Mycotoxin Removal by Lactobacillus spp. and Their Application in Animal Liquid Feed.

The removal of mycotoxins from contaminated feed using lactic acid bacteria (LAB) has been proposed as an inexpensive, safe, and promising mycotoxin decontamination strategy. In this study, viable and heat-inactivated L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells were investigated for their ability to remove aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON) from MRS medium and PBS buffer over a 24 h period at 37 °C. LAB decontamination activity was also assessed in a ZEA-contaminated liquid feed (LF). Residual mycotoxin concentrations were determined by UHPLC-FLD/DAD analysis. In PBS, viable L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells removed up to 57% and 30% of ZEA and DON, respectively, while AFB1 and OTA reductions were lower than 15%. In MRS, 28% and 33% of ZEA and AFB1 were removed, respectively; OTA and DON reductions were small (≤15%). Regardless of the medium, heat-inactivated cells produced significantly lower mycotoxin reductions than those obtained with viable cells. An adsorption mechanism was suggested to explain the reductions in AFB1 and OTA, while biodegradation could be responsible for the removal of ZEA and DON. Both viable LAB strains reduced ZEA by 23% in contaminated LF after 48 h of incubation. These findings suggest that LAB strains of L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T may be applied in the feed industry to reduce mycotoxin contamination.

Dietary exposure of Tunisian adult population aged from 19 to 65 years old to pesticides residues.

Dietary exposure of the Tunisian adult population to pesticide residue was assessed using the Total Diet Study method. In the present study 170 pesticides were searched for in 42 aggregated foods characterised by 64 food samples representing the Tunisian diet. All the food samples were collected, prepared, and analysed for remains of pesticides including organochlorine, organophosphorous, carbamates and pyrethroids. The GC-MS analysis and the LC-MS/MS analysis and some other specific analytical methods were employed for the quantification of the pesticide residues in the food samples. Results revealed that21pesticides reached amounts greater than the LOQ(12.3%) and 149 pesticides reached amounts below the LOD (88%).For the 21 pesticides quantified, the ADI was not exceeded. For the 149 non-quantified pesticides, an interval defined by the lower and upper bounds was defined to assess the dietary exposure of the Tunisian adult population to those pesticides. We conclude that 8 pesticides theoretically exceed the ADI with the 95th percentile of exposure, those are: Diféthialone, Brodifacoum, Bromadiolone, Glufosinate, Heptachlor, Dieldrin Aldrin Oxydemeton-methyl. This study concludes that there is a low dietary exposure to pesticide residue of the Tunisian adult population. In fact, all the cases where the ADI was exceeded were theoretical due to the lower ADI value used.

Effects of Gamma Irradiation on Ochratoxin A Stability and Cytotoxicity in Methanolic Solutions and Potential Application in Tunisian Millet Samples.

Gamma irradiation is a useful technology for degrading mycotoxins. The purpose of this study was to investigate the effect of irradiation on ochratoxin A (OTA) stability under different conditions. OTA was irradiated in methanolic solution and on millet flour at doses of 2 and 4 kGy. Residual OTA concentrations and possible degradation products in irradiated samples were analyzed by high-performance liquid chromatography with fluorescence detection and liquid chromatography coupled to mass spectrometry. The extent of in vitro cytotoxicity of OTA to HepG2 cells, with and without irradiation treatment, was assessed with an MTT assay. OTA was more sensitive to gamma radiation on Tunisian millet flour than in methanolic solutions. After irradiation of naturally contaminated millet flour, the OTA concentration was significantly reduced by 48 and 62% at a dose of 2 and 4 kGy, respectively. However, in the methanolic solution, OTA at concentrations of 1 and 5 µg mL-1 was relatively stable even at a dose of 4 kGy, with no degradation products detected in the chemical analysis. Analytical results were confirmed by cell culture assays. The remaining cytotoxicity (MTT assay) of OTA following irradiation was not significantly affected compared with the controls. These findings indicate that gamma irradiation could offer a solution for OTA decontamination in the postharvest processing chain of millet flour. However, the associated toxicological hazard of decontaminated food matrices needs more investigation.

Effects of static magnetic field on cell growth, viability, and differential gene expression in Salmonella.

In the present study, we investigated the effect of exposure to A static magnetic field (SMF) on cell growth, viability, and gene expression of Salmonella enterica subsp. enterica serovar Hadar. Our results indicated that SMF exposure (200 mT, 13 hours) failed to alter cellular growth but induced a decrease of colony-forming units (CFU) between 3 and 6 hours followed by an increase from 6 to 9 hours. The analysis of the differential expression of rpoA, dnaK, katN, and 16S rRNA genes under SMF exposure (200 mT, 10 hours) showed that the expression level of the 16S rRNA mRNA remained stable during the exposure and can thus be used as a reference gene for the analysis on the differential gene expression of Salmonella Hadar. Interestingly, mRNAs of rpoA, katN, and dnaK genes were over-expressed following 10 hours of SMF exposure (200 mT). These data suggest a possible stress response of Salmonella Hadar to static magnetic field.

Genotoxic potential of Microcystin-LR and nodularin in vitro in primary cultured rat hepatocytes and in vivo in rat liver.

Microcystin-LR (MCYST-LR) and nodularin (NOD) are known as tumor promoters in experimental animals and so present potential health threats for humans. Although their hepatotoxic mechanisms have been very well documented, many other effects of these toxins are relatively undescribed, indeed controversial, notably those related to their genotoxicity. In the present investigation, we examined how these toxins could induce DNA damage using a combination of in vitro and in vivo approaches. We first used the (32)P-postlabeling assay to test hydrophobic adduct formation on DNA from primary cultured rat hepatocytes treated with noncytotoxic concentrations of MCYST-LR and NOD (2 and 10 ng/mL). Analysis of the autoradiograms of DNA digests isolated from the hepatocytes did not show any hydrophobic DNA adduct formation. However, these toxins significantly decreased the amount of hydrophobic endogenous adducts, termed I compounds. We next investigated oxidative DNA damage by using the (32)P-postlabeling assay to analyze 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content as a biomarker of possible DNA lesions. Both MCYST-LR and NOD significantly enhanced 8-oxo-dG in time- and dose-dependent manner in vitro in primary cultured hepatocytes and in vivo in rat liver cells. Thus, it appears that the depletion of endogenous DNA adducts (I compounds) and/or the increase of 8-oxo-dG levels by MCYST-LR and NOD could be involved in the formation of hepatic tumors during long-term exposure to these cyanobacterial hepatotoxins.

Detection by 32P-postlabelling of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA as biomarker of microcystin-LR- and nodularin-induced DNA damage in vitro in primary cultured rat hepatocytes and in vivo in rat liver.

Microcystin-LR (MCYST-LR) and nodularin (NOD) produced by cyanobacteria are potent and specific hepatotoxins. The induction of free-radical formation, reduction of glutathione levels and induction of DNA damage are three major events found in rat hepatocytes treated with these hepatotoxins. However, the mechanism of MCYST-LR- and NOD-mediated induction of oxidative DNA damage has not been fully elucidated. The objective of this study was to determine whether MCYST-LR and NOD increase the formation of a DNA oxidative damage marker such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in vitro in primary rat hepatocytes and in vivo in rat liver cells. Rat hepatocytes were exposed to MCYST-LR or NOD at low doses (2 and 10 ng/ml), at which there is no evidence of morphologically apparent cytotoxic effects, as well as an induced dose- and time-dependent formation of 8-oxo-dG. Moreover, MCYST-LR treatment of rats (50 microg/kg, ip) resulted in a significant increase of 8-oxo-dG in liver DNA, at 24 h after treatment before decreasing at 48 h. However, NOD-induced DNA damage was increased both at 24 and 48 h, in contrast to the MCYST-LR-induced effect. The effects on this oxidative DNA damage marker indicates that MCYST-LR and NOD do evoke oxidative stress, which may contribute, at least in part, to their liver toxicity and carcinogenicity during long-term exposure.

Microcystin-LR and nodularin induce intracellular glutathione alteration, reactive oxygen species production and lipid peroxidation in primary cultured rat hepatocytes.

Microcystin-LR (MCYST-LR) and nodularin (NOD) produced by cyanobacteria are potent specific hepatotoxins. However, the mechanisms of their hepatotoxicity have not been fully elucidated. In the present study the effect of non cytotoxic low concentrations of MCYST-LR and NOD on intracellular reduced glutathione (GSH) alteration, reactive oxygen species (ROS) production and lipid peroxidation was investigated in primary cultured rat hepatocytes. Cell viability was determined by the methylthiazoltetrazolium (MTT) dye assay, reduced GSH was evaluated by enzymatic methods, ROS were evaluated by the dichlorofluorescein diacetate (H2DCF-DA) fluorescent probe and lipid peroxidation by dosing malondialdehyde (MDA) by the thiobarbituric acid method. The 24 h LC50 values of MCYST-LR and NOD were 48 and 62 ng/ml, respectively. Exposure of freshly isolated rat hepatocytes to MCYST-LR or NOD at non cytotoxic low concentrations (2, 10 ng/ml) for 3, 24 and 48 h periods resulted in a significant rise of GSH levels and production of ROS. NOD significantly induced in a time- and concentration-dependent lipid peroxidation. However, MCYST-LR treatment did result in a significant decrease in MDA levels compared with controls. Although MCYST-LR and NOD are closely related in terms of structure and inhibition of protein phosphatases, they induce differently the oxidative stress at non cytotoxic low concentrations. Therefore, the results indicate that oxidative stress mediated by reactive intermediates may be a mechanism by which these cyanotoxins induce their hepatotoxic effect.

Seasonal variation of microcystin concentrations in the Saint-Caprais reservoir (France) and their removal in a small full-scale treatment plant.

At the Saint-Caprais reservoir (France), a mono-specific bloom of Aphanizomenon flos-aquae occurs every year in the autumn months. Levels of microcystin-LR (MCYST-LR) in this reservoir were evaluated by protein phosphatase type 2A (PP2A) inhibition test as MCYST-LR equivalents in both raw and drinking water. Analysis by HPLC of the crude extract of the mono-specific bloom of A. flos-aquae revealed the presence of MCYST-LR with a low concentration of 270.3 +/- 20.4 ng/g wet weight. MCYST-LR equivalent concentrations in raw water were correlated with the cyanobacteria biomass and they varied between 14 and 74 ng/l. The removal of A. flos-aquae and microcystins were evaluated in a small full-scale plant associated with the Saint-Caprais reservoir. Total elimination of cyanobacterial cells and the low concentration of hepatotoxins was achieved through the combined action of pre-ozonation at 0.07 mg/l and adsorption on powdered activated carbon at 20 mg/l. However, pre-chlorination at 0.42 mg/l followed by 20 mg/l of powdered activated carbon removed only 45% of hepatotoxins.