Dynamic culture in multicompartment bioreactor upregulates cytochrome expression in human hepatocytes

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V06 Development of an in vitro sensitization assay based on monocyte-derived dendritic cells Andreas Schepky ∗ , Jochem Spieker, Silke Gerlach, Ludger Kolbe, Walter Diembeck, Wolfgang Pape, Horst Wenck, Klaus-Peter Wittern, Hendrik Reuter Beiersdorf AG, 4228-Toxicology in vitro, Box 682, Hamburg, Germany Dendritic cells, including Langerhans cells, forming a sentinel network for pathogen detection are the most abundant antigen presenting cells in the skin. Through their ability of hapten uptake, processing and presentation to T-cells they play a critical role in the induction of contact allergies. In this process dendritic cells undergo fundamental changes, e.g. surface marker expression. Their observance marks a potential endpoint in the experimental set-up of a predictive in vitro skin sensitization assay. Thus, CD1a-/CD14+ peripheral blood monocytes from donors where purified by density centrifugation and positive selection of anti-CD14-Ig coupled magnetic microbeads. CD1a-/CD14+ monocytes were differentiated into immature dendritic cells by 5-day culture in the presence of IL4 and GM-CSF. Substance treatment for 48 h was followed by FACS analysis of HLA-DR; CD86; CD80; CD14; CD1a and CD83. In this assay all strong sensitizers tested as well as nonsensitizers where identified correctly referring to their allergic potential in the LLNA, whereas moderate sensitizers (according to the LLNA) showed surface marker changes only close to cytotoxic concentrations. Limitations, e.g. donor variability and work intensiveness are widely discussed. However, this assay leads to results that reflect the reaction of a healthy donor population in contrast to single individuals of cell lines. Moreover, after interpretation of comprehensive investigation we assume that a classification of the sensitizing potential of substances may be possible, marking a clear advantage over an all-or-none interpretation of cell line based assays already established. Therefore this assay provides a basic application in assessing the allergic potential of active components. doi:10.1016/j.toxlet.2009.06.197

V07 Cytotoxicity of selected natural substances in human colon carcinoma Caco-2 cells Iva Fojtíková ∗ , Jiˇrí Vrba, Jitka Ulrichová Faculty of Medicine and Dentistry, Palacky University, Department of Medical Chemistry and Biochemistry, Olomouc, Czech Republic The aim of the study was to determine the cytotoxicity of three isoquinoline alkaloids (sanguinarine, chelerythrine, berberine) and three polyphenol compounds (gallic acid, catechin, rutin) in human colon carcinoma Caco-2 cells. Cells were seeded in 96-well plates at 2 × 104 cells/0.1 ml/well and treated with tested compounds for 24 h. The cell viability was evaluated by the MTT reduction assay and neutral red uptake. Our results showed a dose-dependent decline in the viability of Caco-2 cells in response to the treatment with four tested substances whose cytotoxicity decreased in the following order: sanguinarine > chelerythrine > gallic acid > berberine. As determined by the MTT assay and neutral red uptake, sanguinarine for instance at 2 ␮M concentration decreased the cell viability to 2% and 30%, 10 ␮M chelerythrine decreased the viability to 8% and 20%, and 100 ␮M gallic acid reduced the viability to 20% and

21%, respectively. Berberine at the highest tested concentration (200 ␮M) decreased the viability of Caco-2 cells only approximately to 70%, as shown by both methods. After the treatment of Caco-2 cells with catechin (1–1000 ␮M) or rutin (1–500 ␮M), we observed no significant decrease in the cell viability. Mechanisms of cytotoxic action of sanguinarine and chelerythrine in Caco-2 cells will be further investigated. Acknowledgement: The work was supported by grants Nos. MSM 6198959216 and GACR 303/09/H048. doi:10.1016/j.toxlet.2009.06.198

V08 Use of the in vitro model for studying the effects of secondary metabolites produced by indoor fungi Zuzana Kovacikova 1,∗ , Elena Pieckova 1 , Eva Neubauerova 1 , Miroslava Kuricova 1 , Jana Tulinska 1 , Erzsebet Tatrai 2 , Sona Wimmerova 1 1 Slovak Medical University, Bratislava, Slovakia, 2 NIOH, Budapest, Hungary

Presented in vitro experiments were focused to the effects of secondary metabolites produced by indoor microfungi on two types of toxicologically most important lung cells: alveolar macrophages and alveolar epithelial type 2 cells. There are reports about chronic intoxication, allergies or other pulmonary health problems of inhabitants from houses contaminated by microfungi. The majority of studies followed the effect of spores but the microfungi produce secondary metabolites which could be inhaled and their effect cannot be neglected. Aspergillus ustus, Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, isolated from mouldy buildings were cultured and both media and biomass cakes were collected, exo- and endometabolites, respectively were isolated. The isolated rat lung cells were cultured for 20 h with different concentration of the metabolites. After finishing the cultivation the changes on the surface of type 2 cells were evaluated by staining with Maclura pomifera lectin, the antioxidant status of both types of cells was analysed and the concentration of cytokines (TNFalpha, MCP-1) in medium was estimated. The results showed toxic effect of all tested endo- and exo-metabolites even in concentration 0.1 ␮g/ml. The differences between individual isolates were in terms of the extent. Acknowledgement: The study was supported by the APVV 032207 grant. doi:10.1016/j.toxlet.2009.06.199

V09 Dynamic culture in multicompartment bioreactor upregulates cytochrome expression in human hepatocytes Arti Ahluwalia 1,∗ , Bruna Vinci 1 , Duret Cedric 1 , Duret Cedric 2 , Malcolm Wilkinson 3 , Patrick Maurel 2 University of Pisa, Centro Piaggio, Pisa, Italy, 2 Inserm U632/UM-I EA3768, Montpellier, France, 3 Kirkstall Ltd., Sheffield, United Kingdom

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Outline: In vitro liver models for toxicity testing suffer from a number of drawbacks, including short-term viability, and phenotypic changes mainly associated with huge drops in P450 expression of hepatocytes. This has been generally attributed to the fact that the

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

complexity of the physiological environment is not replicated in petridishes or microplates. In fact, all cells are exquisitely sensitive to their microenvironment which is rich with cues from other cells, and from mechanical stimuli due to flow, perfusion and movement. Current methods for investigating cellular responses in vitro are inadequate in this sense, since the complex interplay of mechanical and biochemical factors are absent. Methods: To address these issues we have developed a ‘system on a plate’ modular MultiCompartmental Bioreactor (MCB) array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments. The new system offers mechanical stimuli from flow and biochemical stimuli from cells placed in connected modules, and can be used for assessing the human hepatotoxicity potential of drugs, or for pharmacological or pharmacokinetic studies. Human hepatocytes were cultured in the MCB system, by connecting eight (8) modules in series, and seeding cells in the long-term medium described in Ferrini et al. (1997), three weeks after isolation. The cells were subject to a flow rate of 180 ␮l/min for up to 7 days, and gene expression of a large number of enzymes were quantified with respect to freshly isolated hepatocytes from the same liver sample, as well as cells in control (multiwell) conditions. Results: The results show that most P450 enzymes (CYP 3A4, 2B6, 2C9, 1A2, 3A7, and 1A1) are upregulated in the MCB, as are several phase II enzymes (UGT1A1, GSTA1) as compared with the static controls, and reach about the same levels as found in freshly isolated hepatocytes. Therefore, we were able to maintain expression at high levels after over 24 days of culture. Conclusions: The MCB system is composed of a series of high flow-low shear modular bioreactor chambers designed to mimic crosstalk between cells or tissues. The results show that it is a simple but powerful method for culturing cells and tissues in a more physiological in vitro environment, and could represent a gold standard for in vitro toxicity testing.

Reference Ferrini, J.B., Pichard, L., Domergue, J., Maurel, P., 1997. Long-term primary cultures of adult human hepatocytes. Chemico-Biol Inter. 107, 31–45.

doi:10.1016/j.toxlet.2009.06.200

V10 Development of an in vitro test system to identify compounds with cell transforming activity René Thierbach ∗ , Urte Blume, Pablo Steinberg University of Veterinary Medicine Hannover, Department of Food Toxicology and Replacement/Complementary Methods to Animal Testing, Hannover, Germany The aim of the European Community Regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals is to improve the protection of human health and the environment through the better and earlier identification of the toxic properties of chemical substances, including their carcinogenic potential. In this context thousands of chemical substances have to be investigated in the next few years. Carcinogenicity testing is a very time-consuming and expensive issue. In order to reduce the number of laboratory animals needed, it is imperative to develop an in vitro test system to identify carcinogenic compounds. The aim of the ongoing project is to combine the BALB/c-3T3 cell transformation assay with the soft agar colony formation assay (SAA) in

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order to build up a high throughput transformation test. In a first step an automated SAA was established. In a second step it is now shown that the untransformed cell line BALB/c-3T3 (clone A31-11) can survive at least two weeks in the soft agar without forming colonies and that malignantly transformed A31-1-1 cells do form colonies in soft agar. Furthermore, the optimal time course for the combination of the two assays was determined by using the model compounds 3-methylcholanthrene as a tumour initiator and 12-Otetradecanoyl-phorbol-13-acetate as a tumour promoter. The next steps to be taken are the incorporation of a metabolic bioactivation system and the automation of the combined test. In the end a highly innovative in vitro test system to identify carcinogenic compounds in a high throughput modus will have been developed. doi:10.1016/j.toxlet.2009.06.201

V11 Assessment of apoptosis and cell cycle alterations in human peripheral blood lymphocytes exposed to okadaic acid Vanessa Valdiglesias 1,2,∗ , Blanca Laffon 1 , Julia García-Lestón 1,2 , Eduardo Pásaro 1 , Josefina Méndez 2 1

University of A Coru˜ na, Toxicology Unit, Department Psychobiology, A Coru˜ na, Spain, 2 University of A Coru˜ na, Cell and Molecular Biology, A Coru˜ na, Spain Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are nausea, abdominal pain, diarrhoea and vomiting; illness begins within 4 h and continues for about 3 days, but it does not appear to be fatal. At the molecular level, previous studies have shown that okadaic acid is a tumour promoter and a specific potent inhibitor of protein phosphatases and protein synthesis. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, the effects of different levels of OA on cell cycle and apoptosis have been examined in human peripheral blood lymphocytes. Cells were treated with a range of OA concentrations (5, 10, 20, 50, 100 and 1000 nM) in presence and absence of S9 fraction with the purpose of determining if this compound acts directly or it needs metabolic activation. The OA-induced alterations of cell cycle and the rate of apoptosis/necrosis were evaluated by means of flow cytometry methodologies. Data obtained showed an increase in the percentage of apoptotic cells and mitotic arrest in the highest OA concentrations evaluated when lymphocytes were cultured without S9 fraction. Nevertheless, no effects were found in S9-treated cells for both apoptosis and cell cycle. These results suggest that OA acts as a direct apoptosis inducer and cell cycle disrupter. Acknowledgement: Research supported by Xunta de Galicia (INCITE08PXIB106155PR). doi:10.1016/j.toxlet.2009.06.202