Genomics and Applied Biology 2016, Vol.7, No.2, 1-7
2
The Micronucleus method is a method used in determining detailed chromosomal breakages at
chromosomal/molecular level, demonstrating chromosomal reorganizations, chromosome losses, nondisjunctions,
gene amplifications, necrosis and apoptosis and evaluating genomic instability (Fenech, 2002). Analysis of
micronucleus formation is a simple and sensitive test for radiation damage and has a significant role in biological
dosimetry studies because it has been found that the rate of micronucleus formation increases in a linear manner
with dosage and this feature is an important factor in determining radiation sensitivity (Özalpan, 2001). On the
other hand, micronucleus analysis is the most valid mutation analysis technique in identifying DNA damage
which an agent (genotoxic, cytotoxic or carcinogenic) causes in a eukaryotic cell, mutations and the parameter
that has the potential to contribute to the pathogenity of the other cell. The most important advantage of
cytokinesis-block MN is that scoring is easy and that one can look at for more cells in a short time than the
number of cells looked at for metaphase analysis and make an evaluation (Fenech et al., 1999). Another advantage
of the cytokinesis-block MN method over metaphase analysis is that it can determine chromosome losses in a very
reliable manner. Another cytogenetic method used in demonstrating chromosomal damage is the evaluation of the
frequency of sister chromatid exchange (SCE). Sister chromatid exchange is defined as mutual segment exchange
that occurs between two chromatids in the loci of homologue chromosomes and does not lead to a change in
chromosome morphology (Barch et al., 1991; Hamurcu et al., 2008). In the S phase of SCE cell cycle, the
breakages in the same location in every chromatid in the DNA continue in the order of change in the DNA chain
and repair of these breakages. SCE and MN methods are genotoxicity tests used in investigating clastogeneity,
genotoxicity or genomic instability.
1 Material and Methods
1.1 Patients
In our study, blood samples of 22 patients with breast cancer who applied to N.E.U Meram Medical School
Department of Radiation Oncology in 2011-2012 and were decided to be given adjuvant radiotherapy, and then
were followed and treated, and blood samples of 10 healthy women who applied to Konya Genetikon Laboratory
for various reasons were used. The participants of the study were informed about how the study would be
conducted, what the research method was and how the results would be evaluated and their consent was taken.
They were made to complete the forms stating that they wanted to participate in the study. A data collection form
was prepared for the study. Their age, whether or not they had a serious illness before, whether or not they smoked
and whether or not they were on drugs were questioned. Systemic physical examinations were performed on them.
Also, approval of the Ethical Board of Necmettin Erbakan University Meram Medical School was obtained before
the study was begun.
1.2 SCE
Research techniques
The blood samples taken from the patients and the control group were examined in terms of sister chromatid
exchange, micronucleus and binucleus frequency.
In the SCE test method, in order to be able to observe the sister cell exchange, readymade solutions were used to
obtain metaphase cells undergoing the second cell division (Culture Medium, Stock Bromodeoxyuridine (BrdU)
Solution, Harvest Solutions) and 72-hour culture period was applied.
250 μl (5 drops) peripheric blood and 0.1 ml BrdU were added to the prepared culture medium and cultured in the
drying oven at 37°C. At the 48
th
hour of the culture, 0.1 ml colchicine was added and waited for 20 minutes. It
was centrifuged for 5 minutes at 1 500 rpm. Supernatant was removed and KCl, which had been heated at 37°C,
was added to the pellet and kept in that state for 5 minutes. It was centrifuged again; fixative was added to the
pellet drop by drop and shaken.
The procedure of fixation was repeated 2-3 times. After all these stages, it was spread on glass slides. It was
matured for 2 hours in Pasteur oven at 90°C and then fluorescence-giemsa procedure was begun.
