Citation:

Chorley, B., G. Carswell, G. Nelson, V. Bhat, AND C. Wood. Early MicroRNA Indicators of PPARα Pathway Activation in the Liver. Toxicology Reports. Elsevier B.V., Amsterdam, Netherlands, 7:805-815, (2020). https://doi.org/10.1016/j.toxrep.2020.06.006

Impact/Purpose:

Recent work has highlighted the potential value of short-term transcriptomic-based measurements to identify surrogate points of departure (PODs) for chemical screening. A common challenge in these studies is selecting gene targets indicative of pathway disruption associated with an adverse effect, especially for data-poor chemicals without a known MOA/AOP profile. MicroRNAs (miRNAs) are short, non-coding RNA molecules, usually 22 to 24 nucleotides, that regulate gene expression post-transcriptionally by binding complementary sequences on messenger RNA (mRNA), resulting in degradation of the mRNA or suppression of gene translation. A small number of miRNAs can impact a large number of genes, therefore an alteration of a single miRNA may be indicative of a change of multiple gene networks. Further, miRNAs are acutely responsive to environmental chemical exposures, linked to activation of nuclear receptors and other transcription factors, dose-responsive, and measurable in accessible biofluids, which may enable biosurveillance efforts. Given these characteristics, miRNAs are being widely investigated as biomarkers of both chemical exposure and susceptibility to later adverse health outcomes. In this case study, we evaluated miRNAs associated with induction of peroxisome proliferator-activated receptor alpha (PPARα) activity in the liver. This pathway is a common target of environmental chemicals that mediates a diverse array of metabolic and carcinogenic effects in animal models. We estimated benchmark doses (BMDs) based on sequencing and digital-drop PCR (ddPCR) measurements and compared these values to referent BMDt and apical (BMDa) estimates to assess concordance between liver and serum miRNA targets. Our study suggests that altered miRNAs in mouse liver may serve as early biomarkers of PPARα activity after exposure to agonists and may be less ambiguous about the primary chemical mode of action than global gene transcription measurements. From an Agency perspective, miRNAs should be considered in novel approach methodologies to assess chemical effects in short term assays to reduce ambiguity in transcriptomic measurements that are linking to MOA/AOPs.

Description:

MicroRNAs (miRNAs) are short non-coding RNA species that play key roles in post-transcriptional regulation of gene expression. MiRNAs also serve as a promising source of early biomarkers for different environmental exposures and health effects, although there is limited information linking specific miRNAs to target pathways. In this study, we measured liver miRNA in male B6C3F1 mice exposed to a known chemical activator of the peroxisome proliferator-activated receptor alpha (PPARα) pathway, di(2-ethylhexyl) phthalate (DEHP), for 7 and 28 days at concentrations of 0, 750, 1500, 3000, or 6000 ppm in feed. At the highest dose tested, DEHP altered 61 miRNAs after 7 days and 171 miRNAs after 28 days of exposure, with 48 overlapping miRNAs between timepoints. Analysis of these 48 common miRNAs indicated enrichment in PPARα targets and other pathways related to liver injury and cancer. Four of the 10 miRNAs exhibiting a clear dose trend were linked to PPARα activation: mmu-miRs-125a-5p, -182-5p, -20a-5p, and -378a-3p. Mmu-miRs-182-5p and -378a-3p were subsequently measured using digital drop PCR across a dose range for DEHP and two related phthalates with weaker PPARα activity, di-n-octyl phthalate (DNOP) and n-butyl benzyl phthalate (BBP), following 7-day exposures. Analysis of mmu-miRs-182-5p and -378a-3p by transcriptional benchmark dose analysis correctly identified DEHP as having the greatest potency. Point-of-departure dose estimates for DEHP based on these miRNAs (average 163; range 126-202 mg/kg-day) were higher on average than previously calculated values for PPARα target genes (average 74; range 29-183 mg/kg-day). These findings identify putative miRNA biomarkers of PPARα activation and suggest that early miRNA changes may be used to stratify chemical potency.

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