Acute particulate hexavalent chromium exposure induces DNA double-strand breaks and activates homologous recombination repair in rat lung tissue

Abstract

Hexavalent chromium [Cr(VI)] is an established human lung carcinogen, but the carcinogenesis mechanism is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a major driver of Cr(VI)-induced lung cancer. Unrepaired DNA double-strand breaks are the underlying cause, and homologous recombination repair is the primary mechanism preventing Cr(VI)-induced DNA breaks from causing chromosome instability. Cell culture studies show acute Cr(VI) exposure causes DNA double-strand breaks and increases homologous recombination repair activity. However, the ability of Cr(VI)-induced DNA breaks and repair impact has only been reported in cell culture studies. Therefore, we investigated whether acute Cr(VI) exposure could induce breaks and homologous recombination repair in rat lungs. Male and female Wistar rats were acutely exposed to either zinc chromate particles in a saline solution or saline alone by oropharyngeal aspiration. This exposure route resulted in increased Cr levels in each lobe of the lung. We found Cr(VI) induced DNA double-strand breaks in a concentration-dependent manner, with females being more susceptible than males, and induced homologous recombination repair at similar levels in both sexes. Thus, these data show this driving mechanism discovered in cell culture indeed translates to lung tissue in vivo.

Keywords: DNA double-strand break; chromosome instability; hexavalent chromium; homologous recombination repair; lung.

Graphical abstract

Fig. 1.

Cr accumulates in whole lungs after acute exposure. This figure shows Cr levels in the lung increased with dose after acute zinc chromate exposure. Error bars = Standard error of the mean. The P-values of statistical differences are shown in the graphs. A) Cr levels in whole lungs. B) Cr levels shown with a linear regression line. This figure shows a linear correlation between these 4 variables: saline, 0.2, 0.4, and 0.8 mg/kg zinc chromate. The P-value is <0.0001. C) Cr levels in left and right lungs. D) Cr levels in individual lung lobes and trachea.

Fig. 2.

Zinc levels are unaffected but the iron/chromium rations are decreased in lungs after acute exposure. This figure shows zinc levels were unaffected with dose after acute zinc chromate exposure. It also shows functional iron levels in the lung were reduced. Error bars = Standard error of the mean. The P-values of statistical differences are shown in the graphs. A) Zinc levels in whole lungs. B) Iron levels in whole lungs. C) Iron levels in individual lung lobes. D) Functional iron lung levels.

Fig. 3.

Cr(VI) induces infiltration of inflammatory cells in lung tissue after acute exposure. Error bars = Standard error of the mean. The P-values of statistical differences are shown in the graphs. A) H&E staining of lung tissue. *with arrows, @with arrows, and #with arrows, respectively indicate examples of macrophages, neutrophiles, and lymphocytes. Magnifications are 400×, scale bar = 20um. A.1) Saline exposure—macrophages accumulated in the alveolar region after exposure to saline. A.2) 0.2 mg/kg zinc chromate exposure—neutrophils and lymphocytes accumulated in the alveolar region. A.3) 0.4 mg/kg zinc chromate exposure—many neutrophils and lymphocytes accumulated in the alveolar region. A.4) 0.8 mg/kg zinc chromate exposure—a lot of neutrophils and lymphocytes accumulated in the alveolar region, with pulmonary interstitial edema and capillary congestion. B) Cr(VI)-induced inflammatory cell aggregation in the lung. C) Cr-induced nonmacrophage inflammatory cell aggregation in the lung.

Fig. 4.

Cr(VI) induces DNA double-strand breaks in the lung after acute exposure. Error bars = Standard error of the mean. The P-values of statistical differences are shown in the graphs. A) Representative images of gamma-H2A.X foci captured using confocal microscopy. First column, merged image; second column, DAPI staining; third column, gamma-H2A.X foci staining; fourth column, staining for cytokeratin, a marker of epithelial cells. Scale bar = 2 um. B) Quantification of Cr(VI)-induced DNA double-strand breaks measured as gamma-H2A.X foci.

Fig. 5.

Cr(VI) induces homologous recombination repair in the lung after acute exposure. Error bars = Standard error of the mean. The P-values of statistical differences are shown in the graphs. A) Representative images of RAD51 foci captured using confocal microscopy. First column, merged image; second column, DAPI staining; third column, RAD51 foci staining; fourth column, staining for PMCA ATPase, a marker of the cell membrane. Scale bar = 2um. B) Quantification of Cr(VI)-induced homologous recombination repair measured as RAD51 foci.

Fig. 6.

Cr(VI) induces similar DNA double-strand breaks and homologous recombination repair responses in females compared with males. Error bars = Standard error of the mean. The P-values of statistical differences are shown in the graphs. A) Cr levels in the right lung. Cr levels in the lungs increased in a dose-dependent manner in the female group and male group, respectively. The lower doses (0.2 and 0.4) in both sex groups did not show significant differences from the control, but when we used Student t-test to analyze the data, there were significant differences. B) Nonmacrophage inflammatory cell aggregation. C) DNA double-strand breaks measured as gamma-H2A.X foci in bronchioles. D) Homologous recombination repair measured as RAD51 foci in bronchioles.