Investigating the interactions between nonoparticles and UV-irradiated HaCaT keratinocytes

This study investigated the effects of a range of zinc oxide (ZnO) and titanium dioxide (TiO2) particulates, including those used in nanosunscreens, on the HaCaT human keratinocyte cell line in the presence and absence of UVA and UVB radiation (UVAB radiation). The HaCaT cells were chosen as a model human skin cell line for the in vitro exposure system, and the parameters investigated included cytotoxicity, intracellular Zn2+ and reactive oxygen species (ROS), interleukin-6 (IL-6) cytokine release, and if the mode of cell death was via the apoptotic or necrotic pathway. Cytotoxicity of the particulates to HaCaT cells was determined using the MTS viability assay. ZnO particulates and ZnCl2 were found to be cytotoxic to HaCaT cells, with significant levels of cytotoxicity occurring within 8-12 h of exposure at concentrations ≥ 30 µg/mL. Conversely, TiO2 particulates were found to be well-tolerated by HaCaT cells, even at extremely high doses (5 mg/mL). The effect of UVAB radiation was found to cause a ~50% decrease in cell viability at 24 h post exposure, compared to non-irradiated controls. The presence of particulate exposure with UVAB radiation further contributed to the cytotoxicity induced by UV alone. The proposed paradigm of ZnO particulate cytotoxicity was investigated in this study. The theory suggests that Zn2+ from the dissolution of the particulates themselves are responsible for the cytotoxicity in HaCaT cells. The level of intracellular Zn2+ was determined via flow cytometry using the specific fluorophore, zinquin ethyl ester. In this study, the amount of intracellular Zn2+ generated from particulate exposure increased in a dose-dependent manner. Furthermore, this was found to correlate with the decrease in cell viability observed using the MTS assay. Intracellular ROS production was measured via flow cytometric detection of peroxides and mitochondrial superoxide using DCF-DA and MitoSOX, respectively. Particulate exposure was found to increase mitochondrial superoxide production in a dose-dependent manner, but had no overall effect on peroxide generation. Furthermore, mitochondrial superoxide production was found to correlate with a decrease in cell viability, however peroxide production did not. Keratinocytes can release cytokines when damaged and the production of IL-6 was measured in this study via ELISA. The ability of particulates to stimulate IL-6 release was found to be negligible. In fact, IL-6 was found to be stimulated only by UVAB radiation, which was not altered by co-exposure to particulates. Cell death by apoptosis and necrosis was assessed via flow cytometry using Annexin V-FITC and propidium iodide.<br><br>The mode of cell death induced by ZnO particulates in HaCaT cells was found to be predominantly necrosis at all concentrations tested. However, as this was measured after 24 h of exposure to particulates, it may be possible that early apoptotic events may not have been detected. In conclusion, this study used a variety of techniques in determining the direct effects of ZnO and TiO2 particulates on HaCaT cells, and their interactions with co-exposure to UVAB radiation. Importantly, the concentrations of NPs used in this in vitro study are at least two orders of magnitude higher than would be expected from using a nanosunscreen. This indicates that the particulates used in sunscreens are highly likely to be safe to use by consumers when applied on the skin. However, as this study was performed in immortalised human skin cells, more complex cell models, such as primary cell cultures and human skin explants, would also need to be investigated in order to provide further confirmation of these results.