Impact of di‐ethylhexylphthalate exposure on metabolic programming in P19 ECC‐derived cardiomyocytes

Abstract

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.

Phthalate plasticizers covalently linked to PVC via copper-free or copper catalyzed azide-alkyne cycloadditions

“…The organic layer was separated and the aqueous layer was extracted three times with 10 mL of chloroform. The combined organic layer was washed two times with 10 mL of brine, dried over MgSO 4 and concentrated in vacuo. The resulting red oil (0.8178 g) was purified by silica gel column chromatography with 75:25 hexanes/ethyl acetate as eluent, to give the title compound as a pale yellow oil (0.2414 g, 31.18% yield).…”

“…The organic layer was separated and the aqueous layer was extracted three times with 50 mL of ethyl acetate. The combined organic layer was washed two times with 20 mL of brine, dried over MgSO 4 1-(1-phenylethyl)-1H-1,2,3-triazol-4-yl) 116 mmol) in 10 mL of dimethylformamide was added DEHP-ester 18 (0.7254 g, 1.535 mmol) and stirred at 100 °C for 24 h. The reaction mixture was allowed to cool to room temperature and then poured into 100 mL of methanol, and filtered. The cake was washed three times with 20 mL of methanol and dried to give white solid (0.3256 g).…”

“…1) being the most popular. However, the adverse developmental [3,4], reproductive [5,6], neurological [7] and immune [8] health effects of many phthalates has led to a search for alternative plasticizers. Other common small molecule plasticizers include terphthalates, 1,2-cyclohexane-dicarboxylic acid diisononyl ester (Hexamoll® DINCH®), epoxidized vegetable oils, citrates, mellitates, adipates, benzoates, maleates, succinates, sebacates, phosphates, isosorbide esters.…”

Prenatal Exposure to Phthalic Acid Induces Increased Blood Pressure, Oxidative Stress, and Markers of Endothelial Dysfunction in Rat Offspring

“…It has also been reported that exposure to PA can change the cardiomyocyte metabolic profile and can consequently make heart prone to ischemia and ventricular dysfunctions. In other words, exposure to DEHP alters the expression of genes associated with cellular metabolism of cardiomyocytes [20]. Impaired NO production by coronary artery endothelial cells is one of the major factors causing physiological and morphological changes in cardiovascular systems, which, in turn, would lead to cardiac diseases such as hypertension [21,22].…”

Effects of Di-(2-ethylhexyl) Phthalate on the Hypothalamus–Uterus in Pubertal Female Rats

“…Reverse transcription was performed with 500 ng total RNA in a 10-ìL reaction, and 1 ìL of cDNA was then used for a 25-ìL PCR reaction mixture containing an optimal concentration of primers and SYBR-Green Supermix (SYBR premix Ex Tap II, TaKaRa, Shiga, Japan). The PCR reaction was carried out in 45 cycles of 95 °C for 20 s and 60 °C for 20 s. â-actin was used as an internal control [49]. The primers are listed in Table 1.…”