Development of a method to determine essential oil residues in cow milk.

Over the past few years, the use of essential oils in breeding has been gaining ground because of their wide range of application, especially in terms of antimicrobial activity, and the current emphasis on the use of natural compounds, particularly to replace antibiotics. Among the essential oils and their active components, 4 molecules are of great interest and are studied in this paper: carvacrol, thymol, cinnamaldehyde, and diallyl disulfide. In particular, we examined the transfer of essential oil components from animal feed to milk. Because these components could confer an undesirable odor or taste to milk and derived products or interfere with milk processing, it is necessary to quantify their residues in milk. Milk is a complex biological matrix containing large amounts of water, fat, and protein. As a consequence, the extraction of volatile compounds, which are mainly lipophilic, is often critical. In this work, a method based on solid-phase microextraction coupled with gas chromatography-mass spectrometry was developed and optimized. We evaluated the effect of several parameters on the sensitivity of the method: fiber type, fiber to sample contact, extraction temperature, extraction time, agitation, and salt addition. The best results were obtained using a divinylbenzene-carboxen-polydimethylsiloxane (50/30 µm) fiber in headspace mode at 34.6°C for 32.6 min without agitation or salt addition. Standard calibration curves were constructed, and the limits of detection and quantification and ranges of linear response and repeatability were evaluated. Detection limits ranged from 25 to 50 µg/L, quantification limits from 50 to 75 µg/L, and variation coefficients from 23.5 to 36.6%. Therefore, the optimized methodology was able to evaluate whether significant transfer of essential oil components from feed to milk occurred, and the method is simple and solvent-free. We applied the method to milk samples from cows whose feed included thymol, carvacrol, cinnamaldehyde, or diallyl disulfide as feed additives at the recommended dose and at twice the usual dose. None of the 4 essential oil components were found in milk samples, regardless of the dose administered.

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.

Fusariotoxin transfer in animal.

Mycotoxin fusariotoxins, essentially represented by trichothecenes, zearalenone and fumonisins, are widely scattered in cereals and their products. Human and animals are particularly concerned by toxicity consecutive to oral chronic exposure. Human exposure can be direct via cereals or indirect via products of animals having eaten contaminated feed. As this alimentary risk is considered as a major problem in public health, it is thus of great importance to determine bioavailability, metabolic pathways and distribution of these mycotoxins in animal and human organism. Most studies indicate that fusariotoxins can be rapidly absorbed in the small intestine but the mechanisms involved remain unclear. Except NIV, fusariotoxins can be partly metabolised into more hydrophilic molecules in digestive tract or liver. Fumonisins present different behaviour as they seem very few and slowly absorbed and metabolised. The main part of absorbed fusariotoxins shows a rapid elimination within 24h after ingestion, followed by a slower excretion of small amounts. However, traces of fusariotoxins or their derivates can be found in animal products. This manuscript, reviewing literature published on fusariotoxin transfer, highlights that too little data are available to correctly appreciate fusariotoxin transfer in organism. Further studies focusing on mechanisms involved in the transfer are needed before clarifying risk assessment for human health.

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.

Study of mammary epithelial role in polycyclic aromatic hydrocarbons transfer to milk.

Food would appear to be one of the main routes for animal and human contamination with polycyclic aromatic hydrocarbons (PAH). Many studies have shown the presence of PAH in milk and dairy products, suggesting that these foods can represent a part of this contamination. Our work aimed at defining, in vitro, the mammary barrier role in PAH transfer to milk. MAC T cells were cultivated on permeable filters to measure transepithelial permeability of 14C labeled benzo[a]pyrene (BaP), pyrene (Pyr), and phenanthrene (Phen), which differed in their physicochemical properties. The results showed that only 2 molecules, Phen and Pyr, were able to cross mammary cell layers. Phenanthrene radioactivity appeared more quickly in apical media, and its level after a 6-h exposure was 1.3 times higher than for Pyr and 7.7 times higher than for BaP. These findings suggested that mammary epithelium could play a key role in the selective transfer of PAH from food to milk.