Catha edulis forsk mediates embryotoxic effects in rats: an experimental study / Catha edulis forsk modera los efectos embriotóxicos en ratas: un estudio experimental

Catha edulis Forsk leaves (Khat) is a flowering plant. A high proportion of the adult population in the Arabian Peninsula and the Horn of Africa chews it for its mild stimulant effect. The aim of the current study was to investigate the embryotoxic and teratogenic effects of the Khat extract using 60 female pregnant rats. These were divided to a Khat extract-treated group and a control group. Methanolic extract of Khat was orally given to the treated group 4 days before mating and up to day 16 of pregnancy with a dose of 100 mg/kg. Our results showed that significant number of embryos of the Khat-treated mothers were malformed and different in size and shape compared to embryos from the mothers of the control group. At day 8 of pregnancy, malformed embryos had ill developed primitive layers. By day 10 of pregnancy, neural tube and the somite were not formed compared to the control embryos. At later stages of pregnancy, embryos of the Khat-treated mothers appeared severely abnormal with opened neural groove and visceral pouches. Disrupted normal neural tube development, undifferentiated brain vesicles, incomplete closure of the brain flexures were also observed in these embryos. Highly significant increase in the number of the resorbed embryos of the Khat-treated mothers were observed (P < 0.01). The resorbed embryos appeared as a cellular collection in their placenta with some of their decidua had no visible embryonic tissues. In conclusions, Khat induced embryotoxic effects as well as severely affected the early normal embryonic development in rat.

Catha edulis (Khat) es una planta floreciente. Una alta proporción de la población adulta en la Península Arábiga y el Cuerno de África la mastica por su efecto estimulante. El objetivo del presente estudio fue investigar los efectos embriotóxicos y teratogénicos del extracto de Khat utilizando 60 ratas hembras preñadas. Estas se dividieron en un grupo tratado con extracto de Khat y un grupo control. El extracto metanólico de Khat se administró por vía oral al grupo tratado 4 días antes del apareamiento y hasta el día 16 de preñez con una dosis de 100 mg / kg. Los resultados mostraron que una cantidad significativa de embriones de las madres tratadas con Khat tenían malformaciones y eran diferentes en tamaño y forma en comparación con los embriones de las madres del grupo control. En el día 8 de preñez, los embriones malformados tenían capas primitivas mal desarrolladas. Para el día 10 de preñez, el tubo neural y el somito no se formaron en comparación con los embriones del grupo control. En etapas posteriores de la preñez, los embriones de las madres tratadas con Khat parecían severamente anormales con surcos neurales abiertos y bolsas viscerales. También se observaron alteraciones en el desarrollo normal del tubo neural, vesículas cerebrales indiferenciadas y el cierre incompleto de las flexiones cerebrales en estos embriones. Se observó un aumento altamente significativo en el número de embriones reabsorbidos de las madres tratadas con Khat (P <0,01). Los embriones reabsorbidos aparecieron como una colección celular en su placenta con algunas de sus deciduas sin tejidos embrionarios visibles. Khat indujo efectos embriotóxicos y afectó severamente el desarrollo embrionario normal temprano en la rata.

Differences between NMRI and DBA/2J mice in the development of somites and susceptibility to methylnitrosourea-induced skeleton anomalies

ABSTRACT The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.

Efecto de la administración de ácido retinoico sobre el desarrollo del esqueleto axial en embriones de ratón Mus musculus / Effect of administration of retinoic acid on the development of the axial skeleton in mouse embryos Mus musculus

El déficit y exceso de vitamina A provoca malformaciones congénitas que afectan distintos órganos y sistemas. El objetivo de este estudio fue determinar el efecto que causa la administración de ácido retinoico a distintas dosis sobre la morfogénesis ósea del esqueleto axial en embriones de ratón Mus musculus. Mediante aleatorización simple se distribuyeron hembras recién preñadas en 4 categorías: A, B, C y D. El día 8 post fecundación (p.f), se administró 40 mg/kg de peso de ácido retinoico al grupo A, 20 mg/kg de peso de esta solución al grupo B, 1 ml/kg de peso de dimetil sulfóxido al grupo C, y el grupo D es grupo control. El día 17 de la gestación las hembras y sus fetos fueron anestesiadas y eutanasiadas con sobredosis de pentotal sódico intraperitoneal. Los fetos de cada camada fueron procesados mediante diafanización y tinción con azul de Alcian para destacar cartílago hialino y alizarina para observar tejido óseo. Los resultados se expresaron en porcentajes de malformaciones en los siguientes tres segmentos: 1) cráneo-columna cervical, 2) segmento torácico y abdominal y 3) cintura pélvica, considerándose un 100% cuando la totalidad de los elementos óseos se encontraban comprometidos. Se utilizó la prueba de Fisher para la comparación de frecuencias de malformaciones y se consideró estadísticamente significativo cuando p<0,05. En el grupo A se evidenciaron malformaciones mayores como ausencia de huesos frontales y parietales, exencefalia, defectos en el número de vértebras, y fusiones de costillas; y en el grupo B se observaron malformaciones menores como alteraciones numéricas y fusiones de costillas, existiendo diferencias significativas entre ambos grupos. En los grupos C y D no se consignaron malformaciones. El ácido retinoico administrado intraperitonealmente el dìa 8 p.f en dosis de 40 y 20 mg/kg de peso se comporta como un teratógeno en los embriones de ratón, existiendo además diferencias significativas entre las malformaciones generadas por ambas dosis de ácido retinoico. La primera concentración afecta los huesos de los tres segmentos estudiados (cráneo-cervical, toracoabdominal, y pélvico) y la segunda concentración sólo afecta a dos segmentos (cráneo-cervical y toracoabdominal). Ambos tratamientos afectan los segmentos en una gradiente céfalo caudal, independiente del origen embrionario de las estructuras. Esto se debería a que los cambios en las gradientes de ácido retinoico alteran el comportamiento de células de la cresta neural craneal y el orden de la expresión de genes Hox.

The deficit and excess of vitamin A causes birth defects affecting different organ systems. The objectives of this study are to determine the effect caused by the administration of different doses of retinoic acid on bone morphogenesis of the axial skeleton in embryonic mouse Mus musculus. By simple randomization newly pregnant females were distributed into 4 categories: A, B, C and D. On day 8 post fertilization, 40 mg/kg was administered by weight of retinoic acid to the group A, 20 mg/kg body weight of the group B solution 1 ml/kg body weight of dimethyl sulfoxide and group C. Group D is the control group. On day 17 of gestation the females and their fetuses were anesthetized and euthanized with an overdose of intraperitoneal sodium pentothal. Fetuses from each litter were processed using diaphanization and Alcian blue staining to hyaline cartilage and alizarin to observe bone tissue. The results are expressed as percentages of malformations in the following three segments: 1) cranio-cervical spine, 2) thoracic and abdominal segment and 3) pelvic segment, considering 100% when all the bony elements were compromised. Fisher's exact test for comparison of frequencies of malformations was used and considered statistically significant when p<0.05. In group A, major malformations and defects were evident in the indemnity of the cranial vault, exencephaly, defects in the number of vertebrae, and fusion of ribs. In group B minor malformations as numerical alterations and rib fusions were observed. Significant differences were found between both groups. In groups C and D no malformations were recorded. Retinoic acid administered intraperitoneally at doses of 40 and 20 mg/kg acts as a teratogen in mouse embryos. There are significant differences between the defects induced by concentrations of 40 mg/k and 20 mg/k of retinoic acid. Both concentrations affect the bones of the three segments studied (cranio cervical, thoraco-abdominal and pelvic) in a cephalo caudal gradient, independent of the embryonic origin of the structures. Changes in retinoic acid concentration alter the behavior of cranial neural crest and changing the order of the HOX gene expression in the axial skeleton.

Influence of L-arginine during bovine in vitro fertilization.

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3-/NO2- production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.

Induced maturation of hepatic progenitor cells in vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Development of in vitro produced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.

Evaluación de la embriotoxicidad de misoprostol utilizando el ensayo cultivo de embriones postimplantación / Evaluation of embryotoxicity of misoprostol using the whole embryo culture assay

Background: Approximately 15 percent of misoprostol-induced-abortions may not be successful, leading to in utero exposure to the drug and to the induction of a series of defects including central nervous system, limb and visceral defects. A commonproposal is that the drug causes disruption of the fetal vasculature leading to embryonic or fetal hypoxia. Aim: To evaluate the teratogenicity of misoprostol using the rat post-implantation embryo culture. Material and Methods: Rat embryos were collected at the beginning of organogenesis and cultured in rat serum containing misoprostol at concentrations of 200, 2,000 or 20,000 pg/ml. Functionality, morphology and morphometry parameters were evaluated. Results: Misoprostol induced a dose-dependent embryotoxic effect causing a decrease in embryo viability and function (poor vascular development and survival) and morphometry (alterations in branchial arches, heart and cephalic portions of the neural tube, among others). Conclusions: All the manifestations observed are indicative of the ability of misoprostol to directly induce developmental retardation and alterations.

Assessment of teratogenecity and embryotoxicity of sludge from textile industries at Pali (India) in Swiss albino mice exposed during organogenetic period.

The present investigation was carried out to assess the teratological effects of in-utero exposure of sludge leachate from textile and dyeing industries located in Pali, Rajasthan. Sludge was collected at the combined effluent treatment plant (CETP). Two groups of 10 pregnant Swiss albino mice each, were given sludge leachate of 1/10 and 1/100 dilutions with water ad libitum from 6th day to 15th day of gestation covering the critical period of organogenesis. Cesarean sections were performed on day 18 of gestation and all foetuses were examined for reproductive and teratological tests. Sludge induced maternal toxicity was evidenced by significant increase in leachate consumption, reduction in body weight gain and reduction in fur of the body. Developmental toxicity was evidenced by a significant decrease in foetal weight per litter increase in the number of resorptions and an increase in total number of foetuses showing bone retardation and skeletal variations (specially of skull, sternebrae and vertebrae). The leachate of the sludge that is being dumped in the open areas of the town Pall seems to elicit teratogenic as well as embryotoxic potential as indicated by the findings of the present investigation.

Biological activity predictions, crystallographic comparison and hydrogen bonding analysis of cholane derivatives.

A total of eighteen molecules of cholane derivatives (I-XVIII) (a series of steroids) have been included to predict their pharmacological effects, specific mechanisms of action, known toxicities, drug-likeness, etc, by using the statistics of multilevel neighbourhoods of atoms (MNA) descriptors for active and inactive fragments. The biological activity spectra for substances have been correlated on SAR base (structure-activity relationships data and knowledge base), which provides the different P(a) (possibility of activity) and P(i) (possibility of inactivity). Most of the probable activities have been characterized by P(a) and P(i) values, which depict that all the molecules have high value of teratogen activity. The Lipinski's thumb rule predicts that all the cholane derivatives have stronger preponderance for "cancer-like-drug" molecules and some of their related analogous have entered in the ANCI (American National Cancer Institute) database. Some selected bond distances and bond angles of interest have been taken into account and deviation of bond distances/bond angles, vis-a-vis the substitutional group and X-H...A intra/intermolecular hydrogen bonds has been discussed in detail. X-H...A intra and intermolecular hydrogen bonds in the molecules have been described with the standard distance and angle cut-off criteria. D-theta and d-theta. scatter plots for intra- and intermolecular interactions are presented for better understanding of packing interactions existing among these derivatives. There exists only one C-H...O intramolecular bifurcated hydrogen bond. while high tendency of intermolecular bifurcated hydrogen bonds based on a defined O-H...O has been observed, in which O atom acts as a prototype donor as well as acceptor. The frequency of occurrence of C-H...O hydrogen bonds is predominant (i.e. 85.7%) in intramolecular interactions, whereas in intermolecular interactions, frequency of occurrence for O-H...O interactions is 62.9%. Solvent-solute/solute-solvent interactions have also been investigated to understand more complicated processes that occur for biomolecules in aqueous solutions. The number of hydrogen donors in each derivative is less than 5, except for molecule XVIII and which has 91.3% of drug-likeness, instead of observed range of 96.5-99.39%.

Effects of placental isoferritin on the mouse embryo development in vitro / 华中科技大学学报（医学）（英德文版）

To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.

Superovulation and embryo recovery in rabbits (Oryctolagus cuniculus) using different doses of pregnant mare serum gonadotrophin (PMSG).

Present investigation was conducted to study the ovarian response and embryo recovery using different PMSG dose levels. Six rabbits each were assigned randomly to treatment 1 (PMSG 50 IU + hCG 100 IU), treatment 2 (PMSG 75 IU + hCG 100 IU) and the control group (no hormone administered). PMSG injection (im) was followed 68 hr later by natural mating to a fertile rabbit buck and human chorionic gonadotrophin (hCG) injection iv post coitum. Embryos were recovered 96 hr post coitum by a modified surgical method. Mean number of ovulations in the control group differed significantly from treatment 1 and 2, but no significant difference was observed between treatments 1 and 2. Mean embryo recovery percentage was lowest in treatment 2 and highest in the control group. The higher dose PMSG (treatment 2) was observed to be more disturbing in terms of recovery of embryos as well as their morphology.