Tri-n-butyltin)oxide Research Articles

Immunocytological and biochemical analysis of the mode of action of bis (tri-n-butyltin) tri-oxide (TBTO) in Jurkat cells

Bis (tri-n-butyltin) oxide (TBTO) is one of the organotin compounds known to induce immunosuppression. Previously, we examined the effect of TBTO on whole-genome mRNA expression in the human T lymphocyte cell line Jurkat, which led to the hypothesis that induction of endoplasmic reticulum (ER) stress is the first initiated event, which induces a rise of intracellular calcium levels, activation of NF-kB and NFAT, T cell activation response and oxidative stress together finally resulting in apoptosis. The present study verified this hypothesis with biochemical and cytological experiments. The induction of ER stress was confirmed by the rapid raise in protein levels of ATF3 and DDIT3. Moreover, the impairment of cell viability by TBTO was moderated by the ER stress inhibitor phenyl butyric acid. Real-time fluorescence microscopy confirmed that TBTO increases intracellular calcium levels within 2min of exposure. Furthermore, the involvement of increased calcium levels in the effects of TBTO was evident from the induction of three calcium-dependent events: (1) activation of the protease activity of M-calpain, (2) induction of NF-kB (p65) expression, and (3) activation of NFAT. The induction of oxidative stress was verified by detection of increased levels of reactive oxygen species and a decrease in amount of reduced glutathione. We also showed that TBTO induces cleavage of caspase-3, an event known to mediate apoptosis. Finally, comparative microarray data analysis showed that many of the processes observed in vitro also occur in vivo in thymuses of TBTO-treated mice.

Exposure of Jurkat cells to bis (tri-n-butyltin) oxide (TBTO) induces transcriptomics changes indicative for ER- and oxidative stress, T cell activation and apoptosis

Tributyltin oxide (TBTO) is an organotin compound that is widely used as a biocide in agriculture and as an antifouling agent in paints. TBTO is toxic for many cell types, particularly immune cells. The present study aimed to identify the effects of TBTO on the human T lymphocyte cell line Jurkat. Cells were treated with 0.2 and 0.5 μM TBTO for 3, 6, 12 and 24 h and then subjected to whole genome gene expression microarray analysis. The biological interpretation of the gene expression profiles revealed that endoplasmic reticulum (ER) stress is among the earliest effects of TBTO. Simultaneously or shortly thereafter, oxidative stress, activation of NFKB and NFAT, T cell activation, and apoptosis are induced. The effects of TBTO on genes involved in ER stress, NFAT pathway, T cell activation and apoptosis were confirmed by qRT-PCR. Activation and nuclear translocation of NFATC1 and the oxidative stress response proteins NRF2 and KEAP1 were confirmed by immunocytology. Taking advantage of previously published microarray data, we demonstrated that the induction of ER stress, oxidative stress, T cell activation and apoptosis by TBTO is not unique for Jurkat cells but does also occur in mouse thymocytes both ex vivo and in vivo and rat thymocytes ex vivo. We propose that the induction of ER stress leading to a T cell activation response is a major factor in the higher sensitivity of immune cells above other types of cells for TBTO.

P.3.c.057 Which patients switch antipsychotic medication? Data from the international Antipsychotic Drug Substitution Registry

Chronic toxicity, growth and reproduction were measured in the freshwater gastropod Lymnaea stagnalis exposed to waterborne bis(tri-n-butyltin) oxide (TBTO) over a range of four nominal concentrations (0–10 μg TBT l−1). Egg development was completely inhibited at 10 μg TBT l−1, whilst abnormal embryonic development was observed at 1 μg TBT l−1. For the solvent control and the 0.01 μg TBT l−1treatment group, normal development of L. stagnalis was observed. Survivorship of hatchlings was significantly reduced by TBT at 1 μg l−1 while inhibition of shell growth of L. stagnalis was also observed at this concentration. The data were used to determine intrinsic growth rates (r) using two theoretical approaches (the Euler–Lotka equation and a Leslie Matrix). Both approaches showed that survival, fecundity and population growth rate were reduced at 1 μg TBT l−1. Interestingly, at 0.01 μg TBT l−1 snails showed a higher fecundity and growth rate than in the solvent control. The TBT concentration at which the r would equal zero (ECr0) and the population NOEC (No Observed Effect Concentration) were estimated. The population NOEC was defined as either the lower 95% confidence or lower 95% pointwise percentile limit of the ECr0. Values obtained using the two different approaches were similar and thus a geometric mean was calculated to obtain a final representative population NOEC value for L. stagnalis of 2745 ng TBT l−1. The present data together with chronic toxicity TBT data for freshwater organisms, obtained from peer-reviewed literature, were used to construct a species sensitivity distribution (SSD). A predicted no effect concentration was then derived from the SSD (hazardous concentration at 5%, i.e., HC5 or 95% protection level). This SSD was compared with the SSD derived from saltwater species datasets. The HC5 value for saltwater species (3.55 ng TBT l−1; lower confidence limit: 1.93 ng TBT l−1) was significantly lower than that for freshwater species (30.13 ng TBT l−1; lower confidence limit: 9.23 ng TBT l−1), indicating that saltwater species are probably more susceptible to TBT than their freshwater counterparts.

Proteomic analysis of mouse thymoma EL4 cells treated with bis(tri-n-butyltin)oxide (TBTO)

Here, we report the results of proteomic analysis of the mouse thymoma EL4 cell line exposed to bis(tri-n-butylin)oxide (TBTO), an immunotoxic organotin compound. The objective of the work was to examine whether TBTO affects the expression of proteins in this cell line and to compare the differentially expressed proteins with the corresponding mRNA expression data. The identified proteins were quantified using a label-free quantitative method based on counting the observed peptides as an index of protein abundance. The calculation of the ratio of peptides obtained from exposed and control samples allowed us to evaluate the effect of TBTO on protein expression and to compare these results to those obtained in gene expression profiling studies. Correlation of some of the differentially expressed proteins and their corresponding mRNAs was observed. The analysis of the protein ratios revealed that 12 proteins were significantly affected. These proteins included cytoskeleton proteins myosin-9, spectrin beta 2 and plectin 8. The first two proteins were down-regulated 3-fold, whereas the third was up-regulated 2-fold. Ras-related Rab1, a GTP binding protein and T-complex protein-1 subunit alpha, a chaperonin, were decreased 2- and 3.6-fold, respectively. The ribosomal S10 and eukaryotic translation factor (eIf4G1), which are involved in protein synthesis, were down-regulated 2.6- and 3.7-fold, respectively. Also, proteins involved in splicing of pre-mRNA and in transcription, splicing factor arginine/serine-rich 2 and chromodomain-helicase-DNA binding protein 4 (Chd4), were decreased 2.6- and 4.5 times, respectively. Nuclear RNA helicase II was reduced 2.8-fold. Finally, prothymosin-alpha (ProTα), an essential protein for cell proliferation, and a protein similar to ProTα, (with a molecular weight and a pI (3.54) comparable to that of ProTα) were also down-regulated 6-and 8-fold, respectively. We propose that the observed down-regulation of the expression level of ProTα in the TBTO-exposed cells could account for the previously reported anti-proliferative effect of TBTO.

Histopathological effects of chronic aqueous exposure to bis(tri- n-butyltin)oxide (TBTO) to environmentally relevant concentrations reveal thymus atrophy in European flounder ( Platichthys flesus)

Although the use of tributyltin in antifouling paints has been banned, this compound is still a serious pollutant of the marine environment. This paper describes a unique study in which European flounder ( Platichthys flesus) were chronically (8 months) exposed to bis(tri- n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. Residue levels in selected tissues (liver, muscle) and general health status indices were measured and the effects on several organs (gills, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined histopathologically. Additionally, morphometric analysis of the thymus was performed. The major finding is that exposure of flounder to 5 μg TBTO/l over a period of 8 months, resulting in body burdens comparable to high field levels, induced significant reduction of thymus volume, possibly affecting immunocompetence of the animals. Chronic exposure of European flounder to tributyltin is therefore likely to affect the general health status of this species in heavily polluted aquatic environments.

Overlapping gene expression profiles of model compounds provide opportunities for immunotoxicity screening

In order to investigate immunotoxic effects of a set of model compounds in mice, a toxicogenomics approach was combined with information on macroscopical and histopathological effects on spleens and on modulation of immune function. Bis(tri- n-butyltin)oxide (TBTO), cyclosporin A (CsA), and benzo[a]pyrene (B[a]P) were administered to C57BL/6 mice at immunosuppressive dose levels. Acetaminophen (APAP) was included in the study since indications of immunomodulating properties of this compound have appeared in the literature. TBTO exposure caused the most pronounced effect on gene expression and also resulted in the most severe reduction of body weight gain and induction of splenic irregularities. All compounds caused inhibition of cell division in the spleen as shown by microarray analysis as well as by suppression of lymphocyte proliferation after application of a contact sensitizer as demonstrated in an immune function assay that was adapted from the local lymph node assay. The immunotoxicogenomics approach applied in this study thus pointed to immunosuppression through cell cycle arrest as a common mechanism of action of immunotoxicants, including APAP. Genes related to cell division such as Ccna2, Brca1, Birc5, Incenp, and Cdkn1a (p21) were identified as candidate genes to indicate anti-proliferative effects of xenobiotics in immune cells for future screening assays. The results of our experiments also show the value of group wise pathway analysis for detection of more subtle transcriptional effects and the potency of evaluation of effects in the spleen to demonstrate immunotoxicity.

The in vitro effect of bis(tri- n)butyltin)oxide (TBTO) on gene expression in mouse thymocytes

The in vitro effect of bis(tri- n)butyltin)oxide (TBTO) on gene expression in mouse thymocytes

In vitro immunotoxicity of bis(tri-n-butyltin)oxide (TBTO) studied by toxicogenomics

The biocide and environmental pollutant bis(tri-n-butyltin)oxide (TBTO) causes thymus atrophy in rodents. Whether the depletion of thymic lymphocytes by tributyltin compounds may be the result of inhibition of cell proliferation or induction of apoptosis is subject of debate. We examined gene expression profiles in primary rat thymocytes exposed to TBTO in vitro at dose levels of 0, 0.1, 0.3, 0.5, and 1.0μM. By measuring cell viability and apoptosis, exposure conditions were selected that would provide information on changes in gene expression preceding or accompanying functional effects of TBTO. Several processes related to TBTO-induced toxicity were detected at the transcriptome level. Effects on lipid metabolisms appeared to be the first indication of disruption of cellular function. Many transcriptional effects of TBTO at higher dose levels were related to apoptotic processes, which corresponded to present or subsequent thymocyte apoptosis observed phenotypically. The gene expression profile was, however, not unambiguous since expression of apoptosis-related genes was both increased and decreased. Stimulation of glucocorticoid receptor signaling appeared to be a relevant underlying mechanism of action. These findings suggest that TBTO exerts its toxic effects on the thymus primarily by affecting apoptotic processes, but the possibility is discussed that this may in fact represent an early effect that precedes inhibition of cell proliferation. At the highest dose level tested, TBTO additionally repressed mitochondrial function and immune cell activation. Our in vitro toxicogenomics approach thus identified several cellular and molecular targets of TBTO that may mediate the toxicity towards thymocytes and thereby its immunosuppressive effects.

Chronic toxicity of tributyltin to development and reproduction of the European freshwater snail Lymnaea stagnalis (L.)

Chronic toxicity, growth and reproduction were measured in the freshwater gastropod Lymnaea stagnalis exposed to waterborne bis(tri- n-butyltin) oxide (TBTO) over a range of four nominal concentrations (0–10 μg TBT l −1). Egg development was completely inhibited at 10 μg TBT l −1, whilst abnormal embryonic development was observed at 1 μg TBT l −1. For the solvent control and the 0.01 μg TBT l −1treatment group, normal development of L. stagnalis was observed. Survivorship of hatchlings was significantly reduced by TBT at 1 μg l −1 while inhibition of shell growth of L. stagnalis was also observed at this concentration. The data were used to determine intrinsic growth rates ( r) using two theoretical approaches (the Euler–Lotka equation and a Leslie Matrix). Both approaches showed that survival, fecundity and population growth rate were reduced at 1 μg TBT l −1. Interestingly, at 0.01 μg TBT l −1 snails showed a higher fecundity and growth rate than in the solvent control. The TBT concentration at which the r would equal zero (EC r 0) and the population NOEC (No Observed Effect Concentration) were estimated. The population NOEC was defined as either the lower 95% confidence or lower 95% pointwise percentile limit of the EC r 0. Values obtained using the two different approaches were similar and thus a geometric mean was calculated to obtain a final representative population NOEC value for L. stagnalis of 2745 ng TBT l −1. The present data together with chronic toxicity TBT data for freshwater organisms, obtained from peer-reviewed literature, were used to construct a species sensitivity distribution (SSD). A predicted no effect concentration was then derived from the SSD (hazardous concentration at 5%, i.e., HC5 or 95% protection level). This SSD was compared with the SSD derived from saltwater species datasets. The HC5 value for saltwater species (3.55 ng TBT l −1; lower confidence limit: 1.93 ng TBT l −1) was significantly lower than that for freshwater species (30.13 ng TBT l −1; lower confidence limit: 9.23 ng TBT l −1), indicating that saltwater species are probably more susceptible to TBT than their freshwater counterparts.

Gene Expression Profiling of Bis(tri-n-butyltin)oxide (TBTO)-Induced Immunotoxicity in Mice and Rats

Bis(tri-n-butyltin)oxide (TBTO) is one of the organotin compounds that have been used as biocides and occur as persistent environmental pollutants. Human exposure to these compounds occurs through consumption of meat and fish products in which they accumulate. The most sensitive endpoint of TBTO exposure is immunotoxicity. TBTO causes thymus atrophy and thereby interferes with T-lymphocyte-mediated immune responses. Tributyltin compounds have been found to adversely affect a wide range of cellular components and processes in many species, organ systems, and cell types. Both inhibition of proliferation and induction of apoptosis have been observed in thymocytes. We conducted microarray experiments in mice and rats in order to investigate if the immunosuppressive actions of TBTO could be detected by gene expression profiling, and if so, to elucidate the mechanisms of action. Gene expression changes that were detected in mouse thymuses after exposure to a maximum tolerable dose of TBTO correlated to previously observed effects. Most notably, reduction of expression of cell surface determinants and T-cell receptor chains, suppression of cell proliferation, and a possible involvement of nuclear receptors in interference with lipid metabolism by TBTO were observed. The TBTO-induced thymus involution may therefore primarily be caused by inhibition of thymocyte proliferation. In contrast, in rats only limited effects of a lower dose of TBTO were found at the gene expression level in the thymus, even though thymus involution was observed. Here, most gene expression regulation by TBTO was detected in the liver. These preliminary results indicate that gene expression analysis is able to reveal effects of TBTO and to gain insight into its molecular mechanism of action. It may even be a suitable tool to investigate immunotoxicology in general. However, dose and inter-species differences are apparently clearly reflected in the gene expression profiles.

Use of the local lymph node assay in assessment of immune function

The murine local lymph node assay (LLNA) was originally developed as a predictive test method for the identification of chemicals with sensitizing potential. In this study we demonstrated that an adapted LLNA can also be used as an immune function assay by studying the effects of orally administered immunomodulating compounds on the T-cell-dependent immune response induced by the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). C57Bl/6 mice were treated with the immunotoxic compounds cyclosporin A (CsA), bis(tri- n-butyltin)oxide (TBTO) or benzo[ a]pyrene, (B[ a]P). Subsequently, cell proliferation and interferon-γ (IFN-γ) and interleukin (IL)-4 release were determined in the auricular lymph nodes (LNs) after DNCB application on both ears. Immunosuppression induced by CsA, TBTO and B[ a]P was clearly detectable in this application of the LLNA. Cytokine release measurements proved valuable to confirm the results of the cell proliferation assay and to obtain an indication of the effect on Th1/Th2 balance. We believe to have demonstrated the applicability of an adapted LLNA as an immune function assay in the mouse.

In Vitro Cytotoxicity of the Organotin Compound Bis-(tri-n-butyltin)oxide to FG Cells

The FG cell line derived from the gills of flounder paralichthys olivaceus was used to determine the acute cytotoxic effects of the organotin compound bis-(tri-n-butyltin)oxide (TBTO). Its cytotoxic effects were initially measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay, and cell protein assay. Results indicated that the doses of TBTO ranging from 1.7 × 10− 10 to 1.3 × 10− 7 M were all toxic, and no difference in cytotoxicity was found between the three test systems. The transmission electron microscopic examination of TBTO-exposed cells revealed that their ultrastructures were markedly altered, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear membrane dissolution, and increased level of lysosomes. It is clear that the cells are highly susceptible to TBTO. This renders FG cells one of several choices for rapidly evaluating the acute toxicities of organotin compounds like TBTO. The mode of action of TBTO leading to the cytotoxicity, including the ultrastructural alteration in FG cells, is also proposed.

Toxicogenomics: an emerging discipline.

Toxicogenomics: an emerging discipline.

Ecdysteroid synthesis and imaginal disc development in the midge Chironomus riparius as biomarkers for endocrine effects of tributyltin

Acute effects of the endocrine disruptor bis(tri-n-butyltin) oxide (TBTO) on molting-hormone biosynthesis and imaginal-disc development were investigated in larvae of the midge Chironomus riparius (Meigen). Ecdysteroid synthesis was measured by 24-h incubation of molting-hormone-synthesizing tissues (prothoracic glands) in vitro with or without the addition of TBTO. The amount of ecdysteroids produced was analyzed by radioimmunoassay. Developmental effects in vivo were investigated by determining the developmental phase of the genital imaginal discs before and after a 48-h exposure to TBTO in water. Sex-specific effects were found with both endpoints. Ecdysteroid synthesis was significantly reduced (analysis of variance [ANOVA], p < or = 0.005) in female larvae at all concentrations (TBTO-Sn at 50, 500, and 5,000 ng/L), whereas a significant elevation of the biosynthesis rate occurred in male larvae in the 500-ng/L treatment (ANOVA, p < or = 0.05). In vivo experiments with development of the genital imaginal disc within a 48-h exposure period revealed a significantly slower development in female larvae and a significantly faster development in male larvae (contingency tables, p < or = 0.001) at all concentrations tested (TBTO-Sn at 10, 50, 200, and 1,000 ng/L). These results partly coincided with the in vitro effects on molting-hormone synthesis. The 48-h median lethal concentration (LC50) was 25 microg/L (20-30 microg/L 95% confidence intervals). The combination of in vitro and in vivo methods has proven to be a useful approach for the detection of endocrine effects of TBTO in C. riparius at levels 2,000-fold below the LC50 value. High sensitivity and short test duration suggest that chironomids may have potential as freshwater sentinel organisms for endocrine-disrupting chemicals.

Toxicity of the Tributyltin Compound on the Testis in Premature Mice.

In this study, the toxicity of bis (tri-n-butyltin) oxide (TBTO), a marine pollutant, in premature mouse testes and the correlation of a total tin concentration with toxicity were examined. Mice were treated by oral administration twice a week for four weeks from five weeks of age at doses of 0 mg/kg (control), 0.4 mg/kg, 2.0 mg/kg, or 10 mg/kg, and sacrificed on the day following the final administration. The control mice were administered with distilled water containing 0.2% ethanol as vehicle. Removed testes were used for determination of the testicular sperm head counts and for histological study or determination of the total tin concentration. The sperm head count was significantly decreased in the 2.0 mg/kg and 10 mg/kg administration groups, although the testicular weights of both groups were unchanged compared to that of the control. The histological study revealed that TBTO caused vacuolization of Sertoli cells in several seminiferous tubules that failed to organize, however, the frequencies of occurrence were low. The total tin concentration in the testis increased in a dose-dependent manner in inverse proportion to the reduction of sperm head counts. These results suggest that TBTO is potentially an anti-testicular compound in mice.

Toxicology of environmental chemicals in the flounder ( Platichthys flesus) with emphasis on the immune system: field, semi-field (mesocosm) and laboratory studies

European flounder ( Platichthys flesus) has shown an increased prevalence of liver tumors and lymphocystis disease (a viral infection) that correlated with pollution in field research in Dutch coastal and estuarine waters. Semi-field or mesocosm experiments confirmed the supposed causality. Although these types of research are highly relevant for the feral population, laboratory experiments are necessary to establish causal relationships between specific chemical pollutants and disease. Therefore, the effects on flounder of some of the potentially causative chemicals such as benzo[a]pyrene (BaP), dimethyl-benz[a]anthracene (DMBA), 2,3,7,8 tetrachlorodibenzo- p-dioxin (TCDD), 3,3’,4,4’,5 pentachlorobiphenyl (PCB-126), and bis(tri- n-butyltin)oxide (TBTO) were examined in several laboratory experiments. These effects were evaluated using general toxicological parameters and histopathology. For immune function assessment, attempts to develop an infection model with the lymphocystis virus were made, but appeared unsuccessful and immune function tests are not fully operational at the moment. Flounder has been successfully maintained and exposed to toxic substances in captivity in our laboratory. Short-term aqueous exposure to high levels of BaP or DMBA did not induce marked effects under our experimental conditions. Results of oral exposure of flounder to low levels of TCDD, PCB-126 or harbor sludge extract show significant induction of cytochrome P4501A (CYP1A) in hepatocytes. Oral exposure to high levels of TCDD or PCB-126 also significantly induced CYP1A immunoreactivity in epithelium in mesonephros and digestive tract and in endothelium in several organs. Remarkable was the induction of CYP1A in a distinct population of mononuclear cells in the mesonephros. Moreover, oral exposure to TCDD resulted in an increased mitotic activity and an increase of the hepatosomatic index in the 20 and 500 μg TCDD/kg group respectively. Therefore, exposure to TCDD and related substances may promote the development of liver tumors in the field. Exposure to PCB-126 also significantly reduced the relative thymus volume, but other results indicate that flounder is relatively insensitive to this type of chemicals. Short-term aqueous exposure of flounder to TBTO, in concentrations that were in the same order of magnitude as upper TBT levels measured in the field, caused mortality after 7–12 days associated with gill lesions, and induced reduction of the non-specific resistance and decrease of the relative thymus volume. From these results we therefore conclude that TBTO might play a causal role in, for instance, increased prevalence of lymphocystis virus infections in the field

Some sulfur-containing metabolites of tri-n-butyltin chloride in male rats.

In an attempt to elucidate metabolic destination of TBTO, sulfur-containing metabolites were investigated in the urine. Tri-n-butyltin chloride (TBTC), tri-n-butyltin oxide (TBTO), and their in vitro metabolites in rat liver microsomal enzyme systems, di-n-butyl(3-hydroxybutyl)tin chloride (T3OH), di-n-butyl(3-oxobutyl)tin chloride (T3CO), dibutyltin dichloride (DBTC), and monobutyltin trichloride (MBTC), were intraperitoneally administered to rats. In particular, administration of T3OH and T3CO gave higher amounts of mercapturic acid derivatives, such as N-acetyl-S-(3-oxobutyl)-L-cysteine (3CO-MA) and N-acetyl-S-(3-hydroxybutyl)-L-cysteine (3OH-MA), than TBTC or TBTO. On the other hand, DBTC and MBTC did not yield measurable amounts of 3CO-MA and/or 3OH-MA. The appearance of organotin metabolites in urine indicates that T3OH, T3CO, and hypothesized secondary metabolites, such as n-butyl(3-hydroxybutyl)(3-oxobutyl)tin chloride, n-butyl(3-hydroxybutyl)(4-hydroxybutyl)tin chloride, etc., are subject to the action of glutathione S-transferase to give mercapturic acid derivatives. These sulfur-containing metabolites (3CO-MA and 3OH-MA) were also found in control rat urine.

In vitro exposure effects of cyclosporin A and bis(tri- n-butyltin)oxide on lymphocyte proliferation, cytokine (receptor) mRNA expression, and cell surface marker expression in rat thymocytes and splenocytes

Rat thymocytes and splenocytes were exposed in vitro to the model compounds Cyclosporin A (CsA), an immunosuppressive drug, and bis(tri- n-butyltin)oxide (TBTO), an immunotoxic environmental contaminant. The lymphocyte transformation test (LTT), cytokine (receptor) mRNA expression (RT-PCR and dot blot hybridisation), and flow cytometry were evaluated as assays for in vitro immunotoxicity, at dose levels that did not show effects on viability, this being the aim of the study. LTT and RT-PCR proved useful assays. Lymphocyte transformation was suppressed by both compounds, while IL-2 mRNA expression was suppressed by CsA but not by TBTO, and both compounds suppressed IL-2R mRNA expression in splenocytes but not in thymocytes. Furthermore, the data obtained suggest that antiproliferative effects may be more relevant than apoptosis induction for TBTO induced thymus atrophy.

Risk assessment and immunotoxicology

In general toxicity testing, maximal acceptable concentrations are derived from no-observed adverse effect levels (NOAEL) in rodents. Risk assessment then considers safety factors for the interspecies difference, and intraspecies variability. This approach can be used for assessing maximal acceptable concentrations for chemicals inducing direct immunotoxicity, resulting in e.g. reduced resistance to infections. As for predictive testing of chemicals in terms of sensitization, laboratory animal data are mostly used for risk assessment as well. Generally, the assessment of risk for chemicals that induce contact sensitivity is limited to hazard identification, and risk management is restricted to labeling. An alternative type of evaluation of the risk of adverse effects due to exposure to immunotoxic chemicals may be the so called parallellogram approach. In this parallellogram there are four cornerstones, one of which is the health effect of exposure to a chemical, assessed as an endpoint (e.g. infection model) in experimental animals, and another the quantitative prediction of this endpoint in humans. The other cornerstones are assays of parameters that are relevant to the mechanism of the adverse effect in experimental animals and humans, and are used for species comparison. Species comparisons between the animal species used for hazard identification and humans are crucial for extrapolation of animal data to the human situation. This approach can be used to provide relevant information on the dose-response relationship in humans. In concert with information on actual exposure, such data can then be used for the characterization of risk for adverse health effects in humans. Such approaches have been used for chemicals that exert direct immunotoxic activity (bis(tri-n-butyltin)oxide (TBTO)), and may hold promise for the risk evaluation of chemicals that exert skin sensitizing properties.

Short-term toxicity of bis(tri- n-butyltin)oxide in flounder ( Platichthys flesus): Pathology and immune function

The present study is part of a project that focuses on the relationship between environmental pollution and fish diseases. Field studies in various polluted coastal areas in Europe and the United States of America clearly indicate a relationship between pollution and the increase in prevalence of tumours and infectious diseases in fish. Research under controlled laboratory conditions is necessary to prove causal links between specific xenobiotics and disease prevalence. One of the chemicals of interest in the myriad of xenobiotics found in polluted waters and sediments is the organotin compound tributyltin (TBT), originating mainly from antifouling paints used on the hulls of ships. This report describes a study in which flounders ( Platichthys flesus) were exposed to bis(tri- n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. The effects on several organs (gills, skin, eye, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined using histopathology, and morphometric analysis of the thymus was performed to assess the target organ(s) for TBTO in this fish species. Also the function of the non-specific and specific resistance was studied using ex vivo/in vitro immune function tests. Exposure of flounder to TBTO, in concentrations which were in the same order of magnitude as maximum TBT levels measured in the field (experiment: 17.3 μg TBT/l; field: 7.2 μg TBT/l), caused mortality after 7–12 days, resulted in gill lesions, and induced significant reduction of the non-specific resistance. A significant decrease of the relative thymus volume, but no marked effects on the specific immune system were noted after exposure to TBTO.