A comparison of the distribution, metabolism and excretion of ethylenethiourea in the pregnant mouse and rat

Pregnant mice and rats were treated by stomach intubation on day 15 g of gestation with 240 mg/kg of ethylenethiourea (ETU) made up in part with radiolabeled ETU. Animals were sacrificed at specific times post-treatment, and maternal tissues, fetus, urine and feces were collected for determination of radioactivity. Maternal and fetal tissue levels of ETU were similar at three hours post treatment; thereafter, the mouse (maternal and fetus) showed much less ETU than the rat. The t1/2 of ETU elimination from the maternal blood was 9.4 and 5.5 hours for the rat and mouse, respectively. Analysis of urine by thin-layer chromatography and radiochromatography revealed that the mouse and rat metabolized ETU by different pathways. Furthermore, the mouse is able to metabolize ETU to a greater extent than the rat.     

Proliferation and differentiation in the development of myoneural tissue in birds

Using the alkaline dissociation technique in the ciliary-iris complex of chick embryos 5-19 days of development by mean of 3H-thymidine and 3H-leucine radiography, qualitative cytochemistry and morphometry methods morpho-functional parameters of cell and simplast development in the myoneural tissue have been studied. As a result of divergent differentiation of the stem cells two differons are formed in the myoneural tissue: cellular and simplastic. They both interact in composition of the muscle fiber. The proliferation is specific for the cellular differon, and synthesis of specific proteins takes place in the simplasts. The proliferation and differentiation in the avian myoneural tissue occur on the base of competitive+ type of metabolism, while in the iris pigmentocytes--on the base of non-competitive++ interaction of DNA and specific protein synthesis. A conclusion is made concerning common mechanisms of development for the skeletal muscle and myoneural tissues. They are based on the single type of interaction of proliferation and differentiation processes in histogenesis of the two tissues.     

In vitro development of the embryonic mouse inner ear following exposure to trypsin

Trypsin has been shown to disrupt normal in vitro morphogenesis of embryonic organ rudiments. Otic tissues derived from 11-, 12-, and 13-day-old mouse embryos were exposed to either Ca++- and Mg++-free PBS or 0.25% trypsin dissolved in Ca++- and Mg++-free PBS prior to explanation into organ culture. Trypsin treatment of otic explants disrupted the expression of the normal pattern of inner-ear development in vitro. There was a direct correlation between the embryonic age at time of exposure to trypsin and the severity of dysmorphogenesis of the inner ear. The younger explants showed abnormalities of both vestibular and auditory structures, whereas with increasing embryonic age, abnormalities were confined more to the auditory portion of the inner ear. The results suggest that integrity of the otocyst basal lamina and epitheliomesenchymal tissue interactions are important factors in early otic development. It is postulated that the major effect of trypsin on inner-ear morphogenesis is through disruption of these factors, which may act to regulate the progressive expression of early otic development.     

Ethylene thiourea (ETU). A review of the genetic toxicity studies

Ethylene thiourea (ETU) is a common contaminant, metabolite and degradation product of the fungicide class of ethylene bisdithiocarbamates (EBDCs); as such, they present possible exposure and toxicological concerns to exposed individuals. ETU has been assayed in many different tests to assess genotoxicity activity. While a great number of negative results are found in the data base, there is evidence that demonstrates ETU is capable of inducing genotoxic endpoints. These include responses for gene mutations (e.g. Salmonella), structural chromosomal alterations (e.g. aberrations in cultured mammalian cells as well as a dominant lethal assay) and other genotoxic effects (e.g. bacterial rec assay and several yeast assays).It is important to consider the magnitude of the positive responses as well as the concentrations/doses used when assessing the genotoxicity of ETU. While ETU induces a variety of genotoxic endpoints, it does not appear to be a potent genotoxic agent. For example, it is a weak bacterial mutagen in the Salmonella assay without activation in strain TA1535 at concentrations generally above 1000 μg/plate. Weak genotoxic activity of this sort is usually observed in most of the assays with positive results. Since ETU does not appear very potent and is not extremely toxic to test cells and organisms, it is not surprising to find that ETU does not produce consistent effects in many of the assays reviewed. Consequently, in many instances, mixed results for the same assay type are reported by different investigators, but as reviewed herein, these results may be dependent upon the test conditions in each individual laboratory. A primary shortcoming with many of the reported negative results is that the concentrations or doses used are not high enough for an adequate test for ETU activity. There are also problems with many of the negative assays generally in protocol or reporting, particularly with the in vivo studies (e.g. inappropriate sample number and/or sampling times; inadequate top dose employed).Overall, while ETU does not appear to be a potent genotoxic agent, it is capable of producing genotoxic effects (e.g. gene mutations, structural chromosomal aberrations). This provides a basis for weak genotoxic activity by ETU. Furthermore, based on a suggestive dominant lethal positive result, there may be a concern for heritable effects. Due to the many problems with the conduct and assessment of the in vivo assays, it is worth repeating in vivo     

The development of the immune tissues in marsupial pouch young

Current knowledge of the development of the marsupial immune system, particularly in the context of lymphoid tissue development and the appearance of lymphocytes, has been examined and limitations identified. While primary lymphoid tissues like the thymus have been extensively studied, secondary lymphoid tissues such as the spleen and lymph nodes have been examined to a lesser extent, partly due to the difficulty of macroscopically identifying these structures, particularly in very small neonates. In addition, little research has been conducted on the mucosal‐associated lymphoid tissues; tissues that directly trap antigens and play an important role in the maturity of adaptive immune responses. Research on the development of the marsupial immune tissues to date serves as a solid foundation for further research, particularly on the mechanisms behind the development of the immune system of marsupials. With the recent sequencing and annotation of whole marsupial genomes, the current wealth of sequence data will be essential in the development of marsupial specific reagents, including antibodies, that are required to widen our specific knowledge of the complex marsupial immune system and its development. J. Morphol. 275:822–839, 2014. © 2014 Wiley Periodicals, Inc.     

Effects of thyroid antagonists on rat embryos cultured in vitro

A literature review of individual pregnancies and recent surveys involving large cohorts reveal an association between congenital malformation and maternal hyperthyroidism, suggesting that some aspect of hyperthyroidism or its treatment might compromise the development of the fetus. Experiments have shown that the thyroid antagonist, ethylenethiourea (ETU), causes fetal malformations when administered to pregnant rats, but it is not known whether it is ETU or the imbalance in maternal thyroid hormone which it causes which is the teratogenic agent. Here we employ in vitro culture to determine the possible direct effects on rat embryos of two thyroid antagonists, ETU and methimazole (MMI), the latter being one which is used for treatment of thyrotoxicosis in humans. It was found that ETU can compromise the development of rat embryos in vitro, confirming that ETU has a direct effect on the rat embryo. It was also found that MMI can cause abnormal development of rat embryos in vitro, although the concentration at which MMI disturbs rat embryogenesis is higher than that which is reached in hyperthyroid patients treated with clinical doses of MMI or carbimazole.     

Evaluation of the specificity of lectin binding to sections of plant tissue

Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissues. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus and Ulex lectins for L-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (gamma-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.     

Mechanical design criteria for intervertebral disc tissue engineering

Due to the inability of current clinical practices to restore function to degenerated intervertebral discs, the arena of disc tissue engineering has received substantial attention in recent years. Despite tremendous growth and progress in this field, translation to clinical implementation has been hindered by a lack of well-defined functional benchmarks. Because successful replacement of the disc is contingent upon replication of some or all of its complex mechanical behaviors, it is critically important that disc mechanics be well characterized in order to establish discrete functional goals for tissue engineering. In this review, the key functional signatures of the intervertebral disc are discussed and used to propose a series of native tissue benchmarks to guide the development of engineered replacement tissues. These benchmarks include measures of mechanical function under tensile, compressive, and shear deformations for the disc and its substructures. In some cases, important functional measures are identified that have yet to be measured in the native tissue. Ultimately, native tissue benchmark values are compared to measurements that have been made on engineered disc tissues, identifying where functional equivalence was achieved, and where there remain opportunities for advancement. Several excellent reviews exist regarding disc composition and structure, as well as recent tissue engineering strategies; therefore this review will remain focused on the functional aspects of disc tissue engineering.     

A rheological network model for the continuum anisotropic and viscoelastic behavior of soft tissue

The mechanical behavior of soft tissue demonstrates a number of complex features including nonlinearity, anisotropy, viscoelasticity, and growth. Characteristic features of the time-dependent and anisotropic behavior are related to the properties of various components of the tissue such as fibrous collagen and elastin networks, large proteins and sugars attached to these networks, and interstitial fluid. Attempts to model the elastic behavior of these tissues based on assumptions about the behavior of the underlying constituents have been reasonably successful, but the essential addition of viscoelasticity to these models has been met with varying success. Here, a new rheological network model is proposed using, as its basis, an orthotropic hyperelastic constitutive model for fibrous tissue and a viscoelastic reptation model for soft materials. The resulting model has been incorporated into numerical and computational models, and is shown to capture the mechanical behavior of soft tissue in various modes of deformation including uniaxial and biaxial tension and simple shear.     

Difference in teratogenic potency of ethylenethiourea in rats and mice: relative contribution of embryonic and maternal factors

Ethylenethiourea (ETU) is a potent teratogen in the rat but not in the mouse or any other species tested. Embryotoxic and teratogenic effects are produced in mice only after exposure to 10-40 times the teratogenic dose of ETU in rats. This study was undertaken to determine whether the difference in sensitivity between rats and mice is due to differences within the embryo, to maternal metabolic differences, or both. Comparably staged rat and mouse embryos (gestation day 10.5 and 8.5, respectively) with intact extra-embryonic membranes were maintained under identical conditions in whole embryo culture and exposed to static concentrations of ETU for 48 hours. The teratogenic effects of ETU were qualitatively similar in both species, characterized by excessive fluid accumulations in embryonic structures. The most common abnormalities were distended neural tube, especially in the hindbrain, and clear blisters on the caudal region. At least two times as much ETU was required to produce a similar incidence of abnormalities in mice as in rats. Thus, there is some intrinsic difference in the quantitative response of rat and mouse embryos to ETU, but it is insufficient to account for the in vivo discrepancy. The role of maternal metabolism in modifying the teratogenicity of ETU was assessed by adding hepatic S-9 fractions from Aroclor 1254-induced rats and mice to whole embryo culture. Rat S-9 had no effect on ETU teratogenicity. Mouse S-9 virtually eliminated the formation of abnormalities typical of ETU in both rat and mouse embryos. Decreased exocoelomic fluid osmolality, a physiological effect produced by ETU, also was not observed in embryos exposed to ETU and mouse S-9. ETU-typical defects were observed in embryos exposed to ETU and mouse S-9 which had been treated with carbon monoxide to inactivate its monooxygenase system, indicating that the mouse S-9 was metabolizing ETU. A surprising result was that adding mouse S-9 to embryo cultures containing ETU resulted in the formation of abnormalities (principally open neural tube) that were not observed in in vitro rat or mouse embryos exposed to ETU alone, or in mouse embryos in vivo. We believe that the most likely cause of these abnormalities is a putative ETU metabolite, which is rapidly excreted by the dam in vivo, but accumulates to teratogenic concentrations in vitro.     

Computer simulation for studying calcium dependent abnormalities in firing mechanism of molluscan neurones

Computer modelling technique is proposed to assist in physiological research on invertebrate neuronal membranes. The firing mechanism of a single patch of invertebrate neuronal membrane has been studied in dependence on maximum Ca++ conductance. The calculations are based on modification of Hodgkin-Huxley's data completed by a straight line approximation between experimental points of the kinetic parameters of Ca++ current and early transient potassium current. The time course of conductance changes is assumed to be proportional to m2h for Ca++ current. Three distinct potassium currents are involved into the model, viz. transient potassium current, delayed potassium current and Ca++-dependent potassium current. The modified Euler method run on a digital computer has been used for numerical integration of kinetic equations. Significant effects of Ca++ conductance on spike broadening, plateau development and spike afterhyperpolarization are represented. In the range of small Ca++ conductance an infinite spontaneous activity can be triggered by a short (suprathreshold) current pulse which may be considered a model of pacemaker activity. Plateau development resulting from potassium blocking or decreasing potassium equilibrium is facilitated by Ca++ conductance in the range of greater Ca++ conductance. The effects of voltage sensitivity of the coupling coefficient describing the current of Ca++-dependent K+ channels were studied and compared to the voltage independent case. The coupling coefficient seems to be a crucial factor in broadening the range of Ca++ conductance responsible for pacemaker activity. For greater values of Ca++ conductance, a decrease of the coupling coefficient leads to a transition from prolonged bursting to interruption of burst activity by burst-afterhyperpolarization. The blocking effect of 4-aminopyridine on fast outward current has been studied by the model which has a practical significance considering that aminopyridine is known as a convulsive agent. We suppose that it is reasonable to study the convulsive effects of aminopyridine by the model based on the kinetics of the isolated neuronal membrane. The model may help in understanding the ionic background underlying abnormal network activity during epileptic discharges of mammalian neurones.     

Comparative studies of embryotoxic action of ethylenethiourea in rat whole embryo and embryonic cell culture

The effects of ethylenethiourea (ETU) were investigated using rat (Wistar-imamichi) embryos cultured from days 11 to 13 of gestation or cultured rat embryonic cells extracted on day 11. Malformations in cultured embryos at the concentration of 30 micrograms/ml of ETU were found in the head and tail, which were severely affected, as well as the limb and face. All embryos exposed to 150 and 300 micrograms/ml of ETU had malformed heads, tails, limbs, and facial configurations. Protein contents of the cultured embryos were decreased dose-dependently at the concentrations ranging from 30 to 300 micrograms/ml. In the histological studies of the cultured embryos with ETU, thinner neuroepithelium in head was observed. In the embryonic cells extracted on day 11 of gestation, ETU dose-dependently inhibited the differentiation of midbrain (MB) cells into neurons and that of limb bud (LB) cells into chondrocytes at the concentrations ranging from 30 to 600 micrograms/ml of ETU. The concentrations of ETU that inhibited the production of differentiated foci by 50% (IC50) were 170 micrograms/ml in LB cells of day 11, and greater than 600 micrograms/ml in LB cells on day 12 of development. Therefore, differentiation of MB cells was more sensitive to ETU than the differentiation of LB cells. These results indicated that there was a reasonable correlation of ETU induced changes in cultured whole embryos and embryonic cells.     

In vitro teratogenicity of ethylenethiourea in the rat

We have demonstrated that ethylenethiourea (ETU) is a potent teratogen to the rat embryo developing in vitro. Sprague Dawley rat embryos were explanted on gestation day 10 and cultured for 48 hours in the presence of 40-200 micrograms/ml ETU. This resulted in a dose-related inhibition of growth and differentiation as assessed by crown-rump length, protein and DNA content, and somite number and in an increase in the frequency of abnormalities. A variety of anomalies was produced, including fluid accumulation in the brain (hydrocephalus), decreased mandibular size, decreased telencephalon size, abnormal dorsiflexion, as well as subectodermal blisters on the tail and limb buds and maxilla. Frank malformations have been observed at these same sites--hydrocephalus, brachygnathia, kyphosis, limb and tail defects, cleft palate--in the term fetus in vivo. The presence of abnormal fluid accumulation in the embryos--distended neural tube and subectodermal blisters--suggesting that the osmotic environment of the embryo had been altered by ETU exposure. Osmolality of the exocoelomic fluid (ECF) surrounding the embryo was measured after 48 hours of exposure to a concentration of ETU that caused nearly a 100% incidence of subectodermal blisters. ECF osmolality was found to be significantly lower than that of control embryos. Lowered osmolality would cause water to move out of the ECF, presumably causing the observed fluid accumulation in the embryo. It is speculated that altered osmotic balance and localized edema in the embryo are contributory steps in the formation of defects after ETU exposure.     

Native-mimicking in vitro microenvironment: an elusive and seductive future for tumor modeling and tissue engineering

Human connective tissues are complex physiological microenvironments favorable for optimal survival, function, growth, proliferation, differentiation, migration, and death of tissue cells. Mimicking native tissue microenvironment using various three-dimensional (3D) tissue culture systems in vitro has been explored for decades, with great advances being achieved recently at material, design and application levels. These achievements are based on improved understandings about the functionalities of various tissue cells, the biocompatibility and biodegradability of scaffolding materials, the biologically functional factors within native tissues, and the pathophysiological conditions of native tissue microenvironments. Here we discuss these continuously evolving physical aspects of tissue microenvironment important for human disease modeling, with a focus on tumors, as well as for tissue repair and regeneration. The combined information about human tissue spaces reflects the necessities of considerations when configuring spatial microenvironments in vitro with native fidelity to culture cells and regenerate tissues that are beyond the formats of 2D and 3D cultures. It is important to associate tissue-specific cells with specific tissues and microenvironments therein for a better understanding of human biology and disease conditions and for the development of novel approaches to treat human diseases.     

Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets

Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds.     

Demonstration, kinetic study and solubilization of particular adenylate cyclase from Nocardia restricta

An adenylate cyclase (EC r.6.1.1.) was found in cell-free extracts of several Nocardia species. The enzyme from Nocardia restricta has been specially studied. It is a membrane enzyme which exhibits a strong specific activity, one hundred times greater than that of mammals. It has an optimal pH of 8.5 in Tris buffer and an absolute requirement for divalent ions, Mg++ or Mn++ (Mn++ ions are the most efficient). The kinetic properties of this adenylate cyclase are similar to those that could be expected of an allosteric enzyme having, as a substrate, the ATP-Mg++ complex and, as an activator, free Mn++ ions. Ca++ ions are activators: they set up the maximum velocity without modification of the KM. GTP is a competitive inhibitor (KI = 5.10(-5) M). Fluoride ions have no detectable effect on activity. Non-ionic detergents, Lubrol WX and Triton X 100, are inhibitors of the enzyme which has been partially solubilized by repeated freezing and thawing, following by brief ultra-sonic treatment. Catalytic sites are not modified after the solubilization, but cooperative effects between moles of substrate ATP-Mn++ are diminished: the KM becomes smaller and the sigmoidal shape of the curve v = f (ATP-Mn++) is attenuated.     

Ethylenethiourea-induced hydrocephalus in vivo and in vitro with a note on the use of a constant gaseous atmosphere for rat embryo cultures

Embryos were studied either after direct exposure to ethylenethiourea (ETU) during incubation of embryo cultures or after maternal ETU dosing and subsequent embryonic development in utero with a view to assess the similarity of these two systems to produce hydrocephalus. Ten-day-old rat embryos were incubated with nutrient media containing 0-2.0 mM of ETU in a constant gasseous environment following a newly modified method. The cultured embryos showed hydrocephalus in the form of dilated rhombencephalon and other anomalies at the 1.5 and 2.0 mM of ETU after 26 hours of incubation. No anomalies were seen in the control group. In in vivo studies, dilated rhombencephalon or hydrocephalus was not observed when dams, orally dosed with ETU on gestation day 10, were either killed daily for three postdosing days to examine embryos or killed at term to evaluate fetuses. This discrepancy in dilatation that was incidental to the rhombencephalon in the two systems pointed out that the fourth ventricle of the cranial neural tube responded by dilatation in vitro but remained unaffected in vivo following ETU exposure. ETU dosing of dams on the 12th day of pregnancy, when embryos are known to be sensitive to ETU-induced hydrocephalus, followed by serial gross examination of embryos, suggested that edema occurred in a generalized form but only after the appearance of both hydrocephalus (dilatation primarily in mesencephalon) and, the previously reported, neuroblastic necrosis.     

Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.     

The morphogenesis of myeloschisis in the rat

Pregnant Sprague-Dawley rats were utilized in this study. They were separated into two groups. In the control group, a single intragastric dose of distilled water was given on the 11th day of gestation. In the test group, a single intragastric dose of ethylenethiourea (ETU), 240 mg/kg was given on the same day of gestation. Embryos were recovered 12, 24, 36, and 48 hours after ETU and distilled water administration, and were prepared for scanning electron microscopy and light microscopy. The posterior neuropore of rat fetuses in the control group closed completely on gestation day 12.5. However, the closure of posterior neuropore in ETU-induced fetuses is shown to have been disrupted 12 hours after ETU administration. Marked neural tissue overgrowth in the posterior neuropore resulted in neural fold eversion and finally produced a picture of lumbosacral myeloschisis on day 13 of gestation. Our observation implies that myeloschisis is induced by non-closure of the neural fold, not by reopening after its proper closure.