MELPHALAN-INDUCED CYTOTOXICITY IN THE BONE MARROW OF RATS BY FLOW CYTOMETRY MEASUREMENTS
Background. Bone marrow (BM) that contains hematopoietic cells of various lineages is a sensitive target for a number of cytotoxic agents including chemotherapy drugs.
Objective. Flow cytometry (FCM) was chosen to test cytotoxicity in BM of rats, that received melphalan either intravenously (i.v.) or intraperitoneally (i.p.).
Methods. One group of rats received melphalan i.v. (3 mg/kg) followed by the BM examination on the 3rd and 7th day after drug administration, whereas another group of animals received this drug i.p. in total doses of 9 and 15 mg/kg followed by the BM examination on the next day after the 3rd and 5th injection of the drug. BM cells were stained with acridine orange and analyzed by FCM. Cytotoxicity was assessed by determining the percentage of total nucleated cells (TNC%) among the whole BM cell population and by determining the percentage of polychromatic erythrocytes (PCE%) among the whole population of enucleated erythrocytes.
Results. Regardless of the dose and regimen of melphalan administration, either i.v. or i.p. administered drug caused a significant reduction of TNC%. On the average, the i.p. administered drug resulted in about 2.0-fold decrease of TNC% (P<0.05), while the i.v. administered drug resulted in about 1.3-fold decrease of TNC% (P<0.05). As for enucleated erythrocytes, the i.p. administered drug resulted in about 1.4-fold decrease of PCE% (P<0.05), whereas the i.v. administered drug did not cause any changes in the PCE%.
Conclusions. Under these experimental conditions, i.p. administrated melphalan is considerably more cytotoxic than i.v. administered melphalan. This cytotoxic effect is preferentially due to impaired erythropoiesis.
Hurley LH. DNA and its associated processes as targets for cancer therapy. Nat Rev Cancer 2002; 2: 188-200. doi: 10.1038/nrc749.
Chabner BA, Wilson W, Supko J. Pharmacology and toxicity of antineoplastic drugs. In: Beutler E, Lichtman MA Coller BS, Kipps TJ, Seligsohn U, eds. Williams Hematology, 6th edition. New York: McGraw-Hill, 2001: 185-200.
Damia G, D’Incalci M. Mechanisms of resistance to alkylating agents. Cytotechnology 1998; 27: 165-173. doi: 10.1023/A:1008060720608.
Malhotra V, Perry MC. Classical chemotherapy: Mechanisms, toxicities and the therapeutic window. Cancer Biol Ther 2003; 2 (Suppl. 1): S2-S4. doi: 10.4161/cbt.199.
IARC. Melphalan. IARC Monogr Eval Carcinog Risk Hum 2012; 100A: 107-117.
Wantzin GL, Jensen MK. The induction of chromosome abnormalities by melphalan in rat bone marrow cells. Scand J Haemat 1973; 11: 135-139. doi: 10.1111/j.1600-0609.1973.tb00107.x.
Shelby MD, Gulati DR, Tice RR, Wojciechowski JP. Results of tests for micronuclei and chromosomal aberrations in mouse bone marrow cells with the human carcinogens 4-aminobiphenyl, treosulphan, and melphalan. Environ Mol Mutagen 1989; 13: 339-142. doi: 10.1002/em.2850130410.
Generoso WM, Witt KL, Cain KT, Hughes L, Cacheiro NLA, Lockhart A-MC, et al. Dominant lethal and heritable translocations tests with chlorambucil and melphalan in male mice. Mutat Res 1995; 345: 167-180. doi: 10.1016/0165-1218(95)90052-7.
Ranaldi R, Palma S, Tanzarella C, Lascialfari A, Cinelli S, Pacchierotti F. Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood. Mutat Res 2007; 615: 57-65. doi: 10.1016/j.mrfmmm.2006.10.001.
Sgura A, De Amicis A, Stronati L, Cinelli S, Pacchierotti F, Tanzarella C. Chromosome aberrations and telomere length modulation in bone marrow and spleen cells of melphalan-treated p53+/ mice. Environ Mol Mutagen 2008; 49: 467-475. doi: 10.1002/em.20405.
Ormerod MG (editor). Flow cytometry: a practical approach, 3rd ed. Oxford: University Press; 2000, 296 p.
Traganos F, Darzynkiewicz Z, Sharpless T, Melamed MR. Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J Histochem Cytochem 1977; 25: 46-56. doi: 10.1177/25.1.64567.
Criswell KA, Krishna G, Zielinski D, Urda GA, Theiss JC, Juneau P, et al. Use of acridine orange in: flow cytometric evaluation of erythropoietic cytotoxicity. Mutat Res 1998; 414: 49-61. doi: 10.1016/S1383-5718(98)00041-2.
Suzuki Y, Nagae Y, Li J, Sakaba H, Mozawa K, Takahashi A, et al. The micronucleus test and erythropoiesis. Effects of erythropoietin and mutagen on the ratio of polychromatic to normochromatic erythrocytes (P/N ratio). Mutagenesis 1989; 4: 420-424. doi: 10.1093/mutage/4.6.420.
Von Ledebur M, Schmid W. The micronucleus test: Methodological aspects. Mutat Res 1973; 19: 109-117. doi: 10.1016/0027-5107(73)90118-8.
Shcmid W. The micronucleus test. Mutat Res 1975; 31: 9-15. doi: 10.1016/0165-1161(75)90058-8.
Bolliger AP. Cytologic evaluation of bone marrow in rats: indications, methods, and normal morphology. Vet Clin Pathol 2004; 33: 58-67. doi: 10.1111/j.1939-165X.2004.tb00351.x.
Criswell KA, Bleavins MR, Zielinski D, Zandee JC. Comparison of flow cytometric and manual bone marrow differentials in Wistar rats. Cytometry 1998; 32: 9-17. doi: 10.1002/(SICI)1097-0320(19980501)32:1<3C9::AID-CYTO2>3.0.CO;2-I.
Criswell KA, Bock JH, Wildeboer SE, Johnson K, Giovanelli RP. Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats. Vet Clin Pathol 2014; 43: 125–136. doi: 10.1111/vcp.12132.
Criswell KA, Bleavins MR, Zielinski D, Zandee JC, Walsh KM. Flow cytometric evaluation of bone marrow differentials in rats with pharmacologically induced hematologic abnormalities. Cytometry 1998; 32: 18-27. doi: 10.1002/(SICI)1097-0320(19980501)32:1<18:AID-CYTO3>3.0.CO;2-B.
Criswell KA, Bock JH, Wildeboer SE, Johnson K, Giovanelli RP. Comparison of the Sysmex XT-2000iV and microscopic bone marrow differential counts in Wistar rats treated with cyclophosphamide, erythropoietin, or serial phlebotomy. Vet Clin Pathol 2014; 43: 137-153. doi: 10.1111/vcp.12149.
Rosenthal RL, Pickering BI, Goldschmidt L. A semi-quantitative study of bone marrow in rats following total body X-irradiation. Blood 1951; 6: 600-613.
Peslak SA, Wenger J, Bemis JC, Kingsley PD, Frame JM, Koniski AD, et al. Sublethal radiation injury uncovers a functional transition during erythroid maturation. Exp Hematol. 2011; 39: 434-445. doi: 10.1016/j.exphem.2011.01.010.
Goldenberg GJ, Alexander P. The effects of nitrogen mustard and dimethyl myleran on murine leukemia cell lines of different radiosensitivity in vitro. Cancer Res 1965; 25: 1401-1409.
Hall EJ. Radiobiology for the radiologist, 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2000, 588 p.
Shevchuk OO, Posokhova KA, Sidorenko AS, Bardakhivska KI, Maslenny VM, Yushko LA, et al. The influence of enterosorption on some haematological and biochemical indices of the normal rats after single injection of melphalan. Exp Oncol 2014; 36: 94-100.
Shevchuk OO, Posokhova KA, Todor IN, Lukianova NY, Nikolaev VG, Chekhun VF. Prevention of myelosuppression by combined treatment with enterosorbent and granulocyte colony-stimulating factor. Exp Oncol 2015; 37: 135-138.
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