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: 8734134
• 负责人: DANIEL MORGAN
• 金额: $ 61.38万
• 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/8734134
• 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. 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.

这些研究旨在探讨不溶性铟化合物引起雄性 B6C3F1 小鼠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纤毛运动清除和/或通过巨噬细胞去除。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 颗粒溶解，从而产生游离的铟金属离子（细胞毒性实体）。在本研究中，我们首先表征了 InP 和 ITO 颗粒的溶解度，并通过 ICP-MS 观察到两者在酸性 pH 值 (pH 4) 下均可溶解。然后我们表征了 InP 和 ITO 颗粒对巨噬细胞和肺上皮细胞的体外细胞毒性。用 InP 或 ITO 处理小鼠巨噬细胞系 RAW 264.7 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。 24小时后使用MTT活力和LDH测定法测量细胞毒性。相对于培养基处理的对照，InP 和 ITO 处理的 RAW 264.7 细胞表现出细胞死亡增加。在处理后，BAL 来源的原代小鼠肺泡巨噬细胞摄取颗粒后，观察到类似的细胞毒性作用。用 25 nM 巴弗洛霉素 A1（一种吞噬体酸化的特异性抑制剂）预处理 RAW 264.7 细胞 30 分钟，然后用 InP (100 μg/ml) 或 ITO (300 μg/ml) + 25 nM 巴弗洛霉素 A1 处理 1.5 小时仍然导致颗粒摄取，但 InP 和 ITO 颗粒的细胞毒性降低。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，这两种颗粒都不对 LA-4 细胞产生细胞毒性。这些数据表明，InP和ITO金属颗粒的巨噬细胞细胞毒性需要体外巨噬细胞摄取颗粒后的吞噬体酸化。这些结果也支持这样的假设：颗粒状铟化合物需要溶解才能具有毒性。 为了进一步证实巨噬细胞摄取和分解含铟颗粒是细胞毒性所必需的，用细胞松弛素 D（一种吞噬作用抑制剂）处理 RAW 264.7 细胞。将细胞预处理 30 分钟 +/- 细胞松弛素 D (5 g/mL)，然后用 InP (200 μg/ml) 或 ITO (400 g/ml) +/- 细胞松弛素 D 处理 24 小时。用细胞松弛素处理D 进而阻断颗粒吞噬作用以及颗粒诱导的细胞毒性（通过 MTT 和 LDH 活力测定检测）。 此外，还开发了一种基于原子吸收 (AA) 的方法，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铟金属离子。细胞松弛素 D 可以阻断 InP 颗粒的吞噬作用，从而减少 InP 处理的 RAW 264.7 巨噬细胞培养上清液中游离金属铟离子的产生。这支持了巨噬细胞在摄取后溶解含铟颗粒并随后将游离的铟金属离子释放到细胞外的观点。这也支持了我们的假设，即 InP 对巨噬细胞的细胞毒性需要巨噬细胞（通过吞噬溶酶体途径）摄取和分解 InP 颗粒，然后通过细胞凋亡和/或坏死释放游离的铟金属离子（InP 的细胞毒性实体）。巨噬细胞。初步数据还表明，与颗粒形式的铟相比，可溶性铟对肺源性上皮细胞具有细胞毒性。我们提出的当前模型是，肺泡巨噬细胞在气道暴露后吞噬并溶解含铟颗粒。颗粒溶解在细胞内产生游离的铟金属离子，其对巨噬细胞具有细胞毒性。然后，游离的铟金属离子可能被坏死和/或凋亡的渗漏巨噬细胞（+其他因素或诱导的细胞因子）释放到细胞外，从而驱动颗粒诱导的肺毒性和肺上皮损伤。 有趣的是，我们实验室的体内研究表明，在通过抽吸方式用 InP (1 mg/kg) 治疗前 24 小时对小鼠进行 LPS (2 g/g) 预处理，可以阻止 InP 诱导的肺毒性。尽管机制尚不清楚，但促炎性 LPS 可能会通过以下方式影响肺泡巨噬细胞吞噬和/或分解 InP 颗粒的能力：1) 减少气道/肺组织内可用于颗粒的肺泡巨噬细胞池摄取，2) 诱导抑制巨噬细胞功能的细胞因子（和/或其他因子）和/或 3) 增加气道/肺组织内吞噬中性粒细胞的数量这为颗粒提供了另一种清除机制。