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Background on the Micronucleus Test
The micronucleus (MN) assay is used
to evaluate test articles for their ability to cause DNA damage.
This damage can either be seen as actual breaks in a chromosome
(clastogens), or as the loss of an entire chromosome (aneugens).
If a chromosome break or loss occurs, a micro-nucleus (i.e.,
a ‘small’ nucleus) is formed. Treating precusor cells (stem
cells) with a chemical causing DNA damage will result in more
micronuclei in the daughter cells.
The Creation Of Micronucleated
Cells
These micronuclei can be clearly
seen when their DNA is stained in an otherwise DNA-deficient
population. Measuring MN in Red Blood Cells (RBCs) is ideal
because RBCs expel their main nucleus, leaving behind only
the micronuclei.
Erythrocytes: Young Versus Old
In
order to see the effects of a compound in the peripheral
blood, it is first necessary to differentiate between newly
created RBCs and those that have been in the blood stream
for some time. Micronuclei detected in the youngest cells
(Reticulocytes, or RETs) are indicative of recent damage.
On the other hand, MN found in mature cells (Normochromatic
erythrocytes, or NCEs) indicate damage that was caused at
an earlier time.
The young RETs differ from mature
NCEs in several ways. RETs have a high RNA content, as well
as specific cell surface markers, each of which is degraded
as the cells mature. Rather than detecting RETs by their
RNA content, monoclonal antibodies can be used to specifically
label cell surface markers of these young cells.
In terms of short-term studies,
the youngest cells (RETs) are the most appropriate population
to focus, and the one suggested by regulatory agencies.
With the MicroFlow process, micronuclei are scored in both
the RET and NCE populations.
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