An investigation of the in vitro toxicity of zinc oxide nanoparticles in UV-irradiated skin cells

Zinc oxide (ZnO) nanoparticles (NP) have been incorporated into sunscreens for almost three decades. Compared to larger particles these NP have increased transparency and thereby improve the aesthetical appeal of sunscreens. Sunscreens play an important role in the public health strategies for dealing with the adverse health outcomes associated with ultraviolet (UV) radiation exposure such as sunburn and cancer. However, there is a growing negative public perception associated with the use of NP in sunscreen. Without empirical data, this perception may have an unintended consequence - a decline in the use of sunscreen.

This study investigated the in vitro effects of ZnO NP and chemical UV filters commonly used sunscreens on the function of immortalised and primary skin cells. ZnO particulates (30 nm, 80 nm and 200 nm) were compared to the chemical UV filters: butylmethoxydibenzoylmethane (BMDBM) ethylhexylmethoxycinnamate (EHMC), ethylhexylsalicylate (EHS), homomenthylsalicylate (HMS) and 4-methylbenzylidenecamphor (MBC). Human epidermal keratinocytes (NHEK), epidermal melanocytes (NHEM), dermal fibroblasts (NHDF) and the immortalised keratinocyte HaCaT cell line were used in this study. Cells were exposed to UVB and/or UVA radiation at a dose that is equivalent to fewer than 10 min of sun exposure at noon during Spring to Summer in Sydney, Australia (33.86°S latitude). The cells were also exposed to biological relevant nominal NP concentrations. The parameters investigated included: cytotoxicity, cell death pathways, intracellular Zn2+ and reactive oxygen species (ROS), cytokine release and the effects elicited by antioxidants.

Cytotoxicity was determined using the MTS viability assay. ZnO particulates were found not to be photoprotective. While a hormetic-like response was observed at low concentrations, overall ZnO particulates treatment resulted in a concentration-related increase in cytotoxicity in sham- and UV-irradiated cells. No particle size-related cytotoxicity was observed. Similarities in the concentration-response curves for ZnO particulates and ZnCl2 suggest that Zn2+ may contribute to ZnO NP cytotoxicity. ZnO particulates induced comparable cytotoxicity amongst the primary skin cells, however, near complete cytotoxicity occurred at ZnO particulate concentrations one order of magnitude lower than that for the corresponding immortalised HaCaT cells. In contrast chemical UV filters caused similar cytotoxicity in both primary and immortalised keratinocytes and BMDBM and EHMC were photoprotective in both cell lines. At 4 h, necrosis was the predominant mode of cell death in both sham- and UV-irradiated HaCaT cells and NHEK exposed to Zn particulates.

Intracellular Zn2+ levels were determined via flow cytometry using the fluorophore, zinquin ethyl ester. Elevated Zn2+ levels were observed in sham- and UV-irradiated Zn particulate treated NHEK and HaCaT cells. The shallow concentration-related increase of intracellular Zn2+ observed in NHEK may indicate that a homeostatic response may occur in these cells. Intracellular ROS production was measured via flow cytometric detection of peroxides and mitochondrial superoxide using DCF-DA and MitoSOX™ Red, respectively. A concentration-related increase in peroxide levels was observed following Zn particulate treatment in sham- and UV-irradiated HaCaT cells. Chemical UV filters caused significant increase in peroxide levels in sham-irradiated HaCaT cells but following irradiation levels increased to maximal levels at low concentration and then decreased at higher concentrations. Zn particulate treatment increased peroxide levels in sham-irradiated NHEK. However, when these cells were UV-irradiated peroxide levels decreased. In sham- and UV irradiated NHEK, peroxide levels decreased following UVA or UVB chemical filter treatment but only in the latter after an initial increase at low concentrations.

In sham- and UV-irradiated HaCaT cells and NHEK, Zn particulates generated a significant concentration-related increase in mitochondrial superoxide levels. The chemical UVA filter increased mitochondrial superoxide levels whilst the UVB filter had an opposite effect in HaCaT cells. In sham-irradiated NHEK, the chemical UVA filter reduced mitochondrial superoxide levels while the UVB filter had the opposite effect. In contrast, in UV-irradiated NHEK, the chemical UV filter treatments decreased mitochondrial superoxide generation.

N-acetyl cysteine (NAC) and ascorbic acid were used examine the role of oxidative stress in Zn particulate elicited cytotoxicity. In HaCaT cells, cytotoxicity increased following co-treatment with Zn particulate and antioxidants. In contrast, NHEK cell viability either remained unchanged or increased. It is speculated that these paradoxical results are a consequence not only of the antioxidant properties of NAC and ascorbic acid but also their ability to act as Zn chelators and alter the culture media pH. These experiments and in particular those with ascorbic acid suggest that ZnO ROS generation may be secondary to cytotoxicity.

IL-6 was the principal bioactive molecule secreted by those cells treated with UV filters. It was measured in this study via ELISA. Overall, in sham-irradiated UV filter-treated HaCaT cells and primary skin cells, IL-6 levels tended to increase and decrease, respectively. A significant decrease in IL-6 release was observed in UV-irradiated cells treated with Zn particulates. Attenuation of IL-6 release may be due the anti-inflammatory properties of Zn. In conclusion, this study has shown primary skin cells namely NHEK as the most sensitive and relevant skin cell type to conduct cytotoxicity studies involving ZnO NP used in sunscreens. The data obtained from the immortalised HaCaT cells line differed from that obtained primary skin cells and this may be due absence of functional p53 gene in the former. ZnO NP, at concentrations higher than those normally expected during consumer sunscreen use are cytotoxic to primary skin cells and increased peroxide and mitochondrial superoxide, and decreased IL-6. Chemical UV filters tended to be better tolerated than ZnO particulates. However, as this study was performed in immortalised single human skin cells culture, more complex cell models, such as primary cell co-cultures and human skin explants, would also need to be investigated in order to provide further confirmation of these results