The Toxicologist - Society of Toxicology

individuals. Week 6 results were quantifiable for 5/6 males with a mean of 46.28
pg/mL, while 4/6 females had quantifiable results with a mean of 43.75 pg/mL.
The last samples (Week 7 (fasted)) PTH values were quantifiable for all animals
with means of 82.93 pg/mL and 57.38 pg/mL for males and females, respectively.
Results below LLOQ were only obtained on the occasions when animals had access
to food prior to blood sampling (Day 8 and Week 6), with lower means when compared
to Day 1 and Week 7. In conclusion, increased PTH levels following a 7hour
fasting period relative to the unfasted state provided more quantifiable and
consistent values for data interpretation. This should be considered when developing
a study design involving PTH assessment and, when possible, animals should
be fasted prior to sampling.
2377 EFFECTS OF ACUTE AND CHRONIC ALCOHOL
EXPOSURE ON CELL SIGNALING IN BETA-
ENDORPHIN (βEP) NEURONS OF THE
HYPOTHALAMUS.
L. Louis and S. Dipak. Rutgers University, New Brunswick, NJ.
The endogenous opioid system is known to play a role in different aspects of alcohol
addiction. Specifically, β-endorphin (β-EP) has long been suspected of making
a major contribution to the positive reinforcement and motivational properties of
alcohol. Several studies have indicated that repeated administration of alcohol significantly
attenuate β-EP expression in the hypothalamus and other various limbic
areas, while acute administration of ethanol increases β-EP neurotransmission. In
the rodent central nervous system, β-EP neurons are primarily located in the arcuate
nucleus and the periarcuate area of the medial basal hypothalamus. β-EP produced
in neurons in the arcuate area are derived from the pro-opiomelanocortin
(POMC) peptide which also gives rise to several other peptides in these β-EP neurons
including as alpha melanocyte stimulating hormone and adenocorticotropin
hormone. The cellular mechanisms regulating POMC gene expression and subsequent
β-EP expression under acute and chronic alcohol treatments have not been
well characterized. Using immunohistochemical double staining techniques we
demonstrate that changes in β-EP expression are affected by changes in the calcium
dependant signaling molecules pCAMKII and its downstream molecules c-FOS
and p-CREB after both acute and chronic alcohol treatments. Our findings
demonstrate that under acute alcohol treatment β-EP expressing cells increase in
the hypothalamus while β-EP producing neurons also increase the calcium dependant
signaling molecules pCAMKII, pCREB and CFOS. However under chronic
alcohol treatment β-EP expression in the hypothalamus decreases while β-EP neurons
also expressing pCAMKII, pCREB and CFOS decrease in the hypothalamus.
These results give insight into the possible cell signaling molecules involved in the
regulation of β-EP regulation in the arcuate area of the hypothalamus under the influences
of acute and chronic alcohol exposure.
2378 MIXTURES OF ANTI-ANDROGENS AT
ENVIRONMENTALLY RELEVANT CONCENTRATIONS
DISPLAY SIGNIFICANT ADVERSE EFFECTS ON
HORMONES AND GENE EXPRESSION COMPARED TO
SINGLE EXPOSURES AND THE CONTROL.
J. P. Crago 1 and R. Klaper 2 . 1 Biological Sciences, University of Wisconsin-Milwaukee,
Milwaukee, WI and 2 School of Freshwater Sciences, University of Wisconsin-
Milwaukee, Milwaukee, WI. Sponsor: R. Hutz.
Chemicals used by humans can enter into the aquatic environment through a variety
of pathways. In some cases these chemicals have been shown to be potent agonists
or antagonists of reproductive hormones. Most studies that examined exposures
to these compounds look at single exposures and have no adverse effects on
the organism at concentrations commonly found in the aquatic environment.
There have been few studies that examine the potential additive effects of mixtures
at environmentally relevant concentrations, and specifically effects from compounds
with the same adverse outcome but with different modes of action. The
goal of this study was to assess the effects of anti-androgenic mixtures versus individual
anti-androgens on fish at concentrations commonly found in the aquatic environment.
In this study male fathead minnows (Pimphales promelas) were exposed
to the pesticide linuron (1ppb), di-ethylhexyl phthalate (DEHP) (12ppb), and in a
mixture (linuron and DEHP) for a 28-day period. Gene expression profiles of the
individual chemicals and chemical mixture were determined using cDNA microarrays
followed by qRT-PCR were performed, and plasma testosterone concentrations
and gonad-somatic index (GSI) were measured. The results from this study
show that adverse effects were chemical dependent. Fish exposed to linuron showed
a significantly lower GSI index as compared to control fish, DEHP treated fish, and
fish treated to the mixture. Testosterone levels of fish treated with the mixture were
significantly lower then that of the control, linuron and DEHP exposed fish. Genes
involved in androgen production, such as cytochrome P450 17 and 11a did not differ
amongst treatments, but alternate pathways were modified. These results indicate
that compounds shown to have anti-androgenic effects can have different adverse
outcomes when combined in a mixture.
2379 COMPARATIVE EVALUATION OF THE EFFECTS OF
POLYBROMINATED DIPHENYLETHER (DE-71) AND
ETHYLENE-BIS-TETRABROMOPHTHALIMDE (EBTBP)
ON THYROID HORMONE-MEDIATED ANURAN
METAMORPHOSIS.
D. Fort 1 , R. Rogers 1 , P. Guiney 2 and J. Weeks 2 . 1 Fort Environmental Labs,
Stillwater, OK and 2 S.C. Johnson & Sons, Racine, WI.
An amphibian metamorphosis assay has been proposed by the US EPA in their Tier
1 endocrine disruption screening program (EDSP). Previous studies suggested that
the brominated flame retardant mixture, DE-71 impacts the function of the thyroid
axis during metamorphosis in Xenopus laevis. Since brominated diphenylether
(BDE) flame retardants are currently being replaced by a variety of alternative compounds,
we compared the effects of a potential BDE replacement, ethylene-bistetrabromophthalimide
(EBTBP), and DE-71 on thyroid-mediated metamorphosis.
Tier 1 EDSP amphibian metamorphosis assays were conducted exposing stage
51 X. laevis to 0.0, 0.4, 0.9, 2.1, and 3.8 μg/L DE-71 and 0.0, 4.4, 8.8, 17.5, and
35 μg/L EBTBP from stage 51 for 21-d. Results from studies with DE-71 indicated
that the rate of metamorphosis decreased with increasing concentration with no
marked effect on thyroid gland histology. EBTBP exposure did not alter the rate of
metamorphosis or normal histology of the thyroid gland. Serum thyroxine (T4)
and triiodothyronine (T3), thyroid gland T4, and thyroid hormone receptor beta
(TRβ) expression in tail tissue were measured in stage matched larvae at the conclusion
of exposure. Significantly lower levels of both T4 and T3 were measured in
serum from DE-71 relative to controls, whereas no effect was observed in the
EBTBP-treated larvae. Neither flame retardant significantly altered thyroid gland
T4 level, nor altered the expression of TRβ. Thus, although DE-71 slowed metamorphosis
by altering peripheral mechanisms of thyroid hormone homeostasis,
EBTBP did not alter thyroid-mediated metamorphosis in X. laevis at environmentally
relevant concentrations.
2380 OECD VALIDATION STUDY ON THE
TRANSFERABILITY, INTRA- AND INTER-LABORATORY
REPRODUCIBILITY OF THE ZEBRAFISH EMBRYO
TOXICITY TEST.
F. Busquet 1 , S. Belanger 2 , T. Braunbeck 3 , G. Carr 2 , M. Halder 1 , A. Kleensang 1 ,
A. Lillicrap 4 , S. Walter-Rohde 5 and P. Amcoff 6 . 1 JRC IHCP ECVAM, European
Commission, Ispra, Italy, 2 Procter & Gamble, Cincinnati, OH, 3 University of
Heidelberg, Heidelberg, Germany, 4 NIVA, Oslo, Norway, 5 UBA, Dessau-Rosslau,
Germany and 6 Environment, Health, and Safety Division, OECD, Paris, France.
Sponsor: M. Embry.
The OECD Acute Fish Toxicity Test Guideline (TG 203) is an integral component
in the environmental safety assessment of industrial chemicals, agrochemicals,
pharmaceuticals, feed stuffs, and biocides. One of the most promising alternative
approaches to the acute fish toxicity test is based on the use of zebrafish embryos. In
2005, the German Federal Environment Agency submitted the draft TG on “Fish
embryo toxicity (FET) test” to the OECD Test Guideline Program and a supportive
Background Paper. Subsequently, OECD established the ad hoc Expert Group
on the Fish Embryo Toxicity Test. Based on the outcome of expert meetings,
OECD decided to perform a validation study (coordinated by ECVAM and steered
by a validation management group). The validation study aims to evaluate the
transferability, and the intra/interlaboratory reproducibility of the Zebrafish FET
(ZFET). Newly fertilised zebrafish eggs are exposed for up to 96h to chemicals. 4
apical endpoints are recorded daily as indicators of acute lethality in fish: coagulation
of the egg, lack of somite formation, non-detachment of the tail bud from the
yolk sac and lack of heart-beat. LC50 values are calculated for 48h and 96h exposure.
7 chemicals are tested at 5 different concentrations in 3 independent runs in
at least 4 laboratories with appropriate controls. Stock solutions and test concentrations
of 1 laboratory are confirmed. The poster will give an overview on the validation
study design, the results, next steps and the correlation of the ZFET with acute
fish LC50 data.
“Disclaimer: The opinions expressed and the arguments employed herein are those
of the authors and do not necessarily reflect the official views of the OECD or of
the governments of its member countries’”
SOT 2011 ANNUAL MEETING 511