Endocrine disruptors found in food contaminants enhance allergic sensitization through an oxidative stress that promotes the development of allergic airway inflammation.

In the past few decades, there has been a significant increase in incidence of allergic diseases. The hygiene hypothesis may provide some clues to explain this rising trend, but it may also be attributable to other environmental factors that exert a proallergic adjuvant effects. However, there is limited information on the risks of developing allergic asthma and related diseases through the ingestion of environmental chemicals found in food contaminants. In the present study, we have shown that oral administration of tributyltin, used as a model environmental chemical, induced oxidative-stress status in the bronchial lymph node, mesenteric lymph node and spleen, but not in the lung, where the initial step of allergic asthma pathogenesis takes place. Mice exposed to tributyltin exhibited heightened Th2 immunity to the allergen with more severe airway inflammation. Tributyltin also induced Treg cells apoptosis preferentially over non-Treg cells. All these effects of tributyltin exposure were canceled by the administration of glutathione monoethyl ester. Meanwhile, tributyltin did not affect airway inflammation of mice transferred with allergen-specific Th2 cells. Collectively, these results suggest that tributyltin exerts its pathological effect during the sensitization phase through oxidative stress that enhances the development of allergic diseases. The current study dissects the pathogenic role of oxidative stress induced by oral exposure to an environmental chemical during the sensitization phase of allergic airway inflammation and would be important for developing therapeutics for prevention of allergic diseases.

Tributyltin in blood of marine fish collected from a coastal area of northern Kyushu, Japan.

We investigated levels of the pollutant tributyltin (TBT) in blood of pufferfishes (six species), Japanese sea perch, red sea bream, Japanese common goby, Japanese flounder, rockfish, conger eel, and sea mullet collected off the coast of northern Kyushu, Japan. We found considerable levels of TBT (1.4-190 ng/mL) accumulated in the blood of these fish. Blood TBT concentrations were 1.3-22.5 times liver concentrations and 4.9-78 times muscle concentrations, except in conger eel and mullet. We detected TBT (16-111 ng/mL-blood) in the plasma of the fine-patterned puffer (Takifugupoecilonotus) year-round, without any apparent seasonal trend. These results suggest that fish inhabiting coastal areas of Kyushu, Japan, continue to be contaminated with TBT.

Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder, Paralichthys olivaceus.

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon-intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two alpha-helices and eight beta-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6mug/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.

Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro.

The widespread environmental contamination, bioaccumulation and endocrine disruptor effects of butyltins (BTs) to wildlife are well documented. Although suspected, potential effects of BTs exposure on the immune system of marine mammals have been little investigated. In this study, we assessed the effects of tributyltin (TBT) and its dealkylated metabolites dibutyltin (DBT) and monobutyltin (MBT) on the immune responses of harbour seals. Peripheral blood mononuclear cells isolated from pup and adult harbour seals were exposed in vitro to varying concentrations of BTs. DBT resulted in a significant decrease at 100 and 200 nM of phagocytotic activity and reduced significantly phagocytic efficiency at 200 nM in adult seals. There was no effect in phagocytosis with TBT and MBT. In pups, the highest concentration (200 nM) of DBT inhibited phagocytic efficiency. A reduction of tumor-killing capacity of adult natural killer (NK) cells occurred when leukocytes were incubated in vitro with 50 nM DBT and 200 nM TBT for 24h. In adult seals, T-lymphocyte proliferation was significantly suppressed when the cells were exposed to 200 nM TBT and 100 nM DBT. In pups, the proliferative response increased after an exposure to 100 nM TBT and 50 nM DBT, but decreased with 200 nM TBT and 100 nM DBT. The immune functions were more affected by BTs exposure in adults than in pups, suggesting that other unsuspected mechanisms could trigger immune parameters in pups. The toxic potential of BTs followed the order of DBT>TBT>MBT. BT concentrations of harbour seal pups from the St. Lawrence Estuary (Bic National Park) ranged between 0.1-0.4 ng Sn/g wet weight (ww) and 1.2-13.4 ng Sn/g ww in blood and blubber, respectively. For these animals, DBT concentrations were consistently below the quantification limit of 0.04 ng Sn/g ww in blood and 0.2 ng Sn/g ww in blubber. Results suggest that concentrations measured in pups are considered too low to induce toxic effects to their immune system during first days of life. However, based on our in vitro results, we hypothesize that BTs, and DBT in particular, could pose a serious threat to the immune functions in free-ranging harbour seal adults.

Organotin speciation and tissue distribution in rat dams, fetuses, and neonates following oral administration of tributyltin chloride.

Tributyltin (TBT) is a biocide that contaminates human foodstuffs, especially shellfish. TBT is an endocrine disrupter, producing imposex in several marine gastropods. Previous studies showed that oral dosing of rat dams with TBT chloride leads to abnormal fetal and postnatal development. In this study, the tissue distribution and speciation of organotins in tissues were examined in dams, fetuses, and neonates following dosing of rat dams commencing on gestational day (GD) 8 by oral gavage with TBT in olive oil at 0, 0.25, 2.5, or 10 mg/kg body weight (BW)/d. Dams' body weights were significantly reduced by the 10-mg/kg BW/d TBT treatment. At GD20, there were no significant effects of any TBT treatment on pup weights, litter size, sex ratio, or tissue weights. However, at postnatal day (PND) 6 and 12, neonatal pup weights were reduced by the 10-mg/kg BW/d TBT treatment but tissue weights were unaffected, except for the liver weight of female pups, which was reduced by the 10-mg/kg BW/d TBT treatment. Tissues harvested on GD20 and PND6 and PND12 were extracted for determination of organotins by gas chromatography-atomic emission detection (GC-AED). In most tissues, TBT and its metabolite dibutyltin (DBT) were evident but monobutyltin (MBT) was rarely measured above the detection limit. The livers and brains of fetuses contained TBT and DBT at levels that were approximately 50% of the equivalent tissues in the dams. Furthermore, these tissues appeared to preferentially absorb/retain organotins, since the concentrations were greater than were found for the total loading in whole pups. The placenta also contained relatively large quantities of TBT and DBT. Postnatally, the TBT levels in pups decreased markedly, a probable consequence of the extremely low levels of organotins in rat milk. However, DBT levels in pups livers and brains were maintained, probably due to metabolism of TBT to DBT. Similarly, while dams' spleens contained significant quantities of organotins, the pups' spleens contained smaller quantities, and these decreased rapidly between PND6 and PND12. These results show that organotins cross the placenta and accumulate in fetal tissues but that during lactation, the pups would receive minimal organotins through the milk and during this period, the levels of TBT in pups' tissues decreases rapidly. Consequently, fetuses would be at greater risk of the adverse effects of TBT, but due to the lack of transfer through milk, the risk would be reduced during the lactational period.

Effects of in utero tributyltin chloride exposure in the rat on pregnancy outcome.

Tributyltin, an organotin, is ubiquitous in the environment. The consumption of contaminated marine species leads to human dietary exposure to this compound. Tributyltin is an endocrine disruptor in many wildlife species and inhibits aromatase in mammalian placental and granulosa-like tumor cell lines. We investigated the effects of tributyltin chloride exposure on pregnancy outcome in the Sprague-Dawley rat. Timed pregnant rats were gavaged either with vehicle (olive oil) or tributyltin chloride (0.25, 2.5, 10, or 20 mg/kg) from days 0-19 or 8-19 of gestation. On gestational day 20, dams were sacrificed, and pregnancy outcome was determined. Tributyltin and its metabolites (dibutyltin, monobutyltin) were measured in maternal blood by gas chromatography. Both tributyltin and dibutyltin were present in maternal blood at approximately equal concentrations, whereas monobutyltin contributed minimally to total organotins. Organotin concentrations increased in a dose-dependent pattern in dams, independent of the window of exposure. Tributyltin chloride administration significantly reduced maternal weight gain only at the highest dose (20 mg/kg); a significant increase in post-implantation loss and decreased litter sizes, in addition to decreased fetal weights, was observed in this group. Tributyltin chloride exposure did not result in external malformations, nor was there a change in sex ratios. However, exposure to 0.25, 2.5, or 10 mg/kg tributyltin chloride from gestation days (GD) 0-19 resulted in a significant increase in normalized anogenital distances in male fetuses; exposure from days 8-19 had no effect. There was a dramatic increase in the incidence of low weight (< or =0.75 of the mean) fetuses after exposure to 20 mg/kg tributyltin chloride. Delayed ossification of the fetal skeleton was observed after in utero exposure to either 10 mg/kg or 20 mg/kg tributyltin chloride. Serum thyroxine and triiodothyronine levels were reduced significantly in dams exposed to 10 and 20 mg/kg tributyltin chloride throughout gestation; in dams treated with tributyltin from GD 8-19, serum thyroxine concentrations, but not triiodothyronine, were significantly decreased at both the 2.5 and 10 mg/kg exposures. Thus, maternal thyroid hormone homeostasis may be important in mediating the developmental toxicity of organotins.

Accumulation of tributyltin in the blood of fish: its application for monitoring in the marine environment.

Accumulation of tributyltin (TBT) in serum, liver, muscle, and gill of olive flounder (Paralichthys olivaceus) was examined in a 30-d static-renewal exposure. Tributyltin accumulated rapidly in the serum of the olive flounder and to a greater extent than in the other tissues. The accumulated TBT concentrations in tissues were in the order of serum > gill > liver > muscle on a dry-weight basis. Tributyltin also was detected in the serum of feral fine-spotted flounder, Pleuronichthys cornutus, collected from the coastal area. The mean TBT concentration in serum (2,470 ng Sn/g) of the fine-spotted flounder was about 40 times higher than that in the liver (60 ng Sn/g) and 200 times higher than that in the muscle (27 ng Sn/g) on a dry-weight basis. The TBT concentrations in serum and sediment demonstrated a positive correlation. The percent TBT composition to total butyltin was much higher in the serum (71%) than in the other tissues and sediment (<47%). These results suggest that the analysis of fish blood serum could be a useful tool for monitoring exposure to TBT in the marine environment.

Immunotoxicity of environmentally relevant concentrations of butyltins on human natural killer cells in vitro.

The widespread environmental contamination, bio-accumulation, and toxic effects of butyltins (BTs) in wildlife is well documented, but the role of BTs in debilitating human immune function mediated through natural killer (NK) lymphocytes (a primary immune defense against tumor and virally infected cells) has not been described. In this study, we assessed the effects of in vitro exposure to a range of concentrations (encompassing environmentally relevant concentrations) of MBT, DBT, and TBT on human natural killer lymphocytes obtained from adult male and female donors. TBT inhibited the tumor-killing capacity of NK cells when the NK cells were pretreated in vitro at 200 nM for as little as 1 h. Inhibition of NK cytotoxic function ranged from 40 to greater than 90%. The toxic potential of butyltins followed the order of TBT > DBT > MBT. Conjugation assays revealed that after a 24-h exposure to TBT, there was about a 50% decrease in NK cell binding to tumor cells, indicating alteration of the NK cell receptors for tumor cells. Analysis of whole-blood samples for BTs revealed the presence of detectable concentrations of MBT, DBT, and TBT in all of the donors, indicating possible exposure of NK cells to BTs in the blood. The results of this study provide evidence that butyltin compounds significantly inhibit NK cell function and possible NK cell-mediated immunotoxic potential in humans.

Effect of bis (tributyl tin) oxide on permeability of the blood-brain barrier: a transient increase.

OBJECTIVES: To study the effect of bis (tributyl tin) oxide (TBTO) on permeability of the blood-brain barrier. METHODS: Electron microscopy and an x ray microanalyser with lanthanum chloride as a tracer were used, and blood tin concentrations were determined with an atomic absorption spectrophotometer. Adult male wistar rats received 0.05 ml/kg body weight of TBTO orally. RESULTS: A transient increase in paracellular permeability at the blood-brain barrier was found 2 h after the dose of TBTO. Electron dense lanthanum deposits penetrated tight junctions of the endothelia and permeated the subendothelial space. The x ray microprobe data showed an accumulation of TBTO at the tight junctions at 2 h. Leakage of tracer did not occur at 4 h, but oedematous changes in the surrounding glial cells were prominent between 4 and 8 h and had almost returned to normal by 24 h. By atomic absorption analysis, it was seen that blood tin concentrations rapidly increased at 1 h and rose to a maximum peak at 8 h, then gradually decreased to reach zero at 24 h. CONCLUSIONS: Accumulated TBTO at tight junctions could have caused the temporary replacement of calcium ion by tin, which induces a transient increase in paracellular permeability throughout the blood-brain barrier.

The ultrastructural localization of tri-n-butyltin in human erythrocyte membranes during shape transformation leading to hemolysis.

Scanning electron microscopy, transmission electron microscopy, freeze-fracture, and x-ray energy dispersive spectrometry were used to localize tri-n-butyltin (TBT) in human erythrocytes (RBC's). TBT induced a rapid shape transformation of the RBC discocyte to an echinocyte, which led to hemolysis at concentrations at or above 10 microM TBT. Electron dense spheres or ellipsoids were observed in association with blood cell membranes at or above 10 microM TBT. These structures were visualized initially in thin sections when postfixed with osmium tetroxide. Control cell preparations without TBT did not exhibit these structural densities when fixed with osmium. Freeze-fracture replicas confirmed the presence of TBT aggregates associated with cell membranes as intercalations in the lipid bilayer. In thin sections, these structures measured 71.5 +/- 18.2 nm in diameter. In freeze-fracture replicas of TBT-treated RBC's, particulate structures measuring 60 +/- 18.5 nm in diameter were present on membrane exoplasmic fracture faces and 59.6 +/- 10.8-nm depressions on membrane protoplasmic fracture faces. Qualitative x-ray energy dispersive spectrometry analysis of ultrathin sections of glutaraldehyde-carbohydrazide-embedded samples revealed that the membrane-associated aggregates contained tin. TBT-treated RBC's that were washed with normal saline resulted in a paucity of TBT aggregates associated with the membranes and a reduction in the RBC hemolysis rate. RBC shape transformation occurred at each concentration examined from 0.1 to 100 microM TBT, but was reversible below 1 microM TBT.