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in situ hybridization system as a routine method. Validation of thegene set has to be verified with further compounds.

doi:10.1016/j.toxlet.2011.05.559

Abstracts / Toxicology L

1323igns of cellular senescence induced by,3,7,8-tetrachlorodibenzo-p-dioxin in bovine cells

.E. Granato 1, F. Fiorito 2,∗, R. Ciarcia 1, G. Marfè 3, A. Cantiello 2,. Montagnaro 2, V. Iovane 2, S. Florio 1

Structures, Function and Biological Technologies, University ofaples, Naples, Italy, 2 Pathology and Animal Health, University ofaples, Naples, Italy, 3 Experimental Medicine and Biochemicalciences, University of Rome “Tor Vergata”, Rome, Italy

It is known that tumor-promoting agents such as 2,3,7,8-etrachlorodibenzo-p-dioxin (TCDD) are thought to counteractumor-suppressive mechanisms by altering events that favor pro-iferation while inhibiting differentiation and/or apoptosis andenescence. But it is also shown that TCDD exposure can induceremature cellular senescence, which is a potent anti-cancerechanism controlled by tumor suppressor genes. Previously we

howed that TCDD increased cell viability of the bovine cellsMDBK). Furthermore, TCDD induced an increase of cell prolifer-tion and no signs of apoptosis were observed. In a recent work,e demonstrated that TCDD exposure in MDBK cells caused a

oncomitant IRP1 down-regulation and IRP2 up-regulation thusetermining a marked enhancement of transferrin receptor 1 (TfR-) expression and a response in ferritin content which impairsellular iron homeostasis, leading to changes in the labile iron poolxtent. In general, the iron metabolism dysregulation could induceoxic reactions. Hence, herein we examined in MDBK cells, at differ-nt times of exposure, the effects of TCDD on telomerase activity,ERT and p21. TCDD significantly up-regulates telomerase activityrom 4 until 36 h of exposure and then significantly decreased at thend of exposure. In TCDD exposed groups, densitometric analysisf TERT blots showed the same trend of telomerase activity, while21 protein levels increased as a function of time. Finally, by analyz-

ng cytoskeleton alterations by Giemsa staining, in a large numberf exposed cells the cytoplasm expanded, increased the intercellu-ar spaces and many pycnotic nuclei were observed. These changes

ay be signs of cellular senescence induced by TCDD.

oi:10.1016/j.toxlet.2011.05.557

1324ytotoxicity, biaccessibility and transport by Caco-2 cells ofnniatins and beauvericin

. Font 1,∗, A. Prosperini 2, M.J. Ruiz 2

Laboratory of Toxicology, Faculty of Pharmacy, University ofalencia, Burjassot, Spain, 2 Laboratory of Toxicology, Faculty ofharmacy, University of Valencia, Valencia, Spain

Beauvericin (BEA) and enniatins (ENs) are mycotoxins pro-uced by Fusarium sp. Combination of mycotoxins are commonlyresent in several food commodities worldwide, and they can beound in a variety of products for human and animal consump-ion. The epithelial colorectal adenocarcinoma (Caco-2) cells haveeen widely used for estimating the bioavailability of contaminantss mycotoxins from food. The Caco-2 cell cultures, is used as aodel of intestinal epithelia because they became into enterocytes

n classical culture conditions. This study evaluated cytotoxicity

f mycotoxins and intestinal cell retention and transport pro-esses by Caco-2 cells. To determine cytotoxicity of mycotoxinsn Caco-2 cells, the endpoint evaluated after 24, 48 and 72 h ofxposure was mitochondrial’s integrity by the MTT assay. Reten-

205S (2011) S60–S179 S159

tion and transport experiments were studied with cells grownon polycarbonate membrane filters (transwell inserts) at 15–18days after seeding. These cells were exposed to mycotoxins for4 h. After the incubation time, in order to evaluate mycotoxinretention (cell monolayer) and transepithelial transport (apicaland basal medium) percentages were calculated with respect tothe initial amount of mycotoxins. Analysis was performed byelectrospray-ionization mass spectrometry (ESI-MS). The resultsobtained demonstrated that the cytotoxicity of ENs was three- tofourfold higher than to BEA. In Caco-2 cells, mycotoxin retention,transport, and total uptake (retention + transport) varied depend-ing of mycotoxin tested. Despite the low accessibility of mycotoxins(<50%), contribution from food should be considered in assess-ments of mycotoxins health risk.

This study was financially supported by the projects AGL2010-17024 (Science and Innovation Spanish Ministry).

doi:10.1016/j.toxlet.2011.05.558

P1325Establishment of an in-vitro screening system for the earlydetection of nephrotoxicity

T.C. Fuchs 1, B. Lauer 2,∗, P. Hewitt 1

1 Early, Genetic and Moleculare Toxicology, Toxicology, Darmstadt,Germany, 2 Toxicology, Merck KGaA, Darmstadt, Germany

The European Centre of the Validation of Alternative Meth-ods (ECVAM) advocates the use of Genomics-technologies as wellas cell lines for safety assessment. After the acceptance of thenovel urinary biomarkers for acute rat studies the next logic step,taking into account the 3R-principle and to become more cost effi-cient, is the development and validation of toxicogenomics basedin vitro screening systems for the early identification of compoundswith the potential to injure renal cells. As a first approach wholegenome expression profiles were generated by using the Illumina®

Sentrix® RatRef-12 bead chips, to identify potential biomarkers of3 nephrotoxic compounds (Doxorubicin, Puromycin, and Ampho-tericin B) in NRK-52E cells. The cells were treated with a NOAECand TC20 of each compound and multiple time points. The aimwas to identify specific gene expression patterns and/or singletranscript “biomarkers” which can be used for screening in earlylead discovery of pharmaceuticals and chemicals. 43 Overlappinggenes (p < 0.01, >±1.5-fold) were identified. Subsequently, addi-tional compounds (Cisplatin, Transplatin, and Vancomycin) wereused to validate these biomarkers. To obtain a better mechanisticunderstanding, Acetyl-p-aminophenol and one of its toxic metabo-lites p-aminophenol, were also used for validation the biomarkersas well as the NRK-52 cells. We identified highly predictive genesfor the detection of toxic insult on renal proximal tubules cells. Thenext step will be the establishment of a high content imaging based