Buard A, Beaune P H, Renier A, Jaurand M C, Bignon J, Laurent P
Laboratoire de Pathologie Cellulaire et Moleculaire de l'Environnement, INSERM U. 139, C.H.U. Henri Mondor, Creteil, France.
J Cell Physiol. 1994 Jul;160(1):176-83. doi: 10.1002/jcp.1041600120.
Cultured rat pleural mesothelial cells (RPMC) isolated from male Sprague-Dawley rats have been shown to metabolize polycyclic aromatic hydrocarbons to more oxygenated metabolites. This capacity, which is maintained with passages, suggested the presence of monooxygenase enzymes. In order to clarify the enzymatic pathway, we investigated the expression of cytochromes P450 (CYP) in cultured RPMC by Western and Northern blot analyses. Cells were cultured in Ham's F10 medium supplemented with 10% fetal calf serum. The CYP expression was studied from passage 9 to 16 on different cell strains treated for 48 hours with P450 inducers. CYP1A1 apoprotein expression was very low in untreated cells, but was markedly induced after treatment with 1 microM 3-methylcholanthrene or 22 microM beta-naphthoflavone. CYP1A1 mRNA was not detected in untreated cells and appeared after 3-methylcholanthrene treatment. CYP2E1 apoprotein was constitutively expressed in cultured RPMC, and markedly increased by 170 mM ethanol, and 0.1 microM or 1 microM dexamethasone treatments. Unexpectedly, whereas the amount CYP2E1 mRNA was not modified by ethanol treatment, dexamethasone has a marked inductive effect on CYP2E1 mRNA level. The CYP expression pattern was found similar in RPMC issued from different rats, and not dependent on passage number. The CYP expression and the detection of NADPH-P450 reductase, and of epoxide hydrolase, ascertained that RPMC contain the overall enzymatic pathway required for the biotransformation and activation of procarcinogen compounds, such as polycyclic aromatic hydrocarbons and nitrosamines. Both expression and regulation properties are maintained in long-term cultures of RPMC.
从雄性Sprague-Dawley大鼠分离得到的培养大鼠胸膜间皮细胞（RPMC）已被证明可将多环芳烃代谢为更多的氧化代谢产物。这种随传代而保持的能力提示存在单加氧酶。为了阐明酶促途径，我们通过蛋白质免疫印迹法和Northern印迹分析法研究了培养的RPMC中细胞色素P450（CYP）的表达。细胞在补充有10%胎牛血清的Ham's F10培养基中培养。在不同细胞株上，从第9代到第16代研究了用P450诱导剂处理48小时后的CYP表达。在未处理的细胞中，CYP1A1载脂蛋白表达非常低，但在用1μM 3-甲基胆蒽或22μMβ-萘黄酮处理后显著诱导。在未处理的细胞中未检测到CYP1A1 mRNA，在3-甲基胆蒽处理后出现。CYP2E1载脂蛋白在培养的RPMC中组成性表达，并在170 mM乙醇、0.1μM或1μM地塞米松处理后显著增加。出乎意料的是，虽然乙醇处理未改变CYP2E1 mRNA的量，但地塞米松对CYP2E1 mRNA水平有显著的诱导作用。在来自不同大鼠的RPMC中发现CYP表达模式相似，且不依赖传代数。CYP的表达以及NADPH-P450还原酶和环氧水解酶的检测确定RPMC含有生物转化和激活前致癌物化合物（如多环芳烃和亚硝胺）所需的完整酶促途径。RPMC的长期培养中表达和调节特性均得以保持。
