Macrophage Solubilization and Cytotoxicity of Indium-Containing Particles In Vitro

Abstract

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.

Interferon-γ Inhibits Nonopsonized Phagocytosis of Macrophages via an mTORC1-c/EBPβ Pathway

“…It has been reported that incubation at 4°C or addition of cytochalasin D, an inhibitor of actin assembly and polymerization, decreased the microparticle uptake of macrophages [23,24,25]. PEMs were cultured with or without IFN-γ (1 ng/ml) for 12 h, and then they were treated with cytochalasin D (5 µg/ml) for 30 min followed by co-incubation with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled chicken red blood cells (cRBCs) for 2 h. Additionally, some PEMs were co-incubated with cRBCs at 4°C in a freezer overnight in the presence or absence of IFN-γ.…”

Cytotoxicity and Characterization of Particles Collected From an Indium–Tin Oxide Production Facility

“…RAW cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 2 mM L-glutamine, 10% fetal bovine serum (FBS), and 50 mg/ml penicillin/streptomycin (Invitrogen Life Sciences, Grand Island, NY). RAW cells were selected for these studies because of their potential to react with and engulf particles Gwinn et al, 2013). BEAS-2B cells were grown in DMEM F12 with 10% FBS and 50 mg/ml penicillin/streptomycin.…”

The toxicology of indium tin oxide

“…Ionic indium derived from RAW cells was significantly reduced by treatment with CytoD. These data implicate macrophage uptake and solubilization of InP and UITO via phagolysosomal acidification as requisite for particle-induced cytotoxicity and the release of indium ions (Gwinn et al, 2013).…”

“…The rather low solubilization in the macrophage cell line, RAW 264.7, of less than 0.1% within 24 h, and even lower solubilization in the epithelial cell line LA-4 of 0.05% (Gwinn et al, 2013) could be an explanation for the very long elimination halflives of 142/124 weeks calculated in the studies with intratracheal administration of 3 or 6 mg/kg to hamsters (Tanaka et al, 2010(Tanaka et al, , 2015 and other phenomena to be discussed below.…”