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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment. The exposure of living organisms to TCDD may result in numerous disorders, including reproductive pathologies. By employing two-dimensional fluorescence difference gel electrophoresis we aimed to identify proteins potentially involved in the mechanism of TCDD action and toxicity in porcine granulosa cells. The porcine granulosa cells were treated with TCDD (100 nM) for 3, 12 or 24 h, and afterwards, cytoplasmic proteins were isolated and labeled with cyanines. Next, samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF MS analysis. A total of 75 differentially expressed protein spots (p 0.05 and fold change ≥2.0) were found in granulosa cells treated with TCDD. After 3, 12 and 24 h of TCDD treatment, we were able to identify 29, 34 and 12 spots, respectively. Functional analysis showed that cytoskeletal proteins formed the largest class of proteins significantly affected by TCDD in all time points. We also demonstrated that most of the identified proteins were associated with the "structural constituent of cytoskeleton" and "chaperone binding" Gene Ontology categories. Based on the analysis of the porcine granulosa cell proteome, we demonstrated that TCDD may affect the ovarian follicle fate by the rearrangement of the cytoskeleton and extracellular matrix as well as the modulation of proteins important for the cellular response to stress. The results of the current study present an extended insight into the TCDD mechanism of action in porcine granulosa cells, providing new directions for future functional studies.

Simple Summary According to the available data, colorectal cancer (CRC) is one of the most common malignant neoplasms. Depending on the location, type of cancer or gender, it is ranked 2nd to 4th in terms of incidence in the world. CRC, year by year, shows an increasing tendency in terms of both morbidity and deaths. Many factors may be responsible for the development of this disease, including genetic and environmental factors. Considering all the aspects, we made efforts to systematize the available literature data in terms of epidemiology, risk factors, type and nature of symptoms, development stages, available and future diagnosis of colorectal cancer. Abstract This review article contains a concise consideration of genetic and environmental risk factors for colorectal cancer. Known risk factors associated with colorectal cancer include familial and hereditary factors and lifestyle-related and ecological factors. Lifestyle factors are significant because of the potential for improving our understanding of the disease. Physical inactivity, obesity, smoking and alcohol consumption can also be addressed through therapeutic interventions. We also made efforts to systematize available literature and data on epidemiology, diagnosis, type and nature of symptoms and disease stages. Further study of colorectal cancer and progress made globally is crucial to inform future strategies in controlling the disease’s burden through population-based preventative initiatives.

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.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and /or different activation pathways.

Polychlorinated dibenzo-p-dioxins (PCDDs) are widespread by-products of human industrial activity. They accumulate in tissues of animals and humans, exerting numerous adverse effects on different systems. In living organisms, dioxins are metabolized by enzymes of the cytochrome P450 family, including CYP1A1. Particular dioxin congeners differ in their toxicity level and ability to undergo biodegradation. Since the molecular mechanisms underlying dioxin susceptibility or resistance to biodegradation are unknown, in the present study the molecular interactions between five selected dioxins and porcine CYP1A1 protein were investigated. It was found that the ability of a dioxin to undergo CYP1A1-mediated degradation is associated mainly with the number and position of chlorine atoms in the dioxin molecule. Among all examined congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrated the highest affinity to CYP1A1 and, at the same time, the greatest distance to the active site of the enzyme. Interestingly, in contrast to other dioxins, the binding of the TCDD molecule to the porcine CYP1A1 active site resulted in a rapid and continuous closure of substrate channels. All the information may help to explain the extended half-life of TCDD in living organisms as well as its high toxicity.

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.

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compound is an environmental chemical adversely affecting reproductive processes. Intracellular TCDD effects are mediated via aryl hydrocarbon receptor (AhR). The aim of the current study was to identify genes linking the AhR pathway with phenotypic consequences of TCDD action in granulosa cells of pigs. By applying multifactorial analysis, with TCDD and incubation time as factors, it was possible to determine temporal changes induced by TCDD in the cell transcriptome. Among the identified 144 differentially expressed genes (DEGs; Padjusted

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment. An exposure of living organisms to TCDD may result in numerous disorders, including reproductive pathologies. The aim of the current study was to examine the effects of TCDD on the transcriptome of porcine granulosa cell line AVG-16. By employing next-generation sequencing (NGS) we aimed to identify genes potentially involved in the mechanism of TCDD action and toxicity in porcine granulosa cells. The AVG-16 cells were treated with TCDD (100 nM) for 3, 12 or 24 h, and afterwards total cellular RNA was isolated and sequenced. In TCDD-treated cells we identified 141 differentially expressed genes (DEGs; padjusted