etters

aphbadPalicfmNIfso

i

d

PRc

V

opei

bgfltep

spttpics

O

d

Abstracts / Toxicology L

ffects this process. The knowledge how the alterations of thisrotein expression can alter nervous system development mightave important implications for mechanisms of toxicity inducedy organophosphorous. Methods: We have used a human NT2 ter-tocarcinoma cell line as an in vitro model to study the neuralifferentiation after either NTE inhibition by organophosphates ornpla6 silencing with siRNA. The effects of these treatments werenalyzed at genotypic level (using microarrays) and at phenotypicevel with electrophysiological and morphological (inmunostain-ng and neurite outgrowth quantification) approaches. Results andonclusion(s): Pnpla6 silencing reduced neural electroactivity of dif-erentiated neurons, the neurite outgrowth, changed the neuron

orphology and altered the expression of more than 400 genes.TE inhibition caused no alterations on the assessed end-points.

n conclusion, Pnpla6 is essential for a normal neuronal cell dif-erentiation while inhibition of NTE that causes neurodegenerativeyndrome in adult tissues did not have any effect in gene expressionr changes in morphology.

Acknowledgments: JRC supported David Pamies during his stayn its laboratories.

oi:10.1016/j.toxlet.2012.03.397

13-17ifampicin derivative, Red-Rif, protects against paraquatytotoxicity in RBE4 cells

ânia Vilas-Boas, Renata Silva, Fernando Remião

REQUIMTE, Portugal

A new synthetic rifampicin derivative, Red-Rif, has been devel-ped in order to potenciate the known rifampicin’s P-gp-inducerrofile. This work aimed at evaluating Red-Rif’s effect on P-gpxpression and function and its effect on paraquat (PQ) cytotoxicity,n immortalized rat brain endothelial cells (RBE4).

For that purpose, after evaluating Red-Rif’s cytotoxicity profiley the Neutral Red (NR) uptake and the MTT reduction assays, P-p expression (Western Blot) and activity (Rho 123 efflux assay byow cytometry) were determined 24, 48 and 72 h after exposureo the compound. Furthermore, PQ, a P-gp substrate, was used tovaluate the protective effect of Red-Rif in RBE4 on PQ cytotoxicrofile (assessed by NR) at the predefined time-points.

A significant increase was observed in the transporter’s expres-ion (p < 0.05 at 48 and 72 h) and activity (p < 0.001 at 24 h and< 0.05 at 72 h) in cells exposed to 10 microM Red-Rif, as compared

o untreated cells. The compound induced a significant protec-ive effect against PQ toxicity in RBE4 cells, at 24 and 72 h ofre-exposure (p < 0.05), which may be attributed to the observed

nduction of P-gp. These results indicate that Red-Rif may be a goodandidate to be used as protective agent against neurotoxic P-gpubstrates.

Keywords: P-glycoprotein induction; RBE4 cells; Rifampicin

Acknowledgements: FCT grant (Project PTDC/SAU-

SM/101437/2008).

oi:10.1016/j.toxlet.2012.03.398

211S (2012) S43–S216 S107

P13-18Measuring endocrine activity through an automated in vitroLumicell BG1Luc4E2 assay

Anne Milcamps, Gilles Bories, Juan Casado Poblador, Jean MichelGineste, Elise Grignard, Roman Liska, Luis Saavedra, MauriceWhelan

Joint Research Centre, Italy

In vitro cell based test methods, used for chemical toxicity pro-filing, are traditionally carried out within a single plate format(various concentrations tested in one 96-well plate). Only in recentyears the attention has shifted to automation and high throughputscreening (HTS). The objective of this study is to adapt a standardmanual assay for automation and assess its performance for highthroughput screening.

We report on the automation of the in vitro Lumicell BG1luc4E2assay, which is a reporter gene based assay used for assessmentof chemicals with suspected endocrine activity. Disruption of theendocrine system and subsequent health issues for animals andhumans due to substances in our daily environment has gained anincreased concern. In Europe, efforts are made to validate inter-nationally agreed test methods, and the Lumicell BG1luc4E2 assayis currently proposed as one of the reference test methods of theperformance based test guideline of OECD.

The assay was adapted to automation by using the Hamil-ton STAR platform for cell seeding and cell treatment, applying aquantitative HTS format (qHTS). A collection of 44 chemicals withknown or suspected endocrine activity was simultaneously tested.Our results reveal good intra-laboratory reproducibility and are inline with the results generated in a recent validation study. Theautomated assay performs as well as the standard manual assayand may therefore offer an attractive alternative to the classi-cal one plate experimental design given its automation and highthroughput screening and without compromising the quality of theobtained data.

doi:10.1016/j.toxlet.2012.03.399

P13-19DNA methylation profile of various cancer-related genes in ratcell lines

Sibel Ozden, Goksun Demirel, Buket Alpertunga

Istanbul University, Faculty of Pharmacy, Turkey

DNA methylation is an important epigenetic mechanism thataffects cell function by altering gene expression. Altered DNAmethylation may contribute to carcinogenesis in several ways,including global genomic hypomethylation leading to over-expression of oncogenes and tumor suppressor gene silencingthrough hypermethylation of CpG islands of genes. Cultured celllines have been used in carcinogenesis studies including epigeneticmechanisms.

Objective of this study was to investigate the CpG island pro-moter methylation status of cancer related tumor suppressor genes(p16, p15, VHL, e-cadherin) and oncogene (c-myc) in tumor (C6glioma) and normal cell lines (NRK-52E kidney cells and Clone 9liver cells). We used methylation-specific PCR (MSP) method and

combined bisulfite restriction analysis (COBRA) for the investiga-tion of methylation profile.

CpG island promoter region of e-cadherin, a cell adhesionmolecule, was found to be methylated whereas c-myc and VHL