Pretreatment of low dose radiation reduces radiation-induced apoptosis in mouse lymphoma (EL4) cells

Induction of an adaptive response to ionizing radiation in mouse lymphoma (EL4) cells was studied by using cell survival fraction and apoptotic nucleosomal DNA fragmentation as biological end points. Cells in early log phase were pre-exposed to low dose of γ-rays (0.01 Gy) 4 or 20 hrs prior to high dose γ-ray (4, 8 and 12 Gy for cell survival fraction analysis; 8 Gy for DNA fragmentation analysis) irradiation. Then cell survival fractions and the extent of DNA fragmentation were measured. Significant adaptive response, increase in cell survival fraction and decrease in the extent of DNA fragmentation were induced when low and high dose γ-ray irradiation time interval was 4 hr. Addition of protein or RNA synthesis inhibitor, cycloheximide or 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRFB), respectively during adaptation period, the period from low dose γ-ray irradiation to high dose γ-ray irradiation, was able to inhibit the induction of adaptive response, which is the reduction of the extent DNA fragmentation in irradiated EL4 cells. These data suggest that the induction of adaptive response to ionizing radiation in EL4 cells required both protein and RNA synthesis.     

Reduction of radiation-induced chromosome aberration and apoptosis by dithiothreitol

We have examinedin vitro andin vivo radioprotective effects of a well-known thiol-containing compound, dithiothreitol (DTT). The treatment of both 0.5 and 1 mM of DTT significantly increased clonogenic survival of γ-ray irradiated Chinese hamster (V79-4) cells. In order to investigate the possible radioprotective mechanism of DTT, we measured γ-ray induced chromosome aberration by micronucleus assay. In the presence of 0.5 mM or 1 mM DTT, the frequencies of micronuclei were greatly reduced in all dose range examined (1.5–8 Gy). Slightly higher reduction in micronucleus formation was observed in 1 mM DTT-treated cells than in 0.5 mM DTT-treated cells. In addition, incubation with both 0.5 and 1 mM of DTT prior to γ-ray irradiation reduced nucleosomal DNA fragmentation at about same extent, this result suggests that treatment of DTT at concentrations of 0.5 and 1 mM reduced radiation-induced apoptosis.In vivo experiments, we also observed that DTT treatment reduced the incidence of apoptotic cells in mouse small intestine crypts. In irradiated control group 4.4±0.5 apoptotic cells per crypt were observed. In DTT-administered and irradiated mice, only 2.1±0.4 apoptotic cells per crypt was observed.In vitro andin vivo data obtained in this study showed that DTT reduced radiation-induced damages and it seems that the possible radioprotective mechanisms of action of DTT are prevention of chromosome aberration.     

Dose and Time Dependence of Targeted and Untargeted Effects after Very Low Doses of α-Particle Irradiation of Human Lung Cancer Cells

Understanding the effects to human health resulting from exposure to low doses of ionizing radiation is a persisting challenge. No one questions the deleterious consequences for humans following exposure to high radiation doses; however, in the low dose range, the complex and to some extent unknown cellular responses raise important misgivings about the resulting protective or potentially detrimental effects. Bystander effects are involved in low dose exposures, being characterized by the appearance in unirradiated cells of a cellular damage associated with direct radiation exposure. The purpose of our work was to assess, by using clonogenic and micronuclei assays, the dose and time dependence of the bystander response after cells exposure to very low doses of α-particles and to evaluate its importance in the overall induced damage. The study includes an irradiated cells culture, a medium transfer culture with non-irradiated cells and a culture with irradiated cells after centrifugation. We observed a non-negligible contribution of the bystander effects in the overall cellular damage. Low-dose hyper-sensitivity was observed for medium transfer and irradiated cells after centrifugation cultures. Delayed and earlier cellular damage were similar in almost all experiments, suggesting an effectiveness of irradiated medium to induce a bystander response soon after irradiation.     

TGF-B3 Dependent Modification of Radiosensitivity in Reporter Cells Exposed to Serum From Whole-Body Low Dose-Rate Irradiated Mice

Prior findings in vitro of a TGF-β3 dependent mechanism induced by low dose-rate irradiation and resulting in increased radioresistance and removal of low dose hyper-radiosensitivity (HRS) was tested in an in vivo model. DBA/2 mice were given whole-body irradiation for 1 h at low dose-rates (LDR) of 0.3 or 0.03 Gy/h. Serum was harvested and added to RPMI (4% mouse serum and 6% bovine serum).This medium was transferred to reporter cells (T-47D breast cancer cells or T98G glioblastoma cells). The response to subsequent challenge irradiation of the reporter cells was measured by the colony assay. While serum from unirradiated control mice had no effect on the radiosensitivity in the reporter cells, serum from mice given 0.3 Gy/h or 0.03 Gy/h for 1 h removed HRS and also increased survival in response to doses up to 5 Gy. The effect lasted for at least 15 months after irradiation. TGF-β3 neutralizer added to the medium containing mouse serum inhibited the effect. Serum from mice given irradiation of 0.3 Gy/h for 1 h and subsequently treated with iNOS inhibitor 1400W did not affect radiosensitivity in reporter cells; neither did serum from the unirradiated progeny of mice given 1h LDR whole-body irradiation.     

Protective effects of curcumin against gamma radiation-induced ileal mucosal damage

The major objective of this study was to test curcumin as a potential radioprotectant for the ileum goblet cells of the rat. Wistar albino rats were used in the study. Group A was the control group and group B was the single dose radiation group. Group C was the two dose radiation group (4 days interval). The rats in groups D and E were given a daily dose of 100 mg/kg of curcumin for 14 and 18 days, respectively. During the curcumin administration period, the rats in group D were exposed to abdominal area gamma (γ)-ray dose of 5 Gy on the 10th day and group E was exposed to same dose radiation on the 10th and 14th day. Irradiation and treatment groups were decapitated on the 4th day after exposure to single or two-dose irradiation and ileum tissues were removed for light and electron microscopic investigation. Single or two dose 5 Gy γ-irradiation caused a marked intestinal mucosal injury in rats on the 4th day. Radiation produced increases in the number of goblet cells. Curcumin appears to have protective effects against radiation-induced damage, suggesting that clinical transfer is feasible.     

Nonlinearity in MCF7 Cell Survival Following Exposure to Modulated 6 MV Radiation Fields: Focus on the Dose Gradient Zone

The study of cell survival following exposure to nonuniform radiation fields is taking on particular interest because of the increasing evidence of a nonlinear relationship at low doses. We conducted in vitro experiments using the MCF7 breast cancer cell line. A 2.4 × 2.4 cm² square area of a T25 flask was irradiated by a Varian Novalis accelerator delivering 6 MV photons. Cell survival inside the irradiation field, in the dose gradient zone and in the peripheral zone, was determined using a clonogenic assay for different radiation doses at the isocenter. Increased cell survival was observed inside the irradiation area for doses of 2, 10, and 20 Gy when nonirradiated cells were present at the periphery, while the cells at the periphery showed decreased survival compared to controls. Increased survival was also observed at the edge of the dose gradient zone for cells receiving 0.02 to 0.01 Gy when compared with cells at the periphery of the same flask, whatever the isocenter dose. These data are the first to report cell survival in the dose gradient zone. Radiotherapists must be aware of this nonlinearity in dose response.     

Correlation between cell survival and micronuclei-induction in HeLa cells treated with adriamycin after exposure to various doses of gamma-radiation

The effect of 10 microg/ml of adriamycin (doxorubicin) post-treatment was studied in HeLa cells exposed to 0, 0.5, 1, 2 and 3 Gy of gamma radiation. The survival of HeLa cells declined in a dose dependent manner in both irradiation+PBS and irradiation+ADR groups. Treatment of adriamycin immediately after irradiation resulted in a significant decline in the cell survival. The surviving fraction of HeLa cells reduced to 0.61 after exposure to 0. 5 Gy in the irradiation+ADR group, whereas a similar effect (i.e. surviving fraction of 0.61) was obtained for 3 Gy in the irradiation+PBS group. In contrast, the frequency of micronuclei increased in a dose dependent manner in both irradiation+PBS and irradiation+ADR groups. A significant elevation in the frequency of micronuclei was observed in the latter when compared with the former group. The dose response for both groups was linear quadratic. The cell proliferation indices also showed a dose dependent decline in both the groups. The decline in the cell proliferation was significantly higher in the irradiation+ADR group when compared with the irradiation+PBS group. A close correlation between the cell survival and micronuclei induction was observed in both groups, where the cell survival declined with the elevation in the micronuclei frequency. The relationship between cell survival and micronuclei induction was linear quadratic.     

Anti-apoptotic,anti-inflammatory,and immunomodulatory activities of 3,3′-diselenodipropionic acid in mice exposed to whole body γ-radiation

The present study was designed to evaluate the possible protective effects of 3,3′-diselenodipropionic acid (DSePA), a potent radioprotector, against oxidative organ damage induced by whole body γ-irradiation and explore its mechanistic effects. The mice were subjected to whole body γ-irradiation at 5 Gy for the detection of oxidative stress, apoptosis, and proliferation in the intestinal (jejunum) tissue and at 7 Gy for the examination of intestinal inflammation and immune imbalance. Groups of mice received intraperitoneal injections of DSePA (2 mg/kg/day) or vehicle (phosphate-buffered saline) for 5 consecutive days prior to irradiation. The whole body γ-irradiation of mice led to the induction of oxidative stress and apoptosis in the intestinal tissue, and pretreatment with DSePA significantly reduced both these parameters. It was also found to abrogate the radiation-induced intestinal inflammatory response and augment the proliferation of intestinal cells. Additionally, irradiation-induced polarization of Th1/Th2 immune balance toward the Th2-dominant direction and pretreatment with DSePA ameliorated this shift, which may be beneficial for the recovery from radiation injury. In conclusion, pretreatment with DSePA prevented radiation-induced oxidative damage in small intestine and the underlying mechanisms responsible for this could be attributed to inhibition of oxidative stress, apoptosis, and inflammation.     

The effects of celecoxib, a COX-2 selective inhibitor, on acute inflammation induced in irradiated rats

The potential value of selective and non-selective COX-2 inhibitors in preventing some of the biochemical changes induced by ionizing radiation was studied in rats exposed to carrageenan-induced paw edema and 6-day-old air pouch models. The animals were exposed to different exposure levels of γ-radiation, namely either to single doses of 2 and 7.5 Gy or a fractionated dose level of 7.5 Gy delivered as 0.5 Gy twice weekly for 7.5 weeks. The inflammatory response produced by carrageenan in irradiated rats was markedly higher than that induced in non-irradiated animals, and depended on the extent of irradiation. Celecoxib, a selective COX-2 inhibitor, in doses of 3, 5, 10, and 15 mg/kg was effective in reducing paw edema in irradiated and non-irradiated rats in a dose-dependent manner as well as diclofenac (3 mg/kg), a non-selective COX inhibitor. Irradiation of animals before the induction of the air pouch by an acute dose of 2 Gy led to a significant increase in leukocytic count, as well as in the level of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), LTB₄, PGE₂ (as an index of COX-2 activity), TXB₂ (as an index of COX-1 activity), and the plasma level of MDA. This increase in level of these parameters was more marked than that observed in the non-irradiated animals subjected to the inflammagen. The blood GSH level was not affected by the dose of irradiation used, whereas superoxide dismutase (SOD) activity was suppressed. In many respects, celecoxib (5 mg/kg) was as potent as diclofenac in decreasing the elevated levels of IL-6, IL-1β, TNF-α, LTB₄, PGE₂, but lacked any significant effect on TXB₂ level. Since it is mostly selective for COX-2 with a rare effect on COX-1 enzyme, both drugs at the selected dose levels showed no effect on level of MDA, GSH, and SOD activity.     

Protective role of metallothionein in bone marrow injury caused by X-irradiation

In order to elucidate the role of metallothionein (MT) in preventing the adverse effects of X-ray irradiation, we examined the susceptibility of MT-I/II null mice to bone marrow injury caused by X-irradiation and effects of pretreatment with MT-inducing metals on X-ray injury. Eight-week-old male mice were exposed to a single bout of whole-body X-irradiation at a dose between 0.1 and 6.0 Gy. The numbers of leukocytes, reticulocytes with micronuclei (MNRET) in the blood, and polychromatic erythrocytes with micronuclei (MNPCE) in the bone marrow were determined 24 hr after X-irradiation. X-irradiation significantly decreased the total number of leukocytes in MT-I/II null mice and wild-type mice in a dose-dependent manner, but the total number of leukocytes was significantly lower in MT-I/II null mice than in wild-type mice at a low dose of irradiation, between 0.1 and 1.0 Gy. X-irradiation (0.1 and 0.5 Gy) significantly increased the appearance of MNRET and MNPCE in both strains, but the increase was greater in the MT-I/II null mice than in the wild-type mice. Additional groups of mice were pre-administered bismuth nitrate or zinc sulfate to induce MT in the bone marrow cells prior to X-irradiation; the X-ray injury was prevented by such treatments in wild-type mice only. Thus, the present results suggest that MT plays a protective role against a low dose of X-ray injury.     

AC480, formerly BMS-599626, a pan Her inhibitor,enhances radiosensitivity and radioresponse of head and neck squamous cell carcinoma cells in vitro and in vivo

Purpose: The present study investigated the effect of AC480, a small molecule pan-HER tyrosine kinase inhibitor, on in vitro radiosensitivity and in vivo radioresponse of a human head and neck squamous cell carcinoma cell line. Methods: HN-5 cells were exposed to γ-radiation with and without AC480 and assayed for proliferation, clonogenic survival, apoptosis, cell cycle distribution, and DNA damage. The cells were analyzed by immunoprecipitation and western blotting for proteins involved in apoptosis, cell cycle regulation, and the EGFR pathway. The effect of AC480 on tumor radioresponse was assessed by tumor growth delay assay using HN5 tumor xenografts generated in nude mice. Results: At the molecular level, in HN-5 cells the agent inhibited the expression of pEGFR, pHER2, cyclins D and E, pRb, pAkt, pMAPK, pCDK1 and 2, CDK 6, and Ku70 proteins. The drug also induced accumulation of cells in the G1 cell cycle phase, inhibited cell growth, enhanced radiosensitivity, and prolonged the presence of γ-H₂AX foci up to 24 h after radiation. AC480 did not increase the percentage of cells undergoing radiation-induced apoptosis. The drug given before and during irradiation improved the radioresponse of HN5 tumors in vivo. Conclusion: AC480 significantly enhanced the radiosensitivity of HN-5 cells, expressing both EGFR and Her2. The mechanisms involved in the enhancement included cell cycle redistribution and inhibition of DNA repair. Both in vitro and in vivo data from our study suggest that AC480 has potential to increase tumor response to radiotherapy.     

Susceptibility of mouse splenic cells to oxidative DNA damage by x-ray irradiation

Susceptibility to oxidative stress by X-ray irradiation was examined in splenic cells of BDF1 mouse and fetal human lung fibroblasts, TIG-7. Survival rates of splenic cells irradiated with X-rays were lower than those of TIG-7 cells irradiated similarly. The content of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) immediately after X-ray irradiation in the DNA of splenic cells increased until 2 Gy irradiation, but remained constant above 2 Gy. The 8-oxodG contents rose in proportion to the dose of X-rays in TIG-7 cells. Although the survival rate of splenic cells exposed to 1 Gy irradiation decreased with time, the survival rate of TIG-7 cells remained unchanged. The 8-oxodG content in splenic cells irradiated with X-rays did not decrease even 48 h after irradiation, while that in TIG-7 cells decreased with time, and recovered to the pre-irradiation level after 48 h. A DNA ladder was observed in splenic cells 2 h after X-ray irradiation, but the ladder was not found in fibroblasts. Furthermore, caspase-3 activity increased after X-ray irradiation of splenic cells. These results indicate that splenic cells are sensitive to oxidative stress induced by X-ray irradiation and that splenic cells damaged by even low doses of X-rays are removed through apoptosis rather than by a repair pathway.     

Delayed Mitogenic Stimulation Decreases DNA Damage Assessed by Micronucleus Assay in Human Peripheral Blood Lymphocytes After 60Co Irradiation

While contradictory reports are available on the yield of dicentric chromosomes (DC) in blood samples stored at different temperature and stimulated to enter into cell cycle, various times gap followed by exposure, limited information is available on the micronucleus (MN) assay. As scoring the micronuclei frequency from the blood lymphocytes of exposed individuals is an alternative to the gold standard DC assay for triage applications, we examined radiation induced MN yield in delayed mitogenic stimulation after irradiation of in vitro. Peripheral blood lymphocytes (PBL) were exposed to low LET (⁶⁰Co) radiation dose (0.1 to 5Gy) and incubated at 37°C for 2, 6 and 24 hours. The MN frequency obtained in blood samples stimulated 2 hours post-irradiation showed a dose dependent increase and used to construct the dose-response curve. Further, the results also showed that blood samples stimulated twenty four hours of post-irradiation, a significant reduction (p<0.05) in MN frequencies were obtained when compared to that of blood samples stimulated two hours and six hours after post-irradiation (0.5, 1, 3 and 5Gy). The observed result suggests that the prolonged PBL storage without mitogenic stimulation could lead to interphase cell death and a delayed blood sampling could results in underestimation of dose in biological dosimetry.     

A novel Chk inhibitor,XL-844, increases human cancer cell radiosensitivity through promotion of mitotic catastrophe

Check point kinases (Chk) play a major role in facilitating DNA repair upon radiation exposure. We tested the potency of a novel inhibitor of Chk1 and Chk2, XL-844 (provided by Exelixis Inc., CA, USA), to radiosensitize human cancer cells grown in culture and investigated the underlying mechanisms. HT-29 cells (a human colon cancer line) were exposed to XL-844, radiation, or both, and assessed for clonogenic cell survival. Treatment-dependent effects on phosphorylated forms of Chk proteins were assessed by Western blots. Further mechanistic investigations in HT-29 cells included cell cycle analysis by flowcytometry and assessment of DNA repair kinetics by immuno-cytochemistry (ICC) for nuclear appearance of the phosphorylated form of histone 2AX protein (γ-H2AX) staining. Cells undergoing mitotic catastrophe were identified by irregular pattern of mitotic spindle markers α and γ-tubulin staining by ICC. XL-844 enhanced radiosensitivity in a dose and schedule-dependent manner and the enhancement factor was 1.42 at 0.5 survival fraction. Mechanistically XL-844 abrogated radiation-induced Chk2 phosphorylation, induced pan-nuclear γ-H2AX, and prolonged the presence of radiation-induced γ-H2AX foci, and promoted mitotic catastrophe. In conclusion, our data showed that inhibition of Chk2 activity by XL-844 enhanced cancer cell radiosensitivity that was associated with inhibition of DNA repair and induction of mitotic catastrophe.     

Effect of Low Dose Radiation on Differentiation of Bone Marrow Cells into Dendritic Cells

Low dose radiation has been shown to be beneficial to living organisms using several biological systems, including immune and hematopoietic systems. Chronic low dose radiation was shown to stimulate immune systems, resulting in controlling the proliferation of cancer cells, maintain immune balance and induce hematopoietic hormesis. Since dendritic cells are differentiated from bone marrow cells and are key players in maintaining the balance between immune activation and tolerance, it may be important to further characterize whether low dose radiation can influence the capacity of bone marrow cells to differentiate into dendritic cells. We have shown that bone marrow cells from low dose-irradiated (γ-radiation, 0.2Gy, 15.44mGy/h) mice can differentiate into dendritic cells that have several different characteristics, such as expression of surface molecules, cytokine secretion and antigen uptake capacity, when compared to dentritic cells differentiated from the control bone marrow cells. These differences observed in the low dose radiation group can be beneficial to living organisms either by activation of immune responses to foreign antigens or tumors, or maintenance of self-tolerance. To the best of our knowledge, this is the first report showing that total-body low dose radiation can modulate the capacity of bone marrow cells to differentiate into dendritic cells.     

Minimal Toxicity to Myeloid Progenitor Cells of Weekly24‐Hr Infusion of High‐Dose 5‐Fluorouracil: Direct Evidence from Colony Forming Unit‐Granulocyte andMonocyte (CFU‐GM) Clonogenic Assay

Although very high doses of 5‐fluorouracil was used in the weekly 24‐h infusion, high‐dose 5‐fluorouracil (2600 mg/m²/week) and leucovorin (500 mg/m²/week) protocol, myelosuppression was surprisingly low. The current study was conducted to investigate the possible mechanism underlying the low myelosuppression. To mimic the clinical situation, peripheral blood progenitor cells collected from 12 patients were used for colony forming unit‐granulocyte and monocyte clonogenic assay; and 2 representative modes of 5‐fluorouracil exposure (30 min. versus 24 hr) were examined for cytotoxic effects on human myeloid progenitor cells. Previous pharmacokinetic studies have estimated the concentrations of 5‐fluorouracil in the bone marrow to be 200–400 μM and 1–2 μM for the 30 min. infusion (600–900 mg/m²) and the 24 hr‐infusion (1000–2000 mg/m²) regimens, respectively. The results of our colony‐forming unit‐granulocyte and monocyte clonogenic assay showed that 24‐hr exposure to 5‐fluorouracil (2 μM) and 30 min. exposure to 5‐fluorouracil (100 μM) resulted in 27.2% and 78.2% inhibition of the colony formation, respectively. Our data provided direct evidence which may explain why myelotoxicity is significantly less in weekly 24 hr infusion of fluorouracil than in the conventional bolus regimens.     

Correlation of micronuclei-induction with the cell survival in HeLa cells treated with a base analogue,azidothymidine (AZT) before exposure to different doses of gamma-radiation

The effect of 0.1 microM azidothymidine (AZT) a pyrimidine analogue has been studied on the growth kinetics, cell survival and micronuclei formation in HeLa cells exposed to 0, 0.25, 0.5, 1, 2 and 3 Gy of 60Co gamma-radiation. The AZT pretreatment resulted in a significant decline in the cell growth kinetics, cell survival and cell proliferation indices when compared with the PBS+irradiation group at 20, 30 and 40 h post-irradiation. Conversely, the frequency of micronucleated binucleate cells (MNBNC) elevated in a dose dependent manner in both PBS+irradiation and AZT+irradiation group. This elevation in MNBNC-induction was significantly higher in the latter when compared with the former group at all post-irradiation scoring time periods studied. The dose-response relationship for micronuclei induction for both the PBS+irrradiation and AZT+irradiation groups was linear. The biological response was studied by correlating the cell survival with MNBNC-induction. The cell survival and MNBNC-induction showed a close but inverse relationship and this relationship gave a best fit on the linear quadratic model.     

Effects of sublethal doses of ionizing radiation on schedule-controlled performance in rats

Male rats responded under a fixed-ratio (FR) 50 or a fixed-interval (FI) 120 sec schedule of milk delivery. Separate groups were acutely exposed to 0.5, 1.5, 4.5 or 0 (FI only) Gray (Gy) of cobalt-60 gamma radiation 3 times at 43-day intervals. All rats received an acute dose of 6.5 Gy 64 days after the last of these exposures. One-half and 1.5 Gy did not alter FR or FI performance significantly. After 4.5 Gy, no observable changes in performance occurred within 1 hr of exposure. Maximal reductions in FR response rates occurred 24 hr after exposure and recovery followed over the subsequent 72 hr. Postreinforcement pause was increased and running response rate was decreased by 4.5 Gy. Similar effects were found after each 4.5 Gy exposure. In contrast, FI performance (overall response rate, postreinforcement pause, running response rate, index of curvature) was not altered reliably by 4.5 Gy. Both FR and FI response rates were reduced by 6.5 Gy beginning 24 hr after exposure; FR rates tended to be reduced more than FI rates 24–72 hr after exposure. Response rates under both schedules recovered gradually over 7 weeks. The behavioral effects of 6.5 Gy did not vary as a function of irradiation history. In contrast, irradiation history affected survival in that 4/9 rats previously exposed to 4.5 Gy died during weeks 4–5 after 6.5 Gy, whereas there were no deaths in the rats previously exposed to lower doses. Radiogenic disruption of operant performance was dose-related, reversible, noncumulative and dependent on the schedule of reinforcement.     

Vincristine increases the genomic instability in irradiated cultured human peripheral blood lymphocytes

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.