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105 cells/well in round bottomed 96-well plates) with NBT (spontaneous NBT reduction) or NBT and (100 ng/mL) PMA (stimulated NBT reduction) for 30 minutes. Cell-produced formazan was dissolved with 10% SDS - 0.01N HCl spectrophotometrically and absorbance was
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measured at 540 nm/670 nm by an ELISA 96-well plate reader (GRD, Rome, Italy).
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2.13 Nitric oxide production by peripheral blood polymorphonuclear cells
The concentration of the stable NO oxidation product, nitrite, was measured (as an indicator of
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NO formation) by Griess reaction (Hibbs et al., 1988) in medium conditioned for 48 hours by peripheral blood PMNs freshly isolated after cessation of cadmium treatment (CM). Cells were cultured at 5 × 106/mL in 24-well plates in medium (spontaneous production) or in the presence
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of 100 ng/mL, LPS (LPS-stimulated production). Aliquots (50 μL) of 48-hour CM were mixed with an equal volume of Griess reagent (a mixture of 0.1 % naphtylenediamine dihydrochloride
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in water and 1 % sulphanilamide in 5 % phosphoric acid) and incubated for 10 min at room
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temperature. The amount of nitrite was calculated by the reference to a standard curve
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constructed with known amounts of sodium nitrite. 2.14 Cytokine determination by ELISA Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations were determined in supernatant of cells (5 × 105 cells/well of 96-well plate) cultured for 48 hours in medium solely (spontaneous production) or in the presence of 100 ng/mL of LPS (stimulated production). Enzyme-linked immunosorbent assays (ELISA) for rat TNF-α (eBioscience Inc., San Diego, CA, USA) and rat IL-6 and IL-1β (R&D systems, Minneapolis, USA) were used according to manufacturer instructions. Cytokine titer was calculated by the reference to a standard curve constructed with known amounts of recombinant IL-1β, TNF-α or IL-6. 2.15 Reverse transcription - real time polymerase chain reaction (RT-PCR) Total RNA was isolated from the polymorphonuclear cells or granulocytes immediately after isolation with an RNA Isolator (Metabion, Martinsried, Germany) following the manufacturer’s
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instructions. The isolated RNA was reverse transcribed using random hexamer primers and MMLV (Moloney Murine Leukemia Virus) reverse transcriptase, according to manufacturer's instructions (Fermentas, Vilnius, Lithuania). Prepared cDNAs were amplified by using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the
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recommendations of the manufacturer in a total volume of 20 μL in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Thermocycler conditions comprised an initial
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step at 50°C for 5 minutes, followed by a step at 95°C for 10 minutes and subsequent 2-step PCR program at 95°C for 15 seconds and 60°C for 60 seconds for 40 cycles. The PCR primers were
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as follows: β-actin forward 5’-CCC TGG CTC CTA GCA CCA T-3’, β-actin backward 5’-GAG CCA CCA ATC CAC ACA GA-3’; IL-1β forward 5'-CAC CTC TCA AGC AGA GCA-3', IL1β backward 5'-GGG TTC CAT GGT GAA GTC AAC-3'; TNF-α forward 5’-TCG AGT GAC
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AAG CCC GTA GC-3’, TNF-α backward: 5’-CTC AGC CAC TCC AGC TGC TC-3’; IL-6 forward 5’-GCC CTT CAG GAA CAG CTA TGA-3’. IL-6 backward: 5’-TGT CAA CAA CAT
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CAG TCC CAA G-3’ Accumulation of PCR products was detected in real time and the results were analyzed with 7500 System Software (Applied Biosystems) and calculated as 2−dCt, where
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(-actin).
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dCt was difference between treshold cycle (Ct) values of specific gene and endogenous control
2.16 Data display and statistical analysis
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Results are expressed as means ± standard deviation (S.D.). Statistical analysis was performed by using STATISTICA 7.0 (StatSoft Inc., Tulsa, Oklahoma, USA). Statistical significance was defined by Mann-Whitney U test. P -values less than 0.05 were considered significant. 3. Results
3.1 Cadmium disposition and systemic effects No differences in animal body mass between group (N = 14) that received 1 mg of cadmium per kg (217 ± 13.38 g) and controls (N = 14) (219 ± 6.50 g) were observed. Absolute amounts of cadmium (μmol/kg wet weight) increased in all tissues examined and tissue burden was higher in animals which received cadmium compared to controls (Table 1). Increase (P < 0.001) in both AST (373 ± 96 U/mL, N = 14 compared to 203 ± 60 U/mL in controls, N = 14) and ALT (140 ± 36 U/mL, N = 14 compared to 82 ± 24 U/mL, N = 14) in rats administered with cadmium was
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observed. Histological analysis demonstrated significant presence of neutrophils in liver (Fig. 1A). Infiltration of both mononuclear as well as polymorphonuclear cells in lungs of rats that were administered with cadmium was observed (Fig. 1B) and increased (P < 0.05) myeloperoxidase (MPO) activity was noted in lungs of these animals (4.57 ± 2.34 U/g wet lung
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weight, N = 10 compared to 1.44 ± 1.02 U/g of lungs from control animals, N = 10).
Cadmium administration was associated with an increase in TNF-α concentration in
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plasma (Fig. 2A). Rise in serum levels of haptoglobin and fibrinogen and a decrease in albumin
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levels were observed (Fig. 2B).
3.2 Peripheral blood leukocyte numbers following cadmium administration
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No changes in total cell or differential leukocyte numbers were observed following cadmium administration with significantly higher relative numbers of neutrophils and lower relative numbers of lymphocytes (Table 2). Analysis of bone marrow smears revealed similar numbers of
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metamyelocytes (7.9 ± 5.5 × 103 and 7.7 ± 4.2 × 103), granulocytes (36.3 ± 8.5 × 103 and 35.4 ± 6.7 × 103) and lymphocyte numbers (43.7 ± 7.8 × 103 and 44.8 ± 7.1 × 103) in cadmium-treated
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(N = 14) and control animals (N = 14), respectively.
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3.3 Peripheral blood granular cell expression of CD11b following cadmium administration
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Whole-blood leukocyte flow cytometry analysis revealed an increase in numbers of granular cells that express CD11b molecules in animals that received cadmium (Table 3). Increased density of surface expression of these molecules was observed as well. 3.4 Peripheral blood polymorphonuclear leukocyte viability and survival following cadmium administration
There were no differences between cadmium-treated and control animals in MTT reduction by freshly isolated peripheral blood granulocytes (Fig. 3). Lower (compared to values in freshly isolated cells) capacity of MTT reduction was noted following 24-hour culture by cells from both animal groups but without difference between them. 3.5 The effect of cadmium administration on peripheral blood polymorphonuclear leukocyte oxidative activity
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Cadmium administration resulted in increased levels of intracellular MPO activity in granulocytes from group that received cadmium (Fig. 4A). Granulocytes of both animal groups showed similar levels of spontaneous NBT reduction capacity (Fig. 4B) and responded to stimulation with PMA (higher levels of stimulated compared to spontaneous activity), but the
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levels were significantly higher in cells from rats treated with cadmium (Fig. 4B). There were no differences between groups in spontaneous production of NO. Stimulation with LPS resulted in
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an increase (in comparison to spontaneous activity) of NO release by cells from both animal groups, but higher levels were noted in cultures of granulocytes from rats treated with cadmium
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(Fig. 4C).
3.6 The effect of cadmium administration on peripheral blood polymorphonuclear leukocyte
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cytokines
Measurements of cytokine production by peripheral blood polymorphonuclear cells (Fig. 5A)
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revealed differential effects of cadmium administration when spontaneous cytokine release was measured. While there was no effect on IL-1β and IL-6 release (similar levels in both groups), a
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decrease of spontaneous TNF-α production was observed. When production in response to stimulation with LPS was measured, similar levels of IL-1β produced were observed in both
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groups, while there was lower (compared to control group) production of TNF-α. Although no differences between cadmium and control groups were noted concerning the levels of LPS-
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stimulated IL-6 production, lack of responsiveness to LPS could be observed in cultures of neutrophils from cadmium group.
To correlate granulocyte cytokine production with gene expression in vivo, cytokine mRNA levels were analyzed next (Fig. 5B). As revealed by RT-PCR, a significant decrease of mRNA levels was observed for all cytokines examined (Fig. 5B). 4. Discussion
In this study, the effect of acute cadmium administration on peripheral blood polymorphonuclear cells in rats was examined by measuring changes in numbers of these leukocytes, expression of CD11b molecules relevant for tissue infiltration, their oxidative activity as well as changes in inflammatory cytokine (IL-1β, TNF-α and IL-6) production. Data were obtained that showed differential effects of cadmium administration on various aspects of PMN activity.
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Infiltration of neutrophils in liver is in line with numerous data that showed presence of inflammatory cell infiltration in liver in acute intoxication with cadmium (Kayama et al., 1995a; Rikans and Yamano, 2000; Yamano et al., 1998). Increase in serum levels of liver enzymes, particularly ALT (the enzyme with highest amounts in liver), might have resulted from injurious
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mechanisms of these cells as proposed (Rikans and Yamano, 2000). Presence of neutrophils in lung tissue 48 hours following cadmium administration is in line with our previous data that
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demonstrated infiltration of neutrophils in lungs 24 hours following cadmium administration (Kataranovski et al., 2009a) and show that these cells continue to migrate into this tissue. Owing
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to their effector activities neutrophils can damage lung tissue and their presence in areas of pneumocyte desquamation support such assumption. Lung neutrophil infiltration demonstrated in this study might, however, reflect their recruitment into injured tissue where these cells can take
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part in local healing/reparatory program governed by infiltrating mononuclear cells.
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In line with our previous data that showed that cadmium administration is associated with systemic cytokine response 24 hours later (Kataranovski et al., 1998), increase in plasma levels of TNF-α observed in this study demonstrates that systemic inflammation is evident later as well.
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Rise in circulating levels of TNF-α might have resulted from injured tissues. In this regard,
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tissues of liver and kidneys were shown to produce TNF-α in rats administered with cadmium, respectively (Kayama et al., 1995a, 1995b). Circulating TNF-α might influence remote cell and
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tissue behavior. Relative increase in levels of haptoglobin and fibrinogen, which synthesis in liver cells depends, among other cytokines, on TNF-α (Baumann et al., 1990) illustrate such an influence. A decrease in albumin levels is in line with data that showed that rise of positive acute phase reactants (i.e. haptoglobin and fibrinogen) in rats is associated with a decrease in albumin levels (Mayot et al., 2008).
Shift in relative leukocyte numbers in favor of neutrophils implies proinflammatory effects of cadmium as these cells are known cellular component of inflammation at systemic level (Schwartz and Weiss, 1991). In line with our previous data that showed changes in peripheral blood granulocyte counts 24 hours following cadmium administration to rats (Kataranovski et al., 1998), this study demonstrated protracted effect of cadmium administration on these cells. Increase numbers of peripheral blood neutrophils does not seem to rely on bone marrow, as no changes were noted in either granulocyte or metamyelocyte numbers. Possibly, it
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might be ascribed to peripheral blood neutrophil redistribution. Lungs harbor intravascular reservoirs of leukocytes, predominantly neutrophils (termed as ‘marginated pool’) that constantly exchange with circulating neutrophils both under basal and inflammatory conditions (Kuebler,
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2005). Increased numbers of granular cells that express CD11b molecules (alpha chain of β2 integrin receptors αMβ2), as well as increase in the density of their expression on cell surface,
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point out effects of cadmium administration on activation of these cells (Arnaout, 1990). Observed changes might influence cell’s function, as these molecules are involved in neutrophil
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extravasation and migration to peripheral tissue (Arnaout, 1990) and for their functional activation (Abram and Lowell, 2009). Recent data showed that neutrophils use these molecules
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in the process of recruitment to liver in inflammatory conditions (McDonald and Kubes, 2012). Expression of CD11b molecules on peripheral blood granular cells following cadmium
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administration might bear relevance for neutrophils migration to liver, in the view of data that showed an increase in the expression of adhesion molecule ICAM-1, a major ligand for CD11b (Arnaout, 1990) on sinusoidal endothelial cells in liver of rats administered with cadmium
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(Moussa, 2004). This process might be enhanced by cadmium itself, as stimulation of CD11b-
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based adherence of human peripheral blood polymorhonuclear cells in vitro was noted in the presence of 1 μM of cadmium (Hernandez and Macia, 1996) (the concentration that correspond
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to those measured in the blood of rats in the present study). Increase in expression of CD11b on peripheral blood leukocytes might have resulted from influences of systemic microenvironment, TNF-α in particular, as this cytokine is shown to be a priming factor for neutrophil adhesion and migration (Montecucco et al., 2008; Nathan, 2006). Cadmium administration resulted not only in quantitative changes in peripheral blood granulocytes but in qualitative changes as well. Increase in intracellular MPO content, which is along with phagocyte oxidase a source of oxidant activity in phagocytes (Finkel, 2003) stresses the effect of cadmium administration on peripheral blood granulocyte oxidative activities. Significantly higher responsiveness of granulocytes from rats administered with cadmium to activation of respiratory burst by granulocyte activator PMA, reflects their priming, state, i.e. the state in which the functional responses to an activating stimulus are potentiated/amplified by a prior exposure to a priming stimuli (Hallet and Lloyds, 1995). The rise of granulocyte NO
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production in response to LPS, reflects their priming state as well. Increase in oxidative activity of PMNs from cadmium administered rats is in line with our previous data that showed priming effect of cadmium on granulocyte respiratory burst 24 hours following administration (Stosic et al., 2010) and show that it is evident later as well. Increase in intracellular MPO content in
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peripheral blood granulocyte as well as their priming for respiratory burst might rely on the increase of TNF-α in plasma, as it is known priming factor for oxidative activities of neutrophils
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(Berkow and Dodson, 1988; Klebanoff et al., 1986). The effect of cadmium itself on oxidative granulocyte activities should not be excluded, as it was shown that 1 μM of cadmium stimulate
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production of H2O2 by human peripheral blood polymorphonuclear cells (Zhong et al., 1990). In contrast to increase in oxidative activity of peripheral blood granulocytes, cadmium
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administration resulted in a decrease in mRNA for IL-1β, TNF-α and IL-6. Cytokine genes are target for inhibition by cadmium in vitro as shown by data for human mononuclear cell’s IL-1β
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(Marth et al., 2001; Theocharis et al., 1994) and TNF-α (Boscolo et al., 2005; Marth et al., 2001), but there are no data, as far as we know, concerning the effect of cadmium administration on peripheral blood granulocytes. Down-regulation of genes for TNF-α and IL-6 was responsible
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for a decrease in spontaneous and stimulated production of TNF-α and, probably, for negligible
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responsiveness of IL-6 production to LPS stimulation. Lack of the effect on spontaneous production of IL-1β and IL-6 might be explained by the effect of cell culture, as adherence was
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shown to stimulate cytokine mRNA expression during culture (Haskill et al., 1988). Cadmiuminduced suppression of responsiveness of granulocyte TNF-α and IL-6 production to LPS stimulation might reflect an attempt of host to restrain the activity of these cells. Cytokines, such as TNF-α, a known potent stimulator of (deleterious) effector activities of neutrophils (and other cells) and as mediator that can amplify tissue inflammation, might be especially important target for down regulation. In corroboration, in a separate experiment (unpublished) we have noted a decrease (P < 0.05) in both spontaneous (37.5 ± 22.4 pg/mL) as well as LPS-stimulated production (60.8 ± 34.2 pg/mL) of TNF-α by peripheral blood granulocytes 24 hours following administration of 1 mg/kg of cadmium in rats (N = 4) , compared to 185.8 ± 114.15 pg/mL and 181.7 ± 9.4 pg/mL (for spontaneous and LPS-stimulated production, respectively) in controls (N = 4).
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In conclusion, we showed in this study differential effects of cadmium administration on peripheral blood granulocyte activity in rats. Data that illustrated granulocyte activation and priming for oxidative activity, add further to the knowledge of proinflammatory activities of cadmium. Down-regulation of inflammatory cytokine gene expression as well as differential
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effects of cadmium administration on their production is novel finding and point out the manifold relevance of polymorphonuclear cells in cadmium-induced inflammatory and
Conflict of interest statement
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The authors declare that there are no conflicts of interest.
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immunomodulatory processes.
Acknowledgments
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This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant #173039. The authors thank Vesna Subota, M.Sc. for help in
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some experiments and Veljko Blagojevic, M.Sc. for help in the processing of manuscript.
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33. Moussa, S.A., 2004. Expression of adhesion molecules during cadmium hepatotoxicity. Life
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42. Yamano, T., Shimizu, M., Noda, T., 1998. Age-related change in cadmium-induced hepatotoxicity in Wistar rats: role of Kupffer cells and neutrophils. Toxicol. Appl. Pharmacol. 151, 9-15. 43. Zhong, Z., Troll, W., Koenig, K.L., Frenkel, K., 1990. Carcinogenic sulfide salts of nickel
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and cadmium induce H2O2 formation by human polymorphonuclear leukocytes. Cancer Res.
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50, 7564-7570
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Figure captions Fig. 1 Microscopical appearance of (A) liver and (B) lungs 48 hours following cadmium administration. (A) Neutrophils (arrows) in liver sinusoids. Two neutrophils adhere to acidophyllic necrotic binuclear hepatocyte. Intravascular accumulation of neutrophils (insert).
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(B) Perivascular predominantly mononuclear leukocyte infiltration (arrows). Pneumocyte desquamation into alveolar spaces (star). Pneumocytes (star) and neutrophils (arrowheads) that
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adhere to alveolar wall (upper insert). Intravascular accumulation of neutrophils (lower insert).
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Fig. 2 Cytokine and acute phase protein concentrations in circulation. (A) Plasma TNF-α and IL6, (B) Serum fibrinogen, haptoglobin and albumin levels. Data are expressed as mean values ± and ** P < 0.01 vs controls (cadmium 0 mg/kg).
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S.D. from two experiments each with four to six animals per group. Significance at * P < 0.05;
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Fig. 3 Peripheral blood granulocyte viability and survival. Data are expressed as mean values ± S.D. from two experiments each with four to six animals per group. Significance at # P < 0.05 vs
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MTT reduction by freshly isolated granulocytes (0h).
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Fig. 4 Peripheral blood granulocyte oxidative activity. (A) Intracellular MPO activity (B) Spontaneous and PMA-stimulated NBT reduction (C) Spontaneous and LPS-stimulated NO
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production. Data are expressed as mean values ± S.D. from three experiments each with four to six animals per group. Significance at * P < 0.05 and ** P < 0.01 vs controls (cadmium 0 mg/kg). Significance at # P < 0.05 and ## P < 0.01 vs spontaneous production. Fig. 5 Peripheral blood granulocyte inflammatory cytokine (A) production and (B) gene expression. (A) Spontaneous and LPS-stimulated production of IL-1β, TNF-α and IL-6 (B) mRNA expression for IL-1β, TNF-α and IL-6. Data are expressed as mean values ± S.D. from two to three experiments, each with four to six animals per group. Data for mRNA are expressed as percentages of mRNA in PMN from cadmium administered rats relative to mRNA in cells from control rats. Significance at * P < 0.05; ** P < 0.01 and *** P < 0.001 vs controls (cadmium 0 mg/kg). Significance at # P < 0.05 and ### P < 0.001 vs spontaneous production.
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Blood
0
0.09 ± 0.09
0.05 ± 0.02
0.05 ± 0.01
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201.3 ± 21.4*
4.7 ± 0.7*
1.1 ± 0.2*
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Values are given as mean values ± S.D. from 8 animals per group.
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Table 1. Cadmium liver, lungs and blood content Cadmium concentration (μmol/kg wet weight) Cadmium dose Liver Lungs (mg/kg)
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Significance at * P < 0.05 vs controls (cadmium 0 mg/kg)
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37.3 ± 5.7 3.7 ± 1.2
55.1 ± 15.2* 10.0 ± 8.3
Lymphocytes
(%) (×109/L)
58.8 ± 5.8 5.9 ± 2.1
40.5 ± 16.1* 5.5 ± 0.8
Monocytes
(%) (×109/L)
2.6 ± 0.9 0.3 ± 0.1
2.6 ± 0.9 0.4 ± 0.2
Eosinophils
(%) (×109/L)
0.8 ± 0.4 0.1 ± 0.03
0.7 ± 0.3 0.1 ± 0.01
Basophils
(%) (×109/L)
0.4 ± 0.1 0.04 ± 0.02
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(%) (×109/L)
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Neutrophils
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Table 2. Total and differential peripheral blood cell counts Parameter Cadmium dose (mg/kg) 0 1 White blood cells (×109/L) 10.0 ± 3.4 16.3 ± 9.1
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0.3 ± 0.1 0.06 ± 0.06
Data are expressed as mean values ± S.D. from two experiments each with four to six animals per group.
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Significance at * P < 0.05 vs controls (cadmium 0 mg/kg)
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Data are expressed as mean values ± S.D. from two experiments each with four to six animals.
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Table 3. CD11b expression in peripheral blood granular cell population Cadmium dose (mg/kg) 0 1 Number of 91.8 ± 2.5 97.8 ± 3.6** positive cells (%) Mean Fluorescence 2703.0 ± 543.0 3651.0 ± 191.0* Intensity (MFI)
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Significance at * P < 0.05; and ** P < 0.01 vs controls (cadmium 0 mg/kg)
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S1382-6689(13)00267-6 http://dx.doi.org/doi:10.1016/j.etap.2013.11.026 ENVTOX 1887
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
9-9-2013 22-11-2013 28-11-2013
Please cite this article as: Djokic, J., Ninkov, M., Mirkov, I., Aleksandrov, A.P., Zolotarevski, L., Kataranovski, D., Kataranovski, M.,DIFFERENTIAL EFFECTS OF CADMIUM ADMINISTRATION ON PERIPHERAL BLOOD GRANULOCYTES IN RATS, Environmental Toxicology and Pharmacology (2013), http://dx.doi.org/10.1016/j.etap.2013.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Effect of cadmium in vivo on peripheral blood polymorphonuclears (PMN) in rats. Neutrophil infiltration in liver and lungs 48 h following administration observed.
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Inflammation in circulation and rise in neutrophil number noted at that time point. Increase in CD11b+ granular cells and oxidative activity imply their activation.
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Decreased mRNA levels of granulocyte cytokines, but differential production noted.
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DIFFERENTIAL EFFECTS OF CADMIUM ADMINISTRATION ON PERIPHERAL BLOOD GRANULOCYTES IN RATS
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J. Djokica, M. Ninkova, I. Mirkova, A. Popov Aleksandrova, L. Zolotarevskib, D. Kataranovskia,c, M. Kataranovskia,d
a
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Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia b
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Institute of Pathology, Military Medical Academy, University of Belgrade, Crnotravska 17, 11000 Belgrade, Serbia c
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Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia d
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Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
Corresponding author:
Prof. Milena Kataranovski, PhD
Department of Ecology, Institute for Biological Research “Siniša Stanković” Bulevar Despota Stefana 142, 11000 Belgrade, Serbia Tel: +381 11 2078 375 Fax: +381 11 2761 433 E-mail: [email protected]
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ABSTRACT Infiltration of circulatory inflammatory cells is a common histopathological finding in target organs following cadmium administration, but there is paucity of data concerning their activity. In this study, the effects of sublethal (1 mg/kg) cadmium on peripheral blood polymorphonuclear
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(PMN) cells were examined 48 hours following administration in rats, when tissue (liver and lung) infiltration of these cells was observed. Cadmium administration resulted in systemic
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inflammatory cytokine and acute phase response with an increase in circulatory neutrophil numbers and cells that express CD11b molecules. Rise in basic aspects of oxidative activity
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including intracellular myeloperoxidase (MPO), reactive oxygen (nitroblue tetrazolium/NBT cytochemical assay) and nitrogen (Griess assay) species production was observed in PMNs from
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cadmium-administered rats. A decrease in levels of mRNA for IL-1β, TNF-α and IL-6 was noted, but production of these cytokines was affected differentially. Described effects of cadmium on PMNs add further to the understanding of inflammatory potential of this
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environmental contaminant.
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Keywords
Intraperitoneal cadmium administration; Rat; Systemic inflammatory cytokine and acute phase
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response; Peripheral blood polymorphonuclear leukocyte (PMN); Oxidative activity;
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Inflammatory cytokines (IL-1β, TNF-α, IL-6);
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1. Introduction Cadmium is among the most toxic metals in the environment. It adversely affects the number of organs and tissues including the kidneys, liver, lungs, testes, bone, brain and blood (WHO, 1992; US department of Health and Human Services, 1997). Systemic toxicity of cadmium is
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expressed primarily in liver and kidneys (Kayama et al., 1995a, 1995b) but in other distal organs, including lungs and brain, as well (Manca et al., 1991, 1994). Oxidative stress and inflammation
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are the underlying mechanisms of toxicity to these tissues (Kataranovski et al., 2009b; Kayama et al., 1995b; Manca et al., 1991, 1994; Rikans and Yamano, 2000). A common histopathological
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finding in liver, kidneys and lungs of animals following cadmium administration is the infiltration of leukocytes, primarily neutrophils, during the acute phase (Kataranovski et al.,
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2009a; Kayama et al., 1995a,1995b). Activation of neutrophils in tissue has been suggested as the main mechanism of secondary injury of liver tissue (Rikans and Yamano, 2000). The involvement of neutrophil infiltration in cadmium-induced hepatotoxicity was suggested by data
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that showed the lower intensity of damage of liver tissue following reduction of circulating neutrophil numbers by cyclophosphamide pretreatment of rats (Yamano et al., 1998). Release of
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various inflammatory mediators including reactive oxygen species and hydrolytic enzymes from
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neutrophils is proposed as underlying mechanisms responsible for liver damage (Rikans and Yamano, 2000). Pronounced neutrophil infiltration to liver was observed early (24 hours) after
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administration of single high cadmium doses (3 mg/kg b.m. to 6 mg/kg b.m.) to rats (Yamano et al., 1998), but also after longer (4 consecutive days) time period of administration of lower (2 mg/kg) cadmium doses in mice (Kayama et al., 1995a). Cadmium-induced inflammatory processes in liver progressed with time judging by gradual increase in expression of adhesion molecules (relevant for recruitment of neutrophils from circulation) in sinusoidal endothelial cells after administration of a single (3 mg/kg) cadmium dose (Moussa, 2004). Injury of lung tissue induced by cadmium increased with time as well, judging by an increase in gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) content in lung homogenates as indicators of cadmium toxicity (Manca et al., 1991) though it is not known whether cell-based mechanisms are involved. Despite the evidence of involvement of circulatory neutrophils in hepatotoxicity, the activity of these cells in settings of cadmium administration in vivo (which might be important
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for their behavior in peripheral tissues) is virtually unknown. Cadmium might affect neutrophil functionality, as suggested by investigations in vitro. Using human peripheral blood polymorphonuclear cells, generally stimulatory effects of cadmium in vitro were observed. Increase in oxidative activity (Amoruso et al., 1982; Zhong et al., 1990) and adhesive properties
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of PMNs (Hernandez and Macia, 1996) were demonstrated, though inhibition of some aspects of activity (i.e. phagocytic capacity) of cadmium-exposed human PMNs was shown as well
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(Baginski, 1985).
Our previous investigations showed increase in circulatory levels of bioactive
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inflammatory cytokines TNF-α and IL-6 in rats 24 hours following intraperitoneal administration of sublethal cadmium doses (0.5 mg/kg and 1 mg/kg) and at 2 mg of Cd per kg of body mass,
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when lethal outcome was observed in 25%-33% individuals (Kataranovski et al., 1998). Beside these soluble factors, cellular indicators of inflammation including increase in numbers of
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circulating neutrophils and their activity (activation during in vitro adhesion to noncellular matrix and migration to lungs) were observed at 1 mg/kg and 2 mg/kg (Kataranovski et al., 1998). Our later studies with sublethal cadmium doses confirmed increased activity of peripheral
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blood neutrophils (in vivo aggregability as well as priming for oxidative burst) 24 hours
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following administration of 1 mg of cadmium per kg b.m. (Kataranovski et al., 2009b) and showed activation of granulocytes recovered from lungs of administered animals (Kataranovski
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et al., 2009a; Stosic et al., 2010).
In the view of our above cited data concerning coincidence of effects of acute (24-hour) cadmium administration on circulating granulocytes and granulocyte infiltration and activation in lungs and with the knowledge that systemic cadmium toxicity to tissues progresses with time, we examined changes in peripheral blood granulocytes of rats 48 hours following administration of sublethal dose (1 mg/kg) of cadmium. Beside numerical changes and priming for respiratory burst/reactive oxygen species release (examined in our previous studies), several additional aspects of peripheral blood granulocyte activity were examined and included: expression of CD11b molecules (responsible for leukocyte extravasation and migration to peripheral tissues) and production of molecules involved in effector activity of these cells (intracellular myeloperoxidase activity and reactive nitrogen species release). As granulocytes are also a source of cytokines that might modulate local tissue inflammatory reaction (Cassatella, 1999),
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changes in inflammatory cytokine (IL-1β, TNF-α and IL-6) production were measured as well. Circulating levels of acute phase proteins (haptoglobin and fibrinogen) and inflammatory cytokines (TNF-α and IL-6) were measured to see if granulocyte activity is a part of systemic inflammatory reaction. Data were obtained that showed both stimulatory and suppressive effects
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of cadmium administration on peripheral blood granulocyte activity in rats.
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2. Materials and methods 2.1 Chemicals
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Cadmium chloride (Serva, Feinbiochemica, Germany), lypopolysaccharide (LPS), three-(4,5dimethyl-thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT), N-(1-naphtyl) ethylenediamine
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dihydrochloride, sulfanilamide (p-aminobenzenesulfonoamide), hexadecyltrimethylammonium bromide (HTAB), o-dianisidine dihydrochloride, myeloperoxidase (MPO) and phorbol 12myristate 13-acetate (PMA) (all purchased from Sigma, Sigma Aldrich., St. Louis, MO, USA),
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sodium nitrite (Fluka Chemika, Switzerland) and hydrogen peroxide (H2O2) from Zorka Farma, Sabac (Serbia) were used in experiments. LPS, MTT and PMA were dissolved in culture
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medium and phosphate buffered saline (PBS, pH 7.2), respectively. All solutions for cell culture
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experiments were prepared under sterile conditions and sterile filtered (Minisart, pore size 0.20 µm; Sartorius Stedim Biotech, Goettingen, Germany) before use. Dextran T-500 (Sigma Aldrich,
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St. Louis, MO, USA) 6 % solution was prepared in apyrogenic saline and autoclaved (1100 C). Culture medium RPMI-1640 supplemented with 2 mM glutamine (PAA laboratories, Austria), 20 μg/mL gentamycine (Galenika a.d., Serbia), 5% (v/v) heat inactivated fetal calf serum (PAA laboratories, Austria) was used in cell culture experiments. Monoclonal antibody OX-42 (mouse anti-rat CD11b/CD11c) and FITC-conjugated F(ab')2 goat anti-mouse IgG were purchased from Serotec Ltd, Bicester, UK. Lysis buffer (eBioscience Inc., San Diego, CA, USA) was used for red blood cell lysis. OptiPrep (Nycomed AS, Oslo, Norway) was used for peripheral blood granulocyte isolation. 2.2 Animals and cadmium treatment Animal treatment was carried out in adherence to the guidelines of the Ethical Committee of the Institute for Biological Research "Siniša Stanković" (IBISS), Belgrade, Serbia. Male Dark Agouti (DA) rats 12-14 weeks old, weighing 200 - 240 g were used in experiments. Animals
Page 6 of 30
were conventionally housed at IBISS, in a controlled environment (24°C with a 60% relative humidity and a 12-hr light:dark cycle) and had access to standard rodent chow and water ad libitum throughout the study. Four to six animals were assigned to each treatment group in at least two independent experiments. Sterile filtered cadmium chloride prepared in pyrogen-free
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saline was administered i.p. in a dosing volume of 0.5 mL at a concentration at which animals received 1 mg of cadmium/kg body mass (b.m.). Pyrogen-free saline was administered as
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control. All measurements were carried out 48 hours following cadmium administration in animals anesthetized by i.p. 40 mg/kg b.m. of thiopental sodium (Rotexmedica, Tritau,
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Germany). Tissue specimens were processed on ice and were subsequently subjected to freezing (for cadmium burden measurements), or to functional studies or preparation for histological
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analysis. 2.3 Cadmium determination
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Cadmium content in tissues and peripheral blood was determined by atomic absorption spectrometry (AAS; SpectrAA-50, Varian, Inc, Palo Alto, Ca, USA) after ashing the lyophilized
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tissue or blood samples at 5500C and dissolving the ash in 1M HCl. The concentrations were
2.4 Clinical biochemistry
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expressed as μmol of Cd per kg of wet tissue or blood weight ± SD.
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Serum aspartate amino transferase (AST) and alanine amino transferase (ALT) were determined with an autoanalyzer (Ciba Corning Express, Oberline, OH, USA) using commercially available reagents. Plasma fibrinogen was measured by Siemens-Dade Behring-BCT analyzer using Multifibren U test for quantitative determination in plasma. Haptoglobin and albumin were measured in serum by BN (Dade Behring) immunochemical system for human blood proteins measured by Siemens BNII (Dade Behring) BCT analyzer. Cross-reactivity with rat blood proteins was checked using serum obtained from turpentine-induced inflammation in rat, known inflammatory model of acute phase reaction in these animals (Giffen et al., 2003). Relative plasma or serum protein levels are expressed as percent changes with respect to the mean value obtained in control animals (considered as 100 %). 2.5 Histology
Page 7 of 30
Liver and lungs were cut out and immediately fixed in 4% formaldehyde (pH 6.9). After processing, tissue was embedded in paraffin wax for sectioning at 5 m. Hematoxylin and eosin (H&E)-stained histology slides were subsequently analyzed by a certified histopathologist in a
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blinded manner using a Coolscope digital light microscope (Nikon Co, Tokyo, Japan). 2.6 Lung tissue myeloperoxidase (MPO) activity
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The myeloperoxidase (MPO) activity was measured in homogenates of the lung tissue as described (Goldblum et al., 1985). Lungs were removed, cleared of blood and homogenized by
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IKA T18 Basic Homogenizer (IKA Works Inc, Wilmington NC, USA) on the ice in the phosphate buffered saline contaning 1 mM phenylmethanesulfonyl fluoride (PMSF). To one of
crude
homogenate
two
volumes
of
three
times
concentrated
an
volume
hexadecyltrimethylammonium bromide (HTAB) in potassium phosphate buffer (50 mM, pH 6.5) were added to achieve the final concentration of 0.5 % HTAB. The homogenates were subjected
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to three cycles of freezing and thawing, sonicated for 15-20 sec (Bandelin electronic, UW 2070, Berlin, Germany) at 50 % of maximum intensity amplitude and centrifuged (at 1000 x g at 4°C in
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cooling centrifuge). The MPO was evaluated by the addition of 33 μL of homogenate supernatant to 966 μL of substrate solution (0.167 mg/mL o-dianisidine dihydrochloride and
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0.0005% H2O2 in 50mM potassium phosphate buffer, pH 6.0). The absorbance was read at 450 nm at three-minute intervals up to ten minutes against the standard of myeloperoxidase. The
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values were expressed as MPO units per gram of the lung tissue. 2.7 Peripheral blood and bone marrow leukocyte counts Total and differential peripheral blood leukocyte counts were determined automatically using Siemens ADVIA 120 flow cytometer (Terytown, N.Y., USA). Total bone marrow counts were determined using an improved Neubauer hemocytometer. Differential bone marrow cell counts were determined by differentiating at least 1000 cells from air-dried bone marrow smears stained according to the May Grünwald-Giemsa (MGG) protocol. 2.8 Flow cytometry Adhesion molecule CD11b expression was achieved by flow cytometry analysis of leukocytes from whole-blood following lysis of erythrocytes. Obtained peripheral blood cells were
Page 8 of 30
incubated on ice with mouse anti-rat CD11b antibody. After 30-min incubation, cells were washed twice and stained with FITC-conjugated F(ab')2 goat anti-mouse IgG for 30 min. After washing, the cells were fixed and assayed for fluorescence intensity on an CyFLOW SPACE (Partec, Munich, Germany). Cells in histogram with position characteristic for granular cells
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were analyzed for percentages of CD11b positive cells and mean fluorescence intensity (MFI).
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2.9 Peripheral blood granulocyte isolation
Cells were isolated from heparinized blood by dextran sedimentation followed by centrifugation
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of leukocyte-rich plasma over OptiPrep (Nycomed AS, Oslo, Norway) density separation medium. Polymorphonuclear cells (granulocytes) were obtained from the pellet fraction following lysis of erythrocytes with lysis bufer. The purity of cells, determined morphologically
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after MGG staining, was ≥ 95 %.
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2.10 MTT assay for granulocyte viability and survival
A quantitative colorimetric assay described for human neutrophils (Oez et al., 1990) in which tetrazolium salt MTT is metabolically reduced by cell’s mitochondrial dehydrogenases
to
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coloured end product, formazan, was used as a measure of metabolical viability of freshly
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isolated peripheral blood leukocytes as well as of survival of cells cultured for 24 hours. Cells were added to wells of a 96-well plate (0.25 × 106 cells/well) and incubated with 500 μg/mL of
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MTT (added immediately or following 24 hours in culture) for 3 hours. Formazan produced by cells was dissolved by overnight incubation in 10% SDS-0.01 N HCl. Absorbance was measured spectrophotometrically at 540 nm/650 nm, by an ELISA 96-well plate reader (GRD, Roma, Italy).
2.11 Peripheral blood granulocyte intracellular myeloperoxidase (MPO) Myeloperoxidase activity was assesssed on the basis of the oxidation of o-dianisidine dihydrochloride by cells (Bozeman et al., 1990). Briefly, 33 μL of PMN cell lysate was added to 966 μL of substrate solution (0.167 mg/mL o-dianisidine dihydrochloride and 0.0005 % H2O2 in 50 mM potassium phosphate buffer, pH 6.0). Absorbance was read at 450 nm (ELISA 96-well plate reader, GRD, Rome, Italy) at three-minute intervals up to ten minutes against the standard of myeloperoxidase.
Page 9 of 30
2.12 Peripheral blood granulocyte respiratory burst Cytochemical assay for the respiratory burst (that measures intracellular reduction of nitroblue tetrazolium, NBT) (Choi et al., 2006) was employed. Peripheral blood PMNs were cultured (2 ×
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105 cells/well in round bottomed 96-well plates) with NBT (spontaneous NBT reduction) or NBT and (100 ng/mL) PMA (stimulated NBT reduction) for 30 minutes. Cell-produced formazan was dissolved with 10% SDS - 0.01N HCl spectrophotometrically and absorbance was
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measured at 540 nm/670 nm by an ELISA 96-well plate reader (GRD, Rome, Italy).
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2.13 Nitric oxide production by peripheral blood polymorphonuclear cells
The concentration of the stable NO oxidation product, nitrite, was measured (as an indicator of
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NO formation) by Griess reaction (Hibbs et al., 1988) in medium conditioned for 48 hours by peripheral blood PMNs freshly isolated after cessation of cadmium treatment (CM). Cells were cultured at 5 × 106/mL in 24-well plates in medium (spontaneous production) or in the presence
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of 100 ng/mL, LPS (LPS-stimulated production). Aliquots (50 μL) of 48-hour CM were mixed with an equal volume of Griess reagent (a mixture of 0.1 % naphtylenediamine dihydrochloride
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in water and 1 % sulphanilamide in 5 % phosphoric acid) and incubated for 10 min at room
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temperature. The amount of nitrite was calculated by the reference to a standard curve
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constructed with known amounts of sodium nitrite. 2.14 Cytokine determination by ELISA Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations were determined in supernatant of cells (5 × 105 cells/well of 96-well plate) cultured for 48 hours in medium solely (spontaneous production) or in the presence of 100 ng/mL of LPS (stimulated production). Enzyme-linked immunosorbent assays (ELISA) for rat TNF-α (eBioscience Inc., San Diego, CA, USA) and rat IL-6 and IL-1β (R&D systems, Minneapolis, USA) were used according to manufacturer instructions. Cytokine titer was calculated by the reference to a standard curve constructed with known amounts of recombinant IL-1β, TNF-α or IL-6. 2.15 Reverse transcription - real time polymerase chain reaction (RT-PCR) Total RNA was isolated from the polymorphonuclear cells or granulocytes immediately after isolation with an RNA Isolator (Metabion, Martinsried, Germany) following the manufacturer’s
Page 10 of 30
instructions. The isolated RNA was reverse transcribed using random hexamer primers and MMLV (Moloney Murine Leukemia Virus) reverse transcriptase, according to manufacturer's instructions (Fermentas, Vilnius, Lithuania). Prepared cDNAs were amplified by using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the
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recommendations of the manufacturer in a total volume of 20 μL in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Thermocycler conditions comprised an initial
cr
step at 50°C for 5 minutes, followed by a step at 95°C for 10 minutes and subsequent 2-step PCR program at 95°C for 15 seconds and 60°C for 60 seconds for 40 cycles. The PCR primers were
us
as follows: β-actin forward 5’-CCC TGG CTC CTA GCA CCA T-3’, β-actin backward 5’-GAG CCA CCA ATC CAC ACA GA-3’; IL-1β forward 5'-CAC CTC TCA AGC AGA GCA-3', IL1β backward 5'-GGG TTC CAT GGT GAA GTC AAC-3'; TNF-α forward 5’-TCG AGT GAC
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AAG CCC GTA GC-3’, TNF-α backward: 5’-CTC AGC CAC TCC AGC TGC TC-3’; IL-6 forward 5’-GCC CTT CAG GAA CAG CTA TGA-3’. IL-6 backward: 5’-TGT CAA CAA CAT
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CAG TCC CAA G-3’ Accumulation of PCR products was detected in real time and the results were analyzed with 7500 System Software (Applied Biosystems) and calculated as 2−dCt, where
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(-actin).
d
dCt was difference between treshold cycle (Ct) values of specific gene and endogenous control
2.16 Data display and statistical analysis
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Results are expressed as means ± standard deviation (S.D.). Statistical analysis was performed by using STATISTICA 7.0 (StatSoft Inc., Tulsa, Oklahoma, USA). Statistical significance was defined by Mann-Whitney U test. P -values less than 0.05 were considered significant. 3. Results
3.1 Cadmium disposition and systemic effects No differences in animal body mass between group (N = 14) that received 1 mg of cadmium per kg (217 ± 13.38 g) and controls (N = 14) (219 ± 6.50 g) were observed. Absolute amounts of cadmium (μmol/kg wet weight) increased in all tissues examined and tissue burden was higher in animals which received cadmium compared to controls (Table 1). Increase (P < 0.001) in both AST (373 ± 96 U/mL, N = 14 compared to 203 ± 60 U/mL in controls, N = 14) and ALT (140 ± 36 U/mL, N = 14 compared to 82 ± 24 U/mL, N = 14) in rats administered with cadmium was
Page 11 of 30
observed. Histological analysis demonstrated significant presence of neutrophils in liver (Fig. 1A). Infiltration of both mononuclear as well as polymorphonuclear cells in lungs of rats that were administered with cadmium was observed (Fig. 1B) and increased (P < 0.05) myeloperoxidase (MPO) activity was noted in lungs of these animals (4.57 ± 2.34 U/g wet lung
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weight, N = 10 compared to 1.44 ± 1.02 U/g of lungs from control animals, N = 10).
Cadmium administration was associated with an increase in TNF-α concentration in
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plasma (Fig. 2A). Rise in serum levels of haptoglobin and fibrinogen and a decrease in albumin
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levels were observed (Fig. 2B).
3.2 Peripheral blood leukocyte numbers following cadmium administration
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No changes in total cell or differential leukocyte numbers were observed following cadmium administration with significantly higher relative numbers of neutrophils and lower relative numbers of lymphocytes (Table 2). Analysis of bone marrow smears revealed similar numbers of
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metamyelocytes (7.9 ± 5.5 × 103 and 7.7 ± 4.2 × 103), granulocytes (36.3 ± 8.5 × 103 and 35.4 ± 6.7 × 103) and lymphocyte numbers (43.7 ± 7.8 × 103 and 44.8 ± 7.1 × 103) in cadmium-treated
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(N = 14) and control animals (N = 14), respectively.
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3.3 Peripheral blood granular cell expression of CD11b following cadmium administration
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Whole-blood leukocyte flow cytometry analysis revealed an increase in numbers of granular cells that express CD11b molecules in animals that received cadmium (Table 3). Increased density of surface expression of these molecules was observed as well. 3.4 Peripheral blood polymorphonuclear leukocyte viability and survival following cadmium administration
There were no differences between cadmium-treated and control animals in MTT reduction by freshly isolated peripheral blood granulocytes (Fig. 3). Lower (compared to values in freshly isolated cells) capacity of MTT reduction was noted following 24-hour culture by cells from both animal groups but without difference between them. 3.5 The effect of cadmium administration on peripheral blood polymorphonuclear leukocyte oxidative activity
Page 12 of 30
Cadmium administration resulted in increased levels of intracellular MPO activity in granulocytes from group that received cadmium (Fig. 4A). Granulocytes of both animal groups showed similar levels of spontaneous NBT reduction capacity (Fig. 4B) and responded to stimulation with PMA (higher levels of stimulated compared to spontaneous activity), but the
ip t
levels were significantly higher in cells from rats treated with cadmium (Fig. 4B). There were no differences between groups in spontaneous production of NO. Stimulation with LPS resulted in
cr
an increase (in comparison to spontaneous activity) of NO release by cells from both animal groups, but higher levels were noted in cultures of granulocytes from rats treated with cadmium
us
(Fig. 4C).
3.6 The effect of cadmium administration on peripheral blood polymorphonuclear leukocyte
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cytokines
Measurements of cytokine production by peripheral blood polymorphonuclear cells (Fig. 5A)
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revealed differential effects of cadmium administration when spontaneous cytokine release was measured. While there was no effect on IL-1β and IL-6 release (similar levels in both groups), a
d
decrease of spontaneous TNF-α production was observed. When production in response to stimulation with LPS was measured, similar levels of IL-1β produced were observed in both
te
groups, while there was lower (compared to control group) production of TNF-α. Although no differences between cadmium and control groups were noted concerning the levels of LPS-
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stimulated IL-6 production, lack of responsiveness to LPS could be observed in cultures of neutrophils from cadmium group.
To correlate granulocyte cytokine production with gene expression in vivo, cytokine mRNA levels were analyzed next (Fig. 5B). As revealed by RT-PCR, a significant decrease of mRNA levels was observed for all cytokines examined (Fig. 5B). 4. Discussion
In this study, the effect of acute cadmium administration on peripheral blood polymorphonuclear cells in rats was examined by measuring changes in numbers of these leukocytes, expression of CD11b molecules relevant for tissue infiltration, their oxidative activity as well as changes in inflammatory cytokine (IL-1β, TNF-α and IL-6) production. Data were obtained that showed differential effects of cadmium administration on various aspects of PMN activity.
Page 13 of 30
Infiltration of neutrophils in liver is in line with numerous data that showed presence of inflammatory cell infiltration in liver in acute intoxication with cadmium (Kayama et al., 1995a; Rikans and Yamano, 2000; Yamano et al., 1998). Increase in serum levels of liver enzymes, particularly ALT (the enzyme with highest amounts in liver), might have resulted from injurious
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mechanisms of these cells as proposed (Rikans and Yamano, 2000). Presence of neutrophils in lung tissue 48 hours following cadmium administration is in line with our previous data that
cr
demonstrated infiltration of neutrophils in lungs 24 hours following cadmium administration (Kataranovski et al., 2009a) and show that these cells continue to migrate into this tissue. Owing
us
to their effector activities neutrophils can damage lung tissue and their presence in areas of pneumocyte desquamation support such assumption. Lung neutrophil infiltration demonstrated in this study might, however, reflect their recruitment into injured tissue where these cells can take
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part in local healing/reparatory program governed by infiltrating mononuclear cells.
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In line with our previous data that showed that cadmium administration is associated with systemic cytokine response 24 hours later (Kataranovski et al., 1998), increase in plasma levels of TNF-α observed in this study demonstrates that systemic inflammation is evident later as well.
d
Rise in circulating levels of TNF-α might have resulted from injured tissues. In this regard,
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tissues of liver and kidneys were shown to produce TNF-α in rats administered with cadmium, respectively (Kayama et al., 1995a, 1995b). Circulating TNF-α might influence remote cell and
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tissue behavior. Relative increase in levels of haptoglobin and fibrinogen, which synthesis in liver cells depends, among other cytokines, on TNF-α (Baumann et al., 1990) illustrate such an influence. A decrease in albumin levels is in line with data that showed that rise of positive acute phase reactants (i.e. haptoglobin and fibrinogen) in rats is associated with a decrease in albumin levels (Mayot et al., 2008).
Shift in relative leukocyte numbers in favor of neutrophils implies proinflammatory effects of cadmium as these cells are known cellular component of inflammation at systemic level (Schwartz and Weiss, 1991). In line with our previous data that showed changes in peripheral blood granulocyte counts 24 hours following cadmium administration to rats (Kataranovski et al., 1998), this study demonstrated protracted effect of cadmium administration on these cells. Increase numbers of peripheral blood neutrophils does not seem to rely on bone marrow, as no changes were noted in either granulocyte or metamyelocyte numbers. Possibly, it
Page 14 of 30
might be ascribed to peripheral blood neutrophil redistribution. Lungs harbor intravascular reservoirs of leukocytes, predominantly neutrophils (termed as ‘marginated pool’) that constantly exchange with circulating neutrophils both under basal and inflammatory conditions (Kuebler,
ip t
2005). Increased numbers of granular cells that express CD11b molecules (alpha chain of β2 integrin receptors αMβ2), as well as increase in the density of their expression on cell surface,
cr
point out effects of cadmium administration on activation of these cells (Arnaout, 1990). Observed changes might influence cell’s function, as these molecules are involved in neutrophil
us
extravasation and migration to peripheral tissue (Arnaout, 1990) and for their functional activation (Abram and Lowell, 2009). Recent data showed that neutrophils use these molecules
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in the process of recruitment to liver in inflammatory conditions (McDonald and Kubes, 2012). Expression of CD11b molecules on peripheral blood granular cells following cadmium
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administration might bear relevance for neutrophils migration to liver, in the view of data that showed an increase in the expression of adhesion molecule ICAM-1, a major ligand for CD11b (Arnaout, 1990) on sinusoidal endothelial cells in liver of rats administered with cadmium
d
(Moussa, 2004). This process might be enhanced by cadmium itself, as stimulation of CD11b-
te
based adherence of human peripheral blood polymorhonuclear cells in vitro was noted in the presence of 1 μM of cadmium (Hernandez and Macia, 1996) (the concentration that correspond
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to those measured in the blood of rats in the present study). Increase in expression of CD11b on peripheral blood leukocytes might have resulted from influences of systemic microenvironment, TNF-α in particular, as this cytokine is shown to be a priming factor for neutrophil adhesion and migration (Montecucco et al., 2008; Nathan, 2006). Cadmium administration resulted not only in quantitative changes in peripheral blood granulocytes but in qualitative changes as well. Increase in intracellular MPO content, which is along with phagocyte oxidase a source of oxidant activity in phagocytes (Finkel, 2003) stresses the effect of cadmium administration on peripheral blood granulocyte oxidative activities. Significantly higher responsiveness of granulocytes from rats administered with cadmium to activation of respiratory burst by granulocyte activator PMA, reflects their priming, state, i.e. the state in which the functional responses to an activating stimulus are potentiated/amplified by a prior exposure to a priming stimuli (Hallet and Lloyds, 1995). The rise of granulocyte NO
Page 15 of 30
production in response to LPS, reflects their priming state as well. Increase in oxidative activity of PMNs from cadmium administered rats is in line with our previous data that showed priming effect of cadmium on granulocyte respiratory burst 24 hours following administration (Stosic et al., 2010) and show that it is evident later as well. Increase in intracellular MPO content in
ip t
peripheral blood granulocyte as well as their priming for respiratory burst might rely on the increase of TNF-α in plasma, as it is known priming factor for oxidative activities of neutrophils
cr
(Berkow and Dodson, 1988; Klebanoff et al., 1986). The effect of cadmium itself on oxidative granulocyte activities should not be excluded, as it was shown that 1 μM of cadmium stimulate
us
production of H2O2 by human peripheral blood polymorphonuclear cells (Zhong et al., 1990). In contrast to increase in oxidative activity of peripheral blood granulocytes, cadmium
an
administration resulted in a decrease in mRNA for IL-1β, TNF-α and IL-6. Cytokine genes are target for inhibition by cadmium in vitro as shown by data for human mononuclear cell’s IL-1β
M
(Marth et al., 2001; Theocharis et al., 1994) and TNF-α (Boscolo et al., 2005; Marth et al., 2001), but there are no data, as far as we know, concerning the effect of cadmium administration on peripheral blood granulocytes. Down-regulation of genes for TNF-α and IL-6 was responsible
d
for a decrease in spontaneous and stimulated production of TNF-α and, probably, for negligible
te
responsiveness of IL-6 production to LPS stimulation. Lack of the effect on spontaneous production of IL-1β and IL-6 might be explained by the effect of cell culture, as adherence was
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shown to stimulate cytokine mRNA expression during culture (Haskill et al., 1988). Cadmiuminduced suppression of responsiveness of granulocyte TNF-α and IL-6 production to LPS stimulation might reflect an attempt of host to restrain the activity of these cells. Cytokines, such as TNF-α, a known potent stimulator of (deleterious) effector activities of neutrophils (and other cells) and as mediator that can amplify tissue inflammation, might be especially important target for down regulation. In corroboration, in a separate experiment (unpublished) we have noted a decrease (P < 0.05) in both spontaneous (37.5 ± 22.4 pg/mL) as well as LPS-stimulated production (60.8 ± 34.2 pg/mL) of TNF-α by peripheral blood granulocytes 24 hours following administration of 1 mg/kg of cadmium in rats (N = 4) , compared to 185.8 ± 114.15 pg/mL and 181.7 ± 9.4 pg/mL (for spontaneous and LPS-stimulated production, respectively) in controls (N = 4).
Page 16 of 30
In conclusion, we showed in this study differential effects of cadmium administration on peripheral blood granulocyte activity in rats. Data that illustrated granulocyte activation and priming for oxidative activity, add further to the knowledge of proinflammatory activities of cadmium. Down-regulation of inflammatory cytokine gene expression as well as differential
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effects of cadmium administration on their production is novel finding and point out the manifold relevance of polymorphonuclear cells in cadmium-induced inflammatory and
Conflict of interest statement
an
The authors declare that there are no conflicts of interest.
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immunomodulatory processes.
Acknowledgments
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This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant #173039. The authors thank Vesna Subota, M.Sc. for help in
d
some experiments and Veljko Blagojevic, M.Sc. for help in the processing of manuscript.
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42. Yamano, T., Shimizu, M., Noda, T., 1998. Age-related change in cadmium-induced hepatotoxicity in Wistar rats: role of Kupffer cells and neutrophils. Toxicol. Appl. Pharmacol. 151, 9-15. 43. Zhong, Z., Troll, W., Koenig, K.L., Frenkel, K., 1990. Carcinogenic sulfide salts of nickel
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and cadmium induce H2O2 formation by human polymorphonuclear leukocytes. Cancer Res.
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50, 7564-7570
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Figure captions Fig. 1 Microscopical appearance of (A) liver and (B) lungs 48 hours following cadmium administration. (A) Neutrophils (arrows) in liver sinusoids. Two neutrophils adhere to acidophyllic necrotic binuclear hepatocyte. Intravascular accumulation of neutrophils (insert).
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(B) Perivascular predominantly mononuclear leukocyte infiltration (arrows). Pneumocyte desquamation into alveolar spaces (star). Pneumocytes (star) and neutrophils (arrowheads) that
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adhere to alveolar wall (upper insert). Intravascular accumulation of neutrophils (lower insert).
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Fig. 2 Cytokine and acute phase protein concentrations in circulation. (A) Plasma TNF-α and IL6, (B) Serum fibrinogen, haptoglobin and albumin levels. Data are expressed as mean values ± and ** P < 0.01 vs controls (cadmium 0 mg/kg).
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S.D. from two experiments each with four to six animals per group. Significance at * P < 0.05;
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Fig. 3 Peripheral blood granulocyte viability and survival. Data are expressed as mean values ± S.D. from two experiments each with four to six animals per group. Significance at # P < 0.05 vs
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MTT reduction by freshly isolated granulocytes (0h).
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Fig. 4 Peripheral blood granulocyte oxidative activity. (A) Intracellular MPO activity (B) Spontaneous and PMA-stimulated NBT reduction (C) Spontaneous and LPS-stimulated NO
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production. Data are expressed as mean values ± S.D. from three experiments each with four to six animals per group. Significance at * P < 0.05 and ** P < 0.01 vs controls (cadmium 0 mg/kg). Significance at # P < 0.05 and ## P < 0.01 vs spontaneous production. Fig. 5 Peripheral blood granulocyte inflammatory cytokine (A) production and (B) gene expression. (A) Spontaneous and LPS-stimulated production of IL-1β, TNF-α and IL-6 (B) mRNA expression for IL-1β, TNF-α and IL-6. Data are expressed as mean values ± S.D. from two to three experiments, each with four to six animals per group. Data for mRNA are expressed as percentages of mRNA in PMN from cadmium administered rats relative to mRNA in cells from control rats. Significance at * P < 0.05; ** P < 0.01 and *** P < 0.001 vs controls (cadmium 0 mg/kg). Significance at # P < 0.05 and ### P < 0.001 vs spontaneous production.
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Blood
0
0.09 ± 0.09
0.05 ± 0.02
0.05 ± 0.01
1
201.3 ± 21.4*
4.7 ± 0.7*
1.1 ± 0.2*
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Values are given as mean values ± S.D. from 8 animals per group.
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Table 1. Cadmium liver, lungs and blood content Cadmium concentration (μmol/kg wet weight) Cadmium dose Liver Lungs (mg/kg)
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Significance at * P < 0.05 vs controls (cadmium 0 mg/kg)
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37.3 ± 5.7 3.7 ± 1.2
55.1 ± 15.2* 10.0 ± 8.3
Lymphocytes
(%) (×109/L)
58.8 ± 5.8 5.9 ± 2.1
40.5 ± 16.1* 5.5 ± 0.8
Monocytes
(%) (×109/L)
2.6 ± 0.9 0.3 ± 0.1
2.6 ± 0.9 0.4 ± 0.2
Eosinophils
(%) (×109/L)
0.8 ± 0.4 0.1 ± 0.03
0.7 ± 0.3 0.1 ± 0.01
Basophils
(%) (×109/L)
0.4 ± 0.1 0.04 ± 0.02
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(%) (×109/L)
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Neutrophils
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Table 2. Total and differential peripheral blood cell counts Parameter Cadmium dose (mg/kg) 0 1 White blood cells (×109/L) 10.0 ± 3.4 16.3 ± 9.1
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0.3 ± 0.1 0.06 ± 0.06
Data are expressed as mean values ± S.D. from two experiments each with four to six animals per group.
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Significance at * P < 0.05 vs controls (cadmium 0 mg/kg)
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Data are expressed as mean values ± S.D. from two experiments each with four to six animals.
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Table 3. CD11b expression in peripheral blood granular cell population Cadmium dose (mg/kg) 0 1 Number of 91.8 ± 2.5 97.8 ± 3.6** positive cells (%) Mean Fluorescence 2703.0 ± 543.0 3651.0 ± 191.0* Intensity (MFI)
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Significance at * P < 0.05; and ** P < 0.01 vs controls (cadmium 0 mg/kg)
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