etters

gesnatbotbaam

ao

gtM

d

PCc

E

1

P

pt

iCdgs

w(lbt1fi

dm

iCo

cf

amwE

d

Abstracts / Toxicology L

ranuloma formation, thickened alveolar septa and bronchiolarpithelium exfoliation. Type II pneumocytes, containing scarcelyurfactant-lamellated bodies, were also detected. Apoptotic phe-omena enhanced as following Cd-SiNP > CdCl2 > SiNP. Parallelly,significant increase of PCNA-immunoreactive cells was detected

ogether with high mitotic activity. Cellular localization and distri-ution of IL-6, IP-10 and TGFbeta1 revealed an increased expressionf these cytokines evidencing an enhanced cellular inflamma-ory response. CYP450-immunoreactivity was also enhanced, atronchiolar (e.g. Clara cells) and alveolar (e.g. macrophages) levelfter both Cd-SiNP and CdCl2. These overall effects were observedcutely and lasted until 30 days, with Cd-SiNP producing the mostarked effects.Collagen-immunolabelling changed particularly 7 and 30 days

fter Cd-SiNP, when a strong stromal fibrogenetic reactionccurred.

The present findings suggest that Cd-SiNPs produce significantlyreater pulmonary alterations than either SiNP and CdCl2 underhe present experimental conditions (Research Grants from Italian

inistries of Health, Research, Education).

oi:10.1016/j.toxlet.2012.03.733

33-24admium-doped silica nanoparticles toxicity on pulmonaryells: an in vitro study

lisa Roda 1, Uliana De Simone 1, Luigi Manzo 2, Teresa Coccini 1

IRCCS Salvatore Maugeri Foundation, Pavia, Italy, 2 University ofavia, Italy

Cadmium-containing silica nanoparticles (CdNPs) show greatromise for targeted drug delivery, however risk associated withheir exposure needs to be examined.

A battery of in vitro tests was applied to evaluate the cytotox-city of engineered CdNPs, versus silica nanoparticles (SiNP) anddCl2 (1–100 microg/ml), in A549 cells by evaluating (i) mitochon-rial dysfunction, (ii) membrane integrity/cell morphology, (iii) cellrowth and proliferation, (iv) apoptotic pathway and (v) oxidativetress.

(i, ii) MTT data: a dose-dependent strong cytotoxicity of CdNPsas detected at 24 h, further exacerbated for longer incubation time

48 h-exposure). Even CdCl2-exposure caused dose-dependent celloss, however with minor extent. Cell viability was not affectedy SiNP at any concentration after 24 h-exposure; a slight cyto-oxicity (about 10%) was detected after 48 h-exposure at 50 and00 microg/ml. Experiments using Calcein/PI staining totally con-rm MTT data.

(iii) Clonogenic assay: CdNP-exposure for longer period (10ays) caused dose-dependent changes in colony number, size andorphology, more markedly at 50 and 100 microg/ml.(iv) Immunocytochemistry: dose-dependent caspase-3

mmunolabelling was detected after 24 h-exposure to bothdNPs and CdCl2, while a complete lack of immunopositivity wasbserved after SiNP-exposure at any concentration.

(v) GSH evaluated at both time-points was modified by all theompounds; particularly, it was increased in a dose-dependentashion after CdNPs and decreased by SiNP.

These data demonstrated that both CdNPs and CdCl2 affectedll investigated endpoints in a dose-dependent manner, morearkedly after CdNP, while, SiNP influenced oxidative stress path-

ay only. (Grants from Italian Ministries of Health, Research,

ducation – PRIN 2007).

oi:10.1016/j.toxlet.2012.03.734

211S (2012) S43–S216 S205

P33-25Genotoxicity of carbon nanomaterials in bronchial epithelialcells in vitro

Hanna Lindberg, Penny Nymark, Julia Catalán, Satu Suhonen,Hilkka Järventaus, Hannu Norppa

Finnish Institute of Occupational Health, Finland

Genotoxicity data of carbon nanomaterials is still quitelimited. We have examined the genotoxicity of two typesof multiwall carbon nanotubes (MWCNT) – long, needle-likeMWCNT (Mitsui-7, >50 nm × 13 �m) and long, tangled MWCNT(Cheaptubes, 8–15 nm × 10–50 �m) – and graphite (Sigma,80–200 nm × 0.5–20 �m) in human bronchial epithelial BEAS 2Bcells.

The single cell gel electrophoresis (comet) assay (24-h expo-sure) was applied to detect DNA damage and the cytokinesis-blockmicronucleus (MN) assay (48-h exposure) to show chromosomaldamage.

All of the nanomaterials induced DNA damage in BEAS 2Bcells after the 24-h exposure. Long, needle-like MWCNT increasedthe level of DNA damage already at low doses (5, 10, 200 and250 �g/cm2) with a marginal dose-dependency. Graphite induced aclear dose-dependent increase in DNA damage at the highest dosestested (50, 100, 200 and 250 �g/cm2). The DNA-damaging effect oflong, tangled MWCNT was weak, with a significant increase in DNAdamage only at the highest dose examined (250 �g/cm2), despitea dose-dependency. None of the nanomaterials tested inducedmicronuclei in BEAS 2B cells after the 48-h exposure. However, asignificant negative dose response was observed in the MN assay forlong, needle-like MWCNT, with a similar trend also for the long, tan-gled MWCNT. Long, needle-like MWCNTs and graphite induced theclearest effects in the comet assay showing a significant increase inDNA damage already at relatively low doses. None of the materialsinduced micronuclei.

Supported by the European Commission, NANODEVICE, CP-IP211464-2.

doi:10.1016/j.toxlet.2012.03.735

P33-26Microbial biosensors for profiling of toxicity mechanisms ofmetallic nanoparticles

Olesja Bondarenko, Angela Ivask, Anne Kahru

NICPB, Akadeemia tee 23 Tallinn, Estonia

The worldwide market for nano-enabled products is expo-nentially increasing and there is a concern regarding safety ofnanoparticles (NPs). Since 2006, the research of our lab in NICPB,Estonia has focused on developing of bio-analytical approachesfor the profiling of (eco)toxicological properties of synthetic NPscombining exotoxicological tests, recombinant biosensors and ana-lytical techniques. The toxicity of metallic NPs mostly correlateswith their ability to produce reactive oxygen species (ROS) whereasthe nature of ROS is still unclear: are the ROS produced solely byheavy metal ions dissolved from metallic NPs or also by particlesthemselves. For the cost-efficient solving of this scientific problemwe introduced a suite of recombinant microbial biosensors that

specifically recognize already at subtoxic level (i) superoxide radi-cals, (ii) hydrogen peroxide (iii) DNA damaging agents and (iv–iiv)soluble Cu/Ag, Zn or Cd, allowing to reveal the mechanisms of toxiceffects of metallic NPs. We showed that ZnO, CuO and Ag NPs were