Integrated Genomic and Bioinformatics Approaches to Identify Molecular Links between Endocrine Disruptors and Adverse Outcomes

Exposure to Endocrine Disrupting Chemicals (EDC) has been linked with several adverse outcomes. In this review, we examine EDCs that are pervasive in the environment and are of concern in the context of human, animal, and environmental health. We explore the consequences of EDC exposure on aquatic life, terrestrial animals, and humans. We focus on the exploitation of genomics technologies and in particular whole transcriptome sequencing. Genome-wide analyses using RNAseq provides snap shots of cellular, tissue and whole organism transcriptomes under normal physiological and EDC perturbed conditions. A global view of gene expression provides highly valuable information as it uncovers gene families or more specifically, pathways that are affected by EDC exposures, but also reveals those that are unaffected. Hypotheses about genes with unknown functions can also be formed by comparison of their expression levels with genes of known function. Risk assessment strategies leveraging genomic technologies and the development of toxicology databases are explored. Finally, we review how the Adverse Outcome Pathway (AOP) has exploited this high throughput data to provide a framework for toxicology studies.

Assessing the Endocrine Disrupting Effects of Chemicals on Invertebrates in the European Union

Evidence from both laboratory and field studies has shown that currently used synthetic and naturally occurring chemical substances may potentially disrupt invertebrate endocrine systems, although the extent of this in field populations remains unclear. Translating concerns about potential endocrine disrupting chemicals (EDCs) into practical and effective regulatory action is currently hampered by the breadth of invertebrate endocrinology when compared to the better understood vertebrate systems, a lack of fundamental knowledge about the endocrinology of many invertebrate groups, and the resulting uncertainty when making regulatory decisions. This paper i) outlines the breadth of invertebrate endocrine pathways for which European Union regulation of potential EDCs may be relevant, ii) reviews the extent to which current knowledge meets regulatory requirements for invertebrates, including an assessment of the suitability of current invertebrate test guidelines for detecting endocrine modes of action; and iii) proposes a roadmap towards the regulation of potential EDCs with greater confidence, based on the Adverse Outcome Pathway (AOP) concept and a focus on identifying Molecular Initiating Events (MIEs) within AOPs. We propose that the most important research need is compilation of a comprehensive list of endocrine-related MIEs across invertebrate taxa via use of high throughput ‘omics in combination with bioinformatics reverse engineered analyses. Although tractable, such an approach would require significant resource investment for development and implementation.

An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure

Adverse outcome pathways (AOPs) help to organize available mechanistic information related to an adverse outcome into key events (KEs) spanning all organizational levels of a biological system(s). AOPs, therefore, aid in the biological understanding of a particular pathogenesis and also help with linking exposures to eventual toxic effects. In the regulatory context, knowledge of disease mechanisms can help design testing strategies using in vitro methods that can measure or predict KEs relevant to the biological effect of interest. The AOP described here evaluates the major processes known to be involved in regulating efficient mucociliary clearance (MCC) following exposures causing oxidative stress. MCC is a key aspect of the innate immune defense against airborne pathogens and inhaled chemicals and is governed by the concerted action of its functional components, the cilia and airway surface liquid (ASL). The AOP network described here consists of sequences of KEs that culminate in the modulation of ciliary beat frequency and ASL height as well as mucus viscosity and hence, impairment of MCC, which in turn leads to decreased lung function.

Juvenile African Clawed Frogs (Xenopus laevis) Express Growth, Metamorphosis, Mortality, Gene Expression, and Metabolic Changes When Exposed to Thiamethoxam and Clothianidin

Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis, were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles (n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57–66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.

Lessons learned in integrating traditional and NAM-based evidence to assess skin sensitization potential

Classification of chemicals as skin sensitizers have traditionally relied on a small set of in vivo tests. Difficult to test substances, such as poorly soluble, mildly irritating, or those of Unknown or Variable Composition Complex reaction products or Biological Materials (UVCBs), producing weak or borderline results in Local Lymph Node Assay (LLNA), often benefit from additional data integration in a weight of evidence (WOE) approach. Advances in multiple testing and non-testing methodologies (in vivo, in vitro, and in silico) can now provide clarity and confidence in concluding on skin sensitization potential. Here we present several case studies using a WOE approach with difficult to test substances and highlight the utility of Toxicological Prioritization IndexTM (ToxPi™) as a comparative visualization and integration tool of toxicology studies. The three test chemicals chosen represent two poorly soluble substances, tetrakis (2-ethylbutyl) orthosilicate and decyl palmitate, and one UVCB substance, alkylated anisole. Data from in vivo and in vitro assays representing multiple key events within the skin sensitization adverse outcome pathway (e.g., direct peptide reactivity assay, human cell line activation test, GARD®Skin, LLNA) were either gathered from publicly available sources or specifically generated. Incorporating the data on our test chemicals as well as chemicals of a known sensitization class (sensitizer, irritating non-sensitizer, non-sensitizer) into ToxPi™ revealed biological activity profiles which were used to support class prediction for the three test chemicals. Using this method, the biological activity profiles for all three test chemicals were most consistent with a non-sensitizing class. This paper demonstrates that visualizing the WOE using mechanistic data maximizes the value of all data to the overall assessment of skin sensitization potential by reducing the uncertainty associated with any one individual assay.