Introduction

Fluorine is an active non-metal that occurs in the environment and that is used in industry and medicine (diagnostics, prevention) [1]. The daily exposure of our organisms to fluorine mainly depends on the geographical region we inhabit. The most important factor contributing to the exposure is the content of fluorine in drinking water and, to a lesser extent, in air and food [2, 3]. Moreover, this element is commonly used in the prevention of dental caries due to its effectiveness and the low costs of manufacture of products for oral care [2, 4–6].

In the organisms of infants and children, about 80–90 % of the absorbed fluorine is accumulated. A smaller amount is stored in the organisms of adults (60 %). Of the received fluorine, 75 %–90 % undergoes absorption in the stomach and intestines, and 99 % of the fluorine that gets to the circulatory system is transported to tissues rich in calcium (mainly to hard tissues). Retrospective studies showed that the symptoms of fluorosis (the disorder of the physiology of bones and teeth and the damage to soft tissue) appeared when the supply of fluorine was over 0.15 mg/kg/24 h [2, 3, 6–9]. In recent years, scientists have been focusing on the toxic influence of this element on the nervous system. Prolonged exposure to fluorine in the prenatal and postnatal stages of development has a toxic influence on the metabolism and physiology of neurons and glia which results in disorders in the processes connected with memory and learning [4, 10, 11]. Epidemiological studies carried out in geographical regions in which fluorine content in drinking water is high showed that children who live in those areas have a statistically significant decreased level of intelligence in comparison to children from regions not contaminated with fluorine [10, 12, 13]. Fluorine exposure in the prenatal and neonatal periods is dangerous because this element has the ability to penetrate through the placenta and it is able to cross the blood-brain barrier. Young individuals are less resistant to the toxic influence of fluorine due to the fact that their defensive mechanisms are not fully developed and the permeability of their blood-brain barrier is higher than among adults [2, 14–16]. This phenomenon was confirmed by a research carried out on rats. The animals were exposed to high levels of fluorine (10, 25, 50 mg/L) for 8 months. The content of fluorides in the rats’ brains was even 250 times higher than in the control group [9]. However, the exact mechanisms by which fluorine decreases cognitive and learning abilities and causes memory loss were not clearly defined. So far, the element has been studied in terms of its influence on neurotransmission, the synthesis of proinflammatory factors, free-radical processes and the apoptosis of cells of the central nervous system [17].

Cytological Changes within Neurons

Microtubules consisting of compact heterodimer tubulins form the cell cytoskeleton in which organelles are suspended. Depending on the type of cells, microtubules can reach the length of even a few millimetres, and their elasticity and the ability to adjust the length through building or degrading heterodimers are of particular importance for the physiology of cells [18]. A proper construction of the cytoskeleton is important for the functioning of neurons. It was observed that the disorders in the construction of microtubules influence the deterioration of dendrites, the degeneration of axons and the decrease in the number of Purkinje cells [9, 19]. Among adult mice exposed to fluorine, a decrease in the expression of tubulins forming the heterodimers (Tuba1 and TubB2a) in the hippocampus was observed (the content of fluorine in drinking water, 100 mg/L). The disorders in the synthesis of tubulins are important in relation to such processes as the maturing or the division of cells because they might lead to the creation of malfunctioning neurons without the ability of signal transmission [9].

The accumulation of fluorine in the brain also influences the content of Nissl bodies, which are concentrations of ribosomes and RNA in neurons. These concentrations are responsible for the characteristic colour of grey matter. Among adult rats exposed to relatively low concentrations of fluorine, a significant decrease of the content of this Nissl substance was observed (the concentration of fluorine in drinking water, 2.1 and 10 mg/L). These values are calculated in the active neurons. Their content decreases in cells that are growing old and degenerating [1].

Neuron Activity and the Transportation of Ions

The research carried out on adult specimens showed a negative influence of fluorine on the volume of neurons. The regulation of the volume of cells and the concentration of ions have a significant influence on the preservation of homeostasis in the nervous system [4, 20]. The stimulation of a nervous synapse is accompanied by the increase of the cell’s volume by 4–30 % of the initial volume. Such changes influence neuron activity because they are related to the changes in the flow of ions and water from the cytoplasm to the extracellular space and vice versa [21–23]. Fluorine (5 mM) causes disorders in the homeostasis of neurons in the hippocampus of adult rats and mice by increasing the outflow of chloride ion from cells and by changing the activity of proteins from the MAP kinase family. This leads to the decrease of the volume and activity of neurons [4]. MAP kinases, i.e., mitogen-activated protein kinases, are a family of proteins that take part in the regulation of the response to extracellular factors such as mitogens. The proteins ERK and JNK and the isoforms of protein p58 belong to the family of serine-threonine kinases. These proteins influence the regulation of the growth and differentiation of cells, the regulation of apoptosis and the expression of genes [24]. However, recent analysis proved that they also take part in the regulation of the activity of membrane transporters. Fluorine, through its influence on the activity of Ras protein, activates a cascade of reactions that leads to the activation of ERK. This influences the membrane ion channels and leads to changes in the flow of ions (including an increased outflow of chloride ion) and in the nervous cell volume causing disorders in cell metabolism, in cell functioning and, most of all, in the transmission of nerve impulses [25, 26].

The Energy Metabolism of Neurons

The activity of mitochondria is a very important factor which influences numerous processes and the lifespan of neurons. Due to their limited glycolytic capabilities, these cells depend on the processes of oxidative phosphorylation which is the main source of energy in the central nervous system. The energy created by mitochondria is used for the activity of membrane ion channels and for the transmission of impulses through synapses, and the dysfunction of these organelles is observed in neurodegenerative illnesses [27, 28]. One of the important factors influencing the energy metabolism of neurons is the transportation, absorption and transformation of glucose, because it serves as the main source of energy for neurons [29]. It is common knowledge that providing proper amounts of glucose to an organism significantly influences the improvement of cognitive functions, and numerous analyses confirmed that disorders in glucose metabolism may be the cause of the death of neurons [35]. Another study carried out on mice confirmed that fluorine influences the activity of complexes of the respiratory chain and of enzymes of the citric acid cycle. A decrease in the activity of complexes I, II, III and IV; isocitrate dehydrogenase and succinate dehydrogenase was observed in the cerebral cortex, cerebellum and hippocampus of mice exposed to high concentrations of fluorine (the concentration of fluorine in drinking water, 270 mg/L) [27].

A decrease in the activity of the respiratory chain complexes influences the increase in the synthesis of ROS which activate the pathways leading to the degradation of mitochondria as well as the entire cell [27]. ROS and the products of lipid peroxidation are also responsible for the formation of compounds that block the active area of isocitrate dehydrogenase, consequently inhibiting the oxidative decarboxylation of the isocitrate in the Krebs cycle and influencing the activity of this pathway. Fluorine itself influences the activity of many enzymes through its ability to break the hydrogen bonds in proteins—e.g. in the enzyme active centre [36, 37]. Furthermore, the increase in the synthesis of free radicals in mitochondria leads to the initiation of oxidative stress and the degradation of mitochondrial DNA. The result of these processes forms another phenomenon that takes place in the mitochondria—the disorder of the expression of enzymes necessary for the synthesis of ATP and the decrease of ATP concentration. This, in consequence, leads to the activation of the processes that cause the death of the cell [33].

Oxidative Stress and the Activity of Anti-Oxidative Enzymes

The analysis carried out with the usage of experimental animal models more than once confirmed that the accumulation of fluorine in the central nervous system initiates inflammatory and degenerative processes through the activation of oxidative stress in both young and adult specimens. Oxidative stress is caused by the disturbance in the balance between the synthesis of ROS and the activity of anti-oxidative enzymes. The increasing concentration of ROS leads to metabolism disorders, the initiation of inflammatory states and the disorders of the differentiation, maturing and division of cells. This, in consequence, causes tissue damage [17, 38]. ROS do not only cause disorders in the signal pathways of cells, but if their concentration is high, membrane lipid release and oxidation take place. The products of this process might be further transformed into physiological and pharmacological active inflammatory compounds [39].

Numerous analyses carried out on cell cultures and animal models confirmed that fluorine accumulation in the brain leads to the increase of the concentration of ROS, the decrease of the activity of antioxidative enzymes and the increase of the intensity of lipid peroxidation. In the neuron cultures isolated from the hippocampus, after 48 h of fluorine incubation (concentration, 40 and 80 mg/L), several phenomena were observed: the increase in the synthesis of ROS and the derivatives of lipid peroxidation, malondialdehyde (MDA), the decrease of the activity of antioxidative enzymes, superoxide dismutase (SOD) and glutathione peroxidise (GPx), and the decrease of the concentration of glutathione [42]. An increase in the concentration of the products of lipid oxidation was also observed among adult rats that were exposed to 10 mg/L of fluorine concentration in drinking water. In the case of these animals, SOD activity was also smaller [17].

The analyses carried out so far show that one of the mechanisms by which fluorine influences the disorders in brain functioning is the increase of the synthesis of ROS and the weakening of the defensive mechanisms against their activity (the decrease in the activity of antioxidative enzymes) [43]. In the central nervous system, the intensity of the processes that utilize oxygen is very high. Moreover, there are high concentrations of easily oxidizable fatty acids, and the activity of antioxidative enzymes is relatively small in comparison to other tissues [7, 44]. Long-lasting oxidative stress causes the “wear” of enzymes responsible for the removal of free radicals. Their increasing concentration in cells causes lipid peroxidation and the oxidation of proteins and nucleic acids [7, 79].

Neural cell adhesion molecules (NCAM) are membrane glycoproteins that are responsible for the adhesion and migration of cells of the nervous system, the development of axons and synapses and the activation of signal pathways [80]. The disturbances in the expression of the isoforms NCAM-120, NCAM-140 and NCAM-180 influence the cognitive functions of the nervous system, whereas the presence of NCAM-180 significantly influences the plasticity of neurons in the hippocampus. Nerve cells isolated from the hippocampus show a decreased expression of NCAM after incubation with fluorine (the applied fluorine concentrations, 20, 40 and 80 mg/L). The application of the 80-mg/L concentration resulted in a decrease in the amount of all of the three aforementioned isoforms, whereas with lower fluorine concentrations the expression of the isoform NCAM-180 also decreased significantly [94]. Depending on its level of expression and the pathways it influences, it may protect cells from apoptosis or initiate the process [95, 96]. A research carried out on neurons isolated from the hippocampus of a rat incubated 24 h with fluorine (40 and 80 mg/L) indicated a significantly increased frequency of damage to the DNA and an increase in the synthesis of NF-kB [42]. Numerous reports concerning the occurrence of endemic fluorosis lead to the establishment of an accepted concentration of fluorine in drinking water by the World Health Organization (WHO) at a level of which the element does not accumulate excessively in the human organism and does not cause adverse effects. The current value is set at 1.5 mg/L [1, 2]. However, recent findings concerning the toxic influence of this element on the nervous system, especially dangerous in relation to develo** organisms, lead to higher restrictions in countries where fluorosis occurs frequently.