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Research Article - Journal of Veterinary Medicine and Allied Science (2017) Volume 1, Issue 1

Individual approach to treatment and cobalt - a new look at an old problem.

Natalia Kurhaluk1*, Halyna Tkachenko1, Piotr Kaminski2, Fedir Muzyka3

1Department of Zoology and Animal Physiology, Institute of Biology and Environment Protection, Pomeranian University, Poland

2Department of Ecology and Environment Protection, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, Poland

4Research and Development Department, Padua, Italy

*Corresponding Author:
Natalia Kurhaluk
Pomeranian University
E-mail: [email protected]

Accepted Date: February 06, 2017

Citation: Kurhaluk,Tkachenko,Kaminski and Muzyka. Individual approach to treatment and cobalt - a new look at an old problem.. J Vet Med Allied Sci 2017;1(1):1-22.

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An individual approach in the cobalt-induced stress dependently hypoxic states properties was analyzed. Effects of cobalt (Co) chloride treatment (30 mg kg bw, 3 h) on metals content (Na, K, Ca, Fe, Zn, Mn, Mg, Co, Cd and Pb) in femur, plasma, and liver Wistar rats with individual resistance to hypoxia (low resistance - LR and high resistance - HR) were studied. Effects of cobalt chloride were mediated by individual response to hypoxia factor and reduction of iron, lead and cobalt in the long bones with subsequent accumulation in liver tissue. Regression analysis showed a high dependence of the individual organism’s reactivity from a low content of free Co. Possibly, a parameter of low content of Co in the blood can be used as a test system for assessing individual resistance to hypoxia and subsequent pharmacological correction. Effects of Co in our investigation cause the cadmium accumulation in bones and liver. Effects Co show substantial reduction of erythrocyte membranes resistance to the action of hemolytic agents (osmotic, peroxide and acid) with retaining a high level of total antioxidant capacity in erythrocytes and plasma, but not in liver. Individual constitutional resistance to low oxygen tension may be an important criterion for an individual approach in pharmacotherapy


Cobalt, bones, heavy metals, antioxidant defense, different resistance to hypoxia, oxidative stress.


Oxygen is needed for the implementation of oxygen-dependent processes in the mitochondria by production of cellular energy through ATP synthesis. From the other hand, reactive oxygen species (ROS) generated as byproducts of energy generation and have been implicated in the genesis of many diseases [1-3] and heavy metals impact. Therefore, eukaryotic cells have evolved elaborated oxygen-sensing mechanisms decreases in oxygen levels defined as hypoxia [4]. A decrease in ROS production, such as during hypoxia or anoxia, may protect cells from death or injury caused by different free radicals. Conditions that cause hypoxia or different levels of tissue hypoxia are very common in the human and animal population. High level of ROS and the disruption of the antioxidant system in the tissues are among the leading causes of morbidity, disability, and mortality [5]. The molecular pathophysiology of hypoxia states and its potential effects on organism’s health are important issues at the forefront of biomedical research [6].

The states of moderate hypoxia or hypoxia/reoxygenation alternation have a great importance in the formation of body adaptation mechanisms. Cellular mechanisms of adaptation to hypoxia are regulating the expression of more than one hundred genes involved in various biological processes [7]. It has been shown that the adaptation to hypoxia leads to a set of hematological shifts and an increase in blood oxygen capacity, as well as activates a multifactorial cascade of the antioxidant defense systems, mitochondria functioning, improves muscle performance, glucose transport, utilization of lipids etc. [8-10].

Adaptation to periodic hypoxia has effectively been used in different fields of clinical and preventive medicine and sports physiology [11]. There has been a renewed interest in the role of cellular responses to hypoxia specific metabolic stimulus which undoubtedly belongs to, as shown by recent studies, cobalt chloride. Mitochondria and oxygen-dependent processes are a key target of cobalt toxicity [12]. Cobalt interferes with cellular respiration leading to oxygen deprivation [13]. Mitochondrial dysfunction can lead to functional and metabolic pathology due to dose-dependent ATP depletion and may involve of a number of mechanisms. However, using cobalt chloride as a well-established hypoxia mimetic factor and inducer of hypoxia-like responses [14,15] was connected with doping tests in sports. Cobalt chloride administration is an alternative and dangerous blood doping technique, which is virtually undetectable by anti-doping testing [16,17].

Main effectors of oxygen homeostasis are Hypoxia-Inducible Factors (HIF) that enhancement of oxygen delivery (angiogenesis) and cellular energy metabolism [18-20]. HIF-α is constitutively expressed in cells but is post inactivated under normoxic conditions. Exposing cells to hypoxic conditions are due to HIF-stabilization with next gene transcription and association with hypoxia response elements [21]. Co causes gene modulation at the HIF pathway to stimulate the erythropoietin (EPO) transcription or its recombinant form, and increase its levels in blood [17,22]. Erythropoietin using for controlling erythropoiesis [23] in bone marrow, therefore similar stimulating agents often was used in athletes [16,24]. Although these doping may possibly have some physiological effects on tissues, there are significant risks associated with the illicit use of these substances in athletes [25]. Often in literature provides data on the high cobalt content in racehorses [15]. In the some countries there have been reports of unexplained deaths in horses that were found to have elevated blood levels of cobalt chloride [15].

Recently cobalt salts have medical applications for the treatment of anaemia as well-known haematopoietic system agent; it is an essential micronutrient that is important for the formation of the vitamin B12 complex [26]. Co acts as a coenzyme to catalyse various metabolic reactions [27]. Co realizes effects into three classes of enzymes: isomerases, methyl transferases and reductive dehalogenases. These enzymes participate in reactions essential to DNA synthesis, fatty acid synthesis and energy production [28].

But cobalt in high doses can be highly toxic [29]. It exerts neurotoxic effects, cardiomyopathy and ischaemic heart disease, a variety of toxic effects on the nervous system as well as the respiratory system, skin, heart and thyroid [30]. High doses of Co in patients exposed to abnormal levels from damaged hip prostheses induce some negative effects because promote secretion of cytokines from osteoblasts, which leads to inflammation [31-33]. High serum Co has been associated with optic atrophy and retinal dysfunction as possible complications of cobaltism [34].

It has been showed that the bone is a dynamic organ with wellregulated turnover mediated by bone resorption by osteoclasts and bone formation by osteoblasts [35]. For this reason, the study of the pattern of systemic responses of the bones and tissues has an important prognostic value for assessing the functional reserves of the human and animal body, monitoring the process of adaptation of athletes and horses during intense trainings, prediction of the working capacity under extreme living conditions, and prediction of the incidence of risk [36]. However, the pattern of heavy-metal distribution and levels of heavy metals in various tissues of rodents are similar to those found in humans. Therefore, in research rodents frequently serve as mammalian surrogates for humans [37].

ROS generated in the bones by extra- or intra-osteoclast have been shown to act as signaling molecules that expedite the differentiation of osteoclasts, and enhance bone resorption [38,39]. Therefore, deregulation of the redox balance by excessive ROS levels under different toxic agents can accelerate bone resorption and lead to bone fragility [40,41]. On the other hand, ROS play a critical role in the pathogenesis of ageing and osteoporosis. In the old mouse, loss of bone mass and decreases in bone formation and osteoblast function are associated with an increase in ROS levels simultaneously with decreasing of antioxidant defense system in bone. Investigation by [41] study shows that progressive, age-related bone loss correlates with increased oxidative stress in osteoblasts or osteoclasts. Key role in these processes is associated with dihydrogene peroxide in bones [42-44].

It has been investigated that the effects of adaptation to hypoxia depend in many respects on individual sensitivity to it, which, provides an individual reaction of mitochondria functioning, biotransformation of xenobiotics, the sensitivity of target cells to nitric oxide (NO), and formation of individual cell resistance to oxygen deficiency [6,7]. Some known mechanisms of question of sensitivity/resistance to hypoxia is determined by the phenotypic and adaptive-acquired features of the body manifesting themselves at the level of systemic, local, tissue, and molecular-cellular properties [11]. Individual constitutional resistance to low oxygen tension may be an important criterion for an individual approach in pharmacotherapy diseases, sport and early prevention diseases complications [6].

Different toxic and pharmacological agent influences may impact opposite changes in metabolism pathways (oxidative stress first) in tissues of animals with genetically determined differences in the sensitivity to hypoxia. Genetic and phenotypic mechanisms providing the resistance of organs and tissues to hypoxia was shown [9,10]. Individual resistance to hypoxia provides an individual reaction of differences in the parameters of hepatocytes functioning [8], mitochondrial transport, biotransformation of xenobiotics, drug-metabolizing system and hepatic cytochrome P450 activities, properties of mitochondrial enzymes and energy metabolism [8], state of mitochondrial respiration and calcium capacity in liver [9].

The rats highly resistant (HR) and low resistant (LR) to hypoxia differ in the intensity of lipid peroxidation (LPO) and activity of the antioxidant system [9]. In our earlier investigation, we observed that rats with low and high resistance to hypoxia had a different level of capacity. Animals with a high resistance, endured the physical training better than rats with a low resistance, which represented the explored index of dynamic endurance. L-arginine as precursor NO and modulator at hypoxic states [10] increased dynamic endurance to the physical training of rats with LR to the level of HR in the control. This data suggest individual resistance to hypoxia/ stress may be a serious criterion for individual approach in pharmacotherapy of diseases and metabolic ways corrections.

The aim of this study was to evaluate the effect of cobalt impact on elucidate physiological and heavy-metal contents in long bones, plasma and liver, the resistance of erythrocytes to haemolytic agents and oxidative stress tissues’ ability in animals with different predisposition to hypoxia factor.

Materials and Methods

Animals and experimental design

The experiments were conducted with the Guidelines of the European Union Council and the current laws in Poland, according to the Ethical Commission. Male white Wistar rats (180-220 g) were used in the study. The rats were housed at a constant temperature of 20 ± 2oC. The animals had free access to feed and water throughout the experiments. Previously, the animals were divided into two groups: rats with low resistance and high resistance to hypoxia. Resistance of rats to hypoxia was evaluated as survival time (min) in the altitude chamber 11,000 m above sea level. Survival time was measured after achieving the altitude. Cessation of breathing served as the criterion for resistance to hypoxia. Animas were used in experiments after two weeks from this dividing. Animals with middle resistance to hypoxia weren’t used in this study. To eliminate diurnal rhythm changes, all examinations started at 10 and ended at 12 am.

Experimental groups

The rats were randomly assigned into four groups (divided into two subgroups) : untreated control group (I) consisting eight rats with low resistance and rats with high resistance to hypoxia; Co group (II) - rats with low resistance (n = 8) and high resistance to hypoxia (n = 8) receiving 30 mg of cobalt chloride/kg b.w. for 3 hours. Before the experiment a group of control animals was injected 1 ml of saline.

Tissue isolation

The liver, brain, kidney, lung, heart were removed from rats after their decapitation. One rat was used for each homogenate preparation. Briefly, the excised liver were weighed, washed in ice-cold buffer, and minced. The minced tissues were rinsed with cold isolation buffer to remove blood and homogenised in a glass Potter-Elvehjem homogenising vessel with a motordriven Teflon pestle on ice. The isolation buffer contained 120 mM KCl, 2 mM K2CO3, 10 mM HEPES, and 1 mM EDTA; pH of 7.2 was adjusted with KOH.


We collected 2,5 mL blood samples in tubes with EDTA. After centrifugation, plasma samples were frozen at -20°C and stored until analysis. For isolation of erythrocytes, blood samples were centrifuged at 3,000g for 10 min. The plasma was removed; the erythrocytes were washed three times with five volumes of saline solution and centrifuged at 3,000g for 10 min. Plasma was used for the determination of total antioxidant capacity.

Resistance of erythrocytes to haemolytic reagents

Acid resistance of erythrocytes. The acid resistance of erythrocytes was measured spectrophotometrically following the method with 0.1 N HCl [45]. The method is based on the measuring of the dynamics of erythrocytes disintegration under haemolytic reagent action. Disintegration of erythrocytes (%) for every period of time was expressed in curve. The time of haemolytic reagent action serves as the measure of erythrocytes resistance. The absorbance was read at 540 nm in every 30 second after addition of HCl till the end of haemolysis. Difference of absorbance at the beginning and at the end of haemolysis was determined as 100%.

Osmotic resistance of erythrocytes. The osmotic resistance of erythrocytes was measured spectrophotometrically at the wave length of 540 nm as described Kamyshnikov [46]. The method is based on the determination of differences between osmotic resistance of erythrocytes to a mixture with a different concentration of saline solution and urea.

Peroxide resistance of erythrocytes. The peroxide resistance of erythrocytes was determined spectrophotometrically as described by Gzhegotskyi et al. [47] at 540 nm by monitoring the rate of erythrocytes disintegration with hydrogen peroxide. The peroxide resistance of erythrocytes was expressed in %. Absorbance of mixture contained erythrocytes and phosphate buffer without hydrogen peroxide was determined as 100%.

Analyses of chemical element concentration

Samples of left femora and liver tissue were taken for analyses of chemical element concentration (Na, K, Ca, Fe, Zn, Mn, Mg, Co, Cd and Pb). These were preceded by mineralization of samples, which was done using the Berghof Speedwave MWS-2 system (microwave pressure digestion unit with builtin in situ temperature measurement) and using 1.5ml of nitric acid 65% pure for analysis by Sigma-Aldrich to receive a clear solution. The contents of elements (ppm dry weight) were then determined according to Weltz [48] using a ‘‘Perkin-Elmer AAnalyst 800’’ atomic absorption spectrophotometer. Standard curves were prepared using standardized Merck samples for each of the element.

Biochemical assays

Total antioxidant capacity (TAC) assay

The TAC level in the plasma and tissues was estimated spectrophotometrically at 532 nm following the method with Tween 80 oxidation by measuring TBARS level [49]. Plasma inhibits Fe2+/ascorbate-induced oxidation of Tween 80, resulting in a decrease of TBARS level. The absorbance of the obtained solution was measured at 532 nm. Absorbance of blank was determined as 100%. The level of TAC in sample (%) was calculated in respect to the absorbance of blank.

Statistical analysis

Results are expressed as mean ± S.E.M. All variables were tested for normal distribution using the Kolmogorov-Smirnov and Lilliefors tests (p>0.05). Homogeneity of variance was checked using the Levene’s test. Significance of differences in the chemical element concentration between control and cobalt group depending individual resistance of hypoxia was examined using Mann-Whitney U test according to Zar [50]. Differences were considered significant at p<0.05. In addition, the relationships between data of all individuals were evaluated using Pearson's correlation analysis. Multivariate linear regression adjusted for high and low resistance to hypoxia of bones, blood and liver of rats was performed to evaluate the effect of metals content on resistance parameters function. All statistical calculation was performed on separate data from each individual with Statistica 8.0 software (StatSoft Inc., Poland).


Concentration of metals in bone, plasma and liver

Concentration of selected metals (Na, K, Ca, Cu) in the femora, plasma and liver during cobalt treatment dependent resistance to hypoxia are listed in Table 1. Lowering concentration of Na and higher for Ca (for HR animals) was detected in the liver of rats at cobalt treatment.

Table 1. Concentration of selected metals (Na, K, Ca and Cu) in the femora, plasma and liver of rats with different resistance to hypoxia at cobalt treatment.

Parameters Control Co
Na, mg/g LR 26.01 ± 0.29 24.92 ± 0.03*
HR 26.01 ± 0.29 24.93 ± 0.025*
LR 29.15 ± 1.76 24.99 ± 0.10
HR 25.35 ± 0.06# 24.94 ± 0.09
LR 25.54 ± 0.28 25.54 ± 0.49
HR 25.26 ± 0.84 25.51 ± 0.80
K, mg/g bone
LR 4.99 ± 0.001 4.99 ± 0.00
HR 4.99 ± 0.000 4.99 ± 0.00
LR 7.49 ± 0.06 4.99 ± 0.01
HR 4.99 ± 0.004# 4.99 ± 0.004
LR 5.00 ± 0.02 5.01 ± 0.21
HR 5.00 ± 0.12 5.00 ± 0.12
Ca, mg/g bone
LR 11.11 ± 0.37 11.15 ± 0.04
HR 10.20 ± 0.01 10.66 ± 0.06*
LR 11.93 ± 0.12 10.58 ± 0.11*
HR 10.98 ± 1.74 9.93 ± 0.67
LR 47.35 ± 3.77 54.62 ± 3.79
HR 88.34 ± 9.14# 145.03 ± 24.64*
Cu, µg/g bone
LR 27.08 ± 0.07 27.21 ± 0.04
HR 26.98 ± 0.13 27.07 ± 0.02
LR 29.42 ± 1.87 25.43 ± 2.97*
HR 24.91 ± 3.60# 25.28 ± 3.07
LR 33.91 ± 0.01 33.93 ± 0.01
HR 33.94 ± 0.01 33.95 ± 0.01

Co effects in bone, plasma and liver for selected elements (Mg, Fe, Zn and Mn) was presented in Table 2. We observed statistically differences in Fe content between control and Co treated HR animals in bones. For these gropes of rats, we observed higher concentration of Mn in femur vs. control too.

Table 2. Concentration of selected metals (Mg, Fe, Zn and Mn) in the femora, plasma and liver of rats with different resistance to hypoxia at cobalt treatment.

Parameters Control Co
Mg, µg/g LR 1160 ± 0.001 1157 ± 0.89*
HR 1148 ± 1.34 1150 ± 0.89
LR 1165 ± 2.97 1143 ± 2.69
HR 1009 ± 8.84 1149 ± 5.33
LR 852.84 ± 33.99 950.68 ± 20.45
HR 950.65 ± 2.84 879.46 ± 34.72
Fe, µg/g bone
LR 699.15 ± 28.64 460.4 ± 104.8
HR 682.9 ± 80.72 246.25 ± 13.21*
LR 637.11 ± 33.03 415 ± 20.02*
HR 561.56 ± 12.21 283.34 ± 14.23*
LR 692.41 ± 37.16 809.74 ± 24.21*
HR 657.74 ± 43.14 682.05 ± 16.66
Zn, µg/g bone
LR 239.85 ± 11.11 250.9 ± 2.73
HR 245.35 ± 1.99 236.7 ± 5.37
LR 220.85 ± 10.01 230.1 ± 20.03
HR 220.35 ± 15.09 221.4 ± 15.04
LR 455.56 ± 8.03 450.95 ± 3.58
HR 456.46 ± 8.98 452.52 ± 12.80
Mn, µg/g bone
LR 13.1 ± 0.24 13.27 ± 0.17
HR 13.15 ± 0.16 13.87 ± 0.03*
LR 15.06 ± 3.22 12.18 ± 3.38
HR 11.62 ± 4.01 12.48 ± 3.48
LR 17.37 ± 0.01 17.59 ± 0.06
HR 17.40 ± 0.03 17.37 ± 0.01

Co, Cd and Pb in the femora, plasma and liver of animals with different resistance to hypoxia at Co treatment was also investigated (Table 3). Co administration was changed levels these three selected elements and dependent on individual sensitivity to hypoxic factor. So, we observed statistically decreased level of Co in bone and increased ones in liver. Toxic effects of Co at Co treatment were accompanied by statistically increased level of Cd in bone and liver for HR animals. Similar tendencies were observed for LR animals at Co treatment too; however, these differences were not statistically significant. Co effects reflected also on Pb level. Lower concentration of Pb level in bone at Co impact for HR animals was detected. In these conditions we observed the tendencies to accumulation of Pb in liver HR rats.

Table 3. Concentration of selected heavy metals (Co, Cd and Pb) in the femora, plasma and liver of rats with different resistance to hypoxia at cobalt treatment.

Parameters Control Co
Co, mg/g LR 1.55 ± 0.53 0.96 ± 0.03
HR 0.82 ± 0.10 0.37 ± 0.079*
LR 5.23 ± 0.06 1.03 ± 0.35*
HR 2.72 ± 0.24# 2.81 ± 0.71
LR 0.10 ± 0.001 0.13 ± 0.02*
HR 0.10 ± 0.02 0.20 ± 0.001*
Cd, µg/g bone
LR 3.54 ± 0.27 4.85 ± 0.56
HR 3.26 ± 0.07 4.64 ± 0.42*
LR 6.65 ± 0.21 3.02 ± 1.65*
HR 2.80 ± 0.99# 3.17 ± 0.93
LR 0.32 ± 0.07 0.43 ± 0.05
HR 0.32 ± 0.06 0.43 ± 0.04*
Pb, µg/g bone
LR 3.12 ± 0.001 3.80 ± 0.45
HR 5.45 ± 0.26 4.17 ± 0.06*
LR 5.62 ± 0.11 3.20 ± 1.87*
HR 3.47 ± 1.81 3.16 ± 0.98
LR 0.18 ± 0.05 0.09 ± 0.001
HR 0.19 ± 0.004 0.40 ± 0.09

Thus, the effects of cobalt in different tissues (bone, plasma, liver) were dependent on the physiological levels of elements redistribution, especially heavy metals. The cobalt effects were dependent of the level of physiological reactivity to hypoxia. Several correlations between elements in the bones of rats with different resistance to hypoxia were found (Tables 4 and 5). We observed that increased Co level modifies physiological and heavy metals content and participated in significant elementelement relations depending on congenital resistance to hypoxic states.

Table 4. Analysis of correlations between metals level in the plasma of rats with different resistance to hypoxia at cobalt treatment

Parameters Correlative coefficient, p Parameters Correlative coefficient, p
Low Resistance
Control Co
Na-Zn -0.993 (0.000) Na-Mg -0.944 (0.005)
Mg-Ca 0.992 (0.000) Mg-Co -889 (0.018)
Ca-Cd -0.841 (0.037) Zn-Cu 0.857 (0.029)
Fe-Co 0.890 (0.019) Zn-Mn 0.851 (0.023)
Fe-Pb 0.991 (0.000) Cu-Mn 0.969 (0.001)
Co-Pb 0.920 (0.011) Co-Mg -0.889 (0.018)
High Resistance
Control Co
Ca-Mg -0.884 (0.020) K-Cu 0.844 (0.033)
Zn-Cu 0.926 (0.008) K-Mn 0.895 (0.016)
Zn-Mn 0.948 (0.005) Ca-Pb 0.954 (0.003)
Cu-Mn 0.983 (0.012) Zn-Cu 0.849 (0.033)
Cd-Cu 0.909 (0.012) Zn-Mn 0.834 (0.039)
Cd-Mn 0.886 (0.019) Cu-Mn 0.988 (0.000)
Pb-Cu 0.815 (0.048)

Table 5. Analysis of correlations between metals level in the liver of rats with different resistance to hypoxia at cobalt treatment.

Parameters Correlative coefficient, p Parameters Correlative coefficient, p
Low Resistance
Control Co
Na-K 0.783 (0.021) Ca-Cu -0.840 (0.009)
Cu-Mn -0.945 (0.001) Ca-Mn -0.876 (0.005)
Cu-Co 0.981 (0.001) Ca-Cd 0.865 (0.004)
Cu-Cd 0.980 (0.000) Ca-Pb 0.839 (0.009)
Cu-Pb 0.956 (0.000) Cu-Pb 0.835 (0.008)
Co-Cd 0.978 (0.000)
High Resistance
Control Co
Fe-Cu 0.731 (0.032) K-Zn 0.858 (0.006)
Fe-Mn 0.729 (0.040) Cu-Pb -0.987 (0.000)
Fe-Co - 0.739 (0.033) Co-Pb -0.978 (0.001)
Fe-Cd -0.726 (0.023)
Co-Cu - 0.745 (0.024)
Co-Cd 0.987 (0.000)
Co-Pb 0.976 (0.001)

Multiple linear regression analysis is a statistical tool for determining the relationship between single depended variable and a set of independent variables to best represent a relationship in a population. We used regression analysis as a way of predicting an outcome variable from a predictor variable individual organism’s reactivity and metals content in tissues (bones, blood, liver). Stepwise linear multiple regression method was applied to find the dominant factors influencing resistance to hypoxia forming. Our data represented in Table 6 shows dependencies between resistance to hypoxia and metals content in the plasma. Table content the prediction equation, where are dependent from metals variable, the different set of independent variables, the regression coefficient (R2), the corrected regression coefficient (R2 corr), the random error, and probability (p). Multiple linear regression analysis fit a model to our data metal content in the plasma (Na, K, Cu, Co, Cd) in individual resistance to hypoxia in rats. Statistical model used to describe to resistance and predict a value of the dependent variable from set of independent variables. In this study, a research model is based on the selected metals in different tissues to explain the individual resistance factor.

Table 6. Multiple linear regression equation for metal concentrations in plasma rats with different resistance to hypoxia.

Metals Regression equation (Resistance) R2 R2 corr Significance of regression, p
Cu -1.127Cu+143.213 ± 2.868 0.426 0.368 0.021
K 1.247K - 120.97 ± 1.323 0.516 0.468 0.009
Cd -0.963Cd+103.883 ± 1.498 0.665 0.631 0.001
Na 1.899Na - 166.46 ± 1.402 0.688 0.656 0.001
Co -1.126Co+118.982 ± 1.334 0.774 0.751 0.000
Mg 24.45 Mg - 1616.83 ± 81.458 0.292 0.241 0.031
Ca 2.482Ca - 987.678 ± 19.799 0.551 0.519 0.001

Coefficient of determinations (R2) as so as corrected coefficient of determinations (R2corr) for selected (only statistical important) data shows dependencies of metals in model resistance to hypoxia. R2 values (Table 6) revealed that Cu, K, Cd, Na, and Co contents explained the variability of the metal in plasma individual organism’s reactivity to hypoxia to the extent of 42.6% (36.8%), 51.6 % (46.8%), 66.5% (63.1%), 68.8% (65.6%) and 77.4% (75.1%) respectively, and indicated a significant relationship (p<0.05). Regression model in this Table indicated inverse higher relationships for Co content in plasma dependent other metals. This suggests that decrease in blood Co content may lead to increase in resistance factor (Tables 7,8).

Table 7. Analysis of correlations between metals level in the bones of rats with different resistance to hypoxia at cobalt treatment 

Parameters Correlative coefficient, p Parameters Correlative coefficient, p
Low Resistance
Control Co
Na-Co 0.937 (0.006) Ca-Mg -0.884 (0.020)
Fe-Zn 0.821 (0.045) Zn-Cu 0.926 (0.008)
Fe-Cu 0.834 (0.039) Zn-Mn 0.943 (0.005)
Fe-Cd 0.841 (0.036) Cu-Cd 0.909 (0.012)
Fe-Pb 0.856 (0.030) Cu-Pb 0.815 (0.048)
Zn-Mn 0.859 (0.030) Cd- Mn 0.886 (0.019)
Cu-Mn 0.978 (0.001)
Cd-Mn 0.848 (0.033)
Cd-Pb 0.880 (0.021)
High Resistance
Control Co
Ca-Mg -0.884 (0.020) K-Cu 0.844 (0.034)
Zn-Cu 0.926 (0.008) K-Mn 0.895 (0.016)
Zn-Mn 0.943 (0.005) Zn-Cu 0.849 (0.033)
Cu-Cd 0.909 (0.012) Zn-Mn 0.834 (0.039)
Cu-Pb 0.815 (0.048) Pb-Ca 0.954 (0.003)
Mn-Cd 0.886 (0.019)

Table 8. Total antioxidant status (%) in the blood and tissues of rats with different resistance to hypoxia at cobalt treatment.

Parameters Control Co
Plasma LR 23.64 ± 4.52 32.11 ± 6.37
HR 16.59 ± 1.99 41.39 ± 3.33**
Erythrocytes LR 14.12 ± 1.23 43.30 ± 4.44**
HR 18.48 ± 2.02* 46.13 ± 5.98**
Brain LR 10.91 ± 1.34 12.82 ± 3.17
HR 12.48 ± 2.14 12.02 ± 2.45
Kidney LR 7.71 ± 1.12 7.94 ± 1.45
HR 4.42 ± 0.89* 5.18 ± 0.97
Lung LR 10.21 ± 3.11 11.58 ± 3.45
HR 12.48 ± 3.23 8.93 ± 3.12
Liver LR 23.05 ± 2.36 16.89 ± 2.58**
HR 16.04 ± 0.25* 8.77 ± 0.25**
Heart LR 6.83 ± 0.94 4.63 ± 0.70
HR 8.99 ± 1.28 7.25 ± 0.79

Resistance of erythrocytes to haemolytic reagents

Erythrocytes are the one of the most important indicators of the internal exposure of an individual to increased generation of reactive oxygen species and intensification of lipid peroxidation. Therefore, the next goal of our investigation was the measurement of erythrocytes’ resistance to haemolytic reagents in the blood rats at cobalt treatment (Figures 1-3). Using different methods for determining the resistance of red blood cells to hemolytic agents has allowed us to identify the main differences of the cobalt effects dependent of the individual hypoxia reaction.


Figure 1: Osmotic resistance of erythrocytes (% of hemolysated erythrocytes in different concentration of urea) of rats with different resistance to hypoxia at cobalt treatment (n = 8). Horizontal axis: different urea concentration (1 - 0.12, 2 - 0.135, 3 - 0.15, 4 - 0.165, 5 - 0.18, 6 - 0.195, 7 - 0.3 mol/L).


Figure 2: Resistance of erythrocytes to peroxide of rats with different resistance to hypoxia at cobalt treatment (n = 8). * - the significant change was shown as p<0.05 when compared two groups rats with different resistance to hypoxia; ** - significant difference (p<0.05) between Co-treated group rats with low and high resistance to hypoxia.


Figure 3: Acid resistance of erythrocytes (% of hemolysated erythrocytes per min) of rats with different resistance to hypoxia during cobalt treatment (n = 8).

Osmotic resistance of erythrocytes is shown in Figure 1. Percent of hemolysated erythrocytes under incubation with different concentration of urea was lower in the blood of LR (part A) and was higher for HR (part B) at cobalt treatment.

Resistance of erythrocytes to hydrogen peroxide is shown in Figure 2. Resistance of erythrocytes to hydrogen peroxide was found to be lower in LR rats at Co impact and has not been changed at simple condition in HR animals compared to the control group.

Our work clearly establishes that erythrocytes of LR rats at Co impact subjected to the haemolytic agents undergo high level of haemolysis compared to control group due to the oxidizing effect of the acid, urea and hydrogen peroxide. Erythrocyte haemolysis is associated with peroxidation of erythrocytes membranes by Co-induced oxidative stress only for HR animals. This may confirm the participation of cobalt metabolism in the formation in individual hypoxia resistance at blood level.

Total antioxidant status in the blood and tissues

For the purpose of concept proof for activating oxidative stress under the influence of cobalt in various rat tissues, we determined in the blood and tissues total antioxidant properties.

These data are presented in Table 6. Total antioxidant status (TAS) of tested tissues (plasma, erythrocytes, brain, kidney, lung, liver and heart) was significantly affected by Co and changed dependently on tissue specify and basic resistance to hypoxia. It was shown that Co treatment led to the TAS increasing in plasma and erythrocytes and decreasing in liver. Acid resistance of erythrocytes (Figure 3) was significantly lower at Co impact independently resistance to hypoxia. Percent of hemolysated erythrocytes per first min of initiation of haemolysis was higher in LR animals at Co treatment.


In the present work we focused on the effects of Co-associated changers of element content and biomarkers of oxygen metabolism damage pathways in different tissues dependent to physiological predisposition to hypoxia.

Oxidative stress, such as lipid peroxidation, is induced by an imbalance between reactive oxygen species (ROS) production and antioxidant defense capacity [51-52]. Various model organisms diseases hypothesized that oxidative stress can play a role in the regulation of important processes heavy metals and biotransformation of xenobiotics through influencing synthesis of antioxidant enzymes, modulation of signal pathways, inflammation, apoptosis, and cell proliferation, and thus oncogenic transformation [23].

In our study, Co impact was changed levels Na, Ca, Fe, Mn, Co, Cd and Pb elements and dependent on individual sensitivity to hypoxic factor. So, we observed statistically decreased level of Co in bone and increased ones in liver. Selected metals belong to the group of the most important physiological and heavy metals. They are predominantly bivalent metals, which are also included as a coenzyme in the active centers of antioxidant enzymes. Toxic effects of Co at Co treatment were accompanied by statistically increased level of Cd in bone and liver for HR animals. Co effects in bone and liver was focused at Fe and Mn level changers. However, some authors [53] reported that iron ions are themselves free radicals, and ferrous ions can take part with molecular oxygen in electron transfer reaction. It was important that generation of superoxide in the presence of high Fe ions induced to the formation of more reactive hydroxyl radicals by Fenton chemistry. Hydroxyl radicals can initiate lipid peroxidation by hydrogen abstraction. Using different scavengers system was studied superoxide-dependent Fenton reaction. Recently shows that superoxide providing the hydrogen peroxide and also reducing Fe3+ to Fe2+. Another iron complex can stimulate peroxidation by lipid decomposition reactions too [54].

Our results suggest higher sensitivity of erythrocytes membranes to hemolytic agent (peroxide, acid and osmotic) treatment at Co impact. The hemolytic activity at cobalt chloride administration was predominantly associated with ROS production by iron ions [55] realized by heme modification and hemoglobin. Our data was demonstrated higher Co level vs. control group animals. Similar to these observations cobalt ions can penetrate into the liver cells and modify intracellular heme and heme proteins. Increased contents of free heme or its analog in liver cells induced HO-1 [53].

High doses of cobalt exert a variety of dangerous effects including cardiotoxicity in man and in animals [13]. The cobalt (II) ion has recently been reported to react with hydrogen peroxide and to produce reactive oxygen intermediates such as superoxide radicals and hydroxyl radicals [44,56]. The liver and the heart have a higher uptake of cobalt compared to other organs [29]. Our data demonstration that bone’s physiological and heavy metals level was important affected by Co too. This cobalt effects mediated by lowering erythrocytes membranes functioning as suggest our data with different hemolytic agents using. The mechanisms of this toxicity are not entirely clear. The cobalt (II) ion has recently been reported to react with hydrogen peroxide under physiological conditions to form reactive oxygen intermediates [57-59]. Such of them, hydroxylate aromatic compounds and degrade deoxyribose can involve formation of oxygen free radicals [60].

However, some data revealed in vitro and in vivo that cobalt influences bone resorption and bone formation by modulating bone cell metabolism [61]. Dermience et al. [62] showed that cobalt ions affect osteoblast proliferation, size, and shape. Co influences on proliferation and function of human osteoblastlike cells, osteoblastic activities decreases by alkaline phosphatase levels and calcium accretion, which inhibits release of osteocalcin and collagen type 1 proteins [57,63-65]. Co ions effects may be mediate by oxidative stress state in osteoblast-like cells by an unknown mechanism [59,66]. Co leads to inflammation and osteoclast differentiation, maturation, and stimulation by cytokines (TGF-β1, TNF-α, IL- β1, IL-6) secretion [65,67]. In contrast, some studies [68,69] on cobalt-chromium wear particles indicate antagonistic actions, such as induction of apoptosis in osteoclasts and decreased secretion of some inflammatory factors such as prostaglandin E2 and interleukin-6. Effects cobalt-chromium interaction are associated with oxidative stress, oxidation and nitration of proteins, and misregulation of antioxidant enzyme expression [57,59,65].

Cobalt toxicity [33] has been an emerging problem of recent years with the introduction of metal-on-metal hip replacements [70-73]. Direct toxicity by Co and Cr ions represents some data of metal-on-metal hip prostheses, made of cobalt-chromium (Co-Cr) alloys [74-76]. CoCr alloys have widespread and extensively use in the orthopaedics industry as an alternative to titanium alloys due to their superior stiffness and corrosion resistance. Recent studies have shown that Co and Cr particles can enter the bloodstream and accumulate both in tissues and organs of patients. Toxic effects of these ions may cause hypersensitivity to these metals, led to chromosome aberrations and changers of lymphocytes proportions [66,76]. Cobalt nanoparticles could induce primary DNA damage in a concentration-dependent manner. The International Agency for Research on Cancer (IARC) has classified soluble Co2+ as possible carcinogenic and implanted orthopedic alloys as unclassifiable carcinogenic [61].

Our results shows higher level TAC in blood and plasma animals at Co treatment may be mediated by as non-classical antioxidant enzyme heme oxygenase-1 (HO-1). In bones similar mechanism was described by Nishishita and Tsukuba [77] in osteoclasts. ROS was identified as an additional essential factor for the osteoclasts differentiation by cobalt protoporphyrin functioning. Cobalt protoporphyrin was known as metalloprotoporphyrin and inducer of heme oxigenase-1 (HO-1) in cells. Recent studies these authors have demonstrated that induction of HO-1 inhibited differentiation and activation of osteoclasts cells. Induction of HO-1 connected with cobalt protoporphyrins has been shown as effective mechanism to protect apoptosis in the liver from ischemia/reperfusion or in human cardiac cells.

Effects of cobalt nanoparticles on human T cells in vitro was connected with significant decreasing in the SOD, GPX, and CAT activities, inducing oxidative stress and next cytotoxicity and genotoxicity [61]. These authors suggested that nanometer cobalt particles, generated from the articular surfaces metal-onmetal hip prostheses could gradually enter the immune cells, disturb the antioxidant balance, and next induce tissue damage. Similar data of oxidant-antioxidant balance and induced a depletion of cerebral and cerebellar antioxidant activities at Co effects was represented Garoui et al [78] too.

Some studies indicated important role of plasma proteins in toxic cobalt effects and individual’s particular albumin binding capacity. It’s connected with developing next influences in patients with high level cobalt blood concentration such as liver pathology, kidney disease and cancer, trauma, scleroderma and diabetes as well as poor protein diet [71,79]. However, cobalt can result in chemically induced hypoxiamimicking conditions by stimulating hypoxia-inducible factors and modify oxidative phosphorylation [80].

The published data [11] and early results of our studies [10] partially answer the question about the causes of individual variability in the hypoxic resistance and show the contribution of individual functional systems to the oxygen supply of tissues. These two types of animal’s models (low and high resistance to hypoxia) are described by two different functional and metabolic patterns not only hypoxia factor but stress and diseases predominance. They are associated with typical differences in activity of central nervous system and neurohormonal regulation, connected stress-activating and stress-limiting systems, oxygen-transporting function of the blood, mitochondrial functioning, Crebs cycle metabolites functioning [4]. Moreover, these parameters are coupled with energy metabolites exchange and functional activity of the mitochondrial respiratory change in animal tissues [9].

Low individual resistance to hypoxia was due with initially predominant activity sympathetic nervous system, with an even greater increase in the sympathetic activity during the development of the stress response. High individual resistance to hypoxia connected with domination of the parasympathetic activity in the autonomic regulation. Its reserve of the heart rate variability range and was used for a more adequate compensation as showed study [11].

Concluding, the results presented here demonstrate the importance of division animals at two different groups with hypoxia predominance in cobalt impact in conjunction with metal analysis and oxidative stress responses. Effects of cobalt chloride were associated with the formation of individual response to hypoxia and reduction of iron, lead and cobalt in the long bones with subsequent accumulation in liver tissue. Effects of cobalt in our investigation cause the cadmium accumulation in bones and liver. Effects cobalt show substantial reduction of erythrocyte membranes resistance to the action of hemolytic agents with a high level of total antioxidant capacity in erythrocytes and plasma, but not in liver.


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