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Vincristine increases the genomic instability in irradiated cultured human peripheral blood lymphocytes
Toxicology Letters 126 (2002) 179– 186 www.elsevier.com/locate/toxlet
Vincristine increases the genomic instability in irradiated cultured human peripheral blood lymphocytes Ganesh Chandra Jagetia *, Manjeshwar Shrinath Baliga Department of Radiobiology, Kasturba Medical College, Manipal 576 119, Karnataka, India Received 28 May 2001; received in revised form 19 September 2001; accepted 24 September 2001
Abstract Increase in the genomic instability by 10 ng of vincristine (VCR) pretreatment was studied in cultured human peripheral blood lymphocytes exposed to 0, 0.5, 1, 2, and 3 Gy gamma radiation by the micronucleus assay. The frequency of micronucleated binucleate lymphocytes (MNBNC) increased in a dose dependent manner after exposure to different doses of gamma radiation and the dose response was linear. The pre-treatment of lymphocytes with 10 ng/ml VCR caused a further elevation in the frequency of MNBNC and this was significantly greater than that of the concurrent PBS pre-treated irradiated lymphocytes. The dose response for VCR treated group was linear up to 1 Gy irradiation. A further increase in the radiation dose resulted in a decline in the induction of MNBNC in VCR pretreated irradiated group, although it was significantly higher than the PBS treated irradiated group. VCR pretreatment not only increased the frequency of MNBNC with one micronuclei but also the frequency of MNBNC with 2, 3 and 4 MN significantly, indicating the increase in the multiple sites of damage to DNA by VCR pre-treatment. This increase in multiple MNBNC was also dose dependent, however, the dose response was linear quadratic. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vincristine; Micronuclei; Human lymphocytes; Radiation
1. Introduction Vincristine sulphate (VCR), a dimeric alkaloid isolated from the periwinkle plant Catheranthus roseus, is used for the treatment of several forms of malignancies (Avila, 1997). Vincristine has been reported to possess anti-tumour activity against experimental animal tumours and exhibits
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cytotoxic effects both in vivo and in vitro (Johnson et al., 1963). VCR due to its comparatively mild myelosuppressive action is a standard component of regimens for treating paediatric leukemias and solid tumours and is frequently used in adult lymphoma treatment (Johnson et al., 1963). Vincristine has been found to be active against the hematological malignancies, Hodgkins and non-Hodgkins lymphoma, Wilm’s tumour, neuroblastoma, brain tumours, rhabdomyosarcoma, carcinomas of the breast, bladder and the male and female reproductive systems (Dorr and Fritz, 1980).
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The chemotherapeutic drugs used in the treatment of cancer are usually DNA damaging agents and these drugs invariably result in the DNA strand breakage, chromosome breaks and loss or gain of chromosomes (aneuploidy). In fact, DNA damaging agents have been reported to cause phenotypic abnormalities like spontaneous abortion, congenital malformations and malignant transformations (Sandberg, 1980; Hecht and Hecht, 1987). Vincristine is frequently combined with the other chemotherapeutic drugs to get complete remission of certain neoplastic disorders (Dorr and Fritz, 1980). In several instances, it is also combined with radiation to achieve better therapeutic gains (Vietti et al., 1970). The reports regarding the use of vincristine in combination with radiation in experimental system are lacking. However, other vinca alkaloids like vinblastine has been reported to enhance the frequency of MN in mouse bone marrow exposed to different doses of gamma radiation (Jagetia and Jacob, 1994). Similarly, vindesine, a semi-synthetic derivative of vinblastine sulphate has also been reported to enhance the frequency of micronuclei and decrease in the cell survival in the V79 cells exposed to different doses of gamma radiation (Jagetia and Adiga, 2000). Vinorelbine, a semisynthetic vinca alkaloid, has also been reported to enhance the effect of radiation in NCI-H460 and A549 cells (Edelstein et al., 1996). Paclitaxel, another agent interfering with the dynamics of microtubules has been reported to enhance the frequency of irradiated micronuclei in mice bone marrow and cultured V79 cells in vitro (Jagetia and Nayak, 1996; Jagetia and Adiga, 1997). The patients receiving combination treatment are at an increased risk of developing secondary neoplasia in comparison to those receiving a single treatment modality. The UNSCEAR report (1982) has rightfully emphasised that the mode of interaction between chemicals and radiation should be tested in diverse biological systems. Due to its inherent sensitivity to even small doses of radiation and chemicals, the in vitro system is suitable for testing drugs for their radio modifying properties. In combination therapy, usually low doses of chemotherapeutic agents and radiation are used to avoid or minimise the adverse toxic
side effects of the combination treatment. Therefore, the aim of the present study was to evaluate the effect of 10 ng/ml of vincristine in the cultured human peripheral blood lymphocytes exposed to different doses of gamma radiation on the micronucleus formation. 2. Materials and methods
2.1. Drugs and chemicals VCR (Cytocristine, Cipla, Bangalore, India) was dissolved in phosphate buffered saline (PBS) and diluted in such a way so as to get 1 ng/ml of VCR. Cytochalasin-B (Sigma cat. no. C-6762) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml, stored at − 80 °C and diluted with phosphate buffered saline (PBS) immediately before use. Cytochalasin-B, MEM, Lglutamine, gentamycin sulphate, fetal calf serum and DMSO were procured from Sigma Chemical Co., St. Louis, USA.
2.2. The lymphocyte culture The whole blood was collected from a nonsmoking healthy donor in the heparinised vacutainers (Becton Dickinson, USA). The details of the procedure are given elsewhere (Jagetia et al., 2001). Briefly, the erythrocytes were allowed to sediment and the buffy coat containing nucleated cells was used for lymphocyte culture. Usually, 106 nucleated cells were inoculated into each culture tube containing RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), L-glutamine and phytohemagglutinin (PHA) as the mitogen. The cultures were divided into the following groups.
2.2.1. PBS + irradiation This group of cultures was treated with 10 ml/ml of phosphate buffered saline before exposure to various doses of g-irradiation. 2.2.2. VCR + irradiation The cultures of this group were treated with 10 ng/ml of VCR before exposure to different doses of g-irradiation.
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2.3. Irradiation Thirty minutes after PBS or VCR treatment, the cells of PBS + irradiation or VCR+ irradiation groups were exposed to 0, 0.5, 1, 2 and 3 Gy of 60Co g-radiation from a Tele Cobalt therapy source (Gammatron, Siemens Germany) at a dose rate of 1.16 Gy/min. Immediately after exposure (within 5 min), the cell cultures were transferred to a CO2 incubator and allowed to grow for another 72 h at 37 °C.
2.4. Micronucleus assay The micronuclei were prepared according to the method of Fenech and Morley (1985), and the protocol described by Kirsch-Volders et al. (2000) for in vitro micronucleus assay was followed strictly. Briefly, 5 mg/ml of cytochalasin-B was added to the each culture tube 44 h after the irradiation of the cultures to inhibit cytokinesis. The cultures were harvested 72 h after irradiation by centrifugation. The lymphocytes were subjected to a mild hypotonic treatment, centrifuged and fixed in Carnoy’s fixative (3:1 methanol, acetic acid). The cells were centrifuged again, resuspended in a small volume of fixative and spread on to precleaned coded slides to avoid observer’s bias. Triplicate cultures were used for each drug concentration for each group and the micronuclei were scored. The slides containing cells were stained with 0.125% acridine orange (BDH, England, Gurr cat. no. 34001 9704640E) in Sorensen’s buffer (pH 6.8), and washed twice in the buffer. The buffer mounted slides were observed under a fluorescent microscope equipped with 450– 490 nm BP filter set with excitation at 453 nm (Carl Zeiss Photomicroscope III, Germany) using a 40× neofluar objective for the presence of micronuclei (MN) in the binucleate lymphocytes (BNC). A minimum of 2000 BNC with well-preserved cytoplasm was scored from each culture and the frequency of micronucleated binucleate cells (MNBNC) was determined. The micronuclei identification was done according to the criteria of Countryman and Heddle (1976) and Kirsch-Volders et al. (2000).
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The statistical significance between both the groups was determined using Fisher’s exact test and one-way ANOVA. The data were fitted on to linear Y= h+ iD or linear quadratic Y= C + hD+ iD 2 to ascertain the dose response, if any. The Solo 4 statistical package (BMDP Inc, USA and Ireland) was used for the statistical analysis.
3. Results The results are expressed as the frequencies of MNBNC per 1000 9 S.E.M. and are presented in Table 1 and Fig. 1.
Fig. 1. Effect of vincristine sulphate on the induction of MNBNC in the cultured human peripheral blood lymphocytes exposed to different doses of g radiation. Closed symbols PBS+ irradiation and open symbols VCR +irradiation. (a) Total MNBNC; (b) BNC with one MN; and (c) squares BNC with two MN; Circles BNC with three NW; and diamonds with four MN.
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Exposure Frequency of MNBNC per 1000 9 S.E.M. dose (Gy)
0 0.5 1 2 3 r
BNC with one MN
BNC with two MN
BNC with three MN
BNC with four MN
Total MNBNC
PBS+IR
VCR+IR
PBS+IR
PBS+IR
PBS+IR
PBS+IR
19.9990.83 49.4591.43 76.6791.46 117.819 0.51 124.279 1.34 0.96
75.25+0.74a 186.86 91.58a 235.48 94.37a 173.48 91.72a 173.33 91.10a 0.97
0 1.88 90.71c 0 22.35 91.14a a 12.29+1.10 39.46 91.40 5.99 9 0.84 10.46 9 0.93 25.90 91.12 94.72+0.32a 14.71 90.61 14.75 90.64 41.88 91.72 115.66 90.92a 19.93 91.53 26.27 9 1.01 67.06 92.05 102.90 91.06a 36.64 91.60 42.64 9 0.89c 0.997 0.97 0.99 0.997
VCR+IR
VCR+IR
VCR+IR
VCR+IR
0 0 19.9990.83 99.49 9 3.40a 0.639 0.63 2.37 9 0.90 68.36 9 2.81 239.14 94.0la 3.52 9 0.31 6.35 9 0.61b 120.809 1.15 351.30 9 4.51a 4.47 9 0.63 10.58 9 0.70c 184.109 3.94 325.98 9 2.88a 18.6091.01 22.469 0.62c 246.579 4.56 341.33 9 1.87a 0.97 0.99 0.99 0.99
IR, irradiation; PBS, phosphate buffered saline; and VCR, vincristine; PBS+IR group compared with VCR+IR group: aPB0.0001; bPB0.01; cPB0.03; and no symbols, non-significant.
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Table 1 Effect of vincristine treatment on the micronuclei formation in the cultured human peripheral blood lymphocytes exposed to different doses of g-radiation
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The frequency of MNBNC increased in a dose dependent manner in the PBS+ irradiation group and the dose response was linear. The pretreatment of lymphocytes with vincristine before exposure to different doses of gamma radiation resulted in a significant elevation in the frequency of MNBNC. This increase was dose dependent up to a dose of 1 Gy, thereafter the frequency of MNBNC registered a significant decline after 2 Gy (PB 0.01) irradiation and remained unaltered up to 3 Gy exposure (Fig. 1a). Treatment of lymphocytes with VCR before exposure to different doses of g-radiation increased the frequency of MNBNC by 3.5- and 3-fold for 0.5 and 1 Gy, respectively, when compared with the concurrent PBS + irradiation group. However, with the further increase in irradiation dose, this increase also declined and it was 1.7- and 1.4-fold for 2 and 3 Gy, respectively (Table 1). The frequencies of MNBNC with one, two, three and four MN were scored separately. The frequency of MNBNC with one MN elevated in a dose dependent manner in the PBS+irradiation group and the dose response was linear (Fig. 1b). VCR pretreatment resulted in a significant elevation in the MNBNC with one MN and the dose response was linear only up to a dose of 1 Gy irradiation, thereafter, the frequency of MNBNC with one MN declined significantly at 2 Gy (P B 0.0002) when compared with the 1 Gy VCR+ irradiation and remained unaltered up to 3 Gy exposure (Fig. 1b). A 3.8-fold elevation in the frequency of MNBNC with one MN was observed for 0.5 and 1 Gy, respectively, thereafter this increase was lesser. The frequency of MNBNC with one MN was significantly higher in VCR +irradiation group when compared with the concurrent PBS +irradiation group. The MNBNCs with two, three and four MN were conspicuous by their absence for PBS+ (0 Gy) sham-irradiation (Table 1), although the frequency of MNBNC with 2 MN was greatly enhanced after VCR+sham-irradiation (Table 1). The irradiation of lymphocytes resulted in a drastic but dose dependent increase in the frequency of MNBNC with 2 MN (Fig. 1c). Treatment of lymphocytes with VCR resulted in 3.2, 3.7 and 2.8-fold elevation in the frequency of MNBNC
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with 2 MN for 0.5, 1 and 2 Gy, respectively, and this increase was significantly higher when compared with the concurrent PBS+ irradiation group (Table 1). The increase in the frequency of MNBNC with 2 MN was only 1.5-fold greater after 3 Gy irradiation in the VCR+ irradiation group. The dose response for both PBS+ irradiation and VCR+ irradiation groups was linear quadratic. The frequency of MNBNC with 3 and 4 MN increased in a dose dependent manner in the PBS + irradiation group (Fig. 1c). The treatment of lymphocytes with VCR resulted in a significant elevation in the frequency of MNBNC with 3 and 4 MN when compared with the PBS+ irradiation group. The dose response for both PBS+ irradiation and VCR+ irradiation groups was also linear quadratic (Fig. 1c). In spite of a dose related elevation in the frequency of MNBNC with 4 MN, the efficiency of induction of MNBNC with 4 MN declined with the exposure dose. The highest elevation in MNBNC with 4 MN per unit dose was observed at 0.5 Gy irradiation (3.8-fold) in the VCR+ irradiation group when compared with PBS + irradiation group (Table 1).
4. Discussion The combination of chemotherapy and radiotherapy has been used with a remarkable success, especially in cases where either therapy alone has proved ineffective. The radiotherapy and chemotherapy are applied concomitantly or alternatively to overcome the higher clinical toxicity, where low doses of both the agents are used. The use of these regimens has resulted in the increased survival of patients receiving the treatment. However, chemotherapeutic agents and radiation cause DNA damage not only to the malignant cells, but the normal cells also suffer damage to their genome. This damage could be enhanced when both cytotoxic drugs and radiation are combined for therapeutic purposes. This may result in the reduction of the latency period for neoplastic transformation. The assessment of chromosomal damage helps to predict the mutagenicity and carcinogenicity of these agents. The aim of the
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present investigation was to study the genotoxic effects of 10 ng/ml VCR in combination with radiation in the cultured human peripheral blood lymphocytes. Micronucleus assay is used to predict the chromosomal instability and carcinogenicity in various experimental models. The irradiation of lymphocytes to different doses of gamma radiation not only increased the MNBNC with one MN but also the cells bearing 2, 3 and 4 MN and the VCR pre-treatment further increased their frequency significantly. Irradiation has been reported to increase the frequency of cells with 1, 2, 3 and 4 MN in lymphocytes and various cultured cell lines (Catena et al., 1997; Jagetia and Adiga, 1997; Jagetia and Adiga, 2000; Belyakov et al., 1999; Jagetia and Nayak, 2000). Treatment of V79 cells with taxol and vindesine has been reported to enhance the frequency of cells with one and multiple MN (Jagetia and Adiga, 1997, 2000). The inductions of multiple micronuclei in a cell indicate to the multiple sites of irreparable damage suffered by the cell genome, which is subsequently expressed as micronuclei after cell division. As a result of the loss of a large part of the genome, these cells may not be able to survive long. However, this may not be the case for the cells with one MN, where the probability of adaptation of cells to survive with the deletion of a small part of genome is higher when compared with those cells that have lost a sizeable part of genome in the form of multiple micronuclei. The continued survival and replication of these mutated cells may result in the development of neoplasia in due course of time. Therefore, the cells, which have suffered less damage to their genome, may be more prone to the induction of mutagenesis and carcinogenesis than the cells, which have suffered extensive damage in their genome in the form of multiple micronuclei. Due to the heavy damage to their genome, the cells with multiple MN will not be able to divide and will be relegated from the system without any consequence. The genomic instability observed after the treatment of lymphocytes with VCR before irradiation indicates to the increased
probability of mutagenesis and carcinogenesis significantly. The frequency of MNBNC increased up to 1 Gy in VCR + irradiation group and declined thereafter. A similar effect has been reported in mice bone marrow treated with vinblastine, another vinca alkaloid before irradiation. (Jagetia and Jacob, 1994). The decline in MNBNC after 1 Gy irradiation in the VCR pre-treated group may be owing to the increase in the clastogenic and or aneugenic effects of radiation by VCR, that would have resulted into the failure of cell division of the affected cells and consequently, such cells may not be able to express micronuclei. Since a cell division is required after any treatment for micronuclei to appear in the daughter cells (Heddle, 1973). Vinca alkaloids are potent microtubule-inhibiting agents that act by depolymerisation of tubulin resulting in the inhibition of mitosis (Howard et al., 1980; Jordan et al., 1985). Since, G2 + M cells are highly sensitive to the effect of radiation the presence of VCR before and after irradiation may have increased the effect of radiation resulting in an elevation in the micronuclei. The formation of MN after VCR treatment may be owing to the increase in radiation induced chromosome breaks or loss of whole chromosome. This may be the reason for the elevation of MN in VCR+irradiation group when compared with the PBS+ irradiation group. VCR alone has been reported to induce a wide spectrum of chromosomal aberrations (Johnson et al., 1963; Jordan et al., 1985), in a variety of in vitro and in vivo test systems (Yamamoto and Kikuchi, 1980; Gonzalez-Cid et al., 1999). It has been reported that VCR like colchicine has anugenic, as well as, clastogenic effect in the mouse bone marrow erythrocytes (Grawe et al., 1994). A similar effect cannot be ruled out in the present study. Further, vinca alkaloids have been reported to affect a number of cellular systems like the DNA and RNA synthesis (Creasy, 1968; Bernstam et al., 1980; Dustin, 1984), lipid biosynthesis, cyclic neucleotide metabolism (Howard et al., 1980; Sheppard, 1980), glutathione metabolism (Beck, 1980), and calmodulin dependent Ca2 + trans-
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port ATPase (Geitzen et al., 1980), which may also be responsible for higher damage in the VCR + irradiation group. Our study demonstrates that combination of VCR and radiation has increased the genomic instability in the lymphocytes, as measured by micronucleus assay. This increase in the genomic instability in the cells may result in transformation of cells into the altered phenotype leading to neoplasia of the normal cells. The combination of VCR and radiation may have higher probability of fixing the mutagenic damage in the cell genome as a result of which the frequency of development of second malignancies may be increased when VCR is combined with radiation. Alternatively, the combination treatment may shorten the latency period as a result the secondary neoplastic diseases may appear early in the survivors of combination treatment. It has been reported that the patients who have been treated with chemotherapeutic drugs for Hodgkin’s diseases, multiple myeloma, and ovarian tumours develop secondary malignancies after a few years of completion of the treatment (Rosner and Grunwald, 1974; Canellos et al., 1975; Bergsagel et al., 1979; Coltman and Dixon, 1982; Henne and Schmahl, 1985; Blayney et al., 1987; Tucker et al., 1988; Pedersen-Bjergaard and Rowley, 1994). Treatment of antineoplastic drugs is also responsible for later complications not only in the patients, but also in the health care workers administering these agents to patients (Oestreicher et al., 1990; Krepinsky et al., 1990; Roth et al., 1994). The combination of VCR with ionising radiation may cause clastogenesis and mutagenesis of cell genome, which in turn may be transformed into the altered phenotype leading to the enhancement in the development of neoplasia.
Acknowledgements We are thankful to Dr M.S. Vidyasagar, Professor and Head, and Dr J. Velmurugan, Department of Radiotherapy and Oncology, Kasturba Medical College Hospital, Manipal, for providing the necessary irradiation facilities and for dosimetric calculations, respectively.
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Vincristine increases the genomic instability in irradiated cultured human peripheral blood lymphocytes Ganesh Chandra Jagetia *, Manjeshwar Shrinath Baliga Department of Radiobiology, Kasturba Medical College, Manipal 576 119, Karnataka, India Received 28 May 2001; received in revised form 19 September 2001; accepted 24 September 2001
Abstract Increase in the genomic instability by 10 ng of vincristine (VCR) pretreatment was studied in cultured human peripheral blood lymphocytes exposed to 0, 0.5, 1, 2, and 3 Gy gamma radiation by the micronucleus assay. The frequency of micronucleated binucleate lymphocytes (MNBNC) increased in a dose dependent manner after exposure to different doses of gamma radiation and the dose response was linear. The pre-treatment of lymphocytes with 10 ng/ml VCR caused a further elevation in the frequency of MNBNC and this was significantly greater than that of the concurrent PBS pre-treated irradiated lymphocytes. The dose response for VCR treated group was linear up to 1 Gy irradiation. A further increase in the radiation dose resulted in a decline in the induction of MNBNC in VCR pretreated irradiated group, although it was significantly higher than the PBS treated irradiated group. VCR pretreatment not only increased the frequency of MNBNC with one micronuclei but also the frequency of MNBNC with 2, 3 and 4 MN significantly, indicating the increase in the multiple sites of damage to DNA by VCR pre-treatment. This increase in multiple MNBNC was also dose dependent, however, the dose response was linear quadratic. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vincristine; Micronuclei; Human lymphocytes; Radiation
1. Introduction Vincristine sulphate (VCR), a dimeric alkaloid isolated from the periwinkle plant Catheranthus roseus, is used for the treatment of several forms of malignancies (Avila, 1997). Vincristine has been reported to possess anti-tumour activity against experimental animal tumours and exhibits
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cytotoxic effects both in vivo and in vitro (Johnson et al., 1963). VCR due to its comparatively mild myelosuppressive action is a standard component of regimens for treating paediatric leukemias and solid tumours and is frequently used in adult lymphoma treatment (Johnson et al., 1963). Vincristine has been found to be active against the hematological malignancies, Hodgkins and non-Hodgkins lymphoma, Wilm’s tumour, neuroblastoma, brain tumours, rhabdomyosarcoma, carcinomas of the breast, bladder and the male and female reproductive systems (Dorr and Fritz, 1980).
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The chemotherapeutic drugs used in the treatment of cancer are usually DNA damaging agents and these drugs invariably result in the DNA strand breakage, chromosome breaks and loss or gain of chromosomes (aneuploidy). In fact, DNA damaging agents have been reported to cause phenotypic abnormalities like spontaneous abortion, congenital malformations and malignant transformations (Sandberg, 1980; Hecht and Hecht, 1987). Vincristine is frequently combined with the other chemotherapeutic drugs to get complete remission of certain neoplastic disorders (Dorr and Fritz, 1980). In several instances, it is also combined with radiation to achieve better therapeutic gains (Vietti et al., 1970). The reports regarding the use of vincristine in combination with radiation in experimental system are lacking. However, other vinca alkaloids like vinblastine has been reported to enhance the frequency of MN in mouse bone marrow exposed to different doses of gamma radiation (Jagetia and Jacob, 1994). Similarly, vindesine, a semi-synthetic derivative of vinblastine sulphate has also been reported to enhance the frequency of micronuclei and decrease in the cell survival in the V79 cells exposed to different doses of gamma radiation (Jagetia and Adiga, 2000). Vinorelbine, a semisynthetic vinca alkaloid, has also been reported to enhance the effect of radiation in NCI-H460 and A549 cells (Edelstein et al., 1996). Paclitaxel, another agent interfering with the dynamics of microtubules has been reported to enhance the frequency of irradiated micronuclei in mice bone marrow and cultured V79 cells in vitro (Jagetia and Nayak, 1996; Jagetia and Adiga, 1997). The patients receiving combination treatment are at an increased risk of developing secondary neoplasia in comparison to those receiving a single treatment modality. The UNSCEAR report (1982) has rightfully emphasised that the mode of interaction between chemicals and radiation should be tested in diverse biological systems. Due to its inherent sensitivity to even small doses of radiation and chemicals, the in vitro system is suitable for testing drugs for their radio modifying properties. In combination therapy, usually low doses of chemotherapeutic agents and radiation are used to avoid or minimise the adverse toxic
side effects of the combination treatment. Therefore, the aim of the present study was to evaluate the effect of 10 ng/ml of vincristine in the cultured human peripheral blood lymphocytes exposed to different doses of gamma radiation on the micronucleus formation. 2. Materials and methods
2.1. Drugs and chemicals VCR (Cytocristine, Cipla, Bangalore, India) was dissolved in phosphate buffered saline (PBS) and diluted in such a way so as to get 1 ng/ml of VCR. Cytochalasin-B (Sigma cat. no. C-6762) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml, stored at − 80 °C and diluted with phosphate buffered saline (PBS) immediately before use. Cytochalasin-B, MEM, Lglutamine, gentamycin sulphate, fetal calf serum and DMSO were procured from Sigma Chemical Co., St. Louis, USA.
2.2. The lymphocyte culture The whole blood was collected from a nonsmoking healthy donor in the heparinised vacutainers (Becton Dickinson, USA). The details of the procedure are given elsewhere (Jagetia et al., 2001). Briefly, the erythrocytes were allowed to sediment and the buffy coat containing nucleated cells was used for lymphocyte culture. Usually, 106 nucleated cells were inoculated into each culture tube containing RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), L-glutamine and phytohemagglutinin (PHA) as the mitogen. The cultures were divided into the following groups.
2.2.1. PBS + irradiation This group of cultures was treated with 10 ml/ml of phosphate buffered saline before exposure to various doses of g-irradiation. 2.2.2. VCR + irradiation The cultures of this group were treated with 10 ng/ml of VCR before exposure to different doses of g-irradiation.
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2.3. Irradiation Thirty minutes after PBS or VCR treatment, the cells of PBS + irradiation or VCR+ irradiation groups were exposed to 0, 0.5, 1, 2 and 3 Gy of 60Co g-radiation from a Tele Cobalt therapy source (Gammatron, Siemens Germany) at a dose rate of 1.16 Gy/min. Immediately after exposure (within 5 min), the cell cultures were transferred to a CO2 incubator and allowed to grow for another 72 h at 37 °C.
2.4. Micronucleus assay The micronuclei were prepared according to the method of Fenech and Morley (1985), and the protocol described by Kirsch-Volders et al. (2000) for in vitro micronucleus assay was followed strictly. Briefly, 5 mg/ml of cytochalasin-B was added to the each culture tube 44 h after the irradiation of the cultures to inhibit cytokinesis. The cultures were harvested 72 h after irradiation by centrifugation. The lymphocytes were subjected to a mild hypotonic treatment, centrifuged and fixed in Carnoy’s fixative (3:1 methanol, acetic acid). The cells were centrifuged again, resuspended in a small volume of fixative and spread on to precleaned coded slides to avoid observer’s bias. Triplicate cultures were used for each drug concentration for each group and the micronuclei were scored. The slides containing cells were stained with 0.125% acridine orange (BDH, England, Gurr cat. no. 34001 9704640E) in Sorensen’s buffer (pH 6.8), and washed twice in the buffer. The buffer mounted slides were observed under a fluorescent microscope equipped with 450– 490 nm BP filter set with excitation at 453 nm (Carl Zeiss Photomicroscope III, Germany) using a 40× neofluar objective for the presence of micronuclei (MN) in the binucleate lymphocytes (BNC). A minimum of 2000 BNC with well-preserved cytoplasm was scored from each culture and the frequency of micronucleated binucleate cells (MNBNC) was determined. The micronuclei identification was done according to the criteria of Countryman and Heddle (1976) and Kirsch-Volders et al. (2000).
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The statistical significance between both the groups was determined using Fisher’s exact test and one-way ANOVA. The data were fitted on to linear Y= h+ iD or linear quadratic Y= C + hD+ iD 2 to ascertain the dose response, if any. The Solo 4 statistical package (BMDP Inc, USA and Ireland) was used for the statistical analysis.
3. Results The results are expressed as the frequencies of MNBNC per 1000 9 S.E.M. and are presented in Table 1 and Fig. 1.
Fig. 1. Effect of vincristine sulphate on the induction of MNBNC in the cultured human peripheral blood lymphocytes exposed to different doses of g radiation. Closed symbols PBS+ irradiation and open symbols VCR +irradiation. (a) Total MNBNC; (b) BNC with one MN; and (c) squares BNC with two MN; Circles BNC with three NW; and diamonds with four MN.
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Exposure Frequency of MNBNC per 1000 9 S.E.M. dose (Gy)
0 0.5 1 2 3 r
BNC with one MN
BNC with two MN
BNC with three MN
BNC with four MN
Total MNBNC
PBS+IR
VCR+IR
PBS+IR
PBS+IR
PBS+IR
PBS+IR
19.9990.83 49.4591.43 76.6791.46 117.819 0.51 124.279 1.34 0.96
75.25+0.74a 186.86 91.58a 235.48 94.37a 173.48 91.72a 173.33 91.10a 0.97
0 1.88 90.71c 0 22.35 91.14a a 12.29+1.10 39.46 91.40 5.99 9 0.84 10.46 9 0.93 25.90 91.12 94.72+0.32a 14.71 90.61 14.75 90.64 41.88 91.72 115.66 90.92a 19.93 91.53 26.27 9 1.01 67.06 92.05 102.90 91.06a 36.64 91.60 42.64 9 0.89c 0.997 0.97 0.99 0.997
VCR+IR
VCR+IR
VCR+IR
VCR+IR
0 0 19.9990.83 99.49 9 3.40a 0.639 0.63 2.37 9 0.90 68.36 9 2.81 239.14 94.0la 3.52 9 0.31 6.35 9 0.61b 120.809 1.15 351.30 9 4.51a 4.47 9 0.63 10.58 9 0.70c 184.109 3.94 325.98 9 2.88a 18.6091.01 22.469 0.62c 246.579 4.56 341.33 9 1.87a 0.97 0.99 0.99 0.99
IR, irradiation; PBS, phosphate buffered saline; and VCR, vincristine; PBS+IR group compared with VCR+IR group: aPB0.0001; bPB0.01; cPB0.03; and no symbols, non-significant.
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Table 1 Effect of vincristine treatment on the micronuclei formation in the cultured human peripheral blood lymphocytes exposed to different doses of g-radiation
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The frequency of MNBNC increased in a dose dependent manner in the PBS+ irradiation group and the dose response was linear. The pretreatment of lymphocytes with vincristine before exposure to different doses of gamma radiation resulted in a significant elevation in the frequency of MNBNC. This increase was dose dependent up to a dose of 1 Gy, thereafter the frequency of MNBNC registered a significant decline after 2 Gy (PB 0.01) irradiation and remained unaltered up to 3 Gy exposure (Fig. 1a). Treatment of lymphocytes with VCR before exposure to different doses of g-radiation increased the frequency of MNBNC by 3.5- and 3-fold for 0.5 and 1 Gy, respectively, when compared with the concurrent PBS + irradiation group. However, with the further increase in irradiation dose, this increase also declined and it was 1.7- and 1.4-fold for 2 and 3 Gy, respectively (Table 1). The frequencies of MNBNC with one, two, three and four MN were scored separately. The frequency of MNBNC with one MN elevated in a dose dependent manner in the PBS+irradiation group and the dose response was linear (Fig. 1b). VCR pretreatment resulted in a significant elevation in the MNBNC with one MN and the dose response was linear only up to a dose of 1 Gy irradiation, thereafter, the frequency of MNBNC with one MN declined significantly at 2 Gy (P B 0.0002) when compared with the 1 Gy VCR+ irradiation and remained unaltered up to 3 Gy exposure (Fig. 1b). A 3.8-fold elevation in the frequency of MNBNC with one MN was observed for 0.5 and 1 Gy, respectively, thereafter this increase was lesser. The frequency of MNBNC with one MN was significantly higher in VCR +irradiation group when compared with the concurrent PBS +irradiation group. The MNBNCs with two, three and four MN were conspicuous by their absence for PBS+ (0 Gy) sham-irradiation (Table 1), although the frequency of MNBNC with 2 MN was greatly enhanced after VCR+sham-irradiation (Table 1). The irradiation of lymphocytes resulted in a drastic but dose dependent increase in the frequency of MNBNC with 2 MN (Fig. 1c). Treatment of lymphocytes with VCR resulted in 3.2, 3.7 and 2.8-fold elevation in the frequency of MNBNC
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with 2 MN for 0.5, 1 and 2 Gy, respectively, and this increase was significantly higher when compared with the concurrent PBS+ irradiation group (Table 1). The increase in the frequency of MNBNC with 2 MN was only 1.5-fold greater after 3 Gy irradiation in the VCR+ irradiation group. The dose response for both PBS+ irradiation and VCR+ irradiation groups was linear quadratic. The frequency of MNBNC with 3 and 4 MN increased in a dose dependent manner in the PBS + irradiation group (Fig. 1c). The treatment of lymphocytes with VCR resulted in a significant elevation in the frequency of MNBNC with 3 and 4 MN when compared with the PBS+ irradiation group. The dose response for both PBS+ irradiation and VCR+ irradiation groups was also linear quadratic (Fig. 1c). In spite of a dose related elevation in the frequency of MNBNC with 4 MN, the efficiency of induction of MNBNC with 4 MN declined with the exposure dose. The highest elevation in MNBNC with 4 MN per unit dose was observed at 0.5 Gy irradiation (3.8-fold) in the VCR+ irradiation group when compared with PBS + irradiation group (Table 1).
4. Discussion The combination of chemotherapy and radiotherapy has been used with a remarkable success, especially in cases where either therapy alone has proved ineffective. The radiotherapy and chemotherapy are applied concomitantly or alternatively to overcome the higher clinical toxicity, where low doses of both the agents are used. The use of these regimens has resulted in the increased survival of patients receiving the treatment. However, chemotherapeutic agents and radiation cause DNA damage not only to the malignant cells, but the normal cells also suffer damage to their genome. This damage could be enhanced when both cytotoxic drugs and radiation are combined for therapeutic purposes. This may result in the reduction of the latency period for neoplastic transformation. The assessment of chromosomal damage helps to predict the mutagenicity and carcinogenicity of these agents. The aim of the
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present investigation was to study the genotoxic effects of 10 ng/ml VCR in combination with radiation in the cultured human peripheral blood lymphocytes. Micronucleus assay is used to predict the chromosomal instability and carcinogenicity in various experimental models. The irradiation of lymphocytes to different doses of gamma radiation not only increased the MNBNC with one MN but also the cells bearing 2, 3 and 4 MN and the VCR pre-treatment further increased their frequency significantly. Irradiation has been reported to increase the frequency of cells with 1, 2, 3 and 4 MN in lymphocytes and various cultured cell lines (Catena et al., 1997; Jagetia and Adiga, 1997; Jagetia and Adiga, 2000; Belyakov et al., 1999; Jagetia and Nayak, 2000). Treatment of V79 cells with taxol and vindesine has been reported to enhance the frequency of cells with one and multiple MN (Jagetia and Adiga, 1997, 2000). The inductions of multiple micronuclei in a cell indicate to the multiple sites of irreparable damage suffered by the cell genome, which is subsequently expressed as micronuclei after cell division. As a result of the loss of a large part of the genome, these cells may not be able to survive long. However, this may not be the case for the cells with one MN, where the probability of adaptation of cells to survive with the deletion of a small part of genome is higher when compared with those cells that have lost a sizeable part of genome in the form of multiple micronuclei. The continued survival and replication of these mutated cells may result in the development of neoplasia in due course of time. Therefore, the cells, which have suffered less damage to their genome, may be more prone to the induction of mutagenesis and carcinogenesis than the cells, which have suffered extensive damage in their genome in the form of multiple micronuclei. Due to the heavy damage to their genome, the cells with multiple MN will not be able to divide and will be relegated from the system without any consequence. The genomic instability observed after the treatment of lymphocytes with VCR before irradiation indicates to the increased
probability of mutagenesis and carcinogenesis significantly. The frequency of MNBNC increased up to 1 Gy in VCR + irradiation group and declined thereafter. A similar effect has been reported in mice bone marrow treated with vinblastine, another vinca alkaloid before irradiation. (Jagetia and Jacob, 1994). The decline in MNBNC after 1 Gy irradiation in the VCR pre-treated group may be owing to the increase in the clastogenic and or aneugenic effects of radiation by VCR, that would have resulted into the failure of cell division of the affected cells and consequently, such cells may not be able to express micronuclei. Since a cell division is required after any treatment for micronuclei to appear in the daughter cells (Heddle, 1973). Vinca alkaloids are potent microtubule-inhibiting agents that act by depolymerisation of tubulin resulting in the inhibition of mitosis (Howard et al., 1980; Jordan et al., 1985). Since, G2 + M cells are highly sensitive to the effect of radiation the presence of VCR before and after irradiation may have increased the effect of radiation resulting in an elevation in the micronuclei. The formation of MN after VCR treatment may be owing to the increase in radiation induced chromosome breaks or loss of whole chromosome. This may be the reason for the elevation of MN in VCR+irradiation group when compared with the PBS+ irradiation group. VCR alone has been reported to induce a wide spectrum of chromosomal aberrations (Johnson et al., 1963; Jordan et al., 1985), in a variety of in vitro and in vivo test systems (Yamamoto and Kikuchi, 1980; Gonzalez-Cid et al., 1999). It has been reported that VCR like colchicine has anugenic, as well as, clastogenic effect in the mouse bone marrow erythrocytes (Grawe et al., 1994). A similar effect cannot be ruled out in the present study. Further, vinca alkaloids have been reported to affect a number of cellular systems like the DNA and RNA synthesis (Creasy, 1968; Bernstam et al., 1980; Dustin, 1984), lipid biosynthesis, cyclic neucleotide metabolism (Howard et al., 1980; Sheppard, 1980), glutathione metabolism (Beck, 1980), and calmodulin dependent Ca2 + trans-
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port ATPase (Geitzen et al., 1980), which may also be responsible for higher damage in the VCR + irradiation group. Our study demonstrates that combination of VCR and radiation has increased the genomic instability in the lymphocytes, as measured by micronucleus assay. This increase in the genomic instability in the cells may result in transformation of cells into the altered phenotype leading to neoplasia of the normal cells. The combination of VCR and radiation may have higher probability of fixing the mutagenic damage in the cell genome as a result of which the frequency of development of second malignancies may be increased when VCR is combined with radiation. Alternatively, the combination treatment may shorten the latency period as a result the secondary neoplastic diseases may appear early in the survivors of combination treatment. It has been reported that the patients who have been treated with chemotherapeutic drugs for Hodgkin’s diseases, multiple myeloma, and ovarian tumours develop secondary malignancies after a few years of completion of the treatment (Rosner and Grunwald, 1974; Canellos et al., 1975; Bergsagel et al., 1979; Coltman and Dixon, 1982; Henne and Schmahl, 1985; Blayney et al., 1987; Tucker et al., 1988; Pedersen-Bjergaard and Rowley, 1994). Treatment of antineoplastic drugs is also responsible for later complications not only in the patients, but also in the health care workers administering these agents to patients (Oestreicher et al., 1990; Krepinsky et al., 1990; Roth et al., 1994). The combination of VCR with ionising radiation may cause clastogenesis and mutagenesis of cell genome, which in turn may be transformed into the altered phenotype leading to the enhancement in the development of neoplasia.
Acknowledgements We are thankful to Dr M.S. Vidyasagar, Professor and Head, and Dr J. Velmurugan, Department of Radiotherapy and Oncology, Kasturba Medical College Hospital, Manipal, for providing the necessary irradiation facilities and for dosimetric calculations, respectively.
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