Project description:There is a need to generate hazard information for chemicals already in production. This can be costly and time consuming. Higher-throughput techniques offer a method to effectively contextualize and define the hazard potential posed by chemicals and facilitate risk assessment activities in a rapid manner. Transcriptomic points of departure (tPODs), derived from the concentration at which global gene expression significantly differs from controls, are expected to precede apical toxicity. Therefore, concentrations below that elicit modulation of gene expression would be unlikely to cause adverse effects, suggesting tPODs could serve as an alternative point of departure for use in screening level hazard assessment. The present study extends previous work with a larval fathead minnow high-throughput transcriptomics assay but employed a targeted transcriptome rather than whole transcriptome approach to generate tPODs for 22 chemicals (pesticides and pharmaceuticals), expanding the diversity of chemical modes of action evaluated. One day post hatch fathead minnows were exposed to eight concentrations of two piscicides, three herbicides, four insecticides, seven fungicides, two statins, and four PPAR agonists for 24 h. Following exposures, whole body homogenates were sent for targeted RNA sequencing using TempO-Seq. Concentration response modeling, using measured concentrations where feasible, was applied to all sequencing results to estimate benchmark concentrations and derive tPODs. The derived tPODs were lower than concentrations that impacted apical endpoints in fish collected from the ECOTOX Knowledgebase in most cases. However, this was not the case when compared to apical effects in other taxa (crustaceans, insects, algae). To investigate the relationships of chemicals within the same mode of action (MoA), exploratory principal component analysis was used as was examining differentially expressed genes between chemicals within modes of action. Our results showed that the individual genes impacted by chemical exposure rarely clustered within modes of action. The results of this study provide additional data that can be used to examine the applicability of high-throughput assay for possible use in ecological hazard screening

Project description:There is a need to move from binary hazard assessment to more quantitative assessment of genotoxicity to better inform human health risk assessment and understand the relevance of positive in vitro genotoxicity findings. New approach methodologies (NAMs), including transcriptomic biomarkers combined with high-throughput technologies, enable the testing of a broad concentration range, which allows quantitative assessment of in vitro results. Initial work with the transcriptomic GENOMARK and TGx-DDI biomarkers demonstrate their potential use for hazard identification and chemical prioritization; however, no study has evaluated the concordance and complementarity of GENOMARK and TGx-DDI. The overall aim of this study is to examine if a combined approach of integrating both transcriptomic biomarkers for genotoxicity in human relevant HepaRGTM cells increases the certainty in hazard calls and potency rankings of chemicals. A sub-aim is to investigate whether GENOMARK is applicable to the TempO-Seq® high-throughput sequencing technology, to collect concentration-response data to rapidly perform hazard classification and potency ranking. Therefore, HepaRGTM cells were exposed to 10 chemicals (i.e. eight known in vivo genotoxicants and two in vivo non-genotoxicants) in increasing concentrations over 72h. TempO-Seq® was used to obtain concentration-response data for both biomarkers. Benchmark concentration (BMC) modelling of chemicals that were classified positive was conducted to obtain BMCs and transcriptomic points of departure (tPODs) for potency ranking. The results confirm that GENOMARK is applicable to TempO-Seq® since it achieved 100% predictive accuracy. In addition, a high concordance was observed in the hazard classifications and potency rankings between both biomarkers. Overall, our findings show that in vitro transcriptomic data can be used to rapidly and effectively identify genotoxic hazards while simultaneously providing additional insights on potency that is more informative in a modern hazard assessment paradigm. The results of this case study support the high value of integrating these NAMs in a weight of evidence evaluation of genotoxicity using the important human-liver cell line.

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Beyond hazard identification: Discovering mechanisms of action from a ToxCast chemical screen in zebrafish

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