A Short-Term *In Vivo* Screening System for Endocrine Disruptors
Utilizing Mosquitofishes

**(*Gambusia affinis* and *G. holbrook*)**

Project Scope

Female mosquitofish can be useful bioindiators in helping to identify
compounds that effect endocrine systems at environmentally-relevent
concentrations. Because mosquitofish are small, easily bred, and have
short life and reproductive cycles, they can be used to develop
short-term in vivo and in vitro tests for hormonal activity that are
fast, reliable, and inexpensive.

Several studies have demonstrated the usefulness of characterizing the
responses of female mosquitofish to substances with androgenic activity
and have provided basic dose-response data on the masculinizing effects
of various androgens on female mosquitofish.

The purpose of this research was to establish and document the use of
mosquitofish (*Gambusia affinis* and *G. holbrooki*) as highly sensitive
bioindicator organisms for use in screening substances with suspected
endocrine activity, either singly or in complex mixtures. Expression of
the (normally female-limited) vitellogenin gene in males was used to
test chemicals for estrogenic activity. A morphological trait (modified
anal fin called a gonopodium) was used to test for androgenic activity.
The gonopodium is not found in normal females, but is readily induced to
develop in females exposed to chemicals with androgenic activity. In
addition to their utility as laboratory test organisms, these fish were
shown to be suitable as sentinel species for the detection of endocrine
disruptors in a variety of fresh- and warm salt-water environments.

To address these objectives the project sought to: 1) develop an assay
for vitellogenin (VTG) that could be used to detect this egg yolk
protein in the serum of male mosquitofish; 2) develop a quantitative
index of the effects of androgenic chemicals on the anal fin morphology
of females; 3) perform dose-response experiments on the effects of
estrogenic organochlorine insecticides (singly, and in mixtures) on VTG
expression in male mosquitofish; 4) perform dose-response experiments on
the effects of androgenic chemicals on the anal fin morphology of
females; 5) investigate the effects of prior hormone exposures on
reproductive function of female mosquitofish; and 6) perform laboratory
and field tests of selected potential endocrine disruptors.

Project Results and Implications

[Vitellogenin as a Biomarker for Estrogens]{.underline}. Vitellogenin
(VTG) was first identified and purified and then an anti-VTG immume
serum was produced and characterized using Western blot. Analysis
revealed that antibodies in the immume serum bound specifically to the
protein previously identified as VTG, but did not bind to any other
protein in male or female blood. Also, when blood samples were probed
with pre-immune serum, no binding was observed.

To determine if this immunoblotting would be useful for monitoring the
effect of estrogenic compounds on vitellogenesis, Western blotting was
coupled with densitometric scanning to assess the dose- and
time-dependency of 17a-ethynylestradiol (EE~2~)-induced vitellogenesis
in male mosquitofish. VTG immunoreactivity was detected in blood samples
from fish treated with EE~2~ doses as low as 10 ug/g of food. The assay
has been used to demonstrate that the effect of EE~2~ on vitellogenin
synthesis in male *G. affinis* is both concentration- and
time-dependent.

[Assay for Gonopodium Development in Females]{.underline}. A
quantitative morphological study and dose-response experiment of
gonopodium development in normal males and in females treated with doses
of 11-ketotestosterone was completed using computer image analysis
techniques. The length and number of anal fin rays were used to quantify
the degree of gonopodial development, both in normal males and
androgen-treated females, and results of doses ranging from 20 to 100 ug
of hormone/g of food were examined. The morphological results showed
that the length and width ratios of anal fins are useful in that they
can be determined with high precision and are not restricted to a
limited size range. Dose-response results show that at the higher doses
(60, 80, and 100 ug/g), the androgen-treated females all developed
gonopodium-like characters in the anal fins, whereas at the lower doses
(20 and 40 ug/g), one of the four fish at each concentration did not
respond at all. The rapidity of response and extent of elongations
differed substantially among the doses given, but the response was
largely all-or-none (i.e., the fish either did or did not exhibit
changes in the anal fins), especially at the lower doses.

[Identifying the Androgenic Components of Paper Mill
Effluent]{.underline}. A toxicity identification and evaluation approach
was used to identify the androgenic components of paper mill effluent in
the water column and sediment downstream from a mill in Florida. The
river was chosen because of the occurrence of masculinized mosquitofish.
For the water samples, the approach consisted of fractionating water
extracts, performing in vitro assays to locate bioactive fractions, and
identifying compounds using mass spectrometry. For the sediment, the
approach involved solid phase extraction and high pressure liquid
chromatography purification, androgen receptor transcription assays, and
liquid chromatography mass spectroscopy.

All of the female fish collected (*G. holbrooki* mosquitofish, sailfin
molly, and killfish) from the river containing effluent from a nearby
paper mill were masculinized. Androstenedione was determined to be a
major component of the river water, at a concentration of 0.14 nM.
Androstenedione was found in the sediment at a concentration of 2.4 nM
or 17 times greater than measured in the water column, indicating that
androgens that are measured at low concentrations in the water have the
potential to contain much high sediment concentrations. In addition, a
biosynthetic precursor of androstenedione, progesterone, was measured in
the sediment at 155 nM, which is a concentration 65 times greater than
androstenedione in the sediment and 1000 times greater than that found
in the water for progesterone. It is therefore hypothesized that the
phytosteriods in the paper mill effluent tend to accumulate in the
sediment, where microbes convert them first into progesterone and then
into androstenedione and other bioactive steroids. Also, it was found
that unpolluted water bodies not downstream from mill effluent and with
much lower organic matter still contain progesterone, but at
substantially lower levels.

[Reproductive Characteristics of Male Mosquitofish]{.underline}.
Sexually mature male western mosquitofish (*G. affinis*) were collected
downstream of a large wastewater treatment plant near Birmingham,
Alabama to determine effects of exposure to estrogens. Although testes
and liver weights were higher in the effluent-exposed population, no
histological changes in the testes or liver were observed in the study
that would indicate exposure to estrogens and no detectable levels of
VTG were found in the blood of the male mosquitofish.

In addition to the work on the project already described, the following
is planned:

-   Dose-Response Assays With Androgenic Chemicals. These studies to
    characterize the relative sensitivity of the female anal fin rays to
    a variety of steroidal and nonsteroidal chemicals will continue.

-   Reproductive Effects of Masculinization in Females. Female
    mosquitofish of reproductive age are being masculinized by exposure
    to methyltestosterone. Then, the effects of the androgen exposure on
    their reproductive ability will be measured to examine the effects
    on ovarian weight and morphology, histology, female fecundity (brood
    frequency and size), and survivorship of the young after birth.

-   Effects of Masculinizing Chemicals on Embryos. Pregnant females will
    be treated with masculinizing chemicals, exposing the embryos in
    utero. The young will be raised to maturity so that the effects of
    the chemical treatments on sex ratio and fertility can be assessed.

Investigators

Robert Angus -- University of Alabama, Birmingham

Paul Blanchard -- Samford University

W. Mike Howell -- Samford University

R. Douglas Watson - University of Alabama, Birmingham

For More Information

<http://www.uab.edu/uabbio/angus.htm>

**NCER Project Abstract and Reports:**

[http://cfpub2.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/173/report/F](http://cfpub2.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/167/report/0)