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

QDJ：SD大鼠是一种突变株，缺乏苯巴比妥(PB)介导的CYP2B2诱导。肝脏细胞色素P450 2 B蛋白含量和睾酮16β羟基酶活性的个体间差异表明，QDJ：SD大鼠的繁殖群体包括正常(+/+)和中间(+/-)表型以及突变(-/-)型大鼠。对睾酮和4-羟基联苯葡萄糖醛酸化的区域选择性代谢的分析表明，在QDJ：SD(-/-)大鼠中，与其他形式的P450相关的催化活性正常，包括CYP2A、2C和3A，以及PB诱导的UGT-葡萄糖醛酸基转移酶。用逆转录-聚合酶链式反应检测到经苯巴比妥(PB)治疗的QDJ：SD大鼠肝脏中有CYP2B2mRNA的表达，但其含量远低于表型正常的对照动物CRJ：SD大鼠。QDJ：SD(-/-)大鼠的CYP2B2基因与野生型(+/+)大鼠的相同，其长度包括所有外显子/内含子和5‘-上游至-4.7kBP。在外显子中没有观察到停止密码子形成等恶性突变，在含有PB反应增强模块(PBREM)的区域也没有检测到突变。用PB处理动物后，~(32)P标记的PBREM与肝核蛋白的结合在QDJ；SD(-/-)和CRJ：SD大鼠中均没有增加，但在小鼠中观察到了结合的增加。这些结果有力地表明，QDJ：SD(-/-)大鼠细胞色素P450-2B2的诱导受损是由于该基因的基础表达受损所致。这种表达受损的机制可能是1)不同于PBREM和外显子的区域发生突变，或者2)不同于PBREM结合蛋白的反式作用因子(S)的表达缺失或表达降低。

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.