The Toxicologist - Society of Toxicology

317 THE HUNT FOR MISLEADING POSITIVES: CAN NON-
GENOTOXIC REGULATION OF GADD45A INTERFERE
WITH GREENSCREEN HC?
C. Topham 2 and R. Walmsley 1, 2 . 1 Life Sciences, University of Manchester,
Manchester, United Kingdom and 2 Gentronix Ltd., Manchester, United Kingdom.
The GADD45a-GFP ‘GreenScreen HC’ genotoxicity assay (GSHC) has high
specificity (95%) and sensitivity (87%) in the prediction genotoxic carcinogenicity.
Accordingly, the frequency of misleading positive GSHC results is low when compared
with other mammalian in vitro genotoxicity assays. The GSHC assay employs
the response of the DNA damage-inducible gene GADD45a as a marker of genotoxic
stress, using a GADD45a-GFP reporter containing key regulatory elements.
In order to rigorously assess the specificity of the GSHC assay in light of the current
knowledge of the regulation of GADD45a, chemicals with the potential to induce
a GADD45a response in the absence of genotoxic stress were sought in order to expose
the assay to challenging new biological mechanisms. Examples of such chemicals
included activators of signalling pathways known either to positively regulate
GADD45a, such as p53. or inhibitor negative regulators of GADD45a, such as
NF-κB and Bcl-2 family members. A third group included chemicals known to activate
apoptosis by various cytotoxic stress signalling pathways. Stimulation of an
apoptotic response by these chemicals was assessed by monitoring of levels of caspase
3/7 activation, annexin V binding and DNA fragmentation via TUNEL.
Six proprietary and 17 non-proprietary compounds were assessed with GSHC.
Published comparative genotoxicity data for these chemicals were either sparse or
non-existent, so genotoxicity data were generated in this study using the in vitro
micronucleus test (MNT) and the comet assay, as well as in silico alerts from the
DEREK for Windows SAR software. Chemicals with positive GSHC data were
also positive in either the MNT or Comet assay. These data confirm validation
studies which demonstrate that compounds producing a unique positive response
in the GADD45α-GFP assay are rare, even amongst a group of compounds inducing
apoptosis through non-genotoxic mechanisms, which might be considered high
risk for the generation of ‘false’ positives in this assay.
318 COMPARATIVE DNA DAMAGE AND CHRONIC LIVER
TOXICITY OF BENZO[A]PYRENE IN TWO
POPULATIONS OF THE ATLANTIC KILLIFISH
(FUNDULUS HETEROCLITUS) WITH DIFFERENT
EXPOSURE HISTORIES.
R. Di Giulio 1 , D. Jung 2, 1 and L. Wills 3, 1 . 1 Nicholas School of the Environment,
Duke University, Durham, NC, 2 Department of Physiology, Dartmouth Medical
School, Lebanon, NH and 3 Medical University of South Carolina, Charleston, SC.
The Atlantic Wood Industries Superfund site on the Elizabeth River (ER) in
Portsmouth, VA is contaminated with polycyclic aromatic hydrocarbons (PAH) derived
from creosote. Embryos and larvae of ER killifish (Fundulus heteroclitus) are
refractory to the induction of enzymes regulated by the aryl hydrocarbon receptor
(AHR) including cytochrome P4501A (CYP1A) and are resistant to PAH-induced
lethality and teratogenicity. However, adult ER killifish collected from this site
show a greater prevalence of hepatic and pancreatic tumors compared to those from
reference sites. This study examined the relative sensitivities of laboratory-reared
offspring from the two populations to DNA damage and chronic liver exposure following
exposures to benzo[a]pyrene (BaP). Larvae from the ER and a reference site
on King’s Creek (KC) were subjected to two 24 hr aqueous exposures to BaP (0-400
μg/L). At various time points, larvae were analyzed for CYP1A activity, BaP concentrations,
nuclear and mitochondrial DNA damage, and liver pathology. CYP1A
was induced by BaP in KC but not ER larvae, and KC larvae demonstrated a
greater reduction in whole body concentrations of BaP over time. Mitochondrial
and nuclear DNA lesion frequency increased significantly in BaP exposed KC larvae,
but not in those from the ER. Nine months post-exposure, KC juveniles exhibited
significantly more hepatic foci of cellular alteration (FCA), and only KC juveniles
developed hepatocellular carcinomas. Thus, in addition to acquiring the
heritable resistance to the acute teratogenic effects of PAHs, ER fish appear to have
concomitantly developed resistance to chronic effects, including cancer.
319 DNA DAMAGE CAUSED BY TRICYCLIC
ANTIDEPRESSANTS.
E. A. Korobkova and A. K. Williams. Science, John Jay College of Criminal Justice,
New York, NY. Sponsor: B. Shane.
Tricyclic antidepressants were discovered in the 1950s and were used for many years
in the treatment of mood disorders. The antidepressants and their metabolites can
be very genotoxic in living cells. The planar structures of the drugs can insert be-
68 SOT 2011 ANNUAL MEETING
tween DNA bases forming stacking complexes. The metabolism of antidepressants
may lead to the DNA bases modifications or DNA strand breaks. We studied the
effect on DNA of three tricyclic anatidepressants, imipramine, amitriptyline, and
opipramol. We focused on the drug-DNA binding and DNA damage aided by peroxidase
catalysis. As a model of peroxidase we used HRP (Horseradish peroxidase).
We performed ethidium bromide fluorescence quenching experiments and determined
drug concentrations at 50% fluorescence quenching, C50. The value of C50
ranged from 1 mM for opipramol to 5 mM for imipramine and amitriptyline.
Agarose gel electrophoresis studies showed that DNA disappears in the reaction
mixtures containing imipramine and HRP/H2O2. Phenol:chlorophorm:iso-amyl
alcohol extraction from the mixtures containing DNA and imipramine in the presence
of HRP/H2O2 indicated that DNA degrades in the reaction. UV-Vis studies
showed that both imipramine and opipramol are the substrates for HRP. At pH 7,
reaction between HRP and excess of H2O2 and imipramine led to the formation of
a broad spectrum with a peak at 522 nm. The intensity of the spectrum increased
with time. The position of the maximum shifted to the longer wavelengths as the
pH decreased reaching 650 nm at pH 2. These spectra are associates with
imipramine radical. GC-MS analysis of the brown precipitate produced in the mixture
of imiprmaine and HRP/H2O2 indicated the dealkylation process and the formation
of iminodibenzyl. Thus all three antidepressants bind DNA possibly by intercalation,
opipramol exhibiting a greater affinity compared to imipramine and
amitriptyline. DNA degrades in the presence of imipramine and HRP/H2O2 at
the drug concentration of 2 μM. The damage to DNA is caused by imipramine reactive
intermediate.
320 EVALUATION OF DIRECT AND INDIRECT DNA
DAMAGE INDUCED BY OCHRATOXIN A IN CHO AND
TK6 CELLS USING THE COMET ASSAY.
R. Ali 1, 2 , J. G. Shaddock 2 , W. Ding 2 , J. A. Bhalli 2 , Q. M. Khan 1 and R. H.
Heflich 2 . 1 Environmental Biotechnology Division, National Institute for
Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan and 2 Division
of Genetic and Molecular Toxicology, National Center for Toxicological Research /U.S.
FDA, Jefferson, AR. Sponsor: M. Moore.
The fungal toxin, Ochratoxin A (OTA), a common contaminant in human food
and animal feed, has been reported to be mutagenic, clastogenic, and aneugenic in
mammalian cells. The present study assessed the direct and indirect (oxidative)
DNA damage induced by OTA in two mammalian cell lines, CHO-K1-BH4
Chinese hamster ovary cells and TK6 human lymphoblastoid cells. DNA damage
was evaluated using the Comet assay, with and without digestion by formamidopyrimidine-DNA
glycosylase (fpg) to cleave at oxidized purine adducts. The cells were
treated with 5, 10, 20, 30, 40 and 50 μM OTA for 4 hr; the treatments resulted in
>70% viability, as estimated by Trypan Blue dye exclusion. The 4 hr exposure to
OTA did not induce direct DNA damage [as measured by Tail Intensity % (TI%)]
in CHO cells, but it did induce a significant dose-responsive increase in direct
DNA damage in TK6 cells. In addition, OTA exposure resulted in a dose-responsive
induction of fpg-sensitive sites in both cell lines, indicative of the induction of
oxidative DNA damage. Significant induction of fpg-sensitive sites was detected at
OTA concentrations of >20 μM for CHO cells and >30 μM for TK6 cells. The results
of this study suggest that oxidative DNA damage is involved in the genotoxicity
produced by OTA in mammalian cells.
321 THE IN VIVO RAT COMET ASSAY OF
DIETHYLNITROSAMINE: RELEVANCE BETWEEN
CARCINOGENESIS AND DNA DAMAGE IN THE
TARGET ORGANS.
Y. Uematsu, C. Hirogaki, H. Adachi, K. Kijima, T. Yamada, Y. Michimae, I.
Matsumoto, T. Koujitani, H. Oishi, H. Funabashi and T. Seki. Safety Research
Laboratory, Dainippon Sumitomo Pharmacology Co., Ltd., Suita-city, Osaka, Japan.
Diethylnitrosamine (DEN) is a DNA alkylating N-nitroso compound which induces
tumors in the rat liver, esophagus and kidney. Especially in the kidney, tumors
were reported to be induced specifically in the cortical epithelium. In this
study, to understand the relationship between carcinogenesis and the induction of
DNA damage, we investigated the DNA damage of the target organs using the single
cell gel electrophoresis assay (Comet assay). DEN was administered orally once
a day 3 times to male 8 week-old SD rats. Based on the results of the preliminary
study, we set 200 mg/kg as the highest dose group. In the Comet assay, rats were euthanized
3 hours after the last dosing. The liver, kidney cortex, kidney medulla,
grandular stomach and small intestine were obtained and isolated cells of each
organ were analyzed in the Comet assay. The % tail DNA of comet images was analyzed
for 100 nuclei per organ. A small portion of each organ was fixed in 10%
phosphate buffered formalin, embedded in paraffin, and sectioned. The sections
were stained with hematoxylin and eosin and subjected to histopathological examination.
The % tail DNA in the liver and kidney cortex peaked in 50 mg/kg/day
group and decreased gradually in higher dose groups. On the other hand, the % tail