Satyamitra, M. M., Uma Devi, P., Murase, H. and Kagiya, V. T. In Vivo Postirradiation Protection by a Vitamin E Analog, α-TMG. Radiat. Res. 160, 655–661 (2003).
The water-soluble vitamin E derivative α-TMG is an excellent radical scavenger. A dose of 600 mg/kg TMG significantly reduced radiation clastogenicity in mouse bone marrow when administered after irradiation. The present study was aimed at investigating the radioprotective effect of postirradiation treatment with α-TMG against a range of whole-body lethal (8.5–12 Gy) and sublethal (1–5 Gy) doses of radiation in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from micronuclei and chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 600 mg/kg TMG within 10 min of lethal irradiation increased survival, giving a dose modification factor (DMF) of 1.09. TMG at doses of 400 mg/kg and 600 mg/kg significantly reduced the percentage of aberrant metaphases, the different types of aberrations, and the number of micronucleated erythrocytes. DMFs of 1.22 and 1.48 for percentage aberrant metaphases and 1.6 and 1.98 for micronuclei were obtained for 400 mg/kg and 600 mg/kg TMG, respectively. No drug toxicity was observed at these doses. The effectiveness of TMG when administered postirradiation suggests its possible utility for protection against unplanned radiation exposures.
INTRODUCTION
A variety of chemical and biological agents, including food products (1), metal chelators (2), cytokines like interleukin 1 and tumor necrosis factor, and plant extracts and their isolated chemical components (3, 4), have been studied for their ability to reduce the deleterious effects of radiation. Most of them are potent radioprotectors when administered prior to irradiation (2–4). There are a few agents that protect cells against radiation damage when administered postirradiation (5–9). Among them is vitamin E, a singlet oxygen and superoxide anion radical scavenger (10, 11). This lipophilic antioxidant is especially effective in the biological membranes, but it is unlikely to scavenge the active oxygen that may be generated in the aqueous phase (12). 2-(α-d-Glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol, or α-TMG, a water-soluble analog of vitamin E, has demonstrated a similar protection profile as the parent compound and excellent antioxidant activity in both aqueous and lipid phases (12). A preliminary study showed that an intraperitoneal (i.p.) injection of α-TMG within 10 min of irradiation significantly reduced radiation-induced clastogenicity in mouse bone marrow at an optimum dose of 600 mg/kg body weight (13). Therefore, a detailed investigation was done to study the in vivo protection by α-TMG against acute lethal and sublethal doses of ionizing radiation.
MATERIALS AND METHODS
Animals
All experiments were carried out on random bred Swiss albino mice of both sexes from the animal colony of the Department of Radiobiology that were 6–8 weeks old and weighed 25 ± 5 g (14). The colony was maintained under controlled conditions of temperature (23 ± 2°C) and humidity (50 ± 5%) and a 12-h light–dark cycle. The animals were housed in sanitized polypropylene cages containing autoclaved paddy husk as bedding. They had free access to standard mouse food and acidified water.
Irradiation
Mice were placed in well-ventilated Perspex boxes (23.5 × 23.5 × 3.5 cm, partitioned into 3 × 3 × 11-cm cells for each animal) and exposed to whole-body γ rays from a 60Co Gammatron teletherapy unit (Siemens, Germany) at a dose rate of 1.6 Gy/min and an SSD of 90 cm. The irradiation facility was provided by the Department of Radiotherapy and Oncology, S.S.B. Cancer Hospital, Manipal.
Chemicals
TMG, a thermo- and photosensitive drug, was obtained in powder form. It was synthesized by Dr. H. Murase (Gifu, Kyoto, Japan) (15). Colchicine and Giemsa were purchased from Sigma Chemicals (St. Louis, MO) and acridine orange from BDH (England). The rest of the chemicals were purchased from local firms (India) and were of the highest purity grade.
Administration of TMG
TMG was weighed in the dark and dissolved in double-distilled water immediately before use. The animals were injected i.p. with a single dose of 600 mg/kg for survival studies and 400 or 600 mg/kg TMG for cytogenetic studies within 10 min of irradiation.
Experimental Protocol
All the studies were conducted according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.
Survival Studies
Groups of 30 animals each were irradiated with 8.5, 9, 9.5, 10, 11 or 12 Gy followed by a single i.p. injection of double-distilled water/600 mg/kg TMG. The animals were monitored daily for 30 days, and mortality was recorded (4). Actuarial survival curves were drawn by plotting data as percentage survival as a function of postirradiation day using Origin version 6.0. The dose modification factor (DMF) was calculated from the graph plotted with trend of probit survival as a function of radiation dose.
Cytogenetic Studies in Mouse Bone Marrow
Cytogenetic analysis was carried out on groups of five mice each. The mice received an acute dose of 0, 1, 2, 3, 4 or 5 Gy followed by a single i.p. injection of 400 or 600 mg/kg TMG within 10 min of exposure. One group was sham-irradiated and injected with double-distilled water and served as the control. Twenty-two hours after irradiation, the animals were injected i.p. with 0.3 ml/mouse of 0.025% colchicine and killed humanely 2 h later by cervical dislocation. Both femurs were dissected out and cleaned free of adhering tissue. Bone marrow from one femur each was used for chromosome preparation and for the micronucleus assay.
Chromosomal Analysis
The metaphase plates were prepared by the routine air-drying method (16). Briefly, the bone marrow from femurs was aspirated and washed in saline, treated hypotonically (0.559% KCl), fixed in 3:1 methanol:acetic acid, dried and stained with 4% Giemsa. Chromosomal aberrations were scored under a light microscope (AO Reichert). Chromatid and chromosome breaks, fragments, rings and dicentrics, cells showing 10 or more aberrations (severely damaged cells), and polyploids were scored separately (17). Data are presented as means ± standard errors (SE) for five mice.
Micronucleus Study
The micronucleus assay was carried out according to the method of Schmid (18). The bone marrow was flushed out into 0.84% NaCl and processed as described earlier (19). The slides were stained with 0.125% acridine orange in Sorenson's buffer and screened under a fluorescence microscope (Carl Zeiss photomicroscope) using a 40× objective. Polychromatic erythrocytes and normochromatic erythrocytes were scored in a total of 10,000 erythrocytes from each animal. The micronucleated polychromatic erythrocytes and normochromatic erythrocytes were counted and expressed as micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes/1000 cells. The polychromatic erythrocyte/normochromatic erythrocyte ratio (P/N ratio) was also calculated.
Statistical Analysis
Overall animal survival was compared by the log-rank method from the actuarial survival curves (4) using Microcal Origin 6.0.
Statistical evaluation in cytogenetic studies was done by one-way ANOVA using GraphPAD InStat software. A value of P < 0.05 was considered significant. Homogeneity of variance was tested. Radiation dose–response curves were fitted with linear (Y = C + αD) and linear-quadratic (Y = C + αD + βD2) models. The graphs were fitted using Microcal Origin, Version 2.8.
RESULTS
Survival Studies
No mortality was seen after 8.5 Gy radiation alone; an increase in the radiation dose resulted in a steep rise in mortality. Forty-three percent mortality was observed after 9 Gy, rising to 57% after 9.5 Gy and 80% after 10 Gy (Fig. 1A). All animals irradiated with 11 and 12 Gy died within 2 weeks after exposure. Radiation sickness symptoms were observed in the 11- and 12-Gy groups from day 2 after irradiation; lethargy, followed by decreased food and water intake, was seen in 60–75% of the animals. Ruffling of fur and facial edema appeared on day 4 in 13% of the 11-Gy animals and 20% of the 12-Gy animals. Weight loss in the 11-Gy and 12-Gy mice reached a nadir of 45% compared to the controls. The first deaths occurred on day 4, and more than 50% of the animals died (53% and 65% for 11 and 12 Gy, respectively) within 8 days, with none surviving beyond day 14 postirradiation. In the 10- and 9–9.5-Gy groups, facial edema and diarrhea started on days 6 and 7, respectively, with a significant weight loss (30–40% of control). The animals that survived to 30 days showed recovery from the symptoms and a gradual increase in weight, although they remained 10–15% lighter than the controls.
Treatment with TMG significantly increased the survival of animals in the 9–11-Gy groups (Fig. 1B); survival was 86% after 9 Gy, 77% after 9.5 Gy, 40% after 10 Gy, and 13% after 11 Gy compared to 53, 43, 20 and 0%, respectively, in the corresponding radiation-alone groups. Even at 12 Gy, 10% of the animals survived beyond 14 days, although all animals died by 30 days. The severity of radiation sickness was reduced in the drug-treated groups, with fewer animals showing facial edema and diarrhea (4% in 9.5-Gy, 7% in 10-Gy, and 8% in 11-Gy irradiated groups treated with TMG compared to 9, 12 and 15% in the corresponding radiation-alone groups). Weight loss was observed in the 9.5-, 10- and 11-Gy groups (15–30%); animals whose weight loss exceeded 40% in the 10- and 11-Gy groups died. The remaining animals survived to 30 days, with an increase in activity, food and water intake, and weight. Statistical analysis showed that an acute administration of 600 mg TMG significantly (P < 0.05) increased the overall survival. Probit analysis of survival showed a linear dose-dependent decrease (r2 = 0.99 for curves for radiation and radiation + TMG). The LD50/30 for radiation alone was 9.2 Gy and increased to 10.1 Gy with TMG post-treatment, giving a DMF of 1.09.
Chromosomal Aberration Analysis
Radiation produced a significant dose-dependent increase in the percentage of aberrant metaphases and different types of aberrations compared to the sham-treated controls. The percentage of aberrant cells increased from 7% at 1 Gy to 61% at 5 Gy (Fig. 2). The most common aberrations were breaks, fragments, rings and dicentrics; at doses of 3–5 Gy, cells with multiple aberrations and polyploids increased significantly (P < 0.001) above the control levels (Table 1).
Post-treatment with either dose of α-TMG resulted in a significant (P < 0.001) decrease in the percentage of aberrant metaphases 24 h post-treatment (Fig. 2). However, the protective effect of 600 mg TMG was significantly (P < 0.001) greater than that of 400 mg TMG. Qualitatively, post-treatment with 400 mg TMG significantly reduced the frequency of the major aberrations like breaks and fragments at all radiation doses and the frequency of rings and dicentrics and severely damaged cells at doses of 3–5 Gy. The reduction in polyploids was significant (P < 0.05) at the higher radiation doses (4–5 Gy). The higher drug dose of 600 mg was more effective and significantly lowered all types of aberrations as well as polyploids and severely damaged cells at all doses of radiation. TMG alone produced a slight increase in the percentage of aberrant cells, but it was not significantly different from the control value.
The dose–response curves for all groups fitted both linear (r2 = 0.98–0.99) and linear-quadratic (r2 = 0.99) models equally well. The linear dose–response curves were used to calculate the slopes. The values of the slopes (means ± SD) were 13.1 ± 0.9 (95% CI: 14.9–11.3) for radiation alone, 9.8 ± 0.85 (95% CI: 11.5–8.1) for radiation + 400 mg TMG, and 8.1 ± 1.2 (95% CI: 10.4–5.7) for radiation + 600 mg TMG. The slopes of the curves for the 400- and 600-mg TMG post-treatments were significantly (P < 0.01–0.001) lower than that of radiation alone. The DMF was calculated as the ratio of the radiation dose required for inducing 50% of the maximum damage observed in the radiation-alone group to that required for the drug + radiation groups. The DMFs were 1.22 for 400 mg and 1.48 for 600 mg TMG.
Micronucleus Assay
Irradiation resulted in a significant dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes and a significant decrease in the P/N ratio (Table 2). Both doses of TMG significantly decreased the micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes; this effect was dependent on dose (Fig. 3). As observed for chromosomal aberrations, the maximum reduction was shown with 600 mg TMG, which significantly decreased the frequency of micronucleated polychromatic erythrocytes compared to both radiation alone and radiation + 400 mg TMG at all doses of radiation. Post-treatment with TMG also reduced the number of radiation-induced micronucleated normochromatic erythrocytes, the decrease being significant at radiation doses above 1 Gy.
As in the case of aberrant metaphases, the dose–response curves for micronucleated polychromatic erythrocytes fit linear (r2 = 0.98) and linear-quadratic (r2 = 0.99) models equally well. The slopes calculated from the linear dose–response curves were 16.36 ± 0.73 (95% CI: 14.9–17.8) for radiation alone, 10.8 ± 0.6 (95% CI: 9.7–11.9) for radiation + 400 mg TMG, and 9.3 ± 0.6 (95% CI: 8.2–10.4) for radiation + 600 mg TMG. The slopes of the curves for the drug post-treatment groups were significantly (P < 0.001) lower than that of radiation-alone group. The DMFs were calculated to be 1.6 for 400 mg and 1.98 for 600 mg TMG. Comparison of the effect of 400 mg/kg and 600 mg/kg TMG post-treatment groups show that the increase in drug dose significantly increased the protection, as seen in both chromosomal aberrations and micronucleated polychromatic erythrocytes.
TMG increased the P/N ratio at both doses, with 600 mg TMG producing a greater effect (Table 2). The P/N ratio increased to normal levels at 1 Gy and was significantly above the latter at 2–5 Gy. TMG increased the P/N ratio significantly (P < 0.001) above the control level to 1.2 at 400 mg and 1.5 at 600 mg in the sham-irradiated animals.
DISCUSSION
The radioprotective effect of α-TMG was studied both at the cellular level and in the whole animal. The current results for radiation sickness and mortality in mice after lethal whole-body exposure follow a trend similar to that observed in earlier studies (4). At doses of 9–10 Gy, no deaths occurred before 8 days, but animals began to die at day 9, and most deaths took place between days 13 and 22, which shows that the major cause of death was bone marrow syndrome. As the radiation dose was increased to 11 and 12 Gy, more than 50% of the animals died by 8 days, suggesting that death was predominantly due to gastrointestinal (GI) syndrome. TMG post-treatment increased 30-day survival at 9–10 Gy, indicating significant protection against bone marrow syndrome. Even at 11 Gy and the supralethal dose of 12 Gy that caused the GI syndrome, survival rates of 70 and 48%, respectively, were observed at day 8, which implies that the drug gave some protection to the intestinal mucosa. However, the protection was not great, since all animals in the 12-Gy group died by day 16 and only 13% in the 11-Gy drug-treated group survived to 30 days.
Thus the results demonstrate that TMG is effective in protecting against bone marrow syndrome and related mortality, but it is not very effective against the GI syndrome. A similar effect has been reported for the antioxidant aminothiol MPG (20) and the plant flavonoids orientin and vicenin (4). At doses causing GI damage, both the GI system and the hemopoietic system are extensively damaged, with an ensuing decrease in immunity (21). It has been reported that enhanced susceptibility to infections due to hematopoietic damage progresses in parallel with radiation-induced damage of the epithelial lining of the intestines, allowing for an influx of the normal intestinal flora into the bloodstream (22). Deaths occurring between 9–12 days are usually attributed to bacterial infection. This can complicate the effect by allowing bacterial infiltration through the damaged mucosa, causing death due to infection. The TMG post-treatment groups exposed to 8–10 Gy showed significantly higher survival during this period than the corresponding radiation-alone groups, suggesting that TMG may contain the infection by reducing damage to the intestinal lining. Therefore, at doses causing bone marrow syndrome, protection by TMG is more pronounced than at doses causing GI syndrome. This is further supported by our findings that TMG significantly decreased chromosomal damage in bone marrow at high sublethal doses (discussed later).
The DMF of 1.09 obtained with TMG for a range of lethal doses (8.5–12 Gy) does not differ greatly from the DMF of 1.11 reported for vitamin E at 8.5 Gy (8). This shows that the synthetic, water-soluble analog has a protective effect similar to that of the parent vitamin E against radiation-induced mortality in mice. These DMFs are low compared to those of 1.6–1.8 observed for different thiols (23). In general, DMFs lower than 1.4 for 30-day survival have been reported for naturally occurring radioprotectors like the Ocimum flavonoids, polysaccharides and cytokines (4, 24, 25). However, TMG, like vitamin E (8), is effective when administered postirradiation and does not exhibit any detectable toxicity at the dose used.
Cytogenetic studies further confirm the protection of bone marrow by TMG against sublethal radiation injury. Radiation damage to chromosomes is manifested mainly as breaks and fragments; radiation-induced micronuclei originate from acentric fragments (13, 26). Increases in chromosomal aberrations and micronucleus frequency were reported in the bone marrow of irradiated mice (14, 16, 27), which is supported by the present data. The protective effect of TMG is evident in both the myeloid (chromosomal aberrations) and the erythroid (micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes) lineages. Post-treatment with both doses of TMG decreased radiation-induced chromosomal aberrations and micronuclei. The higher dose used, 600 mg, reduced both aberrant metaphases and micronucleated erythrocytes to 60–65% of the irradiated control. This is similar to the effect reported for vitamin E, which decreased radiation-induced bone marrow chromosome damage in mice to 60% of the levels in irradiated controls (28).
TMG was highly effective in reducing simple aberrations, indicating significant protection against single-strand breaks. This was also evident in the reduced micronucleus count. A significant decrease in the complex aberrations like rings and dicentrics and severely damaged cells at the higher radiation doses suggests that TMG also gives good protection against double-strand breaks and decreases the proportion of cells with multiple DNA lesions.
The P/N ratio is an indicator of the rate of proliferation. A decrease in the P/N ratio 24 h postirradiation may be attributed to the suppression of erythropoiesis, resulting from the effect of radiation on the cell cycle (27). TMG increased the P/N ratio to near normal levels at the lower radiation doses and to levels significantly above that with radiation alone at higher doses. This may reflect the mitogenic effect of the drug, since the drug by itself resulted in a significant increase in the P/N ratio. A similar effect was observed in an earlier study in which an increase in the drug dose from 200 mg to 800 mg resulted in a significant elevation of the P/N ratio (13). An elevated mitogenic response has been reported in in vitro irradiated splenic lymphocytes treated with vitamin E (29).
The DMF for 30-day survival was 1.09, while at the same drug dose, the DMFs for percentage aberrant metaphases and micronuclei were 1.44 and 1.98, respectively. This suggests that TMG is far more effective against sublethal doses of radiation than against lethal doses. Similar observations have been reported for antioxidant protectors like MPG (20) and the natural flavonoids, orientin and vicenin (4). The 30-day survival of mice after lethal whole-body irradiation can be correlated with hemopoietic recovery and regeneration. Survival after exposure to high doses of radiation depends on survival of a critical number of hemopoietic stem cells and the ability of these cells to generate enough cells of multiple lineages to repopulate the depleted hematopoietic compartment (30). The main cause of death is the severe depletion of the hemopoietic stem cells, which are more sensitive to radiation than the committed progenitors and mature blood cells (31). The mitogenic effect of TMG may help in the faster regeneration of these cells and thereby in the recovery of the hemopoietic compartment at sublethal radiation doses. Enhanced recovery of bone marrow also has been suggested in radioprotection by vitamin E (9).
TMG differs from vitamin E in that a glucopyranosyl moiety replaces the linear phytyl side chain in the latter (15), thereby making it hydrophilic. The similar protective effects of TMG and vitamin E against radiation-induced mortality and cytogenetic damage (discussed earlier) shows that this substitution does not alter the radioprotective effect of TMG. Instead, the structural modification may make the molecule readily available at the crucial sites. The ready water solubility of α-TMG also decreases the complications arising from emulsifiers and other ingredients present in the vehicle required to solubilize vitamin E (8).
The synthetic thiols and natural compounds like the Ocimum flavonoids exert their protective effect when administered prior to irradiation (3, 4). Their use is therefore limited to those instances where exposure can be anticipated, as in radiotherapy, but not in unplanned exposures like nuclear accidents. With its significant protective effect when administered postirradiation, α-TMG appears to have a potential use against unanticipated radiation exposures.
Our previous study (13) showed a dose-dependent increase in protection against radiation clastogenicity in mouse bone marrow from 200 mg to 600 mg/kg TMG. These doses did not show any toxic effect in mice. However, a further increase in the TMG dose to 800 mg/kg resulted in less protection than at 600 mg/kg; moreover, this dose produced chromosomal toxicity (increased percentage aberrant metaphase cells) in unirradiated mice. A dose of 750 mg/kg TMG resulted in 20% mortality within 30 days (13). Thus the maximum tolerated single dose of TMG is about 60% of its LD50. This is comparable to the well-established synthetic thiol protector Amifostine (WR-2721), the maximum tolerated dose for which is 500 mg/kg, i.e. two-thirds of its LD50 (32). Therefore, 600 mg/kg of TMG appears to be a safe dose for radioprotection when a single administration is needed. However, when the drug has to be administered daily with fractionated radiotherapy, such a high dose may not be tolerated. In the case of WR-2721, a single dose of 500 mg/kg body weight (>60% of LD50) is tolerated in mice, but the daily dose that could be safely given for 10 days is only 40–50% of the maximum tolerated dose (33). Lower doses of 200 mg/kg and 400 mg/kg of TMG have been found to give significant protection of chromosomes in mouse bone marrow (13) and may be better tolerated in daily doses. Further studies are in progress to establish a safe dose for daily administration and its effectiveness in ameliorating short- and long-term tissue reactions.
Acknowledgments
This work was supported by a grant from the Health Research Foundation, Japan. This work was carried out at the Department of Radiobiology, Kasturba Medical College, Manipal. The authors are grateful to Prof. P. L. N. Rao, former Dean, Kasturba Medical College, Manipal, India, for providing the facilities.
REFERENCES
FIG. 1.
Actuarial survival curves for mice exposed to different doses (8.5–12 Gy) of whole-body γ radiation with and without post-treatment with 600 mg/kg TMG. Panel A: Radiation alone. Panel B: Radiation + TMG. 8.5 Gy (▪), 9 Gy (•), 9.5 Gy (▴), 10 Gy (▾), 11 Gy (♦), 12 Gy (★)
FIG. 2.
Effect of TMG on γ-ray (1–5 Gy)-induced chromosomal aberrations in mouse bone marrow at 24 h post-treatment. (▪), Radiation alone, slope = 13.1 ± 0.9; (•), radiation + 400 mg TMG, slope = 9.8 ± 0.9, DMF = 1.22; (▴), radiation + 600 mg TMG, slope = 8.0 ± 1.2, DMF = 1.48. The error bars fall within the symbols; therefore, they are not shown
FIG. 3.
Effect of 400/600 mg TMG on γ-ray (1–5 Gy)-induced micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes in mouse bone marrow, observed at 24 h post-treatment. Micronucleated polychromatic erythrocytes: (▪), radiation alone, slope = 16.4 ± 0.7; (•), radiation + 400 mg, slope = 10.8 ± 0.6, DMF = 1.6; (▴), radiation + 600 mg, slope = 9.32 ± 0.55, DMF = 1.98. Micronucleated normochromatic erythrocytes: (□), radiation; (○), radiation + 400 mg; (▵), radiation + 600 mg. The error bars fall within the symbols; therefore, they are not shown
