Mutation Research, 301 (1993) 143-147
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00
R a d i o p r o t e c t i v e effect o f vitamins C and E Soheir M. E1-Nahas a Fathy E. Mattar b and Amal A. Mohamed a a Cell Biology, National Research Centre, Dokki, Cairo, Egypt and b Faculty of Medicine, AI-Azhar University, Cairo, Egypt
(Received 30 June 1992) (Revision received 27 Septemb.er 1992) (Accepted 9 October 1992)
Keywords: Vitamin C; Vitamin E; Radioprotection
Summary Albino rats were treated with aqueous vitamin C solution and vitamin E solution dissolved in olive oil at t~vo concentrations, 100 and 300 m g / k g / d a y , for 6 months. Some of the animals were then subjected to whole-body irradiation. Chromosomal aberrations and mitotic activity in non-irradiated and irradiated groups were recorded. Both vitamins were found to be non-mutagenic. Vitamin C exerted a radioprotective effect but vitamin E was not radioprotective and it suppressed the radioproteetion otherwise produced by olive oil.
Vitamins C and E are two natural antioxidant compounds (Shamberger et al., 1979). Being antioxidants they are expected to help in restricting the propagation of chain reactions initiated by free radicals which are produced either naturally or as a result of environmental factors. Vitamin E was reported to exert a protective effect when administered with chemical mutagens (Dion et al., 1982; Reddy et al., 1983). On the other hand, it showed a slight coclastogenic effect (Gebhart et al., 1985); however, no mutagenic effect has yet been reported. As for vitamin C, it was reported to exert both genotoxic and antimutagenic effects depending on the conditions and system used (Shamberger, 1984). The role of vitamins C and E as protectors against chromosomal damage in-
duced by y-radiation in mammalian somatic cells is investigated.
Materials and methods Animals
Male albino rats (6 months old) were provided by the animal house of the National Research Centre. Food and water were provided ad libiturn. Chemicals
Vitamin C (ascorbic acid) and vitamin E (atocopherol) were produced by Cid and Nile Company of Pharmaceutical and Chemical Industries, Cairo, Egypt, respectively. Dosage and treatment
Correspondence: Dr. S.M. E1-Nahas, Cell Biology, National Research Centre, Dokki, Cairo, Egypt.
Rats were divided into different groups. They received, via a stomach tube, a daily treatment of
144 vitamin C (dissolved in water) or vitamin E (dissolved in olive oil) for 6 months at two different dose levels, 100 and 300 m g / k g / d a y . Control groups were treated with water or olive oil. After treatment was terminated, half the treated and control animals were subjected to whole-body irradiation (400 rad for 13 s) 6 h before they were killed,
Slide preparation and analysis Two hours before they were killed the rats were injected with 6 m g / k g colchicine. The bone marrow cells were aspirated and processed according to standard cytogenetic techniques. Fifty m e t a p h a s e s / a n i m a l were analyzed for structural chromosomal aberrations. In irradiated animals it was difficult to obtain 50 metaphases, therefore a minimum of 25 metaphases was analyzed. The numbers of cells with sticky chromosomes and of pulverized cells (chromosomes were badly fragmented) were recorded in 1000 cells per animal. The obtained results were analyzed statistically using the 2 × 2 contingency table. Results
Effects of vitamin C Effect on rats before exposure to radiation. Chromosomal aberrations in treated and control rats are presented in Table 1. The percentages of aberrant cells in animals treated with 100 and 300 m g / k g vitamin C were found to be not significantly different from those in control animals.
The numbers of sticky and pulverized cells were also not significantly different from those of the control.
Effect of pretreatment on irradiated rats, As expected, animals exposed to radiation without pretreatment with a protector showed a high percentage of cells (49.88%) with structural aberrations. Aberrant cells recorded in animals pretreated with 100 m g / k g vitamin C were significantly reduced ( P < 0 . 0 5 ) . The percentage of aberrant cells was 26% lower than in irradiated animals. A reduction of 14% was found in animals pretreated with 300 m g / k g (Table 1) which was not statistically different from that reported with 100 m g / k g . Treatment of irradiated animals with 100 and 300 m g / k g vitamin C did not cause any significant change in the frequency of sticky or pulverized cells. Effect ofvitamin E Effects on rats before exposure to irradiation. Chromosomal aberrations in animals treated with olive oil (solvent) were not found to be different from those in water-treated animals. Unirradiated animals treated with 100 and 300 m g / k g vitamin E showed a decrease in the percentage of aberrant cells below the control receiving only the solvent (Table 2). The decrease was statistically significant ( P < 0.05) after treatment with 300 m g / k g . No statistically significant differences in numbers of sticky cells were found between control animals and vitamin E-treated ones. The
TABLE 1 CHROMOSOMAL ABERRATIONS IN BONE MARROW CELLS OF IRRADIATED AND NON-IRRADIATED RATS TREATED WITH VITAMIN C Treatment
Control 100 mg/kg 300 mg/kg Control + radiation 100 mg/kg + radiation 300 mg/kg + radiation
Number of rats examined
6 5 4 4 4 4
Numberof cells examined
300 258 217 137 163 161
Cells with structural aberrations Breaks End to end Exchanges associations N % N % N % 0 0.00 2 0.67 8 2.67 // 0.00 1 I).40 2 0.80 3 1.47 0 0.00 2 0.88 1.35 14 10.10 76 53.14 2 75 43.53 3 1.17 15 8.52 83 50.38 6 3.87 13 8.30
Total N 10 3
5 70 63 71
% ±SD 3.33+__3.01 1.20 +_1.79 2.35 +_1.92 49.88+_10.72 37.06+_5.56 42.96 + 11.83
145 TABLE 2 CHROMOSOMAL ABERRATIONS IN BONE MARROW CELLS OF IRRADIATED AND NON-IRRADIATED RATS TREATED WITH VITAMIN E Treatment
Control 100 mg/kg 300 mg/kg Control + radiation 100 mg/kg + radiation 300 mg/kg + radiation
Number of rats examined
Numberof cells examined
5 6 7 4 4 4
251 321 353 146 122 129
Cells with structural aberrations Breaks End to end Exchanges associations
8 7 2 51 36 80
3.18 2.05 0.56 35.88 29.64 63.75
0 0 0 0 0 6
0.00 0.00 0.00 0.00 0.00 4.55
2 0 1 9 5 17
0.40 0.00 0.28 6.27 4,27 13.54
9 7 3 38 35 71
3.58_+0.89 2.05 _+1.36 0.84 _+1.54 26.62_+10.24 28.97-+4.78 56.11 + 12.98
numbers of pulverized cells decreased significantly ( P <0.01) in animals treated with 300 mg/kg.
Effect of pretreatment on irradiated rats. The percentage of cells with chromosomal aberrations in irradiated animals pretreated with olive oil alone was not different from that in animals pre~reated with 100 m g / k g vitamin E. However, pre~:reatment with 300 m g / k g vitamin E in olive oil caused a significant increase ( P < 0.001) in aberrant cells (Table 2). No statistical differences in numbers of sticky or of pulverized ceils between irradiated animals receiving the solvent (control) and those irradiated after pretreatment with vitamin E were observed. Discussion
The mutagenic potential of L,itamins C and E In the present in vivo study, vitamin C (100 and 300 m g / k g ) did not cause any significant increase in cells with chromosomal aberrations. Previous investigations on in vivo exposure to vitamin C gave mainly negative mutagenic responses as tested by induction of micronuclei and sister-chromatid exchanges, inhibition of D N A synthesis and induction of sperm abnormality (reviewed by Shamberger, 1984; Hartman, 1990; Gulati et al., 1989). Contrary to in vivo studies, in vitro investigations have indicated that ascorbic acid at high
concentrations was mutagenic and cytotoxic (Gulati et al., 1989; Stich et al., 1976, 1979; Omura et al., 1978; Galloway and Painter, 1979). The mutagenic action of vitamin C in vitro probably arises from the hydrogen peroxide and free radicals resulting from the oxidation of vitamin C in the presence of oxygen and metal ions (Shamberger,1984; Weitberg, 1987). Hydrogen peroxide and free radicals can cause damage to D N A and other cellular components (Fridovich, 1976). In in vivo investigations, the high intracellular concentrations of glutathione as well as other catalases and superoxide dismutase seem to protect the cells against damage induced by hydrogen peroxide and free radicals (Shamberger, 1984; Spelt et al., 1980). The protective role of glutathione has been proven (Winkler, 1987). Vitamin E when administered to rats for 6 months did not cause any significant increase in cells with chromosomal aberrations. This was also found to be true in the results reported by Gebhart et al. (1985). When vitamin E was administered at a dose level of 300 m g / k g , the percentage of cells with aberrations was less than in the controls. The decrease may be due to the protective role played by vitamin E, especially at this high dose, which may have prevented or decreased the normal level of oxidative deterioration of cellular unsaturated lipids, thus maintaining or improving the structural and functional integrity at the subcellular level (Kazuo et al., 1984; Gloriay et aI., 1985).
Radioprotection by vitamins C and E Animals exposed to irradiation exhibited a significant increase in the percentage of ceils with chromosomal aberrations and stickiness. During irradiation both hydrogen peroxide and hydroxyl free radicals which can directly or indirectly cause chromatid damage are formed within the cell (Parshad et al., 1980; Leodon and Ward, 1981; Chen and Bernstein, 1987; Cunnigham et al., 1987). Chromosomal stickiness a n d / o r clumping also results from radiation (Casarett, 1968). The present study reports a radioprotective effect of vitamin C but not of vitamin E. Vitamin C has been previously reported to have a protective effect against radiation in Allium (Selimbekova, 1969) and in barley seeds (Conger,1975). The radioprotective effect of ascorbic acid seems to result from the interaction of ascorbic acid with radiation-induced free radicals (Duschesne et al., 1975). In the present investigation, vitamin E, unlike vitamin C, did not play any radioprotective role. This may be attributed to the fact that vitamin C is water-soluble whereas vitamin E is fat-soluble. Fat-soluble antioxidants are the main free radical scavengers in biological autoxidation processes whereas water-soluble antioxidants hold the corresponding function in radiation damage (Konings and Oosterloo, 1980). Hydrogen peroxide and hydroxyl radicals produced by radiation causing cellular damage are not scavenged by vitamin E, which is a singlet oxygen scavenger (Parshad et al., 1980). Vitamin E, besides being non-radioprotective, seems to suppress the radioprotection offered by olive oil (used to dissolve vitamin E) by binding to it. Olive oil has been reported to bind to steroids (Siddique et al., 1980). Irradiated animals pretreated with olive oil showed a significant decrease in chromosomal aberrations compared with those pretreated with water. It seems that free radicals produced by irradiation cause peroxidation of the olive oil thus decreasing or preventing peroxidation of the cellular lipid and consequently reduce cellular damage. In addition, the formed peroxides may act as a source of energy for repair of cellular damage (Smith and Leith, 1977). It should be noted, however, that although
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