Group 13 Chemistry - American Chemical Society

Chapter 19

Aluminofluoride Complexes: A Useful Tool in Laboratory Investigations, but a Hidden Danger for Living Organisms? Downloaded by CORNELL UNIV on September 24, 2016 | http://pubs.acs.org Publication Date: June 3, 2002 | doi: 10.1021/bk-2002-0822.ch019

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Anna Strunecka and Jiri Patocka

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Charles University Prague, Faculty of Sciences, Department of Physiology and Developmental Biology, 128 43 Prague 2, Vinicna 7, Czech Republic M i l i t a r y Medical Academy, Department of Toxicology, 500 01 Hradec Kralove, Simkova 878, Czech Republic 2

Aluminofluoride complexes are used in many laboratory investigations of guanine nucleotide binding proteins (G proteins). Reflecting on many studies, a new view on the toxicity of aluminum and fluoride can be suggested. The hidden danger of their long-term synergistic action is not fully recognized at this point.

Fluoride anions, generally introduced as NaF solutions, have long been known to influence the activity of various enzymes and the purified guanine nucleotide-binding regulatory component of adenylate cyclase (/). Sternweis and Gilman (2) have reported that fluoride activation of adenylate cyclase depends on the presence of trace aluminum. This fact had at first been ignored because aluminum is a normal component of glass from which it is etched by a solution with fluoride. The requirement for aluminum is highly specific: of 28 other metals tested, only beryllium promoted activation of the guanine nucleotide-binding regulatory component of adenylate cyclase by fluoride (2,3).

© 2002 American Chemical Society

Shapiro and Atwood; Group 13 Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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In aqueous solutions, the fluoride anions bind to the metal cation and are exchangeable with free fluoride or hydroxyl ions. The complexes are not permanent; and the proportions of multifluorinated species such as A1F , A1F (OH) and AIF4" depend on the excess concentration of free fluoride ions and on the pH of the solution (4). Further studies demonstrated that slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretation of results. The idea that aluminofluoride complexes act as a high affinity analogue of the terminal phosphate of GTP was suggested (3, 5). Guanine nucleotide binding proteins (G proteins) take part in an enormous variety of biological signaling systems, helping control almost all important life processes (6). Moreover, aluminofluoride complexes also influence the activity of a variety of phosphatases, phosphorylases and kinases (5). Reflecting on many studies, which utilize aluminofluoride complexes, the effects of fluoride in the presence of aluminum on various cells and tissues as observed, can be reviewed. A knowledge of mechanism of action of aluminofluoride complexes on the molecular and cellular level will draw us nearer to an understanding of the detrimental effects of aluminum and fluoride combinations in the environment. 3

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Downloaded by CORNELL UNIV on September 24, 2016 | http://pubs.acs.org Publication Date: June 3, 2002 | doi: 10.1021/bk-2002-0822.ch019

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Aluminofluoride Complexes The exact structure of the activator complex able to simulate a phosphate group in many biochemical reactions has been disputed. Aluminofluoride complexes are easily soluble in water. Calculations based on existing equilibrium data (4) predicted that in the millimolar range of fluoride concentration and physiological pH the major species is A l F a . According to the model of Bigay et al. (3) and Chabre (5), the proposed biologically active state resulted from the binding of tetrahedral A1F ". Martin (7) recalculated the equilibrium of aluminofluoride complexes and suggested that the predominant species are the neutral complex A1F and the mixed complex A1F (0H). These complexes should be hexacoordinated, with water molecules occupying the free sites; only the hydroxylated and the ternary fluorohydroxylated complexes would be tetrahedral. For aluminum, it is uncertain whether the complex that enters the site is an AIF that becomes tetrahedral by losing its three bound water molecules and contracting a fourth bond with the β-phosphate oxygen, or i f it is an already tetrahedral AIF (OH)~that exchanges its hydroxyl for the β-phosphate oxygen or an A l F ^ t h a t exchanges a fluorine. Many of the crystal structures of nucleotide binding proteins complexed with AIF show AIF* "^). It seems that the different coordination numbers originate mainly from the difference in pH at which the enzymes were crystallized. The bound A1F occurs as A1F at pH 7.5 8 .5 but as AIF4 " at pH below 7 (7, 8). 1

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AIF : The Phosphoryl Transfer Transition State Analog


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Transfer of phosphate groups is the basic mechanism in the regulation of the activity of numerous enzymes, energy metabolism, cell signaling, movement, and regulation of cell growth. Phosphate is an important component of phospholipids in the cell membranes. Analogies between a phosphate group and aluminofluoride complex consist in atomic and molecular similarities. The fluorine atom has the same size and the same valence orbitals as oxygen. O f course, fluorine is more electronegative than oxygen and with a similar capacity for forming hydrogen bonds. Aluminum is close to phosphorus in the periodic table, and their valence electrons are in the same shell. An A l - F bond is the same length as a Ρ - Ο bond in phosphate, i.e. 1.5 to 1.6 Â (5). Like phosphorus, aluminum has possible coordination numbers of 1 - 6, due to the possible hybridization of its outer shell 3p electrons with the 3d orbitals. These complexes can bind to proteins by hydrogen bonds to the fluorine atoms just as oxygen atoms of a phosphate ion. However, an important functional difference between a phosphate group and the structurally analogous A1F complexes exist (3,5). In phosphate, oxygen is covalently bound to the phosphorus and does not exchange with oxygen from solvent. In A1F , ionic bonds are formed between the electropositive aluminum and the highly electronegative fluorine. While the reaction of a bound phosphate compound with orthophosphate is endergonic and slow, the corresponding reaction with A1F is rapid and spontaneous. A1F bind ionically to the terminal oxygen of GDP β-phosphate. Enzyme-bound G D P or A D P could therefore form a complex with A1F that imitates A T P or G T P in its effect on protein conformation. This effect often causes a structural change that locks the site and prevents the dissociation of the trisphosphate. X

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A1F in Laboratory Studies X

The low cost and availability of these fluorometallic complexes has probably contributed to their widespread use as a tool in laboratory studies (5, 9). They have been used as evidence for involvement of a G protein in a system. The phosphate-analogue models of A1F action have been accepted for G proteins but may be extended to all enzymes that bind phosphate or nucleosidepolyphosphate (5). Phosphoryl transfer reactions are involved in processes such as energy transduction, regulation of cell growth, activation of metabolites, and cytoskeletal protein assembly. It has been reported that aluminofluoride complexes impair the polymerization-depolymerization cycle of tubulin (5). Shape changes and disorganization of the spectrin network were observed after addition of 1 m M NaF and 10 μΜ A l C b in human red blood cells (10). Rapid and dynamic changes of the cytoskeletal network are of vital importance for many cells. A T P generation in mitochondria requires the association of an Fi subunit with an F transmembrane subunit transporting protons. The binding of A D P X

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and inorganic phosphate in a catalytic site of Fi triggers conformational changes, which lock both of them into the site and induce the formation of pyrophosphate bonds by eliminating a water molecule. So can arise an aluminofluoride analog of pyrophosphate, R - 0 - P O - 0 - A l F i , which may be bound at the site for the γphosphate. The inhibition of mitochondria ATPase activity in the presence of A1F " was reported (//). This inhibition was not reversed by elution of fluoride from solution or by the addition of strong aluminum chelators. No significant release of the complex occurred over a period of days. Aluminofluoride complexes inhibit many ATPases, phosphatases, and phosphorylases. The intervention of aluminofluoride complexes in the energy transformation processes may thus affect the energy metabolism of the entire organism (9). The description of laboratory investigations using aluminofluoride complexes during the past decade would involve hundreds of references. They bring evidence that aluminofluoride complexes influence most cells and tissues of the human body with powerful pharmacological efficacy. It is surprising that numerous laboratory findings of adverse effects of trace amounts of aluminum in the presence of fluoride have not been reported until recently. Aluminofluoride complexes affect all blood elements and blood circulation,, endothelial cells, the function of the immune system, bone cells, fibroblasts, keratinocytes, ion transport, processes of neurotransmission, growth and differentiation of cells, protein phosphorylation, and cytoskeletal protein assembly (9). Enormous possibilities for multiple molecular interactions of aluminum and fluoride exist in the brain and clearly warrant further investigation. Regarding the role of phosphates in cell metabolism and life processes, we can predict hundreds of reactions which might be influenced. The endocrine glands such as the parathyroid gland, the thyroid, the pituitary gland, and the pineal gland, are extremely sensitive to aluminofluoride complexes. Regarding the crucial role of the thyroid in regulation of growth, development, and metabolism of many tissues, A1F might influence the proper function of the entire human body. A phosphoryl transfer transition state analog might thus represent a useful tool for laboratory investigations, but also a strong potential danger for living organisms including humans. :


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AIF : The Messenger of False Information X

Laboratory investigations support the hypothesis that G proteins are potential fluoride and aluminum targets. The heterotrimeric G proteins mediate the transfer of information from heptahelical receptors to effector molecules. Physiological agonists of G protein-coupled receptors include neurotransmitters and hormones, such as dopamine, epinephrine, norepinephrine, serotonin, acetylcholine, glucagon, vasopressin, melatonin, TSH, neuropeptides, opioids, excitatory amino acids, prostanoids, purines, photons and odorants (6). Aluminofluoride complexes may therefore clone or potentiate the action of many neurotransmitters, hormones and growth factors.


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In the liver, for example, the effects of submaximal doses of A1F were potentiated by submaximal doses of glucagon, vasopressin, angiotensin II, and (Xi-adrenergic agonists (12). Using phorbol myristate acetate, the activator of protein kinase C, the conclusion was made that aluminofluoride complexes mimic the effects of Ca mobilizing hormones by activating the G protein which couples the hormone receptor to phospholipase C. Fluoride anions in the presence of aluminum thus affect the liver as an organ involved in glycogenolysis, fatty acid oxidation, and lipolysis. The aluminofluoride complex acts as the first messenger triggering processes of neurotransmission and potentiating the action of various hormones. Numerous laboratory results demonstrate that micromolar A1C1 in the presence of fluoride affect the levels of the second messenger molecules, such as c A M P , inositol phosphates, and cytosolic free calcium ions, in various cells and tissues, as shown in Table I. Such biochemical changes induce various functional responses. The false information of A1F is greatly amplified. The principle of amplification of the initial signal during its conversion into the functional response has been a widely accepted tenet in cell physiology. The discoveries of receptor diversity, numerous G proteins, and phospholipase C families broadens enormously the possibilities of interactions of signal transduction pathways. The diversity of molecules involved in these processes is manifested at all levels of molecular signalling. Understanding the role of G proteins in cell metabolism allows an acceptance of the fact that fluoride together with reactive aluminum now found in the environment, water, and food chain, can evoke numerous pathophysiological symptoms. Every molecule of A1F is the messenger of false infomation. X

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Aluminofluoride Complexes in Ecosystems Aluminum, the most abundant metal in the earths lithosphère, is everywhere: in soil, water sources, air, plants and animals. The natural barrier systems have for aluminum and various physiological ligands are efficient buffers, preventing the increased intake of this metal in natural conditions. Until relatively recently, it existed in forms not generally available to living organisms, and was therefore regarded as non-toxic. With the appearance of acid rain and the massive use of aluminum in industry, there has been an increase in


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Group 13 Chemistry - American Chemical Society

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