RESUMO
The first rotameric monoterpenoid indole alkaloids (MIAs), 1a and 1b, and two unusual dimeric MIAs, 2 and 3, with new dimerization patterns, together with their putative biosynthetic intermediates 4-7, were isolated from the roots of Gelsemium elegans. Compounds 2 and 3 represent the first natural aromatic azo- and the first urea-linked dimeric MIAs, respectively. Their structures and absolute configurations were elucidated by means of NMR spectroscopy, single-crystal X-ray diffraction, and electronic circular dichroism data analyses. The interconverting mechanism of rotamers 1a and 1b was studied by density functional theory computation. Compounds 2 and 3 showed moderate cytotoxic activity against MCF-7 and PC-12 cells, respectively. In addition, a plausible biosynthesis pathway for the new alkaloids was proposed on the basis of the coexistence of their biosynthetic precursors.
Assuntos
Medicamentos de Ervas Chinesas/isolamento & purificação , Gelsemium/química , Alcaloides de Triptamina e Secologanina/isolamento & purificação , Animais , Cristalografia por Raios X , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Células PC12 , Raízes de Plantas/química , Ratos , Alcaloides de Triptamina e Secologanina/química , Alcaloides de Triptamina e Secologanina/farmacologia , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinéticaRESUMO
Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP.
Assuntos
Apoptose , Proliferação de Células , Doença Enxerto-Hospedeiro/sangue , Doença Enxerto-Hospedeiro/terapia , Ativação Linfocitária , Fotoferese/métodos , Sais de Tetrazólio/farmacocinética , Feminino , Humanos , Linfoma de Células T/sangue , Linfoma de Células T/terapia , MasculinoRESUMO
BACKGROUND: Preexposure of brain to isoflurane, a commonly used anesthetic, induces ischemic tolerance. This phenomenon is called isoflurane preconditioning. However, it is not known whether isoflurane application after ischemia provides neuroprotection. METHODS: Corticostriatal slices (400 microm) freshly prepared from adult male Sprague-Dawley rats were subjected to a 15-min oxygen-glucose deprivation (OGD; to simulate ischemia in vitro). Isoflurane was applied after OGD. Brain slices were harvested 2 h after OGD for measuring 2,3,5-triphenyltetrazolium chloride (TTC) conversion to quantify cell injury. Adult male Sprague-Dawley rats were also subjected to middle cerebral arterial occlusion for 90 min and then treated with or without 2% isoflurane for 60 min started at the onset of reperfusion. The infarct volumes, neurologic deficit scores, and performance on rotarod were evaluated at 24 h after the onset of reperfusion. RESULTS: Isoflurane applied immediately after the 15-min OGD for 30 min dose-dependently reversed the OGD-induced decrease of TTC conversion. The TTC conversion was 34 +/- 16% and 58 +/- 28% of the control, respectively, for OGD alone and OGD plus 2% isoflurane (P < 0.05, n = 12). Application of 2% isoflurane for 30 min started at 10 min after the OGD also reduced the OGD-decreased TTC conversion. The presence of 0.3 microm glibenclamide, a general adenosine 5'-triphosphate-sensitive potassium channel blocker, or 500 microm 5-hydroxydecanoic acid, a mitochondrial adenosine 5'-triphosphate-sensitive potassium channel blocker, during the application of 2% isoflurane abolished the isoflurane preservation of TTC conversion. Application of isoflurane during reperfusion also improved neurologic outcome after brain ischemia. CONCLUSIONS: The results suggest that isoflurane administrated after OGD or brain ischemia provides neuroprotection. Mitochondrial adenosine 5'-triphosphate-sensitive potassium channels may be involved in this protection.
Assuntos
Anestésicos Inalatórios/farmacologia , Encéfalo/irrigação sanguínea , Hipóxia-Isquemia Encefálica/prevenção & controle , Isoflurano/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Ácidos Decanoicos/farmacologia , Relação Dose-Resposta a Droga , Glibureto/farmacologia , Hidroxiácidos/farmacologia , Hipóxia-Isquemia Encefálica/etiologia , Hipóxia-Isquemia Encefálica/patologia , Infarto da Artéria Cerebral Média/complicações , Masculino , Artéria Cerebral Média/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/prevenção & controle , Sais de Tetrazólio/metabolismo , Sais de Tetrazólio/farmacocinéticaRESUMO
Cryopreservation has the potential to improve availability of donor parts for composite tissue allotransplantation and may reduce their antigenicity. This study investigates whether the component tissues of composite flaps remain viable after cryopreservation. Forty-one epigastric flaps were harvested from Lewis rats. Twenty-one flaps were perfused with DMSO/trehalose, frozen by controlled cooling to -140 degrees C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylin & eosin and factor VIII staining. An epithelial viability index was calculated for 10 fresh and 11 cryopreserved flaps using the MTT assay. In all cryopreserved samples, hematoxylin & eosin, and factor VIII staining revealed a well-preserved cellular architecture, which was indistinguishable from fresh specimens. The viability index for the cryopreserved samples was 10.90 +/- 2.09 compared with 12.15 +/- 1.32 for fresh flaps (P = 0.123). Results suggest that the skin, adipose, and vascular endothelial cells of composite tissue flaps retain their viability after cryopreservation and thawing.
Assuntos
Criopreservação/métodos , Retalhos Cirúrgicos , Animais , Sobrevivência Celular , Criopreservação/instrumentação , Endotélio/metabolismo , Endotélio/transplante , Desenho de Equipamento , Indicadores e Reagentes , Masculino , Ratos , Ratos Endogâmicos Lew , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinética , Coleta de Tecidos e Órgãos , Transplante HomólogoRESUMO
In vitro responses of potential target cell types to air pollutants under physiological conditions may be useful in understanding the health effects of air pollution exposure. The study evaluated responses of human primary airway epithelial cells to diesel exhaust (DE). Cultures of cells from 3 donors, differentiated by culture on membranes with the apical surfaces exposed to the atmosphere, were exposed to filtered air or DE. Some exposure-related effects were similar among donors, whereas others were affected by the donor, consistent with human population heterogeneity. This model may be useful for mechanistic and comparative toxicology studies.
Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais/efeitos dos fármacos , Mucosa Respiratória/citologia , Emissões de Veículos/toxicidade , Aerossóis , Ar , Fosfatase Alcalina/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Impedância Elétrica , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Microesferas , Muco/metabolismo , Permeabilidade , Propriedades de Superfície , Sais de Tetrazólio/farmacocinética , ÁguaRESUMO
A widespread, rather general, definition of contrast-induced nephropathy (CIN) is an impairment in renal function occurring within 3 days following the intravascular administration of contrast media (CM) and the absence of an alternative aetiology. In spite of the vast clinical importance of CIN, its understanding and the pathophysiology behind CIN remain incomplete. Many studies have been performed; however, they have provided no widely accepted conclusion so far. Here the possible mechanisms underlying CIN are outlined, which span from altered rheological properties, perturbation of renal haemodynamics, regional hypoxia, auto-, and paracrine factors (adenosine, endothelin, reactive oxygen species) to direct cytotoxic effects. Although these potential mediators of CIN will be discussed separately, several factors may act in concert to perturb kidney function after exposure to contrast media. From the current knowledge of the mechanisms causing CIN, it is not possible to recommend a certain class of contrast media, except to avoid large doses of CM of the first generation. From a pathophysiological perspective, volume expansion is effective in avoiding CIN, since water permeability of the collecting ducts will decrease and enhance fluid excretion. Hence, CM in the distal portions of the tubular system is diluted, which implies reduced fluid viscosity and a lower risk of obstruction.
Assuntos
Meios de Contraste/efeitos adversos , Nefropatias/induzido quimicamente , Túbulos Renais Distais/fisiopatologia , Corantes/farmacocinética , Meios de Contraste/administração & dosagem , Humanos , Túbulos Renais Distais/efeitos dos fármacos , Mitocôndrias/fisiologia , Concentração Osmolar , Espécies Reativas de Oxigênio/metabolismo , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinéticaRESUMO
BACKGROUND AND OBJECTIVES: Dosimetry and treatment frequency are controversial phototherapy issues. Efficacy of dose fractionation on photobiomodulation was evaluated in vitro. STUDY DESIGN/MATERIALS AND METHODS: Human HEP-2 and murine L-929 cell lines were cultured in complete DMEM media. Photoradiation (670 nm, 5 J/cm2/treatment, 50 J/cm2 total energy delivery), was performed varying treatments per 24 hour period: Group I (Controls)-0, Group II-1/d, Group III-2/d, Group IV-4/d. Cell proliferation was measured using Cyquant (fluorescent DNA content) and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasolium bromide) assays for 240 hours post therapy. A proliferation index: PI = (#Cells Experimental(t) / #Cells Control(t)) was computed. RESULTS: MTT assay results demonstrated maximal response in Group III (P < 0.05, n = 3). Cyquant maxima occurred in HEP-2 Groups II and III (P < 0.045) and L-929 Group III (P < 0.091). CONCLUSIONS: Cellular response to dose frequency varies. More frequent treatments (2/24 hours) increased metabolism and proliferation in both cell lines. Further investigation of dose fractionation in phototherapy is warranted.
Assuntos
Proliferação de Células/efeitos da radiação , Células do Tecido Conjuntivo/efeitos da radiação , Fracionamento da Dose de Radiação , Células Epiteliais/efeitos da radiação , Fototerapia , Animais , Linhagem Celular , Corantes/farmacocinética , Células do Tecido Conjuntivo/fisiologia , Células Epiteliais/fisiologia , Humanos , Camundongos , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinética , Fatores de TempoRESUMO
MTT is taken up by cells by endocytosis and reduced to formazan in the endosomal/lysosomal compartment. Formazan is deposited intracellularly as blue granules and is later exocytosed as needle-like formazan crystals. The present study involves an analysis of the pattern of exocytosis of MTT in different cell types showing clearcut differences in the response that can be associated to their ability to phagocytose. To further assess the characteristics of the exocytic mechanism of MTT/formazan, different experimental conditions were assayed. When culture medium with decreasing serum concentration was used as a metabolic modulator no variations were observed in the proportion of cells with formazan crystals. Conversely, the markedly sensitivity of phagocytic cells to increasing concentrations of genistein constituted a remarkable difference with non-phagocytic cells. These results must be considered when the modulation of MTT exocytosis is used as a signal of the progress of human diseases.
Assuntos
Exocitose/fisiologia , Formazans/farmacocinética , Macrófagos/citologia , Fagócitos/citologia , Sais de Tetrazólio/farmacocinética , Animais , Neoplasias da Mama , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultura , Endossomos/fisiologia , Endossomos/ultraestrutura , Feminino , Genisteína/farmacologia , Humanos , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos , Fagócitos/fisiologiaRESUMO
Huperzine A (HupA) and donepezil, two novel selective acetylcholinesterase inhibitors available for Alzheimer's disease, were tested for their ability to alleviate injury from oxygen-glucose deprivation (OGD) in the rat pheochromocytoma line PC12 cells. OGD for 30 min triggered death in more than 50% of cells, along with major changes in morphology and biochemistry including elevated levels of lipid peroxide, superoxide disamutase activity and lactate. Cells pretreated for 2 h with HupA or donepezil showed improved survival and reduced biochemical and morphologic signs of toxicity (statistically significant over the range from 10 microM down to 1.0 and 0.1 microM, respectively). Our results indicated that HupA and donepezil protected PC12 cells against OGD-induced toxicity, most likely by alleviating disturbances of oxidative and energy metabolism.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/farmacologia , Glucose/deficiência , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Indanos/farmacologia , Fármacos Neuroprotetores/farmacologia , Piperidinas/farmacologia , Sesquiterpenos/farmacologia , Alcaloides , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Donepezila , Relação Dose-Resposta a Droga , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Células PC12/citologia , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , Ratos , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinéticaRESUMO
Mutations in the beta-amyloid precursor protein (APP) gene cause familial Alzheimer's disease (AD). Although amyloid beta peptide (Abeta) is the principal constituent of senile plaques in AD, other cleavage products of APP are also implicated in playing a role in the pathogenesis of AD. C-terminal fragments of APP (APP-CTs), that contain complete Abeta sequence, are found in neuritic plaques, neurofibrillary tangles and the cytosol of lymphoblastoid cells obtained from AD patients. Our previous report demonstrated that APP-CT105 causes death of differentiated PC12 cells and cultured rat cortical neurons (Kim and Suh [1996] J. Neurochem. 67:1172-1182) and induces strong inward currents in Xenopus oocyte (Fraser et al., [1996] J. Neurochem. 66:2034-2040). In the present study, to investigate which domain of APP-CT105 is responsible for the neurotoxicity, we have made deletion mutants of APP-CT105 without Abeta and transmembrane domain (TM) or without NPTY domain, a putative endocytosis signaling sequence, using the PCR-amplified strategy and the recombinant GST-fusion protein strategy. The effect on cell survival of the deletion mutants of APP-CT105 (8 microM) was then determined by the LDH and MTT assay. We found that C-terminal fragment without NPTY significantly causes cell death in NGF-differentiated PC12 cells and cultured rat cortical neurons. This finding suggests that NPTY may not play an important role in APP-CT105 mediated neurotoxicity. We found, however, that C-terminal fragment without Abeta and TM significantly induces neuronal cell death. Our results suggest that in addition to Abeta, C-terminal fragment of APP without Abeta and TM domain itself may also participate in the neuronal degeneration in AD.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/toxicidade , Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Estrutura Terciária de Proteína/genética , Precursor de Proteína beta-Amiloide/isolamento & purificação , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Corantes/farmacocinética , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/enzimologia , Células PC12 , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinéticaRESUMO
We investigated the function of estrogen receptor-alpha in global myocardial ischemia and reperfusion injury in male estrogen receptor-alpha knockout (ERKO) and wild-type mice. Mouse hearts were subjected to 45 min of global ischemia followed by 180 min of reperfusion. The hearts were excised, cannulated, and maintained in a chilled (4 degrees C) cardioplegia solution until warm (37 degrees C) oxygenated Krebs-Henseleit bicarbonate buffer was perfused through the coronary arteries. ERKO hearts started beating later and had a higher incidence of ventricular fibrillation and/or tachycardia than control hearts. Coronary flow rate was significantly lower in ERKO hearts during the 90- and 120-min periods of reperfusion. Ca(2+) accumulation was significantly greater following 30, 90, 120, 150, and 180 min of reperfusion in ERKO hearts. Nitrite production was significantly less in ERKO hearts following 90, 120, and 150 min of reperfusion. Myocardial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was significantly lower in experimental ERKO hearts. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of ischemia-reperfused ERKO hearts but not in control tissues. Hematoxylin-basic fuchsin-picric acid-stained sections from experimental ERKO hearts had fewer viable myocytes compared with controls. Transmission electron microscopy revealed swollen and fragmented mitochondria with amorphous and granular bodies, loss of matrix, and rupture of cristae in experimental ERKO hearts. This is the first demonstration that estrogen receptor-alpha plays a cardioprotective role in ischemia-reperfusion injury in males.
Assuntos
Isquemia Miocárdica/metabolismo , Receptores de Estrogênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Cálcio/metabolismo , Circulação Coronária , Edema Cardíaco/patologia , Receptor alfa de Estrogênio , Frequência Cardíaca/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Miocárdio/metabolismo , Miocárdio/patologia , Nitritos/metabolismo , Receptores de Estrogênio/deficiência , Traumatismo por Reperfusão/patologia , Sais de Tetrazólio/metabolismo , Sais de Tetrazólio/farmacocinética , Tiazóis/metabolismo , Tiazóis/farmacocinéticaRESUMO
Heat-shock proteins (HSPs) are an important family of endogenous protective proteins, which increase in response to myocardial ischemia and other stresses. Overexpression of HSP72 is cardioprotective. We were interested in the regulation of heat-shock factor (HSF), the transcription factor for HSP genes. Previously we have observed that the inflammatory cytokine tumor necrosis factor-alpha increases HSP72 levels and postulated that dexamethasone might effect the heat shock response. In the adult rat cardiac myocyte we found that treatment with either low (10 microM)- or high (100 microM)-dose dexamethasone activated HSF by 2-6 h as determined by gel shift assay without evidence of cytotoxicity. Although HSF activation is a key step in expression of HSP72, this may not result in an increase in HSP72. We found that 10 microM dexamethasone increased HSP72 38%, and 100 microM dexamethasone increased HSP72 62% (P < 0.05). HSP27 and HSP60 were unchanged. The selective increase in HSP72 was associated with protection of the cardiac myocytes from hypoxia and reoxygenation. We conclude that dexamethasone is a novel inducer of the heat shock response.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Proteínas de Choque Térmico/biossíntese , Fibras Musculares Esqueléticas/enzimologia , Animais , Western Blotting , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Chaperonina 60/análise , Chaperonina 60/biossíntese , Corantes/farmacocinética , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico/análise , L-Lactato Desidrogenase/metabolismo , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinéticaRESUMO
Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response. Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells. The cells that have been implicated in TD-induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV-2 microglial cell lines, were cultured in either thiamine-depleted media or in normal culture media with amprolium, a thiamine transport inhibitor. The activity levels of a key mitochondrial enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons. The unique distribution of the activity did not account for the selective response to TD. TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells. Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation product. On the other hand, chronic lipopolysaccharide-induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells. The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.
Assuntos
Encéfalo/metabolismo , Neurônios/fisiologia , Estresse Oxidativo/fisiologia , Aldeídos/metabolismo , Amprólio , Animais , Apoptose , Encéfalo/citologia , Morte Celular/fisiologia , Células Cultivadas , Corantes , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/classificação , Neurônios/enzimologia , Neurônios/metabolismo , Oxirredutases/metabolismo , Sais de Tetrazólio/farmacocinética , Deficiência de Tiamina/induzido quimicamente , Deficiência de Tiamina/enzimologia , Tiazóis/farmacocinéticaRESUMO
The A549 cell line was used to assess the ability of diisopropylglutathione (DIPE) to protect against a 100 microM challenge dose of sulphur mustard (HD) using gentian violet (GV), thiazolyl blue (MTT) and neutral red (NR) assays as indicators of cell culture viability. As part of a continuing study of the efficacy of protective nucleophiles as candidate treatments for HD poisoning, several different combinations of protectant and HD were used to determine the optimal means of protecting A549 cells from the effects of HD. It was found that DIPE (4 mM) could protect cells against the effects of HD though for optimal effect, DIPE had to be present at the time of HD challenge. Cultures protected with DIPE were up to 2.9-fold more viable than HD exposed cells 48 h after HD challenge when using the GV, MTT and NR assays to assess viability. Observations by phase contrast microscopy of GV stained cultures confirmed these findings. Pretreating A549 cultures with DIPE for 1 h followed by its removal prior to HD challenge did maintain cell viability, though at a relatively low level (only up to 1.4-fold more viable than HD only exposed cells). DIPE was also able to protect HD exposed A549 cultures when added to cell cultures at intervals of up to 12 to 15 min after the initial HD exposure, though viability tended to decrease over this period, so that at 1 h, addition of DIPE did not maintain the viability of the cultures. This is the first such report of the anti-HD protectant properties of DIPE in A549 cells. It is concluded that the protection observed against HD is probably largely due to extracellular inactivation of HD by DIPE.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Glutationa/farmacologia , Gás de Mostarda/toxicidade , Oligopeptídeos/farmacologia , Linhagem Celular , Corantes/farmacocinética , Violeta Genciana/farmacocinética , Glutationa/análogos & derivados , Humanos , Microscopia de Contraste de Fase , Vermelho Neutro/farmacocinética , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinética , Fatores de TempoRESUMO
We compared three techniques, the MTT tetrazolium assay, cell counting, and tritiated thymidine ([3H]TdR) incorporation assay to measure the antiproliferative effect of cyclosporin A (CsA) and interferon-gamma (IFN-gamma) on normal human skin keratinocyte cultures (NHK) used at the second passage and human papilomavirus type 16- and 18-transformed cell lines (EK16 and EK18) exposed continuously to the drugs for 3 days. The three techniques showed that under CsA (0.5 and 8 micrograms/ml) and IFN-gamma (5 and 160 U/ml) treatments the cells remained viable and that the growth of keratinocytes was inhibited. For IFN-gamma, the MTT colorimetric assay consistently underestimated its growth inhibitory activity as compared to cell counting or [3H]TdR incorporation, whatever the cells used. For high doses of CsA, MTT and cell counting gave similar percentages, of inhibitory activity whatever the cells; MTT underestimated this activity as compared to [3H]TdR incorporation only in NHK and EK18 cells, whereas similar results were obtained with EK16 cells. In conclusion, this investigations shows that MTT sensitivity differed with the drug and also according to the keratinocyte cultures. The MTT test is clearly not appropriate for study of IFN-gamma treatment whatever the keratinocytes used. Such discrepancies indicate that the MTT test should be done with care on cultures to measure the effects of drugs on cell growth; the growth inhibition should be carefully considered and it would be best if two different methods were used.
Assuntos
Divisão Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Inibidores do Crescimento/farmacologia , Interferon gama/farmacologia , Queratinócitos/efeitos dos fármacos , Adulto , Contagem de Células , Linhagem Celular Transformada , Sobrevivência Celular , Células Cultivadas , Corantes/farmacocinética , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Papillomaviridae , Reprodutibilidade dos Testes , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinética , Timidina/farmacocinética , TrítioRESUMO
RATIONALE AND OBJECTIVES: Radiologic contrast media (CM) are hyperosmotic compounds injected undiluted into a patient's blood, in which they contact endothelial cells. For some types of cultured cells, the application of a hyperosmotic stimulus may cause intracellular pH (pHi) acidification that is related to the extent of hyperosmolality and that ultimately influences cellular function. Accordingly, endothelial and kidney cells, two types of cells known to be exposed to CM effects, were treated at relevant iodine concentrations with various CM (320-1500 mOsm) to determine whether cell exposure to CM can disturb the pH(i) and to examine the contribution of CM to cellular cytotoxicity. METHODS: Ionic (n = 3) and nonionic (n = 3) CM were compared. Changes in the pH(i) of human vascular endothelial and kidney cell lines were monitored by use of a pH-sensitive fluorescent dye (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester). The viability of cells treated with CM was determined by measuring the reduction of a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenlytetrazolium bromide) to violet formazan, a reaction that requires the activity of mitochondrial dehydrogenase; this measurement was made with a microplate reader. RESULTS: The pH(i) of endothelial and kidney cells exposed to 40-60 mg of iodine per milliliter of CM showed acidification (approximately 0.2 pH unit). Within minutes, gradual pH(i) alkalinization to baseline values occurred. The return to baseline values was slower with ionic compounds than with nonionic CM (P < 0.001). Nonionic agents caused less cellular damage than did ionic CM. CONCLUSIONS: The pH(i) is involved in the immediate intracellular transduction of CM effects in vitro. The exposure of cells to ionic CM is more detrimental than is exposure to nonionic CM, as demonstrated by disturbances in the cytosolic pH and by long-term effects on cell viability.
Assuntos
Meios de Contraste/toxicidade , Endotélio Vascular/efeitos dos fármacos , Rim/efeitos dos fármacos , Adenocarcinoma , Linhagem Celular , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Rim/citologia , Rim/metabolismo , Neoplasias Renais , Concentração Osmolar , Oxirredução/efeitos dos fármacos , Sais de Tetrazólio/farmacocinética , Células Tumorais Cultivadas , Cordão UmbilicalRESUMO
The secosteroid 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) is the major biologically active metabolite of vitamin D. Antitumor activity of this hormone has been observed on several cell lines and on breast cancer in vivo. The purpose of this in vitro study was to determine the possible effect of 1,25(OH)2D3 on glioma cells. Two glioma cell lines from rat (C6) or human (GHD) origin were cultured in the presence of 1,25(OH)2D3. The sensitivity of these cells to 1,25 (OH)2D3 was assessed with a colorimetric MTT assay. A cytotoxic effect of 1,25(OH)2D3 was detected at concentrations around 10(-8) M. A lag period of 3 days was required between the onset of the treatment and the observation of the effects. However, the continuous presence of 1,25(OH)2D3 is not required since cell death occurred even when C6 cells were challenged for 24 hr with 1,25(OH)2D3 and then cultured in the absence of the hormone. In addition, 1,25(OH)2D3 regulates the expression of its own receptors in C6 glioma. These results provide to our knowledge the first evidence for a cytotoxic effect of 1,25(OH)2D3 on rat and human glioma cells and could offer both an experimental model to study a programmed cell death in a brain-derived cell line and a new strategy for the inhibition of glioma growth in vivo.
Assuntos
Neoplasias Encefálicas/patologia , Calcitriol/toxicidade , Glioma/patologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/imunologia , Glioma/tratamento farmacológico , Humanos , RNA Neoplásico/biossíntese , Ratos , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinética , Células Tumorais CultivadasRESUMO
An in vitro cell proliferation assay was developed to measure the capacity of substances to overcome multi-drug resistance (MDR). The assay is a modification of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The inclusion of cell titration curves for each concentration of the resistance modifier (RM) allows the IC50 of the RM to be calculated and provides empirical correction of the cell survival curves for the effect of the RM when it is combined with a standard cytotoxic drug, vincristine. The resistance modification index (RMI) is defined as the ratio of the IC50 of vincristine obtained in control cultures divided by that measured in the presence of RM and is linearly related to the dose of RM. The RMI0.1, the RMI at a one-tenth the IC50 of the RM, provides a relative comparison between the activities of different RMs at non-toxic doses. The results obtained using the MDR cell line, KB-8-5, show that l-(-)-verapamil is approximately 4 times more active than d-(+)-verapamil in modifying MDR. The racemic mixture has an intermediate activity. A similar comparison between the epimers quinidine and quinine shows that, at equimolar doses, quinine has a higher RMI but, because it is more toxic, the RMI0.1 is about one-half of that of quinidine. These results demonstrate the importance of comparing the resistance-modifying activities of different compounds at doses relative to their own toxicity.
Assuntos
Neoplasias/tratamento farmacológico , Quinidina/toxicidade , Verapamil/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Corantes/farmacocinética , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Quinidina/uso terapêutico , Estereoisomerismo , Sais de Tetrazólio/farmacocinética , Tiazóis/farmacocinética , Verapamil/uso terapêutico , Vincristina/antagonistas & inibidores , Vincristina/uso terapêutico , Vincristina/toxicidadeRESUMO
Mesenteric arteries supplying an intestinal segment were occluded for 5 minutes and then released. During reperfusion, two series of measurements were made with various substances topically applied to the extraluminal surface. In the first series, reduced nitro blue tetrazolium (NBT) was extracted from tissue and measured spectrophotometrically, as an index of oxidative damage. In the second series, mucosal and serosal surface pH was measured as an index of the functional ability to maintain ion gradients. In control conditions, NBT deposition averaged 55-63 micrograms/g tissue. After 60 and 120 minutes of reperfusion, NBT was elevated to 446-479 micrograms/g, which was approximately half as large as the NBT increment (846 micrograms/g) produced by a 15-minute application of xanthine plus xanthine oxidase to well-perfused tissue. As expected, NBT levels were significantly lower (299 micrograms/g) in tissue that was continuously suffused with superoxide dismutase (SOD) plus catalase (CAT) before occlusion and during reperfusion. Similar NBT levels (274 micrograms/g) were observed after reperfusion in animals that were fed a diet supplemented with the antioxidant vitamin E for 4-6 weeks. These observations affirm that some, but not all, NBT deposition after reperfusion can be attributed to oxyradicals. However, with exogenous adenosine (ADO) applied for the first 30 minutes after occlusion, NBT was elevated to 174 micrograms/g after 60 minutes, which was only half as large as the increment with SOD plus CAT, even though those substances were continuously applied. The opposite effect was produced by an ADO receptor antagonist, 8-phenyltheophylline; NBT was increased to 516 micrograms/g.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenosina/farmacologia , Hidrogênio/metabolismo , Nitroazul de Tetrazólio/farmacocinética , Traumatismo por Reperfusão/metabolismo , Sais de Tetrazólio/farmacocinética , Animais , Catalase/farmacologia , Concentração de Íons de Hidrogênio , Intestinos/irrigação sanguínea , Masculino , Ratos , Ratos Endogâmicos , Superóxido Dismutase/farmacologiaRESUMO
The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cells, and this reaction is used as the end point in a rapid drug-screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantitative relationship is established between cell number and MTT-formazan production. We have shown that reduction of MTT to MTT-formazan by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan production differs widely between cell lines. The absorption spectrum of MTT-formazan varies with cell number and with pH. At a low cell density or a high pH, the absorption maximum is at a wavelength of 560 to 570 nm. However, at a high cell density or a low pH, there are two absorption maxima; one at 510 nm and a second at about 570 nm. Measurements of absorbance at 570 nm underestimate MTT-formazan production and, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a buffer at pH 10.5 to the solubilized MTT-formazan product can overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MTT is used and the pH of the MTT-formazan product is controlled, dye reduction can be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.