HPLC assay of zearalenone and reduced metabolites in S9 fractions of duck liver

HPLC analysis of zearalenone (ZEA), zearalenols (-ZOL and ß-ZOL) and zearalanols (-ZAL and ß-ZAL) was developed, in order to obtain a sensitive and reproducible method to quantify ZEA and its reduced metabolites in subcellular fractions of animal livers (S9 samples). Optimal in vitro metabolism was observed by incubating 5 mg S9 proteins with 0.016 μmol. ZEA. Acetonitrile and diethylether/chloroform mixture were compared for extraction, as well as different mobile phases and two detection modes in HPLC analysis. Extracted samples were eluted with water/acetonitrile (55:45, v/v) at a flow-rate of 1.0 ml/min-1, resulting in well separated peaks between ZEA and the metabolites. The limits of detection ranged from 0.5 to 2 ng/mg S9 proteins using UV, and from 0.04 to 4 ng/mg S9 proteins, using fluorescence detection. Fluorescence showed a ten-fold higher sensitivity than UV detection for ZEA and -ZOL. Repeatability (10 assays) was 2.7% to 6.99% for zearalenols. Day-by-day coefficients of variation for zearalenone and zeranols with UV detection were 3.3 to 8.5 %, and 2.5 to 4.3 %, respectively. This analysis applied to S9 samples from ducks after 30 min of ZEA incubation allowed to demonstrate that -ZOL is the main reduced metabolite in the duck. The present method is particularly adapted for studying in vitro metabolism of ZEA and inter-species variations

Variations in zearalenone activation in avian food species

Zearalenone (ZEA), a widely distributed oestrogenic fusariotoxin, constitutes a potential risk for human and animal health. ZEA is metabolised to the main metabolites identified in vitro and in vivo: alpha-zearalenol (α-ZOL) and beta-zearalenol (β-ZOL). The efficiency to produce alpha-reduced metabolites appears of particular interest in risk assessment as alpha-reduced metabolites constitute activated forms whereas beta-reduced metabolites are less oestrogenic than ZEA. In this study ZEA activation was compared in avian food species. ZEA and its reduced metabolites were quantified in subcellular fractions of six avian species and rat livers. The α-ZOL/β-ZOL ratio in rats was 19. The various avian food species cannot be considered to be equivalent in terms of ZEA reduction (P<0.001). Quails represented high “beta reducers”, with α-ZOL/β-ZOL ratio less than two. Weak “beta reducers” included on one part ducks and chickens showing α-ZOL/β-ZOL ratio greater than 3 and up to 5.6 and on a second part geese, showing a lower production of α-ZOL than other poultry. Comparisons of enzyme kinetics in ducks and in quails show that these variations can be explained by the action of various isoforms of dehydrogenases. These results are relevant to food safety, in the context of frequently inevitable contamination of animal feed

Comparison of two extraction methods for ergosterol determination in vegetal feeds

Ergosterol is the principal sterol of fungi in which it plays an essential role in cell membrane and other cellular constituents. This sterol is considered as a good marker of fungal contamination and of mycotoxin production. After validation of ergosterol quantification by HPLC-UV system (linearity range: 0.2 to 20.0 mg/ml, repeatability: 3.27%, between day precision: 4.75%), 2 extraction methods of ergosterol from 3 vegetal matrixes (maize, barley and wheat) were compared: the first one, normalized by the AFNOR, is based on solid phase extraction (SPE), while the other is based on liquid/liquid extraction (LLE). The LLE procedure allowed ergosterol extraction gains of around 20% for high initial sterol contents (3 to 5 mg/kg) in naturally contaminated matrixes or in spiked samples, and of 86% for low initial sterol contents (1-2 mg/kg) in maize. Moreover, the precision of ergosterol determination was comparable for the 2 methods even if it was slightly lower using LLE and was more affected by the initial ergosterol contents in vegetal matrix than by its nature. These results suggest that ergosterol contents in vegetal feeds would be underestimated with the official method (SPE) and emphasize the importance of the extraction step

Production and purification of fumonisins from a highly toxigenic Fusarium verticilloides strain

Fumonisins are the major mycotoxins produced by Fusarium verticilloides and F. proliferatum fungi which are widely found as contaminants in corn and corn screenings. These molecules are hepatotoxic and nephrotoxic for several species and carcinogenic in rodents. Moreover their consumption was linked to high prevalence of human oesophageal cancer in certain geographic areas. The aim of this work was to improve FB1 production and purification procedures in laboratory conditions in order to produce large quantities of semi-purified toxin that may be used in experimental intoxications of farm animals. We used a highly toxigenic strain of Fusarium verticilloides (NRRL-3428) isolated from feeds. Influence of substrate, temperature, water content, culture recipient size and screen analysis of the substrate on fumonisin production was tested. Optimal production was obtained when strain was grown on coarsely cracked corn with 50% water content at 21°C during 5 weeks. This allowed the production of 3 to 4 g of fumonisin B1 per kg of culture material. The composition of the extracts was found to be as follow : 54% FB1, 8% FB2, 9% FB3 and 29% of pigments coming from corn. The ratio observed between FB1 and FB2 is comparable to the one reported in naturally contaminated corn. Further purification of these extracts on SAX columns led to the removal of pigments and to obtain of fumonisins extracts pure enough to be used for intra-venous or intra-peritoneal injection

Toxicokinetics of fumonisin B1 in turkey poults and tissue persistence after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds

The kinetic of fumonisin B1 (FB1) after a single IV and oral dose, and FB1 persistence in tissue were investigated in turkey poults by HPLC after purification of samples on columns. After IV administration (single-dose: 10 mg FB1/kg bw), serum concentration–time curves were best described by a three-compartment open model. Elimination half-life and mean residence time of FB1 were 85 and 52 min, respectively. After oral administration (single-dose: 100 mg FB1/kg bw) bioavailability was 3.2%; elimination half-life and mean residence time were 214 and 408 min, respectively. Clearance of FB1 was 7.6 and 7.5 ml/min/kg for IV and oral administration respectively. Twenty four hours after the administration of FB1 by the intravenous route, liver and kidney contained the highest levels of FB1 in tissues, level in muscle was low or below the limit of detection (LD, 13 µg/kg). The persistence of FB1 in tissue was also studied after administration for nine weeks of a feed that contained 5, 10 and 20 mg FB1+FB2/kg diet. Eight hours after the last intake of 20 mg FB1+FB2/kg feed (maximum recommended concentration of fumonisins established by the EU for avian feed), hepatic and renal FB1 concentrations were 119 and 22 µg/kg, level in muscles was below the LD

Effect of fumonisins and Salmonella on digestive flora profiles assessed using a molecular tool (CE-SSCP).

Fumonisins (FB) are mycotoxins frequently found in vegetal feedstuffs, especially in maize used for pig feeding. Among fumonisins, FB1 was the better described toxin. It caused pulmonary and hepatic damages as well as immune response disorders in pigs that were recognised as especially sensitive to FB Intoxication. The FB1 immunosuppressor induced a higher susceptibility of pigs to gut pathogens such as E coli. Effects on Salmonella have poorly been studied despite the frequent asymptomatic carnage in pigs and the presumptive role of nora equilibrium on prevention of Salmonella excretion or re-excretion. To determine the influence of Salmonella carriage, fumonisins or both on digestive flora equilibrium, the use of a molecular technique CE-SSCP (Capillary-Electrophoresis Single Strand Conformation Polymorphism) appeared a good complement to the conventional bacteriological techniques. The objective was to assess the perturbation of nora associated with co-exposition in experimental conditions in absence of clinical sign

Gastric stimulation: influence of electrical parameters on gastric emptying in control and diabetic rats

BACKGROUND: The aim of this study was to test the effect of different pulse frequencies and amplitudes during gastric stimulation (GS) on gastric emptying in the rat. METHODS: GS was performed in 2 groups of laparotomized rats: healthy control animals, and rats with acute diabetes. The effects of four pulse frequencies (0.5, 1, 10, 20 Hz) and three pulse amplitudes (5, 20, 40 mA) were tested. The volumes emptied from the stomach after the oro-gastric instillation of a nutrient solution were compared to those obtained in animals without GS. Intragastric pH values were assessed under basal conditions and after GS. RESULTS: In both groups, GS increased emptied volumes compared to conditions without stimulation (p < 0.05) for pulse frequencies above 0.5 Hz. Increases in pulse frequencies accelerated gastric emptying (p < 0.01) with a plateau at around 10 Hz. The increase in pulse amplitudes resulted in larger emptied volumes only when the pulse frequency was 1 Hz (p < 0.04) while the opposite effect was observed at 20 Hz (p < 0.04). The most effective combinations to enhance gastric emptying compared to baseline conditions were 10 Hz with 5 or 20 mA. The overall effect of GS on gastric emptying compared to baseline conditions without stimulation, was greater in diabetic than in controls rats (p < 0.05). During stimulation, intragastric pH values were not different from basal conditions during fasting or after a meal in control and diabetic rats. CONCLUSIONS: Although both pulse frequency and amplitude should be considered during GS, frequency appears to be the most critical point. The possibility of increasing gastric emptying by electrical stimulation in diabetic rats suggests potential clinical applications for this method

Lolitrem B and Indole Diterpene Alkaloids Produced by Endophytic Fungi of the Genus Epichloë and Their Toxic Effects in Livestock

Different group of alkaloids are produced during the symbiotic development of fungal endophytes of the genus Epichloë in grass. The structure and toxicity of the compounds vary considerably in mammalian herbivores and in crop pests. Alkaloids of the indole-diterpene group, of which lolitrem B is the most toxic, were first characterized in endophyte-infected perennial ryegrass, and are responsible for “ryegrass staggers.” Ergot alkaloids, of which ergovaline is the most abundant ergopeptide alkaloid produced, are also found in ryegrass, but generally at a lower rate than lolitrem B. Other alkaloids such as lolines and peramine are toxic for crop pests but have weak toxicological properties in mammals. The purpose of this review is to present indole-diterpene alkaloids produced in endophyte infected ryegrass from the first characterization of ryegrass staggers to the determination of the toxicokinetics of lolitrem B and of their mechanism of action in mammals, focusing on the different factors that could explain the worldwide distribution of the disease. Other indole diterpene alkaloids than lolitrem B that can be found in Epichloë infected ryegrass, and their tremorgenic properties, are presented in the last section of this review

Lolitrem B and Indole Diterpene Alkaloids Produced by Endophytic Fungi of the Genus Epichloë and Their Toxic Effects in Livestock

Different group of alkaloids are produced during the symbiotic development of fungal endophytes of the genus Epichloë in grass. The structure and toxicity of the compounds vary considerably in mammalian herbivores and in crop pests. Alkaloids of the indole-diterpene group, of which lolitrem B is the most toxic, were first characterized in endophyte-infected perennial ryegrass, and are responsible for “ryegrass staggers.” Ergot alkaloids, of which ergovaline is the most abundant ergopeptide alkaloid produced, are also found in ryegrass, but generally at a lower rate than lolitrem B. Other alkaloids such as lolines and peramine are toxic for crop pests but have weak toxicological properties in mammals. The purpose of this review is to present indole-diterpene alkaloids produced in endophyte infected ryegrass from the first characterization of ryegrass staggers to the determination of the toxicokinetics of lolitrem B and of their mechanism of action in mammals, focusing on the different factors that could explain the worldwide distribution of the disease. Other indole diterpene alkaloids than lolitrem B that can be found in Epichloë infected ryegrass, and their tremorgenic properties, are presented in the last section of this review