Tributyltin in male mice disrupts glucose homeostasis as well as recovery after exposure: mechanism analysis.

Organotin compounds such as tributyltin (TBT) and triphenyltin can induce diabetes and insulin resistance. However, the development of diabetes caused by organotins and its underlying mechanisms remain unclear. In the present study, male KM mice were orally administered with TBT (0.5, 5, and 50 µg/kg) once every 3 days for 45 days. Their body weights increased and reached a significant difference compared to the control, and the fasting blood glucose levels were significantly elevated. The fasting levels of serum insulin and adiponectin increased, while glucagon levels decreased in the animals treated with TBT. The expression of the insulin receptor (IR) signaling cascade, including IR, IR substrate, phosphatidylinositol 3-kinase, Akt, and glucose transporter 4, was inhibited both in the skeletal muscle and the liver, which might be a main reason for the hyperglycemia and hyperinsulinemia. After removing the TBT stress for 60 days, the animals which had received exposure to TBT could recover normoglycemia, accompanied with a recovery of the suppressed IR signal pathway and fasting insulin levels. However, the fasting levels of serum adiponectin and glucagon were lower than that of the control mice, which would remain a potential risk inducing the disruption of glucose homeostasis.

Tributyltin exposure at noncytotoxic doses dysregulates pancreatic ß-cell function in vitro and in vivo.

Tributyltin (TBT) is an endocrine disruptor. TBT can be found in food and in human tissues and blood. Several animal studies revealed that organotins induced diabetes with decreased insulin secretion. The detailed effect and mechanism of TBT on pancreatic ß-cell function still remain unclear. We investigated the effect and mechanism of TBT exposure at noncytotoxic doses relevant to human exposure on ß-cell function in vitro and in vivo. The ß-cell-derived RIN-m5F cells and pancreatic islets from mouse and human were treated with TBT (0.05-0.2 µM) for 0.5-4 h. Adult male mice were orally exposed to TBT (25 µg/kg/day) with or without antioxidant N-acetylcysteine (NAC) for 1-3 weeks. Assays for insulin secretion and glucose metabolism were carried out. Unlike previous studies, TBT at noncytotoxic concentrations significantly increased glucose-stimulated insulin secretion and intracellular Ca2+ ([Ca2+]i) in ß-cells. The reactive oxygen species (ROS) production and phosphorylation of protein kinase C (PKC-pan) and extracellular signal-regulated kinase (ERK)1/2 were also increased. These TBT-triggered effects could be reversed by antiestrogen ICI182780 and inhibitors of ROS, [Ca2+]i, and PKC, but not ERK. Similarly, islets treated with TBT significantly increased glucose-stimulated insulin secretion, which could be reversed by ICI182780, NAC, and PKC inhibitor. Mice exposed to TBT for 3 weeks significantly increased blood glucose and plasma insulin and induced glucose intolerance and insulin resistance, which could be reversed by NAC. These findings suggest that low/noncytotoxic doses of TBT induce insulin dysregulation and disturb glucose homeostasis, which may be mediated through the estrogen receptor-regulated and/or oxidative stress-related signaling pathways.

Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats.

Tributyltin chloride (TBT) is an organometallic pollutant that is used as a biocide in antifouling paints. TBT induces several toxic and endocrine-disrupting effects. However, studies evaluating the effects of TBT on renal function are rare. This study demonstrates that TBT exposure is responsible for improper renal function as well as the development of abnormal morphophysiology in mammalian kidneys. Female rats were treated with TBT, and their renal morphophysiology was assessed. Morphophysiological abnormalities such as decreased glomerular filtration rate and increased proteinuria levels were observed in TBT rats. In addition, increases in inflammation, collagen deposition and α-smooth muscle actin (α-SMA) protein expression were observed in TBT kidneys. A disrupted cellular redox balance and apoptosis in kidney tissue were also observed in TBT rats. TBT rats demonstrated reduced serum estrogen levels and estrogen receptor-α (ERα) protein expression in renal cortex. Together, these data provide in vivo evidence that TBT is toxic to normal renal function and that these effects may be associated with renal histopathology complications, such as inflammation and fibrosis.

Disruption of thyroid hormone functions by low dose exposure of tributyltin: an in vitro and in vivo approach.

Triorganotins, such as tributyltin chloride (TBTCl), are environmental contaminants that are commonly found in the antifouling paints used in ships and other vessels. The importance of TBTCl as an endocrine-disrupting chemical (EDC) in different animal models is well known; however, its adverse effects on the thyroid gland are less understood. Hence, in the present study, we aimed to evaluate the thyroid-disrupting effects of this chemical using both in vitro and in vivo approaches. We used HepG2 hepatocarcinoma cells for the in vitro studies, as they are a thyroid hormone receptor (TR)-positive and thyroid responsive cell line. For the in vivo studies, Swiss albino male mice were exposed to three doses of TBTCl (0.5, 5 and 50µg/kg/day) for 45days. TBTCl showed a hypo-thyroidal effect in vivo. Low-dose treatment of TBTCl exposure markedly decreased the serum thyroid hormone levels via the down-regulation of the thyroid peroxidase (TPO) and thyroglobulin (Tg) genes by 40% and 25%, respectively, while augmenting the thyroid stimulating hormone (TSH) levels. Thyroid-stimulating hormone receptor (TSHR) expression was up-regulated in the thyroid glands of treated mice by 6.6-fold relative to vehicle-treated mice (p<0.05). In the transient transactivation assays, TBTCl suppressed T3 mediated transcriptional activity in a dose-dependent manner. In addition, TBTCl was found to decrease the expression of TR. The present study thus indicates that low concentrations of TBTCl suppress TR transcription by disrupting the physiological concentrations of T3/T4, followed by the recruitment of NCoR to TR, providing a novel insight into the thyroid hormone-disrupting effects of this chemical.

Supramolecular assemblies based on complexes of nonionic amphiphilic cyclodextrins and a meso-tetra(4-sulfonatophenyl)porphine tributyltin(IV) derivative: potential nanotherapeutics against melanoma.

Amphiphilic cyclodextrin (ACyD) provides water-soluble and adaptable nanovectors by modulating the balance between the hydrophobic and hydrophilic chains at both CyD sides. This work aimed to design nanoassemblies based on nonionic and hydrophilic ACyD (SC6OH) for the delivery of a poor-water-soluble organotin(IV)-porphyrin derivative [(Bu3Sn)4TPPS] to melanoma cancer cells. To characterize the porphyrin derivatives under simulated physiological conditions, a speciation was performed using complementary techniques. In aqueous solution (≤ 20 µM), (Bu3Sn)4TPPS primarily exists as a monomer (2 in Figure 1), as suggested by the low static anisotropy (ρ ≈ 0.02) with a negligible formation of porphyrin supramolecular aggregates. MALDI-TOF spectra indicate the presence of moieties (i.e., [(Bu3Sn)3TPPS](-)) that are derivatives of the monomeric species. Spectrofluorimetry coupled with potentiometric measurements primarily assesses the presence of the hydrolytic [(Bu3Sn)4TPPS (OH)4](4-) species under physiological conditions. Nanoassemblies of (Bu3Sn)4TPPS/SC6OH were prepared by dispersion of organic films in PBS at pH 7.4 and were investigated using a combination of spectroscopic and morphological techniques. The UV-vis and emission fluorescence spectra of the (Bu3Sn)4TPPS/SC6OH reveal shifts in the peculiar bands of the organotin(IV)-porphyrin derivative due to its interaction with the ACyD supramolecular assemblies in aqueous solution. The mean size was within the range of 100-120 nm. The ξ-potential was negative (-16 mV) for the (Bu3Sn)4TPPS/SC6OH nanoassemblies, with an entrapment efficiency of approximately 67%. The intracellular delivery, cytotoxicity, nuclear morphology and cell growth kinetics were evaluated via fluorescence microscopy on A375 human melanoma cells. The delivery of (Bu3Sn)4TPPS by ACyD with respect to free (Bu3Sn)4TPPS increases the internalization efficiency and cytotoxicity to induce apoptotic cell death and, at lower concentrations, changes the cellular morphology and prevents cell proliferation.

Effects of low dose treatment of tributyltin on the regulation of estrogen receptor functions in MCF-7 cells.

Endocrine disrupting chemicals are the natural/synthetic compounds which mimic or inhibit the actions of endogenous hormones. Organotin compounds, such as tributyltin (TBT) are typical environmental contaminants and suspected endocrine-disrupting chemical. The present study evaluates the estrogenic potential of this compound in vitro in ER (+) breast adenocarcinoma, MCF-7 cell line. Our data showed that tributyltin chloride (TBTCl) had agonistic activities for estrogen receptor-α (ER-α). Its estrogenic potential was checked using cell proliferation assay, aromatase assay, transactivation assay, and protein expression analysis. Low dose treatment of TBTCl had a proliferative effect on MCF-7 cells and resulted in up-regulation of aromatase enzyme activity and enhanced estradiol production in MCF-7 cells. Immunofluorescence staining showed translocation of ER-α from cytoplasm to nucleus and increased expression of ER-α, 3ß-HSD and aromatase on treatment with increasing doses of TBTCl. Further, to decipher the probable signaling pathways involved in its action, the MCF-7 cells were transfected with different pathway dependent luciferase reporter plasmids (CRE, SRE, NF-κB and AP1). A significant increase in CRE and SRE and decrease in NF-κB regulated pathway were observed (p<0.05). Our results thus showed that the activation of SRE by TBTCl may be due to ligand dependent ER-α activation of the MAPK pathway and increased phosphorylation of ERK. In summary, the present data suggests that low dose of tributyltin genomically and non-genomically augmented estrogen dependent signaling by targeting various pathways.

Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts.

The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1-10nM TBT, 0.01-1nM TCDD or 10nM TBT+ 1nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption.

Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland.

The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman's reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues.

Induction of tributyltin-binding protein type 2 in Japanese flounder, Paralichthys olivaceus, by exposure to tributyltin-d27.

In this study, individual Japanese flounder were intraperitoneally injected with 2 µg tributyltin-d27 (TBT-d27) fish⁻¹. Blood samples were collected on day 7 after injection. TBT-binding protein types 1 and 2 (TBT-bp1, -bp2) in the blood serum were quantified by western blotting analysis. As a result, the concentration of TBT-bp2 in TBT-d27 treated group increased to 220% of that in the solvent control, whereas the TBT-bp1 concentration decreased to 65% of that in the solvent control. Additionally, a positive relationship between the concentrations of TBT-bp2 and TBT was observed in blood sera of wild and cultured flounder. We suggest that TBT-bp2 is produced in response to TBT exposure and may play an important role in fish physiology.

Peripubertal exposure to low doses of tributyltin chloride affects the homeostasis of serum T, E2, LH, and body weight of male mice.

Previous studies have shown that tributyltin could act as an endocrine disruptor in mammals. However, the data on the low-dose effect of tributyltin in animals are still lacking. The objective of this study was to demonstrate the endocrine disruption induced by low levels of tributyltin chloride (TBTCl) in male KM mice. The animals were treated with 0.05 or 0.5 mg TBTCl/kg body weight/3 days from postnatal days (PNDs) 24 to 45, and killed on PNDs 49 and 84, respectively. Mice treated with 0.5 mg TBTCl/kg exhibited decreased serum and intratesticular testosterone (T) levels on PND 49 and then followed by an obvious recovery on PND 84. Furthermore, mice treated with 0.05 mg TBTCl/kg showed reduced serum 17ß-estradiol (E2) levels on PND 49. However, treatments with TBTCl resulted in a dose-dependent increase in serum E2 concentration of the mice on PND 84. Administration of TBTCl also decreased levels of serum luteinizing hormone and intratesticular E2 on PND 84. In addition, mice exposed to 0.05 mg/kg TBTCl exhibited an increase in body weight in the late stage of the experiment. These results indicate that treatment with low doses of TBTCl could disturb hormone homeostasis and body weight gain in rodents, and exposure to different levels of TBTCl might have different effects on changing some physiologic parameters.

Interspecies differences in the accumulation of tributyltin and its metabolites under dietary exposure in sea perch, Lateolabrax japonicus, and red sea bream, Pagrus major.

Interspecies differences in the accumulation of dietary tributyltin (TBT) between sea perch, Lateolabrax japonicus, and red sea bream, Pagrus major, were studied. Although TBTs in both species reached steady-state condition in low-concentration group (L-group, 200 ng/g nominal concentration) by 1 week, it increased up to the end of exposure in high-concentration group (H-group, 3000 ng/g nominal concentration). In H-group, the accumulation rate of TBT in sea perch from 1 to 2 weeks was much higher than in red sea bream, which were 2.4-fold for sea perch and 1.7-fold for red sea bream, although TBT concentrations were similar between sea perch and red sea bream in L-group. Furthermore, in the H-group, the concentrations of TBT at 1 and 2 weeks were about 1.3- and 1.9-fold, respectively, higher in the sea perch than in the red sea bream. On the other hand, DBT residue in red sea bream was about 1.4-fold higher in sea perch for the L-group but concentrations were similar in both fishes for the H-group. These results suggest that red sea bream could metabolize faster the TBT to DBT than sea perch. This study also reveals that fish probably could absorb TBT through the food chain. The uptake of TBT by fish should be regarded in the real environment, because TBT concentration in seawater has been decreasing and now already at significantly low level.

Acute exposure to tributyltin induces c-fos activation in the hypothalamic arcuate nucleus of adult male mice.

Tributyltin (TBT) is a largely diffused environmental pollutant, banned from paints in the European Union from 2003. However, the level of TBT (and other organotins) in food, particularly fish and shellfish, remains still high. Several studies demonstrated that TBT is involved in the development of obesity, via peripheral action, but currently, there are only a few data illustrating effects of TBT on the nervous system. In the present study, we tested the hypothesis that acute exposure to TBT may directly activate brain cells in particular, in those hypothalamic nuclei regulating the food intake. To this purpose, TBT was orally administered at a single dose (10 mg/kg/body weight) to two groups of adult male mice: regularly fed or fasted for 24 h. Mice were sacrificed 90 min after the TBT administration and perfused by 4% paraformaldehyde. Brains were quickly dissected, frozen and sectioned for immunocytochemical detection of c-fos, a common marker of cell activation. In both, fed or fasted mice, exposure to TBT induced a significant increase of c-fos expression in the arcuate nucleus in comparison to control mice. The other nuclei involved in the control of feeding behavior did not show any significant increase. These data are the first in vivo demonstration that TBT has not only peripheral effects, but also may activate elements in the brain, in particular in a crucial region for the regulation of food intake like the arcuate nucleus.

Mechanism underlying the olfactory disturbance induced by an intraperitoneal injection of tributyltin chloride in rats.

Acute intoxication by tributyltin compounds has been known to induce olfactory disturbances, although the underlying mechanism remains unclear. This study investigates the acute effect of tributyltin chloride (TBTC) on the olfactory bulb in rats. The time-course characteristics of the intra-olfactory concentration of TBTC, the histopathological changes of the olfactory bulb and the olfactory function were examined for 96 h after a single intraperitoneal injection of 2.5 mg/kg of TBTC. The olfactory function was evaluated by the discriminating ability for a cycloheximide solution which has an unpleasant odor for rats. The concentration of TBTC in the olfactory bulb, which was measured using gas chromatography/mass spectrometry, quickly increased to a peak value within 24 h and then decreased. The viable cell number significantly decreased after the TBTC administration in the mitral cell layer and granule cell layer of the olfactory bulb, while apoptotic cells significantly increased in these areas at the same time. Hyposmia was evident 96 h after the TBTC injection, although olfactory testing could not been performed until that time because of anorexia. These results suggest that intraperitoneally injected TBTC was promptly transferred to the olfactory bulb through the blood-brain barrier, induced apoptosis of the cells in the olfactory bulb and finally elicited the olfactory disturbance.

Innate immune parameters and haemolymph protein expression profile to evaluate the immunotoxicity of tributyltin on abalone (Haliotis diversicolor supertexta).

The immunotoxicity of tributyltin (TBT) on marine gastropods has been comparatively little studied although risks to wildlife associated with this compound are well known. In this study, a 30-day trial was conducted to evaluate the immunotoxic effects on abalone (Haliotis diversicolor supertexta) by exposing a range of doses of TBT (0, 2, 10, and 50 ng/L). Innate immune parameters, including phagocytic ability (PA), lysozyme activity, phenoloxidase (PO) level and superoxide dismutase (SOD) activity were monitored at intervals of 5, 15 and 30 days. Haemolymph protein expression profile was also examined at the end of the experiment. The results showed that PA value, lysozyme activity and PO level significantly decreased compared with the controls (P < 0.05), which indicated that TBT exposure markedly suppressed non-specific immune competence. Exposure to TBT also caused variation in protein expression patterns of haemolymph. Among the protein spots of differential expressions, seven proteins from the haemolymph of TBT-treated abalone were successfully identified by MALDI-TOF-MS analysis. Three protein spots increased and were identified as carrier-like peptide, peroxidase 21 precursor and creatine phosphokinase. These proteins are believed to up-regulate in expression as a response to detoxification and antioxidative stress mechanisms. The other four protein spots that down-regulated in TBT-treated groups were identified as aromatase-like protein, protein kinase C, ceruloplasmin and microtubule-actin crosslinking factor 1, and these proteins play an important role in endocrine regulation and immune defense. Taken together, the results demonstrate that TBT impair abalone immunological ability and is a potential immune disruptor.

Modulation of acute steroidogenesis, peroxisome proliferator-activated receptors and CYP3A/PXR in salmon interrenal tissues by tributyltin and the second messenger activator, forskolin.

There are uncertainties regarding the role of sex steroids in sexual development and reproduction of gastropods, leading to the recent doubts as to whether organotin compounds do inhibit steroidogenic enzymes in these species. These doubts have led us to suspect that organotin compounds may affect other target molecules, particularly signal transduction molecules or secondary mediators of steroid hormone and lipid synthesis/metabolism. Therefore, we have studied the effects of TBT exposure through food on acute steroidogenesis, PPARs and CYP3A responses in the presence and absence of a cyclic AMP (cAMP) activator, forskolin. Two experiments were performed. Firstly, juvenile salmon were force-fed once with diet containing TBT doses (0.1, 1 and 10mg/kg fish) dissolved in ethanol and sampled after 72h. Secondly, fish exposed to solvent control and 10mg/kg TBT for 72h were transferred to new tanks and exposed to waterborne forskolin (200microg/L) for 2 and 4h. Our data show that juvenile salmon force-fed TBT showed modulations of multiple biological responses in interrenal tissues that include, steroidogenesis (cAMP/PKA activities; StAR and P450scc mRNA, and plasma cortisol), and mRNA for peroxisome proliferator-activated receptor (PPAR) isoforms (alpha, beta, gamma), acyl-CoA oxidase-1 (ACOX1) and CYP3A/PXR (pregnan X receptor). In addition, forskolin produced differential effects on these responses both singly and also in combination with TBT. Overall, combined forskolin and TBT exposure produced higher effects compared with TBT exposure alone, for most of the responses (cortisol, PPARbeta, ACOX1 and CYP3A). Interestingly, forskolin produced PPAR isoform-specific effects when given singly or in combination with TBT. Several TBT mediated toxicity in fish that includes thymus reduction, decrease in numbers of lymphocytes, inhibition of gonad development and masculinization, including the imposex phenomenon have been reported. When these effects are considered with the present findings, it suggests that studies on mechanisms of action or field studies may reveal endocrine, reproductive or other effects of TBT at lower concentrations than those reported to date from subchronic tests of fishes. Since the metabolic fate of organotin compounds may contribute to the toxicity of these chemicals, the present findings may represent some new aspects of TBT toxicity not previously reported.

Analysis of mechanisms of cell death of T-lymphocytes induced by organotin agents.

Organotin compounds are known to cause thymic atrophy and an accompanying deficiency of cell-mediated immunity. The study reported here focused on cell death in the thymus as a contributing factor in the induction of thymic atrophy following exposure to dibutyltin (DBTC) and tributyltin (TBTC). In an in vivo study, a reversible thymic atrophy was induced in rats by a single intraperitoneal administration (2.0 mg/kg) of DBTC or TBTC; the magnitude of this effect over a 4-d post-treatment period differed between the two agents. In in vitro studies, T-lymphocytes were isolated from thymuses of naïve rats and then exposed to 1 microM DBTC or TBTC for varying periods of time. Analysis by flow cytometry showed that DBTC induced primarily necrosis, while TBTC induced apoptosis, of the cells. Activities of caspase-8, -9, and -3 were also measured; TBTC exposure caused marked increases in the activities, while DBTC exposure did not cause any significant change. TBTC exposure also appeared to induce expression of CAD (which fragments DNA), but had minimal effect on levels of the CAD inhibitor, ICAD. In contrast, DBTC exposure resulted in a larger level of ICAD expression. WST-8 and JC-1 assays were used to evaluate mitochondrial function, since a strong activation of caspase-9 by TBTC suggested mitochondrial involvement. The involvement of caspase in the activation was examined using cytochrome c expression; cytochrome expression and the loss of mitochondrial function occurred within 10 min of TBTC exposure. DBTC exposure affected the mitochondria less. These results indicated that effects on mitochondria likely played an important role in the induction of apoptosis by TBTC. The results of this study show that DBTC and TBTC induce necrosis and apoptosis of T-lymphocytes, respectively, by apparently indicating different mechanisms of cell death. It follows that these increases in cell death induced by these organotin compounds likely contributed to the thymic atrophy observed in the rats here.

Tri-n-butyltin increases intracellular Zn(2+) concentration by decreasing cellular thiol content in rat thymocytes.

Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn(2+) concentration was tested in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow cytometer and appropriate fluorescent probes. TBT at concentrations of 30 nM or more (up to 300 nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+) concentration, under external Ca(2+)- and Zn(2+)-free condition. Chelating intracellular Zn(2+) completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn(2+) from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indicator for cellular thiol content, were correlated with a coefficient of -0.962. Result suggests that the intracellular Zn(2+) release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn(2+) potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn(2+) concentration may be a type of stress responses to protect the cells.

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.

Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria.

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus.

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing-polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.