Inhibition, Deiodinase 1 leads to Reduced, Posterior swim bladder inflation

The two major thyroid hormones are thyroxine (T4) and the more biologically active triiodothyronine (T3), both iodinated derivatives of tyrosine. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable of converting T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 tot T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively (Darras and Van Herck, 2012).

Since swim bladder development and/or inflation is regulated by thyroid hormones, decreased T3 levels are expected to result in impaired posterior chamber inflation. Because of the high Km and preference for rT3 as a substrate, the importance of DIO1 in activating T4 to T3 in a physiological situation is likely limited. The role of inhibition of DIO1 in decreasing T3 levels and impairing posterior chamber inflation may therefore be limited.

In chemico DIO1 inhibition assays were performed using potential thyroid disrupting compounds (Stinckens et al., 2018). Using dose-response curves, the IC50 values were determined and used to caterogize the chemicals according to their potency (strong, weak, uncertainty zone). For DIO1, 7 potent DIO1 inhibitors were selected, of which 6 caused an effect on posterior chamber inflation in zebrafish embryos, resulting in only 1 false positive prediction. One out of two compounds in the uncertainty zone impaired posterior chamber inflation. Finally, 2 out of 5 weak DIO1 inhibitors caused impaired posterior chamber inflation, resulting in 2 false negative predictions. The latter false negative predictions are potentially due to the fact that multiple AOPs can result in impaired swim bladder inflation.

Taxonomic: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass) and physostomous (e.g., zebrafish and fathead minnow). Physostomous fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). Much of the evidence for impaired posterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2018; Cavallin et al., 2017; Wang et al., 2020), but this KE is plausibly applicable across fish species with swim bladders, both physostomous and physoclistous.

Sex: This KE/KER is plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. In Medaka, sex can be morphologically distinguished as soon as 10 days post fertilization. Females appear more susceptible to thyroid‐induced swim bladder dysfunction compared with males (Godfrey et al., 2019). In zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KE/KER. Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the posterior chamber inflates around 5 days post fertilization in zebrafish, when sex differentiation has not started yet, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the posterior chamber inflates around 6 days post fertilization (1 dph) in fathead minnows, sex differences are expected to play a minor role in the current AOP.

Life stage: This KER is only applicable to early embryonic development, which is the period where the posterior swim bladder chamber inflates.