Abstracts / Toxicology Letters 189S (2009) S57–S273 S61

M13Development of a quantitative detection system for cereulide,the emetic Bacillus cereus toxin

Tobias Bauer 1,∗, Timo Stark 2, Thomas Hofmann 2, MonikaEhling-Schulz 1,3

1 Technische Universität München, Lehrstuhl für MikrobielleÖkologie, Freising, Germany, 2 Technische Universität München,Lehrstuhl für Lebensmittelchemie und Molekulare Sensorik, Freising,Germany, 3 University of Veterinary Medicine, Clinic for Ruminants,Food Microbiology Unit, Vienna, Germany

The toxin producing Bacillus cereus causes two different types ofgastroenteric diseases: diarrhoea and emesis. The pathogenity ofthe emetic B. cereus arises from the small dodecadepsipeptidecereulide. This cyclic toxin is resistant to heat, proteolysis as well asto acid and basic conditions. Because of these properties cereulidepreformed in food will not be destroyed in the intestinal tract orby reheating of food. Due to its lipophilic character it could rapidlybe absorbed from the gut and may have neurotoxic and immuno-modulating effects.

In recent years three detection methods of cereulide have beendescribed: LC/MS analysis; cytotoxicity assay and a boar sperm-based motility assay. Hitherto all assays were performed usingthe depsipeptide antibiotic valinomycin as surrogate standard.However, it cannot be ruled out that valinomycin behaves differ-ently than cereulide in complex matrices and/or in biochemicalassays. Due to changing lifestyles and eating habitats, emetic B.cereus is gaining increasing prominence as an emerging food bornepathogen and an appropriate standard for a conclusive detectionand quantification of the emetic toxin cereulide is urgently needed.Therefore, a protocol for in vivo production of cereulide in highyields has been established and a HPLC-based purification systemwas developed. The quality and purity of the toxin was monitoredby cell culture assays, LC–MS/MS and 1D/2D-NMR analysis. Nowthe chemically pure toxin can be used as an internal standard inbiological as well as chromatographic analysis to detect cereulidemore specifically in complex matrices as, e.g. food and feeds.

doi:10.1016/j.toxlet.2009.06.303

M14ECG data acquisition by external telemetry for toxicology (ET2)in freely moving dogs: Validation by comparison with invasivetelemetry

Stéphane Milano, Jean-Paul Briffaux ∗, Estelle Chalencon,Christophe Bory, Stephane Baudet, Philippe Lege

MDS Pharma Services, Drug safety Assessment 329, Saint Germainsur Arbresle, France

The reliability of surface ECG investigations collected during regula-tory toxicology studies in non-rodent species may be compromisedby the stress generated by implementation of the recording pro-cedure. Although invasive telemetry is considered as the goldstandard for recording long-term, high quality ECG data, theresources required are often incompatible with the objectives of aregulatory toxicology study. JETTM (DSI) technology represents anattractive alternative to record and transmit surface ECG in a largeset of freely moving and socialised animals.

We have thus compared surface ECG data recorded withthe external telemetry for toxicology (ET2) system that we

recently implemented with those obtained using invasive teleme-try.

Six dogs were surgically equipped for telemetry to record leadII ECG and with the JETTM setup connected to cutaneous patchelectrodes to record a six lead ECG. These data were analyzedby automatic recognition of ECG waveforms but without reposi-tioning of markers. ECGs were collected simultaneously from bothdevices for 24 h before and after oral administration of 30 mg/kgof sparfloxacin. The duration of the RR, PR and QT intervals andof the QRS complex were measured with each device at specifictime-points.

An excellent correlation of key ECG parameters (RR, PR and QTinterval durations) automatically detected by the software of eachsystem (invasive vs. ET2) was demonstrated.

Our recently installed external telemetry for toxicology ET2

system may prove to be a powerful tool for the detection of ECG-modifying properties of new drugs in regulatory toxicology studies(e.g. QT prolongation).

doi:10.1016/j.toxlet.2009.06.304

M15Insulin-producing cells can be achieved in vitro by direct trans-fection of mouse pdx-1 into rat mesenchymal stem cells

Mehdi Kadivar 1,∗, Neda Memari 1,2, Kazem Parivar 2, PejmanFard-Esfehani 1

1 Pasteur Institute of Iran, Biochemistry, Tehran, Islamic Republic ofIran, 2 Science and Research Branch, Islamic Azad University, Tehran,Islamic Republic of Iran

Objective: Mesenchymal stem cells (MSCs) have the ability of self-renewal and multi-directional differentiation. This ability wouldprovide a potentially unlimited source of cells for transplanta-tion. Recent reports showed that these cells could differentiateinto insulin-producing cells or IPCs by direct transfection of someinducer genes such as pancreas and duodenal homeobox factor-1(pdx-1). This gene plays a crucial role in pancreas development, �-cells differentiation into IPCs, and maintenance of mature �-cellsfunction. In this investigation, we showed that rat bone marrow-derived mesenchymal stem cells can be produce islet-like cells withinsulin secretion potential by transfection of a construct containingmouse pdx-1 gene into them.

Methods: Rat MSCs were isolated from femur of Wistar rats andcultured. Passage 3 of MSCs were induced to direct differentiationinto islet-like cells by temporary transfection of a plasmid contain-ing mouse pdx-1 gene into BMSCs by using polyfect as trasfectionreagent. Transfected cells were monitored daily by inverted micro-scope to detection of their morphological changes. After 14 days oftranfection the medium were collected for C-peptide test and cellswere fixed for immunocytochemical stainings.

Results: Immunocytochemical studies showed that differenti-ated cells have the specific markers for �-cells and C-peptide testrevealed the insulin secretion potential of them.

Conclusions: Our data suggested that the temporary expressionof mouse pdx-1 gene into rat MSCs, can lead to islet-like cells withmorphological and functional properties similar to real �-cells. Asthese differentiated cells have the ability of insulin secretion, theresults of this study may be used in the future for diabetes therapyby islet differentiation and transplantation.

Keywords: Mesenchymal stem cells; pdx-1 Gene; Insulin-producingcells

doi:10.1016/j.toxlet.2009.06.305