DNA damage in exfoliated buccal cells and antioxi

作者：Revathi P Shenoy*， Anumeha Vatsa*， Swagatika Sahoo*， Rajashree Rana*， Guruprasad Nayak*， Poorna Bhat*， Guruprasad Kalthur**， Anjali Rao

【摘要】 The present investigation was carried out in exfoliated buccal cells and saliva collected from preoperative brain tumour patients. The DNA damage in these cells was assessed by alkaline comet assay and micronucleus (MN) assay. Salivary flow rate， pH， osmolality， total antioxidant activity (AOA) and vitamin C levels were also assessed in unstimulated whole saliva of these patients. In the comet assay a significant increase in the tail length (P&<0.02) was observed when control and malignant groups were compared. A significant (P&<0.02) difference in tail length was also noted between benign and malignant groups. Non significant results were found when control and benign groups were compared. Further， a marked increase in % MN (P&<0.002) was observed when control and benign groups were compared. A significant increase in % MN (P&<0.029) was also observed in benign cases when compared to malignant tumours. No significance was obtained when % MN in control and malignant cases was compared. Moreover， salivary flow rate and pH was significantly decreased and osmolality was markedly increased in brain tumour patients. The AOA levels in saliva were markedly decreased in brain tumours and vitamin C levels exhibited no change when compared to controls. Thus， as noted above susceptibility to free radical induced DNA damage also exists in the exfoliated buccal cells conducive to the lowered salivary antioxidant status of brain tumour patients.

【关键词】 Buccal cells; Comet assay; Micronucleus assay; Total antioxidant activity; Saliva; Brain tumours

　　Introduction

　　Obtaining an ideal source of cell for analyzing the role of genetic and biochemical factors on the integrity of genomic DNA in various pathological conditions is very critical. Even though peripheral lymphocytes are an excellent source of large amounts of genomic DNA， most of the study subjects are reluctant to provide a blood sample. It is an additional stress on patients to draw the blood especially in patients suffering from dreadful diseases such as cancer. Exfoliated buccal epithelial cells and other cells found in saliva are a very promising alternative source of DNA since they can be obtained using selfadministered， noninvasive and relatively inexpensive techniques[1，2]. In our earlier study we have observed that lymphocytes of the brain tumor patients carry a high level of basal DNA damage and are highly susceptible to exogenous clastogenic challenge[3]. In the present investigation we have tried to gauge the malignancy induced magnitude of DNA damage in exfoliated buccal cells obtained from preoperative brain tumour patients. Further， it is now well established that oxidative stress is involved in the etiopathogenesis of cancer. Oxidative stress induced changes in tissue and biological fluids of cancer patients have been demonstrated by earlier studies[4-9]. In the present investigation we correlated the extent of DNA damage in buccal cells with biochemical changes in the salivary secretion.

　　Material and Methods

　　Patients with brain tumours， admitted to the Department of Neurological Sciences， Kasturba Medical College and Hospital， Manipal (tertiary care hospital) during the period July 2007–August 2008 (convenient sampling) were recruited for the study. The study was approved by the Institutional Ethical Committee and informed consent was taken from all the subjects. Patients were categorized later as benign/malignant after postoperative histopathological confirmation. The study subjects were pided into the following groups:

　　a. Control: Healthy inpiduals who visited the hospital for a routine health checkup served as control group (mean age 41 years， n= 20， male10， female10).

　　b. Benign tumor: Preoperative benign tumor patients with meningiomas， cellular schwannoma and craniopharyngioma were included in this group (mean age 41 years， n= 21， male15， female6).

　　c. Malignant tumor: Preoperative malignant tumor patients with glioblastoma， astrocytoma and oligocytoma were included in this group (mean age 47 years， n= 19， male10， female9).

　　Exclusion criteria: All the patients and control subjects were asked to fill a proforma and subjects with diabetes or any other illness， alcoholics， smokers， postoperative cases， subjects under medication and subjects with unhealthy oral cavity were excluded from the study.

　　Sample collection and preparation: The subjects were asked to rinse their mouth thoroughly with plain water. Buccal scrapings were obtained from the subjects prior to breakfast by the method described by earlier studies[10]. Briefly， the subjects were asked to scrape either side of their inner cheek with the help of blunt wooden sticks with smooth surface. After 5 to 6 gentle strokes the sticks were dipped in sterile phosphate buffered saline (PBS， pH 7.4). The cell suspension was pided into two parts and processed separately for DNA damage analysis using comet assay and micronucleus (MN) assay.

　　The saliva samples were always collected in restful and quiet circumstances. Unstimulated whole saliva was collected for 5 minutes by asking the subject to lean forward and spit the saliva into a graduated container. The salivary flow rate was calculated. The pH of the saliva was noted immediately using pH paper strips and comparing the color change to that of the standard color bar provided (range 210). The salivary osmolality was measured using cryoscopic osmomometer (Osmomat 030，Gonotec GmBH， Germany).

　　Alkaline comet assay: The buccal cells were processed for alkaline comet assay as described by Singh et al with minor modifications[3]. Briefly， the buccal cell suspension was mixed with 1% low melting point (LMP) agarose in 1:1 ratio (vol: vol). The cell suspension was layered on a slide precoated with 1% normal melting point (NMP) agarose. A third coat of NMP was layered on the top of the second layer sandwiching the cell suspension between two layers of NMP. Once the agarose was set， the cells were lysed by keeping the slides overnight at 4℃ in freshly prepared lysing solution ( 2.5 M NaCl， 100 mM Na2EDTA， 10mM Tris HCl， 5% DMSO and 1% Triton X100， pH 10). The slides were then immersed in freshly prepared electrophoresis buffer (300 mM NaOH， 1 mM Na2EDTA， pH&>13) for 30 minutes in cold room to unwind the liberated DNA fragments followed by electrophoresis for 7 minutes at 1.2 volts/cm (300 mAmp) under alkaline conditions (pH&>13) in a horizontal gel electrophoresis unit. The slides were immersed in the neutralizing buffer (0.4 M Tris HCl， pH 7.4) for 5 minutes twice to neutralize the excess alkali and stained using ethidium bromide (0.02mg/mL). The slides were then washed with neutralizing buffer， covered with a cover slip and visualized under 400× magnification using fluorescent microscope. The comet images were captured and the tail length was measured using LQ software (Leica， Germany). At least 500 cells were scored per slide. The length of tail DNA which represents the extent of DNA damage was calculated and expressed as median， IQR.

　　Revathi P Shenoy et al. DNA damage in exfoliated buccal cells and antioxidant status of saliva in brain tumour patients

　　Micronucleus (MN) assay: The MN assay was carried out according to the method of Tolbert et al[11]. Briefly， the cell suspension was washed by centrifugation at 2000 rpm for 5 minutes twice. The cells were fixed with freshly prepared Carnoy′s fixative (methanol: glacial acetic acid in a ratio of 5:1) and kept at 4℃ for at least 30 min for proper fixation of the cells. The cell suspension was later centrifuged at 2000 rpm for 5 minutes and the pellet was resuspended with fresh fixative. The cells were later spread on clean prechilled slides. The air dried slides were stained with acridine orange (0.002% in Sorenson′s buffer， pH 6.8) and scored under fluorescent microscope at 400× magnification using the criteria described by Fenech and Morley. At least 400 cells were scored from each slide and expressed as percent micronucleated cells.

　　Determination of Antioxidant activity :

　　The assay used was developed by Koracevic et al[12]. This assay measured the capacity of the antioxidants in saliva to inhibit the production of thiobarbituric acid reactive substances (TBARS) from sodium benzoate under the influence of the free oxygen radical generated from the Fenton′s reaction. A solution of 1 mmol/litre uric acid was used as standard.

　　Estimation of Ascorbic acid:

　　The ascorbic acid / vitamin C level was measured by the spectrophotometric method. Salivary ascorbic acid / vitamin C is oxidized by cupric ions to form dehydroascorbic acid which reacts with acidic 2，4dinitrophenyl hydrazine to form a red bishydrazone which is measured at 520 nm.

　　Statistical analysis

　　The results were expressed as median， IQR. A P value &<0.05 was considered statistically significant. Statistical analysis was performed by the nonparametric Mann Whitney U test using the SPSS package version11.

　　Results

　　DNA damage in buccal cells: The exfoliated buccal cells of control group had a median tail length of 38.65μm which was almost similar in benign group (38.23μm， Fig. 1a). However， the malignant group exhibited a significantly higher level of DNA damage compared to control and benign group (P&<0.02). Further， control group had a median of 0.32% micronucleated buccal cells. Almost two times higher percentage (0.63%) of micronucleated buccal cells were observed in benign group which was significantly higher then control (P&<0.002). However， in the malignant group the percentage micronucleated cells were significantly lower than benign group (P&<0.029， Fig. 1b).

　　Figure 1 DNA damage in exfoliated buccal cells of brain tumour patients， Median， Interquartile range (IQR).

　　Biochemical changes in saliva: The salivary flow rate， pH and osmolarity was significantly decreased in benign and malignant brain tumour patients when compared to that of controls (P=0.001， Fig. 2a， 2b， 2c). However， between benign and malignant group none of these parameters differed significantly.

　　Figure 2 Biochemical changes in saliva of brain tumour patients， Median (IQR).

　　Antioxidant activity and ascorbic acid level in saliva: The salivary antioxidant activity (Fig. 3a ) was significantly decreased in brain tumour patients (both benign and malignant) when compared to that of controls (P= 0.00). However， saliva from both benign and malignant group did not differ in their free radical scavenging activity. Saliva of the control group had 1.4mg/dL of Vitamin C which was almost 50% lower in both benign and malignant group compared to control (Fig. 3b).

　　Figure 3 Antioxidant status of saliva in brain tumour

　　patients， Median (IQR).

　　Discussion

　　Exfoliated buccal cells have been used as a source of DNA to study the genetic integrity in various pathological conditions[13，14]due to the patient friendly and minimally invasive collection protocol. In the present investigation we studied the DNA integrity in buccal cells of brain tumor patients. Earlier studies have observed that exfoliated buccal cells carry a high degree of DNA damage in malignant conditions

　　[15-17]. The result of comet assay suggests that the buccal cells of patients with brain tumor carry significantly higher amount of DNA damage compared to benign group and control. The result of our investigation agrees with earlier reports in various other types of malignancies[16，17]. However， in micronucleus assay， the benign group had higher percentage of cells with MN than control and malignant group. This could be due to a very high percentage of apoptotic cells in benign group compared to other groups (data not shown). In a previous study conducted in our laboratory[3]， the lymphocytes of brain tumor patients (benign and malignant) showed a significantly higher degree of baseline DNA damage compared to healthy inpiduals. Similarly， in the present study a significant increase in tail length of the comets was observed in the buccal cells of brain tumor patients showing a marked increase in DNA damage which could be due to increased oxidative stress， low level of cellular antioxidants and poor DNA repair ability.

　　When the control group was compared to benign cases and when benign and malignant groups were compared， a significant increase in the MN% was obtained . Moreover， an increase in the MN% was also noted in the malignant group when compared to controls. However this increase was not significant. Increase in MN% in brain tumour cases provides more evidence for oxidative damage to DNA. In a study carried out by Bonassi et al[18] it was found that an increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Another study evaluated micronuclei frequency in the cultured peripheral lymphocytes of the cancer patients before and after radiation treatment where a significant increase in MN frequency was noted after exposure to radiation.

　　The role of reactive oxygen species (ROS) in these pathological conditions is well characterized. Elevated lipid peroxidation and depleted level of antioxidant enzymes especially glutathione reductase in erythrocytes of brain tumor patients has been observed by Rao et al and Seetaramaiah et al[19，20]. In addition， alterations in the level of protein carbonyl， which is an indicator of oxidative stress mediated protein damage and also depletion of protein thiols has been observed in these patients[21]. The ROS load may increase along with depletion of cellular antioxidant level with the progression of pathological condition which is confirmed by our findings. Free radicals generated are highly unstable which attack crucial cellular targets such as nucleus， cell membrane and subcellular organelles resulting in DNA damage， membrane damage and loss of mitochondrial function. Further， it is well documented that malondialdehyde a major product of lipid peroxidation has mutagenic property in mammalian cells[22] which can initiate the pathogenesis. Depletion of antioxidant and increased ROS load may partly explains the elevated level of DNA damage observed in in buccal cells of brain tumor patients of malignant type compared to controls.

　　Oxidative stress experienced by the body during disease states can be assessed by the measurement of the antioxidant activity in human saliva[12]. It has already been observed that the higher prevalence of oral carcinoma in older people is due to the reduction in the salivary antioxidant activity or due to an increase in reactive oxygen species or reactive nitrogen species causing DNA aberrations[23].

　　Further， salivary antioxidant activity was seen to increase in rheumatoid arthritis patients[24]. Earlier reports also proved that free radical toxicity could be one of the factors involved in the pathogenesis of oral squamous cell carcinoma[25]. Uric acid is the major antioxidant in saliva (80%). It scavenges free radicals once they are produced and forms a stable complex with iron ions. However， there are no reports on salivary antioxidants in brain tumour patients. Therefore， as observed in the present study， the total antioxidant activity in saliva was significantly decreased in brain tumour patients when compared to that of controls which is conclusive of the fact that oxidative stress is well established in these patients. Our earlier studies carried out in blood of such patients[19-21] strongly support this observation. Since， the total antioxidant activity methodology adapted in this study does not measure levels of vitamin C， an assessment of the status of this antioxidant vitamin was carried out in brain tumour patients.

　　A protective effect on lipid peroxidation by vitamin C has been well established[26]. Vitamin C may help to reduce the free radical damage and atheroma formation in the blood vessels. A deficiency of vitamin C causes oxidative damage and impairs immune response. However， the role of vitamin C in health and disease is still controversial[27]. Significantly reduced vitamin C level has been reported in astrocytoma. In the present study， no significant change in the salivary ascorbic acid was noted. However， this does not overrule the possibility of alternation of the vitamin at the site of tumour. Further， studies carried out in our laboratory earlier， also reported no significant difference in plasma antioxidant vitamin levels including vitamin C in brain tumour patients when compared to controls[28].

　　Conclusion

　　Thus， as observed in the present study in exfoliated buccal cells of brain tumour patients there is enough evidence of a prooxidant milieu leading to free radical toxicity in such cases which causes increased DNA damage even in the cells of the buccal mucosa. This fact is strongly supported by the observation of significantly low levels of total antioxidant activity in saliva of such patients.

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