Suzuki T, Imagawa M, Yamada R, Yokoyama K, Kondo S, Itakura K, Muramatsu M
Department of Biochemistry, Faculty of Medicine, Saitama Medical School, Japan.
Carcinogenesis. 1994 Aug;15(8):1759-61. doi: 10.1093/carcin/15.8.1759.
We constructed a complementary DNA (cDNA) library from mRNAs of rat liver induced by an initiating dose of a chemical carcinogen, N-nitrosodiethylamine (DEN). Using a differential hybridization with cDNA probes prepared from mRNAs of control and DEN-treated rat liver, eight cDNAs of which expression was altered by an acute single dose of DEN were cloned. Colony hybridization and nucleotide sequencing demonstrated six independent cDNA clones. These were known genes encoding liver-specific proteins such as microsomal epoxide hydrolase (mEH; epoxide hydrolase, EC 3.3.2.3), albumin, transthyretin, CYP2B7, CYP1A2 (microsomal cytochrome P450, EC 1.14.14.1) and argininosuccinate synthetase (EC 6.3.4.5). Quantitative Northern blot hybridization was carried out to measure the mRNA content of DEN-initiated rat liver at various times after DEN injection. We also analyzed the expression of glutathione transferase P (GST-P; glutathione transferase, EC 2.5.1.18), c-jun and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; glyceraldehyde-phosphate dehydrogenase, EC 1.2.1.12). A single injection of DEN increased the mRNA levels of mEH, beta-actin and c-jun markedly and those of GST-P and GAPDH moderately, but decreased the mRNA levels of CYP2B7, CYP1A2, albumin and argininosuccinate synthetase. Transthyretin mRNA content was not changed, indicating that it was a false-positive clone picked up by chance. These dramatic changes in liver gene expression after acute exposure to DEN are discussed in terms of acute reactions to the massive damage to the DNA and self-defense mechanisms against toxic xenobiotics.
我们用化学致癌物N-亚硝基二乙胺（DEN）的起始剂量诱导大鼠肝脏的mRNA构建了一个互补DNA（cDNA）文库。通过用对照和DEN处理的大鼠肝脏的mRNA制备的cDNA探针进行差异杂交，克隆了8个其表达因急性单剂量DEN而改变的cDNA。菌落杂交和核苷酸测序鉴定出6个独立的cDNA克隆。这些是已知的编码肝脏特异性蛋白的基因，如微粒体环氧化物水解酶（mEH；环氧化物水解酶，EC 3.3.2.3）、白蛋白、转甲状腺素蛋白、CYP2B7、CYP1A2（微粒体细胞色素P450，EC 1.14.14.1）和精氨琥珀酸合成酶（EC 6.3.4.5）。进行定量Northern印迹杂交以测量DEN注射后不同时间DEN启动的大鼠肝脏的mRNA含量。我们还分析了谷胱甘肽转移酶P（GST-P；谷胱甘肽转移酶，EC 2.5.1.18）、c-jun和甘油醛-3-磷酸脱氢酶（GAPDH；甘油醛-3-磷酸脱氢酶，EC 1.2.1.12）的表达。单次注射DEN显著增加了mEH、β-肌动蛋白和c-jun的mRNA水平，适度增加了GST-P和GAPDH的mRNA水平，但降低了CYP2B7、CYP1A2、白蛋白和精氨琥珀酸合成酶的mRNA水平。转甲状腺素蛋白mRNA含量没有变化，表明它是偶然挑选出的假阳性克隆。本文从对DNA大量损伤的急性反应和针对有毒外源性物质的自我防御机制方面讨论了急性暴露于DEN后肝脏基因表达的这些显著变化。
