title: Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test.
abstract: The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay.
authors.affiliation: Dow Chemical Company, Toxicology and Environmental Research and Consulting, Midland, Michigan 48674, USA. mjlebaron@dow.com
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: LeBaron
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Schisler
authors.forename: Dorothea K
authors.initials: DK
authors.lastname: Torous
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Dertinger
authors.forename: B Bhaskar
authors.initials: BB
authors.lastname: Gollapudi
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D023381:Endpoint Determination; D005434:Flow Cytometry / Q000379:methods*; D017408:Guidelines as Topic; D051379:Mice; D008815:Mice, Inbred Strains; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D011990:Receptors, Transferrin / Q000096:biosynthesis; D017701:Reticulocyte Count; D012156:Reticulocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013045:Species Specificity
chemical_list: D015703:Antigens, CD; C068126:CD71 antigen; D009153:Mutagens; D011990:Receptors, Transferrin