Oral Presentation 6th Australian Health and Medical Research Congress 2012

ZnO nanoparticles (NPs) have been widely formulated in sunscreens, raising public and scientific concerns about the safety of certain nanomaterial applications. Although it has been reported that Zn²⁺ release from ZnO NPs is related to their cytotoxic potential, it is still unclear whether the cytotoxicity is induced by either intracellular or extracellular dissolution of NPs, with subsequent release of Zn²⁺. To address this question, we exposed human THP-1 monocytes for 24 hr to a broad concentration range of ZnO NPs, or the dialysate generated from ZnO NPs incubated in medium during a prior 24 hr period. We found that whilst direct exposure to ZnO NPs induced cytotoxicity at the relatively high concentrations of ≥25 µg/mL (comparable to ZnCl₂ as the zinc ion control), the NP-free dialysates were not cytotoxic, even from extreme initial ZnO NP concentrations (5 mg/mL). Using the specific dye (zinquin ethyl ester) for intracellular Zn²⁺ levels, we found a strong correlation with ZnO NP-induced cytotoxicity (R²=0.945), suggesting the requirement of NP dissolution for cytotoxicity. Also, intracellular superoxide levels (by MitoSOX Red) were strongly correlated with both intracellular Zn²⁺ levels (R²=0.906) and cytotoxicity (R²=0.826). We also co-exposed antioxidants (α-tocopherol or L-ascorbic acid, ≤10 µg/mL) with ZnO NPs in our cell system and found that intracellular superoxide generation was slightly inhibited by both antioxidants, but cell viability was not improved. This suggests that the intracellular superoxide levels were not the sole cause of ZnO NP-induced cytotoxicity in this test system. These experiments show that ZnO NPs must be at least in contact with the cells, resulting in their uptake and intracellular dissolution, for the cytotoxic response.