Abstract

The alkaline single cell gel electrophoresis assay was performed on peripheral blood lymphocytes of lepromatous and tubercloid leprosy patients (untreated and those undergoing treament) in order to ascertain whether differential damage to DNA occurs. The study group included 28 male and 2 female patients and 15 healthy males who were matched for age and socio-economic status. The results revealed DNA damage in all patients, with a mean DNA migration length of 29.88 ± 3.39 µm and 38% of their cells damaged when compared with the respective values obtained in healthy controls (1.28 ± 0.40 µm, 5%). Multiple regression analysis for effects of confounding factors revealed antibiotic treatment in patients and alcohol consumption in controls as the only variables influencing DNA damage. In lepromatous and tubercloid patients, both untreated and those undergoing treatment, DNA damage increased significantly from that observed in control individuals, with greater increased damage in lepromatous patients. An increase in treatment time increased DNA damage linearly. Furthermore, an arbitrary classification of damaged cells (categories I–IV) was made based on observed tail lengths in leprosy patients (5.00–225.00 µm). The number of damaged cells in untreated patients was lower than in those undergoing treatment; the latter also had more cells with greater DNA migration lengths. There were no category III or IV cells in the control group. The results of the study therefore reveal that patients undergoing therapy had significantly greater DNA damage than untreated patients, indicating bacterial infection and drug therapy as the causal factors, since lepromatous-type disease is the more severe form with the patients having lower resistance to Mycobacterium leprae and requiring heavier and prolonged dosage of antibiotics. The study also corroborates that the assay offers an opportunity for correlating levels of therapy-induced DNA damage with administered dose and for modulating the dose-schedule so as to achieve lower levels of genotoxic damage.

Introduction

Leprosy (Hansen's disease), a severe, chronic infectious disease caused by Mycobacterium leprae may be lepromatous (LL), tubercloid (TT) or indeterminate, depending on its clinical symptoms, and may be graded as paucibacillary or multibacillary. The problems of patients either failing to collect their treatment or not responding to single drug treatment because of bacterial drug resistance and bacterial persistence led a WHO study group in 1982 to recommend multidrug therapy (MDT) for the treatment of leprosy (cited in Bryceson and Pfaltzgraff, 1990). Chemotherapy for leprosy generally includes dapsone, rifampicin and clofazimine, which themselves may affect genetic damage in treated leprosy patients.

In fact, both cytogenetic damage in leprosy patients and genotoxicity of anti-leprotic drugs have been reported in the literature (Chaudhuri et al., 1984; D'Souza and Thomas, 1988; Roy and Dass, 1988; Dash et al., 1991; D'Souza et al., 1991; Kaur et al., 1999; Aly and Donya, 2002), however, measurements of damage to DNA such as strand breaks and alkali labile sites are scarce (Kalaiselvi et al., 2002). Hence, in the present study an effort has been made to assess DNA damage in peripheral blood lymphocytes (PBLs) of some leprosy patients using the alkaline single cell gel electrophoresis (SCGE) assay and to ascertain whether differential damage to DNA occurs in lymphocytes of lepromatous and tubercloid leprosy patients (untreated and those undergoing treatment). LL is characterized by a severe course and the patients show very low resistance to M.leprae. TT is favourable and benign as to its manifestations and general course; the patients have rather high resistance to M.leprae infection and the disease in this stage is not very contagious.

The SCGE assay is a sensitive and simple technique by which DNA damage at the individual cell level can be assessed (Singh et al., 1988; Tice et al., 1992; Collins et al., 1997). The assay has found diverse applications in population monitoring, genotoxicity evaluation and in modulation of the diseased condition in humans (Rajeswari, 1995; Udumudi et al., 1998; Sardas et al., 2001; Maluf and Erdtmann, 2001). The alkaline SCGE technique was developed by Singh et al. (1988) in order to detect the presence of single-strand breaks and alkali-labile damage in individual cells.

Materials and methods

A total of 30 leprosy patients (28 males and 2 females) visiting the Guru Nanak Dev and Old Civil Hospitals, Amritsar and 15 controls were studied for DNA damage in their peripheral blood lymphocytes. The control individuals were non-exercising healthy volunteers without recent infections who were matched for age, sex and socio-economic status and can be considered representative of normal population individuals. The nature of the research work was explained to each individual and written informed consent was obtained. In order to minimize the presence of confounding factors, age, sex, smoking habit, alcohol consumption and diet were used as the criteria for selection of populations and information on exercise, health status and recent infections was recorded before blood sampling. The protocol for the SCGE technique as given by Singh et al. (1988) with alterations suggested by Ahuja and Saran (1999) was followed except for an increase in lysis time from 1 to 7–8 h in the present study and silver staining of comets was used instead of with ethidium bromide (Delincee, 1995), using locally available chemicals. Approximately 200 µl of finger-prick blood from each individual was collected in a sterilized, Eppendorff tube containing EDTA and a few drops of phosphate-buffered saline (PBS). The blood samples were transported to the laboratory on ice and were processed for assay within 2–4 h. Briefly, the blood in PBS was mixed with low melting point agarose (LMPA) and applied to slides precoated (in lieu of frosted slides) with 1% normal melting point agarose (NMPA). Another layer of LMPA followed this and the slides were subjected to lysis followed by electrophoresis and staining with silver nitrate (AgNO3) solution. DNA migration length and scoring of comets and normal cells was done under a binocular microscope at 40× using an ocular micrometer calibrated with the help of a stage micrometer. Two slides were made from each sample and 50 cells (25 per slide) were scored. The samples were coded and scored blind. Cells were measured as they came into the field. A quantitative value for DNA damage (DNA migration length, tail length from the trailing edge of the nucleus to the leading edge of the tail) of each cell was calculated as the difference between length of the comet and diameter of the comet head. Apoptotic cells (with more damage in the tail and forming special figures) were not observed either in the patient samples or in those of the control individuals. Random evaluation of the coded slide preparations was also carried out by another evaluator to assess the quality of slide preparations, to inspect varying degrees of cell damage and to check for apoptotic figures. Statistical analysis of data on tail length was carried out using regression analysis and Student's t-test. Multiple regression analysis was performed to assess the reltionship of various independent variables that could be confounding factors (age, smoking and alcohol consumption) for induction of DNA damage in both patients and controls.

Results

Characteristics of the patients and of the control sample are given in Table I. Occupation and income of the patients revealed their low and middle socio-economic status, with 16 patients living in mud houses and 14 in cement types. The leprosy patients, working in diverse occupations comprised those on MDT (n = 14, including the two females) and those who were yet to start treatment (n = 16). There were no cases of infertility or any other aberrant reproductive outcomes among the married patients (n = 18) or controls (n = 12). The pedigrees of the patient and control groups also did not reveal any familial diseases.

Table I.

Characteristics of the leprosy patients and control individuals

Group Sex
 
 Age range (years)(mean) SES
 
 Dietary habits
 
 Smoking history
 
 Alcohol consumption
 
 Untreated MDT
 
 DNA migration length (µm) meana ± SE 

 
Male
 
Female
 

 
Middle
 
Low
 
Veg
 
Non-veg
 
Smokerb
 
Non-smoker
 
Yes
 
No
 

 
D + R
 
D + R + C
 

 
Patients 28 17–65 (41) 16 14 22 18 12 19 11 16 Untreated 13.29 ± 3.01c 
               Treated 48.85 ± 3.92c 
               Total 29.88 ± 3.39c 
Controls 15 18–60 (39) 10 11    1.28 ± 0.40 
Group Sex
 
 Age range (years)(mean) SES
 
 Dietary habits
 
 Smoking history
 
 Alcohol consumption
 
 Untreated MDT
 
 DNA migration length (µm) meana ± SE 

 
Male
 
Female
 

 
Middle
 
Low
 
Veg
 
Non-veg
 
Smokerb
 
Non-smoker
 
Yes
 
No
 

 
D + R
 
D + R + C
 

 
Patients 28 17–65 (41) 16 14 22 18 12 19 11 16 Untreated 13.29 ± 3.01c 
               Treated 48.85 ± 3.92c 
               Total 29.88 ± 3.39c 
Controls 15 18–60 (39) 10 11    1.28 ± 0.40 

MDT, multidrug therapy; D, dapsone; R, rifampicin; C, clofazimine; SES, socio-economic status.

a

Calculated as an average of individual DNA migration lengths in that group.

b

15 patients both smoked [5–10 cigarettes or beedis (Indian rolled cigarettes)/day] and took alcohol [mostly beer (3–12 l/week) and/or wine (1–4 l/week)]; 2 control individuals similarly both smoked and took alcohol.

c

Statistically significant from total control group and also UT (on treatment group) from UNT (untreated group) group (P ≤ 0.01, P ≤ 0.001, Student's t-test).

Single cell gel electrophoresis analysis on leprosy patients revealed DNA damage in all patients, with a mean DNA migration tail length of 29.88 ± 3.39 µm and 38% of their cells damaged. Since the patient group comprised only two females and the tail lengths of their PBLs were within the range observed in the male patients, the data on DNA damage for females and males were pooled. In some control individuals (n = 7, 47%) DNA damage (1.28 ± 0.40 µm) was also observed, but with only 5% of their cells damaged.

Multiple regression and multivariate analyses were performed in order to assess the possible relationship of various independent variables that may be confounding factors (age, smoking and alcohol consumption) for induction of DNA damage (dependent variable) in both patients and controls. It was observed that in treated and untreated leprosy patients age (r = 0.170, F ratio 0.868), smoking (r = 0.263, F ratio 2.157) and alcohol consumption (r = 0.145, F ratio 0.622) did not influence DNA damage, presumably because of the small sample size. Rather, cells of those who were under MDT had significantly higher DNA tail lengths as compared with those who were untreated, indicating that the increase in DNA damage was probably due to the antibiotic treatment (dapsone, r = 0.712, F ratio 13.36; rifampicin, r = 0.772, F ratio 19.19; clofazimine, r = 0.978, F ratio 129.07). In the healthy individuals alcohol consumption, however, was the only variable influencing DNA damage (r = 0.614, F ratio 8.51).

On the basis of the observed range in tail lengths in leprosy patients (5.00–225.00 µm), an arbitrary classification of damaged cells was made placing the comets into four categories (Table II). Collins et al. (1995, 1997), by visual inspection and subsequently by image analysis, had given each comet a value of 0–4 according to the degree of damage depending on tail intensity. In the present study the overall percentage of damaged cells was least (∼5%) in control individuals (categories I and II) while the treated LL patients had almost 50% of their cells damaged. The spectrum of damaged cells was different in those being treated and the untreated leprosy patients. It was generally observed that the percentage of cells in category I (normal cells) was maximum in all the leprosy cases except for those being treated for LL, where almost equal numbers of damaged and undamaged cells were observed. The number of damaged cells in untreated TT patients was lower as compared with the number of damaged cells in TT patients taking treatment. Similarly, untreated LL patients showed fewer damaged cells as compared with those having treatment. The control group lacked category III and IV cells, while only category IV cells were absent from untreated (both TT and LL) patients. In the case of untreated TT and LL patients more cells showed DNA damage at the level of category II. The trend was different in the treated TT and LL patients, where maximum numbers of cells were in category III, with some in category IV.

Table II.

Gradation of comets among the damaged cells of leprosy patients and controls

Group Treated (T) or untreated patients (UnT) No. of normal cells (%) No. of damaged cells in different categories (%)a
 
  No. of damaged cells/total cells scored (%) 

 

 
I
 
II
 
III (%)
 
IV (%)
 

 
TT UnT (n = 8) 297 (74) 101 (25) 2 (1)  103/400 (26) 
 T (n = 7) 191 (55) 53 (15) 98 (28) 8 (23) 15/350 (45) 
LL UnT (n = 8) 266 (67) 113 (28) 21 (5)  134/400 (34) 
 T (n = 7) 174 (50) 45 (13) 110 (31) 21 (6) 176/350 (50) 
Controls (n = 15)  714 (95) 36 (5)   36/750 (5) 
Group Treated (T) or untreated patients (UnT) No. of normal cells (%) No. of damaged cells in different categories (%)a
 
  No. of damaged cells/total cells scored (%) 

 

 
I
 
II
 
III (%)
 
IV (%)
 

 
TT UnT (n = 8) 297 (74) 101 (25) 2 (1)  103/400 (26) 
 T (n = 7) 191 (55) 53 (15) 98 (28) 8 (23) 15/350 (45) 
LL UnT (n = 8) 266 (67) 113 (28) 21 (5)  134/400 (34) 
 T (n = 7) 174 (50) 45 (13) 110 (31) 21 (6) 176/350 (50) 
Controls (n = 15)  714 (95) 36 (5)   36/750 (5) 

TT, tubercloid patients; LL, lepromatous patients.

a

Damaged cells were distributed throughout the group; I (normal halo, <5.00 µm); II (5.00–78.30 µm); III (>78.00–151.66 µm); IV (>151.66–225.00 µm).

The WHO MDT regimen for the treatment of tubercloid leprosy comprises 100 mg/day dapsone and 600 mg/month rifampicin for 6 months. For the lepromatous type, 100 mg/day dapsone, 600 mg/month rifampicin and 50 mg/day and 300 mg/month clofazimine for 12 months. Such treatment schedules were followed by the patient group under study (Table III). The untreated LL patients had more damaged cells and a higher mean DNA migration length as compared with untreated TT patients. The case of LL patients undergoing treatment was similar, with them showing more DNA migration and damaged cells than TT patients on MDT. Statistical analysis revealed this damage in the sub-groups to be significantly increased from that observed in the control (untreated TT, tcal = 8.89, ttab = 3.83, df = 21, P < 0.001; untreated LL, tcal = 17.82, ttab = 3.83, df = 21, P < 0.001; treated TT, tcal = 39.98, ttab = 3.85, df = 20, P < 0.001; treated LL, tcal = 65.90, ttab = 3.85, df = 20, P < 0.001).

Table III.

Mean DNA damage in untreated and treated leprosy patients

Patients Leprosy type No. of individuals Treatment
 
  Age range years (mean) Total cells scored No. of damaged cells (%) DNA migration length (µm) mean ± SEa 

 

 

 
Months
 
Drugs
 
No. ofindividuals
 

 

 

 

 
Untreated TT    21–65 (43.00) 400 103 (26) 9.03 ± 0.86b 
 LL    18–58 (38.00) 400 134 (34) 17.50 ± 0.93b 
 Total 16    18–65 (41.50) 800 237 (30) 13.29 ± 1.24c 
Treated TT 1–3 D [100 mg/day or 25–30 (27.50) 150 59 (39) 40.08 ± 0.69b 
   >3–6 3.0–3.1 g/month] 4d 28–42 (35.00) 200 100 (50) 45.71 ± 0.83b 
    R [600 mg/month]      
 Total 2–6   25–42 (33.50) 350 159 (46) 43.30 ± 1.19c 
 LL 1–3 D [100 mg/day or 37 (37.00) 50 24 (48) 51.50 ± 8.25b 
   >3–6 3.0–3.1 g/month] 2d 35–55 (45.00) 100 49 (49) 52.82 ± 0.40b 
   >6–9 R [600 mg/month] 38–52 (45.00) 100 43 (43) 55.25 ± 0.45b 
   >9–12 C [50 mg/day and 300 mg/month] 17–38 (27.50) 100 60 (60) 56.60 ± 0.28b 
 Total 2–12  17–55 (36.00) 350 176 (50) 54.40 ± 0.73c 
 Total TT + LL 14     700 335 (48) 48.85 ± 1.64c 
 Total leprosy patients 30     1500 572 (38) 29.88 ± 3.39c 
Controls  15     750 36 (5) 1.28 ± 0.40 
Patients Leprosy type No. of individuals Treatment
 
  Age range years (mean) Total cells scored No. of damaged cells (%) DNA migration length (µm) mean ± SEa 

 

 

 
Months
 
Drugs
 
No. ofindividuals
 

 

 

 

 
Untreated TT    21–65 (43.00) 400 103 (26) 9.03 ± 0.86b 
 LL    18–58 (38.00) 400 134 (34) 17.50 ± 0.93b 
 Total 16    18–65 (41.50) 800 237 (30) 13.29 ± 1.24c 
Treated TT 1–3 D [100 mg/day or 25–30 (27.50) 150 59 (39) 40.08 ± 0.69b 
   >3–6 3.0–3.1 g/month] 4d 28–42 (35.00) 200 100 (50) 45.71 ± 0.83b 
    R [600 mg/month]      
 Total 2–6   25–42 (33.50) 350 159 (46) 43.30 ± 1.19c 
 LL 1–3 D [100 mg/day or 37 (37.00) 50 24 (48) 51.50 ± 8.25b 
   >3–6 3.0–3.1 g/month] 2d 35–55 (45.00) 100 49 (49) 52.82 ± 0.40b 
   >6–9 R [600 mg/month] 38–52 (45.00) 100 43 (43) 55.25 ± 0.45b 
   >9–12 C [50 mg/day and 300 mg/month] 17–38 (27.50) 100 60 (60) 56.60 ± 0.28b 
 Total 2–12  17–55 (36.00) 350 176 (50) 54.40 ± 0.73c 
 Total TT + LL 14     700 335 (48) 48.85 ± 1.64c 
 Total leprosy patients 30     1500 572 (38) 29.88 ± 3.39c 
Controls  15     750 36 (5) 1.28 ± 0.40 
a

Calculated as an average of individual DNA migration lengths in that group.

b

Statistically insignificant when compared with each other but significant when compared with the control from total control group (P ≤ 0.001, Student's t-test).

c

Statistically significant from total control group (P ≤ 0.001, Student's t-test).

d

Includes one female in each sub-group.

D, dapsone; R, rifampicin; C, clofazimine.

Among the patients themselves, there was almost twice the damage in untreated LL as compared with that in untreated TT patients (tcal = 6.29, ttab = 4.14, df = 14, P < 0.001). Furthermore, it was also higher in those being treated for TT (tcal = 21.10, ttab = 4.22, df = 13, P < 0.001) and LL (tcal = 28.45, ttab = 4.22, df = 13, P < 0.001) as compared, respectively, with patients yet to be treated. The overall damage observed for all patients on treatment was also statistically significant as compared with the mean value in untreated patients (tcal = 16.95, ttab = 3.62, df = 28, P < 0.001). An increase in treatment time also increased DNA damage linearly, in those being treated for both TT (1–3 versus 3–6 months, tcal = 4.22, ttab = 4.03, df = 5, P < 0.01) and LL (1–3 versus 3–6 months, tcal = 0.129, ttab = 12.73, df = 1; 3–6 versus 6–9 months, tcal = 2.88, ttab = 4.30, df = 2; 6–9 versus 9–12 months, tcal = 1.824, ttab = 4.30, df = 2).

Discussion

The results of the alkaline SCGE assay performed on PBLs of untreated and treated lepromatous and tubercloid leprosy patients revealed significant DNA damage, which was greater in lepromatous patients receiving treatment. An increase in treatment time increased DNA damage linearly. Multiple regression analysis for effects of confounding factors revealed antibiotic treatment in patients and alcohol consumption in controls as the only variables influencing DNA damage. An arbitrary classification of damaged cells based on observed tail lengths in leprosy patients revealed more cells with greater DNA migration lengths in patients receiving treatment.

These findings find some parallels in the literature. Kalaiselvi et al. (2002) also reported that age, smoking and alcohol consumption, as well as sex, did not influence DNA or chromosomal damage in treated leprosy patients. However in normal males Dhawan et al. (2001) reported that DNA damage depended on age, with the extent of DNA damage increasing with smoking and eating habits. Betti et al. (1994) also reported that DNA migration increased significantly in healthy individuals who smoked: it was greater in men than in women and DNA migration was similar in the young and older people. On the other hand, sister chromatid exchange (SCE) frequency was not affected by smoking, age or sex in the same population. These observations reflect the results of the present study, DNA damage appears to be caused by M.leprae itself in untreated leprosy patients. D'Souza and Thomas (1988), D'Souza et al. (1991) and Kaur et al. (1999) also reported increased chromosomal aberrations (CA) and SCE in untreated leprosy patients. There was almost twice the damage in untreated LL than in untreated TT patients in our study. This may well tally with the fact that the LL type follows a severe course, with the patients showing very low resistance to M.leprae and so possibly being more susceptible to its damaging effects, while the TT form is more favourable and benign as to its manifestations and general course and the patients have rather high resistance to M.leprae infection (Gupta and Minocha, 2000). D'Souza and Das (1994) also reported that the increase in CA and SCE was higher in untreated multibacillary (lepromatous) patients. Other bacterial infections have also been documented as inducing genetic damage in the host, as observed for Mycobacterium tuberclosis (Gopal Rao et al., 1990; Masjedi et al., 2000). DNA damage was reported to be significantly higher in Helicobacter pylori-infected patients (Ladeira et al., 2004). A number of viruses, including those for hepatitis A and B (Chatterjee and Gosh, 1989), have also been observed to cause CAs in human lymphocytes. Infection of cells with human cytomegalovirus during the S phase of the cell cycle resulted in two specific chromosome 1 breaks at positions 1q42 and 1q21 (Fortunato et al., 2000).

The patients on MDT showed significantly greater DNA damage as compared with untreated leprosy patients, probably indicating that both M.leprae and the anti-leprotic drugs are manifesting effects. Support for this is also evident from the literature. Tice et al. (1992) documented longer tail lengths in non-cryopreserved PBLs of breast cancer patients treated with anti-neoplastic drugs (cyclophosphamide and cisplatin) as compared with patients before treatment. Betancourt et al. (1995) reported elevated comet lengths in various categories of well-nourished and malnourished children, with and without infections (respiratory, gastrointestinal, tuberculosis, sepsis, etc.) and with and without antibiotic (including rifampicin) treatment. They reported damage to DNA in PBLs of malnourished children with severe infections to be twice that observed in well-nourished children with severe infections. The tail lengths increased with antibiotic treatment, indicating the DNA-damaging effects of these drugs. There is also evidence of genotoxicity of anti-leprotic drugs in different test organisms. CAs have been reported in dapsone-treated human cells (Beiguelman et al., 1975; Hackel and Beiguelman, 1985), although dapsone, rifampicin and clofazimine, among others, were negative in the Ames Salmonella/microsome assay, with or without metabolic activation, while sulphide and sulphoxide analogues of dapsone were reported to be mutagenic only with metabolic activation (Peters et al., 1983). Rifampicin induces chromatid-type aberrations (Chaudhari et al., 1984) and CA (Chaudhari et al., 1984; Aboul-Ela, 1995). Clofazimine significantly increases CA and micronuclei (Dass and Roy, 1990; Roy and Dass, 1990) and elevates SCE frequencies (Dash et al., 1991). In contrast, combinational anti-leprotic drug therapy was not clastogenic, as there was no increase in CA in treated leprosy patients (D'Souza et al., 1991; D'Souza and Das, 1994). Rather, these researchers and Peters et al. (1983) observed a reduction in CA in treated multibacillary patients, proposing a remedial action of the drugs. However, a DNA-damaging effect of MDT cannot be ruled out, as observed in the present study and by Kalaiselvi et al. (2002, 2003), who reported both DNA damaging and clastogenic actions.

The data of our study further indicate that leprosy patients on MDT had more cells with longer DNA migration lengths than cells of untreated patients. In the case of untreated patients, the damage is probably caused by M.leprae only, as explained above. In patients on MDT, both the bacterium and the drug therapy probably induced the DNA damage, as also suggested by Kaur et al. (1999) and Kalaiselvi et al. (2002, 2003). The presence of varying comet lengths on the same slide preparation may be explainable by the behaviour of DNA fragments during electrophoresis. The alkaline comet assay detects single-strand breaks, double-strand breaks and alkali-labile sites, with the tail length reflecting the amount of breakage in a cell (Singh et al., 1988). The rate of migration of each DNA fragment is inversely proportional to its size, i.e the smaller fragments move with greater speed. Gedick et al. (1992), among others, observed that as few as 0.1 DNA breaks/109 Da are detected by the SCGE assay, i.e. tail length is constant over a range of DNA break frequencies of 1–14 per 1010 Da, but tail intensity increases with damage. McKelvey-Martin et al. (1993) have stated that the comet tail (length and intensity) appears to consist of a series of fragments that retain some higher order structure connected by single-stranded regions. As per Collins et al. (1997), the alkaline comet assay resolves break frequencies up to a few thousands per cell and so the distances between breaks are of the order of 109 Da. The length of the comet is a few hundredths of a millimetre and the comet tail is made up of relaxed loops with the number of loops in the tail (tail intensity) indicating the number of breaks, while tail length is primarily determined by the lengths of the loops.

The presence of different categories of comets on the same slide indicates that neither length of time for unwinding nor time of electrophoretic run could have influenced tail length (Singh et al., 1988). The presence of higher categories of comets in patients on MDT suggests that both M.leprae and MDT are producing more damaged cells with more DNA breaks leading to longer DNA loops, i.e. longer comets. On the other hand, in untreated leprosy patients the damage due to M.leprae leads to more damaged cells in the lower categories, i.e. in which DNA loops are shorter because of fewer DNA breaks.

Besides the DNA damage induced by M.leprae and MDT in leprosy patients, it may also be induced by other mechanisms. Toxic radicals such as superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radicals released from M.leprae infections may also cause DNA damage (Chatterjee, 1992; Bandyopadhyay et al., 1999), since these reactive oxygen radicals can react with cellular DNA and cause strand breaks and/or alkali-labile lesions (Joenje, 1989). An excess of strong oxidizing agents such as molybdenum and copper and low blood levels of antioxidants such as selenium and vitamin E (Rao and Saha, 1988) may further correlate with an increased oxidation state in leprosy patients.

An indirect action of the anti-leprotic drugs may also induce DNA damage, namely rifampicin could have an amplifying effect on phagocytosis and subsequently on oxidative processes when it enters into phagocytes. Clofazimine and other auto-oxidants may inhibit the template function of the DNA strand when they bind along the minor groove of DNA and reduce the serum levels of the antioxidants vitamins A and E in leprosy patients (Rao et al., 1986). Protective roles of vitamins A and C against the clastogenic effects of clofazimine (Sahu and Das, 1994) and of vitamins C and E against rifampicin (Aly and Donya, 2002) have been documented in bone marrow cells of mice, probably by removing the free radicals produced by the drugs. Hence, vitamin supplements may also prove protective against DNA damage in leprosy patients on MDT. It is to be recalled that the socio-economic status of the patients was middle or low, indicating a probable lack of these nutrients in their diets and/or as prescribed supplements.

The results of the present study therefore 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. The higher level of damage in treated and untreated LL patients reflects the fact that the lepromatous type is the more severe form, with the patients probably having lower resistance to M.leprae and requiring heavier and more prolonged dosage with antibiotics. The study also corroborates that the SCGE assay can offer an opportunity for correlating levels of therapy-induced DNA damage with administered dose and for modulating the dose schedule so as to achieve lower levels of genotoxic damage (Tice et al., 1992) at which treatment efficacy is not compromised.

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