OTSUKI Akira TOKYO UNIVERSITY OF FISHERIES, DEPARTMENT OF OCEAN SCIENCES, PROFESSOR, 水産学部, 教授 (30101041)
HASOBE Masahide TOKYO UNIVERSITY OF FISHERIES, DEPARTMENT OF AQUATIC BIOSCIENCES, ASSOCIATE PROFESSOR, 水産学部, 助教授 (10218464)
HARA Akihiko UNIVERSITY OF HOKKAIDO, FACULTY OF FISHERIES, PROFESSOR, 水産学部, 教授 (40091483)
|Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥2,900,000 (Direct Cost: ¥2,900,000)
We have investigate the seasonal changes of serum VTG, as a biomarker of the effect of environmental estrogens, in wild male flounder (Pleuronectes yokohamae) collected from Tokyo Bay, and compare these levels to those in male flounder from a reference site along the coast of Japan. Our reference station was located off Shiriuchi in Hokkaido, where sewage and wastewater inputs to the marine environment were considered to be negligible. Pleuronectes yokohamae from Tokyo Bay, aged approximately 1 to 6 yr. were caught between January 1997 and May 1998 (male: n=130, female: n=143), covering an entire reproductive season. Reference specimens were collected off Shiriuchi, in the Strait of Tsugaru, between March 1997 and June 1998 (male: n= 62, female: n=64). Mean serum levels of VTG for female flounder from Tokyo Bay and Hokkaido suggest that no obvious difference (elevated or depressed) of VTG of females was observed between the two sites. At the reference site off Shiriuchi, the mean VTG l
evels for male flounder was < 50 ng mlィイD1-1ィエD1. In contrast, male flounder in Tokyo Bay showed elevated levels of VTG almost throughout the year (mean: 204 ng mlィイD1-1ィエD1; ranging from < 50 ng ml ィイD1-1ィエD1 to 2200 ng mlィイD1-1ィエD1). The VTG levels in male flounder in Tokyo Bay (n=130) were significantly higher (p <0.001) compared to those of the reference fish from Hokkaido (n=62). The considerable variation in serum VTG concentration observed in individual males from Tokyo Bay could be due to individual differences in sensitivity to or degree of accumulation of estrogenic compounds.
The relationship between VTG in male flounder from Tokyo Bay and other factors such as standard length, hepatosomatie index (HSI), 17β-estradiol (EィイD22ィエD2), testosterone (T), and gonadosomatic index (GSI) was investigated. We found no significant relationship between VTG and standard length which indicates that induction of VTG occurs among male flounder of all ages above one year. Furthermore, no relationship was observed between VTG and HSI, EィイD22ィエD2 or T levels. However, high concentrations of serum VTG were observed more often in male flounder whose GSI was lower than 2% in Tokyo Bay. The GSI is therefore an additional factor that should be considered when male flounder is deployed as biomonitor of environmental estrogens.
The development of a very sensitive method for the determination of very low levels of plasma VTG in male fish allows, a) detection of trace amounts of estrogenic compounds and mainly, b) analysis of highly diluted serum samples, which can avoid a critical problem of the ELISA technique, the unspecific interference with serum compounds at high concentrations. We developed and optimized a luminometric immunoassay that was able to detect trace amounts of VTG in the serum of male flounder collected from a reference site, located off Shiriuchi in Hokkaido, where sewage and wastewater inputs to the marine environment were considered negligible. This method can be applied successfully to monitor seasonal changes in baseline VTG levels in serum samples obtained from a reference site. Such results will be useful in interpreting the effects of environmental estrogens in ecosystems.
Both EィイD22ィエD2 and T in the sera of male and female fish were high during winter, when VTG levels in female fish and the GSI of both sexes were also high. Estrogen has been found in the blood of wild males at levels as high as 10% of those normally seen in mature females in winter. The detectable levels of VTG in the blood of wild male flounder at the reference site in winter may be a consequence of the presence of physiologically active levels of estrogen. Perhaps this species expresses VTG in both sexes in response to "natural" circulating EィイD22ィエD2.
Histological analysis revealed that the gonads of about 15% of male flounder (3 out of 20) collected off Haneda in Tokyo Bay contained female gametes (intersexual gonads contained predominately male tissue). The degree of intersexuality was slight, with a few primary oocytes scattered within apparently normal testicular tissue. No abnormalities were seen in the testes of male flounder collected at the reference site in Hokkaido or in the ovaries of females from either site. Less