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Effects of TCDD on gene expression in hepatocytes isolated from WT and PkmΔDRE mice.

ABSTRACT: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reprograms central carbon metabolism by switching pyruvate kinase expression from isoform M1 (Pkm1) to M2 (Pkm2) mediated by aryl hydrocarbon receptor (AhR) binding to a dioxin response element (DRE) located between exon 3 and 4 within the Pkm locus. To further investigate the consequences of Pkm isoform switching in TCDD elicited hepatotoxicity, we examined gene expression in primary hepatocytes isolated from mice with the Pkm locus DRE excised (PkmΔDRE). Wild-type and PkmΔDRE hepatocytes were treated with 10 nM TCDD for 2, 4, 8, 12, 24, 48, 72, 96 and 120 hours. Central carbon metabolite changes were also assessed in WT and PkmΔDRE mice treated with 30 µg/kg TCDD every 4 day for 28 days. While AHR target genes were comparably induced, some genes exhibited divergent expression patterns in PkmΔDREmice compared to wild-types following treatment with TCDD. Notably, antioxidant gene expression was delayed in PkmΔDRE hepatocytes. Metabolomic analysis also revealed differences in glycolytic, TCA cycle and pentose phosphate pathway metabolite levels in TCDD treated WT and PkmΔDRE liver extracts. In addition, amino acid metabolism and serine/glycine synthesis were also elevated, especially in PkmΔDRE. These findings indicate PKM2 induction affects the transcriptional and metabolic coordination of hepatic responses to TCDD.

ORGANISM(S): Mus musculus

PROVIDER: GSE309637 | GEO | 2025/12/09

REPOSITORIES: GEO

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Project description:Dioxin-like chemicals are well-known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We conducted a thorough investigation to address the following questions: (1) does AHR genotype itself affect constitutive expression of microRNAs? (2) does TCDD affect microRNA levels and, if so, is this response dependent on the AHR? (3) does TCDD affect microRNA levels differently in animals that are sensitive to dioxin toxicity versus those that are dioxin-resistant? We used the Exiqon miRNA array platform as well as quantitative RT-PCR to measure miRNA levels in wildtype vs. Ahr-null mice. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in mouse livers and those changes that were statistically significant were of modest magnitude. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD M-bM-^@M-^S only a few miRNAs differed in expression between Ahr-null mice compared to mice with wildtype AHR. It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult mouse liver. Manuscript Submitted: Moffat ID, Boutros PC, Celius T, Pohjanvirta R & Okey AB. Micro-RNAs in rodent liver are refractory to dioxin treatment. Toxicological Sciences May, 2007. Keywords: miRNA expression, gene knockout, response to xenobiotics, genetic modification This was a two-factor, two-level design, the factors being genotype Ahr+/+ (WT) or Ahr-/- (KO) and treatment TCDD-treated (T) or vehicle control (C). Each total-RNA sample was separately labeled either with a Hy3- or a Hy5-fluorophore (Exiqon). Hy3- and Hy5-labeled samples were co-hybridized to an array. Dye-reversal was performed to eliminate dye bias. We tested 3 TCDD-treated and 3 control mice in the Ahr-/- groups and 4 TCDD-treated and 4 control mice in the Ahr+/+ groups. Technical replication of 1 TCDD-treated and 1 control mice in the Ahr-/- groups was performed. A loop design was used.

Project description:Dioxin-like chemicals are well-known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We used the Exiqon miRNA array platform as well as quantitative RT-PCR to measure miRNA levels in dioxin-sensitive Long-Evans (Turku/AB; L-E) rats vs. dioxin-resistant Han/Wistar(Kuopio; H/W) rats. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in rat livers and those changes that were statistically significant were of modest magnitude. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD – only a few miRNAs differed in expression between between L-E rats (that have wildtype AHR) compared to H/W rats (whose AHR has a large deletion in the transactivation domain). It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult rodent liver. We conducted a thorough investigation to address the following questions: (1) does AHR genotype itself affect constitutive expression of microRNAs? (2) does TCDD affect microRNA levels and, if so, is this response dependent on the AHR? (3) does TCDD affect microRNA levels differently in animals that are sensitive to dioxin toxicity versus those that are dioxin-resistant? We assessed the in vivo effect of TCDD on microRNA levels in liver at multiple time points after TCDD treatment using microRNA arrays along with quantitative RT-PCR. The cumulative results of our experiments indicate that downregulation of mRNA levels by dioxins in adult rodent livers is very unlikely to involve microRNAs. Manuscript Submitted: Moffat ID, Boutros PC, Celius T, Pohjanvirta R & Okey AB. Micro-RNAs in rodent liver are refractory to dioxin treatment. Toxicological Sciences May, 2007. Keywords: miRNA expression, Time course, response to xenobiotics, genetic modification, comparative genome hybridization We examined strain differences in miRNA expression independent of TCDD: 19-h vehicle-treated dioxin-resistant H/W AHRH/W/H/W rats (HC19) vs. vehicle- control dioxin-sensitive L-E AHRWT/WT rats (LC19). In the dioxin-sensitive rats we compared miRNA expression levels 3 h (LT3) or 19 h (LT19) post-TCDD treatment vs. LC19. In the dioxin-resistant rats we compared miRNA levels 3 h (HT3), 19 h (HT19), or 96 h (HT96) post-TCDD treatment vs. HC19.

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Effects of TCDD on gene expression in hepatocytes isolated from WT and PkmΔDRE mice.

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