RESUMO
BACKGROUND: Several metal-based molecules that display cytotoxicity against multiple cell lines have been pursued in an attempt to fight against cancer and to overcome the typical side effects of drugs like cisplatin. In this scenario, ruthenium complexes have been extensively studied due to their activity in both in vitro and in vivo biological systems, including various cancer cell strains. OBJECTIVE: We aimed to develop a method to synthesize novel [Ru(NO)(bpy)2L2]2+ complexes containing amino acid ligands by using an alternative Click Chemistry approach, namely the copper azide-alkyne cycloaddition reaction (CuAAC reaction), to construct nitrosyl/nitrite complexes bearing a modified lysine residue. METHODS: We synthesized a new ligand by Click Chemistry approach and new compounds bearing the unprecedented ligand. Cytotoxicity was assessed by the classical MTT colorimetric assay. MCF-7 and MDAMB- 231 cells were used as breast cancer cell models. MCF-10 was used as a model of healthy cells. RESULTS: Amino acid ligands related to N3-Lys(Fmoc) and the new pyLys were successfully synthesized by the diazotransfer reaction and the CuAAC reaction, respectively. The latter reaction involves coupling between N3-Lys(Fmoc) and 3ethynylpyridine. Both N3-Lys(Fmoc) and the new pyLys were introduced into the ruthenium bipyridine complex I, or cis-[RuII(NO)(NO2)(bpy)2]2+, to generate the common nitro-based complex III, which was further converted to the final complex IV. Results of the MTT assay proved the cytotoxic effect of cis- [RuII(NO)(pyLysO-)(bpy)2](PF6)2 against the mammalian breast cancer cells MCF-7 and MDA-MB231. CONCLUSION: The viability assays revealed that complex IV, bearing a NO group and a modified lysine residue, was able to release NO and cross tumor cell membranes. In this work, Complex IV was observed to be the most active ruthenium bipyridine complex against the mammalian breast cancer cells MCF-7 and MDA-MB231: it was approximately twice as active as cisplatin, whilst complexes I-III proved to be less cytotoxic than complex IV. Additional tests using healthy MCF 10A cells showed that complexes II-IV were three- to sixfold less toxic than cisplatin, which suggested that complex IV was selective against cancer cells.
Assuntos
2,2'-Dipiridil/farmacologia , Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Complexos de Coordenação/farmacologia , Óxido Nítrico/farmacologia , Rutênio/farmacologia , 2,2'-Dipiridil/química , Aminoácidos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Teoria da Densidade Funcional , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Ligantes , Estrutura Molecular , Óxido Nítrico/química , Rutênio/química , Relação Estrutura-AtividadeRESUMO
Ideal drugs to cure cancer leave normal cells unharmed while selectively turning tumor cells unviable. Several copper complexes have been able to selectively slow down tumor proliferation. We hypothesized that Cu(smz)2(bipy)·H2O (1)-a copper-complex that has two ligands capable of interacting with DNA-would outperform Cu(smz)2(OH2)·2H2O (2), and also that supporting 1 on mesoporous silica spheres would decrease even further tumor cell viability in vitro. After exposing osteosarcoma cells (MG-63) and normal phenotype cells of bone origin (MC3T3-E1) to either complex, we studied their toxic effect and mechanisms of action. We determined cell viability (MTT assay) and quantified formation of reactive oxygen species (oxidation of DHR-123 to rhodamine). Moreover, we assessed genotoxicity from (i) formation of micronucleus (MN assay) and (ii) damage of DNA (Comet assay). After the exposure of 1 supported on silica spheres, we tested cell viability. Our results confirm our hypotheses: inhibition of tumor cells follows: supported 1 > dissolved 1 > 2. Future work that enhances the load of the complex exclusively in mesopores may improve the ability of 1 to further inhibit tumor cell viability.
Assuntos
2,2'-Dipiridil/farmacologia , Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Cobre/farmacologia , Microesferas , Osteossarcoma/genética , Osteossarcoma/patologia , Sulfametazina/farmacologia , 2,2'-Dipiridil/química , Células 3T3 , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Estrutura Molecular , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Tamanho da Partícula , Porosidade , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/química , Relação Estrutura-Atividade , Sulfametazina/química , Propriedades de SuperfícieRESUMO
Appropriate treatment of pain requires analgesics and anti-inflammatory drugs generally associated with undesirable side effects and not fully effective in a significant proportion of patients. Organoselenium compounds elicit a plenty of pharmacological effects in different animal models. Among these compounds, the 2,2`-dipyridyl diselenide (DPD) has a potent antioxidant effect and low toxicity. In this way, the aim of this study was to investigate the possible DPD antinociceptive effect and its mechanism of action, as well as the safety of the compound. Female Swiss mice were treated with vehicle or DPD (0.01-50â¯mg/kg) intragastrically. Dose-response curve and time-course of the antinociceptive effect of DPD were performed on formalin and tail immersion tests. Morphine (2.5â¯mg/kg, subcutaneous, 15â¯min earlier) was used as a positive control in behavioral tests. The results showed that DPD presents a rapid antinociceptive effect in low doses, without changing the spontaneous locomotor activity and parameters of toxicity in mice. The DPD antinociceptive effect was also confirmed in male Swiss mice in both formalin and tail immersion tests. In addition, DPD reduced the paw edema induced by 2.5% formalin and ear edema induced by 2.5% croton oil. l-arginine (600â¯mg/kg, intraperitoneally) reduced the DPD antinociceptive effect in the first phase of the formalin test. Moreover, DPD attenuated the increase in iNOS, NF-κB and JNK phosphorylation in the spinal cord of mice injected with formalin. These results showed that DPD exerts peripheral and central nociceptive actions associated with anti-inflammatory effect and this organoselenium compound could be an interesting alternative therapy for pain treatment.
Assuntos
2,2'-Dipiridil/farmacologia , Analgésicos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Compostos Organosselênicos/farmacologia , Medula Espinal/efeitos dos fármacos , 2,2'-Dipiridil/administração & dosagem , 2,2'-Dipiridil/química , Analgésicos/administração & dosagem , Analgésicos/química , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/química , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/tratamento farmacológico , Feminino , Formaldeído , Masculino , Camundongos , Compostos Organosselênicos/administração & dosagem , Compostos Organosselênicos/química , Fosforilação/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Breast cancer cells may exhibit changes in iron homeostasis, which results in increased labile iron pool (LIP) levels. Several studies highlight the crucial role of high LIP levels in the maintenance of tumor cell physiology. Iron chelators have been tested in anticancer therapy in combination with chemotherapeutic agents, to improve drug efficacy. Thus, the aim of this study was to evaluate the effect of 2,2'-dipyridyl (DIP), a Fe2+ chelator, in combination with doxorubicin (DOX) in breast tumor cells. The maximum concentration of DIP that did not significantly reduce the viability of MDA-MB-231 cells was 10µM and for MCF-7 cells was 50µM. We observed that MCF-7 had higher LIP levels than MDA-MB-231 cells. DIP alone increased ROS generation in MCF-7 cells, and DIP pretreatment reduced ROS generation induced by DOX treatment. In conclusion, the increase in MCF-7 cell viability induced by DIP pretreatment in DOX-treated cells seems to be related to an increase in the cellular antioxidant capacity and the iron chelator did not improve drug efficacy in the two breast tumor cell lines analyzed.
Assuntos
2,2'-Dipiridil/farmacologia , Antibióticos Antineoplásicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/toxicidade , Quelantes de Ferro/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , NADPH Oxidases/biossíntese , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismoRESUMO
Dopamine release and phase-amplitude cross-frequency coupling (CFC) have independently been implicated in prefrontal cortex (PFC) functioning. To causally investigate whether dopamine release affects phase-amplitude comodulation between different frequencies in local field potentials (LFP) recorded from the medial PFC (mPFC) of behaving rats, we used RuBiDopa, a light-sensitive caged compound that releases the neurotransmitter dopamine when irradiated with visible light. LFP power did not change in any frequency band after the application of light-uncaged dopamine, but significantly strengthened phase-amplitude comodulation between delta and gamma oscillations. Saline did not exert significant changes, while injections of dopamine and RuBiDopa produced a slow increase in comodulation for several minutes after the injection. The results show that dopamine release in the medial PFC shifts phase-amplitude comodulation from theta-gamma to delta-gamma. Although being preliminary results due to the limitation of the low number of animals present in this study, our findings suggest that dopamine-mediated modification of the frequencies involved in comodulation could be a mechanism by which this neurotransmitter regulates functioning in mPFC.
Assuntos
Ritmo Delta/efeitos dos fármacos , Dopamina/farmacologia , Ritmo Gama/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Vigília , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Animais , Ritmo Delta/fisiologia , Dopamina/metabolismo , Eletroencefalografia , Ritmo Gama/fisiologia , Masculino , Compostos Organometálicos/farmacologia , Estimulação Luminosa/métodos , Ratos , Ratos Wistar , Sono REM/efeitos dos fármacos , Sono REM/fisiologia , Análise Espectral , Fatores de Tempo , Raios UltravioletaRESUMO
Singlet molecular oxygen ((1)O2) contributes to protein damage triggering biophysical and biochemical changes that can be related with aging and oxidative stress. Serum albumins, such as bovine serum albumin (BSA), are abundant proteins in blood plasma with different biological functions. This paper presents a kinetic and spectroscopic study of the (1)O2-mediated oxidation of BSA using the tris(2,2'-bipyridine)ruthenium(II) cation [Ru(bpy)3](2+) as sensitizer. BSA quenches efficiently (1)O2 with a total (chemical+physical interaction) rate constant kt(BSA)=7.3(±0.4)×10(8)M(-1)s(-1), where the chemical pathway represented 37% of the interaction. This efficient quenching by BSA indicates the participation of several reactive residues. MALDI-TOF MS analysis of intact BSA confirmed that after oxidation by (1)O2, the mass protein increased the equivalent of 13 oxygen atoms. Time-resolved emission spectra analysis of BSA established that Trp residues were oxidized to N'-formylkynurenine, being the solvent-accessible W134 preferentially oxidized by (1)O2 as compared with the buried W213. MS confirmed oxidation of at least two Tyr residues to form dihydroxyphenylalanine, with a global reactivity towards (1)O2 six-times lower than for Trp residues. Despite the lack of MS evidences, kinetic and chemical analysis also suggested that residues other than Trp and Tyr, e.g. Met, must react with (1)O2. Modeling of the 3D-structure of BSA indicated that the oxidation pattern involves a random distribution of (1)O2 into BSA; allowing also the interaction of (1)O2 with buried residues by its diffusion from the bulk solvent through interconnected internal hydrophilic and hydrophobic grooves.
Assuntos
Envelhecimento/metabolismo , Estresse Oxidativo , Soroalbumina Bovina/química , Oxigênio Singlete/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Envelhecimento/patologia , Complexos de Coordenação , Interações Hidrofóbicas e Hidrofílicas , Cinética , Oxirredução , Ligação Proteica , Soroalbumina Bovina/metabolismo , Oxigênio Singlete/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptofano/química , Triptofano/metabolismoRESUMO
Neuronal death in Parkinson's disease (PD) is often preceded by axodendritic tree retraction and loss of neuronal functionality. The presence of non-functional but live neurons opens therapeutic possibilities to recover functionality before clinical symptoms develop. Considering that iron accumulation and oxidative damage are conditions commonly found in PD, we tested the possible neuritogenic effects of iron chelators and antioxidant agents. We used three commercial chelators: DFO, deferiprone and 2.2'-dypyridyl, and three 8-hydroxyquinoline-based iron chelators: M30, 7MH and 7DH, and we evaluated their effects in vitro using a mesencephalic cell culture treated with the Parkinsonian toxin MPP+ and in vivo using the MPTP mouse model. All chelators tested promoted the emergence of new tyrosine hydroxylase (TH)-positive processes, increased axodendritic tree length and protected cells against lipoperoxidation. Chelator treatment resulted in the generation of processes containing the presynaptic marker synaptophysin. The antioxidants N-acetylcysteine and dymetylthiourea also enhanced axodendritic tree recovery in vitro, an indication that reducing oxidative tone fosters neuritogenesis in MPP+-damaged neurons. Oral administration to mice of the M30 chelator for 14 days after MPTP treatment resulted in increased TH- and GIRK2-positive nigra cells and nigrostriatal fibers. Our results support a role for oral iron chelators as good candidates for the early treatment of PD, at stages of the disease where there is axodendritic tree retraction without neuronal death.
Assuntos
Antioxidantes/farmacologia , Quelantes de Ferro/farmacologia , Intoxicação por MPTP/tratamento farmacológico , Fibras Nervosas/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/antagonistas & inibidores , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 2,2'-Dipiridil/farmacologia , Animais , Deferiprona , Desferroxamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/biossíntese , Hidroxiquinolinas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Neuritos/metabolismo , Neuritos/patologia , Cultura Primária de Células , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley , Sinaptofisina/agonistas , Sinaptofisina/biossíntese , Tirosina 3-Mono-Oxigenase/biossínteseRESUMO
There is an increasing number of compounds developed to target one or more pathways involved in vasodilation. Some studies conducted with azaindole and indazole derivatives showed cardiovascular activity associated with these compounds. Fast and easy structural modification of these organic molecules can be achieved using metal complexes promoting a much larger spatial change than organic strategies, potentially leading to novel drugs. Here, we have prepared a series of complexes with a formula cis-[RuCl(L)(bpy)(2)]PF(6), where L = 7-azaindole (ain), 5-azaindole (5-ain), 4-azaindole (4-ain), indazole (indz), benzimidazole (bzim) or quinoline (qui), which were characterized by spectroscopic and electrochemical techniques (CV, DPV). These compounds showed reasonable stability exhibiting photoreactivity only at low wavelength along with superoxide scavenger activity. Cytotoxicity assays indicated their low activity preliminarily supporting in vivo application. Interestingly, vasodilation assays conducted in rat aorta exhibited great activity that largely improved compared to free ligands and even better than the well-studied organic compound (BAY 41-42272), with IC(50) reaching 55 nM. These results have validated this strategy opening new opportunities to further develop cardiovascular agents based on metallo-bicyclic rings.
Assuntos
2,2'-Dipiridil/análogos & derivados , Benzimidazóis/química , Indazóis/química , Indóis/química , Compostos Organometálicos/química , Quinolinas/química , Rutênio/química , Vasodilatadores/química , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Compostos Aza/química , Compostos Aza/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular , Humanos , Indazóis/farmacologia , Indóis/farmacologia , Compostos Organometálicos/farmacologia , Quinolinas/farmacologia , Ratos , Rutênio/farmacologia , Superóxidos/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Cancer is the second leading cause of death worldwide and there is epidemiological evidence that demonstrates this tendency is emerging. Naringenin (NGEN) is a trihydroxyflavanone that shows various biological effects such as antioxidant, anticancer, anti-inflammatory, and antiviral activities. It belongs to flavanone class, which represents flavonoids with a C6-C3-C6 skeleton. Flavonoids do not exhibit sufficient activity to be used for chemotherapy, however they can be chemically modified by complexation with metals such as copper (Cu) (II) for instance, in order to be applied for adjuvant therapy. This study investigated the effects of Cu(II) and 2,2'-bipyridine complexation with naringenin on MDA-MB-231 cells. We demonstrated that naringenin complexed with Cu(II) and 2,2'-bipyridine (NGENCuB) was more efficient inhibiting colony formation, proliferation and migration of MDA-MB-231 tumor cells, than naringenin (NGEN) itself. Furthermore, we verified that NGENCuB was more effective than NGEN inhibiting pro-MMP9 activity by zymography assays. Finally, through flow cytometry, we showed that NGENCuB is more efficient than NGEN inducing apoptosis in MDA-MB-231 cells. These results were confirmed by gene expression analysis in real time PCR. We observed that NGENCuB upregulated the expression of pro-apoptotic gene caspase-9, but did not change the expression of caspase-8 or anti-apoptotic gene Bcl-2. There are only few works investigating the effects of Cu(II) complexation with naringenin on tumor cells. To the best of our knowledge, this is the first work describing the effects of Cu(II) complexation of a flavonoid on MDA-MB-231 breast tumor cells.
Assuntos
2,2'-Dipiridil/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/prevenção & controle , Complexos de Coordenação/farmacologia , Cobre/farmacologia , Flavanonas/farmacologia , 2,2'-Dipiridil/química , 2,2'-Dipiridil/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimioprevenção , Complexos de Coordenação/uso terapêutico , Cobre/química , Cobre/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Flavanonas/uso terapêutico , HumanosRESUMO
The water-soluble and visible luminescent complexes cis-[Ru(L-L)2(L)2](2+) where L-L = 2,2-bipyridine and 1,10-phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of cis-[Ru(phen)2(ImH)2](2+) and human serum albumin occurs via noncovalent interactions with K(b) = 9.8 × 10(4) mol(-1) L, ΔH = -11.5 ± 0.1 kcal mol(-1), and TΔS = -4.46 ± 0.3 kcal mol(-1). High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of cis-[Ru(phen)2(ImH)2](2+) against proliferation of HCT116p53(+/+) and HCT116p53(-/-) shows IC50 values of 0.1 and 0.7 µmol L(-1). Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53(+/+). The complex activates proapoptotic PARP in p53(-/-), but not in p53(+/+). A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested.
Assuntos
Antineoplásicos/síntese química , Complexos de Coordenação/síntese química , Substâncias Luminescentes/síntese química , Rutênio , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Histamina/análogos & derivados , Histamina/síntese química , Histamina/farmacologia , Humanos , Imidazóis/síntese química , Imidazóis/farmacologia , Substâncias Luminescentes/farmacologia , Fenantrolinas/síntese química , Fenantrolinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Albumina Sérica/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismoRESUMO
Alkylating agents are used in anti-tumor chemotherapy because they bind covalently to DNA and generate adducts that may lead to cell death. Bifunctional (HN2) and monofunctional (HN1) nitrogen are two such agents, and HN2 was the first drug successfully employed in anti-leukemia chemotherapy. Currently, HN2 is used either alone or combined with other drugs to treat Hodgkin's disease. It is well known that several crosslinking agents require metabolic activation via reactive oxygen species (ROS) to exert their lethal effects. The objective of this work was therefore to determine whether the abovementioned mustards would also require metabolic activation to exert lethal action against Escherichia coli. For this purpose, we measured survival following exposure to HN2 in E. coli strains that were deficient in nucleotide excision repair (uvrA NER mutant), base excision repair (xthA nfo nth fpg BER mutant) or superoxide dismutase (sodAB mutant) activity. We also performed the same experiments in cells pretreated with an iron chelator (2,2'-dipyridyl, DIP). The NER and BER mutants were only sensitive to HN2 treatment (survival rates similar to those of the wild-type were achieved with 5-fold lower HN2 doses). However, wild-type and sodAB strains were not sensitive to treatment with HN2. In all tested strains, survival dropped by 2.5-fold following pretreatment with DIP compared to treatment with HN2 alone. Furthermore, DIP treatment increased ROS generation in both wild type and sodAB-deficient strains. Based on these data and on the survival of the SOD-deficient strain, we suggest that the increased production of ROS caused by Fe(2+) chelation may potentiate the lethal effects of HN2 but not HN1. This potentiation may arise as a consequence of enhancement in the number of or modification of the type of lesions formed. No sensitization was observed for the non-crosslinkable HN2 analog, HN1.
Assuntos
2,2'-Dipiridil/farmacologia , Antineoplásicos Alquilantes/farmacologia , Quelantes/farmacologia , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Mecloretamina/farmacologia , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Mutação , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Iron is an essential element for the survival of microorganisms in vitro and in vivo, acting as a cofactor of several enzymes and playing a critical role in host-parasite relationships. Leishmania (Viannia) braziliensis is a parasite that is widespread in the new world and considered the major etiological agent of American tegumentary leishmaniasis. Although iron depletion leads to promastigote and amastigote growth inhibition, little is known about the role of iron in the biology of Leishmania. Furthermore, there are no reports regarding the importance of iron for L. (V.) braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the effect of iron on the growth, ultrastructure and protein expression of L. (V.) braziliensis was analyzed by the use of the chelator 2,2-dipyridyl. Treatment with 2,2-dipyridyl affected parasites' growth in a dose- and time-dependent manner. Multiplication of the parasites was recovered after reinoculation in fresh culture medium. Ultrastructural analysis of treated promastigotes revealed marked mitochondrial swelling with loss of cristae and matrix and the presence of concentric membranar structures inside the organelle. Iron depletion also induced Golgi disruption and intense cytoplasmic vacuolization. Fluorescence-activated cell sorting analysis of tetramethylrhodamine ester-stained parasites showed that 2,2-dipyridyl collapsed the mitochondrial membrane potential. The incubation of parasites with propidium iodide demonstrated that disruption of mitochondrial membrane potential was not associated with plasma membrane permeabilization. TUNEL assays indicated no DNA fragmentation in chelator-treated promastigotes. In addition, two-dimensional electrophoresis showed that treatment with the iron chelator induced up- or down-regulation of proteins involved in metabolism of nucleic acids and coordination of post-translational modifications, without altering their mRNA levels. CONCLUSIONS: Iron chelation leads to a multifactorial response that results in cellular collapse, starting with the interruption of cell proliferation and culminating in marked mitochondrial impairment in some parasites and their subsequent cell death, whereas others may survive and resume proliferating.
Assuntos
2,2'-Dipiridil/farmacologia , Quelantes/farmacologia , Ferro/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/crescimento & desenvolvimento , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Morte Celular , Membrana Celular/fisiologia , Permeabilidade da Membrana Celular , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , Fragmentação do DNA , Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Humanos , Marcação In Situ das Extremidades Cortadas , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas de Protozoários/biossínteseRESUMO
Stenotrophomonas maltophilia is an emerging nosocomial pathogen. Despite the broad spectrum of syndromes associated with S. maltophilia infections, little is known about its virulence factors, including siderophore production. The aims of this work were to detect S. maltophilia siderophores and to determine their chemical nature. We studied 31 S. maltophilia isolates from device-associated infections, recovered over the period 2006-2011 at Hospital de Clínicas José de San Martín, Buenos Aires, Argentina, and the strain K279a, whose genome has been fully sequenced. The production of siderophores was screened by the chrome azurol S (CAS) agar assay, previously modified to detect siderophores in this species. When grown on modified CAS agar plates, all the clinical isolates and K279a were CAS-positive for siderophore production. In order to determine the chemical nature of siderophores, the Csáky (hydroxamate-type) and Arnow (catechol-type) assays were used. All S. maltophilia isolates produced catechol-type siderophores, but hydroxamate-type siderophores were not detected.
Assuntos
Catecóis/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Sideróforos/isolamento & purificação , Stenotrophomonas maltophilia/química , 2,2'-Dipiridil/farmacologia , Argentina/epidemiologia , Bacteriemia/microbiologia , Técnicas Bacteriológicas , Líquido da Lavagem Broncoalveolar/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Colorimetria , Corantes , Doenças Transmissíveis Emergentes/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Infecções por Bactérias Gram-Negativas/epidemiologia , Humanos , Indicadores e Reagentes , Ferro/análise , Quelantes de Ferro/farmacologia , Pneumonia Associada à Ventilação Mecânica/microbiologia , Stenotrophomonas maltophilia/isolamento & purificação , Traqueia/microbiologia , Cateterismo Urinário/efeitos adversosRESUMO
The aim of this study was to investigate the in vitro antioxidant activity of 2,2'-dipyridyl diselenide (e) by comparing this effect with m-trifluoromethyl-diphenyl diselenide (a), p-fluor-diphenyl diselenide (b), p-chloro-diphenyl diselenide (c), and p-methoxyl-diphenyl diselenide (d) in rat liver homogenate. We also investigated if the mechanisms involved in the antioxidant property of 2,2'-dipyridyl diselenide are the same that of other diselenides. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC) levels were determined in rat liver homogenate, as indicators of antioxidant activity. Dehydroascorbate (DHA) reductase- and glutathione S-transferase (GST)-like activities, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities and the protection against the oxidation of Fe(2+) were determined to better understand the antioxidant property of compounds. δ-Aminolevulinic dehydratase (δ-ALA-D) activity was also carried out in rat liver homogenates, as a toxicological parameter. Compound e showed the highest potency in reducing TBARS (order of IC(50) values: e < b ≤ a < d ≤ c) and PC (order of IC(50) values: e < c ≤ b ≤ a < d) levels and lower potency in inhibiting δ-ALA-D activity than other diselenides. Compound e at all concentrations tested had no enzyme-mimetic property, but had radical-scavenging activity (≥5 µM) and protected against the oxidation of Fe(2+) (50 µM); while compounds a-d showed GST and DHA-mimetic activities and protected against the oxidation of Fe(2+), but had not radical-scavenging activities. This study indicates that (i) 2,2'-dipyridyl diselenide (e) had better in vitro antioxidant effect than other diselenides and lower inhibitory effect on δ-ALA-D activity, (ii) the presence of pyridine ring is responsible for the best antioxidant effect of this compound, and (iii) 2,2'-dipyridyl diselenide acts by different mechanisms of other diselenides.
Assuntos
2,2'-Dipiridil/análogos & derivados , Antioxidantes/farmacologia , Compostos Organosselênicos/farmacologia , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Animais , Antioxidantes/química , Ácido Ascórbico/química , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Ácido Desidroascórbico/química , Compostos Ferrosos/química , Glutationa/química , Glutationa Transferase/química , Peroxidação de Lipídeos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Compostos Organosselênicos/química , Compostos de Organossilício/química , Compostos de Organossilício/farmacologia , Oxirredução , Oxirredutases/química , Sintase do Porfobilinogênio/antagonistas & inibidores , Sintase do Porfobilinogênio/metabolismo , Carbonilação Proteica , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Extratos de TecidosRESUMO
Iron is an essential nutrient for sustaining bacterial growth; however, little is known about the molecular mechanisms that govern gene expression during the homeostatic response to iron availability. In this study we analyzed the global transcriptional response of Enterococcus faecalis to a non-toxic iron excess in order to identify the set of genes that respond to an increment of intracellular iron. Our results showed an up-regulation of transcriptional regulators of the Fur family (PerR and ZurR), the cation efflux family (CzcD) and ferredoxin, while proton-dependent Mn/Fe (MntH) transporters and the universal stress protein (UspA) were down-regulated. This indicated that E. faecalis was able to activate a transcriptional response while growing in the presence of an excess of non-toxic iron, assuring the maintenance of iron homeostasis. Gene expression analysis of E. faecalis treated with H(2)O(2) indicated that a fraction of the transcriptional changes induced by iron appears to be mediated by oxidative stress. A comparison of our transcriptomic data with a recently reported set of differentially expressed genes in E. faecalis grown in blood, revealed an important fraction of common genes. In particular, genes associated to oxidative stress were up-regulated in both conditions, while genes encoding the iron uptake system (feo and ycl operons) were up-regulated when cells were grown in blood. This suggested that blood cultures mimic an iron deficit, and was corroborated by measuring feo and ycl expression in E. faecalis treated with the iron chelating agent 2,2-dipyridil. In summary, our group identified an adaptive transcriptional mechanism in response to metal ion stress in E. faecalis, providing a foundation for future in-depth functional studies of the iron-activated regulatory network.
Assuntos
Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Ferro/farmacologia , 2,2'-Dipiridil/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Testes de Sensibilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da PolimeraseRESUMO
Nitric oxide (NO)-donors are pharmacologically active substances that in vivo or in vitro release NO. Their most common side effect is headache caused by cerebral vasodilatation. We previously demonstrated that the new NO-donor Ru(terpy)(bdq)NO](3+) (Terpy), synthesized in our laboratory, induces relaxation of rat aorta. This study aimed to verify the effect of Terpy and sodium nitroprusside (SNP) in basilar artery. We conducted vascular reactivity experiments on endothelium-denuded basilar rings. The concentrations of iron (Fe) and ruthenium (Ru) complex were analyzed in basilar artery lysates after incubation with NO donors by mass spectrometry. We also evaluated the NO released from SNP and Terpy by using confocal microscopy. Interestingly, Terpy did not induce relaxation of the basilar artery. SNP induced relaxation in a concentration-dependent way. NO donors cross the membrane of vascular smooth muscle and entered the cell. In spite of its permeability, Terpy did not release NO in the basilar artery. Otherwise, SNP released NO in the basilar artery cells cytoplasm. Taken together, our results demonstrate that the new NO donor (Terpy) failed to release NO and to induce relaxation in the basilar artery. The NO donor SNP induces vascular relaxation due to NO release in the vascular smooth muscle cells.
Assuntos
2,2'-Dipiridil/farmacologia , Artéria Basilar/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Vasodilatação/efeitos dos fármacos , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Animais , Artéria Basilar/fisiologia , Complexos de Coordenação/química , Endotélio Vascular/efeitos dos fármacos , Masculino , Microscopia Confocal , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/química , Ratos , Ratos Wistar , Rutênio/químicaRESUMO
AIMS: The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases. METHODS AND RESULTS: The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea. CONCLUSIONS: Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.
Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Ferro/metabolismo , 2,2'-Dipiridil/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Linhagem Celular , Quelantes/farmacologia , Humanos , Ferro/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacosRESUMO
Pretreatment of Escherichia coli cultures with the iron chelator 2,2'-dipyridyl (1 mM) protects against the lethal effects of low concentrations of hydrogen peroxide (<15 mM). However, at H(2)O(2) concentrations equal to or greater than 15 mM, dipyridyl pretreatment increases lethality and mutagenesis, which is attributed to the formation of different types of DNA lesions. We show here that pretreatment with dipyridyl (1 mM) prior to challenge with high H(2)O(2) concentrations (>or=15 mM) induced mainly G:C-->A:T transitions (more than 100X with 15 mM and more than 250X with 20 mM over the spontaneous mutagenesis rate) in E. coli. In contrast, high H(2)O(2) concentrations in the absence of dipyridyl preferentially induced A:T-->T:A transversions (more than 1800X and more than 300X over spontaneous mutagenesis for 15 and 20 mM, respectively). We also show that in the fpg nth double mutant, the rpoB gene mutation (RifS-RifR) induced by 20 mM H(2)O(2) alone (20X higher) was increased in 20 mM H(2)O(2) and dipyridyl-treated cultures (110X higher), suggesting additional and/or different lesions in cells treated with H(2)O(2) under iron deprivation. It is suggested that, upon iron deprivation, cytosine may be the main damaged base and the origin of the pre-mutagenic lesions induced by H(2)O(2).
Assuntos
2,2'-Dipiridil/farmacologia , Quelantes/farmacologia , Dano ao DNA , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Citosina , Escherichia coli/genética , Metaloproteínas , Testes de MutagenicidadeRESUMO
Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.
Assuntos
Bacteriocinas/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Ferro/farmacologia , Xylella/genética , 2,2'-Dipiridil/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Quelantes/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulon/genética , Regulon/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xylella/efeitos dos fármacos , Xylella/crescimento & desenvolvimento , Xylella/metabolismoRESUMO
The base excision repair pathway of Saccharomyces cerevisiae possesses three DNA N-glycosylases, viz. Ogg1p, Ngt1p and Ntg2p, involved in the repair of oxidative DNA damage. It was previously reported that inactivation of any of these activities, in most cases, did not generate a sensitive mutant phenotype to a variety of oxidative agents. Only the ntg1 mutant appeared to be more sensitive to hydrogen peroxide (H2O2) than a wild-type (WT) strain. In the present study we evaluated the role of S. cerevisiae OGG1 and NTG2 genes in the repair of oxidative lesions induced by high H2O2 concentrations (5-100 mM for 20 min), followed by catalase treatment (500 IU/ml). In these conditions, the ogg1 mutant was more sensitive than the WT strain to H2O2 (concentration 40-60 mM). Unexpectedly, the inactivation of NTG2 in an ogg1 background was able to suppress both sensitivity and mutagenesis induced by H2O2. Indeed, even the ntg2 single mutant was more resistant than the WT (60-100 mM H2O2). The use of metal ion chelators dipyridyl and neocuproine allowed us to evaluate the participation of iron and copper ions in the production of lethal and mutagenic lesions during H2O2 treatment in different DNA repair-deficient S. cerevisiae strains. The roles of OGG1 and NTG2 genes in the repair of lethal and mutagenic oxidative lesions induced by H2O2 and their relationships with iron and copper ions are discussed.