Molecular Mechanism Of Brain Impairment Caused By Drinking-Acquired Fluorosis And Selenium Intervention

Fluorine is an essential trace element in the body, but prolonged excessive fluoride intake can produce adverse effects called endemic fluorosis. The effects of fluorosis on skeleton and teeth is well documented, and neurotoxicity of fluoride has also been confirmed wherein fluorosis results in systemic diseases. However, the pathogenesis of fluorosis remains unclear.Selenium, an element demonstrating antioxidant properties and which exerts curative effect as an anti-fluorine agent, has recently attracted the attention of researchers. The present study further explores subchronic toxicity of fluoride based on our previous experiments, where we concluded that 1.5 mg/L is the optimum concentration for selenium to antagonize subchronic fluorosis to observe the learning ability and memory of rats, which are indicators of brain function. This study aimed to determine the rate of nerve cell apoptosis in the CA3 area of the hippocampus and the expression levels of the apoptosis-related factors’ genes and proteins in the mitochondria. The molecular mechanism of brain impairment caused by drinking-acquired fluorosis and the intervention of selenium were investigated, and the key target molecules were screened. Eighty newly weaned male Sprague-Dawley rats, each weighing 70-90 g. The rats were randomly divided into four groups, each group containing 20 rats. The first group served as control group and received tap water. The fluoride-treated group (F) was administered with aqueous sodium fluoride (NaF,100 mg/L). In the selenium only group (Se), selenium was used as sodium selenite at 1.5 mg/L concentration mixed in drinking water. The last group (F+Se) received NaF (100 mg/L)+Se (1.5 mg/L). All of the rats were fed with a standard pellet diet and were provided with water ad libitum for three and six months. Then determined blood/urine fluoride, the ability of behavior and learning memory, the nerve-cell apoptosis rate in the CA3 area of the hippocampus and the expression levels of apoptosis-related genes and proteins in the mitochondria. Results were as follows:(1) Results in animal models of fluorosis:Exposed three and six months later, the rats in the control and Se groups displayed evenly light yellowish teeth with smooth surface, indicating the absence of dental fluorosis. Compared with the control group, the F group displayed yellow and white teeth with clear colored chalk-like stripes, indicating dental fluorosis. Moreover, the F+Se group displayed yellow and white teeth with stripes, indicating mild dental fluorosis.(2) Blood/urine fluoride:Compared with the control group, the blood/urine fluoride were both significantly higher in the F group than in the control group (P<0.01); in addition, the urinary fluoride in the F+Se group was significantly increased (P<0.01). The blood/urine fluoride were also significantly reduced in the F+Se group compared with the F group (P<0.01). Compared with the three months, the urinary fluoride in the F group and F+Se group was significantly increased (P<0.05), while the blood fluoride showed increasing trend respectively (p>0.05).(3) Determination of behavior and learning memory:Compared with the control group, the numbers of grooming, buttresses, standing times,1 minute running grids, the frequency by which the platform was crossed and the duration of stay in the platform in F group was significantly reduced (P<0.01), while the escape latency prolonged. Compared with the F group, the parameters above in F+Se showed an opposed trend.(4) Detection of apoptosis:The hippocampus of the control groups showed rare apoptotic cells. By contrast, the number of apoptotic cells increased significantly in the F group, whereas that in the F+Se decreased. The Se groups also showed some apoptotic cells. Compared with the three months, the apoptotic cells in the F group and F+Se group showed increasing and decreasing trend respectively.(5) PCR test:The mRNA expression levels of Cytc, Caspase-3 and Caspase-9 were significantly increased in the F and F+Se groups compared with the control group (p<0.01); Caspase-9 mRNA expression levels were also significantly increased in the Se group (p<0.05). Moreover, the Cytc,Caspase-3 and Caspase-9 mRNA expression levels were significantly reduced in the F+Se group compared with the F group (p<0.01). Compared with the three months, The mRNA expression levels of Cytc in the F and Se groups and the mRNA expression levels of Caspase-9 in the F groups were significantly increased (p<0.05), and Caspase-3 mRNA expression levels were significantly reduced in the F+Se group (p<0.05).(6) Western Blot test:Compared with the control group, Cytc protein expression levels were significantly increased in the F and F+Se groups while Caspase-9 and Caspase-3 protein expression levels were significantly decreased (p<0.01); in addition, the expression levels of Caspase-3 protein were significantly increased in Se groups (p<0.05). Cytc protein expression levels was also significantly increased in the Se groups of six months (p<0.01). Compared with the F group, Caspase-9 and Caspase-3 protein expression levels were significantly increased whileCytc protein expression levels were significantly decreased in the F+Se group (p<0.01). Moreover, compared with the three months, Cytc protein expression levels were significantly reduced in six months F and six months Se groups respectively(p<0.01 and p<0.05), and in the F+Se groups Cytc and.Caspase-3 protein expression levels were significantly decreased respectively (p<0.01 and p<0.05).In summary, Drinking-water type fluorosis changed the blood/urine fluoride concentrations of rats and increased brain cell apoptosis,and then affected the brain development, reduced the ability of learning memory. It suggests the mitochondrial CytC-Caspase-9-Caspase-3 apoptosis pathway in the hippocampus was one of the mechanisms leading to fluorosis-induced brain damage. Furthermore, the Cytc signaling molecules were possibly the key target molecules in fluorosis-induced apoptosis. On the other hand, the same concentration of selenium have different biological effects under different exposure mode, it exhibited toxic effects on the body when exposed alone, while it could alleviate fluorosis-induced brain injury and this intervention might become more significant as time increasing.