Macrophage solubilization and cytotoxicity of indium-containing particles as in vitro correlates to pulmonary toxicity in vivo.

Indium is widely used in the semiconductor and microelectronics industries often in particulate form with examples being indium phosphide (InP) and indium-tin oxide (ITO). ITO, which typically consists of approximately 90% indium oxide (In2O3) and 10% tin oxide (SnO2) by weight, is used in the manufacture of flat panel displays (Guo et al., 2011) and accounts for a large percentage of the total indium demand in Japan (Tanaka et al., 2010). ITO can be sintered or non-sintered; although the sintered form is most commonly used for industrial applications. Concerns have been raised over the safety and health of industry workers exposed to particulate aerosols when working with materials containing these compounds. Inhalation exposure to indium-containing particles (ICPs) within an occupational setting, mostly ITO, has been associated with increased concentrations of indium measured in the blood, serum, and urine of workers and in the development of interstitial lung disease (Chonan et al., 2007; Hamaguchi et al., 2008; Homma et al., 2005; Liu et al., 2012; Miyaki et al., 2003; Nakano et al., 2009) as well as pulmonary alveolar proteinosis (Cummings et al., 2010; Xiao et al., 2010). In fact, inhalation exposure to ITO in an occupational setting has resulted in two fatalities (Cummings et al., 2010; Homma et al., 2003). The American Conference of Governmental Industrial Hygienists (ACGIH) has recommended an occupational exposure limit of 0.1 mg/m3 (threshold limit value-time weighted average) for all indium compounds (ACGIH, 2007). A critical issue to be addressed is whether the toxicity of ICPs can be predicted based on the total amount of indium present in the compound, the other constituents (eg, tin in ITO), and/or the solubilization potential. Indium ions released from the parent compound by solubilization are likely the main toxic constituents of some ICPs. Subchronic and chronic inhalation exposure of rats and mice to InP and ITO particles resulted in pulmonary toxicity including severe alveolar proteinosis, chronic inflammation, interstitial fibrosis, and alveolar epithelial hyperplasia/metaplasia (Gottschling et al., 2001; Nagano et al., 2011a,b,c; NTP, 2001). In addition, carcinogenicity was observed after chronic inhalation exposure to InP and ITO in rodents and included increased incidences of alveolar/bronchiolar adenomas and carcinomas as well as squamous cell carcinoma of the lung (Gottschling et al., 2001; Nagano et al., 2011c; NTP, 2001). Administration of InP or ITO particles via oropharnygeal aspiration (Kirby et al., 2009; Lison et al., 2009) or intratracheal instillation (Tanaka et al., 2002, 2010; Yamazaki et al., 2000) also induced pulmonary injury, inflammation, and pleural effects. Upon deposition within the lung, alveolar macrophages (AMs) and likely epithelial cells initially phagocytoze the InP and ITO particles which are insoluble at physiological pH; however, the mechanism(s) responsible for pulmonary toxicity is unclear. We recently showed in vitro that uptake of InP and ITO by macrophages (RAW 264.7 cells) and solubilization through the phagolysosomal acidification pathway after 24 h of treatment resulted in particle-induced cytotoxicity and the extracellular release of ionic indium (Gwinn et al., 2013), presumably the primary cytotoxic constituent of InP and ITO. In this previous study, we developed a cell-based assay using graphite furnace-atomic absorption spectroscopy (GF-AAS) to measure the solubilization of ICPs and release of “free” ionic indium by macrophages. Lung epithelial (LA-4) cells were also shown to phagocytoze InP and ITO; however, unlike macrophages, these cells did not solubilize the particles and were resistant to particle-induced cytotoxicity after 24 h of treatment (Gwinn et al., 2013). The InP and ITO used in this previous study were of very different particle diameters and thus a valid direct comparison proved problematic with regards to relative particle solubilization and cytotoxicity. In the current study, we compared the macrophage solubilization (release of ionic indium) and cytotoxicity of InP and ITO with similar particle diameters (∼1.5 µm) and then determined if relative differences in these in vitro parameters correlated with pulmonary toxicity in vivo. Our results showed that macrophage cytotoxicity and release of ionic indium in vitro as well as pulmonary toxicity in vivo were much greater for InP compared with ITO. This suggests that macrophage solubilization and particle-induced cytotoxicity of some ICPs in vitro are capable of predicting pulmonary toxicity in vivo. Furthermore, these differences in particle toxicity were observed despite the two compounds having similar indium content suggesting that particle solubilization, not total indium content, better reflects the toxic potential of some ICPs. Finally, we also demonstrated that soluble InCl3 containing equivalent amounts of indium to that of insoluble particulate InP was more cytotoxic than InP to macrophages and lung epithelial cells in vitro suggesting that ionic indium is the primary cytotoxic constituent of InP.