Phytochemical Constituent Analysis of Phyllanthus emblica L. Fruit Nanoherbals by LC-HRMS and Their Antimutagenic Activity and Teratogenic Effects.

Pregnant women must be wary of using traditional medicines due to the possibility of their having oxytoxic effects. Indonesia is rich in plants containing antioxidants. One of these plants is Phyllanthus emblica L. This study aims to determine the phytochemical constituents of Phyllanthus emblica L. fruit nanoherbals by LC-HRMS analysis and their antimutagenic activity and teratogenic effects. The study commenced by producing nanoherbal extracts from P. emblica fruit. The phytochemical composition of these extracts was then analyzed using LC-HRMS. The nanoherbal extracts were also tested for their ability to prevent mutations, as indicated by a reduction in micronuclei observed in mouse femur bone marrow smear preparations. The teratogenicity test involved administering the P. emblica fruit nanoherbal at 100, 500, and 1000 mg/kg BW doses. The data were analyzed using SPSS. The phytochemical constituents of the P. emblica fruit nanoherbal include flavonoids, phenols, vitamins, and alkaloids. The P. emblica fruit nanoherbal exhibits antimutagenic activity, as evidenced by a statistical analysis that indicated a significant decrease in the quantity of micronuclei per 200 PCE compared to the negative control (p < 0.05). The administration of the P. emblica fruit nanoherbal at a dosage of 1000 mg/kg BW resulted in a teratogenic impact during the organogenesis stage, as shown by hemorrhage and anomalies in the sternum.

Detrimental impacts and QSAR baseline toxicity assessment of Japanese medaka embryos exposed to methylparaben and its halogenated byproducts.

Despite the theoretical risk of forming halogenated methylparabens (halo-MePs) during water chlorination in the absence or presence of bromide ions, there remains a lack of in vivo toxicological assessments on vertebrate organisms for halo-MePs. This research addresses these gaps by investigating the lethal (assessed by embryo coagulation) or sub-lethal (assessed by hatching success/heartbeat rate) toxicity and teratogenicity (assessed by deformity rate) of MeP and its mono- and di-halogen derivatives (Cl- or Br-) using Japanese medaka embryos. In assessing selected apical endpoints to discern patterns in physiological or biochemical alterations, heightened toxic impacts were observed for halo-MePs compared to MeP. These include a higher incidence of embryo coagulation (4-36 fold), heartbeat rate decrement (11-36 fold), deformity rate increment (32-223 fold), hatching success decrement (11-59 fold), and an increase in Reactive Oxygen Species (ROS) level (1.2-7.4 fold)/Catalase (CAT) activity (1.7-2.8 fold). Experimentally determined LC50 values are correlated and predicted using a Quantitative Structure Activity Relationship (QSAR) based on the speciation-corrected liposome-water distribution ratio (Dlipw, pH 7.5). The QSAR baseline toxicity aligns well with (sub)lethal toxicity and teratogenicity, as evidenced by toxic ratio (TR) analysis showing TR < 10 for MeP exposure in all cases, while significant specific or reactive toxicity was found for halo-MeP exposure, with TR > 10 observed (excepting three values). Our extensive findings contribute novel insights into the intricate interplay of embryonic toxicity during the early-life-stage of Japanese medaka, with a specific focus on highlighting the potential hazards associated with halo-MePs compared to the parent compound MeP.

Physiopathologic Bases of Moebius Syndrome: Combining Genetic, Vascular, and Teratogenic Theories.

Moebius syndrome (MBS) is a congenital cranial dysinnervation disorder (CCDD) characterized by a bilateral palsy of abducens and facial cranial nerves, which may coexist with other cranial nerves palsies, mostly those found in the dorsal pons and medulla oblongata. MBS is considered a "rare" disease, occurring in only 1:50,000 to 1:500,000 live births, with no gender predominance. Three independent theories have been described to define its etiology: the vascular theory, which talks about a transient blood flow disruption; the genetic theory, which takes place due to mutations related to the facial motor nucleus neurodevelopment; and last, the teratogenic theory, associated with the consumption of agents such as misoprostol during the first trimester of pregnancy. Since the literature has suggested the existence of these theories independently, this review proposes establishing a theory by matching the MBS molecular bases. This review aims to associate the three etiopathogenic theories at a molecular level, thus submitting a combined postulation. MBS is most likely an underdiagnosed disease due to its low prevalence and challenging diagnosis. Researching other elements that may play a key role in the pathogenesis is essential. It is common to assume the difficulty that patients with MBS have in leading an everyday social life. Research by means of PubMed and Google Scholar databases was carried out, same in which 94 articles were collected by using keywords with the likes of "Moebius syndrome," "PLXND1 mutations," "REV3L mutations," "vascular disruption AND teratogens," and "congenital facial nerve palsy." No exclusion criteria were applied.

Prenatal Care Initiation and Exposure to Teratogenic Medications.

Importance: With new legal abortion restrictions, timing of prenatal care initiation is critical to allow for discussion of reproductive options among pregnancies exposed to teratogenic medications. Objective: To investigate the prevalence of prenatal exposure to teratogenic medications and prenatal care initiation across gestational weeks. Design, Setting, and Participants: This descriptive, population-based cross-sectional study used health encounter data from a national sample of individuals with employer-sponsored health insurance. A validated algorithm identified pregnancies among persons identifying as female that ended with a live or nonlive outcome between January 2017 and December 2019. Data were analyzed from December 2022 to December 2023. Exposures: Prenatal exposure to any of 137 teratogenic medications, measured via pharmacy and medical claims. Measurement of prenatal care initiation was adapted from the Children's Health Care Quality Measures. Main Outcomes and Measures: Prevalence of prenatal exposure to teratogens and prenatal care initiation by gestational week. Timing of prenatal teratogenic exposure was compared with timing of prenatal care initiation and legal abortion cutoffs. Results: Among 639 994 pregnancies, 472 472 (73.8%; 95% CI, 73.7%-73.9%) had a live delivery (mean [SD] age, 30.9 [5.4] years) and 167 522 (26.2%; 95% CI, 26.1%-26.3%) had a nonlive outcome (mean [SD] age, 31.6 [6.4] years). Of pregnancies with live deliveries, 5.8% (95% CI, 5.7%-5.8%) were exposed to teratogenic medications compared with 3.1% (95% CI, 3.0%-3.2%) with nonlive outcomes. Median time to prenatal care was 56 days (IQR, 44-70 days). By 6 weeks' gestation, 8186 pregnancies had been exposed to teratogenic medications (25.2% [95% CI, 24.7%-25.7%] of pregnancies exposed at any time during gestation; 1.3% [95% CI, 1.3%-1.3%] of all pregnancies); in 6877 (84.0%; 95% CI, 83.2%-84.8%), prenatal care was initiated after 6 weeks or not at all. By 15 weeks, teratogenic exposures had occurred for 48.9% (95% CI, 48.4%-49.5%) of all teratogen-exposed pregnancies (2.5% [2.4-2.5] of all pregnancies); prenatal care initiation occurred after 15 weeks for 1810 (16.8%; 95% CI, 16.1%-17.5%) with live deliveries and 2975 (58.3%; 95% CI, 56.9%-59.6%) with nonlive outcomes. Teratogenic medications most used within the first 15 gestational weeks among live deliveries included antiinfectives (eg, fluconazole), anticonvulsants (eg, valproate), antihypertensives (eg, lisinopril), and immunomodulators (eg, mycophenolate). For nonlive deliveries, most antihypertensives were replaced by vitamin A derivatives. Conclusions and Relevance: In this cross-sectional study, most exposures to teratogenic medications occurred in early pregnancy and before prenatal care initiation, precluding prenatal risk-benefit assessments. Prenatal care commonly occurred after strict legal abortion cutoffs, prohibiting consideration of pregnancy termination if concerns about teratogenic effects arose.

Embryos assist morphogenesis of others through calcium and ATP signaling mechanisms in collective teratogen resistance.

Information for organismal patterning can come from a variety of sources. We investigate the possibility that instructive influences for normal embryonic development are provided not only at the level of cells within the embryo, but also via interactions between embryos. To explore this, we challenge groups of embryos with disruptors of normal development while varying group size. Here, we show that Xenopus laevis embryos are much more sensitive to a diverse set of chemical and molecular-biological perturbations when allowed to develop alone or in small groups, than in large groups. Keeping per-embryo exposure constant, we find that increasing the number of exposed embryos in a cohort increases the rate of survival while incidence of defects decreases. This inter-embryo assistance effect is mediated by short-range diffusible signals and involves the P2 ATP receptor. Our data and computational model emphasize that morphogenesis is a collective phenomenon not only at the level of cells, but also of whole bodies, and that cohort size is a crucial variable in studies of ecotoxicology, teratogenesis, and developmental plasticity.

Topiramate ban in women of childbearing potential with idiopathic generalized epilepsy: Does effectiveness offset the teratogenic risks?

Regulatory agencies have recently discouraged the prescription of topiramate (TPM) to women of childbearing potential with epilepsy due to growing evidence of the teratogenic and neurodevelopmental risks associated with its use during pregnancy. It remains, however, unclear whether the use of TPM in this population can be supported to some extent by its high effectiveness. In this multicenter, retrospective, cohort study performed at 22 epilepsy centers, we investigated the comparative effectiveness of TPM and levetiracetam (LEV) given as first-line antiseizure medication in a cohort of women of childbearing potential with idiopathic generalized epilepsy (IGE). A total of 336 participants were included, of whom 24 (7.1%) received TPM and 312 (92.9%) LEV. Women treated with TPM had significantly higher risks of treatment failure and treatment withdrawal and were less likely to achieve seizure freedom at 12 months compared to women treated with LEV. In conclusion, this study highlighted a low tendency among clinicians to use TPM in women of childbearing potential with IGE, anticipating the recently released restrictions on its use. Furthermore, the available data on effectiveness do not appear to support the use of TPM in this population.

The Future of the Teratogenicity Testing.

The purpose of this review is to examine the importance, possible advantages and disadvantages of teratogenicity tests, and their future. For this purpose, numerous sources have been scanned in the field of teratogenicity. Although there are many methods related to teratogenic studies and very important studies have been made in this field, there are still serious deficiencies. There are advantages and disadvantages of in vitro and in vivo classical tests that have been used so far. The current status of in vivo tests is a matter of debate, especially due to the use of experimental animals. However, in vitro tests that do not perform the distribution and metabolism of chemicals also raise doubts in determination of teratogenicity. Despite the modern approaches of molecular biology and genetics and the best diagnostic techniques, the real cause of more than half of congenital diseases is still not understood. In this sense, the importance and necessity of teratogenic tests are understood once again. It is necessary to develop faster, reliable, and inexpensive techniques to replace traditional in vivo tests. It is important to disseminate harmless and reliable imaging techniques such as micro-CT. The use of European Center for the Validation of Alternative Methods (ECVAM) scientifically validated and approved in vitro tests such as embryonic stem cell test (EST), micro mass test (MM), and whole embryo culture (WEC) tests in routine screening can provide a solution in a shorter time than the classical tests. Improving these tests and developing new tests can help to solve the problem permanently.

Development Features on the Selection of Animal Models for Teratogenic Testing.

Today, the use of animal models from different species continues to represent a fundamental step in teratogenic testing, despite the increase in alternative solutions that provide an important screening to the enormous quantity of new substances that aim to enter the market every year. The maintenance of these models is due to the sharing of similar development processes with humans, and in this way they represent an important contribution to the safety in the use of the compounds tested. Furthermore, the application of advances in embryology to teratology, although hampered by the complexity of reproductive processes, continues to prove the importance of sensitivity during embryonic and fetal development to detect potential toxicity, inducing mortality/abortion and malformations.In this chapter, essential periods of development in different models are outlined, highlighting the similarities and differences between species, the advantages and disadvantages of each group, and specific sensitivities for teratogenic testing. Models can be divided into invertebrate species such as earthworms of the species Eisenia fetida/Eisenia andrei, Caenorhabditis elegans, and Drosophila melanogaster, allowing for rapid results and minor ethical concerns. Vertebrate nonmammalian species Xenopus laevis and Danio rerio are important models to assess teratogenic potential later in development with fewer ethical requirements. Finally, the mammalian species Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus, phylogenetically closer to humans, are essential for the assessment of complex specialized processes, occurring later in development.Regulations for the development of toxicology tests require the use of mammalian species. Although ethical concerns and costs limit their use in large-scale screening. On the other hand, invertebrate and vertebrate nonmammalian species are increasing as alternative animal models, as these organisms combine low cost, less ethical requirements, and culture conditions compatible with large-scale screening. Their main advantage is to allow high-throughput screening in a whole-animal context, in contrast to the in vitro techniques, not dependent on the prior identification of a target. Better knowledge of the development pathways of animal models will allow to maximize human translation and reduce the number of animals used, leading to a selection of compounds with an improved safety profile and reduced time to market for new drugs.

A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs.

Animal-based test systems have traditionally been used to screen for the potential teratogenic activity of drugs. Still, their deficits in predicting precise human-specific outcomes and ethical concerns have led to a need for alternative approaches. In vitro, teratogenicity testing using cell cultures or other in vitro systems is a potential alternative. Of the different in vitro platforms, the mouse embryonic stem cell test (mEST) is currently the most widely used and validated in vitro test for assessing the potential effects of teratogens on early embryonic development. The mEST involves exposing mouse embryonic stem cells to the test compound and monitoring their differentiation for several days.Nevertheless, its predictive ability was comparatively lower when distinguishing weak developmental toxicants from non-toxic substances. Since then, several modifications and adaptations of the mEST protocol have been developed. This chapter describes an alternative method based on molecular approaches to predict embryotoxicity. This method, originated from the mEST, analyzes the expression of differentiation genes involved in the development of mesoderm, endoderm, and stoderm and allows screening embryo-toxicants with different mechanisms of action. The hanging drops embryoid bodies used in the original mEST protocol have been replaced with monolayer culture, and thus the process has been shortened. In general, the method shows higher predictability compared with the traditional ones.

Machine Learning to Predict Teratogenicity: Theory and Practice.

Machine learning (ML) is a subfield of artificial intelligence (AI) that consists of developing algorithms that can automatically learn patterns and relationships from data, without being explicitly programmed. It continues to advance with the development of more sophisticated algorithms, increased computational power, and larger datasets, leading to significant advancements in AI technology. With the significant progress made in ML, the need to apply these systems in the area of teratogenicity is growing. It is sought as robust boosting methods to overcome many limitations and restrictions facing the experimental studies. By performing tasks such as classification, regression, clustering, anomaly detection, and decision systems, ML can be used to assess whether an agent is teratogen or not or to determine its teratogenic potential. It may also be used for the purpose of deciding on the use of medicinal products. In this chapter, we describe how ML can be used to investigate teratogenicity.

Teratogenic Effects of Drugs on Primary Lymphocytes Assessed by Flow Cytometry.

Peripheral blood lymphocytes as primary cells can be isolated from human, animal, fetus, and placenta. These cells are an excellent cellular model for the assessment of cytotoxicity, genotoxicity, oxidative stress, and mitochondrial and lysosomal dysfunction induced by drug and chemicals. Moreover, peripheral blood lymphocytes are an easily available source of primary cells appropriate for basic research and in cellular studies regarding teratogenic, genotoxic, and cytotoxic effect of drugs and chemicals. Most drugs and other chemicals that produce birth defects, known as teratogenic agents, produce reactive oxygen species (ROS) formation and mitochondrial and lysosomal dysfunction. It seems that there is an important mechanistic link between oxidative stress, mitochondrial damages, lysosomal integrity, and teratogenic drug-induced birth defects. One of the most sensitive periods in the embryo is transition from an important developmental event to another such as transition from proliferation to differentiation. Mitochondria, lysosomes, and cellular ROS have an important role in proliferative, differentiative, and apoptotic activities during the development. Therefore, disruption of the function of mitochondria, lysosomes, oxidative stress, and redox imbalance leads to cellular dysfunctions and subsequently poor developmental outcomes in the fetus. In this chapter, we will focus on evaluation of mitochondrial/lysosomal functions and estimation of ROS formation using flow cytometry methods in isolated lymphocytes and their isolated mitochondria.

Exploration of Teratogenic and Genotoxic Effects on Model Organism Drosophila melanogaster.

Drosophila melanogaster is one of the crucial in vivo models in terms of analyzing the toxicity of various unknown chemicals. Every part of the fly serves as a model in metabolic and therapeutic approaches. Genotoxic and teratogenic compounds are exposed to Drosophila through the oral route. Further, the toxicity of genotoxic compounds is analyzed in Drosophila's gut, hemolymph, and phenotype. The toxicity of teratogen compounds is also analyzed using a Drosophila embryo. The current chapter summarizes several techniques that are used to detect the genotoxicity and teratogenicity of any unknown compound in this model.

Daphnia magna as a Model Organism to Predict the Teratogenic Effect of Different Compounds.

For aquatic ecosystem Daphnia magna is evolving as a model organism to check the teratogenicity of numerous compounds. D. magna can be easily cultured in the laboratory, and the teratogen effect of several compounds can be easily studied. The developmental stages are well studied in D. magna. All the developmental stages are transparent so the defect can be easily accessed. So, the postembryonic developmental changes can be easily studied after the exposure with teratogen. More importantly, D. magna also have a swimming behavioral phenotype. The behavioral defect can be easily accessed after teratogen exposure. The current chapter summarizes numerous protocols associated with embryo and adult staining and adult behavioral assays that can be used to access the teratogenicity of any unknown compound.

Caenorhabditis elegans as an In Vivo Model Organism to Elucidate Teratogenic Effects.

Exogenous teratogens contribute to approximately 10% of the human abnormality with exposure occurrence during the prenatal and fetal period. However, the assessment methods and underlying mechanism remain unclear. The nematode Caenorhabditis elegans has been recognized as one of the ideal model animals for toxicologic research as convenient culture, low cost, and complete phenotypes and genomic profiling. This chapter describes the protocols about the estimations on the teratogenic effects using nematodes as model organisms, including the growth, development, behavior, reproduction, energy balance, and transgenes.

Biochemical Studies to Understand Teratogenicity and Lethality Outcomes in Modified-FETAX.

The frog embryo teratogenesis assay-Xenopus (FETAX) is a standardized test used to assess the toxic and teratogenic effects of xenobiotics. With this test, toxic and/or teratogenic concentrations of xenobiotic substances can be determined using morphological parameters such as lethality, length, and malformations in stage 8-11 Xenopus laevis embryos after 96 h exposure. These parameters enable the determination of the median lethal and effective concentrations (LC50 and EC50), minimum concentration to inhibit growth (MCIG), and teratogenic index of the tested chemical to reveal the short-term effects of relatively high concentrations. On the other hand, although FETAX provides quantitative and qualitative data on teratogenicity and toxicity, the biochemical and molecular mechanisms of these effects cannot be explained. Recent studies have tried to elucidate the mechanisms causing malformations and to explain the underlying causes of toxicity and teratogenicity by biochemical marker analysis. This chapter describes methods to analyze modified-FETAX and some detoxification and oxidative stress-related biomarkers during the early embryonic development of X. laevis.

High-Resolution Respirometry for the Assessment of Teratogenic Chemicals.

Pesticides are often used in agriculture and residential areas to mitigate pests and weeds. These chemicals can enter aquatic ecosystems via runoff and rain events, exerting negative effects on aquatic species. In rapidly developing fish embryos, metabolic disruption can alter the developmental trajectory and alter ATP levels. Therefore, it is important to quantify mitochondrial integrity in organisms following exposure to pesticides. To achieve this, a high throughput method to assess pesticide effects on oxidative phosphorylation and mitochondria has been optimized for fish embryos. Fish embryos are first exposed to pesticides for 24 or 48 h, and oxygen consumption rates are measured using the Seahorse XFe24/96 Flux Analyzer (formerly Seahorse Biosciences, now Agilent). The assay utilizes a single embryo and precisely measures oxygen consumption and extracellular acidification. Based upon these measurements, characteristics related to mitochondrial bioenergetics are calculated to provide information on mitochondrial integrity. Using this approach, one can identify pesticides affecting the electron transport chain and ultimately ATP production. In this chapter, we describe the mitochondrial stress test to understand mitochondrial dysfunction and metabolic shifts within the fish embryo.

Protocol of Geometric Morphometrics for Teratogenicity Testing.

Geometric morphometrics (GM) enables a quantitative study of shapes and forms allowing the identification and characterization of teratogenic malformations. The GM methodology offers several advantages in comparison to traditional biometric methods, such as higher detail and precision analysis. In this chapter, we describe the recent application of the Procrustes method with ImageJ and MorphoJ programs in the characterization of developmental malformations. With this methodology, we are a step closer to being able to assign molecular pathways or unique signatures to a specific teratogen according to the produced phenotypes or to cluster unknown compounds.

Teratogenic and neuro-behavioural toxic effects of bisphenol A (BPA) and B (BPB) on Xenopus laevis development.

Bisphenol A (BPA) is a plastic additive with endocrine disruptive activity, classified in 2017 by EU ECHA as substance of very high concern. A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species, including the amphibian Xenopus laevis. Among BPA analogues, bisphenol B (BPB) is used as alternative in different not-EU countries, including US, but seems to share with BPA its endocrine disruptor properties. Aim of the present work is the evaluation of the effects of BPB versus BPA exposure in a X. laevis developmental model. A windowed exposure (R-FETAX method) was applied covering the developmental phylotypic period (teratogenicity window), or the late tailbud stages (neuro-behavioural toxicity window, corresponding to the spontaneous swimming acquisition period). Samples were monitored for lethal effects during the full test period. External morphology evaluation and deglutition functional test were applied in any group. Abnormal tadpoles were also processed for cartilage staining. In groups exposed during neuro-behavioural toxicity window the swimming test was also applied. Lethality and malformations were obtained only in samples exposed during the teratogenicity window; these data were modelled using PROAST software and BPB relative potency resulted about 3 times higher than BPA. The day-by-day evaluation revealed that lethality was correlated to embryonic abnormal development of gills and apoptosis in gill primordia. Teratogenicity was never detected in groups exposed during the neuro-behavioural toxicity window, where some significant neuro-behavioural deficits were detected in tadpoles exposed to the highest tested concentrations of BPA and BPB.

Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline.

Zebrafish is a popular toxicology model and provides an ethically acceptable small-scale analysis system with the complexity of a complete organism. Our goal is to further validate this model for its regulatory use for reproductive and developmental defects by testing the compounds indicated in the "Guideline on detection of reproductive and developmental toxicity for human pharmaceuticals" (ICH S5(R3) guideline.) To determine the embryotoxic and developmental risk of the 32 reference compounds listed in the ICH S5(R3) guideline, the presence of morphological alterations in zebrafish embryos was analyzed at two different stages to calculateLC50 and EC50 values for each stage. Teratogenic Indexes were established as the ratio between LC50 and EC50 critical for the proper compound classification as teratogenic when it is ≥ 2. A total of three biological replicates have been conducted to study the reproducibility of the assay. The chemicals' concentration in the medium and internally in the zebrafish embryos was evaluated. In this study, the 3 negative compounds were properly categorized while 23 compounds out of the 29 reference ones (sensitivity of 79.31%) were classified as teratogenic in zebrafish. The 6 that had false-negative results were classified 4 as inconclusive, 1 as not toxic, and 1 compound resulted toxic for zebrafish embryos under testing conditions. After the bioavailability experiments, some of the obtained inconclusive results were refined. The developmental defects assay in zebrafish gives an accuracy of 89.66%, sensitivity of 88.46%, specificity and repeatability of 100% compared to mammals; therefore, this is a well-integrated strategy using New Alternative Methods, to minimize the use of animals in developmental toxicity studies.