| Objective:Because of the excellent physical and chemical properties, titanium dioxide nanoparticle (Nano-TiO2) are widely used in medicine, waste water treatment, painting, sterilization, cosmetics, food additives, ceramics and other biomedical fields. Our human can be exposed to nanoparticles which cause health damage. The main routes of Nano-TiO2 entering human body are respiratory, skin exposure and digestive tract, among which inhalation is the most common way. After acting on the organism, a large number of free radicals duing to the nanoparticle surface with high chemical reactivity can cause oxi dative stress, increased production of Reactive oxygen species (ROS), decreased antioxidant capacity, resulting in damage both of the DNA nucleobases and the sugar phosphate backbone and then cause DNA damage. The oxidative stress caused by Nano-TiO2 is highly associated with their toxic, but the targets and regulation mechanism is still unclear. In the present study, the Nano-TiO2 was through intratracheal injection in rat, which might mimic the physiological responses elicited by the common route of exposure in humans. The oxidative stress and DNA damage levels and mechanisms regulated by the FOXO3a signal pathway were studies.Methods:160 healthy male SD rats,weighing 180-220g were randomly divided--into-four-groups-of-15. The rats were exposed on 25nm TiO2 through once intratracheal injection at a dose of 0,0.2,1,5g/kg body weight respectively. After injection for 1 day,3 days and 7 days,5 animals were sacrificed in each group. Specimens from liver, kidney, lung tissue were frozen in-80℃ refrigerator for subsequent assay2 Animals were weighed before sacrificed. And then the liver, kidney and lung tissue removed and weighed after being killed through electronic weighing scales to calculate the organ coefficient.3 Detection of Ti in liver, kidney and lung of rats:The Ti contents in liver, kidney and lung tissue of rats after Nano-TiO2 exposed through microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS)4 Histopathologic observation:Pathological changes of liver, kidney and lung were observed by light microscopy after hematoxylin-eosin staining.5 Detection of oxidative stress indicators:The MDA levels SOD, GSH-Px activities in liver, kidney and lung of rats were detected by commercial kits.6 Detection of DNA damage:Single cell gel electrophoresis assay (SCGE) was used to detect the DNA damage in liver, kidney and lung of rats.7 Detection of FOXO3a signal related protein contents:the expression of FOXO3a, ChK2, GADD45a and XRCC1 protein were detected by Western blot.Results:1 The general condition of ratsAll rats in each group survived. After intratracheal administration, the rats appeared to reduction of food intake, the action slow, less activity and apathetic. On the next day the rats were in good spirits, returned to normal drink and intake, moreover, general activities of rats were in good condition.2 Ti content changes of each tissue in ratsAfter Nano-TiO2 exposed, Ti content in liver, kidney and lung tissues were significantly increased. Compared with the control group, Ti content significantly increased in liver kidney and lung of rats in the high-dose group (P<0.05). Compared with high-dose Nano-TiO2 exposure for 1 day, there was no significant change of Ti content in liver and kidney tissue of rats. Whereas there was a significant reduction of Ti content in lung tissue of rats after high-dose Nano-TiO2 exposure for 3 or 7 days(P<0.05).3 Organ coefficient of ratsCompared with the control group, there was no significant difference in organ coefficient of rats in each exposure group.4 Histopathologic changes of liver, kidney and lung tissue in rats after Nano-TiO2 exposure for 7 days.After Nano-TiO2 exposure, there appeared histopathologic changes in the liver, kidney and lung tissue of rats in different degree.Liver:In the control group, the central vein was surrounded by liver cells, which is streak rule neatly arranged, and we can also observe the pink cytoplasm and dyeing uniformity; In the low dose group, the cells disordered, distal cells of central vein were stained darkly; In the middle dose group, the vacuoles were observed in cytoplasm and punctate necrosis appeared in the inflammatory cells; In the high-dose group, the necrosis flocks were observed.Kidney:In the control group, the nucleus were round with abundant cytoplasm, glomeruli and tubules were tightly packed; In the low-dose group, the renal tubular gap increased; In the middle dose group, the renal tubular was edema accompany with part necrosis; In the high-dose group, slight contractions in the glomerular on the basis of necrosis were observed.Lung:In the control group, we can observe the neat bronchial epithelium and regular lumen, intact alveolar ducts and alveolar sacs, and normal elastic fibers and reticular fibers of alveolar septa with capillary network abound is normal dyed; In the low dose group, we can observe inflammatory cell infiltration in the bronchus and irregular bronchial lumen; In the middle dose group, aggravating irregular bronchial lumen, thickening alveolar wall and alveolar septal were observed; In the high dose group, the bronchial wall was ruptured and connective tissue was increased.5 The SOD, GSH-Px activities and MDA contentLiver:Compared with the control group, SOD, GSH-Px activities were significantly decreased (P<0.05), MDA content was significantly increased (P <0.05). Compared with that on 1 days after Nano-TiO2 exposure, SOD activity was significantly increased (P<0.05) in middle dose group on 3 days and 7 days after Nano-TiO2 exposure; SOD activity was significantly increased in high dose group on 7 days after Nano-TiO2 exposure (P<0.05); GSH-Px activity was significantly increased in each group on 3 days and 7 days after Nano-TiO2 exposure, (P<0.05); MDA content was no significantly change in each group on 3 days after Nano-TiO2 exposure, whereas MDA content was significantly decreased in each group on 7 days after Nano-TiO2 exposure, (P <0.05).Kidney:Compared with the control group, SOD, GSH-Px activities were significantly decreased (P<0.05), MDA content was significantly increased (P<0.05). Compared with that on 1 days after Nano-TiO2 exposure, SOD activity was significantly increased in low, middle dose group on 3 days and 7 days after Nano-TiO2 exposure(P<0.05), SOD activity was significantly increased in high-dose group on 7 days after Nano-TiO2 exposure (P< 0.05); GSH-Px activity was significantly increased in each dose on 3 days and 7 days after Nano-TiO2 exposure(P<0.05); MDA content was no significant change, in each dose on 3 days after Nano-TiO2 exposure, MDA content was significantly decreased in low and middle dose on 7 days after Nano-TiO2 exposure (PO.05).Lung:Compared with the control group, SOD, GSH-Px activities were significantly decreased (P<0.05), MDA content was significantly increased (P <0.05). Compared with that on 1 days after Nano-TiO2 exposure, SOD activity was significantly increased in low-dose group on 7 days after Nano-TiO2 exposure (P<0.05), SOD activity had no significant change in high-dose group on 3 days and 7 days Nano-TiO2 exposure; there was no significant change in GSH-Px activity in each dose group on 3 days and 7 days after Nano-TiO2 exposure; MDA content was significantly decreased in low-dose group on 7 days after Nano-TiO2 exposure (P<0.05), MDA content was no significant change in the middle and high dose groups on 3 days and 7 days after Nano-TiO2 exposure.6 Changes of OTM and Tail DNA% in liver, kidney and lung cellsCompared with the control group, OTM and Tail DNA% in liver, kidney and lung cells of rats in each Nano-TiO2 treatment group were significantly increased (P<0.05); compared with that on 1 day after Nano-TiO2 exposure, OTM and Tail DNA% in liver, kidney and lung tissue of each Nano-TiO2 treatment group were no significant differences on 3 days and 7 days after Nano-TiO2 exposure.7 The expression of FOXO3a, XRCC1, ChK2 and GADD45a protein in lung tissueCompared with the control group, the expression of XRCC1 and FOXO3a protein in lung tissue of rats was significantly decreased (P<0.05)in Nano-TiO2 treatment groups. ChK2 protein expression was significantly increased in the low-dose group (P<0.05), whereas significantly decreased in the high dose group (P<0.05); GADD45a protein expression was significantly increased in the low-dose group (P<0.05), whereas significantly decreased in the middle and high dose groups (P<0.05).Conclusion:1 After Nano-TiO2 exposure, Ti can distribute in main tissues of rats.2 After Nano-TiO2 exposure, main organ had Histopathologic changes.3 After Nano-TiO2 exposure, cells of rats had oxidative stress.4 After Nano-TiO2 exposure, cells in rats had DNA damages. The DNA damages can’t recover within 7 days.5 After Nano-TiO2 exposure, the expression of FOXO3a protein signal correlation were inhibited. |
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