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Dried blood spots (DBSs), a micro-sampling technique whereby a drop of blood is collected on filter paper has multiple advantages over conventional blood sampling regarding the sampling itself, as well as transportation and storage. This is the first paper describing the development and validation of a method for the determination of 23 mycotoxins and phase I metabolites in DBSs from pigs and broiler chickens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The targeted mycotoxins belong to groups for which the occurrence in feed is regulated by the European Union, namely, aflatoxins, ochratoxin A and several Fusarium mycotoxins, and to two groups of unregulated mycotoxins, namely Alternaria mycotoxins and Fusarium mycotoxins (enniatins and beauvericin). The impact of blood haematocrit, DBS sampling volume and size of the analysed DBS disk on the validation results was assessed. No effects of variation in size of the analysed disk, haematocrit and spotted blood volume were observed for most mycotoxins, except for the aflatoxins and β-zearalanol (BZAL) at the lowest haematocrit (26%) level and for the enniatins (ENNs) at the lowest volume (40 µL). The developed method was transferred to an LC-high resolution mass spectrometry instrument to determine phase II metabolites. Then, the DBS technique was applied in a proof-of-concept toxicokinetic study including a comparison with LC-MS/MS data from plasma obtained with conventional venous blood sampling. A strong correlation (r > 0.947) was observed between plasma and DBS concentrations. Finally, DBSs were also applied in a pilot exposure assessment study to test their applicability under field conditions.
Marianne Lauwers; Siska Croubels; Siegrid De Baere; Milena Sevastiyanova; Eva Maria Romera Sierra; Ben Letor; Christos Gougoulias; Mathias Devreese. Assessment of Dried Blood Spots for Multi-Mycotoxin Biomarker Analysis in Pigs and Broiler Chickens. Toxins 2019, 11, 541 .
AMA StyleMarianne Lauwers, Siska Croubels, Siegrid De Baere, Milena Sevastiyanova, Eva Maria Romera Sierra, Ben Letor, Christos Gougoulias, Mathias Devreese. Assessment of Dried Blood Spots for Multi-Mycotoxin Biomarker Analysis in Pigs and Broiler Chickens. Toxins. 2019; 11 (9):541.
Chicago/Turabian StyleMarianne Lauwers; Siska Croubels; Siegrid De Baere; Milena Sevastiyanova; Eva Maria Romera Sierra; Ben Letor; Christos Gougoulias; Mathias Devreese. 2019. "Assessment of Dried Blood Spots for Multi-Mycotoxin Biomarker Analysis in Pigs and Broiler Chickens." Toxins 11, no. 9: 541.
Applying post-harvest control measures such as adding mycotoxin detoxifying agents is a frequently-used mitigation strategy for mycotoxins. EFSA states that the efficacy of these detoxifiers needs to be tested using specific biomarkers for exposure. However, the proposed biomarkers for exposure are not further optimized for specific target species. Hence, the goal of this study was a) to evaluate the most suitable biomarkers for deoxynivalenol (DON) and zearalenone (ZEN) in porcine plasma, urine and feces; and DON, aflatoxin B1 (AFB1) and ochratoxin A (OTA) in plasma and excreta of broiler chickens and b) to determine the efficacy of a candidate detoxifier, as a proof-of-concept study. Therefore, a mixture of mycotoxins was administered as a single oral bolus with or without detoxifying agent. In accordance with literature AFB1, OTA, and DON-sulphate (DON-S) proved optimal biomarkers in broilers plasma and excreta whereas, in pigs DON-glucuronide (DON-GlcA) and ZEN-glucuronide (ZEN-GlcA) proved the optimal biomarkers in plasma, DON and ZEN-GlcA in urine and, ZEN in feces. A statistically significant reduction was seen between control and treatment group for both AFB1 and DON in broiler plasma, under administration of the mycotoxin blend and detoxifier dose studied suggesting thus, beneficial bioactivity.
Marianne Lauwers; Siska Croubels; Ben Letor; Christos Gougoulias; Mathias Devreese. Biomarkers for Exposure as A Tool for Efficacy Testing of A Mycotoxin Detoxifier in Broiler Chickens and Pigs. Toxins 2019, 11, 187 .
AMA StyleMarianne Lauwers, Siska Croubels, Ben Letor, Christos Gougoulias, Mathias Devreese. Biomarkers for Exposure as A Tool for Efficacy Testing of A Mycotoxin Detoxifier in Broiler Chickens and Pigs. Toxins. 2019; 11 (4):187.
Chicago/Turabian StyleMarianne Lauwers; Siska Croubels; Ben Letor; Christos Gougoulias; Mathias Devreese. 2019. "Biomarkers for Exposure as A Tool for Efficacy Testing of A Mycotoxin Detoxifier in Broiler Chickens and Pigs." Toxins 11, no. 4: 187.
A reliable and practical multi-method was developed for the quantification of mycotoxins in plasma, urine, and feces of pigs, and plasma and excreta of broiler chickens using liquid chromatography–tandem mass spectrometry. The targeted mycotoxins belong to the regulated groups, i.e., aflatoxins, ochratoxin A and Fusarium mycotoxins, and to two groups of emerging mycotoxins, i.e., Alternaria mycotoxins and enniatins. In addition, the developed method was transferred to a LC-high resolution mass spectrometry instrument to qualitatively determine phase I and II metabolites, for which analytical standards are not always commercially available. Sample preparation of plasma was simple and generic and was accomplished by precipitation of proteins alone (pig) or in combination with removal of phospholipids (chicken). A more intensive sample clean-up of the other matrices was needed and consisted of a pH-dependent liquid–liquid extraction (LLE) using ethyl acetate (pig urine), methanol/ethyl acetate/formic acid (75/24/1, v/v/v) (pig feces) or acetonitrile (chicken excreta). For the extraction of pig feces, additionally a combination of LLE using acetone and filtration of the supernatant on a HybridSPE-phospholipid cartridge was applied. The LC-MS/MS method was in-house validated according to guidelines defined by the European and international community. Finally, the multi-methods were successfully applied in a specific toxicokinetic study and a screening study to monitor the exposure of individual animals.
Marianne Lauwers; Siegrid De Baere; Ben Letor; Michael Rychlik; Siska Croubels; Mathias Devreese. Multi LC-MS/MS and LC-HRMS Methods for Determination of 24 Mycotoxins including Major Phase I and II Biomarker Metabolites in Biological Matrices from Pigs and Broiler Chickens. Toxins 2019, 11, 171 .
AMA StyleMarianne Lauwers, Siegrid De Baere, Ben Letor, Michael Rychlik, Siska Croubels, Mathias Devreese. Multi LC-MS/MS and LC-HRMS Methods for Determination of 24 Mycotoxins including Major Phase I and II Biomarker Metabolites in Biological Matrices from Pigs and Broiler Chickens. Toxins. 2019; 11 (3):171.
Chicago/Turabian StyleMarianne Lauwers; Siegrid De Baere; Ben Letor; Michael Rychlik; Siska Croubels; Mathias Devreese. 2019. "Multi LC-MS/MS and LC-HRMS Methods for Determination of 24 Mycotoxins including Major Phase I and II Biomarker Metabolites in Biological Matrices from Pigs and Broiler Chickens." Toxins 11, no. 3: 171.
The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalenone-14-glucoside (ZEN14G) and zearalenone-14-sulfate (ZEN14S), including (pre)systemic hydrolysis in pigs. Cross-over pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analysed to unravel the site of hydrolysis, and urine samples were analysed to determine urinary excretion. Results demonstrate ZEN14G and ZEN14S to be (completely) presystemically hydrolysed to ZEN and demonstrate high oral bioavailability for all administered compounds, with further extensive first pass glucuronidation. Conclusively, the modified ZEN forms α-ZEL, β-ZEL, ZEN14G and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken in account for the risk assessment.
Amelie Catteuw; Nathan Broekaert; Siegrid De Baere; Marianne Lauwers; Elke Gasthuys; Bart Huybrechts; Alfons Callebaut; Lada Ivanova; Silvio Uhlig; Marthe De Boevre; Sarah De Saeger; Ronette Gehring; Mathias Devreese; Siska Croubels. Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, β-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs. Journal of Agricultural and Food Chemistry 2019, 67, 3448 -3458.
AMA StyleAmelie Catteuw, Nathan Broekaert, Siegrid De Baere, Marianne Lauwers, Elke Gasthuys, Bart Huybrechts, Alfons Callebaut, Lada Ivanova, Silvio Uhlig, Marthe De Boevre, Sarah De Saeger, Ronette Gehring, Mathias Devreese, Siska Croubels. Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, β-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs. Journal of Agricultural and Food Chemistry. 2019; 67 (12):3448-3458.
Chicago/Turabian StyleAmelie Catteuw; Nathan Broekaert; Siegrid De Baere; Marianne Lauwers; Elke Gasthuys; Bart Huybrechts; Alfons Callebaut; Lada Ivanova; Silvio Uhlig; Marthe De Boevre; Sarah De Saeger; Ronette Gehring; Mathias Devreese; Siska Croubels. 2019. "Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, β-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs." Journal of Agricultural and Food Chemistry 67, no. 12: 3448-3458.
Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility. The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro. Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex. Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.
Afrouz Yousefi; Marianne Lauwers; Reka Nemes; Thijs Van Holten; Negar Babae; Mark Roest; Gert Storm; Raymond Schiffelers; Enrico Mastrobattista. Hemocompatibility Assessment of two siRNA Nanocarrier Formulations. Pharmaceutical Research 2014, 31, 3127 -3135.
AMA StyleAfrouz Yousefi, Marianne Lauwers, Reka Nemes, Thijs Van Holten, Negar Babae, Mark Roest, Gert Storm, Raymond Schiffelers, Enrico Mastrobattista. Hemocompatibility Assessment of two siRNA Nanocarrier Formulations. Pharmaceutical Research. 2014; 31 (11):3127-3135.
Chicago/Turabian StyleAfrouz Yousefi; Marianne Lauwers; Reka Nemes; Thijs Van Holten; Negar Babae; Mark Roest; Gert Storm; Raymond Schiffelers; Enrico Mastrobattista. 2014. "Hemocompatibility Assessment of two siRNA Nanocarrier Formulations." Pharmaceutical Research 31, no. 11: 3127-3135.