The involvement of CYP1A2 in biodegradation of dioxins in pigs.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most harmful chemicals showing resistance to biodegradation. The majority of TCDD effects is mediated by the aryl hydrocarbon receptor (AhR) pathway. TCDD binding to AhR results in the activation of cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP1B1) involved in dioxin biodegradation. The goal of the study was to explore the potential role of CYP1A2 in the metabolism of TCDD. We investigated a molecular structure of CYP1A2 and the binding selectivity and affinity between the pig CYP1A2 and: 1/ DiCDD or TCDD (dioxins differing in toxicity and biodegradability) or 2/ their selected metabolites. pCYP1A2 demonstrated higher affinity towards DiCDD and TCDD than other pCYP1 enzymes. All dioxin-pCYP1A2 complexes were found to be stabilized by hydrophobic interactions. The calculated distances between the heme oxygen and the dioxin carbon nearest to the oxygen, reflecting the hydroxylating potential of CYP1A2, were higher than in other pCYP1 enzymes. The distances between the heme iron and the nearest dioxin carbon exceeded 5 Å, a distance sufficient to allow the metabolites to leave the active site. However, the molecular dynamics simulations revealed that two access channels of CYP1A2 were closed upon binding the majority of the examined dioxins. Moreover, the binding of dioxin metabolites did not promote opening of channel S-an exit for hydroxylated products. It appears that the undesired changes in the behavior of access channels prevail over the hydroxylating potential of CYP1A2 towards TCDD and the favorable distances, ultimately trapping the metabolites at the enzyme's active site

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

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

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells

Background 2,3,7,8-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. Methods Porcine 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. Results The 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). Conclusions TCDD 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