Dragan Kataranovski

Gender Differences in Acute Cadmium-Induced Systemic Inflammation in Rats

OBJECTIVE To examine the presence of gender differences in pro-inflammatory potential of cadmium in rats by comparing systemic inflammatory response to acute cadmium intoxication in animals of the two sexes. METHODS Basic aspects of this response were evaluated, including plasma levels of inflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) and of major rat acute phase protein alpha 2-macroglobulin (alpha2-M), as soluble indicators of inflammation, and the number and activity of peripheral blood leukocytes, as cellular indicators of inflammation. RESULTS Differential increases of IL-6 and alpha2-M (higher in males than in females) in peripheral blood cell counts and types (leukocytosis and shift in the ratio of granulocytes to lymphocytes more pronounced in males vs females) and in levels of neutrophil priming (higher in males vs females) were noted. CONCLUSION The data document a more intense inflammatory response to cadmium administration in males. The sex differences in inflammatory effects of cadmium might be taken into consideration in studying the toxicity of this heavy metal.

Acute cadmium administration to rats exerts both immunosuppressive and proinflammatory effects in spleen.

Conflicting data (both suppression and augmentation as well as lack of the effect) exist in respect to cadmium (Cd) and splenic T cell-based immune cell activity. Spleen is also the site of innate immune responses but impact of Cd on this type of immunity has been less explored. In the present study the effects of acute Cd administration on basic aspects of both T cell-based and innate immune spleen cell activity were examined in rats. Intraperitoneal injection of 1mg of Cd/kg resulted in decrease in concanavalin A (ConA) induced proliferation which seems to be more related to altered spleen cells responsiveness to IL-2 than to apoptosis. Differential effects on proinflammatory T cell derived cytokines were observed (decreases of IFN-γ gene expression and ConA-stimulated production, but increases in IL-17 mRNA levels with no effect on concentrations of protein product). Reduction of IFN-γ production seemed not to rely on IL-4 and IL-10, but at least partly on nitric oxide (NO). Increased activity relevant for innate immunity (granulocyte and CD11b(+) cell accumulation in the spleen, inducible nitric oxide synthase/iNOS expression and NO production by spleen cells) was observed, but there was a decrease in respiratory burst (dihydrorhodamine/DHR oxidation and nitroblue tetrazolium/NBT reduction). Increases of TNF-α and IL-1β gene expression and IL-1β protein product were noted as well. Administration of 0.5mg Cd/kg resulted in less pronounced (ConA-induced proliferation) or lack of the effect (IFN-γ production) on spleen T cell activities and on innate activities (granulocyte accumulation, NO production) as well. However, increases of spleen cell respiratory burst activity and IL-1β production were observed. Effects of lower cadmium doses (5ppm and 50ppm) on several aspects of spleen cell immune activity were observed in intermediate period of exposure (30 days, oral intake) as well. Differential effects of Cd on immune activities of spleen cells might contribute to our understanding of the complexity of immunomodulatory effects of this metal.

Toxicity of oral cadmium intake: Impact on gut immunity

Gastrointestinal tract is one of the main targets of cadmium (Cd), an important food and drinking water contaminant. In the present study, the effect of subchronic (30 days) oral (in water) intake of 5ppm and 50ppm of cadmium on immune responses in the gut was examined in rats. Cadmium consumption resulted in reduction of bacteria corresponding to Lactobacillus strain, tissue damage and intestinal inflammation [increases in high mobility group box 1 (HMGB1 molecules), superoxide dismutase (SOD) and catalase (CAT) activity and proinflammatory cytokine (TNF, IL-1β, IFN-γ, IL-17) content]. Draining (mesenteric) lymph node (MLN) stress response was observed [elevation of MLN glutathione (GSH) and metallothionein (MT) mRNA levels] and stimulation of both adaptive [cellularity, proliferation, proinflammatory (IFN-γ and IL-17) MLN cell cytokine responses] as well as innate immune activity (increases in numbers of NK and CD68(+) cells, oxidative activities, IL-1β). In contrast to proinflammatory milieu in MLN, decreased or unchanged antiinflammatory IL-10 response was observed. Stimulation of immune activities of MLN cells have, most probably, resulted from sensing of cadmium-induced tissue injury, but also from bacterial antigens that breached compromised intestinal barrier. These effects of cadmium should be taken into account when assessing dietary cadmium as health risk factor.

Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats.

Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-α (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans.

Toxoplasmosis in Naturally Infected Rodents in Belgrade, Serbia

To assess the role of synanthropic rodents in the epidemiology of urban toxoplasmosis, Toxoplasma gondii infection was examined in 144 rats (Rattus norvegicus) and 12 mice (Mus musculus) captured using live animal traps in three locations in Belgrade city characterized by poor housing and degraded environment. In rats, specific IgG antibodies were detected by modified agglutination test in 22 (27.5%) of the 80 blood samples available. Toxoplasma brain cysts were microscopically detected in 11 (7.6%), and Toxoplasma DNA by real-time polymerase chain reaction was demonstrated in 15 (10.4%) animals. Of these, both cysts and Toxoplasma DNA were detected in five (3.5%) rats. In mice, cysts were observed in 3 (25%), but Toxoplasma DNA was detected in even 10 (83.3%) animals, including all 3 with morphologically recognized cysts. Being a link in the chain of Toxoplasma infection, the existence of urban rodent reservoirs of infection represents a public health risk.

Intestinal helminths infection of rats (Ratus norvegicus) in the Belgrade area (Serbia): the effect of sex, age and habitat

Gastrointestinal helminths of Norway rat (Rattus norvegicus) from the Belgrade area were studied as a part of a wider ecological research of rats in Serbia (data on the distribution, population ecology, economic and epizoothiological-epidemiological importance, and density control). Rats were captured from May 2005 to July 2009 at both urban and suburban-rural sites. Of a total of 302 trapped rats 48% were males and 52% females, with 36.5% and 38.8% of juvenile-subadult individuals, per sex respectively. Intestinal helminth infection was noted in 68.5% of rats, with a higher prevalence in male hosts and in adult individuals. Higher numbers of infected juveniles-subadults were noted in suburban-rural habitats, while an opposite tendency was noted in adult rats. Seven helminth species were recovered, of which five were nematode (Heterakis spumosa, Nippostrongylus brasiliensis, Capillaria sp., Trichuris muris and Syphacia muris) and two cestode species (Hymenolepis diminuta and Rodentolepis fraterna). The most prevalent parasites were Heterakis spumosa (36.7%) and Hymenolepis diminuta (30.5%). Sex and habitat-related differences were noted in the prevalence of infection with Capillaria sp. and Trichuris muris, while there were no age-related differences in the prevalence of infection with any individual helminth species. Significantly higher prevalence of infection was noted in summer as compared to spring or winter, with a tendency to be higher in autumn as compared to spring. The only significant difference in the prevalence of infection between habitat-related was noted during spring. H. spumosa was most prevalent in summer, while H. diminuta and N. brasiliensis in autumn. The mean intensity of infection with H. spumosa, R. fraterna, S. muris and T. muris was higher in autumn than in the other seasons, while N. brasiliensis and Capillaria sp. occured in winter. No more than four helminth species were found in one host.

Gender Differences in Pulmonary Inflammation Following Systemic Cadmium Administration in Rats

OBJECTIVE To examine the presence of gender differences in pulmonary inflammation evoked by acute systemic cadmium administration in rats. METHODS Presence of basic indicators of lung inflammation (inflammatory cytokine lung content, leukocyte infiltration and activity of cells recovered from lungs by enzyme digestion) was analyzed and compared in animals of the two sexes. RESULTS Intraperitoneal administration of cadmium (1.0 mg/kg) resulted in higher cadmium content in lungs of female rats. Higher tumor necrosis factor (TNF) content was noted in lung homogenates of male rats, while interleukin-6 (IL-6) content was slightly, but significantly greater in lungs of female rats. Increased leukocyte infiltration was observed in lungs of male rats, mainly due to neutrophils. Increased responsiveness to phorbol myristate acetate (PMA) stimulation was noted in cells recovered from lungs of male rats. Rise in intracellular content of myeloperoxidase (MPO) was noted in lung cells from cadmium-treated rats of both sexes, but higher in cells from male rats. CONCLUSIONS Presented data documented a more intense pulmonary inflammatory response to systemic cadmium administration in males, with higher IL-6 levels in lungs of female individuals. These sex differences in proinflamatory activity of cadmium in lungs should be taken into consideration in studying the remote toxicity of this heavy metal.

Immunotoxicity of epicutaneously applied anticoagulant rodenticide warfarin: evaluation by contact hypersensitivity to DNCB in rats

The immunotoxicity of epicutaneously administered anticoagulant rodenticide warfarin (WF) was examined in this work by using experimental contact hypersensitivity (CHS) reaction to hapten dinitrochlorobenzene (DNCB). WF (0.05 and 0.5 mg/kg) administration 24 h before the induction of CHS does not change expression of CHS evaluated by ear swelling assay. Regional draining lymph node response during sensitization phase was characterized by decreased cellularity but increased spontaneous and IL-2 stimulated proliferation of draining lymph node cells (DLC). No changes in IL-2 production and in numbers of CD25(+) cells were noted and even decreased proliferative index (ratio of IL-2 stimulated to unstimulated DLC proliferation) was detected. Increase in granulocyte activity (MTT reduction and adhesion to plastic) was noted following application of WF solely with further increase following subsequent application of DNCB, when granulocyte activation (NBT reduction) was noted also. Access of WF into general circulation might be responsible for observed changes, what was supported by ex vivo changes in DLC and granulocyte functions assessed before initiation of sensitization and by in vitro effect of exogenous WF as well. Differential effects of WF on lymphocytes and granulocytes noted in this study highlight the need for simultaneous testing of both cell type activity what might constitute a more integrated approach in immunotoxicity studies.

Contact allergic response to dinitrochlorobenzene (DNCB) in rats: Insight from sensitization phase

Contact hypersensitivity (CHS) is a T-cell-mediated skin inflammatory reaction to cutaneous exposure to small sensitizing chemicals, haptens. Majority of CHS studies were conducted in mice and there is paucity of data in other experimental animals. In the present study, characteristics of contact hypersensitivity reaction to dinitrochlorobenzene (DNCB) were determined in Th1-prone Dark Agouti (DA) rats by evaluating sensitization phase as a function of time-dependent changes in draining lymph nodes (DLN). Apart from basic indices of DLN activity (cellularity and proliferation), the production of cytokines relevant for CHS induction, interleukin-6 (IL-6), interferon-γ (IFN-γ), interleukin-17 (IL-17) and interleukin-4 (IL-4) was analyzed. Anti-inflammatory cytokine interleukin-10 (IL-10) production by DLN cells was determined as well. Highest production of IL-6, IFN-γ and IL-17 in sensitized animals was observed at day 3 after DNCB application, with a decrease at day 5. Increased messages for IFN-γ and IL-17 were noted at this time point. In contrast to inflammatory cytokines, anti-inflammatory cytokine interleukin-4 (IL-4) was undetectable during the entire sensitization phase. Differential pattern (IL-6 and IFN-γ) and level (IFN-γ and IL-17) of inflammatory cytokine production was noted in sensitized Th2-prone Albino Oxford (AO) rats. Similarly to DA rats, no changes in IL-4 were noted in AO rats. Strain-dependent differences in inflammatory cytokine production seem to be based on anti-inflammatory cytokine interleukin-10 (IL-10). Production of IFN-γ concomitantly with undetectable IL-4 in both strains classify rat CHS to DNCB as Th1/type 1 reaction. Detection of IL-17 in sensitized DLN cells points to the involvement of T(IL-17) cells in rat contact hypersensitivity.

Lungs: Remote inflammatory target of systemic cadmium administration in rats

Pulmonary inflammation is a biological response to cadmium entering the body via the respiratory route. Systemic administration of this metal revealed the lungs as a significant site of its disposition. In this study, the presence of basic indicators of lung inflammation (leukocyte infiltration and activity of cells recovered from lungs by enzyme digestion) was analyzed in the rat model of acute systemic cadmium intoxication. Intraperitoneal administration of both cadmium doses (0.5mg/kg and 1.0mg/kg) resulted in increased numbers of neutrophils. Signs of spontaneous activation of lung cells including the capacity of reduction of nitroblue tetrazolium (NBT), increase in myeloperoxidase (MPO) intracellular content and increase in interleukin-6 (IL-6) production were noted at both cadmium doses. Increased lung cell responsiveness to stimulation in vitro was noted at the higher cadmium dose. The presence of pulmonary inflammatory parameters in rats administered intraperitoneally with cadmium revealed the lungs as remote inflammatory targets of this metal.