Copper oxide nanoparticles (CuO NPs) are considered for various technological and consumer applications, leading to growing concerns for potential human hazards resulting from inhalation. Recent studies have revealed a dose-dependent toxicity of CuO NPs in rats following short-term inhalation exposure. Here, we utilized transcriptomics approaches to further investigate the responses in rats exposed via inhalation for five consecutive days to two doses of CuO NPs, 3.3 (low dose, LD) and 13.2 (high dose, HD) mg/m3, with collection of lung tissues on days 6 (1 day post-exposure) and 28 (i.e., after a 22-day post-exposure recovery period). Histopathology confirmed an acute inflammatory response that was resolved during the post-exposure phase. Global gene expression analyses yielded about 1,000 differentially expressed genes in HD rats and 200 in LD on day 6, and less than 20 after the recovery period. Pathway analysis indicated cell proliferation/survival and inflammation as the main processes triggered by exposure and identified epithelial cell transforming protein 2 (Ect2) as a potential gene of interest implicated in cell proliferation. Indeed, ECT2 was upregulated in exposed lungs and was localized in the cytoplasm of alveolar epithelial cells in HD rats, in hyperplasic foci identified based on the Ki67 antigen, a marker of cell proliferation. Monocyte chemoattractant protein 1 (MCP-1, also known as CCL2), with an important role in the regulation of inflammatory processes and cell proliferation, was also upregulated and this was confirmed immunohistochemically. Finally, we could not find any evidence for aberrant DNA methylation of inflammation-associated genes in response to CuO NP exposure. In summary, the current findings suggest that airborne CuO NPs cause an acute response that translates into inflammation and cellular proliferation in bronchoalveolar epithelium, with upregulation of neoplasia-related factors even after a short-term exposure.
Overall design: This experiment examined lung tissues from Wistar Unilever outbred rats (strain HsdCpb:WU) exposed to 6 hour equivalent doses of 3.3 and 13.2 mg NPs / m3 of copper oxide nanoparticles by nose only inhalation according to the OECD test Guideline 403 for Acute Inhalation Toxicity Test. Rats were exposed to five consecutive days and lung tissues were sampled on day 1 after the last exposure (referred to as T6) and day 22 post-exposure (referred to as T28). Control animals were exposed to air only. Each treatment group consisted of 5 animals. Exposure and sample collection were conducted at the National Institute for Public Health (RIVM) at Bilthoven, The Netherlands, under permit number 201300190. Experimental procedures were in accordance with the European directive on the protection of animals for scientific purposes (Directive 2010/63/EU).
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