Analysis of human fetus-specific cytochrome P450 : Evaluation of its function (s) by using transgenic mice and estimation of regulation mechnism (S).

人类胎儿特异性细胞色素P450的分析：通过使用转基因小鼠评估其功能并估计调节机制（S）。

• 批准号: 06454593
• 负责人: KAMATAKI Tetsuya
• 金额: $ 4.48万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454593/
• 关键词: P450; CYP; fetus-specific; transgenicmouse; bioactivation; 発現調節; チトクロームP450; バキュロウイルス発現系; 大量発現系

The human fetus-specific P450, CYP3A7, was expressed in C57BL/6N mice by microinjection into the mouse fertilized eggs of a CYP3A7 cDNA fused to a murine metallothionein-I promoter. Six transgenic lines of mice carrying different copies of the MT-I-CYP3A7 transgene were established. In M10 line of mice, the expression of the CYP3A7 at mRNA and/or protein levels were detected in the liver, testis, and other tissues, while in M2 line of mice the expression of CYP3A7 was found in the kiney and many other tissues. The expression of the CYP3A7 was induced by the treatment with zinc sulfate, an inducer of MT-I promoter. The CYP3A7 was expressed perinatally in the M10 mice, beginning on the day 15 of gestation. The expression level was low in fetal life and increased markedly in the livers of neonates 3 days after birth. The CYP3A7 protein expressed in the livers of M10 neonates as well as in the adults was functionally active in terms of midazolam 1'-hydroxylation in vivo. Using M2 and M10 mice as an animal model, the involvement of human CYP3A7 in the bioactivation of AFB_1 in vivo was further evidenced by a significantly higher AFB1-N^7-Guanine adduct level as well as DNA damage in the liver and kidney of M10 and M2 mice, respectively. These give the first evidence that human CYP3A7 plays a role in the bioactivation of AFB_1 in vivo. This transgenic animal model is the first to carry a human P450 gene and provides the first successful example of assessing a human P450 gene in rodent by transgenic technique.

人胎胎胎特异性P450，CYP3A7在C57BL/6N小鼠中通过对CYP3A7 cDNA的小鼠受精卵融合到鼠金属蛋白I-I-I启动子的小鼠受精卵中表达。建立了六只携带MT-I-CYP3A7转基因不同副本的小鼠的转基因线。在小鼠的M10系中，在肝脏，睾丸和其他组织中检测到CYP3A7在mRNA和/或蛋白质水平上的表达，而在M2小鼠的M2系中，CYP3A7的表达在Kiney和许多其他组织中发现。 CYP3A7的表达是由MT-I启动子的诱导剂硫酸锌处理诱导的。从妊娠第15天开始，在M10小鼠中表达了CYP3A7。出生后3天，在新生儿肝脏中，胎儿寿命的表达水平较低，明显增加。在M10新生儿肝和成年人中表达的CYP3A7蛋白在体内的咪达唑仑1'-羟基化方面具有功能活跃。使用M2和M10小鼠作为动物模型，人类CYP3A7参与体内AFB_1的生物活化进一步证明了AFB1-N^7-甲胺加合物水平，以及M10和M2小鼠的肝脏和肾脏中的DNA损伤。这些给出了第一个证据，表明人CYP3A7在体内AFB_1的生物活化中起作用。这种转基因动物模型是第一个携带人类P450基因的模型，并提供了第一个成功的示例，即通过转基因技术评估啮齿动物中的人类P450基因。

项目成果

Miyuki Shimoji, Susumu Itoh, Kenji Toide, Kazuo Sasaki, Kazuo Nakayama, Shinichi Aizawa and Tetsuya Kamataki: "Application of primary hepatocytes from p53-Knockout mice for studies of expression of cyp3a" J.Biochem. 120. 42-48 (1996)

Miyuki Shimoji、Susumu Itoh、Kenji Toide、Kazuo Sasaki、Kazuo Nakayama、Shinichi Aizawa 和 Tetsuya Kamataki：“p53 敲除小鼠的原代肝细胞在 cyp3a 表达研究中的应用”J.Biochem。

Yong Li, Tsuyoshi Yokoi Ryuji Kitamura, Makoto Sasaki, Masae Gunji, Motoya Katsuki and Tetsuya Kamataki: "Establishment of transgenic mice carrying human fetus-specific CYP3A7" Archs, Biochem.Biophys. 329. 235-240 (1996)

Yong Li、Tsuyoshi Yokoi Ryuji Kitamura、Makoto Sasaki、Masae Gunji、Motoya Katsuki 和 Tetsuya Kamataki：“携带人类胎儿特异性 CYP3A7 的转基因小鼠的建立”Archs，Biochem.Biophys。

S.Itoh et al.: "Isolation of a promoter region in mouse cytochrome P450 3A (Cyp3a16) gene and its transcriptional control." Biochim.Biophys.Acta. (in press). (1996)

S.Itoh 等人：“小鼠细胞色素 P450 3A (Cyp3a16) 基因启动子区域的分离及其转录控制。”

S.Itoh et al.: "A novel form of mouse cytochrome P450 3A(Cyp 3a-16):Its cDNA cloning and expression in fetal liver." Eur.J.Biochem.226. 877-882 (1994)

S.Itoh 等人：“小鼠细胞色素 P450 3A (Cyp 3a-16) 的新型形式：其 cDNA 克隆和在胎儿肝脏中的表达。”

Yong Li, Tsuyoshi Yokoi, Motoyo Katsuki, Jia-Sheng Wang, John D.Groopman and Tetsuya Kamataki: "In vivo activation of aflotoxinB1 in C57BL/6N mice carrying a human fetus-specific CYP3A7 gene" Cancer Res.57. 641-645 (1997)

Yong Li、Tsuyoshi Yokoi、Motoyo Katsuki、Jia-Sheng Wang、John D.Groopman 和 Tetsuya Kamataki：“携带人类胎儿特异性 CYP3A7 基因的 C57BL/6N 小鼠中黄曲霉毒素 B1 的体内激活”Cancer Res.57。