title: New DNA probes to detect aneugenicity in rat bone marrow micronucleated cells by a pan-centromeric FISH analysis.
abstract: When characterizing the genotoxicity of chemicals that induce micronuclei, it is practical to be able to classify the chemicals as aneugens or clastogens. This classification gives information on the mechanistic properties of chemicals and is indispensable for setting the threshold safety margins for genotoxicity in pharmaceutical development. A widely used method for detecting aneugens is fluorescence in situ hybridization (FISH) but, even though the rat is an experimental animal generally used in preclinical studies in drug development, DNA probes that hybridize to all the centromeres of rat chromosomes have not yet been established. In the present study, in addition to the previously known satellite I sequence, we identified two novel satellite sequences, satellite II and satellite III, from the rat genome database. DNA probes with a mixture of these satellite DNA sequences were used to establish a FISH method for pan-centromeric staining of rat chromosomes. To confirm the feasibility of the method, vinblastine (VBS) and mitomycin C (MMC) were administered to rats as a typical aneugen and clastogen, respectively. Micronucleated polychromatic erythrocytes (MNPCE) from bone marrow were enriched by sorting in flow cytometry and subjected to the FISH method. As a result, the ratio of centromere-positive MNPCE increased in VBS-treated rats but not in MMC-treated ones. Since the FISH method using the novel DNA probes clearly discriminates the aneugens from the clastogens, we suggest this method as a useful tool for providing mechanistic information for micronucleus induction in vivo.
authors.affiliation: Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan. takeiriakr@chugai-pharm.co.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Takeiri
authors.forename: Shigeki
authors.initials: S
authors.lastname: Motoyama
authors.forename: Kaori
authors.initials: K
authors.lastname: Matsuzaki
authors.forename: Asako
authors.initials: A
authors.lastname: Harada
authors.forename: Junko
authors.initials: J
authors.lastname: Taketo
authors.forename: Chiaki
authors.initials: C
authors.lastname: Katoh
authors.forename: Kenji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Masayuki
authors.initials: M
authors.lastname: Mishima
mesh_terms: D024422:Aneugens / Q000633:toxicity*; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity; D001483:Base Sequence; D001853:Bone Marrow / Q000187:drug effects*; D002503:Centromere / Q000187:drug effects* / Q000235:genetics; D002875:Chromosomes / Q000235:genetics; D003078:Colchicine / Q000633:toxicity; D015342:DNA Probes; D004276:DNA, Satellite / Q000032:analysis / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D005434:Flow Cytometry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects*; D015162:Micronucleus Tests; D016685:Mitomycin / Q000633:toxicity; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D050257:Tubulin Modulators / Q000633:toxicity; D014747:Vinblastine / Q000633:toxicity
chemical_list: D024422:Aneugens; D000903:Antibiotics, Antineoplastic; D015342:DNA Probes; D004276:DNA, Satellite; D009153:Mutagens; D050257:Tubulin Modulators; D016685:Mitomycin; D014747:Vinblastine; D003078:Colchicine
keywords: Aneugen; Centromere; FISH; Rat; Satellite DNA