RESUMO
This study aimed to evaluate the cytotoxicity and genotoxicity from Inga laurina leaves extracts and fractions and obtain their chemical profile. The chemical profile of the crude extract from I. laurina leaves and its fractions was investigated through 1H NMR, RP-HPLC-PDA by co-injection with authentic standards and HPLC-MS. The quinone reductase induction as a biomarker for cancer chemoprevention was evaluated in murine hepatocellular carcinoma line, whereas the cytotoxicity was evaluated by sulforhodamine B assay (SRB) using HepG2 cell line and genotoxicity was evaluated by comet assay. The phytochemical analysis of the leaves crude extract and its fractions showed the presence of 2-hydroxyethyl-dodecanoate and the phenolic compounds: gallic acid, methyl gallate, p-coumaric acid, cinnamic acid, myricetin-3-O-(2â³-O-galoyl)-α-rhamnopyranoside, proanthocyanidin A-2 and myricetrin. All the fractions tested were not considered cytotoxic against the selected human cancer cell lines, they did not cause genotoxic in some concentrations damage and induced the enzyme quinone reductase.
Assuntos
Fabaceae/química , Mutagênicos/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ensaio Cometa , Dano ao DNA , Indução Enzimática/efeitos dos fármacos , Células Hep G2 , Humanos , Camundongos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
Testicular Leydig cells (LC) are modulated by several pathways, one of them being the histaminergic system. Heme oxygenase-1 (HO-1), whose upregulation comprises the primary response to oxidative noxae, has a central homeostatic role and might dysregulate LC functions when induced. In this report, we aimed to determine how hemin, an HO-1 inducer, affects LC proliferative capacity and whether HO-1 effects on LC functions are reversible. It was also evaluated if HO-1 interacts in any way with histamine, affecting its regulatory action over LC. MA-10 and R2C cell lines and immature rat LC were used as models. Firstly, we show that after a 24-h incubation with 25 µmol/L hemin, LC proliferation is reversibly impaired by cell cycle arrest in G2/M phase, with no evidence of apoptosis induction. Even though steroid production is abrogated after a 48-h exposure to 25 µmol/L hemin, steroidogenesis can be restored to control levels in a time-dependent manner if the inducer is removed from the medium. Regarding HO-1 and histamine interaction, it is shown that hemin abrogates histamine biphasic effect on steroidogenesis and proliferation. Working with histamine receptors agonists, we elucidated that HO-1 induction affects the regulation mediated by receptor types 1, 2 and 4. In summary, HO-1 induction arrests LC functions, inhibiting steroid production and cell cycle progression. Despite their reversibility, HO-1 actions might negatively influence critical phases of LC development and differentiation affecting their function as well as other androgen-dependent organs. What's more, we have described a hitherto unknown interaction between HO-1 induction and histamine effects.
Assuntos
Heme Oxigenase-1/metabolismo , Histamina/farmacologia , Células Intersticiais do Testículo/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Hemina/farmacologia , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Mitose/efeitos dos fármacos , Ratos Sprague-Dawley , Esteroides/biossínteseRESUMO
The induction of cytochrome P450 (P450) enzymes in response to drug treatment is a significant contributing factor to drug-drug interactions, which may reduce therapeutic efficacy and/or cause toxicity. Since most studies on P450 induction are performed in adults, enzyme induction at neonatal, infant, and adolescent ages is not well understood. Previous work defined the postnatal ontogeny of drug-metabolizing P450s in human and mouse livers; however, there are limited data on the ontogeny of the induction potential of each enzyme in response to drug treatment. Induction of P450s at the neonatal age may also cause permanent alterations in P450 expression in adults. The goal of this study was to investigate the short- and long-term effects of phenytoin treatment on mRNA and protein expressions and enzyme activities of CYP2B10, 2C29, 3A11, and 3A16 at different ages during postnatal liver maturation in mice. Induction of mRNA immediately following phenytoin treatment appeared to depend on basal expression of the enzyme at a specific age. While neonatal mice showed the greatest fold changes in CYP2B10, 2C29, and 3A11 mRNA expression following treatment, the levels of induced protein expression and enzymatic activity were much lower than that of induced levels in adults. The expression of fetal CYP3A16 was repressed by phenytoin treatment. Neonatal treatment with phenytoin did not permanently induce enzyme expression in adulthood. Taken together, our data suggest that inducibility of drug-metabolizing P450s is much lower in neonatal mice than it is in adults and neonatal induction by phenytoin is not permanent.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fenitoína/farmacologia , Animais , Indução Enzimática/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
Hormones regulate hepatic gene expressions to maintain metabolic homeostasis. Ectonucleotide pyrophosphatase/phosphodiesterase 1 has been thought to interfere with insulin signaling. To determine its potential role in the regulation of metabolism, we analyzed its gene (Enpp1) expression in the liver of rats experiencing fasting and refeeding cycles, and in primary rat hepatocytes and human hepatoma HepG2 cells treated with insulin and dexamethasone using northern blot and real-time PCR techniques. Hepatic Enpp1 expression was induced by fasting and reduced by refeeding in the rat liver. In primary rat hepatocytes and HepG2 hepatoma cells, insulin reduced Enpp1 mRNA abundance, whereas dexamethasone induced it. Dexamethasone disrupted the insulin-reduced Enpp1 expression in primary hepatocytes. This is in contrast to the responses of the expression of the cytosolic form of phosphoenolpyruvate carboxykinase gene to the same hormones, where insulin reduced it significantly in the process. In addition, the dexamethasone-induced Enpp1 gene expression was attenuated in the presence of 8-Br-cAMP. In conclusion, we demonstrated for the first time that hepatic Enpp1 is regulated in the cycle of fasting and refeeding, a process that might be attributed to insulin-reduced Enpp1 expression. This insulin-reduced Enpp1 expression might play a role in the development of complications in diabetic patients.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fígado/enzimologia , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , RNA Mensageiro/efeitos dos fármacos , Animais , Indução Enzimática/efeitos dos fármacos , Jejum/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Resistência à Insulina , Masculino , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/efeitos dos fármacos , Pirofosfatases/biossíntese , Pirofosfatases/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
KEY MESSAGE: The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Ácido Glutâmico/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Raízes de Plantas/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/biossíntese , Proliferação de Células/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Meristema/efeitos dos fármacos , Meristema/enzimologia , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Tirosina Fosfatases/biossíntese , Fatores de Transcrição/metabolismoRESUMO
Hormones regulate hepatic gene expressions to maintain metabolic homeostasis. Ectonucleotide pyrophosphatase/phosphodiesterase 1 has been thought to interfere with insulin signaling. To determine its potential role in the regulation of metabolism, we analyzed its gene (Enpp1) expression in the liver of rats experiencing fasting and refeeding cycles, and in primary rat hepatocytes and human hepatoma HepG2 cells treated with insulin and dexamethasone using northern blot and real-time PCR techniques. Hepatic Enpp1 expression was induced by fasting and reduced by refeeding in the rat liver. In primary rat hepatocytes and HepG2 hepatoma cells, insulin reduced Enpp1 mRNA abundance, whereas dexamethasone induced it. Dexamethasone disrupted the insulin-reduced Enpp1 expression in primary hepatocytes. This is in contrast to the responses of the expression of the cytosolic form of phosphoenolpyruvate carboxykinase gene to the same hormones, where insulin reduced it significantly in the process. In addition, the dexamethasone-induced Enpp1 gene expression was attenuated in the presence of 8-Br-cAMP. In conclusion, we demonstrated for the first time that hepatic Enpp1 is regulated in the cycle of fasting and refeeding, a process that might be attributed to insulin-reduced Enpp1 expression. This insulin-reduced Enpp1 expression might play a role in the development of complications in diabetic patients.
Assuntos
Humanos , Animais , Masculino , Ratos , Pirofosfatases/genética , RNA Mensageiro/efeitos dos fármacos , Dexametasona/farmacologia , Diester Fosfórico Hidrolases/genética , Glucocorticoides/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fígado/enzimologia , Pirofosfatases/biossíntese , Pirofosfatases/efeitos dos fármacos , Resistência à Insulina , RNA Mensageiro/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Jejum/metabolismo , Ratos Sprague-Dawley , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/efeitos dos fármacos , Células Hep G2 , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cancer chemoprevention involves prevention/delay/reverse of the carcinogenic process through administration of cancer chemopreventive agents (CCA). Compounds which are able to induce detoxification-enzymes, especially monofunctional phase II enzymes, have become in excellent approaches for new CCA. Herein, we report the synthesis of new furoxanyl chalcone-like hybrid compounds as CCA. In vitro studies showed that phenylfuroxanyl derivatives 6 and 9 displayed the best activities being 9 the greatest monofunctional-inducer. Additionally, compounds were non-mutagenic against TA98 Salmonella typhimurium strain (Ames test) and could be used in the prevention of the progression of pre-malignant lesions for their cytotoxic activity against tumoral cells. In vivo proof of concept showed increment on phase II-enzymes activities in liver, colon and mammary gland having derivative 9 the best induction profiles. We probed Nrf2 nuclear translocation is operative for both compounds allowing to exert protective effects via expression of downstream phase-II enzymes.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Chalcona/farmacologia , Glutationa Transferase/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxidiazóis/farmacologia , Animais , Antineoplásicos/síntese química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalcona/síntese química , Chalcona/química , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Indução Enzimática/efeitos dos fármacos , Feminino , Células HT29 , Humanos , Células MCF-7 , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Ratos , Ratos Wistar , Salmonella typhimurium/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Occupational toxicology and clinical pharmacology integration will be useful to understand potential exposure-drug interaction and to shape risk assessment strategies in order to improve occupational health. The aim of the present study was to evaluate the effect of exposure to ethanol fuel on in vivo activities of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2C and CYP2D by the oral administration of the probe drugs verapamil, ibuprofen and fluoxetine. Male Wistar rats exposed to filtered air or to 2000 ppm ethanol in a nose-only inhalation chamber during (6 h/day, 5 days/week, 6 weeks) received single oral doses of 10 mg/kg verapamil or 25 mg/kg ibuprofen or 10 mg/kg fluoxetine. The enantiomers of verapamil, norverapamil, ibuprofen and fluoxetine in plasma were analyzed by LC-MS/MS. The area under the curve plasma concentration versus time extrapolated to infinity (AUC(0-∞)) was calculated using the Gauss-Laguerre quadrature. Inhalation exposure to ethanol reduces the AUC of both verapamil (approximately 2.7 fold) and norverapamil enantiomers (>2.5 fold), reduces the AUC(0-∞) of (+)-(S)-IBU (approximately 2 fold) and inhibits preferentially the metabolism of (-)-(R)-FLU. In conclusion, inhalation exposure of ethanol at a concentration of 2 TLV-STEL (6 h/day for 6 weeks) induces CYP3A and CYP2C but inhibits CYP2D in rats.
Assuntos
Biocombustíveis/toxicidade , Indutores das Enzimas do Citocromo P-450/toxicidade , Inibidores das Enzimas do Citocromo P-450/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Etanol/toxicidade , Exposição por Inalação/efeitos adversos , Testes de Toxicidade Crônica/métodos , Poluentes Ocupacionais do Ar/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/metabolismo , Câmaras de Exposição Atmosférica , Biomarcadores/sangue , Biotransformação/efeitos dos fármacos , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/sangue , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/química , Indução Enzimática/efeitos dos fármacos , Fluoxetina/sangue , Fluoxetina/farmacocinética , Ibuprofeno/sangue , Ibuprofeno/farmacocinética , Limoneno Hidroxilases/antagonistas & inibidores , Limoneno Hidroxilases/metabolismo , Masculino , Ratos Wistar , Verapamil/análogos & derivados , Verapamil/sangue , Verapamil/química , Verapamil/farmacocinéticaRESUMO
AIM: The etiopathogenesis of inflammatory bowel disease (IBD) is unclear and further understanding of the mechanisms that regulate intestinal barrier integrity and function could give insight into its pathophysiology and mode of action of current drugs used to treat human IBD. Therefore, we investigated how intestinal inflammation affects Map kinase gene expression in rats, and if current intestinal anti-inflammatory drugs (sulphasalazine, prednisolone and azathioprine) act on these expressions. MATERIAL AND METHODS: Macroscopic parameters of lesion, biochemical markers (myeloperoxidase, alkaline phosphatase and glutathione), gene expression of 13Map kinases, and histologic evaluations (optic, electronic scanning and transmission microscopy) were performed in rats with colonic inflammation induced by trinitrobenzenesulphonic (TNBS) acid. KEY FINDINGS: The colonic inflammation was characterized by a significant increase in the expression of Mapk1, Mapk3 and Mapk9 accompanied by a significant reduction in the expression ofMapk6. Alterations inMapk expression induced by TNBS were differentially counteracted after treatment with sulphasalazine, prednisolone and azathioprine. Protective effects were also related to the significant reduction of oxidative stress, which was related to increase Mapk1/3 expressions, which were reduced after pharmacological treatment. SIGNIFICANCE: Mapk1, Mapk3,Mapk6 and Mapk9 gene expressionswere affected by colonic inflammation induced by TNBS in rats and counteracted by sulphasalazine, prednisolone and azathioprine treatments, suggesting that these genes participate in the pharmacological response produced for these drugs.
Assuntos
Anti-Inflamatórios/farmacologia , Azatioprina/farmacologia , Colite/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Prednisolona/farmacologia , Sulfassalazina/farmacologia , Animais , Colite/induzido quimicamente , Colite/enzimologia , Colite/patologia , Indução Enzimática/efeitos dos fármacos , Expressão Gênica , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , Ratos Wistar , Ácido TrinitrobenzenossulfônicoRESUMO
BACKGROUND: Cancer chemoprevention involves the carcinogenic process prevention, delay or reverse by the administration of chemopreventive agents, which are able to suppress or block the carcinogen metabolic activation/formation. The increased activity of phase II detoxification enzymes such as quinone-reductase (QR) and glutation-S-transferase (GST) correlates with the protection against chemically-induced carcinogenesis. It has been shown that synthetic chalcones and 3H-[1,2]-dithiole-3-thiones promote expression of genes involved in chemoprevention. MATERIALS & METHODS: Herein, the induction of phase II enzymes by designed Michael acceptor-dithiolethione hybrids was studied. RESULTS & DISCUSSION: Hybrids 5 and 7 displayed the induction of quinone-reductase and glutation-S-transferase in vitro in the same order on the wild-type mouse-hepatoma Hepa 1c1c7 and on the aryl-hydrocarbon-nuclear-translocator (Arnt)-defective mutant BPrc1 cells indicating that 7 displays the best chemopreventive potential.