Development and testing of gene expression biomarkers for gonadal dysgenesis in conjunction with the US EPA endocrine disruptor screening program's Tier 2 larval amphibian growth and development assay

Abstract

The Endocrine Disrupter Screening Program of US EPA has recently developed a Tier 2 testing guideline using model amphibian species Xenopus laevis. The Larval Amphibian Growth and Development Assay assesses a chemical's endocrine-related effects in vivo and generates concentration-response information for ecological risk-assessment. Currently, the assay relies on histopathological evaluations for identifying endocrine-related reproductive effects. However, histopathology can seldom define the chemical mode of action and is not easily interpreted in the context of risk assessment when the effects are minimal to moderate. This study explores the use of gene expression biomarkers in the gonad that could potentially inform a chemical's mode of action and detect adverse reproductive effects that are otherwise uncharacterized by histopathology. To identify candidate biomarkers, global expression was analyzed in differentiating ovary and testis tissues of Xenopus tropicalis. Genetic programs responsible for reproductive maturation in gonad tissues were characterized and provided a foundation from which specific genes could be selected proximal to a model chemical's known mode of action. Four genes were selected within the putative androgen molecular network to evaluate as biomarkers of the anti-androgen mode of action characteristic of a common fungicide, prochloraz. Following continuous exposure to prochloraz throughout embryonic, larval and juvenile development in Xenopus laevis, assessments of growth, liver and kidney pathology, and reproductive development were made. To evaluate the predictive capabilities of the candidate genes, one gonad was kept in situ for histopathological evaluations while the other was processed for mRNA analyses. Results indicate that prochloraz exposure caused metabolic toxicity in the liver and kidney; it caused testis degeneration coincident with the onset of androgen-mediated spermatogenesis and inhibited regression of Müllerian ducts. Two of the four candidate genes showed increases in expression at the high test concentration and appeared to be predictive of an anti-androgen-induced adaptive response. The behavior of these biomarkers stimulated valuable discussion and generated testable hypotheses to better understand the evolution of molecular mechanisms driving gonad development in a cross-species context. This study provides a model for expression-based biomarker development in endocrine tissues and offers direction toward enhanced ecological risk-assessment.