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.

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Gwinn, William M; Qu, Wei; Bousquet, Ronald W et al. (2015) Macrophage solubilization and cytotoxicity of indium-containing particles as in vitro correlates to pulmonary toxicity in vivo. Toxicol Sci 144:17-26
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