Skin Penetration Of Nano-TiO₂ And Potential Skin Damage Under UV Irradiation

As a new inorganic nanomaterial, nano titanium dioxide (nano-TiO2) has been widely used in cosmetic creams, costume, and environmental protection. Owing to the existence of nano-TiO2 in environment and sunscreen products, skin is inevitable to become potential targets. However, nano-TiO2 has high photochemical activity, its skin safety has attracted attention widely. At present, the investigation on the toxicities of nano-TiO2 is focued on its toxicities in vitro and animal short term exposure, while the study on nano-TiO2 skin penetration and potential skin damage under UV irradiation is limited. In the present study, we explored the penetration of nano-TiO2 in vitro and in vivo with different sizes (4～90 nm) and different crystal forms (anatase, rutile and anatase/rutile). The cytotoxicity and oxidative stress induced by nano-TiO2 under UV irradiation in human keratinocytes (HaCaT) cells were investigated. Furthermore, the potential of N-acetylcysteine (NAC) in protecting the HaCaT cells from nano-TiO2 induced toxicities was also studied. The main results are as followings:(1) Horizontal diffusion chamber, sucking pig and Bulb/c nude mice were used to investigate skin penetration of nano-TiO2 in vitro and in vivo. The study in vitro indicated that nano-TiO2 could not penetrate through stratum corneum and mostly locate in skin pores and folds. However, study in vivo indicated that after 30 days skin exposure of sucking pig ear, nano-TiO2 could penetrate through horny layer and locate in cytoplasm of cells. Cells damages, such as vacuole around nucleus, desmosomes disappearance were also observed. Further more, the study in mice obtained a more clearly presentation on its tissue distribution and potential toxicities after skin exposure for 60 days. The most important pathological changes were inflammatory infiltrates and foci of necrosis. The skin of Bulb/c nude mice showed more wrinkles, loose and lacking brightness with the significant changes in SOD and MDA levels, which suggested that these lesions are likely to be mediated with oxidative stress by the deposited nano-TiO2. Accordingly, the content of collagen expressed as HYP was also reduced in mice skin. It indicates that topically applied nano-TiO2 on skin with a prolonged period could induce skin aging.(2) MTT assay was used to evaluate the cytotoxicity of nano-TiO2. Flow cytometry and fluorospectrophotometer were used to evaluate the apoptosis, ROS level and mitochondrial membrane potential. Transmission electron microscope(TEM) was used to observe the distribution of nano-TiO2 in HaCaT cells. The results of toxicities study in vitro on HaCaT cells showed that nano-TiO2 could significantly reduce cell viability in a dose-dependent manner. The ROS generated by nano-TiO2 produced oxidative stress in HaCaT cells, the mitochondrial membrane potential of cells was declined significantly while the apoptosis rate was increased. Nano-TiO2 was appeared to enter the cells by endocytosis and stay in cytoplasm as aggregates and/or agglomerates.(3) The way of nano-TiO2 generating ROS and the cytotoxicity mechanism were also investigated by adding the inhibitors of respiratory chain and permeability transition pore, uncouplers of mitochondrial oxidative phosphorylation. Results showed that pretreatment inhibitors of mitochondrial respiratory and the uncouplers of mitochondrial oxidative phosphorylation resulted in obvious changes in the production of intracellular ROS induced by nano-TiO2. The contribution of extramitochondrial ROS source to ROS overproduction induced by nano-TiO2 were also examined. The ROS sources of lipoxygenase, cyclooxygenase and NAD(P)H oxidase had no significant effect on the ROS production induced by nano-TiO2. To some extent, NO synthase had effect on the ROS production. Cyclosporin A could reduce the cytotoxicity of nano-TiO2. These results indicated that mitochondrial respiratory chain maybe the main source of intracellular ROS production induced by nano-TiO2. The mitochondrial permeability transition plays an important role in the cytotoxicity of nano-TiO2.(4) The potential of NAC in protecting HaCaT cells from nano-TiO2 induced oxidative stress and apoptosis under UV irradiation was also investigated. The results showed that NAC could prevent nano-TiO2 inducing oxidative stress and apoptosis in HaCaT cells, maintain mitochondrial membrane potential and protect mitochondrion. NAC inhibited the suppression of nano-TiO2 on Keratine6 mRNA expression. These results indicated that the inhibitory mechanism of NAC on nano-TiO2 induced apoptosis involves the modulation of the intracellular ROS level and the suppression of oxidative stress.In conclusion, the safty of nano-TiO2 on skin was evaluated systematically in this paper. The skin penetration of nano-TiO2 in vivo was observed in sucking pig and Bulb/c nude mice for the first time. Results showed that nano-TiO2 could penetrate through horny layer, exhibite tissue distribution and potential toxicities after long skin exposure. The generation intracellular ROS induced by nano-TiO2 and potential damage in HaCaT cells under UV irradiation were also investigated. Nano-TiO2 could significantly enhance ROS level and injury cell membrane and mitochondrion and induce cell apoptosis. These results indicated that mitochondrial respiratory chain maybe the main source of intracellular ROS production. The mitochondrial permeability transition plays an important role in the cytotoxicity of nano-TiO2. NAC could prevent the oxidative damage and apoptosis inducing by nano-Ti02 in HaCaT cells by mediating the intracellular oxidative stress.