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 miRNA changes to specific target pathways. In this study, we measured liver miRNAs 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-related targets and other pathways related to liver injury and cancer. Four of the 10 miRNAs exhibiting a clear dose trend were linked to the PPARα pathway: 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 and n-butyl benzyl phthalate, 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. However, benchmark dose estimates for DEHP based on these miRNAs (average 163; range 126-202 mg/kg-day) were higher on average than values for PPARα target genes (average 74; range 29-183 mg/kg-day). These findings identify putative miRNA biomarkers of PPARα pathway activity and suggest that early miRNA changes may be used to stratify chemical potency.
Keywords: AIC, Akaike Information Criterion; ALT, alanine aminotransferase; AOP, adverse outcome pathway; AST, aspartate aminotransferase; Acox1, acyl-Coenzyme A oxidase 1; Adverse outcome pathway (AOP); AhR, aryl hydrocarbon receptor; BBP, n-butyl benzyl phthalate; BMD, benchmark dose; BMDA, apical-based benchmark dose; BMDL, BMD lower confidence interval; BMDT, transcriptional-based benchmark dose; BMR, benchmark response; BROD, benzyloxyresorufin O-debenzylation; Benchmark dose (BMD); Biomarkers; CAR, constitutive androstane receptor; DEGs, differentially expressed genes; DEHP, di (2-thylhexyl) phthalate; DEmiRs, differentially expressed miRNAs; DNOP, di-n-octyl phthalate; EPA, U.S. Environmental Protection Agency; EROD, ethoxyresorufin O-dealkylation; GEO, Gene Expression Omnibus; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; Hepatocellular carcinoma; IPA, Ingenuity Pathway Analysis; Liver toxicity; MOA, mode of action; MicroRNAs; Mode of action (MOA); Nrf2, nuclear receptor erythroid 2-like 2; POD, point-of-departure; PPARα, peroxisome proliferator-activated receptor alpha; PROD, pentoxyresorufin O-depentylation; PXR, pregnane X receptor; Peroxisome proliferator-activated receptor alpha (PPARα); Phthalate; SDH, sorbitol dehydrogenase; TMM, trimmed mean of M-values; ddPCR, droplet digital polymerase chain reaction; mRNA, messenger RNA; miRNAs, microRNAs; mtDNA, mitochondrial; rRNA, ribosomal RNA; smallRNA-seq, small RNA sequencing; tRNA, transfer RNA.