Regulation of uterus and placenta remodeling under high estradiol levels in gestational diabetes mellitus models†.

The present study aimed to investigate the regulation of placentas and uterus remodeling and involvement of estradiol in gestational diabetes mellitus. To achieve this, we established in vitro and in vivo models for gestational diabetes mellitus placentas by culturing human placental choriocarcinoma cells (BeWo) under hyperglycemic concentration and treating pregnant rats with streptozotocin. We evaluated the expression of angiogenesis-related proteins. The expression of the anti-angiogenic factor, excess placental soluble fms-like tyrosine kinase 1 was increased in our in vitro gestational diabetes mellitus model compared with the control. Moreover, the expressions of placental soluble fms-like tyrosine kinase 1 and the von Willebrand factor were also significantly elevated in the placenta of streptozotocin-treated rats. These data indicate the disruption of angiogenesis in the gestational diabetes mellitus placentas. The expression levels of connexin 43, a component of the gap junction and collagen type I alpha 2 chain, a component of the extracellular matrix, were decreased in the gestational diabetes mellitus uterus. These results suggest that uterus decidualization and placental angiogenesis are inhibited in gestational diabetes mellitus rats. Our results also showed upregulation of the expression of genes regulating estradiol synthesis as well as estrogen receptors in vivo models. Accordingly, the concentration of estradiol measured in the culture medium under hyperglycemic conditions, as well as in the serum and placenta of the streptozotocin-treated rats, was significantly elevated compared with the control groups. These results suggest that the dysregulated remodeling of the placenta and uterus may result in the elevation of estradiol and its signaling pathway in the gestational diabetes mellitus animal model to maintain pregnancy.

Protective effects of Korean Red Ginseng against toxicity of endocrine-disrupting chemicals.

Several chemicals have been developed owing to the progression of industrialization, among which endocrine-disrupting chemicals (EDCs; essential for plastic production) are used as plasticizers and flame retardants. Plastics have become an essential element in modern life because they provide convenience, thus increasing EDCs exposure to humans. EDCs cause adverse effects such as deterioration of reproductive function, cancer, and neurological abnormalities by disrupting the endocrine system and hence are classified as "dangerous substances." Additionally, they are toxic to various organs but continue to be used. Therefore, it is necessary to review the contamination status of EDCs, select potentially hazardous substances for management, and monitor the safety standards. In addition, it is necessary to discover substances that can protect against EDC toxicity and conduct active research on the protective effects of these substances. According to recent research, Korean Red Ginseng (KRG) exhibits protective effects against several toxicities caused by EDCs to humans. In this review, the effects of EDCs on the human body and the role of KRG in protection against EDC toxicity are discussed.

Cytotoxicity evaluation and mechanism of endocrine-disrupting chemicals by the embryoid body test.

Endocrine-disrupting chemicals (EDCs) are a structurally diverse class of synthetic and natural compounds. EDCs can cause non-communicable diseases such as obesity, type 2 diabetes, thyroid disorders, neurodevelopmental disease, hormone-dependent cancers, and reproductive disorders. The embryoid body test (EBT) is a developmental toxicity test method that determines the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The present study used the EBT to perform cytotoxicity evaluations of 10 EDCs and assessed the mechanistic relationship between endoplasmic reticulum (ER) stress and cytotoxicity. According to the statistical analysis and prediction model results, methylparaben, butylparaben, propylparaben, ethylparaben, triclosan, octylphenol, methoxychlor, bisphenol A, and diethylstilbestrol were classified as cytotoxic, but trichloroacetic acid was non-toxic. Classification accuracy was 90%. The mechanistic study showed that the cytotoxicities of butylparaben, propylparaben, octylphenol, and triclosan were induced by ER stress. The mRNA expressions of BiP, CHOP, and ATF4 were significantly higher following treatments with four EDCs compared to those after the control treatment. Compared to the control treatment, the mRNA levels of XBP1u and XBP1s increased significantly after butylparaben and propylparaben treatments, but did not increase with octylphenol and triclosan treatments. These results indicate that the EBT can be applied as an alternative toxicity test when evaluating the cytotoxicity of EDCs.

Amylin Protein Expression in the Rat Brain and Neuro-2a Cells.

The localization and expression of amylin protein in the rodent brain and mouse neuroblastoma Neuro-2a (N2a) are less widely known. Thus, this study investigated the expression distribution of amylin in the rat brain and N2a treated with steroid hormones. Amylin protein was identified in the olfactory bulb, cerebral cortex, dentate gyrus, thalamus, hypothalamus, ventral tegmental area (VTA), cerebellum, and brain stem in the rat brain. Additionally, the amylin protein was localized with the mature neurons of the cerebral cortex and dopaminergic neurons of the VTA. Progesterone (P4) and dexamethasone (Dex) significantly decreased, and 17ß-estradiol (E2) increased the amylin protein level in the cerebral cortex. The P4 receptor antagonist RU486 significantly influenced the effects of P4 and Dex, and the E2 receptor antagonist ICI 182,780 slightly changed E2's effect. Amylin protein expression was significantly reduced in the VTA by P4 and Dex, and its expression was changed only following P4 plus RU486 treatment. It was confirmed for the first time that amylin protein is strongly expressed in the cytoplasm in N2a cells using immunofluorescent staining. P4 increased the levels of amylin, and RU486 treatment decreased them. Dex significantly increased the levels of amylin protein. RU486 treatment reversed the effects of Dex. Therefore, amylin protein is expressed in the cerebral cortex neurons and dopaminergic neurons of the VTA of the immature rat brain. P4 and Dex influence the expression of amylin protein in the rat brain and N2a cells.

Novel chemical inhibitor against SOD1 misfolding and aggregation protects neuron-loss and ameliorates disease symptoms in ALS mouse model.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu, Zn-superoxide dismutase (SOD1) causing the gain of its toxic property are the major culprit of familial ALS (fALS). The abnormal SOD1 aggregation in the motor neurons has been suggested as the major pathological hallmark of ALS patients. However, the development of pharmacological interventions against SOD1 still needs further investigation. In this study, using ELISA-based chemical screening with wild and mutant SOD1 proteins, we screened a new small molecule, PRG-A01, which could block the misfolding/aggregation of SOD1 or TDP-43. The drug rescued the cell death induced by mutant SOD1 in human neuroblastoma cell line. Administration of PRG-A01 into the ALS model mouse resulted in significant improvement of muscle strength, motor neuron viability and mobility with extended lifespan. These results suggest that SOD1 misfolding/aggregation is a potent therapeutic target for SOD1 related ALS.

Prenatal Octamethylcyclotetrasiloxane Exposure Impaired Proliferation of Neuronal Progenitor, Leading to Motor, Cognition, Social and Behavioral Functions.

Cyclic siloxane octamethylcyclotetrasiloxane (D4) has raised concerns as an endocrine-disrupting chemical (EDC). D4 is widely used in detergent products, cosmetics, and personal care products. Recently, robust toxicological data for D4 has been reported, but the adverse effects of D4 on brain development are unknown. Here, pregnant mice on gestational day 9.5 were treated daily with D4 to postnatal day 28, and the offspring mice were studied. The prenatal D4-treated mice exhibited cognitive dysfunction, limited memory, and motor learning defect. Moreover, prenatal D4 exposure reduced the proliferation of neuronal progenitors in the offspring mouse brain. Next, the mechanisms through which D4 regulated the cell cycle were investigated. Aberrant gene expression, such as cyclin-dependent kinases CDK6 and cyclin-dependent kinase inhibitor p27, were found in the prenatal D4-treated mice. Furthermore, the estrogen receptors ERa and ERb were increased in the brain of prenatal D4-treated mice. Overall, these findings suggest that D4 exerts estrogen activity that affects the cell cycle progression of neuronal progenitor cells during neurodevelopment, which may be associated with cognitive deficits in offspring.

Loss of Nckx3 Exacerbates Experimental DSS-Induced Colitis in Mice through p53/NF-κB Pathway.

Inflammatory bowel diseases (IBDs) comprises a range of chronic inflammatory conditions of the intestinal tract. The incidence and prevalence of IBDs are increasing worldwide, but the precise etiology of these diseases is not completely understood. Calcium signaling plays a regulatory role in cellular proliferation. Nckx3, a potassium-dependent Na+/Ca2+ exchanger, is not only expressed in the brain but also in the aortic, uterine, and intestinal tissues, which contain abundant smooth muscle cells. This study investigated the role of Nckx3 in intestinal inflammation. Microarray analyses revealed the upregulation of the innate immune response-associated genes in the duodenum of Nckx3 knockout (KO) mice. The Nckx3 KO mice also showed an increase in IBD- and tumorigenesis-related genes. Using dextran sodium sulfate (DSS)-induced experimental colitis mice models, the Nckx3 KO mice showed severe colitis. Furthermore, the pathways involving p53 and NF-κB signaling were significantly upregulated by the absence of Nckx3. Overall, Nckx3 plays a critical role in the innate immune and immune response and may be central to the pathogenesis of IBD.

4-tert-Octylphenol Exposure Disrupts Brain Development and Subsequent Motor, Cognition, Social, and Behavioral Functions.

The endocrine-disrupting chemical 4-tert-octylphenol (OP) is a widespread estrogenic chemical used in consumer products such as epoxy resins and polycarbonate plastic. However, the effects of OP on brain development are unknown. The present study examined the effects of OP on neuron and neurobehavioral development in mice. By using primary cortical neuron cultures, we found that OP-treated showed a decreased length of axons and dendrites and an increased number of primary and secondary dendrites. OP reduced bromodeoxyuridine (BrdU), mitotic marker Ki67, and phospho-histone H3 (p-Histone-H3), resulting in a reduction of neuronal progenitor proliferation in offspring mouse brain. Moreover, OP induced apoptosis in neuronal progenitor cells in offspring mouse brain. Furthermore, offspring mice from OP-treated dams showed abnormal cognitive, social, and anxiety-like behaviors. Taken together, these results suggest that perinatal exposure to OP disrupts brain development and behavior in mice.

Data on cytotoxicity of plant essential oils in A549 and Detroit 551 cells.

To secure the safety for industrial applications of plant essential oils, it is necessary to determine the inhibitory concentration and inhibitory mechanism of cell proliferation in skin cells and lung cells. Considering inhalation through the respiratory system and skin contact of humans with essential oils, we used human lung cancer cells A549 and human skin fibroblasts Detroit 551 cells for all experiments. In this study, we examined IC50 values and protein levels of cell cycle markers (cyclin A, cyclin B, cyclin D, and cyclin E) and apoptosis marker (caspase-3) after exposure to 10 plant essential oils, including Dendranthema indicum (L.) Des Moul, Peucedanum japonicum Thunb, Dendranthema zawadskii var. latilobum (Maxim.) Kitam, Agastache rugosa (Fisch.&Mey.) Kuntze, Vitex rotundifolia L.f, Pinus rigida Mill; Orixa japonica Thunb, Pinus strobus L, Chamaecyparis pisifera (Siebold et Zucc.) Endl. var. filifera Beissn. et Hochst, and Citrus sunki Hort. ex Tanaka. After the treatment of A549 and Detroit 551 cells to varying concentrations of the 10 plant essential oils, IC50 values were determined by CCK analysis, whereas protein expressions of the four cyclins and caspase-3 were identified by Western blotting analysis. We believe that by examining the degree and mechanism of cell proliferation inhibition exerted by essential oils on skin and lung cells of humans, data obtained in this study can provide guidelines for the industrial application of plant essential oils.

Dexamethasone Treatment Increases the Intracellular Calcium Level Through TRPV6 in A549 Cells.

This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.

The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain.

The cytosolic calcium concentration is regulated by calcium-processing proteins such as transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodium-calcium exchanger 1 (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). Those calcium-processing proteins are important for physiological functions in the brain. The effects of steroid hormones on calcium-processing protein expressions in the brains are unknown. Thus, the effects of steroid hormones on the distribution, localization, and expressions of calcium-processing proteins in the brain were analyzed. Immature female rats were injected with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182,780 and RU486). We found that TRPV5 and TRPV6 proteins were highly expressed in the cerebral cortex (CT), hypothalamus (HY), and brain stem (BS) compared to that in the olfactory bulb (OB) and cerebellum (CB). Also, the NCX1 protein was highly expressed in CT and BS compared to that in OB, HY, and CB, and PMCA1 protein was highly expressed in CT compared to that in other brain regions. Furthermore, expression levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by E2, P4, and/or DEX in the CT and HY. In summary, calcium-processing proteins are widely expressed in the immature rat brain, and expressions of calcium-processing proteins in CT and HY indicated that they may regulate by E2, P4, and/or DEX and can be attenuated by antagonist treatment. These results indicate that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may serve as a critical regulator of cytosolic calcium absorption and release in the brain.

Obovatol inhibits NLRP3, AIM2, and non-canonical inflammasome activation.

BACKGROUND: Obovatol, a biphenolic chemical originating from Magnolia obovata, has been utilized as a traditional medicine for the treatment of inflammatory diseases. Inflammasome induces maturation of inflammatory cytokines in response to intracellular danger signals, and its dysregulation induces inflammatory diseases. PURPOSE: The effect of obovatol on inflammasome activation has not been reported, although its anti-inflammatory properties have been studied. STUDY DESIGN/METHODS: Obovatol was treated to macrophages with inflammasome triggers, and secretions of interleukin (IL)-1ß, IL-18, and caspase-1 were measured as readouts of inflammasome activation. In addition, Asc pyroptosome formation, caspase-1 activity, and mitochondrial reactive oxygen species (ROS) production were analyzed in mechanical studies. Anti-inflammasome properties of obovatol were confirmed in an animal model. RESULTS: Obovatol inhibited NLRP3, AIM2, and non-canonical inflammasomes through inhibition of Asc pyroptosome formation and mitochondrial ROS generation. In addition, obovatol disrupted the priming step of inflammasome activation and inhibited transcription of inflammatory cytokines. In mice, obovatol attenuated serum IL-1ß elevation in response to monosodium urate crystals. CONCLUSION: Obovatol is suggested as an inhibitor of NLRP3, AIM2, and non-canonical inflammasomes.

Distribution of and steroid hormone effects on calbindin-D9k in the immature rat brain.

Calbindin-D9k (CaBP-9k), one of the major calcium-binding and calcium-buffering proteins, is important in the physiological functioning of organs. The neuroanatomical localization of CaBP-9k in the rodent brain has not been reported; thus, this study investigated the neuroanatomical distribution of CaBP-9k and the regulation of CaBP-9k expression on steroid hormones in the immature rat brain. To confirm the influence of steroid hormones on CaBP-9k expression, immature female rats were injected for 5 days with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182, 780 and RU 486). The localization and expression of the CaBP-9k protein in brain regions were identified by immunofluorescence and western blot assays, respectively. We observed that CaBP-9k expression was especially strong in hypothalamus, cerebellum, and brain stem. In addition, CaBP-9k was colocalized with mature-, GABAergic, dopaminergic, and oxytocinergic neurons. We also observed that the CaBP-9k protein level was significantly increased by P4 and reversed by antagonist RU 486 treatment in immature rat brain. In summary, CaBP-9k positive cells have a wide distribution in the immature rat brain, and CaBP-9k expression is regulated by P4. We suggest that CaBP-9k expression regulated by steroid hormone may serve as an important regulator of cytosolic calcium concentration in the brain.

Dietary intake of genistein suppresses hepatocellular carcinoma through AMPK-mediated apoptosis and anti-inflammation.

BACKGROUND: Women have a lower risk of hepatocellular carcinoma (HCC) than men, and the decreased possibility of HCC in women is thought to depend on estrogen levels. As a soybean-isoflavone product, genistein has estrogenic activity in various reproductive tissues, because it mimics 17ß-estradiol and binds the estrogen receptor. Though genistein is a known liver cancer suppressor, its effects have not been studies in long-term experiment, where genistein is fed to a female animal model of HCC. METHODS: Mice were treated with diethylnitrosamine (DEN) to induce HCC at 2 weeks of age and fed with supplemental genistein for 5 months, from 40 to 62 weeks of age. RESULTS: The dietary intake of genistein decreased the incidence of HCC and suppressed HCC development. Genistein induced phospho-AMPK in total liver extracts, Hep3B cells, and Raw 264.7 cells, and phospho-AMPK promoted apoptosis in liver and Hep3B cells. Moreover, phospho-AMPK down-regulated pro-inflammatory responses and ameliorated liver damage. A suppressed pro-inflammatory response with increased mitochondrial respiration was concomitantly observed after genistein treatment. CONCLUSIONS: Genistein-mediated AMPK activation increases hepatocyte apoptosis through energy-dependent caspase pathways, suppresses the inflammatory response in resident liver macrophages by increased cellular respiration, and consequently inhibits the initiation and progression of HCC.

Pregnenolone as a potential candidate for hormone therapy for female reproductive disorders targeting ERß.

Many steroid hormones such as estrogen (E2) bind to their receptors for the regulation of biological processes. Pregnenolone (P5) is the precursor form of almost all steroid hormones and is often used to treat skin disorders and neurological complications. However, the mechanism and physiological function of P5 in reproductive organs are not well established. In this study, we investigated the effects of P5 on activation and expression of E2 receptor (ER) in the uteri and ovaries. To study the mechanism of P5 directly, Ishikawa cells were transfected with E2 response element (ERE)-luciferase plasmid and isoforms of ER. ERE-luciferase activity induced by P5 was similar to that induced by E2, and P5 showed high activity for ERß without any relevance to P5-metabolizing hormones such as progesterone (P4) and E2. In an animal study, immature female rats treated with P5 showed upregulation of ERα and downregulation of ERß in the uteri, which is the main organ expressing ERα. In ERß-expressing organ ovaries, estrogen receptor 1, estrogen receptor 2, and P4 receptor were all downregulated by P5 and E2. Also, a decrease of ovarian cell proliferation and viability was observed in response to P5 relative to the control, suggesting that P5 may be a candidate for antiproliferative hormone of ovarian cancer. These findings suggest that P5 stimulates ERE promoter by ERß-mediated signaling in the uteri and ovaries. Activation of ERß by P5 may help in understanding the mechanism of ER-related female reproductive diseases such as endometriosis and ovarian cancer.

Nitric oxide prevents H2O2-induced apoptosis in SK-N-MC human neuroblastoma cells.

Nitric oxide (NO) is a cellular signaling molecule in many physiological and pathological processes including neuroprotector. Here we examined the antiapoptotic effect of NO in SK-N-MC cells. H2O2 treatment (10-200 µM) induced cell death in a dose-dependent manner and pretreatment of cells with 100 µM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, attenuated the occurrence of H2O2-induced cell death. DAPI staining showed H2O2-induced nuclear fragmentation and NO treatment suppressed it. NO inhibited the proteolytic activation of caspase-3 and mitochondrial cytochrome c release. Treatment of soluble guanylyl cyclase inhibitor ODQ decreased the protective effect of SNAP on H2O2-treated cells and increased caspase 3-like enzyme activity and activation, cytochrome c release, PARP cleavage, and DNA fragmentation, indicating that cGMP is a key mediator in NO-mediated antiapoptosis. The cGMP analog 8-Br-cGMP blocked H2O2-induced apoptotic cell death; reduction of caspase-3 enzyme, cytochrome c release, and caspase-8 and -9. These preventive effects of SNAP and 8-Br-cGMP were suppressed by PKG inhibitor KT5823. Levels of PKGI, PKGII, and p-VASP proteins were increased by SNAP and 8-Br-cGMP and suppressed by KT5823 treatment. These results indicate that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Akt activation was inhibited the PI3K inhibitors LY294002 and Wortmannin, resulting in the inhibition of cell viability and increase of cytochrome c release. SNAP induced phosphorylation of Akt and Bad and then increased the interactions between 14-3-3ß and p-Bad. These data suggest that the NO suppresses H2O2-induced SK-N-MC cell apoptosis by suppressing apoptosis signal mediating the interaction between 14-3-3ß and Bad phosphorylation via PKG/PI3K/Akt.

Effects of Bisphenol A and 4-tert-Octylphenol on Embryo Implantation Failure in Mouse.

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all human miscarriage cases. Calcium ion has been shown to be involved in many cellular signal transduction pathways as well as in the regulation of cell adhesion, which is necessary for the embryo implantation process. Exposure to endocrine-disrupting chemicals (EDs) during early gestation results in disruption of intrauterine implantation and uterine reception, leading to implantation failure. In this study, ovarian estrogen (E2), bisphenol A (BPA), or 4-tert-octylphenol (OP), with or without ICI 182,780 (ICI) were injected subcutaneously from gestation day 1 to gestation day 3 post-coitus. The expression levels of the calcium transport genes were assessed in maternal uteri and implantation sites. The number of implantation sites was significantly low in the OP group, and implantation sites were absent in the E2, ICI and EDs + ICI groups. There were different calcium transient transport channel expression levels in uterus and implantation site samples. The levels of TRPV5 and TRPV6 gene expression were significantly increased by EDs with/without ICI treatment in utero. Meanwhile, TRPV5 and TRPV6 gene expression were significantly lower in implantation sites samples. NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Compared to vehicle treatment in the uterus, both the MUC1 mRNA and protein levels were markedly high in all but the BPA group. Taken together, these results suggest that both BPA and OP can impair embryo implantation through alteration of calcium transport gene expressions and by affecting uterine receptivity.

Bisphenol A and octylphenol exacerbate type 1 diabetes mellitus by disrupting calcium homeostasis in mouse pancreas.

In pancreatic ß cells, which produce and secrete insulin, Ca2+ signals contribute to insulin production and secretion. Bisphenol A (BPA) and octylphenol (OP) are reported to increase plasma insulin levels and insulin transcription factors, but regulation of plasma glucose levels did not decrease proportionally to the insulin increase. We hypothesized that BPA and OP disrupt calcium homeostasis resulting in insulin resistance through induction of endoplasmic reticulum (ER) stress. BPA and OP treatment leads to survival of pancreatic ß cells against streptozotocin, but despite an increased insulin level, serum glucose regulation is not properly regulated. The expression of genes involved in transporting calcium ions to the cytosol and ER decreased while the expression of those affecting the removal of calcium from the cytosol and ER increased. Depletion of calcium from the ER leads to ER stress and can induce insulin resistance. Insulin resistance is also confirmed by insulin-responsive gene, such as glucose transporter 4 (GLUT4) and IRS2, expression. Taken together, these results imply that disruption of calcium homeostasis by BPA and OP induces ER stress and leads to insulin resistance, especially in a streptozotocin (STZ) -induced type 1 diabetes mellitus model.

The Regulation of Fatty Acid Oxidation in Human Preeclampsia.

Preeclampsia (PE) is a pregnancy disorder characterized by high blood pressure, placental oxidative stress, and proteinuria. In a GeneFishing experiment using human preeclamptic placenta, expression of acyl-coenzyme A dehydrogenase very long chain (ACADVL), which is involved in fatty acid ß-oxidation (FAO), was detected. To investigate the correlation between PE and FAO, this study subjected in vitro BeWo cells and in vivo pregnant mice to oxidative stress induced by hypoxia. Hypoxic condition, which oxygen supply is insufficient in cells and placenta, created a similar state to placental oxidative stress in PE, as evidenced by increased hypoxic (oxoguanine DNA glycosylase 1, hypoxia inducible factor 1 alpha subunit) and preeclamptic markers (soluble fms-like tyrosine kinase 1) both in vitro and in vivo. Increased expression of FAO-related genes (ACADVL, enoyl-coenzyme A hydratase/3-hydroxyacyl coenzyme A dehydrogenase) was observed in these models as well as in cases of preeclamptic preterm labor. In the in vivo liver model, messenger RNA expression of gluconeogenesis-related genes increased. Consequently, these results suggest that expression of FAO-related genes is regulated by hypoxic conditions and onset time of PE and affects maternal gluconeogenesis during pregnancy in patients with PE.

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells.

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA-4, TRA-1-60 and TRA-1-81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5-fluorouracil, indomethacin and non-embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down-regulated in a dose-dependent manner after treatment with embryotoxic chemicals. After treatment with 5-fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up-regulation of development, cell cycle and apoptosis-related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development-, cell cycle- and apoptosis-related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage.