Austrian Society Of Toxicology Research Articles

PI3Kδ: a double-edged sword in leukemia formation

The PI3Kδ isoform is a candidate drug target in leukemia. Here, we explored its role in Abelson-induced leukemia. Frank leukemia emerges if the tumor cells have managed to outwit the immune system. The absence of PI3Kδ affected both the tumor cells and the NK cells. Abelsontransformed PI3Kδ-/cells induced leukemia in RAG2-/animals with a significantly increased latency, implicating PI3Kδ in tumor progression. NK cell function, however, was also contingent on PI3Kδ. PI3Kδ-/NK cells failed to lyse target cells. Capacitance measurements revealed the underlying defect: in PI3Kδ-/NK cells lytic granules did not fuse with the cell membrane. Accordingly, transplanted leukemic cells killed PI3Kδ-/animals more rapidly, both in syngeneic (PI3Kδ-/-) or immunocompromised (RAG2-/PI3Kδ-/-) animals. Our observations define a dual function of PI3Kδ in leukemia and document that the action of PI3Kδ in the NK compartment is as relevant to the survival of the mice as the delayed tumor progression. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Role of fibroblast growth factors in hepatocarcinogenesis of humans and rats

The fibroblast growth factors (FGF) FGF-1 and FGF-2 are often deregulated in hepatocellular carcinoma (HCC). Information is missing on the role of other FGF-family members for hepatocarcinogenesis. We therefore determined the function of FGF-8, FGF-17 and FGF-18 in the development and progression of liver cancer. Half of the rat liver tumors studied showed enhanced expression of FGF-18. In about 50% of human HCC, FGF-8, FGF-17 and FGF-18 and the corresponding FGF-receptors 3 and 4 were found to be upregulated. For functional studies we chose primary co-cultures of normal and premalignant (GSTp+) rat hepatocytes. DNA synthesis was significantly higher in GSTp+ than in normal hepatocytes indicating an inherent growth advantage of the premalignant cell population. FGF-8 and FGF-18 stimulated DNA preferentially in GSTp+ hepatocytes, while FGF-17 exhibited no effect. In HCC-derived cell lines FGF-8, FGF-17 and FGF-18 stimulated growth, which involved phosphorylation of ERK1/ 2 and S6. Conclusions: FGF-8 and FGF-18 induced preferential growth of (pre)malignant hepatocytes and are highly upregulated in liver tumors indicating autoand/or paracrine stimulation during formation and progression of the tumors. The results also show considerable similarities in the role of these FGFs in rat and human hepatocarcinogenesis implying that the molecular mechanisms underlying chemically induced rat hepatocarcinogenesis and human hepatocarcinogenesis are often identical. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Role of mesenchymal liver cells in mediating hepatic toxicity and carcinogenesis

Mesenchymal liver cells (Kupffer cells, KC; sinusendothelial cells, EC) are considered to play a role in the response of the liver to pro-inflammatory stimuli. We studied whether genotoxic and non-genotxic carcinogens are also capable to activate these mesenchymal liver cells. Liver cell suspensions were separated into hepatocytes, KC and EC. Cells were incubated with lipopolysaccharid (LPS), the genotoxic N-nitrosomorpholine (NNM) or non-genotoxic carcinogens like nafenopin, cyproterone acetate, phenobarbital, or arsenic. LPS and NNM incuced a release of TNF-α and superoxide while the other compounds showed mostly minor effects on KC and EC. To assess the impact of activated mesenchymal cells on hepatocarcinogenesis a co-culture model of unaltered and preneoplastic hepatocytes was used. DNA synthesis was significantly higher in preneoplastic than unaltered cells and was further increased by supernatant of LPS-stimulated KC and EC. The supernatant effect was greatly abrogated by antisera neutralizing heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF itself was a potent inducer of DNA synthesis and mitosis preferentially in the preneoplastic hepatocytes. In conclusion, KC and EC, activated by pro-inflammatory stimuli, may contribute to carcinogenesis via release of growth factors for preneoplastic hepatocytes. Whether this cell activation may be caused by non-genotoxic carcinogens requires further investigations. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

MTORC1 inhibition with rapamycin or LY294002 alone but not in combination leads to AKT phosphorylation via mTORC2 in melanoma cells

Inhibition of mTOR complex 1 (mTORC1) with rapamycin leads to phosphorylation of AKT in some cancer cells with unknown biological consequences. A mechanism involving insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K) leading to increased AKT phosphorylation has been described. For melanoma, it is unknown, whether this feedback loop plays a role, although preliminary clinical data indicate poor activity of rapalogues in melanoma. Here, we report that treatment of melanoma cells with rapamycin resulted in strong and long lasting AKT phosphorylation, but had little or no effects on cell viability, cell cycle arrest or apoptosis. Combined PI3K/mTOR inhibition with LY294002 had strong effects on these parameters, but also led to increased phospho-AKT levels after prolonged treatment. In contrast to the single treatments, combination of rapamycin plus LY294002 was able to suppress AKT phosphorylation even after prolonged treatment. Inhibition of mTOR complex 2 (mTORC2) using RNAi led to reduced levels of p-AKT, also under conditions when mTORC1 was inhibited. PTEN mutant melanoma cells showed slightly increased levels of AKT phosphorylation compared to PTEN wild-type cells. Thus, our results indicate that mTORC1 inhibition with specific inhibitors such as rapamycin as well as with multi-target inhibitors such as LY294002 can lead to AKT phosphorylation in melanoma cells via mTORC2, but without significant influence on treatment efficacy in vitro. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Mapping the interaction sites of the viral encoded chemokine receptor US28 and the sorting protein GASP-1

US28 is a chemokine receptor encoded by the human cytomegalovirus (hCMV). It has been shown to be crucial for viral propagation. For instance, it is present on the viral envelope where it may serve as a cell-to-cell attachment factor through binding to the membrane-bound chemokine fractalkine. Most notably, US28 is a constitutively signaling and endocytosing receptor, thus playing a key role in the early reprogramming of the host cell. It has further been suggested that during viral propagation, US28 is integrated into the viral envelope in the lysosomes of host cells. A candidate protein for sorting many G protein-coupled receptors to lysosomes is the G protein-coupled receptor-associated protein-1 (GASP-1). Here we show that GASP-1 interacts with US28 in vitro and set out to map the interaction sites of US28 and GASP-1 by using in vitro translation binding assays. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Lipid hydroperoxide, an intermediate product of oxidative stress, induces tumour progression-associated genes in hepatocarcinoma cells

Hepatocellular carcinoma often develops in the liver under chronic inflammation. Oxygen radicals, produced during inflammation, attack membrane lipids and form a number of oxidized metabolites including lipid hydroperoxides. The addition of linoleic acid hydroperoxide (LOOH) to the medium of recently established human hepatocarcinoma cell line (HCC-1.2) caused dosedependent cell loss and enhanced LDH-release. Under subtoxic conditions LOOH induced intracellular hydrogen peroxide production and a decrease of cellular GSH content. Elevated expression of protooncogene c-myc and a catalytic subunit of telomerase hTERT were observed under LOOH exposure. Myc activation is sufficient to induce cell cycle entry in the absence of growth factors. Accordingly, the cells were pushed into the Sand G2/Mphase by LOOH. An increased expression of c-fos, c-jun, the antiapoptotic enzyme heme oxygenase 1 (HO-1) and the proinflammatory angiogenic interleukin-8 (IL-8) was detected under LOOH exposure. Pre-treatment of cells with antioxidant N-acetylcystein or with selenite, which induces the LOOH-detoxifying enzyme glutathione peroxidase, partially inhibited the expression of LOOHinduced genes implicating the involvement of oxidative stress. Application of SnPPIX, a HO-1 inhibitor, decreased the viability of HCC-1.2 cells indicating the protective role of HO-1 induction. These results show that lipid hydroperoxides may be an important driving force for carcinogenesis in the liver. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Toxicological investigation of nanoparticles: effects on human cells

The aim of the present study was the establishment of an in vitro test system to reveal the potential risk to human health of nanoparticles at the workplace. The essential advantage of in vitro investigations is to be non-invasive, the employees don't have to be bothered and the work routine doesn't have to be intercepted. At occupational settings test cells on Transwell® inserts were exposed to the workplace atmosphere or to particle filtered air for 1 to 3 hrs using a CULTEX® System. 2 types of co-cultures were tested: In the first type differentiated macrophages were exposed and post-incubated with human lung epithelial cells. In the second type differentiated macrophages were seeded on human lung epithelial cells and the co-culture was exposed. As endpoints for particle exposure cell viability (WST-1 assay), oxidative stress (DHR-Assay) and pro-inflammatory cytokines (BDTM CBA-Assay) were evaluated. Cell viability testing showed a negative effect at high exposure. In cells exposed to the workplace atmosphere an increased oxidative burst was detected compared to cells exposed to particle filtered air. Exposure of co-cultures resulted in significantly enhanced TNF-α, IL-6, IL-1β and IL-8 levels. We could show that our in vitro exposure system is very well adapted for the assessment of adverse effects of nanoparticles at the workplace. Our results indicate that nanoparticles involve an occupational risk and further experiments will be performed to analyse additional endpoints. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Pharmacokinetics of amphotericin B colloidal dispersion in liver failure

Results Three patients with impaired and two patients with normal hepatic function (one patient on day 1, one patient on day 5 of therapy) were enrolled so far. After a single dose of ABCD, the AMB half life was similar in patients with impaired and normal liver function. The AMB clearance was slower in liver failure (0.15 vs. 0.54 L/h/kg for total AMB, 0.22 vs. 0.55 L/h/kg for the liberated AMB fraction and 0.52 vs. 35.6 L/kg for lipid-associated AMB) and the apparent volume of distribution was smaller (2.2 vs. 10.9 L/kg for total AMB, 3.1 vs. 11.2 L/kg for liberated and 8.2 vs. 154.5 L/h/kg for lipid-associated AMB). Conclusion The elimination of ABCD appears to be delayed in cholestatic liver failure. More pharmacokinetic data are required to establish reliable dose recommendations for ABCD in patients with liver failure. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Heterologous expression of membrane proteins in cardiac myocytes

The cardiac isoform of the Na+ channel (NaV1.5) is known to accumulate in the endoplasmic reticulum (ER). This retention presumably reflects quality control in the ER. In order to understand the underlying mechanism, we heterologously expressed the human orthologue of the Na+ channel (NaV1.5) in neonatal primary rat and murine cardiomyocytes, in a cardiomyoblast cell line (H9c2) and in HEK293 cells (internal control). In HEK293 cells, NaV1.5 readily accumulated at the cell surface and gave rise to functional channels with the expected electrophysiological properties. In contrast, in cardiomyocytes and H9c2 cells, the NaV1.5 accumulated in the ER regardless of the transfection method employed (lipofection, nucleofection). As a positive control, we employed G protein-coupled β1-adrenergic, A1 and A2A adenosine receptors. In HEK293 cells, export of the A2A receptor is known to be enhanced by the deubiquinating enzyme USP4. Accordingly, we also co-expressed USP4 and the A2A receptor in the different cardiomyocyte preparations. However, in all instances, the membrane proteins were trapped within intracellular compartments. This was, in particular true for the NaV1.5, which, in many instances, accumulated in circular bodies, which are reminiscent of calnexin-rich organized smooth ER structures. Based on these findings, we conclude that (i) membrane proteins undergo stringent quality control in cardiac myocytes and (ii) ERexport of the NaV1.5 is limited by the availability of additional cardiomyocyte-specific components. Acknowledgements Supported by FWF M989-B09 (MR) and OAW DOC-fFORTE 22255 (IG). from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Antioxidant and free radical scavenging activities of sumac (Rhus coriaria) and identification of gallic acid as its active principle

It is known that certain spices are rich in antioxidants. Sumac (Rhus coriaria) is widely consumed in Middle-Eastern countries. We tested its DNA-protective effects in a human intervention trial. Eight participants consumed 3 g of sumac for 3 days. We found strong protective effects in single cell gel electrophoresis assays (SCGE) with endonuclease III (ENDO III), formamidopyrimidine glycosylase (FPG) and hydrogen peroxide in human peripheral lymphocytes. H2O2-induced DNA migration was reduced by 30%, oxidized pyrimidines 36% and oxidized purines 41%, respectively, after the intervention. Subsequent in vitro experiments indicated that gallic acid (GA) is the active principle of sumac. GA is also contained in certain plants (mango, rhubarb, strawberries). In a subsequent trial, 8 participants consumed GA (0.2 mg/kg BW/d) for 3 days and strong protective effects were observed with this phenolic compound which is very rapidly absorbed in the GI tract. The reduction of DNA migration induced by H2O2 was 40%, ENDO III 58%, FPG 52%. Comparisons show that GA is 50 times more protective than the vitamins C and E. The protective effects of sumac and GA were also investigated in animal experiments. Eight male rats per group were fed 3 days with sumac (0.02 g/kg BW/d) and GA (0.2 mg/kg BW/d). After irradiation in a 60Co source (7.74 Gy/1 min), the animals were killed immediately and protective effects were seen in lymphocytes, brain, liver, colon and lung. Taken together, our findings indicate that GA is a super-antioxidant which protects against ROS-induced DNA-damage. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Monitoring platelet activation with two different shear-dependent function tests: application of PFA-100 and the Impact-R ARA

To analyze the aspirin effect we compared two methods, the PFA-100 and the Impact-R to determine their sensitivity and specificity. In this prospective double-blind single centre trial 37 healthy male volunteers (median age 27, range ± 7) were randomized to take aspirin 160 mg per day for 7–10 days or placebo. The platelet function was determined under close to physiological and high shear. At study entry and after 2 weeks we determined the closure time (CT) of collagen/epinephrine cartridges obtained by the PFA-100 (CEP-CT), and the surface coverage (SC), as a measure of platelet adhesion, with and without pre-incubation with arachidonic acid (ARA). By the PFA-100 CEP-CT was median 210 ± 62 before aspirin, and >300 sec in all individuals taking aspirin for 2 weeks (p < 0.0001). The SC pre-incubated with ARA was median 4.6 ± 2.5 before study entry and median 6.8 ± 2.7 after two weeks of aspirin (p < 0.0001). Changes were not significant in controls. In order to normalize for the broad variation of the SC from one individual to the next, we determined the relative change of the SC due to the addition of ARA. Again, aspirin significantly inhibited ARA induced reduction of adhesion (p = 0.014). Sensitivity and specificity to detect aspirin was 100% and 50% with the PFA-100, 100% and 42% with the Impact-R. Conclusion The PFA-100 (collagen/epinephrine) and the Impact-R are useful to estimate the individual response to aspirin. While a variety of factors may contribute to prolonged CEP-CT by the PFA-100, the Impact-R ARA test is specific for aspirin. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

C-terminal splicing reveals intramolecular gating modulation in CaV1.3 L-type Ca2+ channels

Neuronal excitability and pace-making in the sinoatrial node are controlled by low-voltage activated CaV1.3 Ltype Ca2+ channels. We recently found that in related CaV1.4 channels a highly-structured distal C-terminal motif (CTM) modulates voltageand Ca2+-dependent gating (CDI). In CaV1.3, C-terminal splicing leads to a fulllength (CaV1.3L) and at least 1 short (CaV1.3S) splice form. If a CTM would also modulate CaV1.3 gating it would be present in CaV1.3L but not CaV1.3S variants. We therefore compared the biophysical properties of CaV1.3L or CaV1.3S coexpressed with β3 + α2δ-1 in tsA-201 cells using the whole-cell patch-clamp technique. CaV1.3S channels activated at more negative potentials compared to CaV1.3L (~ -10 mV, p < 0.0001), inactivated faster (p < 0.01) and showed more CDI (p < 0.01). These changes resulted in a decreased window current shifted to more hyperpolarized potentials likely to cause a reduction in the channels' dynamic range. Removal of the C-terminal 158 (CaV1.3Δ1158) or 76 amino acids was sufficient to induce gating properties similar to CaV1.3S. FRET experiments revealed interaction of the last 158 amino acids (C158) to a proximal C-terminal domain in CaV1.3L. Coexpression of C158 with CaV1.3Δ1158 completely restored CaV1.3L gating properties confirming this protein interaction. Thus CaV1.3 channel gating is under control of the distal C-terminus allowing alternative splicing to finetune channel activity and adapt channel function to physiological needs. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Use of human-derived cell lines for the investigation of dietary factors which affect DNA stability

One of the key problems in in vitro genotoxicity testing concerns the adequate representation of xenobiotic drug metabolising enzymes in the indicator cells. Conventional cell lines (V-79, CHO) which are currently used lack most phase I as well as phase II enzymes, which catalyse the activation/detoxification of chemical carcinogens. As shown earlier by our working group, the human cell line HepG2 possesses several phase I/phase II enzymes in an inducible form, but one of the disadvantages of HepG2 cells is their instability and the lack of specific enzymes (e.g. CYP2E1, NAT2, CYP1A2) which are important in the activation of dietary carcinogens (nitrosamines, heterocyclic aromatic amines). Therefore we studies the metabolic capacity of four new isolated human-derived cell lines (HCC1.2, HCC2, HCC3 und NKNT-3) by use of RT-PCR and investigated the sensibility of these lines towards induction of DNA damage by selected representatives of different classes of genotoxic dietary carcinogens which require activation (NDMA, PhIP, Trp-P-1, AFB1, B(a)P). Two of theses cell lines were found to possess a variety of enzymes monitored, including those which are lacking in HepG2, and positive results were obtained in the single cell gel electrophoresis assay with all of the model compounds in the line HCC1.2. Taken together, our results indicate that these cell lines may be highly useful tools for the detection of dietary mutagens and antimutagens. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Spin trapping experiments with ethyl-substituted EMPO derivatives (EEMPO)

Free radicals in biological systems play a major role in the onset of many diseases, e.g. oxygen-centered radicals such as hydroxyl or superoxide radicals. In order to identify and localize these radicals a series of four novel spin traps have been developed and their structure fully characterized by [1H]and [13C]-NMR spectroscopy as well as mass spectrometry. The novel compounds can be described as ethylsubstituted EMPO derivatives, namely 5-ethoxycarbonyl3-ethyl-5-methyl-pyrroline N-oxide (3,5-EEMPO), 5ethoxycarbonyl-4-ethyl-5-methyl-pyrroline N-oxide (4,5EEMPO), 5-ethoxycarbonyl-5-ethyl-3-methyl-pyrroline N-oxide (5,3-EEMPO) and 5-ethoxycarbonyl-5-ethyl-4methyl-pyrroline N-oxide (5,4-EEMPO). Their spin trapping behaviour towards a series of different oxygenand carbon-centered radicals is described. All compounds were obtained in two different stereochemical forms (cis and trans), but only 3,5-EEMPO and 5,3-EEMPO could be separated into the different diastereomers using conventional chromatographic procedures. The cisand transforms exhibited considerably different spectral parameters and stabilities of the respective superoxide adducts (ranging from about 12 to 35 min). In addition, spin adducts obtained from different carbon-centered radicals derived from methanol, ethanol, formic acid and linoleic acid hydroperoxide have also been characterized. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

New method for detecting ATP release from rat hippocampal slices

Chemical signaling has a key role in neural and non-neural function; however, only a few techniques are available to measure the concentrations of neurotransmitters and modulators directly in the extracellular space. A recent advance in this area is the development of the multi-enzymatic microelectrode biosensor technique whereby we can measure the concentration of different neurotransmitters and modulators around single neurons or synaptic structures in the brain with high temporal and spatial resolution. Micro-biosensors are also ideal for detecting realtime neurotransmitter release in the central nervous system in vivo because they are minimally invasive. The ATP biosensor is formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3-phosphate oxidase, which results in the production of electroactive H2O2, detected by amperometry. The null sensor, which possesses the silicate layer but lacks the enzymes, shows near-identical responses to the interferences potentially allowing the use of differential recordings to remove almost all of the interfering signals. We used the ATP microelectrode biosensor to demonstrate the release of ATP from rat hippocampal slices in vitro. The basal ATP levels have been estimated to be as low as a few nmol/L (25 ± 5 pA). The ATP sensor exhibited a rapidly increasing current during K+ depolarization which reached 341 ± 170 pA (peak concentration 0.79 ± 0.2 μmol/L). This effect was significantly decreased by the Na+ channel blocker TTX (0.17 ± 0.08 μmol/L) and by Ca2+free medium (0.22 ± 0.1 μmol/L). Acknowledgements This work was supported by the grant from the Hungarian Research and Development Fund (NKFP1A/002/2004). from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Anticonvulsant and neuroprotective actions of endogenous dynorphin

Anticonvulsant actions of dynorphin were proposed since more than 2 decades, however till now, we do not have information on the functions of endogenous dynorphin. Thus, we investigated prodynorphin knockout (KO) mice in different models of seizures and epilepsy. Seizure threshold was investigated by tail-vein infusion of 100 μg pentylenetetrazole (PTZ)/ml at a rate of 100 μl/min until mice displayed generalized clonic seizures. Wild-type mice showed clonic seizures at 39.2 ± 1.88 (mean ± SEM; n = 5) mg PTZ/kg body weight. KOs displayed a significantly reduced seizure threshold of 32.7 ± 1.17 (n = 6) mg PTZ/kg. This phenotype could be rescued entirely by the κ opioid receptor specific agonist U-50488, but not the μ opioid receptor specific agonist DAMGO. The δ opioid receptor specific agonist SNC80 decreased seizure threshold in both genotypes. Pre-treatment with the κ-selective antagonist GNTI completely blocked the rescue effect of U-50488. After injection of kainic acid into stratum radiatum of CA1 of the dorsal hippocampus, KO mice displayed increased neuronal loss along the rostro-caudal axis of the ipsiand partially the contralateral hippocampus at early time points after treatment. Thus, marked neurodegeneration was seen already 1 week after treatment in KO mice. The differences faded with time, being almost gone 5 weeks after kainate. Our data clearly indicate that endogenous prodynorphin derived peptides have anticonvulsant and neuroprotective properties. Acknowledgements This work was supported by the FWF (P20701-B05 and P18471-B05). from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Differential anti-inflammatory properties of peroxisome proliferator-activated receptors (PPAR) α and γ in experimental pancreatitis

Perfluorooctanoic acid (PFOA) has anti-inflammatory effects in models of cutaneous inflammation, possibly via activation of PPAR-α and PPAR-γ. We have therefore investigated whether PFOA has similar effects in a model of acute oedematous pancreatitis and whether such effects could be explained by agonism at PPAR-α or PPAR-γ. Acute pancreatitis was induced in anesthetized rats by i.v. infusion of the cholecystokinin analogue, caerulein. The PPAR-α agonist clofibrate or the PPAR-γ agonist rosiglitazone were injected s.c. before the experiments. Pancreatic oedema, neutrophil activation and production of prostaglandin (PG) E2 and prostacyclin (via 6-keto-PGF1α) were assessed in the pancreas. Acute pancreatitis caused significant oedema formation, neutrophil activation as assessed by myeloperoxidase activity in the tissue, and increased synthesis of pro-inflammatory prostanoids. Neutrophil activation was unaffected by clofibrate but was abolished by rosiglitazone. In contrast, prostanoid synthesis was unaffected by rosiglitazone but was inhibited by clofibrate. In conclusion, our data demonstrate that activation of PPAR-α and PPAR-γ has differential anti-inflammatory effects in acute interstitial-oedematous pancreatitis. Neutrophil activation is sensitive to inhibition by PPAR-γ activation while the production of pro-inflammatory prostanoids can be attenuated by activation of PPAR-α. Thus, the anti-inflammatory potential of PPAR-α and PPAR-γ ligands should be further investigated. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Safety assessment of melanoidins in cell line studies

The identification of substances capable of inducing mutations has become an important procedure in safety assessment of foods. Gene mutations are readily measured in bacteria and cell systems when they cause a change in the growth requirements of the cell, whereas chromosome damage in mammalian cells is typically measured by observing the cell's chromosomes under magnification for breaks or rearrangements. In particular, free radicals and reactive oxygen species (ROS), which are generated permanently, cause mutagenic alterations resulting in cancer, aging and abnormalities in the nervous system. Several tests in cells (in vitro and in vivo grown cells like lymphocytes) are used, e.g. chromosome aberrations, micronuclei and DNA damage, in order to evaluate the mutagenic potential. Tests on the risk assessment of Maillard reaction products, which are also known as nonenzymatic browning products and mainly formed at heating processes, have started around 25 years ago, being more detailed and sophisticated since around ten years. Most of the tests performed so far are based on cell line and bacteria tests. Summarizing the results obtained so far identified brown coloured end products of the Maillard reaction, which are also called melanoidins, are able to induce some genomic marker. However, when the tests are performed with a metabolic activation system by adding a defined enzyme mixture to the cells in order to simulate body conditions, less information is given so far, which identifies melanoidins as mutagenic or genotoxic. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

DIMBOA and DIBOA, two naturally occuring benzoxazinoides, cause aneuploidy in a human-derived liver cell line (HepG2)

Results In the Ames test DIBOA was positive. Effects in YG1024 were little higher as in TA98. DIMBOA was positive in TA100 only. Addition of S9 led to higher mutagenic activities. The sprout juices were clearly positive, however not due to the BAs. The SCGE assay was negative, but the MN assay was positive (DIMBOA at >2.5 μM, DIBOA at >5 μM). The CP showed >80% centromere positive (C+) micronuclei. Conclusion The slight differences between YG1024 and TA98 indicate that acetylation plays no role in the activation. The BAs can be classified as weak bacterial mutagens. It is unclear why the sprout juices were positive. Due to the results of the assays with HepG2, we tested for aneuploidic effects. This was confirmed by the CP result. Aneuploidy is thought to be a key event in cancer induction and no other aneugenic plant-derived substances of dietary relevance are known. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Daptomycin elimination by continuous venovenous hemofiltration: in vitro evaluation of factors influencing sieving and membrane adsorption

The present in vitro study set out to determine for the first time the continuous venovenous hemofiltration (CVVH) clearance of the novel lipopetide antibiotic, daptomycin, from human whole blood. Factors influencing daptomycin sieving and membrane adsorption were investigated in an in vitro setting. A recirculation model was established and daptomycin was added to the simulated blood circuit at different concentrations and in different solvent systems. The concentration of daptomycin over time in the modelled blood compartment and in ultrafiltrate was measured by HPLC. Mean sieving coefficients (SCs) of daptomycin over time were 1 ± 0.05, 0.3 ± 0.02 and 0.4 ± 0.03 in Ringer lactate, Ringer lactate containing human albumin and plasma, respectively. The CVVH clearance of daptomycin from whole blood exceeded the physiological clearance in an individual with normal renal function. Adsorption of daptomycin to synthetic surfaces proved moderate, resulting in loss of around 20% of the initial dose at 1 hour after the start of CVVH. Since mean SCs of daptomycin in protein-containing media were higher than the free fraction in plasma, our results suggest that besides protein binding, unknown factors such as intracellular drug partitioning influence sieving of daptomycin during hemofiltration. Due to the high in vitro CVVH clearance of daptomycin we determined from human whole blood, we recommend monitoring of daptomycin concentrations in patients undergoing hemofiltration. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007