Abstract
We established test conditions for colony-forming unit assay using rat bone marrow cells in which the colony specimens can be preserved. In our test system, all colonies can be transferred from a petri dish to a slide glass with whole agar medium because the agar medium volume is small and movable, enabling the agar medium to be dried and then fixed on the slide glass. Appropriate conditions for the colony-forming unit granulocyte–macrophage assay were as follows: 10 to 30 ng/ml recombinant murine granulocyte and macrophage colony-stimulating factor, 1 × 105 cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for the colony-forming unit erythroid assay were as follows: 2 IU/ml recombinant human erythropoietin, 0.25 to 1 × 105 cells/dish, and culture period of 2–3 days. We also examined responses of bone marrow toxicants 5-fluorouracil and doxorubicin in these test systems, finding that both compounds decreased the numbers of colonies in a concentration-dependent manner in both assays. These findings suggest that these test systems are useful tools in evaluating bone marrow toxicity of these particular compounds.
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References
Aguiar JLA, Berger MR, Frank N (1987) Locoregional application of 5-fluorouracil to the liver: alteration of pharmacokinetics and bone marrow toxicity in rats with thioacetamide-induced cirrhosis. Toxicol Lett 36:181–188
Badami CD, Livingston DH, Sifri ZC, Caputo FJ, Bonilla L, Mohr AM, Deitch EA (2007) Hematopoietic progenitor cells mobilize to the site of injury after trauma and hemorrhagic shock in rats. J Trauma 63:596–602
Beamonte A, Goldfain-Blanc F, Casadevall N, Bazot D, Bertheux H, Claude N (2005) A case of drug-induced hematotoxicity: from in vivo to in vitro assessment. Comp Clin Pathol 14:61–65
Comereski CR, Peden WM, Davidson TJ, Waener GL, Hirth RS, Frants JD (1994) BR96-doxorubicn conjugate (BMS-182248) versus doxorubicin: a comparative toxicity assessment in rats. Toxicol Pharmacol 22:473–488
Deldar A, Lewis H, Bloom J, Weiss L (1988) Reproducible cloning assays for in vitro growth of canine hematopoietic progenitor cells and their potential applications in investigative hematotoxicity. Am J Vet Res 49:1393–1401
French RA, Broussard SR, Meier WA, Minshall C, Arkins S, Zachary JF, Dantzer R, Kelley KW (2002) Age-associated loss of bone marrow hematopoietic cells is reversed by GH and accompanies thymic reconstitution. Endocrinology 143:690–699
Hannoush EJ, Sifri ZC, Elhassan IO, Mohr AM, Alate WD, Offin M, Livingston DH (2011) Impact of enhanced mobilization of bone marrow derived cells to site of injury. J Trauma 71:283–291
Kubota K, Mizoguchi H, Miura Y, Suda T, Takaku F (1980) A new technique for the cytochemical examination of human hematopoietic cells grown in agar gel. Exp Hematol 8:339–344
Matsumura-Takeda K, Kotosai K, Ozaki A, Hara H, Yamashita S (2002) Rat granulocyte colony-forming unit (CFU-G) assay for the assessment of drug-induced hematotoxicity. Toxicology in Vitro 16:281–288
Moezzi J, Ali-Osman F, Murphy MJ (1986) Rapid method for permanent slide preparation of colonies in soft agar cultures. Int J Cell Cloning 4:368–374
National Research Council (1996) Guide for the care and use of laboratory animals. National Academy Press, Washington
Pannacciulli I, Muzzulini C, Massa G, Bogliolo G (1984) Cytotoxic effect of dose and schedule on normal murine hematopoietic progenitor cells following the administration of 4′-deoxydoxorubicin. Br J Cancer 50:647–656
Parchment RE, Huang M, Erickson-Miller CL (1993) Roles for in vitro myelotoxicity tests in preclinical drug development and clinical trial planning. Toxicol Pathol 21:241–250
Pessina A, Bonomi A, Baglio C, Cavicchini L, Gribaldo L (2009) Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications. ATLA 37:417–425
Pessina A, Bonomi A, Cavicchini L, Albella B, Cerrato L, Parent-Massin D, Sibiril Y, Parchment R, Behrsing H, Verderio P, Pizzamiglio S, Giangreco M, Baglio C, Sisto F, Coccè V, Gribaldo L (2010) Prevalidation of the rat CFU-GM assay for in vitro toxicology applications. ATLA 38:105–117
Reagan WJ, Handy V, Mckamey A, Bloom JC, Deldar A, Stevens C, Chan TCK (1993) Effect of doxorubicin on the canine erythroid and myeloid progenitor cells and bone marrow microenvironment. Comp Haematol Int 3:96–101
Reagan WJ, Irizarry-Rovira A, Poitout-Belissent F, Bolliger AP, Ramaiah SK, Travlos G, Walker D, Bounous D, Walter G (2011) Best practices for evaluation of bone marrow in nonclinical toxicity studies. Toxicol Pathol 39:435–448
Rich IN (1991) The effect of 5-fluorouracil on erythropoiesis. Blood 77:1164–1170
Shifri ZC, Ananthakrishnan P, Wang L, Mohr AM, Hauser CJ, Rameshwar P, Deitch EA, Livingston DH (2006) Bone marrow failure in male rats following trauma/hemorrhagic shock (T/HS) is mediated by mesenteric lymph and modulated by castration. Shock 25:12–16
Sonneveld P, Mulder JA, Bekkum DW (1981) Cytotoxicity of doxorubicin for normal hematopoietic and acute myeloid leukemia cells of the rat. Cancer Chemother Pharmacol 5:167–173
Takamatsu K, Wakata A, Shishido T, Izumisawa S, Hirota R, Matsuzawa T (1998) The time course of leucocyte colony-forming in cultures of bone marrow progenitor cells from rats and dogs. Comp Hematol Int 8:191–196
USDA (1989, 1991) 9 CFR Parts 1 and 2 Final Rules.
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Takamatsu, K., Wakata, A., Tabata, H. et al. Refinement and verification of test conditions for colony-forming unit assay in rat bone marrow cells with preservable colony specimens. Comp Clin Pathol 23, 569–575 (2014). https://doi.org/10.1007/s00580-012-1653-9
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DOI: https://doi.org/10.1007/s00580-012-1653-9