Transcriptome Profiling and Toxicity Following Long-Term, Low Dose Exposure of Human Lung Cells to Ni and NiO Nanoparticles-Comparison with NiCl2

Abstract

Production of nickel (Ni) and nickel oxide (NiO) nanoparticles (NPs) leads to a risk of exposure and subsequent health effects. Understanding the toxicological effects and underlying mechanisms using relevant in vitro methods is, therefore, needed. The aim of this study is to explore changes in gene expression using RNA sequencing following long term (six weeks) low dose (0.5 µg Ni/mL) exposure of human lung cells (BEAS-2B) to Ni and NiO NPs as well as soluble NiCl₂. Genotoxicity and cell transformation as well as cellular dose of Ni are also analyzed. Exposure to NiCl₂ resulted in the largest number of differentially expressed genes (197), despite limited uptake, suggesting a major role of extracellular receptors and downstream signaling. Gene expression changes for all Ni exposures included genes coding for calcium-binding proteins (S100A14 and S100A2) as well as TIMP3, CCND2, EPCAM, IL4R and DDIT4. Several top enriched pathways for NiCl₂ were defined by upregulation of, e.g., interleukin-1A and -1B, as well as Vascular Endothelial Growth Factor A (VEGFA). All Ni exposures caused DNA strand breaks (comet assay), whereas no induction of micronuclei was observed. Taken together, this study provides an insight into Ni-induced toxicity and mechanisms occurring at lower and more realistic exposure levels.

Keywords: RNA sequencing; genotoxicity; long-term exposure; nickel nanoparticles.

Figure 1

Low-dose, long-term exposure to nickel containing nanoparticle: experimental design. BEAS-2B cells were exposed to low doses (0.5 µg/mL) of Ni NPs, NiO NPs and NiCl₂ as ion control over 6 weeks, with cells being split and cells re-exposed twice a week. During the six-week exposure, cell proliferation was assessed on a weekly basis. At the end of the six-week exposure, RNA sequencing was performed together with phenotypic evaluation (cell transformation, cell migration and cell invasion), genotoxicity (comet assay/micronucleus test) and cellular nickel content.

Figure 2

Human lung cells (BEAS-2B) were exposed to Ni NPs, NiO NPs and NiCl₂ (0.5 µg/mL) for 6 weeks. At the indicated time-points Alamar Blue proliferation assay was performed. Results are presented as mean values ± S.D.

Figure 3

Cellular nickel content was quantified by ICP-MS after 3 and 6 weeks of exposure. Results and expressed as pg Ni/cell and are presented as mean values ± S.D. (n = 3). Significant results are indicated with asterisks (* p-value < 0.05).

Figure 4

RNA sequencing analysis of the BEAS-2B cells exposed to Ni NPs, NiO NPs or NiCl₂. (A) Venn diagrams of the differentially expressed genes (genes that have a false discovery rate (FDR)-adjusted p-value < 0.05 are considered differentially expressed). (B) Heatmap of the differentially expressed genes that overlap between the Ni, NiO and NiCl₂ exposures. (C) Combined heatmap of the differentially expressed genes shared between two of the exposures. Color indicates log2(fold change).

Figure 5

Canonical pathways enriched after six-week exposure of BEAS-2B cells to Ni, NiO nanoparticles or NiCl₂. Pathway analysis was performed in IPA on the differentially expressed genes following exposure of BEAS-2B cells to Ni, NiO nanoparticles or NiCl₂. Significantly enriched canonical pathways (−log10 (p-value) > 1.3) are illustrated, ordered according to the statistical significance. Pathways containing less than three differentially expressed genes were excluded. Some pathways are additionally characterized by z-score, a measure of the activation state of the pathway. NA, z-score not available; color coding indicates direction of differential expression, red—upregulation, blue—downregulation.

Figure 6

Genotoxicity and cell cycle analysis. (A) Induction of DNA strand breaks after six-week exposure was investigated using the alkaline comet assay. Results are expressed as % DNA in tail and presented as mean ± S.D. (n = 3). (B–C) Micronuclei formation after six-week exposure was evaluated by flow cytometry. Results are expressed as percentage of micronuclei (B) and hypodiploid nuclei (C) and presented as mean values ± S.D. (n = 3). (D) Cell cycle phase evaluation was performed by flow cytometry after 6 weeks of exposure. Results are presented as mean distribution (%) of the different cell cycle phases (G1, S, G2/M) ± S.D. (n = 3). Positive controls: MMC 24 h, mytomycin C, 0.05 µg/mL 24 h. Significant results are marked with asterisks (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001).

Figure 7

Cell transformation and motility. (A–B) Soft agar cell transformation was determined after 6 weeks of exposure. Results are expressed as total number of colonies (A) as well as the overall transformation frequency (B) in which the colony-forming efficiency was considered (mean ± S.D., n = 3). Cell migration (C) and invasion (D) was evaluated after 6 weeks of exposure and results are expressed as fold change compared to the control presented as mean ± S.D. (n = 3). Positive control: TGFβ, 15 ng/mL 72 h. Significant results are marked with asterisks (* p-value < 0.05).