Study On Joint Effect Of Oxidative Stress And Appoptosis Caused By Nano-TiO₂ And PbAc In Human Derived Fetal Hepatocytes

Titanium dioxide (TiO2) in nano-size is a new technology product which was widely used in cosmetics, paints and in decontamination water. Numerous studies demonstrated that nano-TiO2 in nano-size induced damage to human health and environment. Nano-TiO2 could stimulate inflammatory reaction of the organism, reduce the cell viability, and induce the apoptosis and DNA damage. Nano-TiO2 has some special properties, such as nano-scale effect, surface effect, quantum size effect, macroscopic quantum tunneling effect, photocatalytic effect. Compared with the large particles, nano-TiO2 on the nature and intensity of the biological effects of a qualitative change also occurred. According to the present study, mechanisms of damage induced by nano-TiO2 are mainly related to inflammation and oxidative stress.Lead is widely applied heavy metal in batteries, paint, gasoline, paint, instruments. Lead exists in the environment widespread and accumulates in organism. Lead pollution is a particularly prominent environmental problem in the current situation. Lead pollution causes DNA damage and genetic toxicity, damages the nervous, hematopoietic, reproductive and other systems. Recent studies show that mechanisms of damage induced by lead major related to oxidative stress.A large number of nano-Ti02 has been released into the environment with the widespread use. Nano-TiO2 could absorb a variety of pollutants in the environment because of the special surface effect. Nano-TiO2 has a strong adsorption capacity to metal ions or organic produce in the environment as the special physical and chemical properties. This may generate different effects compared with their own biological effects. Pb2+ exists in the environment can be adsorbed onto the surface of nano-TiO2, and interact with each other, and interact with the body or the environment. So far, the combined effects of Pb2+ and nano-TiO2 on the body were unknown.The content of nano-TiO2 and other pollutants in the environment is very low, and exists in trace. In this study, to understand the combined effect of oxidative stress and apoptosis induced by nano-TiO2 mixed with PbAc, we assessed the cell activity, DNA damage, ROS generation, reduction enzyme changes, apoptosis, etc induced by TiO2 in trace mixed with on human embryo hepatocytes (L02) for a 24 hr treatment without photoactivation. Based on the researches on toxicity of nano-TiO2 and PbAc treated alone, the interaction of the mixtures was studied and potential toxicological mechanisms were also evaluated in this study, further consummated the toxicology evaluation and provided the theory basis of nano-TiO2, in addition to understand the healths effects and provide experimental evidence induced by nano-TiO2 and PbAc in trace.The study is composed of the following four parts:PartⅠThe aggregation effect of nano-TiO2 and the adsorptive capacity of PbAc onto nano-Ti02Objective:To observe the aggregation of nano-TiO2 and adsorption of nano-TiO2 on PbAc in the solution.Methods:The aggregation size of 0.001,0.01,0.1,1,10μg/mL nano-TiO2 in aqueous solution was detected by a Nano-Particle Analyzer. The adsorption of Pb2+ onto nano-TiO2 in solution was detected by infrared scanning. The adsorptive capacity of 1μg/mL Pb2+ onto 0.001,0.01,0.1,1,10μg/mL nano-TiO2 were detected by graphite furnace atomic absorption spectrometry.Results:The average size of aggregation is 298.8 nm and there is no significantly change in the given concentrations (P> 0.05). The absorptive capacity of PbAc by nano-TiO2 analyzed by GFAAS is showed that the mixture containing 0.001 and 0.01μg/mL nano-TiO2 absorbed significantly less PbAc than those of 0.1,1,10μg/mL nano-TiO2 (P< 0.05), and there was a dose-dependent absorption by nano-TiO2. Conclusion:nano-TiO2 treated alone in the absence of surfactants aggregated in the short time in water. In the mixtures, there was a significant adsorption on PbAc by nano-TiO2, and adsorption capacity increases with the concentration of nano-TiO2.PartⅡOxidative stress induced by a joint effect of titanium dioxide and lead acetate in human hepatocytesObjective:To observe the cell viability, ROS generation and Oxidative stress induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.Methods:L02 were exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr (The mixtures of nano-TiO2 with 1μg/mL PbAc in the following descriptions were abbreviated as 10,1,0.1,0.01,0.001μg/mL mixture for convenience). In each experiment a negative control (1‰DMSO) was included. The cytotoxicity induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using MTT assay. The production of ROS induced by nano-TiO2,1μg/mL PbAc and the mixtures were assessed by flow cytometry using DCFH-DA as fluorescent probe. The activities of GSH and SOD were determined meanwhile.Results:Compared with the negative control, PbAc and the other nano-TiO2 treatments, cell viability decreased notably when treated with 10μg/mL nano-TiO2 (P< 0.05).1μg/mL nano-TiO2 induced significantly viability decreased compared with the negative control (P< 0.05). The mixtures from 0.1,1,10μg/mL induced significantly higher cytotoxicity than the negative control (P< 0.05). Mixtures of 1 and 10μg/mL induced significantly higher cytotoxicity than 1μg/mL PbAc treatment (P< 0.05). There were no significant increases in ROS, GSH, SOD levels caused by nano-TiO2 and PbAc-alone treatment (P> 0.05). In comparison with the negative control and 1μg/mL PbAc, the ROS generation significant increases in L02 cells after all mixtures treatment for 24 hr, respectively (P< 0.05). The 0.01,0.1 and 1μg/mL mixtures induced significant increases in GSH level, compared with the negative control (P< 0.05). The 1μg/mL mixture induced significant increases in GSH level compared with the 1μg/mL PbAc treated alone (P< 0.05). Furthermore, there were significant increases in SOD level induced by the 0.1 and 0.01μg/mL mixtures compared with the negative control (P< 0.05). There were dose-dependence patterns of cell viability and ROS generation induced by nano-TiO2 alone and the mixtures. However, there were only dose-dependence patterns of GSH and SOD levels induced by nano-TiO2 treated alone. There were no synergistic effect in cell viability, ROS, GSH and SOD levels caused by nano-TiO2 and PbAc (P> 0.05).Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances oxidative stress and cytotoxicity in the absence of UV in L02 over a 24 hr treatment period. The results suggest that the increased oxidative stress triggered the up regulation of defenses to protect cells at low concentrations but triggered the down regulation at higher concentrations of the mixtures.PartⅢDNA damage and Repair protein expression induced by a joint effect of titanium dioxide and lead acetate in human hepatocytesObjective:To observe the DNA damage, DNA adducts generation and Repair protein expression induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.Methods:L02 was exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr. In each experiment a negative control (1‰DMSO) was included. The DNA damage induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using comet assay. The generation of DNA adducts induced by nano-TiO2,1μg/mL PbAc and the mixtures were assessed by HPLC. The expression of OGG1 induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined using western blotting assay.Results:Compared with the negative control and 1μg/mL PbAc, OTM increased notably when treated with 10μg/mL mixture (P< 0.05).1μg/mL nano-TiO2 mixed with 1μg/mL PbAc induced significantly OTM increased compared with the negative control (P< 0.05). There were no significant increases in 8-OHdG formation and OGG1 expression caused by nano-TiO2 and PbAc-alone treatment (P> 0.05). The production of 8-OHdG induced by the 10 and 1μg/mL mixtures was significantly higher than the negative control (P< 0.05). In addition,10μg/mL mixtures caused higher 8-OHdG levels than 1μg/mL PbAc (P< 0.05). All mixtures exposures resulted in the increased OGG1 levels, except for the 10μg/mL mixtures treatment compared with the negative control and 1μg/mL PbAc, respectively (P< 0.05). Nano-TiO2 and the mixtures induced dose-dependence patterns of OTM,8-OHdG formation and OGG1 expression. Addition of nano-TiO2 with PbAC synergistically enhanced OGG1 expression in this study (P< 0.05).Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances oxidative stress and cytotoxicity in the absence of UV in L02 over a 24 hr treatment period. The results suggest that the increased oxidative stress triggered the up regulation of defenses to protect cells at low concentrations but triggered the down regulation at higher concentrations of the mixtures. Part IVApoptosis induced by a joint effect of titanium dioxide and lead acetate in human hepatocytesObjective:To observe the apoptosis effects induced by a joint effect of nano-TiO2 and PbAc in human hepatocytes.Methods:L02 was exposed to nano-TiO2 in 10,1,0.1,0.01,0.001μg/mL and 1μg/mL PbAc treated alone and mixture for a period 24 hr. In each experiment a negative control (1‰DMSO) was included. The early and late apoptosis induced by nano-TiO2,1μg/mL PbAc and the mixtures were determined by flow cytometry using FITC and PI as fluorescent probe. The expression of Caspase 3 induced by nano-TiO2, 1μg/mL PbAc and the mixtures were determined using western blotting assay.Results:Compared with the negative control,1μg/mL PbAc induced no significantly early apoptosis increased (P> 0.05). There were significant increases in early apoptosis caused by 1 and 10μg/mL TiO2 compared with the negative control,1μg/mL PbAc and other concentrations TiO2 treated alone (P< 0.05). The 0.1μg/mL TiO2 induced notably early apoptosis increase compared with the negative control,1μg/mL PbAc,0.001,0.01μg/mL TiO2 treated alone (P< 0.05). All mixtures exposures resulted in the increased early apoptosis levels compared with the negative control and 1μg/mL PbAc, respectively (P< 0.05). All exposures of nano-TiO2 and 1μg/mL PbAc treated alone resulted in no notably late apoptosis increase compared with the negative control (P> 0.05). But late apoptosis increased notably caused by 10μg/mL mixtures treated alone compared with the negative control, PbAc and other mixtures treatments (P< 0.05). All exposures of nano-TiO2 and 1μg/mL PbAc treated alone resulted in no notably Caspase 3 expression increase compared with the negative control (P> 0.05). Compared with the negative control, the expression of Caspase 3 increased significantly when treated with all mixtures except 0.001μg/mL (P< 0.05). Caspase 3 expression increased significantly caused by 10μg/mL mixtures treated alone compared with the negative control, PbAc and other mixtures treatments (P< 0.05). Nano-TiO2 and the mixtures induced dose-dependence patterns of apoptosis. There was synergistic effect in late apoptosis caused by nano-TiO2 and PbAc (P< 0.05).Conclusion:The joint effect of low (≤10μg/mL) concentrations of nano-TiO2 and PbAc enhances apoptosis and relate key protein expression in L02. The results suggest that the Caspase 3 play a very important role in the apoptosis of the cell.