Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m6A

Abstract

Background: Several epidemiological investigations demonstrated that maternal arsenic (As) exposure elevated risk of fetal growth restriction (FGR), but the mechanism remains unclear.

Objectives: This study aimed to investigate the effects of gestational As exposure on placental and fetal development and its underlying mechanism.

Methods: Dams were exposed to 0.15, 1.5, and $15\mathrm{mg}/L$ ${\text{NaAsO}}_2$ throughout pregnancy via drinking water. Sizes of fetuses and placentas, placental histopathology, and glycogen content were measured. Placental RNA sequencing was conducted. Human trophoblasts were exposed to ${\text{NaAsO}}_2$ ($2\mu M$) to establish an in vitro model of As exposure. The mRNA stability and protein level of genes identified through RNA sequencing were measured. $N^6\text{-Methyladenosine}$ ($m^{6}A$) modification was detected by methylated RNA immunoprecipitation-quantitative real-time polymerase chain reason (qPCR). The binding ability of insulin-like growth factor 2 binding protein 2 to the gene of interest was detected by RNA-binding protein immunoprecipitation-qPCR. Intracellular S-adenosylmethionine (SAM) and methyltransferase activity were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and colorimetry, respectively. In vitro ${\mathrm{As}}^{+3}$ methyltransferase (As3MT) knockdown or SAM supplementation and in vivo folic acid (FA) supplementation were used to evaluate the protective effect. A case-control study verified the findings.

Results: Sizes of fetuses (exposed to 1.5 and $15\mathrm{mg}/L$ ${\text{NaAsO}}_2$) and placentas (exposed to $15\mathrm{mg}/L$ ${\text{NaAsO}}_2$) were lower in As-exposed mice. More ${\text{glycogen}}^{+}$ trophoblasts accumulated and the expression of markers of interstitial invasion was lower in the $15\mathrm{mg}/L$ ${\text{NaAsO}}_2\text{-exposed}$ mouse group in comparison with control. Placental RNA sequencing identified cysteine-rich angiogenic inducer 61 (Cyr61) as a candidate gene of interest. Mechanistically, mice and cells exposed to As had lower protein expression of CYR61, and this was attributed to a lower incidence of Cyr61 $m^{6}A$. Furthermore, cells exposed to As had lower methyltransferase activity, suggesting that this could be the mechanism by which Cyr61 $m^{6}A$ was affected. Depletion of intracellular SAM, a cofactor for $m^{6}A$ methyltransferase catalytic domain, partially contributed to As-induced methyltransferase activity reduction. Either As3MT knockdown or SAM supplementation attenuated As-induced Cyr61 $m^{6}A$ down-regulation. In mice, FA supplementation rescued As-induced defective trophoblastic invasion and FGR. In humans, a negative correlation between maternal urinary As and plasma CYR61 was observed in infants who were small for gestational age.

Discussion: Using in vitro and in vivo models, we found that intracellular SAM depletion-mediated Cyr61 $m^{6}A$ down-regulation partially contributed to As-induced defective trophoblastic invasion and FGR. https://doi.org/10.1289/EHP12207.