| Due to the specific physical structure, nanoparticles exihibit a lot of unique propertieswhich are significantly different from ordinary materials, including the optical, thermal,electrical, magnetic, mechanical and chemical properties. For the widely application ofnanomaterials in agriculture, industry, information industry, environmental protection,energy and biomedical industry, the security of nanoparticles have drawn more attentionand have been researched a lot. Titanium dioxide nanoparticle (nano TiO2) and zinc oxidenanoparticle (nano ZnO) are widely applied in detoxification or remediation of wastewater,sunscreen cosmetics and antibacterial materials because of its excellent performance inphoto-catalytic activity.Recent research suggests that nano ZnO and nano TiO2could generate reactivespecies under the irradiation of ultraviolet. These species would induce damage to humanskin tissue, while the damage depends on the exposed environment. Nitrite (NO2-) existsin human body fluids, such as sweat, saliva, blood, etc. In general, the content of NO2-onhuman skin surface is possible to change with the evaporation of sweat. Besides, as apreservative, NO2-is commonly applied in cosmetic products. Previously researchers havenoticed the skin damage induced by nano TiO2and NO2-under ultraviolet irradiation.However, there is little information on the impact of some important factors. In this paper,in order to investigate the effects and mechanism of the photo-catalytic damage to proteinand the photo-toxicity to skin cell induced by nano ZnO and nanoTiO2under ultravioletirradiation in the presence of NO2-, bovine serum album (BSA) was used as model proteinand human keratinocyte (HaCaT) cells as skin cell model. In addition, the protective effectof ascorbic acid on the photo-damage was investigated on the basis of the results.Themain results are as follows:(1) The UV-vis absorbance, fluorescence intensity and synchronous fluorescence ofeach sample were determined to evaluate the damage of BSA molecules structure and themain damage sites by nano ZnO with different nano sizes under different wavelengths ofultraviolet irradiation in the presence of NO2-. The influence of the concentration of nanoZnO and NO2-, irradiation time, the pH value in the medium were individually discussed. The results showed that nitrite dramatically enhanced the damage in the structure of BSA,and the damage site of BSA was mainly in tryptophan residues, while tyrosine residuesfollowed. The effects of photo-catalytic damage to BSA structure were dependent on theparticle size of nano ZnO and the wavelength of ultraviolet light. Overall,254nm UVirradiation induced more photodamage to BSA than365nm UV irradiation in the sameoutput power, and higher concentration of nano ZnO and NO2-, more ultravioletirradiation time and the higher value of pH in the medium could all enhance thephoto-damage of protein.(2) HaCaT Cell’s viability was assessed by MTT assay, and apoptosis was measuredby morphology examination. The results indicated that smaller sized ZnO induced morecytotoxicity in HaCaT cells. Additionally, nitrite enhanced the photo-toxicity of nano ZnOand nano TiO2, and resulted in apoptosis of human keratinocyte cells.(3) Protein oxidation and protein tyrosine nitration in BSA that photo-damaged bynano ZnO or nano TiO2in presence of nitrite were detected by Western blotting, andimmunofluorescence experiments were performed in photo-damaged HaCaT cells forobserving the distribution of3-nitrotyrosine in HaCaT cells by a fluorescence invertedmicroscope. The results suggested that nitrite increased total protein oxidation level andgenerated protein tyrosine nitration in photo-damaged BSA. In photo-damaged HaCaTcells, a significant increasing level of protein tyrosine nitration was also observed, and thenitrated proteins in HaCaT cells were mainly distributed in the cytoplasm, while a fewnitrated proteins appeared in nucleus, cytoskeleton and plasmodesmata.(4) Immunoprecipitation, protein mass spectrometry analysis and proteinidentification were experimented to search out the nitrated protein in HaCaT cells.Cystatin-A located in the cytoplasm was identified to be the main target for proteintyrosine nitration. Moreover, the nitrated site in cycstatin-A was computational predicted.On the basis of the results presented here as well as reported previously, a mechanism wasproposed to describe the enhancement of nitrite of photo-toxicity induced by nano TiO2.Evidence was provided that nitrite may play dual roles in the photo-toxicity of nano TiO2,first in the caspase-independent apoptosis relates to NO-mediated signaling pathwayowning to NO photolysis by nitrite, and second in the post-translational nitration modification in response to ONOO-which is generated in chemical reactions betweennitrite and TiO2under365nm UV irradiation. The mechanism of cell response in humankeratinocyte cells described here appears to be related to the activation of AIF and theinactivation of cystatin-A, which are both known to be important players in apoptosis ofkeratinocyte cells under ultraviolet irradiation. Therefore, we concluded that nitriteenhanced the photo-toxicity of nano TiO2in HaCaT cells.(5) The protection of ascorbic acid to the photo-damaged BSA, which induced bynano ZnO or nano TiO2in the presence of nitrite, was examined by UV-vis absorbanceand fluorescence intensity detection. It was found that ascorbic acid could reduce the thedamage of protein structure, and significantly depressed the level of protein oxidation andprotein tyrosine nitration. |
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