Tributyltin-induced oxidative stress causes developmental damage in the cardiovascular system of zebrafish (Danio rerio).

Tributyltin (TBT) can be used as an antifouling agent with anticorrosive, antiseptic and antifungal properties and is widely used in wood preservation and ship painting. However, it has recently been found that TBT can be harmful to aquatic organisms. In this study, to gain insight into the effects of TBT with respect to the development of the cardiovascular system in zebrafish embryos, zebrafish embryos were exposed to different concentrations of TBT solutions (0.2 µg/L, 1 µg/L, and 2 µg/L) at 2 h post-fertilization (hpf) TBT exposure resulted in decreased hatchability and heart rate, deformed features such as pericardial edema, yolk sac edema, and spinal curvature in zebrafish embryos, and impaired heart development. Expression of cardiac development-related genes (vmhc, myh6, nkx2.5, tbx5a, gata4, tbx2b, nppa) is dysregulated. Transgenic zebrafish Tg (fli1: EGFP) were used to explore the effects of TBT exposure on vascular development. It was found that TBT exposure could lead to impaired development of intersegmental vessels (ISVs), common cardinal vein (CCV), subintestinal vessels (SIVs) and cerebrovascular. The expression of vascular endothelial growth factor (VEGF) signaling pathway-related genes (flt1, flt4, kdr, vegfa) was downregulated. Biochemical indices showed that ROS and MDA levels were significantly elevated and that SOD and CAT activities were significantly reduced. The expression of key genes for prostacyclin synthesis (pla2, ptgs2a, ptgs2b, ptgis, ptgs1) is abnormal. Therefore, it is possible that oxidative stress induced by TBT exposure leads to the blockage of arachidonic acid (AA) production in zebrafish embryos, which affects prostacyclin synthesis and consequently the normal development of the heart and blood vessels in zebrafish embryos.

Transgenerational Effects and Mechanisms of Tributyltin Exposure on Neurodevelopment in the Male Offspring of Rats.

This study aimed to investigate the transgenerational effects of tributyltin exposure on rat neurodevelopment in male offspring and the potential mechanisms. Neonatal female rats were exposed to the environmental level of tributyltin and then mated with nonexposed males after sexual maturity to produce the F1 generation. The F1 generation (with primordial germ cell exposure) was mated with nonexposed males to produce nonexposed offspring (the F2 and F3 generations). Neurodevelopmental indicators and behavior were observed for the F1, F2, and F3 generations during postnatal days 1-25 and 35-56, respectively. We found premature eye-opening and delayed visual positioning in newborn F1 rats and anxiety and cognitive deficits in prepubertal F1 male rats. These neurodevelopmental impacts were also observed in F2 and F3 males. Additionally, F1-F3 males exhibited increased serotonin and dopamine levels and a loose arrangement of neurons in the hippocampus. We also observed a reduction in the expression of genes involved in intercellular adhesion and increased DNA methylation of the Dsc3 promoter in F1-F3 males. We concluded that tributyltin exposure led to transgenerational effects on neurodevelopment via epigenetic reprogramming in male offspring. These findings provide insights into the risks of neurodevelopmental disorders in offspring from parents exposed to tributyltin.

Organotin mixtures reveal interactions that modulate adipogenic differentiation in 3T3-L1 preadipocytes.

Organotin chemicals (butyltins and phenyltins) are the most widely used organometallic chemicals worldwide and are used in industrial applications, such as biocides and anti-fouling paints. Tributyltin (TBT) and more recently, dibutyltin (DBT) and triphenyltin (TPT) have been reported to stimulate adipogenic differentiation. Although these chemicals co-exist in the environment, their effect in combination remains unknown. We first investigated the adipogenic effect of eight organotin chemicals (monobutyltin (MBT), DBT, TBT, tetrabutyltin (TeBT), monophenyltin (MPT), diphenyltin (DPT), TPT, and tin chloride (SnCl4)) in the 3T3-L1 preadipocyte cell line in single exposures at two doses (10 and 50 ng/ml). Only three out of the eight organotins induced adipogenic differentiation with TBT eliciting the strongest adipogenic differentiation (in a dose-dependent manner) followed by TPT and DBT, as demonstrated by lipid accumulation and gene expression. We then hypothesized that, in combination (TBT, DBT, and TPT), adipogenic effects will be exacerbated compared to single exposures. However, at the higher dose (50 ng/ml), TBT-induced differentiation was reduced by TPT and DBT when in dual or triple combination. We tested whether TPT or DBT would interfere with adipogenic differentiation stimulated by a peroxisome proliferator-activated receptor (PPARγ) agonist (rosiglitazone) or a glucocorticoid receptor agonist (dexamethasone). Both DBT50 and TPT50 reduced rosiglitazone-, but not dexamethasone-stimulated adipogenic differentiation. In conclusion, DBT and TPT interfere with TBT's adipogenic differentiation possibly via PPARγ signaling. These findings highlight the antagonistic effects among organotins and the need to understand the effects and mechanism of action of complex organotin mixtures on adipogenic outcomes.

Low-dose tributyltin triggers human chondrocyte senescence and mouse articular cartilage aging.

Tributyltin (TBT) is known as an endocrine-disrupting chemical. This study investigated the effects and possible mechanisms of TBT exposure on inducing human articular chondrocyte senescence in vitro at the human-relevant concentrations of 0.01-0.5 µM and mouse articular cartilage aging in vivo at the doses of 5 and 25 µg/kg/day, which were 5 times lower than the established no observed adverse effect level (NOAEL) and equal to NOAEL, respectively. TBT significantly increased the senescence-associated ß-galactosidase activity and the protein expression levels of senescence markers p16, p53, and p21 in chondrocytes. TBT induced the protein phosphorylation of both p38 and JNK mitogen-activated protein kinases in which the JNK signaling was a main pathway to be involved in TBT-induced chondrocyte senescence. The phosphorylation of both ataxia-telangiectasia mutated (ATM) and histone protein H2AX (termed γH2AX) was also significantly increased in TBT-treated chondrocytes. ATM inhibitor significantly inhibited the protein expression levels of γH2AX, phosphorylated p38, phosphorylated JNK, p16, p53, and p21. TBT significantly stimulated the mRNA expression of senescence-associated secretory phenotype (SASP)-related factors, including IL-1ß, TGF-ß, TNF-α, ICAM-1, CCL2, and MMP13, and the protein expression of GATA4 and phosphorylated NF-κB-p65 in chondrocytes. Furthermore, TBT by oral gavage for 4 weeks in mice significantly enhanced the articular cartilage aging and abrasion. The protein expression of phosphorylated p38, phosphorylated JNK, GATA4, and phosphorylated NF-κB-p65, and the mRNA expression of SASP-related factors were enhanced in the mouse cartilages. These results suggest that TBT exposure can trigger human chondrocyte senescence in vitro and accelerating mouse articular cartilage aging in vivo.

Embryonic exposure to environmentally relevant levels of tributyltin affects embryonic tributyltin bioaccumulation and the physiological responses of juveniles in cuttlefish (Sepia pharaonis).

Tributyltin (TBT) is a typical organic pollutant that persists in aquatic sediments due to its wide usage as an antifouling fungicide during the past few decades. Despite increased awareness of the serious negative consequences of TBT on aquatic species, studies on the effects of TBT exposure on cephalopod embryonic development and juvenile physiological performance are scarce. To investigate the lasting effects of TBT toxicity on Sepia pharaonis from embryo to hatchling, embryos (gastrula stage, 3-5 h post fertilization) were exposed to four levels of TBT until hatching: 0 (control), 30 (environmental level), 60, and 120 ng/L. Subsequently, juvenile growth performance endpoints and behavioral alterations were assessed over 15 days post-hatching. Egg hatchability was significantly reduced and embryonic development (i.e., premature hatching) was accelerated in response to 30 ng/L TBT exposure. Meanwhile, TBT-induced alterations in embryonic morphology primarily included yolk-sac lysis, embryonic malformations, and uneven pigment distributions. During the pre-middle stage of embryonic development, the eggshell serves as an effective barrier to safeguard the embryo from exposure to 30-60 ng/L TBT, according to patterns of TBT accumulation and distribution in the egg compartment. However, even environmental relevant levels of TBT (30 ng/L) exposure during embryonic development had a negative impact on juvenile behavior and growth, including slowing growth, shortening eating times, causing more irregular movements, and increasing inking times. These findings indicate that after TBT exposure, negative long-lasting effects on S. pharaonis development from embryo to hatchling persist, suggesting that long-lasting toxic effects endure from S. pharaonis embryos to hatchlings.

Biochemical mechanisms of tributyltin chloride-induced cell toxicity in Sertoli cells.

Tributyltin chloride (TBTCL) is a widely used fungicide and heat stabilizer in compositions of PVC. TBTCL has been detected in human bodies and potentially causes harmful effects on humans' thyroid, cardiovascular and other organs. As one of the first examples of endocrine disruptors, the toxicity effects of TBTCL on the male reproduction system have aroused concerns. However, the potential cellular mechanisms are not fully explored. In the current study, by using Sertoli cells, a critical regulator of spermatogenesis as a cell model, we showed that with 200 nM exposure for 24 h, TBTCL causes apoptosis and cell cycle arrest. RNA sequencing analyses suggested that TBTCL probably activates endoplasmic reticulum (ER) stress, and disrupts autophagy. Biochemical analysis showed that TBTCL indeed induces ER stress and the dysregulation of autophagy. Interestingly, activation of ER stress and inhibition of autophagy is responsible for TBTCL-induced apoptosis and cell cycle arrest. Our results thus uncovered a novel insight into the cellular mechanisms for TBTCL-induced toxicology in Sertoli cells.

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation.

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRß and RXRß knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Neurotoxicity and physiological stress in brain of zebrafish chronically exposed to tributyltin.

Tributyltin (TBT), an organotin compound, is hazardous in aquatic ecosystems. However, the mechanisms underlying TBT-induced central nervous system (CNS) toxicity remain to be determined especially in freshwater aquatic vertebrates. The aim of present study was to investigate the effects of chronic exposure to TBT on brain functions in a freshwater teleost the adult wild-type zebrafish (Danio rerio). Fish were exposed to sublethal concentrations of TBT (10, 100 or 300 ng/L) for 6 weeks. The influence of long-term TBT exposure was assessed in the brain of zebrafish with antioxidant related indices including malondialdehyde (MDA) levels and total antioxidant capacity, neurological parameters such as activities of acetylcholinesterase, and monoamine oxidase as well as levels of nitric oxide, dopamine, 5-hydroxytryptamine. In addition indices related to sensitivity of toxic insult such as cytochrome P450 1 regulation and heat shock protein 70 were determined. The regulation of related genes involved in endoplasmic reticulum stress (ERS), apoptosis and Nrf2 pathway were measured. Adverse physiological and biochemical responses were significantly enhanced in a concentration-dependent manner reflecting neurotoxicity attributed to TBT exposure. Our findings provide further insight into TBT-induced toxicity in wild-type zebrafish. and enhance our understanding of the molecular mechanisms underlying TBT-initiated CNS effects.

The ameliorative effect of Moringa oleifera oil on tributyltin-induced brain toxicity in albino rats.

Tributyltin (TBT) is an organotin compound widely used as a biocide in antifouling paints. Moringa oleifera oil (MOO) has a promising antioxidant potential, which necessitates further exploration. This study was conducted to investigate the potential protective effect of MOO against TBT-induced brain toxicity. The 30 rats were grouped into five groups (six each), Group I negative control, Group II positive control (vehicle), Group III MOO (5 ml/kg body weight [b.wt.]), Group IV TBT (10 mg/kg b.wt.), and Group V TBT & MOO. All treatments were given orally for 28 days. Thereafter, brains were exposed to oxidative stress and neurological parameters analyses. Histopathological and immunohistochemical (caspase-3, Bax, Bcl-2) examinations were also carried out. In rats administered TBT, increased malondialdehyde level, decreased reduced glutathione, and low total antioxidant capacity levels were in support of oxidative stress mechanism. Neurotoxicity was indicated by high nitric oxide level and increased acetylcholinestrase activity. Along with the histopathological alterations, the dysregulated expression of caspase-3, Bax, and Bcl-2 were indicative of the apoptotic mechanism mediated by TBT. Co-administration of MOO with TBT ameliorated the aforementioned toxic effects. In conclusion, TBT causes brain toxicity via oxidative, nitrosative, and apoptotic mechanisms. MOO demonstrates protective effect against TBT-induced brain toxicity mostly via potent antioxidant and antiapoptotic properties.

Maternal exposure to tributyltin during early gestation increases adverse pregnancy outcomes by impairing placental development.

Tributyltin (TBT) is a persistent organotin pollutant widely used as agricultural and wood biocides, exhibiting well-documented toxicity to reproductive functions in aquatic organisms. However, the effect of TBT on early pregnancy and placental development has been rarely studied in mice. Pregnant mice were fed with 0, 0.2, and 2 mg/kg/day TBT from gravid day 1 to day 8 or 13. TBT exposure led to an increase in the number of resorbed embryo and a reduction in the weight of fetus at gestational days 13. Further study showed that TBT significantly decreased placental weight and area, lowered laminin immunoreactivity and the expressions of placental development-related molecules including Fra1, Eomes, Hand1, and Ascl2. Moreover, TBT treatment markedly inhibited the placental proliferation and induced up-regulation of p53 and cleaved caspase-3 proteins, and down-regulation of Bcl-2 protein. In addition, TBT administration increased levels of malondialdehyde and H2 O2 and decreased activities of catalase and superoxide dismutase. Collectively, these results suggested TBT-induced adverse pregnancy outcomes during early pregnancy might be involved in developmental disorders of the placenta via dysregulation of key molecules, proliferation, apoptosis, and oxidative stress.

Toll-like receptors (TLRs) respond to tributyltin chloride (TBT-Cl) exposure in the river pufferfish (Takifugu obscurus): Evidences for its toxic injury function.

Tributyltin chloride (TBT-Cl) residual in water body had become a noticeable ecological problem for aquatic ecosystems. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play key roles in detecting nonself antigens and immune system activation. In this study, we explored the effect of TBT-Cl exposure on four TLRs expression in river pufferfish, Takifugu obscurus. The four T. obscurus Toll-like receptors (To-TLRs) contained different types of domains such as leucine-rich repeats (LRRs), leucine-rich repeats, typical subfamily (LRR_TYP) and other special domains. The To-TLRs mRNA transcripts expressed in all tissues, also To-TLR2 was investigated with higher level in kidney, as well as To-TLR3 in kidney, while To-TLR18 in liver and To-TLR22 in intestine. After the acute and chronic exposure of TBT-Cl, To-TLR2 and To-TLR3 mRNA transcripts were significantly down-regulated in gill. However, To-TLR18 and To-TLR22 were significantly up-regulated in gill and liver. Moreover, the histology and immunohistochemistry (IHC) results showed the different injury degrees of TBT-Cl in liver and gill and implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-TLRs to TBT-Cl exposure. All the results indicated that To-TLRs might involve in sensing and mediating innate immune responses caused by TBT-Cl for keeping detoxification homeostasis.

Chemical compounds causing severe acute toxicity in heavy liquids used for intraocular surgery.

Perfluorocarbon liquids (PFCLs) have been considered safe for intraocular manipulation of the retina, but since 2013 many cases of acute eye toxicity cousing blindness have been reported in various countries when using various commercial PFCLs. All these PFCLs were CE marked (Conformité Européenne), which meant they had been subjected to evaluation complying with the International Organization for Standardization (ISO) guidelines. These dramatic events raised questions about the safety of PFCLs and the validity of some cytotoxicity tests performed under ISO guidelines. Samples from toxic batches were analyzed by gas chromatography-mass spectrometry combined with Raman and infrared spectrometry. Perfluorooctanoic acid, dodecafluoro-1-heptanol, ethylbenzene and tributyltin bromide were identified and evaluated by a direct contact cytotoxicity test using ARPE-19 cell line, patented by our group (EP 3467118 A1). Perfluorooctanoic acid at a concentration of >0.06 mM and tributyltin bromide at a concentration of ≥0.016 mM were shown to be toxic, whereas the concentration found in the toxic samples reached 0.48 mM, and 0.111 mM, respectively. These finding emphasized the idea that determination of partially fluorinated compounds are not enough to guarantee the safety of these medical devices.

Tributyltin enhanced anxiety of adult male zebrafish through elevating cortisol level and disruption in serotonin, dopamine and gamma-aminobutyric acid neurotransmitter pathways.

Tributyltin (TBT), a widely and persistently distributed organontin, has been well documented to disrupt reproduction and behaviors in animals due to its anti-aromatase activity. TBT has been also reported to enhance anxiety in several fish species, whereas the mechanism underlying remains largely unknown. To investigate the disruption of TBT on fish anxiety and the mechanisms possibly involved, adult male zebrafish (Danio rerio) were treated with TBT (100 and 500 ng/L) for 28 days and anxiety behavior was further investigated using a novel tank dive test. Result showed that TBT treatment significantly enhanced the total time of the fish spent in the lower half, delayed the onset time to the higher half of the tank and increased the total duration of freezing of the fish, indicating an enhanced anxiety in TBT-treated fish. Accordingly, TBT sharply elevated the cortisol levels in plasma in a concentration-dependent manner, suggesting that the elevated cortisol level might be involved in the enhanced anxiety. Although the expression of crha was significantly increased and crhbp was significantly decreased in the brain of TBT-treated fish which is consistent to the elevated cortisol level, the expressions of actha and acthb were sharply down-regulated. In contrast, the expressions of genes responsible for the synthesis and action of serotonin (5-HT) (pet1, thp2 and htr1aa), dopamine (DA) (th1, slc6a3, drd2a and drd2b) and gamma-aminobutyric acid (GABA) (gad2 and gabrg2) were all significantly inhibited. The down-regulation of these pivotal genes acting in 5-HT, DA and GABA neurotransmitter systems in response to TBT corresponded well with the TBT-enhanced anxiety in fish. It was thus strongly suggested that these neurotransmitters might be also involved in TBT-enhanced anxiety in adult male zebrafish. The present study extended our understanding of the neurotoxicity of TBT on the anxiety control and behavioral modulation in fish.

Tributyltin Induces the Tissue-Specific Stresses in Zebrafish, a Study in Various Tissues of Muscle, Gill and Intestine.

Because the mechanism of tissue-specific toxicity of tributyltin (TBT) in aquatic organisms has not been explained clearly, the aim of this study is to investigate the effect of chronic exposure to TBT on muscle-related energy metabolism, gill-related ATPase enzymatic system and intestine-related digestive enzymes activities in zebrafish. Male zebrafish were exposed to sub-lethal concentrations of TBT (10, 100 and 300 ng/L) for 6 weeks. Multiple biomarkers were measured (such as glucose, lactate, hexokinase, pyruvate kinase, lactate dehydrogenase, ATP content, ATPases, trypsin, lipase and amylase), which reflected more serious physiological stress with increasing TBT concentrations during the experimental period. Through principal component analysis (PCA) and integrated biomarker response (IBR) analysis, the toxic effect of TBT in zebrafish was in a concentration-dependent manner. Shortly, the results of this study can provide new evidence for a comprehensive understanding of the toxic effects of TBT.

Effects of Tributyltin (TBT) on Rat Bone and Mineral Metabolism.

BACKGROUND/AIMS: Tributyltin (TBT) is an organotin (OTs) and biohazard organometallic pollutant. Recently our group has shown that TBT, even in very low doses, has deleterious effects on several tissues most likely due to its role as an endocrine-disrupting molecule. Other studies have confirmed that OT exposure could be responsible for neural, endocrine, and reproductive dysfunctions via in vitro and in vivo models. However, TBT effects on bone lack concise data despite the fact that bone turnover is regulated by endocrine molecules, such as parathormone (PTH), estrogen (E2), etc. Our group has already shown that TBT disrupts adrenal and female gonadal functions. METHODS: We studied the effects of TBT on bone metabolism and structure using DXA, microCT scan, and SEM. We also determined the calcium (Ca²âº) and phosphate (Pi) metabolism in TBT-treated rats as well as some biomarkers for bone formation and resorption. RESULTS: Surprisingly, we found that TBT leads to higher bone mineral density (BMD) although lesions in spinal bone were observed by either microCT scan or SEM. Biomarkers for bone resorption, such as the urinary deoxipyridinolines (DPD) excretion ratio was increased in TBT-treated animals versus mock-treated controls. Osteocalcin (OC) and alkaline phosphatase (AP) are markers of bone formation and are also elevated suggesting that the bone matrix suffers from a higher turnover. Serum Ca²âº (total and ionized) do not changed by TBT treatment although hypercalciuria is observed. CONCLUSION: It is known that Sn atoms have three valence states (Sn²âº, Sn³âº, and Sn4⁺); hence, we hypothesized that Sn (more likely Sn²âº) could be competing with Ca²âº and/or Mg²âº in hydroxyapatite mineral matrix to disturb bone turnover. Further work is needed to confirm this hypothesis.

Multispecies study: low-dose tributyltin impairs ovarian theca cell cholesterol homeostasis through the RXR pathway in five mammalian species including humans.

Tributyltin (TBT), an organotin chemical used as a catalyst and biocide, can stimulate cholesterol efflux in non-steroidogenic cells. Since cholesterol is the first limiting step for sex hormone production, we hypothesized that TBT disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. We investigated TBT's effect on cholesterol trafficking, luteinization, and steroidogenesis in theca cells of five species (human, sheep, cow, pig, and mice). Primary theca cells were exposed to an environmentally relevant dose of TBT (1 or 10 ng/ml) and/or retinoid X receptor (RXR) antagonist. The expression of RXRα in sheep theca cells was knocked down using shRNA. Steroidogenic enzymes, cholesterol transport factors, and nuclear receptors were measured by RT-qPCR and Western blotting, and intracellular cholesterol, progesterone, and testosterone secretion by ELISA. TBT upregulated StAR and ABCA1 in ovine cells, and SREBF1 mRNA in theca cells. TBT also reduced intracellular cholesterol and upregulated ABCA1 protein expression but did not alter testosterone or progesterone production. RXR antagonist and RXRα knockdown demonstrates that TBT's effect is partially through RXR. TBT's effect on ABCA1 and StAR expression was recapitulated in all five species. TBT, at an environmentally relevant dose, stimulates theca cell cholesterol extracellular efflux via the RXR pathway, triggers a compensatory upregulation of StAR that regulates cholesterol transfer into the mitochondria and SREBF1 for de novo cholesterol synthesis. Similar results were obtained in all five species evaluated (human, sheep, cow, pig, and mice) and are supportive of TBT's conserved mechanism of action across mammalian species.

Linking chemical exposure to lipid homeostasis: A municipal waste water treatment plant influent is obesogenic for zebrafish larvae.

Obesity, a risk factor for the development of type-2 diabetes, hypertension, cardiovascular disease, hepatic steatosis and some cancers, has been ranked in the top 10 health risk in the world by the World Health Organization. Despite the growing body of literature evidencing an association between the obesity epidemic and specific chemical exposure across a wide range of animal taxa, very few studies assessed the effects of chemical mixtures and environmental samples on lipid homeostasis. Additionally, the mode of action of several chemicals reported to alter lipid homeostasis is still poorly understood. Aiming to fill some of these gaps, we combined an in vivo assay with the model species zebrafish (Danio rerio) to screen lipid accumulation and evaluate expression changes of key genes involved in lipid homeostasis, alongside with an in vitro transactivation assay using human and zebrafish nuclear receptors, retinoid X receptor α and peroxisome proliferator-activated receptor γ. Zebrafish larvae were exposed from 4 th day post-fertilization until the end of the experiment (day 18), to six different treatments: experimental control, solvent control, tributyltin at 100 ng/L Sn and 200 ng/L Sn (positive control), and wastewater treatment plant influent at 1.25% and 2.5%. Exposure to tributyltin and to 2.5% influent led to a significant accumulation of lipids, with white adipose tissue deposits concentrating in the perivisceral area. The highest in vitro tested influent concentration (10%) was able to significantly transactivate the human heterodimer PPARγ/RXRα, thus suggesting the presence in the influent of HsPPARγ/RXRα agonists. Our results demonstrate, for the first time, the ability of complex environmental samples from a municipal waste water treatment plant influent to induce lipid accumulation in zebrafish larvae.

Aging and retinoid X receptor agonists on masculinization of female Pomacea canaliculata, with a critical appraisal of imposex evaluation in the Ampullariidae.

Ampullariidae are unique among gastropods in that females normally show a primordium of the copulatory apparatus (CApp). The aims of this study were (a) to quantitatively evaluate the development and growth of the female CApp with age; (b) to compare the effects of RXR and PPARγ agonists in adult females of known age and (c) to explore the effect of masculinizing RXR agonists on the expression of RXR in the CApp. It was found that the CApp grows and develops with age. A significant increase in penile sheath length (PsL) and also in a developmental index (DI) was observed in 7-8 months old females, as compared with 4-5 months old ones. A reported endogenous agonist of RXR, 9-cis retinoic acid (9cis-RA), as well as two organotin compounds, tributyltin (TBT) and triphenyltin (TPT) which have been also reported to bind to RXR, were injected and its masculinizing effects were measured. Also, the effect of a PPARγ agonist, rosiglitazone, was studied. All studied RXR agonists, but not the PPARγ agonist, were effective in increasing PsL, penile length (PL) and DI. Finally, the expression of the RXR in the CApp was studied (Western blot) in control, TBT, TPT, and 9cis-RA treated females. A significantly increased expression of RXR was only observed after 9cis-RA treatment. It is concluded that (a) development and growth of the CApp is significantly affected by female age; (b) reported RXR agonists, but not a PPARγ agonist, cause female masculinization of young females. An appraisal of previous studies of female masculinization in the Ampullariidae has also been made and it is emphasized that the masculinizing effect of aging should be considered, particularly when interpreting field data.

Influences of Salinity and Organic Compounds on Embryo Development in Three Medaka Oryzias Congeners with Habitats Ranging from Freshwater to Marine.

To clarify whether Oryzias congeners, including freshwater, brackish water, and marine medaka, would be useful models for evaluating environmental chemical effects in various aquatic ecosystems, we examined the influence of salinity on their embryo development. We also compared the toxicity values of the organotin compounds triphenyltin and tributyltin, which remain pollutants of marine and freshwater ecosystems, between Oryzias latipes (freshwater), Oryzias melastigma (brackish water), and Oryzias javanicus (saltwater). Hatching and survival rates of O. latipes were significantly decreased at a salinity of 34, whereas O. melastigma and O. javanicus were adaptable to various salinities from freshwater to seawater. The lowest observed effect concentrations of organotin compounds for survival and embryo development were the similar in the three species. The similarity of the species' responses to organotin compounds indicated that Oryzias congeners are useful for ecological risk assessment of chemicals in a range of aquatic ecosystems, from freshwater to marine.

Bioaccumulation of TBT and Its Cellular Toxic Effects on the Freshwater Prawn Macrobrachium rosenbergii.

To test the toxic effects of tributyltin (TBT), Macrobrachium rosenbergii were exposed to three concentrations of TBT viz. 10 ng/L, 100 ng/L and 1000 ng/L for 90 days. The bioaccumulation of TBT level varied in hepatopancreas based upon dose dependent manner. Histopathological results revealed the reduction in basement membrane thickness, disruption of the hepatopancreatic tubules and abnormal lumen in hepatopancreas of TBT treated prawns. The ultrastructure of the control prawn showed normal architecture of cellular organelles with prominent nuclei in hepatocytes. On the other hand, many vacuoles, irregular arrangements of microvilli, swollen mitochondria, distorted rough endoplasmic reticulum cisternaes and abnormal nucleus were seen in the TBT treated group. Further, the biochemical and vitellogenin content were altered remarkably due to TBT exposure. It directly indicated that TBT had conspicuously inhibited the vitellogenesis. Therefore, it was inferred that the administration of TBT has considerably affected the hepatopancreatic functions in M. rosenbergii.