Maternal cadmium exposure during late pregnancy causes fetal growth restriction via inhibiting placental progesterone synthesis.

Cadmium (Cd) is a major environmental pollutant. Maternal Cd exposure throughout pregnancy caused fetal growth restriction (FGR). However, the pivotal time window of Cd-evoked FGR and its mechanism are unknown. Here, we will establish a murine model to explore the effects of maternal Cd exposure at different stages of gestation on fetal growth and placental progesterone biosynthesis. Pregnant mice were randomly divided into four groups. For Cd groups, mice were given with CdCl2 (150 mg/L) through drinking water at early (GD0-GD6), middle (GD7-GD12) and late (GD13-GD17) gestation, respectively. The controls received reverses osmosis (RO) water. Results showed that maternal cadmium exposure only in late gestation lowered fetal weight and length. Correspondingly, placental Cd level in late gestational Cd exposure is the highest among three different gestational stages. Although gestational Cd exposure had few adverse effects in the weight and diameter of mouse placenta, placental vascular development, as determined by H&E staining and cluster of differentiation-34 (CD-34) immunostaining, was impaired in mice exposed to Cd during late pregnancy. Additionally, late gestational exposure to cadmium markedly reduced progesterone level in maternal serum and placenta. In line, the expression of key progesterone synthetases, including steroidogenic acute regulatory protein (StAR) and 3ß-hydroxyl steroid dehydrogenase (3ß-HSD), was obviously downregulated in placenta from mice was exposed Cd during late pregnancy. These data suggest that maternal Cd exposure during late pregnancy, but not early and middle pregnancy, induces fetal growth restriction partially via inhibiting placental progesterone synthesis.

High-sensitivity chemiluminescent immunoassay investigation and application for the detection of T-2 toxin and major metabolite HT-2 toxin.

BACKGROUND: T-2 toxin is a widely distributed mycotoxin in cereals. HT-2 toxin is the major metabolite, which is also a contaminant in cereals. T-2 toxin and HT-2 toxin have been identified as having carcinogenic, hepatotoxic, teratogenic and immunotoxic properties. To reduce the risk of contamination, a rapid, highly sensitive and inexpensive assay for the detection is required. RESULTS: In this study a high-sensitivity chemiluminescent enzyme-linked immunoassay (CL-ELISA) of T-2 toxin and HT-2 toxin was developed. With the help of the chemiluminescent substrate, this protocol showed a highly sensitive character with an IC50 as low as 33.28 ng mL-1 and 27.27 ng mL-1 for T-2 and HT-2, respectively. In addition, this method had no cross-reaction with other structurally related mycotoxins. CONCLUSION: These results indicated that the developed CL-ELISA could be applied for the detection of T-2 toxin and HT-2 toxin in actual samples without complicated steps. © 2016 Society of Chemical Industry.

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.

Autophagy in Sertoli cell protects against environmental cadmium-induced germ cell apoptosis in mouse testes.

Cadmium (Cd) was an environmental pollutant, which could result in germ cell apoptosis in testes. Sertoli-germ cell communication was vital for germ cell development and maturity. However, little was known about the effect of Sertoli cell autophagy on Cd-induced germ cell apoptosis. Here, we used male Amh-Cre+/Atg5flox/flox (Atg5-/-) mice, loss of autophagy-related gene 5 (Atg5) in testicular Sertoli cells, to explore the obscure effects. Atg5-/- and Wild-type (WT) mice were given with cadmium chloride (CdCl2, 2.0 mg/kg) for 0-24 h. Our results showed that Cd triggered testicular germ cell apoptosis, as evidenced by the increment of TUNEL-labeled germ cells, cleaved caspase3 and cleaved poly (ADP-ribose) polymerase protein level. Additionally, Cd induced testicular autophagy, as determined by elevating the level of autophagy-related proteins, including Atg5, Atg7, LC3B-II, and the gathering of LC3 puncta. 3-methyladenine, a specific autophagy inhibitor, exacerbated Cd-caused germ cell apoptosis. Inversely, rapamycin, an autophagy inducer, relieved Cd-stimulated germ cell apoptosis. Interestingly, we found that autophagy in Sertoli cells was activated in Cd-treated WT mouse testes as evidenced by the increment of LC3 puncta surrounding SOX9, a specific Sertoli cell marker. More importantly, loss of autophagy in Sertoli cells aggravated Cd-triggered germ cell apoptosis. Taken together, these data indicate that autophagy in Sertoli cells alleviates Cd-triggered germ cell apoptosis in mouse testes.

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.

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.

Cadmium down-regulates 11ß-HSD2 expression and elevates active glucocorticoid level via PERK/p-eIF2α pathway in placental trophoblasts.

Fetal overexposure to active glucocorticoid (GC) is the major cause for fetal growth restriction (FGR). This study investigated the influences of cadmium (Cd) exposure on active GC and its mechanism in placental trophoblasts. Pregnant mice were exposed to CdCl2 (4.5 mg/kg, i.p.). Human JEG-3 cells were treated with CdCl2 (0-20 µM). Prenatal Cd exposure significantly increased active GC level in amniotic fluid and placenta. Similarly, Cd treatment also elevated active GC level in medium. Expectedly, the expression of 11ß-HSD2 protein was markedly downregulated in Cd-exposed placental trophoblasts. We further found that Cd activated the PERK/p-eIF2α signaling pathway in placental trophoblasts. Mechanistically, PERK siRNA pretreatment completely blocked PERK/p-eIF2α signaling, and thereby restoring Cd-downregulated 11ß-HSD2 protein expression in human placental trophoblasts. We further found that N-acetylcysteine, a well-known antioxidant, obviously reversed Cd-downregulated 11ß-HSD2 protein expression by inhibiting p-PERK/p-eIF2α signaling in placental trophoblasts. Overall, our data suggest that Cd activates the PERK/p-eIF2α signaling, down-regulates the protein expression of 11ß-HSD2, and thereby elevating active GC level in placental trophoblast.

Paternal cadmium exposure increases the susceptibility to diet-induced testicular injury and spermatogenic disorders in mouse offspring.

The impairments of gestational cadmium (Cd) exposure on testicular development and male fertility in offspring have been reported. Here, we investigated the effect of paternal low-concentration cadmium exposure on testicular development and spermatogenesis in offspring. Five-week-old male mice were exposed to cadmium chloride (100 mg/L) in drinking water for 20 weeks. Results presented that Cd did not affect the testicular histology and sperm count in mice. After mating with untreated females, pregnant mice and pups were then evaluated. No significant difference in the rate for successful pregnancy and the body weight of pups was observed in Cd-exposed mice compared to the controls. Male offspring were given with a chow and high-fat diet from postnatal day (PND) 35 to PND70. Our data indicated that high-fat diet obviously decreased No. of sperm in epididymides of adult offspring due to paternal Cd exposure. Testicular histology revealed that the percentage of seminiferous tubules in stages IX-XII and the atypical residual bodies positive tubules in CdH (paternal cadmium exposure and pubertal high-fat diet) group were higher than these in CdC (paternal cadmium exposure and pubertal chow diet) group. Further analysis demonstrated that high-fat diet markedly accelerated testicular apoptosis, as determined by TUNEL assay and immunostaining for cleaved caspase-3, in male offspring due to paternal Cd exposure. Collectively, high-fat diet exacerbates the damage of testicular development and spermatogenesis in offspring due to paternal cadmium exposure.

Microfractionation bioactivity-based ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry for the identification of nuclear factor-κB inhibitors and ß2 adrenergic receptor agonists in an alkaloidal extract of the folk herb Alstonia scholaris.

Traditional Chinese medicines (TCMs) are generally considered complementary or alternative remedies in most Western countries. The constituents of TCMs are hard to define, and their efficacy is difficult to appraise. Thus, the development of suitable methods for evaluating the relationship between bioactivity and the chemical makeup of complex TCM mixtures remains a great challenge. In the present work, the bioactivity-integrated fingerprints of alkaloidal leaf extracts of Alstonia scholaris, a folk medicinal herb for chronic respiratory diseases, were established by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF). This method was coupled with two dual-luciferase reporter assay systems to show nuclear factor-κB (NF-κB) inhibition and ß(2) adrenergic receptor (ß(2)AR) activation. Using UPLC-Q/TOF, 18 potential candidates were identified according to unique mass spectrometric fragmentation. After in vitro biological evaluation, several indole alkaloids with anti-inflammatory and anti-asthmatic properties were found, including akuammidine, (E)-alstoscholarine, and (Z)-alstoscholarine. Compared with conventional fingerprints, the microfractionation based bioactivity-integrated fingerprints that contain both chemical and bioactivity details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that dual bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential dual-target lead compounds in complex herbal medicines.

Bioactivity-based liquid chromatography-coupled electrospray ionization tandem ion trap/time of flight mass spectrometry for ß2AR agonist identification in alkaloidal extract of Alstonia scholaris.

Although chromatographic fingerprinting combined with chemometrics, is a rational method for the quality control of traditional Chinese medicine (TCM), chemometrics cannot fully explore the relationship between chemical information and the efficacy of the potential activity. In the present work, a cell-based ß2 adrenergic receptor (ß2AR) agonist functional evaluation model coupled with high-performance liquid chromatography was developed to screen the potential ß2AR agonist components in the alkaloidal extract of Alstonia scholaris leaves. Using a liquid chromatography with ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) system, the potential bioactive compounds in the prescription were identified and deduced based on the mass spectrometric fragmentation patterns, tandem mass spectrometry (MS/MS) data, and relevant literature. Several new ß2AR agonists of indole alkaloids were successfully found, and their activities were confirmed through an in vivo relaxant test on guinea pig tracheal muscles. The developed method is rapid and reliable compared with conventional fingerprinting and showed high sensitivity and resolution for the identification of ß2AR agonists in TCM prescriptions. This strategy clearly demonstrates that bioactivity-integrated fingerprinting is a powerful tool not only in screening and identifying potential lead compounds and in determining the therapeutic material basis of Chinese herbal prescriptions, but also in supplying suitable chemical markers for their quality control.