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Genotoxicity evaluation of pyrazinamide in mice
ELSEVIER
Mutation Research 321 (1994) 1-5
Genetic Toxicology
Genotoxicity evaluation of pyrazinamide in mice B. Anitha a, S. Sudha b, P.M. Gopinath b, G. Durairaj a,, Departments of a Zoology and b Genetics, Universityof Madras, Guindy Campus, Madras 600 025, India
(Received 2 June 1992; revision received 12 February 1993; accepted 8 April 1993)
Abstract
Pyrazinamide, an antituberculosis drug, was investigated for genotoxicity in mice, an in vivo rodent system. Three doses (125, 250 and 500 m g / k g bw corresponding to 5, 10 and 20 times the therapeutic dose respectively) were tested. The mitotic index and the frequency of chromosomal aberrations were analysed at three sample times (3, 6 and 24 h) after a single intraperitoneal treatment. The frequency of sperm shape abnormalities was also examined. The mitotic index showed a decrease in drug-treated animals compared with that recorded in the control set. The cells with chromosomal aberrations ranged from 4% to 8%. The maximum frequency of aberrations was found at the 3-h sample time. The frequency of sperm shape abnormalities showed a dose-related increase. These observations suggest pyrazinamide to be a weak genotoxicant at the doses tested. Key words: Pyrazinamide; Chromosome aberrations; Mitotic index
I. Introduction
Pyrazinamide, an antituberculosis drug, is a hydrazine of pyrazinic acid. The therapeutic effect of pyrazinamide is considered to be similar to that of isoniazid, a drug extensively used against tuberculosis. It is a component in combination chemotherapy along with isoniazid and rifampicin as r e c o m m e n d e d by the W H O for treatment of tuberculosis (Pretet et al., 1983). While the genotoxic effects of various antituberculosis drugs have been extensively investigated, only a few investigators have attempted to test genetic effects of pyrazinamide. Recently, Gopal Rao et al. (1990,
* Corresponding author.
1991) reported an increased incidence of sisterchromatid exchanges and an enhanced frequency of chromosomal aberrations in tuberculosis patients undergoing chemotherapy with a combination of drugs which included pyrazinamide. Suk (1977) reported this drug to be mutagenic in strain TA100 of Salmonella typhimurium on metabolic activation. Caratzali et al. (1972) and R o m a n and Georgian (1977) observed the presence of chromosomal aberrations in cultured human lymphocytes exposed to pyrazinamide. At very high doses, equivalent to 20 times the therapeutic dose or higher, the drug exhibited a clastogenie effect in mice (Bhanumathi, 1983). As there is a paucity of data in mammalian systems in vivo, it was thought desirable to examine whether pyrazinamide is genotoxic by employing an in vivo rodent system.
0165-1218/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1218(93)E0045-P
2
B. Anitha et al./Mutation Research 321 (1994) 1-5
2. Materials and methods
Pyrazinamide (PCT Pharmaceuticals, Bombay, India) was tested for genotoxicity in mice of the Swiss albino strain. The animals, about 3 months old, were procured from the Central Animal Facility, Indian Institute of Science, Bangalore and were acclimatised for 3 weeks in the animal house laboratory and were maintained at 25 + 2°C and 60 + 10% humidity. Food (M/s. Hindustan Lever Ltd., Bombay, India) and water were given ad libitum. The feed was in the form of pellets. The composition of the feed was: wheat flour 22%, roasted bengal gram flour 60%, skimmed milk powder 5%, casein 4%, refined oil 4.8% and the rest consisting of a salt mixture with starch, choline and vitamins. Only male mice were employed for all the tests. The animals were exposed to different doses of pyrazinamide, namely 5, 10 and 20 times the therapeutic dose, which amounted to 125, 250 and 500 m g / k g body weight respectively. The drug was administered intraperitoneally. Control animals received distilled water. The mitotic index and chromosome aberrations were analysed from animals killed by cervical dislocation 3, 6 and 24 h after exposure to the drug. Each mouse received 0.5 ml 0.004% colchicine (Loba Chemicals, Bombay, India) 90 min prior to sacrifice. The procedure to obtain chromosomal preparations from the bone marrow cells of drug-treated and control animals was based on the schedules described by Tjio and Whang (1962) and Evans et al. (1964). The preparations were stained in 4% Giemsa (Gurr) for 10 min, washed in running tap water and dried. The mitotic index was determined by scoring the number of dividing ceils in 5000 ceils from each animal. 100 well-spread metaphases from each mouse were analysed to record chromosomal aberrations, classified on the basis of the reports of Buckton and Evans (1982) and Sankaranarayanan (1982). The procedure employed for the sperm abnormality assay was that described by Wyrobek and Bruce (1978). Animals were injected daily with pyrazinamide (125, 250 and 500 m g / k g bw) for 5 days and were killed 35 days following the first
administration. The cauda epididymis was dissected out and the contents were processed to obtain preparations of sperm, which were stained with eosin Y. Three animals were employed for each dose and 2000 sperm were analysed from each mouse. The cytogenetic and sperm shape abnormalities recorded were subjected to statistical evaluation by regression analysis. Pairwise comparison between treated and control groups were carried out by employing standard normal deviate test. The trend analysis was carried out by taking the dose of the drug as the independent variable X, the dependent variable Y being mutation rate: [% of cells with chromosomal aberrations or % of abnormal sperms or % dividing cells (mitotic index)]. The values of X and Y were transformed appropriately (X = arc sine x) to confirm normality as described by Zar (1974). The regressions were tested for H o : B = 0 using standard normal deviate statistics for computing two-sample proportions.
3. Results
There was no variation observed in the frequency of dividing ceils at different time spans of 3, 6 and 24 h in the control set. This is in agreement with our previous data. The data on mitotic index (MI) and chromosomal aberrations (CA) at different time spans using distilled water are presented below. Post-treatment time (h)
MI (%)
CA (%)
3 6 24
2.2 2.04 2.86
2.66 2.66 2.33
In the light of these observations, the control data presented in the tables pertain to only one experimental point. The data on mitotic index in the bone marrow cells of mice treated with different doses of pyrazinamide and sampled at 3, 6 and 24 h after treatment are presented in Table 1. A dose-de-
B. Anitha et al. / Mutation Research 321 (1994) 1-5 Table 1 Mitotic index in bone marrow cells of mice after intraperitoneal administration of pyrazinamide Time (h)
Dose ( m g / k g bw)
Cells in division
Mitotic index %
+ SE
-
330
2.200
0.119
3
125 250 500
243 287 138
1.620 1.910 0.920
0.103 0.111 0.077
6
125 250 500
210 181 149
1.400 1.210 0.990
0.095 0.089 0.080
24
125 250 500
287 193 31
1.910 1.290 0.206
0.111 0.092 0.037
Control
pendent depression of mitotic index was observed and it was significant at 24 h, but not at 3 h and 6 h (2.301 - 0.0041x + 0.1225, P < 0.05). The mi-
3
totic index was found to be lowered at all doses tested. The types of chromosomal aberrations observed in the bone marrow cells of pyrazinamidetreated mice are recorded in Table 2. The aberrations encountered were mostly chromatid gaps, breaks and fragments. Instances of isochromatid gaps and isochromatid breaks were very few. Statistical analysis revealed no significant dose-dependent trend, though pyrazinamide significantly increased the frequency of chromosomal aberrations at doses of 250 and 500 m g / k g bw. The observations from the sperm abnormality assay provided the data presented in Table 3. The abnormalities observed were sperm with amorphous heads, hookless and banana-shaped heads, rare examples of sperm with double heads and sperm with double tails. The abnormal forms of sperm exhibited a significant dose-dependent increase on exposure to pyrazinamide (0.003 +
Table 2 Chromosomal aberrations in bone marrow cells of mice after intraperitoneal treatment with pyrazinamide Time
Dose
Aberrations
(h)
( m g / k g bw)
Gaps
Aberrant cells
Breaks
Fragments
Including gaps N
Excluding gaps
%
+SE
%
+SE
-
2
2
1
4
1.33
0.660
3
1.00
0.574
3
125 250 500
3 4 6
10 8 13
6 8 6
19 20 23
6.33 6.66 7.66
1.400 1.439 1.535
16 16 19
5.33 5.33 6.33
1.025 1.025 1.400
6
125 250 500
5 4 3
9 8 10
5 4 7
16 16 20
5.33 5.33 6.66
1.025 1.025 1.439
12 12 17
4.00 4.00 5.66
1.130 1.130 1.330
24
125 250 500
2 4 6
3 11 5
5 5 5
10 20 16
3.33 6.66 5.33
0.567 1.439 1.025
8 16 10
2.66 5.33 3.33
0.929 1.025 0.567
Control
N
Table 3 Incidence of sperm shape abnormalities seen in pyrazinamide-treated mice Dose ( m g / k g bw)
Sperm analysed
Types of sperm head abnormalities Amorphous
Banana head
Hookless
Double head
Double tail
N
%
0 125 250 500
8000 6000 8055 6000
62 43 73 120
0 0 2 7
10 21 8 17
0 0 6 4
0 0 0 2
72 64 109 150
0.90 1.06 1.36 2.50
Abnormal sperm
+ SE
0.105 0.132 0.129 0.201
4
B. Anitha et al./Mutation Research 321 (1994) 1-5
0.736x + 0.224, P < 0.05). The increase in sperm shape abnormalities was about threefold on administration of 500 m g / k g body weight of pyrazinamide.
4. Discussion Multiple assays were carried out in mice to examine whether pyrazinamide is genotoxic. Animals treated with the drug exhibited a dose-dependent decrease in the number of dividing cells (Table 1). The observations suggest a cytotoxic effect of pyrazinamide. Exposure of mice to different doses of pyrazinamide for varying periods resulted in the induction of chromosomal aberrations (Table 2). Clastogenicity of pyrazinamide was reported by Bhanumathi (1983) in mice and in human lymphocytes in culture. The maximum incidence of aberrations observed in mice was at 3 h after treatment with pyrazinamide. The response at 3 h corresponds to the half-life of the drug, estimated to be 2-3 h (Bareggi et al., 1987). 1 g of drug produced a peak serum level of 60 i z g / m l in patients 2 h after administration (Steele and Desperez, 1988). Various aberrations recorded were mostly of chromatid type, similar to the observations of Caratzali et al. (1972) and Roman and Georgian (1977) in cultured lymphocytes. According to Roman and Georgian (1977) the drug acts on the S-phase of the cell cycle. They emphasised the need to distinguish between the direct clastogenic action of the drug and the secondary effect brought out by the drug on the other cellular components. The drug is reported to interfere with the biosynthesis of tetrahydrofolic acid, a purine precursor, and this may be responsible for the mutagenic effect. The phenotype of the sperm is determined by the autosomal and sex-linked genes. The sperm shape, the fraction of abnormal sperm and dimensions of individual sperm are characteristic of the genotype. The relevance of sperm morphology for genotoxicity assessment was emphasised by Wyrobek and Bruce (1978). Mice exposed to different doses of pyrazinamide exhibited abnormal sperm morphology and the frequencies were
significantly different from those recorded in the control set of animals. The results thus suggest pyrazinamide to be a weak genotoxic drug. One cannot extrapolate the data obtained from mice subjected to the intraperitoneal administration to humans, as patients are treated with the drug orally and that too with a combination of chemotherapeutic agents. Recent reports on genotoxicity of antituberculosis drugs by Gopal Rao et al. (1990, 1991) are on patients subjected to treatment with a combination of drugs. The only reports available on the individual action of pyrazinamide on mammalian cells are those of Caratzali et al. (1972), Roman and Georgian (1977) and Bhanumathi (1983). These studies were carried out on cultured lymphocytes and they found pyrazinamide to be clastogenic. Bhanumathi (1983) has also examined the effects of very high doses of pyrazinamide in mice and reported it to be clastogenic. The findings in the present study are in agreement with the results of these investigators on cells in culture. In this context, it is of interest to record that Ennever et al. (1987) emphasised the importance of short-term animal assays for the prediction of carcinogenicity and non-carcinogenicity to man.
5. Acknowledgements The first author would like to thank the University Grants Commission for providing the funds to carry out this experiment and Dr. A. Ramesh (Department of Genetics) for helping in the statistical evaluation of the data.
6. References Bareggi, S.R., R. Ceruth, R. Rirola, R. Riva and M. Cisternino (1987) Clinical pharmacokinetics and metabolism of pyrazinamide in healthy volunteers, Arzneim. Forsch./ Drug Res., 37, 849-53. Bhanumathi, J. (1983)Cytogeneticand Flow CytometricStudies on the Effects of Some Antituberculosis Drugs, Ph.D. Thesis, Universityof Madras. Buckton, K.E., and H.J. Evans (1982) Human peripheral blood lymphocyteculture, in: T.C. Hsu (Ed.), An In Vitro
B. Anitha et al. / Mutation Research 321 (1994) 1-5 Introductory Assay for the Cytogenetic Effects of Environmental Mutagens, Oxford and IBH Publishing Co., New Delhi, pp. 183-202. Caratzali, A., I. Constantin and I.C. Roman (1972) Identification of human chromosomes by chromerization in vitro, C.R. Heb. Sceances Acad. Sci. Serd. Sci. Nat. (Paris), 274187, 1198-1199. Ennever, F.K., N.J. Thomas and H.S. Rosenkranz (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans, Mutagenesis, 2, 73-78. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testis, Cytogenetics, 3, 289-294. Gopal Rao, V.V.N., E.V.V. Gupta and I.M. Thomas (1990) Sister chromatid exchanges in the lymphocytes of tuberculosis patients receiving short-term chemotherapy, Tubercle, 71, 173-176. Gopal Rao, V.V.N., E.V.V. Gupta and I.M. Thomas (1991) Chromosomal aberrations in tuberculosis patients before and after treatment with short-term chemotherapy, Mutation Res., 259, 13-19.
5
Pretet, S., J. Marsac and D. Choudat (1983) Value of pyrazinamide in tuberculosis, Presse M~d., 12, 871. Roman, I.C., and L. Georgian (1977) Cytogenetic effects of some antituberculosis drug in vitro, Mutation Res., 48, 215-224. Sankaranarayanan, K. (1982) Genetic Effects of Ionization Radiation in Multicellular Eukaryotes and the Assessment of Genetic Radiation Hazards in Man, Elsevier, Amsterdam, 385 pp. Steele, M.A., and R.M. Desprez (1988) The role of pyrazinamide in tuberculosis chemotherapy, Chest, 94, 845. Suk, W.A. (1977) Oncogenic and mutagenic potential of selected antituberculosis drugs, Diss. Int. Abstr. Biol., 35593560. Tjio, J.H., and J. Whang (1962) Direct chromosome preparation of bone marrow cells without prior in vitro culture or in vivo colchicine administration, Stain Technol., 37, 17-20. Wyrobek, A.J., and W.R. Bruce (1978) Sperm abnormality assay, Chem. Mutagen., 5, 257. Zar, J.H. (1974) Biostatistic Analysis, Prentice-Hall, Engelwood Cliffs, NJ, 620 pp.
Mutation Research 321 (1994) 1-5
Genetic Toxicology
Genotoxicity evaluation of pyrazinamide in mice B. Anitha a, S. Sudha b, P.M. Gopinath b, G. Durairaj a,, Departments of a Zoology and b Genetics, Universityof Madras, Guindy Campus, Madras 600 025, India
(Received 2 June 1992; revision received 12 February 1993; accepted 8 April 1993)
Abstract
Pyrazinamide, an antituberculosis drug, was investigated for genotoxicity in mice, an in vivo rodent system. Three doses (125, 250 and 500 m g / k g bw corresponding to 5, 10 and 20 times the therapeutic dose respectively) were tested. The mitotic index and the frequency of chromosomal aberrations were analysed at three sample times (3, 6 and 24 h) after a single intraperitoneal treatment. The frequency of sperm shape abnormalities was also examined. The mitotic index showed a decrease in drug-treated animals compared with that recorded in the control set. The cells with chromosomal aberrations ranged from 4% to 8%. The maximum frequency of aberrations was found at the 3-h sample time. The frequency of sperm shape abnormalities showed a dose-related increase. These observations suggest pyrazinamide to be a weak genotoxicant at the doses tested. Key words: Pyrazinamide; Chromosome aberrations; Mitotic index
I. Introduction
Pyrazinamide, an antituberculosis drug, is a hydrazine of pyrazinic acid. The therapeutic effect of pyrazinamide is considered to be similar to that of isoniazid, a drug extensively used against tuberculosis. It is a component in combination chemotherapy along with isoniazid and rifampicin as r e c o m m e n d e d by the W H O for treatment of tuberculosis (Pretet et al., 1983). While the genotoxic effects of various antituberculosis drugs have been extensively investigated, only a few investigators have attempted to test genetic effects of pyrazinamide. Recently, Gopal Rao et al. (1990,
* Corresponding author.
1991) reported an increased incidence of sisterchromatid exchanges and an enhanced frequency of chromosomal aberrations in tuberculosis patients undergoing chemotherapy with a combination of drugs which included pyrazinamide. Suk (1977) reported this drug to be mutagenic in strain TA100 of Salmonella typhimurium on metabolic activation. Caratzali et al. (1972) and R o m a n and Georgian (1977) observed the presence of chromosomal aberrations in cultured human lymphocytes exposed to pyrazinamide. At very high doses, equivalent to 20 times the therapeutic dose or higher, the drug exhibited a clastogenie effect in mice (Bhanumathi, 1983). As there is a paucity of data in mammalian systems in vivo, it was thought desirable to examine whether pyrazinamide is genotoxic by employing an in vivo rodent system.
0165-1218/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1218(93)E0045-P
2
B. Anitha et al./Mutation Research 321 (1994) 1-5
2. Materials and methods
Pyrazinamide (PCT Pharmaceuticals, Bombay, India) was tested for genotoxicity in mice of the Swiss albino strain. The animals, about 3 months old, were procured from the Central Animal Facility, Indian Institute of Science, Bangalore and were acclimatised for 3 weeks in the animal house laboratory and were maintained at 25 + 2°C and 60 + 10% humidity. Food (M/s. Hindustan Lever Ltd., Bombay, India) and water were given ad libitum. The feed was in the form of pellets. The composition of the feed was: wheat flour 22%, roasted bengal gram flour 60%, skimmed milk powder 5%, casein 4%, refined oil 4.8% and the rest consisting of a salt mixture with starch, choline and vitamins. Only male mice were employed for all the tests. The animals were exposed to different doses of pyrazinamide, namely 5, 10 and 20 times the therapeutic dose, which amounted to 125, 250 and 500 m g / k g body weight respectively. The drug was administered intraperitoneally. Control animals received distilled water. The mitotic index and chromosome aberrations were analysed from animals killed by cervical dislocation 3, 6 and 24 h after exposure to the drug. Each mouse received 0.5 ml 0.004% colchicine (Loba Chemicals, Bombay, India) 90 min prior to sacrifice. The procedure to obtain chromosomal preparations from the bone marrow cells of drug-treated and control animals was based on the schedules described by Tjio and Whang (1962) and Evans et al. (1964). The preparations were stained in 4% Giemsa (Gurr) for 10 min, washed in running tap water and dried. The mitotic index was determined by scoring the number of dividing ceils in 5000 ceils from each animal. 100 well-spread metaphases from each mouse were analysed to record chromosomal aberrations, classified on the basis of the reports of Buckton and Evans (1982) and Sankaranarayanan (1982). The procedure employed for the sperm abnormality assay was that described by Wyrobek and Bruce (1978). Animals were injected daily with pyrazinamide (125, 250 and 500 m g / k g bw) for 5 days and were killed 35 days following the first
administration. The cauda epididymis was dissected out and the contents were processed to obtain preparations of sperm, which were stained with eosin Y. Three animals were employed for each dose and 2000 sperm were analysed from each mouse. The cytogenetic and sperm shape abnormalities recorded were subjected to statistical evaluation by regression analysis. Pairwise comparison between treated and control groups were carried out by employing standard normal deviate test. The trend analysis was carried out by taking the dose of the drug as the independent variable X, the dependent variable Y being mutation rate: [% of cells with chromosomal aberrations or % of abnormal sperms or % dividing cells (mitotic index)]. The values of X and Y were transformed appropriately (X = arc sine x) to confirm normality as described by Zar (1974). The regressions were tested for H o : B = 0 using standard normal deviate statistics for computing two-sample proportions.
3. Results
There was no variation observed in the frequency of dividing ceils at different time spans of 3, 6 and 24 h in the control set. This is in agreement with our previous data. The data on mitotic index (MI) and chromosomal aberrations (CA) at different time spans using distilled water are presented below. Post-treatment time (h)
MI (%)
CA (%)
3 6 24
2.2 2.04 2.86
2.66 2.66 2.33
In the light of these observations, the control data presented in the tables pertain to only one experimental point. The data on mitotic index in the bone marrow cells of mice treated with different doses of pyrazinamide and sampled at 3, 6 and 24 h after treatment are presented in Table 1. A dose-de-
B. Anitha et al. / Mutation Research 321 (1994) 1-5 Table 1 Mitotic index in bone marrow cells of mice after intraperitoneal administration of pyrazinamide Time (h)
Dose ( m g / k g bw)
Cells in division
Mitotic index %
+ SE
-
330
2.200
0.119
3
125 250 500
243 287 138
1.620 1.910 0.920
0.103 0.111 0.077
6
125 250 500
210 181 149
1.400 1.210 0.990
0.095 0.089 0.080
24
125 250 500
287 193 31
1.910 1.290 0.206
0.111 0.092 0.037
Control
pendent depression of mitotic index was observed and it was significant at 24 h, but not at 3 h and 6 h (2.301 - 0.0041x + 0.1225, P < 0.05). The mi-
3
totic index was found to be lowered at all doses tested. The types of chromosomal aberrations observed in the bone marrow cells of pyrazinamidetreated mice are recorded in Table 2. The aberrations encountered were mostly chromatid gaps, breaks and fragments. Instances of isochromatid gaps and isochromatid breaks were very few. Statistical analysis revealed no significant dose-dependent trend, though pyrazinamide significantly increased the frequency of chromosomal aberrations at doses of 250 and 500 m g / k g bw. The observations from the sperm abnormality assay provided the data presented in Table 3. The abnormalities observed were sperm with amorphous heads, hookless and banana-shaped heads, rare examples of sperm with double heads and sperm with double tails. The abnormal forms of sperm exhibited a significant dose-dependent increase on exposure to pyrazinamide (0.003 +
Table 2 Chromosomal aberrations in bone marrow cells of mice after intraperitoneal treatment with pyrazinamide Time
Dose
Aberrations
(h)
( m g / k g bw)
Gaps
Aberrant cells
Breaks
Fragments
Including gaps N
Excluding gaps
%
+SE
%
+SE
-
2
2
1
4
1.33
0.660
3
1.00
0.574
3
125 250 500
3 4 6
10 8 13
6 8 6
19 20 23
6.33 6.66 7.66
1.400 1.439 1.535
16 16 19
5.33 5.33 6.33
1.025 1.025 1.400
6
125 250 500
5 4 3
9 8 10
5 4 7
16 16 20
5.33 5.33 6.66
1.025 1.025 1.439
12 12 17
4.00 4.00 5.66
1.130 1.130 1.330
24
125 250 500
2 4 6
3 11 5
5 5 5
10 20 16
3.33 6.66 5.33
0.567 1.439 1.025
8 16 10
2.66 5.33 3.33
0.929 1.025 0.567
Control
N
Table 3 Incidence of sperm shape abnormalities seen in pyrazinamide-treated mice Dose ( m g / k g bw)
Sperm analysed
Types of sperm head abnormalities Amorphous
Banana head
Hookless
Double head
Double tail
N
%
0 125 250 500
8000 6000 8055 6000
62 43 73 120
0 0 2 7
10 21 8 17
0 0 6 4
0 0 0 2
72 64 109 150
0.90 1.06 1.36 2.50
Abnormal sperm
+ SE
0.105 0.132 0.129 0.201
4
B. Anitha et al./Mutation Research 321 (1994) 1-5
0.736x + 0.224, P < 0.05). The increase in sperm shape abnormalities was about threefold on administration of 500 m g / k g body weight of pyrazinamide.
4. Discussion Multiple assays were carried out in mice to examine whether pyrazinamide is genotoxic. Animals treated with the drug exhibited a dose-dependent decrease in the number of dividing cells (Table 1). The observations suggest a cytotoxic effect of pyrazinamide. Exposure of mice to different doses of pyrazinamide for varying periods resulted in the induction of chromosomal aberrations (Table 2). Clastogenicity of pyrazinamide was reported by Bhanumathi (1983) in mice and in human lymphocytes in culture. The maximum incidence of aberrations observed in mice was at 3 h after treatment with pyrazinamide. The response at 3 h corresponds to the half-life of the drug, estimated to be 2-3 h (Bareggi et al., 1987). 1 g of drug produced a peak serum level of 60 i z g / m l in patients 2 h after administration (Steele and Desperez, 1988). Various aberrations recorded were mostly of chromatid type, similar to the observations of Caratzali et al. (1972) and Roman and Georgian (1977) in cultured lymphocytes. According to Roman and Georgian (1977) the drug acts on the S-phase of the cell cycle. They emphasised the need to distinguish between the direct clastogenic action of the drug and the secondary effect brought out by the drug on the other cellular components. The drug is reported to interfere with the biosynthesis of tetrahydrofolic acid, a purine precursor, and this may be responsible for the mutagenic effect. The phenotype of the sperm is determined by the autosomal and sex-linked genes. The sperm shape, the fraction of abnormal sperm and dimensions of individual sperm are characteristic of the genotype. The relevance of sperm morphology for genotoxicity assessment was emphasised by Wyrobek and Bruce (1978). Mice exposed to different doses of pyrazinamide exhibited abnormal sperm morphology and the frequencies were
significantly different from those recorded in the control set of animals. The results thus suggest pyrazinamide to be a weak genotoxic drug. One cannot extrapolate the data obtained from mice subjected to the intraperitoneal administration to humans, as patients are treated with the drug orally and that too with a combination of chemotherapeutic agents. Recent reports on genotoxicity of antituberculosis drugs by Gopal Rao et al. (1990, 1991) are on patients subjected to treatment with a combination of drugs. The only reports available on the individual action of pyrazinamide on mammalian cells are those of Caratzali et al. (1972), Roman and Georgian (1977) and Bhanumathi (1983). These studies were carried out on cultured lymphocytes and they found pyrazinamide to be clastogenic. Bhanumathi (1983) has also examined the effects of very high doses of pyrazinamide in mice and reported it to be clastogenic. The findings in the present study are in agreement with the results of these investigators on cells in culture. In this context, it is of interest to record that Ennever et al. (1987) emphasised the importance of short-term animal assays for the prediction of carcinogenicity and non-carcinogenicity to man.
5. Acknowledgements The first author would like to thank the University Grants Commission for providing the funds to carry out this experiment and Dr. A. Ramesh (Department of Genetics) for helping in the statistical evaluation of the data.
6. References Bareggi, S.R., R. Ceruth, R. Rirola, R. Riva and M. Cisternino (1987) Clinical pharmacokinetics and metabolism of pyrazinamide in healthy volunteers, Arzneim. Forsch./ Drug Res., 37, 849-53. Bhanumathi, J. (1983)Cytogeneticand Flow CytometricStudies on the Effects of Some Antituberculosis Drugs, Ph.D. Thesis, Universityof Madras. Buckton, K.E., and H.J. Evans (1982) Human peripheral blood lymphocyteculture, in: T.C. Hsu (Ed.), An In Vitro
B. Anitha et al. / Mutation Research 321 (1994) 1-5 Introductory Assay for the Cytogenetic Effects of Environmental Mutagens, Oxford and IBH Publishing Co., New Delhi, pp. 183-202. Caratzali, A., I. Constantin and I.C. Roman (1972) Identification of human chromosomes by chromerization in vitro, C.R. Heb. Sceances Acad. Sci. Serd. Sci. Nat. (Paris), 274187, 1198-1199. Ennever, F.K., N.J. Thomas and H.S. Rosenkranz (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans, Mutagenesis, 2, 73-78. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testis, Cytogenetics, 3, 289-294. Gopal Rao, V.V.N., E.V.V. Gupta and I.M. Thomas (1990) Sister chromatid exchanges in the lymphocytes of tuberculosis patients receiving short-term chemotherapy, Tubercle, 71, 173-176. Gopal Rao, V.V.N., E.V.V. Gupta and I.M. Thomas (1991) Chromosomal aberrations in tuberculosis patients before and after treatment with short-term chemotherapy, Mutation Res., 259, 13-19.
5
Pretet, S., J. Marsac and D. Choudat (1983) Value of pyrazinamide in tuberculosis, Presse M~d., 12, 871. Roman, I.C., and L. Georgian (1977) Cytogenetic effects of some antituberculosis drug in vitro, Mutation Res., 48, 215-224. Sankaranarayanan, K. (1982) Genetic Effects of Ionization Radiation in Multicellular Eukaryotes and the Assessment of Genetic Radiation Hazards in Man, Elsevier, Amsterdam, 385 pp. Steele, M.A., and R.M. Desprez (1988) The role of pyrazinamide in tuberculosis chemotherapy, Chest, 94, 845. Suk, W.A. (1977) Oncogenic and mutagenic potential of selected antituberculosis drugs, Diss. Int. Abstr. Biol., 35593560. Tjio, J.H., and J. Whang (1962) Direct chromosome preparation of bone marrow cells without prior in vitro culture or in vivo colchicine administration, Stain Technol., 37, 17-20. Wyrobek, A.J., and W.R. Bruce (1978) Sperm abnormality assay, Chem. Mutagen., 5, 257. Zar, J.H. (1974) Biostatistic Analysis, Prentice-Hall, Engelwood Cliffs, NJ, 620 pp.