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Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-Mercaptobenzothiazole Part I: Fathead minnow
Nelson, K., A. Schroeder, G. Ankley, B. Blackwell, C. Blanksma, S. Degitz, K. Jensen, R. Johnson, M. Kahl, D. Knapen, Pat Kosian, R. Milsk, E. Randolph, T. Saari, E. Stinckens, L. Vergauwen, AND Dan Villeneuve. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-Mercaptobenzothiazole Part I: Fathead minnow. AQUATIC TOXICOLOGY. Elsevier Science Ltd, New York, NY, 173:192-203, (2016).
The fish early life stage (FELS) toxicity test (OECD 210; OCSPP 850.1400) is the most widely used chronic ecotoxicity test. There is strong interest in development of alternatives to reduce costs, increase efficiency, and minimize animal use. Development of adverse outcome pathways that link pathway perturbations that could be measured either using in vitro systems or in short-term high throughput fish embryo tests to potential adverse effects on fish growth or survival have been viewed as a means to support reduction, refinement, and/or replacement of FELS testing. The present study contributes to the overall goal, while at the same time addressing the topic of thyroid axis disruption, a critical concern relative to implementation of the endocrine disruptor screening program (EDSP). Specifically, two experiments were conducted to evaluate the potential linkage between thyroid peroxidase (TPO) inhibition and swim bladder inflation in fish. Buoyancy regulation facilitated by proper swim bladder inflation and function has been shown to be important for fish survival outside the laboratory environment. Results show the anterior, but not posterior, swim bladder inflation was impacted by exposure to the environmentally relevant TPO inhibitor 2-mercaptobenzothiazole. While delayed inflation and reduced size of the anterior chamber has not yet been directly linked to impaired growth or survival, the results demonstrate an important life stage dependence on the sensitivity to this particular thyroid-disrupting molecular initiating event (MIE). Results suggest that posterior swim bladder inflation is insensitive to the effects of thyroid hormone synthesis inhibitors due to the presence of maternally-derived thyroid hormone during the stage of development in which the posterior chamber inflates around 6 dpf. In contrast, the anterior chamber, which inflates around 14 days post fertilization, is sensitive to inhibitors of thyroid hormone synthesis. Results provide important evidence which supports the development of formal AOP descriptions linking specific thyroid-disrupting MIEs to significant phenotypic outcomes. Further, they suggest alternative short-term in vivo tests with larval fish that could be used to screen chemicals for thyroid disrupting activity and possibly distinguish various thyroid disrupting modes of action. Results of these studies directly support CSS project 12.01, Task 1.3a, Thyroid-axis related formal AOP development.
Development of adverse outcome pathways linking specific chemical-induced pathway perturbations to adverse outcomes relevant to regulatory decision-making has potential to support the development of alternatives to traditional whole organism toxicity tests, such as the fish early life stage test (OECD 210) that rely on direct measurement of apical outcomes. In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder development was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1 mg MBT/L for up 22 d had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around six 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 14 dpf, was impacted. Specifically, at 14 dpf, approximately 50% of fish exposed to 1 mg MBT/L did not have an inflated anterior swimbladder. In fish exposed through 21 or 22 dpf the anterior swim bladder was able to inflate, but was significantly reduced in size in proportion to the posterior chamber. Both expression of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to MBT’s presumed inhibition of TPO activity. Time-course characterization showed that fish exposed for at least 4 d prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber. These results, along with similar results observed in zebrafish (see part II, this issue) provide strong evidence for the role of thyroid hormone in mediating anterior swim bladder inflation and development in cyprinids. Although anterior swim bladder inflation has not been as directly linked to survival as the posterior swim bladder inflation. Nonetheless, given involvement of the anterior chamber in hearing, potential links to adverse ecological outcomes are at least plausible. Further, results suggest potential utility of short-term experiments with fish early life stages as a viable tool for detecting and distinguishing different types of thyroid disrupting chemicals.