RESUMO
Daily exposure to low doses of 3-methylcholanthrene (3MC) during the pubertal period in rats disrupts both follicular growth and ovulation. Thus, to provide new insights into the toxicity mechanism of 3MC in the ovary, here we investigated the effect of daily exposure to 3MC on selected ovarian genes, the role of the aryl hydrocarbon receptor (AhR) and the level of epigenetic remodeling of histone post-transcriptional modifications. Immature rats were daily injected with 3MC (0.1 or 1 mg/kg) and mRNA expression of genes involved in different ovarian processes were evaluated. Of the 29 genes studied, 18 were up-regulated, five were down-regulated and six were not altered. To assess whether AhR was involved in these changes, we used the chromatin immunoprecipitation assay. 3MC increased AhR binding to promoter regions of genes involved in Notch signaling (Hes1, Jag1), activation of primordial follicles (Cdk2), cell adhesion (Icam1), stress and tumor progression (Dnajb6), apoptosis (Bax, Caspase-9) and expression of growth and transcription factors (Igf2, Sp1). Studying the trimethylation and acetylation of histone 3 (H3K4me3 and H3K9Ac, respectively) of these genes, we found that 3MC increased H3K4me3 in Cyp1a1, Jag1, Dnajb6, Igf2, Notch2, Adamts1, Bax and Caspase-9, and H3K9Ac in Cyp1a1, Jag1, Cdk2, Dnajb6, Igf2, Icam1, and Sp1. Co-treatment with α-naphthoflavone (αNF), a specific antagonist of AhR, prevented almost every 3MC-induced changes. Despite the low dose used in these experiments, daily exposure to 3MC induced changes in both gene expression and epigenomic remodeling, which may lead to premature ovarian failure.
Assuntos
Benzoflavonas/farmacologia , Metilcolantreno/toxicidade , Folículo Ovariano/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Acetilação , Animais , Imunoprecipitação da Cromatina , Regulação para Baixo , Epigênese Genética , Feminino , Histonas/química , Metilação , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.
Assuntos
Benzoflavonas/farmacologia , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Transdução de Sinais , Animais , Células Cultivadas , Galinhas , FemininoRESUMO
In the present study, we investigated the effect of 3-methylcholanthrene (3MC) on sexual maturity and the ability of α-naphthoflavone (αNF) to prevent this action. To this end, immature rats were daily injected intraperitoneally with 3MC (0.1 or 1mg/kg) and/or αNF (80mg/kg). Body weight, vaginal opening and estrous cycle were recorded and ovaries were obtained on the day of estrus. Ovarian weight, ovulation rate (measured by the number of oocytes within oviducts), and follicular development (determined by histology) were studied. No differences were found in body weight, ovarian weight, day of vaginal opening, or the establishment of the estrous cycle among the different groups of rats. However, animals treated with 3MC, at both doses, exhibited a lower number of primordial, primary, preantral and antral follicles than controls. Also, 3MC inhibited the ovulation rate and induced an overexpression of both the Cyp1a1 and Cyp1b1 genes, measured by chromatin immunoprecipitation assay. The daily treatment with αNF alone increased the number of follicles in most of the stages analyzed when compared with controls. Moreover, the αNF treatment prevented completely not only the 3MC-induced decrease in all types of follicles but also the 3MC-induced overexpression of Cyp enzymes and the genetic damage in bone marrow cells and oocytes. These results suggest that (i) daily exposure to 3MC during the pubertal period destroys the follicle reserve and alters the ovulation rate; (ii) the 3MC action seems to be mediated by an aryl hydrocarbon receptor-dependent mechanism; (iii) daily administration of αNF has a clear stimulatory action on the ovarian function; and (iv) αNF may prevent both the systemic and gonadal 3MC-induced toxicity.
Assuntos
Benzoflavonas/administração & dosagem , Benzoflavonas/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Metilcolantreno/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/patologia , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
We investigated the presence and inducibility of CYP1A in suckermouth catfish (Hypostomus affinis and Hypostomus auroguttatus, Loricariidae), tilapia (Oreochromis niloticus, Cichlidae) and mice (Mus musculus, Muridae). Alkoxyresorufin-O-dealkylases (EROD, MROD, PROD and BROD) were detected and proved to be inducible (beta-naphthoflavone, BNF or dimethylbenz[a]anthracene, DMBA, 50 mg/kg bw ip) in liver microsomes from tilapia and mice. In loricariids, alkoxyresorufin-O-dealkylases were either undetectable (MROD/EROD) or very low (PROD/BROD), and so they remained after treatment with BNF or DMBA. Ethoxycoumarin-O-deethylase (ECOD) was recorded in all species and proved not to be inducible by BNF or DMBA. In loricariids and tilapia, ECOD was not depressed by a concentration of alpha-naphthoflavone (CYP1A-inhibitor) that markedly depressed EROD in tilapia. A CYP1A-like protein was detected by a monoclonal antibody in rats, mice and tilapia, but not in loricariids. A polyclonal antibody, however, detected a CYP1A-like protein in liver microsomes of loricariids. Suckermouth catfish, rats, mice and tilapia express a protein reactive with a polyclonal antibody against trout CYP3A. Loricariids and tilapia exhibited marked genotoxic responses (enhanced incidence of micronucleated erythrocytes) following treatment DMBA (50 mg/kg bw ip), a promutagen activated by CYP1A/1B. Therefore, although not exhibiting EROD, a CYP1A-mediated activity, loricariids converted DMBA into its genotoxic metabolites. Our findings suggest that the CYP1A-like protein of locariid catfish recognizes DMBA, but not ethoxyresorufin, as a substrate.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Peixes-Gato/metabolismo , Proteínas de Peixes/metabolismo , Fígado/enzimologia , Tilápia/metabolismo , O-Dealquilase 7-Alcoxicumarina/metabolismo , 9,10-Dimetil-1,2-benzantraceno/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/biossíntese , Benzoflavonas/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas de Peixes/biossíntese , Cinética , Fígado/efeitos dos fármacos , Camundongos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Microssomos Hepáticos/enzimologia , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Oxazinas/metabolismo , Receptores de Hidrocarboneto Arílico/agonistas , Especificidade por Substrato , beta-Naftoflavona/farmacologiaRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that, besides mediating toxic responses, may have a central role in ovarian physiology. Studying the actions of AHR ligands on granulosa cells function, we have found that beta-naphthoflavone amplifies the comitogenic actions of FSH and 17beta-estradiol in a dose-dependent manner. This amplification was even greater in cells that overexpress the AHR and was reversed by cotreatment with the AHR antagonist alpha-naphthoflavone, suggesting that this effect is mediated by the AHR. The estrogen receptor is likewise implicated in this phenomenon, because a pure antiestrogen abolished the described synergism. However, the more traditional inhibitory AHR-estrogen receptor interaction was observed on the estrogen response element-driven transcriptional activity. On the other hand, alpha-naphthoflavone inhibited dose-dependently the mitogenic actions of FSH and 17beta-estradiol. Beta-naphthoflavone induced the expression of Cyp1a1 and Cyp1b1 transcripts, two well-characterized AHR-inducible genes that code for hydroxylases that metabolize estradiol to catecholestrogens. Nevertheless, the positive effect of beta-naphthoflavone on proliferation was not caused by increased metabolism of estradiol to catecholestrogens, because these compounds inhibited the hormonally stimulated DNA synthesis. This latter inhibition exerted by catecholestrogens suggests that these hydroxylases would play a regulatory point in granulosa cell proliferation. Our study indicates that AHR ligands modulate the proliferation of rat granulosa cells, and demonstrates for the first time that an agonist of this receptor is able to amplify the comitogenic action of classical hormones through a mechanism that might implicate a positive cross-talk between the AHR and the estrogen receptor pathways.
Assuntos
Estradiol/farmacologia , Células da Granulosa/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/agonistas , beta-Naftoflavona/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/genética , Benzoflavonas/farmacologia , Células Cultivadas , Citocromo P-450 CYP1A1/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , DNA/biossíntese , DNA/efeitos dos fármacos , Sinergismo Farmacológico , Estrogênios de Catecol/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/metabolismo , Mitógenos/farmacologia , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
The modulation of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA-gated Cl(-) channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors expressed in Xenopus laevis oocytes in the micromolar range. alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by alpha(4)beta(2) neuronal nicotinic acetylcholine, serotonin 5-HT(3A) and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized alpha(1)beta(1)gamma(2s) GABA(A) receptors. Effects of apigenin and quercetin on alpha(1)beta(1)gamma(2s) GABA(A) receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation.