| Fluorine distributes in nature widely, people contact with fluoride through various sources such as water, soil, air and so on. If enviromental fluoride is too high, fluoride will accumulate in body and cause physiological and pathological changes, called endemic fluorosis. Excess fluoride can be accumulated in brain tissue through blood-brain barrier, resulting in adverse effects on nervous system, affecting learning and memory. Low dietary calcium might be the main precipitating and aggravating factor that increased the rate of brain cell apoptosis and aggravated the neurotoxic effects in endemic fluorosis, while high dietary calcium fluoride could alleviate the poisoning causing by fluorosis. So far, studies of fluorosis have had a larger progress on brain development and function, the effects of low dietary calcium has also been studied, but lacking studies in-depth and systematicness. In addition, molecular mechanisms and the targets of fluorosis-induced brain development and the intervention of different conditions on dietary calcium are not clear yet. This study combined epidemiological investigation with sub-chronic animal experiments, to explore the possible interventions of fluorosis affecting children’s mental development and the role of dietary calcium, and explore the molecular mechanisms and the targets of the intervention of dietary calcium, to provide a scientific basis for the prevention and treatment of endemic fluorosis.The survey subjects of epidemiological were8-13-year-old school pupils, who were chosen randomly from Xinhuai and Wamiao in Jiangsu Province, We mainly determinated calcium nutritional status and developmental quotient and IQ testing for local children, and based on the determination of urinary fluoride and dental fluorosis prevalence in2003, evaluation the effectiveness of water improvement measures to reduce fluoride taken after2004.In vivo animal experiments, we selected25early weaning SD male rats for breeding, and75female rats were randomly divided into five groups. Control group (Control) drank tap water, and ate normal diet (calcium content of0.79%); fluoride group (F) drank100mg/LNaF solution, and ate normal diet; low calcium group (LCa) drank tap water, and ate low calcium diet (calcium content of0.063%); low calcium fluoride group (F+LCa) drank100mg/LNaF solution, and ate low calcium diet; high calcium fluoride group (F+HCa) drank100mg/LNaF solution, and ate high calcium diet (calcium content of7%). Male rats drank tap water, and ate standard diet (fluorine content<0.2mg/kg, calcium content of7-10g/kg). After three months, the fluorine content of blood and urine served as the success criteria of sub-chronic fluorosis animal model, and then mated for1:1, taking18days gestation rats,14and28days rats for experimental, determined blood/urine fluoride, blood/urine calcium, the ability of behavior and learning memory, brain synaptic ultrastructure, the expression levels of DCX, p38, and the apoptosis-related genes Bcl-2, Caspase12, JNK. Results were as follows:1. Epidemiological investigation:Results in2003:Compared with Xinhuai, the fluoride content of drinking water in Wamiao was significantly increased (p<0.01), children’s fluoride intake through foods increased significantly (p<0.05), a significant increase ocurred in urinary fluoride content (p<0.01), the prevalence of dental fluorosis defect in children was significantly increased (p<0.01). Results in2013: Calcium intake and IQ of two village children had no significant difference (p>0.05), and in a normal range. However, the rate of children with mental retardation (IQ<69) was significantly higher than that of Xinhuai (P<0.01). There was no significant difference in IQ in children of different genders between Xinhuai and Wamiao (p>0.05). Developmental quotient of children in two villages was in the normal range. Height and head circumference of Wamiao were significant differences with Xinhuai (p<0.01), height of boys was significant difference (p<0.05), female children head circumference og girls was significant difference (p<0.05), and height and birth weight were high significant difference (p<0.01). Head circumference of different genders in Wamiao was significantly different (p<0.05). Height and weight of different genders in Xinhuai was significantly different (p<0.05), and the difference of head circumference was highly significant (p<0.01).2. Sub-chronic animal experiments:(1) Results in animal models of fluorosis:exposed three months later, compared with control group, the mothers’teeth of F group and F+LCa group appeared yellow and white, there was a clear colored chalk stripes, showing obvious dental fluorosis symptoms, F+HCa group appeard yellow and white with stripes, showing symptoms of mild dental fluorosis; fluorine content in blood in F group and F+LCa group were significantly higher (p<0.05), urinary fluoride content in F group, F+LCa group and F+HCa group were significantly increased (p<0.01), which showed the sub-chronic fluorosis model was copied successfully after exposed to100mg/L NaF for three months.(2) Blood/urine fluoride and blood/urine calcium:Compared with the control group, blood fluoride of14and28days in F male group were significantly higher (p<0.05),14and28day in F+LCa group were significantly increased (p<0.01); calcium levels of28day in F+LCa and LCa group were significantly increased (p<0.01); urinary fluoride of28day in F, F+LCa and F+HCa group were significantly increased (p<0.01); calcium levels of28day LCa and F+LCa group were significantly lower (p<0.01). Compared with the F group, blood fluoride content of14day F+LCa group was significantly lower (p<0.01); calcium levels of14and28day F+LCa group were significantly lower (p<0.01); urinary fluoride of28day LCa group F+HCa were significantly lower (p<0.01); calcium levels of28day female LCa and F+LCa group were significantly lower (p<0.01), F+HCa group was significantly increased (p<0.01).(3) Determination of behavior and learning memory:Compared with the control group, the numbers of grooming, buttresses, standing times and1minute,3minutes running grids in14and28day F and F+LCa group show a downward trend; the stayed time on platform in14and28day F and F+LCa group shortened significantly. Compared with the F group, the parameters above in14and28day LCa and F+HCa showed an upward trend, while F+LCa group showed a downward trend; the stayed time on platform in14and28day LCa and F+HCa group prolonged.(4) Synaptic density and structural parameters:Compared with the control group, hippocampal synaptic density in LCa and F+LCa showed a decreasing trend; F and F+LCa group synaptic length and thickness of post-synaptic density (PSD) showed a downward trend and the synaptic gap width rised. Compared with the F group, synaptic density in14and28day F+LCa group showed a decreasing trend, synaptic length in14and28days male F+LCa group showed a downward trend; synaptic length in28day LCa and F+HCa group was significantly elevated (p<0.01); the synaptic gap width of fetal rat in LCa group was significantly lower (p<0.05); the thickness of PSD of fetal rats in14days male F+LCa group showed varying degrees of decrease; the thickness of PSD in14day male F+HCa group and28day female LCa group were significantly increased (p<0.05).(5) Detection of apoptosis:the brain hippocampus of fetal rat in the control and the LCa group showed rare apoptotic cells, apoptotic cells in F and F+LCa group increased significantly, apoptotic cells in F+HCa decreased than F group.(6) Immunohistochemistry test:Compared with the control group, DCX protein levels of fetal rat in F, F+LCa and F+HCa group tended to increase (p>0.05); p38protein levels showed a decreasing trend (p>0.05). Compared with the F group, DCX and p38protein expression levels of F+HCa group showed decreasing and increasing trend respectively (p>0.05).(7) PCR test:Compared with the control group, Bcl-2mRNA expression levels of fetal rats in F and F+LCa group were significantly decreased; Caspase12mRNA expression levels of F group, F+LCa and F+HCa group showed a significant rise. Compared with the F group, the Bcl-2mRNA expression levels of LCa and F+HCa group showed a significant rise; Caspase12mRNA expression levels of LCa and F+HCa group declined. JNK mRNA expression levels of each group had no significant difference (p>0.05).In summary, the calcium nutrition of children in Wamiao and Xinhuai was in good condition, showing no significant differences. Using lower fluoride water source in Wamiao was effective, it showed no difference in IQ compared with Xinhuai, lower IQ in individual children might due to incomplete range of changes in water.In addition, different genders of children’s IQ who were exposed to fluoride showed no significant difference. The development providers of children in two villages are in normal range, but height and head circumference in Wamiao were significant differences with Xinhuai (p<0.01). In addition, there were significant differences height, weight and head circumference in different gender of children may be caused due to genetic or dietary differences. Drinking-water type fluorosis changed the blood/urine fluoride concentrations of offsprings and increased brain cell apoptosis,and then affected the offspring’s brain development, reduced the ability of learning memory.The expression of specific protein DCX related to brain development increased,the expression of synaptophysin p38decreased, and also synaptic interface structure parameters changed due to fluorosis, with the length of synaptic decreased, width of synaptic increased, thickness of PSD reduced, causing a decline in the expression level of Bcl-2mRNA and a rise in the expression level of Caspasel2mRNA. Molecular mechanisms of calcium’s intervention on fluorosis might be that it inhibited brain cells apoptosis of rats by regulating the expression of Bcl-2upward and Caspase12downward. Low calcium diet (0.63g/kg) could produce synergies while feeding with high fluoride, and exacerbated the toxic effects of fluoride poisoning; while high calcium diet (70g/kg) with fluoride solution could produce antagonistic effect, reducing the damage to the body induced by fluorosis. |
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