Cyto-Genotoxicity of Tritiated Stainless Steel and Cement Particles in Human Lung Cell Models

Abstract

During the decommissioning of nuclear facilities, the tritiated materials must be removed. These operations generate tritiated steel and cement particles that could be accidentally inhaled by workers. Thus, the consequences of human exposure by inhalation to these particles in terms of radiotoxicology were investigated. Their cyto-genotoxicity was studied using two human lung models: the BEAS-2B cell line and the 3D MucilAir^TM model. Exposures of the BEAS-2B cell line to particles (2 and 24 h) did not induce significant cytotoxicity. Nevertheless, DNA damage occurred upon exposure to tritiated and non-tritiated particles, as observed by alkaline comet assay. Tritiated particles only induced cytostasis; however, both induced a significant increase in centromere negative micronuclei. Particles were also assessed for their effects on epithelial integrity and metabolic activity using the MucilAir^TM model in a 14-day kinetic mode. No effect was noted. Tritium transfer through the epithelium was observed without intracellular accumulation. Overall, tritiated and non-tritiated stainless steel and cement particles were associated with moderate toxicity. However, these particles induce DNA lesions and chromosome breakage to which tritium seems to contribute. These data should help in a better management of the risk related to the inhalation of these types of particles.

Keywords: BEAS-2B cells; DNA damage; MucilAirTM; cement particles; chromosome damage; cytotoxicity; in vitro testing; micronuclei; stainless steel particles; tritium.

Figure 1

Quantification of tritium release from stainless steel and cement particles in cell culture medium. Tritiated particles were suspended in cell culture medium and incubated at 37 °C under agitation. Samples were collected at several time points and then tritium amount was assessed in the supernatant and in the particle suspension using liquid scintillation counting. Data are presented as mean ± SEM of three independent experiments.

Figure 2

Confocal microscopy observation of BEAS-2B cells after exposure to hydrogenated particles. (A,B) After exposure of BEAS-2B cells to stainless steel (A) or cement (B) particles, staining of plasma membrane (green), nucleus (blue), and cytoskeleton (red) was performed. Images were acquired using confocal microscopy. Particles, visualized by light reflection (pink), are indicated by white arrows. (C,D) Staining of nucleus (blue) and cytoskeleton (red) was performed on untreated cells and cells exposed to stainless steel particles. The SS316L particles (green and indicated by white arrow) seem to form depressions in the cells.

Figure 3

Cytotoxicity of hydrogenated and tritiated particles measured in BEAS-2B cells: (A) SS316L particles, 2h exposure; (B) SS316L particles, 24h exposure; (C) cement particles, 2h exposure; (D) cement particles, 24h exposure; (E) HTO, SS316L and cement particles, 24h exposure. As a positive control, cells were exposed to 9% Triton-X 100 diluted in culture medium. Data are presented as mean ± SEM of three independent experiments, each in triplicate. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparisons test; ** p < 0.01, *** p < 0.001.

Figure 4

Evaluation of DNA damage induced by SS316L and cement particles, by alkaline comet assay. Percentage of tail DNA was quantified after 2 h and 24 h of exposure to hydrogenated and tritiated SS316L (A,B) or cement particles (C,D). As a positive control, cells were exposed to 110 µM hydrogen peroxide (black bars). Cells were also exposed to tritiated water with an activity corresponding to the activity of the highest tested concentration of particles (100 kBq/mL and 8 kBq/mL). Each bar represents the mean ± SEM of two to three independent experiments. Asterisks indicate statistically significant increase compared to untreated cells with p-value of p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Figure 5

Cytokinesis Block Proliferation Index (CBPI) upon exposure to hydrogenated/tritiated SS316L (A,B) and cement (C,D) particles. Cells were also exposed to tritiated water with an activity corresponding to the highest activity of particles (100 kBq/mL and 60 kBq/mL). Each bar represents the mean ± SD of two independent experiments. Asterisks indicate statistically significant difference compared to untreated cells evaluated by one-way ANOVA, with p-value of p < 0.01 (**), or p < 0.001 (***).

Figure 6

Micronuclei frequency in BEAS-2B cells exposed to SS316L and cement particles. MMC (0.1 µg/mL) was used as clastogenic positive control. Cells were also exposed to HTO with an activity corresponding to the highest activity of particles (100 kBq/mL and 60 kBq/mL). Centromere labeling (Crest) was performed to discriminate between MN issued by a whole chromosome loss (MN Crest +) and MN resulting from chromosome breakage caused by DSB (MN Crest −). Each bar represents the mean ± SD of two independent experiments. Asterisks indicate statistically significant increase compared to untreated cells determined by Chi-square, with p-value of p < 0.05 (*), p < 0.01, (**) or p < 0.001 (***).

Figure 6

Micronuclei frequency in BEAS-2B cells exposed to SS316L and cement particles. MMC (0.1 µg/mL) was used as clastogenic positive control. Cells were also exposed to HTO with an activity corresponding to the highest activity of particles (100 kBq/mL and 60 kBq/mL). Centromere labeling (Crest) was performed to discriminate between MN issued by a whole chromosome loss (MN Crest +) and MN resulting from chromosome breakage caused by DSB (MN Crest −). Each bar represents the mean ± SD of two independent experiments. Asterisks indicate statistically significant increase compared to untreated cells determined by Chi-square, with p-value of p < 0.05 (*), p < 0.01, (**) or p < 0.001 (***).

Figure 7

Oxidative stress evaluated by GSH/GSSG ratio in BEAS-2B cells, following SS316L particles exposure. Menadione (20 µM) was used as positive control (C+). Data are presented as mean ± SEM of two independent experiments, each in duplicate. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparisons test, *** p < 0.001.