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

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 15mg/L NaAsO2 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 NaAsO2 (2µM) to establish an in vitro model of As exposure. The mRNA stability and protein level of genes identified through RNA sequencing were measured. N6-Methyladenosine (m6A) 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 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 15mg/L NaAsO2) and placentas (exposed to 15mg/L NaAsO2) were lower in As-exposed mice. More glycogen+ trophoblasts accumulated and the expression of markers of interstitial invasion was lower in the 15mg/L NaAsO2-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 m6A. Furthermore, cells exposed to As had lower methyltransferase activity, suggesting that this could be the mechanism by which Cyr61 m6A was affected. Depletion of intracellular SAM, a cofactor for m6A methyltransferase catalytic domain, partially contributed to As-induced methyltransferase activity reduction. Either As3MT knockdown or SAM supplementation attenuated As-induced Cyr61 m6A 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 m6A down-regulation partially contributed to As-induced defective trophoblastic invasion and FGR. https://doi.org/10.1289/EHP12207.

Association between gestational arsenic exposure and intrauterine growth restriction: the role of folate content.

Gestational arsenic (As) exposure is associated with intrauterine growth restriction (IUGR). This study explored the association among gestational As exposure, IUGR, and reduction of folate content in maternal and umbilical plasma from 530 mother-and-singleton-offspring pairs. Birth weight (BW) was negatively correlated with As in maternal plasma (r=-0.194, P<0.001) and umbilical plasma (r=-0.235, P<0.001). By contrast, a positive correlation was found between BW and maternal folate content (r=0.198, P<0.001). The subjects were divided into As-L and As-H groups. The influence of As-H on small for gestational age (SGA) infants, a marker of IUGR, was evaluated by multivariate logistic regression that excludes interferences of gestational age, infant sex, and other confounding factors. Mothers with As-H had an elevated risk of SGA infants (adjusted OR, 2.370; P<0.05). Interestingly, maternal folate content was lower in subjects with As-H than those with As-L (22.4±10.7 vs 11.2±6.7 nmol/L, P<0.001). Linear correlation models show that As level was negatively correlated with folate content in maternal plasma (r=-0.615, P<0.001) and umbilical plasma (r=-0.209, P<0.001). Moreover, maternal folate reduction has an obvious mediating effect between increased As and decreased BW (ß=-0.078, P<0.05). Our results indicate that folate reduction may be a mediator between gestational As exposure and IUGR.

DNA hydroxymethylation reprogramming of ß-oxidation genes mediates early-life arsenic-evoked hepatic lipid accumulation in adult mice.

The metabolic disorders are becoming an epidemic disease endangering public health in countries. Environmental factors are mainly reason for the growth of metabolic disorders. Previous research suggests that DNA methylation is a potential mechanism. Recently, it has been reported that DNA hydroxymethylation is also a stable marker of epigenetic reprogramming. Hence, the study aims to investigate whether DNA hydroxymehylation mediates early-life environmental stress-evoked metabolic disorder in adulthood. Mice were orally administered with arsenic (As), an environmental stressor, throughout pregnancy. We show that early-life As exposure induces glucose intolerance and hepatic lipid accumulation in adulthood. Early-life As exposure alters epigenetic reprogramming and expression of lipid metabolism-related genes including ß-oxidation-specific genes in adulthood. Of interest, early-life As exposure alters epigenetic reprogramming of hepatic lipid metabolism partially through reducing DNA hydroxymethylation modification of ß-oxidation-related genes in developing liver. Mechanistically, early-life As exposure suppresses ten-eleven translocation (TET) activity through downregulating isocitrate dehydrogenases (Idh) and reducing alpha-ketoglutarate (α-KG) content in the developing liver. In addition, early-life As exposure inhibits TET1 binding to CpG-rich fragments of ß-oxidation-related genes in developing liver. This study provide novel evidence that early-life environmental stress leads to later life metabolic disorders by altering hepatic DNA hydroxymethylation reprogramming.

Gestational arsenic exposure induces anxiety-like behaviors in adult offspring by reducing DNA hydroxymethylation in the developing brain.

Several studies found that reduction of 5-hydroxymethylcytosine (5hmC), a marker of DNA hydroxymethylation highly enriched in developing brain, is associated with anxiety-like behaviors. This study aimed to investigate whether gestational arsenic (As) exposure induces anxiety-like behaviors in adult offspring by reducing DNA hydroxymethylation in the developing brain. The dams drank ultrapure water containing NaAsO2 (15 mg/L) throughout pregnancy. Anxiety-like behaviors were evaluated and developing brain 5hmC was detected. Results showed that anxiety-like behaviors were observed in As-exposed adult offspring. In addition, 5hmC content was reduced in As-exposed fetal brain. Despite no difference on Tet1, Tet2 and Tet3 expression, TET activity was suppressed in As-exposed fetal brain. Mechanistically, alpha-ketoglutarate (α-KG), a cofactor for TET dioxygenases, was reduced and Idh2, a key enzymatic gene for mitochondrial α-KG synthesis, was downregulated in As-exposed fetal brain. Of interest, ascorbic acid, a cofactor for TET dioxygenases, reversed As-induced suppression of TET activity. Moreover, ascorbic acid attenuated As-induced reduction of 5hmC in fetal brain. In addition, ascorbic acid alleviated As-induced anxiety-like behaviors in adult offspring. Taken together, these results suggest that gestational As exposure induces anxiety-like behaviors in adult offspring, possibly at part, by inhibiting DNA hydroxymethylation in developing brain.

Reactive oxygen species-evoked genotoxic stress mediates arsenic-induced suppression of male germ cell proliferation and decline in sperm quality.

This study aimed to investigate whether genotoxic stress mediates arsenic (As)-induced decline in sperm quality. Mice drank ultrapure water containing NaAsO2 (15 mg/L) for 70 days. The mature seminiferous tubules and epididymal sperm count were reduced in As-exposed mice. Cell proliferation, determined by immunostaining with Ki67, was suppressed in As-exposed seminiferous tubules and GC-1 cells. PCNA, a proliferation marker, was reduced in As-exposed mouse testes. Cell growth index was decreased in As-exposed GC-1 cells. Flow analysis showed that As-exposed GC-1 cells were retarded at G2/M phase. CDK1 and cyclin B1 were reduced in As-exposed GC-1 cells and mouse testes. Additional experiment revealed that p-ATR, a marker of genotoxic stress, was elevated in As-exposed mouse testes and GC-1 cells. Accordingly, p-p53 and p21, two downstream molecules of ATR, were increased in As-exposed GC-1 cells. Excess reactive oxygen species (ROS), measured by immunofluorescence, and DNA-strand break, determined by Comet assay, were observed in As-exposed GC-1 cells. γH2AX, a marker of DNA-strand break, was elevated in As-exposed seminiferous tubules and GC-1 cells. NAC alleviated As-evoked DNA damage, genotoxic stress, cell proliferation inhibition and sperm count reduction. In conclusion, ROS-evoked genotoxic stress mediates As-induced germ cell proliferation inhibition and decline in sperm quality.

Erythropoietin-Induced Autophagy Protects Against Spinal Cord Injury and Improves Neurological Function via the Extracellular-Regulated Protein Kinase Signaling Pathway.

The objective of this study was to explore the neuroprotective molecular mechanisms of erythropoietin (EPO) in rats following spinal cord injury (SCI). First, a standard SCI model was established. After drug or saline treatment was administered, locomotor function was evaluated in rats using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. H&E, Nissl, and TUNEL staining were performed to assess the ratio of cavities, number of motor neurons, and apoptotic cells in the damaged area. The relative protein and mRNA expressions were examined using western blot and qRT-PCR analyses, and the inflammatory markers, axon special protein, and neuromuscular junctions (NMJs) were detected by immunofluorescence. Both doses of EPO notably improved locomotor function, but high-dose EPO was more effective than low-dose EPO. Moreover, EPO reduced the cavity ratio, cell apoptosis, and motor neuron loss in the damaged area, but enhanced the autophagy level and extracellular-regulated protein kinase (ERK) activity. Treatment with an ERK inhibitor significantly prevented the effect of EPO on SCI, and an activator mimicked the benefits of EPO. Further investigation revealed that EPO promoted SCI-induced autophagy via the ERK signaling pathway. EPO activates autophagy to promote locomotor function recovery in rats with SCI via the ERK signaling pathway.

The protective effect of obeticholic acid on lipopolysaccharide-induced disorder of maternal bile acid metabolism in pregnant mice.

The disorder of bile acid metabolism is a common feature during pregnancy, which leads to adverse birth outcomes and maternal damage effects. However, the cause and therapy about the disorder of bile acid metabolism are still poor. Microbial infection often occurs in pregnant women, which can induce the disorder of bile acid metabolism in adult mice. Here, this study observed the acute effect of lipopolysaccharide (LPS) on maternal bile acid of pregnant mice at gestational day 17 and the protective effect of obeticholic acid (OCA) pretreatment, a potent agonist of bile acid receptor farnesoid X receptor (FXR). The results showed LPS significantly increased the level of maternal serum and disordered bile acids components of maternal serum and liver, which were ameliorated by OCA pretreatment with obviously reducing the contents of CA, TCA, DCA, TCDCA, CDCA, GCA and TDCA in maternal serum and DCA, TCA, TDCA, TUDCA, CDCA and TCDCA in maternal liver. Furthermore, we investigated the effects of OCA on LPS-disrupted bile acid metabolism in maternal liver. LPS disrupted maternal bile acid profile by decreasing transport and metabolism with hepatic tight junctions of bile acid in pregnant mice. OCA obviously increased the protein level of nuclear FXR and regulated its target genes involving in the metabolism of bile acid, which was characterized by the lower expression of bile acid synthase CYP7A1, the higher expression of CYP3A and the higher mRNA level of transporter Mdr1a/b. This study provided the evidences that LPS disrupted bile acid metabolism in the late stage of pregnant mice and OCA pretreatment played the protective role on it by activating FXR.

Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in D-galactosamine-sensitized mice.

D-Galactosamine (GalN) depletes UTP primarily in liver, resulting in decreased RNA synthesis in hepatocytes. When given together with a sublethal dose of lipopolysaccharide (LPS), GalN highly sensitizes animals to produce apoptotic liver injury with severe hepatic congestion, resulting in rapid death. Melatonin is a cytokine modulator, antioxidant and anti-apoptotic agent. In the present study, we investigated the effect of melatonin on LPS-induced apoptotic liver damage in GalN-sensitized mice. Female CD-1 mice were intraperitoneally (i.p.) injected with melatonin (5.0mg/kg) 30min before GalN/LPS (700mg10microg/kg, i.p.), another two doses of melatonin (2.5mg/kg, i.p.) being administered 1 and 2h after GalN/LPS. Results showed that serum alanine aminotransferase (ALT) activities were markedly increased 8h after GalN/LPS treatment, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. Melatonin significantly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, melatonin distinctly improved GalN/LPS-induced congestion. Additional experiment showed that melatonin significantly attenuated GalN/LPS-induced hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA laddering. Furthermore, melatonin markedly increased hepatic Se-dependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic glutathione (GSH) depletion in GalN/LPS-treated mice. Increases in serum tumor necrosis factor alpha (TNF-alpha), which were observed in GalN/LPS-treated mice, were significantly reduced by melatonin. However, melatonin had no effect on LPS-evoked nitric oxide production in GalN-sensitized mice. Taken together, these results indicate that melatonin protected against LPS-induced liver damage in GalN-sensitized mice through its strong ROS-scavenging, antiinflammatory and antiapoptotic effects.