Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4‐tert‐octylphenol or 17β‐trenbolone exposure in Xenopus laevis from embryo to juvenile

Abstract

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17β-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone‐2 exposure in Xenopus laevis from embryo to juvenile

“…Attempts were made to measure T4 levels in plasma using both the optimized ELISA described in Haselman et al . () and the high‐performance liquid chromatography/inductively coupled plasma‐MS method described in Tietge et al . ().…”

“…All test methods including feeding, NF62 larval subsampling, larval blood thyroxine (T4) analyses, the post‐metamorphosis cull, juvenile sampling and somatic growth measurements were performed as described by Haselman et al . (); genetic sexing of juveniles was performed using methods described by Yoshimoto et al . ().…”

“…The work presented here was performed to aid validation efforts for the initial assay design and contribute to the refinement of the final test guideline. Two other studies contributing to the validation effort are presented in a companion paper (Haselman et al ., ) along with results demonstrating the apical effects of exposure to either 4‐ tert ‐octylphenol or 17β‐trenbolone.…”

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

“…The high conservation of the molecular components of TH signaling among all vertebrate classes permits the use of amphibian metamorphosis as a valid and easily accessible model for studying TH signaling and disruption (8,9,14,16). In this study we have presented a suite of three assays to evaluate the potential of compounds to disrupt TH signaling based upon exogenously-added TH-induced precocious metamorphosis.…”

“…The assay relies heavily on limb development as a primary endpoint for developmental staging along with thyroid histology, yet it does not provide direct mode of action information. Recently, an extension of the AMA was proposed that includes embryonic exposures through spontaneous metamorphosis to juveniles, termed the Larval Amphibian Growth and Development assay (LAGDA)(8,9). We set out to develop a quantitative, accelerated, in vivo assay suite, specifically geared for disruption of TH signaling, that functions at several levels of specificity, has ease-of-use, and is low cost.…”