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Comparative Study
. 2015 Mar;144(1):17-26.
doi: 10.1093/toxsci/kfu273. Epub 2014 Dec 19.

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

William M Gwinn  1 , Wei Qu  2 , Ronald W Bousquet  2 , Herman Price  2 , Cassandra J Shines  2 , Genie J Taylor  2 , Michael P Waalkes  2 , Daniel L Morgan  2
Affiliations
Comparative Study

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

William M Gwinn et al. Toxicol Sci. 2015 Mar.

Abstract

Macrophage-solubilized indium-containing particles (ICPs) were previously shown in vitro to be cytotoxic. In this study, we compared macrophage solubilization and cytotoxicity of indium phosphide (InP) and indium-tin oxide (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. RAW 264.7 macrophages were treated with InP or ITO particles and cytotoxicity was assayed at 24 h. Ionic indium was measured in 24 h culture supernatants. Macrophage cytotoxicity and particle solubilization in vitro were much greater for InP compared with ITO. To correlate changes in vivo, B6C3F1 mice were treated with 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 leukocytes. Cell differentials, lactate dehydrogenase activity, and protein levels were also measured in BAL. All lavage parameters were greatly increased in mice treated with InP compared with ITO. These data suggest that macrophage solubilization and cytotoxicity of some ICPs in vitro are capable of predicting pulmonary toxicity in vivo. In addition, these differences in toxicity were observed despite the two particulate compounds containing similar amounts of indium suggesting that solubilization, not total indium content, better reflects the toxic potential of some ICPs. Soluble InCl3 was shown to be more cytotoxic than InP to macrophages and lung epithelial cells in vitro further suggesting that ionic indium is the primary cytotoxic component of InP.

Keywords: InP; indium; macrophage cytotoxicity; pulmonary toxicity; sintered ITO; solubilization.

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Figures

FIG. 1.
FIG. 1.
InP particles are more cytotoxic to macrophages compared with ITO. RAW cells were treated with InP or ITO particles (50, 100, 200, or 400 µg/ml) or medium alone for 2 h, rinsed once to wash out the remaining extracellular particles and then cultured for 24 h in medium prior to measuring cell death by MTT (A) and LDH (B) assays. Absorbance values from treated cells were divided by the mean corresponding value from untreated (medium alone) control cells and expressed as fraction control (MTT) or fold over control (LDH). Data on both graphs are presented as mean ± SEM (for 2 combined experiments). Unpaired Student’s t test was used to compare InP versus ITO at each concentration with a 2-sided P value < 0.05 considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).
FIG. 2.
FIG. 2.
Particle solubilization within macrophages and extracellular release of ionic indium are greater for InP compared with ITO. RAW cells were treated for 24 h with InP or ITO (100 or 200 µg/ml). The concentration (ppb) of total ionic indium in cell culture medium was then measured by GF-AAS (A). Control groups included supernatants from wells without cells treated with the same amount of particles in medium (background). Mean background indium concentration values were subtracted from the concentration values generated from particle-treated cells. LDH activity was also measured in the RAW cell culture medium after treatment with InP or ITO to confirm cytotoxicity (B). Data on both graphs is presented as mean ± SEM (for one representative experiment). Unpaired Student’s t test was used to compare InP versus ITO at each concentration with a 2-sided P value < 0.05 considered statistically significant (**P < 0.01, ****P < 0.0001).
FIG. 3.
FIG. 3.
Lung injury and protein leakage are greater in mice following treatment with InP compared with ITO. B6C3F1 mice were administered a single dose of InP or ITO (0.5 or 1 mg/kg) or PBS vehicle (50 µl) by oropharyngeal aspiration. On Days 14 and 28, BAL and PL were collected. LDH activity (A, B) and total protein (C, D) were measured in cell-free BAL fluid as markers of cellular injury and protein leakage. Data on graphs are presented as mean ± SEM (for one representative experiment; n = 5–7 mice per group for Day 14, n = 6–7 mice per group for Day 28). 1-way ANOVA with Tukey’s test was used to compare all 3 treatment groups (vehicle, InP and ITO) at each dose (0.5 or 1 mg/kg) with a 2-sided P value < 0.05 considered statistically significant. Only statistics for InP versus ITO at each dose are indicated on the graphs (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
FIG. 4.
FIG. 4.
Pulmonary inflammation is greater in mice following treatment with InP compared with ITO. B6C3F1 mice were administered a single dose of InP or ITO (0.5 or 1 mg/kg) or PBS vehicle (50 µl) by oropharyngeal aspiration. On Days 14 and 28, BAL and PL were collected. Total numbers of leukocytes were counted in BAL (A, B) and PL (E, F). H&E-stained cytopreps were prepared and cell differentials were determined for the BAL samples. Macrophages (MACs) and neutrophils (PMNs) in BAL are shown (C, D). Data on graphs are presented as mean ± SEM (for one representative experiment; n = 5–7 per group for Day 14, n = 6–7 per group for Day 28). 1-way ANOVA with Tukey’s test was used to compare all 3 treatment groups (vehicle, InP, and ITO) at each dose (0.5 or 1 mg/kg) with a 2-sided P value < 0.05 considered statistically significant. Only statistics for InP versus ITO at each dose are indicated on the graphs (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
FIG. 5.
FIG. 5.
Soluble InCl3 is more cytotoxic to macrophages and lung epithelial cells compared with InP. RAW (A, B) or LA-4 (C, D) cells were treated with InP particles (200 or 250 µg/ml) or InCl3 (300 or 400 µg/ml) containing equivalent concentrations of indium (150 or 200 µg/ml) for 24 or 48 h prior to measuring cell death by MTT (A, C) and LDH (B, D) assays. Absorbance values from InP or InCl3-treated cells were divided by the mean corresponding value from untreated (medium only) control cells or from control cells treated with medium containing equivalent levels of citrate buffer only (no indium), respectively, and expressed as fraction control (MTT) or fold over control (LDH). Data on graphs are presented as mean ± SEM (for one representative experiment at 24 h; for 2 combined experiments at 48 h). Unpaired Student’s t test was used to compare InP versus InCl3 at each indium concentration (at 24 h for the RAW cells in A, B; at 24 and 48 h for the LA-4 cells in C, D) with a 2-sided P value < 0.05 considered statistically significant (*P < 0.05, ***P < 0.001, ****P < 0.0001).
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