Department of Obstetrics and Gynecology, New York University School of Medicine, New York, NY, USA.
Reprod Sci. 2013 Aug;20(8):917-28. doi: 10.1177/1933719112468946. Epub 2013 Jan 23.
Intrauterine growth restriction (IUGR) has been associated with exposure to polyaromatic hydrocarbons (PAHs) which are released in the combustion of oil, fuel, gas, garbage, and tobacco. Pregnant women exposed to PAHs are at risk of the effects of these environmental toxins; for example, benzo-α-pyrene (BαP) is able to enter the blood stream and could contribute to IUGR or other developmental abnormalities via effects on the placental cells. Since IUGR has been associated with decreased cord blood concentrations of immunoreactive insulin-like growth factor 1 (ir-IGF-1) and IUGR has been associated with disordered development and fetal programming, we tested the effects of BαP on human placental trophoblast cells in culture.
IGF-1 expression and activation was studied using an immortalized human placental trophoblast cell line (HTR-8). The cells were treated with vehicle control or 1 µmol/L BαP, or 5 µmol/L BαP for 12 hours. RNA was extracted and the exons of IGF-1 were amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). The ir-IGF-1 expression levels were compared using gel electrophoresis. The PCR products were sequenced, and levels of mutation were measured with comparative sequence analysis. A computational protein analysis (computer simulation) was performed in order to assess the potential impact of BαP-associated mutation on IGF-1 protein function.
The IGF-1 expression decreased considerably in BαP-treated cells relative to untreated controls (P < .05), also in a dose-dependent manner. Comparative sequence analysis indicated that the level of BαP exposure correlated with the percentage of base pair mutations in IGF-1 nucleotide sequences for both treatment groups (P < .05). Shifts were observed in the open reading frame, indicating a possible change in the IGF-1 start codon. Protein folding simulation analysis indicated that the base pair changes induced by BαP weakened IGF-1-IGF binding protein (IGFBP) interaction.
In concordance with the previous findings, exposure of human placental trophoblast cells to BαP exposure results in reduction of IGF-1 expression and base pair mutations. The direct action of BαP on the placenta indicates that it may not be necessary for BαP to access other maternal tissues in order for gene abnormalities to occur. Given that PAHs are known to work through aryl hydrocarbon hydrolase (AHH), these results are likely due to the presence of AHH in HTR cells. Computational modeling of BαP actions on IGF1, substrate-ligand binding, supports the biological premise of this work and underlines the need to determine actual biological effects rather than equating immune to bioactivity of IGF1.
宫内生长受限（IUGR）与多环芳烃（PAHs）的暴露有关，这些物质是在石油、燃料、天然气、垃圾和烟草的燃烧中释放出来的。接触多环芳烃的孕妇有这些环境毒素影响的风险；例如，苯并[a]芘（BαP）能够进入血液，并可能通过对胎盘细胞的影响导致 IUGR 或其他发育异常。由于 IUGR 与脐带血中免疫反应性胰岛素样生长因子 1（ir-IGF-1）浓度降低有关，并且 IUGR 与发育紊乱和胎儿编程有关，因此我们在培养的人胎盘滋养层细胞中测试了 BαP 的作用。
使用永生化的人胎盘滋养层细胞系（HTR-8）研究 IGF-1 的表达和激活。将细胞用载体对照或 1 μmol/L BαP 或 5 μmol/L BαP 处理 12 小时。提取 RNA，并使用逆转录酶-聚合酶链反应（RT-PCR）扩增 IGF-1 的外显子。通过凝胶电泳比较 ir-IGF-1 表达水平。对 PCR 产物进行测序，并通过比较序列分析测量突变水平。进行计算蛋白分析（计算机模拟），以评估 BαP 相关突变对 IGF-1 蛋白功能的潜在影响。
BαP 处理的细胞中 IGF-1 的表达相对于未处理的对照明显降低（P <.05），并且呈剂量依赖性。比较序列分析表明，BαP 暴露水平与两组治疗中 IGF-1 核苷酸序列中的碱基对突变百分比相关（P <.05）。在开放阅读框中观察到移位，表明 IGF-1 起始密码子可能发生变化。蛋白折叠模拟分析表明，BαP 诱导的碱基对变化削弱了 IGF-1-IGF 结合蛋白（IGFBP）的相互作用。
与先前的发现一致，人胎盘滋养层细胞暴露于 BαP 会导致 IGF-1 表达和碱基对突变减少。BαP 对胎盘的直接作用表明，BαP 不一定需要进入其他母体组织才能发生基因异常。鉴于多环芳烃已知通过芳烃羟化酶（AHH）起作用，这些结果可能是由于 HTR 细胞中存在 AHH。BαP 对 IGF1 的作用的计算模型、底物-配体结合，支持了这项工作的生物学前提，并强调需要确定实际的生物学效应，而不是将 IGF1 的免疫等同于生物活性。
