Endocrine-disruptor (ED) is an exogenous substance or mixture that alters function(s)
of the endocrine system and consequently produces adverse health effects in an intact
organism, or its progeny, or (sub) populations. Aquatic pollution by EDs, especially
estrogenic EDs, has become one of the most serious environmental problems worldwide.
The objective of this study is to develop a transgenic marine medaka (Oryaizs melastigma)
for accurate and prompt detection of estrogenic EDs contaminated waters and screening
of new EDs.
O. melastigma is scarcely studied and its embryonic development is not reported.
The transparent chorion and embryo allow clear observation of the development of inner
organs under light microscope. According to the diagnostic features used to stage the
freshwater medaka (O. latipes), the embryonic developmental process of O. melastigma
was divided into 39 stages and showed high morphological similarity to that of O. latipes
with minor differences. Moreover, advantageous characteristics including small body size
(3-4 cm), short life cycle (2-3 months), high prolificity (20-30 embryos daily per pair
under proper temperature, light cycle and feeding systems), and especially the habitability
in both fresh water and sea water, make O. melastigma an ideal model for aquatic
toxicology studies.
Teleost choriogenins, precursors of the inner layer subunits of egg envelope, are
regarded as sensitive biomarkers for estrogenic pollutants. To select the more estrogen
sensitive gene for transgenic study, full-length cDNAs - omChgH and omChgL - which
encode the choriogenin H and L forms, respectively, were isolated from O. melastigma
and their induced expression at different developmental stages were analyzed.
17β-Estradiol (E2; 10 μg/L)-dependent expression of omChgH and omChgL was observed
starting at embryonic stage 34 and restricted to the liver. In hatchlings, E2 induction of
omChgH was stronger than that of omChgL. Static exposure of adult fish to E2 (0, 1, 10,
100 and 500 ng/L), 17α-ethinylestradiol (EE2; 0, 1, 10, 100 and 500 ng/L), 4-nonylphenol
(NP; 0, 1, 10, 100 and 200 μg/L) and bisphenol A (BPA; 0, 1, 10, 100 and 200 μg/L) in
artificial seawater for 7 days resulted in dose-dependent induction of both genes in the liver.
In the male livers, the sensitivity of omChgH to these estrogenic compounds was higher
than that of omChgL; the lowest-observed-effect concentrations (LOECs) of E2, EE2, NP
and BPA on omChgH were 10 ng/L, 10 ng/L, 100 μg/L and 100 μg/L, respectively, and on
omChgL were 100 ng/L, 100 nglL, 100 μg/L and 200 μg/L, respectively. All these
observations highlighted the potential of using omChgH expression as a sensitive
biomarker for estrogenic EDs in the developing O. melastigma embryos, juveniles and
male adults.
Thus, omChgH genomic DNA sequence including ca. 5 kb 5'-upstream region and ca.
0.8 kb 3'-flanking region was cloned. Cis-regulatory activity analysis of different sizes of
omChgH 5'-upstream region and the regulation effects of 3'-flanking region were analyzed
using microinjection techniques. Results showed that 750 bp 5'-upstream region from
transcription initiation site had the highest promoter activity and homogenous 3'-flanking
region was important for obtaining high promoter activity in omChgH transgenic studies.
Based on the cis-regulatory activity analysis of omChgH 5' and 3'-flanking region, a
transgenic O. melastigma strain harboring the reporter gene green fluorescence protein
(GFP) gene regulated by 758 bp omChgH 5'-upstream region and flanked by omChgH
3'-flanking region was established. In this strain, GFP transgene was expressed
constitutively in the liver of mature female, but could also be induced from non-expression
liver of embryos (since stage 34), juvenile and male fish in response to 17β-estradiol (E2).
GFP fluorescence quantification analysis using MetaMorph revealed that 0.63 nM E2 or
0.17 nM 17a-ethanylestradiol (EE2) significantly induced GFP expression in the livers of
larvae after 24-h exposure and the responses were dose-dependent. Additionally, this strain
was also observed to express GFP fluorescence after exposure to different estrogenic
compounds at concentrations equal to or higher than 1.8 nM estrone (E1), 1.73 nM estriol
(E3), 2000 nM 4-nonylphenol (NP), 4380 nM bisphenol A (BPA), 3670 nM genistein and
0.25 nM ethinylestradiol 3-methyl ether for 24 h. These results suggested the high estrogen
sensitivity of this transgenic O. melastigma strain and its capability to monitor a wide range
of estrogenic chemicals.
Further preliminary field study found that marine water samples collected from Hong
Kong Victoria Harbor could induce GFP expression in the liver of transgenic larvae after
exposure for 24 h. This result demonstrated the practical applicability of this transgenic O.
melastigma strain for prompt in vivo biomonitoring of estrogenic activity of aquatic
environment directly, and such high estrogen sensitivity has not been reported yet.
To conclude, this study successfully developed the first transgenic marine fish for
biomonitoring estrogenic endocrine-disrupting pollutants. This strain showed quick
response to a wide range of estrogenic compounds including weak EDs (e.g. NP, BPA and
genistein), and could detect estrogenic activity of environmental water samples promptly
and directly. All my findings indicate the great practical potential of this transgenic O.
melastigma as a sensitive sentinel for simple, economic, rapid and accurate screening of
new EDs and identification of estrogenic EDs contaminated waters. While the wide salinity
adaptability to both freshwater and marine environment makes this transgenic strain more
unique and powerful.
| Date of Award | 15 Feb 2008 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Shuk Han CHENG (Supervisor) |
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