A risk assessment of endocrine disrupters using analytical methods for gene expression in the poultry embryo

• Project/Area Number: 14580577
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 環境影響評価(含放射線生物学)
• Research Institution: National Institute for Environmental Studies
• Principal Investigator: TAKAHASHI Shinji National Institute for Environmental Studies, Endocrine Disruptors & Dioxin Research Project, Senior Researcher, 環境ホルモン・ダイオキシン研究プロジェクト, 主任研究員 (20197148)
• Project Period (FY): 2002 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥2,900,000 (Direct Cost: ¥2,900,000); Fiscal Year 2003: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
• Keywords: Endocrine disrupting chemicals / Endocrine disrupters / Avian / Estrogen / Gonad / Sexual differentiation / Gene expression / Sex-determining gene / 内分泌攪乱化学物質

Research Abstract

The present study has been performed to develop a risk assessment of endocrine disrupters for avian species. To elucidate the relationship between gene activation during embryogenesis and reproductive abnormalities in wild birds, we have examined expressions of the genes for proposed sex-determining factors, steroidogenic enzymes and the estrogen receptor in the urogenital system during chicken embryogenesis (days 4-16 of incubation), using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Although we found several physiological differences that expressions of the genes for more downstream enzymes in a steroidogenic pathway were clearly different between the sexes, in ovo treatment of a xenoestrogen, diethyistilbestrol, had no effect on the expressions of all the genes examined here. Therefore, it has been considered that new candidates are necessary for target genes and the mechanisms of reproductive disorder in wildlife.
In order to find unknown target gene s, we used a fluorescence differential display method which could detect the difference in the level of mRNA transcript. Fertilized chicken eggs treated with ethynylestradiol (10 ng-100 μg/egg) were incubated and RNA sample was extracted from the gonads in each embryo on day 12. Twelve and one mRNAs which were increased and decreased by xenoestrogen treatment, respectively, were detected in male embryos. The cDNA samples were collected and followed by DNA sequencing analysis. Eight of the cDNAs were derived from one mRNA which has a significant homology with the chaperonin protein gene in human. In addition, one of the cDNAs increased by xenoestrogen has a significant homology with the kynurenine aminotransferase II gene in the mouse, however, the others show no sequence homology with any known genes. It has been reported that chaperonin mRNA transcript in human breast cancer cell lines (MCF-7) increases in dependence on 178-estradiol (Charpentier et al., 2000). These genes reported here are expected to be candidates of new targets for estrogenic actions.