PROCESSES UNDERLINING THE ACTION OF PESTICIDES ON ECOSYSTEMS AND HUMAN ORGANISM

A brief overview of the works carried out over a long period is presented. The role of adenosine triphosphate (ATP) on the mechanism of action of pesticides and pesticide complexes with metals is shown. The results of investigation of the effect of pesticides and their complexes with metals on the enzyme systems and nucleotides are presented. The results are summarized in a single system of interrelated and interdependent processes developing in vivo in the form of a branched biological mechanism.


Introduction
In 1986 the United Nations Organization conference on the environment and development had to concede that pesticides are predominant contaminants of the environment [1]. The use of "plant protection chemicals," in particular, pesticides and herbicides, contributes to the yield [2,3] and has already led to significant negative consequences.
Pesticides as a whole and herbicides in particular are substances with high biological activity. They can exert a toxic effect on many components of cells: enzymes, structural and functional proteins, lipoproteids, polysaccharides, nucleic acids, and others. The elucidation of the mechanism of the toxic effect is an important challenge, the solution of which would allow one to establish the real and potential danger of application of these or other compounds for human and non-target organisms. Despite the enormous scale of production and use of chemical facilities for cultivated plant protection, many data on the mechanism of their action still remain unknown. It is considered that, probably, each pesticide acts via a unique mechanism. For example, the acting components of pesticides, namely, zenkor, lontrel, roundup, kusagard, setoxidim, basagran, tilt, and tachigaren, belong to different classes of chemical compounds. According to available literature data (Table 1), they interact with various enzymatic systems, have their own specific binding sites, and are characterized by different mechanisms of action.
Much data concerning the influence of herbicides and fungicides on various components of the living cell [15][16][17], in particular, on some enzymes [18][19][20][21] have been reported. For instance, anticholinesterase compounds, organophosphorus pesticides, carbamates and triazines [22] are structurally similar to substrates and competitively inhibit their activity. The effect was evaluated for fries of Mediterranean fishes Dicentrarchus labrax [23] and for rats (Maple amber) fed with soybean after treatment with zenkor and atrazine [18]. It was shown that herbicide basagran suppressed the antiphosphatecholinesterase activity and resulted in an increase in the hydroxylase activity [9].
The growth of fungi Cryptococcus neoformans was suppressed by glyphosate due to the inhibition of 5-enolpyruvylshikimate-3phosphate synthase [24]. A non-productive fourmembered complex is formed between the enzyme, pesticide, and phosphate [25]. The octahedral coordination mode is formed by the metal ion: Co glyphosate enzyme as that in 3-deoxy-D-arabiheptulosonate-7-phosphate synthase localized in cytosols [26].
Oxidative phosphorylation is performed by Zn-containing enzymes. Dinoseb, dichlorodiphenyltrichloroethane, sevin and pentachlorophenol separate oxidative phosphorylation in mitochondria of Palma christi [27] and decrease the ATP content in glycols of soybean [28]. Chlorine-containing organic pesticide endosulfan reacts with glutathione (cofactor of glutathione peroxidase), considerably decreasing the activity of the enzyme. The loss of secretory reactions in thylakoids of adrenocortical steroidogenic cells and changes in the enzyme activity indicate that the pesticide was involved in the oxidative reactions [29].
The formation of complexes of vegetable peroxidase with various substrate-inhibitors was established [30]. Both the direct participation of the metal in the substrate addition to the protein part of the molecule and providing a relationship between the flavine group and apoenzyme under the action of the metal are assumed. The neighborhood of the pyridine nitrogen atom to the carboxyl group in picolinic acid (picloram) is manifested as the ability to complexation and metal removal from enzymes [31].
The tests on human and rat tissues showed that tachigaren and its metabolites (four enzymes synthesizing pyrimidine) inhibit mitochondrial [32]. This results in the changes in the pyridinenucleotide pool that provides the work of immune cells. The reaction is reversible and its mechanism is uncompetitive with respect to the substrate and cofactor ubiquinone [10]. On the other hand, diverse xenobiotics, both pesticides and metals, are abundant in considerable amounts in the nature, namely, in air, soil, and water [10,19,20]. If these xenobiotics get into the human organism, they may cause various diseases [21,24]. In the presence of pesticides with the ligand properties, their combined effect on living organisms can be enhanced or weakened.
The ability of the environment to selfpurification, i.e., decomposition of contaminants, is determined, to a great extent, by the occurrence of enzymatic redox processes in cells of plants and microorganisms.
For a long time, we have studied the mechanism of the effect of the following pesticides: zencor, lontrel, roundup, kusagard, setoxidim, bazagrane, tilt, and tachigaren. The acting components of these preparations belong to various classes of chemical compounds; according to available literature data, they are characterized by different mechanisms of action (Table 1). However, we were able to show that the action of these compounds is multifunctional and is not restricted by the properties listed in Table 1. They exhibit a significantly broader range of activity.

Complex formation between pesticides and metals
Metal complexes of lontrel (L) were not studied by our research group only. Previously, we showed that 3,6-dichloropicolinic acid (DCPA) with the active principle of the herbicide lontrel readily formed complexes with metals, which are the major environmental pollutants and stable under natural conditions [33,34].
We have shown, for the first time [33], that the bidentate complexes, or chelates with microelements, are formed in cells of living organisms ( Figure 1). In all cases, 1 : 2 complexes are formed. The complex is formed via a strong covalent metal-oxygen bond of the type O-M-O and metal-nitrogen bond (M-N). According to IR spectra, the strength of the complexes changes in the order: NiL 2 > FeL 2 > МоL 2 = СоL 2 > СuL 2 > MnL 2 > ZnL 2 > MgL 2 .  [8,9] Tachigaren, Hymexazol selective to grass, sugar beet inhibitor of dehydrogenase (mitochondrial) [10] Tilt, Propiconazole fungicide, selective to grain crops, to rape 7-etoxyrezofurine O-diethylase; inductor glutathione S-transferase [11,12] Lontrel metal complexes a wide spectrum of action inhibitor of NADH-oxidoreductase [13,14] These X-ray diffraction pattern of the lontrel complex with copper shows that the complexes of this type have an octahedral structure with various degrees of distortion of the coordination polyhedron as shown in Figure 1. According to the ESR data [35], under the native conditions, the considered complexes exist as a single whole in the non-dissociated state. They can participate in further complex formation with bioactive ligands due to the filling of the coordination sphere of the metal. They are capable of participating in further complex formation with bioactive ligands due to the filling of the coordination sphere of the metal.

Reactions of the pesticides with adenosine triphosphoric acid and NADH
We have proved for the first time [36] that pesticides themselves and their metal complexes react with mono-and dinucleotides. The structures of the adenosine triphosphoric acid (ATF) and ε-ATF complexes with lontrel and its metal complex are shown in Figure 2. In all cases, two-or three-component complex systems are formed. It was shown that the pesticide complex with ATF is formed due to the protonation of the N-7 nitrogen atom of the adenine heterocycle, and the nitrogen atom of the terminal NH 2 group can simultaneously be bound to the pesticide molecule due to the formation of a hydrogen bond.
The interaction of pesticides (P) with ATP (A) occurs according to the scheme: The value of the stability constants of these complexes were determined by the equation: The stoichiometric coefficient n for all the compounds studied was determined as equal to 1 ± 0.2. One molecule of ATP reacts with one molecule of pesticide. The obtained values for the complex formation constants (K c/form ) are presented in Table 2.
The formation of the pesticide complexes with ATP results in an energy deficiency in the tissues of organisms [37][38][39][40]. The effect of pesticides and their metal complexes induces the energy deficiency of the cell, namely, inhibition of energy metabolism due to the formation of a complex with adenosine triphosphoric acid.
It is known that polynucleotides, particularly, pyridinenucleotides, form complexes of various types, including charge-transfer complexes, and are highly reactive towards a series of metals [41]. However, the introduction of the etheno group does not almost change the electronic structure of the nucleotide fragment of a NADH molecule.  Therefore, the complexation of pesticides with NADH was concluded on the basis of the value of fluorescence quenching of its chemical analog, modified dinucleotide ε-NADH in which the adenine fragment is subjected to etheno modification [42,43]. Figure 3 shows the dependences of the fluorescence intensity of ε-NADH on the concentration of various quenchers. When the concentration of pesticide (or metal complex) increases, the fluorescence quenching of compound ε-NADH is observed, which is not accompanied by a shift of the position of the excitation maximum and fluorescence emission. The absence of spectral changes in all considered cases indicates the absence of changes in the ground and excited levels of the modified compounds formed by the reactions with pesticides. Fluorescence quenching was observed at the concentrations of the pesticide and lontrel metal complexes ranging from 10 -6 to 10 -3 М. Such low concentrations of the quencher exclude the assumption that the quenching proceeds via the Stern-Volmer mechanism due to random collisions. Therefore, the result of quenching is the formation of a covalent bond with the adenine fragment, as it is shown in the scheme of the [NADH-L] complex presented in Figure 4. The mathematical model of the process was considered to refine the mechanism of formation of complexes [ε-NADH-pesticide] and to estimate their stability constants, as the complex formation constants (K c/form ), as well as for the ATP ( Table 2). As can be seen from Table 2, among the synthesized pesticides, zenkor has the lowest complexation constant (K c/form ) for the complex with ε-NADH (K c/form = 2.1·10 4 М -1 ) and tilt has the highest one (K c/form = 4.6·10 2 М -1 ). It is noteworthy that the complexation constant of the lontrel metal complexes with ε-NADH is substantially lower than the corresponding constant for lontrel. It is known than in solution NADH exists predominantly in a folded conformation in which the adenine moiety of the molecule is localized near the nicotine amide moiety of the nucleotide [44]. About 90% of dinucleotide exist in this conformation in solution. The rest 10% exist in solution in the "open" conformation when the nicotine amide moiety is removed from the adenine structure. Therefore, it can be assumed that a decrease in the complexation constants with ε-NADH for the metal complexes compared to lontrel indicates steric hindrances appeared upon the formation of the [NADH-ML 2 ] complex. In addition, we were able to determine a relationship between genotoxicity of the investigated pesticides and their complex formation constants with dinucleotide NADH [14].

Effect of pesticides on the activity of NАDНoxidoreductase as oxidative enzyme
The results of our research regarding the action of the pesticides of the enzyme systems are presented in the works [13,39,43,45]. The effect of pesticides on the activity of NАDНoxidoreductase (NADH-OR) is illustrated in Table 3. The experimental kinetic curves for the rate of NADH-OR oxidation vs. the concentration of the substrate were converted to the Lineweaver-Burk coordinates. All studied pesticides inhibit NADH-OR but show different types of inhibition.
Of all the compounds studied, the highest inhibitory activities were found for zenkor and bazagran. The inhibitory ability of CuL 2 , MoL 2 , and FeL 2 is higher than that of the original lontrel. The Michaelis constants (K m ) calculated without an inhibitor are 6.6•10 -4 and 2.47•10 -3 mol L -1 for NADH and NT, respectively. Lontrel, zenkor, kuzagard, tachigaren and СuL 2 , FеL 2 , MnL 2 , МоL 2 inhibit the reduction of an electron donor in the competitive manner. Apparently, being its structural analog, a pesticide binds to the enzyme in the binding site with the formation of the NADH nonproductive complex. Setoxidim, roundup, tilt, MgL 2 , NiL 2 , ZnL 2 , and CoL 2 inhibit the reduction of NADH-OR from NADH in the uncompetitive manner, apparently, due to the nonspecific interaction with the protein matrix outside the enzyme active site.
In terms of the K i values with respect to NADH, the herbicides and lontrel complex with metals can be arranged in the following activity order: CuL 2 <МоL 2 <zenkor<lontrel<FeL 2 <MnL 2 <ZnL 2 <NiL 2 <MgL 2 <basagran<СоL 2 <kusagard<ta chigaren<roundup<tilt<setoxidim. This series is similar to that of complexation constants of these compounds with NADH given in Table 2.
Lontrel, zenkor, basagran, and roundup inhibit the reduction of NT in the uncompetitive manner, apparently, due to the nonspecific interaction with the protein matrix outside the enzyme active center. This interaction could induce conformational changes around the electron transfer site, resulting in the inhibition of enzymatic activity. Meanwhile, kusagard, setoxidim, tilt, and tachigaren compete with NT for the binding region on the enzyme. These differences can be due to different structures of the examined pesticides.  Mold., 2017, 12(1), [20][21][22][23][24][25][26][27][28] The metal complexes of lontrel are known to exhibit herbicide activities in vivo [39,43]. In addition, as noted above, the complex formed by the herbicide lontrel with the copper ion exhibits a much higher inhibitory activity than the starting lontrel. Therefore, we carried out an additional study of a series of complexes of these pesticides with different doubly charged metal ions, ML 2 , and salts of these metals. Among the lontrel complexes with metal ions, only the complexes with Mg and Mo were proved to be competitive reductase inhibitors with respect to NT. The lontrel complexes with Cu, Ni, and Fe ions inhibit the enzyme noncompetitively, while the lontrel complexes with Mn, Zn, and Co display a mixed type of inhibition. The difference between the inhibition patterns may be related to the difference between the acceptor abilities of the metal ions.
The metal complexes are characterized by the predominant influence of the ligand environment. The pyridine ring has a structure close to NADH, i.e., is able to replace the substrate on the protein, and the nitrogen atom may donate an unshared pair of electrons. A change in the coordination sphere of the metal (ligand environment) leads to fundamental changes in the nature of inhibition. It is known [46,47] that the 2Fe-2S cluster is in the composition of the active center of NADH-OR. Being in the composition of the complex, the metal cannot act as a free cation, since it is significantly affected by the ligand environment. The considered ligand (lontrel) is capable of occupying the site of NADH, donor of two electrons, in the active center of the enzyme. Probably, the interaction with iron of the cluster occurs through the carboxyl of the ligand and due to a high electron density of chloropyridine. As a result, the ligand or complex inhibits the NADHbinding region of the electron-transfer chain. The structures of the complexes allow them to play the role of both the electron donor and electron acceptor. It can be assumed that the complexes form chains in which the ligands act as a "bridge": L-M-L-Fe-ОR and the metal of the complex pulls electrons from the 2Fe-2S cluster of the active center of the enzyme as it shown in Figure 5. Figure 6 schematically shows the direction of the inhibitor attack. The intramolecular electron transfer in the active center of NADH-OR proceeds from flavine adenine dinucleotide (FAD) to the iron-sulfur cluster 2Fe-2S and further to an artificial electron acceptor [25, 26,48]. For competitive inhibition, the pesticide or ML 2 occupies the site of NT and thus breaks the electron transfer chain.

Conclusions
These studies have shown that pesticides are substances with a high ability to form complex compounds of superior chemical stability. These complex compounds show no selective action. Pesticides in a related form are transferred through trophic pathways and enter the body. Pesticides inhibit the biological activity of oxidizing enzymes. As a result, the organism and the whole environment lose the ability of degradation of toxic substances. There is a gradual accumulation of pesticides in the bodies of living beings and the environment. The accumulation of pesticides primarily leads to metabolic failure of energy metabolism due to the occurrence of energy deficiency because of binding of ATP with pesticides.
The sum of these effects is the cause of almost all diseases of modern man, including cancer. It is very necessary to forbid the use of pesticides for safety of our world, ecosystems, and human being and for population health improvement.