Embryotoxic Properties Research Articles

Sensing of p-nitrophenol using highly selective and sensitive Boran, Nitrogen doped quantum dots

p-Nitrophenol (PNP) is a nitroaromatic compound that poses a significant threat to human health and the environment due to its carcinogenic, mutagenic, cytotoxic, and embryotoxic properties at low concentrations. Therefore, the selective and sensitive detection of PNP is crucial for both human health and environmental monitoring. Boron (B) and Nitrogen (N) doped quantum dots (B,NQDs) have been found to be effective as blue-green luminescent materials for this purpose. These B,NQDs were synthesized using a one-step hydrothermal method, resulting in the formation of highly stable quantum dot. The addition of trace amounts of PNP, the luminescence of the B,NQDs was significantly quenched, which was found to be linearly dependent on the PNP concentration in the range of 100pM to 6 μM. Further analysis of steady-state absorption and emission, along with photoluminescence decay dynamics, revealed the formation of both static and dynamic quenching complexes. Our simple fluorimetry-based sensor demonstrated an impressive limit of detection (LOD) of 9.08 nM, making it highly selective and sensitive for the detection of PNP. Additionally, the B,NQDs exhibited exceptional stability with respect to pH, UV exposure, salinity, and storage conditions. Finally, we successfully demonstrated the detection of PNP in real water systems and pesticides.

Influence of drop on the ultrastructure lipid composition of liver hepatocytes of pregnant rats

The influence of a toxic agent on the structure of the liver is inevitable. Of interest are the effects of pesticides on intrauterine formation and their teratogenic and embryotoxic properties. The influence of teratogens may manifest itself at different levels of organization of a living organism, and some aspects of cell metabolism change. Using advanced electron microscopy techniques combined with modern knowledge of membranology and toxicology, we conducted a detailed study of morphological changes in the subcellular architecture of the liver after repeated dropplet exposure. Ultrastructural studies were carried out on pregnant rats and their embryos on the 3rd, 13th, 19th days of pregnancy and embryonic development. Poisoning of animals with the pesticide dropp causes clear degenerative processes in the cellular and subcellular structures of the liver.

The use of diffusion chambers in embryotoxic research

Introduction. Currently, there are no standard methodological approaches that allow assessing the danger to human and animal reproduction of poorly soluble chemicals (industrial waste, products of destruction of organic substances, polymer materials), including substances that form complex poorly soluble systems with environmental objects. It may be impossible or ineffective to use standard routes of administration of such substances and complexes to assess their reproductive hazard. An attempt to find an experimental model for conducting toxicological and embryotoxicological studies that allow assessing the danger of prolonged interaction of poorly soluble compounds directly with tissue fluids was the main aim of the work. The study aims to investigate the possibility of using diffusion chambers to assess the reproductive hazard of poorly soluble chemicals. Material and methods. In experiments with chemicals, the researchers placed samples of dry preparations in the inner cavity of one of the rings. Experts sealed the ring on both sides with millipore membranes. The diffusion chambers obtained in this way were implanted into the abdominal cavity or under the skin in the cervical region of female rats during the most sensitive days of pregnancy to pathogenic effects. Results. When implanting diffusion chambers to animals, the possibility of assessing the embryotoxic properties of poorly soluble chemicals, such as bitumen-salt masses, polymer materials and coatings, etc., as well as soil contamination with substances potentially dangerous to the reproductive function of humans and animals is shown. With the help of diffusion chambers, experts assessed the embryotoxic activity of known dysmorphogens, cyclophosphamide, cadmium sulfate, as well as a number of polymer materials (di-paraxilylene film, acrylic acid copolymer, silloxane rubber), Scientists assessed soil contamination with a known insecticide and acaricide - endosulfan. Limitations. In the work, the experts used diffusion chambers with a volume of 400, 900 or 3000 m3. Conclusion. As a result of the experiments conducted, scientists determined that the presented method can be used to assess the reproductive hazard of poorly soluble chemicals (products of destruction of organic matter, polymer and endoprosthesis materials), when assessing the risk of prolonged presence of foreign bodies in tissues, when assessing soil contamination in places of chemical accidents, rocket crashes, storage and destruction of chemical weapons. Ethics. The study was carried out in compliance with the rules of bioethics approved by the European Convention for the Protection of Vertebrates Used for Experimental and Other Purposes (Protocol No. 4 of 8.11. 2022).

Management methods of nitrate content in potato applicable to the soil-climate conditions of Russia

Determination of potato quality should not be limited only to the usually established list of indicators (marketability, starch content, dry matter content, etc.). The safety of crop products should also be analyzed by the presence of undesirable compounds, most often of anthropogenic origin (nitrates, pesticides, radionuclides, heavy metals). Sometimes a quality indicator that does not have an obvious cost is overlooked – the so-called biological or hygienic quality of the product, which significantly affects human health. The biological quality of the product is more important than the yield although it does not have a commercial price. Product quality management requires careful analysis to identify the causes of excessive accumulation of nitrates during the growing season and, on this basis, to develop methods that eliminate them, and then methods to reduce the concentration of NO3 in already grown tubers. Nitrosamines and nitrosamides formed from nitrates with the participation of nitrites have pronounced carcinogenic, mutagenic and embryotoxic properties. In Russia, according to sanitary and hygienic standards, the MPC of nitrates for potato is 250 mg/kg, and for children and diet food the values are even lower – 80 mg/kg of raw tubers.

Antioxidants reduce reactive oxygen species but not embryotoxicity in the metabolic Danio rerio test (mDarT)

Mammalian liver microsomes are occasionally used as a metabolic activation system (MAS) to compensate for the low CYP-mediated bioactivation of drugs in zebrafish embryos, in the so-called mDarT. However, this MAS is embryotoxic and consequently zebrafish embryos are only exposed during a very limited developmental window. The main aim of this study was to try to reduce the embryotoxic properties of MAS in order to extend the exposure window in the mDarT. Removing the microsomes from the incubation medium prior to exposure of the zebrafish embryos did not reduce embryotoxicity. Free radicals (ROS) in the incubation medium were successfully reduced by antioxidants, but the medium remained embryotoxic. Single dosing of NADPH or omitting toxic components from the MAS preparation did also not reduce embryotoxicity. In conclusion, the exposure window in the mDarT could not be extended by reducing ROS levels, single dosing of NADPH or modifications of the MAS preparation.

Reproductive and developmental toxicity of the Ginkgo biloba special extract EGb 761® in mice

Extracts from leaves of Ginkgo biloba are among the most widely used and best investigated phytopharmaceuticals worldwide. Almost all clinical trials and the majority of preclinical studies have been performed with a specifically defined extract (EGb 761®) standardized to contain confined concentrations of active ingredients and limited quantities of potentially harmful substances. Besides pharmaceutical grade extracts poorly characterized Ginkgo preparations are now increasingly appearing on the market as nutraceuticals. While the safety of EGb 761® has been evaluated in an extensive set of toxicology studies, adverse effects of Ginkgo extracts of non-pharmaceutical quality on reproductive functions in mice have been reported in several publications in recent years. As this species has not previously been used in reproductive toxicity studies with EGb 761®, the present investigation was conducted to examine the influence of EGb 761® (100, 350 and 1225mg/kg/day) on embryo-fetal development in mice during the critical period of organogenesis. During external and internal inspection of the fetuses as well as examination of skeletal and soft tissues no embryotoxic properties were noted. In particular, the incidence of malformations, variations or retardations was not increased and the general condition of dams was not influenced. Thus, the no-observed-effect level (NOEL) was above 1225mg/kg/day for the dams and the fetuses.

Complementary Detection of Embryotoxic Properties of Substances in the Neural and Cardiac Embryonic Stem Cell Tests

In developmental toxicity testing, in vitro screening assays are highly needed to increase efficiency and to reduce animal use. A promising in vitro assay is the cardiac embryonic stem cell test (ESTc), in which the effect of developmental toxicants on cardiomyocyte differentiation is assessed. Recently, we developed a neural differentiation variant of the stem cell test (neural embryonic stem cell test [ESTn]). In both of these models, we have previously performed a series of transcriptomic studies to characterize gene expression changes (1) across time during normal differentiation and (2) in response to a series of developmental toxicants in the ESTn and ESTc. Here, using the cumulative of these studies, we compared gene expression profiles of ESTn and ESTc over time as well as model-specific changes induced by seven compounds, comprising six known in vivo developmental toxicants and one negative control. Time-related gene expression profiles showed similarities between the two EST systems. However, specific genes could be identified changing over time differently in each model related to the two different lineages of differentiation. Interestingly, compound-induced gene expression changes were generally model specific, especially for methylmercury and flusilazole, which were predicted better in ESTn and ESTc, respectively. Valproic acid-induced gene expression changes were most comparable out of the six developmental toxicants between the ESTn and ESTc. Direct transcriptomic comparisons between the ESTn and ESTc indicate that combined transcriptomic analyses support and complement each other. Therefore, a combined approach incorporating ESTc and ESTn may improve developmental toxicant detection over individual assays.

Manual Direct Two-Phase Titration Method for Anionic Surface Active Agents Determination with Use of Solvatochromic Dye. The Modification Towards Toxicity Abatement

Abstract The modification of the manual direct two-phase titration method for anionic surface active agent determination is presented. The purpose was to find such organic solvent which can replace solvents applied in the method i. e. chloroform or dichloromethane since both are harmful, exhibiting carcinogenic, mutagenic and, embryotoxic properties. As a new solvent 1-butanol or isobutyl alcohol was used. The reaction in the examined alcohols caused to be acidic during the titration process. Thus the solvatochromic dye (1) i. e. 5-(3-bromo-4-hydroxy-5-methoxyphenyl)-7,7-dimethyl-7H-indolo[1,2-a]quinolinium perchlorate has been replaced with 1-phenyl-2-[2-(3-nitro-4-hydroxy-5-methoxyphenyl)ethenyl]-3,3-dimethyl-3H-indolium perchlorate (2). The pKa (3.51) of (2) facilitates its dissociation, and has the same color change at the titration endpoint. The modified method was tested on three different concentrations of sodium dodecyl sulfate solution, and the results are in good agreement with the real ones.

Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies

The zebrafish embryotoxicity test (ZET) is a fast and simple method to study chemical toxicity after exposure of the complete vertebrate embryo during embryogenesis in ovo. We developed a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system. For teratogenic effects a separate scoring list was developed. The relative effects of eight glycol ethers and six 1,2,4-triazole anti-fungals were evaluated in this system and results were compared with in vivo developmental toxicity potencies.Methoxyacetic acid and ethoxyacetic acid appeared as the most potent glycol ether metabolites, inducing growth retardation and malformations. Other glycol ethers showed no developmental toxicity. Flusilazole appeared the most potent triazole, followed by hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole, respectively.In general, the potency ranking of the compounds within their class in the ZET was comparable to their in vivo ranking.In conclusion, the ZET with the GMS system appears an efficient and useful test system for screening embryotoxic properties of chemicals within the classes of compounds tested. This alternative test method may also be useful for the detection of embryotoxic properties of other classes of chemicals.

Embryotoxicity of serum from women smoking cocaine base paste (CBP)

Objective Using a mouse embryo bioassay, we examined the embryotoxic properties of serum obtained from eight women who had ingested cocaine base paste (CBP) for a period of 1–3 years. Study design Two-cell stage mouse embryos were cultured in the presence of serum extracted from eight CBP-smoking or control women. After 48 h, embryos were evaluated to determine their developmental stage and then processed by Tarkoswki's technique to determine the proportion of embryos with micronuclei, in order to establish the number of micronuclei/embryo. Results Serum from CBP patients diminished the percentage of embryos progressing to the compacted morula stage, while increasing the proportion of embryos with micronuclei and the corresponding micronuclei/embryo ratio. Conclusion These findings provide the first experimental evidence of embryotoxic compounds in the serum of CBP-smoking women. This study highlights the reproductive risk of chronically ingested CBP and demonstrates the need for further studies.

Potential mutagenicity, embryotoxicity, and teratogenicity of calcium ketopantoyl aminobutyrate

We studied mutagenic, embryotoxic, and teratogenic properties of calcium ketopantoyl aminobutyrate, a preparation proposed as a new drug. Long-term oral administration of calcium ketopantoyl aminobutyrate produced no mutagenic, embryotoxic, and teratogenic effects.

Why does hydrosalpinx reduce fertility? The importance of hydrosalpinx fluid.

The debate on hydrosalpinx and impaired IVF outcome has mainly focused on the best treatment before IVF and on functional surgery as an alternative treatment. We would like to initiate a debate on the possible reasons why the outcome is impaired. We know that salpingectomy is effective in terms of improved birth rates after IVF, but we do not know exactly why. The main focus is on embryotoxic properties of the hydrosalpinx fluid, which include micro-organisms, endotoxins, cytokines, oxidative stress and lack of nutrients. The endometrial receptivity may be reduced as an effect of disturbed expression of the cytokine cascade, which is essential for implantation. The presence of excessive fluid in the uterine cavity may also be a mechanical hindrance to implantation. We believe that the hydrosalpinx fluid is of crucial importance, but the actual mechanism of action needs to be clarified.

Embryotoxicity of 1,2-dibromoethane in chick embryos in ovo: early and late effects.

Embryotoxic effects of 1,2-dibromoethane (DBE), a compound still widely used in industry, have been analyzed using chick embryos in ovo. Administration on embryonic days (ED) 3,4 or 5 induced dose-dependent embryotoxicity, manifested namely as the early embryonic death. A serious disturbance of the vascular system represented probably the main cause of strong embryolethality and growth retardation in the group of survivors. Amniotic bands in the parietal region and defects of brain and aorta prevailed in the malformation spectrum registered on ED 10. The local character of early induced changes suggests a direct effect of DBE itself in the embryotoxic action. This process is probably accomplished through interaction with lipids in cell membranes owing to the hydrophobic character of DBE molecules. The results, however, did not exclude an involvement of reactive metabolites in final embryotoxicity via the formation of DNA-adducts. In any case, a decreasing embryotoxicity of DBE with the age of treated embryos documented that the onset of liver function, assumed to occur on ED 5, did not increase the efficacy of DBE bioactivation. Our results confirmed the short-term embryotoxic properties of DBE reported in rat embryonic cultures. In addition, the in ovo system enabled us to reveal also long-term consequences represented namely by the formation of amniotic bands, not detectable in studies in vitro. The results obtained with the chick embryo in ovo confirmed the suitability of this system for embryotoxicity testing.

The influence of hydrosalpinx on IVF and embryo transfer: a review.

Several retrospective studies have shown an impaired outcome of IVF in the presence of hydrosalpinx. The retrospective data have been compiled and presented in meta-analyses, demonstrating a reduction by half in the probability of achieving a pregnancy in the presence of hydrosalpinx and a doubled rate of spontaneous abortion. The main theories trying to explain the underlying mechanisms have focused on potential embryotoxic properties of the fluid or impaired endometrial receptivity to implantation. Several mouse studies have suggested an embryotoxic effect of the hydrosalpingeal fluid, a finding which is not supported in studies on human embryos. It is believed that the fluid exerts a detrimental effect on the endometrium by altering the receptivity or simply by causing a mechanical hindrance for implantation. Different treatment options would then be tubal ligation, salpingostomy, aspiration of hydrosalpinx fluid or salpingectomy. The effect of aspiration has been studied in a few retrospective trials with contradictory results. Treatment with salpingectomy has entered into clinical practice without proper evidence for its benefit. Concerns have also been raised about the potential hazard of surgical intervention to ovarian circulation and function. A randomized controlled trial on salpingectomy prior to IVF has now been conducted as a multicentre study in Scandinavia. Laparoscopic salpingectomy prior to IVF was shown to be beneficial in patients with large hydrosalpinges that were visible on ultrasound, a result which supports the theory of the fluid being involved in the impaired implantation process.

Embryotoxicity and teratogenicity of some derivatives of chloroethylaminophenylacetic acid.

Embryotoxic and teratogenic properties of Lophenal, Phenalon, Pharanox and Pharanoxi selenate were investigated experimentally. All examined antitumour agents showed embryotoxic effects. Lophenal, Phenalon and Pharanox had teratogenic effects. By modifying the structure of Pharanox with selenium a reduction in teratogenic effect was achieved.

The Bioavailability of Substances Administered to Chick Embryos: The Maximum Effective Route of Administration

Toxicokinetic studies are of key importance in both the design and the interpretation of developmental toxicity studies. The aim of this study was to determine concentrations of test substances within the chick embryo following the administration schedule recommended in the chick embryotoxicity screening test (CHEST). The concentration-time relationships were investigated by using four labelled substances with various physicochemical and embryo-toxic properties ([14C] sodium acetate, [14C] palmitic acid, [3H] Cortisol and [3H] cytosine arabinoside). These labelled chemicals were mixed with cold substances and singly administered at two dose levels to chick embryos on days 2, 3 and 4 of incubation. Extrachorial and subgerminal routes were used on day 2, and extrachorial and intra-amniotic applications were chosen on days 3 and 4. The concentration of labelled chemical present within the embryo was assessed at predetermined intervals by scintillation fluorimetry (from 6 minutes to 96 hours after administration), and used for estimating the concentration curves. Regardless of the substance, dose and application route, the concentration curves exhibited a characteristic pattern, reaching their peaks within the first 6 hours, and dropping down to near zero 48–96 hours after administration. The decrease followed the first order law, demonstrating that, within the CHEST system, the avian embryo does not act as a closed system. With regard to the total amount of substance entering the embryo, extrachorial administration appeared to be superior to subgerminal administration on day 2. Intra-amniotic administration was superior to extrachorial administration on days 3 and 4. These differences were most pronounced after administration of lipid-soluble palmitic acid. The concentrations within embryonic tissues were directly dose-dependent. After consideration of all these findings, we concluded that the CHEST system probably has closer similarity to the toxicokinetics of exposure of mammalian embryos (i.e. reaching a peak and then a gradual decline over time) than any other in vitro test of developmental toxicity, where the chemical is simply added to culture media. Several practical recommendations for improving the CHEST system were derived.

An Interlaboratory Evaluation of Five Pairs of Teratogens and Non-teratogens in Post-implantation Rat Embryo Culture

The usefulness of the post-implantation rat embryo culture method in screening xenobiotic compounds for developmental toxicity was validated in four laboratories with five pairs of compounds. This approach was chosen to provide information on the interlaboratory reproducibility of the results and to compare the effects of chemical analogues in embryo culture. By testing analogous compounds which are known to have different embryotoxic potencies in vivo, the discriminating power of the embryo culture method for the compound classes under study could be optimally assessed. The classes selected for testing were triazole antifungals, phthalic ester metabolites, substituted pyridines, sulphonamides and methylated xanthines. In summary, it was possible to distinguish between the compounds in three of the pairs, it was not possible to discriminate between the compounds of one pair, and it was possible to discriminate between the compounds of the other pair at two out of the four laboratories. The embryo culture results generally show a good correspondence with the embryotoxic properties of the compounds tested in vivo, although the embryo culture method appeared to be able to discriminate between only some of the pairs of chemical analogues. Some discrepancies may have arisen among the laboratories, because of methodological differences. These results suggest that the post-implantation rat embryo culture method may be a useful tool for screening xenobiotics within classes of compounds known to interfere with embryogenesis during the period of development represented in culture.

The use of pre-implantation mouse embryos cultured in vitro in toxicological studies

The preimplantation mouse embryo has been found to be a good model for various toxicological investigations. This communication deals with two examples of research activities carried out in our laboratory to detect the embryotoxic properties of tritium and of 1,2:3,4-diepoxybutene (DEB). Exposures of blastocysts for 24 hr to concentrations as high as 0.296 kBq/ml tritiated amino acids or nucleoside induced a statistically significant reduction in the percentage of embryos that reached the stage of two-layer inner cell mass (ICM). The same quantity of tritiated arginine, but not of thymidine or tryptophan, also induced a lower percentage of differentiating ICM than the control when added to culture medium during the second cleavage division. These findings support the idea that tritium released by nuclear power plants as HT or as tritiated water (HTO) could become a radiotoxicological problem since it can be converted easily into organic compounds by living organisms. DEB was found to be highly embryotoxic in preimplantation mouse embryos in vitro at micromolar concentrations. This compound is formed in mammalian cells by the oxidative metabolism of 1,3-butadiene, a chemical used in rubber industries and present in tobacco smoke. Again, the most sensitive stages of preimplantation development were found to be the two- and the four-cell embryos. These results were confirmed by in vivo measurements.

Diabetic teratogenesis. In vitro evidence for a multifactorial etiology with little contribution from glucose per se.

To determine the extent to which elevated glucose and 3-hydroxybutyrate (3OHB) concentrations contribute to the embryotoxic properties of diabetic serum, we tested the effects of serum from untreated or acutely insulin-treated diabetic rats on the development of mouse embryos during neurulation in vitro. Male Sprague-Dawley rats (n = 143) with streptozocin-induced diabetes for 1 week received infusions of insulin (n = 105) or saline (n = 38) for up to 120 min. The insulin-infused animals were exsanguinated when serum glucose concentrations fell to between 5.6 and 8.3 mM. Saline-infused animals were exsanguinated after a similar duration of infusion. Serum samples were tested for embryotoxic effects on 3-6 somite mouse embryos cultured in vitro for 24 h. Of embryos cultured in serum from untreated diabetic animals (glucose: 24 +/- 1 mM; 3OHB: 2.0 +/- 0.3 mM), 36% (31 of 87) exhibited gross malformations, mostly of the neural tube. Only 16% (10 of 62) of embryos grown in serum from acutely insulin-treated animals (glucose: 7.4 +/- 0.2 mM; 3OHB: 0.20 +/- 0.06 mM) were malformed. This rate that was less than half the rate caused by exposure to diabetic serum (P < 0.01), but a rate that remained much greater than the rate associated with culture in normal serum (0% in this study; < 2% historically). In vitro addition of glucose to serum from insulin-treated animals to re-establish hyperglycemia in the diabetic range (25 mM) resulted in a 17% (12 of 70) malformation rate, nearly identical to the 16% rate caused by normoglycemic serum from insulin-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Some Questions of Validity of Results of Experiments in Applied Teratology. 2. Statistical Analysis of Experimental Data in Case of Single Fetuses with External Anomalies

Assessment of the embryotoxic properties of ftorosan, a blood substitute with an oxygen-transporting function, in experiments with rats revealed three fetuses with phocomelia, micrognathia, and eventration of the frontal abdominal wall in 2 of 22 litters from a group of 23 pregnant females given doses of 25 ml/kg of body weight on days 6-16 of pregnancy. In two repeated experiments (24 litters examined in each experiment), one fetus with similar anomalies was detected in each experiment. These data, along with similar hypothetical versions of the results, were analyzed by means of an independent criteria test and Wald's sequential probability ratio test with a view towards evaluating the feasibility of the use of these two tests to analyze these kinds of data. Evaluation was carried out by comparing decisions made on the basis of the researcher's intuition to the results of statistical analysis. Application of these tests under the conditions of comparing the experimental data to the “historical” data, ra...