The In Vitro Micronucleus Test

The principle of the method is to expose cell cnltures to the test substance in both the presence and absence of an in vitro metabolizing system. After exposure, the cultures are grown for a period sufficient to allow chromosome damage or chromosome loss to lead to the formation of micronnclei in interphase cells (usually 

5-2 normal cell cycles after the start of treatment). Harvested and stained interphase cells are then analyzed microscopically for the presence of micronuclei. If the cytokinesis-block technique is used, micronucleus analysis is restricted to binucleate cells, and at least 1000 lymphocytes per duplicate culture should additionally be classified as mononneleates, binucleates, or multinncleates to estimate the cytokinesis-block prolifaadon index, which is a measure of cell cycle delay. 

Micronuclei formed by aneuploidy induction can be distingnished from those produced by clastogenic activity by the presence of centromeric DNA or kinetochore 

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Positive results from the in vitro micronucleus test indicate that the test substance induces chromosome damage and/or damage to the cell division apparams, in cultured mammahan somatic cells. Immunochemical labehng (FISH fluorescence in sim hybridization) of kinetochores, or hybridization with general or chromosome specific centromeric/telomeric probes can provide useful information on the mechanism of micronucleus formation. Use of cytokinesis block facilitates the acquisition of the additional mechanistic information (e.g., chromosome nondisjunction) that can be obtained by FISH techniques. The micronucleus assay has a number of advantages over metaphase analysis performed to measure chromosome aberrations (see OECD TG 487 draft). 

Flow cytometric assessment generates results that reproduce microscopic methods for the in vivo assessment of micronucleus frequency in peripheral blood micronucleated reticulocytes [30] and the method is increasingly applied in in vitro assessments. Litron Laboratories (Rochester, N.Y., USA) recently launched their MicroFlow In Vitro kit for the in vitro micronucleus test, following a six-compound interlaboratory evaluation [31]. This method permits 50 samples to be analyzed over... 

Vian, L., Bichet, N. Gouy D. (1993) The in vitro micronucleus test on isolated human lymphocytes. Mutat. Res., 291, 93-102... 

MILLER B, POTTER-LOCKER E, SEELBACH A, STOPPER H, UTESCH D and MADLE S (1998) Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay position of the GUM Working Group on the in vitro micronucleus test. Gesellschaft fur Umwelt-Mutations-Forschung, Mutat Res. 410, 81-116. 

Frieauff W, Potter-Locher F, Cordier A, et al. Automatic analysis of the in vitro micronucleus test on V79 cells. Mutat Res. 1998 413(l) 57-68. 

Garriott ML, Phelps JB, Hoffman WP. A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity. Mutat Res 2002 517 123-34. 

In the in vitro micronucleus test usually human peripheral blood lymphocytes are used. To distinguish cells that divided just once in culture, the cultures are treated with cytochalasin-B, a chemical that blocks actin polymerization and, as such, also cytokinesis. After one cell cycle, binucleated cells eventually with one or more micronuclei are obtained (Figure 8.7). It is necessary to distinguish the cells that divided in culture, because cell division is required for the formation of a micronucleus and a very small fraction of lymphocytes may have acquired micronuclei in vivo. Furthermore, micronuclei should be detected in cells that divided only once in culture in order to avoid an underestimation of the micronucleus frequency due to cell death. 

The in vitro micronucleus test (MNvit) represents a reliable and relevant alternative to the CAvit for the assessment of genetic damage to chromosomes, in the form of either clastogenic (chromosome break) or aneugenic (whole chromosome) events, as shown by the EURL ECVAM retrospective validation study [57-60],... 

Zhang LS, Honma M, Hayashi M, Suzuki T, Matsuoka A et al (1995) A comparative study of TK6 human lymphoblastoid and L5178Y mouse lymphoma cell lines in the in vitro micronucleus test. Mutat Res 347 105-115... 

A very recent addition to the collection of OECD Guidelines is a draft guideline on the in vitro micronucleus test (OECD 2006) on the basis of various validation exercises for this test (Corvi et al. 2008 Kirsch-Volders et al. 1997, 2003). There is a great interest in this guideline since this test approach is simpler in evaluation than the cytogenetic evaluation of chromosome aberrations that are normally required by guidelines. 

II. The in vitro micronucleus test is endorsed as an alternative option to the in vitro chromosome aberration test and the mouse l)miphoma tk assay. 

Kirsch-Volders, M., Elhajouji, A., Cundari, E., and Van Hummelen, P. (1997). The in vitro micronucleus test A multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat Res 392, 19-30. 

Lorge, E., Lambert, C., Gervais, V, Becourt-Lhote, N., Delongeas, J. L., and Claude, N. (2007). Genetic toxicity assessment Employing the best science for human safety evaluation. Part II Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay. Toxicol Sci 96, 214-217. 

Until recently, most regulatory guidelines have focused mainly on tests for gene mutations and structural chromosome damage. HowevCT, the validation of the in vitro micronucleus test and the development of an OECD gnideline indicates that its use will become much more widespread (OECD 2007). 

Van Goethem F, Lison D and Kirsch-Volders M (1997) Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gd electrophoresis assay for the detection of DNA dam-aging agents genotoxic ffects of cobalt powder, tungsten carbide and cobalt-tungsten carbide. Mutat Res 392(l-2) 31-43. 

Kirkland, D., Reeve, L., Gatehouse, D., Vanparys, P. (2011). A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutation Research, 721, 27-73. 

Lorge, E., Hayashi, M., Albertini, S., Kirkland, D. (2008). Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test I. Theoretical aspects. Mutation Research, Vol. 655, pp. 1-3, ISSN 0027-5107 Lypez-Otin, C, Matrisian, RM (2007). Emerging roles of proteases in tumour suppression. 

P. van Hummelen, A. Elhajouji and M. Kirsch-Volders, Clastogenic and aneugenic effects of three benzimidazole derivatives in the in vitro micronucleus test using human lymphocytes, Mutagenesis, 1995, 10, 23-29. 

To determine the presence of aneugenes and clastogenes by means of in vivo method (B12-TG 474, OECD, 1997c), the micronuclens in cell lines is a method to partially replace laboratory animals. The purpose of the in vitro micronuclens assay is to identily agents that cause structural and numerical chromosome changes. The in vitro micronucleus test may employ cultures of established cell lines or primary cell cnltnres (Evans, 1976). However, until now OECD test guidelines for this method have not been snbmit-ted, nor has any formal valid method been found yet. 

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