Abstracts / Toxicology Letters 229S (2014) S40–S252 S77

tance. However, the effects were independent of activation of thearyl hydrocarbon receptor (AhR) as such. AhR rather suppressed1-NP-induced CXCL8, whereas 1-NP exposure reduced CYP1A1/-1B1 expression in BEAS-2B cells. Concentration-effect assessmentshowed that 1-NP induced CXCL8 responses in BEAS-2B cells from10 �M and higher. However, in TLR3-primed cells 1-NP exacer-bated CXCL8 release already at 1 �M. The mechanism for thislow-dose effect remains unclear.

Our results suggest that 1-NP induced chemokine responsesare regulated through multiple pathways, and include both activa-tion of cellular receptors and metabolic activation with formationof ROS and/or electrophilic metabolites. Moreover, 1-NP inducessome yet unidentified cellular effects at very low concentrationsthat alone are insufficient to induced chemokine responses, butthat may exacerbate responses induced by other pro-inflammatoryagents.

http://dx.doi.org/10.1016/j.toxlet.2014.06.296

P-1.117Sex-dependent gene expression of kidneytransporters after Ochratoxin A exposure inF344 rats

Laura Pastor ∗, Ariane Vettorazzi, Adela López de Cerain

Universidad de Navarra, Pamplona, Navarra, Spain

Ochratoxin A (OTA) is a mycotoxin that contaminates severalfood commodities such as cereals, nuts and spices. It is consideredas a potent renal carcinogen in rats but its mechanism of action isstill not understood. Moreover, a high male susceptibility to tumorformation has been demonstrated. It has been hypothesized thatdifferent susceptibilities to OTA toxicity might be due to variationsin transport mechanisms in kidney cells. Therefore, the aim of thisstudy was to analyze, by RT-qPCR, renal transporters expression inmale (M) and female (F) F344 rats at basal level and after single oralOTA administration (0.50 mg/kg bw). Temporal profiles (24 h, 48 h,72 h, 96 h, 1 and 2 months) were studied per sex and transporter.

Oatp1 (M>F), Oat2 and Pept2 (F>M) sex-differences were con-firmed at basal level. Moreover, a high Bcrp expression wasobserved in males. After OTA exposure, females showed a generaldecrease in all transporters studied, mainly after 48 h. In males,expression changes were observed after 24 h and mainly in apicalproteins; besides, at 48 h, an increase in Oat2 expression (reab-sorption transporter) and a decrease of Mrp2 and Bcrp (exclusiontransporters) were also observed.

While similar time-profiles were determined for Abc, Oatp(Slco1) and Pept (Slc15) families, the highest sex differencesinvolved Oat (Slc22) transporters. Oat2, Oat3 and Oat5 expressionshowed a significant increase in males while Oat1, Oat2 and Oat5level decreased considerably in females. These molecular changesmight be implicated in the highest male susceptibility to OTA renalcarcinogenesis.

http://dx.doi.org/10.1016/j.toxlet.2014.06.297

P-1.118The toxicity of silica nanoparticles on MRC-5cell line

Sorina Nicoleta Petrache Voicu 1,3,∗, Cornelia Sima 2, AncaHermenean 3,4, Aureal Ardelean 3, Anca Dinischiotu 1

1 Department of Biochemistry and Molecular Biology, University ofBucharest, Faculty of Biology, Bucharest, Romania, 2 LaserDepartment, National Institute of Laser, Plasma and RadiationPhysics, Bucharest-Magurele, Romania, 3 Department ofExperimental and Applied Biology, Institute of Life Science, VasileGoldis Western University of Arad, Arad, Romania, 4 Department ofHistology, Faculty of Medicine, Pharmacy and Dentistry, Vasile GoldisWestern University of Arad, Arad, Romania

In this study, we have investigated the expression of heat shockprotein (Hsp) 27, 60, 70, 90 and autophagic marker LC3II in cul-tured lung human fibroblasts (MRC5 cell line) exposed to silicananoparticles after 24,48 and 72 h. The primary nanoparticle sizedistribution was a lognormal function, in the range 3–14 nm, mostof them being of 5–8 nm. The MTT test was used to assess cell via-bility after MRC5 cell treatment with 6.3 × 105 particle SiO2 for 24,48 and 72 h. The expression of Hsp and LC3II protein was analysedby Western Blotting method.

The viability of the pulmonary cells in the presence of thesenanoparticles decreased after 48 and 72 h, by 10% and 30%, respec-tively, compared to control and was unchanged after 24 h. Theexpression of Hsp 27 was significantly inhibited, at all time inter-vals, compared to control.

The level Hsp 60 significantly decreased by 26%, 44 and 62%after 24, 48 respectively 72 h compared to control, whereas thatof Hsp 70 was not affected. A very significant increase of Hsp 90expression occurred after 24, 48 and 72 h of exposure. Autophagywas identified due to increased levels of LC3II after 24 h, 48 h and72 h by 54%, 162%, respectively 254% compared to untreated cells.

Our results suggested that silica nanoparticles affected espe-cially the mitochondria. The low levels of Hsp 27 could correlatewith increased levels of LC3II. The LC3 protein contributes to bet-ter insights of autophagy under physiological conditions and itsrelationship to apoptotic cell death.

http://dx.doi.org/10.1016/j.toxlet.2014.06.298

P-1.119Evaluation of angiotensin-converting enzymeinhibition by LMRBPP9, a syntheticbradykinin-potentiating peptide from Lachesismuta rhombeata venom

Ernesto Pinheiro-Junior 1,∗, Johara Boldrini-Franca 1, NorivalSantos-Filho 2, Eduardo Cilli 2, Eliane Arantes 1

1 Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil,2 Instituto de Química de Araraquara – Universidade EstadualPaulista “Júlio de Mesquita Filho”, Araraquara, Brazil

Bradykinin-potentiating peptides (BPPs) are important com-ponents present in Lachesis muta rhombeata venom. They actdirectly at renin-angiotensin-aldosterone system, inhibitingthe angiotensin-converting enzyme (ACE), which cleaves theangiotensin I into the vasoconstrictor angiotensin II. The cascadeof responses triggered after this reaction plays a crucial role inthe mechanisms that control the blood pressure, such as tubularsalt reabsorption and retention of water in kidneys, aldosteronesecretion and arteriolar vasoconstriction. However, when they are

S78 Abstracts / Toxicology Letters 229S (2014) S40–S252

not well controlled, these actions can bring severe consequences,such as hypertension, a leading cause of cardiovascular mortality.The isolation and characterization of BPPs found in pit vipersvenom are important to the development of specific drugs capableof controlling hypertension in humans. Hence, this work aimed tosynthesize a BPP found in Lachesis muta rhombeata venom andto perform its in vitro biochemical characterization. The LmrBPP9was obtained using an automated solid-phase peptide synthesizer(PS3TM, Protein Technologies). The synthetic peptide was sub-mitted to a reversed-phase fast protein liquid chromatography(RP-FPLC) to confirm its purity. In vitro assays to evaluate theACE enzymatic activity in the presence of different concentrationsof LmrBPP9 showed that the synthetic peptide is able to inhibitACE even at low concentrations (IC50: 103 nM). These resultsindicate that LmrBPP9 could be used for the development of a newantihypertensive drug.

Keywords: Snake venom toxins; Lachesis muta rhombeata;Bradykinin-potentiating peptides; Angiotensin-convertingenzyme; Antihypertensive drug

Support: FAPESP, CNPq, NAP-TOXAN.

http://dx.doi.org/10.1016/j.toxlet.2014.06.299

P-1.120Aqueous cigarette smoke extract promotes theadhesion of monocytic cells to human coronaryartery endothelial cells in direct andindirect-dependent ways

Carine Poussin ∗, Alexandra Laurent, Julia Hoeng, Manuel C.Peitsch, Hector De Leon

Philip Morris International R&D, Neuchâtel, Switzerland

Cigarette smoke (CS) has been shown to affect the adhesionof monocytes to endothelial cells, a critical step in atherogene-sis. Yet, the underlying molecular mechanisms are complex andremain to be thoroughly investigated. We have developed anin vitro adhesion assay to study the impact of CS on this process.Human coronary artery endothelial cells (HCAECs) were treatedfor four hours either directly with freshly generated aqueous CSextract (smoke-bubbled PBS, sbPBS) or indirectly with supernatantof macrophage-like monocytic MM6 cells pre-exposed to the samedoses of sbPBS for two hours. The adhesion of untreated MM6cells to HCAECs was quantified using a nuclear fluorescence-basedstaining protocol. Both sbPBS exposure modalities, direct and indi-rect, increased the adhesion of MM6 cells to HCAECs significantly.Indirect treatment resulted in a maximum increase (∼1.9 time rel-ative to vehicle control) of cell adhesion using sbPBS doses between0.045 and 0.06 puff/mL. Similar doses of sbPBS applied directly onHCAECs had negligible effects. Direct exposure of HCAECs to sbPBSrequired higher doses (>0.2 puff/mL) for MM6 cells to maximally(∼14 times relative to vehicle control) adhere to HCAECs. This directeffect was accompanied by an enhanced toxicity (HCAEC viabilityreduced by 20–30%). Our data indicate that CS-dependent releaseof potent inflammatory factors by monocytes represents the pri-mary mechanism promoting adhesion of monocytes to the vascularendothelium. Further studies should be aimed at characterizing themolecular inflammatory and oxidative stress pathways involved inthe monocyte-dependent effects induced by CS.

http://dx.doi.org/10.1016/j.toxlet.2014.06.300

P-1.121Mechanisms of pulmonary toxicity developed atlow and high doses of dextran-coatedmagnetite nanoparticles

Mihaela Radu 1,4,∗, Alexandra Gheorghiu 1, Daniela Predoi 2, AncaHermenean 3,4, Aurel Ardelean 4, Anca Dinischiotu 1

1 Faculty of Biology, University of Bucharest, Bucharest, Romania,2 National Institute of Materials Physics, Bucharest-Magurele,Romania, 3 Department of Histology, Faculty of Medicine, Pharmacyand Dentistry, Vasile Goldis Western University of Arad, Arad,Romania, 4 Department of Experimental and Applied Biology,Institute of Life Sciences, Vasile Goldis Western University of Arad,Arad, Romania

In order to check the safety of different surface coatings ofnanomaterials used in biomedical applications, toxicity studies areneeded.

We tested pulmonary cell responses to different doses ofdextran-coated magnetite nanoparticles by viability and accumu-lation assays as well as by the analysis of oxidative stress markersup to 72 h.

Starting with 25 �g iron/mL the viability of MRC-5 cellsdecreased in a time and dose-dependent manner. Under thisconcentration no significant changes was observed. Comparativestudies of toxicity mechanisms induced by a low and a high dose ofdextran-coated magnetite nanoparticles (10 and 100 �g iron/mL)on pulmonary cells were carried out. While intracellular accumula-tion of iron was dependent on dose and time, the loss of membraneintegrity was observed just for the higher concentration startingwith 24 h. The maximum of reactive oxygen species generationand minimum in the intracellular GSH level and glutathione-S-transferase and superoxide dismutase activities after 48 h ofexposure was registered. The level of malondialdehyde was ele-vated at 24 and 48 h. The ability of MRC-5 cells to counteract theoxidative stress was highlighted by the up-regulation of heat shockprotein 60 as well as the redox-sensitive transcription factor Nrf2in treated cells after the first day. After 72 h a recover tendency ofall biochemical parameters was noticed.

Our results suggest that dextran-coated nanoparticles hadinduced toxic effects at pulmonary level in the first 48 h of exposure,with a dose-dependent recovering rate after 72 h.

http://dx.doi.org/10.1016/j.toxlet.2014.06.301

P-1.122Hexachlorobenzene exposure enhances COX-2expression and metalloprotease activities inhuman endometrial stromal cells

Florencia Chiappini 1,5, Juan Ignacio Bastón 2, AgustinaVaccarezza 3, Jose Javier Singla 4, Carolina Pontillo 1, NoeliaMiret 1, Mariana Farina 5, Gabriela Meresman 2, Andrea Randi 1,∗

1 Laboratorio de Efectos Biológicos de Contaminantes Ambientales,Depto de Bioquímica Humana, Facultad de Medicina, Universidad deBuenos Aires, Buenos Aires, Argentina, 2 Laboratorio de FisiopatologíaEndometrial, Instituto de Biología y Medicina Experimental,IBYME-CONICET, Buenos Aires, Argentina, 3 CEBBAD, UniversidadMaimónides, Buenos Aires, Argentina, 4 Servicio de Ginecología,Hospital de Clínicas “José de San Martín”, Buenos Aires, Argentina,5 Laboratorio de Fisiopatología Placentaria, Centro de EstudiosFarmacológicos y Botánicos – CEFYBO, CONICET-UBA, Buenos Aires,Argentina