Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.     

Preparation and determination of optical purity of γ-lysine modified peptide nucleic acid analogues

Peptide nucleic acids (PNAs) are DNA analogues in which the nucleic acid backbone is replaced by a pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. γ-Carbon modification of the PNA structure allows monomers, and subsequently oligomers, with improved properties to be obtained. In this study, we report the convenient synthesis of γ-lysine-modified PNA monomers for pyrimidine bases (thymine and cytosine) with high optical purity (> 99.5%) and direct enantiomer separation of γ-lysine-modified PNA analogs, using chiral HPLC to determine the optical purity.     

肽核酸单体骨架修饰的研究进展

肽核酸(PNA)是DNA模拟物,能与DNA和RNA以序列特异性方式结合,在基因研究和基因治疗方面有广泛的应用前景。为了优化肽核酸的性质(如水溶性、对细胞的透膜能力、杂交专一性),许多骨架修饰的PNA被合成出来。该文综述了近年来肽核酸单体骨架修饰的合成研究进展。指出设计新型的PNA结构是改良PNA性能、拓宽DNA应用的主要途径。     

Peptide-nucleic acids (PNAs): a tool for the development of gene expression modifiers

Peptide nucleic acids (PNAs) represent nucleic acid analogues with unique biochemical properties and of great interest for the development of therapeutic agents. The firstly designed and tested PNAs are molecules in which the sugar-phosphate backbone of DNA was replaced with a pseudopeptide chain constituted by N-(2-aminoethyl) glycine monomers. Nucleobases can be linked to this backbone through a carboxymethyl moiety, which allows to maintain a two atom spacer between the backbone and the bases. Since the first reports on PNAs based on N-(2-aminoethyl) glycine backbone, other PNA analogues have been synthesized, with the main purpose of improve biological activities as well as stability and efficient delivery to target cells. Of great interest are chiral PNAs, PNA analogues bearing phosphate groups (PHONA), PNA-DNA and PNA-peptide chimeras, PNA linked to non-peptide vectors. PNAs hybridize to DNA and RNA with high efficiency following the Watson-Crick hybridization rules, forming highly stable PNA/DNA and PNA/RNA duplexes. In addition, homopyrimidine PNAs, as well as PNAs containing a high pyrimidine:purine ratio, are able to bind to DNA or RNA forming highly stable (PNA)(2)-DNA triple helices. Accordingly, therapeutic PNA and PNA analogues could act as antigéne as well as antisense molecules. In addition, recent studies provide evidences for the possible use of PNA-based therapeutic molecules as artificial promoters, as decoy or ribozyme facilitator. Among the therapeutic applications of PNA-based molecules, the most pomising include anti-cancer and anti-viral experimental strategies, but activity of PNAs against bacteria and medically important parasitic organisms have been also reported.     

Fluorine‐18 labeling of peptide nucleic acids

Peptide nucleic acids (PNAs) are a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double stranded DNA. From a chemical point of view, the deoxyribose phosphate backbone of DNA is replaced by a pseudo‐peptide N‐(2‐aminoethyl)glycyl backbone, while the nucleobases of DNA (adenine, guanine, cytosine and thymine) are retained. Due to the increasing interest in the labeling of peptide nucleic acids (PNAs) as potent diagnostic agents in nuclear medicine, we have used and adapted the reliable methodology developed for the fluorine‐18 labeling of oligonucleotides and have now demonstrated that it is possible to label PNAs in sufficient quantity and with high specific radioactivity for PET studies in a time compatible with the half life of fluorine‐18. Copyright © 2002 John Wiley & Sons, Ltd.     

Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by β-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo.     

Inflammation–coagulation response and thrombotic effects induced by silica nanoparticles in zebrafish embryos

Nowadays, nanotechnology environmental health and safety (nanoEHS) is gaining attention. We previously found that silica nanoparticles (SiNPs) could induce vascular endothelial damage. However, the subsequent toxicologic response to SiNPs-induced endothelial damage was still largely unknown. In this study, we explored the inflammation–coagulation response and thrombotic effects of SiNPs in endothelial cells and zebrafish embryos. For in vitro study, swollen mitochondria and autophagosome were observed in ultrastructural analysis. The cytoskeleton organization was disrupted by SiNPs in vascular endothelial cells. The release of proinflammatory and procoagulant cytokines including IL-6, IL-8, MCP-1, PECAM-1, TF and vWF, were markedly elevated in a dose-dependent manner. For in vivo study, based on the NOAEL for dosimetry selection, and using two transgenic zebrafish, Tg(mpo:GFP) and Tg(fli-1:EGFP), SiNPs-induced neutrophil-mediated inflammation and impaired vascular endothelial cells. With the dosage higher than NOAEL, SiNPs significantly decreased blood flow and velocity, exhibiting a blood hypercoagulable state in zebrafish embryos. The thrombotic effect was assessed by o-dianisidine staining, showed that an increasing of erythrocyte aggregation occurred in SiNPs-treated zebrafish. Microarray analysis was used to screen the possible genes for inflammation–coagulation response to SiNPs in zebrafish, and the JAK1/TF signaling pathway was further verified by qRT-PCR and Western blot assays. For in-deepth study, il6st was knocked down with specific morpholinos. The whole-mount in situ hybridization and qRT-PCR analysis showed that the expression jak1 and f3b were attenuated in il6st knockdown groups. In summary, our data demonstrated that SiNPs could induce inflammation–coagulation response and thrombotic effects via JAK1/TF signaling pathway.     

Peptide nucleic acids (PNAs) as diagnostic devices for genetic and cytogenetic analysis

The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acids analogs, based on peptide-like backbone. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences, and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a large variety of research and diagnostic assays in the field of genetics, including genome mapping and mutation detection. Over the last few years, the use of PNAs has also proven its powerful usefulness in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, spermatozoa as well as on isolated oocytes and blastomeres. Multicolor peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful tool for in situ chromosomal investigation.     

Triclocarban at environmentally relevant concentrations induces the endoplasmic reticulum stress in zebrafish

Triclocarban (TCC) is an antibacterial agent commonly found in environmental, wildlife, and human samples. However, with in‐depth study of TCC, its negative effects are increasingly presented. Toxicological studies of TCC at environmentally relevant concentrations have been conducted in zebrafish embryos and indicated that TCC leads to deformity of development causes developmental deformities. However, the molecular mechanisms underlying the toxicity of TCC in zebrafish embryos have not been entirely elucidated. We investigated whether exposure to TCC at environmentally relevant concentrations induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in zebrafish. Zebrafish embryos were grown to 32 hours post fertilization and exposed to 2.5, 5, and 10 μg/L TCC and used in whole‐mount in situ hybridization to visualize the expression of ER chaperone hspa5 and ER stress‐related apoptosis factor chop. Zebrafish livers were exposed to different concentrations of TCC to elaborate the relationships between fatty degeneration and ER stress. Then, a human hepatic cell line (HL‐7702) was used to test whether TCC induced ER stress in human livers similar to those of zebrafish. In zebrafish embryos, TCC induced high hspa5 expression, which could defend against external stimulations. Furthermore, hapa5, hsp90b1, and chop exhibited ectopic expressions in the neuromast, intestinal tract, and tail tip of zebrafish embryos. On the one hand, significant differences were observed in the mRNA and protein expressions of the ER stress molecular chaperone pPERK‐pEIF2a‐ATF4 and ATF6 pathways in HL‐7702 cells exposed to TCC. On the other hand, lipid droplet accumulation slightly increased in zebrafish livers exposed to 10 μg/L TCC in vitro. These results demonstrate that TCC not only damages the development of zebrafish embryos and structure of zebrafish liver but also influences human hepatic cells by activating ER stress and the UPR signaling pathway.     

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up‐regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 × 10^?6, 40 × 10^?6 and 80 × 10^?6 m of benomyl‐treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase‐based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 × 10^?6 m concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds. Copyright © 2008 John Wiley & Sons, Ltd.     

Is there an association between the effects of clozapine on the production of reactive oxygen metabolites by blood monocytes and clinical outcome in neuroleptic-resistant schizophrenia?

The production of reactive oxygen metabolites (ROM) by monocytes (MO) and polymorphonuclear leukocytes (PMNL) from eight patients with neuroleptic-resistant schizophrenia was measured before and after 3 and 10 weeks of clozapine (CLO) treatment. Although ROM production did not show significant longitudinal trends during the trial, the changes in ROM production by non-stimulated (Mon) and phorbol myristate acetate (PMA)-stimulated MO (MOs) at week 3 (values at base-line minus those at week 3) correlated positively (r=0·743, p=0·035, and r=0·838, p=0·009, respectively) with the changes on the total Positive and Negative Syndrome Scale (PANSS) scores, i.e. a decrease or relatively small increase in the ROM production by MOn and MOs was associated with more favourable clinical outcome than a clear-cut increase in their ROM production. The serum concentrations of CLO at week 3 also correlated positively with the changes in ROM production by MOs at week 3 (r=0·761, p=0·047) and at week 10 (r=0·985, p<0·001). A possible causal relationship between these observations and the mechanism of action of CLO is discussed. © 1998 John Wiley & Sons, Ltd.     

Fluorine‐18 labelling of PNAs functionalized at their pseudo‐peptidic backbone for imaging studies with PET

Peptide nucleic acids (PNAs) form a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double‐stranded DNA, where the deoxyribose phosphate backbone of original DNA is replaced by a pseudo‐peptide N‐(2‐aminoethyl)glycyl backbone, while retaining the nucleobases of DNA. We have previously developed an original method to label oligonucleotide‐based macromolecules with the short‐lived positron‐emitter fluorine‐18 (t_1/2: 109.8 min) using the N‐(4‐[¹⁸F]fluorobenzyl)‐2‐bromoacetamide reagent. Using this method, we herein report the fluorine‐18‐labelling of 13 decameric PNAs ( OLP_1‐13 ), of the same sequence (CTCATACTCT), but presenting selected modification of the pseudo‐peptidic backbone at two or three of the thymine residues (positions 2, 5 and 8). Structural characteristics of these backbone modifications include either an amino acid side chain (L ‐Lys, L ‐Glu, L ‐Leu and L ‐Arg) or a glycosyl moiety (mannose, galactose, fucose, N‐Ac‐galactosamine and N‐Ac‐glucosamine) attached via an appropriate spacer. N‐(4‐[¹⁸F]fluorobenzyl)‐2‐bromoacetamide was synthesized in three radiochemical steps from 4‐cyano‐N,N,N‐trimethylanilinium trifluoromethanesulfonate and HPLC‐purified in 85–90 min (typical production: 3.7–4.8 GBq starting from a batch of 29.6–31.4 GBq of [¹⁸F]fluoride). Conjugation of the fluorine‐18‐labelled bromoacetamide reagent with the PNAs was performed in a mixture of acetonitrile and HEPES buffer (0.1 M, pH 7.9) for 10 min at 60°C and gave the corresponding pure labelled conjugated PNAs ([¹⁸F] c‐OLP_1‐13 ) after RP‐HPLC purification. The whole synthetic procedure, including the preparation of the fluorine‐18‐labelled reagent, provides up to 0.9 GBq (25 mCi) of HPLC‐purified [¹⁸F] c‐OLP_1‐13 in 160 min with a specific radioactivity of 45–65 GBq/µmol (1.2–1.7 Ci/µmol) at the end of synthesis starting from 29.6 to 31.4 GBq (800–850 mCi) of [¹⁸F]fluoride. Copyright © 2004 John Wiley & Sons, Ltd.     

Toxicity assessment and vitellogenin expression in zebrafish (Danio rerio) embryos and larvae acutely exposed to bisphenol A,endosulfan, heptachlor,methoxychlor and tetrabromobisphenol A

Organochlorine pesticides and brominated flame retardants, such as tetrabromobisphenol A and polybrominated diphenyl ethers, pose an environmental hazard owing to their persistence, low solubility and estrogenic effects, and concerns have been raised regarding their effects on aquatic biota. In the present study, zebrafish embryos and larvae were used as a model to investigate the sublethal and lethal effects of three different organochlorine pesticides, namely methoxychlor, endosulfan and heptachlor, as well as the flame retardant tetrabromobisphenol A, and its precursor compound bisphenol A. Preliminary data for chemical exposure tests were obtained by determining the 96 h median effective concentration EC₅₀ (hatching rate) and 96 h median lethal concentration LC₅₀. Quantitative polymerase chain reaction was used to investigate the gene expression levels of the biomarker vitellogenin (vtg1) after 96 h exposures to 10, 25, 50 and 75% of the 96 h EC₅₀ value for embryos and 96 h LC₅₀ value for larvae. The use of vtg1 mRNA induction in zebrafish embryos and larvae was found to be a sensitive biomarker of exposure to these organic compounds, and was helpful in elucidating their adverse effects and setting water quality guidelines. Copyright © 2012 John Wiley & Sons, Ltd.     

Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development

The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo.     

The impact of nucleic acid secondary structure on PNA hybridization

Hybridization of oligonucleotides and their analogues to complementary DNA or RNA sequences is complicated by the presence of secondary and tertiary structure in the target. In particular, folding of the target nucleic acid imposes substantial thermodynamic penalties to hybridization. Slower kinetics for hybridization can also be observed, relative to an unstructured target. The development of high affinity oligonucleotide analogues such as peptide nucleic acid (PNA) can compensate for the thermodynamic and kinetic barriers to hybridization. Examples of structured targets successfully hybridized by PNA oligomers include DNA duplexes, DNA hairpins, DNA quadruplexes and an RNA hairpin embedded within a mRNA.     

Triphenyl phosphate-induced pericardial edema is associated with elevated epidermal ionocytes within zebrafish embryos

Triphenyl phosphate (TPHP) is an organophosphate ester-based plasticizer and flame retardant. The objective of this study was to identify the potential role of epidermal ionocytes in mediating TPHP-induced pericardial edema within zebrafish embryos. Exposure to TPHP from 24 to 72 h post fertilization (hpf) resulted in a significant increase in pericardial edema and the number of ionocytes at 72 hpf relative to time-matched embryos treated with vehicle. In addition, co-exposure of embryos to mannitol (an osmotic diuretic) blocked TPHP-induced pericardial edema and effects on ionocyte abundance. However, knockdown of ATPase1a1.4 – an abundant Na⁺/K⁺-ATPase localized to epidermal ionocytes – mitigated TPHP-induced effects on ionocyte abundance but not pericardial edema, whereas co-exposure of embryos to ouabain – a Na⁺/K⁺-ATPase inhibitor – enhanced TPHP-induced pericardial edema but not ionocyte abundance. Overall, our findings suggest that TPHP may have multiple mechanisms of toxicity leading to an increase in ionocyte abundance and pericardial edema within developing zebrafish embryos.     

去水卫矛醇对斑马鱼神经发育毒性评价

目的 探讨去水卫矛醇(dianhydrogalactitol,DAG)诱导斑马鱼胚胎及幼鱼神经发育毒性作用及其机制。方法 在一般毒性评价的基础上,对斑马鱼胚胎进行相应的分组暴露给药,采用幼鱼的自主运动反应、光照刺激反应等试验观察DAG对斑马鱼神经及行为的影响;通过脑部组织病理学检查、吖啶橙染色,观察DAG对斑马鱼脑部的组织影响;利用实时荧光定量PCR方法测定斑马鱼幼鱼体内多巴胺神经元相关基因(DAT、TH、GCH1)及神经抑/促凋亡相关基因(Bax、Bcl-2)的相对表达量。结果 DAG在20,40,75 mg·mL^-1下能明显抑制斑马鱼的自主运动,且自主运动抑制率呈现明显的浓度相关性;DAG在20,40,75 mg·mL^-1下对斑马鱼反应速度有较明显的抑制作用,反应能力下降率呈现浓度相关性;DAG各浓度组斑马鱼脑组织形态变小,但组织结构均未见显著异常;采用吖啶橙染色检测斑马鱼整体胚胎细胞凋亡情况,发现给药组头部绿色荧光比对照组明显,说明细胞凋亡增多,且细胞凋亡呈剂量依赖性增加,与表观一致;DAG在75,150,300,425,600 mg·L^-1下可导致斑马鱼幼鱼多巴胺能神经元相关基因DAT、TH、GCH1的mRNA相对表达量下调,Bax/Bcl-2的RNA相对表达量随着给药浓度增加而上升。结论 高浓度DAG对斑马鱼胚胎和幼鱼具有神经发育毒性作用,可能与多巴胺能神经元的抑制作用有关。     

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model

A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in beta-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.     

Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish

Aim:

To investigate the embryotoxicity of dihydroartemisinin (DHA), the main active metabolite of artemisinin, in zebrafish, and explore the corresponding mechanisms.

Methods:

The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA. Developmental phenotypes of the embryos were observed. Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope. The expression of angiogenesis marker genes vegfa, flk1, and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays.

Results:

Exposure to DHA (1–10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage. Furthermore, exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP) zebrafish line. Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa, flk1, and flt1 in the embryos. Knockdown of the flk1 protein partially blocked the effects of DHA on embryogenesis.

Conclusion:

DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development, demonstrating the potential embryotoxicity of DHA.