doi: 10.15389/agrobiology.2021.4.763eng

UDC: 619:615.36:575.224.46



S.V. Shabunin, G.A. Vostroilova , P.A. Parshin, D.I. Shabanov,
N.A. Khokhlova

All-Russian Research Veterinary Institute of Pathology, Pharmacology and Therapy, 114-b, ul. Lomonosova, Voronezh, 394087 Russia, e-mail (✉ corresponding author),,,,

Shabunin S.V.
Shabanov D.I.
Vostroilova G.A.
Khokhlova N.A.
Parshin P.A.

Received April 29, 2021


The tissue drug aminoseleton, designed at the All-Russian Veterinary Research Institute of Pathology, Pharmacology and Therapy, was obtained from the spleen of cattle by cryogenic fractionation. Adaptogenic, membrane stabilizing, stress-protective, antioxidant and immunomodulatory properties of aminoseleton have been already shown. In this work, the anticlastogenic effect of the tissue drug aminoseleton on the bone marrow cells of mice exposed to the experimental mutagen was revealed for the first time. In addition, the preservation of cytogenetic stability and mitotic activity in bone marrow cells of healthy animals was shown when using the study drug. The objective of this work was to assess the effect of aminoseleton on the cytogenetic stability of bone marrow cells in healthy mice and mice exposed to the experimental mutagen, as well as to identify the antimutagenic properties of the drug in relation to the genotoxic effect of cyclophosphamide (CP) using a micronucleus test. The experiments were carried out on outbred white mice (Mus albus officinarum), which were divided into six groups subjected to the following treatments: i) intramuscular administration of sterile isotonic sodium chloride solution in a volume of 0.2 ml (negative control, n = 12); ii) intraperitoneal injection of 0.2 ml of CP (Baxter Oncology GmbH, Germany) at a dose of 20.0 mg/kg of body weight (positive control, n = 12); iii) intramuscular single injection of 0.2 ml of aminoseleton at a therapeutic dose of 0.5 ml/kg (n = 12); iv) intramuscular single injection of 0.2 ml of aminoseleton at a tenfold therapeutic dose of 5.0 ml/kg (n = 12); v) intramuscular single injection of 0.2 ml of aminoseleton at a dose of 0.5 ml/kg with intraperitoneal injection of 0.2 ml of CP at a dose of 20.0 mg/kg in 72 h (n = 6); vi) intramuscular fivefold injection of 0.2 ml of aminoseleton at a dose of 0.5 ml/kg with 24 h intervals and intraperitoneal administration of CP is similar to animals of other groups 72 h after the fifth injection (n = 6). To determine the amount of chromosomal aberrations in the bone marrow, 2.5 h before euthanasia, mice were injected intraperitoneally with 0.025 % colchicine (PanEco, Russia). Bone marrow cells were washed out of the femurs using Hanks’ buffer solution (pH 7.4), the cell suspension was incubated in 0.075 molar hypotonic KCl solution, then the cells were fixed with acetoalcohol cooled to 4 °C and stained by Romanowsky-Giemsa procedure. The mitotic index (MI) was assessed by the number of dividing cells per 1000 bone marrow cells. The number of cells with chromosomal aberrations was counted in 100 metaphase plates per animal. Single and paired fragments, exchanges and achromatic gaps, as well as cells with multiple pathologies were counted. To study the frequency of micronuclei (micronucleus test) of polychromatophilic erythrocytes (PCE), the obtained bone marrow cells were added to 1 % albumin solution in Hanks’ buffer solution (pH 7.4) and applied to glass slides, then the samples were dried, fixed with methanol and stained by Romanowsky-Giemsa protocol. The frequency of micronuclei per 1000 PCE was determined; a total of 2000 PCE per animal was studied. The proportion of PCE per 500 normochromic erythrocytes (NE) and PCE was also calculated. The frequency of chromosomal aberrations and micronuclei when administering the drug at the studied doses did not statistically significantly differ from that in animals of their negative control group that was 1.0±0.40 and 0.2±0.06 %, respectively. The administration of aminoseleton also had no effect on the mitotic index of bone marrow cells in experimental animals. The course administration of aminoseleton reduced the clastogenic effect of cyclophosphamide, assessed by the number of micronuclei in polychromatophilic erythrocytes of the bone marrow, from 2.3±0.21 % in mice from the positive control group to 1.0±0.40 % in animals after a course of aminoseleton injections. Thus, the clastogenic activity of cyclophosphamide decreased by 51.3 % that was probably due to the correction of the prooxidant-antioxidant system of the animal body with the studied drug. A decrease in the number of micronuclei induced by cyclophosphamide in polychromatophilic erythrocytes of the bone marrow indicates the presence of an anticlastogenic potential in aminoseleton.

Keywords: aminoseleton, cyclophosphamide, mutagenicity, anticlastogenic properties, micronuclei, chromosomal aberrations, bone marrow, white mice, polychromatophilic erythrocytes.



  1. Uchasov D.S., Yarovan N.I., Sein O.B. Vestnik Orlovskogo gosudarstvennogo agrarnogo universiteta, 2013, 1(40): 129-131 (in Russ.).
  2. Plenina L.V., Soroka N.F., Chudakov O.P., Tret'yak S.I., Bykadorova L.G., Mit'kovskaya N.P., Golynskii A.B., Maksimovich A.V., Evseenko V.M., Ivanovskii G.L., Alikevich I.N., Drazhina L.S., Grak N.N., Skuratovskaya L.I. Retsept, 2008, 1(57): 104-106 (in Russ.).
  3. Fedulova L.V., Vasilevskaya E.R. Myasnye tekhnologii, 2016, 12(168): 37-39 (in Russ.).
  4. Chernenkova M.L., Styazhkina S.N., Valinurov A.A. Zhurnal nauchnykh statei zdorov'e i obrazovanie v XXI veke, 2017, 19(8): 161-163 (in Russ.).
  5. Vostroilova G.A., Khokhlova N.A., Parshin P.A., Cheskidova L.V., Bryukhova I.V., Sashnina L.Yu., Kantorovich Yu.A., Kartashov S.S. Veterinarnyi farmakologicheskii vestnik, 2018, 2(3): 37-41 CrossRef (in Russ.).
  6. Shabunin S.V., Vostroilova G.A., Parshin P.A., Khokhlova N.A., Sashnina L.Yu., Mikhailov E.V., Tyurina E.V. Veterinarnaya patologiya, 2018, 3(65): 39-46 CrossRef (in Russ.).
  7. Rukovodstvo po provedeniyu doklinicheskikh issledovanii lekarstvennykh sredstv. Chast' pervaya /Pod redaktsiei A.N. Mironova [Guidelines for conducting preclinical studies of drugs. Part one. A.N. Mironov (ed.)]. Moscow, 2012 (in Russ.).
  8. Durnev A.D. Fiziologiya cheloveka, 2018, 44(3): 116-137 CrossRef (in Russ.).
  9. Genetic toxicology: principles and methods. J.M. Parry, E.M. Parry (eds.). Springer, New York, 2012 CrossRef
  10. Delarmelina J.M., Dutra J.C.V., Batitucci Mdo C. Antimutagenic activity of ipriflavone against the DNA-damage induced by cyclophosphamide in mice. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association, 2014, 65: 140-146 CrossRef
  11. Shabunin S.V., Shakhov A.G., Vostroilova G.A., Parshin P.A., Ermolova T.G., Khokhlova N.A., Bliznetsova G.N. Dostizheniya nauki i tekhniki APK,2019, 33(7): 71-74 CrossRef (in Russ.).
  12. Emadi A., Jones R.J., Brodsky R.A. Cyclophosphamide and cancer: golden anniversary. Nature reviews. Clinical Oncology, 2009, 6(11): 638-647 CrossRef
  13. Nau H., Spielmann H., Lo Turco Morter C.M., Winckler K., Riedel L., Obe G. Mutagenic, teratogenic and pharmacokinetic properties of cyclophosphamide and some of its deuterated derivatives. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1982, 95(2-3): 105-118 CrossRef
  14. Preston R.J., Dean B.J., Galloway S., Holden H., McFee A.F., Shelby M. Mammalian in vivo cytogenetic assays Analysis of chromosome aberrations in bone marrow cells. Mutation Research/Genetic Toxicology, 1987, 189(2): 157-165 CrossRef
  15. Hayashi M. The micronucleus test—most widely used in vivo genotoxicity test. Genes and Environment, 2016, 38: 18 CrossRef
  16. Agarwal D.K., Chauhan L.K. An improved chemical substitute for fetal calf serum for the micronucleus test. Biotechnic & Histochemistry, 1993, 68(4): 187-188 CrossRef
  17. Lu W., Jia D., An S., Mu M., Qiao X., Liu Y., Li X., Wang D. Calf Spleen Extractive Injection protects mice against cyclophosphamide-induced hematopoietic injury through G-CSF-mediated JAK2/STAT3 signaling. Scientific Reports, 2017, 7(1): 8402 CrossRef
  18. Hartleb M., Leuschner J. Toxicological profile of a low molecular weight spleen peptide formulation used in supportive cancer therapy. Arzneimittel-Forschung, 1997, 47(9): 1047-1051.
  19. Dychko K.A., Ryzhova G.L., Kravtsova S.S., Kir'yanova N.L., Kuvshinov N.N., Gridneva V.I. Sposob polucheniya sredstva s adaptogennym i protivoluchevym deistviem. Patent RU 2142284 C1 (RF) MPK6 A 61 K 35/28. Tomskii gosudarstvennyi universitet (RF). № 95110155/14. Zayavl. 14.06.1995. Opubl. 10.12.1999 [Method of obtaining a substance with adaptogenic and antiradiation effects. Patent RU 2142284 C1 (RF) MPK6 A 61 K 35/28. Tomsk State University (RF). № 95110155/14. Appl. 14.06.1995. Publ. 10.12.1999] (in Russ.).
  20. Khokhlova N.A., Lobodina T.E., Grigor'eva N.A., Topol'nitskaya A.V., Fedorova N.M., Panina T.A. Veterinarnyi farmakologicheskii vestnik, 2018, 1(2): 25-30 CrossRef (in Russ.).
  21. Goncharova R.I., Kuzhir T.D. Ekologicheskaya genetika, 2005, 3(3): 19-32 (in Russ.).
  22. Hosseinimehr S.J., Karami M. Chemoprotective effects of captopril against cyclophosphamide-induced genotoxicity in mouse bone marrow cells. Archives of Toxicology, 2005, 79(8): 482-486 CrossRef
  23. Vijayalaxmi K.K., Venu R. In vivo anticlastogenic effects of L ascorbic acid in mice. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 1999, 438(1): 47-51 CrossRef
  24. Manda K., Bhatia A.L. Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice. Cell Biology and Toxicology, 2003, 19(6): 367-372 CrossRef
  25. De Flora S., Ramel C. Mechanisms of inhibitors of mutagenesis and carcinogenesis. Classification and overview. Mutation Research, 1988, 202(2): 285-306 CrossRef
  26. Bolzán A.D., Lacunza E., Bianchi M.S. Effect of recombinant interferon-alpha on streptonigrin-induced chromosome aberrations and sister-chromatid exchanges in hamster cells. Mutation Research, 2003, 522(1-2): 127-134 CrossRef
  27. Novitskii V.V., KHlusova M.YU., Ternovaya S.V., Saratikov A.S. Antimutagennoe sredstvo. Patent RU 2189232 C2 (RF) MPK7 A 61 K 31/415, A 61 P 43/00. NII farmakologii Tomskogo nauchnogo tsentra RAMN (RF). Sibirskii meditsinskii universitet (RF). № 2000118409/14. Zayavl. 10.07.2000. Opubl. 20.09.2002 [Antimutagenic agent. Patent RU 2189232 C2 (RF) MPK7 A 61 K 31/415, A 61 P 43/00. Research Institute of Pharmacology of the Tomsk Scientific Center of the Russian Academy of Medical Sciences (RF). Siberian Medical University (RF). № 2000118409/14. Appl. 10.07.2000. Publ. 20.09.2002] (in Russ.).
  28. Jia D., Lu W., Wang C., Sun S., Cai G., Li Y., Wang G., Liu Y., Zhang M., Wang D. Investigation on immunomodulatory activity of calf spleen extractive injection in cyclophosphamide-induced immunosuppressed mice and underlying mechanisms. Scandinavian Journal of Immunology, 2016, 84(1): 20-27 CrossRef







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