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doi:10.1016/j.toxlet.2012.03.393

Abstracts / Toxicology L

ary probes and is capable of analytical sensitivity of 10–50 attomolf target with a routine coefficient of variance of less than 15%. Inddition to their high sensitivity, these assays have the ability toistinguish between targets differing by a single base pair provid-

ng extraordinary specificity for the intended target. These testsre being applied in combination with cell culture technologieso evaluate potentially toxic compounds in a high throughput for-

at. The exploitation of genetic endpoints will provide a basis forrapid screening technology allowing accurate assessment of theechanistic action of new drugs/chemicals.

oi:10.1016/j.toxlet.2012.03.390

13-10n vitro evaluation of the hepatotoxic potential of piperazineesigner drugs

arcelo Arbo, Maria de Lourdes Bastos, Helena Carmo

University of Porto, Portugal

Widespread among young people, especially in the dance clubcene and the rave scene. Over the last years, piperazine derivedrugs appeared, mainly in the internet, sold as ecstasy pills ornder the names of “Frenzy”, “Bliss”, “Charge”, “Herbal ecstasy”,A2”, “Legal X”, “Legal E” or, simply, party pills. Although piperazineesigner drugs have the reputation of being safe, several exper-

mental and epidemiological studies indicate risks for humans,specially when these drugs are taken in association. The aim ofhis study was to investigate the cytotoxicity of the designer drugs-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine

TFMPP), 1-(4-methoxyphenyl)piperazine (MeOPP) and 1-(3,4-ethylenedioxybenzyl)piperazine (MDBP) in human hepatomaepG2 cells.

Dose–response curves were obtained after 24 h incubations ofepG2 cells with each drug at concentration range of 40 �M to0 mM. Cell death was evaluated through the reduction of MTT andeutral red incorporation assays.

For the MTT assay, the EC50 values for BZP, TFMPP, MeOPP andDBP were 10542, 736.2, 7620, 6113 �M, respectively. For the neu-

ral red assay, the IC50 values were 5260, 665.7, 5057 and 3321 �M,espectively. It was evident that TFMPP was the most cytotoxicompound in this in vitro model. All other tested piperazine drugsas negligible cytotoxic effects under our experimental conditions.iven that TFMPP and BZP are frequently abused in associationnd have been implicated in human intoxications with piperazineesigner drugs, further studies are needed to understand the mech-nisms involved in the observed cytotoxic effects.

Acknowledgements: MDA thanks Capes Foundation (Brazil) foris PhD grant.

oi:10.1016/j.toxlet.2012.03.391

13-11enotoxicity and cytotoxicity of hexachlorobenzene in human

ntestinal Caco-2 cells

ela Chalouati 1, Laurence Payrastre 2, Moncef Ben Saad 1

Faculté des Sciences de Tunis, Tunisia, 2 UMR 1331,TOXALIM, INRAoulouse, France

Purpose: Hexachlorobenzene (HCB) is one of the most persis-ent environmental pollutants, can cause a wide range of toxicffects. The aim of this study was to investigate the possible effects

211S (2012) S43–S216 S105

of low doses of HCB on DNA integrity, cellular viability, differen-tiation and oxido-reductive status in vitro in human colonic cellline Caco-2. Methods: HCB was dissolved in absolute ethanol andtested at low doses (0.04, 0.4, 4, 40, 400 nM and 2 �M) Cytotoxi-city was evaluated by MTT assay and DNA damage was assessedby comet assay. We also examined the effect of HCB on the activ-ity of antioxidant enzymes (Catalase and Glutathion peroxidase).Data were compared by one way ANOVA and Tukey’s post hoc testwith significance level set at p < 0.05 level. Results and conclusions:We observed that alkaline phosphatase activity of caco-2 cells wasnot changed; suggesting that cell differentiation was not affectedupon pesticide treatment. On the other hand our results clearlyshowed an impact of HCB on cell viability which is associated withan induction of the activity of antioxidant enzymes. This effect wasobserved at 4 nM and was dose dependent. In addition, cell exposedto 0.4 nM and 400 nM of HCB showed a significant increase of thepercentage of tail DNA compared to untreated control cells. Thesefindings indicate that exposure of caco-2 cells in HCB, at low doses,induced cytotoxicity and have a genotoxic risk without affectingcell differentiation. These effects may be related to a pro-oxidativemechanism.

doi:10.1016/j.toxlet.2012.03.392

P13-12In vitro test battery to predict skin sensitizers based on keyevents of sensitization

Tzutzuy Ramirez, Caroline Bauch, Tobias Eltze, Susanne N. Kolle,Annette Mehling, Bennard van Ravenzwaay, Robert Landsiedel

BASF SE, Germany

Skin sensitization is a common health problem caused byrepeated contact with an allergen. Currently, identification ofsensitizers relies on animal testing (OECD 406, 429), and so farno validated/regulatory accepted in vitro methods are available.Herein, we report on our in vitro test strategy for the prediction ofskin sensitizers based on key events occurring during skin sensi-tization. Our integrated approach includes several in vitro assays.From a total of 10 assays and “based on their performance” weselected three assays addressing chemical reactivity towards pro-teins and dendritic cell activation: (1) direct peptide reactivityassay (DPRA) to measure chemical reactivity towards proteins withmodel peptides with nucleophilic amino acid residues, (2) LuSens,a reporter cell line that monitors the Nrf-2 transcription factor acti-vation during sensitization and (3) activation of dendritic cell-likelines: myeloid U937 skin sensitization test (MUSST) or human cellline activation test (h-CLAT). The in-house validation of the in vitroassays was performed with a panel of 59 substances of known sensi-tizing potential including the 22 performance standard substancesof the murine local lymph node assay. Out of the 59 substances54 were applicable to the test methods; 5 were insoluble. A com-bination of DPRA and LuSens addresses chemical reactivity andoffered a sensitivity of 100% (enabling the exclusion of a sensitizingpotential). MUSST addressed dendritic cell activation and offered aspecificity of 100% (proofing a sensitizing potential). The combina-