Next-generation sequencing reveals differential responses to acute versus long-term exposures to graphene oxide in human lung BEAS-2B cells

Numerous studies have addressed the biological impact of graphene-based materials including graphene oxide (GO), yet few have focused on long-term effects. Here, we utilized RNA-sequencing to unearth responses of human lung cells to GO. To this end, the BEAS-2B cell line derived from normal human bronchial epithelium was subjected to repeated, low-dose exposures of GO (1 µg/mL or 5 µg/mL) for 28 days, or to the equivalent, cumulative amount of GO for 48 h. Then, samples were analyzed by using the NovaSeq™ 6000 sequencing system followed by pathway analysis and gene ontology enrichment analysis of the differentially expressed genes. Significant differences were seen between the low-dose, long-term exposures and the high-dose, short-term exposures. Hence, exposure to GO for 48 h resulted in mitochondrial dysfunction. In contrast, exposure to GO for 28 days was characterized by engagement of apoptosis pathways with downregulation of genes belonging to the inhibitor of apoptosis protein (IAP) family. Validation experiments confirmed that long-term exposure to GO affected the apoptosis threshold in lung cells, accompanied by a loss of IAPs. These studies have revealed the sensitivity of RNA-sequencing approaches and showed that acute exposure to GO is not a good predictor of the long-term effects of GO. The BEAS-2B cell line was originally derived from normal human bronchial epithelium obtained at autopsy, and the cells are normally maintained in serum-free, bronchial epithelial cell growth medium supplemented with specific growth factors, as detailed in the experimental section. Using these cells, we performed acute versus long-term exposures to equivalent doses of GO. Hence, we exposed BEAS-2B cells to 1 µg/mL or 5 µg/mL twice per week for up to 4 weeks (in other words, a total dose of 8 µg/mL and 40 µg/mL, respectively). In addition, we exposed the cells to the same, cumulative dose (8 µg/mL and 40 µg/mL) for 48 h. Cells were also exposed to a high dose (80 µg/mL) for 48 h, for comparison. Cells were harvested at 48 h and at 1 week and 4 weeks, respectively, and total cell RNA samples were extracted and RNA-sequencing were performed using NovaSeq™ 6000 sequencing system .