Tributyltin Exposure Research Articles

From the Cover: Tributyltin Alters the Bone Marrow Microenvironment and Suppresses B Cell Development.

Organotins are industrial chemicals and agricultural pesticides, and they contaminate both outdoor and indoor environments. Organotins are detectable in human sera at biologically active concentrations and are immuno-and neuro-toxicants. Triphenyltin, tributyltin (TBT) and dibutyltin activate peroxisome proliferator-activated receptor γ in bone marrow multipotent mesenchymal stromal cells and promote adipogenesis. TBT also has been shown to suppress osteogenesis; osteoblasts not only support bone homeostasis but also support B lymphopoiesis. In addition, developing B cells are highly sensitive to exogenous insults. Thus, we hypothesized that bone marrow B cells may be negatively affected by TBT exposure both directly, through activation of apoptosis, and indirectly, through alterations of the bone marrow microenvironment. TBT activated apoptosis in developing B cells at environmentally relevant concentrations (as low as 80 nM) in vitro, via a mechanism that is distinct from that induced by high dose (μM) TBT and that requires p53. TBT suppressed the proliferation of hematopoietic cells in an ex vivo bone marrow model. Concurrent treatment of stromal cells and B cells or pretreatment of stromal cells with TBT induced adipogenesis in the stromal cells and reduced the progression of B cells from the early pro B (Hardy fraction B) to the pre B stage (Hardy fraction D). In vivo, TBT induced adipogenesis in bone marrow, reduced "aging-sensitive" AA4+CD19+ B cells in bone marrow, and reduced splenic B cell numbers. Immunosenescence and osteoporosis are adverse health effects of aging, we postulate that TBT exposure may mimic, and possibly intensify, these pathologies.

Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae

We investigated the effect of combined exposure to nodavirus infection and TBT on medaka (Oryzias latipes). Medaka larvae were infected by immersion in medium containing nodavirus at titers of 102.5, 103.5, or 104.5 TCID50/mL. Infected fish then were exposed to TBT at 0, 0.17, 0.52, 1.6, or 4.7μg/L. Of the 12 groups exposed to both stressors, the mortalities of 6 (102.5 TCID50/mL+0.52, 1.6, or 4.7μg/L, 103.5 TCID50/mL+4.7μg/L and 104.5 TCID50/mL+1.6 or 4.7μg/L) were significantly higher than that of each TBT control. Specifically, mortality was 46±5.5% in the group exposed to both 102.5 TCID50/mL virus and 0.52μg/L TBT, which represent the lowest observed effective dose and concentration, respectively, among the 6 groups with increased mortalities. Our results suggest that combined exposure to both stressors suppresses antiviral mechanisms in the fish, thus increasing mortality.

Tributyltin exposure at noncytotoxic doses dysregulates pancreatic β-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 β-cell function still remain unclear. We investigated the effect and mechanism of TBT exposure at noncytotoxic doses relevant to human exposure on β-cell function in vitro and in vivo. The β-cell-derived RIN-m5F cells and pancreatic islets from mouse and human were treated with TBT (0.05-0.2μM) for 0.5-4h. Adult male mice were orally exposed to TBT (25μg/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 β-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.

Prenatal Exposure to Tributyltin Decreases GluR2 Expression in the Mouse Brain.

Tributyltin (TBT), a common environmental contaminant, is widely used as an antifouling agent in paint. We previously reported that exposure of primary cortical neurons to TBT in vitro decreased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit glutamate receptor 2 (GluR2) expression and subsequently increased neuronal vulnerability to glutamate. Therefore, to identify whether GluR2 expression also decreases after TBT exposure in vivo, we evaluated the changes in GluR2 expression in the mouse brain after prenatal or postnatal exposure to 10 and 25 ppm TBT through pellet diets. Although the mean feed intake and body weight did not decrease in TBT-exposed mice compared with that in control mice, GluR2 expression in the cerebral cortex and hippocampus decreased after TBT exposure during the prenatal period. These results indicate that a decrease in neuronal GluR2 may be involved in TBT-induced neurotoxicity, especially during the fetal period.

Metabolomic responses of Haliotis diversicolor to organotin compounds

Organotin compounds, especially tributyltin (TBT) and triphenyltin (TPT), are a group of hazardous pollutants in marine environments. Haliotis diversicolor is an important marine model organism for environmental science. In this study, 1H NMR spectroscopy together with pattern recognition methods was used to investigate the responses of hepatopancreas and gill of Haliotis diversicolor to TBT and TPT exposure. It was found that obvious gender-, tissue- and compound-specific metabolomic alterations were induced after a 28-day exposure. TBT and TPT exposure not only caused the disturbance in energy metabolism and osmotic balance in hepatopancreas and gill tissues with different mechanisms, but also induced oxidative stresses. These metabolic alterations were highlighted in the accumulation of aspartate, uridine diphosphate-N-acetylglucosamine, uridine diphosphate glucose, guanosine and the depletion of leucine, isoleucine, valine, malonate, homarine, trigonelline in all exposure gills, as well as in the depletion of ATP, AMP, betaine in male exposure gills and pantothenate in male exposure hepatopancreases. The significant decreased aromatic amino acids (AAAs), lysine and glutamate in gills and increased betaine in hepatopancreases for TPT exposure together with increased glutamate and decreased betaine in gills and increased glutamate and glycine in hepatopancreases for TBT exposure demonstrated their specific metabolic characteristics. Among these characteristic metabolites, AAAs, lysine and glutamate in the gill as well as pantothenate in the hepatopancreas might be identified as potential biomarkers for TPT or TBT exposure in Haliotis diversicolor. The results provide a useful insight into the toxicological mechanisms of organotin compounds on Haliotis diversicolor.

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)-1β, IL2, IL5, IL7, IL12βp40, IL13, IL15, keratinocyte chemoattractant (KC), macrophage inflammatory protein 1β (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-1β, IL-2, IL5, IL12βp40 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 IL1β, IL-12 βp40, 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-1β, RANTES and IL-13. These results from human and murine samples clearly suggest that TBT exposures modulate the secretion inflammatory cytokines.

Histopathological effect and stress response of mantle proteome following TBT exposure in the Hooded oyster Saccostrea cucullata

Tributyltin (TBT), an environmental pollutant in marine ecosystems, is toxic to organisms. Although contamination by and bioaccumulation and toxicity of this compound have been widely reported, its underlying molecular mechanisms remain unclear. In the present study, we exposed the Hooded oyster Saccostrea cucullata to TBT to investigate histopathological effects and proteome stress response. Animals were exposed to three TBT sub-lethal concentrations, 10, 50 and 150 μg/l for 48 h. TBT produced stress leading to histopathological changes in oyster tissues including mantle, gill, stomach and digestive diverticula. TBT induced mucocyte production in epithelia and hemocyte aggregation in connective tissue. Cell necrosis occurred when exposure dosages were high. Comparative proteome analyses of mantle protein of oysters exposed to 10 μg/l and control animals were analyzed by a 2-DE based proteomic approach. In total, 32 protein spots were found to differ (p < 0.05). Of these, 17 proteins were identified which included 14 up-regulated and 3 down-regulated proteins. TBT induced the expression of proteins involved in defensive mechanisms (HSP-78, HSP-70, aldehyde dehydrogenase and catalase), calcium homeostasis (VDAC-3), cytoskeleton and cytoskeleton-associated proteins, energy metabolism and amino acid metabolism. Our study revealed that TBT disturbs calcium homeostasis via VDAC-3 protein in mantle and this probably is the key molecular mechanism of TBT acting to distort shell calcification. Moreover, proteins involved in cell structure (tubulin-alpha and tubulin-beta) and protein synthesis were reduced after TBT exposure. Additionally, differential proteins obtained from this work will be useful as potential TBT biomarkers.

Abstract 934: Tributyltin-induced dysregulation of inflammatory cytokine levels in human and mouse immune cells

Abstract The effects of Tributyltin (TBT), a widespread environmental contaminant, on the induction of in vivo and ex vivo cytokine production and secretion were investigated. Human monocyte-depleted peripheral blood mononuclear cells and different groups of Balb/C mice were stimulated with desired concentrations of TBT (200-2.5 nM for human studies and 200-25 nM for mouse studies). The quantitative determination of IFNγ, TNFα and IL-1β was performed in mouse sera by MagPix analysis and western blot. The levels of these cytokines were measured at different time points: 6, 12, 24 and 48 h after injection. Analysis of IFNγ and TNFα in human cells were conducted by ELISA and western blot. MAPK and NF-κB pathways were inhibited to determine TBT-induced dysregulation of IFNγ and TNFα. Inhibition of the p38 and p44/42 pathways blocked the TBT-induced elevation of IFNγ, while the inhibition of the p38 pathway blocked the TBT-induced elevation of TNFα. Results showed that TBT increased the levels of IFNγ, TNFα and IL-1β in the sera of mice. IFNγ and TNFα secretion from human monocyte-depleted peripheral blood mononuclear cells also increased, showing striking agreement in the response to TBT between the mouse and human systems. These data suggest that TBT exposure can result in an inflammatory environment, which could predispose the host to various immunopathologies. Grant U54CA163066 from the National Institute of Health Citation Format: Shanieek Lawrence, Samuel T. Pellom, Anil Shanker, Margaret Whalen. Tributyltin-induced dysregulation of inflammatory cytokine levels in human and mouse immune cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 934.

Tributyltin affects shoaling and anxiety behavior in female rare minnow (Gobiocypris rarus)

Effects of tributyltin (TBT) on reproduction are well established in many fish species. However, few studies report the effects of TBT on non-reproductive behaviors, which is a novel aspect of endocrine disruption in fish. Thus, the present study used rare minnow (Gobiocypris rarus) to investigate the effects of TBT, at environmental concentrations of 1, 10 and 100ng/L, on shoaling and anxiety behaviors. The results showed that fish exposed to TBT had less group cohesion during the course of the 10-min observation period as compared with the control fish. Further, TBT altered the shoaling in the Novel tank test, where shoaling is determined as the tendency to leave a shoal of littermates trapped behind a Plexiglas barrier at one end of the test tank. Fish exposed to TBT had shorter latency before leaving shoal mates and spent more time away from shoal than control fish. In addition, we also used Novel tanks to study the anxiety behavior as the tendency to stay at the bottom when introduced into an unfamiliar environment. The fish exposed to TBT showed increased anxiety, manifested as increased latency to enter the upper half and decreased time in upper half when compared with the control fish. TBT exposure increased the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid, and decreased the levels of 5-hydroxytryptamine and its metabolite 5-hydroxy indole acetic acid in the brain. Thus, the hypofunction of the dopaminergic system or of the serotoninergic system or the combination of the two may underlie the observed behavioral change, which might affect the fitness of fish in their natural environment.

Role of ERK1/2 and p38 MAPKs in Tributyltin‐stimulated Interleukin 1 Beta Secretion and Production from Human Immune Cells.

Tributyltin (TBT) is an organotin compound that contaminates the environment due to its use as a biocide in a wide variety of applications. TBT has been found in human blood samples at levels as high as 265 nM. Previously, we have demonstrated that TBT interferes with the lytic function of human natural killer lymphocytes and alters the secretion of several pro‐inflammatory cytokines from lymphocytes, including interleukin 1 beta (IL‐1β). IL‐1β regulates cellular growth and immune responsiveness. Elevated levels of IL‐1β have been shown to increase the invasiveness of tumors. Our recent studies showed that TBT exposures of 5–25 nM (levels that are seen in human blood samples) caused increased secretion of IL‐1β from monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). In this study we examine the roles of the ERK1/2 and p38 MAPK pathways in TBT‐induced increases in IL‐1β secretion, protein production, and mRNA levels. MD‐PBMCs were treated with the MEK1/2 inhibitor PD98059 or the p38 inhibitor SB202190 for 1 h prior to exposure to TBT concentrations of 25, 10, and 5 nM for 24 h. Secretion of IL‐1β was measured using ELISA, intracellular protein levels were monitored using western blot, and mRNA expression was determined by RT‐qPCR. Results indicated that the TBT‐induced secretion of IL‐1β requires the ERK1/2 pathway. The increases in secretion are accompanied by increased protein levels in TBT‐exposed cells and this increase in protein production also utilized the ERK1/2 pathway. These data suggest that increased protein production accompanies the increases in secretion of IL‐1β seen in human immune cells exposed to TBT and these increases are dependent on MAPK pathway activation.Support or Funding InformationSupported by NIH grant 5U54CA163066.

Alteration of cytochrome P450 1 regulation and HSP 70 level in brain of juvenile common carp (Cyprinus carpio) after chronic exposure to tributyltin.

Tributyltin (TBT), a toxic contaminant in aquatic environments, has bio-accumulated in aquatic food webs throughout the world and can be found at toxic levels in some biota. However, the molecular mechanisms and effects of TBT are not fully understood. The aim of the present study was to investigate the effect of long-term exposure of TBT on cytochrome P450 (CYP450) 1 regulation and heat-shock proteins (HSPs) profiling in brain of freshwater teleost. The effects of long-term exposure to TBT on mRNA expression of cytochrome P450 (CYP450) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in the brain of common carp were evaluated, as well as HSP 70 level. Fish were exposed to sublethal concentrations of TBT (75 ng/L, 0.75 μg/L and 7.5 μg/L) for 15, 30, and 60 days. Based on the results, long-term exposure (more than 15 days) to TBT could lead to obvious physiological-biochemical responses (based on EROD activity, HSP 70 level and CYP450 1 family genes expression). The mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. Thus, the measured physiological responses in fish brain could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity and could be used as potential biomarkers for monitoring the TBT pollution in the field.

Responses of growth and hemolymph quality in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) to sublethal tributyltin and cadmium.

Responses of growth endpoints and hemolymph constituents in juvenile Chinese horseshoe crab Tachypleus tridentatus under treatments of 0.01 and 0.1 mg/l tributyltin (TBT) and 0.1 and 1 mg/l cadmium (Cd) were examined in a 12-week experiment. A significant decline in final body weight, final prosomal width, percentage of individuals molted and mean molting time was detected under TBT exposures. While morphological abnormalities of the juveniles between TBT treatments and the control were statistically indistinguishable, significantly higher occurrences of carapace erosion and appendage loss were noted under 0.1 and 1 mg/l Cd exposures. Various hemolymph quality indicators, including hemolymph plasma protein level, amebocyte viability and percentage of granular-spherical state of amebocytes of the juveniles exposed to TBT or Cd were significantly lower than the control. Such a decrease in hemolymph quality suggested deleterious effects of metal contaminant-induced stressors on the health status of the juveniles even at low exposure levels (i.e., 0.01 mg/l TBT and 0.1 mg/l Cd). Changes of hemolymph parameters in juvenile horseshoe crabs were more sensitive than growth performance as well as morphological abnormalities in response to metal stressors, and can be used as an indicator to reflect habitat conditions and contaminant levels.

Activation of protein kinase C and protein kinase D in human natural killer cells: effects of tributyltin, dibutyltin, and tetrabromobisphenol A

Up to now, the ability of target cells to activate protein kinase C (PKC) and protein kinase D (PKD) (which is often a downstream target of PKC) has not been examined in natural killer (NK) lymphocytes. Here we examined whether exposure of human NK cells to lysis sensitive tumor cells activated PKC and PKD. The results of these studies show for the first time that activation of PKC and PKD occurs in response to target cell binding to NK cells. Exposure of NK cells to K562 tumor cells for 10 and 30 min increased phosphorylation/activation of both PKC and PKD by roughly 2-fold. Butyltins (tributyltin (TBT), dibutyltin (DBT)) and brominated compounds (tetrabromobisphenol A (TBBPA)) are environmental contaminants that are found in human blood. Exposures of NK cells to TBT, DBT, or TBBPA decrease NK cell lytic function in part by activating the mitogen-activated protein kinases (MAPKs) that are part of the NK lytic pathway. We established that PKC and PKD are part of the lytic pathway upstream of MAPKs and thus we investigated whether DBT, TBT, and TBBPA exposures activated PKC and PKD. TBT-activated PKC by 2–3-folds at 10 min at concentrations ranging from 50 to 300 nM while DBT caused a 1.3-fold activation at 2.5 µM at 10 min. Both TBT and DBT caused an approximately 2-fold increase in phosphorylation/activation of PKC. Exposures to TBBPA caused no statistically significant changes in either PKC or PKD activation.

Effects of Tributyltin Exposures on Interleukin 1 beta Secretion and Production from Human Immune Cells

Interleukin 1 beta (IL‐1β) is a pro‐inflammatory cytokine that promotes cellular growth and immune response regulation. Produced predominately by both monocytes and macrophages, IL‐1β appears to increase the invasiveness of tumors. Tributyltin (TBT) is an organotin compound that has been used as a biocide in a variety of industrial applications and contaminates the environment. It is found in human blood samples, and mammals with TBT exposure have shown increased incidences of tumors. The current study examines whether exposure to TBT modifies the secretion and production of IL‐1β from lymphocytes. We examined whether exposure to TBT concentrations of 2.5 to 200 nM after 10 min, 30 min, 6h, and 24h alters IL‐1β secretion as well as its intracellular levels in a monocyte‐depleted preparation of human peripheral blood mononuclear cells (MD‐PBMCs). IL‐1β secretion was monitored using enzyme‐linked immunosorbent assay (ELISA) and intracellular levels were analyzed by western blot. Results indicated that 24h exposures to TBT elevated secretion of IL‐1β compared to control cells at all concentrations of TBT and intracellular levels were increased at TBT concentrations of 2.5 to 100nM. After 30 min exposures to TBT there was elevated secretion at the lowest concentrations of TBT (10, 5, and 2.5 nM) while intracellular levels of IL1β increased at TBT concentrations of 5 to 100nM. These data indicate that even very low concentrations of TBT increase the production of IL‐1β in MD‐PBMCs and suggest that increased production accompanies the increase in secretion that is seen with TBT exposures. Supported by NIH grant 5U54CA163066‐02.

Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p < 0.05) whole body TBT concentrations with concomitant significant decreases (p < 0.05) in NRRT (R(2) values of 0.85 and 0.971 for lower and higher exposure groups, respectively). The higher exposure group showed a typical dose-response curve. For the control, no TBT was detected and NRRT remained stable. It was concluded that the NRRT assay could be considered as a useful technique, and lysosomal membrane destabilization a useful early warning and cellular biomarker of stress due to TBT exposure in M. galloprovincialis.

Disruptions in aromatase expression in the brain, reproductive behavior, and secondary sexual characteristics in male guppies (Poecilia reticulata) induced by tributyltin

Although bioaccumulation of tributyltin (TBT) in fish has been confirmed, information on possible effects of TBT on reproductive system of fish is still relatively scarce, particularly at environmentally relevant levels. To evaluate the adverse effects and intrinsic toxicological properties of TBT in male fish, we studied aromatase gene expression in the brain, sex steroid contents, primary and secondary sexual characteristics, and reproductive behavior in male guppies (Poecilia reticulata) exposed to tributyltin chloride at the nominal concentrations of 5, 50, and 500ng/L for 28 days in a semi-static exposure system. Radioimmunoassay demonstrated that treatment with 50ng/L TBT caused an increase in systemic levels of testosterone of male guppies. Gonopodial index, which showed a positive correlation with testosterone levels, was elevated in the 5ng/L and 50ng/L TBT treated groups. Real-time PCR revealed that TBT exposure had inhibiting effects on expression of two isoforms of guppy aromatase in the brain, and these changes at the molecular levels were associated with a disturbance of reproductive behavior of the individuals, as measured by decreases in frequencies of posturing, sigmoid display, and chase activities when males were paired with females. This study provides the first evidence that TBT can cause abnormalities of secondary sexual characteristics in teleosts and that suppression of reproductive behavior in teleosts by TBT is due to its endocrine–disrupting action as an aromatase inhibitor targeting the nervous system.

Differential gene expression in the brain of Cuvier, Sebastiscus marmoratus, in response to exposure to tributyltin

Tributyltin (TBT), a ubiquitous marine environmental contaminant, has been reported to affect functioning of the central nervous system. However, the mechanism of its neurotoxicity remains unknown. The effects of TBT exposure on the Sebastiscus marmoratus were investigated using a suppression subtractive hybridisation (SSH) combined with mirror orientation selection (MOS) method. A total of 50 candidate clones from SSH libraries were picked for sequencing and homology searching. The results showed that 23 unique differentially expressed genes were obtained, including 11 up-regulated and 12 down-regulated fragments. The functions with which they are associated include energy-related metabolism, polypeptide translation and location, mitosis and cell proliferation. Expression of 11 candidate genes was analysed in brain by real-time fluorescent quantitative PCR. The present study provided a basis for investigating the response of fish to TBT exposure and allowed the characterisation of new potential neurotoxic biomarkers of TBT contamination in seawater.

Responses of the hepatic glutathione antioxidant defense system and related gene expression in juvenile common carp after chronic treatment with tributyltin.

Recently, residual organotin compounds have generally been recognised as relevant sources of aquatic environmental pollutants. However, the effects of these contaminants on the glutathione (GSH)-antioxidant system of fishes have not been adequately studied. In the current study, the chronic effects of tributyltin (TBT) found within antifouling paints for ships, on the GSH antioxidant system and related gene expression in the liver of juvenile common carp (Cyprinus carpio) were investigated. Fishes were exposed to sub-lethal concentrations of TBT (75ng/L, 0.75 and 7.5μg/L) for 15, 30 and 60days. GSH levels and GSH-related enzymes activities, including glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), were quantified in the fish liver. The levels of malondialdehyde were also measured as a marker of oxidative damage. In addition, the expression levels of gstp1, gr and gpx1 in common carp chronically exposed to TBT were determined. The results of the current study indicate that chronic exposure of TBT results in reactive oxygen species stress in the liver of common carp, and mRNA expression levels are more sensitive than related enzyme levels. In short, the measured GSH-related indices could potentially be used as molecular indicators for monitoring organotin compounds in the aquatic environment.

Effect of tributyltin on the food intake and brain neuropeptide expression in rats.

Tributyltin (TBT) is a largely diffused environmental pollutant. Several studies have demonstrated that TBT is involved in the development of obesity. However, few studies addressing the effects of TBT on the brain neuropeptides involved in appetite and body weight homeostasis have been published. Experiments were carried out on female and male Sprague-Dawley rats. Animals were exposed to TBT (0.5 μg/kg body weight) for 54 days. The hepatic triglyceride and total cholesterol were determined using commercial enzyme kits. The NPY, AgRP, POMC and CART mRNA expression in brains were quantified by real-time PCR. TBT exposure resulted in significant increases in the hepatic total cholesterol and triglyceride concentration of both male and female rats. Interestingly, increases in body weight and fat mass were only found in the TBT-treated male rats. TBT exposure also led to a significant increase in food intake by the female rats, while no change was observed in the male rats. Moreover, the neuropeptides expression was different between males and females after TBT exposure. TBT induced brain NPY expression in the female rats, and depressed brain POMC, AgRP and CART expression in the males. TBT can increase food intake in female rats, which is associated with the disturbance of NPY in brains. TBT had sex-different effects on brain NPY, AgRP, POMC and CART mRNA expression, which indicates a complex neuroendocrine mechanism of TBT.

Evaluation of tributyltin toxicity in Chinese rare minnow larvae by abnormal behavior, energy metabolism and endoplasmic reticulum stress

Tributyltin (TBT) is a ubiquitous contaminant in aquatic environment, but the detailed mechanisms underlying the toxicity of TBT have not been fully understood. In this study, the effects of TBT on behavior, energy metabolism and endoplasmic reticulum (ER) stress were investigated by using Chinese rare minnow larvae. Fish larvae were exposed at sublethal concentrations of TBT (100, 400 and 800ng/L) for 7days. Compared with the control, energy metabolic parameters (RNA/DNA ratio, Na+-K+-ATPase) were significantly inhibited in fish exposed at highest concentration (800ng/L), as well as abnormal behaviors observed. Moreover, we found that the PERK (PKR-like ER kinase)–eIF2α (eukaryotic translation initiation factor 2α) pathway, as the main branch was activated by TBT exposure in fish larvae. In short, TBT-induced physiological, biochemical and molecular responses in fish larvae were reflected in parameters measured in this study, which suggest that these biomarkers could be used as potential indicators for monitoring organotin compounds present in aquatic environment.