The Toxicologist - Society of Toxicology

events and carcinogenesis. Here we introduce the next generation of HTS methodology:
a novel dual-read out Saccharomyces cerevisiae screen DEL-XG. Based on
the DEL assay, DEL-XG simultaneously assesses the compound’s genotoxicity and
cytotoxicity properties. DEL-XG test strain cells contain a lacZ gene sequence
(with a GAL promoter) with a region of homology interrupted by a URA3 gene;
during an exposure to a genotoxic event (carcinogens, ionizing radiation, etc) the
URA3 sequence is excised and the lacZ gene recombines to a functional beta-galactosidase
genotype that with an addition of the X-Gal substrate produces a detectable
indole product. Additionally, the test strain stably expresses a florescent
protein as an indication of survival. Thus, in a single well the assay identifies how
the compound of interest affects survival—changes in florescence emission, and genetic
integrity—accumulation of insoluble blue dye. DEL-XG is a rapid and economical
way to screen large chemical libraries for toxicity properties and enables a
screening “triage”: compounds with higher toxicity profiles will be subjected to additional
screening as a first priority.
341 THE RECONSTRUCTED SKIN COMET ASSAY SHOWS
GOOD INTRA- AND INTER-LABORATORY
REPRODUCIBILITY: RESULTS FROM THREE
LABORATORIES.
T. Downs 1 , A. Reus 2 , K. Reisinger 3 , C. Krul 2 and S. Pfuhler 1 . 1 Procter & Gamble
Company, Cincinnati, OH, 2 TNO Quality of Life, Zeist, Netherlands and 3 Henkel
AG & Co KgaA, Düsseldorf, Germany. Sponsor: G. Daston.
EU policy marks the need for a broad replacement of animal tests in relation to the
7th Amendment to the Cosmetics Directive. For cosmetics ingredients as well as
many chemicals, the skin is the main route of exposure. The currently used standard
in vitro genotoxicity assays, if used in a test battery approach, show a high percentage
of irrelevant positive results. Moreover, there is no follow-up assay available
in the target tissue skin and the use of in vivo assays is not allowed to further evaluate
a positive response obtained from the in vitro assays. Genotoxicity assays using
reconstructed human skin could however serve this purpose. EpiDerm skin
models were topically exposed to vehicle or test compounds for 3h, followed by cell
isolation and measurement of DNA damage using the Comet assay methodology.
Inter-laboratory reproducibility of the 3D skin Comet assay was demonstrated for
MMS and 4NQO and results showed good concordance with in vivo data. In the
next phase of the project, the assay was performed with 3 coded compounds
shipped to 3 different laboratories. The results of the collaborative study showed
good reproducibility within and between laboratories. However, considerable intraand
inter-experimental variability was often observed in the background level of
DNA damage in 3D skin tissues, with solvent control values sometimes greater
than 40%. To resolve these problems we worked on several aspects of the experimental
procedure and promising results were seen with the use of “underdeveloped”
tissues, which are shipped at an earlier stage (5 days before regular shipment) and
are then matured in our laboratories. Results thus far indicate that the comet assay
in reconstructed 3D skin models is a relevant model for the safety assessment of
chemicals with dermal exposure. This work was sponsored by the European
Cosmetics industry (COLIPA) and the European Center on Validation of
Alternative Methods (ECVAM)
342 CHLORPYRIFOS DOES NOT INDUCE RAT BRAIN DNA
DAMAGE OR BONE MARROW MICRONUCLEUS
FORMATION IN A MICRONUCLEUS AND COMET
COMBINATION ASSAY.
Y. Xu, K. Carl and L. Timothy. Covance, Vienna, VA.
Chlorpyrifos was reported to induce single strand DNA breakage in the brain of
rats, but but was not reported to be mutagenic in WHO and FAO evaluations. A
combined micronucleus and Comet assay using Cyclophosphamide (CP) and Ethyl
Methanesulfonate (EMS) as a positive control was performed to confirm the findings.
Differing Comet assay conditions (unwinding time and electrophoresis time)
were tested to verify the reported data. As demonstrated in the mouse micronucleus
assays, Chlorpyrifos did not induced bone marrow micronucleus formation at doses
up to 100 mg/kg in three doses. The treatment of Chlorpyrifos did not induce an
increase of % Tail Intensity or Tail Moment in the cerebral cortex cells of the brain
as published. However, CP/EMS treatment significantly elevated the % Tail
Intensity and Tail Moment in the Comet assay. The negative findings with
Chlorpyrifos were further confirmed by the differing Comet assay conditions.
When unwinding and electrophoresis times were increased from 15/20 minutes to
30/40 minutes, the group mean of % Tail Moment in positive control animals was
increased about 6 fold from 1.35 ± 0.19 to 3.03 ± 1.36. Meanwhile the fold increases
in Chlorpyrifos groups (3 fold at 100 mg/kg and 2.3 fold at 50 mg/kg) approximated
the vehicle control group (2.7 fold). The data supports a non-genetoxic
evaluation of Chlorpyrifos and did not confirm DNA damage effects of
Chlorpyrifos in the cerebral cortex cells of brain.
343 GENOTOXICITY TESTING USING THE
MICRONUCLEUS AND COMET ASSAYS IN NORMAL
HUMAN CELL BASED 3D EPITHELIAL MODELS.
J. DeLuca, Y. Kaluzhny, P. J. Hayden, A. Armento, V. Karetsky and M.
Klausner. MatTek Corp, Ashland, MA.
Safety assessment of new products for human use requires genotoxicity testing to
ensure non-carcinogenicity. Current in vitro assays have low specificity resulting in
a high rate of false positives. To determine the biological relevance of positive in
vitro genotoxicity results, in vivo assays are conducted. However, in vivo genotoxicity
testing was banned in 2009 by the 7th Amendment to the Cosmetics Directive.
3D human tissue models, which have in vivo-like barrier function and metabolism
and which allow for topical exposure, are predicted to have improved biological relevance.
Toward this end, the Reconstructed Skin Micronucleus (RSMN) and
Comet assays (CA) that utilize MatTek’s highly differentiated EpiDerm tissue
model have been adapted for use with tracheal, vaginal, oral, and corneal tissues.
EpiDerm is a 3D normal human cell-based epidermal model that is highly reproducible,
contains an in vivo-like barrier, and possesses in vivo-like biotransformation
capabilities. RSMN assay results show statistically significant dose-dependent,
increases in cells containing micronuclei (MNC) for 9 direct genotoxins and 6
genotoxins that require metabolic activation, and no increases for 4 non-genotoxins.
In addition, CA results show statistically significant increases in % tail DNA
after treatment with a model genotoxin. Utilizing the RSMN protocol with tracheal,
vaginal, oral, and corneal tissue models, statistically significant increases in
MNC (0.3 to 1.2%) were observed after treatment with genotoxins. Similarly, CA
results with tracheal, vaginal, oral, and corneal tissue models showed statistically
significant increases in % tail DNA. Hence, the EpiDerm RSMN and CA assays
can be applied to other in vitro human epithelial tissue models to predict genotoxic
effects following real life exposure conditions. Together, RSMM and Comet assays
for skin, tracheal, vaginal, oral, and corneal tissue models will identify a wide spectrum
of genotoxic hazards and will increase confidence in the veracity of in vitro
test results.
344 COLOCALIZATION OF CANNABINOID RECEPTOR
SUBTYPES IN CULTURED RAT CORTICAL
ASTROGLIAL CELLS.
J. A. Torres 1, 2 and A. Shivachar 2 . 1 Environmental Toxicology, Texas Southern
University, Houston, TX and 2 College of Pharmacy and Health Sciences, Texas
Southern University, Houston, TX.
Discovery of the cannabinoid (CB) receptor subtypes opened a door to our understanding
of Marijuana-derived cannabinoids action. So far, there are two wellknown
subtypes of cannabinoid receptors: CB1 expressed in the brain and in other
tissues, and CB2 expressed mainly in immune cells and brain microglia. Although
the expression of CB1 in astrocytes cells is well established, the expression of CB2
receptors remained somewhat controversial. Since previous studies show a partial
antagonism by CB1 selective SR141716A, it’s speculated that a second CB-like receptor
may exist in these cells. Therefore we hypothesize that CB1 and CB2 are coexpressed
in astroglial cells. Highly enriched rat cortical astrocyte cultures, devoid
of microglial cells, were prepared by shaking and low-density plating. The resulting
protoplasmic astrocytes expressed glial fibrillary acidic protein (GFAP) and CB1 receptors.
Similarly, cells that expressed GFAP were also stained positive for CB2 receptor.
The secondary antibody controls showed no detectable fluorescence indicating
signal-specificity. Furthermore double labeling studies using rabbit anti-CB1
and mouse anti-CB2 antibodies indicated that CB1 and CB2 receptors are co-localized
in the same astroglial cell. Western blot analyses corroborated our immunocytochemistry
results and showed distinct immunoreactive bands at the molecular
mass of CB1 (62 KDa) and CB2 (42KDa). Preincubation with CB2 blocking-peptide,
completely abolished the CB2 immunoreactivity. Together, these results indicate
that astroglial cells constitutively coexpress CB1 and CB2 receptors. Thus, by
coexpressing CB1 and CB2 receptor subtypes, astrocytes may represent a unique in
vitro model system in which to study the CB1 and CB2 receptor cross-talk on
neuro- and immuno- modulatory responses of Marijuana-derived neurotoxicants
(Funding support NCRR-RCMI#G12RR03045-17 and NASA JPFP fellowship).
SOT 2011 ANNUAL MEETING 73