Dataset Information

Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

ABSTRACT: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment and affecting human/animal health and reproduction. Intracellular TCDD action usually involves the activation of aryl hydrocarbon receptor (AhR). The aim of the current study was to examine TCDD-induced changes in the proteome of AhR-silenced porcine granulosa cells. The AhR-silenced cells were treated with TCDD (100 nM) for 3, 12 or 24 h. Total protein was isolated, labeled with cyanines and next, the samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF mass spectrometry (MS) analysis and confirmed by western blotting and fluorescence immunocytochemistry. The AhR-targeted siRNA transfection reduced the granulosal expression level of AhR by 60-70%. In AhR-silenced porcine granulosa cells, TCDD influenced the abundance of only three proteins: annexin V, protein disulfide isomerase and ATP synthase subunit beta. The obtained results revealed the ability of TCDD to alter protein abundance in an AhR-independent manner. This study offers a new insight into the mechanism of TCDD action and provide directions for future functional studies focused on molecular effects exerted by TCDD.

SUBMITTER: Orlowska K

PROVIDER: S-EPMC6777791 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Orlowska Karina K Swigonska Sylwia S Sadowska Agnieszka A Ruszkowska Monika M Nynca Anna A Molcan Tomasz T Zmijewska Agata A Ciereszko Renata E RE

PloS one 20191004 10

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment and affecting human/animal health and reproduction. Intracellular TCDD action usually involves the activation of aryl hydrocarbon receptor (AhR). The aim of the current study was to examine TCDD-induced changes in the proteome of AhR-silenced porcine granulosa cells. The AhR-silenced cells were treated with TCDD (100 nM) for 3, 12 or 24 h. Total protein was isolated, labeled with cyanin ...[more]

PMID: 31584984

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Project description:Background2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical, adversely affecting reproductive processes. The well-characterized canonical mechanism of TCDD action involves the activation of aryl hydrocarbon receptor (AhR) pathway, but AhR-independent mechanisms were also suggested. By applying RNA interference technology and Next Generation Sequencing (NGS) we aimed to identify genes involved in the mechanism of TCDD action in AhR knock-down porcine granulosa cells.MethodsPorcine granulosa cells were transfected with small interfering RNAs targeting mRNA of AhR. After transfection, medium was exchanged and the AhR knock-down cells were treated with TCDD (100 nM) for 3, 12 or 24 h, total cellular RNA was isolated and designated for NGS. Following sequencing, differentially expressed genes (DEGs) were identified. To analyze functions and establish possible interactions of DEGs, the Gene Ontology (GO) database and the Search Tool for the Retrieval of Interacting Genes (STRING) database were used, respectively.ResultsThe AhR gene expression level and protein abundance were significantly decreased after AhR-targeted siRNAs transfection of the cells. In TCDD-treated AhR knock-down cells we identified 360 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change [log2FC] ≥ 1.0). The functional enrichment analysis of DEGs revealed that TCDD influenced the expression of genes involved, among other, in the metabolism of vitamin A, follicular development and oocyte maturation, proliferation and differentiation as well as inflammation, stress response, apoptosis and oncogenesis. The three-time point study demonstrated that TCDD-induced changes in the transcriptome of AhR knock-down porcine granulosa cells were especially pronounced during the early stages of the treatment (3 h).ConclusionsTCDD affected the transcriptome of AhR knock-down porcine granulosa cells. The molecules involved in the AhR-independent action of TCDD were indicated in the study. The obtained data contribute to better understanding of molecular processes induced by xenobiotics in the ovary.

Project description:Mammalian bone has shown a variety of responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in experimental and wildlife studies. Although many responses have been well characterized in the postcranial skeleton, dioxin-induced effects on the cranium are largely unknown. In this study, we investigated the effects of chronic adult exposure to TCDD on cranial size and shape in dioxin-resistant Han/Wistar (H/W) and dioxin-sensitive Long-Evans (L-E) rat strains. Three-dimensional landmark configurations for the face, vault, and base of the cranium were recorded and analyzed using geometric morphometrics (GM) and dose-response modeling. The strongest effects were shown by L-E and H/W rats with daily exposures of 100 and 1000 ng TCDD/kg bw/day, respectively, resulting in significant reductions in centroid size (CS) in all three cranial modules for both strains except for the vault in H/W rats. Consistent with previous evidence of intraspecific variation in TCDD resistance, the benchmark doses (CEDs) for cranial size reduction in L-E rats were roughly 10-fold lower than those for H/W rats. For both strains, the face showed the greatest size reduction from the highest doses of TCDD (i.e., 3.6 and 6.3% decreases in H/W and L-E rats, respectively), most likely related to dose-dependent reductions in limb bone size and body weight gain. However, intrinsic morphological differences between strains were also observed: although the control groups of H/W and L-E rats had vaults and bases of comparable size, the face was 6.4% larger in L-E rats. Thus, although H/W rats possess an altered aryl hydrocarbon receptor (AhR) that appears to mediate and provides some resistance to TCDD exposure, their smaller reductions in facial size may also relate to strain-specific patterns of cranial development and growth. Future research will be aimed at understanding how ontogenetic factors may modulate toxic effects of prenatal and lactational exposure on the mammalian skeleton.

Dataset Information

Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Publications

Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

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