One well-understood effect of radiation is related to MN formation. MN is originated from acentric chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during metaphase or anaphase phase of cell division⁷. They reflect chromosome damage and may thus provide a marker of early-stage carcinogenesis⁸–¹⁰. Moreover, MN test can be used to show both clastogenic and aneugenic effects. A number of studies have been designed to evaluate the potential influence of factors such as gender, age, smoking habit and radiation on the MN frequency⁹. It has been shown that factors such as age, smoking habit, alcohol, genotoxic agents, chemical substances and radiation had a remarkable effect on the frequency of MN¹¹. MN test can be performed for different cell types such as lymphocytes, fibroblast and epithelial cells¹². Consequently, MN formation is a reliable biomarker of exposure to radiation¹³–¹⁵.

The aim of the present study was to evaluate the effects of γ–radiation on morphology of WBC and MN formation in patients with lung cancer receiving RT.

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Table 1: Cancer types in subjects included in the study

Ethical standards: In this study, the methods and techniques applied to patients were carried out according to ethical standards of the local ethical committee of Abdurrahman Yurtarslan Research Hospital (Protocol date: 27.10.2005) and favorable to the guidelines set by the World Health Organization (Geneva, Switzerland). Each patient read and signed an informed–consent form before their participation in the study. This permission is always for the analysis and collection of blood samples from patients, and is not used for purposes other than those for which consent was originally given.

Radiation Treatment: Radiation procedure was carried out using “ATC Cobalt 60 SSD=80 cm” instrument. The dose equivalents were calculated and compared to the doses recommended by International Commission on Radiation Protection (ICRP). Totally 20 patients were treated with 10 Gy γ–radiation for five weeks in 50 Gy total dose. Radiation was applied to thorax area during 30 min. Cytogenetical analyses were performed on WBC of patients exposed to γ–radiation.

Collection of Blood Samples: Peripheral blood samples were collected from 20 patients and 20 healthy donors before and after RT. Blood samples were taken from one of the veins of arm of each individual. Three milliliters (3.0 ml) of venous blood was obtained from each individual, evacuated into sterile heparinized tubes and transported within 3 h to the laboratory.

MN Assay: In the absence of cytochalasin B, mononucleated cells were analyzed for the presence of MN. Peripheral WBC were isolated from heparinized blood tubes. 5 µl drop of blood was spread on a clean glass slide, then cells on slide were fixed with 70% methanol (10 min.) and stained with the May-Grünwald Giemsa stain (5%). From each donor, 1000 cells were examined under a binocular light microscope (Japan, Nicon Elipse E600) at X100 magnification and MN cells were photographed at a magnification of X500. For the scoring of MN the following criteria were adopted from Fenech et al (16). i: The diameter of the MN should be less than one-third of the main nucleus, ii: MN should be separated from or marginally overlap with main nucleus as long as there is clear identification of the nuclear boundary, iii: MN should have similar staining as the main nucleus.

Peripheral Blood Smear Test: Blood smear slides have been prepared for provide information about the number of WBC and determine of the morphological damages in WBC of patients. Slides were formed from a small drop of blood (5µl). Then slides were fixed in 70% ethanol (10 min.) and stained by the May-Grünwald Giemsa stain (5%) for 30 min. Slides were air dried over night at room temperature (25ºC). Dried slides were examined using the same microscope (Japan, Nicon Elipse E600).

Statistical Analysis: “Paired Samples T-Test” was used while comparing the morphological damages and the frequency of MN between patients exposed to γ–radiation and the controls. Differences were considered significant if the P value was less than 0.01.

In this study, a significant difference was observed in the morphologies of WBC of cancer patients receiving RT when compared to the controls. Detailed information about the morphological alterations in WBC and the MN frequency following RT was showed in Figure 1–5 and Table 2. The morphological changes such as dense-vacuolization and deformity (Figure 1a,b), membrane defects (Figure 2a,b), increase of cytoplasmic granulation (Figure 3a,b), dead cells (Figure 4a,b) and MN formation (Figure 5a,b) were observed in WBC. Moreover, the MN frequency was compared with WBC counts. There were a significant increase in the frequency of MN and a significantly reduction in peripheral blood WBC count during RT (Table 2). In all patients, the results showed that the highest frequency of MN was observed at the end of the 5^th week of RT and the lowest frequency of MN was observed before the initiation of RT (Table 2). We also found a significant increase in the MN frequency in lung cancer patients after and before RT compared with the controls.

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Figure 1: The appearance of deformation (a) vacuole formation (b) in leukocyte cells (X500)

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Figure 2: The appearance of cell membrane damages in leucocytes (a,b X500)

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Figure 3: The appearance of dense granulation in leukocyte cytoplasm (a,b X500)

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Figure 4: The appearance of dead cells (a,b X500)

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Figure 5: The appearance of micronucleus (a,b X 500)

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Table 2: The frequency of MN and WBC in the peripheral blood of lung cancer patients receiving RT

In our study, the MN frequency was also compared with WBC count. The MN frequency showed an increase during RT and WBC counts decreased significantly during RT. In all patients, the results showed that the highest frequency of MN was observed at the end of 5^th week of RT and the lowest frequency of MN was observed before the initiation of RT. Moreover, the MN frequency in WBC was also elevated in cancer patients before RT when compared with the controls, and difference was statistically significant. Although these patients were not exposed to γ–radiation before RT, MN was observed in prepared slides. This result may be related to the patients' age, chemotherapeutic drugs taken before RT and cigarette smoking in particular. The effect of cigarette smoking on MN frequency was reported in many of the biomonitoring studies¹⁸–²⁰. Besides, MN level was showed greater frequency in the elderly control group (non-smokers) with a mean age of 52.6 ± 2.9 years than the young controls with a mean age of 23.5 ± 1.3 years (non-smokers). MN formation was not observed in the young controls, but a low frequency of MN (0.05%) was determined in the elderly controls. The observation of MN in these healthy persons without RT and chemotherapy clearly indicates that MN formation is related to donors' age. These findings suggest that age can influence the formation of MN in WBC. The effect of aging on spontaneous MN frequency has been reported by various authors^11,12,14,21–²³. Higher frequencies of MN have previously been reported in females than in males²⁴. However, the effect of sex difference was observed in our study.

If we consider the similar studies, our results are found to be in close agreement with previously published data. For example, Anna et al.²⁵ investigated the level of cytogenetic damage in peripheral blood lymphocytes of patients undergoing chemotherapy. As a result, they showed that the highest level of cytogenetic damage was observed at the end of therapy. They determined the frequencies of increased MN during the first half of therapy and declined thereafter. Moreover, they observed the leukocyte count strongly decreased at the beginning of therapy with an upward trend at the end. Boreham et al.²⁶ reported the relationship between radiation dose and radiation-induced the apoptosis and MN formation. They found that apoptosis and the MN frequency decreased in low dose rate of radiation, but apoptosis and the MN frequency in binuclear cells increased with increasing of applied radiation dose. Hubert et al.²⁷ used MN test to determine the effects of radiation on 99 workers studied in Belgium Doel Nuclear Center. They reported an increase in the frequency of MN with increase in the annual exposure to radiation. In another study, MN formation and cell proliferation in human lymphocytes exposed to 50 Hz magnetic fields for 72 h was investigated. As a result, 50 Hz magnetic fields have no effect on MN formation, and a significant increase in cell proliferation was not observed²⁸. Widel et al.²⁹ investigated the frequency of MN in peripheral blood samples were taken before and after RT in patients with cervical cancer. As a result, they reported a significant increase in the MN frequency compared with the controls. Maes et al.³ reported effect of 2450 MHz microwave on the MN frequency in human blood lymphocytes in vitro. They determined an increase in the MN frequency with increasing of exposure duration. Rosin and Gilbert³⁰ investigated the modulation of genotoxic effects of some chemical agents in humans by using MN test as a biomarker. Besides, Stich and Rosin³¹ reported the MN in exfoliated human cells as a tool for studies in cancer risk and intervention.

In conclusion, RT has a lethal effect on cancer cell. However, it may cause severe structural and genotoxic damages on healthy cells and tissues such as blood. These damages may also induce other disease such as leukemia and anemia³². Therefore, effects of RT applications on healthy cells and tissues must be minimized or alternative methods should be developed.

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