Developing Zebrafish Embryos Research Articles

Effects of low-dose ionizing radiation on the molecular pathways linking neurogenesis and autism spectrum disorders in zebrafish embryos

Prenatal exposure to environmental factors may play an important role in the aetiopathogenesis of autism spectrum disorder (ASD). We aim to investigate the potential effects of low-dose x-rays from dental diagnostic x-rays on neurodevelopment and molecular mechanisms associated with ASD in developing zebrafish embryos. Zebrafish embryos were divided into four groups and exposed using a dental x-ray unit: control, 0.08, 0.15 and 0.30 seconds, which are exemplary exposure settings for periapical imaging. These exposure times were measured as 7.17, 23.17 and 63.83 mSv using optical stimulated luminescence dosimeters. At the end of 72 hours post-fertilization, locomotor activity, oxidant-antioxidant status, and acetylcholine esterase (AChE) activity were analyzed. Expression of genes related to apoptosis (bax, bcl2a, p53), neurogenesis (α1-tubulin, syn2a, neurog1, elavl3) and ASD (eif4eb, adsl2a, shank3) was determined by RT-PCR. Even at reduced doses, developmental toxicity was observed in three groups as evidenced by pericardial edema, yolk sac edema and scoliosis. Deleterious effects of dental x-rays on neurogenesis through impaired locomotor activity, oxidative stress, apoptosis and alterations in genes associated with neurogenesis and ASD progression were more pronounced in the 0.30s exposure group. Based on these results we suggest that the associations between ASD and low-dose ionizing radiation need a closer look.

Requirement of a novel gene, drish, in the zebrafish retinal ganglion cell and primary motor axon development.

During neurogenesis, growing axons must navigate through the complex extracellular environment and make correct synaptic connections for the proper functioning of neural circuits. The mechanisms underlying the formation of functional neural networks are still only partially understood. Here we analyzed the role of a novel gene si:ch73-364h19.1/drish in the neural and vascular development of zebrafish embryos. We show that drish mRNA is expressed broadly and dynamically in multiple cell types including neural, glial, retinal progenitor and vascular endothelial cells throughout the early stages of embryonic development. To study Drish function during embryogenesis, we generated drish genetic mutant using CRISPR/Cas9 genome editing. drish loss-of-function mutant larvae displayed defects in early retinal ganglion cell, optic nerve and the retinal inner nuclear layer formation, as well as ectopic motor axon branching. In addition, drish mutant adults exhibited deficient retinal outer nuclear layer and showed defective light response and locomotory behavior. However, vascular patterning and blood circulation were not significantly affected. Together, these data demonstrate important roles of zebrafish drish in the retinal ganglion cell, optic nerve and interneuron development and in spinal motor axon branching.

A crossover study of antimicrobial capacity and biotoxicity of silver nanoparticles

Silver nanoparticles (AgNPs) have a primordial role in the nanotechnology industry since they have great antimicrobial effects against diverse pathogens. AgNP production, use, and discard have been increasing; still, there is debate about the consequences of exposure to AgNPs for animals and humans. The bioaccumulation and toxicity of AgNPs depend on several factors, including AgNP concentration, physicochemical characteristics, and aggregation. So, complete activity and toxicity profiles for each silver nanoparticle synthesized must be performed. The present work aimed to characterize the behavior of specific AgNPs, then crossover of their antimicrobial capacity against Pseudomonas aeruginosa and Staphylococcus aureus with their potential in vitro cytotoxic effects in murine fibroblasts (3T3) and human keratinocytes (HaCat) cell lines, and in vivo general and organ‐specific toxicity in zebrafish embryos and larvae. A high susceptibility of P. aeruginosa and S. aureus to low concentrations of AgNPs was observed, yet a cytotoxic effect was also detected in 3T3 and HaCat cells. Moreover, these concentrations delayed embryonic development in zebrafish embryos and presented morphological abnormalities, neurotoxicity, and cardiotoxicity in zebrafish larvae. The results highlight the importance of jointly evaluating the antimicrobial activity and the toxicological profile of each nanoparticle manufactured; this study presents a suitable platform for such crossover assessment.

Knockdown of Kmt2d leads to growth impairment by activating the Akt/β-catenin signaling pathway.

The KMT2D variant-caused Kabuki syndrome (KS) is characterized by short stature as a prominent clinical characteristic. The initiation and progression of body growth are fundamentally influenced by chondrocyte proliferation. Uncertainty persists regarding the possibility that KMT2D deficiency affects growth by impairing chondrocyte proliferation. In this study, we used the CRISPR/Cas13d technique to knockdown kmt2d in zebrafish embryos and lentivirus to create a stable Kmt2d gene knockdown cell line in chondrocytes (ATDC5 cells). We also used CCK8 and flow cytometric studies, respectively, to determine proliferation and cell cycle state. The relative concentrations of phosphorylated Akt (ser473), phosphorylated β-catenin (ser552), and cyclin D1 proteins in chondrocytes and zebrafish embryos were determined by using western blots. In addition, Akt inhibition was used to rescue the phenotypes caused by kmt2d deficiency in chondrocytes, as well as a zebrafish model that was generated. The results showed that a knockdown of kmt2d significantly decreased body length and resulted in aberrant cartilage development in zebrafish embryos. Furthermore, the knockdown of Kmt2d in ATDC5 cells markedly increased proliferation and accelerated the G1/S transition. In addition, the knockdown of Kmt2d resulted in the activation of the Akt/β-catenin signaling pathway in ATDC5 cells. Finally, Akt inhibition could partly rescue body length and chondrocyte development in the zebrafish model. Our study demonstrated that KMT2D modulates bone growth conceivably via regulation of the Akt/β-catenin pathway.

Carbon nitride dots do not impair the growth, development, and telomere length of tadpoles

Carbon nanoparticles, or carbon dots, can have many beneficial uses. However, we must consider whether they may have any potential negative side effects on wildlife or the ecosystem when these particles end up in wastewater. Early development stages of amphibians are particularly sensitive to contaminants, and exposure to carbon dots could disrupt their development and cause morbidity or death. Past studies have investigated short-term exposure to certain types of nanoparticles, but if these particles get into wastewater exposure may not be short term. Therefore, we tested whether chronic exposure to different concentrations of carbon dots affects the growth, metamorphosis, and telomere length of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We exposed 12 groups of five tadpoles each to different concentrations of carbon dots and a control for three months and tracked survival, growth and metamorphosis. We used carbon nitride dots approximately 2 nm in size at concentrations of 0.01 mg/ml and 0.02 mg/ml, known to interrupt development in zebrafish embryos. After three months, we measured telomere length from tissue samples. We found no difference in tadpole survivorship, growth, development rate, or telomere length among any of the groups, suggesting that carbon dots at these concentrations do not disrupt tadpole development.

Particulate Matter Induced Adverse Effects on Eye Development in Zebrafish (Danio rerio) Embryos.

Particulate matter (PM) can cause human diseases, particularly respiratory diseases. Since eyes are directly exposed to the air, they might be directly adversely affected by PM. Therefore, we determined the toxicity caused to eye development by PM using zebrafish (Danio rerio) embryos. The PM-induced embryo toxicity was dependent on dose and time and caused significant morphological defects, reducing the total body length and the total eye area. Reactive oxygen species (ROS) overproduction was confirmed in the PM treatment group, and antioxidant genes (cat and sod2), photoreceptor cell development, pigmentation genes (atoh8, vsx1, and rho), eye-embryogenesis genes (pax6a and pax6b), and eye-lens-development genes (cryaa) were downregulated, while eye-development genes (crybb1) were upregulated. In conclusion, PM had a direct adverse effect on the eyes, and zebrafish embryos can be used as a model to evaluate PM-induced eye toxicity in vivo.

Comparison of cardiotoxicity induced by alectinib, apatinib, lenvatinib and anlotinib in zebrafish embryos

Four tyrosine kinase inhibitors, alectinib, apatinib, lenvatinib and anlotinib, have been shown to be effective in the treatment of clinical tumors, but their cardiac risks have also raised concerns. In this study, zebrafish embryos at 6 h post fertilization (hpf) were exposed to the four drugs at concentrations of 0.05–0.2 mg/L until 72 hpf, and then the development of these embryos was quantified, including heart rate, body length, yolk sac area, pericardial area, distance between venous sinus and balloon arteriosus (SV-BA), separation of cardiac myocytes and endocardium, gene expression, vascular development and oxidative stress. At the same exposure concentrations, alectinib and apatinib had little effect on the cardiac development of zebrafish embryos, while lenvatinib and anlotinib could induce significant cardiotoxicity and developmental toxicity, including shortened of body length, delayed absorption of yolk sac, pericardial edema, prolonged SV-BA distance, separation of cardiomyocytes and endocardial cells, and downregulation of key genes for heart development. Heart rate decreased in all four drug treatment groups. In terms of vascular development, alectinib and apatinib did not inhibit the growth of embryonic intersegmental vessels (ISVs) and retinal vessels, while lenvatinib and anlotinib caused serious vascular toxicity, and the inhibition of anlotinib in vascular development was more obvious. Besides, the level of reactive oxygen species (ROS) in the lenvatinib and anlotinib treatment groups was significantly increased. Our results provide reference for comparing the cardiotoxicity of the four drugs.

Effects of 700 and 3500 MHz 5G radiofrequency exposure on developing zebrafish embryos

Telecommunications industries are rapidly deploying the fifth generation (5G) spectrum and there is public concern about the safety and health impacts of this type of Radio Frequency Radiation (RFR), in part because of the lack of comparable scientific evidence. In this study we have used a validated commercially available setting producing a uniform field to expose zebrafish embryos (ZFe) to unmodulated 700 and 3500 MHz frequencies. We have combined a battery of toxicity, developmental and behavioral assays to further explore potential RFR effects. Our neurobehavioral profiles include a tail coiling assay, a light/dark activity assay, two thigmotaxis anxiety assays (auditory and visual stimuli), and a startle response - habituation assay in response to auditory stimuli. ZFe were exposed for 1 and 4 h during the blastula period of development and endpoints evaluated up to 120 hours post fertilization (hpf). Our results show no effects on mortality, hatching or body length. However, we have demonstrated specific organ morphological effects, and behavioral effects in activity, anxiety-like behavior, and habituation that lasted in larvae exposed during the early embryonic period. A decrease in acetylcholinesterase activity was also observed and could explain some of the observed behavioral alterations. Interestingly, effects were more pronounced in ZFe exposed to the 700 MHz frequency, and especially for the 4 h exposure period. In addition, we have demonstrated that our exposure setup is robust, flexible with regard to frequency and power testing, and highly comparable. Future work will include exposure of ZFe to 5G modulated signals for different time periods to better understand the potential health effects of novel 5G RFR.

Toxicokinetics of a developmental toxicity test in zebrafish embryos and larvae: Relationship with drug exposure in humans and other mammals

To study the effects of drugs on embryo/fetal development (EFD), developmental and reproductive toxicity studies in zebrafish (Danio rerio) embryos is expected to be an accepted alternative method to animal studies using mammals. However, there is a lack of clarity in the relationship between the concentration of developmental toxicity agents in whole embryos or larvae (Ce) and that in aqueous solution (Cw), and also between the amount of drug exposure required to cause developmental toxicity in zebrafish embryos or larvae and that required in mammals. Here, we measured Ce for developmental toxicity agents every 24 h starting at 24 h post fertilization (hpf). We found a high correlation (R2: 0.87–0.96) between log [Ce/Cw] and the n-octanol–water distribution coefficient at pH 7 (logD) of each drug at all time points up to 120 hpf. We used this relationship to estimate the Ce values of the 21 positive-control reference drugs listed in ICH guidelines on reproductive and developmental toxicity studies (ICH S5). We then calculated the area under the Ce–time curve in zebrafish (zAUC) for each drug from the regression equation between log [Ce/Cw] and logD and compared it with the AUC at the no-observed-adverse-effect level in rats and rabbits and at the effective dose in humans described in ICH S5. The log of the calculated zAUC for the 14 drugs identified as positive in the zebrafish developmental toxicity test was relatively highly positively correlated with the log [AUC] for rats, rabbits, and humans. These findings provide important and positive information on the applicability of the zebrafish embryo developmental toxicity test as an alternative method of EFD testing. (267 words)

Assessing silver nanoparticle uptake dynamics in young zebrafish using swept source optical coherence tomography

Understanding the uptake and accumulation of nanoparticles in living organisms is crucial for assessing their potential effects on health and the environment. In this study, we employed a Swept Source Optical Coherence Tomography (SSOCT) system to investigate the dynamics of silver nanoparticle (AgNP) uptake during various developmental stages of zebrafish. Silver nanoparticles in the 20 nm range were synthesized and their uptake and accumulation in developing zebrafish embryos were studied over time. Changes in optical attenuation properties observed in SSOCT images allowed identification of nanoparticle uptake and accumulation in the eye, intestine, and gill region of developing zebrafish. The changes were quantified by deriving average local attenuation coefficient values. To validate our findings, inductively-coupled plasma optical emission spectrometry (ICP-OES) analysis was performed.

Identification of distinct vascular mural cell populations during zebrafish embryonic development.

Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells. However, the overlapping and distinct expression patterns of these markers in tandem have not been fully described. Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular cell populations on the cranial, axial, and intersegmental vessels between 1 and 5 days postfertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord-a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. Together, our findings highlight the variability and versatility of tracking both pdgfrb and tagln expression in mural cells of the developing zebrafish embryo and reveal unexpected embryonic cell populations that express pdgfrb and tagln.

In Situ Hybridization to Characterize Neurulation and Midbrain-Hindbrain Boundary Formation in Zebrafish.

Whole-mount in situ hybridization is cable to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain-hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain-hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization.

Exposure to volatile organic compounds induces cardiovascular toxicity that may involve DNA methylation

Volatile organic compounds (VOCs) are common air pollutants and water contaminants. We previously found maternal exposure to VOCs was associated with offspring congenital heart disease (CHD). However, little information is available about the effects of VOCs on cardiovascular development at embryonic stage and the underlying mechanism remains unclear. In this study, we aimed to investigate the effects of a mixture of six VOCs on cardiovascular development in zebrafish embryos. Embryos were exposed to different concentrations of VOCs mixture (32 mg/L, 64 mg/L and 128 mg/L) for 96 h, cardiovascular abnormalities including elongated heart shape, increased distance between sinus venosus and bulbus arteriosus, slowed circulation and altered heart rate were observed in a dose- and time-dependent manner. Meanwhile, VOCs exposure increased global DNA methylation levels in embryos. Analysis identified hundreds of differentially methylated sites and the enrichment of differentially methylated sites on cardiovascular development. Two differentially methylated-associated genes involved in MAPK pathway, hgfa and ntrk1, were identified to be the potential genes mediating the effects of VOCs. By enzyme-linked immunosorbent assay, altered human serum hgf and ntrk1 levels were detected in abnormal pregnancies exposed to higher VOCs levels with fetal CHD. For the first time, our study revealed exposure to VOCs induced severe cardiovascular abnormalities in zebrafish embryos. The toxicity might result from alterations in DNA methylation and corresponding expression levels of genes involved in MAPK pathway. Our study provides important information for the risk of VOCs exposure on embryonic cardiovascular development.

MTOR signaling pathway regulates embryonic development and rapid growth of triploid crucian carp

Triploid crucian carp is a new breed of aquaculture with rapid growth characteristics. However, its growth regulation mechanism has not yet been elucidated. In this study, our results show that the expression levels of mTOR signaling pathway-related genes (AKT1, AKT2, AKT3, mTOR, 4E-BP1 and S6K1) are higher in the muscle and intestine tissues of triploid crucian carp than those of diploid red crucian carp. Embryos of triploid crucian carp exhibit a faster growth rate comparing to red crucian carp. And in muscle effect stage, the mRNA levels of mTOR signaling pathway related genes in triploid crucian carp are higher than red crucian carp, except for AKT2. Inhibition of mTOR activity with rapamycin leads to slower embryonic development and the eliminated expression of mTOR in the embryos of neurula, muscle effect, and eye pigmentation stages. Conversely, overexpression of the upstream gene AKT2 can promote the development of zebrafish embryos and affect the expression of mTOR signaling pathway. These data highlight the significant role of mTOR signaling pathway in regulating fish embryonic development and rapid growth, and might offer new insights and avenues for exploring fish embryonic development, improving fish growth.

Embryonic exposure to decitabine induces multiple neural tube defects in developing zebrafish.

Neural tube defects are severe congenital disorders of the central nervous system that originate during embryonic development when the neural tube fails to close completely. It affects one to two infants per 1000 births. The aetiology is multifactorial with contributions from both genetic and environmental factors. Dysregulated epigenetic mechanisms, in particular the abnormal genome-wide methylation during embryogenesis, have been linked to developmental abnormalities including neural tube defects. The current study investigated the influence of decitabine (DCT), a DNA methylation inhibitor, on embryonic development in zebrafish, with a focus on neural tube formation. The developing zebrafish embryos were exposed to graded concentrations of decitabine (from 13.69 μM to 1 mM) before the onset of neurulation. The developmental process was monitored at regular time intervals post fertilization. At 120 h post fertilization, the developing embryos were inspected individually to determine the incidence and severity of neural tube defects. Using alizarin red staining, the cranial and caudal neural tube morphology was examined in formaldehyde fixed larvae. Anomalies in neural tube and somite development, as well as a delay in hatching, were discovered at an early stage of development. As development continued, neural tube defects became increasingly evident, and there was a concentration-dependent rise in the prevalence and severity of various neural tube defects. 90% of growing embryos in the group exposed to decitabine 1 mM had multiple neural tube malformations, and 10% had isolated neural tube defects. With several abnormalities, the caudal region of the neural tube was seriously compromised. The histopathological studies supported the malformations in neural tube. Our study revealed the harmful impact of decitabine on the development of the neural tube in growing zebrafish. Moreover, these findings support the hypothesis that the hypomethylation during embryonic development causes neural tube defects.

Combined toxicity of aflatoxin B1 and tebuconazole to the embryo development of zebrafish (Danio rerio)

Mycotoxins and pesticides are pervasive elements within the natural ecosystem. Furthermore, many environmental samples frequently exhibit simultaneous contamination by multiple mycotoxins and pesticides. Nevertheless, a significant portion of previous investigations has solely reported the occurrence and toxicological effects of individual chemicals. Global regulations have yet to consider the collective impacts of mycotoxins and pesticides. In our present study, we undertook a comprehensive analysis of multi-level endpoints to elucidate the combined toxicity of aflatoxin B1 (AFB1) and tebuconazole (TCZ) on zebrafish (Danio rerio). Our findings indicated that AFB1 (with a 10-day LC50 value of 0.018 mg L−1) exhibits higher toxicity compared to TCZ (with a 10-day LC50 value of 2.1 mg L−1) toward D. rerio. The co-exposure of AFB1 and TCZ elicited synergistic acute responses in zebrafish. The levels of GST, CYP450, SOD, and Casp-9 exhibited significant variations upon exposure to AFB1, TCZ, and their combined mixture, in contrast to the control group. Additionally, eight genes, namely cat, cxcl-cic, il-1β, bax, apaf-1, trβ, ugtlab, and vtg1, displayed marked alterations when exposed to the chemical mixture as opposed to individual substances. Therefore, further exploration of the underlying mechanisms governing joint toxicity is imperative to establish a scientific basis for evaluating the risk associated with the combined effects of AFB1 and TCZ. Moreover, it is essential to thoroughly elucidate the organ system toxicity triggered by the co-occurrence of mycotoxins and pesticides.

Genomic disturbance of vitellogenin 2 (vtg2) leads to vitellin membrane deficiencies and significant mortalities at early stages of embryonic development in zebrafish (Danio rerio)

The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was produced and effects of genomic disturbance were observed in F2 females and F3 offspring. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5×, and in 1 hpf F3 vtg2-mutant embryos was 3.8× less than Wt (p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2-mutant females (p < 0.05). Hatching rate was significantly higher in F3 vtg2-mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29% and 18% at 24 hpf and 20 dpf, respectively, in F3 vtg2-mutant embryos. The introduced mutation caused vitelline membrane deficiencies, significant mortalities at early embryonic stages, and morphological abnormalities in the surviving F3 vtg2-mutant larvae. Overrepresentation of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2-mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Overall findings imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development.

Effects of Paracetamol on the Development of Zebrafish (Danio rerio)

The misuse of paracetamol is becoming more prevalent worldwide. Due to non-compliance with recommended dosage and regulations, paracetamol consumption can result in serious health issues such as liver necrosis, kidney damage, heart damage, and hematological changes. This study sought to investigate the impact of paracetamol on the development of zebrafish embryos, which are often used as a model for assessing the effect of drug exposure on animals. The results indicated that paracetamol negatively affects the hatching and survival rates of zebrafish. Additionally, paracetamol exposure caused spinal abnormalities, pericardial edema, hypopigmentation, reduced heart rate, and spontaneous movement in zebrafish larvae. The developmental abnormalities in zebrafish were more significant with higher concentrations and longer exposure times. These findings may provide valuable insights into the detrimental impact of paracetamol on aquatic animals.

Synthesis of salicylic acid from wintergreen oil by green chemistry overcomes its cytotoxicity in keratinocyte cells and teratogenicity in zebrafish embryos

Salicylic acid topical is used to treat variety of skin conditions. However, salicylic acid in these products is generated through industrial synthesis and has been shown to negatively impact fetal development and cause congenital abnormalities. We hypothesized that teratogenic effects reported in salicylic acid can be prevented by naturally synthesizing salicylic acid from wintergreen oil using green chemistry method. For this purpose, we investigated the effects of natural salicylic acid (NSA) synthesized from wintergreen oil using green chemistry and synthetic salicylic acid (SSA) on keratinocyte cell (HaCaT) proliferation and zebrafish embryo development. NSA structures were analyzed by 1H NMR, 13C NMR, and GC/MS methods. Percentage inhibition against HaCaT cell was determined by MTS assay. xCelligence system was used for cellular activities. Zebrafish embryos were exposed to NSA and SSA for 72 h post-fertilization. Lipid peroxidation, nitric oxide, sialic acid, glutathione-S-transferase, catalase, and superoxide dismutase were evaluated using biochemical methods. Expressions of nqO1, gfap, bdnf, vtg, egr, cyp1a, and igf2 were determined by RT-PCR as developmental indicators. MTS and RT-cell analysis showed increased cell viability by NSA, whereas SSA decreased cell viability. NSA beneficially affected zebrafish embryo development while SSA exerted deleterious effects through oxidant–antioxidant status, inflammation, and development. Results of our study showed for the first time that synthesis of salicylic acid from wintergreen oil by green chemistry overcomes its cytotoxicity in keratinocyte cells and teratogenicity in zebrafish embryos. This finding is important for drug research on safe topical applications during pregnancy, when preventing exposure to drug and chemical-derived teratogens is vital.

Panoramic dental X-ray exposure leads to oxidative stress, inflammation and apoptosis-mediated developmental defects in zebrafish embryos

Panoramic x-ray units are widely used in dental radiodiagnostics. Patients are exposed to relatively low radiation doses with panoramic imaging, but considering lifetime frequency of exposure, even a small risk can have serious health consequences. Our aim was to assess the effects of panoramic x-rays at two different exposure times on developing zebrafish embryos, focusing on oxidative stress, inflammation, apoptotic pathways, and development. Zebrafish embryos were divided into three groups: control, standard panoramic (SPE, 5.5 s exposure time) and pedodontic panoramic x-ray group (PPE, 4.8 s exposure time). Optically stimulated luminescence dosimeters were used to measure absorbed doses. Mean radiation doses for SPE and PPE were 7.83 mSv and 5.83 mSv respectively. At the end of 96 h post-fertilization, lipid peroxidation (LPO), nitric oxide (NO), reduced glutathione (GSH), glutathione S-transferase and superoxide dismutase were measured in the embryos. Expressions of genes related with inflammation (tnfα, il6, ill15, il21), immunoregulation (ifng) and apoptosis (p53, bax, casp2, casp3, casp8) were determined by RT-PCR. Even at reduced doses at high-speed mode, developmental toxicity was observed in both groups as evidenced by decreased pigmentation, yolk sac oedema, and spinal curvature. While deterioration of oxidant-antioxidant balance, suppression of immune response, induction of inflammation and apoptosis were observed through increased LPO, NO, decreased GSH, ifng, and increased expressions of genes related with inflammation and apoptosis, these effects were more pronounced in the SPE group. These results demonstrate the influence of exposure time and indicate the need for further consideration of optimal panoramic modes from a radiation-induced damage perspective.