S66 Abstracts / Toxicology Letters 189S (2009) S57–S273

V06Development of an in vitro sensitization assay based onmonocyte-derived dendritic cells

Andreas Schepky ∗, Jochem Spieker, Silke Gerlach, Ludger Kolbe,Walter Diembeck, Wolfgang Pape, Horst Wenck, Klaus-PeterWittern, Hendrik Reuter

Beiersdorf AG, 4228-Toxicology in vitro, Box 682, Hamburg, Germany

Dendritic cells, including Langerhans cells, forming a sentinelnetwork for pathogen detection are the most abundant antigenpresenting cells in the skin. Through their ability of hapten uptake,processing and presentation to T-cells they play a critical role in theinduction of contact allergies. In this process dendritic cells undergofundamental changes, e.g. surface marker expression. Their obser-vance marks a potential endpoint in the experimental set-up ofa predictive in vitro skin sensitization assay. Thus, CD1a-/CD14+peripheral blood monocytes from donors where purified by den-sity centrifugation and positive selection of anti-CD14-Ig coupledmagnetic microbeads. CD1a-/CD14+ monocytes were differenti-ated into immature dendritic cells by 5-day culture in the presenceof IL4 and GM-CSF. Substance treatment for 48 h was followed byFACS analysis of HLA-DR; CD86; CD80; CD14; CD1a and CD83.

In this assay all strong sensitizers tested as well as non-sensitizers where identified correctly referring to their allergicpotential in the LLNA, whereas moderate sensitizers (according tothe LLNA) showed surface marker changes only close to cytotoxicconcentrations. Limitations, e.g. donor variability and work inten-siveness are widely discussed. However, this assay leads to resultsthat reflect the reaction of a healthy donor population in contrastto single individuals of cell lines. Moreover, after interpretationof comprehensive investigation we assume that a classification ofthe sensitizing potential of substances may be possible, marking aclear advantage over an all-or-none interpretation of cell line basedassays already established.

Therefore this assay provides a basic application in assessing theallergic potential of active components.

doi:10.1016/j.toxlet.2009.06.197

V07Cytotoxicity of selected natural substances in human colon car-cinoma Caco-2 cells

Iva Fojtíková ∗, Jirí Vrba, Jitka Ulrichová

Faculty of Medicine and Dentistry, Palacky University, Department ofMedical Chemistry and Biochemistry, Olomouc, Czech Republic

The aim of the study was to determine the cytotoxicity of threeisoquinoline alkaloids (sanguinarine, chelerythrine, berberine) andthree polyphenol compounds (gallic acid, catechin, rutin) in humancolon carcinoma Caco-2 cells. Cells were seeded in 96-well platesat 2 × 104 cells/0.1 ml/well and treated with tested compounds for24 h. The cell viability was evaluated by the MTT reduction assayand neutral red uptake. Our results showed a dose-dependentdecline in the viability of Caco-2 cells in response to the treatmentwith four tested substances whose cytotoxicity decreased in the fol-lowing order: sanguinarine > chelerythrine > gallic acid > berberine.As determined by the MTT assay and neutral red uptake, sanguinar-ine for instance at 2 �M concentration decreased the cell viabilityto 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 approximatelyto 70%, as shown by both methods. After the treatment of Caco-2cells with catechin (1–1000 �M) or rutin (1–500 �M), we observedno significant decrease in the cell viability. Mechanisms of cyto-toxic action of sanguinarine and chelerythrine in Caco-2 cells willbe further investigated.

Acknowledgement: The work was supported by grants Nos. MSM6198959216 and GACR 303/09/H048.

doi:10.1016/j.toxlet.2009.06.198

V08Use of the in vitro model for studying the effects of secondarymetabolites produced by indoor fungi

Zuzana Kovacikova 1,∗, Elena Pieckova 1, Eva Neubauerova 1,Miroslava Kuricova 1, Jana Tulinska 1, Erzsebet Tatrai 2, SonaWimmerova 1

1 Slovak Medical University, Bratislava, Slovakia, 2 NIOH, Budapest,Hungary

Presented in vitro experiments were focused to the effects of sec-ondary metabolites produced by indoor microfungi on two typesof toxicologically most important lung cells: alveolar macrophagesand alveolar epithelial type 2 cells. There are reports about chronicintoxication, allergies or other pulmonary health problems ofinhabitants from houses contaminated by microfungi. The majorityof studies followed the effect of spores but the microfungi pro-duce secondary metabolites which could be inhaled and their effectcannot be neglected. Aspergillus ustus, Aspergillus versicolor, Penicil-lium chrysogenum, Stachybotrys chartarum, isolated from mouldybuildings were cultured and both media and biomass cakes werecollected, exo- and endometabolites, respectively were isolated.The isolated rat lung cells were cultured for 20 h with differentconcentration of the metabolites. After finishing the cultivation thechanges on the surface of type 2 cells were evaluated by stainingwith Maclura pomifera lectin, the antioxidant status of both typesof cells was analysed and the concentration of cytokines (TNF-alpha, MCP-1) in medium was estimated. The results showed toxiceffect of all tested endo- and exo-metabolites even in concentra-tion 0.1 �g/ml. The differences between individual isolates were interms of the extent.

Acknowledgement: The study was supported by the APVV 0322-07 grant.

doi:10.1016/j.toxlet.2009.06.199

V09Dynamic culture in multicompartment bioreactor upregulatescytochrome expression in human hepatocytes

Arti Ahluwalia 1,∗, Bruna Vinci 1, Duret Cedric 1, Duret Cedric 2,Malcolm Wilkinson 3, Patrick Maurel 2

1 University of Pisa, Centro Piaggio, Pisa, Italy, 2 Inserm U632/UM-IEA3768, Montpellier, France, 3 Kirkstall Ltd., Sheffield, UnitedKingdom

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