RESUMO
Assessing the safety of inhaled substances in the alveolar region of the lung requires an understanding of how the respired material interacts with both physical and immunological barriers. Human alveolar-like macrophages in vitro provide a platform to assess the immunological response in the airways and may better inform the understanding of a response to an inhaled challenge being adaptive or adverse. The aim of this study was to determine if a morphometric phenotyping approach could discriminate between different inhaled nicotine products and indicate the potential mechanism of toxicity of a substance. Cigarette smoke (CS) and e-liquids extracted into cell culture medium were applied to human alveolar-like macrophages in mono-culture (ImmuONE™) and co-culture (ImmuLUNG™) to test the hypothesis. Phenotype profiling of cell responses was highly reproducible and clearly distinguished the different responses to CS and e-liquids. Whilst the phenotypes of untreated macrophages were similar regardless of culture condition, macrophages cultured in the presence of epithelial cells were more sensitive to CS-induced changes related to cell size and vacuolation processes. This technique demonstrated phenotypical observations typical for CS exposure and indicative of the established mechanisms of toxicity. The technique provides a rapid screening approach to determine detailed immunological responses in the airways which can be linked to potentially adverse pathways and support inhalation safety assessment.
Assuntos
Macrófagos Alveolares , Nicotina , Humanos , Macrófagos Alveolares/metabolismo , Nicotina/metabolismo , Administração por Inalação , Macrófagos/metabolismo , Nicotiana , PulmãoRESUMO
The efficient removal of contaminants from the outer surfaces of the body can provide an effective means of reducing adverse health effects associated with incidents involving the accidental or deliberate release of hazardous materials. Showering with water is frequently used by first responders as a rapid method of mass casualty decontamination (MCD). However, there is a paucity of data on the generic effectiveness and safety of aqueous decontamination systems. To address these issues, we have developed a new in vitro skin diffusion cell system to model the conditions of a common MCD procedure ("ladder pipe system"). The new diffusion cell design incorporates a showering nozzle, an air sampling port for measurement of vapour loss and/aerosolisation, adjustable (horizontal to vertical) skin orientation and a circulating manifold system (to maintain a specified flow rate, temperature and pressure of shower water). The dermal absorption characteristics of several simulants (Invisible Red S, curcumin and methyl salicylate) measured with the new in vitro model were in good agreement with previous in vitro and in vivo studies. Moreover, these initial studies have indicated that whilst flow rate and water temperature are important factors for MCD, the presence of clothing during showering may (under certain circumstances) cause transfer and spreading of contaminants to the skin surface.
Assuntos
Descontaminação/normas , Incidentes com Feridos em Massa , Absorção Cutânea , Banhos , Vestuário , Curcumina , Difusão , Corantes Fluorescentes , Substâncias Perigosas , Humanos , PermeabilidadeRESUMO
The biochemical mechanism of toxicity of the experimental astrocyte neurotoxicant and food contaminant S-3-chloro-1,2-propanediol (3-CPD) has been proposed to be via inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have confirmed this action in liver, which shows inhibition to 6.0+/-0.7% control at the neuropathic dose of 140 mg/kg. However, GAPDH activity in brain only fell to a minimum of 54+/-24% control, and the concentrations of lactate and pyruvate (the downstream products of GAPDH), showed no pre-neuropathic decreases in 3-CPD susceptible brain tissue. There was no inhibition of GAPDH activity in primary astrocyte cultures at sub-cytotoxic exposures. We therefore sought alternative mechanisms to explain its toxicity to astrocytes. We were able to show that 3-CPD is a substrate for glutathione-S-transferase and also that, after bioactivation by alcohol dehydrogenase, it generates an irreversible inhibitor of glutathione reductase. In addition, incubation of brain slices from the 3-CPD-vulnerable inferior colliculus produces a depletion of glutathione and an inhibition of glutathione-S-transferase that is not seen in equivalent slices taken from the 3-CPD-resistant occipital neocortex. A smaller but significant and similarly regionally selective decrease in glutathione content is also seen in vivo. We conclude that 3-CPD does not produce its astrocytic toxicity via energy deprivation, and suggest that selective bioactivation and consequent disruption of redox state is a more likely mechanism.
Assuntos
Colículos Inferiores/efeitos dos fármacos , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/metabolismo , Neurotoxinas/toxicidade , alfa-Cloridrina/toxicidade , Animais , Astrócitos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Metabolismo Energético , Glutationa/metabolismo , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Colículos Inferiores/enzimologia , Colículos Inferiores/metabolismo , Lactatos/metabolismo , Masculino , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/patologia , Piruvatos/metabolismo , Ratos , Ratos Endogâmicos F344 , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Flavonoides/farmacologia , Glucose/metabolismo , Albumina Sérica/efeitos dos fármacos , Albumina Sérica/metabolismo , Sítios de Ligação , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Histidina/química , Humanos , Técnicas In Vitro , Lisina/química , Albumina Sérica/química , Triptofano/químicaRESUMO
Recent reports show a pro-oxidant activity of aminoguanidine. Aminoguanidine is able to generate hydrogen peroxide in the presence of Cu (II). These observations have been confirmed by the present studies in that aminoguanidine is, indeed, able to generate oxidants similar in reactivity to the hydroxyl radical and is also able to fragment BSA in a Cu (II)-dependent manner. Studies on glycated bovine serum albumin show that aminoguanidine can affect a number of parameters associated with the nonenzymatic glycation of protein. This includes an ability to decrease glucose attachment and levels of protein fluorescence termed glycophore, resulting from protein glycation. Aminoguanidine also increases the generation of dicarbonyl compounds by glycated protein. All of these effects on parameters of glycation appear to be Cu (II) dependent. Further studies show that one effect of protein glycation is to decrease its susceptibility to proteolysis. The reverse is true of protein oxidation, which has previously been shown to increase the susceptibility of proteins to proteolytic digestion. Evidence is presented suggesting that aminoguanidine is able to enhance the proteolytic digestion of glycated BSA, a protein shown to be protease resistant. Our observations are discussed within the context of current concepts of protein glycation in the development of diabetic complications and aminoguanidine's potential use as a prophylactic agent in diabetes mellitus.
Assuntos
Guanidinas/farmacologia , Oxidantes/farmacologia , Soroalbumina Bovina/metabolismo , Albumina Sérica/metabolismo , Quimotripsina/metabolismo , Diabetes Mellitus , Endopeptidases/metabolismo , Produtos Finais de Glicação Avançada , Peróxido de Hidrogênio/metabolismo , Pepsina A/metabolismo , Albumina Sérica/química , Soroalbumina Bovina/química , Tripsina/metabolismo , Albumina Sérica GlicadaRESUMO
A prominent feature of human atherosclerosis is the lipid-laden foamy macrophage, which often also contains the insoluble pigment, ceroid. The culture of macrophage-like cells, P388D1s, with artificial lipoproteins composed of cholesteryl linoleate (CL) and bovine serum albumin (BSA) results in foam cell formation with lipoprotein uptake and the intracellular accumulation of ceroid. Ceroid accumulation is accompanied by the oxidation of the cholesterol ester as monitored by gas chromatography. The sodium salt of diethyldithiocarbamic acid (DDC) at 1-5 microM effectively inhibited lipoprotein uptake, cholesteryl linoleate oxidation and ceroid accumulation in cultures of P388D1. Further studies showed that intracellular ceroid accumulation appeared to require the presence of cystine in the medium. Lipoprotein oxidation by this macrophage-like cell therefore appears to involve a mechanism dependent on cystine metabolism which is consistent with previous reports of macrophage-mediated lipoprotein oxidation. Studies on CL/BSA-induced ceroid accumulation in human monocytes also showed that DDC behaved in much the same manner. This inhibitory effect of DDC on foam cell formation, often considered a primary event of atherosclerosis, at concentrations as low as 1 microM, suggests the need for further, more comprehensive, studies on this compound's activities.
Assuntos
Ditiocarb/farmacologia , Células Espumosas/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos/patologia , Albuminas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ceroide , Fluorescência , Células Espumosas/efeitos dos fármacos , Lipoproteínas/farmacologia , Macrófagos/efeitos dos fármacos , CamundongosAssuntos
Guanidinas/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Soroalbumina Bovina/química , Albumina Sérica/química , Radioisótopos de Carbono , Eletroforese em Gel de Poliacrilamida , Glucose , Produtos Finais de Glicação Avançada , Glicosilação , Fragmentos de Peptídeos/análise , Soroalbumina Bovina/efeitos dos fármacos , Tripsina , Albumina Sérica GlicadaRESUMO
We have investigated the potential of adenosine uptake as a marker of chemically induced, cell-selective pulmonary injury using cell-selective toxicants. The administration of alpha-naphthylthiourea (ANTU), an agent which is known to damage the pulmonary endothelium, diminished spermidine and adenosine accumulation. In contrast, paraquat (a toxicant that selectively damages pulmonary epithelial cells) did not reduce adenosine uptake, although uptake of spermidine (a marker of pulmonary epithelial damage) was reduced. Taken together, these findings suggest that adenosine and spermidine are accumulated into different cell types. We characterized adenosine uptake and fate in rat lung slices. Accumulation was time and concentration dependent. In our experiments, the radiolabel retained within the slice comprised mainly nucleotides, primarily ATP. Adenosine-induced ATP elevation was initially a rapid event which reached a maximum. The use of a well characterized enzymatic assay for ATP confirmed the ATP elevation suggested by thin-layer chromatography (TLC). Adenosine uptake proved a more consistent marker of ANTU-induced pulmonary injury than measurement of 5-hydroxytryptamine (5-HT) uptake, which was slightly increased by ANTU administration to rats compared with control animals.
RESUMO
Samples of normal human aorta, atherosclerotic lesions and atheroma (necrotic 'gruel' from the interior of advanced lesions) were obtained at necropsy from subjects with no history of diabetes mellitus. Components of each were extracted by ethanol:diethylether (3:1) and, subsequently, by 10% sodium dodecyl sulphate (SDS). Both fractions, organic and aqueous (SDS), were assessed for their relative fluorescence (excitation: 350 nm/emission: 430 nm). The amount of early products of glycated protein was assessed by affinity chromatography in the SDS-soluble fraction. Age-matched plasma samples, obtained from non-diabetic individuals, were also examined. Material from atherosclerotic lesions appeared to exhibit an inverse correlation between protein glycation and fluorescent material which was best reflected within the organic extract. This was not the case for normal aorta. A linear correlation between fluorescent material in the organic extract and SDS extract existed in the normal aorta alone. The only age-dependent change was found in normal aorta in which there was an increase in SDS-soluble fluorescence with increasing age. In plasma samples alone, protein glycation and protein fluorescence appeared to be linearly correlated. The observations are discussed in the context of the possible contribution of protein glycation to atherogenesis.
Assuntos
Aorta/metabolismo , Arteriosclerose/metabolismo , Fluorescência , Proteínas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aorta/patologia , Arteriosclerose/patologia , Cadáver , Cromatografia de Afinidade , Técnicas de Cultura , Glicosilação , Humanos , Pessoa de Meia-IdadeRESUMO
The exposure of proteins to high concentrations of glucose in vitro is widely considered a relevant model of the functional degeneration of tissue occurring in diabetes mellitus. In particular, the enhanced atherosclerosis in diabetes is often discussed in terms of glycation of low-density lipoprotein (LDL), the non-enzymic attachment of glucose to apolipoprotein amino groups. However, glucose can undergo transition-metal-catalysed oxidation under near-physiological conditions in vitro, producing oxidants that possess a reactivity similar to the hydroxyl radical. These oxidants can fragment protein, hydroxylate benzoic acid and induce lipid peroxidation in human LDL. In this study, glycation of LDL in vitro is accompanied by such oxidative processes. However, the oxidation of LDL varies with glucose concentration in a manner which does not parallel changes in protein glycation. Glycation increases in proportion to glucose concentration, whereas in our studies maximal oxidation occurs at a glucose concentration of approx. 25 mM. The modification of LDL resulting from exposure to glucose alters macrophage ceroid accumulation, a process which occurs in the human atherosclerotic plaque. The accumulation of ceroid in macrophages is shown to be related to LDL oxidation rather than LDL glycation, per se, as it too occurs at a maximum of approx. 25 mM. Oxidative sequelae of protein glycation appear to be a major factor in LDL-macrophage interactions, at least with respect to ceroid accumulation. Our observations are discussed in the context of the observed increase in the severity of atherosclerosis in diabetes.
Assuntos
Arteriosclerose/metabolismo , Ceroide/metabolismo , Angiopatias Diabéticas/metabolismo , Glucose/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Oxirredução , Albumina Sérica/metabolismoRESUMO
The accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid, can be monitored semiquantitatively by staining techniques. However, such methods are crude and give little information about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, alpha-tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and flow cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell-mediated lipid oxidation and its potential antagonists.
Assuntos
Ceroide/análise , Citometria de Fluxo , Macrófagos/química , Animais , Arteriosclerose/metabolismo , Fluorescência , Lipoproteínas/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Espectrometria de Fluorescência , Vitamina E/farmacologiaRESUMO
We have investigated the influence of the elevation of pulmonary glutathione (GSH) levels on the toxicity of the rodenticide alpha-naphthylthiourea (ANTU) to rat lung. Administration of phorone (diisopropylidene acetone; 200 mg/kg i.p.) caused an initial depletion of both pulmonary and hepatic GSH followed after 48 hr by a marked elevation in both tissues, due most probably to a compensatory rebound synthesis. In control rats, ANTU produced a dose-dependent lethality, hydrothorax and loss of ability of lung tissue to accumulate adenosine and spermidine (markers of endothelial and epithelial cell function, respectively). These effects were prevented or markedly ameliorated when ANTU was given 48 hr after pretreatment with phorone. The mechanism of the protection by phorone pretreatment against ANTU-induced pulmonary toxicity is unclear. It may be due, in part, to elevated GSH levels in pulmonary endothelial cells and, in addition, to increased detoxification of ANTU in the liver, resulting in a decreased availability to the lung.