Mechanisms of Pleural and Pulmonary Toxicity of Indium Compounds

铟化合物的胸膜和肺部毒性机制

• 批准号: 8553766
• 负责人: DANIEL MORGAN
• 金额: $ 61.24万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553766
• 关键词: Aftercare; Alveolar; Alveolar Macrophages; Apoptotic; Asbestos; Biological Assay; Breathing; Carcinoma; Cell Death; Cell Line; Cells; Chronic; Cytochalasin D; Data; Deposition; Epithelial; Epithelial Cells; Exhibits; Fiber; Fibrosis; Generations; Goals; Hyperplasia; In Vitro; Indium; Industry; Inflammatory; Inhalation Exposure; Injury; Ions; Lung; Lung Neoplasms; Measures; Mesothelial Hyperplasia; Mesothelium; Metals; Methods; Modeling; Movement; Mus; Necrosis; Particulate; Pathway interactions; Phagocytosis; Phagosomes; Pleural; Relative (related person); Respiratory System; Respiratory tract structure; Severities; Solubility; Structure of parenchyma of lung; Toxic effect; absorption; adenoma; bafilomycin A1; base; carcinogenicity; cytokine; cytotoxic; cytotoxicity; exposed human population; extracellular; in vitro Model; in vivo; indium phosphide; indium tin oxide; inhibitor/antagonist; insight; macrophage; male; man; neutrophil; particle; uptake

These studies were conducted to investigate the mechanism by which insoluble indium compounds cause pulmonary toxicity and pleural fibrosis in male B6C3F1 mice. Insoluble particles deposited in the respiratory tract are typically cleared by ciliary movement and /or removed by macrophages. We hypothesized that phagosome acidification within macrophages after particle uptake results in the solubilization of the InP and ITO particles, which generates free indium metal ions, the cytotoxic entity. In this study, we first characterized the solubility of InP and ITO particles and observed that both were soluble at acidic pH (pH 4) by ICP-MS. We then characterized the in vitro cytotoxicity of InP and ITO particles on macrophages and lung epithelial cells. The mouse macrophage cell line RAW 264.7 was treated with InP or ITO for 1.5 hr to allow particle uptake. The cells were then rinsed with media to remove extracellular particles and cultured for 24 hr. Cytotoxicity was measured after 24 hr using the MTT viability and LDH assays. InP and ITO-treated RAW 264.7 cells exhibited increased cell death relative to media-treated controls. Similar cytotoxic effects were observed after treatment and uptake of particles by BAL-derived primary mouse alveolar macrophages. Pre-treatment of RAW 264.7 cells for 30 min with 25 nM bafilomycin A1, a specific inhibitor of phagosome acidification, followed by treatment for 1.5 hr with InP (100 g/ml) or ITO (300 g/ml) + 25 nM bafilomycin A1 still resulted in particle uptake, but the cytotoxicity of InP and ITO particles was reduced. The mouse lung epithelial cell line LA-4 was also treated with InP or ITO particles; however, neither particle was cytotoxic to LA-4 cells despite being phagocytosed. These data indicate that phagosome acidification after particle uptake by macrophages in vitro is required for the macrophage cytotoxicity of InP and ITO metal particles. These results also support the hypothesis that particulate indium compounds require solubilization in order to be toxic. These studies were conducted to investigate the mechanism by which insoluble indium compounds cause pulmonary toxicity and pleural fibrosis in male B6C3F1 mice. Insoluble particles deposited in the respiratory tract are typically cleared by ciliary movement and /or removed by macrophages. We hypothesized that phagosome acidification within macrophages after particle uptake results in the solubilization of the InP and ITO particles, which generates free indium metal ions, the cytotoxic entity. In this study, we first characterized the solubility of InP and ITO particles and observed that both were soluble at acidic pH (pH 4) by ICP-MS. We then characterized the in vitro cytotoxicity of InP and ITO particles on macrophages and lung epithelial cells. The mouse macrophage cell line RAW 264.7 was treated with InP or ITO for 1.5 hr to allow particle uptake. The cells were then rinsed with media to remove extracellular particles and cultured for 24 hr. Cytotoxicity was measured after 24 hr using the MTT viability and LDH assays. InP and ITO-treated RAW 264.7 cells exhibited increased cell death relative to media-treated controls. Similar cytotoxic effects were observed after treatment and uptake of particles by BAL-derived primary mouse alveolar macrophages. Pre-treatment of RAW 264.7 cells for 30 min with 25 nM bafilomycin A1, a specific inhibitor of phagosome acidification, followed by treatment for 1.5 hr with InP (100 g/ml) or ITO (300 g/ml) + 25 nM bafilomycin A1 still resulted in particle uptake, but the cytotoxicity of InP and ITO particles was reduced. The mouse lung epithelial cell line LA-4 was also treated with InP or ITO particles; however, neither particle was cytotoxic to LA-4 cells despite being phagocytosed. These data indicate that phagosome acidification after particle uptake by macrophages in vitro is required for the macrophage cytotoxicity of InP and ITO metal particles. These results also support the hypothesis that particulate indium compounds require solubilization in order to be toxic. To further confirm that uptake and breakdown of indium-containing particles by macrophages is required for cytotoxicity, RAW 264.7 cells were treated with cytochalasin D, which is an inhibitor of phagocytosis. Cells were pre-treated for 30 min +/- cytochalasin D (5 g/mL) and then treated for 24 hrs with InP (200 g/ml) or ITO (400 g/ml) +/- cytochalasin D. Treatment with cytochalasin D in turn blocked both particle phagocytosis as well as particle-induced cytotoxicity as measured by MTT and LDH viability assays. In addition, an atomic absorption (AA)-based method was developed and used to quantitatively measure free indium metal ions in the culture supernatants of InP-treated RAW 264.7 macrophages. Treatment with cytochalasin D, which blocks InP particle phagocytosis, decreased the generation of free indium metal ions in culture supernatants of InP-treated RAW 264.7 macrophages. This supports the notion that macrophages solubilize indium-containing particles following uptake and subsequently release free indium metal ions extracellularly. This also supports our hypothesis that the cytotoxicity of InP for macrophages requires the uptake and breakdown of InP particles by macrophages (via the phagolysosomal pathway) followed by the release of free indium metal ions (the cytotoxic entity of InP) by apoptotic and/or necrotic macrophages. Preliminary data has also indicated that, in contrast to indium in particulate form, soluble indium is cytotoxic to lung-derived epithelial cells. Our proposed current model is that alveolar macrophages phagocytose and solubilize indium-containing particles following airway exposure. Particle solubilization generates free indium metal ions intracellularly, which are cytotoxic to the macrophage. Free indium metal ions may then be released extracellularly by necrotic and/or apoptotic leaky macrophages (+ other factors or induced cytokines) which drives particle-induced pulmonary toxicity and lung epithelial injury. Interestingly, in vivo studies in our lab have indicated that pre-treatment of mice with LPS (2 g/g) 24 hrs prior to treatment with InP (1 mg/kg) via aspiration blocks InP-induced pulmonary toxicity. Although the mechanism(s) are unclear, it is possible that pro-inflammatory LPS may impact the ability of alveolar macrophages to engulf and/or breakdown InP particles by 1) decreasing the pool of alveolar macrophages within the airways/lung tissue available for particle uptake, 2) inducing cytokines (and/or other factors) which suppress macrophage function and/or 3) increasing the pool of phagocytic neutrophils within the airways/lung tissue which provides an alternative clearance mechanism for the particles.

进行了这些研究，以研究雄性B6C3F1小鼠中不溶性依赖化合物引起肺毒性和胸膜纤维化的机制。沉积在呼吸道中的不溶性颗粒通常通过睫状运动和 /或巨噬细胞清除。我们假设颗粒摄取后巨噬细胞内的吞噬体酸化导致INP和ITO颗粒的溶解，从而产生了自由的鉴别金属离子，即细胞毒性实体。在这项研究中，我们首先表征了INP和ITO颗粒的溶解度，并观察到ICP-MS在酸性pH（pH 4）上都可溶。然后，我们表征了巨噬细胞和肺上皮细胞上INP和ITO颗粒的体外细胞毒性。小鼠巨噬细胞系RAW 264.7用INP或ITO处理1.5小时，以允许颗粒摄取。然后用培养基冲洗细胞以去除细胞外颗粒并培养24小时。使用MTT生存力和LDH分析，在24小时后测量细胞毒性。 INP和ITO处理的RAW 264.7细胞相对于经媒体处理的对照表现出增加的细胞死亡。在治疗和吸收颗粒后，通过巴林衍生的原发性小鼠肺泡巨噬细胞观察到类似的细胞毒性作用。用25 nm bafilomycin A1（一种特异性的吞噬体酸化抑制剂）对RAW 264.7细胞进行预处理30分钟，然后用INP（100 g/ml）或ITO（300 g/ml） + 25 nm bafilomycin a1处理1.5小时，但在粒子摄取中仍导致粒子毒性含量，但粒子含量为cistotottotottotot和cytototot和cytotot ossp and ossp and和consot和nos。小鼠肺上皮细胞系LA-4也用INP或ITO颗粒处理；然而，尽管被吞噬了LA-4细胞，这两个粒子都没有细胞毒性。这些数据表明，INP和ITO金属颗粒的巨噬细胞细胞毒性需要巨噬细胞在体外摄取粒子后的吞噬体酸化。这些结果还支持以下假设：颗粒化合物需要溶解才能有毒。 进行了这些研究，以研究雄性B6C3F1小鼠中不溶性依赖化合物引起肺毒性和胸膜纤维化的机制。沉积在呼吸道中的不溶性颗粒通常通过睫状运动和 /或巨噬细胞清除。我们假设颗粒摄取后巨噬细胞内的吞噬体酸化导致INP和ITO颗粒的溶解，从而产生了自由的鉴别金属离子，即细胞毒性实体。在这项研究中，我们首先表征了INP和ITO颗粒的溶解度，并观察到ICP-MS在酸性pH（pH 4）上都可溶。然后，我们表征了巨噬细胞和肺上皮细胞上INP和ITO颗粒的体外细胞毒性。小鼠巨噬细胞系RAW 264.7用INP或ITO处理1.5小时，以允许颗粒摄取。然后用培养基冲洗细胞以去除细胞外颗粒并培养24小时。使用MTT生存力和LDH分析，在24小时后测量细胞毒性。 INP和ITO处理的RAW 264.7细胞相对于经媒体处理的对照表现出增加的细胞死亡。在治疗和吸收颗粒后，通过巴林衍生的原发性小鼠肺泡巨噬细胞观察到类似的细胞毒性作用。用25 nm bafilomycin A1（一种特异性的吞噬体酸化抑制剂）对RAW 264.7细胞进行预处理30分钟，然后用INP（100 g/ml）或ITO（300 g/ml） + 25 nm bafilomycin a1处理1.5小时，但在粒子摄取中仍导致粒子毒性含量，但粒子含量为cistotottotottotot和cytototot和cytotot ossp and ossp and和consot和nos。小鼠肺上皮细胞系LA-4也用INP或ITO颗粒处理；然而，尽管被吞噬了LA-4细胞，这两个粒子都没有细胞毒性。这些数据表明，INP和ITO金属颗粒的巨噬细胞细胞毒性需要巨噬细胞在体外摄取粒子后的吞噬体酸化。这些结果还支持以下假设：颗粒化合物需要溶解才能有毒。 为了进一步证实，细胞毒性需要巨噬细胞对含酰胺的颗粒的摄取和分解，将RAW 264.7细胞用细胞切拉斯蛋白D处理，后者是吞噬作用的抑制剂。将细胞预处理30分钟+/-细胞切拉蛋白D（5 g/mL），然后用INP（200 g/ml）或ITO（400 g/ml）+/- cytochalasin D进行24小时。在转弯粒子吞噬作用中，用细胞chal蛋白D治疗两种颗粒吞噬作用，并通过测量粒子和cytoxikics cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cyttoss cytoshigy。 此外，开发了一种原子吸收（AA）方法，并用于定量测量INP处理的RAW 264.7巨噬细胞的培养上清液中的游离金属离子。通过阻断INP颗粒吞噬作用的细胞切拉斯蛋白D治疗可减少INP处理的RAW 264.7巨噬细胞的培养上清液中游离金属离子的产生。这支持了这样一个观念，即巨噬细胞在吸收后溶解含酰胺的颗粒，然后在细胞外释放自由的金属离子。这也支持我们的假设，即巨噬细胞的INP的细胞毒性需要巨噬细胞（通过吞噬体途径）对INP颗粒的吸收和分解，然后释放自由鉴别型金属离子（INP的细胞毒性实体INP的细胞毒性实体），通过凋亡和/或或/或或/或或/或或或或或necrotation。初步数据还表明，与颗粒形式的依赖相比，可溶性依赖性对肺衍生的上皮细胞是细胞毒性的。我们提出的当前模型是，气道暴露后肺泡巨噬细胞吞噬并溶解含酰胺的颗粒。粒子溶解化会在细胞内产生自由的依赖金属离子，这对巨噬细胞具有细胞毒性。然后，可以通过坏死和/或凋亡的巨噬细胞（+其他因素或诱导的细胞因子）在细胞外释放游离的金属离子，从而驱动颗粒引起的肺毒性和肺上皮损伤。 有趣的是，我们实验室中的体内研究表明，在用INP（1 mg/kg）通过抽吸阻滞INP INP诱导的肺毒性治疗之前，用LPS（2 g/g）的小鼠预处理小鼠（2 g/g）。尽管机制尚不清楚，但促炎的LP可能会影响肺泡巨噬细胞吞噬和/或分解INP颗粒的能力1）减少在颗粒摄取中可用的气道/肺组织中可用的肺泡巨噬细胞/肺组织的能力，2）诱导细胞因子（和/或其他因素），从而诱导和其他因素，并抑制了p的功能，这些因素和其他功能均为pH。气道/肺组织中的中性粒细胞为颗粒提供了替代的间隙机制。