The search for effective methods to correct human body's condition after having experienced the impact of chlorine-containing ecotoxicants still is of great topicality. Employees at chemical plants of organochlorine synthesis are among those who experience the negative impact most often [11]. It is known that activation of the processes of free-radical lipid peroxidation is one of the main mechanisms of the damaging impact of chlorine-containing ecotoxicants [5,7,8]. A number of authors have revealed the negative impact of dichloroethane (DCE) intoxication on the condition of humoral, cellular component of the immune system, antibody-dependent cellular cytotoxicity, and also the ability to cause apoptosis of immune system cells [3, 4, 13]. The DCE ability to damage the immune system contributes significantly to the development of infectious complications and the death rate associated with such intoxication [3, 4].

It is well-known that polymorphonuclear leukocytes (PMNL) are not just first line of a human body's defence from damaging environmental factors, which provides  anti-infectious and antitumoral resistance, are responsible for immunogenesis processes, take part in neuroendocrinal regulation of adaptive responses, but also react sensitively to the slightest changes in physiological state of the body. This fact allows us to use phagocytosis assay as an integral indicator of the immune system condition [2, 6]. On  the other hand, the oxidative stress developing under the impact of chlorine-containing ecotoxicants can significantly damage their antimicrobial activity, the molecular basis of which are the products of reactive oxygen intermediates [2, 9].

Objectives

The goal of research is to study the impact of a long-term dichloroethane (DCE) injection on some peripheral blood values, the intensity of oxygen-dependent metabolism, PMNL microbicidal activity, myeloperoxidase activity in them.

Study areas and Methods

The research was done on 30 white non-inbred mature male rats with the mass of 180-220g, which were kept in standard vivarium conditions. The animals were divided into 2 groups: 1 group was a control group (intact animals), 2 group - animals injected with DCE. DCE in olive oil was injected intragastrically every day through a special catheter  for 60 days with a total dose of 50 mg/kg (0,1 LD₅₀) [10]. The control group was only getting olive oil.

The animals were kept in standard vivarium conditions with the natural light mode, on a standard diet for laboratory animals (all-Union State Standard for Russia 50258-92), adhering the International recommendations of the European convention on protecting vertebrate animals used in experimental research and also following the rules of good laboratory practice in preclinical research in the Russian Federation (all-Union State Standard З 51000.3-96 and 51000.4-96) and the Order of Healthcare Ministry of the Russian Federation №267 of 19.06.2003 «On approval of the rules for good laboratory practice» (GLP).

We estimated the number of leukocytes, neutrophils, and lymphocytes in peripheral blood, the intensity of oxygen-dependent metabolism, the microbicidal ability of PMNL (in respect of fungi Candida albicans), the activity of myeloperoxidase in them [12]. The results were registered on the next day after having stopped injecting of DCE.

The statistical processing was performed using the variation statistics methods [1] and software package Statistica 8.0. The normality of data distribution was checked by means of  the Shapiro-Wilk criterion. The importance of differences estimation was done by computing the median and interquartile interval. The  analysis of variance was performed using Kruskal-Wallis H-test, for multiple comparisons the Dan Q-criterion was used. The critical level of importance р for statistical criteria was set to 0,05. The data in the paper are presented as a percentage of the control group.

Results

The DCE intoxication having been finished, leukopenia was observed among the animals (with the number of leukocytes decreasing to 71,93% in comparison with the control group). The leukopenia was attributable mostly to the decrease in the number of neutrophils (down to 58,04%) with the drop in the number of lymphocytes as well (down to 72,60%) (fig. 1A, B, C).

 A

 B

 C

Fig. 1. Dichloroethane impact on the content of leukocytes (A), neutrophils  (B) and lymphocytes in experimental animals

When studying microbicidal ability of PMNL in conditions of functioning of oxygen-dependent killing mechanisms, we observed the increasing number of colony-formating units (up to 141,91%, p=0,0009). This goes to prove the inhibition of oxidative mechanisms of PMNL fungicidity. Subsequently, the PMNL inactivation index in DcH group amounted to 74,55%.

We observed the decrease in formation of reactive oxygen intermediates (induced NBT - Nitroblue Tetrazolium test) among the animals intoxicated with DCE (the percentage of active cells reduced to 75,0%, p=0,0153, the activation index - to 72,22%, p=0,0312). Alongside with that the myeloperoxidase activity in the group of animals was not any different from that in the group of the intact rats: the percentage of active cells was 82,35%, p=0,3611, the average cytochemical coefficient was 81,91%, p=0,0821). This proves the primary inhibition of peroxidase-independent oxidative factors of microbicidal activity by DCE.

In conditions of blocking the oxygen-dependent killing mechanisms with sodium azide it was also observed that fungicidal ability of PMNL was inhibited (the inactivation index amounted to 66,49%). Subsequently, the increase in the number of colony-formating units (up to 158,21%, p=0,0003) was observed, which characterized the inhibition of non-oxidative microbicidal mechanisms of PMNL.

Conclusions

As a result, the chronic dichloroethane intoxication of experimental animals leads to leukopenia, the forming of which is determined mostly by the reduction in the number of neutrophils. It is attended by inhibition of both oxidative and non-oxidative microbicidal mechanisms. In the structure of oxygen-dependent killing mostly peroxidase-independent microbicidal factors of neutrophils suffer.

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