RESUMO
The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25â µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.
Assuntos
Antibacterianos/farmacologia , Vermelho Neutro/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Azida Sódica/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Vermelho Neutro/análogos & derivados , Vermelho Neutro/química , Processos Fotoquímicos , Fármacos Fotossensibilizantes/química , Azida Sódica/químicaRESUMO
Sunlight is vital for several biochemical processes of the skin organ. However, acute or chronic exposure to ultraviolet radiation (UVR) has several harmful effects on the skin structure and function, especially in the case of the failing function of antioxidative enzymes, which may lead to substantial tissue damage due to the increased presence of reactive oxygen species (ROS). The aim of this work was to investigate the combined effect of ultraviolet B (UVB) irradiation and oxidative stress on the skin barrier integrity. For this, we employed electrical impedance spectroscopy (EIS) to characterize changes of the electrical properties of excised pig skin membranes after various exposure conditions of UVB irradiation, oxidative stress, and the inhibition of antioxidative enzymatic processes. The oxidative stress was regulated by adding hydrogen peroxide (H2O2) as a source of ROS, while sodium azide (NaN3) was used as an inhibitor of the antioxidative enzyme catalase, which is naturally present throughout the epidermis. By screening for the combined effect of UVB and oxidative stress on the skin membrane electrical properties, we developed a new protocol for evaluating these parameters in a simple in vitro setup. Strikingly, the results show that exposure to extreme UVB irradiation does not affect the skin membrane resistance, implying that the skin barrier remains macroscopically intact. Likewise, exposure to only oxidative stress conditions, without UVB irradiation, does not affect the skin membrane resistance. In contrast to these observations, the combination of UVB irradiation and oxidative stress conditions results in a drastic decrease of the skin membrane resistance, indicating that the integrity of the skin barrier is compromised. Further, the skin membrane effective capacitance remained more or less unaffected by UVB exposure, irrespective of simultaneous exposure of oxidative stress. The EIS results were concluded to be associated with clear signs of macroscopic tissue damage of the epidermis as visualized with microscopy after exposure to UVB irradiation under oxidative stress conditions. Finally, the novel methodology was tested by performing an assessment of cosmetic sunscreen formulations with varying sun protection factor (SPF), with an overall successful outcome, showing good correlation between SPF value and protection capacity in terms of skin resistance change. The results from this study allow for the development of new skin sensors based on EIS for the detection of skin tissue damage from exposure to UVB irradiation and oxidative stress and provide a new, more comprehensive methodology, taking into account both the influence of UVB irradiation and oxidative stress, for in vitro determination of SPF in cosmetic formulations.
Assuntos
Espectroscopia Dielétrica/métodos , Estresse Oxidativo , Fator de Proteção Solar , Raios Ultravioleta , Animais , Catalase/antagonistas & inibidores , Catalase/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Pele/metabolismo , Pele/patologia , Pele/efeitos da radiação , Azida Sódica/química , Azida Sódica/metabolismo , Protetores Solares/farmacologia , SuínosRESUMO
The photodynamic mechanism sensitized by N,N-dimethyl-2-[4-(3-N,N,N-trimethylammoniopropoxy)phenyl]fulleropyrrolidinium (DPC602+) was investigated in Staphylococcus aureus cells. Different experimental conditions were used to detect reactive oxygen species (ROS) in S. aureus cell suspensions. First, a photoinactivation of 4 log decrease of S. aureus viability was chosen using 0.5µM DPC602+ and 15min irradiation. An anoxic atmosphere indicated that oxygen was required for an effective photoinactivation. Also, photoprotection was found in the presence of sodium azide, whereas the photocytotoxicity induced by DPC602+ increased in D2O. The addition of diazabicyclo[2.2.2]octane or d-mannitol produced a reduction in the S. aureus photokilling. Moreover, singlet molecular oxygen, O2(1Δg), was detected by the reaction with 9,10-dimethylanthracene into the S. aureus cells. A decrease in the photoinactivation of S. aureus was observed in the presence of ß-nicotinamide adenine dinucleotide reduced form, which was dependent on the NADH concentration. Therefore, under aerobic condition the photocytotoxicity activity induced by DPC602+ was mediated by mainly a contribution of type II process. Moreover, photoinactivation of S. aureus was possible with DPC602+ in the presence of azide anions under anoxic condition. However, these conditions were not effective to photoinactivate Escherichia coli. On the other hand, the addition of potassium iodide produced an increase in the photokilling of bacteria, depending on the KI concentration and irradiation times. The formation of reactive iodine species may be contributing to inactivate S. aureus cells photoinduced by DPC602+.
Assuntos
Escherichia coli/efeitos dos fármacos , Fulerenos/química , Compostos de Amônio Quaternário/química , Espécies Reativas de Oxigênio/química , Staphylococcus aureus/efeitos dos fármacos , Antracenos/química , Cátions Bivalentes/química , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/patogenicidade , Escherichia coli/efeitos da radiação , Fulerenos/farmacologia , Luz , Oxigênio/química , Oxigênio/metabolismo , Iodeto de Potássio/química , Espécies Reativas de Oxigênio/isolamento & purificação , Azida Sódica/química , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/efeitos da radiaçãoRESUMO
Motivated by the photochemical and photophysical properties of curcumin-based composites, the characteristics of a new curcumin-based water-soluble salt were investigated via absorption and fluorescence spectroscopy. Photobleaching was investigated using a set of LEDs in three different wavelengths (405 nm, 450 nm and 470 nm) to illuminate an aqueous solution of curcumin, evaluating its degradation for five different exposure times (0, 5, 15, 45 and 105 minutes). The results were compared with equivalent measurements of dark degradation and illumination in the presence of a singlet-oxygen quencher. Three solution concentrations (50, 100 and 150 µg/ml) were studied. To measure the fluorescence, it was used low power 405 nm excitation laser source. Time dependent photodegradation of curcumin was observed, as compared to the natural degradation of samples maintained on a dark environment. Two main absorption peaks were detected and their relation responded to both concentration and wavelength of the illumination source. A spectral correlation between absorption of curcumin and the emission bands of the sources showed an optimal spectral overlap for the 450 nm LED. For this source, photobleaching showed a less intense degradation on the presence of singlet oxygen quencher. This last result confirmed singlet oxygen production in vitro, indicating a strong potential of this composite to be used as a blue-light-activated photosensitizer.
Assuntos
Curcumina/química , Fotodegradação , Água/química , Reprodutibilidade dos Testes , Oxigênio Singlete/química , Azida Sódica/química , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Rhenium(V) complexes containing tridentate thiosemicarbazones/thiosemicarbazides (H2L1) derived from N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chlorides with 4,4-dialkylthiosemicarbazides have been synthesized by ligand-exchange reactions starting from [ReOCl(L1)]. The chlorido ligand of [ReOCl(L1)] (4) is readily replaced and reactions with ammonium thiocyanate or potassium cyanide give [ReO(NCS)(L1)] (6) and [ReO(CN)(L1)] (7), respectively. The reaction of (NBu4)[ReOCl4] with H2L1 and two equivalents of ammonium thiocyanate, however, gives in a one-pot reaction [ReO(NCS)2(HL1)] (8), in which the pro-ligand H2L1 is only singly deprotonated. An oxo-bridged, dimeric nitridorhenium(V) compound of the composition [{ReN(HL1)}2O] (11) is obtained from a reaction of (NBu4)[ReOCl4], H2L1 and sodium azide. The six-coordinate complexes [ReO(L1)(Ph2btu)] (12), where HPh2btu is N,N-diphenyl-N'-benzoylthiourea, can be obtained by treatment of [ReOCl(L1)] with HPh2btu in the presence of NEt3. Studies of the antiproliferative effects of the [ReOX(L1)] system (X = Cl−, NCS− or CN−) on breast cancer cells show that the lability of a monodentate ligand seems to play a key role in the cytotoxic activity of the metal complexes, while the substitution of this ligand by the chelating ligand Ph2btu− completely terminates the cytotoxicity.
Assuntos
Complexos de Coordenação/química , Rênio/química , Semicarbazidas/química , Tiossemicarbazonas/química , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/toxicidade , Cristalografia por Raios X , Humanos , Ligantes , Células MCF-7 , Conformação Molecular , Azida Sódica/químicaRESUMO
The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (1O2) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.
Assuntos
Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Espécies Reativas de Oxigênio , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Carotenoides/química , Carotenoides/farmacologia , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli/química , Sequestradores de Radicais Livres/química , Radicais Livres , Oxigênio/química , Azida Sódica/química , Azida Sódica/farmacologia , Transativadores/química , Fatores de Transcrição/química , Raios Ultravioleta , Água/químicaRESUMO
Since fluoride (F-) and aluminum (Al3+) present anticariogenic properties and F- release from ionomeric materials depends on the media used in the evaluation, this study tested the hypothesis whether release of Al3+ also depends on the testing media. The simultaneous release of F- and Al3+ was assessed over 15 days in three media: (i) distilled and deionized water (H2O), (ii) artificial saliva (AS) and (iii) solutions simulating a cariogenic challenge (pH-cycling in demineralizing and remineralizing solutions, De-Re-). Six cylindrical samples of each tested material (Ketac-Fil, Vitremer, Fuji Ortho LC and F 2000) were prepared and suspended individually in 1.0 mL of each solution. All solutions were changed daily. F- and Al3+ were determined by ion-selective electrode and atomic absorption, respectively. ANOVA showed statistical significance for the interaction of material, time, media (p < 0.05), either for F- or Al3+ release. The resin-modified glass ionomer Vitremer released the highest amount of F- and Al3+ in De-Re- solutions compared to the other materials (p < 0.05); differences among the materials in H2O and AS were statistically consistent. The data suggest that the media used to evaluate the simultaneous release of F- and Al3+ should be taken into account when the anticariogenic potential of different dental materials is assessed.
Assuntos
Alumínio/química , Cariostáticos/química , Cimentos Dentários/química , Fluoretos/química , Acetatos/química , Resinas Acrílicas/química , Silicatos de Alumínio/química , Análise de Variância , Soluções Tampão , Fosfatos de Cálcio/química , Compômeros/química , Resinas Compostas/química , Cimentos de Ionômeros de Vidro/química , Humanos , Concentração de Íons de Hidrogênio , Imersão , Maleatos/química , Teste de Materiais , Saliva Artificial/química , Azida Sódica/química , Água/químicaRESUMO
An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
Assuntos
Apirase/metabolismo , Leishmania/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apirase/antagonistas & inibidores , Apirase/isolamento & purificação , Cálcio/química , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Inibidores Enzimáticos/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Leishmania/ultraestrutura , Levamisol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Molibdênio/química , Azida Sódica/química , Especificidade por SubstratoRESUMO
Peroxynitrite, a biological oxidant, can induce lipid peroxidation in biological membranes which leads to the formation of various hydroperoxides. Here, we report the formation of singlet oxygen (1O2) in the reaction of peroxynitrite with tert-butyl hydroperoxide (t-BOOH) used as a model compound for organic hydroperoxides. The formation of singlet oxygen was observed by (i) dimol emission in the red spectral region, (ii) monomol emission in the infrared region at 1270 nm and by (iii) chemical trapping of singlet oxygen with anthracene-9,10-diyldiethyl disulfate (EAS). The emission signal was increased when H2O was replaced by deuterium oxide and was quenched by sodium azide. When singlet oxygen was generated in the reaction of peroxynitrite with t-BOOH, the 1O2 quenching rate constant for sodium azide was estimated from a Stern-Volmer plot as 1.3 x 10(8) M(-1) S(-1) which is in line with known values. The 1O2 generation in the peroxynitrite/t-BOOH reaction was also detected by the formation of the endoperoxide of EAS. These data establish the generation of 1O2 in the reaction of peroxynitrite with t-BOOH and suggest a potential involvement of 1O2 in peroxynitrite-mediated reactions in biological systems.