The Toxicologist - Society of Toxicology

induce mutagenic effects in the germ cells of male mice. Male Big Blue transgenic
mice were administered 1.4 or 7.0 mM of AA or GA in the drinking water for up
to 4 weeks. Testicular cII mutant frequency (MF) was determined 3 weeks after the
last treatment, and the types of the mutations in the cII gene were analyzed by
DNA sequencing. The testes cII MFs in mice treated with either the low or high exposure
concentrations of AA and GA were increased significantly. There was no significant
difference in the cII MFs between AA and GA at the low exposure concentration.
The mutation spectra in mice treated with AA (1.4 mM) or GA (both 1.4
and 7.0 mM) differed significantly from those of controls, but there were no significant
differences in mutation patterns between AA and GA treatments.
Comparison of the mutation spectra between testes and livers showed that the spectra
differed significantly between the two tissues following treatment with AA or
GA, whereas the mutation spectra in the two tissues from control mice were similar.
These results suggest that AA possesses mutagenic effects on testes by virtue of
its metabolism to GA, possibly targeting spermatogonial stem cells, but possibly via
different pathways when compared mutations in liver.
69 1, 3-DINITROBENZENE: REVISITING THE
POSTULATED MODE OF ACTION FOR ITS
TESTICULAR TOXICITY.
S. Ludwig, H. Tinwell, D. Rouquié and R. Bars. Research Toxicology, Bayer SAS,
Sophia Antipolis Cedex, France.
Many nitroaromatic compounds used to manufacture polymers, pesticides and
dyes are known testicular toxicants. One such compound, 1,3-dinitrobenzene
(DNB), causes lesions in the rat testis, decreased sperm number, and abnormal
sperm morphology and motility. As ultrastructural changes were found in the
Sertoli cells before changes in germ cell morphology, the Sertoli cell has been postulated
as the primary target for the toxic action of DNB. We have conducted studies
to better understand the pathogenesis of the testicular effects produced by
DNB, and to investigate its mode of action. Rats were orally exposed to 0.1-4 mg
DNB/kg/day for 4 days, and the resultant effects were investigated using standard
parameters (organ weight, histopathology, testosterone measurements) and molecular
tools (microarray analyses and qPCR). Testosterone concentrations were not
affected at any dose level, but marked histopathological lesions were recorded in
the testes at 4mg/kg/day. Global transcriptomic analysis of rat testes revealed hundreds
of genes differentially expressed following exposure to 4mg/kg/day. The
major biological processes affected were related to cell cycle and cell death. In particular,
we identified an alteration in the expression of genes associated with cell
cycle progression (“mitotic roles of polo-like kinase”). Our molecular data (microarray
and qPCR), can be correlated with our histopathological data and also
with earlier publications (particularly for apoptosis). These observations have been
further investigated in a second study, where rats were sacrificed at 8, 24, 48 and 72
hours after a single oral dose of 4mg/kg DNB. The preliminary molecular data indicate
alterations of common biological processes induced by DNB after a single or
after 4 doses. Surprisingly, in contrast to the 4 day data, plasma testosterone was reduced
48h after a single dose, which was associated with a down-regulation of
genes involved in steroidogenesis. As Sertoli cells and not Leydig cells have been
previously proposed as the primary target for DNB, this observation warrants further
investigation.
70 ITCH PROMOTES MEHP-INDUCED GERM CELL
APOPTOSIS.
J. L. Dwyer 1 , Y. Lin 2 , P. Yao 2 and J. H. Richburg 2, 1 . 1 Institute for Cellular and
Molecular Biology, University of Texas at Austin, Austin, TX and 2 Center for
Molecular and Cellular Toxicology, University of Texas at Austin, Austin, TX.
Spermatogenesis is an intricate process that depends on Sertoli cell (SC) support for
germ cell (GC) development. Exposure to various environmental toxicants can injure
SCs and disrupt this process. Di-ethylhexyl phthalate (DEHP) is used to impart
flexibility during the manufacturing of plastic products, but it leaches out and
is often found throughout the environment. Once in the body, DEHP is rapidly
hydrolyzed to mono-ethylhexyl phthalate (MEHP), a well-described SC toxicant.
Previous studies from our lab revealed that MEHP causes an increase in the expression
of SC-derived FasL, a ligand belonging to the Tumor Necrosis Family of proteins
involved in triggering apoptosis. It is proposed that the increased levels of FasL
after MEHP treatment allows for activation of its receptor on GCs, Fas, triggering
a cascade of caspase cleavage and ultimately apoptosis. The cellular FLICE-Like
Inhibitory Protein (c-FLIP) is also present in GCs, and has been shown to inhibit
apoptosis by preventing caspase-8 cleavage. An important negative regulator of c-
FLIP is the E3 ubiquitin ligase Itch, which targets c-FLIP for proteasomal degradation.
In order to further study the regulation of c-FLIP and its influence on
MEHP-induced GC apoptosis, Itch knockout mice were evaluated. Histological
14 SOT 2011 ANNUAL MEETING
examination of peripubertal mice revealed that they are structurally similar to their
wild type C57/BL6J counterparts. However, an analysis of the number of mature
spermatids in the testis of Itch knockout mice revealed a significant decrease in their
production as compared to C57 mice. Following MEHP exposure, C57 mice show
an increase in GC apoptosis, while the Itch knockout mice appear to be partially
protected from injury. Protein analysis revealed that Itch mice have higher levels of
c-FLIP, and these levels slightly increase following MEHP exposure. Taken together,
these data suggest that c-FLIP and Itch may play an important role during
GC development and in modulating the response to MEHP exposure.
71 COMPARISON OF THE EMBRYONIC STEM CELL TEST,
THE WHOLE EMBRYO CULTURE, AND THE
ZEBRAFISH EMBRYO TEST AS ALTERNATIVE
METHODS FOR DEVELOPMENTAL TOXICITY
TESTING OF TRIAZOLES.
E. de Jong 1, 2 , M. Barenys 3 , S. Hermsen 2, 4 , A. Verhoef 2 and A. H. Piersma 1, 2 .
1 Institute for Risk Assessment Sciences, Utrecht University, Utrecht, Netherlands,
2 National Institute for Public Health and the Environment, Bilthoven, Netherlands,
3 Toxicology Unit, Public Health Department, School of Pharmacy, University of
Barcelona, Barcelona, Spain and 4 Department of Health Risk Analysis and Toxicology,
NUTRIM, University of Maastricht, Maastricht, Netherlands. Sponsor: H. van
Loveren.
The high experimental animal use in developmental toxicity testing has stimulated
the search for less animal intensive alternatives. Three widely studied alternative assays
are the mouse embryonic stem cell test (EST), the zebrafish embryotoxicity test
(ZET) and the postimplantation rat whole embryo culture (WEC). The goal of this
study was to determine their efficacy in predicting the relative developmental toxicity
of six triazole compounds, flusilazole, hexaconazole, cyproconazole, triadimefon,
myclobutanil and triticonazole. Triazoles are antifungal agents used in agriculture
and medicine. They are known to induce craniofacial and limb abnormalities
in rodents. We determined the correlation between the effects of the compounds in
the alternative assays and their in vivo developmental toxicity. Both the ZET and
the WEC showed a general pattern of teratogenic effects after exposure to the triazoles,
mainly consisting of effects on the branchial arches in the WEC and head and
heart anomalies in the ZET, which corresponds to the type of abnormalities found
in vivo. In the EST all triazole compounds inhibited cardiomyocyte differentiation.
In the alternative assays, effects observed were concentration-dependent, allowing
potency ranking and comparison with in vivo ranking. Overall, the ZET gave the
best prediction of the relative developmental toxicity of the tested compounds, followed
by EST and WEC, respectively. These results may be explained by differences
in compound kinetics, developmental stage and complexity between alternative
assays.
72 PREDICTING DEVELOPMENTAL TOXICITY OF
TOXCAST PHASE I CHEMICALS USING HUMAN
EMBRYONIC STEM CELLS AND METABOLOMICS.
N. C. Kleinstreuer 2 , P. R. West 1 , A. M. Weir-Hauptman 1 , A. M. Smith 1 , T. B.
Knudsen 2 , E. L. Donley 1 and G. G. Cezar 1, 3 . 1 Stemina Biomarker Discovery, Inc.,
Madison, WI, 2 ORD/NCCT, U.S. EPA, Durham, NC and 3 University of Wisconsin-
Madison, Madison, WI.
EPA’s ToxRefDB contains prenatal guideline study data from rats and rabbits for
over 240 chemicals that overlap with the ToxCast in vitro high throughput screening
project. A subset of these compounds were tested in Stemina Biomarker
Discovery’s developmental toxicity platform, an in vitro method combining human
embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental
toxicity. The purpose of this pilot study was to perform LC-MS based nontargeted
metabolomic analysis on the supernatant of hES cell cultures dosed with
blinded EPA test chemicals to identify human metabolites subject to chemically induced
alterations and biochemical signatures which may be indicative of potential
human developmental toxicity. Significant fold changes in endogenous human
metabolites were detected for 83 annotated mass features in response to the subset
of ToxCast chemicals. The annotations were mapped to specific human metabolic
pathways with nicotinate and nicotinamide metabolism, pantothenate and CoA
biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways
most affected. Stemina’s predictive DevTox® model, trained on 22 pharmaceutical
agents of known teratogenicity and differing potency, was applied to the
blinded EPA test compound data to test predictivity for mammalian in vivo data.
The model correctly predicted teratogenicity for eight of eleven compounds and an