Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
BMC Genomics ; 24(1): 325, 2023 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-37312040

RESUMO

BACKGROUND: Premenopausal women diagnosed with breast cancer often face aggressive chemotherapy resulting in infertility. Tamoxifen (TAM) is a selective estrogen receptor modulator that was previously suggested as a protective agent against chemotherapy-induced ovarian failure. In the current study, we examined mechanisms of the protective action of TAM in the ovaries of tumor-bearing rats treated with the chemotherapy drug cyclophosphamide (CPA). RESULTS: TAM prevented CPA-induced loss of ovarian follicular reserves. The protective TAM effect in the rat ovary partially resulted from decreased apoptosis. In addition, transcriptomic and proteomic screening also implicated the importance of DNA repair pathways as well as cell adhesion and extracellular matrix remodeling in the protective ovarian actions of TAM. CONCLUSIONS: Tamoxifen shielded the ovary from the side effects of chemotherapy without lessening the tumoricidal actions of mammary cancer treatment.


Assuntos
Neoplasias , Tamoxifeno , Feminino , Animais , Ratos , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Ovário , Proteômica , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Agressão
2.
Chem Res Toxicol ; 36(6): 900-915, 2023 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-37184393

RESUMO

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been associated with the induction of oxidative stress and the progression of steatosis to steatohepatitis with fibrosis. It also disrupts metabolic pathways including one-carbon metabolism (OCM) and the transsulfuration pathway with possible consequences on glutathione (GSH) levels. In this study, complementary RNAseq and metabolomics data were integrated to examine the hepatic transsulfuration pathway and glutathione biosynthesis in mice following treatment with TCDD every 4 days for 28 days. TCDD dose-dependently repressed hepatic cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA and protein levels. Reduced CBS and CTH levels are also correlated with dose-dependent decreases in hepatic extract hydrogen sulfide (H2S). In contrast, cysteine levels increased consistent with the induction of Slc7a11, which encodes for the cystine/glutamate Xc- antiporter. Cotreatment of primary hepatocytes with sulfasalazine, a cystine/glutamate Xc- antiporter inhibitor, decreased labeled cysteine incorporation into GSH with a corresponding increase in TCDD cytotoxicity. Although reduced and oxidized GSH levels were unchanged following treatment due to the induction of GSH/GSSG efflux transporter by TCDD, the GSH:GSSG ratio decreased and global protein S-glutathionylation levels in liver extracts increased in response to oxidative stress along with the induction of glutamate-cysteine ligase catalytic subunit (Gclc), glutathione synthetase (Gss), glutathione disulfide reductase (Gsr), and glutathione transferase π (Gstp). Furthermore, levels of ophthalmic acid, a biomarker of oxidative stress indicating GSH consumption, were also increased. Collectively, the data suggest that increased cystine transport due to cystine/glutamate Xc- antiporter induction compensated for decreased cysteine production following repression of the transsulfuration pathway to support GSH synthesis in response to TCDD-induced oxidative stress.


Assuntos
Fígado Gorduroso , Dibenzodioxinas Policloradas , Camundongos , Animais , Cisteína/metabolismo , Cistina , Dissulfeto de Glutationa/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Ácido Glutâmico , Antiporters , Glutationa/metabolismo
3.
Sci Rep ; 13(1): 4138, 2023 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-36914879

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces the progression of steatosis to steatohepatitis with fibrosis in mice. Furthermore, TCDD reprograms hepatic metabolism by redirecting glycolytic intermediates while inhibiting lipid metabolism. Here, we examined the effect of TCDD on hepatic acetyl-coenzyme A (acetyl-CoA) and ß-hydroxybutyrate levels as well as protein acetylation and ß-hydroxybutyrylation. Acetyl-CoA is not only a central metabolite in multiple anabolic and catabolic pathways, but also a substrate used for posttranslational modification of proteins and a surrogate indicator of cellular energy status. Targeted metabolomic analysis revealed a dose-dependent decrease in hepatic acetyl-CoA levels coincident with the phosphorylation of pyruvate dehydrogenase (E1), and the induction of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphatase, while repressing ATP citrate lyase and short-chain acyl-CoA synthetase gene expression. In addition, TCDD dose-dependently reduced the levels of hepatic ß-hydroxybutyrate and repressed ketone body biosynthesis gene expression. Moreover, levels of total hepatic protein acetylation and ß-hydroxybutyrylation were reduced. AMPK phosphorylation was induced consistent with acetyl-CoA serving as a cellular energy status surrogate, yet subsequent targets associated with re-establishing energy homeostasis were not activated. Collectively, TCDD reduced hepatic acetyl-CoA and ß-hydroxybutyrate levels eliciting starvation-like conditions despite normal levels of food intake.


Assuntos
Fígado Gorduroso , Dibenzodioxinas Policloradas , Camundongos , Animais , Acetilcoenzima A/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Ácido 3-Hidroxibutírico/metabolismo , Fígado/metabolismo , Fígado Gorduroso/metabolismo
4.
J Biol Chem ; 298(9): 102301, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35931118

RESUMO

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.


Assuntos
Acil Coenzima A , Poluentes Ambientais , Fígado Gorduroso , Fígado , Dibenzodioxinas Policloradas , Deficiência de Vitamina B 12 , Vitamina B 12 , Ácido Aconítico/metabolismo , Acil Coenzima A/metabolismo , Animais , Cobalto/metabolismo , Cisteína/metabolismo , Poluentes Ambientais/toxicidade , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Succinatos/metabolismo , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/induzido quimicamente , Deficiência de Vitamina B 12/complicações
5.
Reprod Toxicol ; 104: 143-154, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34363982

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a man-made chemical compound contaminating the environment. An exposure of organisms to TCDD results in numerous disorders. The main mechanism of TCDD action involves the induction of the aryl hydrocarbon receptor (AhR) pathway followed by the increase in the expression and activity of cytochrome P450 family 1 (CYP1) enzymes. The main aim of the present study was to identify, by means of RNA sequencing, transcripts involved in the mechanism of TCDD action in Chinese hamster ovary (CHO) cells, known to not express CYP1A1 enzyme. The CHO cells were treated with TCDD for 3, 12 or 24 h, and total RNA was isolated and sequenced. Thirty six (padjusted < 0.05) or six (padjusted < 0.05, log2FC ≥ 1.0/log2FC≤-1.0) differentially expressed genes (DEGs) were identified in TCDD-treated cells depending on the assumed statistical criteria. The dioxin up- and downregulated the expression of genes associated with ovarian follicle functions, development, cardiovascular system, signal transduction, inflammation and carcinogenesis. TCDD did not affect the expression of any of 522 miRNAs which were identified in the cells. The expression of CYP1A1, CYP1A2 and CYP1B1 was demonstrated neither in control nor in TCDD-treated CHO cells, although the respective genes were found in the cell genome. Twenty two other CYP enzymes were identified in CHO cells, however their expression was also not affected by TCDD.


Assuntos
Dibenzodioxinas Policloradas/toxicidade , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Células CHO , Cricetinae , Cricetulus , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Feminino , MicroRNAs , Folículo Ovariano/metabolismo , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/genética
6.
PeerJ ; 8: e8371, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32002328

RESUMO

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.

7.
PLoS One ; 14(10): e0223420, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31584984

RESUMO

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.


Assuntos
Inativação Gênica , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Proteoma/efeitos dos fármacos , Proteômica , Receptores de Hidrocarboneto Arílico/genética , Animais , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Proteômica/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Hidrocarboneto Arílico/deficiência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
8.
Anim Reprod Sci ; 207: 83-94, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31213330

RESUMO

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<0.05, log2 fold change (FC)≥1), 111 DEGs were classified as sustained genes (FC values changing between 3 and 24 h). Eighty six DEGs were classified as early genes and only nine as late genes (FC changes observed between 3 and 12 h or 12 and 24 h, respectively). The sustained gene category included genes related to TCDD mechanism of action (AHR, ARNTL, CYP1A1), cell proliferation (TGFß3), follicular development and ovulation (PTGS2) as well as stress response (NR3C1). The early gene category contained DEGs associated with cell proliferation (DUSP4, TAB1) and cellular response to stress (DHX34). The CYP1A1 gene was the only DEG classified as an early, late and sustained gene. The multifactorial approach allowed for statistically analyzing TCDD-induced changes over time in the gene expression in granulosa cells of pigs. Changes over time in the granulosal transcriptome profile indicated the involvement of stress related molecules in the cellular response to TCDD and TCDD effects on ovulation. The TCDD effects were particularly evident during the early stage of action by this compound.


Assuntos
Células da Granulosa/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Suínos , Transcriptoma/efeitos dos fármacos , Animais , Células Cultivadas , Poluentes Ambientais/farmacologia , Feminino , Perfilação da Expressão Gênica/veterinária , Células da Granulosa/fisiologia , Análise em Microsséries , Ovulação/efeitos dos fármacos , Ovulação/genética , Suínos/genética , Fatores de Tempo
9.
Biochim Biophys Acta Gen Subj ; 1863(2): 291-303, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30278240

RESUMO

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most difficult to biodegradate and the most toxic dioxin congener. Previously, we demonstrated in silico the ability of pig CYP1A1 to hydroxylate 2,7-dichlorodibenzo-p-dioxin (DiCDD), but not TCDD. To increase our knowledge concerning the low effectiveness of TCDD biodegradability, we analyzed in silico the binding selectivity and affinity between pig CYP1B1 and the two dioxins by means of molecular modeling. We also compared the effects of TCDD and DiCDD on CYP1B1 gene expression (qRT-PCR) and catalytic (EROD) activity in porcine granulosa cells. It was found that DiCDD and TCDD were stabilized within the pig CYP1B1 active site by hydrophobic interactions. The analysis of substrate channel availability revealed that both dioxins opened the exit channel S, allowing metabolites to leave the enzyme active site. Moreover, DiCDD and TCDD increased the CYP1B1 gene expression and catalytic activity in porcine granulosa cells. On the other hand, TCDD demonstrated higher than DiCDD calculated affinity to pig CYP1B1, hindering TCDD exit from the active site. The great distance between CYP1B1's heme and TCDD also might contribute to the lower hydroxylation effectiveness of TCDD compared to that of DiCDD. Moreover, the narrow active site of pig CYP1B1 may immobilize TCDD molecule, inhibiting its hydroxylation. The results of the access channel analysis and the distance from pig CYP1B1's heme to TCDD suggest that the metabolizing potential of pig CYP1B1 is higher than that of pig CYP1A1. However, this potential is probably not sufficiently high to considerably improve the slow TCDD biodegradation.


Assuntos
Citocromo P-450 CYP1B1/metabolismo , Dioxinas/metabolismo , Suínos/metabolismo , Animais , Biocatálise , Citocromo P-450 CYP1B1/química , Dioxinas/química , Modelos Moleculares , Estrutura Molecular
10.
Artigo em Inglês | MEDLINE | ID: mdl-30338064

RESUMO

BACKGROUND: Long non-coding RNAs (lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics. The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, results in reproductive defects in many species including pigs. The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells. RESULTS: One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells. The identified lncRNAs were found to be shorter than mRNAs. In addition, the number of exons was lower in lncRNAs than in mRNAs and their exons were longer. TCDD affected the expression of 22 lncRNAs (differentially expressed lncRNAs [DELs]; log2 fold change  ≥ 1, P-adjusted < 0.05) in the examined cells. Potential functions of DELs were indirectly predicted via searching their target cis- and trans-regulated protein-coding genes. The co-expression analysis revealed that DELs may influence the expression of numerous genes, including those involved in cellular response to xenobiotics, dioxin metabolism, endoplasmic reticulum stress and cell proliferation. Aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1 (CYP1A1) were found among the trans-regulated genes. CONCLUSIONS: These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells. To our knowledge, this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile. These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.

11.
Chemosphere ; 212: 170-181, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30144678

RESUMO

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.


Assuntos
Células da Granulosa/química , Dibenzodioxinas Policloradas/farmacologia , Proteoma/efeitos dos fármacos , Animais , Proteínas do Citoesqueleto , Feminino , Células da Granulosa/efeitos dos fármacos , Espectrometria de Massas , Chaperonas Moleculares/metabolismo , Folículo Ovariano/efeitos dos fármacos , Dibenzodioxinas Policloradas/metabolismo , Proteoma/análise , Suínos
12.
J Genet ; 96(1): 75-85, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28360392

RESUMO

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.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Mapeamento Cromossômico , Variação Genética , Filogenia , RNA Mensageiro/genética , Receptores de Hidrocarboneto Arílico/genética , Substituição de Aminoácidos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/classificação , Frequência do Gene , Fases de Leitura Aberta , Receptores de Hidrocarboneto Arílico/classificação , Análise de Sequência de RNA , Suínos
13.
Chemosphere ; 178: 368-377, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28340459

RESUMO

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 < 0.05 and log2 fold change ≥1.0). The DEGs were assigned to GO term, covering biological processes, molecular functions and cellular components. Due to the large number of genes with altered expression, in the current study we analyzed only the genes involved in follicular growth, development and functioning. The obtained results showed that TCDD may affect ovarian follicle fate by influencing granulosa cell cycle, proliferation and DNA repair. The demonstrated over-time changes in the quantity and quality of genes being affected by TCDD treatment showed that the effects of TCDD on granulosa cells changed dramatically between 3-, 12- and 24-h of cell culture. This finding indicate that timing of gene expression measurement is critical for drawing correct conclusions on detailed relationships between the TCDD-affected genes and resulting intracellular processes. These conclusions have to be confirmed and extended by research involving proteomic and functional studies.


Assuntos
Poluentes Ambientais/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Animais , Feminino , Células da Granulosa/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Masculino , Folículo Ovariano/efeitos dos fármacos , Suínos
14.
Chemosphere ; 168: 205-216, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27783961

RESUMO

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.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Dioxinas/metabolismo , Dibenzodioxinas Policloradas/análise , Animais , Sítios de Ligação , Biodegradação Ambiental , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dibenzodioxinas Policloradas/metabolismo , Conformação Proteica , Suínos
15.
J Mol Graph Model ; 67: 119-26, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27288759

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and natural chemicals, including toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the present study, homology models of the porcine AhR-ligand binding domain (LBD) and the porcine aryl hydrocarbon receptor nuclear translocator-ligand binding domain (ARNT-LBD) were created on the basis of structures of closely related respective proteins i.e., human Hif-2α and ARNT. Molecular docking of TCDD to the porcine AhR-LBD model revealed high binding affinity (-8.8kcal/mol) between TCDD and the receptor. Moreover, formation of the TCDD/AhR-LBD complex was confirmed experimentally with the use of electrophoretic mobility shift assay (EMSA). It was found that TCDD (10nM, 2h of incubation) not only bound to the AhR in the porcine granulosa cells but also activated the receptor. The current study provides a framework for examining the key events involved in the ligand-dependent activation of the AhR.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Dibenzodioxinas Policloradas/química , Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Ligantes , Simulação de Acoplamento Molecular , Multimerização Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Sus scrofa
16.
J Reprod Dev ; 62(1): 103-13, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26568065

RESUMO

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor ß (ERß) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERß and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.


Assuntos
Apoptose , Receptor beta de Estrogênio/metabolismo , Genisteína/química , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , Dibenzodioxinas Policloradas/química , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Densitometria , Estradiol/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Fitoestrógenos/química , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Radioimunoensaio , Reação em Cadeia da Polimerase em Tempo Real , Suínos
17.
Folia Biol (Krakow) ; 63(2): 119-28, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26255463

RESUMO

Genistein is a biologically active isoflavone with estrogenic or antiestrogenic activity which can be found in a variety of soy products. Since in pigs' diet soy is the main source of protein, genistein may affect the reproductive/endocrine systems in these animals. Genistein has been shown to alter porcine ovarian and adrenal steroidogenesis but the mechanism of this action is still not clear. It is known that genistein binds to both estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), although it has a higher affinity to ERß. Moreover, this phytoestrogen was demonstrated to posses the activity of protein tyrosine kinase (PTK) inhibitor. The aim of the study was to examine the in vitro effects of genistein on: (1) progesterone (P4) and estradiol (E2) secretion by porcine luteinized granulosa cells harvested from large follicles, and (2) the mRNA and protein expression of ERa and ERß in these cells. In addition, to verify the role of PTK-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. Genistein significantly inhibited P4 and did not affect E2 secretion by porcine luteinized granulosa cells isolated from large follicles. Lavendustin C did not affect basal steroids secretion by examined cells. Genistein did not alter ERa but increased ERß mRNA levels in the cultured porcine granulosa cells. In contrast to medium follicles, the expression of ERß protein was unaffected by genistein in granulosa cells of large follicles. To conclude, the soy phytoestrogen genistein acts directly on the porcine ovary to decrease progesterone production and to increase the expression of ERß mRNA. Moreover, genistein-induced changes in follicular steroidogenesis and granulosal sensitivity to estrogens in pigs may depend on maturity of the follicles.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Células da Granulosa/metabolismo , Suínos/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Feminino , Genisteína/administração & dosagem , Fitoestrógenos/administração & dosagem , Fitoestrógenos/farmacologia , RNA Mensageiro
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA