Jutta Zagon

Safety assessment of BT 176 Maize in broiler nutrition: Degradation of Maize-DNA and its metabolic fate

Insect resistant Bt 176 maize has been developed by genetic modification to resist European borer infection. In the present investigation, the experiment was conducted to determine the effect of feeding a new hybrid of Bt 176 maize (NX 6262 - Bt 176) on general health condition and performance of broiler chickens. Maize grains and diets were subjected to proximate analysis. Amino and fatty acids investigation were applied for both maize grains before used. To evaluate the degradation of NX 6262 - Bt 176 maize DNA and its metabolic fate in broiler blood, muscles and organs. One-day-old male broilers were fed ad libitum on either an experimental diet containing NX 6262 - Bt 176 or a control diet containing the non-modified maize grains for 35 days. Feed consumption and body weight were recorded weekly during the experimental period. All chickens were subjected to nutritional evaluation period at day 20 of age for 5 successive days, to calculate the percentage of apparent digestible nutrients in both diets. At day 35 samples were collected at several intervals after feed withdrawal. Prior to slaughter blood samples were collected from all birds by heart puncture to prevent DNA cross contamination. Samples from pectoral and thigh muscles, liver, spleen, kidney, heart muscle, bursa and thymus glands were collected. Digesta from different sections of the gastrointestinal tract (GIT) were collected as well. Packed cell volume (PCV) and some serum parameters were investigated. There were no significant differences between control and experimental group concerning chemical composition of feeds, apparent digestible nutrients, and all performance parameters measured (P > 0.05). Furthermore, there were no differences in the PCV and the analysed serum parameters between the control and experimental group. The results of maize DNA digestibility showed that the new variety takes the normal physiological passage along broiler GIT similar to the conventional line. In addition, Bt 176 maize DNA appears to be partially degraded in different parts of GIT comparable to the DNA of the control maize line. Results of the metabolic fate of maize DNA in broiler blood, muscles and organs indicated that only short DNA fragments (199 bp) derived from the plant chloroplast gene could be detected in the blood, skeletal muscles, liver, spleen and kidney, which disappeared after prolongation the fasting time. In heart muscle, bursa of Fabricius and thymus, no plant chloroplast DNA was found. Bt gene specific constructs from Bt 176 maize were not detected in any investigated blood or tissue samples.

Dose dependent molecular effects of acrylamide and glycidamide in human cancer cell lines and human primary hepatocytes

Recently published studies suggest a weak positive correlation between increased dietary acrylamide intake and the increased risk of endometrial and ovarian cancer. However, risk assessment of acrylamide remains difficult because the carcinogenic mechanisms are still unknown and in particular the molecular effects of low level acrylamide exposure as seen by dietary intake are not well understood. Therefore, we analyzed in ovarian and endometrial cancer cell lines as well as in primary hepatocytes the expression of genes involved in cancer development and xenobiotic metabolism after high and low dose exposure (1-0.001mM) of acrylamide and its metabolite glycidamide. In conclusion our in vitro results demonstrate that exposure to high doses of glycidamide/acrylamide - exceeding the dietary exposure of the general population by far - can induce genes with growth promoting potential like the oncogene cMYC and genes involved in the MAPK pathway. However, low-dose exposure seems to activate primarily genes involved in the elimination of the toxicant.

Determination of osteocalcin in meat and bone meal of bovine and porcine origin using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and high-resolution hybrid mass spectrometry.

A method has been developed for determining the origin of meat and bone meal (MBM) by detecting species-specific osteocalcin (OC) using matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) and high-resolution hybrid mass spectrometry (HR-Q/TOF MS). The analysis is based on the detection of typical species-specific OC and its tryptic peptide fragments which differ in mass due to differences in the amino-acid sequences between species. After dissolving the MBM samples in EDTA buffer, purification after ultrafiltration was performed using two methods: solid-phase extraction using Zip-Tip C(18) or size exclusion coupled with reverse-phase chromatography. Fractions containing partially purified intact OC were analyzed using LC-Q/TOF and MALDI/TOF mass spectrometry. Species-specific OC was detected at the typical protonated and doubly protonated molecular ions. Furthermore, typical porcine- and bovine-derived tryptic fragments from MBM were detected after enzymatic digestion. In order to determine the underlying amino-acid sequences and to confirm the assignment to OC-derived peptides, MS/MS analysis was carried out. In conclusion, we were able to detect OC in bovine and porcine MBM with high sensitivity and the MS-based method described here by which total OC mass and marker peptides of digested OC are recorded can be used as an alternative approach to detect genus-specific differences in MBM and can be applied as a confirmatory method to mainly immunological osteocalcin screening methods.

Immunological detection of osteocalcin in meat and bone meal: a novel heat stable marker for the investigation of illegal feed adulteration.

A sandwich ELISA was developed for the detection of bovine meat and bone meal (BMBM) in feed, based on polyclonal rabbit antibodies raised against the synthetic N-terminal amino acid sequence 1–9 (YLDHWLGAP) of bovine osteocalcin. To set up a sandwich ELISA pair, a commercial mouse monoclonal capture antibody binding to a highly conserved epitope in the mid-fragment of the peptide was employed. It is shown that the bone marker osteocalcin is immunologically well detectable in BMBM extracts obtained by a simple EDTA-based procedure even in a sample heated up to 145°C. Furthermore, a genus-specific restriction of the major specificity to cattle and horse was possible. The observed bi-specificity is consistent with theoretical predictions. The assay sensitivity with bovine osteocalcin of 1 ng was sufficient to enable the detection of 0.1% BMBM in compound plant feed or fish meal, for which no cross reaction was observed. In general the quantification of osteocalcin in extracts is possible using a standard curve procedure with pure bovine osteocalcin.

Gene transcription analysis of carrot allergens by relative quantification with single and duplex reverse transcription real-time PCR

Single and duplex real-time polymerase chain reaction (PCR) systems have been developed to quantify specific mRNA transcription of genes coding for the major Daucus carota allergen isoforms Dau c 1.01 and Dau c 1.02. Methods were tested with samples from the local market. Whereas the gene transcription levels for Dau c 1.01 were consistently high in all investigated samples, significant differences for the Dau c 1.02 transcription could be demonstrated in randomly collected market samples. The gene transcription level for the minor Dau c 1.02 variant is about one log below Dau c 1.01. Both formats, single or duplex real-time methods, exhibit ideal cycle threshold (CT) ranges and good reproducibility. In particular, the easily performed duplex real-time PCR system is potentially suited for the selection of hypoallergenic varieties and studying the impact of post-harvesting or environmental conditions.

Nachweisverfahren für Rindfleisch in Lebensmitteln unter Anwendung der TaqManTM-Technologie

ZusammenfassungEs wurde ein TaqManTM-PCR-System entwickelt, mit dem Rindfleisch auch in kleinsten Mengen (0,1%) in prozessierten Lebensmitteln nachgewiesen werden kann. Darüber hinaus ermöglicht ein zweites parallel dazu entwickeltes TaqManTM-PCR-System einen sicheren Ausschluss falsch-negativer Resultate durch den Nachweis von Fleisch verschiedener Säugetiere oder Geflügel im Untersuchungsmaterial. Diese beiden TaqManTM-PCR-Systeme erlauben den eindeutigen Nachweis von Rindfleisch in verschiedenen prozessierten Lebensmitteln (Brüh-, Rind-, Kalbsleberwurst, Kalbfleischsülze, Rindergulasch) und in Mischungen aus Rindfleisch mit Schweinefleisch bzw. Hühner- und Putenfleisch.AbstractA TaqManTM-PCR-assay was developed to detect even traces of beef (0.1%) in processed foods. A second TaqManTM-PCR-System was established in parallel to exclude false-negative results by identification of mammalia and poultry meat in the food samples. Both assays can be used for the analysis of food samples to identify unambiguously beef in highly processed foodstuffs (Frankfurter type sausage-, beef- and calf-liver sausage, jellied calf meat, beef goulash) and in mixtures of beef with pork or chicken and turkey, respectively.

Mass Spectrometry-Based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds

The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR) are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of nonruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides, and a LC-MS/MS-based analysis for peptide quantification using isotope labeled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of 4 orders of magnitude with a detection limit in the range of 0.1% to 1% (w/w).

Species Differentiation and Quantification of Processed Animal Proteins and Blood Products in Fish Feed using an 8-plex Mass Spectrometry-Based Immunoassay

With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.

Anwendung von Methoden zum Nachweis von gentechnisch veränderten Sojabohnen und gentechnisch verändertem Mais in im Handel befindlichen Lebensmitteln

Seit dem 1. September 1998 müssen Lebensmittel, die aus gentechnisch veränderten Sojabohnen oder aus gentechnisch verändertem Mais hergestellt wurden, gekennzeichnet werden, wenn sie entweder aus der gentechnischen Veränderung resultierende neue Proteine oder rekombinante DNA enthalten. Zur Kontrolle dieser Kennzeichnungsvorschrift durch die zuständigen Überwachungsbehörden sind geeignete standardisierte Nachweisverfahren erforderlich. Für transgene Sojabohnen steht inzwischen eine amtliche Nachweismethode zur Verfügung. Eine Methode zum Nachweis der gentechnischen Veränderung in transgenem Mais wird derzeit im Ringversuch evaluiert. Die bisher nur anhand unverarbeiteter transgener Sojabohnen und transgener Maiskörner entwickelten und erprobten Nachweisverfahren wurden im Hinblick auf ihre Eignung zur Untersuchung von Lebensmitteln, die Verarbeitungsprodukte aus transgenen Sojabohnen bzw. transgenem Mais enthalten, überprüft. Beide Methoden haben sich als anwendbar für alle untersuchten Lebensmittel erwiesen. Since September 1, 1998 foods and food ingredients produced from transgenic soybeans or from transgenic maize and containing recombinant DNA or proteins resulting from a genetic modification are subject to labeling. Validated analytical methods are necessary in order to control compliance with these labeling requirements. A validated method to identify the genetic modification of glyphosate tolerant soybeans is already available. The evaluation of a method for the detection of transgenic maize is in progress. Both methods have been developed with raw material. The aim of this study was to examine whether these methods can also be applied for the detection of recombinant DNA in foods containing processed material from transgenic soybeans or from transgenic maize. The results demonstrate that both methods are applicable to detect genetic modifications in a variety of processed foods.