Simultaneous evaluation of clastogenicity, aneugenicity and toxicity in the mouse micronucleus assay using immunofluorescence.
An improved antikinetochore antibody technique was established in the mouse micronucleus assay to simultaneously evaluate toxicity, clastogenicity and aneugenicity induced by various test agents. The procedure involved the use of cellulose column fractionated cytospun slides for analysis. The staining method consisted of sequential treatment of slides with crest serum, fluorosceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, polychromatic erythrocytes (PCEs, dark red), normochromatic erythrocytes (NCEs, green), chromosome(s)/fragments/micronuclei (orange), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, cyclophosphamide, X-rays and vincristine were tested for micronucleus/aneuploidy induction and bone marrow toxicity. The aneugen, vincristine, and clastogens, X-rays and cyclophosphamide, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated PCEs, respectively. At the doses tested, cyclophosphamide caused a slight but statistically significant decrease in PCEs in females, and other agents did not produce any severe bone-marrow toxicity in either male or female mice. These results are comparable with the results reported in the literature on these compounds with various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity and in evaluating toxicity.
Krishna
G
G
Department of Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105.
Fiedler
R
R
Theiss
J C
JC
D000782:Aneuploidy; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects / Q000528:radiation effects; D001854:Bone Marrow Cells; D003520:Cyclophosphamide / Q000633:toxicity; D004912:Erythrocytes / Q000187:drug effects / Q000528:radiation effects; D005260:Female; D005455:Fluorescent Antibody Technique; D008297:Male; D051379:Mice; D015162:Micronucleus Tests / Q000379:methods*; D012737:Sex Factors; D013223:Statistics as Topic; D014750:Vincristine / Q000633:toxicity
D014750:Vincristine; D003520:Cyclophosphamide