The Toxicologist - Society of Toxicology

988 NNK-INDUCED CYTOKINE ALTERATIONS IN LUNG
SLICES FROM LEAN AND DIET-INDUCED OBESE
C57BL/6J MICE.
T. Liberati and M. Randle. Internal Medicine, Southern Illinois University School of
Medicine, Springfield, IL.
Lung cancer is a significant cause of mortality in the U.S., with tobacco use being
the primary risk factor. However, only 10% of smokers develop lung cancer suggesting
that other risk factors (e.g. genetic profile, metabolic status, and pre-existing
lung disease) likely play a role. Studies have indicated that inflammation may contribute
to the initiation of lung cancer. However, the inflammatory milieu that promotes
carcinogenesis has not been elucidated. Obesity is recognized as a “state of
chronic inflammation” due to the presence of abundant mediators in serum and
adipose of obese individuals. To investigate how obesity may alter the inflammatory
profile of lung and its innate immune response to a carcinogen, we evaluated the
impact of NNK, a tobacco carcinogen, on lung slices prepared from C57BL/6J
mice that were maintained on either standard or high-fat diets. Following instillation
with culture medium containing 1% LMP agarose, 750μM slices were placed
on Netwell inserts with supplemented DMEM at 37° C and 10% CO 2 . After 24
h, slices were treated with 0, 10, 50 or 250 μM NNK. Slices were harvested 4 and
24 h later to measure LDH release into the medium and concentrations of cytokines/chemokines
in homogenates and medium. Compared with lean mice, slices
from mice with diet-induced obesity (DIO) showed greater LDH release, regardless
of NNK dosage. In slices not exposed to NNK, cytokine concentrations in homogenates
and media were higher in DIO mice as compared with lean mice. This
was most apparent after 48 h in vitro. Homogenates and media from the lung of
DIO mice had higher concentrations of G-CSF, IL-6, CXCL14 and CXCL1 and
lower concentrations of CXCL10 than did samples from lean mice. After exposure
to NNK for 24 h, homogenates and media from DIO mice had higher concentrations
of IL-6, CXCL9, CXCL14 and CXCL1 than did those from lean mice. We
conclude that lung from DIO mice shows an “inflammatory” profile when maintained
in vitro as lung slices and responds to a known carcinogen in an augmented
manner as compared with lung from lean mice.
989 IRRITATION IN THE LLNA: 2000 MICE EAR
THICKNESS MEASUREMENTS DEPENDENCE ON
VEHICLE AND HCA TREATMENT.
J. L. Moore, M. R. Carathers, D. R. Cerven and G. L. DeGeorge. MB Research
Laboratories, Spinnerstown, PA.
The Local Lymph Node assay (LLNA) is known to be susceptible to false-positives
caused by irritating test substances. We have incorporated ear thickness measurements
on Days 1, 3 and 6 of the LLNA in order to measure dermal irritation-induced
ear swelling. Known LLNA false-positive irritants SLS and BAC caused increases
in ear swelling on Day 6 (when compared to Day 1) of 25% or greater
(>1.25-fold). In addition, the default positive control Hexylcinnamaldehyde
(HCA) has irritation potential, which was confirmed at concentrations above 10%
HCA. Interestingly, all common LLNA vehicles (Acetone:Olive Oil 4:1(AOO),
Acetone, Ethanol, Dimethylacetamide:Acetone:Ethanol 4:3:3 (DAE433), etc.) did
not affect ear swelling through Day 6, with the notable exception of
Dimethylsulfoxide (DMSO). DMSO-treated mouse ear swelling was increased
over 15% during the six-day experimental term. This implicates DMSO as moderately
irritating and possibly a problematic vehicle for the LLNA.
990 THE CYTOTOXIC AND INFLAMMATORY RESPONSE
OF BRONCHIAL EPITHELIAL CELLS EXPOSED TO
CIGARETTE WHOLE SMOKE AND VAPOUR AT THE
AIR-LIQUID INTERFACE.
G. J. Phillips, L. E. Haswell, G. Foss-Smith, K. Hewitt, S. Corke, D.
Azzopardi, M. D. Gaca and C. Meredith. Group R&D, British American Tobacco,
Southampton, United Kingdom.
As part of our approach to harm reduction we are developing potential reduced-exposure
products (PREPs). Currently only outline guidelines for the evaluation of
PREPs exist and as such, a case by case approach to PREP assessment is required.
The early stage in this process includes the biological evaluation of cigarette whole
smoke and vapour, rather than just the particulate content, using in vitro models
that measure some physiological or disease associated endpoint. To achieve this, a
whole smoke exposure system has been developed that allows for the exposure of
cells at the air-liquid interface to be undertaken. In this study, bronchial epithelial
cells (NCI-H292) were initially grown to confluence on semi permeable membranes
(Transwell). The cells were then raised to the air-liquid interface by re-
moval of the covering culture medium and exposed for 30 minutes to cigarette
whole smoke (Reference Cigarette 3R4F) over a range of predetermined dilutions
(1:5 to 1:400). An equivalent set of cells were exposed to the vapour phase component
of whole smoke by removal of the particulate fraction using an inline
Cambridge filter pad. Following a 24 hour recovery period under culture medium,
cytotoxicity and secreted mediators of inflammation (IL-6, IL-8 and MMP1) were
measured using the neutral red assay, ELISA and the Meso Scale Discovery
(MSD) platform. Results demonstrate that vapour constitutes 82% of the total
cytotoxicity derived from whole smoke exposure. At subtoxic dilutions of whole
smoke (>1:250), the concentrations of inflammatory mediators were increased
compared to cells exposed to equivalently diluted vapour. These results show that
vapour is a significant cytotoxic and inflammatory component of cigarette whole
smoke. Further studies are underway to assess the effects of PREPs on this model.
991 THE USE OF IN VITRO TOXICITY DATA AND
PHYSIOLOGICALLY BASED KINETIC MODELING TO
PREDICT DOSE-RESPONSE CURVES FOR IN VIVO
DEVELOPMENTAL TOXICITY OF GLYCOL ETHERS IN
RAT AND MAN.
J. Louisse 1, 2, 3 , E. de Jong 4, 5 , J. J. van de Sandt 2 , B. J. Blaauboer 5 , R. A.
Woutersen 1, 2, 3 , A. H. Piersma 4, 5 , I. M. Rietjens 1, 3 and M. Verwei 2, 3 . 1 Division
of Toxicology, Wageningen University, Wageningen, Netherlands, 2 TNO Quality of
Life, Zeist, Netherlands, 3 WUR/TNO Centre for Innovative Toxicology, Wageningen,
Netherlands, 4 National Institute of Public Health and the Environment (RIVM),
Bilthoven, Netherlands and 5 Institute for Risk Assessment Sciences (IRAS), Utrecht
University, Utrecht, Netherlands.
The regulatory assessment of systemic toxicity is almost solely performed using animal
models, providing in vivo dose-response curves. If in vitro toxicity data are to
be used for human risk assessment, in vitro concentration-response curves should
be translated to in vivo dose-response curves that can be used to set a point of departure
for deriving safe exposure limits. The present study shows an approach to
extrapolate in vitro concentration-response curves to in vivo dose-response curves
for developmental toxicity by combining in vitro toxicity data and in silico kinetic
modeling. A physiologically based kinetic (PBK) model was developed, describing
the kinetics of four glycol ethers and their embryotoxic alkoxyacetic acid metabolites
in rat and man. In vitro toxicity data of these metabolites derived in the embryonic
stem cell test (EST) were used as input in the PBK model to extrapolate in
vitro concentration-response curves to predicted in vivo dose-response curves for
developmental toxicity of the parent glycol ethers in rat and man. The predicted
dose-response curves for rat were found to be in concordance with the embryotoxic
dose levels measured in reported in vivo rat studies. These predicted dose-response
curves for rat might be used to set a point of departure for deriving safe exposure
limits in human risk assessment. Combining the in vitro EST toxicity data with a
human PBK model allows the prediction of dose-response curves for human developmental
toxicity. This approach could therefore provide a means to reduce the
need for animal testing in human risk assessment practices.
992 EVALUATION OF MEDAKA AS AN ANIMAL MODEL TO
STUDY AUTISM SPECTRUM DISORDER.
M. Wu 2, 1 , I. A. Khan 1 and A. K. Dasmahapatra 1, 2 . 1 National Center for Natural
Product Research, University of Mississippi, University, MS and 2 Department of
Pharmacology, University of Mississippi, University, MS.
Autism spectrum disorder (ASD) is an umbrella term used to define a broad spectrum
of atypical social, cognitive and verbal behaviors along with repetitive and
sometimes self-injurious actions. The behavioral criteria used to diagnose ASD are
not sufficient to justify the diagnosis. Moreover, there is no appropriate medicine
available for the treatment of ASD. The psychoactive drug risperidone is prescribed
for ASD patients, however, majority of them are used alternative medicines for the
prevention of this disorder. We predict that two major barriers exist in understanding
the pathogenesis of ASD: (1) the absence of a defined phenotypic marker or
gene product specific to ASD, and (2) the lack of an appropriate animal model that
can display core symptoms of ASD. We are evaluating medaka as an animal model
to identify exogenous phenotypic features of ASD using valproic acid (VPA) as the
inducer. Further we will evaluate the potency of natural products as an alternative
to prevent ASD. Fertilized medaka eggs at specific phase of central nervous system
development (1-3 day post fertilization, dpf) were exposed to different concentrations
of VPA for 48 hours and the mortality was assessed on 10 dpf. The calculated
IC50 as observed on 10 dpf is 10.69 +/- 1.87 mM. The hatchling were used for
craniofacial cartilage staining and observed that several neurocranial cartilages are
smaller in size compared to controls. We predict that studies in craniofacial cartilage
development and differentiation may be able to identify a unique phenotypic feature
that can be used to diagnose ASD exogenously
SOT 2011 ANNUAL MEETING 211