title: An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 1. Studies with cyclophosphamide.
abstract: The mouse micronucleus assay has long been used as an indicator of in vivo genotoxicity. Recently, it was shown that no single protocol is adequate to detect all clastogens. As a first step in developing a potentially more sensitive assay, micronucleus induction by cyclophosphamide (CP) was assessed in an in vivo/in vitro system using rat bone marrow and spleen cells. In each of two independent experiments, two rats/dose were treated i.p. with 0, 20, or 40 mg CP/kg and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested and slides prepared 24 h after initiation, while spleen cells were harvested at 48 h. One thousand cells/tissue/group were scored for cell cycle kinetics and 1000 binucleate (BN) cells were scored for micronuclei. In addition, spleen cells were concurrently assayed for chromosome aberrations. A dose-related cell cycle delay was observed in both tissues in both experiments. Bone marrow showed a 6% average background frequency of micronucleated BN cells, while the low dose induced an average of 20%, and the high dose 31%. For spleen, the average control frequency of micronucleated BN cells was 3%, the low dose induced a 40% average frequency, and the high dose 65%. Also in splenocytes, a dose-dependent increase in chromosome aberrations was observed, with an almost 40-fold increase observed over the control value at the high dose. Thus, the in vivo/in vitro approach described here shows great potential in detecting drug induced genotoxicity. Also, spleen appears more sensitive than bone marrow to CP.
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000187:drug effects / Q000648:ultrastructure; D001854:Bone Marrow Cells; D002453:Cell Cycle / Q000187:drug effects; D002869:Chromosome Aberrations; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D015162:Micronucleus Tests / Q000379:methods*; D009152:Mutagenicity Tests / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure
chemical_list: D003520:Cyclophosphamide