송어 cytochrome P450IAI 유전자 발현에 미치는 upstream DNA sequence의 영향

Abstract

Cytochrome P450IA1 유전자의 발현 조절 기전을 조사하기 위하여 송어 cytochrome P450IA1의 5'-flanking 부위를 분리한 후 chloramphenicol acetyltransferase (CAT) expression vector에 연결하여 reporter 유전자인 CAT의 효소 활성을 측정하였다. 송어 CAT construct와 생쥐 CAT construct를 Hepa-1에 transfection시킨 후, cytochrome P450IA의 강력한 유도제인 3-methylcholanthrene (3MC)를 투여하고, arylhydrocarbon hydroxylase 효소 활성의 직접적인 저해제인 chrysin, hesperetin, kaempferol, morin, myricetin과 같은 flavonoids와 aminopyrine을 각각 3MC와 병용 투여한 후 CAT의 효소 활성을 측정함으로써 cytochrome P450IA1 유전자의 발현 조절 기전을 조사하였다. 송어 CAT construct의 CAT 효소 활성은 3MC 투여시 대조군에 비해 2.81배 증가되었고, 이 증가된 정도는 flavonoids, aminopyrine에 의해 29.2-58.0% 감소되었다. 생쥐 CAT construct의 CAT 효소 활성은 3MC 투여시 대조군에 비해 2.24배 증가되었고, 이 증가된 정도는 flavonoids, aminopyrine에 의해 24.1-50.0% 감소되었다. 이상의 결과에서 flavonoids와 aminopyrine은 arylhydrocarbon hydroxylase 효소 활성의 직접적인 저해작용뿐만 아니라 cytochrome P450IA1의 5'-flanking 부위에도 작용하여 유전자 발현을 억제한다고 사료된다.;To investigate the mechanism of the regulation of cytochrome P450IA1, the 5'-flanking region of a trout cytrochrome P450IA1 was cloned into the CAT basic expression vector at HindⅢ site. This trout cytochrome P450IA1 upstream DNA containing CAT construct was transfected into Hepa-1. After transfection, 3MC was treated. And flavonoids including chrysin, hesperetin, kaempferol, morin, myricetin and aminopyrine which are the direct inhibitor of arylhydrocarbon hydroxylase, were treated concomitantly with 3MC. 48 hours after treatments, cells were disrupted and assayed for CAT activity by ELISA. The CAT activity of transfected trout P450IA1-CAT construct increased CAT activity by 2.81 fold when it was compared with that of control. This increased CAT activity was decreased by concomitantly treated flavonoids and aminopyrine. The rate of decrease was 29.2-58.0% of 3MC stimulated CAT activity. The CAT activity of transfected mouse P450IA1-CAT construct increased CAT activity by 2.24 fold when it was compared with that of control. This increased CAT activity was decreased by concomitantly treated flavonoids and aminopyrine. The rate of decrease was 24.1-50.0% of 3MC stimulated CAT activity. As He result of the above, it has been postulated that flavonoids and aminopyrine did act not only as the direct inhibitor of arylhydrocarbon hydroxylase but also as the inhibitor of gene expression in the 5'-flanking region of cytochrome P450IA1.