Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

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Abstract

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO4 that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.

Introduction

Iron is essential for normal cellular functions. However, iron has the potential to cause significant oxidative damage leading to mutagenesis [1]. The precise mechanism of iron induced oxidative damage to DNA is not known but is believed to involve free iron that catalyzes the formation of hydroxyl radicals by the Fenton reaction [2]. DNA damage may result directly by the oxidation of nucleoside bases or indirectly via the formation of lipid peroxides.

Ascorbic acid is a potent, water-soluble antioxidant that has been demonstrated to be an effective free radical scavenger [3]. Several in vitro and in vivo studies have demonstrated antioxidant and anti-mutagenic effects of ascorbic acid. Ascorbic acid also reduces redox active transition metal ions in the active sites of specific biosynthetic enzymes. Investigations into whether ascorbic acid in the presence of iron can acts as a pro-oxidant have resulted in conflicting conclusions [4].

To examine this issue, our present work was initiated to determine whether dietary ascorbic acid supplementation with or without excessive iron potentiates or protects against the genotoxicity of iron. For this purpose 3 weeks old, mice were fed diets containing either 100 mg Fe/kg diet or 300 mg Fe/kg diet with or without ascorbic acid for 3 weeks and genotoxic effects were assessed using bone marrow micronucleus test.

Section snippets

Animals, housing and diets

All the experiments were carried out with weanling C3H mice. From birth to 3 weeks, the mice were housed in plastic cages with a 12 h light-dark cycle and free access to standard laboratory diet and water. Groups 1 and 2 were fed standard rodent chow (LabDiet 5K52, St. Louis, MO) from weanling until 6 weeks of age. Group 2 mice were injected with a single intraperitoneal dose of cyclophosphamide (40 mg/kg bw) 24 h before sacrifice. All other groups were fed purified diets differing in iron

Statistical analysis

Values are expressed as mean+S.E.M. Comparisons among the groups were tested by one-way ANOVA using GraphPad Prism, version 3.0 (GraphPad Software, San Diego, CA). When the P-value obtained from ANOVA was significant, the Tukey test was applied to test for differences among groups. Significance was considered to be P<0.05.

Results

The frequency of micronuclei in the bone marrow cells of mice treated with cyclophosphamide was significantly higher (P<0.001) when compared to all other groups. The frequency of micronuceli in the both negative (group 1) and positive control (group 2) are consistent with other studies cited in the literature.

Ascorbic acid supplementation tended to increase liver iron concentration for both the 100 and 300 mg Fe/kg diets (Fig. 1). However, only mice fed the 300 mg Fe/kg iron diet with ascorbic

Discussion

The micronuclei in young erythrocytes arise primarily from chromosome fragments that are not incorporated into the daughter nuclei at the time of cell division in the erythropoietic blast cells and changes in the incidence of MnPCEs are considered to reflect chromosomal damage [7]. From the results of the present study, it is evident that high dietary iron increases the frequency of in vivo chromosomal damage. In contrast, ascorbic acid alone did not appear to induce significant DNA damage,

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