| Objective: To observe the toxic effects of fluoride, arsenic and co-exposure on learning and Memory ability, ultrastructural changes and neurotransmitters content of neurons in cerebral coaex in rats. To explore the dffects of fluoride, arsenic and co-exposure on oxidative stress, DNA damage and apoptosis in primary hippocampal neurons of rats. To study on Ras/ERK signaling pathway in the damage of hippocampal neurons induced by fluoride and arsenic and the mechanisms involved.These findings will provide academic evidences on prevention and treament of endemic flurosis, endemic arseniasis and flurosis and arseniasis combined poisoning.Methods: A total of 108 SD rats were randomly divided into 9 groups by tow-factor and three-level factorial experiment design, 12 rats in each group, control group(distilled water), low dose Na As O2(1/50 LD50, 0.75 mg/kg) group, high dose Na As O2(1/25 LD50, 1.5 mg/kg) group, low dose Na F(1/20 LD50, 25 mg/kg) group, high dose Na F(1/10 LD50, 50 mg/kg) group, low dose Na As O2(0.75 mg/kg) and low dose Na F(25 mg/kg) combined group, low dose Na As O2(0.75 mg/kg) and high dose Na F(50mg/kg) combined group, high dose Na As O2(1.5 mg/kg) and low dose Na F(25 mg/kg)combined group, high dose Na As O2(1.5 mg/kg) and high dose Na F(50 mg/kg)combined group. The control group was given distilled water and exposure groups were given different doses of Na As O2 and Na F through drinking water. After 6 months of exposure, learning ability was tested by the Morris water maze and Platform experiment; electron microscope samples were made in order to observe ultrastructural changes of neurons in cerebral cortex by transmission electron microscope; the brain tissue was collected to detect content of acetylcholinesterase(Ach E), dopamine(DA)and 5-hydroxytryptamine(5-HT) by enzyme linked immunosorbent assay(ELISA);content of acetylcholine(Ach), activity of monoamine oxidase(MAO), lactate dehydrogenase(LDH) and succinatedehydrogenase(SDH) were detected with colorimetry. Primary hippocampal neurons of rats in culture were randomly divided into 9 groups, control group(cell culture), low dose Na As O2(2 μmol/L) group, high dose Na As O2(10 μmol/L) group, low dose Na F(10 μmol/L) group, high dose Na F(50μmol/L) group, low dose Na As O2(2 μmol/L) and low dose Na F(10 μmol/L) combined group, low dose Na As O2(2 μmol/L) and high dose Na F(50 μmol/L) combined group,high dose Na As O2(10 μmol/L) and low dose Na F(10 μmol/L) combined group, high dose Na As O2(10 μmol/L) and high dose Na F(50 μmol/L) combined group. MTT test was used to detect survival rates of primary hippocampal neurons cells; contents of amounts of reduced glutathione(GSH) were detected with micro-ELISA; activity of catalase(CAT) was detected with visible light; activity of superoxide dismutase(SOD)was detected with WST-1; contents of malondialdehyde(MDA), total antioxidant capability(T-AOC) and activity of glutathione peroxidase(GSH-Px) were detected with colorimetry; DNA damage was detected by single cell gel electrophoresis(SCGE);the cell apoptosis rate was determined by flow cytometer. Contents of intracellular Ca2+was detected by fluorescence spectrophotometric; the proteins expression of Bax and Bcl-2 in cerebral cortex tissues of rats were detected with western blot; the proteins expression of Ras, Raf-1, p MEK, p ERK1/2, p CREB and BDNF were detected with western blot; m RNA expression levels of Ras, Raf-1, MEK, ERK1, ERK2, CREB,BDNF, C-FOS, C-JUN, DNMT1, DNMT3 a and DNMT3 b in primary hippocampal neurons cells of rats were detected with Real-time PCR. Results: 1) Compared with the control group, the escape latency periods and the first platform time of rats exposed to high-dose Na As O2 group, high-dose Na F group,high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly prolonged in water maze test(P < 0.05). In high-dose Na As O2 group,high-dose Na As O2 high-dose Na F combined group, latency periods were significantly decreased while EN1 and EN2 were increased compared with the control group in platform experiment(P < 0.05). The ultrastructure in cortex neurons in high-dose Na As O2 group, high-dose Na F group, low-dose Na As O2 and high-dose Na F combined group, high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group rats all had pathological changes. There were important changes in cell shrinkage, nuclear deformation, the perinuclear mitochondrial swelling and cavitating, mitochondrial ridge reduced, rough endoplasmic reticulum expanded. Compared with the control group, DA contents of rats cerebral cortex treated with high-dose Na As O2 group, high-dose Na F group,low-dose Na As O2 and high-dose Na F combined group, high-dose Na As O2 and low-dose Na F combined group,high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P<0.05). The interaction effect of fluoride and arsenic was not found in the water maze test, platform experiment, content of neurotransmitters and ezemy in the brain tissue(P > 0.05). 2) Compared with the control group, MTT absorbance value of primary hippocampal neurons cells treated with high-dose Na As O2 group, high-dose Na F group, high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P<0.05). Compared with the control group, CAT contents of hippocampal neurons cells treated with high-dose Na As O2 group, high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P < 0.05); GSH contents of hippocampal neurons cells treated with high-dose Na F group, low-dose Na As O2 and high-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P<0.05); SOD contents of hippocampal neurons cells treated with high-dose Na As O2 group, high-dose Na F group,low-dose Na As O2 and high-dose Na F combined group, high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P < 0.05); GSH-Px contents of hippocampal neurons cells treated with high-dose Na As O2 group, high-dose Na F group, high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P<0.05); T-AOC of hippocampal neurons cells treated with high-dose Na As O2 group, low-dose Na As O2 and high-dose Na F combined group,high-dose Na As O2 and high-dose Na F combined group were significantly decreased(P< 0.05); MDA contents of hippocampal neurons cells treated with high-dose Na F group, low-dose Na As O2 and high-dose Na F combined group, high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly increased(P < 0.05). Compared with the control group, olive tail moment in high-dose Na As O2 group, high-dose Na F group, low-dose Na As O2 and high-dose Na F combined group, high-dose Na As O2 and low-dose Na F combined group,high-dose Na As O2 and high-dose Na F combined group were remarkably elevated(P<0.05). Compared with the control group, the cell apoptosis rate in high-dose Na As O2 group, high-dose Na F group, low-dose Na As O2 and high-dose Na F combined group,high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly increased(P < 0.05). Compared with the control group, Bax protein expression in hippocampal neurons cells treated with high-dose Na As O2 group, high-dose Na F group, high-dose Na As O2 and low-dose Na F combined group, high-dose Na As O2 and high-dose Na F combined group were significantly increased(P<0.05); while Bcl-2 protein expression in high-dose Na As O2 group was decreased(P<0.05). The interaction effect of fluoride and arsenic was not found in cell activty, oxidative stress, DNA damage and cell apoptosis in primary hippocampal neurons cells(P>0.05). 3) Compared with the control group, contents of intracellular Ca2+ was significantly increased in 50μmol/L Na F group(P<0.05).Compared with the control group, the proteins expression of Ras, Raf-1, p MEK and p ERK1/2 treated with 50μmol/L Na F group were all decreased(P<0.05); the m RNA expression of Ras and Raf-1 were also decreased(P < 0.05); while the m RNA expression of MEK, ERK1 and ERK2 had no changed(P>0.05). Compared with the control group, the proteins expression of Ras, Raf-1, p MEK treated with 10μmol/L Na As O2 group had no remarkably changed(P>0.05); but the expression of p ERK1/2protein was significantly decreased(P< 0.05); the m RNA expression of Ras, Raf-1,MEK, ERK1 and ERK2 had no changed(P>0.05). Compared with the control group,the expression of p CREB and BDNF protein treated with 10μmol/L Na As O2 group,50μmol/L Na F group, 10μmol/L Na As O2 and 50μmol/L Na F combined group were significantly decreased(P<0.05). The interaction effects between fluoride and arsenic on BDNF protein expression in primary hippocampal neurons cells might be synergetic(P < 0.05). Compared with the control group, the m RNA expression of C-FOS treated with 10μmol/L Na As O2 group was decreased(P < 0.05); while the m RNA expression of C-JUN treated with 10μmol/L Na As O2 and 50μmol/L Na F had no remarkably changed(P > 0.05). Compared with the control group, the m RNA expression of DNMT1 treated with 10μmol/L Na As O2 group, 10μmol/L Na As O2 and50μmol/L Na F combined group were increased(P<0.05); while NGF could inhibited the expression of DNMT1 m RNA stimulated by Na As O2(P < 0.05). Conclusions:Fluoride and arsenic could passing blood-brain barrier and cumulatived in cerebral cortex of rats. Chronic fluoride, arsenic and fluoride-arsenic combined exposure may affect learning and memory abilities through cell pathological changes and DA contents decrease in cerebral cortex of rats. Fluoride, arsenic and fluoride-arsenic combined exposure might cause oxidative stress, DNA damage and cell apoptosis rate increase in hippocampal neurons cells. These effcts might be related to damaged learning and memory of rats. Fluoride, arsenic and fluoride-arsenic combined exposure might cause the protein expression of p CREB and BDNF decreased, this might play an important role in learning and memory damage of rats. The results showed the interaction in the combination group was not found, it indicate that effects mechanism of fluoride and arsenic might be diffirent. The results also showed epigenetic modifications might influence Ras/ERK signaling pathway in hippocampal neurons cells. |
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