Casp8 Hypomethylation and Neural Tube Defects in Association with Polycyclic Aromatic Hydrocarbon Exposure

Background: Epidemiological studies have found that prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with increased risk for neural tube defects (NTDs). Aberrant DNA methylation, excessive apoptosis, and oxidative stress have been implied as the mechanism underlying the association between PAH exposure and NTDs, respectively. However, the role of DNA methylation aberration of apoptotic initiator CASP8 (caspase-8, apoptosis-related cysteine peptidase) in the formation of NTDs in association with PAH exposure is not known. By combining a case-control study and mouse model, we aimed to explore the full spectrum of the links from PAH exposure, oxidative stress, CASP8 methylation change, caspase-8 activation, apoptosis, to NTD formation.

Results: Hypomethylation of CASP8 promoter was noticed in the microarray profiled by Infinium HumanMethylation450 BeadChip using neural tissues from 10 terminated NTD fetuses and 8 terminated non-malformed fetuses (14 CpG sites, with β difference ranging between 8.8 and 26.3%), and was validated in a larger case-control sample performed with neural tissues from 80 NTD cases and 32 non-malformed fetuses, using the Sequenom MassARRAY system (7 CpG sites). Hypomethylation of CASP8 was a risk factor for NTDs (aOR = 1.11; 95% CI, 1.05-1.17) based on the logistic regression model. According to Pearson's correlation, methylation levels of CASP8 were inversely correlated with PAH concentrations in maternal serum and with oxidative stress markers in fetal neural tissues (p < 0.05). In the animal study, increased NTD rates (13.5% frequency), Casp8 hypomethylation, caspase-8 upregulation, increased caspase-8 cleavage, and excessive apoptosis were found in mouse embryos cultured with benz(a)pyrene (BaP) in vitro. Antioxidant N-acetyl-L-cysteine (NAC) and BaP co-treatment attenuated the changes found in BaP treatment group.

Conclusions: Hypomethylation of Casp8 promoter is associated with the formation of NTDs, and Casp8 hypomethylation may be induced by oxidative stress that resulted from exposure to PAHs.