Effect of Boric Acid on Oxidative Damage in Immunocompetent Organs under Conditions of Potassium Bichromate and Gamma Radiation Exposure

The immune system supports antigenic homeostasis in the body and regulates the processes of proliferation and differentiation of cellular components in hemo-and immunopoiesis. Chromium compounds and ionizing radiation lead to the formation of highly reactive free radicals. Sublethal dose of gamma-irradiation is characterized by a 56% decrease in thymus cellularity and a 22% decrease in lymph nodes against the background of a 44% increase in the number of lymphoid cells in the spleen. Under the combined effect of hexavalent chromium and gamma-radiation, a decrease in the number of lymphoid cells is observed. In thymus the cellularity decreases by 70%, in spleen - by 40%, in lymph nodes - by 42% in comparison with control data. Under the influence of boric acid the number of lymphoid cells in thymus significantly increases by 47%, in lymph nodes - by 14% (p<0.05) compared to the data of irradiated animals. Boric acid administration weakens the development of oxidative stress, lipid peroxidation decreases, and the activity of antioxidant defense enzymes in immunocompetent cells is increased.

Lymphoid cells are very susceptible to radiosensitization [1][2][3] .The immunodeficiency syndrome appears as damage to lymphopoiesis [4][5][6][7] and impaired migration of lymphocytes, loss of their ability to recirculate to a node or other lymphoid unit 8,9 .This is demonstrated by inhibition of cellular factors, depression of the humoral bundle and functional deficiency of the phagocytic component of immunity 10 , caused by a complicated set of structural and metabolic disorders.Consequently, prevention, correction and treatment of disorders developing in the body, including in the immune system, remain important.
Chromium can enter the human body through the skin, mucous membranes, lungs, as well as together with food and water (gastrointestinal tract), but some of its compounds are assimilated easier and faster (this is due to their solubility and valence of the metal in them).Thus, Cr (VI) is assimilated by the body better and faster than Cr (III), which is due to its high solubility at physiological pH values 14 .
In particular, we consider in detail the ways of this trace element's entry into the blood.The entry of the metal into the blood is associated with transfer proteins located in the epithelium of the small intestine and carrying out active (energyconsuming) transport of the element.If chromium particles enter the body through the lungs, then when exhaling with a stream of air they get into the oral cavity, from there into the gastrointestinal tract, and the process occurs as described above.
Once in the blood, a part of Cr (VI) binds to erythrocytes, as well as leukocytes and platelets and is reduced to Cr (III), which in turn forms a strong complex with hemoglobin (located on the surface of blood cells) and thus moves around the body.The remaining (unbound) portion of hexavalent chromium is excreted from the body through the kidneys 15 .
Cr (III) upon entering the blood binds to the protein transferrin (which is found in the free state in the blood plasma).If Cr (III) enters the body in large quantities, it can bind to the protein albumin, globulin and amino acids in the blood plasma.Thus, the transportation of this element in the blood is carried out.It is important to know that the protein transferrin transports iron in the blood, and the trace element chromium competes with it for binding sites.
Moving through the body in a bound state, chromium accumulates in organs such as the lungs, liver, pancreas, and bone marrow.Chromium has a high affinity for these tissues.
When CrVI enters a cell, it is reduced to metastable pentavalent chromium (Cr(V)) and then to trivalent chromium (Cr(III)), which is accompanied by the generation of reactive oxygen species (ROS) and causes damage to cellular structures, including immunocompetent organs 16 .At the same time, oxidative stress plays the main role in the realization of the damaging effect arising from chromium intoxication.As a result of which, there is a depletion of the immune system with a transition to immunodeficiency [17][18][19][20][21] .
In this regard, the search for protective agents is necessary to counteract oxidative damage and increase the capacity of the defense response by activating the immune system.
B administration increases the amount of antioxidants in the body (glutathione and its derivatives), thereby limiting oxidative damage to the cell 35 .Boric acid displayed hepatoprotective, antioxidant, anti-inflammatory and immunomodulatory activities in animals [36][37] .Boron supplementation is believed to have immunostimulatory effects, including on T-cell proliferation and increased levels of natural killer cells 38,39 .A number of studies have shown that B reduced levels of inflammatory biomarkers and modulated cellular response in inflammatory processes [40][41][42][43] , as well as playing a key role as a regulator of immune and inflammatory responses and hematopoiesis [44][45][46][47] .According to a number of scientists [48][49][50][51][52] , B compounds are found to have a protective effect by modulating oxidative stress indicators.
The effect of B on immune system disorders, on the state of lipid peroxidation and antioxidant system in immunocompetent organs under the influence of Cr(VI) and gamma irradiation has not been studied.Therefore, it was the subject of the current experimental study.

Materials and Methods
The experiment was conducted on 70 male Wistar rats weighing 170-190 g.The rats were kept in the vivarium under standard conditions.Lighting was natural, room temperature -in accordance with the approved standards.Nutrition -free availability of food and water.
Animals The animals were randomly divided into 5 groups (14 rats in each group) 10 days after they had been acclimatized.The first group was the control group.Animals of the 3rd and 5th groups at the beginning of the experiment received potassium bichromate with drinking water daily for 4 weeks at a dose of 520 mg/L.Animals of the 4th and 5th groups received boric acid in a dose of 400 mg/l.
Four weeks after the start of the experiment, all rats were subjected to gamma irradiation at a sublethal dose (6 Gy, N = 0.54 Gy/ min; Teragam radiotherapy unit (manufactured by the Czech Republic, in 2008).The distribution distance from the source to the skin was 70 cm.
Immunological parameters.To study the cells of lymphoid organs of the immune system, suspensions were prepared from thymus, spleen and lymph nodes of the small intestine.The total number of lymphoid cells and organ mass were determined.
Lipid peroxidation and antioxidant status.The content of malondialdehyde (MDA) in tissues of thymus, spleen and intestinal lymph nodes was determined spectrophotometrically according to the Draper method modified by Hadley 53 .Superoxide dismutase (SOD) activity was estimated according to the method of Beauchamp and Fridovich 54 .Catalase (CAT) activity was determined according to the method of Korolyuk 55 .Glutathione peroxidase (GPO) activity was determined according to the method of Flohe and Gunzlcer 56 .
Statistical analysis.Statistical data processing was performed using Statistica 10 (StatSoft, Inc., USA) program package.

results and discussions
The study of the effect of boric acid on the cellular composition of lymphoid organs showed the following.At sublethal dose of gammairradiation (Table 1) there is a decrease of thymus cells by 56%, lymph nodes by 22%, and at the same time the number of lymphoid cells in spleen increases by 44%.
Under the combined effect of CrVI and gamma-radiation there is a decrease in the number of lymphoid cells in all studied immunocompetent organs.In thymus the complex of cell composition decreases by 70%, in spleen -by 40%, in lymph nodes -by 42% in comparison with control data.
Under the influence of boric acid in the irradiated organism the number of lymphoid cells significantly increases in thymus by 47%, in lymph nodes -by 14% (p<0,05) compared to the data of animals exposed only to irradiation.The number of cells in the spleen is at the level of the control group data.Prophylactic administration of boric acid under combined exposure to CRVI and ã-irradiation causes an increase in the composition of thymus cells by 75%, spleen by 87%, lymph nodes by 22% compared to the data of positive control animals (CRVI and ã-irradiation).
The effect of CrVI and ã-irradiation on lipid peroxidation and antioxidant status in immunocompetent organs was evaluated, as well as the protective effect of boric acid under these exposure conditions.
Under the action of a sublethal dose of gamma-irradiation there was observed an increase of MDA in lymph nodes by 69%, in spleen and thymus tissues by 40 and 57%, respectively, compared to the control.SOD activity was decreased in lymph nodes, spleen and thymus tissues by 35, 27 and 22%, respectively.The activity of catalase, respectively, by 39, 47 and 53%, the activity of HPO -by 17, 37 and 20%, respectively (Table 2) compared to the data of animals of the control group.
Prophylactic administration of boric acid under conditions of isolated exposure to gammairradiation was accompanied by reduction of MDA content in lymph nodes by 26%, in spleen and thymus tissues -by 19 and 25%.Accordingly, the activity of SOD, GPO and CAT in lymph nodes increased by 41, 31 and 43% respectively, in spleen tissues -by 19, 64 and 200% respectively, in thymus tissues -by 21, 51 and 100% compared to the data of positive control (gamma-irradiation).
The combined effect of CrVI and gammairradiation resulted in the increase of MDA level in lymph nodes, spleen and thymus tissues by 169 and 122, 157%, respectively.
Antioxidant system enzymes -SOD activity in lymph nodes, in spleen and thymus tissues -by 46, 36 and 40% respectively, HPO activity -by 27, 45 and 32% respectively, CAT -by 55, 64 and 62% compared to control.Under the influence of CRVI and gamma irradiation, LPO and AOS disorders were more pronounced (p<0.05).SOD activity in spleen tissues (p>0.05),HPO activity in lymph nodes (p>0.05), in spleen and thymus tissues (p>0.05) were the exceptions (Table 2) compared to the data of gamma-irradiated animals.
In response to extreme radiation exposure, various pathological processes develop, affecting the immune system, which is very sensitive to the action of ionizing radiation 57 and heavy metals 58,59 .Ionizing radiation and chromium cause destruction of lymphoid tissue 60 .
Considering the diverse functions of B compounds as regulators of immune, inflammatory reactions and hematopoiesis 61 , we hypothesized that the positive effect of boric acid may be useful for the prevention of immune system disorders.Especially in the case of oxidative damage of immunocompetent organs under the combined effect of CrVI and gamma radiation.
Another mechanism of action of boric acid may be the antioxidant potential of boron compounds.Boron may counteract the inhibitory effect of oxidative stress on lymphocyte proliferation.In our previous studies [62][63][64] , it was found that boric acid at a low dose (400 mg/L), sodium tetraborate at a dose of 22.5 mg/kg body weight when co-administered with potassium bichromate (700 mg/L) exhibited antioxidant effects and reduced damage to immunocompetent organs, providing an immunoprotective effect.
Application of boric acid in a dose of 400 mg/l daily for 28 days reduces the amount of MDA in lymph nodes, thymus and spleen tissues by 26, 25 and 19%, respectively, as well as by 37, 28 and 35% compared to the data of animals of the corresponding groups of positive control.The activity of antioxidant defense enzymes SOD, GPO and CAT in the studied immunocompetent organs, respectively, significantly (p<0.05)increased both at isolated and combined exposure compared to the data of positive control animals (irradiation, CRVI + irradiation).Under the same experimental conditions, boric acid causes an increase in cellularity of lymph nodes, in spleen and thymus tissues.Apparently, B compounds can promote lymphocyte multiplication by increasing the antioxidant potential of cells.

conclusion
The combined and isolated effect of Cr and gamma-irradiation leads to activation of freeradical oxidation with a decrease in the activity of the antioxidant system in lymphoid organs.This contributes to changes in immunologic and biochemical indices, which leads to depletion of cells in the organs of immunogenesis and development of immunologic suppression.Boric acid administration attenuates the development of oxidative stress, lipid peroxidation decreases, and the activity of antioxidant defense enzymes in immunocompetent cells increases.Boric acid modulates the entire immune response, playing a key role in the regulation of immune and inflammatory reactions.

acknowledgMents
We express our deep gratitude to the staff of the Medical University, the staff of the laboratory and the vivarium.

Conflict of interest
There is no conflict of interest.

Funding Source
The experiment was carried out at the expense of the researchers' own funds.references were kept in standard conditions in the vivarium of the Scientific and Practical Center of the Non-Commercial Joint Stock Company "West Kazakhstan Medical University named after Marat Ospanov" (Aktobe, Republic of Kazakhstan) under natural illumination and maximum normalized temperature and food regime with free access to food and water.The experiment was conducted in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes (Strasbourg, 1986).The experimental program was discussed and approved by the university ethics committee of October 20, 2022, protocol No. 8 (local bioethics committee).

table 1 .
Effects of boric acid on the lymphoid organs (cellularity) under conditions of isolated and combined exposure to potassium bichromate and gamma radiation

table 2 .
The effect of boric acid on the level of malondialdehyde and the activity of enzymes of the antioxidant system in immunocompetent organs with isolated and combined effects of bichromate and gamma irradiation