RESUMO
Astrocytes preserve the brain microenvironment homeostasis in order to protect other brain cells, mainly neurons, against damages. Glial cells have specific functions that are important in the context of neuronal survival in different models of central nervous system (CNS) diseases. Microglia are among these cells, secreting several molecules that can modulate astrocyte functions. Although 1,2-dihydroxybenzene (catechol) is a neurotoxic monoaromatic compound of exogenous origin, several endogenous molecules also present the catechol group. This study compared two methods to obtain astrocyte-enriched cultures from newborn Wistar rats of both sexes. In the first technique (P1), microglial cells began to be removed early 48 h after primary mixed glial cultures were plated. In the second one (P2), microglial cells were late removed 7 to 10 days after plating. Both cultures were exposed to catechol for 72 h. Catechol was more cytotoxic to P1 cultures than to P2, decreasing cellularity and changing the cell morphology. Microglial-conditioned medium (MCM) protected P1 cultures and inhibited the catechol autoxidation. P2 cultures, as well as P1 in the presence of 20% MCM, presented long, dense, and fibrillary processes positive for glial fibrillary acidic protein, which retracted the cytoplasm when exposed to catechol. The Ngf and Il1beta transcription increased in P1, meanwhile astrocytes expressed more Il10 in P2. Catechol decreased Bdnf and Il10 in P2 cultures, and it decreased the expression of Il1beta in both conditions. A prolonged contact with microglia before isolation of astrocyte-enriched cultures modifies astrocyte functions and morphology, protecting these cells against catechol-induced cytotoxicity.
Assuntos
Astrócitos , Microglia , Animais , Astrócitos/metabolismo , Catecóis/toxicidade , Células Cultivadas , Interleucina-10/metabolismo , Microglia/metabolismo , Ratos , Ratos WistarRESUMO
NRAS-mutations arise in 15-20% of all melanomas and are associated with aggressive disease and poor prognosis. Besides, the treatment for NRAS-mutant melanoma are not very efficient and is currently limited to immune checkpoints inhibitors or aggressive chemotherapy. 4-nerolidylcathecol (4-NC), a natural product extracted from Pothomorphe umbellata, induces apoptosis in melanoma cells by ROS production, DNA damage and increased p53 expression, in addition to inhibiting invasion in reconstructed skin. Moreover, 4-NC showed cytotoxicity in BRAF/MEKi-resistant and naive melanoma cells by Endoplasmic Reticulum (ER) stress induction in vitro. We evaluated the in vivo efficacy and the systemic toxicity of 4-NC in a NRAS-mutant melanoma model. 4-NC was able to significantly suppress tumor growth 4-fold compared to controls. Cleaved PARP and p53 expression were increased indicating cell death. As a proof of concept, MMP-2 and MMP-14 gene expression were decreased, demonstrating a possible role of 4-NC in melanoma invasion inhibition. Toxicological analysis indicated minor changes in the liver and bone marrow, but this toxicity was very mild when compared to other proteasome inhibitors and ER stress inductors already described. Our data indicate that 4-NC can counteract melanoma growth in vivo with minor adverse effects, suggesting further investigation as a potential NRAS-mutant melanoma treatment.
Assuntos
Antineoplásicos/farmacologia , Catecóis/farmacologia , GTP Fosfo-Hidrolases/genética , Melanoma/patologia , Proteínas de Membrana/genética , Mutação , Neoplasias Cutâneas/patologia , Animais , Antineoplásicos/toxicidade , Catecóis/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Humanos , Melanoma/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Cutâneas/genética , Testes de Toxicidade Subaguda , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
4-Nerolidylcatechol (1) is a secondary metabolite of plants and is described as a promising anti-inflammatory, antimalarial, antiulcerogenic, analgesic and cytotoxic compound possibly due to its antioxidant profile. In this study, we evaluated the pharmacologic activity and the antioxidant and toxicological profiles of compound (1) and its synthetic analogues (2-6). The synthetic analogues were designed from the lead compound, (1), using a molecular-simplification strategy. Compound 5 showed, by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ß-carotene systems, similar antioxidant activity when compared to compound (1). The oxidative stress in erythrocyte membrane demonstrated the highly protective effect of compounds (4), (5) and (6) and high antioxidant/pro-oxidant activity in relation to the concentrations of compounds (1) and (3). Compounds (2), (4), (5) and (6) were haemobiocompatible. All compounds (1-6) showed cytotoxic effects in 3T3 cells, but compounds (2) and (6) were highly cytotoxic in this lineage when compared to compound (1). Compound (5) had a lower myelosuppressive effect in haematopoietic progenitor cells compared to (1). Both compounds, (1) and (5), showed low genotoxic effects in vitro, on human lymphocyte cells. In addition, these compounds also showed low-toxicity in vivo as defined a LD50 > 2000 mg/kg. In this assay, we did not observe death in the animals exposed to treatment with (1) and (5) compound. In conclusion, the structural design of the analogues as validated once compound (5) was found to have an antioxidant profile that was as potent as the lead compound (1). In addition, considering the safety profile, these compounds are promising as preventive and/or therapeutic agents against oxidative damage.
Assuntos
Antioxidantes/farmacologia , Antioxidantes/toxicidade , Catecóis/farmacologia , Catecóis/toxicidade , Células 3T3 , Animais , Antioxidantes/síntese química , Antioxidantes/química , Catecóis/síntese química , Catecóis/química , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Granulócitos/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Relação Estrutura-AtividadeRESUMO
The exposure to benzene is a public health problem. Although the most well-known effect of benzene is hematopoietic toxicity, there is little information about the benzene and its metabolites effects on the central nervous system (CNS). This study examined the toxic effects of 1,2-dihydroxybenzene (catechol), a benzene metabolite, to human glioblastoma GL-15 cells. GL-15 cell cultures were used as a model to provide more information about the toxic effects of aromatic compounds to the CNS. Catechol induced time- and concentration-dependent cytotoxic effects. Morphological changes, such as the retraction of the cytoplasm and chromatin clumping, were seen in cells exposed to 200 microM catechol for 48 hours. In cells exposed to 600 microM catechol for 48 hours, 78.0% of them presented condensed nuclei, and the Comet assay showed DNA damage. The percentage of cells labeled with annexin V (apoptotic cells) was greater in the group exposed to catechol (20.7%) than in control cells (0.4%). Exposure to catechol at concentrations greater than 100 microM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 microM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Catecóis/toxicidade , Poluentes Ambientais/toxicidade , Glioblastoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta a Droga , Glioblastoma/genética , Glioblastoma/metabolismo , Glutationa/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
4-Nerolidylcatechol (1) was isolated from cultivated Pothomorphe peltata root on a multigram scale using straight-forward solvent extraction-column chromatography. New semi-synthetic derivatives of 1 were prepared and tested in vitro against multidrug-resistant Plasmodium falciparum K1 strain. Mono-O-methyl, mono-O-benzyl, O,O-dibenzyl and O,O-dibenzoyl derivatives 2-8 exhibited IC(50) in the 0.67-22.52 microM range. Mono-O-methyl ethers 6 and 7 inhibited the in vitro growth of human tumor cell lines HCT-8 (colon carcinoma), SF-295 (central nervous system), LH-60 (human myeloblastic leukemia) and MDA/MB-435 (melanoma). In general, derivatives 2-8 are more stable to light, air and pH at ambient temperatures than their labile, natural precursor 1. These derivatives provide leads for the development of a novel class of antimalarial drugs with enhanced chemical and pharmacological properties.
Assuntos
Antimaláricos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Catecóis/farmacologia , Extratos Vegetais/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/toxicidade , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/toxicidade , Catecóis/síntese química , Catecóis/química , Catecóis/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Piperaceae/química , Extratos Vegetais/síntese química , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Raízes de Plantas/química , EstereoisomerismoRESUMO
PURPOSE: The aim of this work was to investigate the hypothesis that catechol inhibits FADH -linked basal respiration in mitochondria isolated from rat liver homogenates. Moreover, catechol ability to induce peroxidation of biomolecules in liver nuclear fractions was also studied. METHODS: Rat liver homogenates were incubated with 1mM 1,2-dihydroxybenzene (catechol) at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate. Nuclear fractions were incubated in the presence of 1 mM catechol for 17 hours at room temperature and the peroxidation of biomolecules was investigated by the reaction with thiobarbituric acid, which was determined spectrophotometrically at 535 nm. RESULTS: Catechol induced a time-dependent partial inhibition of FADH -linked basal mitochondrial respiration, however this substance was unable to induce a direct peroxidation of biomolecules in hepatic nuclear fractions. CONCLUSION: Catechol produced an inhibition of basal respiration associated to FADH2 in isolated liver mitochondria that could lead to cytotoxicity, ROS generation and cell death.
Assuntos
Catecóis/toxicidade , Flavina-Adenina Dinucleotídeo/análogos & derivados , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavina-Adenina Dinucleotídeo/antagonistas & inibidores , Mitocôndrias Hepáticas/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
OBJETIVO: Testar a hipótese do catecol inibir a respiração basal associada ao FADH2 em frações mitocondriais hepáticas de rato. Além disso, estudou-se também a capacidade do catecol de induzir peroxidação de biomoléculas nas frações nucleares. MÉTODOS: Os homogeneizados de fígado de ratos foram incubados com catecol a 1 mM em pH fisiológico. Depois disso, as frações mitocondriais foram isoladas por centrifugação diferencial. O consumo basal de oxigênio foi medido com um eletrodo do tipo Clark após injeção de succinato a 10 mM. Frações nucleares foram incubadas com catecol por 17 horas à temperatura ambiente e a peroxidação de biomoléculas foi investigada pela reação com o ácido tiobarbitúrico e mensurada espectrofotometricamente. RESULTADOS: O catecol induziu uma inibição parcial da respiração basal mitocondrial associada ao FADH2 de forma dependente do tempo, contudo essa substância não induziu peroxidação direta das biomoléculas presentes nas frações nucleares hepáticas. CONCLUSÃO: O catecol produz inibição da respiração basal associada ao FADH2 em mitocôndrias isoladas de fígado, o que pode levar à toxicidade, produção de espécies reativas e morte celular.
Assuntos
Animais , Ratos , Catecóis/toxicidade , Flavina-Adenina Dinucleotídeo/análogos & derivados , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavina-Adenina Dinucleotídeo/antagonistas & inibidores , Mitocôndrias Hepáticas/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
It is known that the exposure to benzene in the petroleum industry causes lympho-haematopoietic cancer among workers. However, there is little data concerning the toxicity of benzene to the central nervous system. Benzene easily penetrates the brain where it is metabolized to catechol. Since catechol autoxidizes in physiological phosphate buffer, we hypothesized that it could be toxic towards glial cells due to the generation of reactive oxygen species and quinones. In this work we studied the cytotoxic properties of catechol towards human glioblastoma cells. We found that catechol was toxic towards these cells after 72 hours and this toxicity was related to the formation of quinones. Catechol at 230µM killed 50% of cells. The catechol-induced cytotoxicity was prevented by the addition of 100U superoxide dismutase, which also inhibited the formation of quinones. These data suggest that catechol induces cytotoxicity via the extracellular generation of superoxide and quinones.
Sabe-se que a exposição de trabalhadores ao benzeno na indústria petrolífera é uma causa de câncer do sistema linfo-hematopoiético. Pouco se sabe, contudo, a respeito da toxicidade do benzeno no sistema nervoso central. O benzeno penetra facilmente no cérebro, onde é metabolizado a catecol. Como o catecol se auto-oxida em tampão fosfato no pH fisiológico, supôs-se que esse composto poderia ser tóxico para células gliais por gerar espécies reativas do oxigênio e quinonas. Nesse trabalho estudou-se a citotoxicidade do catecol para células de glioblastoma humano. O catecol foi tóxico após 72 horas e essa toxicidade relacionou-se com a formação de quinonas. O catecol a 230mM matou metade das células em cultura. A toxicidade do catecol e a produção de quinonas foram inibidas por 100U de superóxido dismutase. Esses dados sugerem que a toxicidade induzida pelo catecol deve-se à produção extracelular de superóxido e quinonas reativas.
Assuntos
Humanos , Benzeno/toxicidade , Sistema Nervoso Central , Catecóis/toxicidade , Glioblastoma/metabolismo , Exposição Ocupacional , Petróleo , Quinonas/análise , Superóxido Dismutase/farmacologia , Superóxido Dismutase/metabolismo , Superóxidos/análiseRESUMO
The degradation of polychlorobiphenyls (PCBs) by diverse bacteria, including Burkholderia sp. LB400, is incomplete with a concomitant accumulation of metabolic intermediates. In this study, the toxicity of diverse (chloro)biphenyls and of their biotransformation into the first two metabolic intermediates of the biphenyl pathway, were determined for the model bacterium Escherichia coli. Recombinant E. coli strains expressing different subsets of bph genes of strain LB400 accumulated metabolic intermediates from (chloro)biphenyls. During biotransformation of these compounds into metabolic intermediates, the viability and metabolic kinetics were determined. The toxicity of biotransformation of (chloro)biphenyls into different metabolic intermediates of (chloro)biphenyls varied. Dihydrodiols and dihydroxybiphenyls are very toxic metabolites for bacteria even after short incubation times, affecting the cell viability much more than (chloro)biphenyls. When bacteria transformed 2-CB into dihydrodiol or dihydroxybiphenyl, a great decrease of intact cells and abundant cell lysis was observed by transmission electronic microscopy. Cell viability of Burkholderia sp. LB400 and of E. coli exposed directly to 2,3-dihydroxybiphenyl decreased also drastically. The toxicity of metabolites generated during oxidation of PCBs may partly explain the recalcitrance to biodegradation of these pollutants. Conversion of less toxic compounds into products with increased toxicity resembles the bioactivation of xenobiotics in higher organisms.