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: 4.44). 12/2007; 634(1-2):235-40. DOI: 10.1016/j.mrgentox.2007.07.010
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Testing new chemical entities for genotoxicity is an integral part of the preclinical drug-development process. Lowering the detection limit and enhancing the sensitivity of genotoxicity assays is required, as the standard test-battery fails to detect some carcinogens (non-genotoxic) and weak genotoxins. One of the mechanisms that affect the detection of weak genotoxins is related with the DNA-repair efficiency of the cell system used. In the present study, 3-aminobenzamide (3-AB, 30 mg/kg body-weight), a poly(ADP-ribose)polymerase inhibitor, was used to evaluate the DNA-damaging potential of zidovudine (AZT, 400 mg/kg bw), doxorubicin (DOX, 5 mg/kg bw) and cyclophosphamide (CP, 50 mg/kg bw, as a positive control) and sucrose (SUC, 3 g/kg bw, as a negative control) in Swiss female mice. The endpoints considered included micronucleus formation, DNA breakage (in peripheral blood lymphocytes, bone marrow and liver; comet assay) and chromosome aberrations, as well as immunohistochemistry of PARP-1 and phosphorylated histone H2AX (γ-H2AX). The results clearly indicate that the genotoxicity of zidovudine (AZT), doxorubicin (DOX) and cyclophosphamide (CP) was significantly increased in the combination treatments (3-AB+AZT, 3-AB + DOX, 3-AB + CP) as compared with the respective controls (treatment with AZT, DOX and CP alone). There was no increase in the genotoxicity per se after treatment with SUC, 3-AB or 3-AB + SUC, compared with the control (saline). Correlation analysis suggests that all genotoxicity parameters are well correlated with each other. The results clearly show that the genotoxicity of weak genotoxins can be enhanced and detected in the presence of 3-AB in mice. Thus, this approach can be used in the pre-clinical genotoxicity screening of weak genotoxins [propose adding this concluding sentence here (copied from Introduction) Ed.].
    Mutation Research/Genetic Toxicology and Environmental Mutagenesis 08/2014; 770. DOI:10.1016/j.mrgentox.2014.04.020 · 2.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BK polyomavirus (BKPyV) is an emerging pathogen whose reactivation causes severe disease in transplant patients. Unfortunately, there is no specific anti-BKPyV treatment available, and host cell components that affect the infection outcome are not well characterized. In this report, we examined the relationship between BKPyV productive infection and the activation of the cellular DNA damage response (DDR) in natural host cells. Our results showed that both the ataxia-telangiectasia mutated (ATM)- and ATM and Rad-3-related (ATR)-mediated DDR were activated during BKPyV infection, accompanied by the accumulation of polyploid cells. We assessed the involvement of ATM and ATR during infection using small interfering RNA (siRNA) knockdowns. ATM knockdown did not significantly affect viral gene expression, but reduced BKPyV DNA replication and infectious progeny production. ATR knockdown had a slightly more dramatic effect on viral T antigen (TAg) and its modified forms, DNA replication, and progeny production. ATM and ATR double knockdown had an additive effect on DNA replication and resulted in a severe reduction in viral titer. While ATM mainly led to the activation of pChk2 and ATR was primarily responsible for the activation of pChk1, knockdown of all three major phosphatidylinositol 3-kinase-like kinases (ATM, ATR, and DNA-PKcs) did not abolish the activation of γH2AX during BKPyV infection. Finally, in the absence of ATM or ATR, BKPyV infection caused severe DNA damage and aberrant TAg staining patterns. These results indicate that induction of the DDR by BKPyV is critical for productive infection, and that one of the functions of the DDR is to minimize the DNA damage which is generated during BKPyV infection.
    PLoS Pathogens 08/2012; 8(8):e1002898. DOI:10.1371/journal.ppat.1002898 · 8.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Black cohosh rhizome (Actaea racemosa) is used as a remedy for pain and gynecological ailments; modern preparations are commonly sold as ethanolic extracts available as dietary supplements. Black cohosh was nominated to the National Toxicology Program (NTP) for toxicity testing due to its widespread use and lack of safety data. Several commercially available black cohosh extracts (BCE) were characterized by the NTP, and one with chemical composition closest to formulations available to consumers was used for all studies. Female B6C3F1/N mice and Wistar Han rats were given 0, 15 (rats only), 62.5 (mice only), 125, 250, 500, or 1000 mg/kg/day BCE by gavage for 90 days starting at weaning. BCE induced dose-dependent hematological changes consistent with a non-regenerative macrocytic anemia and increased frequencies of peripheral micronucleated red blood cells (RBC) in both species. Effects were more severe in mice, which had decreased RBC counts in all treatment groups and increased micronucleated RBC at doses above 125 mg/kg. Dose-dependent thymus and liver toxicity was observed in rats but not mice. No biologically significant effects were observed in other organs. Puberty was delayed 2.9 days at the highest treatment dose in rats; a similar magnitude delay in mice occurred in the 125 and 250 mg/kg groups but not at the higher doses. An additional uterotrophic assay conducted in mice exposed for 3 days to 0.001, 0.01, 0.1, 1, 10, 100 and 500 mg/kg found no estrogenic or anti-estrogenic activity. These are the first studies to observe adverse effects of BCE in rodents.
    Toxicology and Applied Pharmacology 06/2012; 263(2):138-47. DOI:10.1016/j.taap.2012.05.022 · 3.98 Impact Factor

Full-text (2 Sources)

Available from
May 26, 2014