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. 2014 Jan 15:760:33-41.
doi: 10.1016/j.mrgentox.2013.11.001. Epub 2013 Nov 27.

Arsenic is cytotoxic and genotoxic to primary human lung cells

Hong Xie  1 , Shouping Huang  2 , Sarah Martin  3 , John P Wise Sr  2
Affiliations

Arsenic is cytotoxic and genotoxic to primary human lung cells

Hong Xie et al. Mutat Res Genet Toxicol Environ Mutagen. .

Abstract

Arsenic originates from both geochemical and numerous anthropogenic activities. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to high levels of arsenic in drinking water has been linked to bladder, lung, kidney, liver, prostate, and skin cancers. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer and few studies have considered effects in normal human lung cells. The purpose of this study was to determine the cytotoxicity and genotoxicity of arsenic in human primary bronchial fibroblast and epithelial cells. Our data show that arsenic induces a concentration-dependent decrease in cell survival after short (24h) or long (120h) exposures. Arsenic induces concentration-dependent but not time-dependent increases in chromosome damage in fibroblasts. No chromosome damage is induced after either 24h or 120h arsenic exposure in epithelial cells. Using neutral comet assay and gamma-H2A.X foci forming assay, we found that 24h or 120h exposure to arsenic induces increases in DNA double strand breaks in both cell lines. These data indicate that arsenic is cytotoxic and genotoxic to human lung primary cells but lung fibroblasts are more sensitive to arsenic than epithelial cells. Further research is needed to understand the specific mechanisms involved in arsenic-induced genotoxicity in human lung cells.

Keywords: Arsenic; Chromosome aberration; DNA double strand breaks; Genotoxicity; Human lung epithelial cells; Human lung fibroblasts.

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Figures

Figure 1
Figure 1. Cytotoxicity of Sodium Arsenite in Primary Human Lung Cells
This figure shows that sodium arsenite induces a time- and concentration-dependent decrease in relative cell survival in human lung cells. Cells were treated with sodium arsenite for 24 h or 120 h. A) Sodium arsenite decreases relative cell survival in human lung fibroblasts. B) Sodium arsenite decreases relative cell survival in human lung epithelial cells. Data represent four independent experiments ± standard error of mean. *Statistically different from control (p<0.05). ** Statistically different from control (p<0.005).
Figure 2
Figure 2. Sodium Arsenite Induces Chromosome Damage in Primary Human Lung Cells
This figure shows that sodium arsenite induces a concentration-dependent increase in chromosome damage in fibroblasts but not in epithelial cells. Cells were treated with sodium arsenite for 24 h or 120 h. A) Chromosome damage in fibroblasts after 24 h or B) 120 h treatment. C) Chromosome damage in epithelial cells after 24 h and D) 120 h treatment. NM: No metaphase. Data represent the chromosome damage found in 100 metaphases and are the means of three independent experiments ± standard error of mean. *Statistically different from control (p<0.05). ** Statistically different from control (p<0.005).
Figure 2
Figure 2. Sodium Arsenite Induces Chromosome Damage in Primary Human Lung Cells
This figure shows that sodium arsenite induces a concentration-dependent increase in chromosome damage in fibroblasts but not in epithelial cells. Cells were treated with sodium arsenite for 24 h or 120 h. A) Chromosome damage in fibroblasts after 24 h or B) 120 h treatment. C) Chromosome damage in epithelial cells after 24 h and D) 120 h treatment. NM: No metaphase. Data represent the chromosome damage found in 100 metaphases and are the means of three independent experiments ± standard error of mean. *Statistically different from control (p<0.05). ** Statistically different from control (p<0.005).
Figure 3
Figure 3. Arsenic Induces Aneuploidy in Primary Human Lung Fibroblasts
This figure shows that chronic exposure to sodium arsenite increases in aneuploidy in fibroblasts. A) Percent of metaphases with aneuploidy. B) Percent of metaphases that are diploid, hypodiploid, hyperdiploid, or tetraploid after 24 h or C) 120 h exposure to sodium arsenite. Data represent three independent experiments ± standard error of mean.*Statistically different from control (p<0.05).
Figure 3
Figure 3. Arsenic Induces Aneuploidy in Primary Human Lung Fibroblasts
This figure shows that chronic exposure to sodium arsenite increases in aneuploidy in fibroblasts. A) Percent of metaphases with aneuploidy. B) Percent of metaphases that are diploid, hypodiploid, hyperdiploid, or tetraploid after 24 h or C) 120 h exposure to sodium arsenite. Data represent three independent experiments ± standard error of mean.*Statistically different from control (p<0.05).
Figure 4
Figure 4. Sodium Arsenite Induces Mitotic Abnormalities in Primary Human Lung Fibroblasts
This figure shows that sodium arsenite induces premature centromere division and premature anaphase in fibroblasts. Cells were treated with sodium arsenite for 24 or 120 h. A) Percent of metaphases with mitotic abnormalities after 24 h exposure or B) 120 h exposure in lung fibroblasts. C) Representative pictures of normal metaphase (1), premature centromere division (2), premature anaphase (3) and centromere spreading (4). Data represent three independent experiments ± standard error of mean.*Statistically different from control (p<0.05). ** Statistically different from control (p<0.005).
Figure 4
Figure 4. Sodium Arsenite Induces Mitotic Abnormalities in Primary Human Lung Fibroblasts
This figure shows that sodium arsenite induces premature centromere division and premature anaphase in fibroblasts. Cells were treated with sodium arsenite for 24 or 120 h. A) Percent of metaphases with mitotic abnormalities after 24 h exposure or B) 120 h exposure in lung fibroblasts. C) Representative pictures of normal metaphase (1), premature centromere division (2), premature anaphase (3) and centromere spreading (4). Data represent three independent experiments ± standard error of mean.*Statistically different from control (p<0.05). ** Statistically different from control (p<0.005).
Figure 5
Figure 5. Sodium Arsenite Induces DNA Double Strand Breaks in Primary Human Lung Cells
This figure shows that sodium arsenite induces an increase in comet tail intensity measured by neutral comet assay. A) Comet tail intensity after 24 or 120 h sodium arsenite treatment in lung fibroblasts. Control values of 7 for 24 h treatment and 8 for 120 h treatment were subtracted from treated values. B) Comet tail intensity after 24 or 120 h sodium arsenite treatment in lung epithelial cells. Control values of 7 for 24 h treatment and 4 for 120 h treatment were subtracted from treated values. C) Photomicrographs of human lung fibroblasts (top) and epithelial cells (bottom) following 0 and 1 uM sodium arsenite treatment using neutral micro gel eletrophoresis. One hundred cells per data point were analyzed in each experiment. Data represent an average of at least three independent experiments ± standard error of mean.*Statistically different from control (p<0.05).
Figure 5
Figure 5. Sodium Arsenite Induces DNA Double Strand Breaks in Primary Human Lung Cells
This figure shows that sodium arsenite induces an increase in comet tail intensity measured by neutral comet assay. A) Comet tail intensity after 24 or 120 h sodium arsenite treatment in lung fibroblasts. Control values of 7 for 24 h treatment and 8 for 120 h treatment were subtracted from treated values. B) Comet tail intensity after 24 or 120 h sodium arsenite treatment in lung epithelial cells. Control values of 7 for 24 h treatment and 4 for 120 h treatment were subtracted from treated values. C) Photomicrographs of human lung fibroblasts (top) and epithelial cells (bottom) following 0 and 1 uM sodium arsenite treatment using neutral micro gel eletrophoresis. One hundred cells per data point were analyzed in each experiment. Data represent an average of at least three independent experiments ± standard error of mean.*Statistically different from control (p<0.05).
Figure 6
Figure 6. Sodium Arsenite Induces Gamma-H2A.X Foci Formation in Primary Human Lung Cells
This figure shows that sodium arsenite induces an increase in DNA double strand breaks measured as gamma-H2A.X foci formation. A) Quantification of gamma-H2A.X foci after 24 or 120 h treatment with sodium arsenite in lung fibroblasts. Control values of 17% of cells with more than 5 gamma-H2A.X foci for 24 h treatment and 6% for 120 h treatment were subtracted from treated values. B) Quantification of gamma-H2A.X foci after 24 or 120 h treatment with sodium arsenite in lung epithelial cells. Control values of 14% of cells with more than 5 gamma-H2A.X foci for 24 h treatment and 15% for 120 h treatment were subtracted from treated values. C) Photomicrographs of gamma-H2A.X foci (green) in human lung fibroblasts (top) and epithelial cells (bottom) treated with sodium arsenite. Nuclei were stained with DAPI (blue). One hundred cells per data point were analyzed. Foci numbers were counted with ImageJ program. Data represent an average of at least three independent experiments ± standard error of mean. *Statistically different from control (p<0.05).
Figure 6
Figure 6. Sodium Arsenite Induces Gamma-H2A.X Foci Formation in Primary Human Lung Cells
This figure shows that sodium arsenite induces an increase in DNA double strand breaks measured as gamma-H2A.X foci formation. A) Quantification of gamma-H2A.X foci after 24 or 120 h treatment with sodium arsenite in lung fibroblasts. Control values of 17% of cells with more than 5 gamma-H2A.X foci for 24 h treatment and 6% for 120 h treatment were subtracted from treated values. B) Quantification of gamma-H2A.X foci after 24 or 120 h treatment with sodium arsenite in lung epithelial cells. Control values of 14% of cells with more than 5 gamma-H2A.X foci for 24 h treatment and 15% for 120 h treatment were subtracted from treated values. C) Photomicrographs of gamma-H2A.X foci (green) in human lung fibroblasts (top) and epithelial cells (bottom) treated with sodium arsenite. Nuclei were stained with DAPI (blue). One hundred cells per data point were analyzed. Foci numbers were counted with ImageJ program. Data represent an average of at least three independent experiments ± standard error of mean. *Statistically different from control (p<0.05).
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