Lung injury, inflammation and Akt signaling following inhalation of particulate hexavalent chromium

Abstract

Certain particulate hexavalent chromium [Cr(VI)] compounds are human respiratory carcinogens that release genotoxic soluble chromate, and are associated with fibrosis, fibrosarcomas, adenocarcinomas and squamous cell carcinomas of the lung. We postulate that inflammatory processes and mediators may contribute to the etiology of Cr(VI) carcinogenesis, however the immediate (0-24 h) pathologic injury and immune responses after exposure to particulate chromates have not been adequately investigated. Our aim was to determine the nature of the lung injury, inflammatory response, and survival signaling responses following intranasal exposure of BALB/c mice to particulate basic zinc chromate. Factors associated with lung injury, inflammation and survival signaling were measured in airway lavage fluid and in lung tissue. A single chromate exposure induced an acute immune response in the lung, characterized by a rapid and significant increase in IL-6 and GRO-alpha levels, an influx of neutrophils, and a decline in macrophages in lung airways. Histological examination of lung tissue in animals challenged with a single chromate exposure revealed an increase in bronchiolar cell apoptosis and mucosal injury. Furthermore, chromate exposure induced injury and inflammation that progressed to alveolar and interstitial pneumonitis. Finally, a single Cr(VI) challenge resulted in a rapid and persistent increase in the number of airways immunoreactive for phosphorylation of the survival signaling protein Akt, on serine 473. These data illustrate that chromate induces both survival signaling and an inflammatory response in the lung, which we postulate may contribute to early oncogenesis.

Figure 1. Characterization of Basic Zinc Chromate

A) Particle size distribution of basic zinc chromate particles suspended in saline at a concentration of 2.4 mg/mL. Analysis was completed on a Malvern Multisizer 2000 laser diffractor at Particle Technology Labs, Ltd (Downers Grove, IL). The average particle size is 4.73 µm. B) BALB/c mice were exposed to 50 µL of 1.2 mg/mL basic zinc chromate or saline. The Cr content of the right portion of the lung was quantified by ICP-AES. Data are the mean ± SEM for 4 animals. * indicates a statistically significant difference, as compared to saline at p=0.001.

Figure 2. Chromate Induces Apoptosis and Karyorrhexis

Bright field microscopy pictures taken of lung sections obtained from mice at 24 hrs post-intranasal exposure to 50 µL of 1.2 mg/mL basic zinc chromate suspended in saline. Sections were stained with Giemsa and images were taken at an original magnification of 40×. A) Cells in the airways of chromate-exposed animals exhibit apoptosis (red arrows) and karyorrhexis (circled). B) Cropped 40× original magnification of the boxed region in A.

Figure 3. Chromium Induces Changes in Leukocyte Subsets after Acute Cr(VI) Inhalation

BALBc mice were exposed to 50 µL of 1.2 mg/mL basic zinc chromate over a period of 24 hrs. A) The number of living cells present in BAL fluid were quantified for 5 animals in each experiment, and the change in cell number from the 0 hr time point was determined. Data are the mean ± SEM of two independent experiments, n=5–10 animals/time point. * indicates a statistically significant difference from time 0 at p<0.05. B) Representative dot plots of forward scatter/side scatter (FSC/SSC), GR-1/FcεRIα staining, and CD3/CD11c staining from cells in the BAL fluid collected at indicated time points. Blue and red regions on FSC/SSC plots highlight the location of neutrophils and macrophages, respectively. Blue and red boxes corresponding to these same two populations are shown on the fluorescence plots. C) Changes in total neutrophil and D) macrophage cell numbers at 0–24 hrs post-Cr(VI) exposure. Data are the mean ± SEM of 5 mice from a representative experiment. * denotes a statistically significant difference from time 0 at p<0.01. E) Quantification and representative images (40× original magnification) of cells in the BAL fluid and stained with H&E. Macrophages and neutrophils are indicated be * and arrows respectively. Data are the mean ± SEM of 3 Cr(VI)-treated and 2-saline treated animals 24 hrs after exposure * denotes a statistically significant difference from saline at p<0.05.

Figure 4. Increases in Cytokine Levels After Acute Cr(VI) Exposure

A)IL-6 and B)GROα levels were measured in BAL fluid at 0–24 hrs post-Cr(VI) exposure. Data are the mean ± SEM of two independent experiments, n=5–10 animals/time point. * indicates a statistically significant difference from time 0 at p<0.05.

Figure 5. Chromium Induces Injury and Inflammation in the Lung

Bright field microscopy pictures taken of lung sections obtained from mice up to 48 hrs post-Cr(VI) exposure to 50 µL of 1.2 mg/mL basic zinc chromate suspended in saline. Sections were stained with H&E and images were taken at 10× (A, C) and 40× (B, D) original magnification. B) Chromate-exposed animals exhibit proximal and mid-proximal toxic airway mucosal injury with sloughing of airway epithelial cells (*), degenerative changes, and periductal inflammation (red arrows). C) Injury in chromate-exposed animals is primarily located in the central region of the lungs while near-pleural lung regions show less response to chromate treatment at 24 hrs. D) Chromate-exposed animals exhibit parenchymal inflammation with features of alveolar and interstitial pneumonitis and in the central regions of the lungs at 48 hrs post-chromate exposure.

Figure 6. Akt Phosphorylation Increases in Airways Exposed to Cr(VI)

A) Bright field microscopy pictures taken of lung sections obtained from mice at 0–24 hrs post-intranasal exposure to 50 µL of 1.2 mg/mL basic zinc chromate suspended in saline. Lung sections were stained by immunohistochemistry with phosphospecific serine-473 AKT antibody and representative images were taken at 40×, original magnification. B) Quantification of phospho-Akt staining frequency and intensity in lung airways. Staining intensity was scored on a scale of 1–3, where 1 is a low level of staining and 3 is strong staining. Data are the mean ± SEM of 4 mice and * denotes a statistically significant difference from time 0 at p<0.01.