Time-dependent degradation of carbon nanotubes correlates with decreased reactive oxygen species generation in macrophages

Introduction and objective: With the increase in carbon nanotube-based products on the commercial market, public concern regarding the possible toxicity of these nanomaterials has attracted much attention. Although previous studies found no obvious toxicity related to carbon nanotubes (CNTs), their safety has not been established because long-term evaluation is still needed. In vitro assays are used to understand the toxicity of nanomaterials. However, the data published so far were generated in short-term assays in which cells are continuously exposed to CNTs. Therefore, the objective of this study is to quantitatively assess the relative long-term cytotoxicity and degradation of CNTs after uptake by macrophages. Methods: We used macrophage cell line of RAW 264.7 and primary rat Kupffer cells to investigate macrophage uptake of CNTs as well as their quantity changes up to a relatively late time point after uptake (7 days) by measuring optical absorbance in the near infrared region and Raman spectra of CNTs in the cell lysates. The time-dependent cytotoxicity was evaluated by measuring reactive oxygen species (ROS), glutathione, cell viability, and caspase 3/7 activity in 1-7 days. Results: CNTs were degraded by approximately 25-30% within first 4 days after uptake; however, and no additional degradation occurred for the remainder of the 7-day test period. Generation of ROS by macrophages decreased as CNT degradation occurred, returning to control levels by Day 7. In the meantime, the glutathione level gradually recovered over time. There were no changes in cell viability or caspase 3/7 activation during CNT degradation. Conclusion: These results confirm that degradation of CNTs by macrophages is associated with ROS generation. The data also suggest that CNT cytotoxicity decreases as they are degraded.