Pharmaceutical Chemistry Journal, Vol. 47, No. 5, August, 2013 (Russian Original Vol. 47, No. 5, May, 2013)
MUTAGENIC AND ANTIMITOTIC ACTIVITY OF THE IMMUNOMODULATORY DRUG TUBOSAN B. S. Kibrik,1 I. M. Prokhorova,2 and D. S. Pesnya3 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 47, No. 5, pp. 29 – 31, May, 2013. Original article submitted April 28, 2012.
The mutagenic and antimitotic activities of the standard antituberculosis drug isoniazid and the immunomodulatory drug tubosan were compared using the Allium test, which took into account the frequency of chromosome aberrations, chromosome lagging, and micronuclei and allowed the mitotic and phase indices to be determined. It was established that isoniazid at concentrations 10, 60, and 120 mg/L inhibited fully the mitotic activity in tissues (for which the mitotic index was zero), implying that isoniazid exhibited very strong mitotoxic activity. The micronucleus test showed that the mutagenic activity of isoniazid increased with increasing concentration. The micronuclei induced by isoniazid were formed as a result of nuclear budding into the interphase. Isoniazid at a concentration of 300 mg/L led to the death of the test object. Tubosan at concentrations 60, 120, 800, and 1200 mg/L showed neither mutagenic nor antimitotic effects. The phase indices were also the same as those in the control. Thus, tubosan did not exhibit genotoxicity and could be classified as a genetically safe drug in the studied concentration range. Keywords: tubosan, isoniazid, mitotic index, chromosome aberration, micronuclei, Allium test.
Tubosan in the concentration range 60 – 80 mg/mL exhibited an antituberculosis effect on multiple and broadly drug-resistant mycobacterium tuberculosis (MBT) for which 71% of the cases exhibited bacteriostatic activity and 29%, bactericidal activity [1, 2]. The genetic activity of tubosan, which is a chemical derivative of isoniazid, has not been studied. The most dangerous of the negative consequences is a genotoxic effect [3, 4], which can persist in a population by passing from generation to generation [5]. Mutagenic activity of the standard antituberculosis drug isoniazid is well studied. Thus, mutagenic and carcinogenic activity of isoniazid was found in in vivo and in vitro experiments [6, 7]. Experiments in mice established that injection of isoniazid caused lung cancer [7]. An elevated frequency of chromosome mutations in patients to whom isoniazid was administered was observed in clinical trials [6]. However, the action of isoniazid on mitotic processes was not studied.
The proposed study was aimed at finding genotoxic indicators such as a mutagenic effect and antimitotic activity of tubosan and isoniazid. The goal of the present investigation was to study the genotoxic activity of isoniazid and tubosan. The scope of our study included the mutagenic and antimitotic activity of isoniazid at concentrations 10, 60, 120, and 300 mg/L and the investigation of the mutagenic and antimitotic activity of tubosan at concentrations 60, 120, 800, and 1200 mg/L. EXPERIMENTAL PART We used the standard antituberculosis drugs Isoniazid (10%, 5 mL, Semashko Moskhimfarmpreparaty) and tubosan (methyldioxotetrahydropyrimidine sulfonisonicotinoyl hydrazide), State Drug Registry No. LSR-006593/08-140808, which belongs to ATC class L03AX, other immunostimulants [8]. A starting stock solution of isoniazid was diluted sequentially with distilled H2O to produce concentrations 10, 60, 120, and 300 mg/L. Tubosan powder was dissolved in distilled H2O and diluted sequentially to produce concentrations 60, 120, 800, and 1200 mg/L.
1 Yaroslavl State Medical Academy, Yaroslavl, 150000, Russia. 2 Yaroslavl State University, Yaroslavl, 150000, Russia. 3 Institute of Internal Waters Biology, Russian Academy of Sciences, Borok, Yaroslavl Region, Russia.
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TABLE 1. Effect of Isoniazid and Tubosan on A. cepa Meristem Concentration, mg/L
CA + lag, %
MN, %
MEE, balls
MA level
Control 0.8 ± 0.10 Isoniazid solution 10 no mitosis
0.08 ± 0.011*
4
weak
60
no mitosis
0.09 ± 0.027*
4.5
weak
120
no mitosis
7 0.14 ± 0.029* Death of test object
300 Tubosan solution 60 0.8 ± 0.22
0.02 ± 0.008
moderate
0.03 ± 0.015
1
None
120
0.9 ± 0.18
0.03 ± 0.018
1
None
800
1.1 ± 0.85
0.04 ± 0.011
2
None
1200
1.6 ± 0.64
0.05 ± 0.023
2
None
*
differences statistically significant for p < 0.05.
The Allium test was used to study the genetic activity of isoniazid and tubosan. The test allowed different types of chromosome mutations induced by both direct mutagens and promutagens that acquired genetic activity only after metabolic activation in the plant organism to be recorded [3]. The Allium test was recommended by WHO experts as a standard in preclinical drug trials [9, 10] because the results obtained in this test agreed with those obtained in tests on human and animal cell cultures [9 – 11]. The method is currently viewed as an alternative to animal tests [12, 13]. Mutagenic activity of the drugs was determined using a modification of ana-telophase analysis of chromosome aberrations (CA) and lagging (lag) of chromosomes (CA + lag, %) and a micronucleus test to study the frequency of micronuclei (MN, %). The simultaneous use of two tests on a single drug allowed the whole collection of dividing and non-dividing cells to be analyzed. This increased the resolution of the method and gave more reliable results [3]. The degree of the mutagenic effect was assessed from the mutagenic effect expression (MEE) [3, 4]. The MEE was determined as the multiple of exceeding the percent induced
mutations over the control value (spontaneous level) and was expressed in balls. MEE balls ranged over mutagenic effect levels and were classified as strong, medium, weak, or absent [3]. Antimitotic activity of isoniazid and tubosan was also assessed in the Allium test. Antimitotic activity of the drugs was assessed from the change of mitotic index (MI) and phase indices (PI, prophase; MeI, metaphase; AI, anaphase; TI, telophase). The determination of the MI enabled the tissue proliferative activity to be estimated. The analysis of the phase indices enabled the types of mitosis disruptions to be found. Antimitotic activity was related to genotoxic effects because disruption of cell division could affect the number of mutations [4]. The total number of dividing cells and cells at various mitosis stages were calculated separately. Results were processed statistically using the t-test and ANOVA at significance level p = 0.05 (*) in Statistica 8.0 software. The test set-up followed the standard method [3, 4, 11]. A total of 90 micro preparations was prepared (10 for each test version). Mitotic and phase indices were determined for each preparation by analyzing 700 cells. The frequency of CA and lag was determined using 3000 interphases for the MN test and all anaphases and telophases. RESULTS AND DISCUSSION Mutagenic and antimitotic activity of isoniazid at concentrations 10, 60, 120, and 300 mg/L The mutagenic and antimitotic effects of isoniazid on root meristems of A. cepa was studied using solutions of isoniazid in distilled H2O at concentrations 10, 60, 120, and 300 mg/L. Tables 1 and 2 present the results. Isoniazid at concentration 300 mg/L caused the death of the test objects. Because the mitotic activity of the tissue affected by isoniazid was zero and dividing cells were completely absent, it was impossible to use ana-telophase analysis to estimate the frequency of CA and lag. Isoniazid at concentrations 10, 60, and 120 mg/L induced an increased frequency of MN in interphase cells that was statistically significantly greater than the control level. In this instance, the MN were
TABLE 2. Mitotic and Phase Indices for the Action of Isoniazid and Tubosan Concentration, mg/L
Control Isoniazid solution 10 60 120 300 Tubosan solution 60
MI, %
8.8 ± 0.12
PI, %
MeI, %
55.7 ± 3.34 22.5 ± 1.31 No mitosis. Zero tissue mitotic activity
AI, %
TI, %
10.4 ± 1.12
15.3 ± 1.33
18.5 ± 1.10
Death of test object 9.2 ± 0.55
55.6 ± 0.33
25.9 ± 0.96
12.4 ± 1.35
120
8.9 ± 0.31
54.8 ± 1.06
22.5 ± 1.80
14.3 ± 0.37
11.4 ± 1.23
800
8.4 ± 0.78
53.3 ± 0.99
21.4 ± 0.32
12.9 ± 0.61
13.7 ± 2.40
1200
8.1 ± 0.43
52.8 ± 2.40
24.1 ± 0.53
15.8 ± 1.77
12.1 ± 0.59
Mutagenic and Antimitotic Activity
formed as a result of nuclear budding. This phenomenon was related to blockage of mitotic processes, the impossibility of mitosis, and disruption of protein synthesis. This led to destruction of interphase chromatin and apoptosis [14]. Therefore, isoniazid at concentrations 10, 60, and 120 mg/L exhibited mutagenic activity in this test system (caused death of the test object at concentration 300 mg/L). The mutagenic effect was classified as medium level. TABLE 2 shows that isoniazid caused a statistically significant decrease of the mitotic index to zero (0%) compared with the control (8.8 ± 1.2%). Mitosis was not observed. Therefore, isoniazid at concentrations 10, 60, and 120 mg/L had very strong mitotoxic activity. Mutagenic and antimitotic activity of tubosan at concentrations 60, 120, 800, and 1200 mg/L We used tubosan solutions of concentrations 60, 120, 800, and 1200 mg/L for studies of its genotoxic effects. The minimum concentration (60 mg/L) was chosen because the bactericidal activity of tubosan against M. tuberculosis began at this value [2, 3]. Table 1 presents the results of the study. It can be seen that the number of CA and lag did not differ statistically significantly from the control level for all studied tubosan concentrations. The frequency of MN for these conditions also did not differ statistically significantly from the control level. Therefore, these tubosan doses did not have a genotoxic effect. Table 2 presents data for the mitotic and phase indices for the action of tubosan at various concentrations. It can be seen that tubosan at all studied concentrations did not cause a statistically significant decrease of the mitotic index compared with the control. An analysis of the phase indices showed that the number of cells entering prophase (PI, %) of mitosis did not change statistically significantly for all studied concentrations. Therefore, it could be assumed that tubosan at all studied concentrations did not suppress cell processes in preparation for division. The ratio of phase indices during the further course of mitosis corresponded to that of the control. Therefore, the action of tubosan at concentrations 60, 120, 800, and 1200 mg/L did not affect the progression of cells to mitosis in the Allium test.
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Results obtained from the Allium test for various compounds, including the drugs, corresponded qualitatively and quantitatively with results of tests in human cell culture [7]. This was related to the fact that the structure and function (storage, formation, and transfer of genetic information) of the genetic apparatus was identical for all eukaryotes. Therefore, results obtained in this investigation suggested that tubosan at the studied doses was non-hazardous for human cells. REFERENCES 1. B. S. Kibrik and O. G. Chelnokova, Immunochemotherapy of Tuberculosis. Bifunctional Drug Tubosan [in Russian], Moscow (2011), p. 154.; 2. B. S. Kibrik, O. G. Chelnokova, and R. V. Maistat, Antituberculosis and Immunomodulatory Activity of Tubosan in Experimental and Clinical Practice [in Russian], in: Materials of the TsNIIT RAMS Session, Moscow (2011), pp. 93 – 95.; 3. D. S. Pesnya, A. V. Romanovskii, I. M. Prokhorova, T. K. Artemova, et al., Biomed. Radioelektron., No. 4, 34 – 45 (2011).; 4. I. M. Prokhorova, A. N. Fomicheva, M. I. Kovaleva, and O. V. Babanazarova, Biol. Vnutr. Vod, No. 2, 17 – 23 (2008).; 5. V. A. Tarasov, Mutagens and Carcinogens in the Environment. New Approaches to Risk Evaluation for Health [in Russian], St. Petersburg (1998), pp. 15 – 30.; 6. V. V. N. Gopal Rao, E. V. Venkatarama Gupta, and I. M. Thomas, Mutat. Res., 259, 13 – 19 (1991).; 7. H. Takasawa, H. Suzuki, I. Ogawa, and Y. Shimada, Mutat. Res., 698, 30 – 37 (2010).; 8. N. M. Goloshchapov, E. N. Goloshchapova, et al., RU Pat. No. 2,141,322, Nov. 20, 1999.; 9. S. Cotelle, J. F. Masfaraud, and J. F. Ferard, Mutat. Res., 426, No. 2, 167 – 171 (1990).; 10. A. Majewska, E. Sliwinska, M. Wierzbicka, and M. Kuras, Caryologia, 61(1), 26 – 44 (2008).; 11. A. Barberrio, J. C. Voltolini, and M. L. S. Mello, Ecotoxicology, 20(4), 927 – 935 (2011).; 12. M. Ghosh, A. Chakraborty, M. Bandyopadhyay, and A. Mukherjee, J. Hazard. Mater., 197, 327 – 336 (2011).; 13. A. H. Siddiqui, S. Tabrez, and M. Ahmad, Environ. Monit. Assess., 179, 241 – 253 (2011).; 14. H. K. Lindberg, X. Wang, H. Jarventaus, et al., Mutat. Res., 617, 33 – 45 (2007).