A coupled microsomal-activating/embryo culture system: Toxicity of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH)

An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decreases in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1.0 mM NADPH alone, or 0.2 mM NADPH in the presence of microsomes and a glucose-6-phosphate dehydrogenase-based NADPH-generating system, greatly reduced embryo and yolk sac growth in vitro. The toxicity of NADPH was not due to lipid peroxidation. Only minor decreases in final yolk sac protein levels occurred when embryos were grown in media containing male rat microsomes and 1.0 mM NADPH. The protective effect of rat hepatic microsomes on NADPH toxicity does not seem to have been due to the oxidation of NADPH to the less toxic NADP. Although cyclophosphamide alone was not toxic to rat embryos cultured in vitro, in the coupled microsomal-activating/embryo culture system, cyclophosphamide reduced yolk sac and embryo growth and caused abnormal embryonic differentiation. The uses of the coupled microsomal-activating/embryo culture system to study mechanisms in anomalous development, as well as its possible use in embryo toxicity and teratogenicity testing, are discussed.     

Microinjection of cultured rat embryos: a new technique for studies in chemical dysmorphogenesis

The utility of a new technique for exposure of cultured whole rat embryos to potential dysmorphogens was demonstrated with nitrosofluorene (NF), a cytotoxic and mutagenic metabolite of 2-acetylaminofluorene (AAF). At an initial concentration in the culture medium of 41 microM, NF produced a 100% incidence of defects in axial rotation with no significant effect on prosencephalic development, consistent with previous reports. This route of exposure was also associated with a significant decrease in yolk sac vasculature and protein content. However, when 2 to 20 ng of NF was microinjected directly into the amniotic space, the predominant malformation observed was prosencephalic hypoplasia. Injection of 10 ng NF resulted in approximately equivalent decreases in viability as 41 microM NF dissolved in the culture medium, but produced only a 41% incidence of rotation defects and a 27% incidence of open neural tubes in the rhombencephalic region. The protein content of injected conceptuses was significantly reduced in the embryo, but not in the visceral yolk sac. When 10 ng of NF was injected inside the yolk sac but outside the amnion, the incidence of abnormal rotation was increased to 75%, and the severity of prosencephalic hypoplasia as well as the incidence of neural tube abnormalities was attenuated. The protein content of both the embryo and yolk sac was significantly decreased relative to that of the controls. The data are consistent with the suggestion that NF elicits defects in axial rotation primarily via its effects on the visceral yolk sac and demonstrate the capacity of this technique to provide insights into mechanistic aspects of chemical dysmorphogenesis.     

Rat conceptus development in vitro: Comparative effects of alkylating agents

Conceptuses removed from the rats in the eleventh day of gestation were cultured in vitro for 2 days. Growth and differentiation of the major organs of the embryo in vitro resembled those developed in vivo. Embryonic development and organogenesis were markedly affected when the alkylating agents TEM (2,4,6-triethylenimino-1,3,5-triazine) and nitrogen mustard (mechlorethamine hydrochloride) were added to the culture medium. At concentrations of 1 and 5 μg/ml medium, these teratogens were highly embryotoxic and affected both growth and differentiation. DNA and protein content of embryos and yolk sacs was reduced significantly (p < 0.001) from the controls. Development of conceptuses in the culture medium that contained cyclophosphamide (0.35 mm) alone apparently were normal. However, addition of cyclophosphamide (0.35 mm), microsomes (0.5 mg protein/ml), and NADPH (1 mm) to the culture medium induced marked deleterious effects on the conceptus growth and differentiation. DNA and protein contents were significantly (p < 0.001) reduced by the combined treatment indicative of formation of reactive metabolites and their interference with macro-molecular biosynthesis. Aminopyrine which has not been shown to be teratogenic, in equimolar amounts to cyclophosphamide, under identical conditions of culture, did not affect conceptus development.     

Embryotoxicity elicited by inhibition of gamma-glutamyltransferase by Acivicin and transferase antibodies in cultured rat embryos

Acivicin (also known as AT-125) and IgG isolated from goat anti-gamma-glutamyltransferase antiserum were used to inhibit the activity of gamma-glutamyltransferase (GGT, EC 2.3.2.2) in rat conceptuses cultured from Days 10 to 11 of gestation. Inhibition of GGT by either Acivicin or anti-GGT IgG produced embryotoxicity and malformations, although each compound produced a unique spectrum of effects. Acivicin, at an initial concentration in the culture medium of 5 microM, produced a marked decrease in yolk sac vasculature and was associated with embryonic malformations such as neural tube necrosis, microophthalmia, and cephalic edema after 24 hr exposure. These malformations were accompanied by significant decreases in both embryonic and yolk sac protein, yolk sac GGT activity, as well as embryonic glutathione (GSH) levels. In contrast, anti-GGT IgG produced no apparent effects on yolk sac vasculature or protein after exposure of conceptuses to an initial concentration of 50 micrograms IgG/ml culture medium, even though equal inhibition of yolk sac GGT (30%) was achieved by each inhibitor. Exposure to IgG (50 micrograms/ml) for 24 hr was associated with decreased embryonic protein; decreased levels of GSH in the embryo were observed after both 3 and 24 hr. The dichotomy of effects on the yolk sac by the two compounds indicates that Acivicin produced these effects by mechanisms other than by GGT inhibition alone. These results demonstrate that inhibition of GGT in rat embryos undergoing organogenesis can elicit embryotoxic effects and produce alterations in GSH levels. The capacity of the anti-GGT antibody to inhibit the GGT activity in the yolk sac (while having no apparent effect on yolk sac morphology), and yet influence the embryo by decreasing protein and GSH levels, underscores the important role of the yolk sac during the highly sensitive stages of organogenesis.     

乙醇对胚胎发育与卵黄囊超微结构的影响

为研究发育敏感期暴露于乙醇不同时间对胚胎发育和胚胎卵黄囊超微结构的影响 .以体外全胚胎培养和电镜技术研究 0 .2g·L- 1乙醇不同时间作用时对胚胎发育影响及胚胎卵黄囊超微结构改变 .结果表明发育异常与乙醇作用存在时间 效应关系 .0 .2g·L- 18h对胚胎发育和形态分化无明显影响 ,随时间延长除胚胎头长 ,体长 ,卵黄囊直径 ,蛋白质和DNA含量等主要发育指标进一步受抑制外 ,胚胎畸形 ,死亡率明显升高 .卵黄囊超微结构变化程度与染毒时间和胚胎发育状况相一致 ,乙醇可导致卵黄囊细胞内微绒毛和溶酶体数量减少 ,线粒体等部分细胞器内膜肿胀 .上述结果提示卵黄囊结构损伤和破坏在乙醇所致的发育异常中起重要作用     

Teratogenicity of cyclophosphamide in a coupled microsomal activating/embryo culture system

Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produced characteristic morphological lesions as well as a general inhibition of embryo and yolk sac growth. Increasing concentrations of NADPH in the presence of microsomes and cyclophosphamide produced progressively greater responses. These effects did not occur when microsomes and NADPH were present in the serum medium for the first 2 hours of incubation followed by one washing and then culturing of the conceptuses from hr 2 to hr 48 in a medium containing cyclophosphamide alone. Cytochrome P-450-depleted microsomes did not bioactivate cyclophosphamide to teratogenic or toxic metabolites. The results indicate that cytochrome P-450-dependent microsomal metabolism of cyclophosphamide is required for the embryotoxic and teratogenic effects observed in vitro.     

Curcumin affects development of zebrafish embryo

Embryotoxic and teratogenic effects of curcumin on the development of zebrafish embryo were investi-gated in this study. The LD(50) values of curcumin (24-h incubation) were estimated at 7.5 microM and 5 microM for embryos and larvae, respectively. The developmental defects caused by curcumin treatments include bent or hook-like tails, spinal column curving, edema in pericardial sac, retarded yolk sac resorption, and shorter body length. In curcumin-treated larvae, fluorescence signals of curcumin were found in edamae sac and some skin cells. Together, these results indicate that zebrafish are suitable model organisms to study the toxic effects of curcumin.     

褪黑素对苯妥英所致小鼠胚胎体外致畸效应的拮抗作用

采用体外着床后全胚培养模型研究苯妥英诱发的胚胎脂质过氧化和形态异常 ,确定褪黑素 (MT)的胚胎保护作用 .d 8.5的小鼠胚胎经苯妥英单独或与MT联合作用 4 8h后测定胚胎生长 ,形态发育 ,全胚蛋白质含量及脂质过氧化产物 .结果表明苯妥英导致胚胎生长发育迟滞并诱发脂质过氧化产物增高 4倍 .MT 1mmol·L- 1有轻微生长抑制作用 ,但可完全拮抗苯妥英诱发的脂质过氧化作用 ,并降低或完全拮抗苯妥英导致的形态生长发育异常 .表明MT有拮抗苯妥英胚胎致畸效应的作用     

Toxicity effects of captan on different life stages of zebrafish (Danio rerio)

The objective of this study was to evaluate the toxicity and developmental effects of captan on different life stages (embryo and adult) of zebrafish (Danio rerio). The results showed that the 96-h lethal concentration 50 (LC₅₀) values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The results of developmental effect experiment showed that captan can significantly decrease the heartbeats and inhibit the hatching rate and growth of zebrafish embryos. Moreover, captan exposure can induce a series of deformities, including pericardial edema, yolk sac edema, spine curvature, and tail bending, in zebrafish embryos during the developmental period. Among these, the most significant were tail bending and spine curvature.     

Apoptosis in Embryos of Diabetic Rats

Abstract: The aim of the present study was to determine whether maternal diabetes affects rat embryo and yolk sac apoptosis during the postimplantation period. Severely malformed and growth-retarded embryos of gestational day 12 from diabetic rats exhibited pronounced DNA laddering on agarose gels. On the other hand, no DNA laddering could be observed in any of the non-malformed embryos from control and diabetic rats, or in their corresponding yolk sacs. Analysis of embryos of gestational day 10 revealed only a few scattered TUNEL positive cells mainly located in the allantois, the foregut epithelium, the cranial neuroepithelium and in the cranial mesenchyme. Embryonic tissue of gestational day 12 showed numerous aggregates of TUNEL-positive cells, indicating developmental remodelling of multiple organs. Analysis of non-malformed embryos of day 10 and 12 revealed a distribution and frequency of TUNEL positive cells unaffected by the diabetic state of the mother on both days. In vitro incubation (2–8 hr) of normal day-12 yolk sacs resulted in strong DNA laddering, but not in the corresponding embryos. Dispersed yolk sac cells generated higher levels of reactive oxygen species than dispersed embryonic cells. Reactive oxygen species levels in both embryonic and yolk sac cells were unaffected by the diabetic state of the mother. Moreover, immunoblot analysis showed high Bcl-2 and undetectable caspase-1 levels in embryos from both normal and diabetic rats and low Bcl-2 and high caspase-1 levels in the corresponding yolk sacs. Immunohistochemical analysis of embryos demonstrated caspase-1-reactivity in a small subpopulation of cells located in proximity to TUNEL-positive cells. We conclude that the inherent capacity of embryonic cells to enter apoptosis in vitro is low as compared to yolk sac cells, and that wide-spread apoptosis is not likely to play a major role in diabetes-induced dysmorphogenesis but rather in early phases of resorption of severely malformed and developmentally retarded embryos.     

利用小鼠全胚胎培养研究环磷酰胺致畸作用

本文应用小鼠全胚胎培养技术 ,通过妊娠 d8分别 ip5,1 0 ,1 5和 2 0 mg·kg-1环磷酰胺 ( CP) ,研究了该药对器官原基形成期胚胎的致畸作用 ,并对其致畸机理作了初步探索 .给药 4h后取胚胎进行培养 ,于 d 1 0 .5收获胚胎 ,测量其卵黄囊直径 ,头长及颅臀长并记录其大体形态的变化 .结果表明 ,1 5mg· kg-1组尾畸形率最高 ;2 0 mg· kg-1组生长迟缓率最高 .电镜观察所显示的细胞凋亡的形态学变化及 DNA琼脂糖凝胶电泳的结果均提示 CP致畸作用可能与其诱导的细胞凋亡有关 .     

Effect of methylmercury on glutamate-cysteine ligase expression in the placenta and yolk sac during mouse development

The placenta and the yolk sac play critical roles in fetal development, including protection from oxidative stress through the presence of detoxifying enzymes. Glutathione (GSH; gamma-glutamylcysteinylglycine), a crucial molecule in the maintenance of cellular redox status, plays a critical role in development, and it is also protective against methylmercury toxicity. Glutamate-cysteine ligase (GCL), the enzyme that catalyzes the rate-limiting step in GSH synthesis, is widely expressed in the mouse embryo and extraembryonic membranes throughout development. The aim of this study was to investigate the effect of low-level subchronic methylmercury exposure on GCL expression in the mouse placenta and yolk sac, after describing the basal developmental expression of the enzyme in these tissues. We found that basal mRNA expression levels increased dramatically in the placenta and the yolk sac at gd 18, whereas protein levels did not increase in parallel with the mRNA. We also found that methylmercury induced GCLc mRNA expression in the placenta at gd 18 in a dose-dependent manner, suggesting an important role for this enzyme in the response of the placenta to toxicants. These changes in expression may be useful as a biomarker of MeHg exposure during development.     

Activity of aldrinepoxidase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase during the development of chick embryos in ovo

Since the chick embryoin ovo is susceptible to the action of some agents needing metabolic activation we studied the development of the activity of cytochrome P450-dependent monooxygenases in embryo/fetal tissue. The activities of aldrinepoxidase (AE), 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) were measured in whole embryos, liver and yolk sac tissue of the chick embryo during developmentin ovo from day 4 to day 15 of incubation (DI). In yolk sac tissue enzyme activities could be detected from DI 4 on. While EROD activity was only marginally developed, AE and ECOD activities were more pronounced in the earlier developmental period and showed a clear decrease by the time the liver activities rose. With the methods used AE activity could be measured in the homogenate of the whole embryo proper from DI 4 on while EROD and ECOD activity was not detectable before DI 6 or DI 7, respectively. In liver tissue enzyme activities of the three monooxygenases studied developed to a considerable degree from DI 9 on and tended to exhibit maximum values around DI 11–13. Studies on monooxygenase activities in extra-embryonically located tissues have not been published until now. The importance of the yolk sac as a metabolically relevant organ during embryogenesis is pointed out in this study.     

硫酸铝在体外对大鼠胚胎组织谷胱甘肽含量和卵黄囊细胞膜流动性的影响

目的　为探讨铝的发育毒性及机理。方法孕 9.5d大鼠胚胎于体外培养系统中给予不同剂量的硫酸铝 ,培养 4 8h后 ,观察胚胎生长发育和器官形态分化状况 ;应用二硫代双硝基苯甲酸 (DTNB)直接法测定胚胎组织谷胱甘肽 (GSH)含量 ;以 1,6 二苯己三烯为荧光探剂 ,用荧光偏振技术测定卵黄囊细胞膜脂质流动性。结果　当培养液中铝浓度为1.2mg·L- 1时 ,胚胎生长发育和分化明显被抑制 ;3.0mg·L- 1时 ,畸形胚胎发生率明显升高 ,主要有神经管闭合不全 ,脑发育不良和体翻转不全 ;6 .0mg·L- 1时 ,胚胎组织GSH含量和卵黄囊细胞膜脂质流动性显著降低。上述效应均呈现出一定的剂量 效应 (反应 )关系。结论　铝有潜在的致畸性和胚胎毒性 ,胚胎组织GSH含量和卵黄囊细胞膜流动性降低可能在铝致胚胎发育毒性中起重要作用     

Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates

The early identification of teratogens in humans and animals is mandatory for drug discovery and development. Zebrafish has emerged as an alternative model to traditional preclinical models for predicting teratogenicity and other potential chemical-induced toxicity hazards. To prove its predictivity, we exposed zebrafish embryos from 0 to 96 h post fertilization to a battery of 31 compounds classified as teratogens or non-teratogens in mammals. The teratogenicity score was based on the measurement of 16 phenotypical parameters, namely heart edema, pigmentation, body length, eye size, yolk size, yolk sac edema, otic vesicle defects, otoliths defects, body axis defects, developmental delay, tail bending, scoliosis, lateral fins absence, hatching ratio, lower jaw malformations and tissue necrosis. Among the 31 compounds, 20 were detected as teratogens and 11 as non-teratogens, resulting in 94.44 % sensitivity, 90.91 % specificity and 87.10 % accuracy compared to rodents. These percentages decreased slightly when referred to humans, with 87.50 % sensitivity, 81.82 % specificity and 74.19 % accuracy, but allowed an increase in the prediction levels reported by rodents for the same compounds. Positive compounds showed a high correlation among teratogenic parameters, pointing out at general developmental delay as major cause to explain the physiological/morphological malformations. A more detailed analysis based on deviations from main trends revealed potential specific modes of action for some compounds such as retinoic acid, DEAB, ochratoxin A, haloperidol, warfarin, valproic acid, acetaminophen, dasatinib, imatinib, dexamethasone, 6-aminonicotinamide and bisphenol A. The high degree of predictivity and the possibility of applying mechanistic approaches makes zebrafish a powerful model for screening teratogenicity.     

Structural requirements of organophosphorus insecticides (OPI) to inhibit chicken yolk sac membrane kynurenine formamidase related to OPI teratogenesis

This paper provides new information related to the mechanism of OPI (organophosphorus insecticides) teratogenesis. The COMFA (comparative molecular field analysis) and COMSIA (comparative molecular similarity indices analysis) suggest that the electrostatic and steric fields are the best predictors of OPI structural requirements to inhibit in ovo chicken embryo yolk sac membrane kynurenine formamidase, the proposed target for OPI teratogens. The dominant electrostatic interactions are localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-2 and carbon-6 of the pyrimidinyls and/or N-substituents and carbon-3 substituents of crotonamides are the steric structural components that contribute to superiority of those OPI as in ovo inhibitors of kynurenine formamidase.