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Environmental
Health Perspectives Supplements Volume 110, Number 3, June 2002
Aromatase Activity in the Ovary and Brain of the Eastern Mosquitofish (Gambusia holbrooki) Exposed to Paper Mill Effluent
Edward F. Orlando,1,2 William P. Davis,3
and Louis J. Guillette Jr.1
1Department of Zoology, University of Florida, Gainesville,
Florida, USA; 2Biology Department, St. Mary's College of
Maryland, St. Mary's City, Maryland, USA; 3National Health
and Environmental Effects Research Laboratory, Gulf Ecology Division,
U.S. Environmental Protection Agency, Gulf Breeze, Florida, USA
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Full Article in PDF
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Abstract
Studies have shown that female mosquitofish living downstream of a paper
mill located on the Fenholloway River, Florida, have masculinized secondary
sex characteristics, including altered anal fin development and reproductive
behavior. Masculinization can be caused by exposure to androgens in the
water or from an alteration in aromatase activity in the fish. We hypothesized
that aromatase activity would be inhibited by a component(s) of the paper
mill effluent. Aromatase inhibition could masculinize the hormonal profile
and, subsequently, secondary sex characteristics of the exposed females.
Therefore, we predicted that ovarian and brain aromatase activity would
be lower in the female mosquitofish from the Fenholloway River compared
with the reference site, the Econfina River. Adult females were collected
and standard length, body mass, anal fin length, and segment number were
measured. Ovarian and brain aromatase activity were determined using a
tritiated water assay. Fenholloway females had masculinized anal fin development
as indicated by an increase in the number of segments in the longest anal
fin ray (p < 0.0001), yet the length of the ray did not differ
between sites (p = 0.95). Fenholloway females exhibited higher
ovarian (p = 0.0039) and brain (p = 0.0003) aromatase activity
compared with reference site fish. These data do not support aromatase
inhibition as the mechanism for masculinization, suggesting that the masculinization
of the Fenholloway female mosquitofish is due to androgenic contaminants.
Future studies should examine the relationship between aromatase enzyme
activity and exposure to environmental androgens. Key words: altered
development, aromatase, brain, endocrine disruption, gonad, masculinization,
paper mill effluent. Environ Health Perspect 110(suppl 3):429-433
(2002).
http://ehpnet1.niehs.nih.gov/docs/2002/suppl-3/429-433orlando/abstract.html
This article is part of the monograph Impact of Endocrine
Disruptors on Brain Development and Behavior.
Address correspondence to E.F. Orlando, St. Mary's
College of Maryland, Biology Dept., 18952 E. Fisher Road, St. Mary's
City, MD 20686-3001 USA. Telephone: (240) 895-4376. Fax: (240) 895-4996.
E-mail: eforlando@smcm.edu
This research was funded by a STEP award to E.F.O.
from the U.S. EPA and a grant to L.J.G. from the U.S. EPA (CR821437).
We thank D. Bass, L. Catalbiano, R. Miller, T. Edwards, S. Kools, G.
Ankley, and L.E. Gray Jr. for their technical support during this project.
Received 8 January 2002; accepted 8 April 2002.
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Introduction
During the last decade, a growing literature has documented the endocrine-disruptive
effects of various environmental contaminants [for review, see (1-3)].
The focus of many early studies was on the estrogenic and antiandrogenic actions
of various agricultural chemicals such as dichlorodiphenyltrichloroethane (DDT)
and its metabolites, methoxychlor, and vinclozlin and industrial compounds such
as polychlorinated biphenyls (PCBs), alkylphenol ethoxylates (APEs), and phthalates
(4-9). To date, research has focused on the effects of environmental
exposure to these chemicals on reproduction and development in wildlife and/or
investigating the underlying mechanisms for some of the observed effects in
laboratory and environmentally exposed wildlife species.
In laboratory studies, researchers have examined the affinity of environmental
contaminants for the estrogen receptor or a chemical's ability to induce estrogenic
responses such as cell proliferation or vitellogenin synthesis (10,11).
A wide range of chemicals were shown to have an affinity for estrogen receptors
from various species (12). In addition to the estrogenic or antiestrogenic
nature of chemical contaminants, data are currently available documenting antiandrogenic
(8,9), androgenic (13-15), antiprogestogenic (16),
and antithyroidal actions (17-19). The mechanisms behind these actions
appear varied, with receptor binding only one of a number of possible responses.
For example, contaminants also appear to influence the endocrine system by altering
enzymes that enable hormone synthesis (20-22), hormone metabolism
(23,24), and hormone storage on plasma carrier proteins (25,26).
One enzyme whose activity appears to be altered by environmental contaminants
is the steroidogenic enzyme P450 aromatase. Aromatase converts (the androgens)
androstenedione or testosterone to the estrogens estrone or 17ß-estradiol
(E2), respectively (27). This enzyme has been identified in
the gonad, liver, brain, and various peripheral tissues, such as fat cells in
mammals, as well as in the gonad and brain of all other vertebrates including
fishes and the cephalochordate, amphioxus (27-29). At least two
forms of aromatase have been reported in mammals and fishes, with gonadal and
brain forms coded for on two separate genes (30-32). Aromatase expression
is constitutive in some tissues, but aromatase expression and activity can be
altered by temperature, season, and various hormones, such as steroids (33-35).
Exposure of embryonic alligators to the herbicide atrazine induces a significant
increase in aromatase activity in the gonad-adrenal-mesonephros of
neonates (21). Similar responses have been reported after in vitro
exposure of a human adrenal cell line to atrazine (36), and two other
triazine herbicides, simazine and propazine, also induced aromatase activity
in human adrenal and human placental choriocarcinoma cell lines (37).
Such studies indicate that aromatase activity can also be affected by environmental
contaminants.
Two well-documented examples of environmental exposure to endocrine-disrupting
chemicals in fishes are from sewage treatment plant effluent and paper mill
effluent. Researchers in Great Britain and the United States have demonstrated
that exposure to sewage treatment plant effluent causes vitellogenesis, decreased
testis size, and altered sex steroid concentrations in male fishes. These effects
have been associated with naturally occurring estrogens (estrone and E2)
and synthetic estrogens from birth control pills (ethinylestradiol) found in
the effluent (38-40).
Additional studies have documented a suite of alterations in the function
of endocrine and reproductive systems and the morphological development of fishes
exposed to paper mill effluent. White suckers, Catastomus commersoni,
exposed to paper mill effluent in Lake Superior, Canada, are known to exhibit
altered pituitary function, decreased plasma sex steroid concentrations, increased
liver size, elevated hepatic mixed-function oxidase levels, decreased egg and
gonadal size, and delayed age to sexual maturity (41-43).
Beginning in the 1980s, a number of researchers reported masculinized female
fish in rivers below paper mill effluent outfalls (44-46). Early
studies observed female mosquitofish (Gambusia affinis and G. holbrooki)
with gonopodial development and altered reproductive behavioral patterns. Mosquitofish
are viviparous and therefore have internal fertilization. Males transfer the
spermatheca to females using a highly modified anal fin called the gonopodium.
Gonopodial development is androgen dependent and can be induced in females exposed
exogenously to androgens (47,48). It has been hypothesized that bacterial
biotransformation of plant sterols in the effluent generates the androgens responsible
for the observed masculinization of female mosquitofish exposed to paper mill
effluent (49). Recently, it was reported that androstenedione was present
after a chemical characterization of the effluent from a paper mill plant in
Florida (50). Using a number of in vitro receptor binding and
gene expression assays, our laboratory in collaboration with others has not
found sufficient androstenedione concentration in Fenholloway River water samples
to support the reported significant androgenic actions of this effluent, however
(15).
The present study was designed to test a mechanism that would explain observations
of masculinized female mosquitofish (G. holbrooki) living downstream
of the Buckeye Paper Mill, in the Fenholloway River, Florida (impacted site).
The Fenholloway River receives approximately 50 million gallons per day of effluent
from the paper mill. The effluent can comprise up to 80% of the Fenholloway
River volume during the drier months of the year (46,51). In contrast
to the Fenholloway River, the Econfina River (reference site) has no known point
sources of pollution. The rivers share the same headwaters and both terminate
in the Gulf of Mexico (15). The Fenholloway River is greatly altered
both physicochemically and biologically, as evidenced by its increased temperature
and decreased dissolved oxygen, clarity, and species diversity of both plants
and animals compared with the Econfina River.
As described above, mosquitofish collected below paper mills can exhibit masculinized
characteristics, such as gonopodial development. However, not all females in
the population exposed to androgenic paper mill effluent exhibit masculinization.
Further, female mosquitofish showing dramatic gonopodial development are also
pregnant, suggesting that the environmental androgens can alter morphology and
behavior but need not preclude reproductive activity. One could hypothesize
that the masculinized female anal fin was due to immersion in water containing
androgenic substances that diffused across the epithelial layers overlying the
fin, stimulating the cells locally to differentiate. Given the reports of altered
behavior in masculinized female mosquitofish, a more likely hypothesis suggests
that environmental androgens are active systemically, not just peripherally.
Thus, females would respond to exogenous androgens in a manner that could maintain
an internal hormonal environment allowing reproductive cyclicity. One mechanism
by which this could be achieved would be via alterations in aromatase activity
of the gonad and possibly the brain. We hypothesized that a component of the
river water was inhibiting aromatase activity in the female mosquitofish, thereby
resulting in a masculinized hormonal profile. Aromatase inhibition could result
in a decrease in estrogens and a concomitant increase in androgens, which could
masculinize the hormonal profile of a female, thereby masculinizing its secondary
sex characteristics. Therefore, we predicted that ovarian and brain aromatase
activity would be lower in Fenholloway mosquitofish compared with that in the
reference site (Econfina River) mosquitofish.
Methods
Fish Morphometrics
In this study, we tested this hypothesis by measuring P450-aromatase activity
in the ovary and brains of adult female mosquitofish. Morphometrics were obtained
from all 83 fish collected. We measured aromatase activity in a subset of those
fish (n = 66) because 17 lacked follicles in their ovaries. All of the
fish were reproductively active adults, and no site differences were observed
in the reproductive stage of the ovaries. On 15 August 1999, all fish were captured
from the Fenholloway and Econfina Rivers using a 1/8-inch drop net. Fish were
transported in coolers partially filled with fresh water obtained from each
site. At the lab, fish were housed at the same density in their respective river
water and held for 24-72 hr under identical conditions of 14:10-hr light:dark
photoperiod and 28°C water temperature.
All lab work was conducted in full compliance with the guidelines of the University
of Florida Institutional Animal Care and Use Committee (Z951). Fieldwork was
conducted under permit FNE-97011 from the Florida Game and Freshwater Fish Commission.
Ovarian Aromatase Activity
Each day, over a 3-day period, the same numbers of fish were processed from
each site. Fish were anesthetized with MS-222 (150 ppm; A5040; Sigma Chemical
Company, Chicago, IL, USA), and standard length and total body mass were measured
to 0.1 mm and 0.001 g, respectively. Next, fish were sacrificed and then gonad
and brain were immediately removed and weighed to the nearest 0.1 mg. Brains
were snap-frozen in liquid nitrogen and stored at -70°C until they
were assayed, approximately 1 month later (see "Brain Aromatase Activity," below).
The number of oocytes was determined for each ovary, then the ovary was placed
in 488 µL of RPMI 1640 culture media (pH 7.2, sterile filtered) (Gibco
23400-021; Life Technologies, Rockville, MD, USA) in borosilicate glass tubes.
Next, 13 µL of labeled androstenedione (androst-4-ene-3,17-dione, [1ß-3H(N)],
NET-926, specific activity = 24.0 Ci/mM; Dupont NEN Life Science Products, Boston,
MA, USA) was added, and each sample was incubated for 6 hr at 28°C.
After incubation, samples were centrifuged at 1,500g
for 15 min at 4°C, and then 400 µL of supernatant was transferred
to a new borosilicate glass tube. Into each tube, 1.5 mL of chloroform (C 298-4;
Fisher Scientific, Pittsburgh, PA) was added; the mixture was pulse vortexed
and centrifuged as above. Next, 200 µL of supernatant was transferred to
a new tube and combined with 200 µL of charcoal dextran [5% Norit A charcoal
(C170, Fisher), plus 0.5% dextran (D4751, Sigma)], vortexed for 5 sec, then
centrifuged as above. From these tubes, 300 µL of supernatant was transferred
to a 7-mL plastic scintillation vial to which 5 mL of scintillation cocktail
(Scintiverse BD; SX 18-4; Fisher) was added and the mixture vortexed for 5 sec.
Tubes were read on a liquid scintillation counter (model LS5801; Beckman Instruments,
Schaumberg, IL, USA).
Brain Aromatase Activity
Brains were thawed and homogenized on ice in microfuge tubes for 5 sec using
a battery-powered tissue homogenizer. Next, 485 µL of RPMI 1640 culture
media (see above) was added and the brain/culture media mixture was further
homogenized for an additional 5 sec. To determine total protein content, an
80 µL sample was transferred to another tube (see below for total protein
protocol). Then, 11 µL of labeled androstenedione (see above) was added,
and each tube was incubated as described above.
Brain aromatase activity was standardized by dividing aromatase activity by
total protein (milligram per milliliter of bovine serum albumin). Total protein
was determined for each brain after a standard Bradford protocol (52).
Assay Validation
Aromatase activity is proportional to the amount of tritium in the scintillation
vials. It was calculated as a percentage of the total substrate added. After
subtracting the nonspecific tritium release, the disintegrations per minute
(dpm) of the sample tubes were converted to a percentage of the total dpm added.
This percentage was multiplied by the mass of the substrate added. Extraction
efficiency was determined by running tritiated water (NET-100B; Dupont NEN,)
through the same protocol as described and calculating the efficiency as a percentage
of the total counts tubes run in duplicate. After adjusting for the loss occurring
during extraction, the values obtained represent the amount of substrate converted
to tritiated water, which is proportional to the aromatase activity.
Assay was validated as follows: Sensitivity was defined as twice the mean
counts per minute of the blank tubes (0.38 pmol). Specificity of the assay was
determined by incubating ovaries from three reference-site females with an aromatase
inhibitor [4-androsten-4-ol-3,17-dione (A5791; Sigma) dissolved in methanol
(27047-4; Aldrich, Milwaukee, WI,USA ) was added to the RPMI 1640 culture media
to a final concentration of 100 µM]. Mean aromatase activity was decreased
93% in the aromatase inhibitor-exposed ovaries (0.12 pmol/ follicle) compared
with ovaries without inhibitor (1.8 pmol/follicle). Sensitivity (1.9 pmol) and
specificity (mean aromatase activity decreased 70% in the aromatase inhibitor-exposed
brains) for the brain aromatase assay were determined using the same methodology
as described for ovarian aromatase assay.
Statistics
Anal fin length was compared between fish collected from the Fenholloway and
Econfina Rivers using an analysis of covariance (ANCOVA) with standard length
as the covariate and segment number was analyzed by Mann-Whitney U-test.
Ovarian aromatase activity (pmol/follicle) was compared using the Mann-Whitney
U-test, and the number of follicles per ovary was compared with an unpaired
t-test. An ANCOVA for brain aromatase activity as the dependent variable
and total protein as the covariate was used to compare fish from the two rivers.
All analyses were carried out using the statistical software package StatView
5.0 (Abacus, Berkeley, CA, USA). F-tests for homoscedasticity were performed,
and where necessary, data were transformed and rechecked (53). Where
variance was still heterogeneous, after transformation, nonparametric statistics
were used. All data are reported as the mean ± 1 SE, and significance was
determined at p < 0.05. Last, all reported values are nontransformed
data.
Results
Fish Morphometrics
Fenholloway River females were significantly smaller in size as both standard
length (U = 116.5, p < 0.0001) and mass (U = 178, p
< 0.0001) were decreased compared with Econfina fish. Females from the
Fenholloway averaged (±1 SE) 2.44 (±0.05) cm in length and 298.4 (±19.6)
mg in mass, whereas females from the reference river, the Econfina, averaged
3.3 (±0.08) cm in length and 728.97 (±88.46) mg in mass. There was
no mean difference in the number of follicles with Fenholloway females [33.7
(±2.8)] and Econfina females [35.8 (±3.1)] (t = -0.493,
df = 1, 64, p = 0.62). Anal fin length was not different between the
two sites (F = 0.003, df = 3, 79, p = 0.95), whereas Fenholloway
females had more segments than females from the Econfina River (U = 302,
p < 0.0001).
Aromatase Activity
Ovarian aromatase activity was examined in females from both rivers and reported
as picomoles of activity per follicle. Because of the presence of embryos in
the ovaries of these viviparous fish, expressing aromatase activity per milligram
of ovary weight would be inappropriate. An initial F-test for aromatase
activity indicated that the variance between these two data sets was significantly
heteroscedastic (F = 7.98, p < 0.0001). Arcsine transformation
had no effect on the heteroscedastic nature of the variance difference of these
data sets (F = 9.04, p < 0.0001). Thus, the nonparametric Mann-Whitney
U-test was performed to determine whether a significant difference existed
in aromatase activity. Females from the Fenholloway River exhibited elevated
ovarian follicular aromatase activity when compared with females from the Econfina
River (U = 260.0, p = 0.0039 (Figure 1).
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Figure 1. Ovarian aromatase
activity (mean ± 1 SE, pmol/ follicle) in female mosquitofish collected
from the Fenholloway (Fen, n = 27) and Econfina (Econ, n
= 34) Rivers, Florida, USA. Sample size is given at the bottom of each
bar. Populations exhibited a significant difference in ovarian aromatase
activity (p = 0.0039).
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Female fish from the Fenholloway River exhibited elevated brain aromatase
activity when compared with fish from the reference site (F = 14.4, df
= 1, 64, p = 0.0003 (Figure 2). No significant difference in the variance
of brain aromatase activity was observed (p = 0.38) between the two populations.
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Figure 2. Brain aromatase
activity (mean ± 1 SE, pmol/mg protein) in female mosquitofish
collected from the Fenholloway (Fen, n = 32) and Econfina (Econ,
n = 34) Rivers, Florida, USA. Sample size is given at the bottom
of each bar. Populations exhibited a significant difference in brain
aromatase activity (p = 0.0003).
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Discussion
Environmental chemicals of natural and synthetic origin can interact with
the endocrine system and alter development and reproduction in wildlife and
humans. Most of our understanding has come from studies examining receptor-based
estrogenic or antiandrogenic compounds or complex mixtures. Given the plethora
of synthetic agricultural and industrial compounds that are used and eventually
find their way into the environment, it is reasonable to predict that some of
these could interact agonistically or antagonistically in other receptor-based
signaling pathways as well as through other mechanisms such as altering rates
of hormone degradation and storage. Indeed, two examples of environmental androgens
have been documented: the masculinization of a) gastropods via tributyl
tin exposure in Europe and the United States (54-56) and b)
female mosquitofish and other fish exposed to paper mill effluent in Florida,
USA (44,45,57).
In this study we tested the hypothesis that masculinization of female mosquitofish
exposed to paper mill effluent in the Fenholloway River was due to aromatase
enzyme inhibition. Microbial degradation of phytosterols (e.g., ß-sitosterol
and stigmastanol) commonly found in paper mill effluent has induced masculinized
anal fin morphology in laboratory exposures (49,58). Other components
of paper mill effluent include lignans and isoflavonoids (59). In humans,
many biological effects are known for these compounds, including anticarcinogenic,
bacteriocidic, and fungistatic activities for lignans (60). Although
weak, inhibition of placental and preadipocyte aromatase activity in humans
is associated with lignans, isoflavonoids, and flavonoids (61,62). We
hypothesized an inhibition of aromatase activity resulting in decreased estrogen
synthesis and a masculinized internal hormonal milieu for the exposed females.
This hypothesis was not supported because, interestingly, we found elevated
ovarian and brain aromatase activity in the Fenholloway River females compared
with that in the reference-site females.
Results of this study do not support aromatase inhibition as a mechanism of
Fenholloway mosquitofish masculinization. They do suggest, however, that the
masculinization is due to androgenic contaminants in the river. Although the
data on androgen exposure and aromatase activity in fish are somewhat inconclusive
[in one study, medaka (Oryzias latipes) treated with methyl testosterone
exhibited decreased aromatase activity (63)], the exposure to environmental
androgens is not inconsistent with increased aromatase activity. In some teleosts
such as the goldfish (Carrasius auratus), there is an association between
plasma levels of aromatizable androgen, brain aromatase activity, and aromatase
mRNA at the beginning of the reproductive season (64). Further, goldfish
treated with high doses of aromatizable androgen had increased plasma E2,
supporting the relationship between increased plasma androgens and aromatase
activity (65).
Through collaborative studies, we know that there are androgenic substance(s)
in the Fenholloway River (15). The Fenholloway River downstream of the
Buckeye Paper Mill is a highly impacted water body. Female mosquitofish living
there have masculinized anal fin development and are smaller in length and mass.
Although controversial, some research has suggested that exposure to the paper
mill effluent masculinized the behavior of the exposed females (44),
whereas other studies have shown a lower level of aggressive reproductive behavior
in experimentally exposed fish (46). Although smaller in size, masculinized
with respect to anal fin morphology and possibly behavior, and having altered
ovarian and brain aromatase activity, female mosquitofish from the Fenholloway
River still produce embryos. However, we do not know if these fry are viable
or if there are transgenerational effects from exposure to paper mill effluent
in these fish. We suggest that female mosquitofish are struggling to maintain
an internal hormonal milieu while bathed with external environmental androgenic
substances downstream of the paper mill effluent. These female fish exhibit
resilience, which allows them to persist when other species have gone locally
extinct.
Although individuals are known to have differing abilities in reproduction
or responsiveness to environmental perturbation, few studies have examined the
role of variation in maintaining resilience (66). Resilience theory has
great potential in helping us understand the influences of sublethal environmental
contamination (67). Resilience theory has provided a powerful tool for
ecologists to "explain" the stability and persistence of relatively complex
ecosystems. Further, the models have allowed the examination of the relative
stability of "alternate states" and thereby provide a means for determining
levels of resilience in populations (68). It is a given that change will
occur in an organism's physiology as their environment changes, but how those
changes relate to population stability over time is still a question. The mosquitofish
of the Fenholloway River are clearly influenced by their environment and exist
in an altered state. Although the females exhibit masculinization, they survive
and apparently maintain the ability to reproduce. Further studies are required
to determine whether the elevated aromatase reported in this study is enough
to allow these females to overcome an adverse environment and help maintain
a viable population. This study, as with the many others performed during the
last decade, has clearly demonstrated the complexity of trying to predict ecosystem
or population health when wildlife populations experience sublethal but detrimental
impacts of chemical exposure via endocrine disruption.
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