Developmental Toxicity in Mice Following Paternal Exposure to Di-N-Butyl-Phthalate (DBP)

Objective The aim of the present study was to investigate the effects of paternal Di‐N‐butyl‐phthalate （DBP） exposure pre‐ and postnatally on F1 generation offspring,and prenatally on F2 generation offspring.Methods Male mice were exposed to either 500 mg/kg or 2 000 mg/kg of DBP for 8 weeks,and mated with non‐exposed females.Three‐quarters of the females were sacrificed a day prior to parturition,and examined for the number of living and dead implantations,and incidence of gross malformations.Pups from the remaining females were assessed for developmental markers,growth parameters,as well as sperm quantity and quality.Results There were no changes in the fertility of parents and in intrauterine development of the offspring.Pups of DBP‐exposed males demonstrated growth‐retardation.Following paternal exposure to 500 mg/kg bw of DBP,there were almost twice the number of males than females born in the F1 generation.F1 generation females had a 2.5‐day delay in vaginal opening.Paternal exposure to 2 000 mg/kg bw of DBP increased the incidence of sperm head malformations in F1 generation males;however,there were no changes in the fertility and viability of foetuses in the F2 generation.Conclusion Paternal DBP exposure may disturb the sex ratio of the offspring,delay female sexual maturation,and deteriorate the sperm quality of F1 generation males.

Assay for the developmental toxicity of safflower (Carthamus tinctorius L.) to zebrafish embryos/larvae

Objective:To explore the developmental toxicity and the potential toxicological mechanism of safflower (Carthamus tinctorius L.) on zebrafish embryos/larvae.Methods:Mortality,malformations and increased apoptosis induced by safflower were assessed in zebraflsh embryos from 6 to 96 hours post-fertilization.Enzymes and genes in the anti-oxidative and apoptotic pathways were also assayed.Results:The lethal concentration 50 of safflower to zebrafish embryos was 345.6 mg/L.Hatching inhibition,abnormal spontaneous movement,depressed heart rate,pericardial edema,yolk sac edema,abnormal head-trunk angle,inhibition of melanin release,enlarged yolk,and short body length were observed in safflower-treated zebrafish.Additional apoptotic cells mainly appeared around the heart.Safflower exposure changed the activities of defense enzymes (SOD T,CAT↑,MDA↑,GPX without trend),increased MDA content,decreased caspase-3 activity,and altered mRNA levels of related genes (ogg1 ↓,p53↓,Cu/Zn-sod↑,Mn-sod↓,cat↓,gpx↑).Conclusion:Safflower exhibits developmental toxicity for zebrafish embryos/larvae.The developing heart was speculated as the target organ of toxicity.Oxidative stress and increased apoptosis have roles in the developmental toxicity of safflower.This article provides a novel method to research the teratogenicity and possible mechanisms of toxicity of traditional Chinese medicines that are prohibited or contraindicated in pregnant women.

Preliminary Validation of Tumor Cell Attachment Inhibition Assay for Developmental Toxicants With Mouse S180 Cells

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180cells attachment to Concanavalin A-coaed surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration tha reduced attachment by 50% ), of these 2 chemicals was 1.2×10-3mol/L and 1 .0 mol/L, respectively. Anoher developmental toxiant, hydmiortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally sindlar to those obtained with ascitic mouse ovdrian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not lindt attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an altemative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

Toxicity of Graphene Quantum Dots in Zebrafish Embryo

Objective To evaluate the bio-safety of graphene quantum dots （GQDs）, we studied its effects on the embryonic development of zebrafish. Methods In vivo, biodistribution and the developmental toxicity of GQDs were investigated in embryonic zebrafish at exposure concentrations ranging from 12.5-200μg/mL for 4-96 h post-fertilization （hpf）. The mortality, hatch rate, malformation, heart rate, GQDs uptake, spontaneous movement, and larval behavior were examined. Results The fluorescence of GQDs was mainly localized in the intestines and heart. As the exposure concentration increased, the hatch and heart rate decreased, accompanied by an increase in mortality. Exposure to a high level of GQDs （200μg/mL） resulted in various embryonic malformations including pericardial edema, vitelline cyst, bent spine, and bent tail. The spontaneous movement significantly decreased after exposure to GQDs at concentrations of 50, 100, and 200μg/mL. The larval behavior testing （visible light test） showed that the total swimming distance and speed decreased dose-dependently. Embryos exposed to 12.5 μg/mL showed hyperactivity while exposure to higher concentrations （25, 50, 100, and 200μg/mL） caused remarkable hypoactivity in the light-dark test. Conclusion Low concentrations of GODs were relatively non-toxic. However, GQDs disrupt the progression of embryonic development at concentrations exceeding 50 μg/mL.

Study of the Toxicity of 1-Bromo-3-Chloro-5,5-Dimethylhydantoin to Zebrafish

Objective 1-Bromo-3-chloro-5,5-dimethylhydantoin （BCDMH） is a solid oxidizing biocide for water disinfection.The objective of this study was to investigate the toxic effect of BCDMH on zebrafish.Methods The developmental toxicity of BCDMH on zebrafish embryos and the dose-effect relationship was determined.The effect of BCDMH exposure on histopathology and tissue antioxidant activity of adult zebrafish were observed over time.Results Exposure to 4 mg/L BCDMH post-fertilization was sufficient to induce a number of developmental malformations,such as edema,axial malformations,and reductions in heart rate and hatching rate.The no observable effects concentration of BCDMH on zebrafish embryo was 0.5 mg/L.After 96 h exposure,the 50% lethal concentration （95% confidence interval （CI）） of BCDMH on zebrafish embryo was 8.10 mg/L （6.15-11.16 mg/L）.The 50% inhibitory concentration （95% CI） of BCDMH on hatching rate was 7.37 mg/L （6.33-8.35 mg/L）.Histopathology showed two types of responses induced by BCDMH,defensive and compensatory.The extreme responses were marked hyperplasia of the gill epithelium with lamellar fusion and epidermal peeling.The histopathologic changes in the gills after 10 days exposure were accompanied by significantly higher catalase activity and lipid peroxidation.Conclusion These results have important implications for studies on the toxicity and use of BCDMH and its analogs.

Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats

Although numerous studies have examined the neurotoxicity of acrylamide in adult animals,the effects on neuronal development in the embryonic and lactational periods are largely unknown.Thus,we examined the toxicity of acrylamide on neuronal development in the hippocampus of fetal rats during pregnancy.Sprague-Dawley rats were mated with male rats at a 1：1 ratio.Rats were administered 0,5,10 or 20 mg/kg acrylamide intragastrically from embryonic days 6–21.The gait scores were examined in pregnant rats in each group to analyze maternal toxicity.Eight weaning rats from each group were also euthanized on postnatal day 21 for follow-up studies.Nissl staining was used to observe histological change in the hippocampus.Immunohistochemistry was conducted to observe the condition of neurites,including dendrites and axons.Western blot assay was used to measure the expression levels of the specific nerve axon membrane protein,growth associated protein 43,and the presynaptic vesicle membrane specific protein,synaptophysin.The gait scores of gravid rats significantly increased,suggesting that acrylamide induced maternal motor dysfunction.The number of neurons,as well as expression of growth associated protein 43 and synaptophysin,was reduced with increasing acrylamide dose in postnatal day 21 weaning rats.These data suggest that acrylamide exerts dose-dependent toxic effects on the growth and development of hippocampal neurons of weaning rats.

Toxicity Evaluation of Acrylamide on the Early Life Stages of the Zebrafish Embryos (<i>Danio rerio</i>)

Acrylamide is a chemical used mainly in industrial applications and the treatment of drinking and wastewater, making it easy to enter aquatic ecosystems. There are few studies known about the toxicity of acrylamide to aquatic organisms which have shown evidence of a number of histopathological effects. To assess the effects of acrylamide to freshwater fish, Zebrafish (Danio rerio) embryos were exposed to serial concentrations of acrylamide (0, 100, 300, and 500 mg/L) to investigate the acute toxicity effects on teleost embryogenesis. Embryos less than 24 hrs old were exposed under static non-renewal conditions for ten days or until hatching. The toxic endpoints evaluated include: egg/embryo viability, hatchability, and morphological/developmental anomalies during organogenesis. The acute toxicity test resulted in a 48 h-LC50 of 585 mg/L for egg viability. Exposure of embryos significantly reduced hatchability and larval survival, in a concentration dependent manner. Dimethyl sulfoxide (DMSO) was used as a solvent carrier to permeate the uptake of acrylamide through the chorion membrane. No significant damages or complications were observed in embryos exposed to DMSO. At 500 mg/L, the highest test concentration, the survival of embryos was greatly reduced within 24 hrs of exposure. The lower test, 100 mg/L, produced a significant number of developmental anomalies to the Zebrafish that included dorsal tail flexure, severe pericardial edema, facial and cranial defects and decreased heartrate (40 bpm). Premature hatching of embryos and developmental arrest was observed in all concentrations. The severity of these anomalies was concentration-dependent and resulted in low survival rate and high frequency of malformations. These results indicate that acrylamide is teratogenic and provide support for sub-lethal toxicity testing using Zebrafish embryos.

3-66 Toxic Effect of CORM-3 on the Development of Zebrafish Embryos

CORM-3, a water soluble transitional metal carbonyls based around ruthenium can release CO activated bylight[1]. Although CORM-3 has resulted in promising preclinical data, such as anti-inflammatory, anti-apoptosisand vasodilatory effect, they present a poorly understanding on toxicological profile.To evaluate the effects of CORM-3 on the developmentof zebrafish embryos, we selected the zebrafish embryosdeveloping normally at the stage of blastocyst at4 h post fertilization and transferred into 24-well cultureplates with 2 embryos per well and then exposedto freshly prepared CORM-3 solution with E3 (0, 6.25,12.5, 25, 50, 100, 200 and 400 mol/L) for 4144 hpffor a continuing observation period on the developmentof zebrafish. Acute endpoints including hatching rateand embryonic/larval mortality were evaluated every 24h(Fig. 1).

Prenatal amoxicillin exposure induces developmental toxicity in fetal mice and its characteristics

Amoxicillin,a widely used antibiotic in human and veterinary pharmaceuticals,is now considered as an“emerging contaminant”because it exists widespreadly in the environment and brings a series of adverse outcomes.Currently,systematic studies about the developmental toxicity of amoxicillin are still lacking.We explored the potential effects of amoxicillin exposure on pregnancy outcomes,maternal/fetal serum phenotypes,and fetal multiple organ development in mice,at different doses(75,150,300 mg/(kg·day))during late-pregnancy,or at a dose of 300 mg/(kg·day)during different stages(mid-/latepregnancy)and courses(single-/multi-course).Results showed that prenatal amoxicillin exposure(PAmE)had no significant infuence on the body weights of dams,but it could inhibit the physical development and reduce the survival rate of fetuses,especially during the midpregnancy.Meanwhile,PAmE altered multiple maternal/fetal serum phenotypes,especially in fetuses.Fetal multi-organ function results showed that PAmE inhibited testicular/adrenal steroid synthesis,long bone/cartilage and hippocampal development,and enhanced ovarian steroid synthesis and hepatic glycogenesis/lipogenesis,and the order of severity might be gonad(testis,ovary)>liver>others.Further analysis found that PAmE-induced multiorgan developmental and functional alterations had differences in stages,courses and fetal gender,and the most obvious changes might be in high-dose,late-pregnancy and multicourse,but there was no typical rule of a dose-response relationship.In conclusion,this study confirmed that PAmE could cause abnormal development and multi-organ function alterations,which deepens our understanding of the risk of PAmE and provides an experimental basis for further exploration of the long-term harm.

Developmental toxicity assessment of neonicotinoids and organophosphate esters with a human embryonic stem cell-and metabolism-based fast-screening model

In recent years,neonicotinoids(NEOs)and organophosphate esters(OPEs)have been widely used as substitutes for traditional pesticides and brominated fame-retardants,respectively.Previous studies have shown that those compounds can be frequently detected in environmental and human samples,are able to penetrate the placental barrier,and are toxic to animals.Thus,it is reasonable to speculate that NEOs and OPEs may have potential adverse effects in humans,especially during development.We employed a human embryonic stem cell differentiation-and liver S9 fraction metabolism-based fast screening model to assess the potential embryonic toxicity of those two types of chemicals.We show that four NEO and five OPE prototypes targeted mostly ectoderm specification,as neural ectoderm and neural crest genes were down-regulated,and surface ectoderm and placode markers up-regulated.Human liver S9 fraction's treatment could generally reduce the effects of the chemicals,except in a few specific instances,indicating the liver may detoxify NEOs and OPEs.Our findings suggest that NEOs and OPEs interfere with human early embryonic development.

An Overview of Adverse Outcome Pathway Links between PM_(2.5)Exposure and Cardiac Developmental Toxicity

Fine particulate matter(PM_(2.5))is a significant risk factor for birth defects.As the first and most important organ to develop during embryogenesis,the heart’s potential susceptibility to PM_(2.5)has attracted growing concern.Despite several studies supporting the cardiac developmental toxicity of PM_(2.5),the diverse study types,models,and end points have prevented the integration of mechanisms.In this Review,we present an adverse outcome pathway framework to elucidate the association between PM_(2.5)-induced molecular initiating events and adverse cardiac developmental outcomes.Activation of the aryl hydrocarbon receptor(AhR)and excessive generation of reactive oxygen species(ROS)were considered as molecular initiating events.The excessive production of ROS induced oxidative stress,endoplasmic reticulum stress,DNA damage,and inflammation,resulting in apoptosis.The activation of the AhR inhibited the Wnt/β-catenin pathway and then suppressed cardiomyocyte differentiation.Impaired cardiomyocyte differentiation and persistent apoptosis resulted in abnormalities in the cardiac structure and function.All of the aforementioned events have been identified as key events(KEs).The culmination of these KEs ultimately led to the adverse outcome,an increased morbidity of congenital heart defects(CHDs).This work contributes to understanding the causes of CHDs and promotes the safety evaluation of PM_(2.5).

Developmental impacts and toxicological hallmarks of silver nanoparticles across diverse biological models

Silver nanoparticles(AgNPs),revered for their antimicrobial prowess,have become ubiquitous in a range of products,from biomedical equipment to food packaging.However,amidst their rising popularity,concerns loom over their possible detrimental effects on fetal development and subsequent adult life.This review delves into the developmental toxicity of AgNPs across diverse models,from aquatic species like zebrafish and catfish to mammalian rodents and in vitro embryonic stem cells.Our focus encompasses the fate of AgNPs in different contexts,elucidating associated hazardous results such as embryotoxicity and adverse pregnancy outcomes.Furthermore,we scrutinize the enduring adverse impacts on offspring,spanning impaired neurobehavior function,reproductive disorders,cardiopulmonary lesions,and hepatotoxicity.Key hallmarks of developmental harm are identified,encompassing redox imbalances,inflammatory cascades,DNA damage,and mitochondrial stress.Notably,we explore potential explanations,linking immunoregulatory dysfunction and disrupted epigenetic modifications to AgNPsinduced developmental failures.Despite substantial progress,our understanding of the developmental risks posed by AgNPs remains incomplete,underscoring the urgency of further research in this critical area.

Comparison of developmental toxicity of different surface modified CdSe/ZnS QDs in zebrafish embryos

Quantum dots(QDs)are new types of nanomaterials.Few studies have focused on the effect of different surface modified QDs on embryonic development.Herein,we compared the in vivo toxicity of Cd Se/Zn S QDs with carboxyl(-COOH)and amino(-NH 2)modification using zebrafish embryos.After exposure,the two Cd Se/Zn S QDs decreased the survival rate,hatching rate,and embryo movement of zebrafish.Moreover,we found QDs attached to the embryo membrane before hatching and the eyes,yolk and heart after hatching.The attached amount of carboxyl QDs was more.Consistently,the Cd content in embryos and larvae was higher in carboxyl QD-treatment.We further observed that the two QDs caused zebrafish pericardial edema and cardiac dysfunction.In line with it,both carboxyl and amino QDs upregulated the transcription levels of cardiac development-related genes,and the levels were higher in carboxyl QD-treated groups.Furthermore,the chelator of Cd^2+diethylene triamine pentacetate acid could partially rescued the developmental toxicity caused by the two types of QDs suggesting that both the nature of QDs and the release of Cd^2+contribute to the developmental toxicity.In conclusion,the two Cd Se/ZnS QDs have developmental toxicity and affect the cardiac development,and the carboxyl QDs is more toxic possibly due to the higher affinity and more release to embryos and larvae.Our study provides new knowledge that the surface functional modification of QDs is critical on the development on aquatic species,which is beneficial to develop and applicate QDs more safely and environmentfriendly.

Developmental toxicity and programming alterations of multiple organs in offspring induced by medication during pregnancy

Medication during pregnancy is widespread,but there are few reports on its fetal safety.Recent studies suggest that medication during pregnancy can affect fetal morphological and functional development through multiple pathways,multiple organs,and multiple targets.Its mechanisms involve direct ways such as oxidative stress,epigenetic modification,and metabolic activation,and it may also be indirectly caused by placental dysfunction.Further studies have found that medication during pregnancy may also indirectly lead to multi-organ developmental programming,functional homeostasis changes,and susceptibility to related diseases in offspring by inducing fetal intrauterine exposure to too high or too low levels of maternal-derived glucocorticoids.The organ developmental toxicity and programming alterations caused by medication during pregnancy may also have gender differences and multi-generational genetic effects mediated by abnormal epigenetic modification.Combined with the latest research results of our laboratory,this paper reviews the latest research progress on the developmental toxicity and functional programming alterations of multiple organs in offspring induced by medication during pregnancy,which can provide a theoretical and experimental basis for rational medication during pregnancy and effective prevention and treatment of drug-related multiple fetal-originated diseases.

Developmental toxicity of TCBPA on the nervous and cardiovascular systems of zebrafish (Danio rerio): A combination of transcriptomic and metabolomics

Tetrachlorobisphenol A(TCBPA),a widely used halogenated flame retardant,is frequently detected in environmental compartments and human samples.However,unknown developmental toxicity and mechanisms limit the entire understanding of its effects.In this study,zebrafish(Danio rerio)embryos were exposed to various concentrations of TCBPA while a combination of transcriptomics,behavioral and biochemical analyzes as well as metabolomics were applied to decipher its toxic effects and the potential mechanisms.We found that TCBPA could interfere with nervous and cardiovascular development through focal adhesion and extracellular matrix-receptor(ECM-receptor)interaction pathways through transcriptomic analysis.Behavioral and biochemical analysis results indicated abnormal swimming behavior of zebrafish larvae.Morphological observations revealed that TCBPA could cause the loss of head blood vessels.Metabolomic analysis showed that arginine-related metabolic pathways were one of the main pathways leading to TCBPA developmental toxicity.Our study demonstrated that by using omics,TCBPA was shown to have neurological and cardiovascular developmental toxicity and the underlying mechanisms were uncovered and major pathways identified.

T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos

T-2 toxin is one of the most important trichothecene mycotoxins occurring in various agriculture products. The developmental toxicity of T-2 toxin and the exact mechanism of action at early life stages are not understood precisely. Zebrafish embryos were exposed to different concentrations of the toxin at 4-6 hours post fertilization （hpf） stage of development, and were observed for different developmental toxic effects at 24, 48, 72, and 144 hpf. Exposure to 0.20 Ixmol/L or higher concentrations of T-2 toxin significantly increased the mortality and malformation rate such as tail deformities, cardiovascular defects and behavioral changes in early developmental stages of zebrafish. T-2 toxin exposure resulted in significant increases in reactive oxygen species （ROS） production and cell apoptosis, mainly in the tall areas, as revealed by Acridine Orange staining at 24 hpf. In addition, T-2 toxin-induced severe tail deformities could be attenuated by co-exposure to reduced glutathione （GSH）. T-2 toxin and GSH co-exposure induced a significant decrease of ROS production in the embryos. The overall results demonstrate that T-2 toxin is able to produce oxidative stress and induce apoptosis, which are involved in the developmental toxicity of T-2 toxin in zebrafish embryos.

Developmental Toxicity and Potential Teratogenicity of Compound Danshen Tablet, Angong Niuhuang Pill, and Lidan Paishi Tablet in Zebrafish Embryos

Objective Herbal medicines containing toxic herbs or minerals such as Compound Danshen Tablet （CDT）, Angong Niuhuang Pill （ANP）, and Lidan Paishi Tablet （LPT） are avoided or used with caution for pregnant women because of potential teratogenicity. To understand their mechanism, they were chosen as model subjects for the research. Methods Zebrafish embryos were used to evaluate their potential teratogenic risk in vitro. Results All of them showed teratogenic and lethal effects in zebrafish embryos, with the ECs0 values at 351,793, and 220 μg/mL, and LC50 values at 41 7, 596, and 380 μg/mL, respectively. CDT and LPT, displaying week potential teratogenicity as their teratogenicity indexes were greater than 1, induced tail malformation and cardiac edema mainly in zebrafish embryos, respectively. Conclusion The results provide the significant guidance of clinical safety of medication.

A Multi-Generation Schmakeria poplesia Culturing System for Use in Ecotoxicological Study

Crustacean zooplankton form the keystone link between primary producers and fish stocks in marine and estuary, ecosystems. We have established a multi-generation cultivation system for zooplankton ruth which future experiments on the biological effects of pollutants in marine and estuary environments can be better performed. A population of calanoid copepod, Sehmakeria poplesia, was collected in December 2003 and maintained in a static system through all stages （eggs to adults）. The population ex- hinted an average developmental time of 13.6 d in conditions corresponding to the natural environment （water temperature 20~C salinity 15）. A series of experiments were performed to examine copepod egg production and hatching success as functions of food type and feeding concentration. Results in our study showed that Isoehrysis galbana was more favored for the reproduction of cope- pods than Phaeodactylum tricornutum, and 10xl0%ellsmL-1 was the most practical algae concentration. We have demonstrated that the Schmakeria poplesia population can be maintained in the laboratory through multiple generations. In addition, methods to control egg production through changes in food concentration have been established, making it feasible to control the start date of exposure experiments or the timing of the collection of oftspring to initiate a new generation.

Acute Developmental Toxicity of Panax notoginseng in Zebrafish Larvae

Objective:To evaluate toxicity of raw extract of Panax notoginseng(rPN)and decocted extract of PN(dPN)by a toxicological assay using zebrafish larvae,and explore the mechanism by RNA sequencing assay.Methods:Zebrafish larvae was used to evaluate acute toxicity of PN in two forms:rPN and dPN.Three doses(0.5,1.5,and 5.0μg/mL)of dPN were used to treat zebrafishes for evaluating the developmental toxicity.Behavior abnormalities,body weight,body length and number of vertebral roots were used as specific phenotypic endpoints.RNA sequencing(RNA-seq)assay was applied to clarify the mechanism of acute toxicity,followed by real time PCR(qPCR)for verification.High performance liquid chromatography analysis was performed to determine the chemoprofile of this herb.Results:The acute toxicity result showed that rPN exerted higher acute toxicity than d PN in inducing death of larval zebrafishes(P<0.01).After daily oral intake for 21 days,d PN at doses of 0.5,1.5 and 5.0μg/m L decreased the body weight,body length,and vertebral number of larval zebrafishes,indicating developmental toxicity of dPN.No other adverse outcome was observed during the experimental period.RNA-seq data revealed 38 genes differentially expressed in dPN-treated zebrafishes,of which carboxypeptidase A1(cpa1)and opioid growth factor receptor-like 2(ogfrl2)were identified as functional genes in regulating body development of zebrafishes.qPCR data showed that dPN significantly down-regulated the mRNA expressions of cpa1 and ogfrl2(both P<0.01),verifying cpa1 and ogfrl2 as target genes for dPN.Conclusion:This report uncovers the developmental toxicity of dPN,suggesting potential risk of its clinical application in children.