Tributyltin exposure alters cytokine levels in mouse serum.

Tributyltin (TBT), a toxic environmental contaminant, has been widely utilized for various industrial, agricultural and household purposes. Its usage has led to a global contamination and its bioaccumulation in aquatic organisms and terrestrial mammals. Previous studies suggest that TBT has debilitating effects on the overall immune function of animals, rendering them more vulnerable to diseases. TBT (at concentrations that have been detected in human blood) alters secretion of inflammatory cytokines from human lymphocytes ex vivo. Thus, it is important to determine if specified levels of TBT can alter levels of cytokines in an in vivo system. Mice were exposed to biologically relevant concentrations of TBT (200, 100 or 25 nM final concentrations). The quantitative determination of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL2, IL5, IL7, IL12ßp40, IL13, IL15, keratinocyte chemoattractant (KC), macrophage inflammatory protein 1ß (MIP), MIP2 and regulated on activation normal T-cell-expressed and secreted (RANTES) was performed in mouse sera by MAGPIX analysis and Western blot. Results indicated alterations (both decreases and increases) in several cytokines. The pro-inflammatory cytokines IFNγ, TNFα, IL-1ß, IL-2, IL5, IL12ßp40 and IL-15 were altered as were the chemokines MIP-1 and RANTES and the anti-inflammatory cytokine IL-13. Increases in IFNγ and TNFα were seen in the serum of mice exposed to TBT for less than 24 h. Levels of IL1ß, IL-12 ßp40, IL-5 and IL-15 were also modulated in mouse serum, depending on the specific experiment and exposure level. IL-2 was consistently decreased in mouse serum when animals were exposed to TBT. There were also TBT-induced increases in MIP-1ß, RANTES and IL-13. These results from human and murine samples clearly suggest that TBT exposures modulate the secretion inflammatory cytokines.

Impact of tributyltin on immune response and life history traits of Chironomus riparius: single and multigeneration effects and recovery from pollution.

Chironomids play an important role in the detritus cycle and as a component in brackish- and freshwater benthic and terrestrial food webs. If TBT is present in their environment, then they may accumulate tributyltin (TBT) during their juvenile period, which negatively affects many of their life history characteristics. The aim of this experiment is to test the effects of three TBT sediment concentrations (nominal 30, 90, and 180 µg/kg) on life history traits (development time, survival, fecundity, and weight) and immune response (number of hemocytes and phenoloxidase activity) of the nonbiting midge, Chironomus riparius. These responses were recorded immediately after one generation of TBT exposure, and in the long run during five consecutive generations. We also assessed recovery from pollution after four generations of TBT exposure. In a single generation, TBT affected all measured parameters, except phenoloxidase activity, when compared to the control. Long-term-effects of TBT lead to extinction of all treatments after the fifth generation. Again, all measured variables significantly differ from the control, although TBT had varying effects on the measured variables. Most of the effects of TBT on population viability were not evident during recovery, once TBT was removed from the sediment. The effect of previous TBT contamination was observed only in delayed larval development, suggesting that TBT has only limited maternal/epigenetic effects on individual condition. However, altered schedules in the life-cycle can have unexpected ecological impacts. TBT decreases the viability of Chironomus riparius and the effect will become stronger if exposure to TBT continues for many generations. Yet, the harmful effect of TBT disappears quickly as the TBT is removed from the environment.

Tributyltin chloride-induced immunotoxicity and thymocyte apoptosis are related to abnormal Fas expression.

Tributyltin chloride (TBTC), a characteristic organotin compound, is widely used as an agricultural pesticide, as a stabiliser for polyvinyl-chloride plastics and in antifouling paints for ship hulls. Organotin compounds are known to produce toxicity in the immune system, but the mechanism underlying this immunotoxicity remains unclear. In this study, we evaluated the immunotoxic effect of TBTC on the acquired immune response, and we investigated the involvement of thymocyte apoptosis and Fas expression in the observed immunotoxicity of TBTC. Mice were randomly assigned to four groups (10 mice per group) and treated with TBTC at doses of 0, 0.5, 4 and 20 mg/kg by oral gavage for 28 days. Following TBTC administration, animals were sacrificed, and morphologic changes in the thymus and spleen were assessed. Atrophy in both the thymus and spleen was observed in all groups treated with TBTC, and the relative organ weight in the highest TBTC group (20 mg/kg) was significantly lower than that observed in the control group. We also conducted assays to assess the cellular and humoral functional responses such as plaque-forming cell assay (PFC), lymphocyte proliferation test and delayed-type hypersensitivity (DTH) response to SRBC. Our results indicate that at doses of 4 mg/kg and 20 mg/kg, TBTC could significantly suppress both the humoral and cellular immune responses when compared to the control group (p<0.05). In addition, immunohistochemical staining and flow cytometry analysis were carried out to measure the expression of Fas and thymocyte apoptosis, respectively. We observed a dose-dependent increase in thymocyte apoptosis and that Fas expression in the TBTC-treated groups (4 mg/kg and 20 mg/kg) was significantly enhanced when compared to the control group. Correlation analysis demonstrated a positive linear correlation between apoptosis and Fas expression, indicating that TBTC-induced thymocyte apoptosis may be mediated by Fas expression. Taken together, our data clearly demonstrate that TBTC-induced immunotoxicity is associated with thymocyte apoptosis and that this process is mediated by the Fas pathway.

T-cell activation and the development of an apoptosis-resistant CD45RO+ T-cell population.

Growing experimental evidence supports a broadening role for the caspases; not only do they participate in the process of apoptosis but also in the control of the cell cycle and cellular proliferation. The biological role of the caspases in the process of T-cell activation and proliferation is still not defined. In the present study, we propose a potential role, by demonstrating an association of T-cell receptor-mediated caspase activity with the development of an apoptosis-resistant memory CD45RO+ T-cell population. As previously shown by us, a time-dependent induction of caspase activity, in the absence of apoptosis, can be observed in CD3-stimulated human peripheral blood lymphocytes. We here show that a population of CD45RO+ cells, with activated caspase-3 and with resistance to tributyltin-induced apoptosis, develops after 3 days of stimulation. A concomitant expression of the anti-apoptotic protein Bcl-xL accompanied the caspase activity and the development of the apoptosis-resistant phenotype. Finally, upon co-culturing with dexamethasone (DEX), the CD3-induced caspase-3 activity was blocked. During this condition, the expression of the activation marker HLA-DR as well as the cellular proliferative response was strongly suppressed. The development of memory cells with a CD45RO+ phenotype was also blocked. Our data support the hypothesis that caspase-3 activity, observed in CD3-stimulated cells, may be an important component in the proliferation process and, furthermore, might play a role for the development of memory T cells, and DEX inhibits this process.

Immunotoxicity of bis(tri-n-butyltin) oxide in the rat.

Previous studies in the rat have shown that bis(tri-n-butyltin) oxide (TBTO), used as a biocide, was immunotoxic at dose levels that did not affect other organs. In order to determine a no-effect level, weanling rats were treated for at least 28 consecutive days with TBTO at 0, 0.5, 2, 5, or 50 mg/kg of diet. Studies on clinical chemistry, hematology, pathology, and immune function, that is, plaque-forming cell (PFC) assay, delayed-type hypersensitivity (DTH) response, and the splenic clearance of Listeria monocytogenes, were performed at the end of treatment. No treatment-related effects were noted on clinical chemistry and hematology parameters and on PFC and DTH response, whereas thymic atrophy and impaired clearance of L. monocytogenes were noted only at a dietary concentration of 50 mg/kg. These results confirm the thymus as a target organ of TBTO immunotoxicity. Under the conditions of these experiments the dietary concentration of 5 mg/kg, equivalent to a dose of 0.5 mg/kg body weight, represents a no observed effect level (NOEL) for immunotoxicity in the Sprague-Dawley rat.

Immune alterations in rats following subacute exposure to tributyltin oxide.

Adult male Fischer 344 rats were dosed by oral gavage with bis(tri-n-butyltin)oxide (TBTO) in peanut oil for 10 consecutive days, at dosages ranging from 1.25 to 15 mg/kg/day. Other groups of rats were dosed daily for 10 days by oral gavage with cyclophosphamide (CY) at dosages ranging from 0.75 to 6 mg/kg/day. These rats served as positive controls for the immune assays employed. The immune function parameters examined included the following: delayed-type hypersensitivity (DTH) and antibody responses to bovine serum albumin (BSA), primary antibody responses to sheep red blood cells (SRBC) and trinitrophenyl lipopolysaccharide (TNP-LPS) and enumeration of splenic lymphocyte populations. The DTH and antibody responses to BSA were not affected by TBTO exposure; however these responses were suppressed in rats dosed with CY at 6 mg/kg/day. The plaque forming cell (PFC) response to the T cell-dependent antigen SRBC was enhanced in rats dosed with TBTO at from 5 to 15 mg/kg/day. On the other hand, the PFC response to the T cell-independent antigen TNP-LPS was unaffected by TBTO exposure. Rats dosed with CY had suppressed PFC responses to SRBC and TNP-LPS at dosages of 3 and 6 mg/kg/day, respectively. Enumeration of splenic lymphocyte populations from TBTO-exposed rats revealed a reduction in OX8- but not W3/25- or IgG-positive cells. These results, as well as results from an earlier study from this lab, suggest that T lymphocytes are a primary target for TBTO-induced immune alterations and that the enhancement of the PFC response to SRBC in TBTO-exposed rats may be mediated by alterations in the suppressor (OX8-positive) T lymphocyte population.