Marchand A, Barouki R, Garlatti M
Institut National de la Santé et de la Recherche Médicale, Unité-490, 45 rue des Saints-Pères, 75006 Paris, France.
Mol Pharmacol. 2004 Apr;65(4):1029-37. doi: 10.1124/mol.65.4.1029.
The dioxin 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) induces phase I and II xenobiotic metabolizing enzymes (XME) which act sequentially to eliminate different classes of xenobiotics. The transcriptional effects of TCDD are generally mediated by the arylhydrocarbon receptor (AhR). We hypothesized that TCDD could also act indirectly, by increasing the activity of cytochrome P450 1A1 (CYP1A1), a phase I gene, which could then mediate the induction of other XME genes, such as the NAD(P)H:quinone oxidoreductase 1 (NQO1). To test this hypothesis, NQO1 gene expression was monitored after either overexpression of CYP1A1 or siRNA-mediated knock-down of CYP1A1 activity in the hepatoma cell line HepG2. Overexpression of CYP1A1 in the absence of TCDD was carried out using either adenoviral infection or the "Tet-off" system. Recombinant adenoviruses were produced encoding no protein, CYP1A1 (Ad1A1), or a mutated inactive CYP1A1 (Ad1A1mut). In the HepG2 Tet-off cell line, CYP1A1 expression was induced by the removal of doxycycline (dox) from the cell medium. Ad1A1 infection or dox removal induced CYP1A1 activity and H(2)O(2) production similarly to TCDD treatment. Moreover, in both systems, the amount of NQO1 mRNA increased to the same level as after TCDD treatment (approximately 2-fold). The UDP-glucuronosyl transferase 1A6 (UGT1A6) gene is also similarly regulated. NQO1 gene expression was not induced when mutant, inactive CYP1A1 was overexpressed or when the antioxidant N-acetyl cysteine (NAC) was added to Ad1A1. Finally, either NAC or siRNA directed against CYP1A1 mRNA decreased the induction of NQO1 gene expression by TCDD. We conclude that, after exposure to TCDD, the NQO1 gene expression can be controlled by CYP1A1 activity through an oxidative stress mediated pathway.
二噁英2,3,7,8 - 四氯二苯并 - 对 - 二噁英（TCDD）可诱导I相和II相异生物质代谢酶（XME），这些酶依次作用以消除不同种类的异生物质。TCDD的转录效应通常由芳烃受体（AhR）介导。我们推测TCDD也可能通过增加细胞色素P450 1A1（CYP1A1，一种I相基因）的活性而间接发挥作用，然后CYP1A1可介导其他XME基因如NAD(P)H:醌氧化还原酶1（NQO1）的诱导。为验证这一假设，在肝癌细胞系HepG2中，过表达CYP1A1或用siRNA介导降低CYP1A1活性后，监测NQO1基因表达。在无TCDD的情况下，使用腺病毒感染或“Tet-off”系统过表达CYP1A1。制备了编码无蛋白、CYP1A1（Ad1A1）或突变的无活性CYP1A1（Ad1A1mut）的重组腺病毒。在HepG2 Tet-off细胞系中，通过从细胞培养基中去除强力霉素（dox）诱导CYP1A1表达。Ad1A1感染或去除dox诱导CYP1A1活性和H₂O₂产生的情况与TCDD处理相似。此外，在这两种系统中，NQO1 mRNA的量增加到与TCDD处理后相同的水平（约2倍）。尿苷二磷酸葡萄糖醛酸基转移酶1A6（UGT1A6）基因也受到类似调控。当过表达突变的无活性CYP1A1或向Ad1A1中添加抗氧化剂N - 乙酰半胱氨酸（NAC）时，未诱导NQO1基因表达。最后，NAC或针对CYP1A1 mRNA的siRNA均可降低TCDD对NQO1基因表达的诱导。我们得出结论，暴露于TCDD后，NQO1基因表达可通过氧化应激介导的途径由CYP1A1活性控制。
