03-Combined.181719.pdf145.2KB

INTRODUCTION

  Mycotoxins are secondary metabolites of mould; they produce a wide range of adverse and toxic effects in animals in addition to being food borne hazards to humans.

  Among hundreds of known mycotoxins, aflatoxins, OTA and ZEA stand out as the most common contaminants in a variety of food. AFs are produced as secondary metabolites by Aspergillus flavus and Aspergillus parasiticus fungi. AFB1 is considered one of the most powerful human carcinogens. AFB1 is activated by hepatic cytochrome P450 enzyme system to produce a highly reactive intermediate, AFB1-8,9-epoxide, which subsequently binds to nucleophilic sites in DNA (Brahmi et al. 2011). OTA, produced by fungi Aspergillus and Penicillium genus, is mutagenic, teratogenic and nephrotoxic. Its oxidative activity is thought to play a central role in OTA-mediated carcinogenesis and may be divided into direct (covalent DNA adduction) and indirect (oxidative DNA damage) mechanisms of action (Pfohl-Leszkowicz et al. 2007). ZEA is a highly estrogenic mycotoxin produced by Fusarium species. ZEA caused cancer or at least increased its prevalence, although the mechanism of action is unknown. Many papers mentioned that exposure to ZEA results in genotoxicity and DNA damage (Ben Salah-Abbes et al. 2009).

  Often more than one mycotoxin is found on a contaminated substrate. There are literature data on co-occurance of AFs, OTA and ZEA in foods (Šegvi´c Klari´c 2012). However most studies are limited to the toxicology of a single mycotoxin, data on their combined action is very limited.

  The alkaline single-cell gel electrophoresis (comet) assay is a sensitive and powerful method for determining DNA damage in vitro and in vivo (Tice et al. 2000). It was shown that alkaline comet assay is adequate and reliable biomarker of exposure to dietary/environmental compounds, including mycotoxins (Kamp et al. 2005; Zeljezi´c et al. 2006).

  The aim of the present study was to characterize combined genotoxic effects of AFB1, OTA and ZEA in vivo in rat bone marrow and blood leukocytes using the comet assay.

MATERIALS AND METHODS

1. Reagents

  AFB1, OTA and ZEA were purchased from Scientific Research Institute of Nutrition of RAMS (Russia), heparin sodium and all reagents for comet assay were obtained from Sigma Aldrich (St. Louise, MO, USA), NaOH and salts for phosphate buffered saline were purchased from Medisar LLC (Armenia).

2. Animal treatment

  Adult Wistar rats weighing 200±20 g were kept in standard environmental conditions with a 12-h light/dark cycle and at a constant temperature of 24℃, fed a standard diet and had free access to water. The study was approved by the Ethical Committee of the Institute of Molecular Biology of the NAS RA (IRB IORG0003427). The rats were randomly assigned to four groups of 3 animals each receiving mixture of AFB1 (0.13 mg kg^-1 bw per day), OTA (0.064 mg kg^-1 bw per day) and ZEA (0.032 mg kg^-1 bw per day) or solvent only (control group) for 15, 30 and 60 days. The mycotoxins doses selection was based on the literature data about individual genotoxicity of AFB1 (Brahmi et al. 2011), OTA (Kamp et al. 2005; Zeljezi´c et al. 2006) and ZEA (Ouanes et al. 2005) in rodents. Considering that the most studies have observed additive or synergistic effects of mycotoxins ( Šegvi´c Klari´c 2012), for the mixture we have chosen a lower concentration than those that were effective in the individual action. The rats were killed by cervical dislocation; blood and bone marrow were sampled for comet assay.

3. Cell preparation

Bone marrow

  The left femur was removed and the bone marrow at both ends was exposed with bone cutters. Cells were flushed out with 3 mL PBS (pH 7.4) using a needle and syringe, and the cell suspension was filtered through a three layer bolting cloth.

Blood

 Tail vein blood (1 mL) was collected into syringe with heparin (0.3 mL).

4. Comet assay

  The level of DNA damage was evaluated by standard alkaline single-cell gel electrophoresis (comet assay) (Tice et al. 2000). Slides were examined at 250× magnifcation on a fluorescent microscope (ZEISS, Germany). At least 150 cells were scored per animal (50 cells scored per each of three replicate slides). Images of comets were recorded with a video camera with high sensitivity (Variocam, PCO, Germany) and processed on a computer program Comet Assay IV (Version 4.3). The tail intensity and Olive tail moment are used to evaluate the extent of DNA damage. Statistical analysis of the results was performed using SPSS 19 with application of non-parametric Mann-Whitney test (U test).

RESULTS AND DISCUSSION

  In recent years, research efforts have concentrated on the investigation of the individual effects of AFB1 (Brahmi et al. 2011), OTA (Kamp et al. 2005, Zeljezi´c et al. 2006) and ZEA (Ouanes et al. 2005; Pfohl-Leszkowicz et al. 2007). However, considering the coincident production of mycotoxins, mixture toxicity data is needed for an improved and more realistic risk assessment. The in vivo effects of AFB1, OTA and ZEA combination commonly produced by fungi species has to our knowledge, not yet been investigated.

  Combination of AFB1, OTA and ZEA found to be genotoxic to bone marrow and blood cells of rats, treated 15, 30 and 60 days (Figs. 1 and 2). In bone marrow cells the mycotoxins-induced increase of tail intensity was observed after 15, 30 and 60 days of exposure (p<0.05). The increase of Olive tail moment was revealed only after 30 and 60 days of treatment (p<0.05). In leukocytes the significant increase of tail intensity and Olive tail moment was observed after 30 and 60 days (p<0.05). DNA damage, induced  by mycotoxins, was more pronounced in bone marrow cells than in leukocytes. This effect may be due to high metabolic activity of bone marrow cells and requires further elucidation.

Fig. 1. The levels of tail intensity in the bone marrow cells and blood leukocytes of rats treated with mixture of AFB1, OTA and ZEA (*Significant difference (p<0.05) compared with control).

Fig. 2. The levels of Olive tail moment in the bone marrow cells and blood leukocytes of rats treated with mixture of AFB1, OTA and ZEA (*Significant difference (p<0.05) compared with control).

  Our study of combined AFB1, OTA and ZEA genotoxicity in vivo confirmed the genotoxic potential of mycotoxins combination, and show that the severity of DNA lesions correlates with time of treatment and depends on tissue type. The data obtained suggests that prolonged exposure to mycotoxins combination through food consumption can induce DNA damage contributing to the harmful effects in vivo.

  The application of comet assay confirmed its potential value as a sensitive tissue specific biomarker of mycotoxins genotoxicity.

ACKNOWLEDGEMENTS

 The work was supported by State Committee of Science of RA (grant number 11-1f 329).