m6A-methylated Lonp1 drives mitochondrial proteostasis stress to induce testicular pyroptosis upon environmental cadmium exposure.

Cadmium (Cd) is a widely distributed typical environmental pollutant and one of the most toxic heavy metals. It is well-known that environmental Cd causes testicular damage by inducing classic types of cell death such as cell apoptosis and necrosis. However, as a new type of cell death, the role and mechanism of pyroptosis in Cd-induced testicular injury remain unclear. In the current study, we used environmental Cd to generate a murine model with testicular injury and AIM2-dependent pyroptosis. Based on the model, we found that increased cytoplasmic mitochondrial DNA (mtDNA), activated mitochondrial proteostasis stress occurred in Cd-exposed testes. We used ethidium bromide to generate mtDNA-deficient testicular germ cells and further confirmed that increased cytoplasmic mtDNA promoted AIM2-dependent pyroptosis in Cd-exposed cells. Uracil-DNA glycosylase UNG1 overexpression indicated that environmental Cd blocked UNG-dependent repairment of damaged mtDNA to drive the process in which mtDNA releases to cytoplasm in the cells. Interestingly, we found that environmental Cd activated mitochondrial proteostasis stress by up-regulating protein expression of LONP1 in testes. Testicular specific LONP1-knockdown significantly reversed Cd-induced UNG1 protein degradation and AIM2-dependent pyroptosis in mouse testes. In addition, environmental Cd significantly enhanced the m6A modification of Lonp1 mRNA and its stability in testicular germ cells. Knockdown of IGF2BP1, a reader of m6A modification, reversed Cd-induced upregulation of LONP1 protein expression and pyroptosis activation in testicular germ cells. Collectively, environmental Cd induces m6A modification of Lonp1 mRNA to activate mitochondrial proteostasis stress, increase cytoplasmic mtDNA content, and trigger AIM2-dependent pyroptosis in mouse testes. These findings suggest that mitochondrial proteostasis stress is a potential target for the prevention of testicular injury.

NBR1-dependent autophagy activation protects against environmental cadmium-evoked placental trophoblast senescence.

Cadmium (Cd), a well-established developmental toxicant, accumulates in the placentae and disrupts its structure and function. Population study found adverse pregnancy outcomes caused by environmental Cd exposure associated with cell senescence. However, the role of autophagy activation in Cd-induced placental cell senescence and its reciprocal mechanisms are unknown. In this study, we employed animal experiments, cell culture, and case-control study to investigate the above mentioned. We have demonstrated that exposure to Cd during gestation induces placental senescence and activates autophagy. Pharmacological and genetic interventions further exacerbated placental senescence induced by Cd through the suppression of autophagy. Conversely, activation of autophagy ameliorated Cd-induced placental senescence. Knockdown of NBR1 exacerbated senescence in human placental trophoblast cells. Further investigations revealed that NBR1 facilitated the degradation of p21 via LC3B. Our case-control study has demonstrated a positive correlation between placental senescence and autophagy activation in all-cause fetal growth restriction (FGR). These findings offer a novel perspective for mitigating placental aging and placental-origin developmental diseases induced by environmental toxicants.

Maternal prednisone exposure during pregnancy elevates susceptibility to osteoporosis in female offspring: The role of mitophagy/FNDC5 alteration in skeletal muscle.

Maternal exposure to glucocorticoids has been associated with adverse outcomes in offspring. However, the consequences and mechanisms of gestational exposure to prednisone on susceptibility to osteoporosis in the offspring remain unclear. Here, we found that gestational prednisone exposure enhanced susceptibility to osteoporosis in adult mouse offspring. In a further exploration of myogenic mechanisms, results showed that gestational prednisone exposure down-regulated FNDC5/irisin protein expression and activation of OPTN-dependent mitophagy in skeletal muscle of adult offspring. Additional experiments elucidated that activated mitophagy significantly inhibited the expression of FNDC5/irisin in skeletal muscle cells. Likewise, we observed delayed fetal bone development, downregulated FNDC5/irisin expression, and activated mitophagy in fetal skeletal muscle upon gestational prednisone exposure. In addition, an elevated total m6A level was observed in fetal skeletal muscle after gestational prednisone exposure. Finally, gestational supplementation with S-adenosylhomocysteine (SAH), an inhibitor of m6A activity, attenuated mitophagy and restored FNDC5/irisin expression in fetal skeletal muscle, which in turn reversed fetal bone development. Overall, these data indicate that gestational prednisone exposure increases m6A modification, activates mitophagy, and decreases FNDC5/irisin expression in skeletal muscle, thus elevating osteoporosis susceptibility in adult offspring. Our results provide a new perspective on the earlier prevention and treatment of fetal-derived osteoporosis.

Environmental cadmium inhibits testicular testosterone synthesis via Parkin-dependent MFN1 degradation.

Low testosterone (T) levels are associated with many common diseases, such as obesity, male infertility, depression, and cardiovascular disease. It is well known that environmental cadmium (Cd) exposure can induce T decline, but the exact mechanism remains unclear. We established a murine model in which Cd exposure induced testicular T decline. Based on the model, we found Cd caused mitochondrial fusion disorder and Parkin mitochondrial translocation in mouse testes. MFN1 overexpression confirmed that MFN1-dependent mitochondrial fusion disorder mediated the Cd-induced T synthesis suppression in Leydig cells. Further data confirmed Cd induced the decrease of MFN1 protein by increasing ubiquitin degradation. Testicular specific Parkin knockdown confirmed Cd induced the ubiquitin-dependent degradation of MFN1 protein through promoting Parkin mitochondrial translocation in mouse testes. Expectedly, testicular specific Parkin knockdown also mitigated testicular T decline. Mito-TEMPO, a targeted inhibitor for mitochondrial reactive oxygen species (mtROS), alleviated Cd-caused Parkin mitochondrial translocation and mitochondrial fusion disorder. As above, Parkin mitochondrial translocation induced mitochondrial fusion disorder and the following T synthesis repression in Cd-exposed Leydig cells. Collectively, our study elucidates a novel mechanism through which Cd induces T decline and provides a new treatment strategy for patients with androgen disorders.

Multigenerational paternal obesity enhances the susceptibility to male subfertility in offspring via Wt1 N6-methyladenosine modification.

There is strong evidence that obesity is a risk factor for poor semen quality. However, the effects of multigenerational paternal obesity on the susceptibility to cadmium (a reproductive toxicant)-induced spermatogenesis disorders in offspring remain unknown. Here, we show that, in mice, spermatogenesis and retinoic acid levels become progressively lower as the number of generations exposed to a high-fat diet increase. Furthermore, exposing several generations of mice to a high fat diet results in a decrease in the expression of Wt1, a transcription factor upstream of the enzymes that synthesize retinoic acid. These effects can be rescued by injecting adeno-associated virus 9-Wt1 into the mouse testes of the offspring. Additionally, multigenerational paternal high-fat diet progressively increases METTL3 and Wt1 N6-methyladenosine levels in the testes of offspring mice. Mechanistically, treating the fathers with STM2457, a METTL3 inhibitor, restores obesity-reduced sperm count, and decreases Wt1 N6-methyladenosine level in the mouse testes of the offspring. A case-controlled study shows that human donors who are overweight or obese exhibit elevated N6-methyladenosine levels in sperm and decreased sperm concentration. Collectively, these results indicate that multigenerational paternal obesity enhances the susceptibility of the offspring to spermatogenesis disorders by increasing METTL3-mediated Wt1 N6-methyladenosine modification.

The Long-Term Effect of Maternal Iron Levels in the Second Trimester on Mild Thinness among Preschoolers: The Modifying Effect of Small for Gestational Age.

The supplementation of multiple micronutrients throughout pregnancy can reduce the risk of adverse birth outcomes and various diseases in children. However, the long-term effect of maternal multiple micronutrient levels in the second trimester on the overall development of preschoolers remains unknown. Therefore, 1017 singleton mother-infant pairs and 6-year-old preschoolers were recruited based on the China-Wuxi Birth Cohort Study. Meanwhile, information on the demographic characteristics of pregnant women and preschoolers, maternal copper, calcium, iron, magnesium, and zinc levels in whole blood during the second trimester, and neonatal outcomes, were collected. We aimed to investigate the long-term impact of maternal copper, calcium, iron, magnesium, and zinc levels in the second trimester on mild thinness among 6-year-old preschoolers, and the modifying effect of small for gestational age (SGA), within the Chinese population. Multiple logistic regression models revealed that high-level maternal iron in the second trimester reduced the risk of mild thinness [adjusted OR: 0.46 (95% CI: 0.26, 0.80)] among 6-year-old preschoolers. However, no significant association was found for the remaining four maternal essential metal elements. Additionally, the restricted cubic spline function showed that the risk of mild thinness decreased when maternal iron concentration exceeded 7.47 mmol/L in whole blood during the second trimester. Furthermore, subgroup analysis indicated that the long-term protective effect of high-level maternal iron on mild thinness was only observed in SGA infants. Summarily, high-level maternal iron in the second trimester distinctly lowers the risk of mild thinness among 6-year-old preschoolers, especially in preschoolers with birth outcomes of SGA. Our findings offer evidence for the implementation of iron supplementation in the second trimester as a preventive measure against mild thinness in children.

Loss of Atg5 in Sertoli cells enhances the susceptibility of cadmium-impaired testicular spermatogenesis in mice.

Cadmium (Cd), one of the most common contaminants in diet and drinking water, impairs testicular germ cell development and spermatogenesis. Autophagy is essential for maintaining Sertoli cell function and Sertoli-germ cell communication. However, the role of Sertoli cell autophagy in Cd-caused spermatogenesis disorder remains unclear. Here, the mice of autophagy-related gene 5 (Atg5) knockouts in Sertoli cells were used to investigate the effect of autophagy deficiency on Cd-impaired spermatogenesis and its underlying mechanisms. Results showed that Sertoli cell-specific knockout of Atg5 exacerbated Cd-reduced sperm count and MVH (a specific marker for testicular germ cells) level in mice. Additionally, Sertoli cell Atg5 deficiency reduced the number of spermatocytes and decreased the level of meiosis-related proteins (SYCP3 and STRA8) in Cd-treated mouse testes. Loss of Atg5 in Sertoli cell exacerbated Cd-reduced the level of retinoic acid (RA) and retinal dehydrogenase (ALDH1A1 and ALDH1A) in mouse testes. Meanwhile, we found that the level of transcription factor WT1 was significantly downregulated in Atg5-/- plus Cd-treated testes. Further experiments showed that Wt1 overexpression restored Cd-decreased the levels of ALDH1A1 in Sertoli cells. Collectively, the above data suggest that knockout of Atg5 in Sertoli cell enhances the susceptibility of Cd-impaired testicular spermatogenesis. These findings provide new insights into autophagy of Sertoli cell preventing environmental toxicants-impaired testicular spermatogenesis.

Sperm Rhoa m6A modification mediates intergenerational transmission of paternally acquired hippocampal neuronal senescence and cognitive deficits after combined exposure to environmental cadmium and high-fat diet in mice.

Little is currently known about the effect and mechanism of combined paternal environmental cadmium (Cd) and high-fat diet (HFD) on offspring cognitive ability. Here, using in vivo model, we found that combined paternal environmental Cd and HFD caused hippocampal neuronal senescence and cognitive deficits in offspring. MeRIP-seq revealed m6A level of Rhoa, a regulatory gene of cellular senescence, was significantly increased in combined environmental Cd and HFD-treated paternal sperm. Interestingly, combined paternal environmental Cd and HFD markedly enhanced Rhoa mRNA, its m6A and reader protein IGF2BP1 in offspring hippocampus. STM2457, the inhibitor of m6A modification, markedly mitigated paternal exposure-caused the elevation of hippocampal Rhoa m6A, neuronal senescence and cognitive deficits in offspring. In vitro experiments, Rhoa siR significantly reversed mouse hippocampal neuronal senescence. Igf2bp1 siR obviously reduced the level and stability of Rhoa in aging mouse hippocampal neuronal cells. In conclusion, combined paternal environmental Cd and HFD induce offspring hippocampal neuronal senescence and cognitive deficits by promoting IGF2BP1-mediated Rhoa stabilization in offspring hippocampus via elevating Rhoa m6A in paternal sperm.

Activation of Atg5-dependent placental lipophagy ameliorates cadmium-induced fetal growth restriction.

Cadmium (Cd), an environmental contaminant, can result in placental non-selective autophagy activation and fetal growth restriction (FGR). However, the role of placental lipophagy, a selective autophagy, in Cd-induced FGR is unclear. This work uses case-control study, animal experiments and cultures of primary human placental trophoblast cells to explore the role of placental lipophagy in Cd-induced FGR. We found association of placental lipophagy and all-cause FGR. Meanwhile, pregnancy Cd exposure induced FGR and placental lipophgay. Inhibition of placental lipophagy by pharmacological and genetic means (Atg5-/- mice) exacerbated Cd-caused FGR. Inversely, activating of placental lipophagy relieved Cd-stimulated FGR. Subsequently, we found that activation of Atg5-dependent lipophagy degrades lipid droplets to produce free cholesterol, and promotes placental progesterone (P4) synthesis. Gestational P4 supplementation significantly reversed Cd-induced FGR. Altogether, activation of Atg5-dependent placental lipophagy ameliorates Cd-induced FGR.

Gestational cadmium exposure disrupts fetal liver development via repressing estrogen biosynthesis in placental trophoblasts.

Cadmium (Cd), commonly found in diet and drinking water, is known to be harmful to the human liver. Nevertheless, the effects and mechanisms of gestational Cd exposure on fetal liver development remain unclear. Here, we reported that gestational Cd (150 mg/L) exposure obviously downregulated the expression of critical proteins including PCNA, Ki67 and VEGF-A in proliferation and angiogenesis in fetal livers, and lowered the estradiol concentration in fetal livers and placentae. Maternal estradiol supplement alleviated aforesaid impairments in fetal livers. Our data showed that the levels of pivotal estrogen synthases, such as CYP17A1 and 17ß-HSD, was markedly decreased in Cd-stimulated placentae but not fetal livers. Ground on ovariectomy (OVX), we found that maternal ovarian-derived estradiol had no major effects on Cd-impaired development in fetal liver. In addition, Cd exposure activated placental PERK signaling, and inhibited PERK activity could up-regulated the expressions of CYP17A1 and 17ß-HSD in placental trophoblasts. Collectively, gestational Cd exposure inhibited placenta-derived estrogen synthesis via activating PERK signaling, and therefore impaired fetal liver development. This study suggests a protective role for placenta-derived estradiol in fetal liver dysplasia shaped by toxicants, and provides a theoretical basis for toxicants to impede fetal liver development by disrupting the placenta-fetal-liver axis.

Combination of high-fat diet and cadmium impairs testicular spermatogenesis in an m6A-YTHDF2-dependent manner.

Environmental cadmium (Cd) or high-fat diet (HFD) exposure alone are risk factors of male infertility. However, the effect and mechanism of co-exposure to HFD and Cd on sperm quality remain unclear. This study was aimed to explore the combined effects of HFD and Cd on spermatogenesis as well as its m6A-dependent mechanism in vivo and in vitro. As a result, co-exposure of HFD and Cd resulted in a significant decrease in the number of mature testicular seminiferous tubules and epididymis sperm quantity in mice, compared with Cd or HFD exposure alone. Correspondingly, the mRNAs expression of Smc3(spermatocytes marker), Acrv1(round spermatids marker) and Lzumo3(elongated spermatids marker) were downregulated in HFD and Cd group. Furthermore, combined exposure downregulated the expression of meiosis-related proteins (STRA8 and SYCP3), increased the m6A level of Stra8, and upregulated the expression of m6A-related proteins (METTL3 and YTHDF2) in mouse spermatocytes. Mechanistically, the above-mentioned impacts caused by co-exposure were markedly restored by Mettl3 siR and Ythdf2 siR. In addition, RNA stability assay showed that Ythdf2 siR obviously reversed co-exposure-increased Stra8 mRNA degradation rate in actinomycin-D-treated mouse spermatocytes. Meanwhile, excess ROS was observed in combined-exposure group, and a free radical scavenger N-tert-Butyl-α-phenylnitrone (PBN) attenuated co-exposure-upregulated expression of METTL3 and YTHDF2 in mouse spermatocytes. These results suggested that combination of HFD and Cd impaired spermatogenesis by degrading Stra8 in an m6A-YTHDF2-dependent manner via ROS activation.

Environmental cadmium exposure during gestation impairs fetal brain and cognitive function of adult offspring via reducing placenta-derived E2 level.

Early-life exposure to environmental cadmium (Cd) is known to cause developmental disorders, yet the effect and mechanism of gestational exposure to Cd on the offspring's cognitive function remains unclear. Placenta as a well-established target organ for Cd-impaired fetal development, its role in estrogen regulation and offspring cognitive function is unknown. Our in vivo experiments found that gestational Cd exposure impaired cognitive function in adult male offspring, accompanied with lowered 17ß-estradiol (E2) level in the male fetal brain upon Cd exposure. Correspondingly, the expression of synapse-associated proteins including brain-derived neurotrophic factor (BDNF), post-synaptic density protein 95 (PSD95) and synapsin-1 were downregulated, which were reversed when supplemented with E2 hormone during gestation. Further observation showed placental estrogen synthesis inhibition and general control non-derepressible 2 (GCN2) signaling activation upon Cd exposure, whereas placental estrogen synthesis could be restored through inhibiting GCN2 activity. Based on ovariectomy (OVX) of pregnant mice, we confirmed that Cd exposure reduced E2 level in fetal brain via inhibiting placenta-derived estrogen synthesis. The aforementioned Cd-induced fetal brain injury and cognitive impairment in adult offspring were significantly alleviated when pregnant dams were supplemented with anti-stress agent N-Acetyl-l-cysteine. In summary, Cd disrupted placenta-derived estrogen synthesis via activating GCN2 signaling, and thereby caused cognitive impairment in adult offspring mice. Our findings suggest that placenta-derived estrogen may be an effect marker of environmental toxicants-evoked cognitive dysfunction in adult offspring and suggest that environmental toxicants may affect the fetal brain development via placenta-fetal-brain axis.

Low-frequency electrical stimulation alleviates immobilization-evoked disuse muscle atrophy by repressing autophagy in skeletal muscle of rabbits.

BACKGROUND: The study aimed to investigate the effect of low-frequency electrical stimulation (LFES) on disuse muscle atrophy and its mechanism in a rabbit model of knee extension contracture. METHODS: This study involved two experiments. In the time-point experiment, 24 rabbits were randomly divided into 4 groups: Control 1 (Ctrl1 group), immobilization for 2 weeks (I-2 group), immobilization for 4 weeks (I-4 group), and immobilization for 6 weeks (I-6 group). In the intervention experiment, 24 rabbits were randomly divided into 4 groups: Control 2 (Ctrl2 group), electrical stimulation (ESG group), natural recovery (NRG group), and electrical stimulation treatment (ESTG group). All intervention effects were assessed by evaluating the knee joint range of motion (ROM), cross-sectional area (CSA) of the rectus femoris muscle, and expression of autophagy-related proteins. RESULTS: The time-point experiment showed that immobilization reduced the knee ROM, reduced the rectus femoris muscle CSA, and activated autophagy in skeletal muscle. The levels of five autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), autophagy-related protein 7 (Atg7), p62, and microtubule-associated protein light chain 3B-II (LC3B-II)] were significantly elevated in the skeletal muscle of the I-4 group. The intervention experiment further showed that LFES significantly improved the immobilization-induced reductions in ROM and CSA. Additionally, LFES resulted in a significant decrease in the protein expression of mTOR, p-mTOR, Atg7, p62, and LC3B-II in the rectus femoris muscle. CONCLUSIONS: LFES alleviates immobilization-evoked disuse muscle atrophy possibly by inhibiting autophagy in the skeletal muscle of rabbits.

Gestational exposure to environmental cadmium induces placental apoptosis and fetal growth restriction via Parkin-modulated MCL-1 degradation.

Heavy metal cadmium (Cd), a classical environmental pollutant, causes placental apoptosis and fetal growth restriction (FGR), whereby the mechanism remains unclear. Here, our human case-control study firstly showed that there was a positive association of Parkin mitochondrial translocation, MCL-1 reduction, placental apoptosis, and all-cause FGR. Subsequently, Cd was administered to establish in vitro and in vivo models of placental apoptosis or FGR. Our models demonstrated that Parkin mitochondrial translocation was observed in Cd-administrated placental trophoblasts. Meaningfully, Parkin siRNA (siR) dramatically mitigated Cd-triggered apoptosis in placental trophoblasts. Mdivi-1 (M-1), an inhibitor for Parkin mitochondrial translocation, mitigated Cd-induced apoptosis in placental trophoblasts, which further ameliorated the effect of attenuated placental sizes in Cd-exposed mice. Furthermore, the interaction of MCL-1 with Parkin or Ub in Cd-stimulated cells was stronger than that in controls. MG132, an inhibitor for proteasome, abolished MCL-1 degradation in Cd-stimulated cells. Importantly, Parkin siR and M-1 memorably abolished the ubiquitin-dependent degradation of MCL-1 in placental trophoblasts. Interestingly, mito-TEMPO and melatonin, two mitochondria-targeted antioxidants, obviously rescued Cd-caused mitochondrial membrane potential (MMP) decrease, Parkin mitochondrial translocation, MCL-1 degradation, and apoptosis in placental trophoblasts. In conclusion, cadmium induces placental apoptosis and FGR via mtROS-mediated Parkin-modulated degradation of MCL-1.

Environmental cadmium impairs blood-testis barrier via activating HRI-responsive mitochondrial stress in mice.

Cadmium (Cd) is a well-known testicular toxicant. Blood-testis barrier (BTB), a vital part of testes, which has been reported to be damaged upon Cd exposure. However, the detailed mechanism about Cd-mediated disruption of BTB remains unclear. This study aims to investigate the role of Heme-Regulated Inhibitor (HRI)-responsive mitochondrial stress in Cd-mediated disruption of BTB. Male mice are intraperitoneally injected (i.p.) with melatonin (Mel, a cellular stress antagonist, 5.0 mg/kg) before Cd treatment (i.p., 2.0 mg/kg) for 8 h, and then treated with Cd for 0-48 h. Mouse Sertoli cells are pretreated with Mel (10 µM) for 1 h, and then treated with Cd (10 µM) for 0-24 h. We find that Cd damages the BTB and reduces the Occludin protein, a crucial BTB-related protein via activating p38/matrix metalloproteinase-2 (p38/MMP2) pathway and Integrated Stress Response (ISR). Further experiments reveal that the Heme-Regulated Inhibitor (HRI)-responsive mitochondrial stress is triggered in Cd-treated Sertoli cells. Most importantly, Cd-activated p38 signaling and ISR are regulated by HRI-responsive mitochondrial stress in Sertoli cells. Unexpectedly, we find that melatonin rescues the Cd-mediated disruption of BTB through blocking HRI-responsive mitochondrial stress in testes. Overall, these data indicate that environmental cadmium exposure impairs the BTB through activating HRI-responsive mitochondrial stress in Sertoli cells.

miR-6769b-5p targets CCND-1 to regulate proliferation in cadmium-treated placental trophoblasts: Association with the impairment of fetal growth.

Environmental cadmium (Cd) is positively associated with placental impairment and fetal growth retardation. Nevertheless, its potential mechanisms remain unclear. microRNAs (miRNAs) are known to influence placental development and fetal growth. This work was aimed to determine which miRNAs are involved in Cd-impaired placental and fetal development based on the mRNA and miRNA expression profiles analysis. As a result, gestational Cd exposure deceased fetal and placental weight, and reduced the protein level of PCNA in human and mouse placentae. Furthermore, the results of mRNA microarray showed that Cd-downregulated mRNAs were predictively correlated with several biological processes, including cell proliferation, differentiation and motility. In addition, the results of miRNA microarray and qPCR assay demonstrated that Cd significantly increased the level of miR-6769b-5p, miR-146b-5p and miR-452-5p. Integrated analysis of Cd-upregulated miRNAs predicted target genes and Cd-downregulated mRNAs found that overlapping mRNAs, such as CCND1, CDK13, RINT1 and CDC26 were also significantly associated with cell proliferation. Further experiments showed that miR-6769b-5p inhibitor, but not miR-146b-5p and miR-452-5p, markedly reversed Cd-downregulated the expression of proliferation-related mRNAs, and thereby restored Cd-decreased the proteins level of CCND1 and PCNA in human placental trophoblasts. Dual luciferase reporter assay further revealed that miR-6769b-5p directly targets CCND1. Finally, the case-control study demonstrated that increased miR-6769b-5p level and impaired cell proliferation were observed in small-for-gestational-age human placentae. In conclusion, miR-6769b-5p targets CCND-1 to regulate proliferation in Cd-treated placental trophoblasts, which is associated with the impairment of fetal growth. Our findings imply that placental miR-6769b-5p may be used as an epigenetic marker for environmental pollutants-caused fetal growth restriction and its late-onset chronic diseases.

Gestational cadmium exposure impairs placental angiogenesis via activating GC/GR signaling.

Gestational exposure to environmental Cd caused placental angiogenesis impairment and fetal growth restriction (FGR). However, its mechanism remained unclear. This study was to investigate the effects of Cd exposure during pregnancy on placental angiogenesis and its mechanism. Pregnant mice were exposed to CdCl2 (4.5 mg/kg) on gestational day (GD) 8 with or without melatonin (MT) (5.0 mg/kg), an anti-endoplasmic reticulum stress agent, from GD7 to GD15. Human primary placental trophoblasts and JEG-3 cells were stimulated using CdCl2 (20 µM) after MT (1 mM) preprocessing. We firstly found MT treatment obviously mitigated environmental Cd-induced placental angiogenesis disorder and reduction of the VEGF-A level. Mechanistically, MT reversed environmental Cd-downregulated the protein expression of VEGF-A via inhibiting glucocorticoid receptor (GR) activation. Notably, our data showed MT treatment antagonized Cd-activated GC/GR signaling via blocking PERK signaling and thereby upregulated VEGF-A and 11ß-HSD2 protein expression. Based upon the population case-control study, the levels of VEGF-A and 11ß-HSD2 protein in small-for-gestational-age placentae were significantly reduced when compared to appropriate-for-gestational-age placentae. Overall, environmental Cd exposure during gestation impaired placental angiogenesis via PERK-regulated GC/GR signaling in placental trophoblasts. Our findings will provide a basis for prevention and treatment of placental impairments and fetal growth restriction caused by environment toxicants in future.

Environmental cadmium exposure during pregnancy causes diabetes-like phenotypes in mouse offspring: Association with oxidative stress in the fetal liver.

Cadmium (Cd), a noxious heavy metal, is widespread in the living environment. Gestational exposure to Cd at environmental dose has been shown to cause fetal growth restriction (FGR). However, the long-term effects and the mechanisms underlying environmental Cd exposure on glucose metabolism in offspring remain unclear. Here, we established a murine model to study the impacts of gestational exposure to environmental Cd on glucose metabolism at different life stages of offspring. Results demonstrated that the offspring mice developed hyperglycemia in puberty and impaired glucose tolerance in adulthood following maternal Cd exposure during gestation. Further mechanistic investigation showed that Cd exposure upregulated the expression of key proteins in hepatic gluconeogenesis, including p-CREB, PGC-1α and G6PC, in pubertal and adult offspring. In addition, we demonstrated that Cd exposure during pregnancy markedly elevated the level of oxidative stress-related proteins, including NOX2, NOX4 and HO-1, in the fetal liver. The effects of gestational exposure to N-acetylcysteine (NAC), a free-radical scavenging antioxidant, presented that NAC supplementation alleviated hepatic oxidative stress in fetuses, and thereby reversed hyperglycemia and glucose intolerance in mouse offspring. Collectively, our data suggested that gestational exposure to environmental Cd caused diabetes-like phenotypes via enhancing hepatic gluconeogenesis, which is associated with oxidative stress in fetal livers. This work provides new insights into the protective effects of antioxidants on fetal-originated diabetes triggered by environmental toxicants.

Melatonin protects against environmental stress-induced fetal growth restriction via suppressing ROS-mediated GCN2/ATF4/BNIP3-dependent mitophagy in placental trophoblasts.

Gestational exposure to environmental stress induces fetal growth restriction (FGR), and thereby increasing the risk of infant death and chronic noncommunicable diseases in adults. However, the mechanism by which environmental stress induces FGR remains unclear. Based on case-control study, we found that the reduced level of melatonin (MT), a major secretory product from the pineal gland, was observed in placentae of FGR. This work was to investigate the protective effect of MT on environmental stress-caused FGR and its mechanisms. We used cadmium (Cd) as an environmental stressor to stimulate pregnant mice and thereby establishing a FGR model. The data showed that maternal Cd exposure lowered the P4 concentration in maternal sera, placentae and amniotic fluid, and caused FGR. Correspondingly, the expression of CYP11A1, a critical P4 synthase, was markedly downregulated in Cd-treated placentae. Simultaneously, Cd triggered BNIP3-dependent mitophagy in placental trophoblasts, as determined by the degradation of mitochondrial proteins, including HSP60 and COX IV, and the accumulation of puncta representing co-localization of TOM20 with LC3B or BNIP3 with LC3B. Based on our case-control study, we also found that activated BNIP3-dependent mitophagy and P4 synthesis inhibition occurred in SGA placentae. Most importantly, BNIP3 siRNA reversed Cd-induced P4 synthesis suppression in human placental trophoblasts. It is noteworthy that MT alleviated Cd-caused P4 synthesis suppression and FGR via antagonizing BNIP3-dependent mitophagy in placental trophoblasts. Further results confirmed that MT attenuated Cd-triggered BNIP3-dependent mitophagy via blocking GCN2/ATF4 signaling. Amusingly, Cd triggered oxidative stress and then activating GCN2/ATF4 signaling in placental trophoblasts. As expected, MT obviously suppressed Cd-caused reactive oxygen species (ROS) release. In the present study, we propose a neoteric mechanism by which MT protects against environmental stress-impaired P4 synthesis and fetal growth via suppressing ROS-mediated GCN2/ATF4/BNIP3-dependent mitophagy in placental trophoblasts. As above, MT is a potential therapeutic agent antagonizing environmental stress-induced developmental toxicity.

Environmental exposure to cadmium impairs fetal growth and placental angiogenesis via GCN-2-mediated mitochondrial stress.

Cadmium (Cd), a well-known environmental pollutant, can lead to placental insufficiency and fetal growth restriction. However, the underlying mechanism is unknown. The purpose of our study is to explore the effect of Cd on placental angiogenesis and its mechanism using in vitro and in vivo models. Results found that gestational Cd exposure obviously decreased placental weight and impaired placental vascular development in mice. Correspondingly, Cd exposure evidently downregulated the expression of VEGF-A protein (a key indicator of angiogenesis) and progesterone receptor (PR) in placental trophoblasts. Further experiment showed that lentivirus PR overexpression reversed Cd-caused the reduction of VEGF-A level in human placental trophoblasts. In addition, Cd significantly reduced progesterone level, down-regulated the expression of key progesterone synthase (StAR, CYP11A1), and activated mitochondrial stress response and GCN-2/p-eIF2α signaling in placental trophoblasts. Additional experiment showed that GCN-2 siRNA pretreatment markedly alleviated Cd-activated mitochondrial stress response, restored Cd-downregulated the expression of CYP11A1, reversed Cd-reduced the level of progesterone and VEGF-A in human placental trophoblasts. Finally, our case-control study confirmed that impaired placental angiogenesis and reduced progesterone level occurred in all-cause small for gestational age placenta. Taken together, environmental exposure to Cd impairs fetal growth and placental angiogenesis via GCN-2-mediated mitochondrial stress.