From cells to tadpoles: mechanistic insights into ZnO-nanoparticle-induced oxidative stress and developmental toxicity in Bufo bufo.

n-Butanol is extensively used in industrial, food, pharmaceutical, and petrochemical sectors, raising concerns about environmental and developmental exposure. However, evidence regarding its developmental toxicity remains limited. This study investigated the teratogenic and toxicological effects of n-butanol using a chick embryo (Gallus gallus domesticus) model, with emphasis on dose-dependent morphological alterations, feeding, and locomotory behavioral outcomes, oxidative stress responses, and histopathological damage. Fifty fertilized eggs were randomly assigned to five groups (n = 10 per group): a control group (received an equivalent sterile saline) and four treatment groups receiving n-butanol at doses of 10, 12, 14, and 20 μL per egg, via in ovo injection. Embryonic development was evaluated through morphometric measurements, including body length, limb length, head and beak dimensions, crown-rump length, and body weight. Oxidative stress was assessed in cardiac and hepatic tissues by determining DPPH radical scavenging activity and the activities of superoxide dismutase (SOD) and catalase (CAT), complemented by histopathological examination. Exposure to n-butanol resulted in a significant, dose-dependent reduction in most morphometric parameters (p < 0.0001), whereas head circumference remained unaffected. Antioxidant enzyme activities were markedly altered, indicating enhanced oxidative stress at higher exposure levels. Histopathological analysis revealed pronounced structural damage in liver and heart tissues, accompanied by behavioral abnormalities and limb deformities at elevated doses. Overall, these findings demonstrate that n-butanol induces developmental toxicity in chick embryos in a dose-dependent manner, likely mediated through oxidative stress and tissue injury. The chick embryo model proved to be a sensitive and effective system for evaluating developmental and environmental toxicants, highlighting potential risks associated with n-butanol exposure.

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential. Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

Benzo[a]pyrene is a ubiquitous emerging contaminant; however, whether and how it disrupts fish development and skeletal health across multiple generations, particularly at the metabolomic level, remains unclear. The latent effects of embryonic benzo[a]pyrene exposure at environmentally relevant levels (2.5–80 μg/L) on developmental toxicity and skeletal integrity were assessed over three generations (F0–F2) of medaka. The F1 and F2 offspring were reared in clean water to isolate the inherited impacts. Untargeted metabolomic analysis (Variable Importance in Projection &gt; 1.0, <italic>p</italic> &lt; 0.05 in Student's <italic>t</italic>-test, and ratio ≥ 1.5 or ≤ 0.67) of F0 and F2 embryos identified 233 (86) and 254 (752) differentially abundant metabolites in the F0 (F2) embryos exposed ancestrally to benzo[a]pyrene at 2.5 and 20 μg/L. Benzo[a]pyrene exposure induced multigenerational impairments, including reduced survivorship and hatching success in F0 embryos, altered morphometrics (e.g., reduced body length, smaller eye pit, and larger pericardial cavity) in F0–F1 larvae, and elevated skeletal malformation frequency (e.g., craniofacial and spinal deformities) in F0–F2 larvae. Metabolomic perturbations were linked to four major biological processes: (1) metabolic activation and oxidative stress; (2) disruption of energy metabolism and redox balance, evidenced by a triad of elevated 3-hydroxybutyric acid, oxoglutaric acid, and depleted 2-hydroxyglutarate; (3) dysregulation of cell signaling and developmental programming, including energy crisis (elevated AMP), impaired vascular development (reduced prostaglandin I2), and disrupted efferocytosis; and (4) impairment of neurological and sensory functions. These findings demonstrate that F0 embryonic benzo[a]pyrene exposure induces lasting multigenerational toxicity by disrupting metabolic, signaling, and sensory pathways, ultimately leading to developmental impairment and osteotoxicity. While F2 larvae showed partial morphological recovery, this may represent a survival trade-off, where metabolic resources were redirected from skeletal development to support the formation of essential organs. This study identifies the metabolomic basis for the multigenerational developmental and skeletal toxicity induced by embryonic benzo[a]pyrene exposure.

Hinokitiol induces developmental and cardiovascular toxicity in zebrafish larvae and potential mechanisms.

Buprofezin causes early developmental toxicity of zebrafish (Danio rerio) embryos: morphological, physiological and biochemical responses.

Nano-titanium nitride (Nano-TiN) has strong resistance to wear and corrosion, good biocompatibility, and an attractive metallic luster. Nano-TiN is widely used in medical devices, such as orthopedic implants, syringe needles, coronary stents, and long-term dental implants, and also in imitation gold jewelry. Despite its widespread use, there are few reports describing safety evaluations of Nano-TiN. Here, we exposed healthy zebrafish embryos to different concentrations of Nano-TiN solution for five days, starting at about four hours post fertilization, and found that Nano-TiN caused dose- and time-dependent developmental toxicity. With increasing Nano-TiN concentration and length of exposure, mortality, and deformities gradually increased; body length shortened and hatching rate and motility were significantly reduced. We also found that exposure to Nano-TiN affected development of the heart, liver, nerves, and other organs, and led to elevated levels of reactive oxygen species and reduced antioxidant capacity. Exposure to Nano-TiN resulted in downregulation of expression of antioxidant genes, such as nrf2, gclc, gclm, ho-1, and nqo1. Our results showed that exposure to Nano-TiN caused developmental and organ toxicity in zebrafish embryos and that the toxic effects may be mediated through oxidative stress.

Members of the Teratology Society (established in 1960) were involved in the first governmental developmental and reproductive toxicity testing guidelines (1966) by FDA following the thalidomide epidemic, followed by other national and international governmental testing guidelines. The Segment II (developmental toxicity) study design, described in rodents and rabbits, has evolved with additional enhanced endpoints and better descriptions, mechanistic insights, range-finding studies, and toxico/pharmacokinetic ADME information (especially for pharmaceuticals). Society members were also involved in the development of the current screening assays and tests for endocrine disruptors (beginning in 1996) and are now involved with developing new testing guidelines (e.g., the extended one-generation protocol), and evaluating the current test guidelines and new initiatives under ILSI/HESI sponsorship. New initiatives include ToxCast from the U.S. EPA to screen, prioritize, and predict toxic chemicals by high throughput and high-content in vitro assays, bioinformation, and modeling to reduce (or eliminate) in vivo whole animal studies. Our Society and its journal have played vital roles in the scientific and regulatory accomplishments in birth defects research over the past 50 years and will continue to do so in the future. Happy 50th anniversary!

Profiling assay performance in the DevTox germ layer reporter platform.

Antibiotic residues in wastewater treatment plant (WWTP) effluents are a matter of global environmental health concern. This study combined global WWTP monitoring data with epidemiological modeling and zebrafish embryo assays to assess developmental risks. A meta-analysis of 121 studies identified amoxicillin, trimethoprim and norfloxacin as high priority contaminants. Ecological modeling using generalized additive models (GAM) revealed a statistical association between effluent antibiotic levels and neonatal disease incidence. Zebrafish exposed to environmentally relevant concentrations exhibited dose-dependent developmental toxicity, including pericardial edema, oxidative stress, and impaired cardiac function. Molecular analysis indicated dysregulation of the Wnt/β-catenin signaling pathway, with Wnt1 upregulation mediating toxic effects. Functional validation using CRISPR interference targeting wnt1 attenuated cardiac malformations and apoptosis, while docking simulations confirmed strong antibiotic-Wnt1 binding. These findings establish a mechanistic link between environmental antibiotic exposure and developmental toxicity and underscore the potential of Wnt1 as a biomarker for ecological risk assessment. This work supports the need for advanced tertiary treatment strategies and molecular-based monitoring frameworks.

Oxidative stress, unfolded protein response, and apoptosis in developmental toxicity.

Developmental toxicity of Zishen Guchong Pill on the early life stages of Zebrafish

Aged polystyrene microplastics exposure affects apoptosis via inducing mitochondrial dysfunction and oxidative stress in early life of zebrafish

Acetyl-11-keto-β-boswellic acid (AKBA), a triterpenoid from Boswellia serrate, is regarded as an angiogenesis inhibitor. However, its toxicity is unknown. The aim of this study was to examine its developmental toxicity and cardiotoxicity. A developmental toxicity assay in zebrafish embryos/larvae from 4 to 96 hours post-fertilization (hpf) was performed and a cardiotoxicity assay was designed from 48 to 72 hpf. Markers of oxidative stress and related genes were selected to access the possible mechanisms. According to the results, AKBA induced pericardium edema, yolk-sac edema, abnormal melanin, spinal curvature, hatching inhibition and shortened body length. Further, increased SV-BA distance, reduced heart rate, increased pericardium area and decreased blood flow velocity were detected in AKBA treated groups. The inhibition of cardiac progenitor gene expression, such as Nkx2.5 and Gata4, may be related to cardiotoxicity. The activities of antioxidant enzymes were decreased and the content of MDA was increased. In addition, AKBA treatment decreased the expression levels of Mn-Sod, Cat, and Gpx. These results suggested that AKBA induced developmental toxicity and cardiotoxicity through oxidative stress. As far as we know, this is the first report on the toxicity of AKBA. It reminds us to pay attention to developmental toxicity and cardiotoxicity of AKBA.

Background: Epilepsy is one of the most prevalent chronic neurologic disorders. Antiepileptic drugs (AEDs) may impact fetal development, however pregnant women are unable to discontinue using them to avoid seizures that could endanger the mother and the fetus. Forty percent of women need polytherapy to control seizures which make their fetus at a higher risk to have deformities than for those who receive monotherapy. Both Lamotrigine and Gabapentin are among the new AEDs, that are relatively safe however when used combined there is increasing evidence to have adverse reproductive effects especially neurological congenital malformations. Vitamin C, a hydrophilic potent antioxidant, can cross the placental and blood brain barrier. Aim: This research aims to assess the antioxidant effect of VitC on AEDs polytherapy. Material and Methods: Forty-eight pregnant mice were categorized into 3 groups of 8 pregnant mice each: group I (control), group II (Lamotrigine 20 mg/kg and Gabapentin 200 mg/kg), group III (Vitamin C 40mg/kg with Lamotrigine and Gabapentin) from gestational days (GD) 6-15. On GD 18 they were sacrificed and the uteri and placentae examined and blood collected to assess oxidant markers while fetuses were removed for gross, skeletal and histopathological examinations. Results: AEDs polytherapy caused significant signs of developmental toxicity as decreased maternal body weight, decrease in absolute and relative placental weight. Also, it caused significant decrease in number of implantations, live fetuses, fetal weights and increase in number of resorptions. MDA was increased, while SOD decreased thus oxidative stress. Gross examination showed reduced size of fetuses with not well-developed limbs. Stereomicroscopic findings of the skeleton revealed incomplete ossification of parts of the skeleton. Light microscopic examination of the frontal cortex showed loss of neurons with dark pyknotic nuclei and vacuolization. Ultrastructural examination revealed the nucleus with chromatin aggregates, clumps and crescents, vacuolated organelles in cytoplasm. Administration of Vitamin C caused significant increase in maternal and fetal parameters, decrease in oxidative stress, accompanied by marked improvements in gross and histological findings. Conclusion: Vitamin C regimen appeared to be protective against AEDs induced developmental toxicity, therefore intake of Vitamin C as adjunctive therapy to AEDs should be mandatory in pregnant epileptic women. KEY WORDS: Antiepileptic drugs, Vitamin C, Oxidative stress, developmental toxicity, congenital malformations, Electron Microscopy of the brain, Neuroprotection.

Developmental and cardiac toxicity assessment of Ethyl 3-(N-butylacetamido) propanoate (EBAAP) in zebrafish embryos