SPLEEN MORPHOMETRY UNDER THE IM- PACT OF LAPROXIDES IN EXPERIMENT

In subacute experiment were uncovered organometric alterations of the matured male rat’s spleen after the administration of 1/10, 1/100 LD50 of polyether – tryglycidyl ether of polyoxypropylene triol. Rapid technology growth and its implementation in all spheres of the people’s lives dictates the need for thorough study of the influence of different chemicals on human's health. This study was undertaken to elucidate the structural changes that occur in the matured rats’ spleen experimentally induced by selected xenobiotic, exactly the linear dimensions and the weight of the rodents spleen. Were used morphometric, mathematical and statistical methods. Revealed changes of morphologiс data in comparison to control data shows high reactivity of the spleen in response to the induced xenobiotic. These data are useful for understanding the toxicological effects induced by investigated polyether and provide evidence for the mechanism of toxicity of the agent and how it impacts the immune


Introduction
A variety of environmental factors determines a wide range of morphological and functional changes in humans and animals. The chemicals that are commonly considered the term "xenobiotics" have specific effects on organs, tissues, cells and subcellular components. These chemicals include products of human activities, household chemical substances and most of the medicines. Toxicants of different chemical nature to some extent have damaging properties and, under certain conditions, permeate into the body, making a harmful impact on health, including the immune system [1].
As the material for the study was used xenobiotic, which has a marketable name "Laproxides" with regulated physico-chemical property: tryglycidyl ether of polyoxypropylene triol (TEPPT) with molecular weight 303 (L-303). The choice of this group of substances was made on grounds of large volumes of production, extensive contact with the population, the lack of prognostic characteristics of their potential danger for humans and warm-blooded animals [2, 3], and the need to justify pathophysiological mechanisms of structural and metabolic disorders under prolonged intake of subtoxic doses. The organs of the lymphoid system participating in maintaining the constancy of homeostasis, among the first responding to exogenous influences and have the high potential to ensure the adaptation processes while the adverse impact of chemicals.

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According to the scientific literature, detergents have found wide enough tests on laboratory animals; a number of experimental studies demonstrated that they meet the requirements for substances which are constantly in contact with the humans [1,4]. Spleen is one of the recommended organs to evaluate for enhanced histopathology of the immune system [5] as it is the largest secondary lymphoid organ and it is considered the draining site for compounds that are administered intravenously, and, therefore, serves as an important organ to evaluate for treatment-related lesions.
TEPPT is a typical polyether that has characteristics enabling wide use in the production of many materials such as drugs prolongators, ointment ingredients, additives to the household products, coatings, and adhesives. Potential toxic effects attracting wide attention and only a few studies have assessed the effects of TEPPT on the immune system [3, 5, 7-12]. These studies have shown that TEP is a class of environmental pollutant with versatile toxicities.

Material and Methods
The study was conducted on 72 outbred WAG male matured rats with the weight 200±10 g. The control and experimental series consisted of animals of the same age. Animals were divided into 2 series. The first seria -control animals (3 groups, 6 animals in each), were fed a regular diet and received an appropriate amount of water. The second seria was experimental animals. They were randomly divided into 3 groups 6 in each depending on the dose of induced polyether and length of administration: 7 days, 15 days and 30 days. All laboratory animals were maintained in the conventional environment of KhNMU vivarium in a controlled-temperature room t 20±2°C, humidity 65±10%. All rats were treated via gastric gavage during 7, 15, 30 days by aqueous solutions of TEPPT in the doses 1/10 and 1/100 LD 50 in conversion to 5.75 g/kg. At the end of the investigation, changes were observed. Food intake and body weight were measured every 2 days. All rodents were killed by immediate cervical dislocation at the end of the experiment according to European Convention for the Protection of Vertebrate Animals (Strasbourg, 18.03.1986), principles of Ukrainian law №3447-IV about the protection of animals from cruel treatment. Methods: morphometrical, mathematical, statistical.

Results
The available evidence suggests, that spleen weight reflects the functional activity of the organ and dynamic balance of immune system [6,13]. No significant changes were found in body weight of any of the TEPPT-treated rats` groups in comparison to the control group. In the 1 seria of the animals that received TEPPT in dose 1/10LD 50 the spleen's weight during the period of observation increased from 438.33±33.20 mg to 545±22.02 mg. However, the spleen weight due to 1/10LD 50 administration reduced on 18.3% (7 th day), on 13.71% (on the 15 th day), on 11.86% (on 30 th day) in comparison to the control group ( Table 1). All of the indexes were considered to be statistically significant (p<0.05). In the 2 seria of animals that received TEPPT in dose 1/100LD 50 the spleen's weight during the period of observation increased from 528.3±28.81mg to 561.66±22.27mg. Therefore, an impact of 1/100LD 50 was less noticeable when on 7 th day of experiment the spleen weight reduced on 2.17%, on 15 th day on 2.82% and on 30 th day -on 13.82% (Table2). Statistically significant change was observed only on 30 th day (p<0.05). Particular attention was drawn to the fact that the biggest alterations occurred on 7 th day with the 1/10LD 50 dose and on the 30 th day with the dose 1/100LD 50 . In the 1 seria abrupt weight reduction on 18.3% in early time observation (7 days) while in 2 seria gradual spleen weight reduction with the highest number on 30 th day (on 13.82%). Thus, the most noticeable changes are found on 7 th and 30 th day of the experiment. The gross appearance and size of the spleen are variable, depending on the species and the degree of distension, nonetheless, spleen weights can be important in its evaluation [1,7,14]. Different doses of the TEPPT had also an impact on linear dimensions of the spleen. In the 1 seria of animals that received TEPPT in dose 1/10LD 50 the spleens length increased from 32.57±1.21mm to 36.33±1.44mm which is less than control groups on 12.71% (p=0.0076), on 9.67% (p=0.0094), and on 8.75% (p=0.0467) on 7 th , 15 th , 30 th day respectively. The spleens breadth during period of observation increased from 7.10±0.12mm to 7.33±0.13 mm which is on 8.27% (p=0.0173), on 11.81% (p=0.0240), on 10.41% (p=0.0417) lower than the showings of intact animals on 7 th , 15 th and 30 th day. The height of the spleen during the experiment has changed from 3.19±0.15 mm to 3.98±0.19 mm which is lower than the control group indicators on 17.58% (p=0.0216) on 7 th day, on 5.84% (p=0.404) on 15 th day, on 3.4% (p=0.0652) on 30 th day (Fig.1). Generally, the changes in linear dimensions of the However, the alterations of spleen`s linear dimensions under the impact of TEPPT in dose 1/100LD 50 (2 seria) were slightly different. The spleens length increased from 37.22±0.53mm to 37.94±1.21 mm which is less than control groups on 0.11% (p=0.9581), on 1.83% (p=0.4170), and on 4.7% (p=0.1750) on 7 th , 15 th , 30 th day respectively. The spleens breadth during the period of observation increased from 7.46±0.32 mm to 7.81±0.41 mm which is on 1.72% (p=0.7306), on 2.19% (p=0.7872), and on 1.89% (p=0.8148) lower than the showings of intact animals on 7 th , 15 th and 30 th day. The height of the spleen during the experiment has changed from 3.98±0.26 mm to 3.95±0.34 mm where on 7 th day the height increased on 5% (p=0.6232) compared to control group, on 15 th day the indexes were lower on 3.3% (p=0.7646), on 30 th day lower on 1.76% (p=0.9440) (Fig.2). Generally, the changes in linear dimensions of the spleen under this dose of xenobiotic were characterized by statistically insignificant indexes decrease except the height alterations on 7 th day where the indicators increased on 5% (Table 2).

Conclusion
The received and analyzed data demonstrate the morphological changes of the spleen, specifically changes of the linear dimensions and weight of the spleen due to the influence of the different doses of the TEPPT.
Revealed changes suggest: 1. The spleen is very sensitive to the effects of xenobiotics, in this case, polyesters, in particular, TEPPT that is even reflected in its morphological features, like weight and linear dimensions. However, changes in the rats '  2. The spleen actively responds to the administration of above mentioned polyether. Its 1/10LD 50 and 1/100LD 50 induction causes a reduction in weight and linear dimensions of the spleen compared to intact animals, especially pronounced in the early stages of the surveillance, which may be related to its immunosuppressive effect due to its toxicity and сan be associated with immunological sensitivity.
3. However, the induction of 1/10LD 50 has more severe impact rather than 1/100LD 50 , that is apparently explained by the dose, and, hence during investigation was noticed that this dose has statistically significant impact almost on all indicators of weight and linear dimensions where the higher numbers of alterations are observed on 7 th day. On 30 th day the organomethric indexes lower down what can be caused by their normalization, which indicates the ability of spleen to adjust.
4. The impact of 1/100LD 50 characterized by slight statistically insignificant changes where the higher number of organometric indexes alterations were observed on 30 th day which may be approved by the ability of spleen to accumulate. Thus, alteration in spleen weight and linear dimensions are the reliable indicators in toxicity assessment. Weight and gross morphology are the first parameters studied in toxicity assessment and a response to injury is often expressed as a change in tissue size and weight parameters.

Perspectives of the further research
In the following publications we plan to highlight the features of the cells spleen changes on histological level under the influence of tryglycidyl ether of polyoxypropylene triol in different doses.