The Toxicologist - Society of Toxicology

17 BIOASSAY USE IN IDENTIFYING ENDOCRINE
DISRUPTORS IN THE ENVIRONMENT.
D. Schlenk. Environmental Toxicology Program, University of California Riverside,
Riverside, CA.
Advances in analytical chemistry have led to the detection of multiple chemical
agents from numerous sources in surface waters around the globe. As such, the interactions
of these mixtures continue to be an area of great uncertainty with regard
to the potential risk of these “emerging” chemical agents to aquatic organisms, particularly
extremely sensitive endocrine-specific responses. Using receptor-based
models for estrogenic activity, strong correlations are observed with the occurrence
of known estrogen receptor ligands and biological activity facilitating implementation
of an EEQ approach based upon receptor additivity. However, when in vitro
responses are partnered with in vivo activities, poor correlations are observed particularly
in agricultural locations. Studies in the Central Valley of California using a
combined approach of analytical chemistry with in vitro and in vivo bioassays for
estrogenic activity in a Toxicity Identification Evaluation failed to identify concentrations
of estrogen receptor ligands equivalent to either in vitro or in vivo EEQ values.
Of more than a hundred analytes, only a few pesticides, alklyphenols (APs) and
alkylphenol ethoxylates were detected in samples showing estrogenic activity. When
detected compounds were administered in ambient concentrations as individual
compounds, neither in vitro nor in vivo estrogenic activity was observed. However,
when pesticides were combined with surfactants, similar EEQ values to those observed
in field samples were observed in vivo, but not in vitro. Overall, these results
indicate additivity-based models using in vitro methods may underestimate endocrine
responses of mixtures in vivo, particularly when compounds have multiple
targets along the hypothalamus-gonadal-pituitary axis (eg. APs).
18 DIAGNOSTIC ASSESSMENT OF THE ECOLOGICAL
RISK OF EDCS IN COMPLEX MIXTURES.
G. Ankley. Med., NHEERL, ORD, U.S. EPA, Duluth, MN. Sponsor: L. Gray.
Although it is important to be able to forecast the potential endocrine toxicity of
chemical mixtures that could enter aquatic environments, in many instances there
is a need to determine possible effects of endocrine-active chemicals already present
in complex environmental mixtures (e.g., effluents, sediments), as well as discern
specific chemicals causing observed responses in exposed animals such as invertebrates,
fish and amphibians. This presentation will focus on techniques to: (1) detect
the occurrence of endocrine-active chemicals (e.g., estrogen and androgen receptor
agonists) in complex aqueous samples using mechanism-based responses in
cell lines or whole animals, (2) identify specific chemicals responsible for endocrine
toxicity in complex mixtures through biologically-based fractionation procedures,
and (3) use pathway-specific models to assess the potential for adverse effects of
chemical mixtures on individuals and populations. Case examples illustrating these
approaches will focus on the effects of complex municipal, industrial or agricultural
discharges, and chemical components of these wastes
19 MOLECULAR ENDPOINTS AND MIXTURES OF
ENDOCRINE DISRUPTING CHEMICALS IN FISH.
N. D. Denslow 1 , N. Garcia-Reyero 2 , K. J. Kroll 1 , E. F. Orlando 3 , K. H.
Watanabe 4 , M. S. Sepúlveda 5 , D. L. Villeneuve 6 , E. J. Perkins 7 and G. T.
Ankley 6 . 1 Physiological Sciences, University of Florida, Gainesville, FL, 2 Chemistry,
Jackson State University, Jackson, MS, 3 Animal and Avian Sciences, University of
Maryland, College Park, MD, 4 Division of Environmental and Biomolecular System,
Oregon Health & Science University, Beaverton, OR, 5 Forestry and Natural
Resources, Purdue University, West Lafayette, IN, 6 ORD, NHEERL, Med., U.S.
EPA, Duluth, MN and 7 Environmental Laboratory, U.S. Army Engineer Research
and Development Center, Vicksburg, MS.
Microarray technology is a relatively novel tool in ecotoxicology and is beginning to
be used for exposure and/or hazard characterization for ecological risk assessment.
To develop a basis for this type of analysis, fathead minnows (Pimephales promelas)
were treated with two binary mixtures: 17α-ethinylestradiol and the estrogen antagonist,
ZM 189,154; and 17β-trenbolone (androgen) and the androgen antagonist,
flutamide. Concentrations chosen were anchored to levels which affect cumulative
egg production in 21-day fathead minnow reproduction assays. Unique gene
expression fingerprints were identified in endocrine active tissues including brain,
gonad and liver, which point to adverse outcome pathways. In the presence of excess
antagonist, gene expression was reversed, but only for some genes, suggesting
that the model compounds have additional activities beyond binding to soluble sex
hormone receptors. Using the validated microarrays, effluents from water treatment
plants and animal agricultural areas were tested. Expression profiles were unique at
each of the locations, suggesting that they vary in their composition and complex-
4 SOT 2011 ANNUAL MEETING
ity. Expression changes in key genes such as steroidogenic acute regulatory protein
(StAR) and estrogen receptors, among others illustrate the presence of endocrine
disrupting chemicals. Pathway analysis illustrates toxicity pathways that may be of
importance for complex effluents.
20 EFFECTS OF MIXTURES OF PHTHALATES,
PESTICIDES, AND TCDD ON SEXUAL
DIFFERENTIATON IN RATS: A RISK FRAMEWORK
BASED UPON DISRUPTION OF COMMON
DEVELOPING SYSTEMS.
L. E. Gray. Reproductive Toxicology Branch, U.S. EPA, Research Triangle Park, NC.
Since humans are exposed to more than one chemical at a time, concern has arisen
about the effects of mixtures of chemicals on human reproduction and development.
We are conducting studies to determine the 1) classes of chemicals that disrupt
sexual differentiation via different mechanisms of toxicity, 2) effects associated
with in utero phthalate, pesticide and 2,3,7,8 dioxin (TCDD) exposures, and 3)
how mixtures of phthalates behave when combined with pesticides or TCDD. In
the mixture studies, we have examined the postnatal development of male rat offspring
after in utero exposure to 1) pairs of AR antagonists, 2) pairs of phthalates,
3) phthalates with AR antagonists, 4) five phthalates, 5) seven chemicals (four pesticides
and three phthalates), 6) ten chemicals (four pesticides and six phthalates)
and 7) the potent Ah receptor agonist TCDD plus a phthalate. We also have examined
the effects of some of these chemicals on fetal male rat hormone levels and testicular
gene expression levels. Results of these studies demonstrate that dose addition
models generally provide the most accurate predictions of the observed effects
of these mixtures on male rat sexual differentiation. For example, when ten chemicals
were administered in utero, 100% of the males displayed reproductive tract
malformations as predicted by dose addition models, whereas response addition
models predicted that none of the males would be malformed. Our data indicate
that cumulative risk assessments based upon disruption of common fetal targets or
systems during development more accurately predicts mixture effects than does one
based upon mechanisms of toxicity. This abstract does not necessarily reflect EPA
policy. NTP, NIEHS/EPA Interagency Cooperative Research Agreement HHS Y1-
ES-8014-01; EPA RW75922
21 ESTROGENIC CHEMICAL MIXTURES AND
ENDOGENOUS HORMONES – A ROLE IN BREAST
CANCER DEVELOPMENT?
A. Kortenkamp and E. Silva. Centre for Toxicology, The School of Pharmacy,
University of London, London, United Kingdom. Sponsor: C. Rider.
Elevated levels of endogenous steroidal estrogens have been linked to breast cancer
risk, and there is good evidence that pharmaceutically administered steroids (for example
as hormone replacement therapy) also play a role in the disease. The possible
contribution of synthetic environmental estrogens remains controversial, in view of
their low potency compared with estradiol. To assess whether synthetic estrogens
could conceivably add to the internal estrogenic load of women, we have conducted
in vitro experiments with mixtures of steroidal, plant-derived and synthetic estrogenic
chemicals. The agents were combined at the levels reported to occur in
women’s serum, and assessed in the E-Screen assay. We found that the combined effects
could be predicted accurately by using the concept of concentration addition.
With extreme exposure scenarios similar to those encountered in certain areas of
South Europe, there was a significant added contribution of synthetic estrogens,
over and above that of endogenous estrogens. Persistent organochlorine compounds
such as p,p-DDE, frequently measured in breast cancer epidemiology studies,
contributed little to an overall combination effect. The contribution of more
polar phenolic chemicals, such as those used as UV-filter substances, was more pronounced.
Our studies may help to prioritise chemicals for study in breast cancer
epidemiology.
22 ENVIRONMENTAL OXIDATIVE POLLUTANTS-
INDUCED PULMONARY TOXICITY.
L. L. Mantell 1 and J. T. Zelikoff 2 . 1 St. John’s University College of Pharmacy, Queens,
NY and 2 New York University School of Medicine, Tuxedo, NY.
Environmental oxidant toxicants remain a major public health concern in industrialized
cities throughout the world. Population and epidemiological studies have associated
oxidant air pollutant exposures with morbidity and mortality outcomes,
and underscore the important detrimental effects of these pollutants on the lung. A