| ObjectiveTitanium dioxide nanoparticles (Tio2NPs) is a kind of important nano material. TiO2NPs is widely used in chemical, pharmaceutical, food, electronics, cosmetics and other fields for its unique physical and chemical properties. In the past, many studies have verified that TiO2NPs can do harm to various organisms, such as lung, liver, brain and kidney. In recent years, studies have suggested TIO2NPs may cause damage to the nervous system. However, at present domestic and foreign researches of its neurotoxicity are focusing on acute toxicity effect, but the neurotoxic mechanism is still lack of systematic research. So, we choose a lower dose and a longer exposure period to infect rats to observe the central neurotoxicity of Tio2NPs and to discussion its neurotoxic mechanism from multiple approaches under this condition.Method1. Treatment of animals50SPF male Wistar rats were randomly divided into5groups:control group (equal volume of distilled water), three TiO2NPs exposure groups(low dose group (62.5mg/kg), middle-dose group (125mg/kg), high dose group (250mg/kg)) and the ordinary particle size TiO2exposure (250mg/kg) group,10rats in each group. Titanium dioxide was given to rats by oral gavage in the volume of lml/100g.bw for60days,1time a day.2. Determination of conventional observation indexesa. The two kinds of titanium dioxide particles were characterized by transmission electron microscopy in order to observe the size, shape and dispersion.b. Observe the general condition of the rats and record the weight, determine the organ coefficient of total brain, cerebral cortex and hippocampus after the rats were killedc. Separate the hippocampus of rat and get blood samples, then determine the content of titanium in blood and hippocampus by inductively coupled plasma mass spectrometry(ICP-MS).3. Observation of central neurotoxicity effectsa. Morris water maze was used to determine the learn and memory ability of rat. In the Morris water maze experiment, the escape latency, the total distance and the average crossing number were recorded.b.Two rats were randomly selected from each group at the end of the experiment. Then HE staining slices were prepared to observe the changes in cerebral cortex and hippocampus of rats in all groups.c. TUNEL method was used to observe the neuronal apoptosis of cerebral cortex and hippocampus in all groups.4. Investigation of the central neurotoxic mechanism of Tio2NPsa. At the end of the experiment, blood samples and cerebral cortex samples were get after the rats were killed to prepare the serum samples and cerebral cortex homogenates. Oxidative stress Kit was used to determine the T-SOD activity, GSH-Px activity, total antioxidant capacity (T-AOC) and MDA.b. Part of the cerebral cortex of rat was taken to prepare cell suspension. Fura-2/AM fluorescent probe was used to determine the concentration of Ca2+in cerebral cortex. The expression of calpain1in cerebral cortex was determined by Western blot.c. The expression of Bax and Bcl-2in cerebral cortex was also determined by Western blot. Result1. conventional observation indexa. From the transmission electron microscopy we can see that the two types of titanium dioxide particles are both spherical polyhedron and part of the two kinds of particles are form loose aggregate; the diameter of nanometer titanium dioxide particles is about20～60nm and the diameter of ordinary particle size titanium dioxide particles is about100～300nm.b. The general condition of rats in all groups was good. The brain organ coefficient of high-dose group was decreased significantly when compared with the control group (P<0.05). Other indexes of all groups showed no significant changes when compared with the control group (P>0.05).c. Titanium content in blood and hippocampus of all groups was increased significantly when compared with the control group (P<0.05). When compared with the ordinary particle size group, the titanium content of high-dose group in blood was significantly increased (P<0.05), but titanium content in hippocampus showed no significant change (P>0.05).2. Central neurotoxicity of TiO2NPsa. In the place navigation test, compared with the control group, the escape latency of high-dose group was higher in the third and fourth day (P<0.05); the escape latency of middle-dose group was higher in the third day(P<0.05); the total distance of middle-dose and high-dose group were higher than the control group in the third day (P<0.05); the other groups’total distance and escape latency showed no significant changes in all four days. Compared with the ordinary size group, the escape latency of high-dose group was higher in the fourth days(P<0.05), but no significant changes were found in the other indexes of all four days(P>0.05). In the spatial probe test, the average crossing number and surrounding distance high-dose group were lower than the control group (P<0.05), and the surrounding distance of middle-dose group was lower than the control group, too (P<0.05); but there were no significant changes in other groups with other indexes. Compared with the ordinary particle size group, the average crossing number of high-dose group was lower (P<0.05); but there were no significant changes in other indexes (P>0.05).b. From the pathological slices we can see different degrees of nuclear condensation, chromatolytic and decreased cell density in nanometer titanium dioxide exposed groups, and from the high-dose group’s hippocampus we can also see liquefaction necrosis in the gate region. There’re mild injury in the cerebral cortex and hippocampus of ordinary particle size group. Nuclear condensation and chromatolytic phenomenon is relatively light and we can also see liquefaction necrosis in the granular cell layer.c. TUNEL results showed that TUNEL-positive cells were significantly increased in the cerebral cortex and hippocampus of high-dose group when compared with the control group (P<0.05) as well as the hippocampus of ordinary particle size group (P<0.05). Compared with the ordinary size group, the TUNEL-positive cells were significantly increased in the cerebral cortex and hippocampus of high-dose group (P <0.05).3. Central neurotoxic mechanism of TiO2NPsa. We determined the indexes of oxidative stress in cerebral cortex. Compared with the control group, the activity of T-SOD was decreased in high-dose group (P<0.05); the content of MDA was decreased in middle-dose and high-dose groups (p<0.05); other indexes in the rest groups showed no significant changes (p>0.05). Compared with the ordinary size group, the content of MDA was increased in high-dose group (p<0.05); but there were no significant changes in other indexes (P>0.05). The indexes of oxidative stress in serum showed that the activity of T-SOD was decreased in high-dose group when compared with the control group (P<0.05), but the other indexes in the rest groups showed no significant changes (P>0.05). Compared with the ordinary size group, there were no significant changes in high-dose group with all oxidative stress indexes (P>0.05).b. Compared with the control group, the concentration of intracellular calcium was increased in high-dose group (P<0.05) while there were no significant changes in other groups (P>0.05). Compared with the ordinary size group, there was no significant change in the high-dose group (P>0.05). We also determined the expression of calpain1, the result showed that there were no significant changes in all other groups when compared with the control group.(P>0.05).c. Western Blot test results showed that, the expression of Bax was increased in high-dose group and ordinary size group when compared with the control group (P<0.05). The expression of Bcl-2was decreased in high-dose group and ordinary size group when compared with the control group (P<0.05). However, the expression of Bax and Bcl-2showed no significant changes in other groups (P>0.05).Conclusions1. TiO2NPs can enter and accumulate in blood and hippocampus of rat by oral gavage.2. TiO2NPs can make the spatial learning and memory abilities of rats decreased; cause cerebral cortex and hippocampus tissue damage and induction of cerebral cortex and hippocampus nerve cells apoptosis3. TiO2NPs can cause intracellular calcium overload in the cerebral cortex of rat, but the expression of calpain1has no significant change under our experimental conditions.4. The neurotoxicity of TiO2NPs may be associated with the enhancement of oxidative stress, intracellular calcium overload and the exprssion level of apoptosis related proteins (Bcl-2and Bax). |
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