Exploring the Effects of Experimental Parameters and Data Modeling Approaches on In Vitro Transcriptomic Point-of-Departure Estimates
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE249377 | GEO | 2023/12/05
REPOSITORIES: GEO
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Project description:New approach methods (NAMs) can reduce the need for chronic, animal-based toxicity and carcinogenicity bioassays. Here, we apply benchmark dose (BMD) response modeling (BMDExpress 2.3) to transcriptomic data after short-term exposures to three organophosphate pesticides (OPPs) to estimate chronic adverse effect levels in mouse. Specifically, mouse-liver were collected for RNA sequencing after 7-days of exposure, respectively, to several dose-levels of well-known OPPs: fenthion, methidathion and parathion. Transcriptional points-of-departure (TPODs) were compared to chronic, apical points-of-departures (APOD) for each OPP to inform more efficient means of estimating chemical potency and risk. The mouse-liver TPODs for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-day, respectively. These short-term TPODs reflected the relative potencies of the most sensitive, apical effects from the chronic studies with fenthion identified as the most potent OPP. Moreover, the TPODs were 2.3 to 17-fold more sensitive than the chronic APODs for mouse, suggesting utility in using short-term gene response for estimating long-term chemical risk. Further investigation into OPPs’ modes of action identified significant impacts on acetylcholinesterase mRNA abundance (FDR p-value <0.05, |FC|>2) as well as enrichment of canonical pathways (IPA, z-score>|2|) associated with organism death and neurological and immune dysfunctions, which have been known to be affected by OPPs. Despite bioassay-related differences, our results indicate the conservation of key events potentially important to OPPs biological activity and potency across species. While additional research is needed, these results build confidence in using short-term, molecular-based assays for the characterization of toxicity and risk, thereby reducing reliance on chronic, rodent-based studies.
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Project description:The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.
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Project description:Current methods to support chemical hazard assessment face significant challenges given the rapidly growing number of compounds of emerging concern (CEC) requiring assessment. Standard testing strategies rely upon live animal testing, which are time-consuming, expensive, and ethically questionable. These concerns serve as an impetus to develop new approach methodologies (NAMs) that do not rely on live animal tests. This study explored the application of the molecular benchmark dose (BMD) approach in a 7-day embryo-larval fathead minnow (FHM) assay to derive determine transcriptomic points-of-departure (tPODs) which can be useds to predict apical BMDs of fluoxetine (FLX), a highly prescribed and potent selective serotonin reuptake inhibitor frequently detected in surface waters. Fertilized FHM embryos were exposed to graded concentrations of FLX (confirmed at <LOD, 0.19, 0.74, 3.38, 10.2, 47.5 µg/L) for 32 days. Subsets of fish were subjected to omics and locomotor analyses at 7 days post-fertilization (dpf) and to histological and biometric measurements at 32 dpf. Enrichment analyses of transcriptomics and proteomics data revealed significant perturbations in gene sets associated with serotonergic and axonal functions. BMD analysis resulted in tPOD values of 0.56 µg/L (median of the 20 most sensitive gene-level BMDs), 5.0 µg/L (tenth percentile of all gene-level BMDs), 7.51 µg/L (mode of the first peak of all gene-level BMDs) , and 5.66 µg/L (pathway-level BMD). These tPODs are protective of apical locomotor and reduced weight effects (LOEC of 10.2 µg/L) observed in this study and are well within the range of chronic apical BMDs of FLX reported in the literature. Furthermore, the distribution of gene-level BMDs followed a bimodal pattern, revealing disruption of sensitive neurotoxic pathways at low concentrations and metabolic pathway perturbations at higher concentrations. This is one of the first studies to derive tPODs for FLX using a short-term embryo assay at a life stage not considered to be a live animal under current legislations.
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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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