IP03 Avian Endocrine Test Protocols
B113 & B114
1:20 PM - 4:40 PM, Monday
(IP020) Cloning of quail androgen receptor cDNA, mRNA expression and its application for development of an in vitro binding assay.
Shimada , K.1, Saito, N.1, Mizushima, S1, Nishizuka, M2, Kunimoto, M2, Maekawa, S2, Imagawa, M2, 1 Nagoya University, Nagoya, Aichi, Japan2 Nagoya City University, Nagoya, Aichi, Japan
ABSTRACT- Androgen receptor (AR) of cDNA of Japanese quail was isolated to examine the tissue distribution of androgen receptor mRNA expression and it was used to establish an in vitro method to detect androgenic activity of chemical substances that were designated by the Ministry of Environment of Japan. The partial sequence of quail AR cDNA consisted of 1385 bases and contained ligand binding domain (LBD). RT-PCR analysis showed that AR mRNA levels markedly increased in the vas deferens and epididymis after 3-week exposure to continuous light. For in vitro screening, the quail AR was produced in E. coli and competitive enzyme immunoassay was established. The recombinant AR was competitively bound either with free androgen or with test substance and subsequently, free androgen was allowed to compete with horseradish peroxidase (HRP)-labeled androgen to bind to androgen-specific monoclonal antibody. The bound form of HRP-labeled and androgen was measured by optical density and receptor binding of the test substances was calculated. Among 20 substances examined, IC50 of chemicals was within the order of 10-9 Mol for Milbolerone (positive control), 10-5 for 4-Nonylphenol, 10-5 for Benzyl-n-butyl phthalate, 10-5 M for Bisphenol, respectively. The remaining chemicals had no binding capacity. (A part of the present study was supported by the Ministry of the Environment, Japan)
Key words: endocrine disrupters, androgen receptor mRNA, in vitro AR binding assay