Introduction

Breast cancer is the most prevalent malignant tumor among women across the world with the exception of skin cancers and constitutes about 30% of all cancers seen in women (Aslan and Gürkan, 2007; www.cancer.org). Breast cancer also comes second after lung cancer in women in terms of mortality rate. Radiotherapy (RT) has an important place in invasive and non-invasive breast cancer therapy. In ductal carcinoma in situ (DCIS) treatment, the goal is minimal recurrence risk, optimal cosmetic result and treatment of the breast by conserving it. In prospective studies conducted, rates of > 10% local recurrence risk are reported in breast-conserving surgery (BCS) even among low-risk patients in a five-year follow-up period. Invasive recurrence is observed in 31-64% of the cases exhibiting local recurrence. Today, RT following breast conserving surgery is considered to be a standard approach in DCIS treatment. On the other hand, in invasive breast cancer treatment, adjuvant RT is considered to be a standard approach in early stage after BCS and after local mastectomy in advanced stage. It was shown in phase III studies that compared only BCS and tamoxifen and BCS+RT+tamoxifen combination in cases having positive prognostic factors and having positive hormone receptors that tamoxifen could not replace RT. RT after BCS takes place in the form of whole breast 45-50 Gy followed by 10-15 Gy in tumor bed. In a European study that investigated the effect of additional dose to tumor bed on local control, it was shown that treatment with additional dosage made a contribution to local control in all age groups. There is a consensus on the need for adjuvant RT if post-mastectomy 4 axillary lymph node (LN) metastasis is >5 cm tumor, and there is skin-facia involvement. In recent years, as a consequence of the publication of the long-term results of Canadian and Danish studies and their demonstration of local control and survival advantages through adjuvant RT, increasing rates of adjuvant RT have been indicated in cases of with 1-3 LN metastasis (Yıldız, 2008).

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.

PBS readymade solution was used as solution. Standard blood culture was performed from the blood belonging to the patient on whom SCE would be applied. 24 hours after culture, 100 µl BrdU solution (per tube) was added to the culture. Harvest procedures that are performed in 72-hour blood culture were applied here. Preparations made at the end of the harvest were stained with 2% giemsa for 10 minutes.

In preparations, SCE, stained with FPG (Fluorescence + Giemsa technique), the number of SCEs and their distribution to chromosomes were examined using Olympus BX51 light microscope in the Applied Imaging Karyotyping system by selecting the slides according to the appropriate metaphases. Terminal changes were counted as one change whereas interstitial changes were counted as two changes. The total number of SCE counted in a case (patient) was divided by the number of metaphases examined and thus a patient’s average number of SCEs per metaphase was determined. An average of 50 metaphases were counted and photographed in the patients and the controls.

1.3 Micronucleus test

In order to determine the number of MN, the method developed by Fenech (Fenech, 2000) and Kirsch-Volders et al. (Kirsch-Volders et al., 2003) was modified and used. Blood samples taken from 22 women with breast cancer who were about the same age were heparinized at a rate of 1/10 and then 6 drops (0.2 ml) of them were added to the chromosome media in sterile conditions (Rencüzoğulları and Topaktaş, 1991). The cell culture was incubated in the incubator for 68 hours at 37±1°C. At the end of the 68^th hour, which is the duration of culture, culture tubes were centrifuged for 15 minutes at 1 200 rpm, and the supernatant was removed. The 0.5-0.7 ml liquid that remained at the bottom and contained the cells were mixed thoroughly and then the hypotonic solution kept in the drying oven at 37°C was added to the tubes. The addition of this solution was implemented drop by drop and by stirring. After 5 ml hypotonic solution was added to each tube, their caps were replaced and put in the incubator. The cells were treated in hypotonic solution at 37°C for 5 minutes. At the end of the period, the tubes were centrifuged at 1 200 rpm for 15 minutes and supernatant was removed. This time, cold fixative was added, 5 ml to each tube, slowly and by stirring as in the case of addition of hypotonic solution. The fixative was prepared by diluting 1 unit of acetic acid and 5 units of methyl alcohol mixture at a rate of 1/1 with 0.9 NaCl %. The cells that were treated with the fixative at room temperature for 20 minutes were centrifuged at 1 200 rpm for 15 minutes, supernatant was removed and again fixative was added to the tubes. This procedure was repeated twice and fixatives different from the ones used in the first fixative were used (1 unit acetic acid and 5 units methyl alcohol). At the end of the 3^rd treatment with fixative, it was seen that the liquid remaining in the tube had become totally clear. The liquid was centrifuged each time after addition of fixative and the supernatant was removed. After the last centrifuge, when the supernatant was removed so that 0.5-0.7 ml liquid remained at the bottom, preparation procedure was begun. The cells that accumulated at the bottom of the tube were mixed using a Pasteur pipette, thereby homogenizing them. 4-5 drops were drawn into the Pasteur pipette from this cell suspension. Cell suspensions were dripped, 1 drop in each area, on different areas on the sliding glass, which had been cleaned before and kept in a fridge in pure water (4-5 drops on each sliding glass), and thus cells were enabled to spread across the sliding glass. During the dripping of the cell suspension on sliding glasses, it was ensured that the drops did not overlap. The preparations obtained in this way were kept at room temperature for 24 hours to dry. Then, the preparations were colored with giemsa stain. The preparations stained with giemsa were examined under Olympus BX51 light microscope in the Applied Imaging Karyotyping system. 1 000 cells that had not suffered damage and had not been fused with other cells were counted. Micronuclei that had even edges, whose dimensions were one third of the main nucleus and which showed the same coloring characteristics were evaluated.

1.4 Binuclear cell

To obtain binucleated cells, cytochalasin B (6 mg/mL) was added to each culture 44 hours after starting cultures, to inhibit cytokinesis. Cells were allowed to grow for another 28 hours, after which they were collected by centrifugation, resuspended in a prewarmed hypotonic solution (0.075 mol/L KCl) for 15 minutes at 37°C, and fixed in acetic acidemethanol (1: 3 vol/vol). Airdried preparations were stained with 4% Giemsa. Under 100°C magnification, a minimum of 1 000 binucleate cells with well-preserved cytoplasm was scored from each patient and the MN frequency was determined by the total number of MN in all the binucleate cells divided by the total number of binucleate cells counted (Aristei et al., 2009).

2 Results

A significant difference emerged in SCE (p=0.008) and MN (p=0.004) between RT-a and control groups. No statistical difference observed in BNC frequencies in breast cancer patients compared to control group. Compared to the control group there was a significant increase in SCE frequencies in RT-b (p=0.008) and RT-c (p=0.005). There was also a significant increase in SCE frequencies in RT-b (p=0.001) and RT-c (p=0.001) values compared to RT-a measurements. There was not any statistically significant difference in the SCE frequencies between RT-b and RT-c measurements.

The frequencies of MN were also significantly higher in RT-b (p=0.005) and RT-c (p=0.005) than in control group. The MN frequencies were significantly increased in the RT-b compared to RT-a (p=0.001). However, there was not any statistically significant difference in MN frequency between RT-a and RT-c measurements. MN levels decreased to pre-RT levels three months after completion of treatment. MN decreased significantly at RT-c compared to RT-b (p=0.001). No significant difference in BNC was observed between control group and any study group values (Table 1).

3 Discussion

The damages caused by various carcinogenic, cytotoxic, anogenic and genotoxic agents that induce damage in DNA chromosomes were identified by Fenech as biomarkers indicating that a damage has occurred in the DNA (Fenech, 1999). DNA breakages, chromosome aberrations, micronuclei, aneuploidy and telomere shrinkage are some of them (Fell et al., 2000). Anogenic and clastogenic agents are by far the best stimulators known in the formation of MN and apoptosis. Both agents cause genomic instability characterized by direct DNA breakages in dividing cells, acentric fragments, kinetochore anomalies, translocations and in vivo DNA amplification (Gül, 2005).

In a study they conducted, Barret 1993 and Bishop 1991 pointed out that chromosomal breakages, reorganizations among chromozomes and induced DNA damages were fundamental mechanisms that led to the occurrence of various cancer types (Gül, 2005).

In our study, we aimed to determine whether or not RT had a genotoxic effect after RT application for medicinal purposes in patients with breast cancer, and whether or not there were increases in sister chromatid exchange (SCE), micronucleus (MN) and binucleus in human peripheral lymphocytes.

In this study, human peripheral lymphocytes taken from patients with breast cancer who had received RT were treated with Cyt-B at the 72^nd hour and the results obtained from the study were compared with the results of other researchers.

Natarajan and Obe reported in 1980 that 72-hour cultures allowed cells to undergo two cell cycles and that the changes that occurred in the first cycle might undergo cellular repair in the second cycle (Seligmann et al., 2003). Tucker and Preston (Tucker and Preston, 1996) stated that for the frequency of the induced chromosome aberration to be predicted accurately, cytogenetic evaluations should be made within a short time after the treatment while the cells were undergoing their first mitotic division after the treatment. According to this information, aberrations can be repaired in the second cell cycle and aberrations can be seen below their real frequencies.Yoshioka et al. found, in a study they conducted, that radiation, a clastogenic agent, affected mitochondrial DNA, ATP level and MN formation (Gül, 2005).

In a study which Aristei et al. conducted in 2009 on 20 women with breast cancer in early phase I and II (individual and familial cancer patients were excluded from the study), they found an increase in SCE and MN rates compared with the control group. In the same study, an increase was also observed in SCE frequency after receiving chemotherapy (CT). On the other hand, decreases were observed in SCE frequency in the same patient group 6-12 months after CT (Stracci et al., 2009). However, smoking also causes increases in SCE frequency. Increases were observed in MN frequencies in all patients after RT. Age is also correlated with MN frequency. In this study, MN frequency in older patients exhibited a higher increase than in younger patients. This rate in the patients can be related to the DNA repair mechanism (Kutbay, 2001).

In their study, Fenech and Morley reported that X-ray caused chromosomal damage in human lymphocytes and it was an agent that affected aging (Kutbay, 2001).

In many studies, researchers reported that MN frequency increased in direct proportion to radiation dosage (Cole, 1981)

In a study they conducted in 1998, Sönmez et al. evaluated SCE in patients with Behcet’s disease and found significant increases in SCE rate in the patient group in comparison with the control group (p<0.000 1). They concluded that Behcet’s Disease could be related to genetic disorder and damage to DNA (Sönmez et al., 1998).  Sönmez et al. (1997) found increases in SCE frequencies in 15 patients with ankylozing spondylitis (Sönmez et al., 1997).

In an in vivo study which Kasuba et al. conducted in 1999 on nurses who had been in contact with cytotoxic drugs for long periods, no change was found in SCE increase whereas it was reported that the drug caused an increase in MN frequency. An increase in MN was identified in nurses who had been in contact with drugs for long periods compared with the control group. The fact that there was an increase in MN despite a lack of increase in SCE indicated that MN analysis was a more sensitive technique than SCE analysis in mutagenic analyses (Gül, 2005).

Jakopin and Bilban (2001) applied mutageneity tests including chromosomal aberrations (CA), SCE and MN on 30 patients with Hodgkin’s disease after CT and RT treatment. They could not find significant differences between the patients and the control groups before the treatment but after a 6-month treatment, they observed decreases in CA, SCE and MN rates. However, when these rates were compared with the values right after the treatment, it was found that they were not statistically significant. The researchers found significant differences in micronucleus and chromosome aberration frequencies of patients who were treated with radiotherapy in comparison with those who were treated with chemotherapy. When the treatment was completed 6 months later, there was not complete recovery although mitotic activity was almost normal and chromosome damage was observed. They attributed these changes to the possibility that in malignant diseases oncogenes may become activated (Jakopin and Bilban, 2001). In this study, a significant increase was observed in MN frequency in patients in RT-b (p=0.005) and RT-c (p=0.005) groups compared with the control group. When RT-b was compared with RT-a (p=0.001), a significant increase was observed in MN frequency. When RT-a and RT-c measurements were compared, a statistically significant change was not observed. After a three-month treatment, on the other hand, MN level dropped to pre-RT level. No difference was observed between the control groups and the study groups in terms of BNC values. When RT-c and RT-b (p=0.001) groups were compared, a significant decrease was observed in MN. On the other hand, no difference was observed between RT-a and RT-c in terms of MN frequency (Table 1). The results of our study are in parallel with the results of other studies.

MN increases in lymphocytes depending on age and gender (Kutbay, 2001; Fenech, 2007).

SCE is a cytological indicator of segment change among the homologous areas of DNA replication products as a result of DNA breakage and recombination. According to a paper submitted by Tucker et al. in 1993, the interpretation that SCE frequency is an indication of genotoxic effect is based either on doubling of SCE number compared with the control group or a statistical increase in any dose (p<0.05) (Rodriguez-Mercado et al., 2003).

SCE increased in patients with cervical cancer, nasopharyngeal carcinoma, prostate, ovarian carcinoma, acute leukemia and Chronic lymphocytic leukemia (CLL). It was observed in studies on breast cancer that SCE increased in patients and patients with nonmalignant fibroadenoma. In another study, SCE and CA also increased in patients with hereditary breast cancers and their first degree relatives. Perhaps, chromosomal instability will be a guide in patients with hereditary breast cancer (Çefle, 2006).

In a study which Fenech conducted in 1993, it was shown that MN increased in all patients depending on dosage in different patient groups that received RT treatment. In the same study, periodic measurements were taken after a 12-month RT and increases of 91.6% were observed 3 months later, 72% 6 months later and 57% 12 months later (Fenech, 1993). Buckton obtained similar results in patients with ankylosing spondylitis (Buckton, 1983). In our study, a significant increase was observed in SCE frequencies in RT-b (p=0.008) and RT-c (p=0.005) groups when compared with the control group. When RT-b and RT-c groups were compared with RT-a group in terms of SCE, again significant increases were determined in terms of SCE. No difference was observed in SCE when RT-b and RT-c groups were compared between themselves. In conclusion, our study is in parallel with other studies in terms of SCE.

4 Conclusion

Increasing MN and SCE frequencies following radiotherapy is an expected situation. Decrease in MN frequency at 3-month after the completion of RT suggests that expected repair continues. Persistent SCE at the same period suggests that recovery in SCE has not completed yet and a longer period of time is needed.

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