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Elevation of micronuclei frequency in mouse bone marrow treated with various doses of teniposide (VM-26)
Toxicology Letters 104 (1999) 203 – 210
Elevation of micronuclei frequency in mouse bone marrow treated with various doses of teniposide (VM-26) Ganesh C. Jagetia *, R. Aruna Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India Received 25 August 1998; received in revised form 15 October 1998; accepted 19 October 1998
Abstract The effect of various doses (0–10 mg/kg body wt.) of teniposide (VM-26) was studied on the induction of micronuclei at 12, 24 and 36 h post-treatment. The frequency of micronuclei (MPCE and MNCE) increased in a dose-dependent manner up to a dose of 0.3125 mg/kg VM-26, where a peak frequency of micronuclei was observed. A further increase in the drug dose resulted in the reduction in micronuclei frequency in comparison with 0.3125 mg/kg drug dose reaching a nadir at 10 mg/kg. However, it was significantly higher than DDW (double distilled water) treated controls. The pattern of micronuclei induction was similar for all the post-treatment time periods. The frequency of micronuclei also increased with scoring time and the highest frequency of micronuclei was observed at 24 h post-treatment, which declined thereafter without restoration to DDW treated control level. Conversely, the PCE/NCE ratio registered a dose-dependent decline after treatment of mice with various doses of VM-26. A peak decline was observed at a dose of 0.3125 mg/kg, thereafter the decline became consistently less resulting in an elevation in the PCE/NCE ratio in comparison with 0.3125 mg/kg VM-26. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mice; Teniposide; Micronuclei; PCE/NCE ratio; Bone marrow
1. Introduction Podophyllotoxins are used in the treatment of various malignancies. Teniposide 4%-demethylepipodophylotoxin-4-(4,6-O-thenylidine-bD-glucopyranoside) or VM-26 is a semisynthetic * Corresponding author. Tel.: + 91-8252-71200, extn.2122; fax: +91-8252-70062.
derivative of podophyllotoxin resin. Teniposide is an effective anticancer drug used for the treatment of various neoplastic disorders (O’Dwyer et al., 1984). Teniposide has been found to be active against murine leukemias, Lewis lung carcinoma. (Sklansky et al., 1974; Pedersen et al., 1984; Bork et al., 1986) non-Hodgkin’s and Hodgkin’s Lymphoma, hematosarcomas (Mathe et al., 1974) ovarian tumors (Van der Gaast and
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 9 8 ) 0 0 3 6 8 - 3
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Splinter, 1992) and other solid tumors (Dombernowsky et al., 1972; Radice et al., 1979; O’Dwyer et al., 1984). VM-26 is a potent topoisomerase II inhibitor (Long and Minocha, 1983; Ross et al., 1984) and it exerts its cytotoxic effects by acting on a cellular enzyme, topoisomerase II. DNA topoisomerases play a crucial role in basic cellular growth processes, such as DNA replication, recombination and transcription (Gellert, 1981; Wang, 1985). Topoisomerase II is involved in chromosome segregation (Yanagida and Wang, 1987). It has been found to be a major component of the nuclear matrix (Berrios et al., 1985) and mitotic chromosome scaffold (Earnshaw et al., 1985). Topoisomerase has also been found to be localized at the bases radial loop domain of mitotic chromosomes (Earnshaw and Heck, 1985; Gasser et al., 1986). It is also responsible for overwinding, underwinding and catenation, which arise during replication and other DNA processes (D’Arpa and Liu, 1989). Topoisomerase II are ATP dependent enzymes that catalyze the breakage and religation steps in DNA synthesis by forming a transient ‘cleavable complex’ (Gellert, 1981; Wang, 1987; Zhang et al., 1990), before the break is resealed (Pommier et al., 1991). Teniposide interferes with the DNA reunion step, thus stabilizing the otherwise transient covalently linked ‘cleavable complexes’ of topoisomerase II and 5% termini of the DNA molecule (Tewey et al., 1984a,b; Chen et al., 1984; Osheroff, 1986). This interaction mediates the cytotoxicity of the drug. The stabilization of topoisomerase complexes, prevents the rapid turnover of the protein-cross-linked DNA strand breaks resulting ultimately in cell death (Kalwinsky et al., 1983; Chatterjee et al., 1990). Teniposide is a potent clastogenic agent (DeMarini et al., 1987) and it has been reported to induce sister chromatid exchanges in CHO cells. (Singh and Gupta, 1983a). It has also been reported to increase 6-thioguanine and 6-MeMFRresistant mutants in a dose-dependent manner(Singh and Gupta, 1983b). Teniposide has been reported to exert strong mutagenic effects in mammalian cells but not in bacteria (Gupta et al., 1987; DeMarini et al., 1987). VM-26 produced
DNA double strand breaks in mammalian cells. (Kaufmann, 1989; Binaschi et al., 1997). DNA double strands are mainly responsible for chromosome breaks (Obe et al., 1982; Natarajan et al., 1986). Therefore, it was desired to evaluate the effect of various doses of teniposide on the induction of micronuclei in the bone marrow of mouse at various post-treatment time periods.
2. Material and methods Six- to eight-week-old male Swiss albino mice weighing 329 2 g were selected from an inbred colony maintained under controlled conditions of temperature (2392°C), humidity (509 5%) and light (10 and 14 h of light and dark, respectively). The animals had free access to sterile food (prepared in the laboratory following standard formulation) and water. Usually five animals were housed in a sterile polypropylene cage containing sterile paddy husk (procured locally) as bedding material.
2.1. Preparation of drug Teniposide, i.e. VM-26 (Bristol Laboratories, IN) was received in the form of a 5 ml solution containing 10 mg/ml of VM-26. The drug was diluted in sterile double distilled water (DDW) in such a way so as to get a concentration of 10 mg/ml. The drug was further diluted with DDW to get 0.019, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/kg body wt.
2.2. Mode of administration The animals were administered with 0.01 ml/g body wt. of DDW or VM-26 intraperitoneally, irrespective of the drug dose.
2.3. Experimental protocol Five animals were used for each drug dose of VM-26 at each post-treatment time period studied. The animals were divided into two groups as follows:(1) DDW treated group: The animals of this group received 0.01 ml/g body wt. of DDW.
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(2) VM-26 treated group: The animals of this group were injected with 0.019, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/kg body wt. of VM-26. Animals from both groups were killed at 12, 24 and 36 h after DDW or VM-26 treatment. The micronuclei were prepared according to the method of Schmid (1975) with certain modifications described by Jagetia and Jacob (1991). Briefly, the femurs of each animal were dissected out and the bone marrow was separately flushed out into Dulbecco’s modified Eagle’s medium (DMEM). The suspension was centrifuged. A few drops of fetal calf serum (FCS) were added and the pellet was mixed thoroughly. Smears were drawn on to precleaned coded slides using a drop of the resultant suspension in FCS. The slides were air dried and fixed in absolute methanol. The slides were stained with 0.125% acridine orange (BDH, UK, Gurr Cat. No. 34001 9704640E) in Sorensen’s buffer (pH 6.8). The slides were then washed twice in Sorensen’s buffer and mounted in Sorensen’s buffer and observed under a fluorescent microscope (Carl Zeiss Photomicroscope III, Germany) using a 40c Neofluar objective. A minimum of 2000 polychromatic (PCE) and 2000 normochromatic erythrocytes (NCE) were counted for the presence of micronuclei (MPCE and MNCE) for each animal. A total of not less than 10 000 either PCE or NCE were scored for each dose of VM-26. Data regarding the polychromatic and normochromatic erythrocyte ratio (PCE/NCE ratio) were also collected, where a minimum of 4000 erythrocytes per animal were scored. The statistical significance between control and VM-26 groups was calculated using one-way analysis of variance (ANOVA) with application of post-hoc Bonferroni’s test.
3. Results The results are expressed as the frequency of MPCE or MNCE per 10009SEM or PCE/NCE ratio 9SEM (Table 1). Administration of various doses of teniposide to mouse resulted in a significant increase in the frequency of micronucleated polychromatic ery-
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throcytes (MPCE) in mouse bone marrow at 12, 24 and 36 h post-treatment irrespective of the drug dose. The increase in the frequency of MPCE was dose-dependent up to a dose of 0.3125 mg/kg VM-26, where a peak frequency of MPCE was observed. The frequency of MPCE started declining steadily with 0.625 mg/kg in comparison with the preceding dose i.e. 0.3125 mg/kg and this trend continued up to 10 mg/kg VM-26, where MPCE frequency reached a nadir. In spite of reaching nadir at 10 mg/kg, the frequency of MPCE was significantly higher at 10 mg/kg VM26 when compared to DDW treated control. The frequency of MPCE was significantly higher after treatment of mice with 0.039–5 mg/kg VM-26 when compared to 0.019 mg/kg drug dose except 10 mg/kg, where it was significantly lower at all the treatment time periods studied. The frequency of MPCE increased with scoring time and a peak frequency of MPCE was observed at 24 h posttreatment, that declined at 36 h post-treatment, where the MPCE frequency was higher than that of 12 h post-treatment. The trend for the elevation of MNCE after administration of VM-26 was similar to that of MPCE. The frequency of MNCE increased in a dose-related manner up to a dose of 0.3125 mg/kg and this elevation in the MNCE was significantly higher at all the drug dose at 12, 24 and 36 h post-treatment. A further increase in drug dose to 0.625 mg/kg resulted in a consistent decline in the MNCE frequency than that of the preceding i.e. (0.3125 mg/kg) dose and this trend continued up to a dose of 10 mg/kg, where the MNCE frequency was almost similar to DDW treatment. The administration of various doses of VM-26 caused a significant decline in the PCE/NCE ratio at all the post-treatment time periods. This decline in PCE/NCE ratio was dose-dependent up to a dose of 0.3125 mg/kg. With a further increase in drug dose, the decline in PCE/NCE ratio was arrested. The PCE/NCE ratio registered an elevation after administration of 0.625 mg/kg VM-26 in comparison with 0.3125 mg/kg drug dose. This trend was dose-dependent up to a dose of 10 mg/kg, where the highest PCE/NCE ratio was observed among drug-treated groups.
2.709 0.12 5.4090.03d 7.7590.02d 10.7590.03d 27.5990.03d 43.6690.03d 35.1690.04d 13.7990.02d 10.8390.01d 6.6290.03d 4.7890.03d
2.809 0.12 6.55 9 0.02d 9.81 9 0.03d 12.37 90.02d 30.18 90.03d 58.73 90.02d 38.56 90.03d 15.32 90.02d 12.64 90.03d 7.82 9 0.02d 5.57 9 0.02d
2.80 90.12 7.489 0.03d 10.4290.02d 13.6290.05d 34.4990.04d 75.8090.02d 49.4590.02d 26.2790.02d 18.6290.03d 8.509 0.03d 6.769 0.02d 0.6490.11 1.12 9 0.004d 1.42 9 0.004d 1.68 9 0.005d 2.07 9 0.004d 2.63 9 0.004d 2.19 9 0.004d 2.09 9 0.003d 1.70 9 0.004d 1.10 9 0.004d 0.59 9 0.004
12 h
36 h
12 h
24 h
MNCE
MPCE
Frequency of micronuclei per 1000 9 SEM
0.759 0.13 1.94 9 0.004d 2.05 9 0.004d 2.26 9 0.004d 2.62 9 0.004d 3.55 9 0.004d 3.10 9 0.004d 2.80 9 0.004d 2.40 9 0.007d 1.24 9 0.005c 0.88 90.004
24 h 0.6490.11 1.15 9 0.004d 1.45 9 0.004d 1.70 9 0.004d 2.08 9 0.004d 2.65 9 0.004d 2.22 9 0.003d 2.11 9 0.003d 1.72 9 0.004d 1.12 9 0.004d 0.6090.004
36 h
1.06 9 0.004 0.9890.003d 0.9290.002d 0.8590.004d 0.7990.003d 0.7290.005d 0.7590.003d 0.8190.005d 0.8890.002d 0.9590.004d 1.02 9 0.003c
12 h
1.079 0.002 1.0390.003d 0.9490.003d 0.8990.004d 0.8490.005d 0.7890.005d 0.8290.003d 0.8690.003d 0.9390.005d 0.9990.002d 1.0490.003b
24 h
PCE/NCE ratio 9 SEM
1.07 90.002 0.99 90.002d 0.93 90.003d 0.869 0.003d 0.81 90.002d 0.76 90.005d 0.79 90.003d 0.829 0.005d 0.91 90.002d 0.979 0.003d 1.039 0.002c
36 h
a MPCE, micronucleated polychromatic erythrocyte; MNCE, micronucleated normochromatic erythrocyte; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte; 2000 PCE or NCE per animal were scored for the presence of micronuclei; 4000 erythrocytes (PCE and NCE) per animal were scored for the evaluation of PCE/NCE ratio. Non-significant, no symbols; Level of significance was determined using one-way ANOVA. b PB0.05. c PB0.01. d PB0.001.
0 0.019 0.039 0.078 0.156 0.3125 0.625 1.25 2.5 5.0 10
Dose (mg/kg)
Table 1 Effect of various doses of teniposide (VM-26) on the induction of micronuclei in mice bone marrow at different post-treatment time periodsa
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4. Discussion Topoisomerase II inhibiting agents are used in the treatment of cancer. These agents usually cause damage to the DNA. Therefore, long-term effects of these drugs may be of great concern to the patients receiving this type of drugs for the treatment of neoplastic disorders. In the present study an attempt has been made to evaluate the clastogenic effect of different doses of VM-26 in mouse bone marrow by evaluating the frequency of micronuclei. The frequency of micronuclei (MPCE and MNCE) increased in a dose-dependent manner up to a dose of 0.3125 mg/kg VM-26 with a subsequent decline at higher doses when compared to 0.3125 mg/kg VM-26, without reaching normal control level. Topoisomerase II is found to play an important role in the segregation of newly replicated parts of intertwined chromosomes (Holm et al., 1985; Uemura and Yanagida, 1986), condensation and decondensation of chromosomes and supercoiling of intracellular DNA (Yanagida and Wang, 1987; Yanagida and Sternglanz, 1990). DNA topoisomerase II catalyzes the breakage and rejoining of both DNA strands, relaxes the superhelical twist, and catenates or decatenates circular DNA. DNA topoisomerase II performs these topological transformation by transporting one double stranded DNA segment through an enzyme-mediated transient double-stranded break in another. The severed DNA strands are rejoined after completion of the above process (Brown and Cozzarelli, 1979; Liu et al., 1980). Teniposide is a potent DNA topoisomerase II inhibitor and it stabilizes the DNA double strand breaks and does not allow them to rejoin leading to cell death (Pommier et al., 1985; Kaufmann, 1989; Del Bino et al., 1991). These DNA double strand breaks may subsequently lead to chromosome breaks that finally may lead to the production of micronuclei after one cell division. This may be one of the reasons for micronuclei-induction by VM26 in the present investigation. Etoposide (VP-16), an epipodophyllotoxin derivative, has been reported to induce micronuclei in rat spermatids (La¨hdetie et al., 1994). Sim-
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ilarly, another topoisomerase II inhibitor drug, adriamycin has been reported to induce micronuclei in a dose-dependent manner in HeLa cells (Jagetia and Vijayashree, 1996). The frequency of micronuclei increased up to a dose of 0.3125 mg/kg VM-26 and then started declining with increasing doses of VM-26 when compared with 0.3125 mg/kg. However, the frequency of micronuclei was significantly higher than that of DDW treated control at all the doses studied at 12, 24 and 36 h post-treatment. Teniposide has been reported to inhibit the synthesis of DNA in a dose-dependent manner (Del Bino et al., 1991). This may be the plausible reason for the decline in the frequency of micronuclei after doses greater than 0.3125 mg/kg VM-26 in the present study. However, interplay of some other unknown mechanism cannot be ruled out. The PCE/NCE ratio declined in a dose-related manner up to a dose of 0.3125 mg/kg and then showed a constant elevation, compared to 0.3125 mg/kg VM-26. However, it was always significantly lower than the non-drug treated control. A dose-related decline in the cell survival of V79 and human lymphoma cells treated with etoposide, and another topoisomerase II inhibitor, has been reported (Olive et al., 1993; Drewinko and Barlogie, 1976). The activity of topoisomerase II is higher in proliferating cells than quiescent cells (Chow and Ross, 1987, Zwelling et al., 1987, Hsiang and Liu, 1988). The expression of topoisomerase II has been reported to be high during G2 + M phase of the cell cycle (Saijo et al., 1992, Fairman and Brutlag, 1988). Teniposide is a potent topoisomerase II inhibitor (Gellert, 1981; Wang, 1987; Zhang et al., 1990; Pommier et al., 1991) and its presence may have deactivated the enzyme resulting in a decline in the PCE/NCE ratio when compared with the DDW treated control. Further, teniposide has been reported to inhibit the transition of S-phase into G2 and M phase, especially at low concentrations (Del Bino et al., 1991). This action of teniposide may be responsible for the decline in the PCE/NCE ratio. Our study clearly demonstrates that VM-26 brings about genotoxic damage in the normal dividing tissue in vivo. Therefore, the patients
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receiving VM-26 for the treatment of various neoplastic disorders may be at increased risk for the development of secondary malignancies in their later part of life owing to the clastogenic damage inflicted by VM-26 on somatic cells. VP16, another epipodophyllotoxin derivative has been found to increase the risk of secondary leukemia in patients receiving this drug (Kobayashi and Ratain, 1992; Zeimet et al., 1992; Nichols et al., 1993; Bajorin et al., 1993).
Acknowledgements We are thankful to Bristol Laboratories (A Bristol Meyers Squib Co.), IN for the supply of VM-26 throughout the study as a kind gift.
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Elevation of micronuclei frequency in mouse bone marrow treated with various doses of teniposide (VM-26) Ganesh C. Jagetia *, R. Aruna Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India Received 25 August 1998; received in revised form 15 October 1998; accepted 19 October 1998
Abstract The effect of various doses (0–10 mg/kg body wt.) of teniposide (VM-26) was studied on the induction of micronuclei at 12, 24 and 36 h post-treatment. The frequency of micronuclei (MPCE and MNCE) increased in a dose-dependent manner up to a dose of 0.3125 mg/kg VM-26, where a peak frequency of micronuclei was observed. A further increase in the drug dose resulted in the reduction in micronuclei frequency in comparison with 0.3125 mg/kg drug dose reaching a nadir at 10 mg/kg. However, it was significantly higher than DDW (double distilled water) treated controls. The pattern of micronuclei induction was similar for all the post-treatment time periods. The frequency of micronuclei also increased with scoring time and the highest frequency of micronuclei was observed at 24 h post-treatment, which declined thereafter without restoration to DDW treated control level. Conversely, the PCE/NCE ratio registered a dose-dependent decline after treatment of mice with various doses of VM-26. A peak decline was observed at a dose of 0.3125 mg/kg, thereafter the decline became consistently less resulting in an elevation in the PCE/NCE ratio in comparison with 0.3125 mg/kg VM-26. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mice; Teniposide; Micronuclei; PCE/NCE ratio; Bone marrow
1. Introduction Podophyllotoxins are used in the treatment of various malignancies. Teniposide 4%-demethylepipodophylotoxin-4-(4,6-O-thenylidine-bD-glucopyranoside) or VM-26 is a semisynthetic * Corresponding author. Tel.: + 91-8252-71200, extn.2122; fax: +91-8252-70062.
derivative of podophyllotoxin resin. Teniposide is an effective anticancer drug used for the treatment of various neoplastic disorders (O’Dwyer et al., 1984). Teniposide has been found to be active against murine leukemias, Lewis lung carcinoma. (Sklansky et al., 1974; Pedersen et al., 1984; Bork et al., 1986) non-Hodgkin’s and Hodgkin’s Lymphoma, hematosarcomas (Mathe et al., 1974) ovarian tumors (Van der Gaast and
0378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 9 8 ) 0 0 3 6 8 - 3
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Splinter, 1992) and other solid tumors (Dombernowsky et al., 1972; Radice et al., 1979; O’Dwyer et al., 1984). VM-26 is a potent topoisomerase II inhibitor (Long and Minocha, 1983; Ross et al., 1984) and it exerts its cytotoxic effects by acting on a cellular enzyme, topoisomerase II. DNA topoisomerases play a crucial role in basic cellular growth processes, such as DNA replication, recombination and transcription (Gellert, 1981; Wang, 1985). Topoisomerase II is involved in chromosome segregation (Yanagida and Wang, 1987). It has been found to be a major component of the nuclear matrix (Berrios et al., 1985) and mitotic chromosome scaffold (Earnshaw et al., 1985). Topoisomerase has also been found to be localized at the bases radial loop domain of mitotic chromosomes (Earnshaw and Heck, 1985; Gasser et al., 1986). It is also responsible for overwinding, underwinding and catenation, which arise during replication and other DNA processes (D’Arpa and Liu, 1989). Topoisomerase II are ATP dependent enzymes that catalyze the breakage and religation steps in DNA synthesis by forming a transient ‘cleavable complex’ (Gellert, 1981; Wang, 1987; Zhang et al., 1990), before the break is resealed (Pommier et al., 1991). Teniposide interferes with the DNA reunion step, thus stabilizing the otherwise transient covalently linked ‘cleavable complexes’ of topoisomerase II and 5% termini of the DNA molecule (Tewey et al., 1984a,b; Chen et al., 1984; Osheroff, 1986). This interaction mediates the cytotoxicity of the drug. The stabilization of topoisomerase complexes, prevents the rapid turnover of the protein-cross-linked DNA strand breaks resulting ultimately in cell death (Kalwinsky et al., 1983; Chatterjee et al., 1990). Teniposide is a potent clastogenic agent (DeMarini et al., 1987) and it has been reported to induce sister chromatid exchanges in CHO cells. (Singh and Gupta, 1983a). It has also been reported to increase 6-thioguanine and 6-MeMFRresistant mutants in a dose-dependent manner(Singh and Gupta, 1983b). Teniposide has been reported to exert strong mutagenic effects in mammalian cells but not in bacteria (Gupta et al., 1987; DeMarini et al., 1987). VM-26 produced
DNA double strand breaks in mammalian cells. (Kaufmann, 1989; Binaschi et al., 1997). DNA double strands are mainly responsible for chromosome breaks (Obe et al., 1982; Natarajan et al., 1986). Therefore, it was desired to evaluate the effect of various doses of teniposide on the induction of micronuclei in the bone marrow of mouse at various post-treatment time periods.
2. Material and methods Six- to eight-week-old male Swiss albino mice weighing 329 2 g were selected from an inbred colony maintained under controlled conditions of temperature (2392°C), humidity (509 5%) and light (10 and 14 h of light and dark, respectively). The animals had free access to sterile food (prepared in the laboratory following standard formulation) and water. Usually five animals were housed in a sterile polypropylene cage containing sterile paddy husk (procured locally) as bedding material.
2.1. Preparation of drug Teniposide, i.e. VM-26 (Bristol Laboratories, IN) was received in the form of a 5 ml solution containing 10 mg/ml of VM-26. The drug was diluted in sterile double distilled water (DDW) in such a way so as to get a concentration of 10 mg/ml. The drug was further diluted with DDW to get 0.019, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/kg body wt.
2.2. Mode of administration The animals were administered with 0.01 ml/g body wt. of DDW or VM-26 intraperitoneally, irrespective of the drug dose.
2.3. Experimental protocol Five animals were used for each drug dose of VM-26 at each post-treatment time period studied. The animals were divided into two groups as follows:(1) DDW treated group: The animals of this group received 0.01 ml/g body wt. of DDW.
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(2) VM-26 treated group: The animals of this group were injected with 0.019, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/kg body wt. of VM-26. Animals from both groups were killed at 12, 24 and 36 h after DDW or VM-26 treatment. The micronuclei were prepared according to the method of Schmid (1975) with certain modifications described by Jagetia and Jacob (1991). Briefly, the femurs of each animal were dissected out and the bone marrow was separately flushed out into Dulbecco’s modified Eagle’s medium (DMEM). The suspension was centrifuged. A few drops of fetal calf serum (FCS) were added and the pellet was mixed thoroughly. Smears were drawn on to precleaned coded slides using a drop of the resultant suspension in FCS. The slides were air dried and fixed in absolute methanol. The slides were stained with 0.125% acridine orange (BDH, UK, Gurr Cat. No. 34001 9704640E) in Sorensen’s buffer (pH 6.8). The slides were then washed twice in Sorensen’s buffer and mounted in Sorensen’s buffer and observed under a fluorescent microscope (Carl Zeiss Photomicroscope III, Germany) using a 40c Neofluar objective. A minimum of 2000 polychromatic (PCE) and 2000 normochromatic erythrocytes (NCE) were counted for the presence of micronuclei (MPCE and MNCE) for each animal. A total of not less than 10 000 either PCE or NCE were scored for each dose of VM-26. Data regarding the polychromatic and normochromatic erythrocyte ratio (PCE/NCE ratio) were also collected, where a minimum of 4000 erythrocytes per animal were scored. The statistical significance between control and VM-26 groups was calculated using one-way analysis of variance (ANOVA) with application of post-hoc Bonferroni’s test.
3. Results The results are expressed as the frequency of MPCE or MNCE per 10009SEM or PCE/NCE ratio 9SEM (Table 1). Administration of various doses of teniposide to mouse resulted in a significant increase in the frequency of micronucleated polychromatic ery-
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throcytes (MPCE) in mouse bone marrow at 12, 24 and 36 h post-treatment irrespective of the drug dose. The increase in the frequency of MPCE was dose-dependent up to a dose of 0.3125 mg/kg VM-26, where a peak frequency of MPCE was observed. The frequency of MPCE started declining steadily with 0.625 mg/kg in comparison with the preceding dose i.e. 0.3125 mg/kg and this trend continued up to 10 mg/kg VM-26, where MPCE frequency reached a nadir. In spite of reaching nadir at 10 mg/kg, the frequency of MPCE was significantly higher at 10 mg/kg VM26 when compared to DDW treated control. The frequency of MPCE was significantly higher after treatment of mice with 0.039–5 mg/kg VM-26 when compared to 0.019 mg/kg drug dose except 10 mg/kg, where it was significantly lower at all the treatment time periods studied. The frequency of MPCE increased with scoring time and a peak frequency of MPCE was observed at 24 h posttreatment, that declined at 36 h post-treatment, where the MPCE frequency was higher than that of 12 h post-treatment. The trend for the elevation of MNCE after administration of VM-26 was similar to that of MPCE. The frequency of MNCE increased in a dose-related manner up to a dose of 0.3125 mg/kg and this elevation in the MNCE was significantly higher at all the drug dose at 12, 24 and 36 h post-treatment. A further increase in drug dose to 0.625 mg/kg resulted in a consistent decline in the MNCE frequency than that of the preceding i.e. (0.3125 mg/kg) dose and this trend continued up to a dose of 10 mg/kg, where the MNCE frequency was almost similar to DDW treatment. The administration of various doses of VM-26 caused a significant decline in the PCE/NCE ratio at all the post-treatment time periods. This decline in PCE/NCE ratio was dose-dependent up to a dose of 0.3125 mg/kg. With a further increase in drug dose, the decline in PCE/NCE ratio was arrested. The PCE/NCE ratio registered an elevation after administration of 0.625 mg/kg VM-26 in comparison with 0.3125 mg/kg drug dose. This trend was dose-dependent up to a dose of 10 mg/kg, where the highest PCE/NCE ratio was observed among drug-treated groups.
2.709 0.12 5.4090.03d 7.7590.02d 10.7590.03d 27.5990.03d 43.6690.03d 35.1690.04d 13.7990.02d 10.8390.01d 6.6290.03d 4.7890.03d
2.809 0.12 6.55 9 0.02d 9.81 9 0.03d 12.37 90.02d 30.18 90.03d 58.73 90.02d 38.56 90.03d 15.32 90.02d 12.64 90.03d 7.82 9 0.02d 5.57 9 0.02d
2.80 90.12 7.489 0.03d 10.4290.02d 13.6290.05d 34.4990.04d 75.8090.02d 49.4590.02d 26.2790.02d 18.6290.03d 8.509 0.03d 6.769 0.02d 0.6490.11 1.12 9 0.004d 1.42 9 0.004d 1.68 9 0.005d 2.07 9 0.004d 2.63 9 0.004d 2.19 9 0.004d 2.09 9 0.003d 1.70 9 0.004d 1.10 9 0.004d 0.59 9 0.004
12 h
36 h
12 h
24 h
MNCE
MPCE
Frequency of micronuclei per 1000 9 SEM
0.759 0.13 1.94 9 0.004d 2.05 9 0.004d 2.26 9 0.004d 2.62 9 0.004d 3.55 9 0.004d 3.10 9 0.004d 2.80 9 0.004d 2.40 9 0.007d 1.24 9 0.005c 0.88 90.004
24 h 0.6490.11 1.15 9 0.004d 1.45 9 0.004d 1.70 9 0.004d 2.08 9 0.004d 2.65 9 0.004d 2.22 9 0.003d 2.11 9 0.003d 1.72 9 0.004d 1.12 9 0.004d 0.6090.004
36 h
1.06 9 0.004 0.9890.003d 0.9290.002d 0.8590.004d 0.7990.003d 0.7290.005d 0.7590.003d 0.8190.005d 0.8890.002d 0.9590.004d 1.02 9 0.003c
12 h
1.079 0.002 1.0390.003d 0.9490.003d 0.8990.004d 0.8490.005d 0.7890.005d 0.8290.003d 0.8690.003d 0.9390.005d 0.9990.002d 1.0490.003b
24 h
PCE/NCE ratio 9 SEM
1.07 90.002 0.99 90.002d 0.93 90.003d 0.869 0.003d 0.81 90.002d 0.76 90.005d 0.79 90.003d 0.829 0.005d 0.91 90.002d 0.979 0.003d 1.039 0.002c
36 h
a MPCE, micronucleated polychromatic erythrocyte; MNCE, micronucleated normochromatic erythrocyte; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte; 2000 PCE or NCE per animal were scored for the presence of micronuclei; 4000 erythrocytes (PCE and NCE) per animal were scored for the evaluation of PCE/NCE ratio. Non-significant, no symbols; Level of significance was determined using one-way ANOVA. b PB0.05. c PB0.01. d PB0.001.
0 0.019 0.039 0.078 0.156 0.3125 0.625 1.25 2.5 5.0 10
Dose (mg/kg)
Table 1 Effect of various doses of teniposide (VM-26) on the induction of micronuclei in mice bone marrow at different post-treatment time periodsa
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4. Discussion Topoisomerase II inhibiting agents are used in the treatment of cancer. These agents usually cause damage to the DNA. Therefore, long-term effects of these drugs may be of great concern to the patients receiving this type of drugs for the treatment of neoplastic disorders. In the present study an attempt has been made to evaluate the clastogenic effect of different doses of VM-26 in mouse bone marrow by evaluating the frequency of micronuclei. The frequency of micronuclei (MPCE and MNCE) increased in a dose-dependent manner up to a dose of 0.3125 mg/kg VM-26 with a subsequent decline at higher doses when compared to 0.3125 mg/kg VM-26, without reaching normal control level. Topoisomerase II is found to play an important role in the segregation of newly replicated parts of intertwined chromosomes (Holm et al., 1985; Uemura and Yanagida, 1986), condensation and decondensation of chromosomes and supercoiling of intracellular DNA (Yanagida and Wang, 1987; Yanagida and Sternglanz, 1990). DNA topoisomerase II catalyzes the breakage and rejoining of both DNA strands, relaxes the superhelical twist, and catenates or decatenates circular DNA. DNA topoisomerase II performs these topological transformation by transporting one double stranded DNA segment through an enzyme-mediated transient double-stranded break in another. The severed DNA strands are rejoined after completion of the above process (Brown and Cozzarelli, 1979; Liu et al., 1980). Teniposide is a potent DNA topoisomerase II inhibitor and it stabilizes the DNA double strand breaks and does not allow them to rejoin leading to cell death (Pommier et al., 1985; Kaufmann, 1989; Del Bino et al., 1991). These DNA double strand breaks may subsequently lead to chromosome breaks that finally may lead to the production of micronuclei after one cell division. This may be one of the reasons for micronuclei-induction by VM26 in the present investigation. Etoposide (VP-16), an epipodophyllotoxin derivative, has been reported to induce micronuclei in rat spermatids (La¨hdetie et al., 1994). Sim-
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ilarly, another topoisomerase II inhibitor drug, adriamycin has been reported to induce micronuclei in a dose-dependent manner in HeLa cells (Jagetia and Vijayashree, 1996). The frequency of micronuclei increased up to a dose of 0.3125 mg/kg VM-26 and then started declining with increasing doses of VM-26 when compared with 0.3125 mg/kg. However, the frequency of micronuclei was significantly higher than that of DDW treated control at all the doses studied at 12, 24 and 36 h post-treatment. Teniposide has been reported to inhibit the synthesis of DNA in a dose-dependent manner (Del Bino et al., 1991). This may be the plausible reason for the decline in the frequency of micronuclei after doses greater than 0.3125 mg/kg VM-26 in the present study. However, interplay of some other unknown mechanism cannot be ruled out. The PCE/NCE ratio declined in a dose-related manner up to a dose of 0.3125 mg/kg and then showed a constant elevation, compared to 0.3125 mg/kg VM-26. However, it was always significantly lower than the non-drug treated control. A dose-related decline in the cell survival of V79 and human lymphoma cells treated with etoposide, and another topoisomerase II inhibitor, has been reported (Olive et al., 1993; Drewinko and Barlogie, 1976). The activity of topoisomerase II is higher in proliferating cells than quiescent cells (Chow and Ross, 1987, Zwelling et al., 1987, Hsiang and Liu, 1988). The expression of topoisomerase II has been reported to be high during G2 + M phase of the cell cycle (Saijo et al., 1992, Fairman and Brutlag, 1988). Teniposide is a potent topoisomerase II inhibitor (Gellert, 1981; Wang, 1987; Zhang et al., 1990; Pommier et al., 1991) and its presence may have deactivated the enzyme resulting in a decline in the PCE/NCE ratio when compared with the DDW treated control. Further, teniposide has been reported to inhibit the transition of S-phase into G2 and M phase, especially at low concentrations (Del Bino et al., 1991). This action of teniposide may be responsible for the decline in the PCE/NCE ratio. Our study clearly demonstrates that VM-26 brings about genotoxic damage in the normal dividing tissue in vivo. Therefore, the patients
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receiving VM-26 for the treatment of various neoplastic disorders may be at increased risk for the development of secondary malignancies in their later part of life owing to the clastogenic damage inflicted by VM-26 on somatic cells. VP16, another epipodophyllotoxin derivative has been found to increase the risk of secondary leukemia in patients receiving this drug (Kobayashi and Ratain, 1992; Zeimet et al., 1992; Nichols et al., 1993; Bajorin et al., 1993).
Acknowledgements We are thankful to Bristol Laboratories (A Bristol Meyers Squib Co.), IN for the supply of VM-26 throughout the study as a kind gift.
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