Insight into the Mechanisms of Combined Toxicity of Single-Walled Carbon Nanotubes and Nickel Ions in Macrophages: Role of P2X7 Receptor.
Coexistence of nanomaterials and environmental pollutants requires in-depth understanding of combined toxicity and underlying mechanism. In this work, we found that coexposure to the mixture of noncytotoxic level of single-walled carbon nanotubes (SWCNTs) (10 μg/mL) and Ni2+ (20 μM) induced significant cytotoxicity in macrophages. However, almost equal amount of intracellular Ni2+ was detected after Ni2+/SWCNT coexposure or Ni2+ single exposure, indicating no enhanced cellular uptake of Ni2+ occurred. SDS-PAGE analysis revealed 50% more SWCNTs retained in Ni2+/SWCNT exposed cells than that with SWCNT exposure alone, regardless of the exposure sequence (coexposure, Ni2+ pre- or post-treatment), suggesting inhibited SWCNT exocytosis by Ni2+. The increased cellular dose of SWCNTs could quantitatively account for the elevated toxicity of Ni2+/SWCNT mixture to cells. It was then found that agonist (ATP) and antagonist (o-ATP) of P2X7R could regulate intracellular SWCNT amount and the cytotoxicity accordingly. In addition, inhibition of P2X7R by P2X7-targeting siRNA diminished the inhibitory effect of Ni2+. It was therefore concluded that Ni2+ impeded SWCNT exocytosis by inhibiting P2X7R, leading to higher intracellular retention of SWCNTs and elevated cytotoxicity. Our work identified exocytosis inhibition as an important mechanism for SWCNT/Ni2+ toxicity, and revealed the crucial role of P2X7R in mediating such inhibitory effect.