Chronic exposure to the mycotoxin T-2 promotes oral absorption of chlortetracycline in pigs.

The aim of this study was to investigate whether T-2 toxin, a potent Fusarium mycotoxin, affects the oral absorption of the antibiotic chlortetracycline in pigs. Animals were allocated to blank feed without T-2 toxin (controls), feed containing 111 µg T-2/kg feed, T-2-contaminated feed supplemented with a yeast-derived feed additive, or blank feed supplemented solely with the feed additive, respectively. After 21 days, an intragastric bolus of chlortetracycline was given to assess potential alterations in the pharmacokinetics of this commonly used antibiotic. A significantly higher area under the plasma concentration-time curve and maximal plasma concentration of chlortetracycline was observed after intake of T-2-contaminated feed compared with control. Thus, exposure to T-2-contaminated feed can influence the oral bioavailability of chlortetracycline. This effect could have consequences for the withdrawal time of the drug and the occurrence of undesirable residues in edible tissues.

The mycotoxin T-2 inhibits hepatic cytochrome P4503A activity in pigs.

Mycotoxins are toxic metabolites produced by fungi that readily colonize crops. After ingestion, these mycotoxins can compromise intestinal health, and once entering the blood stream, even affect the liver and its metabolizing enzymes. It was therefore the aim of the present study to investigate the effect of T-2 toxin, an emerging and potent Fusarium mycotoxin, on the enzymatic activity of cytochrome P4503A (CYP3A) metabolizing enzymes in the liver of pigs. In addition, a yeast-derived feed additive that claims to bind T-2 toxin was included in the study to evaluate its efficacy. Our results demonstrated that a 14-days intake of T-2 toxin contaminated feed at a dose of 903 µg/kg feed, whether or not combined with the mycotoxin binder, results in a substantial inhibition of the CYP3A activity in the liver of pigs. This result may be of importance for animal health, the pharmacokinetics and the withdrawal time of drugs that are substrate of CYP3A enzymes, and consequently can be a threat for public health with respect to tissue residues of these drugs.

Toxic effects of dietary exposure to T-2 toxin on intestinal and hepatic biotransformation enzymes and drug transporter systems in broiler chickens.

The effects of the mycotoxin T-2 on hepatic and intestinal drug-metabolizing enzymes (cytochrome P450) and drug transporter systems (MDR1 and MRP2) in poultry were investigated during this study. Broiler chickens received either uncontaminated feed, feed contaminated with 68µg/kg or 752µg/kg T-2 toxin. After 3weeks, the animals were euthanized and MDR1, MRP2, CYP1A4, CYP1A5 and CYP3A37 mRNA expression were analyzed using qRT-PCR. Along the entire length of the small intestine no significant differences were observed. In the liver, genes coding for CYP1A4, CYP1A5 and CYP3A37 were significantly down-regulated in the group exposed to 752µg/kg T-2. For CYP1A4, even a contamination level of 68µg/kg T-2 caused a significant decrease in mRNA expression. Expression of MDR1 was not significantly decreased in the liver. In contrast, hepatic MRP2 expression was significantly down-regulated after exposure to 752µg/kg T-2. Hepatic and intestinal microsomes were prepared to test the enzymatic activity of CYP3A. In the ileum and liver CYP3A activity was significantly increased in the group receiving 752µg/kg T-2 compared to the control group. The results of this study show that drug metabolizing enzymes and drug transporter mechanisms can be influenced due to prolonged exposure to relevant doses of T-2.

Hepatic and intestinal CYP3A expression and activity in broilers.

Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT-PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra-acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity.

Development of a liquid-chromatography tandem mass spectrometry and ultra-high-performance liquid chromatography high-resolution mass spectrometry method for the quantitative determination of zearalenone and its major metabolites in chicken and pig plasma.

A sensitive and specific method for the quantitative determination of zearalenone (ZEN) and its major metabolites (α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), α-zearalanol (α-ZAL), ß-zearalanol (ß-ZAL) and zearalanone (ZAN)) in animal plasma using liquid chromatography combined with heated electrospray ionization (h-ESI) tandem mass spectrometry (LC-MS/MS) and high-resolution Orbitrap(®) mass spectrometry ((U)HPLC-HR-MS) is presented. The sample preparation was straightforward, and consisted of a deproteinization step using acetonitrile. Chromatography was performed on a Hypersil Gold column (50 mm × 2.1 mm i.d., dp: 1.9 µm, run-time: 10 min) using 0.01% acetic acid in water (A) and acetonitrile (B) as mobile phases. Both mass spectrometers were operated in the negative h-ESI mode. The method was in-house validated for all analytes: matrix-matched calibration graphs were prepared and good linearity (r≥0.99) was achieved over the concentration range tested (0.2-200 ng mL(-1)). Limits of quantification (LOQ) in plasma were between 0.2 and 5 ng mL(-1) for all compounds. Limits of detection in plasma ranged from 0.004 to 0.070 ng mL(-1). The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values (within-day precision: RSD(max)=2((1-0.5logConc)) x 2/3; between-day precision: RSD(max)=2((1-0.5logConc))). The accuracy fell within -50% to +20% (concentrations <1 ng mL(-1)), -30% to +10% (concentrations between 1 and 10 ng mL(-1)) or -20% to +10% (concentrations >10 ng mL(-1)) of the theoretical concentration. The method has been successfully used for the quantitative determination of ZEN in plasma samples from broiler chickens and pigs. α-ZEL and ß-ZEL were the only metabolites that could be detected, but the concentrations were around the LOQ levels. The intact ZEN-glucuronide conjugate could be detected using the (U)HPLC-HR-MS instrument. A good correlation (r(2)=0.9979) was observed between the results for ZEN obtained with the LC-MS/MS and (U)HPLC-HR-MS instruments. The results prove the usefulness of the developed method for application in the field of toxicokinetic analysis and for exposure assessment of mycotoxins.

Efficacy and safety testing of mycotoxin-detoxifying agents in broilers following the European Food Safety Authority guidelines.

Contamination of feeds with mycotoxins is a worldwide problem and mycotoxin-detoxifying agents are used to decrease their negative effect. The European Food Safety Authority recently stated guidelines and end-points for the efficacy testing of detoxifiers. Our study revealed that plasma concentrations of deoxynivalenol and deepoxy-deoxynivalenol were too low to assess efficacy of 2 commercially available mycotoxin-detoxifying agents against deoxynivalenol after 3 wk of continuous feeding of this mycotoxin at concentrations of 2.44±0.70 mg/kg of feed and 7.54±2.20 mg/kg of feed in broilers. This correlates with the poor absorption of deoxynivalenol in poultry. A safety study with 2 commercially available detoxifying agents and veterinary drugs showed innovative results with regard to the pharmacokinetics of 2 antibiotics after oral dosing in the drinking water. The plasma and kidney tissue concentrations of oxytetracycline were significantly higher in broilers receiving a biotransforming agent in the feed compared with control birds. For amoxicillin, the plasma concentrations were significantly higher for broilers receiving an adsorbing agent in comparison to birds receiving the biotransforming agent, but not to the control group. Mycotoxin-detoxifying agents can thus interact with the oral bioavailability of antibiotics depending on the antibiotic and detoxifying agent, with possible adverse effects on the health of animals and humans.

New bolus models for in vivo efficacy testing of mycotoxin-detoxifying agents in relation to EFSA guidelines, assessed using deoxynivalenol in broiler chickens.

In this study, three new models were developed for efficacy testing of mycotoxin-detoxifying agents in relation to recent European guidelines. In the first model, deoxynivalenol was given to broiler chickens as an intra-crop bolus together with a mycotoxin-detoxifying agent in order to study the plasma concentration-time profile of deoxynivalenol. In the second model, the same oral bolus was given, preceded by an oral bolus of mycotoxin-detoxifying agent, to make sure the detoxifying agent was present in the whole intestinal tract when the mycotoxin was administered. In the third model, the mycotoxin-detoxifying agent was mixed in the feed of broiler chickens, and after 1 week's feeding, deoxynivalenol was given as an oral bolus. In order to evaluate the efficacy of these agents, plasma concentration-time profiles were set up and the main toxicokinetic parameters were compared. Two commercially available mycotoxin-detoxifying agents were tested, but they were not able to lower the oral availability of deoxynivalenol. As a positive control, activated carbon was used. We showed that activated carbon significantly reduces the absorption and oral availability of deoxynivalenol in all three models. Therefore, it can be concluded that these models are able to demonstrate the efficacy of mycotoxin-detoxifying agents in relation to European Food Safety Authority guidelines.

Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and deepoxy-deoxynivalenol in animal body fluids using LC-MS/MS detection.

A sensitive and specific method for the quantitative determination of deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), T-2 toxin (T-2) and HT-2 toxin (HT-2) in animal body fluids (plasma and bile) using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The extraction of plasma consisted of a deproteinization step using methanol, followed by a clean-up using an Oasis HLB solid-phase extraction column. For bile analysis, an extraction using a methanol/water mixture (70/30, v/v), followed by a liquid-liquid extraction using ethyl acetate, was performed. Chromatographic separation was achieved on a reversed-phase Nucleosil (100-5 C18 G100 × 3.0 mm) column. For the analysis of DON and DOM-1, a mixture of 0.1% acetic acid in water and methanol was used as the mobile phase. T-2 and its metabolite HT-2 were separated using 5mM ammonium acetate in a mixture of water/methanol/acetic acid. The mass spectrometer was operated in the negative or positive ESI selected reaction monitoring mode for DON and T-2 analysis, respectively. Calibration graphs (1-250 ng mL(-1)) were prepared for all matrices and correlation and goodness-of-fit coefficients were between 0.9978-1.000 and 2.96-11.77%, respectively. Limits of quantification were between 1 and 2.5 ng mL(-1) for all compounds. Limits of detection ranged from 0.01 to 0.63 ng mL(-1). The results for the within-day precision and accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of DON, DOM-1, T-2 and HT-2 in plasma and the semi-quantitative determination of the same compounds in bile from broiler chickens and pigs, respectively.