Se-Ra Lee

Black ginseng inhibits ethanol-induced teratogenesis in cultured mouse embryos through its effects on antioxidant activity.

Fetal alcohol syndrome is caused by excessive ethanol consumption during pregnancy. We investigated the effect of black ginseng (red ginseng that is subjected to 9 cycles of 95-100 degrees C for 2-3h) on ethanol-induced teratogenesis using an in vitro whole embryo culture system. Postimplantational mouse embryos at embryonic day 8.5 were exposed to ethanol (1 microl/ml) in the presence or absence of black ginseng (1, 10, and 100 microg/ml) for 2 days, and then morphological scoring and real-time PCR analysis were carried out. In ethanol-treated embryos, the total morphological score and individual scores for flexion, heart, fore-, mid-, and hindbrains, otic, optic, and olfactory systems, branchial bars, maxillary and mandibular processes, caudal neural tube, and somites were significantly lower than the control group (p<0.05). Treatment with black ginseng improved most of the morphological scores significantly as compared to ethanol-treated embryos (p<0.05). The mRNA levels of the antioxidant enzymes cytosolic glutathione peroxidase (GPx), phospholipid hydroperoxide GPx, and selenoprotein P were significantly decreased in ethanol-treated embryos, but co-treatment with black ginseng restored the mRNA levels to those of control embryos. These results indicate that black ginseng has a protective effect on ethanol-induced teratogenesis through the augmentation of antioxidative activity in embryos.

The spatio-temporal expression pattern of cytoplasmic Cu/Zn superoxide dismutase (SOD1) mRNA during mouse embryogenesis

The cytoplasmic Cu/Zn-superoxide dismutase (SOD1) represents along with catalase and glutathione peroxidase at the first defense line against reactive oxygen species in all aerobic organisms, but little is known about its distribution in developing embryos. In this study, the expression patterns of SOD1 mRNA in mouse embryos were investigated using real-time RT-PCR and in situ hybridization analyses. Expression of SOD1 mRNA was detected in all embryos with embryonic days (EDs) 7.5–18.5. The signal showed the weakest level at ED 12.5, but the highest level at ED 15.5. SOD1 mRNA was expressed in chorion, allantois, amnion, and neural folds at ED 7.5 and in neural folds, notochord, neuromeres, gut, and primitive streak at ED 8.5. In central nervous system, SOD1 mRNA was expressed greatly in embryos of EDs 9.5–11.5, but weakly in embryos of ED 12.5. At EDs 9.5–12.5, the expression of SOD1 mRNA was high in sensory organs such as tongue, olfactory organ (nasal prominence) and eye (optic vesicle), while it was decreased in ear (otic vesicle) after ED 10.5. In developing limbs, SOD1 mRNA was greatly expressed in forelimbs at EDs 9.5–11.5 and in hindlimbs at EDs 10.5–11.5. The signal increased in liver, heart and genital tubercle after ED 11.5. In the sections of embryos after ED 13.5, SOD1 mRNA was expressed in various tissues and especially high in mucosa and metabolically active sites such as lung, kidney, stomach, and intestines and epithelial cells of skin, whisker follicles, and ear and nasal cavities. These results suggest that SOD1 may be related to organogenesis of embryos as an antioxidant enzyme.

Effects of endocrine disrupting chemicals on expression of phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), an antioxidative selenoprotein, is modulated by estrogen in the testis and oviduct. To examine whether potential endocrine disrupting chemicals (EDCs) affect the microenvironment of the testes, the expression patterns of PHGPx mRNA and histological changes were analyzed in 5-week-old Sprague-Dawley male rats exposed to several EDCs such as an androgenic compound [testosterone (50, 200, and 1,000 µg/kg)], anti-androgenic compounds [flutamide (1, 5, and 25 mg/kg), ketoconazole (0.2 and 1 mg/kg), and diethylhexyl phthalate (10, 50, and 250 mg/kg)], and estrogenic compounds [nonylphenol (10, 50, 100, and 250 mg/kg), octylphenol (10, 50, and 250 mg/kg), and diethylstilbestrol (10, 20, and 40 µg/kg)] daily for 3 weeks via oral administration. Mild proliferation of germ cells and hyperplasia of interstitial cells were observed in the testes of the flutamide-treated group and deletion of the germinal epithelium and sloughing of germ cells were observed in testes of the diethylstilbestrol-treated group. Treatment with testosterone was shown to slightly decrease PHGPx mRNA levels in testes by the reverse transcriptionpolymerase chain reaction. However, anti-androgenic compounds (flutamide, ketoconazole, and diethylhexyl phthalate) and estrogenic compounds (nonylphenol, octylphenol, and diethylstilbestrol) significantly upregulated PHGPx mRNA in the testes (p < 0.05). These findings indicate that the EDCs might have a detrimental effect on spermatogenesis via abnormal enhancement of PHGPx expression in testes and that PHGPx is useful as a biomarker for toxicity screening of estrogenic or antiandrogenic EDCs in testes.

Spatiotemporal expression of the selenoprotein P genein postimplantational mouse embryos

Selenoprotein P (Sepp) is an extracellular glycoprotein which functions principally as a selenium (Se) transporter and antioxidant. In order to assess the spatiotemporal expression of the Sepp gene during mouse embryogenesis, quantitative RT-PCR and in situ hybridization analyses were conducted in embryos and extraembryonic tissues, including placenta. Sepp mRNA expression was detected in all embryos and extraembryonic tissues on embryonic days (E) 7.5 to 18.5. Sepp mRNA levels were high in extraembryonic tissues, as compared to embryos, on E 7.5-13.5. However, the levels were higher in embryos than in extraembryonic tissues on E 14.5-15.5, but were similar in both tissues during the subsequent periods prior to birth. According to the results of in situ hybridization, Sepp mRNA was expressed principally in the ectoplacental cone and neural ectoderm, including the neural tubes and neural folds. In whole embryos, Sepp mRNA was expressed abundantly in nervous tissues on E 9.5-12.5. Sepp mRNA was also expressed in forelimb and hindlimb buds on E 10.5-12.5. In the sectioned embryos, on E 13.5-18.5, Sepp mRNA was expressed persistently in the developing limbs, gastrointestinal tract, nervous tissue, lung, kidney and liver. On E 16.5-18.5, Sepp mRNA expression in the submandibular gland, whisker follicles, pancreas, urinary bladder and skin was apparent. In particular, Sepp mRNA was detected abundantly in blood cells during all the observed developmental periods. These results show that Sepp may function as a transporter of selenium, as well as an antioxidant, during embryogenesis.

Immunohistochemical identification and quantitative analysis of cytoplasmic Cu/Zn superoxide dismutase in mouse organogenesis

Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos.

Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos

Prenatal exposure to alcohol promotes the level of reactive oxygen species within embryos and results in developmental disorders. In this study, we investigated the effect of capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient in red peppers, on ethanol-induced teratogenicity in mouse embryos (embryonic days 8.5-10.5). In response to ethanol administration (1.0 microl/ml), developmental parameters such as yolk sac circulation, allantois, heart, hindbrain, midbrain, forebrain, otic and optic systems, branchial bar, olfactory system, forelimb, hindlimb, and somites decreased significantly in comparison with those of control group (p<0.05). However, the concurrent administration of capsaicin (1 x 10(-8) microg/ml or 1 x 10(-7) microg/ml) and ethanol significantly ameliorated most of the morphological scores excepting yolk sac circulation and hindlimb scores (p<0.05). Furthermore, the levels of superoxide dismutase activity and cytoplasmic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNAs in the ethanol-treated embryos recovered to the levels observed in control embryos by capsaicin co-administration. These results indicate that capsaicin has a protective effect against ethanol-induced teratogenicity via an antioxidative activity.

Protective effect of [6]-gingerol on the ethanol-induced teratogenesis of cultured mouse embryos

Excessive ethanol consumption during pregnancy causes fetal alcohol syndrome. We investigated the effect of [6]-gingerol on ethanol-induced embryotoxicity using a whole embryo culture system. The morphological changes of embryos and the gene expression patterns of the antioxidant enzymes cytosolic glutathione peroxidase (cGPx), cytoplasmic Cu/Zn superoxide dismutase (SOD1), and Mn-SOD (SOD2), and SOD activity were examined in the cultured mouse embryos exposed to ethanol (5 μL/3 mL) and/or [6]-gingerol (1×10−8 or 1×10−7 μg/mL) for 2 days. In ethanol-exposed embryos, the standard morphological score of embryos was significantly decreased compared with those of the control (vehicle) group. However, cotreatment of embryos with [6]-gingerol and ethanol significantly improved all of the developmental parameters except crownrump length and head length, compared with those of the ethanol alone group. The mRNA expression levels of cGPx and SOD2, not SOD1, were decreased consistently, SOD activity were significantly decreased compared with the control group. However, the decreases in mRNA levels of antioxidant enzymes and SOD activity were significantly restored to the control levels by [6]-gingerol supplement. These results indicate that [6]-gingerol has a protective effect against ethanol-induced teratogenicity during mouse embryogenesis.

Expression profiles of extracellular superoxide dismutase during mouse organogenesis.

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.

Developmental expression of plasma glutathione peroxidase during mouse organogenesis

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.