Dietary supplementation with nicotinamide riboside improves fetal growth under hypoglycemia.

Nicotinamide riboside (NR) is considered a super-supplement that prevents obesity and diabetes. While NR has been investigated for various effects depending on nutritional conditions, metabolic research on women and pregnant women has rarely been discussed. In this study, we focused on the glycemic control of NR in females and found the protective role of NR in pregnant animals under hypoglycemic conditions. Metabolic-tolerance tests were performed in vivo under progesterone (P4) exposure after ovariectomy (OVX). NR enhanced resistance to energy deprivation and showed a slight increase in gluconeogenesis in naïve control mice. However, NR reduced hyperglycemia and significantly induced gluconeogenesis in OVX mice. While NR reduced hyperglycemia in the P4-treated OVX mice, it reduced insulin response and substantially increased gluconeogenesis. Similar to animal experiments, NR increased gluconeogenesis and mitochondrial respiration in Hep3B cells. The gluconeogenic function of NR is mediated by tricarboxylic acid cycle (TCA) cycle enrichment, as residual pyruvate could induce gluconeogenesis. NR recovered fetal growth by increasing blood glucose levels when hypoglycemia was induced by diet-restriction during pregnancy. Our study revealed the glucose-metabolic function of NR in hypoglycemic pregnant animals, suggesting NR as a dietary supplement to improve fetal growth. Because diabetic women suffer from hypoglycemia due to insulin therapy, NR has therapeutic potential for use as a glycemic control pill.

The Improvement Effect of D-Chiro-Inositol and Ecklonia cava K. in the Rat Model of Polycystic Ovarian Syndrome.

Introduction: Polycystic Ovarian Syndrome (PCOS) is known to be an endocrine state that is characterized by oligomenorrhea, hyperandrogenism, and highly cystic follicles in the ovaries. The use of food ingredients and traditional medicine in Asian countries is well known, and previous studies have shown that Ecklonia cava K. [Alariaceae] (EC) is able to alleviate PCOS symptoms. D-Chiro-inositol (DCI) administration in pathologies where steroid biosynthesis is a crucial factor, i.e., PCOS, has provided satisfactory results. Methods: Therefore, we studied the synergistic effects of the two previously known active compounds. In rats with letrozole-induced PCOS, we focused on alternative therapies using EC and/or DCI extracts to alleviate ovarian failure. Results: As a nonsteroidal aromatase inhibitor, letrozole inhibits the conversion of testosterone to estrogen and subsequently causes PCOS. We divided 6-week-old female mice into the following six groups and evaluated them: vehicle, PCOS, PCOS + MET (metformin), PCOS + DCI, PCOS + EC, and PCOS + DCI + EC. In our study, PCOS rats treated with EC and DCI had low serum LH and T levels and low serum levels of inflammatory cytokines such as TNFα and IL-6. These treatments also appeared to regulate the production of factors that affect follicle formation and inflammation in the ovaries. Conclusion: We concluded that EC extract and/or DCI administration influenced aromatase production and reduced LH and T stimulation, and cotreatment with EC and DCI consequently restored ovarian dysfunction or anti-inflammatory responses in rats with PCOS-like symptoms.

The aqueous fraction of Castanea crenata inner shell extract reduces obesity and intramuscular lipid accumulation via induction of mitochondrial respiration and fatty acid oxidation in muscle.

BACKGROUND: Skeletal muscle is responsible for free fatty acid (FFA) disposal via mitochondrial respiration and fatty acid oxidation (FAO). Obesity triggers high levels of circulating FFAs, which can cause intramuscular lipid (IMCL) deposition. Diverse phytochemicals, including crude Castanea crenata inner shell extract (CCE), have been shown to possess an anti-obesity effect. PURPOSE: We aimed to demonstrate whether the aqueous fraction of CCE (ACCE) provides an anti-obesity effect with a decrease in plasma FFAs and reduces IMCL. METHODS: High-fat-fed C57BL/6 mice received ACCE via water intake. A204 cells incubated with fatty acids were treated with ACCE. Lipid accumulation and mitochondrial metabolism were assessed using histological and molecular techniques. RESULTS: ACCE possessed a notably higher gallic acid content than CCE among the constituents. ACCE-administered mice exhibited reduced plasma FFA levels, adiposity, and IMCL. Muscle lipotoxicity was suppressed, including apoptosis, ER stress, and inflammation. The anti-lipid effect of ACCE was observed with the induction of mitochondrial respiration and fatty acid oxidation in muscle. CONCLUSIONS: ACCE increases mitochondrial respiration and FAO in skeletal muscle and protects muscle from IMCL and lipotoxicity, reducing plasma FFA and adiposity.

Traditional Medicine (Mahuang-Tang) Improves Ovarian Dysfunction and the Regulation of Steroidogenic Genes in Letrozole-Induced PCOS Rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Mahuang-Tang (MHT) has traditionally been used in Asia to treat a variety of diseases, such as fever without sweating, joint pain, lower back pain, asthma, and gynecological conditions. Polycystic ovary syndrome (PCOS) is a kind of gynecological disease that causes amenorrhea, infertility, and menopausal and urogenital disorders that could benefit from MHT treatment. AIM OF THE STUDY: In this study, we examined the effects of MHT on ovarian hormones and steroidogenic enzymes in female PCOS rats. METHODS AND RESULTS: The PCOS rat model was induced by Letrozole, and an in vivo evaluation of whether the dietary consumption of MHT improved the PCOS-like symptoms was conducted. The luteinizing hormone (LH) level and luteinizing hormone/follicular-stimulating hormone (LH/FSH) ratio increased in PCOS rats but decreased following MHT treatment. In the PCOS rats, the reduced estrogen level was restored to that of normal controls with MHT treatment in serum. The transcription level(s) of gonadotropin receptors (Fshr and Lhr), steroid receptors (Pgr, and Esr1) and steroidogenic enzymes (Cyp19a1, Hsd3b1, Hsd17a1, and Cyp11a1) changed under the PCOS condition, and were regulated by MHT treatment in the ovaries of PCOS rats. The reproductive tissues of Letrozole-induced PCOS rats were restored into estrogenic condition from androgen environments. CONCLUSION: These results suggest that MHT ameliorates the symptoms of PCOS by improving the dysregulation of ovarian steroids and steroidogenic enzymes in PCOS rats.

The Protective Role of Calbindin-D9k on Endoplasmic Reticulum Stress-Induced Beta Cell Death.

Intracellular calcium ion content is tightly regulated for the maintenance of cellular functions and cell survival. Calbindin-D9k (CaBP-9k) is responsible for regulating the distribution of cytosolic free-calcium ions. In this study, we aimed to investigate the effect of CaBP-9k on cell survival in pancreatic beta cells. Six-month-old wildtype CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice were used to compare the pathological phenotypes of calcium-binding protein-deleted mice. Subsequently, the endoplasmic reticulum (ER) stress reducer tauroursodeoxycholic acid (TUDCA) was administered to wildtype and CaBP-9k KO mice. In vitro assessment of the role of CaBP-9k was performed following CaBP-9k overexpression and treatment with the ER stress inducer thapsigargin. Six-month-old CaBP-9k KO mice showed reduced islet volume and up-regulation of cell death markers resulting from ER stress, which led to pancreatic beta cell death. TUDCA treatment recovered islet volume, serum insulin level, and abdominal fat storage by CaBP-9k ablation. CaBP-9k overexpression elevated insulin secretion and recovered thapsigargin-induced ER stress in the INS-1E cell line. The results of this study show that CaBP-9k can protect pancreatic beta cell survival from ER stress and contribute to glucose homeostasis, which can reduce the risk of type 1 diabetes and provide the molecular basis for calcium supplementation to diabetic patients.

Antidepressant Effect of Tetragonia tetragonoides (Pall.) Kuntze Extract on Serotonin Turnover.

Tetragonia tetragonoides (Pall.) Kuntze (TTK) is a groundcover found along coastal areas of the Korean peninsula. TTK is traditionally used to improve women's health and treat gastrointestinal diseases. Use of herbal medicines in the treatment of mood disorders has recently been suggested as an alternative therapeutic strategy. In the present study, we determined that consumption of TTK extract ameliorated progression of depressive-like symptoms in ovariectomized (OVX) rats and further examined the mechanisms involved, i.e., synthesis, release, and reuptake(s) of serotonin (also known as 5-HT). We assessed the mRNA expression levels of tryptophan hydroxylases (TPH-1 and TPH-2) and serotonin transporter (SERT) as well as the reuptake activity of serotonin in RBL-2H3 cells. We also determined whether or not TTK extract regulates the serum level of serotonin and improves depressive-like symptoms in 0.5, 1, and 2% TTK-fed OVX female rats in a forced swimming test. Our results show that the mRNA levels of TPH-1 and SERT were significantly reduced, whereas the mRNA level of TPH-2 was dose-dependently elevated by TTK (50 and 100 µg/mL) in RBL-2H3 cells. TTK significantly inhibited LPS- (lipopolysaccharide-) induced serotonin uptake in RBL-2H3 cells in a dose-dependent manner. The serum level(s) of serotonin was elevated by 1% and 2% TTK treatment in OVX female rats. Moreover, immobility time in the forced swimming test was reduced by 1% and 2% TTK treatment but not altered by 0.5% TTK treatment in OVX female rats. Taken together, these results indicate that TTK may significantly inhibit depressive-like symptoms due to upregulation of serotonin level(s) and regulation of serotonin reuptake activity. Thus, TTK may exert beneficial effects on depression during pre- or/and postmenopausal periods via modulation of serotonin synthesis and metabolism.

Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression.

Curcumae radix is the dry root of Curcuma longa L. (turmeric) that can be used either as a spice or traditional medicine. The aim of this study was to investigate the survival benefits and the anti-metastatic activity of curcumae radix extract (CRE) in MCF7 cells and in MMTV-PyMT transgenic mice-a mouse model of breast cancer metastasis. In vitro wound scratch assay revealed that CRE treatment inhibited cell motility and cell migration in a dose-dependent manner. To investigate the effect of CRE in breast cancer metastasis, MMTV-PyMT transgenic female virgin mice were used and randomly divided into two groups. For survival curve analysis, CRE was administered in a dose of 50 mg/kg to 8⁻20-week-old mice. Interestingly, CRE treatment significantly increased the median and prolonged survival of MMTV-PyMT mice. Furthermore, CRE treatment decreased tumor burden and inhibited cell proliferation in primary breast tumor, and also suppressed mammary tumor-derived lung metastasis. The size of the lung metastases substantially decreased in the CRE-treated group compared with the ones in the control group. Curcumae radix extract showed anti-metastatic activity through regulating the expression of metastasis markers including C-C Chemokine Receptor Type 7, Matrix Metalloproteinase 9 and the proto-oncogenes c-fos and c-jun. We demonstrated that these metastatic regulators were decreased when CCR7 expression was suppressed in MCF7 cells transfected with CCR7 siRNA. The results of this study show that curcumae radix exerts antitumor and anti-metastatic activities, and we suggest that curcumae radix might be a potential supplement for the treatment and prevention of breast cancer metastasis.

Welsh Onion Root (Allium fistulosum) Restores Ovarian Functions from Letrozole Induced-Polycystic Ovary Syndrome.

Polycystic ovarian syndrome (PCOS) is an endocrine, metabolic, and systemic disease. It is mainly characterized by hyperandrogenism, oligomenorrhea, and high levels of luteinizing hormone (LH). There is no obvious therapy for PCOS, so patients have received symptomatic therapy. Welsh onion (Allium fistulosum) is well-known in Asian countries for its usage in food ingredients and traditional medicines. It is also studied for its many effects. These include activation of immune responses, antihypertensive effects, and antioxidant effects. Using letrozole-induced PCOS rats, we focused on herbal therapy using extract of Allium fistulosum (AF; A. fistulosum) roots to improve ovarian functions. As a nonsteroidal aromatase inhibitor, letrozole blocks conversion of testosterone to estrogen and subsequently induces PCOS phenomenon. We divided six-week-old female rats into four groups, including control, letrozole, letrozole + AF extract, and temporary letrozole groups. In our study, treatment with AF extract shows a low plasma LH/FSH ratio, and reveals high estrogen levels, ovarian morphology, folliculogenesis-related genes, and aromatase expression under PCOS mimic conditions. We concluded that AF extract administration influenced aromatase production, enhanced the estrogen steroid synthesis, and consequently restored the estrogenic feedback mechanism on the pituitary-ovary system.

Therapeutic effects of Schisandra chinensis on the hyperprolactinemia in rat.

Prolactin (PRL) is secreted from the pituitary gland in response to eating, mating, and ovulation. Increased serum concentration of PRL during pregnancy contributes to enlargement of the mammary glands of the breasts and prepares for production of milk. However, high PRL levels derived from prolactinoma and hyperprolactinemia induce physiological disorders such as infertility and early menopause. Natural compounds isolated from S. chinensis have been known to possess anti-oxidative, anti-inflammatory and anti-diabetic effects. In the present study, we examined the therapeutic effect of S. chinensis and its single compounds on hyperprolactinemia in the pituitary gland. In rat pituitary cells, PRL expression levels were examined using real-time PCR and western blot assay. Crude S. chinensis extract and its single compound, gomisin N, reduced mRNA and protein levels of PRL in GH3 cells. In addition, cell proliferation and PRL target gene expression in cells were modulated by S. chinensis. Similar to the in vitro experiments, crude S. chinensis extract and gomisin N reduced PRL levels in the pituitary and serum of immature female rats. These results show that S. chinensis and its single compound, gomisin N, are regulators of PRL production and may be candidates for treatment of hyperprolactinemia and prolactinoma.

Establishment of a rapid drug screening system based on embryonic stem cells.

Embryonic stem (ES) cells have the capacity for self-renewal and differentiation into three germ layers following formation of embryonic bodies (EB). To investigate toxicity of pharmaceutical compounds, five toxic chemicals, indomethacin, dexamethasone, hydroxyurea, 5-fluorouracil, and cytosine arabinoside were applied in mouse ES cells during formation of EBs. Using microscopic evaluation, the size of EBs was reduced in a dose-dependent manner by treatment with pharmaceutical chemicals. While apoptosis-related proteins, cleaved caspase-3 and PARP, were decreased in compound-exposed EBs, necrosis-related protein (Hmgb1) was present in culture media of EBs, indicating that detection of Hmgb1 can result in activation of necrosis by pharmaceutical compounds. While pharmaceutical compounds impaired the differentiation of mES cells linked with spontaneous apoptotic cell death, it was determined that cytotoxic cell damage is necrosis-dependent in mES cells. In addition, an apoptotic transcript (Noxa mRNA) in toxicant-exposed EBs was decreased in parallel with apoptosis-related proteins. Following impairment of apoptosis, differentiation-related markers including un-differentiation (Sox2), endoderm (Hnf4), mesoderm (Bmp4), and ectoderm (Pax6) also fluctuated by treatment with pharmaceutical compounds. Taken together, the data imply that exposure to pharmaceutical compounds results in increased cell death hindering the spontaneous apoptosis of cells to undergo differentiation. Using both characteristics of ES cells like self-renewal or cellular pluripotency and potentials of ES cells for evaluation in toxicity of various compounds, the current study was conducted for establishment of a novel drug screening system beyond hidden virtues of the well-known chemicals.

Regulation of tight junction gene expression in the kidney of calbindin-D9k and/or -D28k knockout mice after consumption of a calcium- or a calcium/vitamin D-deficient diet.

BACKGROUND: Calciotropic hormones were thought to facilitate calcium transfer through active transcellular or passive paracellular pathway for calcium homeostasis. While calcium transport proteins such as CaBP-28 k, TRPV5, NCX1, PMCA1b are involved in calcium reabsorption of the renal tubule using transcellular transport, tight junction proteins are known as critically related to calcium absorption through paracellular pathway. The regulation of each pathway for calcium transport was well studied but the correlation was not. It is expected that present study will provide new information about the link between transcellular and paracellular pathway within renal tubules. RESULTS: Transcripts and proteins of tight junction related genes (occludin, ZO-1, and claudins) were examined in CaBP-9 k-and/or-28 k-deficient mice as well as the effect of dietary calcium and/or vitamin D supplementation. With a normal diet, the transcriptional and translational expressions of most tight junction proteins in the kidney was not significantly changed but with a calcium- and vitamin D-deficient diet, and they were significantly increased in the kidney of the CaBP-28 k and CaBP-9 k/28 k double KO (DKO) mice. In these genotypes, the increase of tight junction related transcripts and proteins are referred to as an evidence explaining correlation between transcellular transport and paracellular pathway. CONCLUSIONS: These findings are particularly interesting in evidences that insufficient transcellular calcium transports are compensated by paracellular pathway in calcium or calcium/vitamin D deficient condition, and that both transcellular and paracellular pathways functionally cooperate for calcium reabsorption in the kidney.

Korean red ginseng extracts inhibit NLRP3 and AIM2 inflammasome activation.

Korean red ginseng extract (RGE) is one of the most popular natural herbs modulating the immune system. Although the effects of RGE on immunity have been reported, its effects on inflammasomes, multi-protein complexes that activate caspase-1 to induce maturation of interleukin (IL)-1ß, have not been studied yet. In this study, we elucidated the effect of RGE on inflammasome activation using mouse and human macrophages. In our results, RGE inhibited IL-1ß maturation resulting from NLRP3 inflammasome activation in both in vitro and in vivo models. In addition, RGE strongly attenuated IL-1ß secretion as well as pathogen clearance via pyroptotic cell death by macrophages through inhibition of AIM2 inflammasome activation. Ginsenosides Rg1 and Rh3 were suggested as inhibitors of the inflammasome activation. Thus, we demonstrated that RGE inhibits both NLRP3 and AIM2 inflammasome activation, with predominant involvement of the AIM2 inflammasome.

Alteration of tight junction gene expression by calcium- and vitamin D-deficient diet in the duodenum of calbindin-null mice.

Calcium absorption is regulated by both active (transcellular) and passive (paracellular) pathways. Although each pathway has been studied, correlations between the two pathways have not been well elucidated. In previous investigations, the critical transcellular proteins, calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), were shown to affect other transcellular pathways by buffering intracellular calcium concentrations. The rate of paracellular calcium transport in the duodenum is generally determined by the expression of tight junction genes. In the present study, the effect of dietary calcium and/or vitamin D supplementation on the expression of tight junction genes (occludin, ZO-1 and claudin 2, 10b, 12 and 15) in the duodenum of CaBP-9k- and/or -28k-deficient mice was examined. With a normal diet, the expression of most tight junction genes in the duodenum was significantly increased in CaBP-9k knockout (KO) mice compared to wild-type (WT) animals. With a calcium- and vitamin D-deficient diet, tight junction gene expression was significantly decreased in the duodenum of the CaBP-9k KO mice. These findings suggest that expression of paracellular tight junction genes is regulated by transcellular CaBP proteins, suggesting that active and passive calcium transport pathways may function cooperatively.

Lentinus edodes promotes fat removal in hypercholesterolemic mice.

Lentinus (L.) edodes (shiitake mushroom) is used as a traditional medicine in Asia. One of the components of L. edodes, eritadenine (an adenosine analog alkaloid), has been shown to reduce cholesterol levels. The hypocholesterolemic action of eritadenine appears to be achieved through the modification of hepatic phospholipid metabolism. In the present study, the effects of L. edodes in a mouse model of hypercholesterolemia were investigated. Hypercholesterolemia was induced by the consumption of a high-fat diet (HFD). The animals were divided into six groups, which were fed a normal diet, HFD alone, HFD containing eritadenine [10 mg/kg of body weight (BW)] or HFD with 5, 10 or 20% L. edodes, respectively, for 4 weeks (from 5 to 9 weeks of age). The mice in the six groups had similar BW gains. Total serum cholesterol (T-CHO), low-density lipoprotein (LDL) and triglyceride (TG) levels were increased in the HFD-fed group compared with those in the normal diet group. However, the levels of high-density lipoprotein (HDL) were not significantly altered. In mice treated with L. edodes (5, 10 or 20%), the T-CHO, LDL and TG serum levels were reduced in a dose-dependent manner. The mRNA expression of cholesterol 7-α-hydroxylase 1 (CYP7A1) was decreased in hypercholesterolemic mice and increased by eritadenine and L. edodes (5, 10 and 20%) supplementation. In liver tissues, it was observed that lipid accumulation was reduced by treatment with eritadenine and L. edodes. In addition, it was revealed that the formation of atherosclerotic plaques due to the HFD was also suppressed by eritadenine and L. edodes. The results of the study indicated that the consumption of an HFD may inhibit CYP7A1 expression in the liver by increasing serum T-CHO, LDL and TG levels. L. edodes may help regulate lipid metabolism, suggesting that this fungus ameliorates hypercholesterolemia in mice by regulating CYP7A1 expression in the liver.

Protective effects of the pyrolyzates derived from bamboo against neuronal damage and hematoaggregation.

ETHNOPHARMACOLOGICAL RELEVANCE: Bamboo species are thought to be originally from Central China, but are now found in many temperate and semi-tropical regions around the world. Although the extracts from bamboo may have antioxidant activities and anti-inflammatory effects, their exact biological activities have not been elucidated. AIM OF THE STUDY: Two biological activities of bamboo-derived pyrolyzates were investigated; the protective effects against N-methyl-d-aspartate (NMDA)-induced cell death in primary cultured cortical neuron and the anti-plasmin effects determined by using fibrin and fibrinogen degradation products (FDPs) assay. RESULTS: Treatment of neuronal cells with pyrolyzates of Phyllostachys pubescens, Phyllostachys nigra and Phyllostachys bambusoides resulted in restored cell viability when compared to untreated cells in an NMDA-induced neuronal cell death assay. In addition, cortical neurons treated with Phyllostachys pubescens and Phyllostachys nigra showed a reduction of apoptosis following exposure to NMDA, as determined by Hoechst 33342 staining. In addition, Phyllostachys nigra pyrolyzates also exhibited anti-plasmin action in a FDP assay. It is of interest to note that pyrolyzates exhibited activities of NMDA-receptor antagonist and antifebrin (ogen), since a combination of NMDA receptor antagonists, glucocorticosteroids, GABAergic drugs and heparin are useful for treatment in delayed postischemic injury. CONCLUSION: Our results indicate that the pyrolyzates derived from bamboo may have anti-apoptotic effects, and can be useful as a supplement for ischemic injury treatment.

The essential oils of Chamaecyparis obtusa promote hair growth through the induction of vascular endothelial growth factor gene.

Chamaecyparis obtusa (C. obtusa) is a conifer in the cypress family Cupressaceae, native to northeast Asia. The essential oils of C. obtusa have antibacterial and antifungal effects and several products such as hygienic bands, aromatics, and shampoos contain these oils as a natural source of antimicrobial/antifungal agents. Interestingly, some consumers suffering from baldness and/or other forms of hair loss have reported a hair growth promoting effect of shampoos containing these oils. In the present study, the hair growth promoting effect of C. obtusa oils was elucidated in an animal model. C. obtusa oils promoted the early phase of hair growth in shaved mice. In addition, we examined the molecular effect of C. obtusa oils on the regulation of hair morphogenesis and hair growth using the human keratinocyte cell line HaCaT. In the current study of hair growth regulating genes, the expressions of vascular endothelial growth factor (VEGF), transforming growth factor (TGF beta 1), and keratinocyte growth factor(KGF) have been analyzed by real-time PCR in HaCaT cells. The essential oils of C. obtusa were divided into seven fractions for treatment of HaCaT cells. VEGF transcripts were induced by fractions 6 and 7; however, TGF beta 1 and KGF mRNA levels were unchanged by C. obtusa oils or fractions. Fraction 7 was separated into seven sub-fractions and studied further. Sub-fractions E and D significantly increased VEGF and KGF gene expression without up-regulating the hair growth inhibition factor, TGF beta 1. The components of the two sub-fractions were further analyzed by gas chromatography and mass spectrometry. Cuminol, eucarvone, and calamenene were common to these two sub-fractions, although the effects of these individual components were not determined. Taken together, these results suggest that C. obtusa oils promote hair growth in an animal model and a positive regulator of hair growth, VEGF, was induced by particular components of these oils.

Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals.

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.

Maternal exposure to bisphenol a during late pregnancy resulted in an increase of Calbindin-D9k mRNA and protein in maternal and postnatal rat uteri.

It has been reported that Calbindin-D9k (CaBP-9k) is rapidly and strongly induced by environmental estrogenic compounds, possibly through estrogen receptors (ERalpha) in the uterus of mammals. CaBP-9k can be evaluated as an early gene marker for assaying estrogenic effects of putative environmental chemicals in the rat uterus. This study was undertaken to investigate CaBP-9k mRNA and protein expression in the postnatal rat uterus following maternal exposure to 17beta-estradiol (E2) and bisphenol A (BPA) during the neonatal period. Treatment with a high dose of BPA (600 mg/kg body weight (BW) per day) resulted in a 3-fold increase in CaBP-9k mRNA expression for 3 days, while a single dose of E2 (40 microg/kg BW per day) induced 2-fold increase of this gene in the maternal uterus. In an agreement with maternal CaBP-9k mRNA, postnatal CaBP-9k mRNA in the uterus increased 4-fold when treated with BPA (600 mg/kg BW per day). In addition, treatment with increasing concentrations of BPA resulted in significant increases in CaBP-9k protein in the maternal rat uterus. It is of interest that increasing doses of BPA induced a significant ERalpha mRNA increase in the postnatal uterus. Furthermore, immunohistochemistry revealed that treatment with BPA induced CaBP-9k protein in the maternal uterus. We demonstrated that maternal exposure to BPA during late pregnancy induced CaBP-9k mRNA and protein in maternal and postnatal rat uteri. These results suggest that rapid absorption and distribution of environmental estrogenic compounds occurs in maternal and neonatal rat uteri and these chemicals can easily pass though the placenta during pregnancy to affect postnatal reproductive functions.

Antibacterial and antifungal effects of essential oils from coniferous trees.

Essential oils have potential biological effects, i.e., antibiotic, anticarcinogenic, and sedative effects during stress. In the present study, we investigated the antibacterial and antifungal effects of essential oils extracted from the coniferous species Pinus densiflora, Pinus koraiensis, and Chamaecyparis obtusa, because their biological activities have not been yet elucidated. The essential oils were quantified using gas chromatography and identified in gas chromatography-mass spectrometric analysis. Simultaneously, antibacterial and antifungal assays were performed using the essential oils distilled from the needles of coniferous trees. The major components and the percentage of each essential oil were: 19.33% beta-thujene in P. densiflora; 10.49% alpha-pinene in P. koraiensis; 10.88% bornyl acetate in C. obtusa. The essential oils from P. densiflora and C. obtusa have antibacterial effects, whereas essential oils from P. koraiensis and C. obtusa have antifungal effects. These results indicate that the essential oils from the three coniferous trees, which have mild antimicrobial properties, can inhibit the growth of gram-positive and gram-negative bacteria and fungi.

Induction of calbindin-D9k messenger RNA and protein by maternal exposure to alkylphenols during late pregnancy in maternal and neonatal uteri of rats.

Environmental chemicals are proposed to possess hormone-like properties, such as mimicking natural hormones, inhibiting the action of hormones, and inducing abnormal gene expression. Among environmental chemicals, the alkylphenol products (APs), octylphenol (OP) and nonylphenol (NP), are derived from alkylphenol ethoxylates and have been reported to be environmentally persistent. Thus, in the present study, we examined the effect of two APs, OP and NP, on the expression of Calbindin-D(9k) (CaBP-9k) following maternal exposure during late pregnancy in maternal and fetal uteri. Treatment with a high dose (600 mg/kg body weight [BW]) of OP and NP resulted in an induction of CaBP-9k mRNA at Day 5 of lactation, as did a single treatment with diethylstilbestrol (DES) and 17beta-estradiol (E2) in maternal uteri. The expression of CaBP-9k mRNA was also induced following treatment with a high dose (600 mg/kg BW) of OP, transferred from the mother, exposed to fetuses during late pregnancy, and persisted through Day 5 of lactation. It is of interest that treatments with high doses of OP (400 and 600 mg/kg BW) reduced the expression of maternal estrogen receptor alpha (ERalpha) mRNA, as E2 did. However, all doses of NP resulted in an inhibition of neonatal ERalpha, while only the high does of OP (600 mg/kg BW) induced the reduction of neonatal ERalpha mRNA expression, as E2 did. Parallel to mRNA, the expression of CaBP-9k protein was significantly induced by treatment with a high dose of OP and NP. In conclusion, maternal exposure to APs, OP and NP, during late pregnancy increased the expressions of CaBP-9k mRNA and protein in maternal and neonatal uteri. These results suggest that the absorption and distribution of environmental estrogenic compounds in maternal and neonatal uteri are extremely rapid, and these chemicals can easily pass though the placenta during pregnancy to affect functions of neonatal reproductive tissues.