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ENGLISH VERSION: STATE OF EXCHANGE OF L-TRYPTOPHAN IN THE SUBACUTE EXPERIMENT UNDER INFLUENCE OF OLIGOETHERCYCLOCARBONAT IN THE SUBTOXIC DOSES*
Bagmut I.Yu.
Kharkov Academy of Postgraduate Education, Kharkov, Ukraine
The effects of small doses of sub-toxic xenobiotic - oligoethercyclocarbonat type P-803 at the rate of 1/10; to 1/100 and 1/1000 of the body LD50 investigated were in 40 adult Wistar rats in the subacute experiment. In the blood serum of experimental and control animals the content of L-tryptophan and its metabolites - serotonin, melatonin, 5 - oxigindola-cides acid (5-OIAA), animal indican in liver enzyme activity tryptophan-2,3-dioxygenase (TAR) were were determined. The serum is also one of the end products of oxidation deaminationof amino acids, biogenic amines, purine nitrogenous bases, etc. - ammonia (NH3). It was established that xenobiotic 1/10 and 1/100 of reduced content of LD50 in plasma of melatonin and tryptophan against increase of serotonin, 5-OIAA, indican, ammonia and enzyme activity in liver TAR. The results indicate that oligoethercyclocarbonat 1/10 and 1/100 LD50 leads to profound disruption of protein, neurotransmitter, hormone, cofactor metabolism in rats.
Keywords: xenobiotics, L-tryptophan, serotonin, melatonin, 5 blood serum of rats.
Introduction
Over the past decade synthesized dozens or even hundreds of thousands of new chemicals are often chemically resistant, high-level, with pronounced biotropicality to which a man is not evolutionarily adapted [7, 3]. A significant gap between high ability of modern civilization to create a new chemical potential and limited capacity to neutralize its harmful effects on the flora and fauna originated. This situation provides the real conditions for the formation of various environment-related diseases and conditions. However, the mechanisms of action of many chemical compounds and combinations thereof, in most cases remain unexplored. [4] The need for extensive study pathochemical mechanisms of pathological states, is closely connected with the development of prevention and correction of structural and metabolic abnormalities that occur in long-term effects on the sub-toxic chemicals [8]. Particularly relevant for the task is new and completely unstudied chemical compounds capable of not only the direct toxic effect on the body, but also of shaping the development of possible long-term effects of mutagenesis, carcinogenesis, atherogenesis, immune deficiency, teratogenesis, etc. In this regard, study of pathophysio-logical mechanisms of action on the organism of small subtoxic doses of xenobiotics is the first priority in making a forecast of the potential dangers of new chemical compounds, which are at the stage of pilot production and implementation of the national economy. This fully applies to the new chemical compound industry polyoxy-
- oxigindolacides acid (5-OIAA), animal indican, liver,
propylene - oligoethercyclocarbonat, which is widely used in various industries for epoxy resins, lacquers, enamels, plastics, foams, thermoplastics, etc. [4, 8, 9]. Given the above, the purpose of the work was to study the effect of sub-toxic doses oligoethercyclocarbonat, in long-term uptake, the exchange of essential amino acid L-tryptophan in subacute experience.
Materials and methods
The research program included a subacute toxicology experiment on mature white Wistar rats weighing 180200 g in accordance with the conditions of the experiment the animals daily for 45 days in the morning before feeding with a metal probe exposed to aqueous solutions of oral priming oligoethercyclocarbonat type P-803 based 1/10; To 1/100 and 1/1000 of LD50. The control group received the appropriate volume of drinking water. On the basis of acute toxicity parameters P-803 refers to a low-toxic compounds (Hazard Class 4), non-cumulative properties and species sensitivity. Of the mean dose (LD50) to albino rats was set at 18,75 g/kg of animal weight and cumulation coefficient (CC) at the level of 7,82. Selecting this xenobiotic for research was justified lack of prognostic characteristics of the potential hazard to warmblooded animals and humans, large volumes of production, wide track undelivered xenobiotic population and the need to justify the hygienic standards as maximum allowable concentrations in the environment. Conducting subacute experience accompanied by the observance of
* To cite this English version: Bagmut I.Yu.. State exchange l-tryptophan of the subacute experiment under infuense oligoethercyclocarbonat in the subtoxic doses / / Problemy ekologii ta medytsyny. - 2014. - Vol 18, № 1-2. - P. 53 -55.
Том. 18, N 1-2 2014 р.
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the principles of bioethics and the "European Convention for the Protection of Vertebrate Animals used for research and other purposes" - Strasbourg, 1985 Total was used in the experimental part of 40 white rats, 10 in each experimental and control group.
Investigation included the determination of tryptophan metabolism in the serum of control and experimental animals L-tryptophan content and its metabolites - serotonin, melatonin, 5 - oxigindolacides acid (5-OIAA), animal indican in liver enzyme activity tryptophan-2,3-dioxygenase (TAR ). The serum is also one of the end products of oxidative deamination of amino acids, bio-genic amines, purine nitrogenous bases, etc. - ammonia (NH3). Determination of ammonia in the serum was carried out by ion exchange chromatography on ionity exchangers. After separation of the substrate on ion-exchangers registration NH3 concentration was carried out on an automatic amino acid analyzer T-339 (Czech Republic). Tryptophan and its metabolites exchange -serotonin, 5 - HIAa were determined by Atack C., Mag-nusson T. [10]. Melatonin was investigated by ELISA using monoclonal antibodies. Used for these purposes reagent kit Melatonin ELJSA (Hamburg), Kat-^RE 54021. On the functional state of transformation processes of amino acids in the colon under the influence of microflora and the detoxifying function of the liver judged by the amount of the final product of tryptophan metabolism -indican animal serum conventional method [2]. It is known that L-tryptophan is an enzyme stabilizer TAR. Promoting the formation of stable conformational state, TAR liver has absolute substrate specificity for L-tryptophan and irreversibly catalyzes the key reaction for the catabolism of the amino acid exchange kynureninity its path to form N-formylkynurenine and later end of one of the key metabolites - NAD+. This enzyme accelerates the insertion of molecular oxygen directly into the molecule of L-tryptophan, and their reaction is catalyzed conversion step speedlimity substrate. TAR activity determined by A.A. Badawe - V., Evans M. [11]. Statistical processing of the results of the study were performed using Student's test, Fisher.
Results and discussion
Studying the influence of P-803 oligoethercyclocarbo-nat exchange rate limiting amino acid L-tryptophan found that xenobiotic 1/10 and 1/100 of reduced content of LD50 in plasma melatonin and tryptophan against increase serotonin 5OIUK, indican, ammonia, and the activity of the liver enzyme TAR (Table). Thus, L-tryptophan was reduced by 39.04% and 21,64%, 73,79% by melatonin and 53,23% with serotonin increased by 721,42%, and 560,71%, 5-OIAA at 454,76 % and 326,19%, 269,56% by indican and 179,13%, 128,34% ammonia and 92,3%, respectively, under the influence of 1/10 and 1/100 of LD50. It is known that tryptophan is a source of nicotinamide coenzyme forms (NAD+ and NADP+) of vitamin E - niacin synthesis of biogenic monoamines - serotonin, a hormone - melatonin inducer of cell differentiation and proliferation - 5 - oxiindolacetat that can have a significant ef-
fect on the metabolism of various organs and tissues of the body [5]. Convincingly shows the effect of serotonin on the mediator processes in the nervous system, it acts as a local regulator function of peripheral organs and tissues, is a potent vasoconstrictor and stimulator of smooth muscle tissue.
Approximately 95% of serotonin is produced in the adult intestinal enterochromafin cells. The rest of it is in the mast cells of the connective tissue of the skin, spleen, liver, kidney, lung, pineal gland, brain cortex, hypothalamus, blood platelets, etc. [5]. L-tryptophan and the immediate precursor of serotonin, are substrates for the synthesis of the hormone of the pineal gland, which provides circadian rhythms of metabolic processes in the body - melatonin, which is produced by N-acetylation and the next methylation 5-HT. [6] This hormone regulates puberty, heat, wind, water-salt metabolism, wakefulness, circadian dynamics of metabolic processes in different organs and tissues of the body, depending on the time of day, seasons of the year, an immunomodulator and active substrate having antiradical and antipyroxidant properties, etc. However, L-tryptophan can be converted into gastrointestinal tract under the influence of colonic microflora to indole, skatole, indole etc. cresol and derivatives. The final product of these transformations, is excreted in the urine mainly 5-oksiindolatsetat that acts potent mito-genic factor in differentiation and proliferation of rapidly renewing tissues [5, 6]. These disorders are characterized, and a significant increase of oxidative deamination of amino acids, biogenic monoamines - ammonia (NH3), as well as animal indican (Table). It is known that the main, if not the only mechanism through which TAR activity interferes with the synthesis of serotonin in the body is the change in the level of free L-tryptophan [5,6]. Studies have found increased activity and reduced tar content of L-tryptophan in the blood serum. In these metabolic conditions opens the way of the increased synthesis of coenzyme forms of NAD+ and NADP+ needed to enhance the redox processes, differentiation and proliferation, as a result of protective and adaptive reactions of the organism, which are aimed at reducing the activation of synthesis conditions subtoxicaly toxifications.
TAR regulation is carried out by the feedback end products kynurenine pathway of L-tryptophan-NAD+ and NADP+. Activation of the enzyme is associated with increased levels of substrate oxidation - L-tryptophan. TAR enzyme activators positive ions are Cu2 +, hematin, and ferrigem 6-aminolevulinic acid (6-ALA). Hemin, thus, is a coenzyme TAR. A significant increase in the activity of the tAr to judge cut proteinsynthesizing function of hemoglobin synthesis, resulting in heme is oxidized by oxygen in hemin being coenzyme activator of the enzyme, on the other hand - the oxidized form of heme (hemin) inhibits the activity of the mitochondrial enzyme 6-aminolevulenacintazy catalyzes the first reaction heme synthesis of succinyl CoA from glycine ~ - 6-aminolevulinic acid.
Проблеми екологп та медицини
Table
Influence of oligoethercyclocarbonat P-803 on the state exchange of L-tryptophan under subtoxic long Intake of
Indicators Observation group, LD50, M ± m
Control 1/10 1/100 1/1000
L-tryptophan (mkm/l) 68,4±3,2 41,7±2,8* 53,6±3,5* 49,6±4,3
Serotonin (mkm/l) 0,56±0,04 4,6±0,52* 3,7±0,46* 0,53±0,05
5-OIAA (mkm/l) 0,42±0,02 2,33±0,18* 1,79±0,14* 0,38±0,04
Melatonin (pkg/l) 178,5±7,3 46,8±4,10* 83,5±6,2* 184,7±8,5
Indican (mkm/l) 2,3±0,26 8,5±0,76* 6,42±0,57* 2,17±0,24
Ammonia (nmol/L) 24,7±1,54 56,4±4,3* 47,5±3,3* 26,4±2,5
TAR(nmolkynurenina /mg protein • 1 hour) 36,5±2,7 71,6±5,8* 63,7±4,9* 35,6±3,2
Note: * difference reliable, p <0,05
The observed changes in the exchange of L-tryptophan and activity of TAR may indicate inappropriate conjugate metabolic processes associated with anabolic and catabolic transformations of proteins, neurotransmitters, hormones, inducers of differentiation and proliferation, end metabolites exchange, etc. Much of the metabolite of L-tryptophan, a biogenic amine - serotonin is oxidation deamination with the formation of ammonia, H2O2, aldehyde, 5-OIAA, wherein the conversion of tryptophan may be associated with the synthesis of melatonin levels were significantly lower than that under the influence of 1/10 and 1/100 of oligoethercyclocarbonat LD50. Serotonin is intermediate between hormones and neurotransmitters. This narrows the mediator arterioles and increases blood pressure, increases peristalsis, exerts an antidiuretic effect. In the central nervous system, serotonin functions as a neurotransmitter and a source of synthesis in the pineal gland hormone melatonin. Literary sources indicate that melatonin neonatal development affects the differentiation centers of the brain that controls the function of the gonads and adrenal glands in the "critical" period of development [5, 6]. It inhibits the secretion of gonadotropins in the hypothalamus and causes the antagonistic relationship between this body and the gonads, as a physiological inhibitor of precocious puberty. It played an important role in the mechanism of "biological clock", the frequency of activation and inhibition functions of the body at different times of day, seasons of the year [1, 5, 6].
The results of the dynamics of serotonin and melatonin indicate a serious dysfunction of the neuroendocrine system in the regulation of structural and metabolic processes and mechanisms of pathological conditions. The level of one of the end products of metabolism of L-tryptophan - animal indican was significantly increased under the influence of 1/10 and 1/100 of oligoethercyclo-carbonat LD50, which confirms the increase of formation of the final product toxic decay L-tryptophan - indole. These data suggest inappropriate processes that are associated with digestion of proteins on the background of a possible change in intestinal microbial profile associated with the development and putrefaction dysbiosis [2]. However, studies indicate activation detoxifying liver function was confirmed by the increase in serum indole associated as etheresulfur acid with potassium or sodium (indican). It is known that one of the metabolites of the exchange of L-tryptophan is 3-hydroxyanthranilic acid, which has antioxidant properties. It is characterized by the ability to recover a-tocopherol, associated with low density lipoproteins (LDL). Analysis of the results of exchange of L-tryptophan and increased activity in the TAR oligoethercyclocarbonat toxification is able to make some contribution to the conditions of formation of cooperative interaction between oxidant-antioxidant homeostasis,
which may be associated with increasing free-radical processes, activation of lipid peroxidation, oxidative modification of proteins and development of membrane pathology.
Conclusions
The study of the exchange of L-tryptophan in experimental animals suggests that oligoethercyclocarbonat in 1/10 and 1/100 LD50 leads to profound disruption of protein, neurotransmitter, hormone, cofactor metabolism, accompanied by endotoxemia, which was confirmed by an increase in serum ammonia and indole derivative -indican, which is a measure of the adverse assessment of the homeostatic functions of the body. Studies show that the effect may be accompanied by xenobiotic poly-tropic disorders various organs, systems and functions, which are based on free radical pathology and oxidative stress.
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