Unknown,Transcriptomics,Genomics,Proteomics

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High conservation in transcriptomic and proteomic response of white sturgeon to equipotent concentrations of 2,3,7,8-TCDD, PCB 77, and benzo[a]pyrene

ABSTRACT: Adverse effects associated with exposure to dioxin-like compounds (DLCs) are mediated primarily through activation of the aryl hydrocarbon receptor (AHR). However, little is known about the cascades of events that link activation of the AHR to apical adverse effects. Therefore, this study used high-throughput, next-generation molecular tools to investigate similarities and differences in whole transcriptome and whole proteome responses to equipotent concentrations of three agonists of the AHR, 2,3,7,8-TCDD, PCB 77, and benzo[a]pyrene, in livers of a non-model fish, the white sturgeon (Acipenser transmontanus). A total of 926 and 658 unique transcripts were up- and down-regulated, respectively, by one or more of the three chemicals. Of the transcripts shared by responses to all three chemicals, 85% of up-regulated transcripts and 75% of down-regulated transcripts had the same magnitude of response. A total of 290 and 110 unique proteins were up- and down-regulated, respectively, by one or more of the three chemicals. Of the proteins shared by responses to all three chemicals, 70% of up-regulated proteins and 48% of down-regulated proteins had the same magnitude of response. Among treatments there was 68% similarity between the global transcriptome and global proteome. Pathway analysis revealed that perturbed physiological processes were indistinguishable between equipotent concentrations of the three chemicals. The results of this study contribute towards more completely describing adverse outcome pathways associated with activation of the AHR. Compared transcriptomic as well as proteomic responses to 3 different agonists of the aryl hydrocarbon receptor (AHR) at equipotent doses.

ORGANISM(S): Acipenser transmontanus

SUBMITTER: Jon Doering

PROVIDER: E-GEOD-79624 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

High Conservation in Transcriptomic and Proteomic Response of White Sturgeon to Equipotent Concentrations of 2,3,7,8-TCDD, PCB 77, and Benzo[a]pyrene.

Doering Jon A JA Tang Song S Peng Hui H Eisner Bryanna K BK Sun Jianxian J Giesy John P JP Wiseman Steve S Hecker Markus M

Environmental science & technology 20160425 9

Adverse effects associated with exposure to dioxin-like compounds (DLCs) are mediated primarily through activation of the aryl hydrocarbon receptor (AHR). However, little is known about the cascades of events that link activation of the AHR to apical adverse effects. Therefore, this study used high-throughput, next-generation molecular tools to investigate similarities and differences in whole transcriptome and whole proteome responses to equipotent concentrations of three agonists of the AHR, 2 ...[more]

PMID: 27070345

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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.

Project description:Major toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) result from dysregulation of gene expression mediated by the aryl hydrocarbon receptor (AHR). Dioxin-like chemicals alter expression of numerous genes in liver but the specific genes whose dysregulation leads to toxicities such as wasting, hepatotoxicity and lethality have not been identified. We searched for genes that are most likely to be key to dioxin toxicity by using gene expression arrays to contrast hepatic gene expression after TCDD treatment in dioxin-sensitive rats (that carry wildtype AHR) with gene expression in H/W(Kuopio) rats which are highly resistant to dioxin toxicity due to a major deletion in the AHR's transactivation domain (TAD). The total number of TCDD-responsive genes was smaller in rats with the AHRH/W genotype than in rats with wildtype AHR. However, genes in the classic AH gene battery such as CYP1A1, CYP1A2 and CYP1B1 remained fully responsive to TCDD in AHRH/W rats; thus the TAD deletion selectively interferes with expression of a subset of hepatic genes rather than abolishing global AHR-mediated responses. Genes in the following functional categories differ in response to TCDD between dioxin-sensitive rats and dioxin-resistant rats: fatty acid oxidation, metabolism (xenobiotic, alcohol, amino acid, and fatty acid), phosphate transport, regulation of steroid biosynthesis, nitrogen compound catabolism, and generation of precursor metabolites and energy. Many of these differentially-responsive genes are integral parts of pathways such as: protein degradation and synthesis, fatty acid metabolism and synthesis, cytokinesis, cell growth, and apoptosis which may be part of mechanisms which lead to TCDD-induced wasting, hepatotoxicity, tumors, and death. These differentially-responsive genes are worthy candidates for further mechanistic studies to test their role in mediating or protecting from major dioxin toxicities. Experiment Overall Design: Four strains of rats were either treated with 100 ug/kg TCDD or cornoil vehicle and profiled at 19 hours, each with four replicates. Two strains were also analyzed at 3 hours, each with four replicates.

Project description:Background: The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is activated by xenobiotic chemicals that function as AHR ligands. The response to xenobiotic AHR ligands is toxicity and the induction of drug metabolizing enzymes. The impact of AHR knockdown on gene expression and pathways in human breast cancer cells in absence of xenobiotic AHR ligands has not been investigated on a genome-wide scale. Methods: MCF-7 cells were used as a model of human breast cancer. The AHR was silenced with short interfering RNA against AHR (siAHR). RNA-sequencing coupled with Ingenuity pathway analysis (IPA) was used to determine the impact of AHR knockdown on gene expression and pathways in the absence of xenobiotic AHR ligands. Western blot analysis and recombinant tumor necrosis factor (TNF) was used to investigate the impact of AHR knockdown on TNF induction of MNSOD expression. Results: We found that the AHR is transcriptionally active in MCF-7 breast cancer cells in the absence of xenobiotic AHR ligands. In total, the expression of 634 genes was significantly changed in AHR knockdown cells compared to controls at a false discovery rate of < 10%. The analysis confirmed that drug metabolizing enzymes were AHR targets; however, we found that AHR also promoted the expression of genes that were not directly related to the metabolism of xenobiotics, such as those involved in lipid and eicosanoid synthesis. Gene pathway analysis of the AHR regulated gene dataset predicted TNF activity to be reduced in AHR knockdown cells. Our finding that AHR knockdown inhibited TNF-stimulated increases in MNSOD expression confirmed this IPA prediction. Conclusions: This is the first gene expression profiling study of AHR knockdown breast cancer cells. Several known and novel AHR targets were identified. The results suggest that endogenous AHR regulation impacts eicosanoid synthesis by regulating gene expression. The IPA prediction of reduced TNF activity in AHR knockdown cells was confirmed by showing that TNF-induced increases in MNSOD expression was inhibited in AHR knockdown cells. The requirement of AHR for MNSOD activation is novel and provides a new link by which AHR may impact reactive oxidative species (ROS) signaling. Expression profiles by mRNA sequencing were generated for human MCF-7 cells transfected with cRNAi (6 replicates) or AHR-siRNA (5 replicates) for 36hr. Sequencing was performed on an Illumina HiSeq 1000 using a 2x100 base paired-end strategy.

Dataset Information

High conservation in transcriptomic and proteomic response of white sturgeon to equipotent concentrations of 2,3,7,8-TCDD, PCB 77, and benzo[a]pyrene

Publications

High Conservation in Transcriptomic and Proteomic Response of White Sturgeon to Equipotent Concentrations of 2,3,7,8-TCDD, PCB 77, and Benzo[a]pyrene.

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