The Toxicologist - Society of Toxicology

caused an inhibitory effect on the neurite outgrowth in Neuro-2a cells in the absence
of cell death. AMPK activation induced by retinoic acid in differentiating cells
was blocked by arsenic. Pretreatment with an AMPK specific activator, 5-aminoimidazole-4-carboxamide
riboside (AICAR), or overexpression of a constitutively active
AMPK-α1 plasmid recovered arsenic-induced neurite retraction. The activation
of LKB1, a major AMPK-kinase, was also suppressed by arsenic by inhibiting
the translocation of LKB1 from nuclear to cytoplasm. Antioxidants, such as NAC
and SOD, but not catalase, protected against arsenic-induced inactivation of
LKB1-AMPK pathway, and reversed the inhibitory effect of arsenic on neurite outgrowth.
Conclusions: Neurite retraction induced by arsenic results from deficient
activation of AMPK as a consequence of LKB1 inactivation. Oxidative stress induced
by arsenic, especially excessive superoxide, plays a critical role in the blocking
LKB1-AMPK pathway. Our studies provide an insight into the underlying mechanisms
of arsenic-induced developmental neurotoxicity and could be important in
designing a new strategy for protecting children against this neurotoxic substance.
1394 TARGETING OF DEVELOPING DOPAMINERGIC
NEURONS BY METHYLMERCURY IN THE
PICOMOLAR RANGE.
M. Leist, B. Zimmer, B. Baudis, F. Matt and S. Kadereit. Doerenkamp-Zbinden
Chair for Alternative in vitro Methods, University Konstanz, Konstanz, Germany.
Sponsor: A. Buerkle.
Methylmercury (MeHg) is a huge health concern due to its neurodevelopmental
toxicity at very low concentrations. This is characterized by delayed onset and a
complete absence of any overt signs of neuropathology. We aimed here at setting up
an experimental system that would identify such low dose effects. In order to cover
different steps and mechanisms, we designed a test battery consisting of different
model systems. The following assay systems were used: Murine embryonic stem
cells (mESC) were converted first into neural precursor cells and then into terminally
differentiated neurons or astrocytes, Condi-tionally-immortalized human
neural precursor cells (LUHMES cells) were differentiated within 5 days to a homogeneous
popu-lation with a complex neurite network and typical biochemical
and morphological features of dopaminergic neurons. In both systems cells were exposed
to MeHg in different developmental phases and after differentiation. MeHg
exerted the well described cytotoxicity in the low micromolar range, as detemined
by multiple endpoints, including a quantification of neurite length. However,
murine neural precursors developing to neurons showed a reduced expression of
neural mRNA transcripts upon exposure to 1000-fold lower MeHg concentrations.
This effect was independent of cytotoxicity, but accompanied by downregulation of
neuronal specific betaIII tubulin protein. In particular, dopaminergic (DA) cells
were reduced, and the total activity of dopamine transporter in the cultures was significantly
decreased. We conclude that neurodevelopmental toxicity by MeHg may
be based on imparired differentiation of DA neurons.
1395 INHALATION TOXICITY STUDIES WITH 12
NANOMATERIALS USING DIFFERENT
DOSIMETRIES–NONE FITTED ALL.
R. Landsiedel 1 , Y. Staal 3 , L. Ma-Hock 2 , K. Wiench 1 , S. Treumann 1 , V. Strauss 1
and B. van Ravenzwaay 1 . 1 Experimental Toxicology and Ecology, BASF SE,
Ludwigshafen am Rhein, Germany, 2 Product Safety, BASF SE, Ludwigshafen am
Rhein, Germany and 3 TNO Quality of Life, Zeist, Netherlands.
Several studies demonstrate that identical mass concentrations of nano-particles are
more toxic to the lung than larger particles of similar chemistry. Surface area and
particle count concentration have been discussed as critical factor responsible for
higher pulmonary toxicity of nanoparticles (Oberdoerster et al. 2005 EHP 113,
823-839). We tested the toxicity of 15 substances (12 nano¬materials: SiO2, surface
coated SiO2, TiO2 P25, coated TiO2, CeO2, doted CeO2, ZrO2, BaSO4,
carbon black, MWCNT1, MWCNT2 and – for comparison - 3 micron-scaled materials:
quartz, TiO2, ZnO) after inhalation exposure. All materials were sufficiently
characterized and tested by the well-established short-term inhalation toxicity protocol
for nanomaterials (Ma-Hock et al. 2009, Inhal. Toxicol. 21, 102); total protein
concentration and polymorphonuclear neutrophils in bronchoalveolar lavage
fluide were used as sensitive indicators of effects in the lung. The observed lung toxicity
was related to (a) particle mass, (b) particle volume, (c) particle surface area
and (d) particle count per aerosol volume (aerosol concentration) and per lung surface
(lung burden).
For insoluble nanomaterials the lung toxicity varied over two orders of magnitude
when using mass concentration as dosimetry, and between different materials toxicity
was largely independent of surface area, particle count, volume or mass.
Evidently, there is not one dosimetry for all nanomaterials. Rather, nanomaterials
exert their toxicity by different effects (e.g. ROS generation, ion-release, loading of
macrophages) and hence their lung toxicity is determined by different material
properties.
We propose to use mass concentration as dosimetry for all particles. At the same
time we stress the necessity to fully characterize the tested materials, this will allow
for transformation of mass to other dosimetry parameters. The correct dosimetry
can only be identified by recognizing the very material properties causing the toxic
effect.
1396 OCCUPATIONAL RISK ASSESSMENT OF MULTIWALL
CARBON NANOTUBES.
A. Lecloux 1 , F. Luizi 1 , M. Del Tedesco 3 , D. Brouwer 2 , R. Muir 2 , D. Green 4 , L.
Ma-Hock 4 , K. E. Wiench 4 and R. Landsiedel 4 . 1 Nanocyl S.A., Sambreville,
Belgium, 2 Naneum Ltd., Canterbury, Kent, United Kingdom, 3 TNO, Zeist,
Netherlands and 4 BASF SE, Ludwigshafen, Germany.
Multi Wall Carbon Nanotubes (MWCNT) are of great commercial interest, as they
combine high electrical conductivity, good mechanical strength and excellent thermal
conductivity. It is therefore essential to ensure a sustainable development of
these materials. This requires a risk assessment of production and use of MWCNT
followed by risk management measures. The potential exposure of workers via inhalation
during production, processing and handling of MWCNT NC 7000 from
Nanocyl has been measured. Several methods were used such as ELPI, CPC,
SMPS, etc. A new detector (Nano-ID) combining a cascade impactor and a diffusion
deposition able to identify particles from a few nm up to 30 μm was also used.
In addition, in-situ on-line MWCNT specific counter based upon Nano-ID was
employed. Measurements have been carried out by Nanocyl and several organizations
at Nanocyl facilities. Measurements consistently demonstrated that exposure
can be minimized by applying appropriate handling measures. For hazard assessment,
a 5-day and a 90-day inhalation toxicity study with NC 7000 was performed
according to OECD test guideline 413. Rats inhaled concentrations of 0 (control),
0.1, 0.5, or 2.5 mg/m3 on 6 h/day on 5 days/week. The exposure resulted in no systemic
toxicity, but a concentration-dependent increase of lung weights and granulomatous
inflammation with histiocytic, neutrophilic inflammation and intra-alveolar
lipoproteinosis in the lung and lung-associated lymph nodes at 0.5 and 2.5
mg/m3; at 0.1 mg/m3, there was still minimal granulomatous inflammation. The
data were analyzed by the Uncertainty Factor Method and the Benchmark Dose
Method. The risk assessment on this MWCNT indicates that (i) toxicological data
appear to be sufficient for initial risk estimates for non-cancer lung responses, (ii)
standard methods for hazard and risk assessment appear feasible and (iii) strict industrial
hygiene measures during handling and processing are effective.
1397 MESOTHELIOMA INDUCTION BY MICROMETER-
SIZED MULTI-WALL CARBON NANOTUBE
INTRAPERITONEALLY INJECTED TO P53
HETEROZYGOUS MICE.
J. Kanno 1 , A. Takagi 1 , T. Nishimura 2 and A. HIrose 3 . 1 Division of Cellular and
Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan, 2 Division
of Environmental Chemistry, National Institute of Health Sciences, Tokyo, Japan and
3
Division of Risk Assessment, National Institute of Health Sciences, Tokyo, Japan.
Nanomaterials of carbon origin tend to form various shapes of particles and aggregates
in micrometer dimensions. Among them, certain make of multi-wall carbon
nanotubes (MWCNT) forms fibrous or rod-shaped particles of length around 10
to 20 micrometers with an aspect ratio of more than three. Fibrous particles of this
dimension including asbestos and some man-made fibers are reported to be carcinogenic,
typically inducing mesothelioma. Here we report that such micrometersized
MWCNT induces mesothelioma when administered intraperitoneally to p53
heterozygous mice that have been reported to be sensitive to asbestos. In this study,
as an accidental finding, fullerene which was administered intraperitoneally as negative
control of the study turned out to be distributed systemically. It was histologically
suggested that the surface of fullerene aggregates were corroded by phagocytic
activities, possibly down to nanometer dimensions and brought out from the peritoneal
cavity. Our results indicate that newly made particulate matters of various
sizes have a potential to exhibit known as well as totally unknown toxicity, especially
of chronic nature. To maintain sound activity of industrialization of nanomaterials,
timely transfer of information on the chronic toxicity of the new materials
or the products to the manufacturers would be of great importance. In order to facilitate
such process, cultivation of proper toxicology scientists who are able to predict
such “new” chronic toxicity by making full use of traditional as well as recent
methodologies should be promoted and supported not only by the government but
also by the private sector.
296 SOT 2010 ANNUAL MEETING