Bot. Bull. Acad. Sin. (2005) 46: 301-305

AGAR and ALPSOY — Antagonistic effect of selenium

Antagonistic effect of selenium against aflatoxin G₁ toxicity induced chromosomal aberrations and metabolic activities of two crop plants

Güleray AGAR* and Lokman ALPSOY

Department of Biology, Faculty of Science and Arts, Atatürk University, 25240/Erzurum, TURKEY

(Received November 15, 2004; Accepted April 15, 2005)

Abstract. The antagonistic effect of selenium (Se⁴⁺) against aflatoxin G₁ (AFG₁) toxicity induced chromosomal aberrations and metabolic activities in plants was studied. The results showed that 0.1, 0.2, 0.4 ppm concentrations of aflatoxin G₁ increased chromosomal aberration and mitotic index on total protein, and chlorophyll content decreased. When 0.08, 8, 800 ppm concentrations of Se⁴⁺ were added to AFG₁, the frequencies of chromosomal aberrations decreased and mitotic index, total protein and chloropyll content increased. Also, results suggested that Se⁴⁺ has an antagonistic effect against AFG₁, and that the degree of antagonistism of Se⁴⁺ against AFG₁ is probably related to its concentration ratio.

Keyword: Aflatoxin G₁; Genotoxicity; Selenium; Vicia faba; Zea mays.

Introduction

Aflatoxins, the most toxic of all the mycotoxins, have been described as natural contaminants in many consumables. These have been reported to affect the physiological processes of some crops. Most studies done have focused on the effect of aflatoxin B₁ (AFB₁). Other aflatoxins have received less attention, and very few studies have been carried out with AFG₁.

Previous papers on the effect of AFG₁ in higher plants indicate that it has an effect on total lipid, protein, and carbohydrate content, chlorophyll synthesis, inhibition of seed germination and growth, and chromosomal aberration (Asahi et al., 1969; Crisian, 1973; Abdou et al., 1984; Abdou et al., 1989; El-Zawahri et al., 1990; El-Naghy et al., 1999).

Recently, several studies have demonstrated that certain dietary substances (such as selenium, b carotene and vitamins) provide a protective effect against AFB₁ cytotoxicity in several animal species. The anticarcinogenic and antimutagenic effects of Se⁴⁺ against AFB₁ have been found in several animal studies (Chen, 1982; Chen et al., 1982; Bhattacharya et al.,1987; Gregory et al., 1984; Bronzetti et al., 2001). However, so far no report has shown a protective effect of Se⁴⁺ against AFG₁ toxicity. In the present study, we describe the effect of AFG₁ on total protein, chlorophyll content, and chromosomal aberration and discuss whether Se⁴⁺ has protective effects on these parameters.

Materials and Methods

AFG₁ and sodium selenite were obtained from Sigma Chemical Company, USA. Vicia faba seeds were obtained from the Department of Field Crops, Faculty of Agriculture, Atatürk University (Turkey). Zea mays seeds were obtained from the Department of Field Crops, Faculty of Agriculture, Karadeniz Technical University (Turkey). Vicia faba and Zea mays seeds of equal size were chosen and surface sterilized with 2.5 %/w/v of NaOCl for 3 min. After this treatment the seeds were rinsed with four changes of top water, and dried using sterile filter paper. The seeds were soaked in sterile distilled water for 1 h, and then fifteen seeds were germinated in 15 cm diameter petri dishes on four layers of sterile Whatman number 1 filter paper. Solution containing zero (control), 0.1, 0.2, 0.4 ppm of AFG₁ and the joint concentration of AFG₁ together with 0.8, 8, 800 ppm of Se⁴⁺ were added to each plate as seen Tables 1-4. The dishes were allowed to germinate in the dark at 25°C. When the roots reached 1.5-3.0 cm in length, they were collected and fixed in acetic acid: alcohol (1:3).

Cytological preparations were carried out using Feulgen's squash technique. The percentage of mitotic activity (mitotic index-MI.) was calculated by counting the total number of dividing cells to the total number of cells examined. Chromosomal aberrations (CA) were scored at anaphase and telophase, and the mutagenic effect was estimated as the percentage of cells with bridges and fragments. In addition to bridges and fragments, other forms of anomalies were identified in all mitotic stages.

For physiological studies, after 7 days, the total chlorophyll content of 20 g fresh tissue from shoot systems

*Corresponding author. E-mail: gagar@atauni.edu.tr; agar252001@yahoo.com; Tel: +90-442-2314341; Fax: +90-442-2360948.

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

in animals and plants (Lilly, 1965; Abdou et al., 1984; Abdou et al., 1989; El-Zawahri et al., 1990). The inhibition of mitotic activity and production of several aberrations caused by some fungal metabolites have been explained as being the effect of these compounds on DNA synthesis and their acting as an enzymatic inhibitor of the enzyme system required for the chain reaction of DNA synthesis (Smith and Sullivan, 1960; Taylor et al., 1962; Abdou et al., 1989).

Total chlorophyll and total protein of both plants decreased with increasing concentration of AFG₁ (Tables 3, 4). The decrease was more obvious with increased AFG₁ concentration. Aflatoxins inhibit chlorophyll synthesis, resulting in virescence or albinism in the affected plant (Schoental and White, 1965; Reiss, 1971; Sinha and Kumari, 1990; El-Naghy et al., 1999). Electron microscopic studies revealed the inhibition of grana formation in chloroplast of maize and oat leaves treated with aflatoxin (Slowatizky et al., 1969; El-Naghy et al., 1999).

AFG₁ affected the total proteins, and the protein content decreased with increased of AFG₁ concentration (Table 4). Little information is available on the effect AFG₁ on total protein concentration. Suppression of protein levels by aflatoxins was observed in germinating maize and other crop plants (El-Naghy et al., 1999). Singh et al. (1974) attributed the inhibition of protein synthesis to the non availability of m-RNA, which is necessary for the synthesis of proteins in the cell.

or leaves of corn and broad bean seedling was estimated spectrophotometrically according to Arnon (1949). Quantitative estimation of total protein in the ground tissue of roots, shoot, and leaves systems of both corn and broad bean seedling were estimated according to Lowry et al. (1951).

Each experiment was performed at least three times. Analysis of variance was conducted using a one-way ANOVA test using SPSS 12.0 for Microsoft Windows, and means were compared by Duncan's multiple range test at the 0.001 level at confidence.

Results and Discussion

AFG₁ decreased significantly the mitotic index of Vicia faba and Zea mays root meristems when compared with control as seen in Table 1, and the mitotic index decreased progressively with increased AFG₁ concentration. Such a decrease was found to be statistically significant (P<0.01). All concentrations of AFG₁ used in this study increased the frequencies of abnormalities such as C-mitosis, chromosomal stickiness, and anaphase and telophase bridges involving one or more chromosome micronuclei and fragments (Table 2). Total frequencies of increasing chromosomal abnormalities were found statistically significant. The effects of AFG₁ on the frequencies of total chromosomal aberration and the mitotic index have been earlier reported

AGAR and ALPSOY — Antagonistic effect of selenium

It is apparent that the effects exerted by AFG₁ are similar to those caused by AFB₁ and both are capable of inhibiting the synthesis of chlorophyll, protein, and mitotic division of Vicia faba and Zea mays.

However, these effects of AFG₁ seen at higher levels decreased after treatment with different concentrations of Se⁴⁺. Among all the treatment groups with Se⁴⁺ together with AFG₁, especially the 8 ppm concentration of Se⁴⁺, chromosomal aberrations decreased significantly, and mitotic index, total protein, and total chlorophyll concentration increased (Tables 1-4). Se⁴⁺ has been found to inhibit the mutagenic of effects AFB₁ in several animal studies (Chen, 1982; Chen et al., 1982; Francis et al., 1988; Shi et al., 1994; Shi and Hew, 1995). It is recognized as an antimutagenic and anticarcinogenic substance against the cytotoxicity and genotoxicity of AFB₁. The protective effects of Se⁴⁺ have been reported to be primarily associated with its presence in glutathione peroxidases, which are known to protect DNA and other cellular components from damage by oxygen radicals (Gregory and Edds, 1984; Nakae, 1987; Kodama et al., 1990).

Although the selenium has an antimutagenic and anticarcinogenic effect, its optimal concentration is not known. At high concentrations it is toxic, mutagenic, and carcinogenic while at low concentrations it is antimu

tagenic and anticarcinogenic (Oldifield, 1987; Biswas et al., 1996; Schrauzer, 2000). 10 µM of selenium concentration was observed to be cytotoxic to human blood lymphocytes (Abul Hassan et al., 2004). Moreover, the toxicity of elemental selenium was also obserbed when used in anticancer studies. Even though the recommended daily allowance (RDA) of selenium by the U.S. Food and Drug Administration is 50 µg/day, cancer preventive use of selenium is typically 200 µg daily. It is generally accepted that daily intake should not exceed 600 µg (Bahr et al., 1999).

The protective role of Se⁴⁺ against AFG₁ has not been reported. The antagonistic effect of selenium against cadmium induced chromosomal aberrations and micronuclei on higher plant has been studied. The 8-ppm concentration of Se⁴⁺, in particular, has been shown to reduce the micronuclei and chromosomal aberrations effectively. This protective role of Se⁴⁺ has been attributed to glutathione peroxides (Zhang and Xiao, 1998; Aar and Taplnar, 2003).

In summary, the 8-ppm Se⁴⁺ concentration has been found to be an active inhibitor of the mutagenicity of AFG₁ as observed with AFB₁ and its effect is probably related to its action on the enzymatic activation system since selenium is the essential component of glutathione peroxides. Cleary, information on the biochemistry of an

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

tioxidant protection against aflatoxin toxicity in plants is needed.

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AGAR and ALPSOY — Antagonistic effect of selenium