Chronic Inhalation Exposure to Antimony Trioxide Exacerbates the MAPK Signaling in Alveolar Bronchiolar Carcinomas in B6C3F1/N Mice

Abstract

Antimony trioxide (AT) is used as a flame retardant in fabrics and plastics. Occupational exposure in miners and smelters is mainly through inhalation and dermal contact. Chronic inhalation exposure to AT particulates in B6C3F1/N mice and Wistar Han rats resulted in increased incidences and tumor multiplicities of alveolar/bronchiolar carcinomas (ABCs). In this study, we demonstrated Kras (43%) and Egfr (46%) hotspot mutations in mouse lung tumors (n = 80) and only Egfr (50%) mutations in rat lung tumors (n = 26). Interestingly, there were no differences in the incidences of these mutations in ABCs from rats and mice at exposure concentrations that did and did not exceed the pulmonary overload threshold. There was increased expression of p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) protein in ABCs harboring mutations in Kras and/or Egfr, confirming the activation of MAPK signaling. Transcriptomic analysis indicated significant alterations in MAPK signaling such as ephrin receptor signaling and signaling by Rho-family GTPases in AT-exposed ABCs. In addition, there was significant overlap between transcriptomic data from mouse ABCs due to AT exposure and human pulmonary adenocarcinoma data. Collectively, these data suggest chronic AT exposure exacerbates MAPK signaling in ABCs and, thus, may be translationally relevant to human lung cancers.

Keywords: MAPK pathway; alveolar/bronchiolar carcinoma; antimony trioxide; immunohistochemistry; inhalation exposure; mouse.

Figure 1.

Alveolar/bronchiolar carcinoma, H&E. A solid, densely cellular mass in the lung of a female B6C3F1/N mouse exposed to 30 mg antimony trioxide/m³ by inhalation for 2 years. Please note the scattered black aggregates of antimony trioxide dust within the parenchyma (arrows).

Figure 2.

Principal Component Analysis (PCA) showing the distribution of various sample types (Control, Spontaneous, AT-Treated). Three samples (1 control and 2 Spontaneous) that labeled poorly compared to the other samples were treated as outliers and removed from analysis.

Figure 3.

Hierarchical Cluster Analysis (HCA) comparing standardized global gene expression profiles (all 45,101 probesets on the array) of normal lungs from Control Group, alveolar/bronchiolar carcinomas from the Spontaneous Group, and alveolar/bronchiolar carcinomas from the AT-treated Group, in B6C3F1/N mice.

Figure 4.

Venn Diagram showing transcripts differentially expressed in spontaneous tumors compared to control samples, AT-exposed tumors compared to control samples and the corresponding significant overlap of differential expressed transcripts between the two tumor types.

Figure 5.

Illumina BaseSpace^™ Correlation Engine was used to compare the differentially expressed mouse antimony trioxide (AT)-exposed alveolar/bronchiolar carcinomas (ABCs) tumor data set to the most similar human NSCLC data set (from the Illumina BaseSpace^™ curated data sets as well as National Center for Biotechnology Information’s GEO database). The human NSCLC data set (GSE27262) that closely matches the mouse CMD-ABC tumor dataset was from stage one lung adenocarcinoma from patients compared to adjacent nontumor tissue using Affymetrix Human Genome U133 Plus 2.0 Array.

Figure 6.

Expression of p44/42 MAPK in B6C3F1/N mouse lung. In normal mouse lungs, p44/42 MAPK expression is predominantly in the endothelium (arrows) and a few transmigrating leukocytes (arrowheads) (A, B). However, the neoplastic alveolar/bronchiolar cells stain positive for p44/42 MAPK in the mouse lung tumors. The distribution pattern of p44/42 MAPK expression in neoplastic cells was predominantly nuclear and mainly in neoplastic cells at the periphery of the neoplasm and those infiltrating surrounding parenchyma (C).