Zerilli A, Ratanasavanh D, Lucas D, Goasduff T, Dréano Y, Menard C, Picart D, Berthou F
Equipe de recherche EA-948, Faculté de Médecine, UBO, Brest, France.
Chem Res Toxicol. 1997 Oct;10(10):1205-12. doi: 10.1021/tx970048z.
4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% (+/- 11) and 13% (+/- 16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.
先前的研究表明，在动物和人类中，4-硝基苯酚2-羟基化活性主要由P450 2E1催化。由于这种化合物被广泛用作P450 2E1的体外探针，因此本研究旨在测试其催化特异性。首先，对用不同诱导剂处理的大鼠的肝微粒体和肝细胞培养物进行实验。用吡唑和地塞米松处理的大鼠的肝微粒体使对硝基苯酚羟基化，其代谢率比对照提高了2.5倍和2.7倍。地塞米松处理增加了P450 3A的肝脏含量，但未增加P450 2E1的含量。两种P450 3A催化活性的特异性抑制剂，即酮康唑和三乙酰竹桃霉素（TAO），在用地塞米松处理的大鼠中抑制了高达50%的4-硝基苯酚羟基化，但在对照中未观察到这种抑制作用。用地塞米松处理的大鼠的肝细胞培养物将对硝基苯酚转化为4-硝基儿茶酚的能力比对照高7.8倍。这种催化活性受到TAO的抑制。同样，用吡唑处理的大鼠的肝细胞培养物使对硝基苯酚羟基化，其代谢率比对照提高了约8倍。该反应受到二乙基二硫代氨基甲酸盐和二甲基亚砜的抑制，这两种物质都是P450 2E1的抑制剂。其次，在一组13个人类肝微粒体中检测了除P450 2E1之外的人类P450催化形成4-硝基儿茶酚的能力。二乙基二硫代氨基甲酸盐和酮康唑分别使4-硝基苯酚羟化酶活性降低了77%（±11）和13%（±16）。此外，二乙基二硫代氨基甲酸盐抑制后的残余活性与七种P450 3A4催化活性显著相关。最后，使用经基因工程改造用于表达人类P450的人类细胞系表明，P450 2E1和3A4使4-硝基苯酚羟基化，周转率分别为19.5和1.65 min-1。总之，P450 3A可能对人和大鼠的4-硝基苯酚羟化酶活性有显著贡献。
