Relationships between Malignant Melanoma and Chromosome Damage in Human Peripheral Blood Lymphocytes

Most of deaths from skin cancer is responsible for malignant melanoma and it developes from malignant transformation of melanocytes which are pigment producing cells. Epidemiological studies have revealed that the risk of getting melanoma increases with an increasing age (Helfand et al., 2001; Diepgen et al., 2002; Cho et al., 2005; Mensink et al., 2011; McLean et al., 2012). The one of the significiant effect of aging process is an accumulation of oxidative damage occuring in macromolecules, particularly in the genetical material in the length of lifetime (Oresajo et al., 2012). Damage in the genetic material may then lead to an activation of protooncogens and inactivation of tumor suppressor genes resulting in cancer susceptibility (Miguel et al., 1980; Hagmar et al., 1994; Knowles et al., 2005). Susceptibility to cancer can be associated with structural chromosome alterations (Boveri et al., 1914, Nowell et al., 1960). Cytogenetic alterations, such as CAs may reflect genotoxic exposure. These alterations can determine the individual sensitivity for the particular genotoxic agent and this can be used as a marker in a correlation to cancer. Some epidemiological studies have shown that the individuals with high levels of CAs in their


Introduction
Most of deaths from skin cancer is responsible for malignant melanoma and it developes from malignant transformation of melanocytes which are pigment producing cells.Epidemiological studies have revealed that the risk of getting melanoma increases with an increasing age (Helfand et al., 2001;Diepgen et al., 2002;Cho et al., 2005;Mensink et al., 2011;McLean et al., 2012).The one of the significiant effect of aging process is an accumulation of oxidative damage occuring in macromolecules, particularly in the genetical material in the length of lifetime (Oresajo et al., 2012).
Damage in the genetic material may then lead to an activation of protooncogens and inactivation of tumor suppressor genes resulting in cancer susceptibility (Miguel et al., 1980;Hagmar et al., 1994;Knowles et al., 2005).Susceptibility to cancer can be associated with structural chromosome alterations (Boveri et al., 1914, Nowell et al., 1960).Cytogenetic alterations, such as CAs may reflect genotoxic exposure.These alterations can determine the individual sensitivity for the particular genotoxic agent and this can be used as a marker in a correlation to cancer.Some epidemiological studies have shown that the individuals with high levels of CAs in their Department of Biology, Faculty of Science, Istanbul University, Vezneciler, Istanbul, Turkey *For correspondence: narin_abdullah@ hotmail.com

Relationships between Malignant Melanoma and Chromosome Damage in Human Peripheral Blood Lymphocytes
Abdullah Narin*, Orta Tuncay peripheral blood lymphocytes (PBL) have an increased risk of cancer (Bonassi et al., 2004;Kolusayın et al., 2005;Vodicka et al., 2010a;2010b;Bonassi et al., 2011).This association between a high level of CAs in peripheral blood lymphocytes and the risk of devoloping cancer for an healthy individual is also supported with many other studies (Rossner et al., 2005;Norppa et al., 2006;Boffetta et al., 2007;Bonassi et al., 2008).So the presence of high levels of CAs could be considered as a marker of cancer risk.
The MN assay is the one of the in vitro genotoxicity techniques which measures CAs and evaluates the effects of genotoxic exposures in human (Geller et al., 2002).In this technique, scoring micronuclei in binucleate cells is easier and faster than scoring CAs in metaphase cells.Therefore scoring thousands of cells with using the MN assay enables more statistical certainty (Norppa et al., 2003).Micronuclei are formation of additional nuclei that appeares at mitosis.They may consist of a whole chromosome or an acentric chromosome which are not associated with main nucleus.They have similar shape, structure and painting features with the main nucleus but they are smaller.In the studies with the MN technique, it has been observed that the level of cytogenetic disorder as being measured with an excess amount of micronuclei increase with an increasing age in human peripheral blood lymphocytes (Vanparys et al., 1990;Bolognesi et al., 1997;Cardoso et al., 2001;Iarmarcovai et al., 2008).This relationship is also hold between MN frequency and the risk of cancer.The increased amount of micronuclei has been shown to be a marker of chromosome damage, gene instability, genetic susceptibility and mutations (Hagmar et al., 1994;Iarmarcovai et al., 2008).
In this study with using human PBL, we have investigated a possible relationship between CAs and malignant melanoma by using the MN assay in order to determine if this MN method could be used as being a cytological marker in the diagnosis of the disease besides the classical diagnosing methods.Chromosamal damage and proliferative efficiency of cells were also measured after treating cells with the non-toxic levels of H 2 O 2 in addition to measuring the spontaneous levels of micronuclei and proliferative indexes.

Cell culture
In this study, we have measured micronuclei in the peripheral blood lymphocytes in the group of 15 patients with malignant melanoma and in the group of 15 healthy controls.Control individuals matched the similar gender and age range as in the patient group.This work was approved by the ethical committee of the Cerrahpaşa Medical Faculty of the Istanbul University.Volunteers were properly informed and signed consent documents.
Peripheral blood samples were collected by venous puncture from both the patients and the controls.Blood samples were drawn into two separate 4, 5 ml sterilized lithium-heparin tubes.One tube was left as a control for spontaneous MN frequencies and the other was treated with hydrogene peroxide (H 2 O 2 ).0.5 ml of whole blood was added to culture medium containing 15µg/ml of phytohemagglutinin (Sigma), 0.5 ml of Newborne Calf Serum and 4 ml of RPMI-1640 with glutamine (Sigma) supplemented with 100 µg/ml streptomycin and 100 IU/ ml penicilin.The cultures were incubated at 37 °C for 44 hours.After adding cytochalasine-B (Sigma: 6 µg/ ml) solution, cells were left for incubation for another 28 hours.At the end of 72 and hours cells were centrifuged at 200g for 10 minutes, then treated with hypotonic KCL solution (0.075M).They were then washed and fixed three times with methanol/acetic acid (7/1) solution.Cell suspension that was left at the end of the third wash was dropped on glass microscope slides which were in turn stained by 5% Giemsa solution (prepared in phosphate buffer) for 10 minutes.
Scoring 1000 binucleated cells were analysed per person and MN frequencies were evaulated according to the criteria described by Fenech (Fenech et al., 2003).MN scoring in binucleate cells was made using light microscope at a magnification of 400.Also 1000 magnification (immersion objectives) was used for the comfirmation of MN.Additionally, for each individual, cells including 1,2,3 or 4 nuclei were analyzed for PI.

Statistical analysis
Spontaneous and H 2 O 2 induced MN frequencies and PI for individuals in both malignant melanoma group and matched control group were compared using t-test.

BetPatient group:
The patients consisted of 9 women and 6 men.Their ages ranged from 36-75 with the mean of 53.The type of malignancies in the group of melanoma patients were superficial spreading melanoma (in 8 patients), nodular melanoma (in 3 patients), lentigo maligna melanoma (in 2 patients) and acral-lentiginous melanoma (in 2 patients).Eight patients had grade-1 tumor, six patients had grade-2 tumor and one patient had grade-3 tumor.
The spontaneous MN frequencies of patient group were calculated for each individuals in at least 1000 binucleated cells per culture (Table 1).The results for patients showed that mean value of spontaneous MN frequency was 0.029±0.022(mean±standart deviation) within the range from 0.009-0.084.After H 2 O 2 treatment was applied to cultures of patient groups, MN frequencies decreased significantly, ranging from 0.006-0.059with the mean value of 0.019±0.014(mean±standart deviation).
When these MN results before and after H 2 O 2 treatment were compared by paired t test in the patient group, there was a significiant difference (p<0.01).Spontaneous (p=0.82) and H 2 O 2 induced (p=0.65)MN frequencies were not different between the patients with grade-1 tumor and the patients with grade-2 tumor.
Additionally, cells with one, two, three and four nuclei (M1, M2, M3, M4 respectively) were scored for calculating PI for each individuals as; PI=(M1+2(M2)+3(M3)+4(M4))/N, N represents total number of cells.Spontaneous PI for patients were found to be within the range from 1.390-1.920with the mean value of 1.690±0.045(±standart deviation).After H 2 O 2 treatment was applied to the same group, PI values decreased (mean value:1.503±0.187,range: 1.222-1.820).When we compared the results between spontaneous and H 2 O 2 treated patient groups by paired t test, we obtained very significiant difference (p<0.0001).

Control group:
The control group contained 15 healthy individuals who had similar age and gender with patients.Nine of them were women and six of them were men.Their ages were between 36 and 76.Similar applications were applied to their blood samples as in the patient group.
The results for controls showed that the mean value of spontaneous MN frequency was 0.009±0.003(mean±standart deviation) and MN distribution ranged from 0.005-0.015.After treating control individuals with H 2 O 2 MN frequencies were distributed between 0.004 and 0.013 with the mean MN frequency of 0.008±0.003(Table 2).When we compared the results between the two groups by paired t test, there was no difference (p=0.29) between the two.
The mean value for the spontaneous PI for controls was found to be 1.931±0.324ranging from 1.303-2.340.After H 2 O 2 treatment was applied to the same group, PI values decreased ranging between 1.240 and 2.081 with the mean value of 1.764±0.284.When the comparision was made between the treated and untreated samples, the difference was extremely significiant (p<0.0001).

Comparisions between the two groups
The findings of patients and mated healthy individuals were compared by using unpaired t-test.Spontaneous MN frequencies of the patient group were higher than the frequencies of the control group.The significant difference (p<0.01) was observed by applying unpaired t-test.This difference (p<0.01)remained the same between the H 2 O 2 treated samples of the two groups.
When the same statistical test was applied to compare the PI values between patient and control groups, significiant differences were found for the spontaneous values (p<0.05) and H 2 O 2 treated values (p<0.01).

Discussion
Chromosomal damages which can be measured as translocations, acentric fragments, telomere shortening, nondisjunction, loss of chromosomal material, uneuploidy and micronuclei increases with an increasing age (Countryman et al., 1976;Rossner et al., 2005;Kazimirova et al., 2009).Epidemiological studies have reported that the risk of cancer increases in individiuls who had high level of CAs in their PBL (Bonassi et al., 2004;Vodicka et al., 2010).Studies with MN assay showed increases in the MN frequency in PBL and fibroblast cultures of individuals with familial cutaneous malignant melanoma (Weichenthal et al., 1989;Berg-Drewniok et al., 1997).On the other hand, Widel et al investigated radiosensitivity of 25 malignant melanoma patients with MN assay and found an association between MN sensitivity and radiation responsive tumor cells (Widel et al., 1997).
In this study, we have investigated spontaneous and H 2 O 2 induced MN frequencies and proliferative indexes in the peripheral blood lymphocyte cultures of 15 malignant melanoma patients and appropriately matching 15 healthy controls.Their ages ranged from 36-76 years.In the patient group there was no influence of either sex or having an immunotherapy on the induction of the MN frequencies.We have observed a significant decrease (p<0.01) in the MN frequencies of the patients lymphocytes after H 2 O 2 treatment.But H 2 O 2 treatment didn't change the MN frequencies in healthy individuals giving no difference between the two (p=0.29).Application of H 2 O 2 to the cultures resulted in the decreases of the PI values in both the patient group (p<0.0001) and the control group (p<0.01).
When we compared the groups in terms of MN frequencies, there was a significiant difference (p<0.01) between the patient and control groups.Higher MN frequencies in the patients compared to the healthy individuals in the control group can be associated with the patients having individual sensitivity factors which could lead to cancer as a result of the long term exposure to genotoxic carcinogens (Figure 1).The difference (p<0.01) between the groups was also observed between the H 2 O 2 teated cultures.This difference is due to the decreases in the micronuclei which occured particularly in the patient group after application of H 2 O 2 .Because H 2 O 2 application didn't alter the MN frequency in the control groups.
In this study, increases in the MN frequencies were obtained in the malignant melanoma patients compared to the MN frequencies in the healthy individuals (Figure 1).Increasing amount of damage to DNA that is observed with the increasing amounts of MN frequencies in malignant melanoma patients can be associated with the genetic instability leading to the chromosome instability which may be important in cancer induction and aging processes.This increased number of micronuclei can also be used for cytological marker in identifying malignant melanoma.

Figure 1 .
Figure 1.Comparison of MN frequencies between patient and control group.After H 2 O 2 treatment was applied to the same group, PI values decreased ranging between 1.240 and 2.081 with the mean value of 1.764±0.284.When the comparision was made between the treated and untreated samples, the difference was extremely significiant (p<0.0001).

Table 2 . Results of MN and PI Frequencies in the Control Group
† MN: Micronucleus, ‡ BN: Binucleate, § PI: Proliferative index