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Abstracts / Toxicology L

33-11evelopment of a triple-culture model of the alveolar-capillaryarrier

ennifer Kasper 1, Maria I. Hermanns 2, Christoph Bantz 3, Olgaoshkina 4, Michael Maskos 5, Ronald E. Unger 6, C. Jamesirkpatrick 6

Universitiy Medical Center Mainz, Germany, 2 ikfe GmbH, Institutür klinische Forsch, Germany, 3 Johannes Gutenberg University

ainz, Ins, Germany, 4 BAM Bundesanstalt für Materialforschung,ermany, 5 IMM Institut für Mikrotechnik Mainz GmbH, Germany,University Medical Centre, Institute of Germany, Germany

Compared to conventional monocultures, which are usuallysed to study, e.g. cellular nanoparticle interactions, well designedulticellular culture models more closely mimic the cellular com-

osition in the human body. Therefore, we developed a complexoculture model of the alveolar-capillary barrier consisting of lungpithelial cells (NCI H441) on top and microvascular endothelialells (ISO-HAS-1) on the bottom side of a filter-membrane to studyanoparticle cytotoxicity.

In a further development we added a third cell type, the alveolaracrophage (AM), to our coculture. AMs represent the front-line

f alveolar defense against aspirated pathogens or particles (viahagocytosis and subsequent killing of microbes) and display a cen-ral role in controlling lung immunity. As model we applied theuman acute monocytic leukemia cell line, THP-1 to the epithe-

ial monolayer. On stimulation with PMA (phorbol 12-myristate3-acetate) the THP-1 differentiate to cells with AM features.

8 nM PMA-stimulated (for 4 d) THP-1 did not have an influencen the TER (transepithelial electrical resistance) of our coculture,hereas 100 nM PMA-stimulated THP-1 caused a drastic decrease

f TER. Furthermore, whereas 8 nM PMA-stimulated THP-1 did notause any inflammatory responses in the coculture, 100 nM treatedells elicited marked IL-8 release. LPS (lipopolysaccharide) stimula-ion of the triple-culture using 8 nM PMA-stimulated THP-1 causedsignificantly higher IL-8 release compared to the coculture. Thus,

he triple-culture reacts more sensitively to stimuli compared tohe coculture. In conclusion, this triple-culture model has greatotential to mimic more closely realistic cell interactions withanoparticles in the distal lung.

oi:10.1016/j.toxlet.2012.03.721

33-12Trojan Horse type mechanism: Cellular dose and toxicity ofg and CuO nanoparticles

ontus Cronholm 1, Hanna L. Karlsson 1, Jonas Hedberg 2, Troyowe 2, Karine Elihn 3, Inger Odnevall Wallinder 2, Lennartöller 1

Karolinska Institutet, Sweden, 2 KTH Royal Institute of Technology,weden, 3 Stockholm University, Sweden

Purpose: The aim of this study was to investigate the cellularose and toxicity of Ag and CuO nanoparticles in two human lungell lines, A549 and BEAS-2B. The hypothesis was that the parti-le structure increases the cellular uptake via the so-called Trojanorse type mechanism. Comparisons were therefore made between

he CuO and Ag nanoparticles and their metal ions (AgNO3, CuCl2).ethods: The cellular concentration of Cu and Ag was analyzed

sing atomic absorption spectroscopy (AAS). Particle internaliza-ion was further studied using Transmission Electron Microscopy

211S (2012) S43–S216 S201

(TEM) and Laser Scanning Confocal Microscopy (LSCM). Cell deathwas measured using trypan blue staining and DNA damage usingthe comet assay. Results and conclusion: In both cases, the resultsshowed a higher cellular uptake of nanoparticles compared to ions.TEM images confirmed the cellular uptake and LSCM indicated thatthe Ag nanoparticles could be internalized into the cell nuclei. Ingeneral, BEAS-2B cells were more sensitive compared to A549 whenmeasuring cell death and DNA damage. In BEAS-2B, increased celldeath was observed already after a 4 h exposure of CuO nanopar-ticles at doses of 5 and 10 �g/mL, an effect not observed whenexposed to Cu ions. No cell death or DNA damage were observedafter exposure to Ag nanoparticles after 4 h. However, exposures tosilver ions resulted in cell death after the same time period suggest-ing that extracellular mechanisms are of importance for toxicity ofAg ions. The internalization of Ag nanoparticles into the cell nucleiwarrants further investigations.

doi:10.1016/j.toxlet.2012.03.722

P33-13Mechanism underlying multiwall carbon nanotube-inducedIL-1beta secretion

Tomoko Nishimaki-Mogami, Hongyan Cui, Keiichiro Okuhira,Mikihiko Naito, Kazuhiro Suzuki, Nishimura Tetsuji, AkihikoHirose

National Institute of Health Sciences, Japan

Purpose: Carbon nanotubes (CNTs) are increasingly used invarious industrial fields. However, because of the asbestos-likeshape and size, the potential risk on human health is a problemof great concern. In this study, we tested the ability of mul-tiwall carbon nanotubes (MWCNT) dispersed in phospholipidsto stimulate a proinflammatory cytokine IL-1beta secretion inhuman macrophage-like THP-1 cells, and investigated the mech-anism. Results: MWCNTs with various lengths and diameters haddifferent abilities to induce IL-1beta production, while fullerenehad no effect. Dispersion of MWCNT in a phosphatidylcholine(PC)/phosphatidylglycerol (PG) mixture, a known artificial lungsurfactant, or PC alone enhanced the effect, whereas an anionicphosphatidylserine caused a robust IL-1beta production withoutMWCNT. MWCNT stimulated mature form of IL-1beta secretion,which was accompanied by the cleavage of caspase-1. Caspase-1is auto-cleaved by a signal within multiprotein complexes calledinflammasome. MWCNT-induced IL-1beta secretion was com-pletely blocked by a caspsase-1 inhibitor or 130 mM KCl thatsuppresses NLR pyrin domain containing 3 (NLRP3)-inflammasomeactivation. Furthermore, siRNA knockdown of NLPR3 effectivelyreduced MWCNT-mediated IL-1beta secretion and caspase-1 cleav-age. MWCNT-elicited IL-1beta production is blocked by inhibitorsof phagocytosis, lysosomal acidification, and lysosomal proteasecathepsin B. Conclusions: MWCNT dispersed in lung-surfactantphospholipids induces IL-1beta production in macrophages viaNLRP3 inflammasome activation, suggesting the asbestos-like tox-icity and implication with chronic inflammatory diseases.

doi:10.1016/j.toxlet.2012.03.723

P33-14Toxicity of metal nanoparticles in vitro

Ylva Rodhe, Zuzana Hassan, Lennart Möller

Karolinska Institutet, Sweden

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Purpose: The purpose with this project was to study the tox-city of different metal nanoparticles in vitro. Nanotechnology iseveloping fast and nanoparticles are used within many areas, e.g.lothing, cosmetics and medicine. Concerns of health effects haveeen raised and certain nanoparticles have been shown to increase

evels of oxidative stress, inflammation and DNA damage. Under-tanding of cellular mechanisms behind nanotoxicity is importantor assessment of human health risks.

Methods: Human cell lines (lung and blood cells) have beensed to evaluate toxic effects of a range of metal and metalxide nanoparticles. Various methods have been used to assesshe toxicity in terms of cytotoxicity using trypan blue staining,

itochondrial damage using the fluorescent probe tetramethylrho-amine methyl esther and apoptosis analysis using Annexin-V andI staining.

Results and conclusions: Studies of 38 nanoparticles showhat the cytotoxicity varies greatly, some nanoparticles gener-ted up to 100% cytotoxicity after 18 hours exposure of 80 �g/ml.ose-response curves show that certain nanoparticles are highlyytotoxic with IC50-values varying from 15-90 �g/ml for the mostotent nanoparticles. Differences in toxicity between cell typesre also seen when comparing three leukemia cell lines and lym-hocytes. Necrosis or secondary apoptosis is the main type of celleath. Results from analysis of mitochondrial damage show thathe nanoparticles are diverse in their mitochondrial interaction, inerms of time and dose dependency. Conclusion from this studys that certain metal nanoparticles are highly toxic and that the

echanisms behind the toxicity depend on composition and size.

oi:10.1016/j.toxlet.2012.03.724

33-15etection of nanoparticle-induced DNA oxidation using theomet assay

ohanna Kain, Hanna Karlsson, Lennart Möller

Karolinska Institutet, Sweden

Purpose: One increasingly used method for detection of DNAamage induced by nanoparticles is the comet assay, often usedo measure DNA breaks as well as oxidatively damaged DNA, theatter by addition of specific DNA repair enzymes such as formami-opyrimidine DNA glycosylase (FPG). The aim of this study was to

nvestigate the use of the comet assay for analysis of DNA oxidationy different nanoparticles in the lung cell lines A549 and BEAS-2B,nd to test the hypothesis that nanoparticles present in the cellsuring the assay performance may interact with FPG. Methods: Thebility of nanoparticles (Ag, CeO2, Co3O4, Fe3O4, NiO and SiO2) tonduce DNA breaks and oxidatively damaged DNA was assessedsing the comet assay, intracellular production of reactive oxy-en species (ROS) by 2′,7′-dichlorofluorescin diacetate fluorescencend non-cellular oxidation of the DNA base guanine using HPLC-EC.esults and conclusion: DNA breaks were induced by all nanopar-icles except from SiO2, but no or low levels of FPG-sites could beetected. In contrast, several nanoparticles induced intracellularOS, and especially Ag nanoparticles caused non-cellular oxidationf guanine. Incubation of FPG with the particles led to less FPG activ-ty, particularly with nanoparticles of Ag due to released Ag ions, butlso with CeO2, Co3O4 and SiO2. We conclude that measurement ofxidatively damaged DNA in cells exposed to nanoparticles may be

nderestimated in the comet assay due to interactions with FPG.

oi:10.1016/j.toxlet.2012.03.725

211S (2012) S43–S216

P33-16Toxicity of zinc oxide nanoparticles towards a fish andmammalian cell line

Tobias Lammel 1, Maria-Luisa Fernández-Cruz 1, Mona Connolly 1,Barrado Ana Isabel Conde Estefania 2, Sylvain Derick 3, YolandaPérez 4, Marta Fernández 2, Christophe Furger 3, José MariaNavas 1

1 INIA, Spain, 2 CIEMAT, Spain, 3 NOVALEADS, France, 4 UniversidadRey Juan Carlos, Spain

Purpose: The objective of this study was to assess the toxic effectsthat zinc oxide (ZnO) nanoparticles (NPs) exert at the (sub)cellularlevel. It was further aimed to extend toxicity assessment of ZnONPs to non-mammalian in vitro models. In addition, the applica-bility of a recently developed cytotoxicity assay (DAP) to assessnanomaterial toxicity was tested. Methods: The toxicity of threedifferent ZnO NPs and their bulk counterpart was studied in thefish and the human hepatoma cell lines PLHC-1 and HepG2. Mor-phology and size distribution of particles were characterized byTEM and DLS, respectively. Real concentrations were measuredusing ICP-MS. Cytotoxicity was determined with the MTT, NRR,LDH, and DAP assay. ROS generation was examined using DCFH-DA.Contribution of the dissolved ZnO fraction to toxicity was inves-tigated through exposure to supernatants of centrifuged particlesuspensions. Results and conclusion(s) of the study: All tested ZnOparticles caused a dose-dependent decrease in cell viability, withHepG2 being more sensitive than PLHC-1. The DAP assay was suc-cesfully applied to the fish cells. The dissolved ZnO fraction wasfound to contribute to the cytotoxicity observed in both cell lines.Induction of oxidative stress could not explain ZnO NP cytotoxicity.When taking into account measured concentrations, cytotoxicityappeared to depend only on concentration and not on other factorssuch as NP size. These results stress the importance of determiningreal NP concentrations to avoid erroneous conclusions on cytotoxicpotential.

doi:10.1016/j.toxlet.2012.03.726

P33-17Cytotoxicity of silver nanoparticles on tumoral and normalhuman cells

Alicia Ávalos 1, Ana Isabel Haza 2, Paloma Morales 2

1 Facultad de Veterinaria, UCM, Spain, 2 Facultad de Veterinaria,Spain

An improved understanding of the potential risks of nanomate-rials is necessary to check its toxicity or safety. Therefore, the aimof this study was to evaluate the cytotoxic effects of silver nanopar-ticles (AgNps) of different sizes on tumoral [leukemia (HL-60) andhepatoma (HepG2)] and normal [dermal (NHDF) and pulmonary(HPF) fibroblasts].

MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) reduction and lactate dehydrogenase (LDH) leakagewere used for cytotoxicity assessment.

4.7 nm silver nanoparticles showed a strong decrease in HepG2from 3.36 to 13.45 �g/mL (14%, 5% and 4% of cell survival, respec-tively), in HL-60 at 13.45 �g/mL for 24–72 h (3%, 3.7% and 3.8%

of cell survival, respectively) and increased dramatically the per-centage of LDH leakage (80%, 98% and 99%, respectively) in HepG2and to 100% in HL-60. NHDF cell viability decreased drastically atconcentrations of 6.72 and 13.45 �g/mL (10% and 8% of survival,