Kim S G, Reddy S L, States J C, Novak R F
Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201.
Mol Pharmacol. 1991 Jul;40(1):52-7.
The expression and molecular regulation of the cytochrome P450IA (P450IA) gene subfamily have been examined in rat hepatic tissue after treatment with pyridine. The microsomal ethoxyresorufin O-deethylase activity, which has been shown to be specific for the P450IA subfamily, was increased approximately 2- and 3.5-fold over control values at 10 and 16 hr, respectively, after a single dose of pyridine (100 mg/kg, intraperitoneally). P450IA1 protein expression was also elevated in a time-dependent manner, with a maximal increase in P450IA1 protein being seen at approximately 16 hr after a single dose of pyridine (100 mg/kg, intraperitoneally), as detected by immunoblot analysis using a monoclonal antibody that detects both P450IA1 and P450IA2. The immunochemically detectable level of P450IA1 decreased to that of control at 48 hr after treatment. Oligonucleotide probes specific for P450IA1 and P450IA2 mRNA were used in hybridization analyses to examine mRNA levels of P450IA1 and P450IA2, respectively. The level of P450IA1 mRNA in poly(A)+ mRNA was increased approximately 3- and 2-fold at 5 and 12 hr, respectively, after a single injection of pyridine, as evidenced by both slot blot and Northern blot analyses. A lesser increase (approximately 1.5-2-fold) in P450IA2 mRNA was also seen at 5 and 12 hr after treatment. The P450IA1 and P450IA2 mRNA levels returned to control values at 48 hr after pyridine administration. These results were compared with those produced by 3-methylcholanthrene at 5 hr after treatment. A multiplex polymerase chain reaction assay was also used to monitor simultaneously the changes in P450IA1, P450IA2, and P450IIE1 mRNA levels, and the results showed induction of P450IA1, in agreement with the results of slot and Northern blot analyses. In summary, metabolic activity assays, immunochemical detection, and Northern and slot blot analyses provide evidence to support the conclusion that pyridine modulates the expression of the P450IA gene subfamily and does so by elevating P450IA1 and P450IA2 mRNAs, through either transcriptional activation or increased mRNA stabilization. These results are in sharp contrast to P450IIE1 induction by pyridine, which appears to proceed through increased translational efficiency. Thus, pyridine, which is present in tobacco and tobacco smoke, is capable of simultaneously elevating multiple forms of P450 that are active in carcinogen metabolism.
用吡啶处理大鼠肝脏组织后，对细胞色素P450IA（P450IA）基因亚家族的表达及分子调控进行了研究。微粒体乙氧基异吩恶唑酮O - 脱乙基酶活性已被证明对P450IA亚家族具有特异性，单次腹腔注射吡啶（100 mg/kg）后，在10小时和16小时时，该活性分别比对照值增加了约2倍和3.5倍。P450IA1蛋白表达也呈时间依赖性升高，单次腹腔注射吡啶（100 mg/kg）后，在约16小时时P450IA1蛋白增加至最大值，这是通过使用能同时检测P450IA1和P450IA2的单克隆抗体进行免疫印迹分析检测到的。处理后48小时，免疫化学可检测到的P450IA1水平降至对照水平。分别使用对P450IA1和P450IA2 mRNA特异的寡核苷酸探针进行杂交分析，以检测P450IA1和P450IA2的mRNA水平。单次注射吡啶后，在5小时和12小时时，poly(A)+ mRNA中P450IA1 mRNA水平分别增加了约3倍和2倍，狭缝印迹和Northern印迹分析均证实了这一点。处理后5小时和12小时时，P450IA2 mRNA也有较小程度的增加（约1.5 - 2倍）。吡啶给药后48小时，P450IA1和P450IA2 mRNA水平恢复至对照值。将这些结果与处理后5小时3 - 甲基胆蒽产生的结果进行了比较。还使用多重聚合酶链反应分析同时监测P450IA1、P450IA2和P450IIE1 mRNA水平的变化，结果显示P450IA1被诱导，这与狭缝印迹和Northern印迹分析结果一致。总之，代谢活性测定、免疫化学检测以及Northern和狭缝印迹分析提供了证据，支持吡啶通过转录激活或增加mRNA稳定性来上调P450IA1和P450IA2 mRNA从而调节P450IA基因亚家族表达的结论。这些结果与吡啶诱导P450IIE1的情况形成鲜明对比，后者似乎是通过提高翻译效率来实现的。因此，烟草和烟草烟雾中存在的吡啶能够同时上调多种参与致癌物代谢的P450形式。
