Articles
Alligator Tales: New Lessons
about Environmental
Contaminants from a Sentinel
Species
MATTHEW R. MILNES AND LOUIS J. GUILLETTE JR.
Wildlife species have been recognized as sentinels of environmental health for decades. In fact, ecological data on various wildlife populations
provided the impetus for banning some organochlorine pesticides over the last few decades. Alligators are important sentinels of ecosystem health in
the wetlands of the southeastern United States. Over the last 15 years, a series of studies have demonstrated that environmental exposure to a
complex mixture of contaminants from agricultural and municipal activities alters the development and functioning of alligators’ reproductive and
endocrine systems. Further studies of basic developmental and reproductive endocrinology in alligators and exposure studies performed under
controlled laboratory conditions support the role of contaminants as causal agents of abnormalities in gonadal steroidogenesis and in reproductive
tract development. These studies offer potential insight into environmentally induced defects reported in other wildlife and human populations
exposed to a wide array of endocrine-disruptive contaminants.
Keywords: endocrine disruptors, alligators, environmental contaminants, reproductive endocrinology, ecotoxicology
O
ver the past few decades, the field of toxicology
expanded from a focus on lethality and carcinogenicity to include alterations in development and reproduction
resulting from low-dose exposure to anthropogenic contaminants. As early as 1962, Rachel Carson brought reproductive impairment in wildlife exposed to pesticides to the
public’s attention in Silent Spring (Carson 1962); however, a
multidisciplinary approach to toxicology—popularized by the
publication of Our Stolen Future (Colborn et al. 1996)—did
not become widespread until the past 10 to 15 years. During
this time, a host of man-made chemicals, ranging from
organochlorine (OC) pesticides to phthalates and surfactants to polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (used as flame retardants in various
industrial applications) have been implicated in developmental and reproductive abnormalities in numerous species.
The development and functioning of the reproductive
system is largely dependent on hormonal signaling in a dosedependent fashion. Interruption of hormonal signaling can
lead to the improper development of reproductive organs and
tissues or to the mistiming of reproductive processes. Many
xenobiotics that have shown adverse effects at low doses are
known to interfere with the normal functioning of the endocrine system, and therefore have been designated collectively
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as endocrine-disrupting chemicals (EDCs). Our intent is to
provide a brief overview of the mechanisms of endocrine disruption characterized through controlled laboratory experiments. We will then present a case study from the alligators
of Lake Apopka, Florida, a wildlife population that has experienced low reproductive success in association with exposure to EDCs.
Mechanisms of endocrine disruption
EDCs can mimic or antagonize endogenous hormones, alter
synthesis or degradation, and affect hormone transport in the
bloodstream (figure 1). Recent evidence also suggests that exposure to EDCs can lead to changes in transcriptional activity and gene expression brought about by epigenetic effects.
Many aspects of the endocrine system are highly conserved
across vertebrate evolution, and thus the mechanisms through
which exogenous chemicals affect hormonal signaling in one
Matthew R. Milnes (e-mail: [email protected]) is a scientist at the
Zoological Society of San Diego, Center for Conservation and Research for Endangered Species, in California. Louis J. Guillette Jr. (e-mail: [email protected])
is a professor in the Department of Zoology at the University of Florida,
Gainesville. © 2008 American Institute of Biological Sciences.
December 2008 / Vol. 58 No. 11 • BioScience 1027
Articles
Figure 1. Schematic representation of the hypothalamo-pituitarygonad/thyroid axis. Circulating hormones interact with receptors in
target tissues throughout the body, altering gene expression profiles.
Plasma steroid and thyroid hormone concentrations are also affected
by plasma-binding proteins, synthesized by the liver, as well as hepatic
biotransformation and clearance. It is worth noting that the alligator’s
endocrine system is nearly identical to that of the human and other
mammals, making it an effective sentinel of potential environmental
and human health concerns.
species often hold true for others. That said, organismal response to EDCs does vary among individuals and species.
The use of in vitro models, in which differences as subtle
as single amino-acid substitutions in receptors can be assessed, is elucidating some sources of individual and interspecies variation (see, for example, Katsu et al. 2008).
EDCs are commonly classified in accordance with the
receptor-signaling pathway they are shown to disrupt, as
opposed to chemical structure. For instance, the pesticide
1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (o,p'-DDT)
and the plastic monomer bisphenol A (BPA) are often referred to as environmental estrogens, and in vitro assays
have shown that both chemicals activate estrogen receptor (ER)–dependent pathways (Andersen et al. 1999). It is
important to note, however, that in vitro experiments do
not always predict in vivo effects, as the timing and route
of exposure can also affect the outcome. A case in point:
The production of vitellogenin, or yolk protein, in rainbow
trout is stimulated through the activation of ER in the
liver; a single injection of DDT fails to induce vitellogenin
production in rainbow trout (Andersen et al. 1999), whereas
a similar dose distributed over three injections does induce
it (Donohoe and Curtis 1996). Endocrine disruption can
also occur by inhibiting, rather than activating, receptordependent pathways. The pesticide metabolite p,p'-DDE
(1,1-dichloro-2,2-bis[p-chlorophenyl]ethylene) and a
metabolite of the fungicide vinclozolin act as antiandrogens by competitively binding the androgen receptor (AR)
without activating AR-dependent pathways (Gray et al.
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2001). Classifying EDCs by the signaling pathway
they disrupt has also been applied to complex mixtures in the environment such as sewage effluent.
In such cases, the physiological effects evident in animals environmentally exposed to the effluent are
similar to those seen in animals experimentally exposed to estrogenic or antiandrogenic positive controls (Filby et al. 2007). Some environmental
contaminants have been shown to inhibit specific
enzymes involved in hormone synthesis. The initial and rate-limiting step of de novo steroidogenesis is the transfer of cholesterol from the outer to
the inner mitochondrial membrane by the steroidogenic acute regulatory protein (StAR; Stocco and
Clark 1996). Cholesterol is then converted to pregnenolone by the cytochrome P450 (CYP) enzyme
CYP11A, and pregnenolone converted to other
steroid hormones by a series of hydroxysteroid
dehydrogenases and CYP enzymes (figure 2).
The insecticides lindane (organochlorine) and
dimethoate (organophosphate) inhibit steroidogenesis by disrupting the transcription of StAR
messenger RNA (mRNA; Walsh and Stocco 2000,
Figure 2. A representative model of vertebrate steroidogenesis,
including the initial steps that occur in the mitochondria. A
gonadotropin, such as luteinizing hormone (LH) is delivered via
the blood and binds to a specific membrane receptor (LH-R) that
is associated with a guinine nucleotide-binding protein–adenyl
cyclase (AC) complex. This complex generates a second messenger,
cAMP, that activates a kinase cascade and enzyme activity associated with steroidogenesis. Initial stages of steroidogenesis occur in
the mitochondria following NR5A1-induced production of steroid
acute regulatory (StAR) protein. StAR is essential for the transport
of cholesterol across the mitochondial membrane. Cholesterol is
then modified by enzyme action both within the mitochondria
and in the smooth endoplasmic reticulum of the cytoplasm to generate steroid hormones that are released into the blood. Enzymes:
P450scc, P450 side chain cleavage; 3ßHSD, 3ß hydroxysteroid
dehydrogenase; 17ßHSD, 17ß hydroxysteroid dehydrogenase;
P450arom, P450 aromatase. Abbreviation: ATP, adenosine5'-triphosphate.
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Walsh et al. 2000a), whereas posttranscriptional disruption
of StAR was observed following treatment of MA-10 (mouse
Leydig tumor) cells with the antifungal drugs econazole and
miconazole (Walsh et al. 2000b). Similarly, chronic exposure
to PCBs inhibited CYP11A activity in the testes of bulls, contributing to low serum testosterone concentrations (Machala
et al. 1998).
Hepatic degradation, transport, and clearance of endogenous hormones are also susceptible to alterations following
exposure to xenobiotics (Gunderson et al. 2001). Oxidoreductase enzymes from the CYP2 and CYP3 families, conjugating enzymes such as glutathione S-transferases and
sulfotranserases, and membrane-transport proteins such as
organic anion transport protein are all involved in the breakdown and elimination of steroids and lipophilic xenobiotic
compounds. Transcriptional regulation of these enzymes
and transport proteins is largely under the control of the orphan nuclear receptors, the steroid and xenobiotic receptor,
and the constitutive androstane receptor (Maglich et al.
2002). Each of these receptors is characterized by its broad
ligand specificity relative to related nuclear receptors, and
each has been shown prone to activation by numerous xenobiotics (Kretschmer and Baldwin 2005, Milnes et al. 2008a).
Ligand-dependent activation of these receptors by xenobiotics
typically results in increased metabolism of the compound,
but it also increases the biotransformation and elimination
of endogenous signaling molecules, potentially disrupting
steroid homeostasis (Zhai et al. 2007). Additionally, some
man-made compounds have been shown to interact with
plasma proteins that transport specific hormones and protect
them from hepatic metabolism. PCBs have been implicated
in the disruption of the hypothalamus-pituitary-thyroid axis
(Desaulniers et al. 1999). Cheek and colleagues (1999) found
that hydroxylated PCBs (OH-PCBs) and the thyroid hormone thyroxine (T4) have the same affinity for transthyretin,
a serum thyroid-hormone transport protein. By competitively
binding transthyretin, OH-PCBs could increase the bioavailability of T4 and increase its susceptibility to hepatic
degradation.
Finally, transcriptional and epigenetic effects, in which
gene expression or DNA is modified without altering nucleotide sequence, has been associated with exposure to xenobiotics (reviewed in Tabb and Blumberg 2006, Edwards and
Myers 2007). For example, nuclear hormone receptors, including steroid receptors, are typically degraded through a
ubiquitin-proteosome pathway. Masuyama and Hiramatsu
(2004) found that BPA inhibits ubiquitination and degradation of estrogen receptor ß (ERß), potentially increasing the
transcription of ERß target genes. Another mechanism for epigenetic effects of EDCs involves the sex-specific methylation
patterns of DNA, which can be passed along through the
germ cell line. Reduced spermatogenic capacity has been observed in association with altered DNA methylation patterns
in rats following in utero exposure to vinclozolin and methoxychlor. This phenotype was perpetuated in male offspring for
four generations after exposure (Anway et al. 2005).
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Alligators as a model species for ecosystem health
As a long-lived top predator and a species of aesthetic and
commercial importance in the southeastern United States, the
alligator serves as a sentinel of wetland ecosystem health.
The alligator is an oviparous species that produces a single
clutch of eggs during a synchronous reproductive cycle. This
allows for the coordinated collection of a large number of eggs
enabling adequate sample sizes for ecotoxicological monitoring
and experimental designs. Alligators also exhibit temperaturedependent sex determination (TSD)—that is, the egg incubation temperature during a thermosensitive period of
embryonic development determines the sex of the offspring
(Lang and Andrews 1994). Females are typically produced at
temperatures below 31 degrees Celsius (°C), males are produced at or above 33°C, and a mixed ratio of males and
females are produced at intermediate temperatures (Lang
and Andrews 1994). Although the molecular mechanisms of
sex determination have not been worked out in detail for TSD
species, experimental exposure to natural and synthetic
steroids and some EDCs has been shown to override the
effects of temperature on sex determination in alligators
(Crain et al. 1997, Milnes et al. 2005a). Thus, alligators are
susceptible to experimental manipulations of incubation
temperature and hormonal milieu to facilitate the investigation of temperature and sex-specific developmental patterns
and indices. Although their large size and time to sexual
maturity (10 or more years) makes transgenerational studies problematic, alligators are an excellent model for investigating the effects of chronic exposure to xenobiotic chemicals
on reproductive success and embryonic development.
Lake Apopka, Florida: A case study
in endocrine disruption
The initial indication of reproductive impairment in alligators from Lake Apopka, Florida, was the lack of recruitment
of juveniles into the population. Following nearly 40 years of
pesticide and nutrient runoff from adjacent agricultural operations, Lake Apopka experienced a pesticide spill in 1980
from the Tower Chemical Company site. The spill, which
resulted from the overflow of holding ponds during heavy
rainfall, consisted primarily of dicofol and derivatives of
DDT (USEPA 1994). Unfortunately, data from before the
spill are unavailable, but rates for alligator egg viability and
juvenile population density declined significantly in the years
after the spill (Woodward et al. 1993). Compared with yolk
samples taken from alligator eggs at Lake Griffin, Florida, an
area of intermediate anthropogenic influence (Heinz et al.
1991), samples taken at Lake Apopka in 1985 had higher
concentrations of several OC pesticides and metabolites, including toxaphene, dieldrin, p,p'-DDE, p,p'-dichlorodiphenyl
dichloroethane, p,p'-DDT, cis-chlordane, and trans-nonachlor.
In the following decade, egg viability rates rose but still remained below those at two reference sites, Lake Woodruff
National Wildlife Refuge (NWR) and Orange Lake (Masson
1995, Rice et al. 1999). More recent studies from eggs collected
between 2000 and 2002 show that concentrations of OC
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pesticide residues in Lake Apopka eggs still have not appreciably decreased, and Lake Apopka egg viability and posthatching survival remain low relative to Orange Lake and Lake
Woodruff NWR (Rauschenberger et al. 2007, Milnes et al.
2008b).
Exposure to EDCs does not appear to be an immediate
threat to the existence of alligators in Lake Apopka or elsewhere; however, this population of alligators offers the opportunity to study and better understand the various modes
of action through which environmental exposure to EDCs
affects development and reproduction in a long-lived species,
enabling improved population monitoring and more-informed policy decisions. It is difficult to compare contaminant concentrations across species and tissues, and although
yolk concentrations of OC pesticides in Lake Apopka are
one to two orders of magnitude above concentrations reported
in fish across the southeastern United States (Hinck et al. 2008),
this situation is not unique. For instance, the concentrations
of p,p'-DDE and toxaphene in the visceral fat and liver of freshand saltwater crocodiles (Crocodylus johnstoni and Crocodylus porosus) from the Ord River, Western Australia, are among
the highest ever reported in wildlife, even though it has been
nearly 30 years since pesticides were applied to this drainage
(Yoshikane et al. 2006). Although variation in size and reproductive status among the Ord River sampling sites prevented statistical comparisons of phenotypic effects reported
in Lake Apopka alligators, the study nonetheless illustrates the
bioaccumulative nature and long-lasting legacy of these
chemicals in top predators.
That the reduced egg viability reported in Lake Apopka alligators could be caused by exposure to OC pesticides rather
than industrial chemicals or metals is supported by analyses
of contaminant concentrations in various alligator tissues.
Whereas yolk and serum OC concentrations in samples from
Lake Apopka were elevated in comparison with samples from
Lake Woodruff NWR and Orange Lake (Heinz et al. 1991,
Guillette et al. 1999a), no differences in PCB concentrations
were found among these lakes (Guillette et al. 1999a). Metal
and metalloid concentrations in all three lakes (Burger et al.
2000) were well below suggested toxic levels and levels previously reported in alligators from the Everglades and Georgia (Heaton-Jones et al. 1997).
Other proposed hypotheses to explain the low reproductive success reported in Lake Apopka alligators include nutritional and genetic differences. One as yet unstudied but
plausible explanation is that nutritional differences resulting
from several decades of nutrient and contaminant loading may
affect the composition of prey species. Previous research has
shown dietary differences and altered egg yolk lipid composition to be associated with reduced egg viability in captive alligators (Noble et al. 1993). If the prey species composition
has been drastically altered on Lake Apopka, it is possible that
essential nutrients are lacking in the diet, thus leading to deficiencies in specific fatty acids or nutrients incorporated
into the egg yolk during vitellogenesis.
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Genetic differences resulting from contaminant-induced
selection or natural variation seem an unlikely cause of low
reproductive success on Lake Apopka at this point. The maximum life span of American alligators is not known, but
conservative estimates place the number well beyond 30
years. No mass-mortality events have been reported for adult
alligators at Lake Apopka, suggesting that a portion of the current breeding population hatched before the Tower Chemical Company spill. Given alligators’ long life span and time to
maturation, and the population’s connectivity with other
lakes in the same drainage basin, it seems unlikely that exposure to contaminants has significantly altered allele frequency in Lake Apopka’s alligators relative to those in nearby
reference sites. Indeed, microsatellite DNA analysis revealed
insignificant variation among alligator populations (including Lake Apopka and Lake Woodruff NWR) across Florida
and southern Georgia (Davis et al. 2002).
Field studies. For the purposes of this review, neonates are defined as alligators one month of age or younger; hatchlings
are more than one month but less than or equal to one year
of age; and juveniles are older than one year but less than 90
centimeters (cm) long from snout to vent, the approximate
minimum size at sexual maturity (Wilkinson and Rhodes
1997). All fieldwork described in this review from our laboratory was permitted by the Florida Fish and Wildlife Conservation Commission, and the use of experimental animals
was performed under the guidelines specified by the Institutional Animal Care and Use Committee at the University of
Florida.
Because natural systems are infinitely complex and contain
numerous biotic and abiotic characters that can potentially
influence development, true replication of the scenario at
Lake Apopka cannot be achieved. Rather, our laboratory and
others have used Lake Apopka as a case study of a chronically
exposed alligator population that has experienced poor reproductive success. In many of the comparative studies, Lake
Woodruff NWR was used as a reference population, as it has
been subjected to fewer anthropogenic influences and exhibits
consistently high reproductive success relative to the population at Lake Apopka. It is important to note, however, that
physiological differences described in alligators from Lake
Apopka relative to those from Lake Woodruff NWR are
merely associative, not causative, and complementary experimental studies are required to characterize the specific
effects of EDCs.
The initial indicator that exposure to EDCs could be causing reproductive problems in alligators at Lake Apopka—aside
from the relatively low percentage of eggs hatching there—
was the observation of morphological abnormalities in the
gonads of six-month-old hatchlings. Multioocytic ovarian follicles and multinuclear oocytes (figure 3), which resembled
the gonads of female mice exposed in utero to the synthetic
estrogen diethylstilbestrol, were observed in the ovaries of
captive-raised alligator hatchlings from Lake Apopka (Guillette et al. 1994). Recent studies have reported observations
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4; Crain et al. 1998, Guillette et al. 1999a). Egg-hatch
rates that are higher than Lake Apopka’s and lower than
Lake Woodruff NWR’s have been reported at other
Florida lakes that have experienced agricultural or municipal influences that are intermediate to those of Lake
Apopka and Lake Woodruff NWR. Several of these
lakes—including Lake Okeechobee (Crain et al. 1998,
Gunderson et al. 2004) and Lakes Griffin and Jessup
(Guillette et al. 1999b)—also show depressed plasma
testosterone concentrations in juvenile males. Females
from Lakes Apopka, Griffin, Okeechobee, and Orange exhibit elevated E2 compared with Lake Woodruff NWR juveniles (Guillette et al. 1999b). Seasonal variation has also
been examined in thyroid hormones in juvenile alligators from several Florida lakes, and the overall pattern of
variation was similar to that for sex steroids (Bermudez
Figure 3. Photomicrographs of normal and multioocytic ovarian
et al. 2005). Crain and colleagues (1998) found a negafollicles (a, b) and a multinuclear oocyte (c) from juvenile Ameritive relationship between thyroxine (T4) and trican alligators. Multioocytic (polyovular) follicles have been reiodothyronine (T3) and body size in juveniles from Lake
ported from females alligators from Lake Apopka, Florida, as well
Woodruff
NWR, but no such relationship existed in alas from female caiman treated with various estrogenic contamiligators
from
Lake Apopka.
nants. Abbreviations: o, oocyte; mof, multioocytic follicle; mno,
Because
of
the bioaccumulating and lipophilic namultinuclear oocyte. Photomicrographs: Brandon C. Moore,
ture
of
many
of
the contaminants ubiquitous to Lake
University of Florida.
Apopka, individuals experience two major stages of conof multioocytic follicles in caiman (Caiman latirostris) or
taminant exposure. First, the embryo is exposed to any conmice following embryonic or neonatal experimental exposure
taminants the mother transfers to the embryonic environment
to estradiol, BPA, or phytoestrogens, such as genistein (Jefduring the vitellogenic stage of oocyte maturation. Embryferson et al. 2006, Stoker et al. 2008). Further morphological
onic exposure to hormonally active compounds can have
evidence of chemicals interfering with endocrine signaling was
unique consequences as a result of the profusion of cell proevident when the size of the phallus, an androgen-dependent
liferation and differentiation in the developing embryo (Bern
tissue, was compared among alligators from different lakes:
1992). Second, offspring experience environmental exposure
alligators from Lake Apopka show reduced phallus size when
throughout their growth and reproductive maturation. With
compared with similar-sized animals from less-polluted lakes
this in mind, it is important to distinguish abnormalities
(reviewed in Guillette et al. 2000). Other morphological difpresent in Lake Apopka’s alligators as a result of embryonic
exposure to EDCs from alterations caused by environmenferences noted between juvenile alligators from Lake Apopka
and Lake Woodruff NWR include thymus and spleen histological structure (Rooney et al. 2003) and long-bone
mineral density and composition (Lind et al. 2004)
Just as hormonally active xenobiotics can affect development, alterations in endogenous hormone concentrations can have similar deleterious effects. The
thyroid and steroid hormones of alligators from Lake
Apopka and from reference sites such as Lake Woodruff
NWR have been compared multiple times; the results of
these comparisons vary, depending on many factors such
as age or size and time of year. Juvenile alligators display
seasonal variation in plasma sex steroids (Rooney et al.
2004), with the degree of the response depending on
body size; alligators above a size threshold of approxiFigure 4. Plasma testosterone concentrations in size-matched juvemately 38 cm from snout to vent begin to show pronile male alligators from Lakes Woodruff NWR and Lake Apopka.
nounced seasonal variation in estradiol-17ß(E2) and
Bars represent means ± the standard error of the mean. At all juvetestosterone, suggesting a peripubescent period. Although
nile body sizes represented, males from Lake Apopka have signifiseasonal patterns among lakes are somewhat variable,
cantly lower plasma testosterone concentrations than do those
some trends are evident. Juvenile males living in Lake
from Lake Woodruff NWR. Data are from Guillette and colleagues
Apopka have lower concentrations of circulating testos(1999b) and Crain and colleagues (1998).
terone than males living in Lake Woodruff NWR (figure
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December 2008 / Vol. 58 No. 11 • BioScience 1031
Articles
tal exposure. We have attempted to do this by collecting eggs
from Lake Apopka and Lake Woodruff NWR and raising
the hatchlings in a controlled environment, thus limiting the
cause of developmental differences to embryonic or genetic
origins.
When we compared neonates collected as eggs from Lake
Apopka and from Lake Woodruff NWR, we found that males
and females from Lake Apopka had higher plasma testosterone
concentration (Milnes et al. 2005b). Interestingly, although
plasma testosterone concentrations were higher, the phallus
tip length and cuff diameter were smaller in males from Lake
Apopka (figure 5). In contrast, the plasma testosterone concentration was lower in six-month-old males (Guillette et al.
1994) and nine-month-old females (Crain et al. 1997) from
Lake Apopka than it was in animals of the same age from Lake
Woodruff NWR. The results from the six- and nine-monthold hatchlings are more consistent with our observations of
field-caught juveniles. Current studies in our laboratory are
examining the phenomena of embryonic and posthatching
sexual differentiation and ontogeny of sexual maturation of
the gonads and external genitalia. Recent results suggest that
significant organizational changes within the steroidogenic tissues of the immature gonad take place during the first weeks
to three months of life (Moore et al. 2008).
principle of parsimony), we conducted experiments exposing alligators from Lake Woodruff NWR to individual contaminants relevant to Lake Apopka to reveal specific
contaminant-induced effects. Many of these experiments
used chemicals dissolved in ethanol and applied topically to
the eggshell. The major criticism of this technique is that
the amount of chemical delivered into the egg is variable, and
typically only a small percentage of what was applied, thus invalidating dose-response relationships (Muller el al. 2007). On
the other hand, if proper controls are employed and results
replicated, such as ovarian development at male-producing
temperatures following treatment with natural or synthetic
estrogens, phenotypic and genetic responses can still be attributed to the treatment even if the dose is underestimated.
Two of the OC contaminants found in the highest concentration in either serum or egg yolk samples from Lake Apopka
are p,p'-DDE and toxaphene. Although p,p'-DDE has been
shown to have a weak affinity for the alligator ER (Vonier et
al. 1996), previous studies on mammals indicate that it inhibits
androgen-dependent pathways (Gray et al. 2001). Embyronic exposure to p,p'-DDE did not influence sex determination at temperatures that typically produce all males, but
did cause a higher than expected percentage of females at a
temperature that normally produces both sexes (figure 6;
Milnes et al. 2005a); in ovo exposure to toxaphene had no
Experimental exposure studies. As previously stated, the
observed differences between alligators from Lake Apopka
and Lake Woodruff NWR are associative, and controlled
experimental approaches must be applied to begin to elucidate proximate causes for these. Invoking Occam’s razor (the
Figure 5. Phallus cuff diameter and tip length of male
neonatal alligators from Lake Apopka (AP) and Lake
Woodruff (WO). Bars represent least-square means adjusted for snout-to-vent length ± the standard error of
the mean, and asterisks denote significant differences
between lakes within an incubation temperature. One
asterisk indicates p ≤ 0.05; two asterisks indicate p ≤
0.005. The data are taken from Milnes and colleagues
(2005b).
1032 BioScience • December 2008 / Vol. 58 No. 11
Figure 6. Sex ratios of alligators exposed in ovo to p,p'DDE or estradiol-17ß and incubated at 32°C or 33.5°C.
Treatments are reported in parts per billion wet egg mass.
Asterisks denote a significant difference between treatment and the control and vehicle groups. The data are
taken from Milnes and colleagues (2005a).
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effect on sex determination, but did result in elevated plasma
testosterone in the exposed neonates (Milnes et al. 2004).
Interestingly, gonadal synthesis of testosterone was not affected,
suggesting differences in hepatic degradation as a possible
source of differences in circulating steroid concentrations. Intravenous exposure of juvenile alligators to toxaphene resulted in the up-regulation of CYP3A77 mRNA transcripts,
an enzyme involved in biotransformation of xenobiotics and
endogenous steroids (Gunderson et al. 2006).
In a recent study by Rauschenberger and colleagues (2007),
captive alligators were exposed to an OC pesticide mixture in
their diet to achieve environmentally relevant concentrations of contaminants in the egg yolk. The percentage of eggs
within a clutch that hatched was lower in the treatment group
than in controls, the result of a dramatic increase in nonbanded eggs that were either infertile or experienced very early
embryonic mortality. In the same study, differences in clutch
viability among contaminated and reference populations
(including Lake Apopka and Orange Lake) in the wild were
accounted for by early and late embryo mortality, confirming an earlier study (Masson 1995). The stress of captivity,
however, may have been a confounding factor in the more recent study. Only four of the six control females and three of
the seven treated females produced clutches over the three
years of the study (Rauschenberger et al. 2007), demonstrating the difficulty of captive breeding and transgenerational
studies in this species.
In addition to examining steroidogenesis at the molecular
level through an examination of the enzymes involved in
this process, we have also examined steroid and thyroid receptor expression in various target tissues. In vertebrates, the
steroid and thyroid receptors belong to a superfamily of
nuclear transcription factors that include all other steroid
hormone receptors (such as progestogens, androgens, glucocorticoids, mineralocorticoids), the vitamin D receptor, and
the retinoic acid receptor (Blumberg and Evans 1998). The ancestral condition for the jawed vertebrates (Gnathostomata)
appears to have been the presence of two forms of ER, corresponding to ERα and ERß; two forms of thyroid receptor;
one AR; and one progestin receptor (Thornton 2001). Two
forms of ER have been found in mammals, fish, birds, reptiles, and amphibians. Our group cloned and sequenced ERα
and ERß from the American alligator (Alligator mississippiensis; Katsu et al. 2004). The alligator steroid-receptor sequences can be divided into five domains on the basis of
their sequence homology to other steroid hormone receptors.
We have found that the hormone (ligand)- and DNA-binding domains of the alligator are highly conserved with birds
and mammals, with the DNA-binding domain of the alligator ERa having 100% sequence similarity with the same region in the chicken, mouse, and human ERα (figure 7).
Likewise, the amino acid sequence of the ligand-binding domain is also highly conserved among birds, mammals, and
crocodilians such as the alligator (Katsu et al. 2004). These data
suggest that environmental contaminants that act through the
alligator ER are likely to act through the human ER as well,
which is yet another reason to use wildlife as sentinels of environmental health: they could also forewarn us of dangers
to our own health.
We have recently observed alterations in ERß, NR5A1,
and StAR mRNA expression in testicular tissue from juvenile
Mechanistic studies and future directions. We have begun to
investigate the mechanistic actions of EDCs in alligators at the
cellular and molecular levels. By quantifying gene expression related to endocrine function, we are beginning to understand the genetic attributes underlying the physiological
basis of the abnormalities identified in Lake Apopka’s alligators. Moreover, molecular cloning and the development
of in vitro receptor binding and gene expression assays are
providing a means of conducting predictive experiments
that minimize the use of experimental animals (Kohno
et al. 2008a). We hope that these experiments, in concert
with existing comparative and in vivo studies, will reveal
specific receptor-mediated pathways susceptible to endocrine disruption and reduce the use of experimental
animals.
In a recent study, we observed lower expression of
mRNA for steroidogenic factor-1 (NR5A1) and StAR in
one-year old male alligators from Lake Apopka relative
to those from Lake Woodruff NWR (Milnes et al. 2008b).
A reduction in the expression of NR5A1 would be expected to reduce the synthesis of cholesterol by decreasFigure 7. Comparison of human ESR1 (ERα) protein with estrogen
ing the transcription of steroidogenic enzymes such as
receptors of representative vertebrate species. The functional A/B
CYP11A, and lower expression of StAR could reduce
to F domains are schematically represented with percentage simithe availability of cholesterol to steroidogenic enzymes.
larities of sequence presented. Note the high sequence similarity
These results provide a potential mechanism for previ(values at or close to 100%) between human, mouse, chicken, and
ous studies that found that hatchling and juvenile males
alligator for the DNA-binding (DBD) and ligand-binding (LBD)
from Lake Apopka had lower plasma testosterone condomains.
centrations than did those from Lake Woodruff NWR.
www.biosciencemag.org
December 2008 / Vol. 58 No. 11 • BioScience 1033
Articles
alligators from Lake Apopka (Kohno et al. 2008b). Likewise,
alterations of aromatase and DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X
chromosome, gene 1) mRNA expression in ovarian tissue
could provide further insight into the mechanisms leading
to modified folliculogenesis and ovarian development in
females (Kohno et al. 2008b). Current studies are extending
this work to examine the interaction between steroid hormones and their receptors and thyroid hormone action,
via thyroid hormone nuclear receptors and thyroid hormone–dependent gene expression and ovarian development
and functioning (Helbing et al. 2006). Given the conserved
nature of the molecular mechanisms underlying the biology
of reproduction among vertebrates, these studies have the
potential to expand our understanding of the environmental and genetic interactions that lead to birth defects involving the reproductive system in alligators and other species.
Conclusions
Sublethal exposure to EDCs can have far-reaching consequences for both wildlife and human populations. In addition to its own intrinsic value, each wildlife population is a
critical part of an ecosystem that depends upon interspecies
relationships to maintain the processes that provide humans
with necessary commodities such as clean air, water, soil,
and food. Chemical exposure that reduces reproductive success within a population can upset the balance of processes
that maintain a healthy ecosystem. Although direct extrapolation of the effects of EDCs on wildlife species to humans
cannot be made, many of the molecular, cellular, and physiological processes regulated by the endocrine system are
conserved throughout vertebrate evolution, and a weight-ofevidence approach should be used in guiding future policy
decisions. The data presented here reveal alterations in development and endocrine function in alligators, a long-lived
species that is chronically exposed to a complex mixture of
environmental contaminants in Lake Apopka. The captiveraised alligator studies begin to separate the alterations of
embryonic origin from those of environmental origin, and the
laboratory-based studies identify mechanisms through which
some of the chemicals can act.
The success of any population, human or wildlife, ultimately
depends on survival and successful reproduction, not survival
alone. Alligators have adapted their reproductive strategy to
a type III survivorship curve, meaning they produce a large
number of offspring with a low probability of survival. As individuals attain sexual maturity, the mortality rate decreases.
Alligator populations in central Florida can withstand a loss
of up to 50% of the estimated annual recruitment without a
detectable decrease in population density, as evidenced by the
monitored harvesting of eggs and hatchlings for commercial
purposes on selected lakes (Rice et al. 1999). However, maintaining these populations depends on the production of
viable offspring over succesive generations. The Tower Chemical Company spill on Lake Apopka occurred in 1980. From
1983 to 1986, egg viability was at an all-time low (approxi1034 BioScience • December 2008 / Vol. 58 No. 11
mately 20%) and the juvenile population declined from 1981
to 1987 (Woodward et al. 1993). This time period could indicate the acute toxicological effects of juvenile and adult
contaminant exposure. Since the initial decline, egg viability
and the number of juvenile alligators have gradually increased, and the current level of embryonic and posthatching mortality on Lake Apopka does not appear immediately
detrimental to the population; however, nothing is yet known
about the fecundity of the newly recruited individuals. From
an evolutionary perspective, the cost of altered reproductive
success in a species that takes 10 to 15 years to attain sexual
maturity is difficult to predict. If the currently surviving offspring are selected for traits that confer resistance to contaminants, how do these traits contribute to or detract from
reproductive success? We could be observing the gradual recovery of Lake Apopka’s alligators in terms of population size
and, simultaneously, a reduction in heritable variation and in
the ability to cope with future challenges in a changing environment. As more chemicals that disrupt development are
identified, the charge for the current and future generations
is to evaluate the consequences of altered development on
reproductive fitness. This will undoubtedly be particularly
challenging to do for long-lived species such as alligators and
humans.
Acknowledgments
Research described from the laboratory of L. J. G. was supported by grants from the University of Florida Opportunity
Fund, NICHHD 1 R21 HD047885-01, and NIEHS R21
ES014053-01A1. We are especially grateful to Allan Woodward
and the Florida Fish and Wildlife Conservation Commission for their continued support of this research. We thank
Satomi Kohno for preparing the estrogen-receptor species
comparison figure.
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