Schaedlich Kristina, Schmidt Juliane-Susanne, Kwong Wing Yee, Sinclair Kevin D, Kurz Randy, Jahnke Heinz-Georg, Fischer Bernd
Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Grosse Steinstrasse 52, 06097, Halle, Germany.
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Leicestershire, LE12 5RD, United Kingdom.
J Appl Toxicol. 2015 Jul;35(7):861-9. doi: 10.1002/jat.3085. Epub 2014 Oct 29.
Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml(-1)) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes.
邻苯二甲酸二（2-乙基己基）酯（DEHP）是日常塑料制品中最常见的增塑剂。它是一种普遍存在的环境污染物，主要已知会损害男性性腺发育和生育能力。尽管已证实DEHP可穿过胎盘，但关于产前DEHP暴露对某些疾病的长期影响的研究[健康与疾病的发育起源范式（DOHaD）假说]却很少。在过去几个世纪中，环境污染的加剧与心血管和代谢疾病患病率的上升相吻合。我们研究了在不同发育阶段早期胚胎暴露于DEHP对心肌发生的影响。我们使用了一种体外模型，即小鼠P19胚胎癌细胞系（P19 ECC），它可以模拟早期胚胎阶段直至分化为跳动的心肌细胞。将未分化阶段的P19 ECC暴露于DEHP（5、50、100μg ml⁻¹）5天，随后分化为跳动的心肌细胞。我们通过定量实时PCR（qRT-PCR）分析了代谢（Pparg1、Fabp4和Glut4）、心脏（Myh6、Gja1）和甲基化（Dnmt1、Dnmt3a）标记基因的表达、细胞的跳动率和分化速度。通过焦磷酸测序研究了Pparg1、Ppara和Glut4的甲基化状态。DEHP显著改变了所有研究基因的表达。跳动率和分化速度加快。暴露于DEHP导致Ppara和Pparg1内特定CpG的甲基化有小幅但具有统计学意义的增加，否则这些区域通常是低甲基化的，但Glut4的甲基化未改变。P19 ECC早期暴露于DEHP会改变与细胞代谢相关的基因表达以及心肌细胞的功能特征。
