RESUMEN
Macrophage-solubilized indium-containing particles (ICPs) were previously shown in vitro to be cytotoxic. In this study, we compared macrophage solubilization and cytotoxicity of indium phosphide (InP) and indium-tin oxide (ITO) with similar particle diameters (â¼ 1.5 µm) and then determined if relative differences in these in vitro parameters correlated with pulmonary toxicity in vivo. RAW 264.7 macrophages were treated with InP or ITO particles and cytotoxicity was assayed at 24 h. Ionic indium was measured in 24 h culture supernatants. Macrophage cytotoxicity and particle solubilization in vitro were much greater for InP compared with ITO. To correlate changes in vivo, B6C3F1 mice were treated with InP or ITO by oropharyngeal aspiration. On Days 14 and 28, bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected and assayed for total leukocytes. Cell differentials, lactate dehydrogenase activity, and protein levels were also measured in BAL. All lavage parameters were greatly increased in mice treated with InP compared with ITO. These data suggest that macrophage solubilization and cytotoxicity of some ICPs in vitro are capable of predicting pulmonary toxicity in vivo. In addition, these differences in toxicity were observed despite the two particulate compounds containing similar amounts of indium suggesting that solubilization, not total indium content, better reflects the toxic potential of some ICPs. Soluble InCl3 was shown to be more cytotoxic than InP to macrophages and lung epithelial cells in vitro further suggesting that ionic indium is the primary cytotoxic component of InP.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Indio/toxicidad , Enfermedades Pulmonares/inducido químicamente , Macrófagos/efectos de los fármacos , Fagocitosis , Fosfinas/toxicidad , Compuestos de Estaño/toxicidad , Contaminantes Ocupacionales del Aire/química , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Indio/química , Exposición por Inhalación , L-Lactato Deshidrogenasa/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Tamaño de la Partícula , Fosfinas/química , Células RAW 264.7 , Solubilidad , Factores de Tiempo , Compuestos de Estaño/químicaRESUMEN
Indium-containing particles (ICPs) are used extensively in the microelectronics industry. Pulmonary toxicity is observed after inhalation exposure to ICPs; however, the mechanism(s) of pathogenesis is unclear. ICPs are insoluble at physiological pH and are initially engulfed by alveolar macrophages (and likely airway epithelial cells). We hypothesized that uptake of ICPs by macrophages followed by phagolysosomal acidification results in the solubilization of ICPs into cytotoxic indium ions. To address this, we characterized the in vitro cytotoxicity of indium phosphide (InP) or indium tin oxide (ITO) particles with macrophages (RAW cells) and lung-derived epithelial (LA-4) cells at 24h using metabolic (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) and membrane integrity (lactate dehydrogenase) assays. InP and ITO were readily phagocytosed by RAW and LA-4 cells; however, the particles were much more cytotoxic to RAW cells and cytotoxicity was dose dependent. Treatment of RAW cells with cytochalasin D (CytoD) blocked particle phagocytosis and reduced cytotoxicity. Treatment of RAW cells with bafilomycin A1, a specific inhibitor of phagolysosomal acidification, also reduced cytotoxicity but did not block particle uptake. Based on direct indium measurements, the concentration of ionic indium was increased in culture medium from RAW but not LA-4 cells following 24-h treatment with particles. Ionic indium derived from RAW cells was significantly reduced by treatment with CytoD. These data implicate macrophage uptake and solubilization of InP and ITO via phagolysosomal acidification as requisite for particle-induced cytotoxicity and the release of indium ions. This may apply to other ICPs and strongly supports the notion that ICPs require solubilization in order to be toxic.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Indio/farmacología , Macrófagos/efectos de los fármacos , Animales , Línea Celular , Macrófagos/citología , Ratones , Fagocitosis , Solubilidad , Espectrofotometría AtómicaRESUMEN
The mechanism(s) by which chronic inhalation of indium phosphide (InP) particles causes pleural fibrosis is not known. Few studies of InP pleural toxicity have been conducted because of the challenges in conducting particulate inhalation exposures, and because the pleural lesions developed slowly over the 2-year inhalation study. The authors investigated whether InP (1 mg/kg) administered by a single oropharyngeal aspiration would cause pleural fibrosis in male B6C3F1 mice. By 28 days after treatment, protein and lactate dehydrogenase (LDH) were significantly increased in bronchoalveolar lavage fluid (BALF), but were unchanged in pleural lavage fluid (PLF). A pronounced pleural effusion characterized by significant increases in cytokines and a 3.7-fold increase in cell number was detected 28 days after InP treatment. Aspiration of soluble InCl(3) caused a similar delayed pleural effusion; however, other soluble metals, insoluble particles, and fibers did not. The effusion caused by InP was accompanied by areas of pleural thickening and inflammation at day 28, and by pleural fibrosis at day 98. Aspiration of InP produced pleural fibrosis that was histologically similar to lesions caused by chronic inhalation exposure, and in a shorter time period. This oropharyngeal aspiration model was used to provide an initial characterization of the progression of pleural lesions caused by InP.
Asunto(s)
Indio/toxicidad , Fosfinas/toxicidad , Pleura/efectos de los fármacos , Administración por Inhalación , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Indio/administración & dosificación , Exposición por Inhalación , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Material Particulado/administración & dosificación , Material Particulado/toxicidad , Fosfinas/administración & dosificación , Pleura/patología , Derrame Pleural/etiología , Derrame Pleural/metabolismo , Derrame Pleural/patología , Pleuresia/etiología , Pleuresia/metabolismo , Pleuresia/patología , Factores de TiempoRESUMEN
Nitric oxide (NO) released by activated alveolar macrophages (AM) can mediate effects on target cells and can also react with superoxide anion (O2-) to form peroxynitrite (PN), a highly cytotoxic product. The role of NO and PN in AM cytotoxicity for normal lung cells was investigated using co-cultures of rat lung fibroblasts (FB) and rat AM treated with lipopolysaccharide + interferon-gamma (LI). AM and FB, alone and in co-culture, were treated with LI for 5 days and cell viability measured. The culture media was analyzed for NO, TNF-alpha, O2-, and IL-1beta. A decreased FB viability was correlated with increased NO release by LI-activated AM. Pretreatment of co-cultures with the inducible NOS inhibitor L-NAME caused dose-related decreases in NO release by AM and increases in FB viability. Although TNF-alpha release was increased in co-cultures treated with LI, the viability of FB was not affected when cultures were treated with similar concentrations of TNF-alpha in the absence of AM. O2- could not be detected in the media and addition of superoxide dismutase (SOD) did not protect FB. These data suggest that neither O2- nor PN contributed to the loss of cell viability. Activated AM may kill normal rat lung FB through a NO-mediated pathway that does not involve PN.