Mutation Research, 240 (1990) 101-107
101
Elsevier MUTGEN 01512
Differential sensitivity of peripheral blood lymphocytes of untreated leprosy patients to mitomycin C Doris D'Souza
1, B.C.
Das 2 and I.M. T h o m a s 1
Division of Human Genetics, Department of Anatomy, St. John's Medical College, Bangalore 560034 (India) and 2 Molecular Oncology, Cytology Research Centre (ICMR), Maulana Azad Medical College Campus, New Delhi 110002 (India)
(Received8 June 1989) (Revisionreceived31 August1989) (Accepted28 September1989)
Keywords: MitomycinC; Mutagensensitivity;Leprosy
Summary The effects of a bifunctional alkylating agent mitomycin C (MMC), an effective inducer of chromosome aberrations and sister-chromatid exchanges (SCEs), have been studied in untreated leprosy patients. This was done to study the mutagen sensitivity of the leprosy patients. The frequency of chromosomal aberrations induced by MMC (conc. 0.01 #g/ml) was 2.5% in controls, 3.6% in paucibacillary (PB), and 6.8% in multibacillary (MB) patients. The difference in the frequency of MMC-induced chromosome aberrations between the 3 groups studied was highly significant (p < 0.01). Cultures grown with MMC showed the frequency of SCEs/cell to be 12.70 + 1.19 in controls, 19.97 + 3.51 in PB, and 29.66 + 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs between the 3 groups were found to be highly significant (p < 0.01). The enhanced frequencies of spontaneous and MMC-induced chromosome aberrations and SCEs observed in PB and MB patients indicate a clear differential mutagen sensitivity between PB and MB patients who are known to have different immunological status and thereby differ in the severity of the disease.
Leprosy is a chronic granulomatous mycobacterial disease, primarily affecting the peripheral nerves and secondarily involving skin and other tissues in humans. The bacterium, Mycobacterium leprae, is known to be the causative agent of leprosy which is most prevalent in tropical countries. India, more than any other country, has a very large number of leprosy patients (WHO, 1985). Leprosy is not an inherited disease, though
Correspondence: Dr. D. D'Souza, Patna Women's College, Avila Convent,BayleyRoad, Patna 800001, Bihar(India).
lepromatous patients have depressed cellular immunity to M. leprae (Ridley and Jophng, 1966). Therefore, an increased incidence of malignancy may be anticipated in leprosy patients. However, reports of various studies on leprosy patients and their susceptibility to developing cancer are conflicting (Michalany, 1966; Oleinick, 1969; Purtillo and Pangi, 1975; Kolonel and Hirohata, 1977; Tokudome et al., 1981; Weshler and Sheskin, 1983; Brinton et al., 1984). Various cancer-prone syndromes as well as certain autosomal recessive syndromes which show impaired immunity (Garriga and Crosby, 1959; Lynch et al., 1967; Dosik et al.,
0165-1218/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
102
1970; Bernstein et al., 1971; Robbins et al., 1974; Schroeder and German, 1974) are characterised by chromosomal instability (Hecht et al., 1966; Swift and Hirschhorn, 1966; German, 1972; Schroeder and German, 1974; Harden, 1974; Chaganti et al., 1974), and are highly susceptible to DNA-damaging agents producing various types of chromosomal anomalies (Fugiwara et al., 1977; Natarajan and Obe, 1982; Cerutti, 1982; Natarajan, 1984) and increased frequency of sister-chromatid exchanges (SCEs) (Chaganti et al., 1974; Wolff et al., 1977; De Weerd et al., 1977; Shiraishi and Sandberg, 1979). Recently, we have shown, for the first time, a significant increase in the frequency of spontaneous chromosome aberrations and SCEs in blood lymphocyte cultures of untreated leprosy patients (D'Souza and Thomas, 1988). Taking these findings into consideration the present study was conducted to gain further insight into the genetic instability and mutagen sensitivity of leprosy patients by using the potent mutagen mitomycin C (MMC). MMC, an anticancer antibiotic, is known to act on chromosomal DNA (Mertz, 1961). Upon metabolic activation, the drug is transformed into a reactive bifunctional alkylating agent (Iyer and Szybalski, 1964). It has been shown to cause a dramatic increase in the frequency of chromosome aberrations and SCEs in blood lymphocyte cultures of some autosomal recessive syndromes (Fugiwara et al., 1977; Shiraishi and Sandberg, 1979). In this study, the effects of MMC in inducing chromosome aberrations and SCEs in blood lymphocyte cultures of untreated leprosy patients have been studied. For this study only male subjects were considered in order to avoid the variations induced in females by various other factors (Das, 1988; D'Souza et al., 1988). Materials and methods
Patient selection The patients selected were classified by clinical symptoms and bacterial index (BI) according to the Ridley-Jopling scale (Ridley and Jopling, 1966). The tuberculoid leprosy (TT) and the borderline tuberculoid leprosy (BT) patients were included in the paucibacillary group (PB), while
borderline lepromatous (BL) and lepromatous leprosy (LL) patients were considered as the multibacillary group (MB). Five untreated PB and 5 untreated MB patients were studied. These patients had been carefully selected after ascertaining that they had never undergone treatment for leprosy, and apparently had no other disease. Four age-matched healthy normal individuals who were not under any medication prior to this investigation served as controls. All samples of the study consisted only of males. Lymphocyte cultures Phytohaemagglutinin M (Difco)-stimulated peripheral blood lymphocyte cultures were grown in TC199 medium (Gibco), supplemented with 20% heat-inactivated human AB serum and antibiotics (penicillin and streptomycin) for 48 h and 72 h. MMC (Sigma) was added to the cultures at a final concentration of 0.01 g g / m l at the time of culture initiation. Bromodeoxyuridine (BrdU, Sigma, 5 gg/rnl) was added at the time of initiation of cultures. In each group 2 sets of cultures were set up. 100 first-cycle metaphases from 48-h cultures were studied to score chromosome aberrations for each subject, using ISCN criteria (ISCN, 1978). 20-50 second-cycle metaphases were scored from 72-h cultures for SCEs in each subject, and the values were expressed as the number of SCEs per metaphase or cell. Results
Chromosome aberrations Table 1 shows the mean frequency distribution of spontaneous and MMC-induced chromosome aberrations in controls, PB and MB patients. The frequency of spontaneous chromosome aberrations per 100 metaphases was found to be 1.5 in controls, 3.2 in PB and 5.2 in MB patients. A significant (p < 0.001) difference was found in the frequency of spontaneous chromosome aberrations between controls, PB and MB patients, whereas, when gaps were excluded, only MB patients showed an increase in the frequency of aberrant metaphases. The frequency of chromosome aberrations induced by MMC (0.01 gg/ml) was 2.5% in con-
103 TABLE 1 F R E Q U E N C Y OF CHROMOSOME ABERRATIONS IN LYMPHOCYTES OF U N T R E A T E D LEPROSY PATIENTS, CULT U R E D WITH MMC Group
Number of subjects/ metaphases
Controls Without MMC
4/400
With MMC
4/400
PaucibaciUary Without MMC
5/500
With MMC
5/500
Multibacillary Without MMC
5/500
With MMC
5/500
Chromosome aberrations Meta-
Breaks
Meta-
Gaps
phases with aberrations
Csb
Ctb
phases with breaks
Csg
Ctg
phases with gaps
6 (1.5) 10 (2.5)
-
3 (0.75) 7 (1.75)
3 (0.75) 7 (1.75)
-
2 (0.5) 2 (0.5)
2 (0.5) 2 (0.5)
-
4 (0.80) 4 (0.80)
4 (0.80) 4 (0.80)
11 (2.2) 11 (2.2)
11 (2.2) 10 (2.0)
-
5 (1.00) 10 (2.00)
5 (1.00) 10 (2.00)
14 (2.8) 18 (3.6)
16 (3.2) 16 (3.2)
-
16 * (3.2) 18 ** (3.6)
-
26 * (5.2) 34 ** (6.8)
-
-
-
-
-
2 (0.4) -
Meta-
Cte
Others
a
Aberrant metaphases without gaps
-
2 (0.4) 6 (1.2)
1 c (0.25) 1 a (0.25)
4 (1.0) 8 (2.0)
1 ~ (0.2) 2 f (0.4)
5 (1.0) 8 (1.6)
5 s (1.0) 2 h (0.4)
10 (2.0) 18 (3.6)
Figures in parentheses indicate percentages. Csb, chromosome break; Ctb, chromatid break; Csg, chromosome gap; Ctg, chromatid gap; Cte, chromatid exchanges. a 'Others' includes acentric fragments (AF); dicentric chromosomes (dic); double minutes (DMs); and metaphases with pulverised (shattered) chromosomes (pvz). c 1 D M s ; d 1 A F ; e 1 DMs; f 1 d i c a n d l A F ; S 3 p v z a n d 2 A F ; h 2AF. * Without MMC: significant in chi-square: p < 0.001. * *With MMC: significant in chi-square: p < 0.01.
TABLE 2 F R E Q U E N C Y OF SCEs IN LYMPHOCYTES OF U N T R E A T E D LEPROSY PATIENTS, C U L T U R E D W I T H MMC Group Controls Without MMC With MMC Paucibacillary (PB) Without MMC With MMC Multibacillary (MB) Without MMC With MMC
Number of subjects studied
Number of cells analysed
SCEs/cell
+ SD
4 4
145 200
6.33 12.70
+ 0.85 + 1.19
1-17 3-35
5 5
170 110
8.06 a 19.97 a
+ 1.34 + 3.51
1-28 5-35
5 5
165 150
9.78 b,c 29.66 e,f
+ 2.30 + 5.92
1-22 12-62
Significant in Student's t-test: Without MMC: a PB vs. controls: p < 0.01. b MB vs. controls: p < 0.01. c PB vs. MB: p < 0.01.
With MMC: d PB vs. controls: p < 0.01. e MB vs. controls: p < 0.01. f PB vs. MB: p < 0.01.
SCE range
I
¢ Fig. l(a-d). Partial metaphases showing chromatid exchanges.
trols, 3.6% in PB, and 6.8% in MB patients. There was certainly an increase in the frequency of chromosome aberrations following MMC treatment in patient groups, the difference was found to be non-significant ( p > 0.05) within the groups while the difference was significant ( p < 0.01) between the groups. When gaps were excluded, only MB patients showed an increase in the frequency of aberrant metaphases. The aberrations included mainly chromatid breaks and chromatid gaps; other aberrations induced by MMC were mostly triradial and quadriradial figures (Fig. l a - d ) .
Sister-chromatid exchanges The frequency of spontaneous and MMC-induced SCEs in controls, PB and MB patients is shown in Table 2. The spontaneous frequency of SCEs per cell was found to be 6.33 + 0.85 in
[ ] Controls 4o
[]
PB
[]
MB
~ 35 a
30
~
2O
"6
v..
o
~
o
o
~
o
SCE/cell
Fig. 2. Frequencydistribution of MMC-induced SCEs in blood lymphocytes of controls, untreated paucibacillary (PB) and multibacillary(MB) patients.
105 controls, 8.06 + 1.34 in PB and 9.78 + 2.3 in MB patients. The increase in the frequency of SCEs in PB and MB patients was found to be statistically significant (p < 0.01) compared to that of controis. Cultures grown with MMC gave a frequency of SCEs/cell of 12.7 + 1.19 in controls, 19.97 + 3.51 in PB, and 29.66 + 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs were found to be statistically significant (p < 0.01) in PB and MB patients compared to controls. The difference in the frequency of MMC-induced SCEs between PB and MB patients was also found to be significant (p < 0.01). In PB patients the frequency of SCEs ranges from 5 to 35, whereas in MB it ranges from 12 to 62; this is highly significant when compared to controls (3-35). MB patients showed a more than 2-fold increase in SCE frequency compared to that of controls (see Table 2). On evaluation of the frequency distribution of SCEs, the maximum number of cells in controls shows 11-15 SCEs/ cell, in PB 21-24 per cell, and 26-30 and 31-35 per cell in the MB group (Fig. 2).
Discussion It is well documented that lymphocytes of cancer-prone syndromes with impaired immune system, namely ataxia telangiectasia (AT) and Bloom's syndrome (BS), reveal an increased frequency of spontaneous chromosomal damage (German et al., 1965; Hecht et al., 1966; German, 1972; German et al., 1974; Harden, 1974; Chaganti et al., 1974; Schroeder and German, 1974; Henderson and German, 1978; Shiraishi and Sandberg, 1979). These syndromes are highly susceptible to treatment with different mutagenic/ carcinogenic agents, and show a dramatic increase in the frequency of chromosome aberrations and SCEs (Peterson et al., 1964; Krepinsky et al., 1979; Shiraishi and Sandberg, 1979; Natarajan and Obe, 1982; Cerutti, 1982). Leprosy patients, who have a depressed cellular immunity, also show similar results. An enhanced frequency of spontaneous and MMC-induced chromosome aberrations was observed in MB patients; an increase in the frequency of spontaneous and MMC-induced SCEs was observed in
PB and MB patients. These observations indicate an existence of chromosomal instability. There is a clear differential mutagen sensitivity between PB and MB patients who have a different immunological spectrum, and thereby differ in the severity of the disease. In controls, the MMC-induced SCE frequency peaks at 11-15 per cell, in PB at 21-25 SCEs/cell, while in MB patients it peaks at 26-30 as well as 31-35 SCEs/cell (Fig. 2). This observation indicates a shift to higher SCE levels with increasing severity of the disease and its association with mutagen sensitivity. Results on chromosome aberrations have also shown a similar pattern, though the increase is not found to be statistically significant. This may be because of the very low concentration of MMC (0.01 /~g/ml) used in the present study. In a pilot study it was observed that a higher concentration of MMC (0.02 /~g/ml) is toxic/lethal to the cultures from the leprosy patients, and therefore a dose-response study was not possible. This suggests that in leprosy the analysis of SCEs can serve as a more sensitive bioassay since it needs a much lower concentration of chemicals than is needed to induce chromosome aberrations. One of the most interesting findings of the present study is the observation of the highest frequency of chromosome aberrations and SCEs in MB patients who harbour M. leprae in greater density and in whom cell-mediated immunity (CMI) is lowest or absent. This suggests that the extent of pathogenic bacterial infection along with CMI may in some way influence the genetic or chromosomal stability of the patient. This is in agreement with the earlier observation that bacteria, such as Mycoplasma, can also cause genetic damage (Aula and Nichols, 1967; Ito-Kuwa and Shigeji, 1984). Since genetic instability is a general characteristic of cancer-prone syndromes, it is also not unlikely that leprosy patients have a greater risk of developing cancer. A detailed and prospective population study could throw light on this aspect.
Acknowledgements This work was financially supported by Rameshwardas Birla Smarak Kosh, Bombay (In-
106 dia). W e t h a n k t h e staff of t h e D e p a r t m e n t of D e r m a t o l o g y , St. J o h n ' s M e d i c a l C o l l e g e H o s p i t a l , a n d the s t a f f o f S u m a n a H a l l i R e h a b i l i t a t i o n C e n t r e , B a n g a l o r e , for p r o v i d i n g b l o o d s a m p l e s of the patients.
References Aula, P., and W.W. Nichols (1967) The cytogenetic effects of mycoplasma in human leukocyte cultures, J. Cell. Physiol., 70, 281-290. Bernstein, M.S., R.L. Hunter and S. Yachinin (1971) Hepatoma and peliosis hepatitis developing in a patient with Fanconi's anemia, N. Engi. J. Med., 284, 1135. Brinton, L.A., R. Hoover, R.R. Jacobson and J.F. Fraumeni, Jr. (1984) Cancer mortality among patients with Hansen's disease, J. Natl. Cancer Inst., 72, 109-114. Cerutti, P. (1982) Abnormal oxygen metabolism in Bloom syndrome?, in: A.T. Natarajan, G. Obe and H. Altmann (Eds.), DNA Repair, Chromosome Alterations and Chromatin Structure, Elsevier Biomedical, Amsterdam, pp. 203-214. Chaganti, R.S.K., S. Schonberg and J. German (1974) A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes, Proc. Natl. Acad. Sci. (U.S.A.), 71, 4508-4515. Das, B.C. (1988) Factors that influence formation of sister chromatid exchanges in lymphocyte cultures, CRC Crit. Rev. Toxicol., 19, 43-86. De Weerd-Kastelein, E.A., W. Keijzer, G. Rainaldi and D. Bootsma (1977) Induction of sister chromatid exchanges in Xeroderma pigmentosum cells after exposure to ultraviolet light, Mutation Res., 45, 253-261. Dosik, H., L.Y. Hau, G.J. Todaro, S.L. Lee, K. Hirschhorn, E.S. Selirio and A.A. Alter (1970) Leukemia in Fanconi's anemia: cytogenetic and tumor virus susceptibility studies, Blood, 36, 341-352. D'Souza, D., and I.M. Thomas (1988) Chromosome aberrations and sister chromatid exchanges (SCEs) in peripheral blood lymphocyte cultures of untreated leprosy patients, Lepr. Rev., 59, 121-125~ D'Souza, D., I.M. Thomas and B.C. Das (1988) Variation in spontaneous chromosomal damage as a function of biologic rhythms in women, Hum. Genet., 79, 83-85. Fugiwara, Y., M. Tatsumi and M. Sasaki (1977) Cross-linked repair in human cells and its possible defect in Fanconi's anemia cells, J. Mol. Biol., 113, 635. Garriga, S., and W.H. Crosby (1959) The incidence of leukemia in families of patients with hypoplasia of the marrow, Blood, 14, 1008-1014. German, J. (1972) Genes which increase chromosomal instability in somatic cells and predispose to cancer, Prog. Med. Genet., 8, 61. German, J., R. Archibald and D. Bloom (1965) Chromosomal breakage in a rare and probably genetically determined syndrome of man, Science, 148, 506.
German, J., L.P. Crippa and D. Bloom (1974) Bloom's syndrome. III, Analysis of the chromosome aberration characteristic of this disorder, Chromosoma, 48, 361-366. Harden, D.G. (1974) Ataxia-telangiectasia: cytogenetic and cancer aspects, in: J. German (Ed.), Chromosomes and Cancer, John Wiley and Sons, New York, pp. 619-636. Hecht, F., R.D. Koler, D.A. Rigas, G.S. Dhanke, M.P. Case, V. Tisdale and R.W. Miller (1966) Leukemia and lymphocytes in ataxia telangiectasia, Lancet, ii, 1193. Henderson, E., and J. German (1978) Development and characterization of lymphoblastoid cell lines (LCLS) from 'chromosome breakage syndromes' and related genetic disorders, J. Supramol. Struct. (Suppl.), 2, 83. ISCN (1978) An International System for Human Cytogenetic Nomenclature, Cytogenet. Cell Genet., 21, 309-404. Ito-Kuwa, S., and A. Shigeji (1984) Increased sister chromatid exchanges in human lymphocyte cultures infected with mycoplasma, Microbiol. Immunol., 28, 893-901. Iyer, V.N., and W. Szybalski (1964) Mitomycins and porfiromycin, chemical mechanism of activation and cross linking of DNA, Science, 145, 55-58. Kolonel, L,N., and T. Hirohata (1977) Leprosy and cancer, a retrospective cohort study in Hawaii, J. Natl. Cancer Inst., 58, 1577-1581. Krepinsky, A.B., J.A. Heddle and J. German (1979) Sensitivity of Bloom's syndrome lymphocytes to ethyl methanesulphonate, Hum. Genet., 50, 151-156. Lynch, H., D.E. Anderson, J.L. Smith, J.B. Howell and A.J. K_rush (1967) Xeroderma pigmentosum, malignant melanoma, and congenital ichthyosis, Arch. Dermatol., 96, 625635. Mertz, T. (1961) Effects of mitomycin-C on lateral root tip chromosomes of Viciafaba, Science, 133, 329-330. Michalany, J. (1966) Malignant tumours of the skin among leprosy patients, Int. J. Lepr., 34, 274-286. Natarajan, A.T. (1984) Origin and significance of chromosomal alterations, in: G. Obe (Ed.), Mutations in Man, Springer-Verlag, Berlin, Heidelberg, pp. 156-176. Natarajan, A.T., and G. Obe (1982) in: J.A. Heddle (Ed.), Mutagenicity - New Horizons in Genetic Toxicology, Chapter 7, Academic Press, New York, pp. 171-213. Oleinick, A. (1969) Altered immunity and cancer risk: a review of the problem and analysis of the cancer mortality experience of leprosy patients, J. Natl. Cancer Inst., 43, 775-781. Peterson, R.D.A., W.D. Kelly and R.A. Good (1964) Ataxia telangiectasia. Its association with a defective thymus, immunological deficiency disease, and malignancy, Lancet, i, 1189-1193. Purtillo, D.T., and C. Pangi (1975) Incidence of cancer in patients with leprosy, Cancer, 35, 1259-1261. Robbins, J.H., K.H. Kraemer, M.A. Lutzner, B.W. Festoff and H.G. Coon (1974) Xeroderma pigmentosum. An inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair, Ann. Intern. Med., 80, 221-248. Ridley, D.S., and W.H. Jopling (1966) Classification of leprosy according to immunity of five group system, Int. J. Lepr., 34, 255-273.
107 Schroeder, T.M., and J. German (1974) Bloom's syndrome and Fanconi's anemia: demonstration of two distinctive patterns of chromosome disruption and rearrangement, Humangenetik, 25, 299-306. Shiraishi, Y., and A.A. Sandberg (1979) Effects of chemicals on the frequency of sister chromatid exchanges and chromosome aberrations in normal and Bloom's syndrome lymphocytes, Cytobios, 26, 97. Swift, M.R., and K. Hirschhorn (1966) Inherited susceptibility to chromosome breakage in various tissues, Ann. Intern. Med., 65, 496-503. Tokudome, S., S. Kono, M. Ikeda, M. Huratsune and S.
Kumamaru (1981) Cancer and other causes of death among leprosy patients, J. Natl. Cancer Inst., 67, 285-289. Weshler, Z., and J. Sheskin (1983) Hanseniasis y cancer, Hansenol. Int., 8, 18-21. Wolff, S., B. Rodin and J.E. Cleaver (1977) Sister chromatid exchanges induced by mutagenic carcinogens in normal and xeroderma pigmentosum cells, Nature (London), 265, 347-349. WHO Study Group (1985) Epidemiology of Leprosy in Relation to Control, Tech. Rep. Ser. 716, World Health Organization, Geneva, pp. 11, 21, 26, 29.
101
Elsevier MUTGEN 01512
Differential sensitivity of peripheral blood lymphocytes of untreated leprosy patients to mitomycin C Doris D'Souza
1, B.C.
Das 2 and I.M. T h o m a s 1
Division of Human Genetics, Department of Anatomy, St. John's Medical College, Bangalore 560034 (India) and 2 Molecular Oncology, Cytology Research Centre (ICMR), Maulana Azad Medical College Campus, New Delhi 110002 (India)
(Received8 June 1989) (Revisionreceived31 August1989) (Accepted28 September1989)
Keywords: MitomycinC; Mutagensensitivity;Leprosy
Summary The effects of a bifunctional alkylating agent mitomycin C (MMC), an effective inducer of chromosome aberrations and sister-chromatid exchanges (SCEs), have been studied in untreated leprosy patients. This was done to study the mutagen sensitivity of the leprosy patients. The frequency of chromosomal aberrations induced by MMC (conc. 0.01 #g/ml) was 2.5% in controls, 3.6% in paucibacillary (PB), and 6.8% in multibacillary (MB) patients. The difference in the frequency of MMC-induced chromosome aberrations between the 3 groups studied was highly significant (p < 0.01). Cultures grown with MMC showed the frequency of SCEs/cell to be 12.70 + 1.19 in controls, 19.97 + 3.51 in PB, and 29.66 + 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs between the 3 groups were found to be highly significant (p < 0.01). The enhanced frequencies of spontaneous and MMC-induced chromosome aberrations and SCEs observed in PB and MB patients indicate a clear differential mutagen sensitivity between PB and MB patients who are known to have different immunological status and thereby differ in the severity of the disease.
Leprosy is a chronic granulomatous mycobacterial disease, primarily affecting the peripheral nerves and secondarily involving skin and other tissues in humans. The bacterium, Mycobacterium leprae, is known to be the causative agent of leprosy which is most prevalent in tropical countries. India, more than any other country, has a very large number of leprosy patients (WHO, 1985). Leprosy is not an inherited disease, though
Correspondence: Dr. D. D'Souza, Patna Women's College, Avila Convent,BayleyRoad, Patna 800001, Bihar(India).
lepromatous patients have depressed cellular immunity to M. leprae (Ridley and Jophng, 1966). Therefore, an increased incidence of malignancy may be anticipated in leprosy patients. However, reports of various studies on leprosy patients and their susceptibility to developing cancer are conflicting (Michalany, 1966; Oleinick, 1969; Purtillo and Pangi, 1975; Kolonel and Hirohata, 1977; Tokudome et al., 1981; Weshler and Sheskin, 1983; Brinton et al., 1984). Various cancer-prone syndromes as well as certain autosomal recessive syndromes which show impaired immunity (Garriga and Crosby, 1959; Lynch et al., 1967; Dosik et al.,
0165-1218/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
102
1970; Bernstein et al., 1971; Robbins et al., 1974; Schroeder and German, 1974) are characterised by chromosomal instability (Hecht et al., 1966; Swift and Hirschhorn, 1966; German, 1972; Schroeder and German, 1974; Harden, 1974; Chaganti et al., 1974), and are highly susceptible to DNA-damaging agents producing various types of chromosomal anomalies (Fugiwara et al., 1977; Natarajan and Obe, 1982; Cerutti, 1982; Natarajan, 1984) and increased frequency of sister-chromatid exchanges (SCEs) (Chaganti et al., 1974; Wolff et al., 1977; De Weerd et al., 1977; Shiraishi and Sandberg, 1979). Recently, we have shown, for the first time, a significant increase in the frequency of spontaneous chromosome aberrations and SCEs in blood lymphocyte cultures of untreated leprosy patients (D'Souza and Thomas, 1988). Taking these findings into consideration the present study was conducted to gain further insight into the genetic instability and mutagen sensitivity of leprosy patients by using the potent mutagen mitomycin C (MMC). MMC, an anticancer antibiotic, is known to act on chromosomal DNA (Mertz, 1961). Upon metabolic activation, the drug is transformed into a reactive bifunctional alkylating agent (Iyer and Szybalski, 1964). It has been shown to cause a dramatic increase in the frequency of chromosome aberrations and SCEs in blood lymphocyte cultures of some autosomal recessive syndromes (Fugiwara et al., 1977; Shiraishi and Sandberg, 1979). In this study, the effects of MMC in inducing chromosome aberrations and SCEs in blood lymphocyte cultures of untreated leprosy patients have been studied. For this study only male subjects were considered in order to avoid the variations induced in females by various other factors (Das, 1988; D'Souza et al., 1988). Materials and methods
Patient selection The patients selected were classified by clinical symptoms and bacterial index (BI) according to the Ridley-Jopling scale (Ridley and Jopling, 1966). The tuberculoid leprosy (TT) and the borderline tuberculoid leprosy (BT) patients were included in the paucibacillary group (PB), while
borderline lepromatous (BL) and lepromatous leprosy (LL) patients were considered as the multibacillary group (MB). Five untreated PB and 5 untreated MB patients were studied. These patients had been carefully selected after ascertaining that they had never undergone treatment for leprosy, and apparently had no other disease. Four age-matched healthy normal individuals who were not under any medication prior to this investigation served as controls. All samples of the study consisted only of males. Lymphocyte cultures Phytohaemagglutinin M (Difco)-stimulated peripheral blood lymphocyte cultures were grown in TC199 medium (Gibco), supplemented with 20% heat-inactivated human AB serum and antibiotics (penicillin and streptomycin) for 48 h and 72 h. MMC (Sigma) was added to the cultures at a final concentration of 0.01 g g / m l at the time of culture initiation. Bromodeoxyuridine (BrdU, Sigma, 5 gg/rnl) was added at the time of initiation of cultures. In each group 2 sets of cultures were set up. 100 first-cycle metaphases from 48-h cultures were studied to score chromosome aberrations for each subject, using ISCN criteria (ISCN, 1978). 20-50 second-cycle metaphases were scored from 72-h cultures for SCEs in each subject, and the values were expressed as the number of SCEs per metaphase or cell. Results
Chromosome aberrations Table 1 shows the mean frequency distribution of spontaneous and MMC-induced chromosome aberrations in controls, PB and MB patients. The frequency of spontaneous chromosome aberrations per 100 metaphases was found to be 1.5 in controls, 3.2 in PB and 5.2 in MB patients. A significant (p < 0.001) difference was found in the frequency of spontaneous chromosome aberrations between controls, PB and MB patients, whereas, when gaps were excluded, only MB patients showed an increase in the frequency of aberrant metaphases. The frequency of chromosome aberrations induced by MMC (0.01 gg/ml) was 2.5% in con-
103 TABLE 1 F R E Q U E N C Y OF CHROMOSOME ABERRATIONS IN LYMPHOCYTES OF U N T R E A T E D LEPROSY PATIENTS, CULT U R E D WITH MMC Group
Number of subjects/ metaphases
Controls Without MMC
4/400
With MMC
4/400
PaucibaciUary Without MMC
5/500
With MMC
5/500
Multibacillary Without MMC
5/500
With MMC
5/500
Chromosome aberrations Meta-
Breaks
Meta-
Gaps
phases with aberrations
Csb
Ctb
phases with breaks
Csg
Ctg
phases with gaps
6 (1.5) 10 (2.5)
-
3 (0.75) 7 (1.75)
3 (0.75) 7 (1.75)
-
2 (0.5) 2 (0.5)
2 (0.5) 2 (0.5)
-
4 (0.80) 4 (0.80)
4 (0.80) 4 (0.80)
11 (2.2) 11 (2.2)
11 (2.2) 10 (2.0)
-
5 (1.00) 10 (2.00)
5 (1.00) 10 (2.00)
14 (2.8) 18 (3.6)
16 (3.2) 16 (3.2)
-
16 * (3.2) 18 ** (3.6)
-
26 * (5.2) 34 ** (6.8)
-
-
-
-
-
2 (0.4) -
Meta-
Cte
Others
a
Aberrant metaphases without gaps
-
2 (0.4) 6 (1.2)
1 c (0.25) 1 a (0.25)
4 (1.0) 8 (2.0)
1 ~ (0.2) 2 f (0.4)
5 (1.0) 8 (1.6)
5 s (1.0) 2 h (0.4)
10 (2.0) 18 (3.6)
Figures in parentheses indicate percentages. Csb, chromosome break; Ctb, chromatid break; Csg, chromosome gap; Ctg, chromatid gap; Cte, chromatid exchanges. a 'Others' includes acentric fragments (AF); dicentric chromosomes (dic); double minutes (DMs); and metaphases with pulverised (shattered) chromosomes (pvz). c 1 D M s ; d 1 A F ; e 1 DMs; f 1 d i c a n d l A F ; S 3 p v z a n d 2 A F ; h 2AF. * Without MMC: significant in chi-square: p < 0.001. * *With MMC: significant in chi-square: p < 0.01.
TABLE 2 F R E Q U E N C Y OF SCEs IN LYMPHOCYTES OF U N T R E A T E D LEPROSY PATIENTS, C U L T U R E D W I T H MMC Group Controls Without MMC With MMC Paucibacillary (PB) Without MMC With MMC Multibacillary (MB) Without MMC With MMC
Number of subjects studied
Number of cells analysed
SCEs/cell
+ SD
4 4
145 200
6.33 12.70
+ 0.85 + 1.19
1-17 3-35
5 5
170 110
8.06 a 19.97 a
+ 1.34 + 3.51
1-28 5-35
5 5
165 150
9.78 b,c 29.66 e,f
+ 2.30 + 5.92
1-22 12-62
Significant in Student's t-test: Without MMC: a PB vs. controls: p < 0.01. b MB vs. controls: p < 0.01. c PB vs. MB: p < 0.01.
With MMC: d PB vs. controls: p < 0.01. e MB vs. controls: p < 0.01. f PB vs. MB: p < 0.01.
SCE range
I
¢ Fig. l(a-d). Partial metaphases showing chromatid exchanges.
trols, 3.6% in PB, and 6.8% in MB patients. There was certainly an increase in the frequency of chromosome aberrations following MMC treatment in patient groups, the difference was found to be non-significant ( p > 0.05) within the groups while the difference was significant ( p < 0.01) between the groups. When gaps were excluded, only MB patients showed an increase in the frequency of aberrant metaphases. The aberrations included mainly chromatid breaks and chromatid gaps; other aberrations induced by MMC were mostly triradial and quadriradial figures (Fig. l a - d ) .
Sister-chromatid exchanges The frequency of spontaneous and MMC-induced SCEs in controls, PB and MB patients is shown in Table 2. The spontaneous frequency of SCEs per cell was found to be 6.33 + 0.85 in
[ ] Controls 4o
[]
PB
[]
MB
~ 35 a
30
~
2O
"6
v..
o
~
o
o
~
o
SCE/cell
Fig. 2. Frequencydistribution of MMC-induced SCEs in blood lymphocytes of controls, untreated paucibacillary (PB) and multibacillary(MB) patients.
105 controls, 8.06 + 1.34 in PB and 9.78 + 2.3 in MB patients. The increase in the frequency of SCEs in PB and MB patients was found to be statistically significant (p < 0.01) compared to that of controis. Cultures grown with MMC gave a frequency of SCEs/cell of 12.7 + 1.19 in controls, 19.97 + 3.51 in PB, and 29.66 + 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs were found to be statistically significant (p < 0.01) in PB and MB patients compared to controls. The difference in the frequency of MMC-induced SCEs between PB and MB patients was also found to be significant (p < 0.01). In PB patients the frequency of SCEs ranges from 5 to 35, whereas in MB it ranges from 12 to 62; this is highly significant when compared to controls (3-35). MB patients showed a more than 2-fold increase in SCE frequency compared to that of controls (see Table 2). On evaluation of the frequency distribution of SCEs, the maximum number of cells in controls shows 11-15 SCEs/ cell, in PB 21-24 per cell, and 26-30 and 31-35 per cell in the MB group (Fig. 2).
Discussion It is well documented that lymphocytes of cancer-prone syndromes with impaired immune system, namely ataxia telangiectasia (AT) and Bloom's syndrome (BS), reveal an increased frequency of spontaneous chromosomal damage (German et al., 1965; Hecht et al., 1966; German, 1972; German et al., 1974; Harden, 1974; Chaganti et al., 1974; Schroeder and German, 1974; Henderson and German, 1978; Shiraishi and Sandberg, 1979). These syndromes are highly susceptible to treatment with different mutagenic/ carcinogenic agents, and show a dramatic increase in the frequency of chromosome aberrations and SCEs (Peterson et al., 1964; Krepinsky et al., 1979; Shiraishi and Sandberg, 1979; Natarajan and Obe, 1982; Cerutti, 1982). Leprosy patients, who have a depressed cellular immunity, also show similar results. An enhanced frequency of spontaneous and MMC-induced chromosome aberrations was observed in MB patients; an increase in the frequency of spontaneous and MMC-induced SCEs was observed in
PB and MB patients. These observations indicate an existence of chromosomal instability. There is a clear differential mutagen sensitivity between PB and MB patients who have a different immunological spectrum, and thereby differ in the severity of the disease. In controls, the MMC-induced SCE frequency peaks at 11-15 per cell, in PB at 21-25 SCEs/cell, while in MB patients it peaks at 26-30 as well as 31-35 SCEs/cell (Fig. 2). This observation indicates a shift to higher SCE levels with increasing severity of the disease and its association with mutagen sensitivity. Results on chromosome aberrations have also shown a similar pattern, though the increase is not found to be statistically significant. This may be because of the very low concentration of MMC (0.01 /~g/ml) used in the present study. In a pilot study it was observed that a higher concentration of MMC (0.02 /~g/ml) is toxic/lethal to the cultures from the leprosy patients, and therefore a dose-response study was not possible. This suggests that in leprosy the analysis of SCEs can serve as a more sensitive bioassay since it needs a much lower concentration of chemicals than is needed to induce chromosome aberrations. One of the most interesting findings of the present study is the observation of the highest frequency of chromosome aberrations and SCEs in MB patients who harbour M. leprae in greater density and in whom cell-mediated immunity (CMI) is lowest or absent. This suggests that the extent of pathogenic bacterial infection along with CMI may in some way influence the genetic or chromosomal stability of the patient. This is in agreement with the earlier observation that bacteria, such as Mycoplasma, can also cause genetic damage (Aula and Nichols, 1967; Ito-Kuwa and Shigeji, 1984). Since genetic instability is a general characteristic of cancer-prone syndromes, it is also not unlikely that leprosy patients have a greater risk of developing cancer. A detailed and prospective population study could throw light on this aspect.
Acknowledgements This work was financially supported by Rameshwardas Birla Smarak Kosh, Bombay (In-
106 dia). W e t h a n k t h e staff of t h e D e p a r t m e n t of D e r m a t o l o g y , St. J o h n ' s M e d i c a l C o l l e g e H o s p i t a l , a n d the s t a f f o f S u m a n a H a l l i R e h a b i l i t a t i o n C e n t r e , B a n g a l o r e , for p r o v i d i n g b l o o d s a m p l e s of the patients.
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