The Influence of the Superoxide Dismutase Preparation Rexod® on Survival of CFU–S in С57ВL/6 Mice Exposed to Irradiation

The radioprotective efficiency of the Rexod^® drug (State Research Institute of Highly Pure Biopreparations of the Federal Medical-Biological Agency of Russia) containing recombinant human superoxide dismutase (SOD) has been investigated in terms of survival and proliferative activity of hematopoietic stem cells in male С57Вl/6 inbred mice of the SPF category after acute external γ-irradiation. The animals were subjected to radiation from an IGUR-1M apparatus containing a source of ¹³⁷Cs, and the γ-exposure dose was within the range of 3.0–7.5 Gy, while the absorbed dose-rate was 0.78 Gy/min. A single dose of 180 mg/kg of recombinant SOD in a volume of 1 mL was administered intraperitoneally 15 minutes prior to irradiation. Mice from the control group similarly received the same volume but of normal saline. The radioprotective properties of SOD were assessed based on the amount of CFU–S in the endotest, nucleated cells (NCs) in the bone marrow and thymus, erythrocytes and leukocytes in the peripheral blood, and the weights of the thymus and spleen in the dynamics. It was shown that the use of SOD made it possible to increase the survival of CFU–S after acute sublethal γ-irradiation of mice at a dose of 4Gy. The registered changes in the amount of CFU–S and other hematological parameters might testify to the fact that the administered SOD stimulated hematopoiesis in the spleen. The revealed stimulatory effect of SOD on the erythropoiesis and the number of NCs in the thymus in both irradiated and nonirradiated animals opens up new opportunities for the use of the preparation and requires further studies of the mechanisms of the detected effects.

Keywords: ionizing radiation; superoxide dismutase; hematopoietic stem cells; CFU–S; survival

Copyright comment ISSN 1062-3590, Biology Bulletin, 2021, Vol. 48, No. 11, pp. 2071–2078. © Pleiades Publishing, Inc., 2021. Russian Text © The Author(s), 2021, published in Radiatsionnaya Biologiya. Radioekologiya, 2021, Vol. 61, No. 2, pp. 158–166.

INTRODUCTION

Currently there is significant evidence for the ability of antioxidants to modify the reactions of hematopoiesis to irradiation. The radioprotective effect of antioxidants was shown in experiments in vitro, in vivo, and under clinical conditions [[1]]. The mechanism of the radioprotective action of antioxidants is directly and indirectly related to the inhibition of cytotoxic and mutagenic effects of active radicals by their elimination, inhibition of lipid peroxidation, activation of enzymatic damage reparation, and the reduction of NO synthase activity [[2]]. It was demonstrated that a high radiosensitivity of certain strains was associated with the disturbance of antioxidant activity [[3]].

It is well-known that manganese and copper superoxide dismutases (SOD) are crucial enzymes of antioxidant defense controlling the oxidative processes involving free radicals of oxygen and nitrogen and the formation of products of lipid peroxidation, elevated after irradiation and in numerous pathological conditions accompanied by oxidative stress [[1], [4]]. Despite the fact that SOD is able to improve the survival of human hematopoietic stem cells (HSCs) and animals suffering from bone marrow syndrome, a type of acute radiation syndrome [[6]], the mechanisms of its influence on hematopoiesis remain undetermined. This study investigated the radioprotective action of the drug Rexod®, a recombinant human SOD synthesized at the State Research Institute of Highly Pure Biopreparations, Federal Medical-Biological Agency of Russia. The key active ingredient of Rexod®, characterized by antioxidative, anticytolytic, and anti-inflammatory actions (RU LP-004754-260318), is a human recombinant SOD genetically engineered using a culture of Saccharomyces cerevisiae yeast of Y21341 strain as the producer [[7]].

The purpose of this work was to assess the influence of the drug Rexod® on hematopoiesis in mice, including the analysis of preservation and proliferative activity of hematopoietic stem cells exposed to acute external γ-irradiation.

MATERIALS AND METHODS

The study comprised mature conditional inbred С57В1/6 male mice of the SPF category aged 2.0–2.5 months, weighing 20–23 g, and kept in the Institute of Cytology and Genetics, Novosibirsk.

The laboratory animals were kept under optimal conditions for this species. The air temperature indoor was +22°C with a humidity of 50–60%. The length of daylight hours was maintained at 12 h by artificial light. The animals were fed in the standard manner and had unlimited access to drinking water.

First, the correlation between CFU–S (spleen colony forming cells, endocolony) [[8]] and the dose of external γ-irradiation was estimated to choose the dose applied for the endotest to determine the radioprotective action of SOD. The procedure of the endotest method was performed in strict accordance with the A.E. Pereverzev modification [[9]].

The animals were divided into 15 groups of eight. The survival of CFU–S was estimated after the acute total γ-irradiation of mice at doses of 3.0, 3.25, 3.5, 3.75, 4.0, 4.5, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 7.0, and 7.5 Gy. The irradiation of animals was conducted in an IGUR-1M apparatus, containing four sources of 137Cs. The γ-irradiation intensity of the apparatus was 0.78 Gy/min; the inhomogeneity of the γ-field in the working compartment was less than 5%. The number of CFU–S was calculated in the spleen on the 9th day from the acute total γ-irradiation.

The Rexod® drug was used to assess the radioprotective properties of recombinant human SOD in the intravenous solution at a dose of 3.2 million U (RU LP-004754-260318), produced by the State Research Institute of Highly Pure Biopreparations, Federal Medical-Biological Agency of Russia. The radioprotective properties of the drug were evaluated referring to the parameters characterizing the state of hematopoiesis, particularly, the amount of CFU–S, the number of nucleated cells (NCs) in the bone marrow, the amount of red blood cells (RBCs) and white blood cells (WBCs) in the peripheral blood, and the state of lymphoid organs, i.e., the weight and amount of cells in the thymus and the weight of the spleen. Two independent experiments were conducted to assess the radioprotective action of the drug Rexod®.

In the first experimental session two groups of 20 animals in each were formed. The mice of the experimental group received a single intraperitoneal (i/p) injection of Rexod® at a volume of 1 mL (dose 180 mg/kg) 15 min before irradiation at a dose of 4 Gy, whereas the control mice were injected with the solvent (normal saline) at the same moment and in the same volume 15 min before irradiation.

The mice were sacrificed on the 9th day after acute total γ-irradiation, then the autopsy, macroscopic examination of organs, measurement of the weights of the spleen and thymus, calculation of CFU–S in the spleen and NCs in the bone marrow and thymus, and the blood cell count were conducted.

During the second experimental session, four groups of animals were comprised: group 1 of 20 male mice receiving an i/p injection of 1 mL of Rexod® at a dose of 180 mg/kg 15 min before the 4 Gy irradiation; group 2 (control) of 20 male mice receiving an i/p injection of 1 mL of normal saline 15 min before the 4 Gy irradiation; group 3 of 11 male mice receiving an i/p injection of 1 mL of Rexod®s drug at a dose of 180 mg/kg 15 min without irradiation (unirradiated animals); and group 4 (control) of 11 male mice receiving an i/p injection of 1 mL of normal saline without irradiation (unirradiated animals).

The mice were sacrificed on the 9th day after acute total γ-irradiation, then the autopsy, macroscopic examination of organs, measurement of the weights of the spleen and thymus, calculation of CFU–S in the spleen and NCs in the bone marrow and thymus, and the blood cell count were conducted.

Common statistical tests were applied to analyze the experimental results [[10]]. The mean values and standard error were counted; correlation and regression analyses were used to estimate the correlations between the parameters studied. The distribution analysis with the Kolmogorov–Smirnov test discovered no statistical difference from the normal distribution, hence, the parametric methods. The means were compared with the help of Student's t-test. The differences were statistically important at p ≤ 0.05.

RESULTS

At the first stage, the correlation between the viable CFU–S in C57B1/6 male mice and the dose of the external total γ-irradiation was measured. The results of calculating CFU–S in different dose groups of mice are presented in Fig. 1. The figure demonstrates that the survival of CFU–S was illustrated well by the exponential function (R2 = 0.93; F = 190.95; р = 6.15 × 10–10). The expected D0 value for CFU–S was 0.73 Gy in male C57B1/6 mice. The following investigation of the radioprotective action of Rexod® was conducted at 4 Gy.

Graph: Fig. 1. The number of endocolonies in the spleen of C57B1/6 mice under irradiation at different doses, CFU/spleen.

The results of the first experimental session estimating the radioprotective effect of Rexod® in the experiments with single acute exposure to radiation of C57B1/6 mice are presented in Table 1. The cell count in the peripheral blood did not differ statistically between mice of the experimental and control groups, though the experimental animals showed higher number of red blood cells (RBCs) and white blood cells (WBCs).

Table 1. Results of the first series of experiments, assessing the radioprotective effect of SOD during acute γ-irradiation of C57B1/6 mice at a dose of 4 Gy

Groups	Animal body weight, g	Spleen weight, mg	CFU–S	NCs in the bone marrow, mln /femur	Thymus weight, mg	NCs in thymus, mln/mg	RBCs, mln/µL	WBCs, mln/mL
Experiment SOD + 4 Gy	22.42 ± 0.30	33.45 ± 1.50	8.05 ± 0.90 p = 0.02	18.70 ± 0.60	22.35 ± 0.50 p = 0.05	1.81 ± 0.10	2.35 ± 0.10	2.53 ± 0.10
Control Saline + 4 Gy	22.40 ± 0.20	32.90 ± 1.80	5.45 ± 0.60	19.36 ± 0.70	24.10 ± 0.68	2.02 ± 0.08	2.11 ± 0.14	2.21 ± 0.13

The thymus weight was significantly less in the experimental animals (22.35 ± 0.50 mg, p = 0.05) compared to 24.10 ± 0.68 mg in the control. The number of NCs in the thymus of experimental animals of 1.81 ± 0.10 million NCs/mg did not differ statistically from the control (2.02 ± 0.08 million NCs/mg).

Statistical differences were observed in the number of CFU–S in the experimental and control animals. The amount of CFU–S was 1.5 times higher in the animals injected with Rexod® 15 min before the irradiation (8.05 ± 0.90, p = 0.02) in comparison with the control group (5.45 ± 0.60).

The second experimental session provided the results of studies involving single acute irradiation of C57B1/6 mice to estimate the radioprotective properties of Rexod® (Table 2). The application of Rexod® increased the number of stem cells. The amount of CFU–S in the animals injected with the drug 15 min before the irradiation (10.30 ± 0.98) was 40% higher (p = 0.02) compared to the control group (7.35 ± 0.70).

Table 2. Results of the second series of experiments to assess the radioprotective effect of SOD during acute γ-irradiation of C57B1/6 mice at 4 Gy

Groups	Animal body weight, g	Spleen weight, mg	CFU–S	NCs in the bone marrow, mln /femur	Thymus weight, mg	NCs in thymus, mln/mg	RBCs, mln/µL	Leukocytes, mln/mL
SOD + 4 Gy	23.39 ± 0.30	32.50 ± 1.30	10.30 ± 0.98 p = 0.02	18.19 ± 0.70	27.55 ± 1.0	1.94 ± 0.10 p = 0.0001	5.01 ± 0.20 p = 0.0001	2.25 ± 0.10 p = 0.02
Soline + 4 Gy	23.25 ± 0.17	31.15 ± 0.66	7.35 ± 0.70	18.13 ± 0.89	26.30 ± 1.01	1.47 ± 0.05	3.44 ± 0.16	1.88 ± 0.12
SOD	22.63 ± 0.60	51.50 ± 2.40	–	21.01 ± 0.80	27.17 ± 2.30 p = 0.05	1.60 ± 0.10 p = 0.01	6.31 ± 0.20 p = 0.0001	11.06 ± 0.70
Soline	22.05 ± 0.40	48.27 ± 1.40	–	21.79 ± 1.20	21.64 ± 1.40	0.91 ± 0.20	3.96 ± 0.20	11.51 ± 1.30
The statistical significance is presented for the groups SOD + 4 Gy and Normal Saline + 4 Gy, and SOD and Normal Saline.

The RBC count in irradiated animals receiving Rexod® was 5.01 ± 0.20 million/µL, which was 1.5 times higher (p = 0.0001) than in the irradiated control group. The drug administration by unirradiated mice was also associated with significant elevation of the RBC count in the peripheral blood compared to the unirradiated control (p = 0.0001).

The mean WBC count was 2.25 ± 0.10 million/mL in the animals given Rexod® before the irradiation, which is statistically higher (p = 0.02) than in the irradiated control (1.88 ± 0.12 million/mL).

In the second experimental session, the weight of the thymus of irradiated animals did not differ between groups 1 and 2. However, nonirradiated animals treated with Rexod® had a thymus weight of 27.17 ± 2.30 mg, statistically exceeding (p = 0.05) this parameter in the nonirradiated control (21.64 ± 1.40 mg).

The number of NCs in the thymus was statistically higher in irradiated animals than nonirradiated ones receiving Rexod®.

The methods of regression and correlation analyses were applied to assess the influence of SOD on the response of hematopoiesis to the total acute irradiation, determining the correlations between the parameters studied and the number of CFU–S in irradiated mice receiving Rexod® and the irradiated control.

A positive correlation was seen between the amount of CFU–S and the spleen weight in the group of the irradiated control and the group receiving Rexod® (Fig. 2). The correlation between the spleen weight and the CFU–S number in the control group was linear (R2 = 0.50; F = 38.57; p < 0.001). A similar but more prominent trend was detected in the group of irradiated animals receiving Rexod® (R2 = 0.91; F = 370.35; p < 0.001).

Graph: Fig. 2. The results of regression analysis of the relationship between the number of CFU–S and the spleen weight of control animals (a) and mice treated with Rexod® (b).

The correlation analysis did not detect a statistical correlation between the number of CFU–S and NCs in the bone marrow of mice in the irradiated control group (Fig. 3a). Nevertheless, a statistically negative correlation was registered between the amounts of CFU–S and NCs in the bone marrow of animals influenced by irradiation and Rexod® (Fig. 3b). The irradiated control groups did not demonstrate a statistical correlation with the amount of CFU–S and WBCs in peripheral blood (Fig. 4a), whereas in the irradiated mice receiving Rexod® these parameters were positively correlated (Fig. 4b).

Graph: Fig. 3. The results of correlation analysis of the relationship between the number of CFU–S and the number of NCs in the bone marrow of control animals (a) and mice receiving Rexod® (b).

Graph: Fig. 4. The results of correlation analysis of the relationship between the amount of CFU–S and the number of leukocytes in the peripheral blood of control animals (a) and mice receiving Rexod® (b).

It should be emphasized that NCs in the bone marrow did not correlate with the WBC count in the peripheral blood (Fig. 5).

Graph: Fig. 5. The results of correlation analysis of the relationship between the amount of NC in the bone marrow and the number of leukocytes in the peripheral blood of control animals (a) and mice receiving Rexod® (b).

According to the abovementioned information, the second experimental session (Table 2) revealed that Rexod® positively influenced the recovery of the NC number in the thymus after acute irradiation. The correlation analysis identified a fundamental change in the dependence between the NC number in the thymus and CFU–S in mice treated with Rexod®. Thus, mice of the irradiated control group demonstrated a statistical negative correlation between the NC number in the thymus and the amount of CFU–S (Fig. 6a), while administration of the drug provoked the reverse of this correlation from negative to positive (Fig. 6b).

Graph: Fig. 6. The results of correlation analysis of the relationship between the number of CFU–S and the amount of NCs in the thymus of control animals (a) and irradiated mice receiving Rexod® (b).

DISCUSSION

It is known that irradiation exposure can affect the levels of antioxidant enzymes inside cells even at low doses [[11]]. It was found that redox-sensitive NF–κB was activated by low doses of irradiation, leading to the intensification of gene expression of manganese SOD, its enzymatic activity, and changes in cell radiosensibility [[12]].

Previous experiments and clinical trials have reported the radioprotective role of SOD, administered either before or after irradiation [[4], [6]]. There is evidence that SOD-containing drugs promote a reduction in the amount of morphologically altered cells, thus improving their survival after exposure to high doses of γ-irradiation [[13]]. The treatment with SOD preparations succeeded in managing radiation-induced fibrosis [[14]]. However, the mechanisms of such a wide range of biological action of SOD preparations are still unclear. It can be proposed that the efficacy of SOD in treating a number of diseases of different etiology is associated with its effects on stem cells and the universal mechanism of the radioprotective action of SOD involving the inactivation of active oxygen and nitrogen species that ensues from the preservation of stem cells in different tissues from the oxidative injury.

The high efficiency of the recombinant drug action of Rexod® preserving the stem cell pool from damage of acute irradiation was shown when testing CFU–S. The results of the pilot study demonstrated that SOD preparation increased stem cell survival in C57B1/6 inbred mice after an intraperitoneal injection at a dose of 180 mg/kg 15 min before the acute total γ-irradiation at a dose of 4 Gy in the test of CFU–S evaluation (endotest). The effect was confirmed in two sessions of independent experiments.

The analysis of the correlation between the spleen weight and the CFU–S number allowed interpreting the results from the perspective of proliferative activity of a stem cell forming a colony. Since the coefficient of the regression slope described the mean colony weight, the statistical increase in this coefficient might indicate the proliferative activity of stem cells and improvement of their survival. The present study failed to identify statistical differences in the weight of a single colony in the group of the irradiated control (1.28 ± 0.21 mg) from this parameter in the irradiated group receiving Rexod® (1.41 ± 0.07 mg, p = 0.55). Our results suggested that Rexod® apparently increased the survival of stem cells but did not affect their proliferative activity.

It has been established that the crucial factors of HSC survival during oxidative stress are FoxO genes and their transcription factors. Presumably, they play an important role in the regulation of the cell cycle, supporting the resting state of HSCs and maintaining their self-renewal and repopulation. It was shown that the FoxO3 transcription factor suppressed the formation of active radicals in HSC by stabilizing АТМ gene [[15]].

The results of correlation and regression analysis allow us to propose that spleen hematopoiesis prevailed in irradiated animals receiving Rexod®. The assumption that SOD influenced the redistribution of hematopoiesis in irradiated animals from predominantly medullary to extramedullary hematopoiesis in the spleen could be proven by the correlation analysis of the number of WBCs in the peripheral blood and CFU–S. The negative correlation detected between the amount of CFU–S and NCs in the bone marrow, along with the positive correlation between CFU–S and the WBC count in the peripheral blood of irradiated mice treated with Rexod® as opposed to their absence in the irradiated control, allowed assuming that SOD caused the redistribution of active hematopoiesis from the bone marrow to the spleen in irradiated animals.

The redistribution of active hematopoiesis from the bone marrow to the spleen in mice exposed to irradiation and receiving the drug suggested that SOD affected the hematopoiesis structure. The involvement of SOD in the signaling pathways of hematopoiesis regulation demonstrated the participation of extracellular SOD in the regulation of erythropoietin gene expression [[16]].

The radioprotective action of Rexod® on CFU–S seen in this study implicitly demonstrates the potential efficacy of this drug for stem cells and other organs, such as the bone marrow and thymus. It was found that the drug elevated the amount of NCs in the thymus, RBCs, and WBCs in the peripheral blood in irradiated mice compared to the irradiated control on the 9th day after exposure. The analysis of the RBC and WBC counts in the peripheral blood of animals exposed to sublethal doses of radiation and SOD indicated sufficient activation of hematopoiesis in general. The activation of hematopoiesis is probably related to the improvement of stem cell survival in the spleen, bone marrow, and thymus. The ability of SOD to influence the proliferation and survival of hematopoietic cells was previously demonstrated on the models of knockout animals with combined deficit of Fancc-/- Sod1-/- [[17]].

Apart from the radioprotective action, the stimulating effect of Rexod® was found on erythropoiesis and lymphopoiesis in the thymus of unirradiated animals. The change in the correlation between the CFU–S number and NC from negative in the γ-control to positive in irradiated mice receiving Rexod® allowed us to presume that SOD influenced both hematopoiesis and immunity. It was demonstrated earlier that extracellular SOD–3, a SOD isoform, controlled the immune response by regulating the maturation of dendritic cells, T-cell activation and proliferation, and differentiation of T-helpers 2 (Th2) and regulatory Th17. Most likely, SOD is involved in the regulation of adaptive immunity indirectly by influencing the expression of redox-sensitivity of the Nrf 2 transcription factor [[18]].

CONCLUSIONS

The results of this pilot study demonstrated an increase in survival of hematopoietic stem cells of C57B1/6 mice in the endotest of CFU–S estimation after acute total γ-irradiation of mice with a sublethal dose and intraperitoneal administration of recombinant human SOD produced by State Research Institute of Highly Pure Biopreparations, Federal Medical-Biological Agency of Russia (invented name Rexod®) at a dose of 180 mg/kg 15 min before irradiation. The ability of SOD to regulate immunohemopoiesis was discovered. The experimental results detected that SOD could change the balance between medullar and splenic hematopoiesis and provoke the redistribution of active hematopoiesis from the bone marrow to the spleen. The stimulating effect of Rexod® on erythropoiesis and T-lymphopoiesis was found in unirradiated animals. The abovementioned effects could be related to the SOD-induced increase in the survival of stem cells of the bone marrow and thymus, as well as to changes in the balance of hematopoiesis activity in the organism.

The identified effects of SOD modifying immunohemopoiesis require further investigation to clarify their mechanisms. The experimental results suggest that the drug Rexod® is a promising tool for correcting immunohemopoiesis, also in post-radiation conditions.

COMPLIANCE WITH ETHICAL STANDARDS

Conflict of interest. The authors declare that they have no conflict of interest.

Statement on the welfare of animals. The welfare and feeding conditions of animals corresponded with order no. 742 on the Regulations for the Conduct of Research with Experimental Animals, November 13, 1984, and Good Laboratory Practice (order of the Ministry of Health and Social Development of the Russian Federation no. 708n, August 23, 2010). The regulations on experimental animal welfare were followed in the research, according to the Regulations for the Conduct of Research with Experimental Animals issued by the order of the Ministry of Health of the USSR (annexation to order no. 775 of the Ministry of Health of the USSR, August 12, 1977) and the standards of the Ethical Committee of the Ural Research Center for Radiation Medicine.

Translated by E. Sherstyuk

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By A. V. Akleev; I. A. Shaposhnikova; I. V. Churilova; A. N. Grebenyuk and E. A. Pryakhin

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