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14
TOM 17. N 5-6 2013 P.
English version: LONG-DURATION ACTION OF OXYETHYLIZED NONYLPHENOLS AND THEIR DERIVATIVES ON CONTENT OF IMMUNOGLOBULINS AND CYTOKINES IN THE BLOOD SERUM OF RATS*
Marakushin D.I.
Kharkov national medical university, Kharkov
This research deals with influence of oxyethyiized alkylphenols and their derivatives on content of immunogiobuiins and cytokines in the blood serum of rats during subacute experiment. Examined detergents in doses 1/10 and 1/100 DL50 cause a supression of antibody production, decrease of immunoglobulins A, M, G level, decrease of secretion of IL-1/S, 2, 4, 6, 8, 10 and TNF-a. These changes may serve as addttionai prognostic tests, which characterize adverse action of oxyethyiized alkylphenols and their derivatives on the organism of warm-blooded animals.
Key words: oxyethyiized nonylphenols, immunoglobulins, cytokines, immune system
Introduction
The violation of homeostasis regulatory mechanisms in conditions of intoxication by the xenobiotics is considered as «toxic stress», at which not only nervous and endocrine systems but also immune system interfere in a process [1, 2]. In addition, immune system, carrying out an important role in the adaptation and protective reactions of an organism is one of the first systems undergo to external influence, and tension arising in its activity results in violation of functioning of other systems and provokes pathology [3]. Research of xenobiotics' mechanisms of action, creation of scientifically grounded diagnostic programs, revelation of objective prognostic tests of pathological processes becomes one of priority tasks of modern medicine. The sharp necessity of its decision is related to making progress contamination of environment, worsening of people health, and is based on the scientific ground of criteria which determine the beginning of the state of decompensation of functions of the organism [4]. Oxyethylized nonylphenols (OENP) and their derivatives sodium salts of carboxymethylates of oxyethylized isononylphenols (CM-OENP) are belong to priority contaminants of environment, which have physical and chemical properties and features of structure of molecules like ionogenic detergents. These substances are characterized by considerable output volumes of synthesis, wide usage in different industries of national economy (as basis of industrial issue of plastics, polyure-thanes, cleaning agents, emulsifying agents, anticorrod-ing preparations, hydraulic and cooling fluids, and others like that), entry to the sources of water consumption and due to it possible influence on the organism of man [5, 6]. Mechanisms of action of OENP and their derivatives are studied not enough. Their revelation is the grounds for explanation of measures on the conservation of the environment and health of population.
The purpose of this research was to study the content of immunoglobulins and cytokines in the blood serum of rats in conditions of prolonged peroral influence of OENP and their derivatives in doses 1/10, 1/100 and 1/1000 DL50.
Materials and research methods
The standard compounds with scheduled physical-chemical properties were used in the work: OENP with number of oxyethylized groups 10 (OENP10) and CM-OENP with number of oxyethylized groups 5, 6, 10 (CM-OENP5 -10). The experimental part of the research was
performed on white rats of WAG population, with mass (180-220) g. The experiments on white rats were performed in compliance with the international principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986). Every day before feeding they received orally, through a metal probe, aqueous solutions of xenobiotics with 1/10, 1/100 and 1/1000 of LD50. The duration of subacute influence was 45 days. Half-lethal doses (LD50) were determined at the levels: OENP10 — 4,3 g/kg; CM-OENP5 - 2,8 g/kg; CM-OENP6 - 2,2 g/kg; CM-OENP 10 - 3,2 g/kg of the animal weight. The proper volumes of water were entered to the animals of control group. Research of immunoglobulin and cytokines content was carried out in 45 days after the beginning of experiment. Eevery group made up 15 animals. The slaughter of animals was performed by a decapitation, preliminary using thiopental sodium.
The content of immunoglobulins (Ig) A, M, G in the blood serum is determined by method of radial immunodiffusion in a gel by the instrumentality of monospecific serums against immunoglobulins made by FGOUP «NPO Microgen» (Russia). The level of cytokines was determined by an immunoenzyme method with the use of reagents sets «ProCon IL-1p», «ProCon IL-2», «ProCon IL-4», «ProCon IL-6», «ProCon IL-10», «ProCon TNF-a» («Proteinic contour», Russia), «Interleukin-8-IFA-BEST» («Vector-Best», Russia) and analyzer immunoenzyme Stat Fax 303 Plus. The statistical data analysis was performed with the usage of computer programs for statistical data handling Statistica 6.1 (StatSoft, Inc., the USA). The primary statistical data processing begun from verification of hypothesis about correspondence of sample to Gaussian distribution law, using Shapiro-Wilk test. the rightness of conclusion in relation to normality of sample distribution was additionally controlled by the instrumentality of the asymmetry and excess coefficients. Quantitative signs, that had normal distribution, were described by parametric descriptions - mean value of index (M) and standard deviation (s); in the case of absence of normal distribution - nonparametric: median (Me) and interquartile range. The Student's t-test is used for comparison of two normal distributions. If at least one of distribution was not normal, for comparison of independent samples the Mann-Whitney U test was used. For the critical level of statistical significance at verification of statistical hypotheses took p<0,05.
* To cite this English version: Marakushin D.I. Long-duration action of oxyethylized nonylphenols and their derivatives on content of immunoglobulins and cytokines in the blood serum of rats / / Problemy ekologii ta medytsyny. - 2013. - Vol 17, № 5-6. - P. 42 -45.
Results and their discussions
On 45-day of action OENP and their derivatives in a dose 1/10 DL50 was observed statistically significant (p<0,001), comparatively with the control, reduction of IgA content in the blood serum of rats: for OENP10 and CM-OENP5 at the average in 2 times, for CM-OENPa and CM-OENP10 - almost in 3 times (tabl. 1). The same dynamics of changes of content IgA was determined and for a dose 1/100 DL50, but less expressive (reduction at the average in 1,5 times). The dose 1/1000 DL50 was not effective. Reduction of IgA content testifies about insufficiency of humoral and local immunity. Index of level IgM also significantly (p<0,001), comparatively with the control, decreased on 45-day of peroral introduction of compounds in 1/10 and 1/100 DL50 at the average accord-
ingly in 2,0 and 1,5 times (tabl. 2). At most this level was registered in the case of action CM-OENP5, minimum low - in the case of action OENP10. It should be noted that the protracted influence of CM-OENP5 and CM-OENP10 in a dose 1/1000 DL50 was also accompanied by statistically significant (p=0,003 and p=0,032 accordingly) decreasing of IgM content, while OENP10 and CM-OENP6 in this dose did not affect the level of this index (p=0,163 and p=0,087). As IgM appear on the first stage of immune respond and mainly are found in a vascular bed, they play an important protective role on the early stages of toxemia. The reveal decrease of IgM content also testifies about insufficiency of humoral immunity, disturbances of their synthesis or intensification of catabolism.
Table 1
The content of immunoglobulin A in the blood serum of rats on 45-day under the influence of oxyethylized nonylphenols and their
derivatives (g/l; n=15; Me [25%; 75%] or M±s)
Indices Dose, DL50
1/10 1/100 1/1000
OENP10 0,29±0,057 p<0,001 0,39 [0,36; 0,41] r<0,001 0,53 [0,48; 0,55] p=0,237
CM-OENP5 0,22±0,039 p<0,001 0,33 [0,29; 0,37] p<0,001 0,51±0,031 r=0,69
CM-OENP6 0,19 [0,17; 0,20] p<0,001 0,35 [0,28; 0,37] p<0,001 0,49±0,028 r=0,384
CM-OENP10 0,18 [0,15; 0,22] p<0,001 0,29±0,037 p<0,001 0,46 [0,43; 0,48] r=0,019
Control 0,50±0,053
Note: p - level of statistical significance comparatively with the control
Table 2
The content of immunoglobulin M in the blood serum of rats on 45-day under the influence of oxyethylized nonylphenols and their
derivatives (g/l; n=15; Me [25%; 75%] or M±s)
Indices Dose, DL50
1/10 1/100 1/1000
OENP10 0,52±0,094 0,63±0,069 0,82±0,062
p<0,001 p<0,001 r=0,163
CM-OENP5 0,38±0,043 p<0,001 0,47±0,065 p<0,001 0,7 [0,69; 0,79] r=0,003
CM-OENP6 0,42 [0,38; 0,44] p<0,001 0,52 [0,49; 0,56] p<0,001 0,76±0,037 r=0,087
CM-OENP10 0,46±0,041 p<0,001 0,56±0,046 p<0,001 0,83±0,044 r=0,032
Control 0,81 ±0,028
Note: p - level of statistical significance comparatively with the control
The content of IgG in the blood serum of rats on 45-day of influence OENP and their derivatives in a dose 1/10 DL50 also was significantly below (p<0,001) comparatively with data of the control group (tabl. 3). In this case most substantial decrease of this index is typical for CM-OENP6 (almost in 2 times), minimal - for OENP10 (in 1,4 times). The protracted influence of compounds in an effective dose 1/100 DL50 also resulted in statistically sig-
nificant, comparatively with the control, decreasing of IgG level in blood serum of rats, but it was less expressive (at the average in 1,3 times; p<0,006), than in the case of influence 1/10 DL50. Action of compounds in a dose 1/1000 DL50 did not cause significant changes from the side of IgG. The stable decreasing of IgG level testifies about exhaustion of their protective role.
Table 3
The content of immunoglobulin G in the blood serum of rats on 45-day under the influence of oxyethylized nonylphenols
and their derivatives (g/l; n=15; Me [25%; 75%] or M±s)
Indices Dose, DL50
1/10 1/100 1/1000
OENP10 4,0±0,62 4,5±0,53 5,8 [4,9; 6,5]
p<0,001 r=0,006 r=0,455
cm-oenp5 2,9±0,53 p<0,001 4,0±0,88 p<0,001 5,1±0,95 r=0,283
CM-OENP6 2,6 [2,4; 3,2] p<0,001 3,9±0,71 p<0,001 5,0 [4,8; 6,4] r=0,88
CM-OENP10 3,3±0,65 p<0,001 4,3 [3,6; 5,0] r=0,002 5,4±0,79 r=0,89
Control 5,5±1,05
Note: p - level of statistical significance comparatively with the control
43
An adequate immune response is based on balance of the cellular-mediated and humoral immune reactions, development of which is supported and regulated by cytokines [7-9]. The compounds result in disturbances from the side of cytokinic profile of blood serum of rats (tabl. 4). In the case of their protracted peroral introduction in a dose 1/100 DL50 it is observed, comparatively with the control, decreasing (p<0,003) of level IL-1 p: for CM-OENP5 - almost in 2 times, CM-OENPa and CM-OENP10 - at the average in 1,8 times, OENP10 - only in 1,4 times. The proinflammatory cytokine IL-1p has numeral general effects and assists to development of system character of pathological process by formation of autoantibodies and rise of concentration in the blood of the C-reactive
The content of cytokines in the blood serum of rats on 45-day
protein. The decrease of this index is the important diagnostic criterion of chronicity of inflammatory process in the organism of rats in conditions of protracted influence of OENP and their derivatives. The decrease of level IL-2 almost in 2 times in conditions of influence CM-OENP5 (p<0,001) in 1,8 times - CM-OENPa (p<0,001), in 1,5 times - CM-OENP10 (p<0,001) and in 1,3 times -OENP10 (p=0,003) was marked also on a 45-day in blood serum of rats. IL-2 is considered as a regulator cytokine, able to compensate the displays of immune insufficiency, to proceed in balance T-helpers, that results to regulation of synthesis of anti- and proinflammatory cytokines. The decrease of IL-2 synthesis is confirmation of origin of the T-cell immunodeficiency.
Table 4
;r the influence of oxyethylized nonylphenols and their derivatives in a dose 1/100 DL50, (ng/l; n=15; Me [25%; 75%] or M±s)
Index OENP10 CM-OENP5 CM-OENP6 CM-OENP10 Control
1 L-1 p 40,2 [30,7; 50,5] p<0,001 29,3±6,36 p<0,001 30,3±5,69 p=0,0018 32,5 [29,0; 46,0] p<0,001 56,9±7,05
IL-2 48,3±8,53 p=0,003 30,6±6,12 p<0,001 36,5 [25,8; 40,1] p<0,001 43,9 [32,1; 50,0] p<0,001 64,7±17,1
IL-4 29,5 [26,0; 39,4] p<0,001 20,3 [17,5; 29,0] p<0,001 26,3±8,37 p<0,001 32,0 [27,9; 39,0] p<0,001 40,9 [39,4;60,3]
IL-6 24,4±6,04 p<0,001 18,9 [12,0; 28,5] p<0,001 28,9 [19,2; 30,2] p=0,0015 23,1±5,88 p<0,001 39,8 [35,0; 50,6]
IL-8 31,8±6,58 p<0,001 20,1 [17,0; 27,0] p<0,001 25,1±6,27 p<0,001 30,5 [28,0; 44,5] p<0,001 48,4 [44,0; 59,0]
IL-10 25,0 [19,3; 31,0] p<0,001 20,0 [15,1; 22,5] p<0,001 24,7 [17,0; 29,0] p<0,001 32,7±7,57 p=0,0013 43,9±9,66
TNF-a 110,0 [90,6; 118,6] p<0,001 97,8 [80,5; 108,3] p<0,001 85,8±11,84 p=0,0016 118,0±9,39 p<0,001 136,8 [123,8; 148,4]
Note: p - level of statistical significance comparatively with the control
As a marker of autoimmune and allergic processes IL-4 is considered, which ensures an activation of humoral chain of the immune system, stimulates proliferation and differentiation B-cells, synthesis of IgE, and also inhibits of monocytes and macrophages, synthesis of TNF-a. In the blood serum of rats on 45-day of influence of compounds in a dose 1/100 DL50 it was showed the decrease (p<0,001), comparatively with the control, level of IL-4 at average in 1,6 times. The decrease of secretion IL-4, in this case, can be accompanied by depression of antigen stimulation T- and B-lymphocytes, macrophages, mass cells, basophiles, stromal cells, hemopoietic precursors.
The protracted action OENP and their derivatives result also to the decrease (p<0,0015) of content IL-6 in the blood serum of animals on the average in 1,7 times. A biological role IL-6, at first, consists in induction of restorative mechanisms and activation of immune defence (activation and differentiation T-cells, maturation of B-cells, intensification of hemopoiesis). It is also known an inhibitory action of IL-6 on the inflammatory reaction by inhibition of synthesis of proinflammatory substances, in that number TNF-a. The decrease of IL-6 level in the condition the protracted action of compounds can result in depression of their functional activity and reduction of humoral immune response. Also a tendency to statistically significant (p<0,001), comparatively with the control,
decreasing of IL-8 content is observed. Most substantial it was for protracted action of OENP10 in a dose 1/100 DL50, namely in 2,4 times. Other compounds decrease content of IL-8 at the average in 1,7 times. Cytokine IL-8 is produced by monocytes and macrophages, executes the role of inductor of acute inflammatory reactions, causing expression of molecules of intercellular adhesion and intensifying of adhesion of neutrophils to the endothelial cells and subendothelial matrix proteins, that testifies about its basic role in mediation of inflammatory response. The decrease of its synthesis is one of the reasons of depression of cellular and humoral immunity.
IL-10 refers to cytokines with expressive antiinflammatory effect, which is producted T-cells and examined as an antagonist of row other cytokines. IL-10 depresses the synthesis of proinflammatory cytokines, proliferative response of T-cells on antigens and mitogens, and also diminishes a secretion by activated monocytes of IL-p, TNF-a, IL-6. But simultaneously IL-10 can stimulate the synthesis of IgE. In the result it assists to development of humoral component of immune response. OENP10, CM-OENP5, CM-OENP6 in an operating dose 1/100 DL50 on a 45-day of action resulted in decrease (p<0,001) of IL-10 level at the average in 2 times. For CM-OENP10 the level of IL-10 decrease (p<0,0013) was less expressive, only in 1,3 times. These changes also specify on violation of the functional state of protective systems of the rats organism.
The results of TNF-a content have an important value in metabolic plan. So, the content of this index in the blood serum of rats on 45-day of compounds introduction is statistically decreased (p<0,001), at the average in 1,4 times, comparatively with a control group. TNF-a is produced by both the cells of the immune system and other types of cells (astrocytes, fibroblasts, glial cells, keratino-cytes). Low levels of TNF-a in the conditions of protracted influence of the examined compounds can be related to insufficient stimulation of macrophagic defence of the organism.
In general the decrease of cytokines in the blood of rats in the conditions of protracted influence of OENP is the evidence of deep disturbances of immunity, demonstration of development of the secondary immune insufficiency.
Conclusions
1. In the conditions of protracted influence in doses 1/10 and 1/100 DL50 OENP and their derivatives result in suppression of antibody synthesis in the organism of rats, that is confirmed by decrease in the blood serum of level of immunoglobulins A, M and G. The dose 1/1000 DL50 is inactive on the content of immunoglobulins.
2. OENP and their derivatives in the case of the protracted peroral influence in an operating dose 1/100 DL50 cause substantial disturbances in the system of cytokines - mediators of control of the processes of proliferation, differentiation, apoptosis and functional activity of cellular elements in the homeostatic systems of the organism, that is confirmed by the decrease in the blood serum of rats of secretion IL-1 p, 2, 4, 6, 8, 10 and TNF-a.
3. Changes from the side of imounoglobulins and cy-tokines can serve by additional prognostic tests, that
characterize the unfavorable influence of OENP and their
derivatives in doses 1/10 and 1/100 DL50 on the organism
of warm-blooded animals.
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