J. Biol. Macromol., 5(2), 39-44 (2005)

Effects of tributyltin, triphenyltin and atrazine on plasma vitellogenin
concentration in Japanese medaka fish Oryzias latipes
Sumio Ishijima, Akiko Nochide, Kenji Kawamoto, Dai Shirakawa and Masatake Ohnishi
Graduate School of Agriculture, Kyoto Prefectural University, Kyoto, 606-8522, Japan
Received May 1, 2005; Accepted June 16, 2005
The Japanese medaka fish, Oryzias latipes, was used to determine the influence of exposure tochemicals on plasma vitellogenin (VTG) concentration using anti-medaka VTG antibody. Whenfemales were exposed to tributyltin (TBT) and triphenyltin (TPT) for 7 days, dose-related decreaseof plasma VTG was observed. Significant decrease was observed with 1 ppt TBT and TPT, andTPT was more effective than TBT. Addition of 10 ppt 17β-estradiol to 1 ppt TPT gave no increase,
but rather decrease in female plasma VTG. When males were treated with 1 ppt TBT and TPT or
1 ppt TPT + 10 ppt 17β-estradiol for 7 days, no VTG was detected in plasma. When females were
exposed to 10 and 100 ppt atrazine, plasma VTG tended to be decreased. When males weretreated with 10 and 100 ppt atrazine, no VTG was detected in plasma. The ecological significanceof these findings and implications for the development of the plasma VTG tests using Japanese
medaka fish are important.

Key words: medaka fish, plasma vitellogenin, triburyltin, triphenyltin

Introduction
A number of substances have been
detected in the environment that can affect theendocrine
system[1].
These
endocrine-disrupting chemicals, such asorganotin,
atrazine,
and17β-estradiol
discharged from industries and municipal
sewage treatment plants ultimately reachcoastal areas [2]. These chemicals have been
widely distributed in sediments, fish, andmammals, despite regulation or prohibition of
the use.
Recent studies have noted that
endocrine-disrupting chemicals lead toabnormal physiological response and cause
adverse effects on the reproductive system offish [3-5]. Estrogenic chemicals, such as17β-estradiol and atrazine, induce intersex
conditions, such as the development of oocytes
within testicular tissue (testis-ova)[6-8].
Induction of testis-ova is one indicator of
exposure to estrogenic chemicals in male

medaka.
Organotin compounds, such as
tributyltin (TBT) and triphenyltin (TPT) havebeen widely used as biocides, agriculture
fungicides, wood preservatives, disinfecting
agents in circulating industrial cooling waters,
and as antifouling paints for marine vessels[9,10]. Organotin compounds are a typicalendocrine-disrupting chemical that can induce
imposex in mollusks [11,12]. However, few
studies addressing the effect of organotin
compounds in fish have been reported.
Nakayama et al. administered TBT to medaka
for 3 weeks and indicated that TBT affected
sexual behavior and reproduction in male
medaka [13].
Atrazine (Atz), 2-chloro-4-ethylamino-6-isopropylamino-S-triazine, is probably
the most widely used herbicide in the world andone of the most common contaminants inground and surface water. Recent studies

40

Effects on plasma vitellogenin in Japanese medaka fishindicate that Atz may be a potent
endocrine-disrupting compound in frogs
exposedtopart-per-billion(µg/L)
concentrations [14].
Among populations of fish withgonadal intersex, significant levels ofvitellogenin (VTG) were reported in male fish.
The egg precursor protein VTG has beenfrequently used as a biomarker of exposure ofmale fish to estrogenic substances. VTG isnormally only present in the plasma of mature
female fish, but exposure to estrogenic
substances can induce VTG synthesis in malefish and in juvenile females [15].
Previously we found that exposure ofmale medaka fish to 17β-estradiol induced a200-kDa protein in blood plasma and liver [16].
The 200-kDa protein was supposed to be VTG,
but was not identified. We here measured theplasma VTG level using anti-medaka VTG
antibody, and found that exposure of female
medaka fish to TBT and TPT decreased theplasma VTG. The decrease of the plasma
VTG was also observed with female medaka
fish exposed to Atz.
Materials and methods
Materials
Medaka fish was purchased from
Shimizu Suisan Co., Kyoto and usually kept in
the glass-made 57 liter aquarium (vessel) filledwith natural water (tap water, after leaving for 2days) under a controlled environment asfollows: light 14 h, dark 10 h, watertemperature 26°C, fed with a commercial
fishfood twice a day [16]. TBT chloride, TPT
chloride, and atrazine were obtained from
Wako Pure Chemical Industries, Ltd., and17β-estradiol was from Nacalai Tesque. An
enhanced chemiluminescence (ECL) kit waspurchased from Amersham Biosciences.

Blood sampling
Adult fish was kept in a 200 ml
glass-made beaker filled with the natural watercontaining TBT chloride, TPT chloride,
17β-estradiol and atrazine at concentrations
indicated under the controlled environment.
Tail was cut off from fish body andblood exuded was put into microtubes filledwith a buffer consisting 25 mM Tris-HCl (pH7.2), 150 mM NaCl, and 5 mM EDTA.
Supernatant was obtained by centrifugation ofblood at 12,000×g and 4°C for 15 min. The
protein concentration was determined by theMicro BCA Protein Assay Reagent (Pierce).
Measurement of plasma vitellogenin
The plasma vitellogenin was detectedaround 200 kDa by SDS-PAGE [17]. The
separation gel was prepared with 8%
polyacrylamide, and plasma sample (0.45 µg of
protein) was subjected to electrophoresis.
After equilibration in transfer buffer, proteins
were electrophoretically transferred onto PVDF
membranes. Membranes were blocked in 3%
gelatin in PBS for 1 h, and incubated with
1:500 diluted anti-medaka VTG antibody(Enbio Tec Laboratories), then washed with
PBS containing 0.1% Tween 20, incubatedfurther with horseradish peroxidase-linked
anti-mouse IgG (Amersham Biosciences).
The protein bands were visualized with ECL.
Statistical analysis
All results are expressed as the means± SD. Student’s t-test was used to determine
the statistical significance.
Results
Effect of TBT and TPT on plasma VTG in
female medaka fish

41

S. Ishijima et al.

1

2

3

4

5

(B)

Plasma VTG levels(relative band intensity)

(A)

150k

100k
75k

2.00
1.50

*

**

1.00
0.50
0.00
C

0.1 1
1 0.1 1
ppt
ppt
TBT
TPT

1 ppt TPT+
10 ppt Est

Fig. 1. Relative plasma vitellogenin (VTG) levels in the tributyltin (TBT) chloride- and triphenyltin(TPT) chloride-treated female medaka. Panel A, females were kept with 0.0001% DMSO (lanes 1 and
2) or 1 ppt TBT chloride (lanes 3-5) for 7 days. Plasma samples were separated on a
SDS-polyacrylamide gel, and Western blotting was performed using anti-medaka VTG antibody.
Bands were visualized using the ECL system according to the manufacture’s instructions. Panel B,
females (N=5-9) were kept with 0.0001% DMSO (C), 0.1 and 1 ppt TBT chloride, 0.1 and 1 ppt TPT
chloride, or 1 ppt TPT chloride and 10 ppt 17β-estradiol (Est) for 7 days. Relative amounts ofvitellogenin in plasma were determined by Western blot. * and ** indicate a statistically significant
difference at p < 0.03 and p < 0.003, respectively, compared with the levels in the control.

When females were exposed to TBT
and TPT for 7 days, dose-related decrease ofplasmaVTG
wasobserved(Fig.1).
Significant decrease was observed with 1 ppt
TBT and TPT, and TPT was more effective
1

2

3

4

5

6

than TBT. Addition of 10 ppt 17β-estradiol,which can induce plasma VTG in male medaka
fish, to 1 ppt TPT gave no increase, but rather
decrease in female plasma VTG.
When males were treated with 1 ppt
TBT and TPT or 1 ppt TPT + 10 ppt
17β-estradiol for 7 days, no VTG was detected
in plasma (Fig. 2 and data not shown).

150k

100k
75k

Fig. 2. Relative VTG levels in the TPT
chloride-treated male medaka. Males were
kept with 1 ppt TPT for 7days, and plasma
VTG bands were visualized as in Fig. 1.
Lane 1: control male, lane 2: control female,lanes 3-6: males treated with 1 ppt TPT.

Effect of Atz on plasma VTG in female medakafish
When females were exposed to 10
and 100 ppt Atz, plasma VTG tended to be
decreased (Fig. 3). When males were treatedwith 10 and 100 ppt Atz, no VTG was detected
in plasma (data not shown).
Discussion
The present results indicated thatorganotin compounds, such as TBT and TPT

42

Effects on plasma vitellogenin in Japanese medaka fish

Plasma VTG levels(relative band intensity)

reduced the plasma VTG in female medaka fish.

1.50
1.25
1.00
0.75
0.50
0.25
0.00

It is reported that fertilization successFig. 3. Relative plasma vitellogenin (VTG)
levels in the atrazine (Atz)-treated female
medaka. Females (N=4-5) were kept with
0.0001% DMSO (C), 10 and 100 ppt atrazine
for 7 days. Relative amounts of VTG in
plasma were determined as in Fig 1.
C

10

100
ppt Atz

TPT was effective than TBT, that may becaused by that TBT is more easily metabolized
than TPT, and that net bioaccumulation of TPT
is greater than that of TBT [18]. Exposure to
Atz also tended to produce the decreased
plasma VTG in female medaka fish.
Organotin compounds are underintense debate because they were recognized in
the 1990s for a variety of disastrous effects inthe marine ecosystem. TBT, which is used inhuge amounts in antifouling paints for large
ships, has been called “the most toxic chemical
ever deliberately released into the seas” (World
Wildlife Fund). Organotin compounds havebeen recognized as endocrine-disrupting
chemicals because numerous marine organisms
have been shown to exhibit sexual
abnormalities following exposure to TBT and
TPT. In gastropod mollusks, TBT has been
demonstrated to induce the superimposition ofmale sex organs, a phenomenon known as
imposex. Several reports have suggested that
TBT inhibits the enzyme aromatase whichcatalyzes androgen to estrogen [19,20].
Therefore, it has been theorized that TBT
increases androgen levels through inhibition ofaromatase activity and/or a suppression ofandrogen excretion.
However, no clear
evidence has been provided to verify thishypothesis.

and the numbers of males that performed thesexual behavior such as following and dancing
were significantly decreased in male medaka
exposed to TBT [13], and TBT induced
abnormal development of the eyes, reduced
hatchability and increased swim-up failure in
medaka [21]. Previously we suggested that
TBT induced the expression of a 200 kDa
protein in blood plasma of male medaka, but
the 200 kDa protein was not identified [16].
We here did not detect VTG in plasma of male
medaka exposed to TBT and TPT, using
anti-medaka VTG antibody. Nozaka et al.
also observed no significant VTG response inmale medaka exposed to TBT, using an
enzyme-linked immunosorbent assay [22].
Toxicity of organotin to male medaka should befurther studied. Although the production of
VTG in males and immature females has beensuccessfully exploited as a biomarker of
exposure specific for estrogenic compounds ina variety of fish species recommended by
Organization for Economic Cooperation andDevelopment (OECD) for the validation [23],effects on the VTG response and sexual
behavior in fish may be different depending on
the conditions (e.g. exposure time). It may be
important to clarify whether it is needed toconsider normal physiological rhythms such as
daily and seasonal differences.

S. Ishijima et al.

Previous studies have been suggested
that Atz is an endocrine disruptor. Atz has
been linked to decreased reproductive output inbluegill sunfish, L. macrochirus [24].
Developmental arrest in Japanese medaka
embryos was observed when embryos were
exposed to sublethal concentrations (25-100ppm) of Atz [25]. Atz may affect mammals
and amphibians by altering aromatase activity,
but these effects have been observed in a single
strain of rat or were produced only at highdoses.
We here observed the VTG responsein female medaka exposed to TBT, TPT and
Atz. The mechanisms of the reduced plasma
VTG in females remain unclear, but theecological significance of these findings andimplications for the development of the plasma
VTG tests using Japanese medaka fish areimportant.

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Communicated by Keiko Kitagishi