Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (Impact Factor: 3.68). 12/2007; 634(1-2):235-40. DOI: 10.1016/j.mrgentox.2007.07.010
Source: PubMed


Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.

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Available from: Makoto Hayashi, Jan 09, 2014
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    • "A one-tailed independent t-test was used to verify a significant (P = 0.05) induction of DNA damage by the positive control compound, EMS. MN Assay—Because measurements of MN frequency are obtained from a large number of cells using flow cytometry, it is reasonable to assume that the proportion of micronucleated cells is approximately normally distributed within each sample [Kissling et al., 2007]. The NTP uses Levene's test at α = 0.05 to test for equal variances among the treatment groups. "
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    ABSTRACT: Styrene-acrylonitrile Trimer (SAN Trimer), a by-product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2-year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN-RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose-related increases (P < 0.0001) in MN-RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical-related bone marrow toxicity. Results of the Comet assay showed significant, dose-related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical-related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer.
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    • "However, evidence has been accumulating which suggests that blood can be used effectively to perform in vivo micronucleus assays, even in species with efficient splenic filtration function [8] [9] [10] [11] [12] [13] [14] [15]. The ability of flow cytometry, compared to conventional microscopy, to survey many times more cells for the presence of MN has made it possible to maintain assay sensitivity despite spleen function [16]. The fact that the MN analyses can be restricted to the youngest fraction of reticulocytes (CD71-positive) may also contribute to the retention of sensitivity [17]. "
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    ABSTRACT: Erythrocyte-based micronucleus tests have traditionally been performed with bone marrow specimens, since, in most preclinical animal models, the spleen can efficiently remove aberrant erythrocytes from the circulation. Even so, evidence is mounting that by examining tens of thousands of young (CD71-positive) circulating reticulocytes for the presence of micronuclei via flow cytometry, a sensitive assay of cytogenetic damage is realized. The work described herein was designed to test this hypothesis further, using an important preclinical toxicology model, the beagle dog. In these experiments, purebred male beagles were treated for five consecutive days with cyclophosphamide (0, 6.25, 12.5 or 25mg/m(2)/day) or for two consecutive days with etoposide (0, 1.56, 6.25 or 12.5mg/m(2)/day). Before treatment, and on each day of administration, blood specimens were collected and processed for flow cytometric scoring of micronucleated reticulocyte (MN-RET) frequency. Twenty-four hours after the final administration, blood MN-RET frequencies were determined via flow cytometry, and frequencies of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) were determined using acridine orange and May-Grunwald Giemsa staining. In the case of cyclophosphamide, elevated blood MN-RET frequencies were observed 2 days after treatment began, and the maximal frequency was achieved 1 day later. Similarly, etoposide-induced blood MN-RET were not evident 1 day after administration began, but a robust effect was apparent 2 days after treatments were initiated. Twenty-four hours after the final administrations, dose-related micronucleus responses were evident for both agents and in both blood and bone marrow compartments. Good overall agreement between MN-RET and MN-PCE frequencies was evidenced by high Spearman's correlation coefficients-0.89 for blood flow cytometry versus bone marrow acridine orange staining and 0.83 for blood flow cytometry versus bone marrow May-Grunwald Giemsa staining. Taken together, these results provide further support for the cross-species utility of flow cytometry-based blood MN-RET measurements.
    Full-text · Article · Feb 2012 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
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