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Feminization
of Fish and Analysis of Estrogenic Compounds
Dr. Hiroaki Shiraishi, National Institute of Environmental Studies,
Japan
It is well known that vitellogenin, a precursor protein of egg yolk,
in male fish is useful biomarker to examine estrogenic activity of
anthropogenic contaminants in aquatic environment. It is reported
that vitellogenin levels in wild male flounder in Tokyo Bay are statistically
higher (p<0.001) than these of control site, where little influence
of sewage and wastewater is expected. Vitellogenin can be measured
by enzyme immunoassay. We selected Medaka fish (Oryzias latipes) as
test organism and developed automated Medaka vitellogenin ELISA system
after preparation of a suitable antibody and antigen. But the preparation
of vitellogenin specific antibody, which could be applicable to enzyme-linked
immunosorbent assay (ELISA) takes long time. Quantification of the
biomarker genes, which are expressed in advance of protein induction,
is another sensitive screening technique. If gene sequence of biomarker
mRNA is known, it is detectable in high sensitivity with RT-PCR. Until
recently complicated operation with the extraordinary labor is needed
to quantify mRNA because of narrow dynamic range of RT-PCR, but the
problem have been improved by real-time RT-PCR method. In Medaka,
genes, such as choriogenin H and L, vitellogenin, and an estrogen
receptor, can be used.
To monitor the estrogenic activity in aquatic environments, we have
developed a rapid estrogenicity assay procedure using the yeast two-hybrid
system. The original assay system was implemented through two methods,
the 96-well plate-culture method as a simple procedure and the chemiluminescent
reporter-gene assay method for more sensitive determinations. The
assay system is designed for use both as an agonist test with/without
metabolic activation using rat liver S9 and as an antagonist test.
17$-estradiol (E2), which is considered as responsible substance for
fish feminization, was detected at relatively high concentrations
(> 10 ng l -1 ) in waters of the Tama River, an urban river running
through Tokyo, and Lake Kasumigaura during governmental surveillance
of endocrine-disrupting chemicals. The E2 concentrations at the sampling
stations were near the threshold value that induces vitellogenin,
an egg-precursor protein, in male fish. The analytical methodology
in the national surveillance studies was ELISA. ELISA is highly sensitive,
and has been successfully applied to E2 determination in clinical
studies, but not to environmental samples; it is not possible to confirm
the presence of E2 by ELISA alone. For this purpose, we developed
another sensitive, instrumental analytical method for E2, its metabolite
(estron, estriol) and ethynyl estradiol. Sample waters are spiked
with stable isotope-labeled E2-d4, and are extracted by the solid-phase
extraction method. The extracts are sequentially converted to pentafluorobenzyl
(PFB) derivatives by PFB bromide, and to trimethylsilyl (TMS) derivatives
by TMS imidazole, and finally determined by gas chromatography/negative
chemical ionization mass spectrometry. The detection limit of this
method is about 0.1 ng l -1. The E2 level in Lake Kasumigaura waters
has been monitored by this method and the yeast two-hybrid system
for one year, and has remained less than 1 ng l -1. At present, the
cause of fish feminization is not revealed yet. The sexual / reproductive
function of slightly feminized male fish is unknown. Further studies
must be done to answer the questions.
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