Linkage of genomic biomarkers to whole organism end points in a Toxicity Identification Evaluation (TIE).

Aquatic organisms are exposed to many toxic chemicals and interpreting the cause and effect relationships between occurrence and impairment is difficult. Toxicity Identification Evaluation (TIE) provides a systematic approach for identifying responsible toxicants. TIE relies on relatively uninformative and potentially insensitive toxicological end points. Gene expression analysis may provide needed sensitivity and specificity aiding in the identification of primary toxicants. The current work aims to determine the added benefit of integrating gene expression end points into the TIE process. A cDNA library and a custom microarray were constructed for the marine amphipod Ampelisca abdita. Phase 1 TIEs were conducted using 10% and 40% dilutions of acutely toxic sediment. Gene expression was monitored in survivors and controls. An expression-based classifier was developed and evaluated against control organisms, organisms exposed to low or medium toxicity diluted sediment, and chemically selective manipulations of highly toxic sediment. The expression-based classifier correctly identified organisms exposed to toxic sediment even when little mortality was observed, suggesting enhanced sensitivity of the TIE process. The ability of the expression-based end point to correctly identify toxic sediment was lost concomitantly with acute toxicity when organic contaminants were removed. Taken together, this suggests that gene expression enhances the performance of the TIE process.

Characterization of the Fundulus heteroclitus embryo transcriptional response and development of a gene expression-based fingerprint of exposure for the alternative flame retardant, TBPH (bis (2-ethylhexyl)-tetrabromophthalate).

Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (n = 16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.

Proteomic analysis of a model fish species exposed to individual pesticides and a binary mixture.

Pesticides are nearly ubiquitous in surface waters of the United States, where they often are found as mixtures. The molecular mechanisms underlying the toxic effects of sub-lethal exposure to pesticides as both individual and mixtures are unclear. The current work aims to identify and compare differentially expressed proteins in brains of male fathead minnows (Pimephales promelas) exposed for 72 h to permethrin (7.5 µg/L), terbufos (57.5 µg/L) and a binary mixture of both. Twenty-four proteins were found to be differentially expressed among all three treatments relative to the control using an ANOVA followed by a Dunnett's post hoc test (p ≤0.05). One protein was found to be differentially expressed among all treatment groups and one protein was in common between the terbufos and the mixture group. Fifteen spots were successfully sequenced using LC-MS/MS sequencing. Proteins associated with the ubiquitin-proteasome system, glycolysis, the cytoskeleton and hypoxia were enriched. As a second objective, we attempted to establish protein expression signatures (PES) for individual permethrin and terbufos exposures. We were unable to generate a useable PES for terbufos; however, the permethrin PES was able to distinguish between control and permethrin-exposed individuals in an independent experiment with an accuracy of 87.5%. This PES also accurately classified permethrin exposed individuals when the exposure occurred as part of a mixture. The identification of proteins differentially expressed as a result of pesticide exposure represent a step forward in the understanding of mechanisms of toxicity of permethrin and terbufos. They also allow a comparison of molecular responses of the binary mixture to single exposures. The permethrin PES is the first step in establishing a method to determine exposures in real-world scenarios.