TITANIUM DIOXIDE AND ZINC OXIDE NANOPARTICLES IN SUNSCREENS 229 concentrations from 0% to 100% applied for three consecutive days were not a dermal sensitizer or skin irritant (33). In a 14-d toxicity study, TiO2 NPs applied topically to rat skin (Wistar) induced short- term toxicity at the biochemical level (34). Enzymes for which concentrations increased are lactate dehydrogenase, lipid peroxidase, serum glutamic pyruvic transaminase, and serum glutamic oxaloacetic transaminase. Depletion in the levels of catalase and glutathi- one S-transferase (GST) activity was detected. They concluded that short-term exposure to TiO2 NPs can cause hepatic and renal toxicity in rats. It should be underlined that the doses used in these studies are high (14, 28, 42, and 56 mg/kg) and humans are not ex- posed to those high concentrations (35). INFLUENCE OF TIO2 AND ZNO ON ROS GENERATION AND POTENTIAL CYTOTOXICITY Results of the recent studies provided the information that both ZnO and TiO2 NPs can generate reactive oxygen species (ROS): superoxide anions, hydroxyl radicals, and singlet oxygen (36,37). The mechanism of the reaction is UV-induced photocatalysis. ROS can damage cellular components and macromolecules, and ultimately cause cell death if pro- duced in excess or if they are not neutralized by antioxidant defenses. ROS derived from the photocatalysis of NPs are cytotoxic to a variety of cell types (38). Sayes et al. have investigated the difference between two crystal forms of TiO2 NPs in producing ROS. They reported that anatase NPs generated more ROS than rutile after UV irradiation. It has been concluded that TiO2 anatase has a greater toxic potential than TiO2 rutile. Also, anatase ROS production does not occur under ambient light conditions (39). A study by Lewicka et al. (40) reported a greater generation of ROS by ZnO NPs than TiO2 NPs. The cytotoxicity of TiO2 NPs was demonstrated in keratinocytes, using different tests and exposures, with or without UV exposure, but many in vivo experiments on animals did not confi rm this effect (41–43). Cytotoxicity studies on HaCaT cells gave an important result that TiO2 NPs induce cy- totoxic effects only at very high concentrations after 7 d (44). In vitro toxicity was also observed. Vinaredell et al. used the EpiSkin model, to determine the differences between ZnO and ZnO NPs. Formulations with ZnO and ZnO NPs were fi rst applied for 15 min and for 24 h, but cytotoxic effects were not observed. The per- centage of viability of the treated cells was around 100% for all ZnO materials, regardless of their size (45). Kiss et al. investigated in vivo penetration and effects on cell viability of TiO2 on human skin transplanted to immunodefi cient mice. They demonstrated that with TiO2 NPs, there was no penetration through the skin, but when exposed directly to cell culture in vitro, they have signifi cant effects on cell viability (23). Liu et al. have conducted an important study. During the PC12 cells treatment with different concentrations of TiO2 NPs, the viability of cells was signifi cantly decreased in the peri- ods of 6, 12, 24, and 48 h, showing a signifi cant dose effect and time-dependent manner. The number of apoptotic PC12 cell increased with the increasing concentration of TiO2 NPs (35) (Table IV).
JOURNAL OF COSMETIC SCIENCE 230 GENOTOXICITY TiO2 and ZnO NPs were investigated for their potential genotoxicity in in vitro and in vivo test systems. No genotoxicity was observed in vitro (Ames’ Salmonella gene mutation test and V79 micronucleus chromosome mutation test) or in vivo (mouse bone marrow micro- nucleus test and Comet DNA damage assay in lung cells from rats exposed by inhalation) (46). The SCCS (2012) comprehensive review of ZnO NPs revised both in vitro and in vivo studies on photo-mutagenicity/genotoxicity and concluded that there is no defi nite evi- dence to claim if ZnO NPs pose a mutagenic/genotoxic, phototoxic, or photomutagenic/ genotoxic risk to humans (47). CARCINOGENESIS There are reports that dermal application of noncoated rutile TiO2 does not exhibit a promoting effect on UVB-induced skin carcinogenesis in rats. Xu et al. researched c-Ha- ras proto-oncogene transgenic rats, which are sensitive to skin carcinogenesis, and their wild-type siblings were exposed to UVB radiation. Their back skin is shaved twice weekly for 10 weeks. On the shaved area, a suspension of 100 mg/ml TiO2 NPs was applied. In the observed groups, the tumor incidence was not different (48). Sagawa et al. have reached the same conclusion, after studying the promoting effect of silicone-coated TiO2 NPs suspended in silicone oil and noncoated TiO2 NPs suspended in Pentalan 408 on a two-stage skin chemical carcinogenesis model (49). Newman et al. also suggested that TiO2 NPs are not carcinogenic to the skin. However, the authors emphasized that further studies for the safety evaluation of the TiO2 NPs in sunscreens must be performed to simulate real-world conditions, particularly in sun- burned skin and under UV exposure (50). Table IV Cytotoxicity of TiO2 NPs in vivo Type of NPs Cells Effect Ref. TiO2 NPs 20 nm Human HaCaT and keratinocytes Induction of the mitochondrial “common deletion” in HaCaT cells following exposure to TiO2 NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. (41) TiO2, 25 nm dispersion in serum-free medium Immortalized keratinocyte cells and HaCaT cells Increase production of ROS, the toxicological effects can be simplifi ed into six events (43) TiO2 NPs Keratinocyte cells Alter the calcium homeostasis and induced a decrease in cell proliferation associated with early keratinocyte differentiation, without any indication of cell death. (42) TiO2 NPs (anatase, rutile, and anatase–rutile) sizes (4, 10, 21, 25, and 60 nm) UVA radiation Human keratinocyte and HaCaT cells Induced ROS resulted in oxidative stress in these cells by reducing SOD and increasing MDA levels and damage HaCaT cells (44)
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