Indium-containing particles (ICPs) such as InP and ITO are insoluble and engulfed by alveolar macrophages;however, the mechanism(s) of pathogenesis resulting in pulmonary toxicity is unclear. Previously, in published studies (Gwinn et al., 2013, Toxicological Sciences), we showed in vitro that macrophage solubilization of ICPs via the phagolysosomal acidification pathway resulted in particle-induced cytotoxicity and the release of ionic indium. It is undetermined whether the toxicity of ICPs relates to the total amount of indium in the compound, the partner ion(s) and/or the solubilization potential. The InP and ITO used in these previous studies were of very different particle diameters (1.5 and 0.5 m, respectively) and thus a valid direct comparison proved problematic with regards to relative particle solubilization and cytotoxicity. In additional studies, we compared the macrophage solubilization and cytotoxicity of InP and ITO with similar particle diameters (1.5 m) and determined if relative differences in these in vitro parameters correlated with particle-induced pulmonary toxicity in vivo. We collaborated with RTI and co-authored a manuscript (Levine et al., 2014, Analytical Letters) describing the development of a sonication method to generate ITO with a similar hydrodynamic particle diameter to that of InP. RAW 264.7 macrophages were treated with InP or ITO, which contained similar amounts of indium, and cytotoxicity was assayed at 24 hrs. Particle solubilization (release of ionic indium) was also measured at 24 hrs in culture supernatants by atomic absorption spectroscopy. Macrophage cytotoxicity and particle solubilization in vitro were much greater for InP compared to ITO. To correlate changes in vivo, B6C3F1 mice were treated with 0.5 or 1 mg/kg InP or ITO by oropharyngeal aspiration. On days 14 and 28, bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected and assayed for total numbers of leukocytes. Cell differentials, LDH activity and protein levels were also measured in BAL as additional markers of lung toxicity. All of these BAL and PL parameters were greatly increased in mice treated with InP compared to ITO. These data suggest that macrophage solubilization and cytotoxicity of some ICPs in vitro are capable of predicting pulmonary toxicity in vivo. Furthermore, these differences in toxicity were observed despite the two compounds having similar indium content suggesting that solubilization, not total indium content, better reflects the toxic potential of some ICPs. Finally, soluble InCl3 containing equivalent amounts of indium to that of insoluble particulate InP was shown to be more cytotoxic than InP to macrophages and lung epithelial cells in vitro suggesting that ionic indium is the primary cytotoxic component of InP. A manuscript describing these recent studies will soon be submitted to Toxicological Sciences for publication. Studies are also in progress to determine if prolonged chronic exposure to sub-lethal levels (0.5 and 1 M) of soluble InCl3 can transform human lung epithelial (HPL-1D) cells into malignant cancer cells. If so, potential mechanism(s) of indium carcinogenicity will be addressed using this model. The purpose of this carcinogenicity study is based on previous inhalation data showing that InP and ITO particles are highly carcinogenic in chronically-exposed rodents.
