The effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish

Abstract

A set of adverse outcome pathways (AOPs) linking inhibition of thyroperoxidase and deiodinase to impaired swim bladder inflation in fish has recently been developed. These AOPs help to establish links between these thyroid hormone (TH) disrupting molecular events and adverse outcomes relevant to aquatic ecological risk assessment. Until now, very few data on the effects of TH disruption on inflation of the anterior chamber (AC) of the swim bladder were available. The present study used zebrafish exposure experiments with three model compounds with distinct thyroperoxidase and deiodinase inhibition potencies (methimazole, iopanoic acid and propylthiouracil) to evaluate this linkage. Exposure to all three chemicals decreased whole body triiodotyrosine (T3) concentrations, either through inhibition of thyroxine (T4) synthesis or through inhibition of Dio mediated conversion of T4 to T3. A quantitative relationship between reduced T3 and reduced AC inflation was established, a critical key event relationship linking impaired swim bladder inflation to TH disruption. Reduced inflation of the AC was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose ACs inflated. Together the data provide compelling support for AOPs linking TH disruption to impaired AC inflation in fish.

Graphical abstract

1. Introduction

A number of high-profile environmental pollutants adversely affect the thyroid hormone (TH) system of different species. The thyroid axis is highly conserved among vertebrate taxa¹, and THs play a crucial role in the regulation of vertebrate development, as well as important homeostatic processes related to growth and energy metabolism.² In fish, disruption of the hypothalamic–pituitary–thyroidal (HPT) axis has been demonstrated to cause a wide variety of adverse effects³, including impaired craniofacial development^4,5, fin formation⁶, pigmentation⁷, skeletal development⁸, eye development and visual performance^9–11, as well as swim bladder inflation^12,13.

In the present study, we examined the effects of TH disruption on swim bladder inflation in the zebrafish during late larval development. The swim bladder is a gas-filled sac that in most fish species consists of two distinct but connected chambers, a posterior chamber (PC) and an anterior chamber (AC). In zebrafish, PC inflation occurs at 4–5 days post fertilization (dpf) by gulping air, while AC inflation occurs around 20–21 dpf. Both chambers have a role in regulating buoyancy, and the AC additionally functions as a resonating chamber to assist in hearing in fish species, like the zebrafish, that possess a Weberian apparatus connecting the swim bladder to the auditory system¹⁴. Impaired PC and/or AC inflation is therefore likely to impact swimming performance and hearing, potentially affecting behaviours related to predator avoidance, feeding and spawning, and ultimately survival^14–18.

Based on earlier work and literature data, a set of adverse outcome pathways (AOPs) was recently developed linking inhibition of thyroperoxidase (Tpo) and deiodinase (Dio) to impaired swim bladder inflation in fish through decreased levels of 3,5,3’-triiodothyronine (T3, the biologically active form of TH) in serum ^{12,13,19–22} (AOPs 155–159, www.aopwiki.org). An AOP is a conceptual construct describing the scientific evidence linking perturbation of a molecular target such as Tpo or Dio by a chemical to adverse effects that are considered relevant to risk assessment and regulatory decision-making²³. Notably, AOPs 155–159 are interconnected forming an AOP network which describes the apical effects of chemicals that interact with different molecular targets but impact downstream biological processes common to multiple pathways²¹. An important aspect of AOP and AOP network development involves documentation of the weight of evidence supporting the relationships between various key events. While the link between TH disruption and impaired PC inflation in fish is supported by a large body of evidence^{11–13,19–22,24–26}, comparatively few data on the effects of TH disruption on AC inflation are available^{12,13,19,20,27}. To further strengthen this relatively unsupported part of the AOP network (Figure 1), the present study was designed to empirically test whether Tpo and/or Dio inhibition would result in impaired AC inflation via decreased TH levels, and whether this subsequently impacts swimming performance.

Figure 1:

AOPs (https://aopwiki.org/aops/156,158,159) describing the effect of Tpo and Dio inhibition on TH concentrations, leading to impaired anterior chamber inflation, reduced swimming performance and young of year survival.

To evaluate this hypothesis, we exposed zebrafish embryos to three model compounds with distinct Tpo and Dio¹² inhibition potencies in 32 day Fish Early-Life Stage toxicity tests (FELS test, OECD TG 210²⁸). We used methimazole as a potent Tpo inhibitor²⁹, iopanoic acid as a Dio inhibitor¹², and 6-propylthiouracil, a Tpo inhibitor²⁹ and a possible Dio inhibitor¹². Essential steps and processes along the AOP network were evaluated, including both molecular initiating events (Tpo and Dio inhibition), the main intermediate key events KEs (TH levels and AC inflation), and swimming performance as the adverse outcome. Additionally, we investigated the quantitative relationships between TH levels and AC inflation since this is the critical key event relationship linking impaired swim bladder inflation to TH disruption.

The present dataset supports the proposed AOPs and increases the level of confidence in the linkages between TH disruption, detected via screening assays, and adverse effects on fish development. Knapen et al. (submitted) discuss in more detail how the current AOP network can be used to develop an AOP network-based tiered testing strategy for the assessment of TH disruption.

2. Materials and Methods

2.1. Ethics statement

All experiments of this study exceeded the non-protected animal stage (i.e., 120 hours post fertilization (hpf) for zebrafish³⁰) and were approved by the Ethical Committee for Animals of the University of Antwerp (project IDs 2014–29 and 2016–46). Experiments were carried out in strict accordance with EU Directive 2010/63/EU on the protection of animals used for scientific purposes³¹.

2.2. Fish Early-Life Stage (FELS) tests

2.2.1. Test compounds and exposure concentrations

Anterior chamber inflation was assessed in FELS tests based on OECD TG 210²⁸, exposing zebrafish embryos from 1 hpf until 32 dpf to three chemicals: methimazole (MMI, CAS 60–56–0, Sigma-Aldrich, ≥99% purity), iopanoic acid (IOP, CAS 96–83–3, TCI, >98% purity) and 6-propylthiouracil (PTU, CAS 51–52–5, Sigma-Aldrich, ≥99% purity). Selection of exposure concentrations (MMI: 0, 50 and 100 mg/L; IOP: 0, 0.35 and 1 mg/L; and PTU: 0, 37 and 111 mg/L) was based on three criteria, using available data from previous 120 hpf zebrafish exposures¹² (See also section 1.1 in supporting information, SI): (1) no effect on hatching at 48 hpf, a process required for initial swim bladder inflation, (2) no effect on survival at 120 hpf, since we were interested in sublethal effects, and (3) no inhibition of PC inflation, because this likely inhibits AC inflation since the latter buds from the PC¹⁴. An additional high 2 mg/L IOP treatment (12.5% mortality, 34% effect on PC inflation based on previous 120 hpf zebrafish exposures¹²) was included to assess the effect of impaired PC inflation on survival (Section 1.2 in SI).

Stock solutions (MMI, 500 mg/L; PTU, 888 mg/L) were prepared by directly dissolving chemicals in reconstituted freshwater. To achieve solvent-free test conditions for IOP, a 5 g/L solution was prepared in reconstituted freshwater containing 0.1 M NaOH by sonicating for 5 min (or until solubilized), and diluting using reconstituted freshwater to 100 mg/L IOP (final concentration of 0.002 N NaOH). Stocks were freshly prepared once a week and stored in the dark, at room temperature until use. Working solutions were prepared fresh daily (pH only needed to be adjusted for IOP working solutions, to 7.5 using 0.1 N HCl).

2.2.2. Collection of eggs and exposure

Zebrafish housing and egg production was conducted as described in Stinckens et al.^12,13 Briefly, adult zebrafish (in house wild type zebrafish line) were accommodated in a ZebTEC stand-alone system (Tecniplast, Buguggiate, Italy) with reconstituted freshwater (conductivity of 500 ± 15 µS/cm, pH 7.5 ± 0.3), at a temperature of 28 ± 0.2°C under a 14/10 h light/dark cycle. Embryos were collected from breeding groups consisting of one female and two males placed in breeding tanks.

Exposures were performed as described previously.¹² Briefly, normally shaped fertilized embryos were exposed in pre-saturated polystyrene 24-well plates (sterile tissue culture plates, Greiner Bio-One, Frickenhausen, Germany) with one embryo and 2 mL of test solution per well. Exposures were started within 30–60 minutes after spawning and the stocked wells stored in an incubator (MIR-254-PE, Panasonic, TCPS, Rotselaar, Belgium) under a 14/10 h day/night cycle at 28.5°C. The first row of four chambers on each plate contained internal negative control (untreated) embryos, resulting in 20 chemically-exposed embryos per plate. For MMI, eight plates were used per treatment (n=160), while for IOP and PTU there were 15 plates (n=300). For the additional high 2 mg/L IOP treatment there were six plates (n=120). From 5 dpf onwards, larvae were progressively moved into larger test chambers/volumes (960 mL plastic beakers containing 200, 400, and 750 mL at 5–10, 10–20, and 20–32 dpf, respectively). The number of replicate test chambers each containing 30–35 larvae was five for MMI, seven for IOP and PTU and four for 2 mg/L IOP. From 5–10 dpf fish were fed once a day with paramecia and twice with SDS-100 (Special Diets Services, Tecnilab-BMI, The Netherlands) dry food. From 10–20 dpf fish were fed once with paramecia, once with a combination of Artemia sp. nauplii and SDS-100, and once with SDS-100. From 20–32 dpf fish were fed once with Artemia sp. nauplii, once with a combination of Artemia sp. Nauplii and SDS-100, and once with SDS-100. Test solutions were renewed daily. Oxygen and ammonium concentrations were measured daily from 5 dpf onwards. Dissolved oxygen remained above 60% of the air saturation value and ammonium concentrations below 0.25 mg/L throughout the test. Tests were valid if ≥75% of the negative controls survived, ≥70% successfully hatched, and the minimum mean total length of control fish at the end of the study exceeded 11 mm (OECD TG 210²⁸).

A subset of larvae per time point (32 dpf was always included and complemented by 14 and 21 dpf in selected cases) was euthanized using 1 g/L MS-222 adjusted to pH 7.5 with NaHCO_3, and larvae were rinsed using reconstituted water as described previously.¹³ Larvae were pooled in cryovials, weighed (fresh weight), snap frozen and kept at −80°C until triiodothyronine (T3)/ thyroxine (T4) or test chemical measurements were conducted. An overview of the timing of assessment of endpoints, as well as detailed information regarding number of replicates, etc., is given in Table S1.

2.2.3. Morphological and physiological assessment

In addition to the endpoints listed in Table S1, mortality, hatching (≥ 48 hpf) and PC inflation (≥ 96 hpf) were monitored daily to check the criteria for correct assessment of AC inflation (Section 2.2.1). The proportion of inflated ACs was recorded in a binary fashion (inflated or non-inflated, 4 or 5 replicates of 20–35 larvae each) using a stereomicroscope (Leica S8APO, Leica Microsystems GmbH, Germany). Length and swim bladder surface area were measured by placing larvae on a glass slide in a small drop of exposure medium, which was removed until the fish assumed a lateral position, and immediately photographing them together with a calibrator using a camera (Canon EOS 600D, 18 megapixels) mounted on a stereomicroscope (n per treatment depends on proportion of inflation, Table S1). Larvae were transferred back to the appropriate treatment vessel as soon as possible. From 24 dpf onwards, the light intensity of the microscope was decreased to compensate for developing pigmentation. Larval total length and the surface of the posterior and anterior chamber(s) were determined using the ImageJ software (available at http://rsbweb.nih.gov/ij/). Surface measurements were performed by marking the actual circumference of the PC and AC. Relative chamber surfaces were calculated in order to correct for differences in growth rate^13,32 (Section 1.4 in SI, Figure S1).

Swimming capacity of a subset of larvae (at least 24 per treatment, Table S1) was determined in 24-well plates (5 dpf) or six-well plates (all other time points) using a Zebrabox 3.0 video tracking device (Viewpoint, Lyon, France). Swimming behaviour was recorded during 40 min in light and data were analysed using the ZebraLab software version 3.20.5.104. The sum of all swimming movements (mm) was compared among the various treatments.

2.3. Medium concentrations and tissue residues

Samples (three per treatment) of each exposure solution (500 µL) were taken before and after medium renewal for at least three time points during the study (Table S1). Samples were stored in 1.5 mL glass vials (5182–0716, Agilent Technologies, Palo Alto, CA, USA) at −20°C until analysis. Analytical verification was performed using an Agilent 1260 Series (Agilent Technologies) high performance liquid chromatograph (HPLC) with diode array detection. Additional details on the analysis of each exposure compound are provided in section 1.6 of the SI.

Tissue residues of the test chemicals in larvae were measured as a proxy for dose in pooled whole body larvae (3–4 replicates of 4–5 larvae each per treatment, at least at 32 dpf and sometimes additionally at 14 and 21 dpf, Table S1) using an Agilent HPLC 1200 Series (Agilent Technologies) connected to a triple quadrupole mass spectrometer (Agilent 6410 QqQ, Agilent Technologies). Detailed procedures for sample preparation and HPLC and HPLC-QqQ-MS parameters can be found in section 1.6 of the SI.

2.4. Whole body T3 and T4 levels

Whole body T3 and T4 concentrations were measured in larval fish (3 or 4 replicates of 4–20 larvae each depending on age, at least at 32 dpf and sometimes at 14 and 21 dpf, Table S1) as described by Nelson et al.¹⁹ and Stinckens et al.¹³ (Section 1.7 in SI). Briefly, THs were extracted using ethanol, the lipid removed with hexane, and cleaned-up by solid-phase extraction (SPE, Evolute CX cartridges, Biotage). Quantification was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS).

2.5. Evaluation of Tpo inhibitory potential of the test compounds

We directly measured the Tpo inhibitory potential of the three selected model compounds. To rank the model compounds relative to other potential environmental TH disrupting chemicals, we evaluated a library of 44 chemicals (Table S2). The same chemical library was evaluated for Dio inhibitory potential in a previous study¹². Tpo inhibition was measured in an Amplex UltraRed (AUR®; Life Technologies, cat. no. A36006) assay, according to Paul et al.²⁹, with porcine instead of rat thyroid microsomes. Details on the methods can be found in sections 1.8 in SI.

2.6. Data analysis

Statistical analyses were performed using GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA) and data were considered significantly different when p-values were <0.05. Data normality was assessed using a Shapiro-Wilk test. Data for larval length, condition factor, relative chamber surfaces, distance travelled and T3/T4 measurements were analysed using a one-way analysis of variance (ANOVA) with a Tukey’s multiple comparisons test. The percentage of AC inflation was compared among conditions and time points using a chi-squared contingency table test, with a Tukey’s multiple comparisons test. A Pearson correlation coefficient was calculated between T4/T3 levels and relative chamber surface values.

3. Results and discussion

In this study we empirically tested an AOP network-derived hypothesis stating that Tpo and Dio inhibition (the molecular initiating events, MIEs) reduce TH concentrations (key event 1, KE1), leading to impaired anterior chamber (AC) inflation (KE2) and subsequently to reduced swimming performance (the adverse outcome, AO) in fish. Our main results are shown in Figures 2, 3 and 4 for exposure to MMI, IOP and PTU respectively. Table 1 summarizes the key findings of the current study complemented with corresponding data that were previously published^12,13 (highlighted in grey) for easy reference. The following sections will chronologically discuss the new evidence for each of the building blocks of the AOP network (MIEs → KE1 → KE2 → AO), and the table and figures are structured accordingly. Test chemical concentrations in water and tissue residue measurements (Section 2.1 in SI, Table S3, Figure S3), mortality and hatching (Section 2.2 in SI), length (Figure S4), condition factor (Section 1.5 in SI, Figures S2 and S5) and PC inflation (Section 2.5 in SI), and detailed results from T3/T4 measurements (Figures S6 and S7), relative chamber surface (Figures S8 and S9), KE relationships between both (Figures S10–S15) and swimming distance (Figure S16) are in in the SI.

Figure 2:

Results after MMI exposure in zebrafish aligned along the AOP. In vivo data were observed at 32 dpf. Treatments are color-coded (dark blue: 0 mg/L, light blue: 50 mg/L, red: 100 mg/L MMI). Larvae with (closed symbols/bars) and without (open symbols/bars) inflated anterior chamber (AC) are shown separately if applicable. (A) Mean IC50 values ± SE for Tpo, Dio1 and Dio2 inhibition (MIEs). (B) Mean T4 and T3 concentrations (KE). (C) KE relationship between T3 concentrations and mean relative posterior (circles) and AC surface area (squares). (D) AC inflation (KE) as a function of time and treatment (left), mean relative AC surface area (right). (E) Mean swimming distance (AO). Error bars (B-D) indicate standard deviations. Sample sizes (n) are given between parentheses. Different letters indicate significant differences between treatments (B, D right panel, E) or among concentrations and between different days of exposure (D left panel).

Figure 3:

Results after IOP exposure in zebrafish aligned along the AOP. In vivo data were observed at 32 dpf. Treatments are color-coded (dark blue: 0 mg/L, light blue: 0.35 mg/L, red: 1 mg/L, purple: 2 mg/L IOP). Larvae with (closed bars) and without (open bars) inflated anterior chamber (AC) are shown separately if applicable. (A) Mean IC50 values ± SE for Tpo, Dio1 and Dio2 inhibition (MIEs). (B) Mean T4 and T3 concentrations (KE). (C) KE relationship between T3 concentrations and mean relative posterior (circles) and AC surface area (squares). (D) AC inflation (KE) as a function of time and treatment (left), mean relative AC surface area (right). (E) Mean swimming distance (AO). Error bars (B-D) indicate standard deviations. Sample sizes (n) are given between parentheses. Different letters indicate significant differences between treatments (B, D right panel, E) or among concentrations and between different days of exposure (D left panel).

Figure 4:

Results after PTU exposure in zebrafish aligned along the AOP. In vivo data were observed at 32 dpf. Treatments are color-coded (dark blue: 0 mg/L, light blue: 37 mg/L, red: 111 mg/L PTU). Larvae with (closed bars) and without (open bars) inflated anterior chamber (AC) are shown separately if applicable. (A) Mean IC50 values ± SE for Tpo, Dio1 and Dio2 inhibition (MIEs). (B) Mean T4 and T3 concentrations (KE). (C) KE relationship between T3 concentrations and mean relative posterior (circles) and AC surface area (squares). (D) AC inflation (KE) as a function of time and treatment (left), mean relative AC surface area (right). (E) Mean swimming distance (AO). Error bars (B-D) indicate standard deviations. Sample sizes (n) are given between parentheses. Different letters indicate significant differences between treatments (B, D right panel, E) or among concentrations and between different days of exposure (D left panel).

Table 1:

Overview of the impact of chronic exposure to MMI, PTU, IOP and MBT on the MIEs (Dio and Tpo inhibition), KEs (TH concentrations and swim bladder inflation) and AO (swimming capacity) of the proposed thyroid AOP network. Results from previous publications^12,13 are indicated by grey shading.

	MIEs			KE1				KE2				AO
	Tpo and Dio inhibition			Reduced TH levels				Reduced swim bladder inflation				Reduced swimming capacity
	Dio1	Dio2	Tpo	Expected		Observed		Expected		Observed		Expected	Observed
				T4	T3	T4	T3	PCI	ACI	PCI	ACI
MMI	–	–	+	↓	↓	↓	↓	–	+	–	+	↓	↓
PTU	+a	±	+	↓	↓	↓	↓	+	+	+b	+	↓	↓
IOP	+	+	–	–	↓	–	↓	+	+	+	+	↓	↓
MBT	±	±	+	↓	↓	↓	–	–	+	–	+	↓	↓

PCI: posterior chamber inflation, ACI: anterior chamber inflation

^–: no effect observed

⁺: effect observed

^±: zone of uncertainty in where in vitro Dio inhibition is not certain to cause biological effects by altering TH concentrations in vivo

^↓: significant decrease

^ain vitro Dio inhibition was measured using porcine tissue¹² but Dio inhibitory potential of PTU in fish is uncertain

^bonly observed until 120 hpf, while delayed PC inflation may occur.

3.1. MIEs: Thyroperoxidase and deiodinase inhibition

The three model compounds were selected based on their distinct and well-known TH disrupting properties^12,29. MMI is a Tpo inhibitor, IOP is a Dio inhibitor, and PTU inhibits both Tpo and Dio. MMI and PTU are the most commonly used antithyroid drugs in the world³³. The Tpo inhibitory capacity of our test chemicals relative to the complete library of 44 chemicals can be found in section 2.11, Figure S17 and Table S4 of the SI. MMI is the most potent Tpo inhibitor in the library (Figure 2A and S17), with similar potencies observed for PTU (Figure 4A and S17) and a number of other compounds such as carbimazole, 2,2′−4,4′-tetrahydroxy benzophenone, 2-mercaptobenzothiazole (MBT) and 2-thiouracil. IOP, the most commonly used oral cholecystographic agent with structural similarities to THs, was among the most potent Dio1 and Dio2 inhibitors in our previous study¹² together with, for example, pentachlorophenol, 2-mercaptobenzimidazole and pentadecafluorooctanoic acid. In addition to its strong Tpo inhibitory capacity, PTU was also a potent inhibitor of Dio1 in our porcine enzyme inhibition assay, which is similar to observations in some other vertebrate species^34–37. However, the sensitivity of teleost Dio to PTU remains unclear (see section 3.2)^38,39.

3.2. KE1: Reduced thyroid hormone levels

Tpo inhibition reduces TH synthesis (Figure 1). Consistent with this expectation, both MMI and PTU resulted in reduced whole body T4 at all time points (Figures 2B, 4B and S6). Due to the decreased availability of T4 for conversion to T3 by deiodinases, this subsequently led to decreased T3 concentrations at all time points (Figures 2B, 4B and S6). A positive correlation was found between T4 and T3 concentrations at 21 and 32 dpf for exposures to MMI and PTU (Figure S10). Dio inhibition on the other hand does not directly influence TH synthesis but inhibits activation of T4 to T3. This resulted in decreased T3, but not T4 levels, after prolonged exposure to IOP (decreases were noted at 21 and 32 dpf, but not at 14 dpf, Figures 3B, S6). Overall, exposure to each of the three compounds resulted in decreased whole body T3 concentrations at 21 and 32 dpf. This illustrates the position of reduced T3 levels as a so-called hub KE, a point of convergence in the AOP network²¹, where AOPs with different MIEs lead to the same physiological consequences via different mechanisms.

Severely reduced T4 concentrations were also found in MMI exposed pro-metamorphic Xenopus laevis larvae⁴⁰, rat⁴¹ and 5 d old zebrafish larvae⁴². PTU decreased T4 in the same zebrafish study⁴². Exposure to IOP has previously been shown to result in decreased T3 levels in fathead minnows²⁰, Rana catesbeiana tadpoles⁴³ and humans^44–46. In earlier work, we also observed reduced whole body T4 concentrations after exposure to MBT, an environmental Tpo inhibitor, in zebrafish¹³ and fathead minnow¹⁹, but the TH decreases were less consistent across exposure concentrations and time points. Feedback mechanisms seemed to compensate for decreased TH levels after MBT exposure resulting in recovery at the end of the exposure duration in the fathead minnow, which was not observed in the present study. However, MBT can cause substantial toxicity, probably through additional mechanisms, compared to the antithyroid pharmaceuticals MMI and PTU (see LC50 values, concentration at which 50% mortality occurs, at 120 hpf¹²). The exposure concentrations that were used for MBT (0.1 and 0.35 mg/L in zebrafish, 0.25–1 mg/L in fathead minnow) in our previous studies were therefore much lower compared to the those of MMI (50 and 100 mg/L) and PTU (37 and 111 mg/L) in the present study. Since the Tpo inhibitory potencies of MMI, PTU and MBT are comparable (Figure S17), this could explain why the effects of MBT were less pronounced and feedback mechanisms were more effective at compensating for TH disruption.

While the Tpo inhibitory capacity of PTU is generally accepted, there has been extensive debate on the seeming (in)sensitivity of fish Dio to PTU^38,39,47. The decrease of T3 levels (Figure 4B) could either be caused by Tpo inhibition, by Dio inhibition, or by a combination of both mechanisms. However, like MMI, PTU decreased the T4/T3 ratio (Figure S7). A relative increase of T3 compared to T4 after chemical exposure could be caused by a compensatory induction of Dio activity, increasing the activation of T4 into T3. This suggests that PTU may act primarily as a Tpo inhibitor in the zebrafish.

3.3. KE2: Reduced anterior chamber inflation

There is convincing evidence that inhibition of Dio activity, either through specific gene knockdown/knockout experiments^11,24,26,48 or chemical exposure^{12,13,19,20,49,50}, results in impaired posterior chamber (PC) inflation. Stinckens et al.¹² showed that effects on PC inflation in zebrafish embryos could be predicted based on the Dio1/Dio2 enzyme inhibition potential. A similar role of TH in AC inflation is suggested by increased whole-body T4⁵¹ and Dio1 and Dio2 expression⁵² at the time of AC inflation in normally developing zebrafish. In fathead minnows, both T4 and T3 increased at the time of AC inflation⁵³. However, evidence of the relationship between TH disruption and AC inflation was limited.

In the present study, exposure to all tested compounds impaired AC inflation, but not to the same extent (Figures 2D, 3D and 4D). In all experiments, 100% of control larvae had inflated ACs at 21 dpf. Both PTU and MMI (the Tpo inhibitors) delayed AC inflation by at least several days, and most MMI exposed larvae still had non-inflated ACs at 32 dpf. Godfrey et al.⁴⁹ observed similar effects in MMI exposed zebrafish, with 60% non-inflated ACs at 28 dpf. In previous work, we observed reduced AC inflation in both zebrafish¹³ and fathead minnows¹⁹ exposed to MBT. Chopra et al.²⁷ recently found that deficiency of dual oxidase, an enzyme responsible for producing H₂O₂ that is essential for Tpo activity, resulted in a lack of T4 staining in thyroid follicles and impaired AC inflation in zebrafish. In the present study, at 21 dpf, exposure to 0.35, 1 and 2 mg/L of IOP (the Dio inhibitor) resulted in 35%, 43%, and 61% of the larvae with non-inflated ACs, respectively, and at the end of the experiment, around 25% of IOP exposed larvae still had impaired AC inflation. Cavallin et al.²⁰ also demonstrated delayed AC inflation after exposure of fathead minnows to IOP.

All compounds also affected the surface area of both the AC and the PC (Figures 2D, 3D, 4D, S8 and S9). In all cases where the AC was inflated, its surface area was significantly smaller compared to control larvae, and the PC surface increased with decreasing AC surface (Figure S15). As a result, the sum of both chamber surfaces, reflecting the total amount of gas, was equal to controls for most treatments (Figure S14). On the other hand, while the PC surface was significantly larger as well when the AC remained non-inflated (Figure S8), the total surface of both chambers did, for most treatments, not reach control levels (Figure S14). Similar results, where a smaller AC was associated with a larger PC, were found in both zebrafish and fathead minnow exposed to MBT^13,19 and in fathead minnow exposed to IOP²⁰, suggesting a possible compensatory mechanism. As shown by Stoyek et al.⁵⁴ however, the AC volume is highly dynamic under normal conditions due to a series of regular corrugations running along the chamber wall, and is the main driver for adjusting buoyancy while the basic PC volume remains largely invariable. Therefore, the question remains whether the functionality of the swim bladder is retained when AC inflation is incomplete, even when the PC appears to fully compensate the gas volume of the swim bladder.

The KE relationship leading from decreased serum T3 to reduced AC inflation is a critical component of the AOP network because it links the primary morphological effects to the hub KE of altered hormone levels. We found a strong correlation between whole body T3 levels and AC size for all compounds (Figures 2C, 3C, 4C, S11). The mechanism underlying the link between reduced T3 and reduced AC inflation remains unclear, but several hypotheses exist.^13,18,20 For example, altered gas distribution between chambers could be the result of impaired development of smooth muscle fibers, delayed and/or impaired evagination of the AC, impaired anterior budding through altered Wnt and hedgehog signalling, etc.^14,55–57 Together with the quantitative relationship between T4 and T3 levels (Section 3.2) this type of data could provide the starting point for the development of quantitative AOPs⁵⁸.

3.4. AO: Reduced swimming performance

All three test chemicals reduced both AC inflation and swimming distance, the AO of the AOP network (Figures 2E, 3E, 4E, S16). The current dataset provides evidence for a specific, direct link between AC inflation and reduced swimming performance. First, after 21 d of exposure to 111 mg/L PTU around 30% of ACs were not inflated and swimming distance was reduced (Figures 4D, 4E), while by 32 dpf all larvae had inflated ACs and the effect on swimming distance had disappeared (Figure S16). The most direct way to assess the role of AC inflation in swimming performance, however, is to compare larvae with and without inflated AC at the same time point and within the same experimental treatment. Both in the PTU exposure at 21 dpf (Figure 4E) and in the IOP exposure at 21 and 32 dpf (Figures 3E, S16), swimming distance was clearly reduced in larvae lacking an inflated AC, while the swimming distance of larvae with inflated AC was equal to that of controls. After exposure to 100 mg/L MMI, 95% of the larvae failed to inflate their AC at 32 dpf and swimming distance was reduced (Figure 2E). On the other hand, there was no effect of impaired AC inflation on swimming distance in the MMI exposure of 50 mg/L. A similar result, where non-inflated ACs did not consistently lead to reduced swimming performance, was previously found after exposure to MBT¹³. Also, inflated but smaller ACs did not result in a decreased swimming performance in the present study (Figures 2E, 3E, 4E, S16).

In summary, we simultaneously observed reduced AC inflation and reduced swimming distance for all compounds, but the precise relationship between these two KEs is not easy to determine and may be different for different chemicals. Swimming capacity can be affected via other processes which may or may not depend on the HPT axis, such as decreased cardiorespiratory function, energy metabolism and growth^59–61. For example, Conradsen and McGuigan⁶² found a correlation between natural pelvic-to-anal fin distance (a measure of growth) and swimming speed in adult zebrafish. Furthermore, the role and importance of the swim bladder during early-life stages is currently unclear. It has been suggested that active regulation of buoyancy by the swim bladder only starts past the first month of age¹⁴. Since swimming activity in the absence of stimuli, as measured in the present study, is only one aspect of swimming performance, future research could investigate the impact of reduced swim bladder inflation on more demanding behaviours such as predator avoidance. The impact on hearing could also be further investigated. Hearing ability has been shown to be associated with the size of the AC in cichlids and several species of catfishes^63,64.

Supplementary Material

Sup1

Supporting Information

Supplement to materials and methods

Concentrations for in vivo exposures; effect of 2 mg/L IOP; timing and assessment of endpoints and sampling; correction of swim bladder surface for growth retardation; calculation of the condition factor; medium concentrations and tissue residues; T3 and T4 concentration measurement; Tpo inhibition potential measurements.

Supplement to results and discussion

Medium concentrations and tissue residues; mortality, hatching, length, condition factor, posterior chamber inflation effects; T3 and T4 concentrations; posterior and anterior chamber surface area; swimming capacity; Tpo inhibition potential.