Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances

Abstract

In support of an Organization for Economic Cooperation and Development (OECD) Amphibian Metamorphosis Assay (AMA) Test Guideline for the detection of substances that interact with the hypothalamic-pituitary-thyroid axis, a document was developed that provides a standardized approach for evaluating the histology/histopathology of thyroid glands in metamorphosing Xenopus laevis tadpoles. Here, a consolidated description of histology evaluation practices, core diagnostic criteria and severity grading schemes for the AMA, an atlas of the normal architecture of amphibian thyroid glands over the course of metamorphosis, and the core diagnostic criteria with examples of severity grades is provided. Core diagnostic criteria include thyroid gland hypertrophy/atrophy, follicular cell hypertrophy, and follicular cell hyperplasia. The severity grading scheme is semiquantitative and employs a four-grade approach describing ranges of variation within assigned ordinal classes: not remarkable, mild, moderate, and severe. The purpose of this severity grading approach is to provide an efficient, semi-objective tool for comparing changes (compound-related effects) among animals, treatment groups, and studies. Proposed descriptions of lesions for scoring the four core criteria are also given.

Introduction

The Organization for Economic Cooperation and Development (OECD) has initiated a high-priority activity to develop test guidelines for the screening and testing of potential endocrine active substances (EAS). As part of this effort, the development and validation of the Amphibian Metamorphosis Assay (AMA) was undertaken (OECD 2004a, 2004b, 2007a, 2007b, 2007c, 2008a, 2008b). The AMA is intended to empirically identify substances that may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. It represents a generalized vertebrate model to the extent that it is based on the conserved structure and functions of thyroid systems within vertebrate classes. Additionally, amphibian metamorphosis is a well-studied, thyroid-dependent process that responds to substances active within the HPT axis, and it is currently the only available assay that assesses thyroid activity in an animal undergoing morphological development (Kloas et al. 1999; Lutz and Kloas 1999). For an in-depth description of this assay, please see the OECD Detailed Review Paper on the Amphibian Metamorphosis Assay (OECD 2004a), and for a description of the full histological method, see the Guidance Document on Amphibian Thyroid Histology (OECD 2007b).

The premise of the AMA is that metamorphic development in the African clawed frog (Xenopus laevis) is dependent upon proper synthesis and regulation of thyroid hormone and that xenobiotic perturbation of the HPT axis can lead to measurable developmental effects. The general experimental design of the AMA exposes Nieuwkoop and Faber (Nieuwkoop and Faber 1994) (N&F) stage 51 X. laevis tadpoles to a minimum of three different test chemical concentrations and a dilution water control for twenty-one days. The primary endpoints of the assay are hind limb length, developmental stage, and thyroid histology/histopathology. Hind limb length and developmental stage are morphological indicators of metamorphic development, as described by Nieuwkoop and Faber (1994). Effects on morphological development in treated organisms, in comparison to control organisms, suggest perturbation of normal thyroid hormone homeostasis. Thyroid gland histology is an important diagnostic end-point with several histomorphological features that are modulated by regulatory components of the HPT, most notably thyroid stimulating hormone (TSH), the pituitary hormone that regulates synthesis and release of thyroid hormone. Other endpoints, such as snout-to-vent length, wet weight, and daily observations of mortality and clinical manifestations of overt toxicity, are also measured to help distinguish between thyroid specific effects and generalized toxicity.

During the validation process for the AMA, it was recognized that thyroid histology served as a valuable and sensitive diagnostic end point for detecting a chemical’s ability to interact with the HPT axis, particularly for thyroid system antagonism. Since this assay potentially will be used in various countries, it is important to standardize the histologic analysis of thyroid tissues from X. laevis to maximize comparability among pathologists, thereby reducing inconsistency and bias. Therefore, an OECD guidance document was developed that addresses histopathology reading practices, diagnostic criteria, severity grading, and data reporting and presents an atlas of illustrative reference photomicrographs. This guidance defines the diagnostic criteria for the histologic analysis of the AMA. The criteria are based on pathologists’ experience with the AMA and documented changes of the thyroid glands in response to chemical exposure.

This report provides a consolidated description of the OECD histological guidance provided in the test guideline. It presents an approach to standardized reading practices for the AMA, a description of the core diagnostic criteria and severity grading schemes, and updated photomicrographic examples of the normal thyroid gland architecture during the course of metamorphosis and core diagnostic criteria with representative severity grades. Technical information on morphological sampling and histological preparation procedures is not reviewed here; however, that information can be obtained in the OECD guidance document on thyroid histology (OECD 2007c).

General Recommendations for Histological Evaluation of AMA Studies

Generally, tissues are prepared for histological examination by fixation in Davidson’s solution, paraffin embedding, transverse or frontal sectioning at the largest diameter of the glands, and staining with hematoxylin and eosin (OECD 2007b). Thyroid histology is evaluated using standard approaches for toxicologic pathology, as have been described in the Society of Toxicologic Pathology Best Practices document (Crissman et al. 2004) (e.g., nonblinded). It is recommended that the histological evaluation be performed by experienced toxicological pathologists who are familiar with normal X. laevis thyroid histology, with thyroid gland physiology, and with general responses of the thyroid glands to agonism or antagonism. Over the course of metamorphosis in X. laevis, the needs of developing and remodeling tissues are dynamic. Therefore, the normal histomorphology of the thyroid glands changes over time with the developmental stage of the tadpole. These normal changes must be considered when thyroid histology of tadpoles in different developmental stages is compared.

Normal Progression of the Amphibian Thyroid Glands Over the Course of Metamorphosis

Over the course of metamorphosis, the architecture of the thyroid glands of X. laevis progressively changes to accommodate physiological needs of the developing amphibian (Dodd et al. 1976). The thyroid glands increase in size and volume from proliferation of follicles and an increase in follicular cell size and number. The thyroid glands achieve maximal size at approximately N&F stage 63, and maximum follicular cell height between stages 62 and 64 (Dodd et al. 1976). These changes must be taken into consideration when evaluating relative differences between treatments, so as not to confound developmental stage–specific effects.

To provide a reference atlas of the normal architecture of the developing amphibian thyroid glands as it relates specifically to the AMA, representative examples, by developmental stage, of the histological appearance of the glands in developing X. laevis tadpoles are provided in Figures 1 and 2. Examples were taken from tadpoles sequentially sacrificed beginning with N&F stage 51 and ending with N&F stage 66. The tissues were processed as described above, sectioned at the level of the largest glandular diameter (approximately mid-gland) in the frontal plane, and stained with H&E. Consistent with Dodd et al. (1976), the thyroid glands during this developmental period increase in area, and follicular cells increase in height. Regression of thyroid gland size occurs after metamorphic climax. The maximum area of the glands is achieved close to N&F stage 64, whereas the maximum cell height is achieved between N&F stages 62 and 63.

General Severity Grading Scheme for Histopathological Lesions

For the AMA, quantitative approaches to scoring the grades of severity of diagnostic criteria are not suitable. The core criteria are most effectively evaluated using a semiquantitative, four-grade severity scoring system, ranging between zero and three (Table 1). The descriptors are based on relative differences from thyroid glands in control animals, and/or on the percentage of cells or tissue affected. In addition to the severity grade, qualitative changes associated with the lesions should be documented.

Several validation studies for the AMA were conducted using chemicals that interact with the HPT axis through different modes of action, including iodine uptake inhibition, iodine deficiency, and altered thyroxine metabolism (OECD 2004b, 2007a, 2008b; Opitz et al. 2005; Opitz et al. 2006). Using the information on the typical effects on thyroid gland histology that was collected during these studies, four core diagnostic criteria were determined: thyroid gland atrophy, thyroid gland hypertrophy, follicular cell hypertrophy, and follicular cell hyperplasia. Following are descriptions and photomicrographs of these lesions and approaches to grading the severity of the lesions. The examples used in the photomicrographs are taken from midluminal sections at the widest cross-sectional area of the thyroid glands. The plane of section is either transverse or frontal. After several histological analyses were performed using the AMA protocol, it was found that information from both planes of section was equivalent.

Thyroid Gland Atrophy/Hypertrophy

Decreases (atrophy) or increases (hypertrophy) in the overall size of the thyroid glands are a consequence of changes in follicular cell size and number, and/or in the size and number of follicles. The severity of either thyroid atrophy or hypertrophy is graded on an overall, general appearance of the thyroid glands. Because the diagnosis of hypertrophy or atrophy is dependent on a comparison to thyroid glands from control animals, it is necessary to establish the normal variability of thyroid gland sizes in control tadpoles prior to making determinations on thyroid gland size in treatment/dose groups. Additionally, to make comparable observations, evaluated sections must be representative of the greatest cross-sectional area of the glands. See Tables 2 and 3 for severity grading schemes specific to thyroid gland atrophy and hypertrophy and Figures 3 and 4 for photomicrographs of examples of these lesions.

Follicular Cell Hypertrophy

Hypertrophic follicular cells are graded based on the percentage of the cells exhibiting this feature. For accurate determination of follicular cell size, the tissue section must be in the proper location (ideally mid-luminal). Evaluation of cells that are tangentially sectioned can be misleading. Follicular cells in appropriately sectioned follicles exhibit uniformity in nuclear size and shape. It is recognized that follicular cell hypertrophy may present as a generalized lesion and interpreted as such. Since normal amphibian thyroid glands show heterogeneity in follicular cell shape, ranging from squamous to tall columnar, severity is determined by the change in percentage of cells exhibiting tall columnar structure. See Table 4 for a severity grading scheme specific to follicular cell hypertrophy and Figure 5 for photomicrographs of examples of this lesion.

Follicular Cell Hyperplasia

Follicular cell hyperplasia is diagnosed when there is follicular cell crowding, stratification (multiple layers), and/or papillary infolding of single or multiple layers of follicular cells. The severity grading scheme for follicular cell hyperplasia is based on the percentage of follicles that exhibit hyperplasia, and/or the percentage of tissue that is affected. As with follicular cell size, the section plane should ideally be mid-luminal. See Table 5 for a severity grading scheme specific to follicular cell hyperplasia and Figure 6 for photomicrographs of examples of this lesion.

Additional descriptive criteria include follicular lumen area and colloid quality. Luminal area can be reduced or increased and is indicative of a steady state and/or instantaneous physiological condition of the thyroid gland, such as the release of T4. Severity of effects on luminal area is graded based on the general grading scheme. Changes in colloid quality are generally considered in association with follicular luminal area. Typical descriptions include homogeneous, heterogeneous, lacy, or granular. If present, these findings are reported in a narrative format. The specific significance of changes in colloid quality is not always certain, possibly representing artifacts of tissue processing or reabsorption of thyroglobulin in cases of decreased circulating T4.

Summary

The diagnostic criteria and severity scheme presented are based on the current understanding of the relative changes that occur in the developing tadpole in response to thyroid-active substances. The diagnostic criteria presented in this document are associated with compensatory responses that are modulated by changes in circulating thyroid hormone and TSH concentrations, including, for example, follicular cell hypertrophy and hyperplasia, and colloid depletion. These alterations and hormone changes, when excessive or prolonged, can also result in a toxic developmental effect.