One-Step Organic Synthesis of 18ß-Glycyrrhetinic Acid-Anthraquinone Ester Products: Exploration of Antibacterial Activity and Structure-Activity Relationship, Toxicity Evaluation in Zebrafish.

To combine the activity characteristics of 18ß-glycyrrhetinic acid (18ß-GA) and anthraquinone compounds (rhein and emodin), reduce toxicity, and explore the structure-activity relationship (SAR) of anthraquinones, 18ß-GA-anthraquinone ester compounds were synthesized by one-step organic synthesis. The products were separated and purified by HPLC and characterized by NMR and EI-MS. It was finally determined as di-18ß-GA-3-rhein ester (1, New), GA dimer (2, known), 18ß-GA-3-emodin ester (3, known), and di-18ß-GA-1-emodin ester (4, new). The MIC of three reactants and four products against Escherichia coli and Staphylococcus aureus were detected in vitro. Its developmental toxicity and cardiotoxicity were assessed using zebrafish embryos. The experimental results showed that rhein had the best antibacterial activity against Staphylococcus aureus with MIC50 of 2.4 mM, and it was speculated that -COOH, -OH, and intramolecular hydrogen bonds in anthraquinone compounds would enhance the antibacterial effect, while the presence of-CH3 might weaken the antibacterial activity. Product 1 increased the hatching rate and survival rate of zebrafish embryos and reduced the malformation rate and cardiomyocyte apoptosis. This experiment lays the foundation for further studying the SAR of anthraquinones and providing new drug candidates.

Investigation of the biocompatibility of various pulp capping materials on zebrafish model.

Testing the biocompatibility of commercially available dental materials is a major challenge in dental material science. In the present study, the biocompatibility of four commercially available dental materials Mineral Trioxide Aggregate, Biodentine, Harvard BioCal-CAP and Oxford ActiveCal PC was investigated. The biocompatibility analysis was performed on zebrafish embryos and larvae using standard toxicity tests such as survivability and hatching rates. Comparative toxicity analysis of toxicity was performed by measuring apoptosis using acridine orange dye and whole mount immunofluorescence methods on zebrafish larvae exposed to the dental materials at different dilutions. Toxicity analysis showed a significant decrease in survival and hatching rates with increasing concentration of exposed materials. The results of the apoptosis assay with acridine orange showed greater biocompatibility of Biodentine, Oxford ActiveCal PC, Harvard BioCal-CAP and Biodentine compared to MTA, which was concentration dependent. Consequently, this study has shown that showed resin-modified calcium silicates are more biocompatible than traditional calcium silicates.

Rearing conditions (isolated versus group rearing) affect rotenone-induced changes in the behavior of zebrafish (Danio rerio) embryos in the coiling assay.

Under regulations such as REACH, testing of novel and established compounds for their (neuro)toxic potential is a legal requirement in many countries. These are largely based on animal-, cost-, and time-intensive in vivo models, not in line with the 3 Rs' principle of animal experimentation. Thus, the development of alternative test methods has also received increasing attention in neurotoxicology. Such methods focus either on physiological alterations in brain development and neuronal pathways or on behavioral changes. An example of a behavioral developmental neurotoxicity (DNT) assay is the zebrafish (Danio rerio) embryo coiling assay, which quantifies effects of compounds on the development of spontaneous movement of zebrafish embryos. While the importance of embryo-to-embryo contact prior to hatching in response to environmental contaminants or natural threats has been documented for many other clutch-laying fish species, little is known about the relevance of intra-clutch contacts for zebrafish. Here, the model neurotoxin rotenone was used to assess the effect of grouped versus separate rearing of the embryos on the expression of the coiling behavior. Some group-reared embryos reacted with hyperactivity to the exposure, to an extent that could not be recorded effectively with the utilized software. Separately reared embryos showed reduced activity, compared with group-reared individuals when assessing. However, even the control group embryos of the separately reared cohort showed reduced activity, compared with group-reared controls. Rotenone could thus be confirmed to induce neurotoxic effects in zebrafish embryos, yet modifying one parameter in an otherwise well-established neurotoxicity assay such as the coiling assay may lead to changes in behavior influenced by the proximity between individual embryos. This indicates a complex dependence of the outcome of behavior assays on a multitude of environmental parameters.

Transcriptomic Point of Departure (tPOD) of androstenedione in zebrafish embryos as a potential surrogate for chronic endpoints.

The transcriptomic Point of Departure (tPOD) is increasingly used in ecotoxicology to derive quantitative endpoints from RNA sequencing studies. Utilizing transcriptomic data in zebrafish embryos as a New Approach Methodology (NAM) is beneficial due to its acknowledgment as an alternative to animal testing under EU Directive 2010/63/EU. Transcriptomic profiles are available in zebrafish for various modes of action (MoA). The limited literature available suggest that tPOD values from Fish Embryo Toxicity (FET) tests align with, but are generally lower than, No Observed Effect Concentrations (NOEC) from long-term chronic fish toxicity tests. In studies with the androgenic hormone androstenedione in a Fish Sexual Development Test (FSDT), a significant shift in the sex ratio towards males was noted at all test concentrations, making it impossible to determine a NOEC (NOEC <4.34 µg/L). To avoid additional animal testing in a repetition of the FSDT and adhere to the 3Rs principle (replacement, reduction, and refinement), a modified zebrafish FET (zFET) was conducted aiming to determine a regulatory acceptable effect threshold. This involved lower concentration ranges (20 to 6105 ng/L), overlapping with the masculinization-observed concentrations in the FSDT. The tPOD analysis in zFET showed consistent results with previous FSDT findings, observing strong expression changes in androgen-dependent genes at higher concentrations but not at lower ones, demonstrating a concentration-response relationship. The tPOD values for androstenedione were determined as 24 ng/L (10th percentile), 60 ng/L (20th gene), and 69 ng/L (1st peak). The 10th percentile tPOD value in zFET was 200 times lower than the lowest concentration in the FSDT. Comparing the tPOD values to literature suggests their potential to inform on the NOEC range in FSDT tests.

Melatonin prevents the transgenerational toxicity of nanoplastics in zebrafish (Danio rerio).

As a novel pollutant, microplastic pollution has become a global environmental concern. Melatonin (MT) has a protective effect on the damage caused by pollutants. However, there is still a lack of research on the transgenerational toxicity of microplastics and the alleviation of microplastics toxicity by MT. In this study, the adult zebrafish was exposed to (0, 0.1 and 1 mg/L) polystyrene nanoplastics (PSNP) with or without (1 µM) MT for 14 days, and embryos (F1) were used for experiments. Our study found that long-term exposure of parents to 1 mg/L PSNP reduced fertilization rate and survival rate of offspring, increased the deformity rate and induced embryos to hatch in advance. The growth inhibition of offspring was related to the gene transcription of the growth hormone/insulin-like growth factor axis. Moreover, PSNP caused oxidative stress in offspring, damaged immune system, reduced antioxidant capacity and induced apoptosis. MT supplementation could effectively alleviate the developmental toxicity and oxidative damage of offspring, but the negative effects brought by PSNP could not be completely eliminated. Our research provided a new reference for the protective effect of MT on transgenerational toxicity induced by PSNP.

Chemical toxicity of leachates from synthetic and natural-based spat collectors on the embryo-larval development of the pearl oyster, Pinctada margaritifera.

In French Polynesia, the pearl farming industry relies entirely on collecting natural spat using a shade-mesh collector, which is reported to contribute to both plastic pollution and the release of toxic chemicals. With the aim of identifying more environment-friendly collectors, this study investigates the chemical toxicity of shade-mesh (SM) and alternative materials, including reusable plates (P), a newly developed biomaterial (BioM) and Coconut coir geotextile (Coco), on the embryo-larval development of Pinctada margaritifera. Embryos were exposed during 48 h to four concentrations (0, 0.1, 10 and 100 g L-1) of leachates produced from materials. Chemical screening of raw materials and leachates was performed to assess potential relationships with the toxicity observed on D-larvae development. Compared to the other tested materials, results demonstrated lower levels of chemical pollutants in BioM and no toxic effects of its leachates at 10 g L-1. No toxicity was observed at the lowest tested concentration (0.1 g L-1). These findings offer valuable insights for promoting safer spat collector alternatives such as BioM and contribute to the sustainable development of pearl farming.

Silver Nanoparticles Exposure Impairs Cardiac Development by Suppressing the Focal Adhesion Pathway in Zebrafish.

Introduction: The potential toxic effects of wastewater discharges containing silver nanoparticles (AgNPs) and their release into aquatic ecosystems on aquatic organisms are becoming a major concern for environmental and human health. However, the potential risks of AgNPs to aquatic organisms, especially for cardiac development by Focal adhesion pathway, are still poorly understood. Methods: The cardiac development of various concentrations of AgNPs in zebrafish were examined using stereoscopic microscope. The expression levels of cardiac development-related genes were analyzed by qRT-PCR and Whole-mount in situ hybridization (WISH). In addition, Illumina high-throughput global transcriptome analysis was performed to explore the potential signaling pathway involved in the treatment of zebrafish embryos by AgNPs after 72 h. Results: We systematically investigated the cardiac developing toxicity of AgNPs on the embryos of zebrafish. The results demonstrated that 2 or 4 mg/L AgNPs exposure induces cardiac developmental malformations, such as the appearance of pericardial edema phenotype. In addition, after 72 h of exposure, the mRNA levels of cardiac development-related genes, such as myh7, myh6, tpm1, nppa, tbx5, tbx20, myl7 and cmlc1, were significantly lower in AgNPs-treated zebrafish embryos than in control zebrafish embryos. Moreover, RNA sequencing, KEGG (Kyoto Encyclopedia of Genes) and Genomes and GSEA (gene set enrichment analysis) of the DEGs (differentially expressed genes) between the AgNPs-exposed and control groups indicated that the downregulated DEGs were mainly enriched in focal adhesion pathways. Further investigations demonstrated that the mRNA levels of focal adhesion pathway-related genes, such as igf1ra, shc3, grb2b, ptk2aa, akt1, itga4, parvaa, akt3b and vcla, were significantly decreased after AgNPs treatment in zebrafish. Conclusion: Thus, our findings illustrated that AgNPs could impair cardiac development by regulating the focal adhesion pathway in zebrafish.

Impact of the novel chlorinated polyfluorinated ether sulfonate, F-53B, on gill structure and reproductive toxicity in zebrafish.

6:2 Chlorinated polyfluorinated ether sulfonate, commonly known as F-53B, is widely used as a mist suppressant in various industries and is frequently detected in the environment. Despite its prevalent presence, the adverse effects of F-53B are not well understood and require future investigation. This study utilized zebrafish embryos and adults to examine the toxic effects of F-53B. Our findings revealed that F-53B impaired gill structure and increased erythrocyte numbers in adult zebrafish. Notably, F-53B demonstrated a higher sensitivity for inducing mortality (LC50 at 96 h) in adult zebrafish compared to embryos. Additionally, F-53B disrupted the expression of critical steroidogenic genes and hindered sex hormone production, which negatively affecting egg production. In conclusion, this study underscores the detrimental impact of F-53B on gill structure and reproductive toxicity in zebrafish, providing valuable insights into its overall toxicity.

Developmental Toxicity and Apoptosis in Zebrafish: The Impact of Lithium Hexafluorophosphate (LiPF6) from Lithium-Ion Battery Electrolytes.

With the growing dependence on lithium-ion batteries, there is an urgent need to understand the potential developmental toxicity of LiPF6, a key component of these batteries. Although lithium's toxicity is well-established, the biological toxicity of LiPF6 has been minimally explored. This study leverages the zebrafish model to investigate the developmental impact of LiPF6 exposure. We observed morphological abnormalities, reduced spontaneous movement, and decreased hatching and swim bladder inflation rates in zebrafish embryos, effects that intensified with higher LiPF6 concentrations. Whole-mount in situ hybridization demonstrated that the specific expression of the swim bladder outer mesothelium marker anxa5b was suppressed in the swim bladder region under LiPF6 exposure. Transcriptomic analysis disclosed an upregulation of apoptosis-related gene sets. Acridine orange staining further supported significant induction of apoptosis. These findings underscore the environmental and health risks of LiPF6 exposure and highlight the necessity for improved waste management strategies for lithium-ion batteries.

Effect of High Glucose on Embryological Development of Zebrafish, Brachyodanio, Rerio through Wnt Pathway.

Gestational diabetes mellitus (GDM) is a worldwide pregnancy complication. Gestational diabetes can significantly impact fetus development. However, the effects of high glucose on embryological development post-fertilization are yet to be researched. Danio rerio embryos are a great model for studying embryonic development. In this study, the effects on embryological (morphological and genetic) development were examined in the presence of a high-glucose environment that mimics the developing fetus in pregnant women with GDM. Fertilized zebrafish embryos were treated with normal media and high glucose for 5 days from 3 h post-fertilization (hpf) to 96 hpf, respectively, as control and experimental groups. Morphological changes are recorded with microscope images. Hatch rate and heart rate are compared between groups at set time points. RNA-Seq is performed to examine the gene changes in the experimental group. Glucose delayed the zebrafish embryo development by slowing the hatch rate by about 24 h. The brain, heart, and tail started showing smaller morphology in the glucose group compared to the control group at 24 hpf. Heart rate was faster in the glucose group compared to the control group on days 2 and 3 with a statistically significant difference. Among the zebrafish whole genome, the significantly changed genes were 556 upregulated genes and 1118 downregulated genes, respectively, in the high-glucose group. The metabolic and Wnt pathways are altered under high-glucose conditions. These conditions contribute to significant physiological differences that may provide insight into the functionality of post-embryological development.

Eco-corona enhanced the interactive effects of nanoplastics and 6:2 chlorinated polyfluorinated ether sulfonate in zebrafish embryos.

Nanoplastics (NPs, < 1000 nm) interact with chemicals and biomolecules to produce chemical-/eco-corona, altering the environmental destiny, bioavailability, and toxicity of plastic particles and co-occurring chemicals. This study employs exogenous (humic acid, HA) and endogenous (bovine serum albumin, BSA) natural organic matter (NOM) to investigate the eco-corona formation on NPs and explore the interfacial effects of eco-corona and 6:2 chlorinated polyfluorinated ether sulfonate (Cl-PFESA, commonly named as F-53B) on zebrafish (Danio rerio) after 7 days of exposure. Our results indicated significant changes in growth and developmental indices of zebrafish embryos among all eco-corona groups (p < 0.05). Additionally, NFB (BSA-corona, 1 mg/L NPs + 200 µg/L F-53B + 10 mg/L BSA), NFH (HA-corona, 1 mg/L NPs + 200 µg/L F-53B + 10 mg/L HA) and NFHB (BSA-HA-corona, 1 mg/L NPs + 200 µg/L F-53B + 10 mg/L BSA + 10 mg/L HA) showed elevated bioaccumulation of NPs, ROS generation and induction of apoptosis. Transcriptomic analysis showed the number of differentially expressed genes (DEGs) in the following order: BSA-HA-corona (NFHB (2953) > HA-corona (NFH (2797) > NH (2721) > F-53B (2292) > NF (2033) > BSA-corona (NFB (687) > NB (450)), and no DEGs were detected in the single NP compared to the control. Further, the PI3K-AKT, immune system, endocrine system, digestive system, infectious diseases, and neurovegetative disease pathways showed sensitive responses in the NFH/NFHB groups compared to those in the NFB group. Therefore, the interactive effects of NPs and F-53B on zebrafish embryos were lower in the presence of BSA-corona than those in HA- or HA-BSA-coronas, indicating a relationship between the formation of diverse eco-coronas on NPs by multiple NOM and an associated increase in the interfacial toxicological effects of plastic particles and F-35B in freshwater organisms.

Use of Zebrafish (Danio rerio) for Biosafety Evaluation of Strontium Nanostructured Hydroxyapatite.

Despite the numerous studies on biocompatibility with nano-biomaterials, the biological effects of strontium-substituted HA nanoparticles (nSrHA) need to be better understood. So, we conducted an embryotoxicity test using zebrafish (Danio rerio) according to the OECD 236 guideline, a model that represents a viable alternative that bridges the gap between in vitro and mammalian models. Zebrafish embryos were exposed for 120 h to microspheres containing nSrHA nanoparticles with low and high crystallinity, synthesized at temperatures of 5°C (nSrHA5) and 90°C (nSrHA90). We evaluated lethality, developmental parameters, and reactive oxygen species (ROS) production. The larval behavior was assessed at 168 hpf to determine if the biomaterials affected motor responses and anxiety-like behavior. The results showed that the survival rate decreased significantly for the nSrHA5 group (low crystalline particles), and an increase in ROS was also observed in this group. However, none of the biomaterials caused morphological changes indicative of toxicity during larval development. Additionally, the behavioral tests did not reveal any alterations in all experimental groups, indicating the absence of neurotoxic effects from exposure to the tested biomaterials. These findings provide valuable insights into the biosafety of modified HA-based nanostructured biomaterials, making them a promising strategy for bone tissue repair. As the use of hydroxyapatite-based biomaterials continues to grow, it is crucial to ensure rigorous control over the quality, reliability, and traceability of these materials.

Imatinib­ and ponatinib­mediated cardiotoxicity in zebrafish embryos and H9c2 cardiomyoblasts.

Tyrosine kinase inhibitors (TKIs) offer targeted therapy for cancers but can cause severe cardiotoxicities. Determining their dose­dependent impact on cardiac function is required to optimize therapy and minimize adverse effects. The dose­dependent cardiotoxic effects of two TKIs, imatinib and ponatinib, were assessed in vitro using H9c2 cardiomyoblasts and in vivo using zebrafish embryos. In vitro, H9c2 cardiomyocyte viability, apoptosis, size, and surface area were evaluated to assess the impact on cellular health. In vivo, zebrafish embryos were analyzed for heart rate, blood flow velocity, and morphological malformations to determine functional and structural changes. Additionally, reverse transcription­quantitative PCR (RT­qPCR) was employed to measure the gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), established markers of cardiac injury. This comprehensive approach, utilizing both in vitro and in vivo models alongside functional and molecular analyses, provides a robust assessment of the potential cardiotoxic effects. TKI exposure decreased viability and surface area in H9c2 cells in a dose­dependent manner. Similarly, zebrafish embryos exposed to TKIs exhibited dose­dependent heart malformation. Both TKIs upregulated ANP and BNP expression, indicating heart injury. The present study demonstrated dose­dependent cardiotoxic effects of imatinib and ponatinib in H9c2 cells and zebrafish models. These findings emphasize the importance of tailoring TKI dosage to minimize cardiac risks while maintaining therapeutic efficacy. Future research should explore the underlying mechanisms and potential mitigation strategies of TKI­induced cardiotoxicities.

Acute toxicity of deoxynivalenol and bioremediation of a highly effective deoxynivalenol degrading Achromobacter spanius P-9 on zebrafish embryos and adults.

Deoxynivalenol (DON) is one of the mostly concerned mycotoxins and several microbes showed bioremediation effects on DON toxic effects. In this study, the acute toxicity of a new DON degrading strain Achromobacter spanius P-9 with DON on zebrafish embryos and adults were firstly performed. For zebrafish embryos, bacterial concentrations of 2.5 × 107 CFU/mL and 5.0 × 107 CFU/mL had no significant effects on growth and development. However, at 7.5 × 107 CFU/mL, some effects were observed, and at 10.0 × 107 CFU/mL, the embryo survival rate decreased to 70%, with 3.3% teratogenicity. Higher bacterial concentrations correlated with faster heart rates. DON (100 µg/mL) significantly reduced embryo survival to 36.7% in 96 h. Bacterial solutions at 7.5 × 107 CFU/mL and 10.0 × 107 CFU/mL expanded the zebrafish intestinal tissue wall, while DON at 100 µg/mL negatively impacted intestinal morphology. Liver tissue in zebrafish exposed to Achromobacter spanius P-9 showed no significant differences from the control group. However, exposure to DON solution increased liver fluorescence intensity and caused liver cell changes, including edema, vacuolization, and blurred boundaries. For adult zebrafish, the ROS and 8-OHdG contents in the exposure group increased with the increase of bacterial solution concentration, the SOD enzyme activity, CAT enzyme activity, GST enzyme activity and MDA was not significantly different with the control group. Compared with the control group, the content of ROS, GST enzyme activity, MDA and 8-OHdG after DON treatment showed an upward trend, SOD and CAT enzyme activities showed a decreasing trend. Achromobacter spanius P-9 has no obvious inhibitory effect on the growth and development of zebrafish embryos and has no obvious death and toxicity during the growth of adult fish, providing data support for the future application of this strain in the biodegradation of DON.

Toxic Effects of Biogenic and Synthesized Silver Nanoparticles on Sea Urchin Echinometra lucunter Embryos.

Due to their broad-spectrum antimicrobial action and ease of synthesis, silver nanoparticles (AgNP) are one of the most widely used nanomaterials in different industrial and ecological areas. AgNP are released into marine ecosystems, nevertheless, their ecotoxicological effects have been overlooked. In this study, we evaluated the toxic effects of biogenic and synthesized AgNP (AgNPIBCLP11 and AgNPSINT) on sea urchin Echinometra lucunter embryos and compared them with the metal precursor silver nitrate (AgNO3). Fertilized eggs were exposed to five concentrations of the test compounds and a negative control for 48 h under controlled conditions. The IC50-48 h of AgNPIBCLP11, AgNPSINT and AgNO3 were 0.31, 4.095, and 0.01 µg L-1, evidencing that both AgNP are less toxic than AgNO3, and that AgNPSINT is less toxic than the AgNPIBCLP11. Toxicity to E. lucunter embryos could be explained by the fact that Ag affects DNA replication and induces the formation of pores in the cellular wall, leading to apoptosis.

Assessment of the Aryl Hydrocarbon Receptor-Mediated Effects of Aromatic Sensitizers in Paper Recycling Effluent Employing Zebrafish Embryos and in Silico Docking.

Aromatic sensitizers and related substances (SRCs), which are crucial in the paper industry for facilitating color-forming and color-developing chemical reactions, inadvertently contaminate effluents during paper recycling. Owing to their structural resemblance to endocrine-disrupting aromatic organic compounds, concerns have arisen about potential adverse effects on aquatic organisms. We focused on SRC effects via the aryl hydrocarbon receptor (AHR), employing molecular docking simulations and zebrafish (Danio rerio) embryo exposure assessments. Molecular docking revealed heightened binding affinities between certain SRCs in the paper recycling effluents and zebrafish Ahr2 and human AHR, which are pivotal components in the SRC toxicity mechanism. Fertilized zebrafish eggs were exposed to SRCs for up to 96 h post fertilization; among these substances, benzyl 2-naphthyl ether (BNE) caused morphological abnormalities, such as pericardial edema and shortened body length, at relatively low concentrations (1 µM) during embryogenesis. Gene expression of cytochrome P450 1A (cyp1a) and ahr2 was also significantly increased by BNE. Co-exposure to the AHR antagonist CH-223191 only partially mitigated BNE's phenotypic effects, despite the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin being relatively well restored by CH-223191, indicating BNE's AHR-independent toxic mechanisms. Furthermore, some SRCs, including BNE, exhibited in silico binding affinity to the estrogen receptor and upregulation of cyp19a1b gene expression. Therefore, additional insights into the toxicity of SRCs and their mechanisms are essential. The present results provide important information on SRCs and other papermaking chemicals that could help minimize the environmental impact of the paper industry. Environ Toxicol Chem 2024;43:2176-2188. © 2024 SETAC.

A Quantitative Adverse Outcome Pathway for Embryonic Activation of the Aryl Hydrocarbon Receptor of Fishes by Polycyclic Aromatic Hydrocarbons Leading to Decreased Fecundity at Adulthood.

Quantitative adverse outcome pathways (qAOPs) describe the response-response relationships that link the magnitude and/or duration of chemical interaction with a specific molecular target to the probability and/or severity of the resulting apical-level toxicity of regulatory relevance. The present study developed the first qAOP for latent toxicities showing that early life exposure adversely affects health at adulthood. Specifically, a qAOP for embryonic activation of the aryl hydrocarbon receptor 2 (AHR2) of fishes by polycyclic aromatic hydrocarbons (PAHs) leading to decreased fecundity of females at adulthood was developed by building on existing qAOPs for (1) activation of the AHR leading to early life mortality in birds and fishes, and (2) inhibition of cytochrome P450 aromatase activity leading to decreased fecundity in fishes. Using zebrafish (Danio rerio) as a model species and benzo[a]pyrene as a model PAH, three linked quantitative relationships were developed: (1) plasma estrogen in adult females as a function of embryonic exposure, (2) plasma vitellogenin in adult females as a function of plasma estrogen, and (3) fecundity of adult females as a function of plasma vitellogenin. A fourth quantitative relationship was developed for early life mortality as a function of sensitivity to activation of the AHR2 in a standardized in vitro AHR transactivation assay to integrate toxic equivalence calculations that would allow prediction of effects of exposure to untested PAHs. The accuracy of the predictions from the resulting qAOP were evaluated using experimental data from zebrafish exposed as embryos to another PAH, benzo[k]fluoranthene. The qAOP developed in the present study demonstrates the potential of the AOP framework in enabling consideration of latent toxicities in quantitative ecological risk assessments and regulatory decision-making. Environ Toxicol Chem 2024;43:2145-2156. © 2024 SETAC.

Cannabidiol exposure during embryonic period caused serious malformation in embryos and inhibited the development of reproductive system in adult zebrafish.

Cannabidiol (CBD) is a non-psychoactive component of cannabis with potential applications in biomedicine, food, and cosmetics due to its analgesic, anti-inflammatory, and anticonvulsant properties. However, increasing reports of adverse CBD exposure events underscore the necessity of evaluating its toxicity. In this study, we investigated the developmental toxicity of CBD in zebrafish during the embryonic (0-4 dpf, days post fertilization) and early larval stages (5-7 dpf). The median lethal concentration of CBD in embryos/larvae is 793.28 µg/L. CBD exhibited concentration-dependent manner (ranging from 250 to 1500 µg/L) in inducing serious malformed somatotypes, like shorter body length, pericardial cysts, vitelline cysts, spinal curvature, and smaller eyes. However, no singular deformity predominates. The 5-month-old zebrafish treated with 100 and 200 µg/L of CBD during the embryonic and early larval stages produced fewer offspring with higher natural mortality and malformation rate. Gonadal growth and gamete development were inhibited. Transcriptomic and metabolomic analyses conducted with 400 µg/L CBD on embryos/larvae from 0 to 5 dpf suggested that CBD promoted the formation and transportation of extracellular matrix components on 1 dpf, promoting abnormal cell division and migration, probably resulting in random malformed somatotypes. It inhibited optical vesicle development and photoreceptors formation on 2 and 3 dpf, resulting in damaged sight and smaller eye size. CBD also induced an integrated stress response on 4 and 5 dpf, disrupting redox, protein, and cholesterol homeostasis, contributing to cellular damage, physiological dysfunction, embryonic death, and inhibited reproductive system and ability in adult zebrafish. At the tested concentrations, CBD exhibited developmental toxicity, lethal toxicity, and reproductive inhibition in zebrafish. These findings demonstrate that CBD threatens the model aquatic animal, highlighting the need for additional toxicological evaluations of CBD before its inclusion in dietary supplements, edible food, and other products.

Effects of 2,4,6-Trichloroanisole on the morphological development and motility of zebrafish.

2,4,6-Trichloroanisole (2,4,6-TCA), a compound with a characteristic earthy odor, is a common source of odorous pollutants in drinking water and wine. However, research on its biological toxicity is limited. In this study, we used zebrafish as an indicator model to investigate the effects of 2,4,6-TCA exposure on morphological development, oxidative stress, apoptosis, heart rate, blood flow, and motility. We found that exposure to 2,4,6-TCA resulted in significant spinal, tail, and cardiac deformities in zebrafish larvae and promoted a pronounced oxidative stress response and extensive cell apoptosis, notably in the digestive tract, head, spine, and heart, ultimately leading to significant reductions in zebrafish heart rate, blood flow, and motility. Moreover, these effects became more pronounced with an increase in the concentration of 2,4,6-TCA to which the zebrafish were exposed. Furthermore, qPCR analysis revealed that exposure to 2,4,6-TCA promoted significant changes in the expression levels of genes associated with oxidative stress, apoptosis, cardiac development, and the nervous system, particularly key genes (p53, apaf1, casp9, and casp3) in the mitochondrial apoptotic pathway, which were significantly upregulated. Similarly, we detected significant upregulation of ache gene expression. These findings indicated that exposure to 2,4,6-TCA resulted in the accumulation of reactive oxygen species in zebrafish, induced strong oxidative stress responses, and triggered lipid peroxidation and extensive cell apoptosis. Cellular apoptosis, which mitochondrial signaling pathways may mediate, has been found to lead to malformations in zebrafish embryos, resulting in significant reductions in cardiac function and motility. To our knowledge, this is the first systematic assessment of the toxicity of 2,4,6-TCA, and our findings provide an important reference for risk assessment and early warning of 2,4,6-TCA exposure.

Parental (F0) exposure to Cadmium and Ketoprofen induces developmental deformities in offspring (F1): A transgenerational toxicity assessment in zebrafish model.

In the aquatic environment, the primary pollutants of heavy metals and pharmaceuticals always occur in coexisting forms, and the research about combined impacts remains unclear, especially transgenerational effects. Cadmium (Cd) is a heavy metal that can damage the endocrine reproduction systems and cause thyroid dysfunction in fish. Meanwhile, ketoprofen (KPF) is a nonsteroidal anti-inflammatory drug (NSAID) that can cause neurobehavioral damage and physiological impairment. However, to our knowledge, the combined exposure of Cd and KPF in transgenerational studies has not been reported. In this investigation, sexually mature zebrafish were subjected to isolated exposure and combined exposure to Cd (10 µg/L) and KPF (10 and 100 µg/L) at environmentally relevant concentrations for 42 days. In this background, breeding capacity, chemical accumulation rate in gonads, and tissue morphologies are investigated in parental fish. This is followed by examining the malformation rate, inflammation rate, and gene transcription in the F1 offspring. Our results indicate that combined exposure of Cd and KPF to the parental fish could increase the chemical accumulation rate and tissue damage in the gonads of fish and significantly reduce the breeding ability. Furthermore, these negative impacts were transmitted to its produced F1 embryos, reflected by hatching rate, body deformities, and thyroid axis-related gene transcription. These findings provide further insights into the harm posed by Cd in the presence of KPF to the aquatic ecosystems.