Mono-(2-ethylhexyl) phthalate induces apoptosis through miR-16 in human first trimester placental cell line HTR-8/SVneo.

Phthalates have been linked to adverse pregnancy complications. Mono-(2-ethylhexyl) phthalate, an active metabolite of di-(2-ethylhexyl) phthalate and an endocrine disruptor, has been shown to induce apoptosis in various cell types including placental cells. However, the mechanism of action of MEHP induced apoptosis is still unknown. We hypothesized that apoptosis may be mediated in part through altered microRNA(s) in placenta under MEHP exposure. In the present study, we report that MEHP increases miR-16 expression in a time- and dose-dependent manner (p<0.05), while inducing apoptosis in HTR-8/SVneo. Cells treated with MEHP showed a dose-dependent increase in cytotoxicity and reactive oxygen species along with decreased cell viability. Consistent with significant increase in apoptosis analyzed by flow cytometry, we detected decreased anti-apoptotic BCL-2 at transcriptional and translational levels with MEHP (p<0.05). Knockdown of miR-16 did not decrease the BCL-2/BAX protein expression ratio in the presence of MEHP when compared to negative control demonstrating that MEHP induces apoptosis directly through miR-16. In conclusion, our study demonstrates for the first time that MEHP induces miR-16, which in turn, alters BCL-2/BAX ratio leading to increased apoptosis. This study provides a novel insight into MEHP induced epigenetic regulation in placental apoptosis which may lead to pregnancy complications.

The plasticizer BBP selectively inhibits epigenetic regulator sirtuins.

The plasticizer benzyl butyl phthalate (BBP) is a well-known endocrine disruptor. Widespread human exposure to phthalates has raised substantial public concern due to its detrimental health effects. However, molecular mechanisms of the phthalates effect require elucidation. In this study, we analyzed: 1) the binding interaction of several phthalates and persistent organic pollutants with epigenetic regulator sirtuins and 2) the effect of BBP on the sirtuins in HepG2 cells. AutoDock molecular docking analysis showed that BBP binds to Sirt1 and Sirt3 proteins similarly to the native ligands with shortest binding free energies (ΔGb) of -7.35 and -8.3 kcal/mol, respectively; and inhibition constants (Ki) of 4.07 µM and 0.82 µM, respectively. Furthermore, BBP was superimposed onto the co-crystallized ligands within the least root-mean-square deviation (RMSD) of 0.96Å and 1.55Å for Sirt1 and Sirt3, respectively, and bound into the sites with a sufficient number of hydrogen bonds, implying the best fit compared to other sirtuins. In HepG2 cells, BBP significantly down-regulated Sirt1 and Sirt3 (p<0.05) gene expression at a concentration as low as 10nM; other sirtuins remained unaffected. Consistent with decreased gene expression, Sirt1 and Sirt3 protein levels were significantly decreased at 48 h (p<0.05). In addition, mitochondrial biogenesis regulators PGC-1α, NRF-1, and NRF-2, were decreased (p<0.05). SiRNA studies showed that BBP did not regulate PGC-1α via sirtuin and BBP requires sirtuin's presence to regulate NRF-1 or NRF-2. BBP significantly increased ROS production (p<0.05) and ROS may be chiefly regulated by NRF-1 and NRF-2 in HepG2 cells under Sirt1 and Sirt3 silenced condition. This is the first report to demonstrate that BBP selectively disrupts specific sirtuins in HepG2 cells. In conclusion, our study suggests that BBP can impair two vital epigenetic regulators and mitochondrial biogenesis regulators in liver cells.

Pyrroloquinoline quinone increases the expression and activity of Sirt1 and -3 genes in HepG2 cells.

Sirtuin (Sirt) 1 and Sirt 3 are nicotinamide adenine dinucleotide ((+))-dependent protein deacetylases that are important to a number of mitochondrial-related functions; thus, identification of sirtuin activators is important. Herein, we hypothesize that pyrroloquinoline quinone (PQQ) can act as a Sirt1/Sirt3 activator. In HepG2 cell cultures, PQQ increased the expression of Sirt1 and Sirt3 gene, protein, and activity levels (P < .05). We also observed a significant increase in nicotinamide phosphoribosyltransferase gene expression (as early as 18 hours) and increased NAD(+) activity at 24 hours. In addition, targets of Sirt1 and Sirt3 (peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1 and 2, and mitochondrial transcription factor A) were increased at 48 hours. This is the first report that demonstrates PQQ as an activator of Sirt1 and Sirt3 expression and activity, making it an attractive therapeutic agent for the treatment of metabolic diseases and for healthy aging. Based on our study and the available data in vivo, PQQ has the potential to serve as a therapeutic nutraceutical, when enhancing mitochondrial function.