Application of toxicogenomic approaches to study chemical-induced effects on the hypothalamic- pituitary- gonadal (HPG) axis of the Japanese medaka (Oryzias. latipes)

System models utilizing genomic approaches can be powerful tools for mechanistic toxicological research. This dissertation describes the development and validation of a real time polymerase chain reaction (RT-PCR) array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG PCR array combines the quantitative performance of SYBRRTM Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG PCR array was evaluated by examining effects of five model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2), the anabolic androgen, 17beta-trenbolone (TRB), the aromatase inhibitor, fadrozole (FAD), the imidozole-type fungicides, prochloraz (PCZ) and ketoconazole (KTC) on the HPG axis of the Japanese medaka. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. Four-month-old medaka were exposed to different concentration of chemicals for 7 d in a static renewal exposure system and the exposure concentrations were EE2 (5, 50, 500 ng/L), or TRB (50, 500, 5000 ng/L) or PCZ (3, 30, 300 mug/L) or KTC (3, 30, 300 mug/L) or 50mug FAD/L. TRB, PCZ, KTC or FAD caused time-dependent reductions in fecundity by Japanese medaka, but EE2 did not. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH R I and testicular CYP17. Despite their different biochemical properties, TRB or FAD caused similar responses in Japanese medaka, such as lesser fecundity and down-regulation of VTG and CHG genes in the liver of females. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH R II and ovary CYP19A. Exposure to FAD for 8 h resulted in an 8-fold and 71-fold down-regulation of expression of estrogen receptor alpha (ER-alpha) and CHG L , respectively in female liver. TRB caused similar down-regulation but the effects were not observed until 32 h of exposure. These results support the hypothesis that FAD reduces plasma E2 more quickly by inhibiting aromatase enzyme activity than does TRB, which inhibits production of the E2 precursor testosterone. Exposure to KTC or PCZ significantly down-regulated expression of ER-alpha and egg precursors in livers of males and females. However, PCZ was more potent than KTC both in modulating transcription and in causing lesser fecundity. Correlation analysis indicated that ER-alpha plays a primary role in the transcription of VTG and CHG genes in livers. The mRNA level of the five egg precursors and ER-alpha in livers of females was log-log related to the ecologically relevant endpoint, fecundity. Overall, the organ- gender- and concentration-specific gene expression profiles derived by the Japanese medaka HPG axis RT-PCR array provides a powerful tool to not only delineate chemical-induced modes of action, but also to quantitatively evaluate chemical induced adverse effects on reproduction.