Compartmentalized Regulation Of NAD+ By Di（2-ethyl-hexyl） Phthalate Induces DNA Damage

Objective:Di（2-ethyl-hexyl）phthalate（DEHP）is an environmental endocrine disruptor widely used as a plasticizer in PVC materials,which is easy to release into the environment and into the human body.DEHP can cause DNA damage by interfering with the DNA repair pathway and inhibit placental development.However,the specific mechanism of interfering with DNA repair is unclear.Nicotinamide adenine dinucleotide（NAD+）is an important substrate of PARP1 and is widely involved in DNA repair pathways.In this study,from the perspective of NAD+,we explored its role in the inhibition of placental trophoblast proliferation induced by DEHP.Methods:Part I:ICR mice were used to establish a DNA damage model in pregnant mice with DEHP（0,5,50,200 mg/kg/d）from GD 0 to 15.Western Blotting detected phosphorylatedγH₂AX,PCNA protein expression.HTR-8 cells were treated with MEHP（0,20,200,500μM）for in vitro validation by neutral comet assay.Changes in the largest longitudinal section of the placenta and the area of the labyrinth trophoblast were evaluated by HE staining.Part II:Detection of NAD+level during pregnancy.The serum and placenta NAD+of pregnant mice were detected by WST-8 assay.NAMPT,CD38 and CD157 protein expressions were detected by Western Blotting.Part III:The mechanism of DEHP-induced the inhibition of placental trophoblast proliferation.The expressions of PARP1 and PARP-PAR were detected by western blotting.In order to understand the regulation of NAD+by DEHP,the method of separating placental nucleus,cytoplasm and mitochondria was used to detect NAD+.The expression of NMNAT1/2/3 proteins were detected simultaneously.Changes in mitochondrial numbers and damage were detected by transmission electron microscopy,mitochondrial DNA copy number and cellular immunofluorescence.Seahorse detected the changes of ATP in HTR-8 cells.Detection of ACPM and OXPHOS protein expression to explore placental cell respiratory chain damage.Part IV:To investigate whether NAD+precursor supplementation can alleviate placental DNA and mitochondrial damage.A pregnant mouse model of NAM intervention during pregnancy was constructed,and the experiment was divided into four groups:control group,NAM group（500 mg/kg/d）,DEHP group（50 mg/kg/d）,and NAM+DEHP group.DNA damage was detected by the above-mentioned Western Blotting,neutral comet assay.Mitochondrial damage was observed by transmission electron microscope.Results:It was found that there was a dose-dependent increase between the expression of placental phosphorylatedγH₂AX protein and the concentration of DEHP.The neutral comet assay observed an increase in the comet tail distance of HTR-8 cells,indicating that DEHP can cause placental DNA damage.The expression of PCNA was decreased.The maximum longitudinal section of the placenta and the area of the placental trophoblast decreased,indicating that the proliferation of placental trophoblasts was inhibited under the action of DEHP.NAD+detection showed that the NAD+decreased in serum,placenta,and HTR-8 cells.CD38,CD157 expression was elevated,and NAMPT was unchanged.This suggests that DEHP may reduce placental NAD+by enhancing the expression of NAD+hydrolase.Under the action of DEHP,PARP1protein expression had little effect,but its activity was reduced.The expression of NMNAT1/2 was decreased,and the expression of NMNAT3 was increased,suggesting that DEHP reduced the concentration of NAD+in the placental nucleus by reducing the level of NAD+synthase NMNAT1.Large amounts of NAD+are synthesized in mitochondria to maintain cell survival.Transmission electron microscopy and mitochondrial DNA copy number assays revealed that DEHP reduced placental mitochondrial numbers and disrupted mitochondrial structure.In vitro fluorescence experiments also confirmed this.OXPHOS and Seahorse results suggest that the placental mitochondrial respiratory chain is blocked,reducing ATP production.Supplementation of NAD+precursor NAM can rescue placental DNA and mitochondrial damage caused by DEHP to some extent.Conclusion:This study explored the mechanism of DEHP induced placental cell proliferation inhibition cause by DNA damage from the perspective of NAD+and ATP.On the one hand,DEHP reduced the NAD+of the placenta by regulating the distribution of NAD+and the expression of synthetic hydrolase.On the other hand,DEHP reduces the production of ATP by destroying the placental mitochondrial structure,reducing the number of mitochondria,and hindering the electron transfer in the mitochondrial respiratory chain.Under the combined effect of NAD+and ATP depletion,PARP1 activity is inhibited,causing accumulation of DNA damage and a reduction in placental trophoblast area,ultimately.The damaging effects of DEHP on DNA and mitochondrial structure can be alleviated by appropriate supplementation of NAD+precursor NAM.