GENOTOXICITY : THE 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 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 (added 44 hours after the start of cultivation), an inhibitor of actins, 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

Do not score:

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. Moreover, 24 hour post-PHA time-point may be the right time to count apoptotic/necrotic cells (Kirsch-Volders et al, 2001)

For the scoring of micronuclei the following criteria were adopted from Fenech et al, 2003:

Characteristics of the MN-test

Mononucleated cells   damage accumulated before cultivation of the cells

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

Advantages/ Disadvantages

MN-assay

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
  • Cytoxicity of cytochalasin B itself varies between cell types and sometimes even between subtypes of the same cell type

Confounding factors: 

age
gender
smoking?

 


References: