The Toxicologist - Society of Toxicology

clastogenic effects, a number of samples did affect hematological parameters, providing
empirical evidence of target tissue involvement. More limited studies using
in vitro protocols produced similar results. These in vitro results suggest that the
negative results in vivo are not a consequence of the organ being evaluated but an
indication that the substances being tested lack in vivo clastogenic potential. It was
concluded that petroleum-derived materials are unlikely to produce clastogenic effects
in bone marrow assays regardless of test or sample conditions.
1123 DNA DAMAGING AND CLASTOGENIC EFFECTS OF
PHYTOCHEMICALS PARTIALLY ISOLATED FROM
CRUDE EXTRACT OF GLINUS LOTOIDES.
J. D. Kimmo 1, 2 , E. Engidawork 2 , T. Leta 3 , U. Goranson 3 and B. Hellman 1 .
1
Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, Uppsala,
Uppland, Sweden, 2 Pharmacology, School of Pharmacy, Addis Ababa University,
Addis Ababa, A. A., Ethiopia and 3 Medicinal Chemistry, Division of Pharmacognosy,
Uppsala University, Uppsala, Uppland, Sweden. Sponsor: M. Stigson.
A methanolic extract of Glinus lotoides, a medicinal plant used in Africa and Asia
for various therapeutic purposes, was recently shown to cause DNA damage in
vitro. To further explore the potential genotoxicity of this plant, fractionation of the
crude extract was performed using reverse solid-phase extraction and a stepwise gradient
elution of methanol in water. Four fractions were collected and subsequently
analyzed for their DNA damaging and cytogenetic effects in mouse lymphoma cells
using an alkaline version of the comet assay and a cytokinesis-blocked version of the
micronucleus assay, respectively. To identify potential genotoxic and non-genotoxic
principles, each fraction was subjected to LC-MS and LC-MS/MS analyses, followed
by database and literature search. While fractions containing flavonoids and
oleanane-type saponins or thier mixture produced neither DNA nor chromosomal
damage, those containing hopane-type saponins exhibited a DNA damaging effect
leaving the chromosomes unaffected. The fact that the DNA was damaged but the
chromosomes remained intact suggests that the hopane-type saponin-induced
DNA damage is efficiently repaired. This study presents evidence that hopane-type
of saponins are endowed with a DNA damaging ability and could be cited as a culprit
for the previously reported genotoxicity of the crude extract.
1124 DISCRIMINATION OF CLASTOGENIC AND
ANEUGENIC COMPOUNDS IN HUMAN
LYMPHOCYTES BY IMMUNOFLOURESCENT
TECHNIQUES IN THE CB MICRONUCLEUS TEST.
A. H. Poth and S. Bohnenberger. Harlan Cytotest Cell Research, Rossdorf, Germany.
Sponsor: R. Fautz.
The micronucleus assay in human lymphocytes was developed as a short term
screening test for the detection of both clastogenic and aneugenic chemicals. For
human lymphocytes it is recommended to score micronuclei by the cytokinesis
block (CB) method using cytochalasin B. The original method developed by
Fenech and Morley, 1985, focusses exclusively on binucleated cells. However, recent
studies suggest that micronuclei in mononucleated cells could provide complementary
information. Results obtained with aneugenic compounds show a dosedependent
increase of micronuclei in mononucleated cells. At present, the
underlying mechanism has not been clearly indentified. In order to obtain more information
two immunofluorescence techniques were employed involving CREST
analysis for detection of kinetochore proteins and staining of phosphorylated histone
H2AX (yH2AX). The CREST analysis reveals whether micronuclei in
mononucleated cells contain chromosomal fragments or whole chromosomes. The
yH2AX staining detects phosphorylation of histone H2AX at serine 139 rapidly occurring
at sites flanking DNA double strand breaks. Our results suggest that micronuclei
in mononucleated cells can be used to investigate the aneugenic activity
of chemicals in a fast and easy way, and can be included in the CB assay with
human lymphocytes.
1125 GADD45 INDUCTION IN THE GREENSCREEN HC
INDICATOR ASSAY DOES NOT OCCUR
INDEPENDENTLY OF CYTOTOXICITY.
A. J. Olaharski 1 , S. Albertini 2 , L. Mueller 2 , A. Zeller 2 , M. Struwe 2 , E. Gocke 2
and K. Kolaja 1 . 1 Nonclinical Safety, Hoffmann-La Roche, Nutley, NJ and
2
Nonclinical Safety, Hoffman-La Roche, Basel, Switzerland.
Mammalian chromosomal integrity assays are influenced by cytotoxicity, a phenomenon
which impacts data interpretation, assay specificity and regulatory testing
guidelines. Concordance of the GADD45α GreenScreen HC indicator assay to established
in vitro and in vivo genetic toxicological assays has previously been described,
yet a detailed description in the manner by which cytotoxicity influences
its performance has not. Here we present a post-hoc analysis of a previously tested
set of 91 proprietary and non-proprietary compounds investigating the interaction
between GADD45α induction and cytotoxicity as well how varying assay threshold
criteria influences concordance. GADD45α induction strongly correlates with cytotoxicity
for the majority (72%) of compounds causing a positive GADD45α response.
Furthermore, modification of the GADD45α induction and cytotoxicity
threshold criteria resulted in an increased assay sensitivity (from 30 to 68%) and
concordance (from 55 to 68%), though a concomitant decrease in specificity is observed
(from 97 to 68%). Additionally, an analysis of Roche proprietary compounds
tested in the micronucleus test demonstrates micronucleus induction is also
influenced by cytotoxicity, albeit in an attenuated manner if compared to the
GreenScreen HC indicator assay. We conclude that GADD45α induction in the
GreenScreen HC indicator assay is influenced by cytotoxicity and that assay performance
can be improved if different assay criteria are implemented.
1126 SIMULATED SPACEFLIGHT INCREASES CHROMATE-
INDUCED GENOTOXICITY.
J. P. Wise 1, 2 , S. Wise 1, 2, 3 , J. Wise 1, 2 , J. McKay 1, 2 , M. Browne 4 , K. Joyce 1, 2 ,
M. Braun 1, 2 , C. Wise 1, 2 , R. Duffy 1, 2 , E. Estelle 4 , J. Brown 4 , C. Gianios 1, 2 , M.
Mason 4 , T. Shehata 5 , D. Hammond 6 and J. P. Wise 1, 2, 3 . 1 Wise Laboratoey of
Environmental and Genetic Toxicology, University of Southern Maine, Portland, ME,
2
Maine Center of Toxicology and Environmental Health, University of Southern
Maine, Portland, ME, 3 Department of Applied Medical Sciences, University of
Southern Maine, Portland, ME, 4 Department of Biological and Chemical
Engineering, University of Maine, Orono, ME, 5 Maine Space Grant Consortium,
Portland, ME and 6 NASA Johnson Space Center, Houston, TX.
CONSTELLATION is NASA’s next mission to explore the surface of the moon.
Under this program, NASA plans to send manned missions back to the Moon by
the year 2020. Thus it is essential to determine the effects of altered gravity on cellular
morphology and metabolism to make long-term space travel safer for the astronauts
involved. We hypothesized that altered gravity changes normal cell function
resulting in an increase in chemical-induced genotoxicity. We conducted our
experiments aboard NASA’s Weightless Wonder, a C9-B plane that simulates environments
of microgravity (0 g) and hypergravity (2 g). We exposed human lung fibroblast
cells to sodium chromate during fight along with a parallel experiment
conducted simultaneously on the ground. We found that, after a 4 h exposure, altered
gravity increased the amount of chromosomal damage. We further found that
altered gravity decreased the amount of chromium ion uptake indicating that differential
uptake was not the underlying mechanism. These data indicate that altered
gravity can significantly increase the potency of genotoxic agents, which suggests
that risks of exposure to astronauts in space are greater than on earth. Future research
is aimed at understanding the relative contributions of hyper- and microgravity
to these effects and investigating the underlying mechanisms. This project
was supported by the Reduced Gravity Flight Opportunities Program at the
Johnson Space Center and the Maine Space Grant Consortium.
1127 CHEMICAL EXPOSURE AND THE GENERATION OF
COPY NUMBER VARIANTS (CNVS).
J. L. Freeman and S. Peterson. Health Sciences, Purdue University, West Lafayette, IN.
Until recently, single nucleotide polymorphisms (SNPs) were thought to be the predominant
form of genomic variation and to account for much of the normal phenotypic
variation. Recent developments and applications of genome-wide technologies
have lead to the discovery of thousands of copy number variants (CNVs:
defined as a gain or loss of DNA sequence measuring 1 kilobase and larger in size)
in the human genome. Human genomic copy number variation has been studied
for a number of years, but it was assumed that CNVs were few in number, had a
relatively limited impact on the total amount of genetic variation, and were mainly
associated with highly penetrant disease phenotypes. CNVs that did not result in
early-onset, highly penetrant genomic disorders were presumed to be neutral in
function, but the role of CNVs in complex diseases is now becoming increasingly
more appreciated. At this time, the factors and mechanisms that generate spontaneous
CNVs are not well defined, including the potential role of exposure to chemical
stressors. To investigate the role of chemical exposure in the generation of
CNVs, an assay utilizing the zebrafish model system was developed. Use of the zebrafish
model system in this assay presents many strengths including the fact that
the zebrafish genome, similar to the human genome, is also copy number variable.
To elucidate the role of chemical exposure in the generation of spontaneous CNVs,
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