Influence of triphenyltin on morphologic abnormalities and the thyroid hormone system in early-stage zebrafish (Danio rerio)

Highlights

• Exposure to triphenyltin disrupted thyroid hormone signaling in zebrafish.

• Larvae with exposure-associated morphologic abnormalities died soon after hatching.

• No difference in triphenyltin toxicity between fed and fasted groups.

• Triphenyltin increased expression of trα and trβ mRNA.

Abstract

In the present study, we assessed the negative effects of triphenyltin (TPT) on zebrafish (Danio rerio) by exposing embryos and early-stage larvae to various concentrations of TPT from 2 h after fertilization (haf) until 30 days after hatching (dah). Whether test groups were fed or fasted during ecotoxicity studies using fish models has varied historically, and whether this experimental condition influences test results is unknown. Here, we confirmed that the lethal concentration of TPT to embryo and early-stage larvae (i.e., 3 dah or younger) showed in fed (lowest observed effect concentration (LOEC); 6.34 μg/L) and fasted (LOEC; 6.84 μg/L) groups. In addition, 84% and 100% of the larvae in the 2.95 and 6.64 μg/L exposure groups, respectively, had uninflated swim bladders; all affected larvae died within 9 dah. This finding suggests that morphologic abnormalities in early larval zebrafish are useful as endpoints for predicting the lethality of chemical substances after hatching. We then assessed the expression of several genes in the thyroid hormone pathway, which regulates swim bladder development in many fish species, including zebrafish. Larvae exposed to 6.64 μg/L TPT showed significant increases in the mRNA expression levels of thyroid hormone receptor α (trα) and trβ but not of thyroid stimulating hormone β subunit. These findings suggest that TPT disrupts the thyroid system in zebrafish.

Introduction

Zebrafish (D. rerio) are widely used for ecotoxicity testing since they are small (<5 cm in length), easy to keep, and have a short generation cycle (~3 months). Numerous studies have used zebrafish to investigate the influence of chemicals on mortality (Horie et al., 2017a; Wang et al., 2019), embryonic development (Horie et al., 2017c; Sarasquete et al., 2018), growth (Horie et al., 2020; Kovács et al., 2016; Memmert et al., 2013), reproduction (Kwon et al., 2016; Oliveira et al., 2020), sexual differentiation (Baumann et al., 2013), and effect on multigenerations (Constantine et al., 2020). Recently, attention of animal welfare consideration for current ecotoxicity testing is increasing. One such ecotoxicity testing is short-term toxicity test using embryo and sac-fry stages (OECD TG 212). Test animals typically are not fed throughout the test when the effects of chemical substances are examined in this toxicity test (OECD, 1998). Therefore, this test has been termed by some as the “fish starvation test” (ENV/JM/MONO (2012)). This suggests that the short-term toxicity test should include feeding from the view of animal welfare. However, whether ecotoxicity values differ between fed and fasted zebrafish is unknown.

The organotin compound triphenyltin (TPT) is used worldwide as a biocide in marine anti-fouling coatings to prevent the attachment and growth of marine organisms (Blunden and Evans, 1989; Snoeij et al., 1987). However, many adverse effects of TPT on aquatic organisms have been reported, including inhibition of reproduction in freshwater gastropods (Lymnaea stagnalis; Giusti et al., 2013) and Japanese medaka (O. latipes; Zhang et al., 2008); growth inhibition in marine medaka (O. melastigma; Yi and Leung, 2017) and Japanese medaka (O. latipes; Horie et al., 2017b); and abnormal sexual development in false kelpfish (Sebastiscus marmoratus; Sun et al., 2011) and calenoid copepods (Acartia tonsa; Watermann et al., 2013). Therefore, the use of TPT has been limited in various countries, including Japan.

The potential for thyroid hormone-disrupting chemical contamination in ecosystems is garnering increased attention worldwide, owing to thyroid hormone's key roles in maintaining normal physiology and in brain development, metabolism, and growth (Bernal, 2005; Casals-Casas and Desvergne, 2011). In a previous study, TPT induced increased expression of thyroid stimulating hormone β subunit (tshβ) but inhibited thyroid hormone receptor α (trα) and thyroid hormone receptor β (trβ) expression in zebrafish larvae at 7 dah (Li et al., 2019), whereas another group reported upregulation of both tshβ and trβ expression in zebrafish larvae at 7 and 14 days after fertilization (daf) (Yao et al., 2020). Together these data suggest that TPT disrupts the thyroid hormone system in zebrafish, but the precise effects of TPT exposure on the expression of tshβ or tr remain unclear because results differ between studies.

Recently, swim bladder inflation has become a potential biomarker for the detection of thyroid hormone disruptors because swim bladder development in fish is controlled by thyroid hormone systems (Spaan et al., 2019). In fact, perfluorooctanoic acid (PFOA), perfluorobutyric acid (PFBA), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP), all of which are thought to act as thyroid disruptors, induced delays in or the absence of swim bladder inflation in zebrafish (Godfrey et al., 2017). However, it is still unclear whether TPT exposure induces delays in or the absence of swim bladder inflation.

Therefore, we addressed the following questions in the present study. First, does the ecotoxicity of TPT on zebrafish larvae differ between fed and fasted group during testing? Second, does TPT exposure affect expression of TSH-related genes (tshβ and tr)? Third, does TPT exposure induce delays in or the absence of swim bladder inflation?