Environmental Health Perspectives Volume
103, Supplement 4, May 1995
[Citation
in PubMed] [Related
Articles]
Vitellogenin Induction by Xenobiotic Estrogens in the Red-eared Turtle
and African Clawed Frog
Brent D. Palmer and Sylvia K. Palmer
Laboratory of Reproductive Ecology, Department of Biological Sciences,
College of Osteopathic Medicine, Ohio University, Athens, Ohio
Abstract
Many environmental pollutants have estrogenic activity in animals. Xenobiotic
estrogens include many pesticides and industrial chemicals that bioaccumulate.
The impact of these common pollutants on the reproductive success of wildlife
may be considerable, particularly in threatened or endangered species. This
research examined the use of plasma vitellogenin in males as a biomarker
for estrogenic xenobiotics in reptiles and amphibians. Adult male turtles
(Trachemys scripta) and frogs (Xenopus laevis) were given
ip injections of estradiol-17ß (E2), diethylstilbestrol
(DES), or o,p'-DDT (1-chloro-2-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene)
daily for 7 days, and plasma was collected on day 14. The estrogenic activity
of each compound was determined by measuring the induction of plasma vitellogenin.
Vitellogenin was identified by precipitation, electrophoresis, Western blot,
and enzyme-linked immunosorbant assay (ELISA). In both species, estradiol
and DES treatments induced the most vitellogenin, whereas DDT treatments
induced smaller amounts of vitellogenin in a dose-dependent fashion. These
data indicate that induction of plasma vitellogenin in males may be a useful
biomarker of xenobiotic estrogen activity in wild populations of reptiles
and amphibians. -- Environ Health Perspect 103(Suppl 4):00-00 (1995)
Key words: estrogen, xenobiotic, diethylstilbestrol, DDT, turtle,
frog, vitellogenin
This paper was presented at the Conference on Environmentally
Induced Alterations in Development: A Focus on Wildlife held 10-12 December
1993 in Racine, Wisconsin.
We acknowledge the efforts of Dr. Malcolm Powell for assistance
with polyclonal antibody production; Dr. Kenneth Goodrum for assistance
with ELISAs; Dr. Kyle W. Selcer for discussions and comments; Biotechnologies
for the Ecological, Evolutionary and Conservation Sciences Program for
plasma radioimmunoassays; and Jane Perkins and Sue Simon for assisting
with animal care, blood collection, and data analyses. This research was
supported in part by Ohio University College of Osteopathic Medicine, the
Department of Biological Sciences, the Office of Animal Research, and a
grant from Ohio University Research Committee.
Address correspondence to Dr. Brent D. Palmer, Laboratory
of Reproductive Ecology, Department of Biological Sciences, College of
Osteopathic Medicine, Ohio University, Athens, OH 45701-2979.
Introduction
Xenobiotic estrogens in the environment pose an insidious risk to both
wildlife and humans. By disrupting reproduction and developmental processes,
they may impair not only the individuals exposed, but have lasting influence
on their offspring as well. Xenobiotic estrogens include many herbicides,
fungicides, insecticides, and industrial chemicals. At least 45 chemicals
or their metabolites have been identified as endocrine disrupters (1).
The impact of these common pollutants on the reproductive success of wildlife
may be considerable. Due to their lipophilic nature and persistence in the
environment, many of these compounds bioaccumulate (2). Therefore,
those most affected will be those in the upper food chain or aquatic species
that are exposed to high doses due to toxic accumulation in aquatic ecosystems.
A rapid, sensitive, and inexpensive test to screen wildlife for the effects
of estrogenic chemicals is greatly needed. This test should be one that
demonstrates not merely the presence of a compound in the tissues or body
fluids, but measures a biological effect. An ideal test should meet several
criteria, specifically a) have widespread applicability across many
animal groups; b) require a small and easily obtainable sample without
causing undue harm or distress to the animals; c) measure a physiological
response to xenobiotic compounds; d) measure a response through known
biochemical pathways; e) be responsive to a large class of estrogenic
chemicals; and f) be quantifiable.
One of the most important and sensitive responses to estrogen is the
induction of protein transcription and translation (3,4). Particularly
well known among these responses is estrogenic induction of the lipoprotein
vitellogenin, expressed in all oviparous and ovoviviparous vertebrates (5,6).
This makes testing for vitellogenin useful as an indicator of estrogenic
activity over a wide range of vertebrate groups. Further, the mechanism
has been studied in detail as a model of estrogen action (4-13).
Vitellogenin is produced by cells in the liver in response to estrogen.
The response is only elicited if an estrogenic compound binds to and activates
estrogen receptors in hepatocytes. In females, vitellogenin is transported
in the circulatory system to the ovaries, where it is incorporated into
the developing ovarian follicles as yolk.
Males normally have no detectable production of vitellogenin due to their
normally low levels of endogenous estrogens (13-18). However, their
liver is capable of synthesizing and secreting vitellogenin into the blood
in response to exogenous estrogen stimulation (6,17-25). The response
is not as rapid or as strong as in females that are exposed to the same
concentrations of estrogen (18). However, since males normally have
no vitellogenin, the expression of any vitellogenin serves as an ideal biomarker
for xenobiotic estrogenic stimulation. Vitellogenin expression has been
used successfully for identification of exposure to environmental estrogens
in fish, including wild populations (26,27), and under laboratory
(28) and in vitro conditions (29,30).
This study was undertaken to determine if plasma vitellogenin is inducible
by xenobiotic estrogens in male turtles and frogs, therefore making it a
potential biomarker of xenobiotic estrogen exposure in reptiles and amphibians.
Turtles and frogs were exposed to a) natural estrogen, estradiol-17ß
(E2); b) a well-known artificial estrogen, diethylstilbestrol
(DES); and c) an important environmental estrogen, the pesticide
o,p´-DDT. Measurable vitellogenin was induced in both
turtles and frogs, indicating that vitellogenin may be useful as a biomarker
for xenobiotic estrogen exposure in amphibians and reptiles.
Materials and Methods
Species
Adult red-eared turtles (Trachemys scripta) were purchased from
a commercial dealer (Lemberger; Oshkosh, WI). The turtles were housed in
large Rubbermaid tubs (1.25 m long x 0.75 m wide x 0.75 m deep) at 21°C.
Each tub was supplied with a basking ramp mounted 0.3 m below 150 watt flood
lamps. This allowed the animals to thermoregulate their body temperature.
Food (Wardley Pond-Ten; Secaucus, NJ) was provided ad libitum 3 days
per week.
Adult male African clawed frogs (Xenopus laevis) were purchased
from a commercial dealer (Sullivan; Nashville, TN). The frogs were divided
randomly into groups of 5 and housed in 5 gal glass aquaria in 10 cm of
water maintained at room temperature (21°C). Food (Wardley Pond-Ten)
was provided 3 days per week, and the water was changed the following day.
Chemicals
Estradiol-17ß (E2; Sigma, St. Louis, MO), diethylstilbestrol
(DES; Sigma), and o,p´-DDT (1-chloro-2-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene;
AccuStandard, New Haven, CT) were dissolved in corn oil. Placebos consisted
of pure corn oil. Volumes of all injections were equilibrated across treatment
groups for each species.
Production of Turtle Vitellogenin for Antibody Production
Six adult female turtles were injected daily with 1 µg/g E2
for 7 days, and on alternate days until day 21. Blood was collected from
the caudal vein of each turtle using a 1-ml syringe and a 26-gauge needle
(31). Whole blood was centrifuged in heparinized tubes for 2 min
at 14,000g, and the plasma was separated and frozen at -20°C.
Vitellogenin was purified by precipitation and checked for purity by electrophoresis.
Purification of Vitellogenin
The protocol for purification of vitellogenin was modified from that
described by Wiley (32). Two hundred fifty µl of plasma sample
were gently mixed with 1 ml of 20 mM Na2EDTA and 80 µl
of 0.5M MgCl2 and centrifuged at 2500g for 15 min. The
supernatant was discarded, and the pellet containing vitellogenin was redissolved
in 150 µl of 1 M NaCl, 50 mM Tris-HCl (pH 7.5) and then centrifuged
at 2500g for 30 min. The supernatant was removed to another centrifuge
tube, and then vitellogenin was precipitated with 1.24 ml distilled H2O
and centrifuged at 2500g for 15 min. The resulting pellet of purified
vitellogenin was redissolved in 1M NaCl, 50 mM Tris-HCl (pH 7.5) and stored
at -20°C. Purification of frog vitellogenin was performed using 50 µl
of plasma and proportionately reduced quantities of other reagents. Quantification
of vitellogenin was accomplished using Lowry reagent (BioRad, Melville,
NY).
Polyclonal Antibody Production and Purification
Turtle (T. scripta) vitellogenin was purified as above and used
to produce polyclonal antibodies in rabbits. Turtle vitellogenin (0.50 mg
in 1 ml of 1 M NaCl, 50 mM Tris-HCl, pH 7.5) was mixed with 1 ml of Freund's
complete adjuvant and injected intradermally at four to six sites along
the back of rabbits using a 20-gauge 1.5-inch needle. Using the above protocol
the rabbits were boosted twice with Freund's incomplete adjuvant at 3-week
intervals. The rabbits were bled by cardiac puncture under deep anesthesia.
The blood was allowed to clot at 4°C overnight to separate the serum,
which was then stored at -80°C.
Antivitellogenin antibodies were purified using a procedure modified
from Harlow and Lane (33). A 1-ml column was prepared by swelling
0.3 g Sepharose-4B-CNBr in 1 mM HCl for 15 min, rinsing with 70 ml of 1
mM HCl, and washing 3 times with 1 ml coupling buffer (0.5 M NaPO4,
pH 7.5). Precipitation-purified vitellogenin was dissolved in coupling buffer
(20 mg/2 ml), added to the Sepharose column, agitated overnight, and rinsed
twice with coupling buffer. The column was washed once with 1 M NaCl, 0.05
M NaPO4 (pH 7.5) and incubated overnight, with gentle rocking,
at room temperature with 10 vol of blocking buffer (100 mM ethanolamine,
pH 7.5). The column was washed twice with phosphate-buffered saline (PBS;
140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)
and merthiolate added to 0.01% and stored at 4°C until use.
Before use, the column was rinsed with 10 ml of 10 mM Tris buffer (pH
7.5) and flushed with 10 ml of 100 mM glycine buffer, pH 2.5, followed by
10 ml of 10 mM Tris buffer pH 8.8. The column was then washed with 10 ml
of 100 mM triethylamine (pH 11.5), followed by 10 ml of 10 mM Tris, pH 7.5.
Following column preparation, polyclonal antiserum specific to vitellogenin
was diluted to 10% in 10 mM Tris, pH 7.5 and passed through the column 3
times with a flow rate of 1 ml/min. The column was washed with 20 ml of
10 mM Tris, pH 7.5 and then 20 ml of 500 mM NaCl, 10 mM Tris-buffered saline
(pH 7.5). The column was eluted with 20 ml of 100 mM glycine buffer, pH
2.5, and the antibodies collected in a tube containing 1 ml of 1 M Tris,
pH 8.8. The column was washed with 10 mM Tris, pH 8.8, the column eluted
again with 10 ml of 100 mM triethylamine, pH 11.5. Antibodies were collected
in a tube containing 1 ml of 1 M Tris, pH 8.8. The vitellogenin antibody
fractions were combined and dialyzed against PBS with 0.02% sodium azide.
Specificity of purified antivitellogenin antibody was tested against
whole plasma, precipitation-purified vitellogenin, a protein of 214 kDa
(the presumptive vitellogenin identified electrophoretically) electroeluted
from 5% PAGE, and bovine serum albumin (BSA). Antivitellogenin antibodies
were tested by enzyme-linked immunosorbant assay (ELISA) and Western blot
to determine cross-reactivity. Controls included omission of protein antigen,
omission of primary antibody, and omission of secondary antibody. Only batches
of purified antivitellogenin antibody that exhibited high affinity for vitellogenin
and minimal cross-reactivity with other antigens were used in immunodetection.
One-dimensional Polyacrylamide Gel Electrophoresis
Whole plasma or purified vitellogenin samples were solubilized in 10
mM Tris buffer (pH 7.4) containing 2% (w/v) sodium dodecyl sulfate (SDS)
and 10% (v/v) 2-mercaptoethanol and separated by molecular weight using
one-dimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE). Separation
gels were made from 5% T (total acrylamide) to facilitate analyses of high
molecular weight proteins (34). Plasma samples or purified vitellogenin
were loaded on to a discontinuous PAGE apparatus, then run at 200 V for
35 min or until complete. The gels were fixed and stained with Coomassie
blue (34) and silver (35). Molecular weights were determined
using BioRad high molecular weight standards and calculation of Rf
values (36). Vitellogenin was quantified from 1D-SDS-PAGE of plasma
using a BioRad GS-670 imaging densitometer to digitize the images. The concentrations
of vitellogenin (band densities) were calculated by plotting the band absorbance
and integrating the area under the curve for vitellogenin (BioRad Molecular
Analyst software). Comparisons of treatment groups were performed using
Kruskal-Wallis one-way analysis of variance on ranks (ANOVA: p<0.05).
Multiple means comparisons were performed using the Student-Newman-Keuls
method (37).
Western Blotting
To identify vitellogenin immunologically, whole plasma proteins or precipitation
purified vitellogenin were transferred to polyvinylidene difluoride (PVDF)
membranes immediately following 1D-SDS-PAGE. Briefly, the gels were equilibrated
in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol) (38)
immediately following electrophoresis, and the proteins were transferred
to a PVDF membrane under a 15V electrical field for 1 hr. After transfer,
the PVDF membrane was equilibrated in Tris-buffered saline (TBS; 25 mM Tris,
0.3M NaCl, pH 7.4) for 15 min, blocked with 5% (w/v) powdered milk in TBS
(2 hr), and incubated overnight with primary antibodies (diluted 1:50,000
in 5% (w/v) powdered milk in TBS) specific for vitellogenin. The transfer
membrane was washed in TBS (3 x 5 min) and incubated for 2 hr with goat
anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Sigma) diluted
1:1,000 in 5% powdered milk in TBS. The PVDF membrane was washed again in
TBS (3 x 5 min), and incubated at 37°C in 0.1 g/l nitro blue tetrazolium
(NBT), 0.05 g/l 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 2 mM MgCl2
in 0.1M Tris buffer (pH 8.8 for 15 min) for localization of reactive proteins.
Induction of Vitellogenin in Turtles with E2, DES, and
o,p´-DDT
Adult male turtles were ranked by mass and divided into experimental
and control groups to equalize specimen sizes among treatments. The treatment
groups consisted of animals treated with 1 µg/g E2, 1 µg/g
DES, 250 µg/g o,p´-DDT, 1 µg/g o,p´-DDT,
and oil only as control. Each compound was dissolved in 0.1 ml corn oil
and injected ip. The treatments were given daily for 7 days. Plasma was
collected on day 14 from the caudal vein of each turtle using a 1-ml syringe
and a 26-gauge needle (31). Whole blood was spun in heparinized tubes
for 2 min at 14,000g, and plasma was separated and frozen at -20°C.
This treatment protocol with exogenous E2 has been shown to produce
circulating concentrations of serum E2 in turtles of approximately
333 pg/ml (Selcer and Palmer, unpublished data), which is equivalent to
E2 concentrations in females during natural vitellogenesis.
Induction of Vitellogenin in Frogs with E2, DES, and o,p´-DDT
Twenty-five adult male frogs of matched size were randomly assigned into
treatment groups of five and housed in separate 5 gal aquaria with 10 cm
of water. The treatment groups consisted of animals treated with 1 µg/g
E2, 1 µg/g DES, 250 µg/g
o,p´-DDT, 1 µg/g o,p´-DDT, and oil only
as control. Compounds were dissolved in 40 µl of corn oil and injected
ip. Injections were given daily for 7 days, and frogs were bled by cardiac
puncture on day 14. Whole blood was centrifuged in heparinized tubes for
2 min at 14,000g, and plasma separated and frozen at -20°C.
ELISA
ELISA (enzyme-linked immunosorbant assay) was used to determine relative
levels of vitellogenin in plasma samples. Ten- microliter plasma samples
and 10 µl excess BSA were each diluted with 40 µl TBS (25 mM
Tris, 0.3M NaCl, pH 7.4). The diluted plasma solutions were added to individual
wells of a polystyrene microtiter plate, and the BSA solution was added
to a single well as a control; and the plate was incubated overnight at
4°C. Plates were washed three times with TBS-Tween (TBS plus 0.2% Tween
20) and blocked with 150 µl Blotto-Tween (5% nonfat dry milk, 0.2%
Tween 20, and 0.02% sodium azide in TBS) for 2 hr. The plates were washed
three times with TBS-Tween, and 50 µl antivitellogenin antibodies
diluted 1:20,000 in Blotto-Tween were added to each well. The plates were
then incubated for 2 hr at room temperature. The plates were thoroughly
washed four times with Blotto-Tween and incubated for 2 hr with goat anti-rabbit
immunoglobulin conjugated to alkaline phosphatase (Sigma) diluted 1:1,000
in Blotto-Tween. After thoroughly washing each well with TBS-Tween 4 times,
the plates were developed in 10% p-nitrophenyl phosphate (PNPP) in
developing buffer (0.1 M NaCl, 0.1 M Tris, 5 mM MgCl2, pH 9.5)
and incubated for 15 min at room temp. The reaction was stopped with 50
µl of stop buffer (20 mM Tris, 5 mM EDTA, pH 7.5), and the plate densities
were read on a Bio Tek Microplate Reader at 405 nm. The well coated with
BSA served as the reagent blank, and its absorbance was subtracted from
that of the other wells. Statistical comparisons among treatment groups
were performed using Kruskal-Wallis one-way analysis of variance on ranks(ANOVA:
p<0.05) Multiple means comparisons were performed using the Student-Newman-Keuls
method (37).
Results
Induction of Vitellogenin in Turtles
A protein of 214 kDa was extractable from E2, DES, and both
o,p´-DDT treatment groups. This protein exhibited cross-reactivity
with antivitellogenin antibodies in Western blots (Figure 1) and was identified
as vitellogenin. ELISA analyses of day 14 turtle plasma indicated significant
(p<0.001) induction of vitellogenin production from E2,
DES, and both DDT treatments (Figure 2). E2 and DES, a well known
artificial estrogen, induced the highest concentrations of vitellogenin.
The persistent pesticide o,p´-DDT induced smaller amounts of
vitellogenin in a dose-dependent manner. No vitellogenin was extractable
from the control plasma or detectable using 1D-SDS-PAGE and Western blot
analyses, indicating that the slight absorbance of control plasma in ELISA
analyses is attributed to nonspecific binding of the polyclonal antibody
(Figure 2).
Figure 1. Western
blot of plasma from a 1D-SDS-PAGE of control (lane A) and estrogen-treated
(lane B) male turtles (Trachemys scripta) using antivitellogenin
antibody. A protein of 214 kDa (arrow), vitellogenin, is present in plasma
of estrogen-treated specimens, but not in controls.
Figure 2. Vitellogenin
induction in male turtles treated for 7 days with 1 µg/g estradiol-17ß,
1 µg/g DES, 250 µg/g o,p'-DDT, or 1 µg/g o,p'-DDT.
Plasma was collected on day 14 of treatment. Vitellogenin was measured using
ELISA with purified polyclonal antibody to turtle (T. scripta) vitellogenin.
Treatments are significantly different at p<0.001. Treatment groups
with different letter designations are statistically different by the Student-Newman-Keuls
method.
Induction of Vitellogenin in Frogs
A protein of approximately 200 kDa was extractable from the plasma of
frogs treated with E2, DES, and both DDT regimes (Figure 3),
but was not extractable from the plasma of control specimens. This protein
was purified as described above and identified as vitellogenin. However,
this protein demonstrated no cross-reactivity with the antiturtle vitellogenin
antibodies. Computerized image analysis of ID-SDS-PAGE was used to quantify
relative concentrations of vitellogenin induced by the treatment regimes.
The pattern of vitellogenin production in frogs mirrored that seen in turtles,
with significant variation (p<0.001) in relative quantities of
vitellogenin produced by treatments (Figure 4). E2 induced the
most vitellogenin, followed by DES and o,p´-DDT in a dose-dependent
fashion. No extractable vitellogenin was detectable by 1D-SDS-PAGE from
control samples.
Figure 3. Coomassie
blue-stained 5% 1D-SDS-polyacrylamide gel of vitellogenin extracted from
plasma from control (lane A) and DDT-treated (lane B) male frogs (Xenopus
laevis). Induction of 200 kDa protein, vitellogenin (arrow), in males
is present in plasma of estrogen-treated specimens, but not in controls.
Figure 4. Vitellogenin
induction in male frogs (Xenopus laevis) treated for 7 days with
1 µg/g estradiol-17ß, 1 µg/g DES, 250 µg/g o,p'-DDT,
or 1 µg/g o,p'-DDT. Plasma was collected on day 14 of treatment.
Vitellogenin concentration was quantified from 1D-SDS-PAGE by computerized
image analysis. Treatments are significantly different at P<0.001.
Treatment groups with different letter designations are statistically different
by Student-Newman-Keuls method.
Discussion
DES was developed as a synthetic estrogen that was used to prevent premature
births in women beginning in 1948. Over 1 million women took DES between
1960 and 1970 (1). DES was shown to have significant detrimental
effects on embryonic development in humans and animal models, and was subsequently
banned from use by pregnant women in 1971. Daughters of women who took DES
suffer reproductive organ dysfunction, reduced fertility, abnormal pregnancies
(39), and increased incidence of vaginal clear-cell adenocarcinomas
(40). Similar reproductive and developmental problems have been seen
in female laboratory animals (39,41-43). Exposure of male humans
and male laboratory animals to DES during development leads to lesions of
the reproductive system (44,45) and an increased incidence of reproductive
tract cancers in laboratory animals (46). The mechanism of DES action
has been well studied. It is an agonist of estrogen receptors, binding to
them and eliciting transcription of estrogen-induced proteins (39,47).
The use of the organochlorine pesticide DDT was restricted in the United
States in 1972; however, elevated levels of DDT and its metabolites are
still encountered in the tissues of wildlife in this country (48).
Although some of this can be due to DDT's long half-life (57.7 years) in
temperate soils (49), this does not entirely account for the continuing
high levels. A likely source is the continued manufacture and use of DDT
in developing countries (1). DDT used in developing countries is
readily vaporized and transported globally via the atmosphere (50,51),
providing a continuing rain of DDT throughout the world. This indicates
that DDT is still an important environmental hazard and serves as a good
model for physiological studies due to the extensive studies on its mechanism
of action.
DDT is well known for its effects on reproduction in avian species, most
notably causing eggshell thinning (52). However, the role of DDT
as an estrogen has been more insidious (53). DDT has two isomers,
p,p´-DDT and o,p´-DDT, which break down into their
respective metabolites, p,p´-DDE and o,p´-DDE
(53). The o,p´ isomers of DDT and DDE bind to estrogen
receptors agonistically, mimicking endogenous estrogen (54,55).
The induction of vitellogenin by a compound indicates that it is an agonist
of hepatic estrogen receptors, stimulating them to induce transcription
of the vitellogenin genes. Both the artificial estrogen, DES and the pesticide
DDT were able to stimulate male turtles and frogs to produce significant
levels of vitellogenin. DES has been shown to bind to nuclear estrogen receptors
in turtles, inhibiting 96.7% of specifically bound E2 (56).
In the environment, DDT is known to accumulate in large concentrations in
turtles and frogs. The softshell turtle has been recorded with 732 ppm DDT
in adults (57). Adult toads have exhibited 0.13 ppm DDT (57).
An insidious aspect of lipophilic compounds is that they are also deposited
in eggs during vitellogenesis. It is likely that as lipids are mobilized
from body stores, lipophilic contaminants also are mobilized and become
incorporated into the growing ovarian follicles. Thus the adult's burden
of lipophilic contaminants may be passed on to their offspring, where they
can influence the fertilizability of the egg and development of the embryo,
the most sensitive period of the life cycle. Eggs of loggerhead sea turtles
exhibited approximately 0.1 ppm DDE (58), equal to the highest levels
detected in the adult turtles (59). In the snapping turtle, up to
864 ppb DDT has been reported in their eggs (60). The levels of organochlorine
contaminants in reptilian eggs correlates with increases in deformities
and lowered rates of hatching success (61). In species that exhibit
temperature-dependent sex determination (TSD), such as many turtles, the
implications for effects by environmental estrogens are enormous. The sex
of the offspring in species that exhibit TSD is regulated by levels of sex
steroids (62,63). By increasing levels of perceived estrogen, xenobiotic
estrogens may adversely affect sexual development in these species. It has
been shown that exposure of turtle eggs to xenobiotic estrogens induces
ovary-like characteristics in hatchlings incubated at male-producing temperatures
(64).
Vitellogenin may prove useful as a biomarker for exposure to xenobiotic
estrogens due to its numerous advantages. First, it is applicable to a wide
range of species, potentially any oviparous or ovoviviparous vertebrate.
The criterion for using vitellogenin as a biomarker in a particular species
is that males do not normally express vitellogenin, but are capable of doing
so in response to estrogenic compounds. Second, the test is relatively noninvasive,
requiring only small (microliter) quantities of plasma or serum. This eliminates
the necessity of acquiring tissue biopsies of organs such as liver, muscle,
and fat. Third, it indicates a physiological response to an environmental
challenge, indicating that the animal is being influenced by environmental
estrogens. This eliminates the confusion often encountered when contaminants
are detected in wildlife, but without knowing if the contaminant is causing
any physiological effects. Fourth, the expression of vitellogenin is through
known physiological and biochemical pathways. Indeed, the induction of vitellogenin
has been studied intensively as a model for steroid regulation of gene expression
and protein synthesis (4-13). Fifth, the induction of vitellogenin
is sensitive to any estrogenic contaminant. In fact, the vitellogenic response
has been used to screen unknown compounds for estrogenic properties (29,30).
Sixth, the response is quantifiable. Finally, the assay for vitellogenin
can be performed relatively easily and inexpensively, especially compared
to the exorbitant cost of screening for a wide range of specific contaminants.
The vitellogenic response has been used to test for exposure to estrogenic
xenobiotics in fishes (26-30).
There are, however, several drawbacks regarding vitellogenin analysis
that must be considered. It is unlikely that the production of vitellogenin
in males represents a seriously deleterious physiological response. This
assay also provides no direct information regarding the female or developing
embryo. However, if estrogen receptors are being stimulated in the liver
of males, receptors in other organs such as the testes and prostate gland
of males and reproductive tissues of females and embryos may likewise be
affected. In fact, since females and embryos often show greater responses
to estrogen, they may be affected equally or even more so. Further, vitellogenin
production does not indicate what compound may be causing the effect. However,
it may be used as a rapid, sensitive, and economical initial screen, followed
(as indicated by positive vitellogenic responses) by more costly screens
to identify the specific contaminating compounds. This assay assumes that
males do not produce vitellogenin but are capable of doing so. This requires
that some physiology of the species be known, such as comparing contaminated
populations to clean control populations. These limitations considered,
vitellogenin may serve as an excellent biomarker for xenobiotic estrogen
exposure in reptiles and amphibians or other oviparous and ovoviviparous
vertebrates.
Increasingly more evidence of the estrogenic effects of contaminants
on wildlife is appearing. In the late 1970s, male herring gull embryos and
chicks from Lake Ontario showed the development of oviducts and ovarylike
gonads. Laboratory studies have since shown that estrogenic pesticides such
as dicofol, kelthane, and methoxychlor produce these effects in California
gulls, western gulls, and kestrels (65). Herring gull populations
contaminated with estrogenic compounds such as DDT, DDE, and PCBs have been
shown to form female-female pairs (66). In Florida, the occurrence
of demasculinized and feminized male alligators is associated with the presence
of the estrogenic pesticides (67). Hermaphroditic fish populations
have been reported in the lagoons of sewage treatment works (27).
Are these examples of wildlife effects isolated incidents or just the
tip of the iceberg? It is unclear at this point. Some populations, such
as bald eagles surrounding the Great Lakes, are still in trouble. Adult
bald eagles that migrate to the lake shore develop reproductive difficulties
associated with contaminated food (48). The Great Lakes is acting
as a sink for bald eagles migrating from reproductively fit inland populations.
Alligators in Lake Apopka, Florida, have drastically reduced reproductive
success (67,68). These local populations of long-lived species may
be heading for extinction, because reproduction on the site is insufficient
to maintain the population size, requiring migration from surrounding populations
to sustain them. It is possible that the decline of other species around
the globe, such as amphibians (69-74), is partially linked to reproductive
impairment by endocrine-disrupting compounds (BD Palmer, unpublished data).
The assay of vitellogenin in males of these and other species in the wild
would indicate whether they are being physiologically influenced by the
estrogenic properties of environmental contaminants.
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