Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|TPO inhib alters metamorphosis||AdverseOutcome|
|NIS inhib alters metamorphosis||AdverseOutcome|
|IYD inhib alters metamorphosis||AdverseOutcome|
|DIO1 inhib alters metamorphosis||AdverseOutcome|
|DIO2 inhib alters metamorphosis||AdverseOutcome|
|DIO3 inhib alters metamorphosis||AdverseOutcome|
|Pendrin inhib alters metamorphosis||AdverseOutcome|
|DUOX inhib alters metamorphosis||AdverseOutcome|
|Hepatic nuclear receptor activation alters metamorphosis||AdverseOutcome|
|African clawed frog||Xenopus laevis||High||NCBI|
Key Event Description
Vertebrate metamorphosis is a biological transformation process that transitions an organism from one life stage to another; it is defined by growth of new tissues, programmed death of other tissues and physiological transformation of yet other tissues (Laudet, 2011; Brown and Cai, 2007). In the case of most amphibians, metamorphosis mediates the transition from aquatic to terrestrial life, while in bony and jawless fish, metamorphosis mediates transitions between life stages that offer various advantages for survival and reproduction. In vertebrates, metamorphosis is orchestrated by the hypothalamus-pituitary-thyroid (HPT) axis involving complex timing of gene expression/repression within various tissues, whereas in some cases across taxonomic classes, metamorphosis has been shown to be controlled very differently by the HPT axis.
Thyroid hormone-mediated amphibian metamorphosis can be characterized by three phases during larval development: (1) pre-metamorphosis, (2) pro-metamorphosis and (3) metamorphic climax. All three of these phases coincide with activity states of the HPT axis. Pre-metamorphosis is characterized by a fully aquatic organism with low-level function of the thyroid gland and very low circulating levels of thyroid hormone. Pro-metamorphosis is characterized by the onset of full thyroid axis function and the initiation of rising levels of thyroid hormone in the plasma, with consequential changes in anatomy and physiology defining the transition from aquatic to terrestrial life. Metamorphic climax occurs when circulating thyroid hormone levels peak, which subsequently decrease to levels maintained homeostatically as adults. This climax period also represents the time at which all anatomical and physiological changes induced by thyroid hormone have either been initiated or are already completed. Detailed descriptions of these processes are reviewed by Brown and Cai (2007).
Altered metamorphosis occurs when these thyroid hormone-mediated processes are perturbed, primarily during pro-metamorphosis and metamorphic climax. These perturbations can lead to either, delayed/arrested development, accelerated development or asynchronous development depending on the xenobiotic mode of action or MIE. Genetic defects or xenobiotic exposure that reduce thyroid hormone synthesis can delay metamorphosis, and in extreme cases, can completely arrest development. The most profound impacts on TH-mediated metamorphosis have be demonstrated through inhibition of key proteins in the TH synthesis pathway including the sodium-iodide symporter (Tietge et al., 2005, 2010; Hornung et al., 2010) and thyroperoxidase (Degitz et al., 2005; Tietge et al., 2010, 2013; Hornung et al., 2010, 2015). Alternatively, agonism of the thyroid axis through inhibition of negative feedback at the level of the hypothalamus-pituitary, or premature activation of thyroid receptor-mediated transcription can accelerate metamorphosis (Degitz et al., 2005), which can lead to asynchronous development due to errors in gene expression timing across the various metamorphic tissues. Asynchronous development can also occur due to inhibition of deiodinase (DIO) enzymes in peripheral tissues. DIO enzymes are responsible for activation and catabolism of TH; when dio gene expression profiles are altered, or the enzymes themselves undergo chemical inhibition, the imbalance of prohormone (T4), active hormone (T3) and inactive hormone (rT3, T2) can cause aberrant tissue development.
How It Is Measured or Detected
Rates of metamorphosis in model amphibian species, Xenopus laevis, are measured multiple ways, both of which rely on a developmental staging atlas developed by Nieuwkoop and Faber (NF)(1994). The method utilized within the 21 d Amphibian Metamorphosis Assay regulatory test guideline (OECD, 2009; US EPA 2009) relate the distribution of developmental stage of control larvae to the distributions of developmental stages of treated/exposed larvae. These data are typically analyzed for differences from control using non-parametric statistical approaches such as the Kruskal-Wallis test followed by Dunn's test for pairwise comparisons. The method utilized within the Larval Amphibian Growth and Development Assay regulatory test guideline (OECD, 2015; US EPA 2015) relate the number of days to reach metamorphic climax (NF stage 62) in control larvae to the number of days to NF stage 62 in treated/exposed larvae. These data are typically analyzed for differences from control using a Cox mixed-effects proportional hazard model.
Asynchronous development is identified as disruption of the relative timing of morphogenic milestones and/or somatic development within a single larvae undergoing metamorphosis. The inability to identify an organism's developmental stage based on accepted criteria, such as outlined in Nieuwkoop and Faber (1994) for Xenopus sp. or Gosner (1960) for anurans, constitutes evidence of asynchronous development and would be counted as an incidence. Evaluations of severity are possible but the accuracy and resolution of the results would depend on the experience of the observer. One possible statistical approach for analyzing these data collected from a regulatory test guideline (OECD, 2009, 2015) would be a Rao-Scott-Cochran-Armitage by slices test (Green et al., 2014), as is often used for analysis of histopathology incidence and severity data.
Domain of Applicability
Evidence for Perturbation by Stressor
Methimazole exposure during prometamorphosis in Xenopus laevis causes delayed/arrested metamorphosis.
Propylthiouracil exposure during prometamorphosis in Xenopus laevis causes delayed/arrested metamorphosis.
Mercaptobenzothiazole exposure during prometamorphosis in Xenopus laevis causes delayed/arrested metamorphosis.
Perchlorate exposure during prometamorphosis in Xenopus laevis causes delayed/arrested metamorphosis.
Regulatory Significance of the Adverse Outcome
Altered metamorphosis is a critical apical endpoint evaluated as part of regulatory test guideline studies (OECD, 2009, 2015; US EPA 2009, 2015). Measurable effects on metamorphic rates can be an indication of endocrine disruption, and more specifically thyroid disruption, due to the requirement of thyroid hormone for amphibians to undergo metamorphosis. Although this outcome is evaluated at the level of the individual organism, delayed or arrested metamorphosis can have implications toward population-level effects; however, significant effects on metamorphic rates are typically considered in a weight-of-evidence evaluation to determine a chemical's potential to cause thyroid disruption.
Brown, D.D. and Cai, L., 2007. Amphibian metamorphosis. Developmental biology, 306(1), pp.20-33.
Degitz, S.J., Holcombe, G.W., Flynn, K.M., Kosian, P.A., Korte, J.J. and Tietge, J.E., 2005. Progress towards development of an amphibian-based thyroid screening assay using Xenopus laevis. Organismal and thyroidal responses to the model compounds 6-propylthiouracil, methimazole, and thyroxine. Toxicological sciences, 87(2), pp.353-364.
Gosner, K.L., 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica, 16(3), pp.183-190.
Green, J.W., Springer, T.A., Saulnier, A.N. and Swintek, J., 2014. Statistical analysis of histopathological endpoints. Environmental toxicology and chemistry, 33(5), pp.1108-1116.
Hornung, M.W., Degitz, S.J., Korte, L.M., Olson, J.M., Kosian, P.A., Linnum, A.L. and Tietge, J.E., 2010. Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicological Sciences, 118(1), pp.42-51.
Laudet, V., 2011. The origins and evolution of vertebrate metamorphosis. Current Biology, 21(18), pp.R726-R737.
Nieuwkoop, P.D. and Faber, J., 1994. Normal Table of Xenopus laevis (Daudin) Garland Publishing. New York, 252.
OECD. (2009). Test No. 231: Amphibian Metamorphosis Assay, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris.
OECD. (2015). Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA), OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris.
Tietge, J.E., Butterworth, B.C., Haselman, J.T., Holcombe, G.W., Hornung, M.W., Korte, J.J., Kosian, P.A., Wolfe, M. and Degitz, S.J., 2010. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. Aquatic Toxicology, 98(1), pp.44-50.
Tietge, J.E., Holcombe, G.W., Flynn, K.M., Kosian, P.A., Korte, J.J., Anderson, L.E., Wolf, D.C. and Degitz, S.J., 2005. Metamorphic inhibition of Xenopus laevis by sodium perchlorate: effects on development and thyroid histology. Environmental Toxicology and Chemistry, 24(4), pp.926-933.
Tietge, J.E., Degitz, S.J., Haselman, J.T., Butterworth, B.C., Korte, J.J., Kosian, P.A., Lindberg-Livingston, A.J., Burgess, E.M., Blackshear, P.E. and Hornung, M.W., 2013. Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole. Aquatic toxicology, 126, pp.128-136.
U.S. EPA. (2009). OCSPP 890.1100: Amphibian Metamorphosis Assay (AMA), Endocrine Disruptor Screening Program Test Guidelines, 890 Series. Available at: www.regulations.gov, ID: EPA-HQ-OPPT-2009-0576-0002. Accessed March 20, 2020.
U.S. EPA. (2015). OCSPP 890.2300: Larval Amphibian Growth and Development Assay (LAGDA), Endocrine Disruptor Screening Program Test Guidelines, 890 Series. Available at: www.regulations.gov, ID: EPA-HQ-OPPT-2014-0766-0020. Accessed March 20, 2020.