Kling David E, Cavicchio Amanda J, Sollinger Christina A, Schnitzer Jay J, Kinane T Bernard, Newburg David S
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Birth Defects Res B Dev Reprod Toxicol. 2010 Jun;89(3):223-32. doi: 10.1002/bdrb.20247.
Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling.
To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response-element (RARE) was transfected into HEK-293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace-cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets.
Nitrofen-induced dose-dependent declines in RARE-reporter gene expression. However, similar reductions were observed in control-reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen-induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes.
The observed declines in nitrofen-associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition.
除草醚是一种二苯醚，可在啮齿动物中诱发先天性膈疝（CDH）。其作用机制被推测为抑制视黄醛脱氢酶（RALDH），从而导致视黄酸信号传导减少。
为了确定除草醚是否抑制RALDH酶，将含有视黄酸反应元件（RARE）的报告基因构建体转染到HEK-293细胞中，并在视黄醛（视黄醇）存在的情况下用不同浓度的除草醚处理。通过半胱天冬酶切割微孔板分析和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）分析来表征细胞死亡。使用活/死染色法对胎鼠肺外植体进行细胞活力的体外分析。使用荧光免疫组织化学法，用磷酸化组蛋白和活化的半胱天冬酶抗体对外植体组织进行细胞增殖和凋亡评估。尼罗红染色用于识别细胞内脂滴。
除草醚诱导RARE报告基因表达呈剂量依赖性下降。然而，在对照报告基因构建体中也观察到了类似的下降，这表明除草醚损害了细胞活力。观察到的细胞活力下降是由细胞死亡增加导致的，并通过两种独立的分析得到证实。体外分析表明，间充质细胞特别容易受到除草醚诱导的凋亡影响，而胎鼠肺外植体中的上皮细胞增殖则显著减少。用除草醚处理这些外植体还显示出明显的脂质重新分布，主要是向吞噬细胞的重新分布。
观察到的除草醚相关视黄酸信号传导的下降似乎与RALDH抑制无关，可能是由除草醚诱导的细胞死亡/凋亡导致的。这些结果支持了CDH发展的细胞凋亡机制，独立于RALDH抑制。
