Authors would like to thank Swamy Vivekanandha college of pharmacy, Elayampalayam, Tiruchengode and Department of Animal Genetics and Breeding, Veterinary College and Research Institute, Namakkal, Tamilnadu, for providing facilities to carry out the research work.
Study results strongly indicate that antitubercular drugs cause cellular damage in TB patients and the extent of damage is more pronounced in alcoholics and smokers. The high level of concordance of the result obtained in the comet assay showed that the comet assay is not only sensitive enough to detect low levels of DNA damage in human lymphocytes, but also highly specific to detect DNA damage.
Tuberculosis (TB) is a highly contagious infection caused by the bacterium called Mycobacterium tuberculosis. TB can persist for decades in infected individuals in the latent state as an asymptomatic disease and can emerge to cause active disease at a later stage. There was an evidence that M.tuberculosis cells are exposed to DNA damaging agents such as reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) generated by host macrophages1 . The repair of DNA damage is expected to be particularly important to intracellular pathogens such as M. tuberculosis, and so it is of interest to examine the response of M. tuberculosis to DNA damage. The expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M. Tuberculosis2 . In pulmonary tuberculosis patients, little is known about peripheral DNA damage, although increased oxidative stress is a well documented entity. Therefore, we aimed to investigate DNA damage in pulmonary tuberculosis patients. DNA damage was assessed by Comet assay3 . The SCGE, also known as comet assay, is one of the recent methods established in order to detect different types of DNA damage. The comet assay has been established as a simple, rapid, cheap, flexible and, most importantly, sensitive method to detect DNA damage4 . DNA strand breaks allow DNA to extend from lysed and salt-extracted nuclei, nucleoids, to form a comet like tail on alkaline electrophoresis. Cells undergoing active cell death or apoptosis demonstrate highly fragmented DNA. Progression of cell death results in the extensive formation of double strand breaks and is readily detected using alkaline electrophoretic conditions5 . To the best of our knowledge, no study has expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M. Tuberculosis2 . In pulmonary tuberculosis patients, little is known about peripheral DNA damage, although increased oxidative stress is a well documented entity. Therefore, we aimed to investigate DNA damage in pulmonary tuberculosis patients. DNA damage was assessed by Comet assay3 . The SCGE, also known as comet assay, is one of the recent methods established in order to detect different types of DNA damage. The comet assay has been established as a simple, rapid, cheap, flexible and, most importantly, sensitive method to detect DNA damage4 . DNA strand breaks allow DNA to extend from lysed and salt-extracted nuclei, nucleoids, to form a comet like tail on alkaline electrophoresis. Cells undergoing active cell death or apoptosis demonstrate highly fragmented DNA. Progression of cell death results in the extensive formation of double strand breaks and is readily detected using alkaline electrophoretic conditions5 . To the best of our knowledge, no study has
Low melting agarose, Normal melting agarose, Triton X-100 and Phosphate buffer saline (PBS; Ca++ , Mg++ free) were purchased from HiMedia pvt. Laboratories (Mumbai). All other chemicals were of the highest purity available. 2.2 Subject selection and collection of blood samples DNA damage is an indication of cellular damage. Comet assay is widely regarded as a quick and reliable method for analyzing DNA damage in individual cells. 2ml of venous blood samples were collected in ETDA tube from subjects of Control group (G0), newly diagnosed TB patients (G1), three months treated TB patients (G2) and six months treated TB patients (G3). Institutional ethical committee of Swamy Vivekanandha College of pharmacy, Tiruchengodu, approved the protocol for the present study. All the patients were given verbal and written information about the study prior to withdrawal of blood sample. Three months treated TB patients were receiving Rifampacin, Isoniazid, Pyrazinamide and Ethambutol. Six months treated TB patients were receiving Rifampacin and Isoniazid. Newly diagnosed TB patients were recruited into the present study (n=25 for each group). All the study groups were compared with the 25 volunteers who constituted control group. 2.3 Single gel electrophoresis Supernatant liquid is discarded after centrifugation of blood. A small amount of remaining cells are placed in the glass slide with the help of micropipette. Half frosted slides were dipped into a chromic acid solution and then 100 % methanol is used to remove particulate matters. Half-frosted slides were dipped into 1% normal melting agarose (NMA), underside of the slide was wiped and slide was laid on flat surface to dry (First layer). To the coated slide, 75 ?L 0.5% low-melting point agarose (LMPA) (prepared in PBS; Ca++, Mg++ free) was added to prepare second layer. Third agarose layer with 80 ?L of 0.5% LMPA then followed (Third layer). Slides were kept in the lysing solution (2.5M NaCl, 100mM EDTA and 10 mM Trizma Base, 1 % Triton X-100 and 10 % DMSO were added freshly) at 40 C overnight. After lysis, slides were kept in electrophoresis chamber containing electrophoresis buffer (30 ml 10N NaOH, 5 ml 200mM EDTA q.s. 1000 ml, pH>13). Slides were allowed to sit in alkaline buffer for 20 mins to allow unwinding of DNA and the expression of alkali labile damage, and then electrophoresed for 30 mins (24 volts, 300 milliamperes). Slides were then coated with the neutralization buffer (0.4 M Tris in dH2O, pH 7.5). Each cell had the appearance of a comet, with a brightly fluorescent head and a tail to one side formed by the DNA containing strand breaks that were drawn away during electrophoresis. Numbers of comet parameters were calculated with TriTek CometScore TM Freeware version 1.5. Samples were run in duplicate, and 50 cells were randomly analyzed per slide for a total of 100 cells per sample. For quantitative evaluation, undamaged cells (C0), mild and moderate damaged cells (C1), highly damaged cells (>C1) cells were taken into account. It has been documented that any change in the level of DNA damage reflect most accurately in these three parameters (i.e) tail length, %DNA in tail and Olive tail moment10 . 2.4 Statistical analysis The statistical calculation were done using Graph pad Instat software version 3.01. The results are expressed as mean ? S.E.M. Difference between diseased (TB) and control subjects were assessed using one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test. P<0.05 was considered as statistically significant. 3. Results 3.1 Qualitative analysis of cellular damage The number of cells >C1 were found increased in Three months treated TB patients (G2) and Six months treated TB patients (G3). Newly diagnosed patients (G1) did not show any significant change in >C1 when compared with Control (G0) group at p<0.0001 (Table I). There was a linear increase in mild to moderately damaged cells (C1) with respect to increase in the time duration of treatment (Figure I). 3.2 Quantitative analysis of cellular damage Comet assay of white blood cells of study subjects show extremely significant increase in comet length in TB patients, when compared to control group. (G3=193.37; G2=175.42; G1=117.61; G0=80.47; P=0.0005) Similarly a significant increase in Comet intensity, Comet mean intensity, Head Area, Head mean intensity, Tail Area, tail mean intensity, %DNA in tail and Olive tail moment were found in TB patients when compared to control (Table II). When a comparison is made among the TB patients ( G1, G2, G3), Six months Anti-tubercular drug treatment exhibited significantly extensive damage in DNA than 3 months treated patients and newly diagnosed patients. Some comet parameters like Comet height, Comet Area, head diameter, head intensity, %DNA in head, Tail length, Tail intensity, Tail moment showed increase but not statistically significant 3.3 Comparison of some commonly used comet metrics: Results of three different parameters namely, tail length, %DNA in tail and Olive tail moment have been presented in (Table III) (Figure II). 3.3.1 Tail length: Tail length is the distance of DNA migration from the body of the nuclear core, which is related directly to the fragment size and it is expected to be proportional to the extent of DNA damage. Newly diagnosed TB patients demonstrated non significantly increased tail length values (7.832 ? 0.613) when compared to control subjects and drug treated patients (5.81 ? 1.12; 7.486 ? 0.77; 5.494 ? 0.675). during electrophoresis. Numbers of comet parameters were calculated with TriTek CometScore TM Freeware version 1.5. Samples were run in duplicate, and 50 cells were randomly analyzed per slide for a total of 100 cells per sample. For quantitative evaluation, undamaged cells (C0), mild and moderate damaged cells (C1), highly damaged cells (>C1) cells were taken into account. It has been documented that any change in the level of DNA damage reflect most accurately in these three parameters (i.e) tail length, %DNA in tail and Olive tail moment10 . 2.4 Statistical analysis The statistical calculation were done using Graph pad Instat software version 3.01. The results are expressed as mean ? S.E.M. Difference between diseased (TB) and control subjects were assessed using one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test. P<0.05 was considered as statistically significant.
3.1 Qualitative analysis of cellular damage The number of cells >C1 were found increased in Three months treated TB patients (G2) and Six months treated TB patients (G3). Newly diagnosed patients (G1) did not show any significant change in >C1 when compared with Control (G0) group at p<0.0001 (Table I). There was a linear increase in mild to moderately damaged cells (C1) with respect to increase in the time duration of treatment (Figure I). 3.2 Quantitative analysis of cellular damage Comet assay of white blood cells of study subjects show extremely significant increase in comet length in TB patients, when compared to control group. (G3=193.37; G2=175.42; G1=117.61; G0=80.47; P=0.0005) Similarly a significant increase in Comet intensity, Comet mean intensity, Head Area, Head mean intensity, Tail Area, tail mean intensity, %DNA in tail and Olive tail moment were found in TB patients when compared to control (Table II). When a comparison is made among the TB patients ( G1, G2, G3), Six months Anti-tubercular drug treatment exhibited significantly extensive damage in DNA than 3 months treated patients and newly diagnosed patients. Some comet parameters like Comet height, Comet Area, head diameter, head intensity, %DNA in head, Tail length, Tail intensity, Tail moment showed increase but not statistically significant 3.3 Comparison of some commonly used comet metrics: Results of three different parameters namely, tail length, %DNA in tail and Olive tail moment have been presented in (Table III) (Figure II). 3.3.1 Tail length: Tail length is the distance of DNA migration from the body of the nuclear core, which is related directly to the fragment size and it is expected to be proportional to the extent of DNA damage. Newly diagnosed TB patients demonstrated non significantly increased tail length values (7.832 ? 0.613) when compared to control subjects and drug treated patients (5.81 ? 1.12; 7.486 ? 0.77; 5.494 ? 0.675).3.3.2 %DNA in tail: It gives an idea regarding the damaged DNA content in individual cells, measured as the total intensity of ethidium bromide in each comet tail, verified by DNA leached out of the cell when exposed to alkaline electrophoretic conditions. It is defined as the ?ratio of tail optical intensity to the sum of tail and head optical intensity?, multiplied by 100. Very significant increase in %DNA in tail in newly diagnosed TB patients were observed (5.328 ? 0.634) when compared to control group. Three months treated TB patients showed significant difference (5.09 ? 0.4283) when compared with control group (P =0.0049). 3.3.3 Olive tail Moment (OTM): It is defined as the fraction of tail DNA multiplied by the distance between the profile centres of gravity for DNA in head and tail. OTM incorporates a measure of both the smallest detectable size of migrating DNA (reflected in the comet tail length) and the number of relaxed/ broken pieces (represented by the intensity of DNA in the tail). OTM was observed to be least in controls and highest in six months treated group, and the difference was significant (0.098 ? 0.0124; 0.162 ? 0.0174; 0.234 ? 0.0457; 0.242 ? 0.0538) (P =0.0449). 3.4 Impact of Alcoholism on comet length and its significance Comparison of both the Alcoholic and nonalcoholic group, comet length shows very significant difference in both three months treated TB patients and in six months treated TB patients when compared with control group (P<0.0010). Non-alcoholic group didn?tderived units are based. The percentage DNA in tail is directly proportional to the amount of damaged DNA11. Olive tail moment (OTM) is the tail moment the product of the tail length and the fraction of total DNA in the tail. Tail moment incorporates a measure of both the smallest detectable size of migrating DNA (reflected in the comet tail length) and the number of relaxed / broken pieces (represented by the intensity of DNA in the tail). A significant difference in (P=0.0449), control group when compared with newly diagnosed, three months, and six months was observed. Any change in the level of DNA damage will be reflected most accurately by Olive tail moment measurements. Increase in Olive tail moment in our study confirms the role of Mycobacterial infection and anti-TB drugs on DNA damage. The mean? S.E.M value of comet length in control group of patient was 80.476 ? 3.624, for newly diagnosed TB patients it was 117.61 ? 13.689 for Three months treated patients it was 175.426 ? 27.587 and for Six months treated patients 193.374? 10.027 which was found to be extremely significant (P= 0.0005). Comet length showed extremely significant difference among the cases when compared with control subjects indicating increased DNA damage among cases and this is in consistent with other studies12 . The comet length also showed significant increase in Alcoholics and smokers, it is greater in males taking anti-tubercular drug treatment when compared with females, which clearly indicates the influence of alcohol induced oxidative stress on cell damage. Oxidative stress and DNA damage are increased in pulmonary tuberculosis patients. Increased oxidative stress associated DNA damage may be one of the pathogenetic mechanisms involved in the disorders suggested to be associated with pulmonary tuberculosis3 . Certain drugs are known to induce DNA damage in healthy cells and potentiate the oxidative stress generated during cellular events13. It was observed that six months treated Tuberculosis patients taking antitubercular drug treatment of Rifampacin and Isoniazid showed an extremely significant increase in cellular damage. Three months treated TB patients taking antitubercular drugs like Rifampacin, Isoniazid, Pyrazinamide, and Ethambutol also showed a significant difference in cellular damage. The co-administration of Rifampacin and antioxidants (Vit.C & Vit. E) has protective effect on the damaging potentials of Rifampacin on the DNA. It may then be recommended that the clinician may incorporate antioxidants in the regimen of patients with tuberculosis so as to reduce the possible adverse effect on the DNA9 . Thus, in the light of our observation, it is suggested that Tuberculosis patients showed increased DNA damage as significant differences were detected between Control, newly diagnosed TB patients, Three months treated TB patients and Six months treated TB patients in terms of frequencies of damaged cells. The result of the present study reveal that patients undergoing therapy had significantly greater DNA damage as compared with untreated patients, indicating that bacterial infection and drug therapy are causal factors.
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