Aryl hydrocarbon receptor mediates the cardiac developmental toxicity of EOM from PM2.5 in P19 embryonic carcinoma cells.

Ambient fine particulate matter (PM2.5) has been found to be associated with congenital heart defects, but the molecular mechanisms remain to be elucidated. Our previous study revealed that extractable organic matter (EOM) from PM2.5 exerted cardiac developmental toxicity in zebrafish embryos. The aim of the current study is to explore the effects of EOM on cardiac differentiation of P19 mouse embryonic carcinoma stem cells. We found that EOM at 10 µg/ml (a non-cytotoxic dose level) significantly reduced the proportion of cardiac muscle troponin (cTnT) positive cells and the percentage of spontaneously beating embryoid bodies, indicating a severe inhibition of cardiac differentiation. Immunofluorescence and qPCR data demonstrated that EOM increased the expression levels of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1A1 and diminished the expression level of ß-catenin. Furthermore, EOM treatment significantly upregulated cell proliferation rate and elevated the percentage of γH2A.X positive cells without affecting apoptosis. It is worth noting that the EOM-induced changes in gene expression, cellular proliferation and DNA double strain breaks were attenuated by the AhR antagonist CH223191. In conclusion, our data indicate that AhR mediates the inhibitory effects of EOM (from PM2.5) on the cardiac differentiation of P19 cells.

Crosstalk between AhR and wnt/ß-catenin signal pathways in the cardiac developmental toxicity of PM2.5 in zebrafish embryos.

Recent studies have shown an association between congenital heart defects and air fine particle matter (PM2.5), but the molecular mechanisms remain elusive. It is well known that a number of organic compounds in PM2.5 can act as AhR agonists, and activation of AhR can antagonize Wnt/ß-catenin signaling. Therefore, we hypothesized that PM2.5 could activate AhR and then repress the expression of wnt/ß-catenin targeted genes essential for cardiogenesis, resulting in heart defects. To test this hypothesis, we investigated the effects of extractable organic matter (EOM) from PM2.5 on AhR and Wnt/ß-catenin signal pathways in zebrafish embryos. We confirmed that EOM could cause malformations in the heart and decreased heart rate in zebrafish embryos at 72hpf, and found that the EOM-induced heart defects were rescued in embryos co-exposed with EOM plus AhR antagonist CH223191 or ß-catenin agonist CHIR99021. We further found that EOM had increased the expression levels of AhR targeted genes (Cyp1a1, Cyp1b1 and Ahrra) and reduced the mRNA levels of ß-catenin targeted genes (axin2, nkx2.5 and sox9b). The mRNA expression level of Rspo2, a ß-catenin upstream gene, was also decreased in embryos exposed to EOM. Supplementation with CH223191 or CHIR99021 attenuated most of the EOM-induced expression changes of genes involved in both AhR and wnt/ß-catenin signal pathways. However, the mRNA expression level of AhR inhibitor Ahrrb, which did not change by EOM treatment alone, was increased in embryos co-exposed to EOM plus CH223191 or CHIR99021. We conclude that the activation of AhR by EOM from PM2.5 might repress wnt/ß-catenin signaling, leading to heart defects in zebrafish embryos. Furthermore, our results indicate that the cardiac developmental toxicity of PM2.5 might be prevented by targeting AhR or wnt/ß-catenin signaling.

[Seasonal Variation and Source Analysis of the Water-soluble Inorganic Ions in Fine Particulate Matter in Suzhou].

A total of 87 daily PM2.5 samples were collected in the urban area of Suzhou city during 2015, representing spring, summer, autumn, winter, respectively. Mass concentration of PM2.5 was analyzed gravimetrically. Water-soluble inorganic ions, including F-, Cl-, NO3-, SO42-, Na+, NH4+, K+, Mg2+and Ca2+, were determined by ion chromatography. The average mass concentration of PM2.5 was (74.26±38.01) µg·m-3. The seasonal variations of PM2.5 concentrations decreased in the order of winter > spring > autumn > summer. The average total mass concentrations of 9 ions was (43.95±23.60) µg·m-3, and the order of concentration of ions was NO3- > SO42- > NH4+ > Na+ > Cl- > K+ > Ca2+ > F- > Mg2+. Seasonal variation of ion concentrations was significant, with the highest concentration observed in winter and the lowest in summer. The secondary inorganic species, including SO42-, NO3- and NH4+ (SNA) were the major components of the water-soluble ions in PM2.5. SNA's correlations with each other were significant. SO42-, NO3- and NH4+ were probably in the form of NH4NO3 and (NH4)2SO4. The [NO3-]/[SO42-] ratio approaching to 1 implied that mobile sources were as important as stationary sources. Ion balance calculations indicated strong correlations between anion and cation equivalents. The PM2.5 was acidic. Industrial emission, combustion process, secondary formation and fugitive dust were the major sources of the water-soluble ions in PM2.5..