Application of the frog (Rana rugosa) characterized by two types of sex-determining mechanisms to invivo assay for screening endocrine disruptors -Assessment of estrogenicity in four kinds of pesticides-

• Project/Area Number: 14580125
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 家政学一般(含衣・住環境)
• Research Institution: Hiroshima prefectural Women's University
• Principal Investigator: ICHIKAWA Youko Hiroshima prefectural Women's University, Department of Health Science, Assistant, 生活科学部, 助手 (20084163)
• Project Period (FY): 2002 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥2,800,000 (Direct Cost: ¥2,800,000); Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
• Keywords: endocrine disruptor / in vivo assay / amphibian / gonadal sex-differentiation / pesticide / estrogenicity / meiosis / in vivo / 生殖腺の分化 / カルバリル / 内分泌攪乱化学物質 / アトラジン / エンドスルファン / トリフルラリン

Research Abstract

The frog Rana rugosa has two types of sex-determining mechanisms, XX/XY and ZW/ZZ. The crossing between XX-females and ZZ-males produces genetically all-male XZ-tadpoles that are estrogen-sensitive during days 20-22 after fertilization. Ovaries are distinguishable from testes in the 40-day-old tadpoles based on an ovarian cavity and the wealth of meiotic germ cell (MGC). These properties enabled the author et al. to establish an in vivo XZ-tadpole gonadogenesis assay for screening estrogenlike chemical substances.
This study was designed to evaluate the estrogenicity of pesticides endosulfan(END), carbaryl(CAR), trifluralin(TRI) and atrazine(ATR) from that assay, and to develop a much-improved assay in sensitivity by using ZZ-tadpoles from crossing between ZZ parents. ZZ-mothers were produced from ZZ-males by exposure to 10 μM 17 β-estradiol(E2).
About 30 XZ-tadpoles were exposed to END solutions at concentrations of 0.1,1 and 5 nM, with the other solutions at 0.01,0.1 and 1 μM. As posit ive and vehicle controls, 0.01,0.1 and 1 μM E2, and 0.01% ethanol were used, respectively. The gonads of all 40-day-old tadpoles were examined and classified into ovary, testis and hermaphrodite. All those exposed to 1 μM E2 developed into females, while all of the vehicle control developed into males. XZ-tadpoles exposed to END, CAR and TRI developed into neither female nor hermaphrodite, but those exposed to TRI formed testes with many MGCs. ATR caused 3.4% hermaphrodites and 19.5% males having many MGCs.
Statistical evaluation by XZ-tadpole assay detected the estrogenicity of ATR and TRI, but never that of EDN and CAR.
Contrarily, in the ZZ-tadpole assay, exposures to 0.01-1 μM CAR caused 1.9% females and 4.8% hermaphrodites, denoting the estrogenicity of CAR.
Consequently, the ZZ-tadpole assay showed higher sensitivity to estrogenlike chemical substances than the XZ-tadpole assay. This result suggests the possibility of an improved in vivo ZZ-tadpole assay for screening chemical substances.