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Mutagenesis, Vol. 17, No. 4, 309-312, July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press

Chromosomal aberration, micronucleus and Comet assays on peripheral blood lymphocytes of leprosy patients undergoing multidrug treatment

K. Kalaiselvi3, P. Rajaguru, M. Palanivel, M.V. Usharani1 and G. Ramu2

Department of Environmental Science, PSG College of Arts and Science, Coimbatore 641 014, India, 1 Department of Environmental Science, Bharathiar University, Coimbatore, India and 2 GKNM Hospital Quarters, Coimbatore, India


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
To evaluate the genetic damage in leprosy patients, we carried out the alkaline Comet assay and chromosomal aberration (CA) and micronucleus (MN) tests in peripheral blood lymphocytes of 50 leprosy patients receiving multidrug treatment (MDT) and 50 healthy individuals. The Comet assay showed statistically higher mean values for length to width ratios of DNA mass (P < 0.01) and for mean frequencies of tailed cells (P < 0.001) in cells of leprosy patients than in those of controls. Similarly, the mean frequencies of micronucleated cells (per 1000 cytochalasin B-induced binucleated cells) were significantly greater (P < 0.001) in leprosy patients (19.92 ± 2.564) than in controls (1.6 ± 0.231). A statistically significant 10-fold increase in the frequency of CAs (11.16 ± 0.411) was observed in leprosy patients compared with controls (1.28 ± 0.242). In multiple regression analyses, when patients and controls were considered together, disease factor alone significantly influenced the genotoxicity markers. In the control group, age and alcohol consumption significantly influenced MN and length to width ratios and CA frequency, respectively. However, in MDT-treated leprosy patients none of the other confounding factors (sex, age, smoking and alcohol drinking) significantly affected the extent of genetic damage.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Leprosy, a chronic mutilating disease, is caused by Mycobacterium leprae. Leprosy remains endemic in over 10 countries. India is one of them, which accounts for almost 61% of the global incidence of leprosy (Noordeen, 2001Go). Multidrug therapy (MDT) is the WHO recommended method of treatment for leprosy. In MDT, dapsone and rifampicin are the effective chemotherapy, followed by other front line drugs like clofazimine and ofloxacin. Despite the reported evidence of possible genotoxicity of the components of MDT (Beigulman et al., 1975Go; Peters et al., 1983Go; Roy and Dass, 1988Go; D'Souza et al., 1991Go; Aboul-Ela-El, 1995Go), no comprehensive cytogenetic investigation has been conducted on leprosy patients.

Therefore, with the aim of assessing possible genetic damage in leprosy patients, we carried out a cytogenetic study involving leprosy patients and age-matched controls living in the same area. Peripheral blood lymphocytes of the patients and control subjects were analyzed for the presence of DNA damage, using single cell gel electrophoresis (the Comet assay), and for cytogenetic parameters [chromosomal aberrations (CA) and micronucleus (MN) frequency]. The use of CA and MN as markers of early biological effects is well established in genotoxicity studies, while the Comet assay is a more innovative technique.

Moreover, to our knowledge, no report is available on the cytogenetic effects of leprosy in patients determined using MN and the Comet assay. Therefore, we report the results of the MN and the Comet assay for the first time, along with structural CAs, in peripheral blood lymphocytes of leprosy patients.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects
Fifty randomly selected patients who underwent MDT treatment at Coimbatore Urban Leprosy Eradication Scheme (CULES) hospital and 50 healthy individuals resident in the same area, matched to the age and sex of the patients, as controls were included in the study after obtaining their informed consent. Each person was interviewed for possible confounding factors such as smoking, alcohol drinking, medicine intake and exposure to radiation and/or chemical mutagens. Heparinized peripheral venous blood samples were collected from the donors and processed for CA, MN and Comet assays.

Comet assay
The Comet assay was performed on whole blood under alkaline conditions following the procedure of Ward et al.(1997) with minor modifications. All the steps of the Comet assay were conducted under yellow light to prevent the occurrence of additional DNA damage. Briefly, 5 µl blood samples were diluted with 50 µl of phosphate-buffered saline (PBS) and mixed with 150 µl of 1% low melting point agarose (37°C) prepared in PBS and pipetted onto a 1% normal melting point agarose precoated slide, which had been dried overnight, and covered with a coverslip. After keeping the slide on a chilled plate for 10 min, the coverslip was removed and the slide lowered into freshly made ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10) and kept at 4°C in the dark for 60 min. After draining the lysis solution, the slide was rinsed with distilled water for 15 min. After repeating the distilled water wash twice, the slide was placed in a horizontal electrophoresis tank containing freshly made buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 30 min. Electrophoresis was performed in the same buffer for 20 min by applying an electric field of 25 V (0.8 V/cm) and adjusting the current to 300 mA by slowly changing the buffer level in the tray. After electrophoresis the slide was rinsed gently with 0.4 M Tris–HCl buffer (pH 7.5) for 5 min. This step was again repeated. Then the slide was dried at room temperature and kept in a refrigerator in a sealed container until analysis. Duplicate slides were prepared for all the samples.

The slides were immersed in distilled water for 30 min, then stained with 100 µl of ethidium bromide (5 µg/ml) and read at 250x using a Zeiss fluorescence microscope equipped with a green excitation filter and a 590 nm barrier filter. All slides were coded and examined blind. Routinely 100 cells (50 cells/slide) were screened per sample. In selecting cells for measurements, straight line scanning of a slide was begun at an arbitrary point and cells were measured as they came into the field, provided there was no overlap with patterns from other cells. The length and width of the DNA mass was measured using an ocular micrometer disk. Under these conditions a DNA pattern with a ratio of one has a DNA length of ~25 µm and a ratio of four has a DNA length of ~100 µm. The length:width ratios of the DNA mass and the frequency of tailed (damaged) cells were used in all the comparisons.

Chromosomal aberration and micronucleus test
Whole-blood 72 h cultures were set up for cytogenetic analyses in patients and control lymphocytes. Culture procedures, cell collection and staining of all slides were done using routine methods (Fenech and Morley, 1985Go; IAEA, 1986Go). For each sample 100 metaphases were screened for CAs. In this study the frequency of aberrant cells was evaluated excluding gaps. The induction of MN was evaluated by scoring a total of 2000 binucleated lymphocytes with preserved cytoplasm for each subject. Microscopic screening was performed on coded slides by two investigators.

All chemicals used in this study were obtained from Himedia, India, unless stated otherwise.

Statistical analysis
Statistical tests were performed using SPSS v.8.0. The mean values of biomarker data in leprosy patients and controls were compared using a one-tailed Student's t-test. To estimate the possible influence of various confounding factors, the data were tested using multiple regression analysis. All the statistical tests were performed at an {alpha} level of 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The average ages of the leprosy patients and the control group were 35.2 ± 8.6 and 30.6 ± 6.3 years, respectively. The prevalence of smoking in the leprosy patients and controls was similar (18/50 versus 20/50), while alcohol consumption was slightly higher among the patients (20/50 versus 11/50). All the leprosy patients were treated with MDT drugs. None of the controls reported to have recent exposure to medication or radiation.

To assess the possible relationship between biomarkers of genotoxicity, simple correlation analyses were performed between the different biological end points measured in the total study population (Table IGo). Significant correlation was found between different parameters of genotoxicity (length/width ratios of DNA mass, L:W; tailed cell percentage, TC; MN frequency per 1000 binucleated cells, MN; CA frequency per 100 metaphase cells, CA).


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  		Table I. . Simple correlations for MDT-treated leprosy patients and controls (n = 100)
 
The results of the Comet assay and MN and CA analyses of the MDT-treated leprosy patients and control groups are shown in Table IIGo. The mean length to width ratio of the DNA mass observed in the leprosy patients was significantly greater (P < 0.01) in comparison with the control group. Similarly, the mean frequency of tailed cells was 23.12 ± 1.767 among the leprosy patients and was significantly different (P < 0.001) compared with the controls (7.96 ± 1.012). Figure 1Go illustrates the distribution of L:W and TC in controls and leprosy patients under MDT treatment. For both parameters the median values were higher in leprosy patients than in controls. The distribution of damaged cells was also wider in leprosy patients. Similarly, the lengths of the boxes for leprosy were greater, indicating larger interquartile ranges for MDT-treated leprosy patients than controls. In the case of L:W, for the control group the median line is close to the top edge of the box, indicating tailing towards smaller values. In the leprosy group the tail was towards higher values. In both groups, since the median lines are in the middle of the boxes, the distribution of TC was more or less symmetrical.


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  		Table II. . Biological parameters of the study groups
 


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  		Fig. 1. . Box-and-whisker plot of the distribution of DNA damage in MDT-treated leprosy patients and controls. The boxes include 50% of the data. The inner line marks the median value and whisker lines extending from the box represent the minimum and maximum values. Small circles represent outliers.

 
The mean frequency of cells with MN in peripheral blood lymphocytes of leprosy patients (19.92 ± 2.564) was significantly higher (P < 0.001) than in controls (1.6 ± 0.231). The frequency distribution of lymphocytes with MN shows that among the control group 44% were found to have no micronucleated lymphocytes, while in 56% it was in the range 1–4 per 1000 binucleated cells. All the leprosy patients were found to have micronucleated lymphocytes. The median MN frequency and interquartile ranges were greater in leprosy patients (Figure 2Go). The position of the median line in the box for leprosy patients indicates that the MN frequency was skewed towards larger values. Variability in MN frequency was extensive among leprosy patients compared with the control values.



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  		Fig. 2. . Box-and-whisker plot of the distribution of micronucleated lymphocytes in MDT-treated leprosy patients and controls. The boxes include 50% of the data. The inner line marks the median value and whisker lines extending from the box represent the minimum and maximum values. Small circles represent outliers.

 
The observed CAs consisted of chromatid gaps, breaks, fragments and dicentrics. A statistically significant (P < 0.001) 10-fold increase in the mean frequencies of CAs (excluding gaps) in leprosy patients was observed relative to that of controls. Figure 3Go illustrates a wider distribution of larger values of CA frequencies in leprosy patients. The median value was higher and close to the bottom edge of the box for MDT-treated leprosy patients.



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  		Fig. 3. . Box-and-whisker plot of the distribution of chromosomal aberrations in MDT-treated leprosy patients and controls. The boxes include 50% of the data. The inner line marks the median value and whisker lines extending from the box represent the minimum and maximum values. Small circles represent outliers.

 
The influence of various independent variables on genotoxicity parameters was examined by multiple regression analysis in the study group (leprosy patients and controls). The influence of the following independent variables was tested on each biomarker: disease (leprosy patient or control), age, sex (male or female), smoking (smoker or non-smoker) and alcohol drinking (consumer or non-consumer). When both controls and patients were considered together (Table IIIGo) disease factor alone influenced the genotoxicity markers (L:W, TC, MN and CA). In this analysis the disease term explained 48, 53, 51 and 90% of the variance of L:W, TC, MN and CA, respectively. When the analysis was limited to controls (Table IVGo), MN frequency and L:W ratio were significantly influenced by age and CA frequency was influenced by alcohol consumption. In leprosy patients other independent factors (age, sex, smoking and alcohol consumption) were found to have no significant influence on genotoxic parameters.


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  		Table III. . Determinants of genotoxicity parameters for MDT-treated leprosy patients and controls
 

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  		Table IV. . Determinants of genotoxicity parameters for controls
 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The results of all three assays used in this study clearly indicate a significantly higher incidence of DNA damage in the peripheral blood lymphocytes of MDT-treated leprosy patients compared with controls. D'Souza and Thomas (1988) also reported a higher incidence of CA in leprosy patients. Other similar infections caused by Mycobacterium tuberculosis (Gopal et al., 1990Go; Masjedi et al., 2000Go) and a number of viruses, including both hepatitis A and B (Chatterjee and Gosh, 1989Go), were also demonstrated to cause CA in human lymphocytes.

In the standard MDT regime for leprosy, dapsone, rifampicin and clofazamine are important anti-leprosy drugs recommended by the WHO study group on chemotherapy for leprosy. These drugs are reported to induce cytogenetic damage in different test systems. Intraperitoneal doses of rifampicin were found to increase SCE frequency in bone marrow cells and structural CA frequency in germ cells of mice (Aboul-Ela-El, 1995Go). Similarly, Roy and Dass (1988) reported significantly higher incidences of MN and CA in dapsone-treated mice. Conversely, various anti-leprosy drugs (clofazimine, ethionamide, prothionamide, prothionamide s-oxide, rifampicin, dapsone and N-acetylated and N-hydroxylated derivatives of dapsone) were found to be negative with or without metabolic activation in the Ames Salmonella/microsome assay. The sulfoxide and sulfide analogs of dapsone were found to be mutagenic with metabolic activation (Peters et al., 1983Go).

D'Souza et al.(1991) indicated higher incidences of CA and SCE in leprosy patients treated with MDT. Similar structural chromosomal abnormalities were observed in cultures of skin fibroblasts of leprosy patients treated with dapsone (Hachel and Beigulman, 1985Go) and in human lymphocytes in vitro (Beigulman et al., 1975Go). These findings suggest that the higher level of DNA damage observed in the peripheral blood lymphocytes of leprosy patients may be due to the activities of M.leprae and/or caused by the drugs given in the treatment of leprosy.

Some or all of the mechanisms listed below may be responsible for the induced cytogenetic damage in leprosy patients observed in this study.

  1. Mycobacterium leprae infection may result in the release of toxic radicals [superoxide anion (Oo–), hydrogen peroxide (H2O2), singlet oxygen (O) and hydroxyl radicals (OHo–)] (Chatterjee, 1992Go; Bandyopadhyay et al., 1999Go). Reaction of these reactive oxygen species with cellular DNA results in strand breaks and/or alkali-labile lesions (Joenje, 1989Go).
  2. Low blood levels of antioxidants, such as selenium and vitamin E (Rao and Saha, 1988Go), along with an excess of the strong oxidizing agents molybdenum and copper in leprosy patients may correlate with an increased oxidation state.
  3. Alternatively, the anti-leprosy drugs used in the MDT regime may be capable of inducing DNA damage as: (i) dapsone has been reported to induce chromosomal abnormalities in both in vivo and in vitro studies; (ii) drugs such as clofazimine are auto-oxidants and may bind along the minor groove of the DNA molecule and inhibit template function of the DNA strand and reduce serum levels of vitamin A and E in leprosy patients (Rao et al., 1986Go); (iii) rifampicin, included in the MDT regime, enters phagocytes and could have an amplifying effect on phagocytosis and subsequent oxidative processes.

The present study was conducted on patients undergoing the MDT regime. Therefore, to clarify the possible role of M.leprae and of components of the MDT regime in inducing the observed DNA damage in leprosy patients, further studies involving patients without prior exposure to MDT or other drugs are currently being conducted in our laboratory.


   Acknowledgments
 
The authors wish to thank all persons who agreed to be a subject in this study and acknowledge the staff of CULES for their cooperation.


   Notes
 
3 To whom correspondence should be addressed. Tel: +91 422 573386; fax: +91 422 575622. Email: kkalaiselvi{at}rediffmail.com Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    Aboul-Ela-El (1995) Cytogenetic effects of rifampicin in somatic and germinal cells of the mouse. J. Appl. Toxicol., 15, 325–326.[Web of Science][Medline]

    Bandyopadhyay,U., Das,D. and Banerjee,R.K. (1999) Reactive oxygen species: oxidative damage and pathogenesis. Curr. Sci., 77, 658–666.

    Beigulman,B., Pisani,R.C. and Ei Guirdy,M.M. (1975) In vitro effects of dapsone on human chromosomes. Int. J. Lepr., 43, 41–44.

    Charterjee,B.R. (1992) Antioxidants, Meny Bergel, Dapsone and downgrading of Hanson's disease. The Star, 51, 2–4.

    Charterjee,B. and Gosh,P.K. (1989) Chromosome damages in viral hepatitis. Mutat. Res., 210, 49–57.[Web of Science][Medline]

    D'Souza,D. and Thomas,I.M. (1988) Chromosome aberration and sister chromatid exchanges (SCEs) in peripheral blood lymphocyte cultures of untreated leprosy patients. Lepr. Rev., 59, 121–125.[Web of Science][Medline]

    D'Souza,D., Das,B.C. and Thomas,I.M. (1991) Cytogenetic studies in leprosy patients before and after chemotherapy. Hum. Genet., 87, 665–670.[Web of Science][Medline]

    Fenech,M. and Morley,A.A. (1985) Measurement of micronuclei in lymphocytes. Mutat. Res., 147, 29–36.[Web of Science][Medline]

    Gopal Rao,V.V.N., Venkataraman Gupta,E.V. and Thomas,I.M. (1990) Chromosome damage in untreated tuberculosis patients. Tubercle, 71, 169–172.[Web of Science][Medline]

    Hachel,C. and Beigulman,B. (19859) Chromosomal aberration in cultures of skin fibroblasts of leprosy patients. Int. J. Lepr., 53, 533–539.

    IAEA (1986) Biological Dosimetry: Chromosomal Aberration Analysis for Assessment, IAEA Technical Reports Series no. 260. IAEA, Vienna, Austria.

    Joenje,H. (1989) Genetic toxicology of oxygen. Mutat. Res., 219, 193–208.[Web of Science][Medline]

    Masjedi,M.R., Heidary,A., Mohammdi,F., Velayati,A.A. and Dokouhaki,P. (2000) Chromosomal aberrations and micronuclei in lymphocytes of patients before and after exposure to anti-tuberculosis drugs. Mutagenesis, 15, 489–494.[Abstract/Free Full Text]

    Noordeen,S.K. (2001) Global alliance to eliminate leprosy, The First Meet at Delhi, 30–31 January, 2001. Bull Lepr. El. Al., 1, 1–4.

    Peters,J.H., Gordon,G.R., Murray,J.E.Jr and Simmon,V,F. (1983) Mutagenic activity of antileprotic drugs and their derivatives. Int. J. Lepr., 51, 45–53.

    Rao,K.N. and Saha,K. (1988) Under nutrition and lepromatous leprosy: serum vitamin A and E levels in leprosy spectrum. Int. J. Lepr., 60, 66–70.

    Rao,K.N., Lakshmi,V. and Saha,K. (1986) Under nutrition in lepromatous leprosy Part I. Is it associated with poverty or with disease? Lepr. Rev., 57, 299–309.[Web of Science][Medline]

    Roy,B. and Dass,R.K. (1988) Cytogenetic effects of dapsone an antileprotic drug in the mouse in vivo system. Int. J. Lepr., 56, 574–579.

    Ward,T.H., Butler,J., Shakbakhti and Richards,J.T. (1997) Comet assay studies on the activation of two diazinidinylbenzo quinones in K562 cells. Biochem. Pharmacol., 53, 1115–1121.[Web of Science][Medline]

Received on August 10, 2001; accepted on February 1, 2002.


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Re: Chromosomal aberration, micronucleus and Comet assays on peripheral blood lymphocytes of leprosy patients undergoing multidrug treatment (Mutagenesis, 17, 309–312, 2002)
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