The thyroid hormone system disrupting potential of resorcinol in fish.

Environmental pollutants capable of interfering with the thyroid hormone (TH) system increasingly raise concern for both human and environmental health. Recently, resorcinol has received attention as a compound of concern due to its endocrine disrupting properties. It is a known inhibitor of thyroperoxidase (TPO), an enzyme required in TH synthesis, and therapeutic use of resorcinol exposure has led to hypothyroidism in humans. There is limited evidence concerning ecotoxicologically relevant effects of resorcinol in fish. A set of adverse outcome pathways (AOPs) has recently been developed linking thyroid hormone system disruption (THSD) to impaired swim bladder inflation and eye development in fish. In the present study, these AOPs were used to provide the background for testing potential THSD effects of resorcinol in zebrafish eleutheroembryos. We exposed zebrafish eleutheroembryos to resorcinol and assessed TH levels, swim bladder inflation and eye morphology. As a TPO inhibitor, resorcinol is expected to affect TH levels and eye morphology but not swim bladder inflation during embryonic development. Indeed, thyroxine (T4) levels were significantly decreased following resorcinol exposure. In contrast to our hypothesis, swim bladder inflation was impaired at 5 days post fertilization (dpf) and no effects on eye morphology were detected. Therefore, in vitro assays were performed to identify potential additional thyroid hormone system disruption-related mechanisms through which resorcinol may act. Two new mechanisms were identified: TH receptor (TR) antagonism and transthyretin (TTR) binding inhibition. Both of these mechanisms can plausibly be linked to impaired swim bladder inflation and could, therefore, explain the observed effect. Overall, our study contributes to the knowledge of the THSD potential of resorcinol both in vivo in the zebrafish model as well as in vitro.

Natural pyrethrins induced developmental toxicity of zebrafish swim bladder in vivo and genotoxicity of lung cells in vitro.

Natural pyrethrins (NPs) are insecticidal compounds isolated and extracted from pyrethrum flowers and are primarily use to control sanitary pests. The lungs become the main target after exposure, and its use may pose potential hazards to respiratory health. Therefore, in this paper, the toxic effects of NPs on human lung cells A549 were investigated and the risk of respiratory toxicity of NPs was studied using zebrafish swim bladder as a model. The results showed that NPs induced cytotoxicity, caused oxidative DNA damage and triggered mitochondria-mediated apoptosis. In addition, exposure to NPs decreased zebrafish embryo survival, hatchability, and heartbeat, and may inhibit normal swim bladder development by disrupting Wnt and Hedgehog signaling pathways. In conclusion, our results suggest that NPs can induce cytotoxicity in A549 in vitro and developmental toxicity in zebrafish in vivo. This study provides a conceptual basis for understanding the mechanisms of toxicity of NPs and assessing respiratory health risks in humans.

Exposure to thiourea during the early stages of development impedes the formation of the swim bladder in zebrafish larvae.

Thiourea, a widely used agrochemical, is known to inhibit the activity of thyroid peroxidase, a key enzyme in the biosynthetic pathway of thyroid hormones. Thyroid insufficiency compromises the basal metabolic rate in warm-blooded organisms and embryonic development in vertebrates. In this study, we looked for developmental defects by exposing the zebrafish embryos to an environmentally relevant dose of thiourea (3 mg/mL). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to validate thiourea's presence in the treated zebrafish embryos. Structural anomalies like bent tail and pericardial edema were noticed in 96-h post-fertilization (hpf) larvae. On histological examination, underdeveloped swim bladder was noticed in 96 hpf larvae exposed to 3 mg/mL thiourea. The treated larvae also failed to follow the characteristic swimming behavior in response to stimuli due to defective swim bladder. Swim bladder being homologous to the lung of tetrapod, the role of Bmp4, a major regulator of lung development, was studied along with the associated regulatory genes. Gene expression analysis revealed that thiourea administration led to the downregulation of bmp4, shh, pcna, anxa5, acta2, and the downstream effector snail3 but the upregulation of caspase3. The protein expression showed a similar trend, wherein Bmp4, Shh, and Pcna were downregulated, but Cleaved Caspase3 showed an increased expression in the treated group. Therefore, it is prudent to presume that exposure to thiourea significantly reduces the expression of Bmp4 and other key regulators; hence, the larvae fail to develop a swim bladder, a vital organ that regulates buoyancy.

Parental exposure to Cypermethrin causes intergenerational toxicity in zebrafish offspring.

Cypermethrin (CYP), a synthetic pyrethroid pesticide, has been detected in agriculture and aquaculture. However, there is limited knowledge about the transgenerational impacts. This study aimed to investigate the developmental toxicity of CYP on F1 larvae offspring of adult zebrafish exposed to various CYP concentrations (5, 10, and 20 µg/L) for 28 days. The results indicated that CYP accumulated in parental zebrafish, and CYP was below the limit of quantification in offspring. Paternal exposure impacted the hatching rate and heart rate of the F1 generation. Furthermore, CYP significantly impacted the development of swim bladders in progeny and dysregulated the genes relevant to swim bladder development. The neutrophil migrated to the swim bladder. The mRNA levels of the inflammatory factors were also significantly elevated. According to network toxicology, PI3-AKT may be the signaling pathway for CYP-influenced bladder development. Subsequent molecular docking and Western blot analysis showed CYP affected the PI3-AKT signaling pathway. Notably, MK-2206, a specific Akt inhibitor, rescued the CYP-induced damage of swim bladder development in offspring. The present study highlights the potential risks of CYP on the development of offspring and lasting impact in aquatic environments.

Effect of short-term exposure to the strobilurin fungicide dimoxystrobin: Morphofunctional, behavioural and mitochondrial alterations in Danio rerio embryos and larvae.

Strobilurins, among the most used fungicides worldwide, are considered non-toxic to mammals and birds, but there is growing evidence that these compounds are highly toxic to aquatic species. Dimoxystrobin has been included in the 3rd Watch List of the European Commission, and it has been classified as very toxic to aquatic life. However, previous studies focused on acute toxicity and only two reports are available on its impact on fish, and none on its effects during the early life stages. Here, we evaluated for the first time the effects induced on zebrafish embryos and larvae by two dimoxystrobin sublethal concentrations (6.56 and 13.13 µg/L) falling in the range of predicted environmental concentrations. We demonstrated that short-term exposure to dimoxystrobin may exert adverse effects on multiple targets, inducing severe morphological alterations. Moreover, we showed enhanced mRNA levels of genes related to the mitochondrial respiratory chain and ATP production. Impairment of the swim bladder inflation has also been recorded, which may be related to the observed swimming performance alterations.

Iopanoic acid alters thyroid hormone-related gene expression, thyroid hormone levels, swim bladder inflation, and swimming performance in Japanese medaka.

Disruption of the thyroid hormone system by synthetic chemicals is gaining attention owing to its potential negative effects on organisms. In this study, the effects of the dio-inhibitor iopanoic acid (IOP) on the levels of thyroid hormone and related gene expression, swim bladder inflation, and swimming performance were investigated in Japanese medaka. Iopanoic acid exposure suppressed thyroid-stimulating hormone ß (tshß), tshß-like, iodotyronin deiodinase 1 (dio1), and dio2 expression, and increased T4 and T3 levels. In addition, IOP exposure inhibited swim bladder inflation, reducing swimming performance. Although adverse outcome pathways of thyroid hormone disruption have been developed using zebrafish, no adverse outcome pathways have been developed using Japanese medaka. This study confirmed that IOP inhibits dio expression (a molecular initiating event), affects T3 and T4 levels (a key event), and reduces swim bladder inflation (a key event) and swimming performance (an adverse outcome) in Japanese medaka.

Particle size-dependent effects of silver nanoparticles on swim bladder damage in zebrafish larvae.

Particle size-dependent biological effects of silver nanoparticles (AgNPs) are of great interest; however, the mechanism of action of silver ions (Ag+) released from AgNPs concerning AgNP particle size remains unclear. Thus, we evaluated the influence of particle size (20, 40, 60, and 80 nm) on the acute 96-h bioaccumulation and toxicity (swim bladder damage) of AgNPs in zebrafish (Danio rerio) larvae, with a focus on the mechanism of action of Ag+ released from differently sized AgNPs. The 40- and 60-nm AgNPs were more toxic than the 20- and 80-nm versions in terms of inflammation and oxidative damage to the swim bladder, as indicated by inhibition of type 2 iodothyroxine deiodinase enzyme activity, mitochondrial injury, and reduced 30-50% adenosine triphosphate content. Furthermore, up-regulation and down-regulation of swim bladder development-related gene expression was not observed for pbx1a and anxa5, but up-regulation expression of shha and ihha was observed with no statistical significance. That 20-nm AgNPs were less toxic was attributed to their rapid elimination from larvae in comparison with the elimination of 40-, 60-, and 80-nm AgNPs; thus, less Ag+ was released in 20-nm AgNP-exposed larvae. Failed inflation of swim bladders was affected by released Ag+ rather than AgNPs themselves. Overall, we reveal the toxicity contribution of Ag+ underlying the observed size-dependent effects of AgNPs and provide a scientific basis for comprehensively assessing the ecological risk and biosafety of AgNPs.

The agrochemical S-metolachlor disrupts molecular mediators and morphology of the swim bladder: Implications for locomotor activity in zebrafish (Danio rerio).

Metolachlor herbicides are derived from the chloroacetamide chemical family of which there are the S- and R-metolachlor isomers. S-metolachlor is a selective herbicide that inhibits cell division and mitosis via enzyme interference. The herbicide is used globally in agriculture and studies report adverse effects in aquatic organisms; however, there are no studies investigating sub-lethal effects of S-metolachlor on swim bladder formation, mitochondrial ATP production, nor light-dark preference behaviors in fish. These endpoints are relevant for larval locomotor activity and metabolism. To address these knowledge gaps, we exposed zebrafish embryos/larvae to various concentrations of S-metolachlor (0.5-50 µM) over early development. S-metolachlor affected survival, hatching percentage, and increased developmental deformities at concentrations of 50 µM and above. Exposure levels as high as 200 µM for 24 and 48 h did not alter oxygen consumption rates in zebrafish, and there were no changes detected in endpoints related to mitochondrial oxidative phosphorylation. We observed impairment of swim bladder inflation at 50 µM in 6 dpf larvae. To elucidate mechanisms related to this, we measured relative transcript abundance for genes associated with the swim bladder (smooth muscle alpha (α)-2 actin, annexin A5, pre-B-cell leukemia homeobox 1a). Smooth muscle alpha (α)-2 actin mRNA levels were reduced in fish exposed to 50 µM while annexin A5 mRNA levels were increased in abundance, corresponding to reduced swim bladder size in larvae. A visual motor response test revealed that larval zebrafish exhibited some hyperactivity in the light with exposure to the herbicide and only the highest dose tested (50 µM) resulted in hypoactivity in the dark cycle. Regression analysis indicated that there was a positive relationship between surface area of the swim bladder and distance traveled, and the size of the swim bladder explained ~10-14% in the variation for total distance moved. Lastly, we tested larvae in a light dark preference test, and we did not detect any altered behavioral response to any concentration tested. Here we present new data on sublethal endpoints associated with exposure to the herbicide S-metolachlor and demonstrate that this chemical may disrupt transcripts associated with swim bladder formation and morphology, which could ultimately affect larval zebrafish activity. These data are expected to contribute to further risk assessment guidelines for S-metolachlor in aquatic ecosystems.

Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation.

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV-V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1ß induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.

The effects of exposure to crude oil or PAHs on fish swim bladder development and function.

The failure of the swim bladder to inflate during fish development is a common and sensitive response to exposure to petrochemicals. Here, we review potential mechanisms by which petrochemicals or their toxic components (polycyclic aromatic hydrocarbons; PAHs) may affect swim bladder inflation, particularly during early life stages. Surface films formed by oil can cause a physical barrier to primary inflation by air gulping, and are likely important during oil spills. The act of swimming to the surface for primary inflation can be arduous for some species, and may prevent inflation if this behavior is limited by toxic effects on vision or musculature. Some studies have noted altered gene expression in the swim bladder in response to PAHs, and Cytochrome P450 1A (CYP1A) can be induced in swim bladder or rete mirabile tissue, suggesting that PAHs can have direct effects on swim bladder development. Swim bladder inflation failure can also occur secondarily to the failure of other systems; cardiovascular impairment is the best elucidated of these mechanisms, but other mechanisms might include non-inflation as a sequela of disruption to thyroid signaling or cholesterol metabolism. Failed swim bladder inflation has the potential to lead to chronic sublethal effects that are as yet unstudied.

The synthetic pyrethroid deltamethrin impairs zebrafish (Danio rerio) swim bladder development.

Deltamethrin (DM) is a widely used insecticide and reveals neural, cardiovascular and reproductive toxicity to various aquatic organisms. It has been known that DM negatively affects motion of zebrafish (Danio rerio). However, little is known in relation to the impacts of DM on development of swim bladder, which is a key organ for motion. In the present study, zebrafish embryos were exposed to 20 and 40 µg/L DM. The changes of swim bladder morphology were observed and transcription levels of key genes were compared between DM treatments and the control. The results showed that DM treatments significantly blocked the formation of progenitor and tissue layers in swim bladder of zebrafish embryos, leading to failed inflation of swim bladder. Compared with the control, the key genes (pbx1, foxA3, mnx1, has2, anxa5b, hprt1l and elovl1a) responsible for swim bladder development also showed decreased levels in response to DM treatments, suggesting that DM might specifically affect swim bladder development. Moreover, transcription levels of genes in the Wnt (wnt5b, tcf3a, wnt1, wnt9b, fzd1, fzd3 and fzd5) and Hedgehog (ihhb, ptc1 and ptc2) signaling pathways all decreased significantly in response to DM treatments, compared with the control. Considering the importance of Wnt and Hedgehog pathways in development of swim bladder, these results suggested that DM might affect swim bladder development through inhibiting the Wnt and Hedgehog pathways. Overall, the present study reported that swim bladder might be a potential target organ of DM toxicity in zebrafish, which contributed more information to the evaluation of DM's environmental risks.

Sex-specific endocrine-disrupting effects of three halogenated chemicals in Japanese medaka.

Several halogenated chemicals are found in an array of products that can cause endocrine disruption. Human studies have shown that endocrine responses are sex specific, with females more likely to develop hypothyroidism and males more likely to have reproductive impairment. The objective of this study was to assess sex differences on thyroid and estrogenic effects after exposure of Japanese medaka (Oryzias latipes, SK2MC) to halogenated compounds. This strain is an excellent model for these studies as sex can be determined non-destructively a few hours postfertilization. Medaka embryos were exposed to sublethal concentrations of Tris(1,3-dichloro-2-propyl) phosphate (TDCPP, 0.019 mg/L), perfluorooctanoic acid (PFOA, 4.7 mg/L) and its next generation alternative, perfluorobutyric acid (PFBA, 137 mg/L). Methimazole (inhibits thyroid hormone synthesis) and the thyroid hormone triiodothyronine served as reference controls. Fish were exposed throughout embryo development until 10 days postfertilization. Females displayed significantly larger swim bladders (which are under thyroid hormone control) after exposure to all chemicals with the exception of triiodothyronine, which caused the opposite effect. Females exposed to TDCPP and PFOA had increased expression of vitellogenin and exposure to PFOA upregulated expression of multiple thyroid-related genes. Upregulation of estrogenic-regulated genes after exposure to TDCPP, PFOA and methimazole was only observed in males. Overall, our results suggest that females and males show an estrogenic response when exposed to these halogenated chemicals and that females appear more susceptible to thyroid-induced swim bladder dysfunction compared with males. These results further confirm the importance of considering sex effects when assessing the toxicity of endocrine-disrupting compounds.

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.

Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish.

Endocrine disrupting chemicals (EDCs) can alter thyroid function and adversely affect growth and development. Halogenated compounds, such as perfluorinated chemicals commonly used in food packaging, and brominated flame retardants used in a broad range of products from clothing to electronics, can act as thyroid disruptors. Due to the adverse effects of these compounds, there is a need for the development of safer next generation chemicals. The objective of this study was to test the thyroid disruption potential of old use and next generation halogenated chemicals. Zebrafish embryos were exposed to three old use compounds, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) and two next generation chemicals, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxdie (DOPO) and perfluorobutyric acid (PFBA). Sub-chronic (0-6days post fertilization (dpf)) and chronic (0-28dpf) exposures were conducted at 1% of the concentration known to kill 50% (LC50) of the population. Changes in the surface area of the swim bladder as well as in expression levels of genes involved in the thyroid control of swim bladder inflation were measured. At 6dpf, zebrafish exposed to all halogenated chemicals, both old use and next generation, had smaller posterior swim bladder and increased expression in the gene encoding thyroid peroxidase, tpo and the genes encoding two swim bladder surfactant proteins, sp-a and sp-c. These results mirrored the effects of thyroid hormone-exposed positive controls. Fish exposed to a TPO inhibitor (methimazole, MMI) had a decrease in tpo expression levels at 28dpf. Effects on the anterior swim bladder at 28dpf, after exposure to MMI as well as both old and new halogenated chemicals, were the same, i.e., absence of SB in ∼50% of fish, which were also of smaller body size. Overall, our results suggest thyroid disruption by the halogenated compounds tested via the swim bladder surfactant system. However, with the exception of TBBPA and TDCPP, the concentrations tested (∼5-137ppm) are not likely to be found in the environment.

Copper impairs zebrafish swimbladder development by down-regulating Wnt signaling.

Copper nanoparticles (CuNPs) are used widely in different fields due to their attractive and effective abilities in inhibiting bacteria and fungi, but little information is available about their biological effects and potential molecular mechanisms on fish development. Here, CuNPs and copper (II) ions (Cu2+) were revealed to inhibit the specification and formation of three layers of zebrafish embryonic posterior swimbladder and impair its inflation in a stage-specific manner. CuNPs and Cu2+ were also revealed to down-regulate Wnt signaling in embryos. Furthermore, Wnt agonist 6-Bromoindirubin-3'-oxime (BIO) was found to neutralize the inhibiting effects of CuNPs or Cu2+ or both on zebrafish swimbladder development. The integrated data here provide the first evidence that both CuNPs and Cu2+ act on the specification and growth of the three layers of swimbladder and inhibit its inflation by down-regulating Wnt signaling in a stage-specific manner during embryogenesis.

Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid.

Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 d postfertilization (dpf), at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6-dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole-body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 messenger ribonucleic acid (mRNA) was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. Environ Toxicol Chem 2017;36:2942-2952. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Perturbation of metabonome of embryo/larvae zebrafish after exposure to fipronil.

The escalating demand for fipronil by the increasing insects' resistance to synthetic pyrethroids placed a burden on aquatic vertebrates. Although awareness regarding the toxicity of fipronil to fish is arising, the integral alteration caused by fipronil remains unexplored. Here, we investigated on the development toxicity of fipronil and the metabolic physiology perturbation at 120h post fertilization through GC-MS metabolomics on zebrafish embryo. We observed that fipronil dose-dependently induced malformations including uninflated swim bladder and bent spine. Further, the "omic" technique hit 26 differential metabolites after exposure to fipronil and five significant signaling pathways. We speculated that changes in primary bile acid synthesis pathway and the content of saturated fatty acid in the chemical-related group indicated the liver toxicity. Pathway of Aminoacyl-tRNA biosynthesis changed by fipronil may relate to the macromolecular synthesis. Concurrently, methane metabolism pathway was also identified while the role in zebrafish needs further determination. Overall, this study revealed several new signaling pathways in fipronil-treated zebrafish embryo/larval.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow.

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted. Specifically, at 14dpf, approximately 50% of fish exposed to 1mg MBT/L did not have an inflated anterior swim bladder. In fish exposed to MBT through 21 or 22dpf, the anterior swim bladder was able to inflate, but the ratio of the anterior/posterior chamber length was significantly reduced compared to controls. Both abundance of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to the presumed inhibition of TPO activity by MBT. Time-course characterization showed that fish exposed to MBT for at least 4 days prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber, compared to controls. These results, along with similar results observed in zebrafish (see part II, this issue) are consistent with the hypothesis that thyroid hormone signaling plays a significant role in mediating anterior swim bladder inflation and development in cyprinids, and that role can be disrupted by exposure to thyroid hormone synthesis inhibitors. Nonetheless, possible thyroid-independent actions of MBT on anterior swim bladder inflation cannot be ruled out based on the present results. Overall, although anterior swim bladder inflation has not been directly linked to survival as posterior swim bladder inflation has, potential links to adverse ecological outcomes are plausible given involvement of the anterior chamber in sound production and detection.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish.

Disruption of the thyroid hormone (TH) system, an important mode of action, can lead to ecologically relevant adverse outcomes, especially during embryonic development. The present study characterizes the effects of disruption of TH synthesis on swim bladder inflation during zebrafish early-life stages using 2-mercaptobenzothiazole (MBT), a thyroid peroxidase (TPO) inhibitor. Zebrafish were exposed to different MBT concentrations until 120/168h post fertilization (hpf) and 32days post fertilization (dpf), in two sets of experiments, to investigate the effects of TPO inhibition on posterior and anterior swim bladder inflation respectively, as well as whole body thyroid hormone concentrations (triiodothyronine (T3) and its prohormone, thyroxine (T4)). At 120hpf, MBT did not directly impair posterior chamber inflation or size, while anterior chamber inflation and size was impaired at 32dpf. As previously shown in amphibians and mammals, we confirmed that MBT inhibits TPO in fish. Whole-body T4 decreased after MBT exposure at both time points, while T3 levels were unaltered. There was a significant relationship between T4 levels and the anterior chamber surface at 32dpf. The absence of effects on posterior chamber inflation can possibly be explained by maternal transfer of T4 into the eggs. These maternally derived THs are depleted at 32dpf and cannot offset TPO inhibition, resulting in impaired anterior chamber inflation. Therefore, we hypothesize that TPO inhibition only inhibits swim bladder inflation during late development, after depletion of maternally derived T4. In a previous study, we showed that iodothyronine deiodinase (ID) knockdown impaired posterior chamber inflation during early development. Our findings, in parallel with similar effects observed in fathead minnow (see part I, this issue) suggest that thyroid disruption impacts swim bladder inflation, and imply an important distinction among specific subtypes of TH disrupting chemicals. However, the existence of another - yet unknown - mode of action of MBT impacting swim bladder inflation cannot be excluded. These results can be helpful for delineating adverse outcome pathways (AOPs) linking TPO inhibition, ID inhibition and other TH related molecular initiating events, to impaired swim bladder inflation in fish during early life stages. Such AOPs can support the use of in vitro enzyme inhibition assays for predicting reduced survival due to impaired posterior and anterior chamber inflation.

Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae.

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3-3000ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300 ng/L and 3000 ng/L) as well as growth impairment and delayed development (100 ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169 ng/L) was similar to the LC50 value at 8 dpf (188 and 202 ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species.