RESUMO
Zearalenone (ZEN) is a non-steroid estrogen mycotoxin produced by numerous strains of Fusarium which commonly contaminate cereals. After oral administration, ZEN is reduced via intestinal and hepatic metabolism to α- and ß-zearalenol (αZEL and ßZEL). These reduced metabolites possess estrogenic properties, αZEL showing the highest affinity for ERs. ZEN and reduced metabolites cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring, suggesting a fetal exposure to these contaminants. In our previous work, we have suggested the potential impact of ZEN on placental cells considering this organ as a potential target of xenobiotics. In this work, we first compared the in vitro effects of αZEL and ßΖΕL on cell differentiation to their parental molecule on human trophoblast (BeWo cells). Secondly, we investigated their molecular mechanisms of action by investigating the expression of main differentiation biomarkers and the implication of nuclear receptor by docking prediction. Conversely to ZEN, reduced metabolites did not induce trophoblast differentiation. They also induced significant changes in ABC transporter expression by potential interaction with nuclear receptors (LXR, PXR, PR) that could modify the transport function of placental cells. Finally, the mechanism of ZEN differentiation induction seemed not to involve nuclear receptor commonly involved in the differentiation process (PPARγ). Our results demonstrated that in spite of structure similarities between ZEN, αZEL and ßZEL, toxicological effects and toxicity mechanisms were significantly different for the three molecules.
Assuntos
Estrogênios não Esteroides/toxicidade , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Zearalenona/toxicidade , Zeranol/análogos & derivados , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Trofoblastos/metabolismo , Zeranol/toxicidadeRESUMO
The placenta is a key organ in fetal growth and development because it controls maternal-to-fetal exchanges of nutrients and hormones. It also interferes with drug delivery to the fetus by expressing active membrane transporters and xenobiotic metabolism enzymes. Developing strategies to understand the role of the placenta in drug delivery is a challenge in toxicology. Despite common physiological functions, the placentas of different species are heterogeneous in their morphology and in their expression of membrane transporters and metabolizing proteins. These characteristics raise the difficulty of obtaining a good representative model of human placental transfer. To date, different in vitro, in vivo, and ex vivo tools have been used to elucidate transport and metabolism processes in the human placenta. This study recapitulates the typical features of human placenta and then presents the placental enzymes of xenobiotic metabolism, ATP-binding cassette transporters, solute carrier transporters, and their role in fetal exposure to xenobiotics. The study also compares the characteristics of different models of human placenta, in terms of membrane localization of transporters, and the expression of xenobiotic metabolism enzymes. The use of these models for toxicological studies, in particular xenobiotic transfer, is described, and the advantages and limits of each model are summarized.
Assuntos
Troca Materno-Fetal , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Placenta/metabolismo , Xenobióticos/farmacocinética , Transporte Biológico , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Placenta/enzimologia , GravidezRESUMO
The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10-200 microM ZEA showed efficacious metabolism of the toxin. alpha-zearalenol and beta-zearalenol were the measured preponderant metabolites (respectively 40.7+/-3.1% and 31.9+/-4.9% of total metabolites, after a 3h exposure to 10 microM ZEA), whereas ZEA-glucuronide and alpha-zearalenol glucuronide were less produced (respectively 8.2+/-0.9% and 19.1+/-1.3% of total metabolites, after a 3h exposure to 10 microM ZEA). Cell production of reduced metabolites was strongly inhibited by alpha-and beta-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited alpha-hydroxysteroid dehydrogenase type II and beta-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, alpha-zearalenol was preponderantly found at the basal side, whereas beta-zearalenol and both glucuronides were preferentially excreted at the apical side. As alpha-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.
Assuntos
Estrogênios não Esteroides/metabolismo , Micotoxinas/metabolismo , Zearalenona/metabolismo , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Transporte Biológico , Células CACO-2 , Humanos , Neoplasias Intestinais/metabolismoRESUMO
Mycotoxin nivalenol (NIV) is a natural contaminant of various cereal crops, animal feed and processed grains throughout the world. Human and animal contamination occurs mainly orally, and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. In this study, we investigated the mechanisms involved in NIV transepithelial transfer. The human intestinal Caco-2 cell line showed a basal-to-apical polarized transport of NIV. Using metabolic inhibitors and temperature-dependent experiments, we demonstrated that basolateral-apical (BL-AP) transfer of NIV involved an energy-dependent transport whereas apical-basolateral (AP-BL) transfer was governed by passive diffusion. NIV efflux was significantly decreased in the presence of the P-glycoprotein (P-gp) inhibitor valspodar, the multi-drug resistance-associated proteins (MRPs) inhibitor MK571, but was not modified by the breast cancer resistance protein (BCRP) inhibitor Ko143. Intracellular NIV accumulation was investigated using epithelial cell lines transfected with either human P-glycoprotein or MRP2. This accumulation was significantly decreased in LLCPK1/MDR1 and MDCKII/MRP2 cells, compared to wild-type cells, and this effect was reversed by valspodar and MK571, respectively. These in vitro results suggested that NIV was a substrate for both P-glycoprotein and MRP2. This interaction may play a key role in weak intestinal absorption of NIV and the mainly predominant excretion of NIV in faeces in animal studies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Micotoxinas/metabolismo , Tricotecenos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/fisiologia , Transporte Biológico Ativo/efeitos dos fármacos , Células CACO-2 , Linhagem Celular , Cromatografia Gasosa , Eletroquímica , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Humanos , Imuno-Histoquímica , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Propionatos/farmacologia , Quinolinas/farmacologia , Temperatura , Sais de Tetrazólio , TiazóisRESUMO
Deoxynivalenol (DON) is a major mycotoxic contaminant of cereal grains in Europe and North America. Human and animal contamination occurs mainly orally, and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. This study investigates the mechanisms of DON transepithelial transfer. Investigations using the human intestinal Caco-2 cell line showed a basal-to-apical polarized transport of the toxin. Both apical-basolateral (AP-BL) and basolateral-apical (BL-AP) transfers were time- and concentration-dependent, and not saturable between 5 and 30 microM DON. Arrhenius plot analysis revealed that transfer of 10 microM DON was temperature-dependent, with apparent activation energy E(a)=3.2 kcal mol(-1) in the AP-BL direction, and E(a)=10.4 kcal mol(-1) in the BL-AP direction. Intracellular DON accumulation was increased and DON efflux was decreased by ATP depletion, by P-glycoprotein inhibitor valspodar and by MRP2 inhibitor MK571, but not by BCRP inhibitor Ko143. Intracellular DON accumulation was then investigated using epithelial cell lines transfected with human P-glycoprotein or MRP2. This accumulation was decreased in LLCPK1-MDR1 and MDCKII-MRP2 cells, compared to wild-type cells, and the decrease could be reversed by valspodar or MK571. Taken together, these results suggest that DON is a substrate for both P-glycoprotein and MRP2.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Tricotecenos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico Ativo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ciclosporinas/farmacologia , Interpretação Estatística de Dados , Cães , Epitélio/metabolismo , Citometria de Fluxo , Humanos , Células LLC-PK1 , Proteínas de Membrana Transportadoras/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Propionatos/farmacologia , Quinolinas/farmacologia , Suínos , Temperatura , TransfecçãoRESUMO
Organophosphate insecticide diazinon is widely used in agricultural practices, submitting farmers to repeated exposure. Because efflux pumps, as P-glycoprotein (P-gp), serve both as natural defense mechanisms and influence the bioavailability and disposition of drugs, we analyzed the ability of diazinon to act as efflux modulator. Oral administration of diazinon (2-20 mg/kg, 5 days, or 10 mg/kg, 2-12 days) increased intestinal mdr1a mRNA of rats, in both dose- and time-dependent manner, and increased the expression of intestinal P-gp. Using the intestinal cell-line Caco-2, we found that 100 microM diazinon significantly inhibited digoxin and vinblastine secretive flux through the cell monolayers, whereas digoxin and vinblastine absorptive flux increased. The 25 microM diazinon was transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. The efflux rate significantly decreased in the presence of metabolic inhibitors sodium azide and 2-deoxy-d-glucose, P-gp inhibitors cyclosporin A and valspodar, but not in the presence of MRPs inhibitor MK571. Repeated exposure of Caco-2 cells to diazinon increased P-glycoprotein expression and activity. These results suggested the involvement of P-gp in the transfer of diazinon, leading to potential consequences for xenobiotic interactions, and showed that repeated exposure to low doses of pesticide may lead to up-regulated P-gp functions in the intestine of mammals.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Diazinon/toxicidade , Inseticidas/toxicidade , Intestinos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Animais , Células CACO-2 , Ciclosporinas/farmacologia , Diazinon/farmacocinética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Inseticidas/farmacocinética , Mucosa Intestinal/metabolismo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Mycotoxin zearalenone (ZEN) is a cereal contaminant produced by various species of Fusarium fungi. When interacting with estrogen receptors, ZEN leads to animal fertility disturbances and other reproductive pathologies. Few data are available on the effects of perinatal exposure to ZEN, particularly in the blood-testis barrier. The aim of this study was to assess the impact of ZEN in adult rats exposed neonatally. We focused on the expression and cellular localization of major ABC transporters expressed in adult rat testis, comparing ZEN effects with those of Estradiol Benzoate (EB) neonatal exposure. Dose-dependent and long term modulations of mRNA and protein levels of Abcb1, Abcc1, Abcg2, Abcc4 and Abcc5 were observed, along with Abcc4 protein cellular delocalization. ZEN exposure of SerW3 Sertoli cells showed modulation of Abcb1, Abcc4 and Abcc5. Comparison with EB exposure showed similar modulation profiles for Abcg2 but differential modulations for Abcb1, Abcc1, Abcc4 and Abcc5 in vivo, and a similar profile for Abcb1 modulation by ZEN and EB, but differential modulation for Abcc4 and Abcc5 in vitro. ZEN and EB effects were inhibited by in vitro addition of the pure anti-estrogen ICI 182.780, suggesting the at least partial implication of ZEN estrogenic activity in these modulations. These results suggested that ZEN neonatal exposure could affect the exposure of testis to ABC transporter substrates, and negatively influence spermatogenesis and male fertility.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Estrogênios não Esteroides/toxicidade , Testículo/efeitos dos fármacos , Zearalenona/toxicidade , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Testículo/metabolismoRESUMO
The mycotoxin zearalenone (ZEN) is produced by a variety of Fusarium fungi and contaminates numerous cereals, fruits and vegetables. Interacting with the estrogen receptors, ZEN and reduced metabolites zearalenols cause hormonal effects in animals. Few data are available on the effects of repeated exposure to ZEN, particularly during pregnancy. The aim of our work was to assess the impact of this toxin on the expression of ABC transporters and nuclear receptors in fetal liver and pregnant rats that were exposed daily (gestation day 7-20) to 1 mg/kg ZEN. Significant variations were observed, depending on the tissue type, the tissue origin (maternal or fetal), and the time of analysis after the last exposure to ZEN (4 h or 24 h). The modulations of expression were independent of the magnitude of tissue impregnation by ZEN and its metabolites. The maternal uterus was the most sensitive tissue: Abcb1a, Abcb1b and Abcg2 mRNA and protein expressions were induced at both times, while Abcc1, Abcc3 and Esr1 mRNA and protein expressions were inhibited then induced 4 h and 24 h after exposure, respectively. In the fetal liver, Abcb1a and Esr1 protein expression was inhibited at both times, while mRNA expression was induced 24 h after the last exposure to ZEN. These results suggested that ZEN exposure could impact maternal and fetal exposure to ABC transporters substrates, and influence fetus development through nuclear receptor modulation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Estrogênios não Esteroides/toxicidade , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Zearalenona/toxicidade , Animais , Western Blotting , Receptor alfa de Estrogênio/biossíntese , Receptor beta de Estrogênio/biossíntese , Feminino , Indicadores e Reagentes , Fígado/embriologia , Gravidez , RNA/biossíntese , RNA/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores de Progesterona/biossíntese , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
The mycotoxin zearalenone, produced by Fusarium species, is a worldwide contaminant of concern in cereals and other plant products. Due to its estrogenic activity, zearalenone (ZEA) is known to have toxicological effect in animals on reproductive system and the placental transfer of ZEA was suggested by in vivo studies. Although passive diffusion is the principal transport mechanism across the placenta, several carrier-mediated transport protein such as ABC transporter (P-gp, MRP1, MRP2, BCRP) have been identified in the placenta. In this work, we have investigated the effect of ZEA on trophoblast differentiation and ABC transporter expression by using an in vitro model of transplacental barrier, the BeWo cell line. In the presence of 10 microM ZEA morphological (syncytium formation) and biochemical (hCG secretion) differentiation of BeWo cells were observed after a 48h exposure. Results were compared to 17beta-estradiol (E2) and an inducer of syncytialisation (forskolin). The influence of cell differentiation and ZEA exposure on expression profiles of major ABC transporters was investigated in BeWo cells: expression of mRNA MRP1, MRP2 and BCRP was induced after 24h of ZEA exposure. Induction of P-gp, MRP1, and MRP2 protein was observed after 48h of ZEA exposure. Similar results were obtained after forskolin exposure. Our study reported for the first time the implication of a food contaminant in biological effect and ABC transporter expression modulation in human choriocarcinoma cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Diferenciação Celular/efeitos dos fármacos , Estrogênios não Esteroides/farmacologia , Trofoblastos/fisiologia , Zearalenona/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Colforsina/farmacologia , Corantes/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Gravidez , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Fatores de Tempo , Trofoblastos/metabolismoRESUMO
The myco-estrogene zearalenone (ZEA) is a worldwide cereal contaminant, implicated in reproductive disorders in animals and humans. Intestinal cells constitute a first barrier to mycotoxins exposure, since they express membrane ABC transporters that may affect the bioavailability of food xenobiotics. In this study, we investigated the mechanisms involved in the transepithelial transfer of ZEA and its major metabolites alpha- and beta-zearalenols (ZOLs), first using human intestinal Caco-2 cells. When exposed to ZEA, alpha-ZOL or beta-ZOL either in the apical (AP) or basolateral (BL) compartment, cells showed asymmetry in the AP-BL and BL-AP transfer of mycotoxins. Metabolic inhibitors increased ZEA, alpha-ZOL and beta-ZOL intracellular accumulation. Caco-2 cells apically exposed to ZEA produced metabolites (ZOLs and glucuronides) whose distribution between AP, BL and intracellular compartments was significantly modified by ABCCs inhibitor MK571. ABCB1-, ABCC1-, ABCC2 and ABCC3-transfected cells were used for studies of intracellular accumulation of ZEA, alpha-ZOL and beta-ZOL with or without specific inhibitors, and for competitive studies using fluorescent substrates. The results showed that ZEA, alpha-ZOL and beta-ZOL were substrates for ABCC2. ABCC1 was also involved in ZEA and alpha-ZOL transport, whereas ABCC3 only interacted with beta-ZOL. These specific interactions suggest a role for ABCC1-3 transport proteins in zearalenone exposure and its resulting risk for human health.