Intestinalni i sistemski imunski efekti oralnog unosa kadmijuma kod pacova
Intestinal and systemic immune effects of oral cadmium intake in rats
Doctoral thesis (Published version)
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Cadmium (Cd) is a heavy metal which is found in every part of the environment, it does not have any known biological function and has an adverse effect upon the living systems. The most common way of Cd exposure is orally, through contaminated water and food, where the prime target of this metal toxicity is the gastrointestinal tract. It is known that Cd causes damage to intestinal tissue and disrupts the epithelial barrier function which is necessary for maintaining immune homeostasis, however, the immunotoxicity mechanisms in this region have not been examined sufficiently. Additionally, it is known that Cd toxic effects may depend also on genetic background / strain of experimental animals, but the strain-dependent differences in intestinal toxicity of oral Cd intake have not been examined to date. This dissertation was aimed at characterization of the effect of subchronic oral Cd administration on rat’s intestinal immune system. Rats were, for 30 days, orally (in drinking water) exposed to Cd in the form of cadmium chloride (CdCl2) at concentration of 5 ppm (5 mg Cd/l) and 50 ppm and (50 mg Cd/l) of Cd, which corresponds to the doses present in the environment. Within the local immunomodulatory Cd effect, basic parameters of immune response in duodenum (region of greatest Cd absorption) and in mesenteric lymph nodes (MLN) which drain intestine, were investigated. The indicators of tissue damage, oxidative stress and inflammatory changes were tested in duodenum, whereas in the MLN basic phenotype characteristics and parameters of this lymph tissue cells' activities (cellularity, proliferation, cytokine responses, and innate-immune cell activity) were tested. Besides the local, the systemic response to oral Cd intake was tested as well, including humoral and cellular parameters of the inflammatory reaction in blood (changes in hematological parameters, presence of inflammatory mediators and oxidative stress), as well as oxidative stress and basic characteristics of the innate and adaptive immune response in the spleen, a lymph organ in which immune response to blood borne antigens are generated. Aiming to test the contribution of genetic background to intestinal and systemic immunotoxicity of oral Cd exposure, local and systemic effects were analyzed in two rat strains, Dark Agouti (DA) and Albino Oxford (AO), which establish qualitatively and/or quantitative different immune response to the same stimuli. The study has shown that oral Cd treatment leads to dose-dependent accumulation of this metal in intestine, MLN and spleen, similarly in DA and AO rats, which demonstrates that genetic background has no effect on the metal deposition levels. Despite similar Cd concentrations in the intestine of both rat strains, more pronounced mononuclear leukocytes infiltrates in intestinal tissue, higher degree of enterocytes necrosis [measured by increased necrosis marker HMGB1 (High mobility group box 1) molecule] and the increased production of proinflammatory cytokines (tumor necrosis factor/TNF, interferone-gamma/IFN-γ, interleukin-17/IL-17) were shown in intestinal tissue of DA rats compared to rats of AO strain. The change in activity of basic enzymes of anti-oxidative defense (superoxide dismutase/SOD and catalase/CAT), in intestine of DA rats and SOD in AO rats, which depicts host tissue attempt to counterattack reactive oxygen species (ROS) and limit tissue damage, as well as glutathione-s-transferase/GST (the enzyme responsible for electrophilic substances binding, such as Cd, to the reduced form of glutathione, GSH), was also more expressed in rats of DA strain. Such type of changes is probably in the background of the increased level of lipid peroxides (measured by the changes of malondialdehyde/MDA) in AO rats. Oral Cd treatment results in reduced prevalence of lactobacilli in DA rats' intestine which may contribute to the development of proinflammatory cytokine response to Cd, whereas relative preservation of commensal flora in AO rats (along with significant increase in prevalence of L. johnsonii and L. Murinus) might be of significance for preservation of gut barrier integrity and absence of the local inflammatory cytokine response in this strain of rats. Oral Cd treatment affected the MLN activity in both strains of rats (increase of MLN mass and cellularity, ROS production). Nevertheless, Cd induced metalotioneinas/MT gene expression as well as proinflammatory immune response (MLN cells' proliferative activity, oxidative activities, Th1/Type1 and Th17/Type17 response) and inhibited anti-inflammatory response (interleukine-10/IL-10 gene expression and production) only in MLN of DA rats. Higher degree of intestinal epithelium damage and necrosis observed in DA treated rats (compared to AO rats) is the most likely factor which stimulated proinflammatory response in MLN of DA rats. The changes in MLN of DA rats indicate that Cd disrupts the tolerogenic immune environment in the gut by induction of both innate and the acquired immune responses. Although the investigation of the systemic effect of orally administered Cd indicated, in general, slight effect of the metal on the basic hematological and biochemical parameters in the peripheral blood, the effect on anti-oxidative activities of erythrocytes in both strains of rats was noticed. The increased levels of HMGB1 molecule in blood of DA rats only, suggests higher degree of organs damage and inflammatory character of immune response to Cd in this strain of rats. Although the same quantity of Cd is deposited in MLN and spleen, the response in these two immune organs differs, showing that the micro environment of the examined tissue is also of great importance in observing Cd effects. Unlike MLN, the oral Cd administration did not affect spleen mass and cellularity but it resulted in reduced viability and reduced proliferative activity of spleen cells, whereas, similar as in MLN, the increased oxidative activity of spleen cells (activity of myeloperoxidase/MPO, nitrogen-oxide/NO production) was noted and the increase of non-stimulated production of proinflammatory cytokines (interleukine-1 beta/ IL-1ß, IFN-γ, IL-17) was noted, only in DA rats. This proinflammatory cytokine response of spleen cells in DA rats may be associated with increased level of HMBG1 molecule in the spleen. More pronounced proinflammatory response to oral Cd administration in gut as well as in MLN and spleen of DA rats in comparison to AO rats (in which similar concentrations of deposited Cd were found) show that DA rats are more susceptible to oral subchronic administration of this metal. The results of this study contribute to the understanding of the mechanisms which underlie the toxic Cd effects on immune response in intestine, for the first time showing the effect of this metal on homeostasis of innate and adaptive components of immune response in MLN. At the same time, this study has also shown the significance of tissue in expressing Cd toxicity. Also, for the first time, this study has indicated the effect of genetic background on orally administered Cd effects, suggesting the significance of careful selection of experimental animals to be used in the analysis of immunotoxic effects of this metal.
Keywords:Oral cadmium administration; Intestinal immune response; Mesenteric lymph nodes; Spleen; DA and AO rats; Strain differences; Immunotoxicity
Source:University of Belgrade, Faculty of Biology, 2016, 1-139
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