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Inhibitory Effects of Cadmium on Peripheral Blood Mononuclear Cell Proliferation and Cytokine Release Are Reversed by Zinc and Selenium Salts Paolo Boscolo,1 Luca Di Giampaolo,1 Niu Qiao,2 Marcella Reale,3 Maria Luisa Castellani,3 Isabella Lucci,3 Paola Travaglini,1 Mary Kouri,1 Nicola Verna,1 Anna Rita Volpe,4 Marco Carmignani,4 Roberto Paganelli,1 and Mario Di Gioacchino1 Departments of 1Medicine and Sciences of Ageing and of 3Oncology and Neurosciences, University G. D’Annunzio, Chieti, Italy; 2Department of Occupational Health, Shanxi Medical University, Taiyuan, China; and 4Department of Basic and Applied Biology, University of L’Aquila, Italy Abstract. Zinc (Zn) and selenium (Se) exert regulatory activities on immune functions, while cadmium (Cd) is an immunotoxic agent. The object of this study was to detect effects of 10-4, 10-5, and 10-6 M Cd sulphate, Zn sulphate, and sodium selenite, and their combinations on human peripheral blood mononuclear cell (PBMC) proliferation and IFN-γ and TNF-α production. Only 10-5 M Zn sulphate significantly enhanced spontaneous PBMC proliferation, which was unaffected by the other salts. At 10-4 and 10-5 M, Cd sulphate exerted a dose-response inhibitory action on phytohemagglutinin- (PHA-) stimulated PBMC proliferation and cytokine release, while 10-4 M and 10-5 M Zn sulphate and 10-5 M sodium selenite induced a stimulatory effect on both proliferation and cytokine release; 10-4 M sodium selenite enhanced only the PBMC proliferation; at 10-6 M, none of the salts changed the PHA-stimulated immune activity. Moreover, 10-4 and 10-5 M Zn and 10-5 M Se strongly upregulated IFN-γ (a Th1 cytokine) release, even in presence of 10-5 M Cd, and reduced the inhibitory effects of Cd on PBMC proliferation and TNF-α release. This study confirms that Zn and Se both strongly enhance cytokine release induced by mitogenic stimulation, showing also that Zn acts with a broader range of concentrations than Se. This suggests that dietary excess of Se may not have beneficial effects. (received 4 February 2005; accepted 9 March 2005) Keywords: zinc, selenium, cadmium, lymphocyte proliferation, cytokine, immunotoxicity Introduction Cd exposures induce cancer and malformations and affect the reproductive and cardiovascular systems, as well as renal function and bone metabolism [14]. Immunotoxicity of Cd is also recognized; high levels of Cd salts exert inhibitory and suppressive effects on peripheral blood mononuclear cells (PBMC), whereas low Cd levels have stimulatory actions [5]. The mechanisms of Cd toxicity include inhibition of the electron transfer chain and enhanced production of reactive oxygen species in Address correspondence to Professor Paolo Boscolo, Occupational Medicine, G. D’Annunzio University, Via dei Vestini, 66013 Chieti Scalo, Italy; tel 39 0871 355 6704; fax 39 0871 355 6747; e-mail
[email protected].
mitochondria [6] and the activation of apoptosis by modulating Ca ion metabolism and signal transduction pathways [7]. Zn is an essential element that plays important roles in immune function regulation. Zn deficiency alters antibody production, T lymphocyte and thymic hormone activity, and cell mediated cytotoxicity [8,9]. Immune effects of Zn include protection against factors that induce apoptosis [10]. Importance of Zn for immune functions in humans was shown by significant relationships between the Zn levels and blood lymphocyte subsets [11]. Metallothionein plays a physiological role in Zn homeostasis and may donate or sequester this metal in enzymes and transcription factors [12]. Cd competes with Zn not only on metallothionein
0091-7370/05/0200-0115. $1.50 © 2005 by the Association of Clinical Scientists, Inc.
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binding but also on its synthesis. Cd interacts with Zn finger proteins involved in gene transcription and nucleotide and base excision repair [13]. Selenium (Se) is a component of the active center of about 20 eukaryotic proteins with roles in the prevention of inflammatory and degenerative diseases [14]. Se-proteins with redox functions (eg, glutatione peroxidases and thioredoxin reductase) are involved in preventing radical-induced DNA damage [14,15]. Moreover, Se counteracts the toxic effects of Hg by binding this metal to selenoprotein P [16]. Anti-tumorogenic activity of Se has been demonstrated in several animal studies, while epidemiological studies have linked body Se status to the risk of cancer [14,15]. Dietary Se improves immune responses that enhance lymphocyte proliferation and B-cell and T-cell functions [17]. The aim of this study was to evaluate the in vitro effects of various concentrations of Se and Zn salts on Cd inhibition of lymphocyte proliferation and cytokine release. Material and Methods Cd, Zn, and Se salts. Solutions of 10-3 M Cd sulphate (CdSO4), zinc sulphate (ZnSO4), and sodium selenite (Na2SeO3) (Alfa, Milan, Italy) were prepared by dissolving the salts in distilled water. The solutions of 10-3 M salts were diluted to 10-4, 10-5, and 10-6 M in the culture media. Subjects. Healthy male volunteers (N = 9, mean age 32 yr, range 24-54 yr) were recruited for this study. They were not taking any drugs; their routine blood analyses, including white blood cell count (WBC), were within the normal ranges. Fasting samples of EDTA-treated whole blood were obtained from each subject at 8 am and PBMC were purified by FicollHypaque (BioSpa, Milan, Italy) density gradient centrifugation (20 min, 400 x g). After 3 washings with Hanks’ balanced salt solution (HBSS), PBMC were resuspended in RPMI 1640 medium that containied 10% fetal calf serum, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 µg/ ml streptomycin (Sigma Chemical Co., St Louis, MO, USA). This is designated as complete medium.
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PBMC proliferation. PBMC were suspended at 106 cells/ml in complete medium. Aliquots (100 µL) of cell suspension were placed in each well of a 96-well microtiter plate (Falcon, Oxnard, CA, USA) under the following conditions: (a) no other reagent added (control sample); (b) with 20 µg/ml PHA (Sigma); (c) with 10-4, 10-5, or 10-6 M CdSO4, ZnSO4, or Na2SeO3; (d) with 10-4, 10-5, or 10-6 M CdSO4, ZnSO4, or Na2SeO3 in the presence of PHA; (e) with 10-5 M CdSO4 and addition of 10-4, 10-5, or 10-6 M ZnSO4 or Na2SeO3 in the presence of PHA. The cells were incubated for 72 hr at 37°C in a humidified atmosphere with 5% CO2. Enzymatic immunoassay of PBMC proliferation. Proliferation was evaluated by a 5’-bromo-2’-deoxyuridine (BrdU) cell proliferation assay (Oncogene Research Products, Darmstadt, Germany). BrdU was added to wells of microtiter plates during the final 24 hr of culture. Cells were fixed and permeabilized, and their DNA was simultaneously denaturated by treatment for 30 min at room temperature with a fixative/denaturing solution. Anti-BrdU monoclonal antibody was pipetted into the wells and incubated for 1 hr. Unbound antibody was washed away and horseradish peroxidaseconjugated goat anti-mouse antibody was added for 30 min at room temperature. Contents of wells were removed by inversion over a sink and tapping on paper towels. Chromogenic substrate solution, tetramethylbenzidine, was added to each well and incubated in the dark at room temperature for 15 min. A stop solution was added to each well in the same order as the previously added substrate solution. All reagents were provided with the kit and were used according to the manufacturer’s protocol. Assays were performed in triplicate. The absorbance of the contents of each well was measured using a spectrophotometric plate reader at dual wavelengths (450 and 540 nm). Color intensity was proportional to the cellular incorporation of BrdU and also to the degree of cell proliferation. Production and measurement of cytokines. Cultures were established in 1-ml wells of 24-well Costar plastic plates, using 0.8 ml of PBMC (containing 106 cells) in complete medium as follows: (a) no
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Zn and Se reduce Cd immunotoxicity in vitro
Fig. 1. Percent (mean ± SD) of PHA-stimulated PBMC proliferation in presence of CdSO4, ZnSO4, and Na2SeO3. **p
