レビュー論文：フッ素が中枢神経系の恒常性障害に与える影響(The Influence of Fluorine on the Disturbances of Homeostasis in the Central Nervous System)

出典：https://link.springer.com/article/10.1007/s12011-016-0871-4
doi: 10.1007/s12011-016-0871-4
PMCID: PMC5418325  PMID: 27787813
他の参考：フッ素による人体と環境への過剰負荷の化学的側面
https://pmc.ncbi.nlm.nih.gov/articles/PMC8945431/

©Biological Trace Element Research
Author:
K Dec, A Łukomska, D Maciejewska, K Jakubczyk, I Baranowska-Bosiacka, D Chlubek, A Wąsik, I Gutowska

Received 2016 Aug 24;
Accepted 2016 Oct 11;
Issue date 2017.

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Abstract

Fluorides occur naturally in the environment, the daily exposure of human organism to fluorine mainly depends on the intake of this element with drinking water and it is connected with the geographical region. In some countries, we can observe the endemic fluorosis—the damage of hard and soft tissues caused by the excessive intake of fluorine. Recent studies showed that fluorine is toxic to the central nervous system (CNS). There are several known mechanisms which lead to structural brain damage caused by the excessive intake of fluorine. This element is able to cross the blood-brain barrier, and it accumulates in neurons affecting cytological changes, cell activity and ion transport (e.g. chlorine transport). Additionally, fluorine changes the concentration of non-enzymatic advanced glycation end products (AGEs), the metabolism of neurotransmitters (influencing mainly glutamatergic neurotransmission) and the energy metabolism of neurons by the impaired glucose transporter—GLUT1. It can also change activity and lead to dysfunction of important proteins which are part of the respiratory chain. Fluorine also affects oxidative stress, glial activation and inflammation in the CNS which leads to neurodegeneration. All of those changes lead to abnormal cell differentiation and the activation of apoptosis through the changes in the expression of neural cell adhesion molecules (NCAM), glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF) and MAP kinases. Excessive exposure to this element can cause harmful effects such as permanent damage of all brain structures, impaired learning ability, memory dysfunction and behavioural problems. This paper provides an overview of the fluoride neurotoxicity in juveniles and adults.

フッ素は環境中に自然に存在し、ヒトのフッ素への毎日の曝露は主に飲料水によるこの元素の摂取に依存し、地理的地域と関係している。一部の国では、私たちは風土病のフッ素症を観察できます-フッ素の過剰摂取によって引き起こされる硬組織と軟部組織の損傷である。最近の研究は、フッ素が中枢神経系(CNS)に有毒であることを示した。フッ素の過剰摂取によって引き起こされる構造的脳損傷につながるいくつかの既知のメカニズムがある。この元素は血液脳関門を通過することができ、細胞学的変化、細胞活性およびイオン輸送(例えば塩素輸送)に影響を及ぼすニューロンに蓄積する。さらに、フッ素は非酵素的糖化最終産物(AGE)の濃度、神経伝達物質の代謝(主にグルタミン酸作動性の神経伝達に影響を及ぼす)、およびグルコース輸送体の障害であるGLUT1によるニューロンのエネルギー代謝を変化させる。また、呼吸鎖の一部である重要なタンパク質の活性を変化させ、機能不全を引き起こす可能性もある。フッ素はまた、神経変性につながるCNSにおける酸化ストレス、グリア活性化および炎症に影響する。これらの変化の全ては、神経細胞接着分子(NCAM)、グリア線維性酸性蛋白質 (GFAP)、脳由来神経栄養因子(BDNF)およびMAPキナーゼの発現の変化を介して、異常な細胞分化およびアポトーシスの活性化をもたらす。この元素への過剰な曝露は、すべての脳構造の永続的な損傷、学習能力の障害、記憶障害および行動上の問題などの有害な影響を引き起こす可能性がある。この論文では、小児および成人におけるフッ化物神経毒性の概要を提供する。

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].

フッ素は環境中に存在する活性非金属であり、工業や医療(診断、予防)で使用されている [1]。私たちの生物のフッ素への毎日の暴露は、主に私たちが居住する地理的地域に依存する。暴露に寄与する最も重要な因子は、飲料水中のフッ素含有量であり、程度は低いが、大気中および食物中のフッ素含有量である[2, 3]。さらに、この元素は、その有効性および口腔ケア用製品の製造コストの低さのために、虫歯の予防に一般的に使用されている[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].

乳児および小児の生体では、吸収されたフッ素の約80–90%が蓄積される。より少量が成人の生体に貯蔵される(60%)。摂取されたフッ素の75%~90%は胃や腸で吸収され、循環系に到達したフッ素の99%はカルシウムを豊富に含む組織(主に硬組織)に輸送される。遡及的研究はフッ素症の症状(骨や歯の生理機能の障害および軟組織の損傷)がフッ素の供給が0.15mg/kg/24時間を超えると出現することを示した[2, 3, 6~9]。近年、科学者たちはこの元素が神経系に及ぼす毒性影響に注目している。出生前および出生後の発達段階におけるフッ素への長期曝露は、ニューロンおよびグリアの代謝および生理学に毒性影響を及ぼし、記憶および学習に関連する過程の障害をもたらす[4, 10, 11]。飲料水中のフッ素含有量が高い地理的地域で実施された疫学調査では、これらの地域に住む小児は、フッ素に汚染されていない地域の小児と比較して、知能レベルが統計的に有意に低下していることが示された[10, 12, 13]。胎児期および新生児期のフッ素曝露は、この元素が胎盤を透過する能力を有し、血液脳関門を通過できるため危険である。若年者は、防御機構が十分に発達しておらず、血液脳関門の透過性が成人より高いため、フッ素の毒性影響に対する抵抗性が低い[2, 14~16]。この現象はラットで行われた研究で確認された。その動物は高レベルのフッ素(10、25、50mg/L)に8か月間曝露された。ラットの脳内のフッ化物含有量は、対照群の250倍も高かった [9]。しかし、フッ素が認知および学習能力を低下させ、記憶喪失を引き起こす正確なメカニズムは明確には定義されていない。これまで、この元素は、神経伝達、炎症性因子の合成、フリーラジカルプロセスおよび中枢神経系の細胞のアポトーシスに対する影響に関して研究されてきた [17]。

Cytological Changes within Neurons(ニューロン内の細胞学的変化)

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].

コンパクトなヘテロ二量体チューブリンからなる微小管は細胞小器官が浮遊する細胞骨格を形成する。細胞の種類によっては、微小管は数mmの長さにも達することがあり、その弾力性とヘテロ二量体の構築または分解によって長さを調節する能力は、細胞の生理学にとって特に重要である [18]。細胞骨格の適切な構築はニューロンの機能にとって重要である。微小管構築の障害は樹状突起の劣化、軸索の変性およびPurkinje細胞数の減少に影響した[9, 19]。フッ素に曝露された成体マウスでは、海馬におけるヘテロ二量体 (Tuba1およびTubB2a) を形成するチューブリンの発現低下が観察された(飲料水中のフッ素含有量100mg/L)。チューブリンの合成における障害は、細胞の成熟や分裂などの過程に関連して重要である、なぜなら、これらの障害は、シグナル伝達の能力を欠いた機能不全のニューロンの生成につながる可能性があるからである [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].

脳内のフッ素の蓄積は、ニューロンのリボソームとRNAの濃度であるニッスル小体の含有量にも影響する。これらの濃度は灰白質の特徴的な色の原因となる。比較的低濃度のフッ素に暴露された成体ラットでは、ニッスル物質の含有量の有意な減少が観察された(飲料水中のフッ素濃度、2.1mg/Lおよび10mg/L)。これらの値は、活動中のニューロンで計算されます。それらの含量は、老化して退化する細胞で減少する [1]。

Neuron Activity and the Transportation of Ions(ニューロンの活動とイオンの輸送)

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].

成体標本で行われた研究は、ニューロンの体積に対するフッ素の負の影響を示した。細胞の体積とイオン濃度の調節は、神経系の恒常性の維持に重要な影響を及ぼす[4, 20]。神経シナプスが刺激されると、細胞の体積は最初の体積の4~30%増加する。このような変化は、細胞質から細胞外へのイオンと水の流れの変化、およびその逆に関連しているため、ニューロンの活動に影響を及ぼす[21–23]。フッ素(5mM)は、細胞からの塩化物イオンの流出を増加させ、MAPキナーゼファミリーのタンパク質の活性を変化させることによって、成体ラットおよびマウスの海馬のニューロンの恒常性に障害を引き起こす。これはニューロンの体積と活動の減少につながる[4]。MAPキナーゼ、すなわちマイトジェン活性化プロテインキナーゼは、マイトジェン(分裂促進因子)のような細胞外因子に対する応答の調節に関与するタンパク質ファミリーである。タンパク質ERKとJNKおよびタンパク質p58のアイソフォームはセリントレオニンキナーゼファミリーに属する。これらのタンパク質は、細胞の成長と分化の調節、アポトーシスの調節、および遺伝子の発現に影響を及ぼす [24]。しかしながら、最近の解析により、それらが膜輸送体の活性の調節にも関与していることが証明された。フッ素はRasタンパク質の活性に影響を与え、ERKの活性化につながる一連の反応を活性化する。これは膜イオンチャネルに影響を与え、イオンの流れの変化(塩化物イオンの流出の増加を含む)と神経細胞の体積の変化を引き起こし、細胞代謝、細胞機能、そして何よりも神経インパルスの伝達に障害を引き起こす[25, 26]。

The Energy Metabolism of Neurons(ニューロンのエネルギー代謝)

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 [30–32]. Among rats exposed to fluorine, decreased glucose usage was observed (the concentration of fluorine in drinking water, 50 and 100 mg/L) as well as a decrease in the expression of the main receptor responsible for the glucose uptake in the nervous system, GLUT1 (the concentration of fluorine in drinking water, 25, 50 and 100 mg/L), in the cerebral cortex and hippocampus [30].

ミトコンドリアの活動は、ニューロンの数多くの過程と寿命に影響する非常に重要な因子である。解糖能力が限られているため、これらの細胞は中枢神経系の主要なエネルギー源である酸化的リン酸化のプロセスに依存している。ミトコンドリアによって作られたエネルギーは、膜イオンチャネルの活性とシナプスを介したインパルスの伝達に使われ、これらの器官の機能不全は神経変性疾患で観察される[27, 28]。ニューロンのエネルギー代謝に影響する重要な因子の１因は、グルコースの輸送、吸収および変換である。グルコースはニューロンの主要なエネルギー源であるからである[29]。生体に適切な量のグルコースを供給することが認知機能の改善に大きく影響することは周知の事実であり、グルコース代謝の障害がニューロンの死の原因である可能性があることが多くの解析によって確認されている [30–32]。フッ素に曝露されたラットでは、グルコース使用量の減少(飲料水中のフッ素濃度、50および100mg/L)、ならびに神経系におけるグルコース取り込みに関与する主要受容体であるGLUT1(飲料水中のフッ素濃度、25、50および100mg/L)の大脳皮質および海馬における発現の減少が観察された [30]。

An equally important factor for the functioning of mitochondria is oxidative stress. Oxidative stress caused by the excessive production of reactive oxygen species (ROS) significantly influences the functioning of neurons through its influence on the activity of mitochondrial enzymes. Increased ROS synthesis in the mitochondria of nervous cells, disorders in integrity and changes in the potential of mitochondrial membrane were observed among rats which received fluorine in the proportion of 13 mg/kg/24 h [33]. Prenatal and postnatal exposure of mice to high concentrations of fluorine in drinking water (150 mg/L) caused disorders in the energy metabolism of the cerebral cortex of the animals. One of the observed facts was the increased activity of one of the subunits of ATP synthase—ATP5h. This enzyme consists of several subunits that form two dimers—F1 and F0. F1 is the catalytic part, whereas F0 is a membrane subunit which takes part in the transportation of protons. The disorders in the functioning of these subunits lead to changes in the energy balance (ATP/ADP) within the cell. The analysis showed the hyperactivity of subunit ATP5h, a part of the content of F0, which leads to disorders in the activity of ATP synthase [15, 34]. The exposure to fluorine also caused a decrease in the activity of NADH-ubichinon oxidoreductase which is a part of respiratory chain complex 1. It is involved in the transportation of ions in the chain and in the creation of sodium gradient necessary in the process of ATP synthesis. The aforementioned changes in the activity of ATP synthase and NADH-ubichinon oxidase lead to a significant decrease of ATP synthesis in the mitochondria of nervous cells [15, 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].

ミトコンドリアの機能にとって同様に重要な因子は酸化ストレスである。活性酸素種(ROS)の過剰産生によって引き起こされる酸化ストレスは、ミトコンドリア酵素の活性に対する影響を介してニューロンの機能に有意に影響する。13mg/kg/24時間の割合でフッ素を投与したラットでは、神経細胞のミトコンドリアにおけるROS合成の増加、ミトコンドリア膜の完全性の障害およびミトコンドリア膜電位の変化が観察された[33]。高濃度(150mg/L)のフッ素飲料水への出生前および出生後のマウスの暴露は、動物の大脳皮質のエネルギー代謝に障害を引き起こした。観察された事実の1個は、ATPシンターゼのサブユニットの1個であるATP5hの活性の増加であった。この酵素は、F1とF0という2個の二量体を形成するいくつかのサブユニットからなる。F1は触媒部分であり、F0はプロトン輸送に関与する膜サブユニットである。これらのサブユニットの機能障害は細胞内のエネルギーバランス(ATP/ADP)の変化をもたらす。その結果、F0のコンテンツの一部であるサブユニットATP5hの活性亢進が認められ、ATP合成酵素の活性に異常が生じていた[15, 34]。フッ素曝露は呼吸鎖複合体1の一部であるNADH‐ユビキノン酸化還元酵素の活性低下を引き起こした。それは鎖中のイオンの輸送とATP合成過程に必要なナトリウム勾配の生成に関与している。前述のATP合成酵素とNADH-ユビキノン酸化酵素の活性の変化は、神経細胞のミトコンドリアにおけるATP合成の有意な減少をもたらす[15, 35]。マウスで実施された別の研究では、フッ素が呼吸鎖の複合体およびクエン酸回路の酵素の活性に影響を及ぼすことが確認された。複合体I、II、IIIおよびIVの活性の低下；高濃度フッ素(飲料水中のフッ素濃度270mg/L)に暴露されたマウスの大脳皮質、小脳及び海馬においてイソクエン酸脱水素酵素及びコハク酸脱水素酵素が認められた [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].

呼吸鎖複合体の活性低下は、細胞全体と同様にミトコンドリアの分解につながる経路を活性化するROSの合成の増加に影響する[27]。ROSと脂質過酸化産物は、イソクエン酸デヒドロゲナーゼの活性領域を遮断する化合物の生成にも関与し、その結果、クレブス回路におけるイソクエン酸の酸化的脱炭酸を阻害し、この経路の活性に影響を及ぼす。フッ素自身は、タンパク質の水素結合を切断する能力を通じて、多くの酵素の活性に影響を与える - 例えば、酵素活性中心において[36, 37]。さらに、ミトコンドリアにおけるフリーラジカル合成の増加は、酸化ストレスの開始とミトコンドリアDNAの分解をもたらす。これらのプロセスの結果、ミトコンドリアで起こる別の現象が形成される - ATP合成に必要な酵素の発現の障害とATP濃度の低下。その結果、細胞死を引き起こすプロセスが活性化される[33]。

Oxidative Stress and the Activity of Anti-Oxidative Enzymes(酸化ストレスと抗酸化酵素の活性)

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].

実験動物モデルを用いて実施した解析により、中枢神経系におけるフッ素の蓄積が、若齢および成体標本の両方において酸化ストレスの活性化を介して炎症および変性プロセスを開始することが確認された。酸化ストレスは、ROSの合成と抗酸化酵素の活性との間のバランスの乱れによって引き起こされる。ROS濃度の増加は代謝障害、炎症状態の開始および細胞の分化、成熟および分裂の障害をもたらす。その結果、組織損傷を引き起こす[17, 38]。ROSは細胞のシグナル経路に障害を引き起こすだけでなく、濃度が高いと膜脂質放出と酸化が起こる。このプロセスの産物はさらに生理的および薬理学的に活性な炎症性化合物に変換される可能性がある[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 [40]. Increased activity of catalase (CAT) was observed among young rats exposed to fluorine, which might point to the activation of protective mechanisms against the harmful activity of oxygen free radicals in the organism (the concentration of fluorine in drinking water, 30 and 100 mg/L) [33, 41]. In relation to adult rats that received 20 mg of fluorine per kilogram of body mass every 24 h, it was observed that the concentration of glutathione in the brain decreased, the production of the radicals OH and NO increased and the activity of antioxidative enzymes CAT, SOD, GPX and glutathione reductase (GR) was smaller [5]. Moreover, increased intensity of oxidation of lipids and proteins was observed in the cerebral cortex, cerebellum and medulla oblongata (the concentration of fluorine in drinking water, 50 and 150 mg/L). These results were confirmed by another analysis in which, among rats exposed to fluorine in the prenatal and postnatal periods, increased concentrations of fluorine in the serum and the brain were observed as early as on the 14th day of life, which resulted in the decreased activity of SOD and higher intensity of lipid peroxidation in the brains of the analysed animals (the concentration of fluorine in drinking water, 20 mg/L) [33, 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].

細胞培養および動物モデルで実施された多数の分析は、脳におけるフッ素蓄積がROS濃度の増加、抗酸化酵素活性の低下および脂質過酸化の強度の増加をもたらすことを確認した。海馬から分離したニューロン培養において、48時間のフッ素インキュベーション(濃度、40および80mg/L)後、いくつかの現象が観察された：ROSおよび脂質過酸化の誘導体、マロンジアルデヒド(MDA)の合成の増加、抗酸化酵素、スーパーオキシドジスムターゼ (SOD)およびグルタチオンペルオキシダーゼ(GPx)の活性の減少、およびグルタチオン濃度の減少[40]。カタラーゼ(CAT)活性の増加がフッ素に曝露された若齢ラットで観察され、これは生命体における酸素フリーラジカルの有害な活性に対する保護機構の活性化を示す可能性がある(飲料水中のフッ素濃度、30および100mg/L)[33, 41]。体重1kgあたり20 mgのフッ素を24時間ごとに投与した成体ラットに関連して、脳内グルタチオン濃度が低下し、OHおよびNOラジカルの産生が増加し、抗酸化酵素CAT、SOD、GPXおよびグルタチオンレダクターゼ(GR)の活性が低下することが観察された[5]。さらに、脂質および蛋白質の酸化強度の増加が大脳皮質、小脳および延髄で観察された(飲料水中のフッ素濃度、50および150mg/L)。これらの結果は、出生前および出生後にフッ素に曝露されたラットにおいて、生後14日目という早期に血清中および脳中のフッ素濃度の上昇が観察され、その結果、分析された動物の脳においてSOD活性の低下および脂質過酸化の強度の上昇が生じた別の分析によって確認された(飲料水中のフッ素濃度20mg/L) [33, 42]。飲料水中のフッ素濃度10mg/Lに暴露した成体ラットでも、脂質酸化生成物濃度の上昇が観察された。これらの動物の場合、SOD活性も低かった[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 [40, 45]. The changes in the content of membrane phospholipids in neurons, caused by their release and oxidation, result in the changes in the fluidity, stability and permeability of the cell membrane [46, 47]. Furthermore, by activating different signal paths, oxidative stress leads to the decrease in the lifespan of cells, the disorders in growth and differentiation and the initiation of apoptosis [7, 48, 49].

これまでの解析から、フッ素が脳機能障害に影響を与えるメカニズムの１個として、ROSの合成増加とその活性に対する防御機構の弱体化(抗酸化酵素の活性低下)が挙げられる[43]。中枢神経系では、酸素を利用するプロセスの強度が非常に高い。さらに、高濃度の容易に酸化できる脂肪酸が存在し、抗酸化酵素の活性は他の組織と比較して比較的小さい[7, 44]。長期にわたる酸化ストレスは、フリーラジカルの除去に関与する酵素の「摩耗」を引き起こす。細胞内のフリーラジカルの濃度が上昇すると、脂質の過酸化やタンパク質および核酸の酸化が起こる[40, 45]。ニューロンにおける膜リン脂質の放出と酸化による含有量の変化は、細胞膜の流動性、安定性、透過性の変化をもたらす[46, 47]。さらに、異なるシグナル経路を活性化することにより、酸化ストレスは細胞寿命の減少、成長と分化の障害およびアポトーシスの開始をもたらす[7, 48, 49]。

The Advanced Glycation End Products(終末糖化産物)

The advanced glycation end products (AGEs) of proteins and lipids are created spontaneously in living organisms in a multi-stage process that does not undergo enzymatic catalysis. In physiological conditions, AGEs fulfil regulatory functions, including the inhibition of cell differentiation. A high content of glycation products was observed in foetal stem cells. Their intensive degradation ensues when they begin to differentiate. The increase in the synthesis and the concentration of AGEs in the organism is observed in pathological states [43, 50]. In properly functioning organisms, AGEs are quickly degraded in proteasomes. However, in some products, cross-linking occurs, which influences the physicochemical properties of these cells and leads to the creation of insoluble aggregates. AGEs are bound by specific receptors. Currently, there are five known types of receptors that bind the products of non-enzymatic glycation: MSR-1 (macrophage scavenger receptor), AGE-R1, AGE-R2 (which binds phosphoproteins), AGE-R3 (which recognizes galactosidic groups) and RAGE (the receptor for AGEs). While the receptors AGE-R1–3 and MSR-1 take part in the removal of the products of glycation from circulation, RAGE acts as a signal receptor activating the processes connected with the synthesis of ROS, transcription factors and the proinflammatory particles such as nuclear factor kB (NF-kB) and the previously mentioned MAP kinases (MAPK) [43, 50, 51]. RAGE demonstrates expression on macrophages, T lymphocytes, cardiomyocytes, endothelial cells, smooth muscle cells, neurons and dendritic cells [52]. The accumulation of AGEs in neurons increases the synthesis of ROS, disturbs the transmission of nervous impulses and stimulates the atrophy of nerve fibres. Moreover, it correlates with the increased expression of proteins that regulate the apoptosis and differentiation of cells [43, 53].

蛋白質と脂質の高度糖化最終産物(AGE)は、酵素触媒を介さない多段階プロセスで生体内で自然に生成される。生理学的条件において、AGEは細胞分化の阻害を含む調節機能を果たす。胎児幹細胞では糖化産物の含量が高かった。それらは分化し始めると集中的に分解される。生体内でのAGEの合成と濃度の増加は病理状態で観察される[43, 50]。正常に機能している生体では、AGEはプロテアソーム中で速やかに分解される。しかしながら、いくつかの産物では架橋が起こり、それがこれらの細胞の物理化学的性質に影響を与え、不溶性の凝集体の生成につながる。AGEは特異的な受容体に結合する。現在、非酵素的糖化産物に結合する受容体が5種類が知られている：MSR-1 (マクロファージスカベンジャー受容体)、AGE-R1、AGE-R2 (リンタンパク質に結合する)、AGE-R3 (ガラクトシド基を認識する)、RAGE (AGEs受容体)。受容体AGE-R1–3とMSR-1は循環からの糖化産物の除去に関与するが、RAGEはROS、転写因子、および核因子kB(NF-kB)や前述のMAPキナーゼ (MAPK)などの炎症性粒子の合成に関連するプロセスを活性化するシグナル受容体として作用する[43, 50, 51]。RAGEは、マクロファージ、Tリンパ球、心筋細胞、内皮細胞、平滑筋細胞、ニューロンおよび樹状細胞に発現を示す[52]。ニューロンにおけるAGEの蓄積はROSの合成を増加させ、神経インパルスの伝達を妨害し、神経線維の萎縮を刺激する。さらに、それは細胞のアポトーシスと分化を調節する蛋白質の発現増加と相関する[43, 53]。

In vivo research carried out on adult rats showed that fluorine increases the concentration of products of advanced glycation of proteins in cells (the concentration in drinking water, 50 mg/L) [43]. A significant increase in the expression of RAGE and NADPH oxidase 2 (NOX2) was also observed among specimens exposed to fluorine for 6 months (the concentration in drinking water, 5 and 50 mg/L). In order to determine the influence of NOX2 on the activation of AGE/RAGE, simultaneous research with cell cultures was carried out. SH-SY5Y cells originating from human neuroblastoma were incubated with various concentrations of fluorine for 48 h (the applied concentrations, 0.5, 5 and 50 mg/L). The following phenomena were observed in the culture with the concentration of 50 mg/L: a significant increase in ROS and MDA after 6 h of incubation, increased expression of NOX2 after 12 h of incubation and RAGE after 24 h of incubation and an increased concentration of AGE in cells after 36 h of incubation (the concentrations of the analysed particles positively correlated with the time of incubation). The analysis confirmed that one of the mechanisms that lead to the degeneration of neurons is the activation of the AGE/RAGE complex. An additional analysis carried out in vitro confirmed that one of the mechanisms that lead to the activation of AGE/RAGE is the increase in the activity of NOX2 [43]. An increased intensity of non-enzymatic glycation of proteins is predominantly observed when the activity of oxidative factors becomes stronger and, at the same time, when the antioxidative mechanisms are inhibited. As mentioned before, fluorine strengthens the synthesis of free radicals and decreases the activity of antioxidative enzymes in the central nervous system which may strengthen the synthesis of AGEs and the expression of RAGE on the membranes of neurons. This, in consequence, may cause pathological accumulation of AGEs in the cells of the nervous system [43, 52]. The accumulation of AGEs in neurons activates proinflammatory transcription factors—NF-kB initiating the inflammatory state and MAP kinases which change the permeability of the membrane of nerve cells and may activate the apoptosis pathway. These processes may initiate the demyelination of dendrites and the degeneration of neurons.

成体ラットで実施されたin vivo研究は、フッ素が細胞内のタンパク質の高度糖化産物の濃度を増加させることを示した(飲料水中濃度50mg/L)[43]。RAGEとNADPHオキシダーゼ2(NOX2)の発現の有意な増加も、6か月間フッ素に曝露した標本で観察された(飲料水中の濃度、5および50mg/L)。AGE/RAGEの活性化に対するNOX2の影響を調べるために、細胞培養との同時研究を行った。ヒト神経芽細胞腫由来SH‐SY5Y細胞をさまざまな濃度のフッ素と48時間培養した(適用濃度0.5、5および50mg/L)。次の現象が50 mg/Lの濃度の培養で観察された：培養6時間後のROSとMDAの有意な増加、培養12時間後のNOX2の発現増加と培養24時間後のRAGEの発現増加、および培養36時間後の細胞におけるAGE濃度の増加(分析された粒子の濃度は培養時間と正に相関)。神経細胞の変性を引き起こす機構の一つがAGE/RAGE複合体の活性化であることを確認した。in vitroで実施された追加解析では、AGE/RAGEの活性化につながるメカニズムの１個がNOX2の活性の増加であることが確認された[43]。
蛋白質の非酵素的糖化の強度の増加は、酸化因子の活性が強くなると同時に、抗酸化機構が阻害されると主に観察される。前述のように、フッ素はフリーラジカルの合成を強化し、中枢神経系における抗酸化酵素の活性を低下させ、AGEsの合成とニューロンの膜上でのRAGEの発現を強化する可能性がある。その結果、神経系の細胞にAGEが病的に蓄積する可能性がある[43, 52]。神経細胞にAGEが蓄積は炎症性転写因子を活性化する - 炎症状態を引き起こすNF-kBや神経細胞膜の透過性を変化させ、アポトーシス経路を活性化する可能性のあるMAPキナーゼ。これらのプロセスは樹状突起の脱髄とニューロンの変性を開始する。

The Synthesis of Proinflammatory Factors(炎症誘発因子の合成)

One of the factors indicating the initiation and development of an inflammatory state in all organs is the change in the concentration of interleukins. Interleukins belong to cytokines—proteins that take part in the intercellular signalling and the regulation of immunological response [54]. Cytokines in the nervous system influence the regulation of sleep and the processes connected to memorizing. They also take part in neurodegenerative processes, and they help keep the integrity of the blood/brain barrier [55]. Interleukin 6 (Il-6) is secreted by macrophages, B-lymphocytes, microglia, neurons, adipocytes, myocytes and fibroblasts, but only a few cells show the expression of receptors for Il-6—some of the leukocytes and microglial cells. In the nervous system, astrocytes and oligodendrocytes do not show the expression of receptors for Il-6 [54]. In physiological conditions, a low concentration of Il-6 in the nervous system is observed [56]. In low concentrations, Il-6 may have a neuroprotective effect—it influences the differentiation of oligodendrocytes, acts as a neurotrophic factor and takes part in the regeneration of neurons and, in relation to mice among which the Il-6 gene is blocked, the activation of microglia is decreased. The rise in the concentration of Il-6 appears in inflammatory states and neurodegenerative illnesses [57, 58]. Among illnesses with an inflammatory basis and neurodegenerative diseases, an increased concentration of other cytokines such as Il-1B and TNF-α is also observed. These cytokines are responsible for the initiation of inflammatory states and the death of neurons [59]. Research shows that the exposure of adult rats to fluorine causes the activation of microglia in the hippocampus and cerebral cortex and that it initiates an inflammatory state through the synthesis of proinflammatory cytokines—Il-1B, Il-6 and TNF-α (the concentration in drinking water, 60 and 120 mg/L) [60].

すべての臓器における炎症状態の開始と進展を示す因子の１個は、インターロイキン濃度の変化である。インターロイキンはサイトカインに属する - 細胞間シグナル伝達と免疫応答の調節に関与するタンパク質である[54]。神経系のサイトカインは、睡眠の調節や記憶に関連するプロセスに影響を与える。また、神経変性プロセスにも関与し、血液/脳関門の完全性を維持するのに役立つ[55]。インターロイキン6(Il‐6)はマクロファージ、Bリンパ球、ミクログリア、ニューロン、脂肪細胞、筋細胞および線維芽細胞によって分泌されるが、Il‐6受容体 - 白血球およびミクログリア細胞の一部である - の発現を示す細胞はごく少数である。神経系では、アストロサイトとオリゴデンドロサイトはIl-6に対する受容体の発現を示さない[54]。生理学的条件下では、神経系における低濃度のIl-6が観察される[56]。低濃度では、Il-6は神経保護作用を有する可能性がある - すなわち、Il-6はオリゴデンドロサイトの分化に影響を及ぼし、神経栄養因子として作用し、ニューロンの再生に関与し、Il-6遺伝子が遮断されたマウスでは、ミクログリアの活性化が低下する。Il‐6濃度の上昇は炎症状態や神経変性疾患に現れる[57, 58]。炎症性疾患や神経変性疾患では、Il‐1BやTNF‐αなどの他のサイトカイン濃度の上昇も認められる。これらのサイトカインは炎症状態の開始とニューロンの死に関与する[59]。成体ラットをフッ素に曝露すると、海馬および大脳皮質のミクログリアが活性化され、炎症性サイトカイン - Il-1B、Il-6およびTNF-α - の合成を介して炎症状態が引き起こすことを研究が示す(飲料水中の濃度、60および120mg/L)[60]。

The Activation of Glial Cells and the Migration of Lymphocytes to CNS(グリア細胞の活性化とリンパ球の中枢神経系への移動)

Despite the big numbers of glial cells located in the nervous system (it is estimated that there are 10 times more of these cells than neurons), they do not take part in the information transfer, but their task is to support the activity of neurons. We can divide them into macroglia that include astrocytes, oligodendrocytes and Schwann cells, and microglia. Astrocytes fulfil functions crucial to maintaining the homeostasis and the proper functioning of the nervous system. Among other roles, they are responsible for the metabolism and the preservation of the proper concentration of potassium, the regulation of pH, the metabolism and transport of neurotransmitters and the regulation of the strength of stimulation [17, 61]. The rise in the number of glial fibrillary acidic protein (GFAP) cells indicates the activation of astrocytes and the initiation of defensive mechanisms against harmful factors, such as fluorine. GFAP is a protein specific for astrocytes. Its expression increases when the cells of the nervous system become damaged or when they show disorders in metabolism. It stimulates their proliferation in order to minimize and repair the damage [17, 62]. Microglia are cells differentiating from macrophages, which are “settled” in the nervous system. As in the case with astrocytes, their task is to preserve the balance in the nervous system. However, their influence and activity are homological to the activity of macrophages. They are responsible for the absorption of the products of nerve tissue breakdown. Therefore, their proliferation is stimulated by external factors such as improper proteins that find their way into intercellular space or substances released from dying neurons [63]. The activation of microglia might occur when the pathway associated with the activity of MAP–ERK kinase is initiated. Oxidative stress in the nervous system causes an increase in the expression of ERK in glial cells which initiates the synthesis of proinflammatory substances [64, 65]. The migration of B lymphocytes to the nervous system is observed in pathological states. They take part in the inhibition of damage progression and in the repair processes engaging, among others, microglia [17, 34].

神経系には多数のグリア細胞が存在するが(これらの細胞はニューロンの10倍と推定されている)、情報伝達には関与せず、ニューロンの活動を支援することを任務としている。星状細胞、オリゴデンドロサイト、シュワン細胞を含むマクログリアとミクログリアに分けることができる。星状細胞は、恒常性と神経系の適切な機能の維持に不可欠な機能を果たす。他の役割の中でも、カリウムの代謝と適切な濃度の維持、pHの調節、神経伝達物質の代謝と輸送、刺激の強さの調節に関与している[17, 61]。グリア線維性酸性蛋白質(GFAP)細胞数の増加は星状細胞の活性化とフッ素のような有害因子に対する防御機構の開始を示す。GFAPは星状細胞に特異的な蛋白質である。神経系の細胞が損傷を受けたり、代謝障害を起こしたりすると、その発現が増加します。それは損傷を最小限にし、修復するためにそれらの増殖を刺激する[17, 62]。ミクログリアは神経系に「定着」するマクロファージから分化する細胞である。星状細胞の場合と同様に、その仕事は神経系のバランスを保つことである。しかしながら、それらの影響と活性はマクロファージの活性と相同性がある。それらは神経組織の分解産物の吸収に関与している。したがって、ミクログリアの増殖は、細胞間空間に入り込む不適切なタンパク質や、死にかけたニューロンから放出される物質などの外的要因によって刺激される[63]。ミクログリアの活性化は、MAP-ERKキナーゼの活性に関連する経路が開始されたときに起こる可能性がある。神経系における酸化ストレスは、炎症性物質の合成を開始するグリア細胞におけるERKの発現増加を引き起こす[64, 65]。Bリンパ球の神経系への遊走は病的状態で観察される。それらは、損傷の進行の阻害と、とりわけミクログリアに関与する修復プロセスに関与する[17, 34]。

Immunohistochemical analyses carried out on the material collected from adult rats exposed to fluorine showed that the activation of astrocytes takes place in the cerebral cortex (the concentration of fluorine in drinking water, 10 mg/L). In the studied material, an increased immunoreactivity of GFAP was observed, which is specifically connected to astrocytes. The research also confirmed that the activation of microglia and the migration of B cells took place in the brains of the exposed rats. The research consisted of antibodies anti-CD68 which are specific to cells originating from the line of macrophages, including microglia, and of antibodies anti-CD20 specific to B cells [17].

フッ素に曝露された成体ラットから採取した材料で実施した免疫組織化学分析は、星状細胞の活性化が大脳皮質で起こることを示した(飲料水中のフッ素濃度10mg/L)。研究試料では、星状細胞に特異的に結合したGFAPの免疫反応性の増加が観察された。また、曝露されたラットの脳では、ミクログリアの活性化とB細胞の移動が起こっていることが確認された。その研究は、ミクログリアを含むマクロファージ系由来の細胞に特異的な抗CD68抗体と、B細胞に特異的な抗CD20抗体から構成された [17]。

The Metabolism of Neurotransmitters(神経伝達物質の代謝)

Another mechanism by which fluorine may influence the disorders in the functioning of neurons is the change in the concentration of neurotransmitters. Prolonged exposure to fluorine causes a decrease in the concentration of glutamate in the brain. Glutamate constitutes about 30 % of all neurotransmitters in the central nervous system and is the main stimulating transmitter. It is secreted in high amounts in the hippocampus, which is responsible for the processes of memorizing and learning [66–68]. This amino acid is supplied to the organism with the diet. However, only a small amount of it passes through the blood/brain barrier. This is a protective mechanism against excessive inflow of this neurotransmitter to the brain which could cause the depolarization and damaging of neurons [69, 70]. Therefore, in the central nervous system, there has to be a balance between the synthesis of the endogenic glutamate and its loss. In the synthesis of glutamate, aspartate transaminase (AST) and alanine aminotransferase (ALT) take part. Their activity is inhibited by an excessive supply of fluorine [69, 71]. This element also increases the activity of glutamate decarboxylase (GAD) which results in the transformation of glutamate into γ-aminobutyric acid (GABA)—the main inhibiting transmitter in the nervous system. These processes lead to the decrease in the pool of glutamate in the brain leading to dysfunctions in synaptic transmission and the disorders in cognitive functions.

フッ素がニューロンの機能障害に影響する別のメカニズムは、神経伝達物質の濃度変化である。フッ素に長期間さらされると、脳内のグルタミン酸濃度が低下します。グルタミン酸は中枢神経系の全神経伝達物質の約30%を占め、主要な刺激伝達物質である。記憶と学習の過程をつかさどる海馬に多量に分泌される[66–68]。このアミノ酸は食餌とともに生体に供給される。しかしながら、血液/脳関門を通過するのはごく少量である。これは、この神経伝達物質の脳への過剰な流入から神経細胞の脱分極と損傷を防ぐための防御機構である[69, 70]。したがって、中枢神経系では、内因性グルタミン酸の合成とその喪失の間にバランスがなければならない。グルタミン酸の合成には、アスパラギン酸トランスアミナーゼ(AST)とアラニンアミノトランスフェラーゼ(ALT)が関与する。それらの活性はフッ素の過剰供給によって阻害される[69, 71]。この元素はまた、グルタミン酸デカルボキシラーゼ(GAD)の活性を増加させ、その結果、グルタミン酸がγ-アミノ酪酸(GABA) - 神経系における主要な阻害伝達物質 - に変換される。これらのプロセスは、脳内のグルタミン酸プールの減少につながり、シナプス伝達の機能障害や認知機能の障害につながる。

Bergmann glial cells (BGC) are abundantly present in the cerebellum. They participate in the transportation and metabolism of neurotransmitters, the preservation of the balance of potassium and the correct pH in the nervous system. One of the more important functions of BGC is the metabolism of glutamate. These cells are one of the main sources of this neurotransmitter, and to a large extent, they are responsible for glutamatergic stimulation [6, 72]. The incubation of BGC cells (isolated from the cerebellum of rats) with fluorine in concentrations of 1 and 2 mM caused a significant decrease in the lifespan of cells, causing disorders in the metabolism of glutamate [6, 73]. Other research showed a decrease in the concentration of glutamate in the serum, hippocampus and cerebral cortex of the studied animals (the concentration of fluorine in drinking water, 120 mg/L), an increase in the activity of glutamic acid decarboxylase and a decrease in the activity of asparagine transferase and alanine transferase—the enzymes participating in the metabolism of glutamate (the concentration of fluorine in drinking water, 150 mg/L) [66, 69, 74]. Glutamate participates in memory processes through the stimulation of specific ionotropic and metabotropic (mGluRs) receptors. One of the things that the receptors of the mGluR I group (mGluR1 and mGluR5) are responsible for is the preservation of the correct plasticity of synapses. They show expression in the hippocampus, cerebral cortex and cerebellum. The inhibition of the expression of mGluR5 among test animals causes significant disorders in spatial orientation and “spatial learning”. Fluorine causes an insignificant decrease in the expression of mGluR5 [66, 75]. Glutamatergic stimulation is strongly related to memory (mainly long-term memory) and learning, so all the disturbances of the synthesis and transportation of glutamate negatively influence those two processes [6, 76].

ベルクマングリア細胞(BGC)は小脳に豊富に存在する。神経伝達物質の輸送と代謝、神経系におけるカリウムバランスと正しいpHの維持に関与している。BGCのより重要な機能の１個はグルタミン酸の代謝である。これらの細胞はこの神経伝達物質の主要な供給源の１個であり、グルタミン酸作動性刺激の大部分を担っている[6, 72]。ラットの小脳から単離したBGC細胞を1mMおよび2mMの濃度のフッ素で培養すると、細胞寿命が有意に減少し、グルタミン酸の代謝障害を引き起こした[6, 73]。他の研究では、試験動物の血清、海馬および大脳皮質におけるグルタミン酸濃度の減少(飲料水中のフッ素濃度120 mg/L)、グルタミン酸デカルボキシラーゼ活性の増加およびグルタミン酸の代謝に関与する酵素であるアスパラギン酸トランスフェラーゼおよびアラニントランスフェラーゼの活性の減少が示された(飲料水中のフッ素濃度150 mg/L) [66, 69, 74]。グルタミン酸は特異的なイオンチャネル型および代謝調節型(mGluRs)受容体の刺激を介して記憶プロセスに関与する。mGluR I群の受容体(mGluR1とmGluR5)が担っていることの１個は、シナプスの正しい可塑性の維持である。
これらは海馬、大脳皮質、小脳で発現する。試験動物におけるmGluR5の発現阻害は、空間定位および「空間学習」における有意な障害を引き起こす。フッ素はmGluR5の発現をわずかに減少させた[66, 75]。グルタミン酸作動性刺激は記憶(主に長期記憶)と学習に強く関係しているので、グルタミン酸の合成と輸送のすべての障害はこれら２個のプロセスに負の影響を与える[6, 76]。

Fluorine also causes changes in the secretion of neurotransmitters such as serotonin, dopamine, norepinephrine, acetylcholine and epinephrine (the concentration of fluorine in drinking water, 20, 40 and 60 mg/L) [77]. Adult rats that received drinking water with 100 mg/L concentration of fluorine had a higher concentration of noradrenalin and serotonin in the hippocampus, striatum and cerebral cortex [78].

フッ素はまた、セロトニン、ドーパミン、ノルエピネフリン、アセチルコリン、エピネフリンなどの神経伝達物質の分泌に変化をもたらす(飲料水中のフッ素濃度、20、40、60mg/L)[77]。100mg/Lのフッ素濃度の飲料水を与えられた成体ラットは、海馬、線条体および大脳皮質におけるノルアドレナリンおよびセロトニンの濃度が高かった[78]。

The Expression of Proteins that Regulate the Maturing, Differentiation and Proliferation of Neurons(ニューロンの成熟、分化、増殖を制御するタンパク質の発現)

Constant exposure of young specimens to fluorine in the prenatal and postnatal periods initiates processes that lead to the degeneration of neurons through the influence on the expression of regulatory proteins. Among other phenomena, changes in the demyelinating character, a decrease in the number of Purkinje cells and the degradation of dendrites are observed in the histopathological image of animals that received fluorine for a long period of time [79].

出生前および出生後の期間における若い標本のフッ素への継続的曝露は、調節蛋白質の発現への影響を介してニューロンの変性につながるプロセスを開始する。他の現象の中で、脱髄性の変化、プルキンエ細胞数の減少、樹状突起の分解が、長期間フッ素を投与された動物の病理組織像で観察されている[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 [40]. The decrease in the expression of NCAM influences the plasticity of neurons and causes disturbances in cognitive functions. This phenomenon is confirmed by experiments carried out on animals with the NCAM gene blocked, as they showed that the animals had problems with spatial learning [40, 81].

神経細胞接着分子(NCAM)は、神経系の細胞の接着と移動、軸索とシナプスの発達、およびシグナル経路の活性化に関与する膜糖タンパク質である[80]。アイソフォームNCAM‐120、NCAM‐140およびNCAM‐180の発現の障害は神経系の認知機能に影響するが、NCAM‐180の存在は海馬におけるニューロンの可塑性に有意に影響する。海馬から単離した神経細胞はフッ素(適用フッ素濃度20、40、80mg/L)との培養後にNCAMの発現低下を示す。80mg/Lの濃度では、前述の３個のアイソフォームの量がすべて減少したが、低いフッ素濃度では、アイソフォームNCAM-180の発現も有意に減少した[40]。NCAMの発現低下はニューロンの可塑性に影響し、認知機能の障害を引き起こす。この現象は、NCAM遺伝子をブロックした動物で行われた実験で確認されており、動物は空間学習に問題があることが示された[40, 81]。

Animals exposed to fluorine suffered from chronic pain and had a higher sensitivity to pain. Furthermore, there was an increase in the expression of brain-derived neurotrophic factor (BDNF) and a decrease in the expression of GFAP in the cerebral cortex and hippocampus of those animals (the concentration of fluorine in drinking water, 50 and 150 mg/L) [30]. The expression of the BDNF is regulated, among others, by serine-threonine kinases, so the changes in the concentration of BDNF in nerve cells may be caused by the activation of proteins from the MAP kinase family by fluorine [4]. BDNF is a protein belonging to neurotrophins—a group of neurotrophic factors that includes substances supporting the differentiation and survivability of neurons [82]. BDNF regulates the growth and regeneration of neurons, and it influences their plasticity. The increase in its synthesis accompanies such processes as the damaging of tissue or ageing [83–86]. GFAP is a protein specific for astrocytes, and the changes in its expression influence the maturing of neurons and glial cells [87]. The increased concentration of BDNF in the nervous system with the simultaneous decrease in the concentration of GFAP indicates disorders in the maturing of nerve cells and the activation of repair processes aiming at the neutralization of the damage caused by the toxic influence of fluorine [11].

フッ素に曝露された動物は慢性疼痛に苦しみ、疼痛に対する感受性が高かった。さらに、これらの動物の大脳皮質及び海馬において、脳由来神経栄養因子(BDNF)の発現の増加及びGFAPの発現の減少が認められた(飲料水中のフッ素濃度、50および150mg/L) [30]。BDNFの発現は、とりわけセリン-トレオニンキナーゼによって調節されているので、神経細胞におけるBDNF濃度の変化は、フッ素によるMAPキナーゼファミリーのタンパク質の活性化によって引き起こされる可能性がある[4]。BDNFはニューロトロフィンに属するタンパク質である - ニューロトロフィンは神経栄養因子のグループであり、神経細胞の分化と生存をサポートする物質を含む[82]。BDNFはニューロンの成長と再生を調節し、その可塑性に影響する。その合成の増加は、組織の損傷や老化などの過程に伴う[83–86]。GFAPは星状細胞に特異的なタンパク質であり、その発現の変化はニューロンやグリア細胞の成熟に影響を及ぼす[87]。神経系におけるBDNF濃度の上昇と同時にGFAP濃度の低下は、神経細胞の成熟における障害と、フッ素の毒性影響によって引き起こされた損傷の中和を目的とした修復プロセスの活性化を示している[11]。

The Influence of Fluorine on the Initiation of Apoptosis in the Central Nervous System(中枢神経系におけるアポトーシスの開始に対するフッ素の影響)

ROS in cells function as signalling particles and, in physiological concentrations, influence the activity of metabolic pathways. However, if their concentration in cells is too high, it leads to the oxidation of nucleic acids, including the oxidation of DNA and the braking of α-helix. The accumulation of many such changes in the DNA, detected by repair mechanisms, leads to the activation of the apoptosis pathway [88–90]. There was a significant increase in the number of apoptotic cells in the culture of nerve cells isolated from the hippocampus after 48 h of incubation with fluorine. The following concentrations of fluorine were used in the research—20, 40 and 80 mg/L. However, the increase in the concentrations of apoptotic cells appeared in the case of 40 and 80 mg/L [40].

細胞中のROSはシグナル伝達粒子として機能し、生理的濃度では代謝経路の活性に影響する。しかし、細胞内の濃度が高すぎると、DNAの酸化やαヘリックスの破壊など、核酸の酸化を引き起こす。修復機構によって検出されるこのような多くの変化がDNAに蓄積すると、アポトーシス経路が活性化される[88–90]。海馬から分離した神経細胞をフッ素で48時間培養すると、アポトーシス細胞数が有意に増加した。この研究では、20、40、80 mg/Lの濃度のフッ素が使用された。しかしながら、アポトーシス細胞の濃度の増加は40および80 mg/Lの場合に現れた[40]。

Nuclear factor kappa B (NF-kB) is a transcription factor that participates in the processes related to cell growth, the regulation of cell cycle, the development of an inflammatory state and the response to stress [91–94]. 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 [48]. Among animals exposed to lower concentrations of fluorine (30 mg/L), there was an increase in the expression of NF-kB which correlated with an increased concentration of calcium ions in the studied cells. It is widely known that fluorine increases the synthesis of ROS in neurons, which causes damage to the cell membrane. Calcium ions move through the damaged membrane to nerve cells causing, among others, an increase in the expression of NF-kB. Consequently, they influence the initiation of programmed death [95, 97].

核因子κB(NF-kB)は、細胞増殖、細胞周期の調節、炎症状態の発生およびストレスへの応答に関連するプロセスに関与する転写因子である[91–94]。その発現レベルとそれが影響する経路に応じて、それはアポトーシスから細胞を保護したり、プロセスを開始したりする[95, 96]。ラットの海馬から分離したニューロンをフッ素(40および80mg/L)で24時間培養した研究では、DNA損傷の頻度が有意に増加し、NF-kBの合成が増加した[48]。低濃度のフッ素(30mg/L)に暴露した動物では、NF-kBの発現が増加し、これは研究した細胞におけるカルシウムイオン濃度の増加と相関していた。フッ素はニューロンにおけるROSの合成を増加させ、細胞膜に損傷を引き起こすことが広く知られている。カルシウムイオンは損傷した膜を通って神経細胞に移動し、とりわけNF-kBの発現を増加させる。その結果、プログラムされた死の開始に影響を及ぼす[95, 97]。

The analyses carried out so far indicate that the apoptosis of neurons observed in chronic fluorosis may be activated by the mitochondrial pathway. It was proved that the processes related to nerve cell degeneration are influenced by MAP kinases, signal pathways with the participation of G proteins, calcium ions, the p38 protein and Jun N-terminal kinase (JNK) [98–100]. The 6-month exposure of rats to both low (5 mg/L) and high (50 mg/L) concentrations of fluorine in drinking water caused a significant increase in the number of apoptotic cells in the brain and in the content of the phosphorylated form of JNK. In the case of the total content of JNK, the changes were insignificant. The analysis suggests that fluorine stimulates apoptosis through the activation of JNK because in the brains of the exposed animals there is an increase in the content of the active (phosphorylated) form of this protein, and not its total content, in comparison to the animals from the control group [98]. JNK kinases influence apoptosis through the activation of caspases and the changes in the expression of genes associated with this process [101]. Furthermore, among adult rats supplied with fluorine in drinking water, there was an increased expression of the proapoptotic protein Bax and a decreased expression of the apoptosis inhibiting Bcl-2. The analysis carried out by means of the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method confirmed the increased intensity of apoptotic processes in brain structures of the animals (the concentration in drinking water, 60 and 120 mg/L) [60].

これまでの解析は、慢性フッ素症で観察されるニューロンのアポトーシスがミトコンドリア経路によって活性化されることを示している。神経細胞変性に関わるプロセスにはMAPキナーゼ、G蛋白質、カルシウムイオン、p38蛋白質、Jun N末端キナーゼ(JNK)が関与するシグナル経路が影響することが証明された[98–100]。ラットを低濃度(5mg/L)および高濃度(50mg/L)のフッ素飲料水に6ヶ月間暴露すると、脳のアポトーシス細胞数およびリン酸化型JNKの含量が有意に増加した。JNKの総量の場合、その変化はわずかであった。この分析は、フッ素がJNKの活性化を介してアポトーシスを刺激することを示唆している、なぜならこれは、暴露された動物の脳では、対照群の動物と比較して、このタンパク質の総含量ではなく、活性型(リン酸化型)の含量が増加しているからである[98]。JNKキナーゼは、カスパーゼの活性化およびこのプロセスに関連する遺伝子発現の変化を介してアポトーシスに影響を及ぼす[101]。さらに、飲料水中のフッ素を与えられた成体ラットでは、アポトーシス促進蛋白質Baxの発現増加とアポトーシス阻害Bcl‐2の発現減少があった。Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)法による解析により、動物の脳構造におけるアポトーシス過程の強度の増加が確認された(飲料水中の濃度、60および120 mg/L)[60]。

The results achieved in vivo were confirmed by in vitro studies. In the culture of cells of the SH-SY5Y line, after 48 h of incubation with fluorine (40 and 80 mg/L), there was an increase in the concentration of caspase-3 and in the expression of Fas, Fas-L, caspase-3 and caspase-8. The activity of caspase-3 is regulated by the changes in the expression and activity of other proapoptotic proteins, including caspase-8 and caspase-9 that are activated in the mitochondrial pathway. The damage of cells caused by the toxic influence of fluorine leads to the activation of the mitochondrial pathway of apoptosis through the activation of procaspase-8 by Fas and the activation of caspase-3 which eventually initiates the degradation of neurons [102].

in vivoで得られた結果をin vitro研究で確認した。SH‐SY5Y系細胞の培養において、フッ素(40および80mg/L)との48時間の培養後、カスパーゼ‐3の濃度およびFas、Fas‐L、カスパーゼ‐3およびカスパーゼ‐8の発現の増加があった。カスパーゼ‐3の活性は、ミトコンドリア経路で活性化されるカスパーゼ‐8およびカスパーゼ‐9を含む他のアポトーシス促進蛋白質の発現および活性の変化によって調節される。フッ素の毒性影響によって引き起こされる細胞の損傷は、Fasによるプロカスパーゼ-8の活性化と、最終的にニューロンの分解を開始するカスパーゼ-3の活性化を介して、アポトーシスのミトコンドリア経路の活性化につながる[102]。

Summary(まとめ)

Previous studies on fluorine neurotoxicity showed that one of the main mechanisms that lead to the disturbances in central nervous system homeostasis is oxidative stress. ROS act as mediators in many processes and in high concentrations. They are able to initiate cell damage and metabolism disorders [38]. Fluorine is responsible for both—increase in ROS synthesis and lipid peroxidation and decrease in anti-oxidative enzyme activity in neurons and glia [43]. An excessive intake of fluoride is also responsible for the increase in AGE synthesis in CNS which leads to synthesis of transcription factors and proinflammatory substances including NF-kB, interleukins and MAP kinases [43]. Moreover, fluorine causes glial cell activation which is involved in inflammation in the brain and change in the expression of proteins which regulate neuron differentiation and proliferation and initiate apoptosis such as NCAM, GFAP, BDNF, JNK, Bax and Bcl2 [17, 30, 60, 61, 101]. The accumulation of this element in CNS causes cytological changes within neurons (changes in tubulin expression and concentration of Nissl bodies) and changes in neuron activity and their energy metabolism [1, 4, 9, 15, 30]. The accumulation of fluorine in the nervous system influences the synthesis of neurotransmitters, the activity of enzymes, the expression of receptors and the plasticity of neurons [105–107]. Numerous analyses carried out in in vivo and in vitro models confirmed that prolonged exposure to high concentrations of fluorine leads to the degeneration of neurons [1, 60].

フッ素の神経毒性に関する以前の研究は、中枢神経系の恒常性の障害につながる主要なメカニズムの１個が酸化ストレスであることを示した。ROSは多くのプロセスにおいて高濃度で仲介者として作用する。それらは細胞損傷と代謝障害を引き起こすことができる[38]。フッ素は、ニューロンとグリアにおけるROS合成と脂質過酸化の増加と抗酸化酵素活性の低下の両方に関与している[43]。フッ化物の過剰摂取はまた、転写因子およびNF-kB、インターロイキンおよびMAPキナーゼを含む炎症性物質の合成につながるCNSにおけるAGE合成の増加の原因となる[43]。さらに、フッ素は脳の炎症に関与するグリア細胞活性化と、NCAM、GFAP、BDNF、JNK、BaxおよびBcl2のようなニューロンの分化と増殖を調節し、アポトーシスを開始する蛋白質の発現の変化を引き起こす[17, 30, 60, 61, 101]。CNSにおけるこの元素の蓄積は、ニューロン内の細胞学的変化(チューブリンの発現とニッスル小体の濃度の変化)、およびニューロンの活動とそのエネルギー代謝の変化を引き起こす[1, 4, 9, 15, 30]。神経系におけるフッ素の蓄積は、神経伝達物質の合成、酵素の活性、受容体の発現およびニューロンの可塑性に影響を及ぼす[105–107]。in vivoおよびin vitroモデルで実施された多数の解析により、高濃度のフッ素への長期暴露がニューロンの変性を引き起こすことが確認された[1, 60]。

The central nervous system during development is highly sensitive to the influence of fluorine due to its weakened protective mechanisms. In the childhood period, exposure to this element may cause permanent damage to the functions of all brain structures [103, 104]. Among both young and adult specimens exposed to the toxic influence of high doses of fluorine, we can observe impaired ability to learn, disturbances in memory and information processing and behavioural problems. All of these cause a decrease in the quality of life [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 developing organisms, lead to higher restrictions in countries where fluorosis occurs frequently.

発達中の中枢神経系は、防御機構が弱まっているため、フッ素の影響に非常に敏感である。小児期にこの元素に暴露すると、すべての脳構造の機能に永続的な損傷が生じることがあります[103, 104]。高用量のフッ素の毒性影響に曝露された若年および成人の両方の標本において、学習能力の障害、記憶および情報処理の障害および行動上の問題を観察することができる。これらはすべて生活の質を低下させる[42]。風土病のフッ素症の発生に関する多くの報告により、世界保健機関(WHO)は飲料水中のフッ素の許容濃度を、ヒトの生体内に過剰に蓄積せず、有害作用を引き起こさないレベルに設定した。現在の値は1.5mg/Lに設定されている [1, 2]。しかし、神経系に対するこの元素の毒性影響に関する最近の知見、特に発生中の生物との関連で危険であることから、フッ素症が頻繁に発生する国ではより高い規制が必要となっている。

翻訳者より補足：日本の水道水について

水道水質基準：https://www.env.go.jp/water/water_supply/kijun/kijunchi.html#01
PFAS：https://www.env.go.jp/water/pfas.html
PFAS　Q＆A：https://www.env.go.jp/content/000242834.pdf
他の参考：フッ素による人体と環境への過剰負荷の化学的側面
https://pmc.ncbi.nlm.nih.gov/articles/PMC8945431/

日本の環境省より、日本の水道水質基準では0.8mg/L(≒0.8ppm)となっている。それに加えてPfas(有機フッ素化合物のこと。問題になっているのがペルフルオロアルキル化合物及びポリフルオロアルキル化合物)が難分解性、高蓄積性のため、日本では2020年に50ng/Lと別途制限基準を設けている。
詳しく知りたければ他の参考にPFASについて半減期が推定3.5年か4.8年とか書かれてるので各自勝手に調べてね。

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