Assessment of deoxynivalenol (DON) adsorbents and characterisation of their efficacy using complementary in vitro tests.

Deoxynivalenol (DON) is a prevalent and resistant mycotoxin found in cereals and related products. Adsorbents appear to provide an opportunity to decrease DON absorption in animals but, due to their specificity, it is very difficult to evaluate their actual efficacy. It is pointless to extrapolate results obtained with one mycotoxin to another and even to extrapolate results obtained in vitro in buffer to an in vivo situation. We carried out experiments to characterize the properties of potential DON adsorbents. Initial tests in buffer pH 7 allowed us to focus on six adsorbents: activated charcoal, cholestyramin, Saccharomyces cerevisiae mannans, algal beta-glycan, fungal beta-glycan and leguminous plant. The use of equilibrium sorption models suggested a non-saturated phenomenon and involved variable mechanisms according to the specific material. Subsequent tests with a Caco-2 cell model showed a high reduction in DON cytotoxicity on proliferative intestinal cells and DON absorption by differentiated intestinal cells when adsorbent was added (except for cholestyramin). Otherwise, values were not always in accordance with those obtained in buffer. Our work allowed us to identify five potential DON adsorbents and to propose a complementary in vitro test allowing improved determination of adsorbent properties.

Diazinon cytotoxicity and transfer in Caco-2 cells: Effect of long-term exposure to the pesticide.

The purpose of this work was to investigate the effect of prolonged exposure to diazinon (widely used organophosphorus pesticide) on the intestinal cell-line Caco-2. Cytotoxicity of the pesticide (50µM-6mM) significantly decreased in long-term exposed (20µM, 2 months) cells, compared to untreated control cells. In long-term exposed cells, the resistance to diazinon cytotoxicity was reversed in the presence of PSC-833, a P-glycoprotein (P-gp) inhibitor, but not in the presence of MK 571, a Multidrug Resistance Protein (MRP) inhibitor. Cell exposure to 25µM diazinon showed a secretory-directed transport of the molecule, which increased in long-term exposed cells. This efflux decreased significantly, for both long-term and non-exposed cells, in the presence of verapamil and PSC-833, but not MK 571. Furthermore, the total amount of P-gp increased in long-term exposed cells. These results suggest that ABC transporter P-gp is involved in the intestinal transfer of diazinon, and that repeated exposure to low doses of diazinon could strengthen the activity of ABC transporters in intestinal cells, thus increasing cell resistance to pesticide cytotoxicity.

Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha.

The effects of metal mixture (Cd+Cu) versus single-metal exposure on total MT response and bioaccumulation were investigated in the freshwater bivalve Dreissena polymorpha. A two-month exposure period, including two levels of contamination, was chosen for each of the two metals: 5, 10 microg/L for Cu, and 2, 20 microg/L for Cd, with mixtures of, respectively, 5 microg/L Cu+2 microg/L Cd, 5 microg/L Cu+20 microg/L Cd, 10 microg/L Cu+2 microg/L Cd, and 10 microg/L Cu+20 microg/L Cd. Total MT contents were assessed by an Ag-saturation method, and metals contents were determined by atomic absorption spectrometry. Results at the whole-organism level showed a significant and early increase of total MT biosynthesis after exposure to Cd. This increase was significantly correlated with Cd bioaccumulation. By contrast, Cu did not modify total MT response, and mussels limited Cu bioaccumulation. The mixture either did not influence or only weakly influenced metal accumulation and MT response to Cu and Cd after long-term exposure. Our results suggest that the form of MT existing in D. polymorpha was not Cu-inducible. This could limit the use of MT in D. polymorpha as a biomarker of heavy metal pollution in freshwater ecosystems.

Assessment of the bioavailability of PAHs in rats exposed to a polluted soil by natural routes: induction of EROD activity and DNA adducts and PAH burden in both liver and lung.

In order to assess bioavailability of polycyclic aromatic hydrocarbons (PAHs) present in soils, male laboratory rats were exposed to litters of control and polluted soils. After 88+/-2 h of exposure, several biomarkers were measured in both liver and lung. When rats were exposed to SIV soil, contaminated by a mixture of at least 13 PAHs, (1) only 2 or 3 PAH compounds were detected in liver and lung; (2) cytochrome P450-dependent monooxygenase activity, followed by 7-ethoxyresorufin O-deethylase (EROD) activity measurement, was highly induced in liver (13-fold-induction) and lung (up to 78-fold); and (3) DNA adducts were significantly increased. For what concerns soil artificially contaminated by only one PAH (phenanthrene or B[a]P), EROD activity was not or fully induced, respectively. These results demonstrate the occurrence of a high bioavailability of PAHs to mammals in natural conditions of exposure. First results concerning DNA adducts must be profound, but they already show that a short exposure of mammals to PAH-polluted soils can lead to potential genotoxic effects. EROD activity can be used as a sensitive biomarker in both liver and lung of rats maintained on litters of soils in the laboratory, and such a test can be used routinely to contribute to risk assessment.

Rapid analysis of ergovaline in ovine plasma using high-performance liquid chromatography with fluorimetric detection.

A rapid high-performance liquid chromatographic method for the determination of the mycotoxin ergovaline in ovine plasma is described here. Ergotamine was used as an internal standard. A simple extraction procedure with diethyloxide was carried out, before chromatography on a C8 column, with the excitation and emission wavelengths fixed at 250 and 420 nm respectively, on a fluorimetric detector. The method, which was found to be linear between 3.5 and 15 ng/ml, had good specificity, precision and accuracy. The limit of quantification and the limit of detection were 3.5 and 1.2 ng/ml, respectively. A preliminary application of the described assay to a plasma kinetic study, after intravenous administration of a single dose of ergovaline (17 micrograms/kg body mass) to four sheep, showed a very rapid decrease of the plasma ergovaline levels. The terminal half-life and the total clearance of the mycotoxin were found to be 23.6 min and 0.020 l/min kg-1 body mass, respectively.

Identification of a benzhydrolic metabolite of ketoprofen in horses by gas chromatography-mass spectrometry and high-performance liquid chromatography.

A benzhydrolic metabolite of ketoprofen, formed by reduction of the keto group of the drug, has been identified by gas chromatography-mass spectrometry in equine plasma and urine. After partial synthesis, its structure has been confirmed by UV, IR and 1H NMR spectroscopy. The kinetics of ketoprofen and this metabolite have been monitored in plasma by high-performance liquid chromatography. The two products were quantified in plasma up to 4 and 3 h, respectively, and were detected in urine up to 72 and 24 h, respectively, after a single intravenous administration to horses at the dose of 2.2 mg/kg. Simultaneous detection of both compounds increases the reliability of antidoping control analysis.