Roles Of Neural Cell Adhesion Molecules In Neurotoxicity Of Fluoride In The Primary Rat Hippocampal Neurons

Endemic fluorosis which seriously impairs human health is prevalent in the world. Fluorosis can damage central nervous system. Previous epidemiological studies have demonstrated that the intelligence of children who ingestided high level of fluoride was seriously impaired, appearing the learning ability and rememberance decreased. Previous animal experimental results have shown that hippocampus was a neurotoxical target site attacked by fluoride. Apoptosis playing some roles in the toxicological mechanisms induced by fluoride has gotten attention. Free radi theory is one of apoptosis sources.However, the effects of fluoride on oxidative stress and apoptosis are rarely reported. Synaptic plasticity is the basic of formation and stabilization of learning and memory. Furthermore, neural cell adhesion molecules (NCAM) play an important role in formation of synapse and regulation of plasticity. NCAM has three isoforms, NCAM-180, NCAM-140 and NCAM-120 by alternative splicing of the pre-mRNA encoded by a single gene. NCAM mediates cell adhesion through homophilic as well as heterophilic interactions, and influences cell migration, neurite extension, and possibly formation of synapses in the brain. NCAM is important for neuronal plasticity in the adult brain associated with neural development and some major brain functions such as formation, stabilization and regeneration of learning and memory. Thus we surmised fluoride would cause neurotoxical effects via influencing the expression of NCAM in hippocampus.We treated primary rat hippocampal neuron as target and did some experiments in vitro to explore effects on oxidative stress, DNA damage, apoptosis and expression levels of NCAM mRNA and NCAM protein by low-dose fluoride. Whether apoptosis or NCAM plays a certain role in neurotoxical mechanism such as learning and memory disorder impaired by fluoride is primarily explained. It is the first research using the model of primary rat hippocampal neurons to investigate neuraltoxicology of fluoride and its effect on the expression of NCAM on genetic transcriptional and translational levels in hippocampus.PartⅠEstablishment of primary rat hippocampal neuron culture and find suitable fluoride-exposed dosesObjective To establish the method of primary rat hippocampal neuron culture, investigate survival of hippocampal neuron after incubated and determine the fluoride-exposed doses. Methods The axon prolification, dendron outgrowth and neuron survival were observed, and the rate of cellular survivors was measured by methylthiazolyltetrazolium (MTT) assay after the neurons were incubated with 20, 40, 80 and 160μg/ml sodium fluoride for 24 hours in vitro. Results Compared with control group, the length and number of neurites were significantly decreased in 80 and 160μg/ml fluoride-treated groups. The rates of cellular survivors in 80 and 160μg/ml fluoride-treated groups were remarkably lower than that in the control group (P<0.05). The survivor rate in 160μg/ml fluoride-treated group was less than 50%. Conclusion Fluoride can inhibit survival of rat hippocampal neuron. A certain-dose fluoride can restrain growth of rat hippocampal neuron. The impairment of hippocampus induced by fluoride may be a neuro-toxicological situs.PartⅡEffects of fluoride on oxidative stress, DNA damage and apoptosis in primary rat hippocampal neuronsObjective To explore the effects of fluoride on oxidative stress, DNA damage, and apoptosis in primary rat hippocampal neurons, and to explore the involved role of reactive oxygen species (ROS) on apoptosis. Methods The level of intracellular ROS, the activities of intracellular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), the contents of intracellular malondialdehyde (MDA) and glutathione (GSH), the activity of extracellular lactate dehydrogenase(LDH), DNA damage, and apoptosis were detected after the neurons were incubated with 20, 40 and 80μg/ml sodium fluoride for 24 hours in vitro. Results Compared with control group, The ROS level, MDA content, activity of extracellular LDH and percentage of apoptosis in 40 and 80μg/ml fluoride-treated groups were significantly higher (P<0.05, P<0.05, P<0.05, P<0.05, respectively), the activity of GSH-Px and the content of GSH were significantly decreased in all fluoride-treated groups (P<0.05, P<0.05, respectively), the activity of SOD in 80μg/ml fluoride-treated group was remarkably lower (P<0.05). The Olive tail moments in all fluoride-treated group were remarkably elevated (P<0.05). The significant relationship of positive correlation between ROS content and percentage of apoptosis was also demonstrated in identical fluoride-treated groups (r=0.913, P<0.05). Conclusion Fluoride can impair membranes of hippocampal neurons and induce oxidative stress, DNA damage, and apoptosis in rat hippocampal neurons. The oxidative stress may play an important role in the apoptosis induced by fluoride.PartⅢEffects of fluoride on mRNA and protein expression levels of neural cell adhesion molecules (NCAM) in primary rat hippocampal neuronsObjective To observe the effects of NCAM expression levels on the genetic transcriptional and translational levels induced by fluoride in primary rat hippocampal neurons, and to explore the dose-effect relationship of NCAM expression induced by fluoride. Methods The mRNA and protein expression levels of NCAM were detected by reversed transcriptive polymerase chain reaction (RT-PCR) and Western blot after the rat hippocampal neurons were incubated with 20, 40 and 80μg/ml sodium fluoride for 24 hours in vitro. Results Compared with control group, the levels of NCAM mRNA expression in 40 and 80μg/ml fluoride-treated groups were significantly decreased (P<0.05). NCAM-180 expression levels in 40 and 80μg/ml fluoride-treated groups, NCAM-140 expression levels in all fluoride-treated groups, and NCAM-120 expression level in 80μg/ml fluoride-treated group were decreased (P<0.05, P<0.05, P<0.05, respectively). Conclusion Fluoride can provoke the underexpressions of NCAM mRNA and three major NCAM isoforms: NCAM-180, NCAM-140, and NCAM-120.