Hazardous impacts of silver nanoparticles on mouse oocyte maturation and fertilization and fetal development through induction of apoptotic processes

Silver nanoparticles (AgNPs) are antibacterial materials widely used in numerous products and medical supplies. Previously, we showed that AgNPs trigger apoptotic processes in mouse blastocysts, leading to a decrease in cell viability and impairment of preimplantation and postimplantation embryonic development in vitro and in vivo. In the present study, we further investigated the hazardous effects of AgNPs on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent preimplantation and postimplantation development in vitro and in vivo. Data from in vitro experiments revealed that AgNPs impair mouse oocyte maturation, decrease IVF rates, and induce injury effects on subsequent embryonic development to a significant extent. In an animal model, intravenous injection of AgNPs (5 mg/kg body weight) led to a significant decrease in mouse oocyte maturation and IVF concomitant with impairment of early embryonic development in vivo. Importantly, pretreatment with N‐acetylcysteine effectively prevented AgNP‐triggered reactive oxygen species (ROS) production and apoptosis, clearly suggesting a critical role of ROS as an upstream initiator or key regulator of AgNP‐induced hazardous effects on oocyte maturation and sequent embryonic development. Furthermore, preincubation of oocytes with Ac‐DEVD‐cho, a caspase‐3‐specific inhibitor, effectively prevented hazardous effects, highlighting the potential involvement of caspase‐dependent apoptotic signaling cascades in AgNP‐mediated events. Expression levels of p53 and p21 of blastocysts were upregulated upon preincubation of mouse oocytes with AgNPs. Our collective results imply that cell apoptosis in mouse blastocysts derived from the AgNP‐pretreated oocytes via intracellular ROS generation, which is further mediated through p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms.     

Effects of ochratoxin a on mouse oocyte maturation and fertilization,and apoptosis during fetal development

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1–10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked these OTA‐triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase‐dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA‐pretreated oocytes, indicating that such cells undergo apoptosis via p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724–735, 2016.     

Dosage‐related beneficial and deleterious effects of ginsenoside Rb1 on mouse oocyte maturation and fertilization and fetal development

Ginsenoside Rb1 (GRb1), the major saponin component of ginseng root, has a wide range of therapeutic applications for various diseases. Previously, our group showed that GRb1 triggers ROS‐mediated apoptotic cascades in mouse blastocysts, leading to decreased cell viability and impairment of pre‐ and postimplantation embryonic development, both in vitro and in vivo. In this study, we further found that GRb1 exerted dose‐dependent effects on oocyte maturation and sequent development in vitro. Oocytes preincubated with 25 μg/mL GRB1 displayed significantly enhanced maturation and in vitro fertilization (IVF) rates, along with progression of subsequent embryonic development. In contrast, treatment with 50 and 100 μg/mL GRB1 led to impairment of mouse oocyte maturation, decreased IVF rates, and injurious effects on subsequent embryonic development. In vivo, intravenous injection of 1 mg/kg body weight GRb1 significantly promoted mouse oocyte maturation, IVF, and early‐stage embryo development after fertilization while administration of 5 mg/kg body weight GRb1 led to a marked decrease in oocyte maturation and IVF rates concomitant with impairment of early embryonic development in our animal model. In terms of the mechanisms underlying the regulatory effects of GRb1 demonstrated increased intracellular reactive oxygen species (ROS) production and apoptosis in the 100 μg/mL GRb1 treatment group. However, we observed a significant decrease in total intracellular ROS content and inhibition of apoptosis events in the 25 μg/mL GRb1 treatment group, signifying that the intracellular ROS content serves as a key upstream regulator of GRb1 that influences its dose‐dependent beneficial or deleterious effects on oocyte maturation and sequent embryonic development. For further clarification of the mechanisms underlying GRb1‐triggered injurious effects, oocytes were pretreated with Ac‐DEVD‐CHO, a caspase‐3‐specific inhibitor, which effectively blocked injury to oocyte maturation, fertilization, and sequent development. In sum, study findings highlight the potential involvement of p53‐, p21‐, and caspase‐3‐dependent regulatory signaling cascades in GRb1‐mediated apoptotic processes.     

Berberine impairs embryonic development in vitro and in vivo through oxidative stress‐mediated apoptotic processes

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H₂O₂‐induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine—triggering or preventing apoptosis—has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5‐10 μM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine‐induced injury of mouse blastocysts appeared to be attributable to oxidative stress‐triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

Impairment of preimplantation and postimplantation embryonic development through intrinsic apoptotic processes by ginsenoside Rg1 in vitro and in vivo

Ginsenoside Rg1, which is the most abundant compound found in Asian ginseng (Panax ginseng), has demonstrated various pharmacological actions, including neuroprotective, immune‐stimulatory, and antidiabetic effects. Pregnant women, especially in the Asian community, consume ginseng as a nutritive supplement. Thus, the effects of ginsenoside‐Rg1 on embryonic development need to be investigated, such as in a mouse model. As previous investigations have found that ginsenoside Rg1 appears to either trigger or prevent apoptosis in different cell lines, the effects of this agent on apoptosis remain to be clarified. In this study, we investigated whether ginsenoside Rg1 exerts a hazardous effect on mouse blastocysts and/or affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25–100 μM ginsenoside Rg1 exhibited significant induction of apoptosis and a corresponding decrease in the inner cell mass (ICM) cell number. Importantly, the implantation rate was lower among ginsenoside Rg1‐treated blastocysts compared to untreated controls. Moreover, embryo transfer assays revealed that blastocysts treated with 100 μM ginsenoside Rg1 exhibited increased resorption of postimplantation embryos and decreased weight among surviving fetuses. In vivo, intravenous injection of mice with ginsenoside Rg1 (2, 4, or 6 mg/kg body weight/day) for 4 days was associated with increased apoptosis of blastocyst‐stage embryos and negatively impacted early embryonic development. Further experiments revealed that these effects may reflect the ability of ginsenoside Rg1 to trigger oxidative stress‐mediated intrinsic apoptotic signaling. Our in vitro results indicate that ginsenoside Rg1 treatment increases intracellular oxidative stress, decreases mitochondrial membrane potential, increases the Bax/Bcl‐2 ratio, and activates caspase‐9 and caspase‐3, but not caspase‐8. Taken together, our study results strongly suggest that ginsenoside Rg1 induces apoptosis and impairs the early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

Resveratrol protects against methylglyoxal‐induced apoptosis and disruption of embryonic development in mouse blastocysts

Methylglyoxal (MG) is a glucose metabolite. Diabetic patients have increased serum levels of MG, and MG is also implicated in tissue injury during embryonic development. In the present work, we show that MG induces apoptosis in the inner cell mass of mouse blastocysts and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape‐derived phytoalexin with known antioxidant and anti‐inflammatory properties. MG‐treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented MG‐induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that MG directly promotes reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (MMP), and activation of caspase‐3, whereas resveratrol effectively blocks MG‐induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that MG triggers the mitochondrion‐dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents MG‐induced toxicity. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 431–441, 2013.     

Retinoic acid influences the embryoid body formation in mouse embryonic stem cells by induction of caspase and p38 MAPK/JNK‐mediated apoptosis

Although all‐trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development, high levels are teratogenic in many species. RA results in immediate effects on the preimplantation embryo and on blastocyst development in vitro and in vivo. To further elucidate the cellular mechanisms of early postimplantation embryo development induced by RA, we present an embryonic cell line, B5, as a candidate system for the investigation of these processes. We used undifferentiated ES cells as the model, which is from the undifferentiated status to differentiated status [embryoid body (EB) formation] mimicking postimplantation embryo development (egg‐cylinder stage of embryo formation) to clarify the cellular mechanism of action of RA in the implanted blastocysts and cell apoptosis following the series of exposures to differing RA concentrations. Using an in vitro model, we identified the impact of RA on undifferentiated embryonic stem (ES) cells, including inhibition of cell proliferation and induction of cell apoptosis. JNK, P‐38 and caspase activation were shown in the nature of RA‐triggered apoptotic signaling in ES cells. The carry‐on influences of RA on the ES cell were shown in the formation of EB from the pretreated ES cells. RA resulted in apparent impact on undifferentiated ES cells in vitro, with increased numbers of apoptotic cells initially and inhibited cell proliferation, which led to decreased size of EB. The process of EB formation (mimicking the early postimplantation embryo development) is regulated by RA‐induced apoptosis through the activation of caspase and P38 MAPK/JNK pathway. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Hazardous effects of sanguinarine on maturation of mouse oocytes,fertilization, and fetal development through apoptotic processes

Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti‐oxidant, anti‐inflammatory and pro‐apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 μM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked sanguinarine‐triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase‐dependent apoptotic mechanism. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 946–955, 2015.     

Emodin induces embryonic toxicity in mouse blastocysts through apoptosis

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin inhibits cell proliferation and induces caspase 3-dependent apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized as yet. In the current study, we examined the cytotoxic effects of emodin on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer. Blastocysts treated with 25-75 μM emodin exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with emodin was lower than that of their control counterparts. Moreover, in vitro treatment with 25-75 μM emodin was associated with increased resorption of post-implantation embryos and decreased fetal weight. With the aid of an in vivo mouse model, we showed that consumption of drinking water containing emodin led to apoptosis and decreased cell proliferation, and inhibited early embryonic development to the blastocyst stage. Our findings support a degree of selective inhibition of retinoic acid receptors in blastocysts treated with emodin. In addition, emodin appears to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively indicate that emodin has the potential to induce embryonic cytotoxicity.     

Oxidative stresses‐mediated apoptotic effects of ginsenoside Rb1 on pre‐ and post‐implantation mouse embryos in vitro and in vivo

Ginsenoside Rb1, the major saponin component of ginseng root, has a wide range of therapeutic application. Previous studies have established that ginsenoside Rb1 inhibits the cell cycle and induces apoptosis. However, its side‐effects, particularly those on embryonic development, have not been well characterized to date. In the current study, we examined whether ginsenoside Rb1 exerts a cytotoxic effect on mouse embryos at the blastocyst stage, and affects subsequent embryonic development in vitro and in vivo . Blastocysts treated with 25–100 μg mL^?1 ginsenoside Rb1 exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with ginsenoside Rb1 was lower than that of their control counterparts. Moreover, in vitro treatment with 25–100 μg mL^?1 ginsenoside Rb1 was associated with increased resorption of post‐implantation embryos and decreased fetal weight. In an in vivo model, intravenous injection with ginsenoside Rb1 (1, 3, 5 mg kg^?1 body weight/day) for 4 days resulted in apoptosis of blastocyst stage embryos and early embryonic developmental injury. In addition, ginsenoside Rb1 appeared to induce injury in mouse blastocysts through oxidative stresses‐triggered intrinsic apoptotic signaling processes to impair sequent embryonic development. The collective results strongly indicate that ginsenoside Rb1 induces apoptosis and retards early pre‐ and post‐implantation development of mouse embryos, both in vitro and in vivo . © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1990–2003, 2017.     

Impact of chronic low-dose ethanol ingestion during sexual maturation of female mice on in-vitro and in-vivo embryo development

Little is known of the consequences of ethanol intake prior to fertilization on preimplantation embryo development. Recently we showed that chronic 10 and 5% w/v ethanol intake by young female mice reduces in vitro fertilization (IVF) rates. The purpose of the present work was to investigate whether the adverse effects of preconceptional low-dose chronic ethanol intake by sexually maturing female mice affects preimplantation embryo growth in vitro or in vivo in subsequent pregnancy. Prepubertal female mice were given 5% ethanol in their drinking water for 30 days. On day 27 and 29 of the ethanol treatment, females were superovulated. IVF-derived cultured embryos (in vitro development) or embryos obtained from oviducts and uteri (in vivo development) were evaluated. Whether analyzed on a per embryo or per dam basis, ethanol treatment was associated with a significant decrease in progression through embryo stages during the seven days of in vitro development and with an increase in morphologically abnormal embryos. Progression through embryo stages during four days of in vivo development was also inhibited by ethanol pretreatment of dams At 99 h post-hCG of in vivo development, there were fewer total, hatched, and expanded blastocysts, and a complete absence of implanting blastocysts among females treated with ethanol. In summary, low-dose chronic ethanol consumption of sexually maturing female mice prior to conception has adverse effects on preimplantation embryo development, both under in vitro and in vivo conditions, manifested as retarded development, embryo anormalities, and a reduction in expansion and hatching of the preimplantation blastocyst.     

Effects of citrinin on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. An earlier study by our group shows that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Here, we further investigate the effects of CTN on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. CTN induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryo development. Treatment of oocytes with 5 microM CTN during in vitro maturation (IVM) led to increased resorption of postimplantation embryos, and decreased placental and fetal weight. Using an in vivo mouse model, we show that consumption of drinking water containing 5 microM CTN results in decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. To our knowledge, this is the first study investigating the impact of CTN on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Effects of melamine on pregnant dams and embryo‐fetal development in rats

There are worldwide concerns regarding the potential adverse effect of melamine. This study investigated the potential effects of melamine on pregnant dams and embryo‐fetal development in Sprague–Dawley rats following maternal exposure on gestational days (GD) 6–20. Melamine was administered to pregnant rats by gavage at doses of 0, 200, 400 and 800 mg kg^?1 per day (n = 8–10 for each group). All dams were subjected to a Caesarean section on GD 21 and their fetuses were examined for morphological abnormalities. With administration of melamine at 800 mg kg^?1 per day, maternal toxicity manifested as increased incidences of clinical signs and death, lower body weight gain and food intake, and increases in heart, adrenal gland and kidney weights. Histopathological examinations revealed an increase in incidences of congestion, tubular necrosis/degeneration, crystals, casts, inflammatory cells in tubules, tubular dilation and tubular hyaline droplets in the maternal kidneys, while fetal kidneys (one fetus/litter) did not show any histopathological changes. Developmental toxic effects included a decrease in fetal weight, an increase in the incidence of skeletal variations and a delay in fetal ossification. No treatment‐related maternal or developmental effects were observed at doses ≤400 mg kg^?1 per day. These results show that 15‐day repeated oral dosing of melamine is embryo‐/fetotoxic at a maternotoxic dose, but not teratogenic in rats. The no‐observed‐adverse‐effect level of melamine for pregnant dams and embryo‐fetal development is considered to be 400 mg kg^?1 per day. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of genistein on mouse blastocyst development in vitro

AIM: To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro. METHODS: Mouse blastocysts were incubated in medium with or without genistein (25 or 50 micromol/L) or daidzein (50 micromol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis. RESULTS: TUNEL staining and Annexin-V/PI staining showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment decreased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis. CONCLUSIONS: Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein on mouse blastocysts.     

Effect of citrinin on mouse embryonic development in vitro and in vivo

Citrinin (CTN), a mycotoxin that is often found as a natural contaminant in foodstuffs and animal feeds, has been demonstrated to have cytotoxic and genotoxic effects on various mammalian cells. In this study, we examined the cytotoxic effects of CTN on mouse blastocysts and subsequent early development in vitro and in vivo. Blastocysts treated with 15 or 30 microM CTN showed significant increases in apoptosis and significant decreases in total cell number. In addition, CTN-pretreated blastocysts showed a significantly lower implantation success rate. Treatment with 30 microM CTN was associated with increased resorption of postimplantation embryos and decreased fetal weight. Our results collectively indicate that CTN-induced apoptosis in the mouse blastocyst reduced cell number and retarded early postimplantation development. The extent to which CTN may have teratogenic potential in early human development is not known.     

Neurotoxic effects of alcohol and acetaldehyde during embryonic development

Alcohol drinking during pregnancy results in abnormal fetal development, including fetal alcohol syndrome (FAS) in humans and experimental animals. FAS is characterized by two major effects, including central nervous system (CNS) dysfunction and multiple anomalies recognizable mainly as a typical face. However, the mechanisms of alcohol-induced embryotoxicity have not been clearly demonstrated. The aim of the present study was to investigate the possible mechanisms underlying ethanol-induced FAS in the developing embryo. First, ethanol-induced developmental abnormalities were investigated in vitro. Postimplantation embryos at gestation day (GD) 9.5 were cultured for 48 h and observed for morphological changes. Ethanol-mediated changes in proteins regulated apoptosis (p53 and bcl-2), antioxidant (vitamin E and catalase) activities, generation of reactive oxygen species (ROS), and oxidative DNA damage shown as 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in embryonic midbrain cells. Alcohol or acetaldehyde significantly induced cytotoxicity in cultured rat embryonic midbrain cells. The levels of p53, bcl-2, and 8-OHdG were concomitantly changed by alcohol and acetaldehyde treatment in midbrain cells. Injured cells induced by ROS were increased by alcohol or acetaldehyde treatment in midbrain cells. Cotreatment with alcohol or acetaldehyde and catalase decreased cytotoxicity in midbrain cells. In postimplantation embryo culture, alcohol or acetaldehyde-treated embryos showed retardation of embryonic growth and development in a concentration-dependent manner. These results indicate that alcohol and its metabolite acetaldehyde induce fetal developmental abnormalities by disrupting cellular differentiation and growth. Data demonstrate that some antioxidants can partially protect against the alcohol-induced embryonic developmental toxicity.     

Molecular events involved in ochratoxin A induced mitochondrial pathway of apoptosis,modulation by Bcl‐2 family members

In this study, we looked for the role of the mitochondrion in the cytotoxicity of ochratoxin A (OTA), which is one of the most abundant food‐contaminating mycotoxins in the world. In different human carcinoma cell lines, OTA triggered a mitochondria‐dependent apoptotic process, which is characterized by opening of the mitochondrial permeability transition pore (PTPC), loss of mitochondrial transmembrane potential (ΔΨ_m), increase in O₂[chemp]^– production, mitochondrial relocalization of Bax, release of cytochrome c, and caspase activation. However, studies performed on purified organelles suggested that OTA does not directly target the mitochondrion. In addition, we showed that mitochondrial alterations induced by this mycotoxin are favored by the proapoptotic protein Bax, but not Bak. These alterations are prevented by the antiapoptotic proteins, Bcl‐2 and to a lesser degree by Bcl‐X_L. Taken together, these data indicate that although mitochondria, PTPC members and proteins of Bcl‐2 family play a pivotal role in OTA‐induced apoptosis, they do not constitute real targets to overcome its toxicity. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

Injury effects of ginkgolide B on maturation of mouse oocytes,fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1–6 μM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3–6 μM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts

Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3-1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca(2+) and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells.