Influence of Boron Compounds on Chromium-induced Hemorheology Disorders in Rats

The authors of this study evaluated the protective influence of boric acid on chromiuminduced blood rheology disorders. The experiment was carried out on Wistar rats, which were divided into three groups: Group 1 the control group; Group 2 rats with simulated chromium-induced hemorheology disorders; Group 3 rats, in which boric acid was administered throughout 10 days on the background of chromium-induced hemorheology disorders. In rats with simulated chromiuminduced hemorheology disorders, one could observe decrease in deformability of red blood cells and in hematocrit level on the background of increased aggregation, peroxide hemolysis and osmotic fragility of red blood cells. In the third group, corrective application of boric acid inhibited development of blood rheology disorders, i.e., its protective action was revealed.


INTRODUCTION
Quantitative and qualitative changes in blood rheology occur under the impact of industrial chemicals and these changes reflect their chemical profile (Krivokhizhina, et al., 2006).Since red blood cells make 93% of formed elements, changes in their physico -chemica properties hinder implementation of their main function -transport of oxygen in microvasculature, which leads to the development of tissue hypoxia (lack of oxygen transfer or termination of its delivery, decrease in redox activity, development of structural and qualitative changes in cell membranes (Khetsuriani and Kipiani, 2002; Lukyanova, 2003)), increased risk of cardio -vascular diseases and relevant complications (Lowe, et al., 1997) as well as ischemic cerebrovascular diseases (Szapary, et al., 2004).Rheological properties of blood depend on many factors (Ormotsadze and Nadareishvili, 2002); they may change under the impact of various stress factors (Gyawali, et al., 2015) and chemicals (Zairova et al., 2006 Chromium is a self-existing microelement.Its valence Cr +3 or Cr +6 influences absorption.The oxidation degree and solubility of chromium compounds determine their toxicity.The impact of hexavalent chromium Cr +6 has a number of negative effects, including neurotoxicity, hepatotoxicity, cardiotoxicity, renal toxicity, genotoxicity, carcinogenicity, immunotoxicity as well the development of microcytic hypochromic anemia (Kawanish et al., 2002;O'Brien et al., 2003;Bazarbekova, 2002;Sudha et al., 2011;Stout et al., 2009).Once inside the cell, Cr +6 is restored to Cr +3 ; this is accompanied by the generation of reactive oxygen intermediates, which cause oxidation of macromolecules such as DNA and lipids (Aruldhas et al., 2005;Wise et al., 2008;Wang et al., 2011;Iztleuov, 2003;Iztleuov et al., 2011) and induce tissue damages in a number of organs, such as liver, pancreas, kidneys and the blood-vascular system (Stout et al., 2009;Solis-Heredia et al., 2000;Bagchi et al., 2002;Fatima et al., 2005).Different people are exposed to high concentrations of Cr +6 professionally, ecologically or internally (Mamyrbayev, 2012).
The damaging impact of Cr +6 -induced oxidative stress is caused mainly by a hydroxyl radical, which damages macromolecules, forms protein crosslinks promoting protein denaturation and aggregation.Besides, it causes formation of secondary radicals by reacting with low-molecular compounds (Iztleuov, 2004).Oxidative stress develops when the content of antioxidants is reduced (Tapiero et al., 2004).Antioxidants can protect cells from free radicals in the presence of metal-induced oxidative stress (Valko et al., 2005).Once inside the body, boric acid (boron compounds) enhances the prooxidant -antioxidant balance (Bolanos et al., 2004;Turkez, 2008) and increases activity of antioxidant enzymes, thereby neutralizing reactive oxygen intermediates, eliminating and preventing oxidative damage of cell membranes in macromolecules.However, protective action of boron compounds in the presence of chromium-induced hemorheology disorders.
The aim of this study was to evaluate the protective influence of boric acid on chromiuminduced blood rheology disorders.

MATERIALS AND METHOD
Experiments were performed on 24 male Wistar rats weighing 190 -220 g.The animals were kept in plastic cages in observance of a certain light regime (12- Ten days after acclimatization, the animals were randomly divided into three groups (eight rats in each group): the first group comprised intact rats (control), the second and the third group comprised animals with simulated chromium-induced hemorheology (diselementosis) caused by a single intraperitoneal injection of potassium dichromate K 2 Cr 2 O 7 , purchased from LLP "Chemistry and Technology" (Kazakhstan) at the rate of 14 mg / kg of body weight (0,5LD 50 ).In contrast to the second group, boric acid H 3 BO 3 purchased from OJSC "Farmak" (Ukraine) was administered orally after K 2 Cr 2 O 7 in rats from the third group -5.0 mg / kg of body weight during 10 days.Selection of doses, routes of administration and duration were substantiated in the previous studies (Iztleuov et al., 2011) and chosen according to relevant literature data (Moore, 1997;Pahl et al., 2005).
Experimental animals were under ether anesthesia.Blood was collected from the heart using siliconed needles.Rheological properties of red blood cells was assessed by erythrocyte deformability index (EDI), erythrocyte aggregation coefficient (EAC), by erythrocyte peroxide hemolysis (EPH), hematocrit (Ht) as well as by erythrocyte osmotic fragility (EOF).
Erythrocyte deformability index was determined using the method developed by Zakharova N.B., Tselik N.I., and Klyachkin M.L. (1989).This method implies preparing the 60% erythrocyte suspension and 0.02 µl (20 µl) of the suspension is applied onto a filter paper with pore diameter of 4±1 µl.After 60 seconds, the stain diameter (D 1 ) is measured.Next, after 60 seconds, 20 uL (0.02 µl) of a 60% suspension is applied again to the center of the obtained spot and the spot diameter (D 2 ) is measured again.Erythrocyte deformability index is calculated using the following formula: EDI=D 1 /D 2 where D 1 and D 2 -diameters of stains.
Erythrocyte aggregation coefficient (EAC) was determined using the method developed by V.A. Lapotnikov and L.M. Kharash (1982).This method implies the following: erythrocyte aggregates in a blood sample are fixed by formalin.The difference in weight of erythrocyte aggregates fixed by formalin and single cells causes changes in erythrocyte sedimentation rate (ESR) as compared to the control sample without formalin.In order to determine erythrocyte aggregation, the authors used 0.1M phosphate buffer with pH 7.4, 0.077 M EDTA (ethylenediaminetetraacetate) 4% clarified formalin.These reagents were used to prepare working solutions: solution No.1 -3 ml of 0.077 M EDTA, 5.0 µl of 4% clarified formalin, 12.0 µl of 0.1M phosphate buffer, pH -7,4.Solution No.2 -3,0 µl of 0.077 M EDTA, 17 µl of 0.1 M phosphate buffer, pH -7,4.Sequence of procedures: using siliconed needles, blood by 0.5 µl (500 µl) is poured in two centrifuge tubes containing 2 µl of solution No. 1 and No. 2, respectively.After blood was mixed with both solutions, ESR was determined in each sample using the incubator at 37 0 C. Determination of peroxide hemolysis of erythrocyte membranes was performed according to the method developed by A.A. Pokrovskiy and A.A. Abrarov (1964), which is a modified macromethod developed by Gyorgy P., Cogan G., Rose C.S (1952).
Modification implied spectrophotometric (l = 543 nm) determination of erythrocyte content, hemolyzed in standard conditions under the impact of hydrogen peroxide (H 2 O 2 ).The use of ultramicroanalysis was a specific feature of this modified method, which significantly reduced the volume of blood (20 µl, 0,02 µl) required for this experiment.
The level of hematocrit was measured by using the automated hematological analyzer CELL-DYN Ruby (Abbott, USA).
Erythrocyte osmotic fragility was determined by the Daisy method (Todorov, 1968).
C e l l h e m o l y s i s w a s c a l c u l a t e d percentagewise as related to 100% hemolysis induced by 0,1% NaCl solution.

RESULTS AND DISCUSSION
Analysis of the obtained data shows blood rheology disorders in the presence of chromiuminduced diselementosis.This is displayed by significant reduction of erythrocyte deformability index and hematocrit level against the backdrop of increase in erythrocyte aggregation coefficient, erythrocyte peroxide hemolysis (Table 1) and in erythrocyte osmotic fragility (EOF).EOF is significantly increased (p <0,05) in the presence of 0.4; 0.45; 0.5; 0,55 and 0,85% NaCl (Table 2).Intake of boric acid on the background of chromium-induced hemorheology disorders leads to the improvement of blood rheological properties (pd" 0,05).Chromium causes a wide range of toxicological effects and biochemical dysfunctions that imply serious health risks (Mamyrbayev, 2012;Bielicka et al., 2005).Some studies show that Cr +6 and its compounds do not directly generate free radicals; however, reduction of Cr +6 to Cr+3, as well as the effect of Haber -Weiss and Fenton mechanisms (Lloid et al., 1998), imply the emergence of different radicals that cause damages characteristic of oxidative stress (Pritchard et al., 2000; Barrera et al., 2003).Consequently, one of the possible basic approaches used for prevention (correction) of K 2 Cr 2 O 7 -induced damage implies using agents (elements) with powerful antioxidant properties.Recent studies have shown that boron and its compounds displayed significant protective effects against damages induced by metals, such as aluminum and arsenic (Turkez et al., 2011;Kucukkurt et al., 2015).Erythrocytes are sensitive to the impact of heavy metals, including chromium compounds (Ryspekova et al., 2013); they present a convenient model to assess cytotoxicity of chemicals.Hemolysis and its value may serve as a stability test for cell membranes of erythrocytes (Pagano and Faggio, 2015).The latter are characterized by certain rheological properties (deformation and flow), due to which they have a lenticular shape, high flexibility, elasticity and deformability.Deterioration of their rheological properties trigger the occurrence of hemorheology disorders in the presence of various diseases (Muraviov and Tikhomirova, 2009;Sharapova, 2012;Baev et al., 2013), including the cardio -vascular (Vaya et al., 2013) and cerebrovascular diseases (Szapary et al., 2004); this may complicate the course of these diseases (Plotnikov et al., 2005).This experiment was conducted with a view to show the impact of potassium dichromate on blood rheology and to explore the impact of boric acid on the morphofunctional disturbances of erythrocyte membranes, induced by hexavalent chromium (K 2 Cr 2 O 7 ).In this study, EDI decrease and EAC increase, peroxide hemolysis and erythrocyte osmotic fragility reflects deterioration of blood rheological properties.Intake of boric acid in the presence of chromiuminduced diselementosis improves blood rheological properties (EDI, Ht, EAC, EPG and EOF).Perhaps, changes in blood rheological properties is associated with antioxidant activity of boron compounds (indirect action).Thus, a number of scientists found that boron compounds (boric acid, borax, etc.) could be used for correcting metal-induced (arsenic, bismuth, cadmium, mercury, lead) oxidative stress effects (Turkez et al., 2007;Kucukkurt et al., 2015;Pawa and Ali, 2006).Indeed, oxidative stress occurs at the decreased level of antioxidants (Tapiero et al., 2004).Boric acid intake contributes to the preservation of pro-oxidant -antioxidant balance (Bolanos et al., 2004;Turkez, 2008;Inse et al., 2010).
Possibly, there is another mechanismthe direct and immediate impact, which implies hematopoiesis disorders under the influence of boron compounds, which, in turn, influence physical and chemical blood properties (Oto et al., 2015).Boron in low doses (40 and 80 mg / l) can stimulate erythropoiesis and hemoglobin synthesis (Feng et al., 2014) having protective effect in the presence of metal-induced hemorheology disorders (Turkez et al., 2012).Boron in high doses (160 -640 mg / l) can inhibit hematopoiesis and hemoglobin synthesis; i.e., it can be toxic (Feng et al., 2014).
Thus, the authors of the present study first found that boric acid in the presence of chromiuminduced hemorheology disorders prevents (hinders) blood rheology disorders in rats (showing protective action).Apparently, taken in certain doses, boric acid is a promising remedy in the case of chromiuminduced hemorheology disorders and suggests new dimensions of subsequent studies related to the biological effects of boron compounds.In the presence of chromium-induced disorders (diselementosis) in rats, one can observe changes in blood rheological properties -erythrocyte deformability index and the level of hematocrit are reduced on the background of increase in EAC, peroxide hemolysis and erythrocyte osmotic fragility.Oral administration of boric acid on the background of chromium-induced changes in the blood rheological properties inhibits the development of blood cell disorders (protective action).

CONCLUSION
Implications and recommendations for future studies are as follows: Some studies show that Cr +6 and its compounds do not directly generate free radicals; however, reduction of Cr +6 to Cr +3 , as well as the effect of Haber -Weiss and Fenton mechanisms (Lloid et al., 1998), imply the emergence of different radicals that cause damages characteristic of oxidative stress (Pritchard et al., 2000; Barrera et al., 2003).Consequently, one of the possible basic approaches used for prevention (correction) of K 2 Cr 2 O 7 -induced damage implies using agents (elements) with powerful antioxidant properties.Recent studies have shown that boron and its compounds displayed significant protective effects against damages induced by metals, such as aluminum and arsenic (Turkez et al., 2011;Kucukkurt et al., 2015).Erythrocytes are sensitive to the impact of heavy metals, including chromium compounds (Ryspekova et al., 2013); they present a convenient model to assess cytotoxicity of chemicals.Hemolysis and its value may serve as a stability test for cell membranes of erythrocytes (Pagano and Faggio, 2015).The latter are characterized by certain rheological properties (deformation and flow), due to which they have a biconcave shape, high flexibility, elasticity and deformability.Deterioration of their rheological properties trigger the occurrence of hemorheology disorders in the presence of various diseases (Muraviov and Tikhomirova, 2009;Sharapova, 2012;Baev et al., 2013), including the cardio -vascular (Vaya et al., 2013) and cerebrovascular diseases (Szapary et al., 2004); this may complicate the course of these diseases (Plotnikov et al., 2005).This experiment was conducted with a view to show the impact of potassium dichromate on blood rheology and to explore the impact of boric acid on the morphofunctional disturbances of erythrocyte membranes, induced by hexavalent chromium (K 2 Cr 2 O 7 ).In this study, EDI decrease and EAC increase, peroxide hemolysis and erythrocyte osmotic fragility reflects deterioration of blood rheological properties.Intake of boric acid in the presence of chromiuminduced diselementosis improves blood rheological properties (EDI, Ht, EAC, EPG and EOF).