Fertile Hen's Egg Research Articles

A Comparative Study on In vitro Anti-cancer and In vivo Anti-angiogenic Effects of TRPC Blockers Pyr-3 and SKF-96365

Introduction: Angiogenesis is involved in many physiological and pathological conditions including cancer. A number of TRP channels induce angiogenesis, promote cell proliferation or induce apoptosis in several types of human cancers. Therefore, TRP channels may be considered potential pharmacological targets for therapeutic options of disorders caused by insufficient angiogenesis or aberrant vascularization. Aims: This study aimed to comparatively investigate in vitro anti-cancer and in vivo anti-angiogenic effects of TRPC blockers Pyr-3 and SKF-96365. Methods: For anti-cancer effects, four cancer cell lines (MDA-MB-231, A549, PC-3, and HCT-116) were used. In vivo anti-angiogenic effects were investigated by employing in vivo CAM assay of fertilized hen eggs. Results: Pyr-3 affected cell viability in a dose-dependent manner, all concentrations of SKF-96365 significantly reduced cell viability in all cell lines. Pyr-3 and SKF-96365 at concentrations of 2.5 µg/pellet and 50 µg/pellet, respectively inhibited in vivo angiogenesis significantly. Conclusion: The concentration of 2.5 µg/pellet caused no irritation, whereas 50 µg/pellet produced some slight irritation. Apart from their anti-cancer effects, our findings indicate that Pyr-3 and SKF-96365 may be promising anti-angiogenic agents for the treatment of angiogenesis-related disorders.

Formation mechanism of high-viscosity gelatinous egg white among “Fenghuang Egg”: Phenomenon, structure, and substance composition

“Fenghuang Egg” is a special egg product incubated for 12 days by fertilized hen eggs. Its egg white contains high-viscosity and excellent thermal gel strength. A comparative study on the differences in gel properties, structure, and substance composition between fresh egg white (FEW) and “Fenghuang egg” gelatinous egg white (GEW) was carried out. Experimental results showed GEW had better apparent viscosity, as well as the hardness, cohesiveness and water holding capacity (WHC) of thermal gel; the content and size of aggregate structure increased significantly in GEW, and a fibrous dense network composed of numerous spherical nanoparticles connected in series was formed after heating. In addition, it also discovered that more water molecules in GEW existed in the form of bound water. A total of 41 proteins changed significantly in FEW and GEW, Mucin 6 might be the main reason for the enhanced viscosity of GEW, and OVA might be the dominant protein differentiating the thermal gel properties between FEW and GEW. This study revealed that the differences in gel properties and structures between FEW and GEW were closely related to the content of highly glycosylated globular proteins, laying a theoretical foundation for the application of high-viscosity egg whites.

Double-PEGylated Cyclopeptidic Photosensitizer Prodrug Improves Drug Uptake from In Vitro to Hen's Egg Chorioallantoic Membrane Model.

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.

Purification and Identification of a Natural Antioxidant Protein from Fertilized Eggs.

Fertilized hen eggs are rich in a variety of bioactive ingredients. In this study, we aimed to obtain an antioxidant protein from fertilized eggs and the radical scavenging abilities on 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (•OH), superoxide anion (O2−•) were used to evaluate the antioxidant activity of the purified protein. During 20 d of incubation, the radical scavenging ability of protein extracted from fertilized eggs exhibited significantly differences and the protein on day 16 showed higher antioxidant capacity. Based on this, the antioxidant protein of the samples on day 16 were isolated for the follow-up study. With a molecular weight 43.22 kDa, the antioxidant protein was purified by Diethylaminoethyl cellulose −52 (DEAE-52) column and Sephadex G-100. The LC-MS analysis showed that the purified protein molecular weight was 43.22 kDa, named D2-S. The sequence of amino acids was highly similar to ovalbumin and the coverage reached to 84%. The purified protein showed a radical scavenging rate of 52.34±3.27% on DPPH and 63.49±0.25% on •OH, respectively. Furthermore, the C-terminal amino acid sequence was NAVLFFGRCVSP, which was consistent with the sequence of ovabumin. These results here indicated that purified protein may be a potential resource as a natural antioxidant.

Biological evaluation of 4,5-diarylimidazoles with hydroxamic acid appendages as novel dual mode anticancer agents.

New (4-aryl-1-methylimidazol-5-yl)cinnamoylhydroxamic acids were prepared as potential dual mode anticancer agents combining the antivascular effect of the 4,5-diarylimidazole moiety and the histone deacetylases (HDAC) inhibition by the cinnamoyl hydroxamate. Their antiproliferative activity against a panel of primary cells and cancer cell lines was determined by MTT assays and their apoptosis induction by caspase-3 activation. Their ability to reduce the activity of HDAC was measured by enzymatic assays and Western blot analyses of cellular HDAC substrates. Additional effects on cancer cell migration were ascertained via immunofluorescence staining of cytoskeleton components and three-dimensional migration assays. The chorioallantoic membrane assay was used as an in vivo model to assess their antiangiogenic properties. The 4-phenyl- and 4-(p-methoxyphenyl)-imidazole derivatives had a greater antiproliferative and apoptosis inducing effect in a variety of cancer cell lines when compared with the approved HDAC inhibitor SAHA, and most distinctly so in non-malignant human umbilical vein endothelial cells. Like SAHA, both compounds acted as pan-HDAC inhibitors. In 518A2 melanoma cells, they led to hyperacetylation of histones and of the cytoplasmic HDAC6 substrate alpha-tubulin. As a consequence, they inhibited the migration and invasion of these cells in transwell invasion assays. In keeping with its pronounced impact on endothelial cells, the 4-phenyl-imidazole derivative also inhibited the growth and sprouting of blood vessels in the chorioallantoic membrane of fertilized hen eggs. The 4-phenyl- and 4-(p-methoxyphenyl)-imidazole compounds combine the antivascular effects of 4,5-diarylimidazoles with HDAC inhibition by cinnamoyl hydroxamates and show additional antimetastatic activity. They are promising candidates for pleiotropic HDAC inhibitors.

Purification and characterization of a natural antioxidant peptide from fertilized eggs

Fertilized hen eggs are traditionally considered as dietary supplements in many Asian countries. This work aimed to obtain information on antioxidant peptides derived from fertilized eggs. Antioxidant activities were evaluated by measuring reducing power, DPPH radical scavenging activity and inhibition of pyrogallol autoxidation. During 15days of incubation, the antioxidant activity of peptides increased with increasing incubation time. The peptides on day 15 were employed for isolation of antioxidant peptide. An antioxidant peptide, HLFGPPGKKDPV (MW: 1291.51Da), was purified by consecutive chromatographic methods. The purified peptide was a novel peptide corresponding to the fragment 302–313 of ovotransferrin. The conformational prediction and Fourier transform infrared spectroscopy suggested that the peptide existed in β-sheet. Furthermore, the peptide showed an inhibition ratio of 60.20% on linoleic acid autoxidation and an inhibitory effect on ABTS radicals (IC50: 312μM). These results suggested that fertilized eggs could be explored as a source of antioxidant peptides.

Evaluation of the chicken embryo chorioallantoic membrane model for laryngeal tumor transplantation

The laryngeal squamous cell carcinoma is the second common malignant tumor of the respiratory tract and together with recurrent respiratory papillomas represents the most common tumors of the larynx. Many experimental models are used to study the morphology of malignant tumors. The chicken chorioallantoic membrane (CAM) model is one of them. The CAM has all the nutrition needed for the piece of the transplanted tumor to survive. The aim of this study was to investigate whether the laryngeal papilloma and the laryngeal squamous cell carcinoma tissues transplanted on the chick CAM survive with their main histological features, and to determine the morphological changes of the CAM with different transplants. For the preparation of the CAM, fertilized hen eggs were put into an incubator for 3 days. Then the windows in the shell were opened. The fresh samples of tumors were transplanted on the CAM on the 7th day of incubation. After 3 days after transplantation the CAM with onplants were excised and fixed in the 10% formalin solution. Morphological changes in the control CAM and in the CAM with tumor onplants were observed using the digital camera on the OLYMPUS microscope. The results showed that the CAM with the laryngeal squamous cell carcinoma onplant was distinctly thicker than that of the control group and than the CAM with the papilloma onplant; the chorionic epithelium was thickened and appeared stratified of up to 5–6 layers and in some locations squamous keratinized; the mesenchymal cells were densely arranged under the tumor transplants. We observed that morphological changes in the thickness of the CAM and the chorionic epithelium were more obvious in the CAM under the carcinoma transplants. After 72 hours of the tumor tissue transfer onto the membrane, the tumor cells retained their vitality and also their influence on the CAM tissues could be observed.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

New generation of cell culture assay for smallpox vaccine potency.

The potency of smallpox vaccines produced in the 1970s was tested by titration onto chorioallantoic membranes of fertilized hen eggs (CAM assay). The potency specification commonly approved for these vaccines was a titer above 10(8) pock-forming units per milliliter. We developed and validated a cell culture titration assay to have a more reliable potency test. The cell titration assay and the CAM assay were tested in parallel on 34 first-generation smallpox vaccine lots. These allowed us to demonstrate that a correlation does exist between the two titration techniques and to determine a new in-house specification for the cell titration method. This in vitro potency assay will allow us to test first-generation smallpox vaccines produced on the skin of living animals but will also give a hint of the potency specification that should be assigned for new generations of cell-derived smallpox vaccines.

Irritation test of tissue adhesives for facial prostheses

Statement of problem. Adhesives are commonly used to improve the retention of a facial prosthesis to the skin. Although no requirement exists for facial prosthetic adhesives, an adhesive should be nonirritating and nontoxic. Purpose. This study assessed the irritative potential of facial prosthetic adhesives by using an in vitro technique for detection of eye-irritating chemicals. Material and methods. Ten adhesives were evaluated by the hens egg test chorioallantoic membrane method. Adhesives were applied to the chorioallantoic membrane in fertilized hen eggs, and the membrane examined by a photomacroscope for injury to the blood vessels. The average irritation score was calculated from the recorded times for the debut of hemorrhage, lysis, and coagulation, and the products were classified as being non, slight, moderate, or strong irritants, based on the irritation score. Results. The predominant injury to the membrane was coagulation of blood vessels, and the exposure time needed to initiate the reaction was dependent on the composition of the product. Four products were classified as strong irritants, 1 as moderate, and the remaining 5 as slight or nonirritant. Conclusion. On the basis of a test for eye irritation, the irritant potential of tissue adhesives varied from non to severe. The most severe reactions were mainly seen in products containing the solvent ethyl acetate. (J Prosthet Dent 2000;84:453-7)

Antiinfluenza virus effect of extracts from marine algae and invertebrates.

Sixty products, derived from marine organisms, typical of the Bulgarian Black Sea coast, were examined for inhibitory activity on the reproduction of influenza viruses in tissue cultures. The antiviral effect was investigated by the reduction of virus infectivity. Using representative strains of influenza virus it was shown that apparently the inhibitory effect was strain-specific. The most effective products were further studied in fertile hen's eggs and in experimental influenza infection in white mice.

Assessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study

The chorioallantoic membrane vascular assay (CAMVA) is an alternative to the Draize rabbit eye irritation method. The CAMVA employs the vascularized membrane of a fertile hen's egg to assess eye irritation potential. This irritation potential is a function of alterations in the vasculature following the administration of test material. Because of the history of use of the CAMVA it was selected as one of the methods for a validation study organized and sponsored by COLIPA. For this validation study mathematical prediction models (PMs) were developed to convert the CAMVA results into predicted Draize eye irritation scores known as a modified maximum average Draize score (MMAS). These predicted scores were statistically compared with the observed scores to assess the relevance of the CAMVA. The assay was conducted on the same set of test materials by two independent laboratories. These two sets of data were compared to assess the interlaboratory reproducibility of the assay. The results of this validation study of the CAMVA show that for test materials with MMASs in the 0 to 5 range or the 55 to 110 range, the CAMVA did not give a good prediction. The predictions were better for samples of mild to moderate irritation (MMAS 5–55). The difficulty in predicting at the low end of the irritation scale appears to be due to the biological variability of the test system and the subjective nature of the CAMVA evaluation. For those samples with an MMAS above 55, the CAMVA appeared to be limited in demonstrating the more severe response. This may be due to the fact that the PMs were developed using historical data sets of test materials with MMASs below this range. Two approaches for improving the CAMVA for eye irritation prediction are (1) to decrease the variability at the low end by reducing the subjectivity in the scoring and (2) to develop better prediction models using more data in the range of severe irritants.

Morphological comparison of in ovo derived and naturally occurring flukes using Microphallus similis, M. pygmaeus and Maritrema arenaria

AbstractThis investigation deals with the evaluation of the in ovo technique for the cultivation of trematodes. The technique has previously been used for the maturation of digenetic flukes and the establishment of life cycles in the laboratory but it was not known if the adult flukes produced were morphologically comparable to those found in their natural hosts. Three species of microphallids, Microphallus similis, M. pygmaeus and Maritrema arenaria were used. Metacercariae of each species were inoculated into fertile hen's eggs which were incubated at 41°C for 120 hours. The adult flukes recovered were stained and mounted and morphonietric measurements obtained were compared to definitive descriptions of the three microphallid species. The results showed that the in ovo technique provided adult flukes which were indistinguishable from those occurring in natural hosts.

Effects of 2,4-dichlorophenoxyacetic acid butyl ester on chick liver

Fertilized hen eggs were externally treated with 3.1 mg 2,4-dichlorophenoxyacetic acid butyl ester (2,4-D ester) before starting incubation. Liver lipid composition, (14C)2,4-dichlorophenoxyacetic acid (14C-2,4-D) subcellular distribution and covalent binding to proteins and lipids of chicks hatched from these eggs were studied. Furthermore, catalase, glutathione S-transferase and glucose 6-phosphatase activities as well as reduced glutathione content were determined. The herbicide produced a significant decrease on the total lipids, specially on phospholipids, both in total liver and microsomes. (14C-2,4-D) subcellular distribution in liver demonstrated that the herbicide passed through the plasmatic membrane and it was present in all the studied fractions. Herbicide bound covalently to hepatic proteins and lipids, being the binding to proteins tenfold higher than to lipids. Because these results might be of potential relevance to the understanding of the 2,4-D toxicity, further studies on the mechanisms of these reactions are necessary. On the other hand, the microsomal and cytosolic glutathione S-transferases activities remained unchanged, even though in vitro studies 2,4-D ester caused a decrease of the enzyme activities with an I50 value of 0.2 mM. No significant change in reduced glutathione content between control and treated livers was observed. The catalase activity increased two-fold whereas glucose 6-phosphatase activity decreased 46% with respect to the corresponding control values. These results indicate that 2,4-D ester may have effects on the metabolism of xenobiotics.

Esterase activities during chick embryonic development and its relationship with the metabolism of 2,4-dichlorophenoxyacetic acid butyl ester.

2,4-Dichlorophenoxyacetic acid (2,4-D) is an organic chemical developed in the mld-1940"s as a herbicide for the control of broad leaf weeds. This compound was formulated for use as butyl ester which is more volatile than the parent acid and therefore easier to apply in aerial spraying (Sanders 1981). Taking into account the importance of knowing the fate in the environment and the toxicological effects of herbicides on living organisms, Dullard et al. (1982) demonstrated that external application of 2,4-D butyl ester on fertile hen eggs on day 0 of incubation resulted in a 50% of mortality. Chicks hatched from these treated eggs exhibited postural and motor dlsfunctlone and other alterations as hypomyelination and changes in the nervous system lipid composition (Mori et al. 1985, 1986). The same embryotoxic effects of 2,4-D butyl ester were found when the herbicide was applied on day 5 and i0 of incubation, but the 15 day group improved the hatching percentage (Dullard et al. 1987a).

In ovo cultivation of Microphallus primas (Trematoda: Microphallidae) metacercariae to ovigerous adults and the establishment of the life-cycle in the laboratory

This investigation reports for the first time the establishment of a trematode life-cycle in the laboratory using in ovo cultivation of the adult stage. Microphallus primas metacercarial cysts were removed from Carcinus maenas crabs and chemically excysted. Using a modification of the technique developed by Fried (1962) the released metacercariae were grown in fertile hen's eggs to ovigerous adult flukes. These were removed from the under surface of the chorio-allantoic membrane of chick embryos 5 days post-infection and the fluke eggs recovered were used to infect laboratory-reared Hydrobia ulvae snails. When challenged against parasite-free crabs the cercariae released from the experimentally infected snails gave rise to metacercarial cysts that were again identified as M. primas.

Evaluation of the vascular components of the chorioallantoic membrane assay as a model for eye irritation potential: II

AbstractPrevious research has shown that the chorioallantoic membrane (CAM) of a fertile hen's egg can be a useful model for predicting eye irritation potential. However, the specific test procedure utilized will affect the degree of correlation between the CAM response and the results from the standard Draize rabbit eye irritation test. We studied how various components of a standard CAM assay, such as age of CAM and the endpoint evaluated, have an impact on the relationship of the CAM response to the in vivo eye response. These studies suggested the basis for a new CAM assay procedure that improves the correlation with the in vivo test. Herein is described an assay method that involves treating a 14-day-old CAM and evaluating the vascular changes that occur 30 min following treatment. This method is referred to as the CAM vascular assay (CAMVA). The CAMVA is shown to produce fewer false-positive responses than previously described CAM procedures and is especially effective at differentiating irritants f...

Quantitative evaluation to predict the eye irritancy of chemicals: Modification of chorioallantoic membrane test by using trypan blue

The chorioallantoic membrane (CAM) test using fertile hen's eggs has been reported as a promising alternative method to predict the eye irritancy of chemicals. However, the scoring system of the original CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Furthermore, the test requires skill when the effects of test substances are to be graded. To overcome these disadvantages, a more objective means of judging the injurious effects of chemicals was examined as follows: after treatment with the test chemical, the CAM was stained with trypan blue and the amount of pigment adsorbed was measured spectrophotometrically. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the in vivo eye irritation test scores ( r = 0.89). The findings suggest that the trypan blue staining method is very simple and reproducible, and thus would be a promising alternative method for predicting the eye irritancy of chemicals. However, further studies will be required to validate this test method with a wide variety of chemicals and formulations.

Biochemical alterations in skeletal muscle induced by 2,4-dichlorophenoxyacetic butyl ester during chick embryonic development

2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) was applied on fertile hen eggs before starting the incubation. Chicks hatched from treated eggs showed motor dysfunctions, postural troubles and edematous muscles. The electromyography revealed muscular weakness, prolonged motor distal latency, and myotonia. The biochemical composition of leg and complexus muscles from 1-day-old chicks was determined. A significant diminution (24%) in the glycogen level of leg muscles was produced by the treatment. There was a small increase (15%) in sarcoplasmic proteins from leg muscles and an increase of a 20 kD protein in the myofibrillar proteins from complexus muscles. Even though total lipid content was not changed, 2,4-D b.e. treatment produced a diminution of sterol esters (20%) and phosphatidylcholine (11%) and an increase of phosphatidylserine (61%), triglycerides (37%) and free fatty acids (FFA) (448%) in leg muscles. Increases of phosphatidylethanolamine (16%), sterols (58%) and FFA (267%) were detected in complexus muscles. A remarkable increase (700–1500%) of unsaturated FFA, e.g. oleic, linoleic and arachidonic acids, was observed. Considering the avian embryo lipid metabolism, it is proposed that FFA and triglycerides were accumulated because they could not be metabolized in the mitochondria. Since FFA are potent cytotoxic compounds, their increase may be a key factor in the 2,4-D b.e. toxic action in muscle and other tissues during embryonic development.

Ca 2+ Homeostasis alterations induced by 2,4-dichlorophenoxyacetic butyl ester and 2,4-dichlorophenoxyacetic acid on avian skeletal muscle

Fertilized hen eggs were treated externally with 2,4-dichlorophenoxyacetic butyl ester (2.4-D b.e.) (3.1 mg/egg) before the start of the incubation. Actomyosin and sarcoplasmic reticulum adenosine triphosphatase (ATPase) activities from leg and complexus muscles of chicks hatched from treated eggs were measured. No significant variations were detected in the ATPase activities of actomyosin, but the sarcoplasmic reticulum Mg 2+-activated ATPase and Ca 2+, Mg 2+-activated ATPase were inhibited 50 and 38% respectively. 45Ca 2+ uptake into soleus muscle was increased by the 2,4-D b.e. treatment. The compartmental analysis of 45Ca 2+ uptake kinetics showed increases in Ca 2+ fluxes in sarcolemma and mitochondria and in the mitochondrial calcium pool. Isolated soleus muscles were treated with 2,4-dichlorophenoxyacetic acid (2.4-D) or 2,4-D b.e. [ 14C]2,4-D reached it highest level in these muscles after 1 hr of treatment. The in vitro treatment with 2,4-D or 2,4-D b.e. increased 45Ca 2+ uptake into the muscles. 2,4-D b.e. produced greater alterations than 2,4-D. The compartmental analysis of the 45Ca 2+ uptake kinetics also showed increases of the mitochondrial Ca 2+ pool and Ca 2+ fluxes through sarcolemma and mitochondria. These results led to a hypothesis based on Ca 2+ permeability alterations for explaining the myopathic actions of these phenoxyherbicides.