Vitamin E reduces reactive oxygen species mediated damage to bio-molecules in leprosy during multi-drug therapy
R.Vijayaraghavan1* C.S. Suribabu1, P.K.Oommen1 and C. Panneerselvam2
1 Central Leprosy Teaching and Research Institute,
Chengalpattu Pin code # 603001, India
2Department of Medical Bio-Chemistry,
Postgraduate Institute of Basic Medical Sciences, University of Madras ,
Taramani Campus, Madras (Chennai) - 600 113, India.
*For Correspondence: firstname.lastname@example.org
Current Trends in Biotechnology and Pharmacy , October-2009-3(4)
Chronic infectious disease process of leprosy caused by the intracellular parasitic pathogen Mycobacterium leprae involves accumulation of highly reactive oxygen species (ROS). The present treatment method available for leprosy is MDT (Multi Drug Therapy) involves combination of Rifampicin, Dapsone and Clofazimine, although MDT disinfect leprosy patients, it has limited impact on the increased ROS production and decreased antioxidant status in affected individuals. In the present study we have investigated the causative bacillary load, lipid peroxidation, DNA damage in single cells using single cell gel electrophoresis (alkaline comet assay), oxidative stress indices in multi bacillary leprosy patients. The mean values plasma lipid peroxidation, erythrocyte oxidative stress indices in untreated leprosy cases were higher than the controls, this further increases significantly (p<0.05) with MDT. Upon supplementation with 400 IU antioxidant vitamin E we noticed a significant decrease was noticed in plasma lipid peroxidation, oxidative stress indices, and DNA damage. Hence we conclude that MDT along with vitamin E supplementation reduces the oxidative stress mediated insult to cells and bio-molecules during the chronic course of treatment in leprosy.
MDT : Multi Drug Therapy, ROS : Reactive oxygen species, LPO: Lipid Peroxidation , TBARS: Thio Barbutric Acid Reactive Substances, DNA: Deoxy Ribo nuclic Acid, MDA: Malondialdehyde, DMSO: Dimethyl Sulphoxide, WBC: White Blood Cell, SOD: Superoxide Dismutase, NBT: Nitroblue tetrazolium, EDTA: Ethelene Diamine Tetra Acetic acid.
Hansen’s disease “historically known as leprosy” is a mutilating, debilitating, devastating and deforming disease of mankind. The causative intra cellular germ Mycobacterium leprae mediate strong inflammatory response and cause gross destruction of tissues during the chronic course of infection in affected individuals. Patients with leprosy are treated with Multi Drug Therapy (MDT) consisting of Rifampicin, Dapsone and Clofazimine. Despite of large-scale implementation of MDT by the world health organization, the incidence of the disease is still cause of concerned in several hyper-endemic countries(1). MDT although disinfect leprosy patients, it has limited impact on the increased ROS production and decreased antioxidant status in affected individuals (2,3,4). In our earlier studies, we reported apparent involvement of oxidative stress together with a significant decrease in the antioxidant status in leprosy cases (5)
In leprosy affected persons, the exact mechanism by which intracellular M leprae resist destructions of bactericidal activity of the host cell still remains poorly understood. Possible mechanism of bactericidal activity is believed to include the effects of toxic oxygen derivatives or ROS (Reactive Oxygen Species) such as superoxide anion (O2·-), hydroxyl radical. (OH) or hydrogen peroxide (H2O2) derived from the phagocytic cell respiratory burst (6). Other mechanism that mediate ROS production is the drugs used in MDT in releasing ROS during the mode of action (4,7,8). Recent evidences reveal the possible association of ROS in causing injury to cell and damaging effect on DNA such as oxidation of purine and pyramidine bases, break in double strand and single strands (9). Apart from the severity caused by the infectious agent, malnutrition often seen in leprosy patients aggravates infection, while infection with intracellular Mycobacterium leprae adversely affect the nutritional status(10). Earlier studies shown that in the malnourished, the severity of the disease and susceptibility disease is relatively higher (11). Malnutrition usually co-exists with the depletion of micronutrients and antioxidant vitamins (12). Under normal condition the production of ROS is kept in control by the antioxidant defense system but in leprosy cases this antioxidant defense system is affected and leprosy patients are exposed for oxidative stress which affect the cell structures and bio-molecules (13,14).
Several intervention studies support the long-term supplementation of antioxidant vitamin E results in declining in the oxidative damage to DNA during the chronic pathology caused by Mycobacterium (15). Earlier studies reveal reduction of clastogenic effect was noticed upon treatment with vitamin A and vitamin C in murine models infected with M.leprae (16). Apparent renal toxicity owning to anti leprosy drug administration is also reported in recent years (17). Low levels of antioxidants as seen in patients with leprosy could expose the leprosy patients to oxidative stress mediated insult through ROS.
Hence exogenous supplementation of micronutrients and intervention of antioxidants is an attractive approach in the treatment of leprosy. In the present study we have investigated the causative bacillary load, lipid peroxidation, DNA damage in single cells using single cell gel electrophoresis (alkaline comet assay), oxidative stress indices in multi bacillary leprosy patients and we have supplemented 400 IU of antioxidant vitamin E to evaluate the protective role against the oxidative stress mediated changes to cells and bio-molecules during the chronic course of infection and anti leprosy chemotherapy (MDT).
Materials and Methods
Laboratory diagnosis of leprosy is made on the basis of the skin smear examination and the causative bacillary load is recorded as (BI) Bacteriological index (18). A total of 80 untreated multi bacillary (severe) type of leprosy patients attending the OPD of Central Leprosy Teaching and Research Institute in the age group of 25 to 40 years of both the gender were enrolled in this study. Of the 80 diagnosed leprosy cases only 50 cases fully cooperated for the study and they are enrolled as Group II (remaining 30 cases did not fully co-operate for this study). Enrolled 50 cases in the Group II are further divided in to Group II a and Group II b. Group II a (n=25) leprosy cases are treated with MDT and Group II b (n =25) consisted of leprosy patients treated with MDT + antioxidant vitamin E 400 IU/ L supplementation. Group I (n=25) consist of control subjects and they are age matched healthy without any signs and symptoms of the disease. Informed consent from the participants and clearance of ethical bodies of the institution was obtained. Persons with systemic ailments having the habit of smoking, consuming alcohol, taking treatment for any other ailments were excluded from the study. No study subject consumed vitamin E in the last 6 months.
All diagnosed leprosy patients were treated with anti leprosy chemotherapy (MDT) and patients in group II b is treated with vitamin E 400 IU along with MDT. Following diagnosis, anti coagulated blood samples were collected from the control and experimental subjects (group II at the time of diagnosis) and group II a and group II b (after the treatment period) were processed for the separation of plasma, WBC (Using Ficoll Hypaque) and red blood cells and haemolysate. Lipid peroxidation assay was estimated using plasma samples. Detection of DNA damage in single cells (WBC) were estimated using the separated WBC fraction, erythrocyte and haemolysate was used for the estimation of oxidative stress indices. Haemolysate was prepared according to standard bio-chemical method (19). Skin smear grading is used to understand the grading of bacterial load in a leprosy patient (20). Mean standard deviation values are tabulated and when found significant ANOVA tests is performed for inter group comparison at P<0.05 significance level.
Oxidative stress indices
Superoxide radical assay
Superoxide radical is a very labile radical and analysis was made in all samples processed identically from the collection site. The blood samples were cooled during the transport from the collection site during the transport and the nitro blue tetrazolium (NBT) reduction was measured exactly at the end of one hour after blood collection to obtain uniformity. For each sample of blood superoxide was measured as NBT reduction in two sets of tubes of which SOD was inhibited in one set. The difference gave the superoxide radicals present at the time of assay. A stock solution containing 100 mM of NBT was prepared in water and diluted to make a working standard of 10mM/ml of NBT served as standard for the assay of superoxide radicals. Superoxide levels in erythrocytes were expressed in terms of milli moles of NBT reduced/ 10 12 cells/ 10 minutes (21).
Hydroxyl radical assay
Hydroxyl radicals in the haemolysate were estimated by the method of Gutteridge by their reaction (hydrogen abstraction) from 2-deoxyribose, resulting in the formation of thio barbutric acid and reactive substances. 2.0 ml of 0.5 % hemolysate, 2.0ml of deoxyribose was added and incubated at 37◦C for one hour. Then 0.5 ml of TBA (thiobarbituric acid solution) and 0.5 ml of trichloroacetic acid solution were added and heated in a boiling water bath for 15minutes. The mixture was cooled and the reading was taken at absorbance at 530 nm. Blank were included without 2-deoxy ribose to assess the basal thiobabuturic acid reactive substance in the hemolysate for each blood samples analyzed. Standards in the range of 5 to 30 nanomoles were also developed. Amount of hydroxyl radicals present in the erythrocytes were expressed in terms of nanomoles of MDA/1012 cells/ hour (22).
Hydrogen peroxide assay
Hydrogen peroxide in the erythrocytes were estimated by the method of Wolf (23). Hydrogen peroxide oxidizes ferrous iron to ferric iron selectively and the resultant Fe3+ can be determined by sensitive dye xylenol orange (O-cresol sulfonapthalene, 3’3’-bis methylamino diacetic acid which is highly sensitive to Fe3+ to form a blue-purple complex measured at 560 nm. Fresh blood samples containing heparin and 1mM sodium azide a catalase inhibitor was used in this assay. Solution containing 1-5µM of hydrogen peroxide were treated in a similar way is used a standard. Erythrocyte hydrogen peroxide levels were expressed as µ moles/ 10 12 cells.
Lipid peroxidation in the plasma was estimated using TBA (Thiobarbutric acid) reaction. Standard MDA 50 mM solution of malondialdehyde was prepared in distilled water using 1,1,3,3 tetrahydroxypropane. This was stored in 4 °C and diluted just before use such that working standard contains 50nM/ml. Plasma TBARS values were expressed as n moles of MDA/ L (24).
Detection of DNA damage in single cells (Comet assay)
DNA damage in the separated portion of white blood cells (WBC’s) was carried out by the method of detection of DNA damage in single cells (25). Comet assay: single cell gel electrophoresis is a technique which detects DNA damage and repair in individual cells like WBC. 100 µl of normal melting agarose in phosphate buffer was dropped on a frosted slide, immediately covered with a cover slip and kept for 10 minutes in a refrigerator to solidify. Then, cover slips were removed and 100 µl low melting agarose containing 75 µl of WBC in PBS and 100 µl low melting point agarose) were added to the slides. Again the cover slips were replaced and slides were kept in refrigerator for another 10 minutes. After this, the cover slips were removed and the top layer of 100 µl low melting agarose was added and cooled again for another 10 minutes. After this step, the slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA and 10 mM Tris-Hydrochloric acid, pH adjusted to 10.0 with NaOH; 1% Triton X – 100 and 10% DMSO were added freshly) and slides were kept in dark at 4°C for at least 1 hr to prevent additional DNA damage the following procedures were carried out under dim light. The slides were removed from lysing solution and placed on a horizontal electrophoresis tank. The electrophoresis unit was filled freshly made electrophoresis buffer (300 mM NaOH and 1mM EDTA, pH 13.0) to a level of 0.25 cm above the slides. The cells were exposed to alkali for 20 minutes to allow unwinding of DNA. An electric current of 25 volt and 300mA (high voltage electrophoresis) was applied for 20 minutes. After electrophoresis, slides were placed horizontally and neutralized with Tris-HCl. Finally, 50µl of ethidium bromide was added to each slide and covered with cover slip again and analyzed under the high power magnification of fluorescent microscope with a calibrated ocular. Images of 50 randomly selected cells were analyzed from each sample. For each cell, the length of the image (diameter of the nucleus plus migrated DNA) was measured. The intact DNA appear like a spot while the damage in DNA appear as comet with tail. The tail length is directly proportional to the DNA damage in single cell (WBC). The damage is represented by an increase of DNA fragments that have migrated out of the cell nucleus in the form of a characteristic streak similar to the tail of a comet.
Table 1: depicts the levels of toxic metabolites of oxygen derived free radicals like superoxide radicals, hydroxide radicals, and hydrogen peroxide in controls and experimental subjects, Superoxide and hydroxyl levels were estimated in the haemolysates prepared from erythrocytes and the levels of hydrogen peroxide were estimated in the erythrocytes of control and experimental subjects. Nearly two fold increase in the levels of oxygen-derived free radicals noticed in group II. The levels of oxygen derived free radicals increase further upon treatment with MDT (compare group II verses group II a). Upon co-supplementation of vitamin E oxidative stress decreases
Figure 1: represents the bacterial load scored after examining 100 oil-immersion fields by number ranging from 0 to 4. 5 + each representing, on the average of 10 times as many bacilli as the smaller number is 1 +. Untreated MB leprosy cases had a high bacillary index of 4. 5 + on Ridley’s logarithmic scale. Upon treatment with MDT the BI fall from 4.5 + to 2 +. Group II (untreated leprosy) patients showed a profound increase in the BI which may be due to the lack of immunity against the invading intra cellular M.leprae. Treatment with MDT showed a progressive reduction in the bacterial index from 4 + to 2 + (compare group II vs group II a). There is no significant change in the bacterial index in group II b (vitamin E supplemented subjects) which means vitamin E supplementation did not either induce or reduce the bacterial load in leprosy patients.
Figure 2: depicts the levels of plasma lipid peroxidation in control and experimental subjects. Lipid peroxidation is an organic expression of free radical mediated changes to lipid and the end product of lipid peroxidation (LPO) is malondialdehyde which can be measured in plasma samples by their reaction with thiobarbitutaric acid reactive substances. We noticed a sharp increase in the LPO in plasma was observed in leprosy patients when compared to control (compare group I vs group II), LPO levels further increased in leprosy subjects undergoing MDT (compare group II a vs group II). Upon supplementation with antioxidant vitamin E (group II b) the LPO levels decreased (compare group II a Vs group II b).
Figure 3: depicts the extent of DNA damage in single cells (WBC) in control and leprosy affected persons. The tail length of the comet is directly proportional to the extent of DNA damage in single cells. Single cells of multi bacillary leprosy cases shows a significantly increased tail length (P<0.05) indicates the increased DNA damage in response to the infection,
Fig 4. There was a significant protection against the DNA damage in single cells upon treatment with antioxidant vitamin E.
Fig. 1. Bacteriological index of experimental subjects
Values expressed as mean ± S.D
Group I (not applicable)
a -group II compared with group I
b- group II a compared with group II
a,b statistically significant at p<0.05
cNS - not significant at p<0.05 (group II b compared with group II a)
Fig. 2. Lipid peroxidation products (TBARS) in the plasma of control and experimental subjects
Values expressed as mean ± S.D
a -group II compared with group I
b- group II a compared with group II
c- group II b compared with group II a
a,b and c statistically significant at p<0.05
Fig. 3. Demonstration of DNA damage in single cells (White Blood Cells) by the alkaline comet assay
stained with Ethidium bromide fluorescent staining technique in control group I , untreated group II, MDT
treated (group II a ) and MDT + vitamin E co-supplementation ( group II b ). Magnification X 400 times.-
Fig. 4. DNA migration (comet tail length) control and experiental subjects.
Values expressed as mean ± S.D
a -group II compared with group I
b- group II a compared with group II
c- group II b compared with group II a
a,b and c statistically significant at p<0.05
The greatest paradox of aerobic respiration is oxygen,which is essential for energy production may also be detrimental,because it leads to the production of reactive oxygen species (26). Oxidative stress is a condition in which theelevated levels of ROS damage cells, tissues and internal organs (27,28). Oxygen derived free radicals are produced as a result of metabolism of oxygen biradical during reduction reactions (29). Superoxide radicals are unique in that it can lead to the formation of many other reactive oxygen species including hydroxyl radical (HO2). Superoxide radicals also reacts with Hydrogen peroxide to generate the singlet oxygen molecule (30). The hydroxyl radicals are most potent oxidant encountered in the biological system, because they readily react with almost all biological substrates (31). Hydrogen peroxide is not a radical by definition but it remains most extensively as they are formed as a secondary product of one-electron transfer oxidation of oxygen biradical during reduction reaction (32). Oxidative DNA damage refers to the functional or structural alterations of DNA resulting from the insults of ROS (33). DNA base modification and DNA strand breaks are two of the major forms of oxidative DNA damage caused by ROS. DNA also has the ability to repair itself against the toxic substances and deleterious effects of reactive oxygen species (34,35). When the oxidative stress override the antioxidant status, a physiological imbalance is produced in multi bacillary leprosy patients, such imbalance expose the DNA to persistent attack of ROS.
At molecular level, damage to DNA caused by ROS, oxidize the purine and pyrimidine bases, there by result in gross changes in DNA such as single strand breaks, sister chromatid exchanges and the formation of micronuclei during the chronic infectious disease process and treatment with drugs used in MDT. They are produced continuously in the cells either as accidental by-products of metabolism or deliberately during the pathogenesis of chronic infectious, inflammatory diseases. It is difficult to block theoxidative stress-induced injury to cells or tissues because ROS are continuouslyproduced by cellular aerobic metabolism (36). Oxidative stress may be limited by using chain-breakingantioxidants such as vitamin E which neutralizes hydroxyl, superoxide,and hydrogen peroxide radicals and prevents oxidative stress (37). In addition, it also helps in membrane stability and recycling of vitamin C. In our earlier studies we have reported free radical mediated oxidation of lipids and proteins in untreated and MDT treated leprosy patients (38,39).
Dietary antioxidants form an essential part of the human antioxidantdefense system. Fruits and vegetables as well as daily dietarysupplements constitute the potential sources of various antioxidants (38). The daily requirement of vitaminE varies from 50 to 800 mg. The ‘antioxidant hypothesis’ proposes that antioxidants like vitamin C, vitamin E, carotenoids present in fruits and fresh leafy vegetables afford protection against the oxidative damage to cells and bio-molecules (39). But leprosy patients do not get antioxidant rich diet like fruits and fresh leafy vegetables everyday and moreover, the deranged liver function affects the homeostasis of in vivo antioxidant status in affected individuals (40). Agnihotri et al, reported higher plasma LPO with decreased activity of renal brush border membrane in M.leprae infected mouse models (41). The elevation of LPO in the plasma is considered as a cause for degeneration of organs and tissues in leprosy patients. Lipid peroxides formed at the primary site could be transferred through blood circulation to other organs and tissues provoke damage by lipid peroxidation(42).
Mycobacterium leprae does not affect all the individuals who are exposed to it, nor does it produce the same degree of illness in those who become infected by it. A variety of risk factors have been invoked to explain this variation in disease susceptibility and morbidity of which nutrition plays a key role in determining the type of disease. Leprosy afflicted patients are devoid of balanced food for various reasons like: lack of job opportunity, loss of social status, deformity, disfigurement, social stigma and further the deranged liver function as seen in leprosy patients could affect the homeostasis of micronutrients. Further, the drugs used in MDT (multi drug therapy) for treatment of leprosy is very effective in controlling the proliferation of M.leprae but, during the mode of action produce copious amount of oxygen-derived free radicals this process could affect the functional integrity of the cells. In the present study we have noticed a decline in the oxidative stress indices upon supplementation of antioxidant vitamin E which compel us to examine more and more on oxidative stress mediated changes to cells and bio-molecules in affected individuals. Advances in understanding of the oxidative stress related changes to biomolecules like lipid and DNA may lead to tools for use of specific drugs used in control programmes in leprosy and will provide new insights on Mycobacterium leprae virulence, pathogenecity and treatment of leprosy. This study offers an opportunity for correlating levels of MDT therapy-induced DNA damage with administered dose and modulating effect of exogenous supplementation of antioxidant vitamin E in genotoxicity during the chronic course of the disease and anti-leprosy chemotherapy.
Author acknowledge Dr. R. K. Jeevan Ram, Health and Safety Division, IGCAR, Kalpakkam, Tamil Nadu, India for providing training in detection of DNA damage in single cells. Author also thanks the Director, Central Leprosy Teaching and Research Institute, Chengalpattu, Tamilnadu, India for permitting to collect samples from leprosy patients. Author thanks the leprosy patients for providing the vital benchmarks to mount the findings on.
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