MICRONUCLEUS TEST

A micronucleus (MN) is formed during the metaphase/anaphase transition of mitosis (cell division). It may arise from a whole lagging chromosome (aneugenic event leading to chromosome loss) or an acentric chromosome fragment detaching from a chromosome after breakage (clastogenic event) which do not integrate in the daughter nuclei.

Scoring of micronuclei can be performed relatively easily and on different cell types relevant for human biomonitoring: lymphocytes, fibroblasts and exfoliated epithelial cells, without an extra in vitro cultivation step. MN observed in exfoliated cells are not induced when the cells are at the epithelial surface, but when they are in the basal layer.

An ex vivo / in vitro analysis of lymphocytes in the presence of cytochalasin-B, an inhibitor of actins (added 44 hours after the start of cultivation), allows to distinguish easily between mononucleated cells which did not divide and binucleated cells which completed nuclear division during in vitro culture. Indeed, in these conditions the frequencies of mononucleated cells provide an indication of the background level of chromosome/genome mutations accumulated in vivo and the frequencies of binucleated cells with MN a measure of the damage accumulated before cultivation plus mutations expressed during the first in vitro mitosis.

The combination of the micronucleus assay with fluorescence in situ hybridisation (FISH) with a probe labeling the (peri-)centromeric region of the chromosomes (FISH assay) allows discrimination between micronuclei containing a whole chromosome (centromere positive micronucleus) and an acentric chromosome fragment (centromere negative micronucleus).

The criteria for selecting binucleated cells to score are the following:
Score binucleated cells with

main nuclei that are separate and of approximately equal size,
main nuclei that touch and even overlap as long a nuclear boundaries are able to be distinguished, and
main nuclei that are linked by nucleoplasmic bridges

Do not score:

trinucleated, quadranucleated, or multinucleated cells, or
cells where main nuclei are undergoing apoptosis (because MN may be gone already or may be caused by apoptotic process)

In the absence of cytochalasin B, mononucleated cells are analyzed for the presence of micronuclei.
In the presence of cytochalasin B, mononucleated cells are recommended to be harvested at 24 hours post-PHA stimulation as there can be no doubt at this time-point that MN within such a cell are a result of in vivo rather than ex vivo division. Binucleated cells are recommended to be harvested at 72 hours post-PHA stimulation. Moreover, 24 hour post-PHA may be the right time to count apoptotic/necrotic cells (Kirsch-Volders et al., 2001)

For the scoring of micronuclei the following criteria were adapted from Fenech et al., 1997:

the diameter of the MN should be less than one-third of the main nucleus
MN should be separated from or marginally overlap with the main nucleus/nuclei as long as there is clear identification of the nuclear boundary
MN should have similar staining as the main nucleus/nuclei

Characteristics of the MN-test

Biomarker of effect: relevant for risk assessment of cancer
Endpoint: identification of chromosome + genome mutations
Expression of MN requires cell division
MN contain either a whole chromosome or an acentric fragment
Discrimination between mononucleated cells and binucleated cells in the cyto-B assay

Mononucleated cells à damage accumulated before cultivation of the cells

Binucleated cells à damage accumulated before cultivation of the cells + damage expressed during cultivation

Advantages/ Disadvantages

	Advantages	Disadvantages
With/without cyto-B	Cell/cell approach Simultaneous detection of chromosome + genome mutations Discrimination between clastogen/ aneugen Possible co-detection of apoptosis/necrosis Applicable on many cell types Rapidity Cheap Simplicity Potential for automation Statistical power	Does not detect all structural chromosome aberrations (only acentric fragments) Requires cell division for expression of MN
With cyto-B	Discrimination between cells which underwent nuclear division and cells which did not Enables detection of dicentric bridges as nucleoplasmic bridges Assessment of cell proliferation (% binucleated cells)	Possible interference of cyto-B with test chemical; like spindle poisons Possible interference with other inhibitors of cytokinesis Cytotoxicity of cytochalasin B itself varies between cell types and sometimes even between subtypes of the same cell type

Confounding factors:

age
smoking?

References:

Kirsch-Volders M, Fenech M. "Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes." Mutagenesis, 2001, 16(1):51-58
Fenech M, Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E; HUman MicronNucleus project. "HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures." Mutat Res., 2003, 534(1-2):65-75