Professional Documents
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doi:10.1093/toxsci/kfq329
Advance Access publication October 25, 2010
1
To whom correspondence should be addressed at Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building,
Midland, MI 48674. Fax: (989) 638-9863. E-mail: mmarty@dow.com.
The endocrine system is one of the body’s major toxicity, followed by coverage of key historical events in
homeostatic control systems whose aim is to maintain normal basic science, and then bring the two together to discuss
functions and development in the face of a constantly changing implications of the science on testing both now and for the
environment. Working in tandem with the nervous system, future.
which mainly is responsible for rapid and immediate responses,
the approximately 30 different glands comprising the endocrine
system tend to act in a slower and more sustained manner to EARLY HISTORY OF TOXICITY TESTING FOR
regulate processes as diverse as the female reproductive cycle, ENDOCRINE-MEDIATED EFFECTS
bone growth, cell proliferation, and psychosocial behavior.
Multiple endocrine glands also work in concert with one A casual observer might easily be led to believe that the
another to form complex feedback loops, which tightly regulate ability of exogenous substances to interfere with the endocrine
critical physiological processes. system was unknown until relatively recently, but in fact, this is
Like all homeostatic control systems, the capacity to far from true. Even the ancients were familiar with the actions
maintain physiological parameters within normal bounds is of various herbal preparations to modulate what we now know
finite, and when this capacity is exceeded by chemical to be hormonally regulated processes. For centuries, farmers
exposures, drugs, or environmental stressors, adverse have observed reproductive problems in female sheep and
consequences can ensue. In the mid-1990’s, concerns about cows grazing on pastures rich in certain clover species, which
the potential for environmental chemicals, drugs, and other later were found to contain estrogenic compounds such as
stressors to alter endocrine physiology rapidly mounted and coumestrol (Adams, 1995). As early as the 1930’s, the ability
received a great deal of attention within the toxicology of both natural and synthetic chemicals to interact with
community as well as in the public media. These circumstances endogenous hormone receptors was already well established
spurred a flurry of research on mechanisms of toxicity, new (Schueler, 1946; Sluczewski and Roth, 1948; Walker and
questions about how to deal with cumulative exposures to Janney, 1930).
endocrine-active compounds (EACs) from man-made, dietary, Starting around the 1940’s, steroidal compounds began to be
and endogenous sources, and the creation of major chemical used in the livestock industry to modulate reproductive cycles
screening and testing programs focused on endocrine-mediated and to enhance rate and efficiency of body weight gain, while
modes of toxic action. compounds such as ethinyl estradiol, mestranol, norethynodrel,
In this review, we will trace the path of knowledge regarding and megestrol acetate were being developed as contraceptive
the effects of exogenous agents on the endocrine system, the agents and to treat specific disease conditions in humans
events which gave rise to the explosion of interest in this topic (Newburgh, 1975). In parallel with these developments,
in the mid-1990’s, and how it led to a fundamentally different toxicologists began to investigate these compounds for po-
paradigm in toxicity testing as well as a great deal of basic tential adverse effects.
research on mechanisms of endocrine-mediated toxicity. We Diethylstilbestrol (DES), a ‘‘synthetic estrogen’’ with similar
first cover the history of testing for endocrine-mediated potency to 17b-estradiol, was first prescribed in 1938 to
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S94 MARTY, CARNEY, AND ROWLANDS
prevent miscarriage and premature births. This drug was We Threatening Our Fertility, Intelligence and Survival?—
particularly significant as the daughters of women who took A Scientific Detective Story written by Colborn et al. (1996)
DES were found to have an increased incidence of clear-cell with a forward penned by then Vice President Al Gore. These
carcinoma, a rare vaginal cancer. As a result, in 1971, the Food and other authors proposed that many EACs elicited effects at
and Drug Administration (FDA) advised physicians to stop doses far lower than toxicities caused by other modes of
prescribing DES to pregnant women. Further research has action (MOAs) and thus required special regulation. Also
revealed an increased risk for breast cancer in DES mothers around the same time were a number of more specific reports
and also possible links with reproductive tract abnormalities in alleging associations between endocrine-disrupting chemicals
DES sons and daughters (Giusti et al., 1995). It has been and declining sperm counts (Carlsen et al., 1992; Sharpe and
estimated that 5–10 million Americans received DES during Skakkebaek, 1993), increases in breast cancer (Davis et al.,
pregnancy or were exposed to the drug in utero. 1993), cryptorchism and hypospadias (Barlow et al., 1999),
Somewhat unwittingly, the new emphasis on endocrine and antagonists, as well as compounds which act indirectly.
disruption also marked the beginning of a fundamental change Indirect mechanisms are plentiful and include the inhibition of
away from the traditional ‘‘outside-in’’ approach to toxicology, steroid biosynthesis or modulation of the hypothalamic-
in which adverse outcomes are identified first followed by pituitary-gonadal (HPG) axis. An even more complex example
mechanistic research to understand these effects. With begins with activation of the Ah receptor (AhR), which can
endocrine disruption came a reversed approach in which ultimately oppose estrogen signaling.
identification of mechanism came first followed by research to Ironically, some compounds bearing the very properties
define possible consequences of activating these mechanisms. that are the targets of screening can actually be beneficial. Studies
The implications of this change are quite significant and are of endocrine-disrupting AhR ligands have led to the development
discussed later in this review. of selective AhR modulators, such as ring-substituted
diindolylmethanes (DIMs), for chemotherapy of breast,
Testing and Assessment (EDTA) Task Force and the however, the weanling model did not detect weak antiandrogens
Validation Management Group (VMG)—Mammalian. This as consistently as the castrated adult model (Freyberger and
validation process, led by Japanese representatives on the Schladt, 2009) and therefore was not included in either the EPA
EDTA, included a detailed background document prepared by or the OECD test guideline.
the OECD (2003) and multiphase intra- and interlaboratory Initially, the 20-day female pubertal assay was recommen-
validation studies involving approximately 20 laboratories to ded, and the 20-day male pubertal assay was an alternate EDSP
show transferability, reproducibility, and reliability. This screen. Prior to initiation of the interlaboratory validation work,
validation program tested a potent estrogen (ethinyl estradiol), the exposure period for the pubertal male assay was increased
weak estrogen agonists in both a coded (blinded) and dose- from 20 to 30 days, and ultimately, both assays were adopted
response manner to evaluate sensitivity, and negative into the Tier I EDSP battery. One of the strengths of the male
compounds to verify specificity. This work culminated in and female pubertal assays is that these assays look for changes
hydroxyatrazine results reported in Laws et al., 2003). With (i.e., advanced, delayed, or asynchronous development), thyroid
few negative compounds included in the pubertal assay histopathology is an important endpoint to confirm a specific
validation, it is unclear whether the male and female pubertal thyroid effect. To facilitate interpretation, laboratories are
assays are prone to nonspecific effects, which will produce encouraged to include positive control compounds to verify
a high frequency of false-positive results. assay performance and build historical data. Thus, the
One alternative assay proposed as part of the EDSP was the amphibian metamorphosis data should be considered in
15-day intact adult male rat assay. Approximately 30 EACs, a weight-of-evidence approach to determine potential thyroid
across a variety of MOAs, have been evaluated using the activity.
15-day intact male assay including estrogen agonists/antagonists, Originally, the fish gonadal recrudescence assay was
androgen agonists/antagonists, steroid biosynthesis inhibitors, proposed as an EDSP Tier I screening assay. In this assay,
thyroid-active compounds, and prolactin modulators. The fish were examined for compound effects on maturation from
among some toxicologists. The National Toxicology Program relevant dose levels. Although risk assessment approaches for
(NTP), with support from the U.S. EPA, organized a panel to cumulative and aggregate risk have been in place for many
evaluate the scientific evidence on the low-dose effects of years (reviewed in Lipscomb et al., 2010), interest in
endocrine disruptors (Melnick et al., 2002), defining low-dose cumulative exposures to multiple EACs has received increased
effects as ‘‘biologic changes that occur in the range of human attention in recent years (Carney et al., 2010; Hotchkiss et al.,
exposures or at doses lower than those typically used in the 2008; Kortenkamp, 2008). Mixtures have been reported to
standard testing paradigm of the U.S. EPA for evaluating have additive effects (e.g., Gennings et al., 2004), less than
reproductive and developmental toxicity.’’ The panel additive effects (e.g., Charles et al., 2007), greater than additive
concluded that (1) there was evidence for low-dose effects in effects (e.g., Laetz et al., 2009), or even counteracting effects
laboratory animals, although in some cases, these effects have (e.g., Tanida et al., 2009). Overall, data indicate that the
not been replicated; (2) the shape of the dose-response curve interactions of chemicals in mixtures are dose dependent.
nature of an NR being a trans-acting transcriptional regulator effect obtained for one response to another response is
allows for their exogenous expression in numerous cell types questionable. Moreover, the problem of confounding is even
and cell lines to study the effects of potential ligands as more problematic when considering that measured parameters of
agonists, antagonists, etc. These studies have led to the in vitro high-throughput screening systems are by nature dif-
adoption of some assays into the EDSP battery of tests and ferent from those found for receptor systems in vivo. Therefore,
many others that are used in the ToxCast panel of assays. The the results from these in vitro screening systems may seldom
cloning of these receptors has also facilitated the development accurately reflect the intrinsic efficacies of NR ligands in vivo
of NR knockout (KO) mouse models that provided important and challenge the validity for extrapolating observations to
confirmatory evidence for the physiological role of hormones humans. Even if a single in vitro system was reflective of
in development, reproduction, and normal physiology as well a human response under certain conditions, the fact that the
as some unexpected observations. intrinsic efficacy of an EAC is dependent upon the cellular
exposures to hormonally active agents can be addressed in highlighted the tremendous impact of hepatic first pass
a scientifically rigorous and efficient manner. metabolism in converting these compounds to their
This movement toward evaluating more chemicals is also biologically inactive glucuronide forms (Teeguarden and
being combined with a fundamental change toward mechanism- Barton, 2004; Teeguarden et al., 2005). Therefore, route of
based testing (inside out) versus the traditional adverse effects- exposure can have a dramatic effect on the percentage of free,
based system (outside in) as described in the landmark report biologically active compound and consequently the effects
by the U.S. NRC Committee on Toxicity Testing and 1365 produced. The key point is that as we delve deeper into the
Assessment of Environmental Agents, Board of Environmental details of individual mechanisms, it is essential that these new
Studies and Toxicology, Institute for Laboratory Animal findings be interpreted in a holistic, physiological framework.
Research (2007) report entitled ‘‘Toxicity Testing in the 21st Fortunately, new tools in exposure modeling, physiologically
Century: A Vision and a Strategy.’’ Within this report, based pharmacokinetic modeling, and systems biology are
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