A novel approach for the mechanism of CYP2B induction: A study using mutant rats that lack response to the PB-mediated induction of CYP2B2 and the analyses of the gene structure and transcription factors

CYP2B诱导机制的新方法：使用对PB介导的CYP2B2诱导缺乏反应的突变大鼠进行的研究以及基因结构和转录因子的分析

• 批准号: 12672173
• 负责人: YAMADA Hideyuki
• 金额: $ 2.11万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672173/
• 关键词: Cytochrome P450; CYP2B subfamily; Phenobarbital; Mutant rats with abnormal inducubility; Qdj:SD rats; Inducer-responsive genomic sequence; CYP2B; 誘導不全; Odj:SDラット

The Qdj:SD rat is a mutant strain lacking in phenobarbital (PB)-mediated induction of CYP2B2. Presence of inter-individual differences in the hepatic content of CYP2B proteins and testosterone 16β-hydroxylase activity demonstrated that the breeding colony of Qdj :SD rats involves normal (+/+)-and intermediate (+/-)- phenotypes as well as mutant (-/-)-type rats. Analysis of regioselective metabolism of testosterone and 4-hydroxybiphenyl glucuronidation demonstrated normal catalytic activities associated with other forms of P450s, including CYP2A, 2C and 3A, as well as PB-inducible UGT-glucuronosyltransferase in Qdj:SD (-/-) rats. Hepatic CYP2B2 mRNA was detectable by reverse transcription - polymerase chain reaction in the Qdj:SD rats treated with phenobarbital (PB), but the content was far lesser (<1/10) than that in the drug-untreated Crj:SD rat, a reference animal with normal phenotype. The CYP2B2 gene in Qdj:SD (-/-) rats was same as those of the wild-type (+/+) rats in its length of the region containing all exon/introns and 5'-upstream up to -4.7 kbp. Malignant mutation such as stop codon formation was not observed in the exons, and no mutation was detected in the region containing the PB-responsive enhancer module (PBREM). The binding between 32P-labelled PBREM and hepatic nuclear proteins was not increased both in Qdj;SD (-/-) and Crj:SD rats by treatment of animals with PB, although PB-mediated increase in the binding was observed in mice. These results strongly suggest that the impaired induction of CYP2B2 in Qdj:SD (-/-) rats is due to the damage in basic expression of the gene. The impaired machinery for the expression is assumed to be either 1) mutation at the region different from PBREM and exons, or 2) absence or lowered expression of trans-acting factor(s) different from PBREM binding proteins.

Qdj:SD 大鼠是缺乏苯巴比妥 (PB) 介导的 CYP2B2 诱导的突变品系，CYP2B 蛋白的肝脏含量和睾酮 16β-羟化酶活性存在个体间差异，这表明 Qdj:SD 大鼠的繁殖群体。涉及正常（+/+）-和中间（+/-）-表型以及突变（-/-）型大鼠的睾酮和区域选择性代谢分析。 4-羟基联苯葡萄糖醛酸化显示出与其他形式的 P450（包括 CYP2A、2C 和 3A）以及 Qdj:SD (-/-) 大鼠肝脏 CYP2B2 mRNA 的 PB 诱导型 UGT-葡萄糖醛酸基转移酶相关的正常催化活性。 - 用苯巴比妥 (PB) 治疗的 Qdj:SD 大鼠中的聚合酶链反应，但是其含量远低于未经药物治疗的正常表型参考动物Crj:SD大鼠的含量(<1/10)。Qdj:SD(-/-)大鼠的CYP2B2基因与野生动物相同。 -型(+/+)大鼠在其包含所有外显子/内含子的区域长度和5'-上游至-4.7kbp的外显子中未观察到终止密码子形成等恶性突变，并且没有突变。在含有 PB 反应增强子模块 (PBREM) 的区域中检测到 32P 标记的 PBREM 与肝核蛋白之间的结合在 Qdj;SD (-/-) 和 Crj:SD 大鼠中均未通过用 32P 处理而增加。 PB，尽管在小鼠中观察到 PB 介导的结合增加，这些结果强烈表明 Qdj:SD (-/-) 大鼠中 CYP2B2 的诱导受损是由于损伤。表达的受损机制被认为是 1) 与 PBREM 和外显子不同的区域的突变，或 2) 与 PBREM 结合蛋白不同的反式作用因子的表达缺失或降低。

项目成果

Yamada, H., Matsunaga, H., Tsuji, K., Matsumoto, S., Yamamoto, M., Ishii, Y., Omiecinski, C.J., and Oguri, K.: "Sequence analyses of CYP2B genes and catalytic profiles for P450s in Qdj:Sprague-Dawley rats that lack response to the phenobarbital-mediated i

Yamada, H.、Matsunaga, H.、Tsuji, K.、Matsumoto, S.、Yamamoto, M.、Ishii, Y.、Omiecinski, C.J. 和 Oguri, K.：“CYP2B 基因的序列分析和催化谱

Yamada, H., Matsunaga, H., Tsuji, K., Matsumoto, S., Yamamoto, M., Ishii, Y., Omiecinski, C.J., Oguri, K.: "Sequence analyses of CYP2B genes and catalytic profiles for P450s in Qdj : Sprague-Dawley rats that lack response to the phenobarbital-mediated ind

Yamada, H.、Matsunaga, H.、Tsuji, K.、Matsumoto, S.、Yamamoto, M.、Ishii, Y.、Omiecinski, C.J.、Oguri, K.：“CYP2B 基因的序列分析和 P450 的催化谱

H.YAMADA 他7名: "Seguence Analyses of CYP2B Genes and Catalytic Profiles for P450s in Qdj : Sprague-Dawley Rats That Lack Response to the……"J.Phormacol.Exp.Ther.. 295(3). 986-993 (2000)

H.YAMADA 和其他 7 人：“Qdj 中 CYP2B 基因和 P450 催化谱的序列分析：对……缺乏反应的 Sprague-Dawley 大鼠”J.Phormacol.Exp.Ther.. 295(3)。 (2000)

Yamada, H., Matsunaga, H., Tsuji, K., Matsumoto, S., Yamamoto, M., Ishii, Y., Omiecinski, C.J., Oguri, K.: "Sequence analyses of CYP2B genes and catalytic profiles for P45Os in Qdj : Sprague-Dawley rats that lack response to the phenobarbital-mediated ind

Yamada, H.、Matsunaga, H.、Tsuji, K.、Matsumoto, S.、Yamamoto, M.、Ishii, Y.、Omiecinski, C.J.、Oguri, K.：“CYP2B 基因的序列分析和 P45O 的催化谱