The Toxicologist - Society of Toxicology

372 EVALUATING THE BIOLOGICAL INTERACTIONS OF
NANOMATERIALS USING FLUORESCENCE BASED
TECHNOLOGY.
L. Braydich-Stolle, A. Castle, E. Maurer and S. Hussain. RHPB, Air Force
Research Labs, Wright Patterson, OH.
Currently there is no standardized method for making nanomaterials or evaluating
their toxicity. Studies are showing great variability and lack of reproducibility in results,
with the discrepancies partially as a result of the assays used to evaluate exposure
toxicity. While the traditional biochemical methods for evaluating toxicity
have been employed there is an issue of how the nanomaterials themselves interact
with the dyes. For example, the MTS assay is read at a wavelength of 490nm and
this wavelength corresponds to the spectral signature for silver nanomaterials and
therefore can give false stimulatory effects. The current methodologies need to be
evaluated to determine the toxicity of the nanomaterials and ensure there is no interaction
between the nanomaterials and the reagents used to complete the assays.
This current study evaluated mechanisms of nanomaterial toxicity using mitochondrial
function, loss of mitochondrial membrane potential, and formation of reactive
oxygen species as indicators of toxicity, and compared the data obtained on the
BD Pathway 435 confocal microscope with HCA analysis with data obtained from
a BioTek plate reader. Furthermore, nanoparticle uptake and localization was evaluated
using the 3D imaging capabilities on the BD Pathway 435. These studies
demonstrated that the fluorescence based assays analyzed on the BD Pathway 435
provided more sensitive detection for the cellular based assays in comparison to a
plate reader alone. Furthermore, organelles such as the mitochondria and the lysosome,
were stained with organelle dyes and demonstrated co-localiztion of
nanoparticles within the organelles verifying uptake and cellular interactions.
373 METAL OXIDES INFLUENCE CELLULAR
HOMEOSTASIS VIA MULTIPLE INTERCONNECTED
SIGNALING PATHWAYS.
Y. Huang, C. Huang, Y. Xu and R. S. Aronstam. Biological Sciences, Missouri
University of Science and Technology, Rolla, MO.
Influence of cellular homeostasis was studied using various nanoparticles (NPs) of
metal oxides. NPs induced oxidative stress (OS). An inverse correlation between
OS and cell viability was observed (p < 0.05). Biochemical analyses and gene expression
studies revealed that OS leads to cell death via DNA injury, membrane depolarization,
loss of membrane integrity, and induction of death-related genes. NPs
elevated intracellular calcium levels ([Ca2+]in) in a time- and concentration-dependent
fashion. An inverse relation between [Ca2+]in and cell viability was identified
(p < 0.05). [Ca2+]in can be partially attenuated by antioxidant N-acetylcysteine
indicating a link between calcium modulation and OS. Nifedipine partially
attenuated the increase in [Ca2+]in, suggesting that a portion of the influx involved
L-type calcium channels. Direct membrane lipid peroxidation resulting in leaky cytoplasmic
membrane and the release of calcium from intracellular stores may participate
in calcium influx as well. Initiation of OS and intracellular calcium modulation
via multiple mechanisms is discussed. Finally, a diagram depicts relationships
between NPs, OS, [Ca2+]in and cell death.
374 EFFECTS OF QUANTUM DOTS ON CELLULAR STRESS
MARKERS IN HEPG2 CELLS.
W. E. Smith 1, 3 , J. H. Tracy 1, 3 , T. Lin 1, 3 , C. C. White 1, 3 , X. Hu 2 , X. Gao 2 , D.
L. Eaton 1, 3 and T. J. Kavanagh 1, 3 . 1 Departments of Environmental and
Occupational Health Sciences, University of Washington, Seattle, WA, 2 Department
of Bioengineering, University of Washington, Seattle, WA and 3 Center for Ecogenetics
and Environmental Health, University of Washington, Seattle, WA.
Quantum dots (Qdots) are nanometer-sized crystals with photochemical and photophysical
properties that offer distinct advantages over organic dyes and fluorescent
proteins, such as high levels of brightness and photostability. Qdots are generally
constructed of a core metal composition and surrounded with various coatings
specific to the intended use. The core composition of Qdots consists of Cd, Se and
Hg. While metals utilized for this core composition are known to be toxic, very little
is known regarding potential toxicities elicited by various surface coatings.
Previously, we found that poly(maleic anhydride-alt-1-tetradecene), tri-noctylphosphineoxide
(PMAT-TOPO) co-polymer CdSe Qdots appear to colocalize
with cultured HepG2 cells in a dose-dependant manner over a period of 24 hours.
In addition to Qdot uptake, our preliminary results indicate that this particular
class of stable Qdots has no adverse effects on HepG2 cells under the conditions assessed
with the MTT cytotoxicity assay. To further assess for more sensitive markers
of cellular stress, we measured various endpoints (uptake, glutathione content and
mitochondrial membrane potential) by flow cytometric analysis after 24 hours of
exposure to Qdots. Consistent with our previous studies, measurements by flow cytometry
indicate a dose-dependent increase in uptake. Next, we measured for
changes in mitochondrial membrane potential with nonylacridine orange. No
changes in membrane potential were evident over the range of concentrations
tested. Interestingly, levels of glutathione appear to be slightly reduced at higher
doses of Qdots with PMAT-TOPO coating. Work is currently ongoing to more
thoroughly characterize effects on cellular functioning elicited by Qdots. This work
was supported by NIEHS grants 1R01ES016189, P30ES07033 and T32ES07032.
375 COMPARATIVE TOXICITY OF LUNAR, MARTIAN, AND
EARTH DUSTS IN HUMAN SKIN FIBROBLAST CELLS.
J. Wise 1 ,R.Duffy 1 ,H.Xie 1 ,C.LaCerte 1 ,M.Mason 2 , A. Jeevaragen 3 ,W.
Wallace 3 , D. Hammond 3 , T. Shehata 4 and J. P. Wise 1 . 1 Department of Applied
Medical Science, University of Southern Maine, Portland, ME, 2 Department of
Chemical and Biological Engineering, University of Maine, Orono, ME, 3 NASA
Johnson Space Center, Houston, TX and 4 Maine Space Grant Consortium, Augusta, ME.
NASA has plans to build a permanent structure on the Moon by 2020; therefore
they are concerned about the potential toxicity of lunar dust. NASA also hopes to
visit Mars, and thus are also concerned about Martian dust. In our study we compared
the cytotoxic and genotoxic effects of two lunar dust simulants (JSC-1AVF,
JSC-1AF) to Earth dust (urban particulate matter), and a Mars dust simulant
(MARS-1AF) in human skin fibroblast cells (BJhTERT). Comparing the cytotoxicity
of these chemicals shows that the Mars dust simulant is the most cytotoxic after
a 24 h and a 120 h. The JSC-1AVF lunar dust simulant is more cytotoxic than the
JSC-1AF, which is possibly due to the AVF particles being smaller. The cytotoxicity
of Earth dust is similar to Mars dust. The 120 h genotoxicity shows that all four
simulants show similar effects. This work is supported by the Maine Center for
Toxicology and Environmental Health, NASA grant EP-08-01, and the Maine
Space Grant Consortium.
376 EFFECTS OF FENTANYL CITRATE ON FERTILITY AND
EARLY EMBRYONIC DEVELOPMENT TO
IMPLANTATION IN RATS.
K. H. Denny 2 and A. S. Faqi 1 . 1 Toxicology, MPI Research, Mattawan, MI and
2
Drug Safety and Disposition, Cephalon, Inc., West Chester, PA.
The objective of this study was to determine the fertility effects of fentanyl citrate in
male and female rats. Four treatment groups of 25 male and 25 female [(Crl:CD®
(SD)] rats, received Fentanyl Citrate subcutaneously at respective dose levels of
12.5, 25, 50 (increased to 100 in females on day 8), and 300μg/kg/day. Two groups
of 25 animals per sex served as the control and received the vehicle, 0.9% Sodium
Chloride for Injection, USP. Six additional groups of 25/sex were untreated and utilized
for breeding with each assigned treatment group. An additional high dose of
300μg/kg/day was later added in the study at the request of a regulatory agency.
Males and females were treated for 28 and 14 days prior to mating, respectively.
Dosing continued in males through mating and postmating: females were dosed
through gestational day 7. Mortality was observed at 300 μg/kg/day in male and female
rats and at 50/100 μg/kg/day in female rats. Exaggerated pharmacological effects
including decreased activity, prostration, loss of righting reflex and slow
breathing were observed at doses >25μg/kg/day. Decrements in body weight parameters,
and food consumption were observed at 50/100 and 300 μg/kg/day.
Sexual and accessory sexual organ weights were significantly decreased at
300μg/kg/day in comparison to the controls. Fertility and fecundity indices were
significantly reduced in naïve females mated with males receiving 300μg/kg/day.
Likewise, uterine weights were significantly reduced in naïve females mated with
males at 300μg/kg/day. The uterine weight reduction correlated with a significant
decrease in implantation sites and viable embryos, and with increased post-implantation
loss. Based on the results of this study, the NOAEL for general toxicity was
12.5μg/kg/day for both sexes. The NOAEL for reproductive toxicity in treated
male and female rats were 50/100μg/kg/day and 300μg/kg/day, respectively.
377 EVALUATION FOR OVARIAN TOXICITY OF SODIUM
VALPROATE USING RAT CULTURED OVARIAN
FOLLICLES.
H. Inada, K. Chihara, C. Fukuda, A. Yamashita, Y. Tateishi, T. Yamada, J.
Kimura, H. Funabashi and T. Seki. Safety Research Laboratories, Dainippon
Sumitomo Pharmacology Co., Ltd., Osaka, Japan.
Sodium valproate (VPA), an antiepileptic drug, is known to induce endocrine side
effects including characteristic symptoms of polycystic ovary syndrome in women
with epilepsy, and cystic follicles in the ovaries have been reported in VPA-treated
SOT 2010 ANNUAL MEETING 81