Abstracts / Toxicology Letters 205S (2011) S60–S179 S165 P1342 Bridging the gap between Caco-2 cells and human carboxylesterases J. Lamego 1,* , A. Coroadinha 2 , A. Simplicio 2 1 IBET/ITQB-UNL, Oeiras, Portugal, 2 IBET, Oeiras, Portugal In early drug development, it is important to have in vitro models predictive of intestinal drug absorption in order to accel- erate the identification of promising or troublesome compounds while avoiding the use of animals. Caco-2 cells express the main active transporters and, at the same time correlate well to pas- sive in vivo absorption, and therefore are widely accepted as a model for screening permeability. However, Caco-2 cell line misses the correct levels of expression of carboxylesterases (CESs). Taking into account that the absorption of ester-containing drugs, such as aspirin, cocaine or meperidine, may be affected by pre-systemic esterase hydrolysis, the lack of expression of specific CESs has to be an important limitation for accurate in vitro prediction of the absorption of this type of drugs. We envisioned developing an improved Caco-2 cell line that better mimics the processes occur- ring at the intestinal epithelial barrier in terms of CES metabolism. This model will be a major contribution to the in vitro toxicology field, providing potentially more accurate results in the predic- tion of permeability of ester compounds and ultimately delays the need for animal models. We already applied molecular and cell biology techniques to obtain a Caco-2 cell line over-expressing carboxylesterase 2. In order to evaluate its expression levels, and since Caco-2 also expresses other CESs, we developed a powerful, fast and repeatable analytical methodology enabling to distinguish and quantify specific carboxylesterases activities with low sample consumption. We have successfully applied this methodology to monitor CESs activities, during Caco-2 differentiation process. doi:10.1016/j.toxlet.2011.05.576 P1343 CYP3A4 inhibition by six herbs commonly used in pregnancy A.J. Langhammer * , O.G. Nilsen Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway Purpose: Concurrent use of conventional and herbal medications during pregnancy is rather widespread and opens for herb-drug metabolic interactions. The CYP3A4 inhibition potential of six fre- quently used herbs taken for pregnancy-related conditions was evaluated with respect to IC 50 -metabolic inhibition constants and possible clinical significance. Methods: Black elderberry, cranberry, fennel, ginger, horsetail, and raspberry leaves were extracted from commercially available herbal products. Separate herbal extracts or the positive inhibition control ketoconazole were incubated with cDNA baculovirus-expressed CYP3A4, cofactors, and testosterone as substrate. After a 5 min preincubation period, the metabolic process was initiated by addition of a NADPH regeneration sys- tem and stopped on ice with methanol after 10 min. The formation of the metabolite 6-hydroxy-testosterone was determined by a validated HPLC methodology. IC 50 -inhibition constants were esti- mated from a CYP3A4 activity inhibition plot by using non-linear regression. Possible clinical significance was related to recom- mended single or daily dosages of the different herbs. Results of the study: The IC 50 -inhibition constants ± SD, 40 ± 4, 81 ± 11, 600 ± 28, 1025 ± 62, 2064 ± 155, and 3007 ± 118 g/ml, were obtained for fennel, raspberry leaves, ginger, cranberry, horsetail, and black elderberry, respectively. After one single recommended dose, fennel, raspberry leaves, and ginger will most probably exceed their IC 50 -concentrations in the small intestine. Related to recom- mended daily dosages, all herbs except black elderberry might have a potential to approach their IC 50 -concentrations. In both cases fennel and raspberry leaves showed the highest potencies for a possible clinical relevant inhibition of intestinal CYP3A4. doi:10.1016/j.toxlet.2011.05.577 P1344 Profiling the toxicity of new drugs: a non animal-based approach integrating toxico-dynamics and biokinetics B. Lauer * , G.L. Truisi, S.O. Mueller, P.G. Hewitt Toxicology, Merck KGaA, Darmstadt, Germany The increasing costs of drug development and the decreasing numbers of new drug approvals have forced the pharmaceutical industry to maximize the effectiveness of their preclinical R&D pro- grams. Thus, the overall goal of the EU-funded project Predict-IV is to develop non-animal based in vitro strategies to improve the assessment of drug safety in early stages of drug development. The focus is on liver, kidney and CNS, as major target organs of drug induced toxicity. In the liver work package, primary rat and human hepatocytes in sandwich culture, as well as the human HepaRG cell line, were treated with 11 pharmaceuticals with liver specific adverse effects in humans. Negative controls lacking significant human hepatotoxicity were also included. A whole genome gene expression study was performed after one, three and 14 days of treatment at one cytotoxic and one non cytotoxic dose. Along with time matched proteomic and metabolomic analyses the dose and time dependency of cytotoxic effects could be shown. Furthermore, a set of genes and proteins was identified to be linked with rele- vant compound specific in vivo toxicities. The combination of gene, protein and/or functional biomarkers with kinetic modelling tools will enable the prediction of safe human doses in clinical phases. The goal of our studies is to reduce attrition of drug candidates by front loading these predictive test systems to the non-clinical test phases. This would also minimize animal use and would represent a major improvement in line with the 3R principle. Funded by EU project Predict-IV, grant 202222. doi:10.1016/j.toxlet.2011.05.578 P1345 Old and novel pathways of benzo[a]pyrene toxicity in epithelial cell models M. Machala 1,* , P. Simeckova 1 , P. Hulinkova 1 , L. Umannova 1 , E. Hruba 1 , P. Krcmar 1 , J. Neca 1 , J. Vondracek 2 1 Department of Toxicology, Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic, 2 Department of Cytokinetics, Institute of Biophysics AS CR, Brno, Czech Republic The activation of aryl hydrocarbon receptor (AhR) plays a promi- nent role in the toxicity and carcinogenicity of benzo[a]pyrene (BaP), which has been related mainly to the induction of CYP1A/1B bioactivating enzymes. Nevertheless, the activation of AhR can lead to deregulation of a much broader spectrum of other genes involved in regulation of cell survival mechanisms, cell-to-cell communica- tion, adhesion and differentiation. Disruption of these processes