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Sublethal exposure causes abnormalities in bones of the F0 zebrafish head. Zebrafish larvae were exposed to TCDD at 50 pg/ml at 3 and 7 wpf and collected at 1 year of age for alizarin red staining as described in the Materials and Methods section. Photographs show lateral views of the live fish and the same view after digestion and staining, revealing the bones. Arrows point to the shortened jaw structures and to the shortened operculum in the live fish and to the altered underlying bone in the stained specimen.
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The acute effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been well documented in many vertebrate species. However, less is known about the consequences in adulthood from sublethal exposure during development. To address this, we exposed zebrafish to sublethal levels of TCDD (1 h; 50 pg/ml), either in early embryogenesis (day 0)...
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... exposure at 3 and 7 wpf also produced craniofacial mal- formations similar to those noted in other studies (Hornung et al., 1999;Xiong et al., 2008). The cartilages and bones mak- ing up the jaw were reduced in size, making the mouth smaller (Fig. 3). In this figure, images of individual control and exposed fish are shown together with their digested and stained skulls, showing the underlying bone structure. From this, it can be seen that the branchial arches and the operculum are shortened in the TCDD-exposed fish, whereas the bone at the top of the skull is thicker than normal. These effects produce a charac- teristic bump at the top of the head and expose the gills, both visible in the live ...
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... According to one study, malformations and increased mortality were identified in the subsequent offspring of zebrafish fish during an embryogenesis experiment. Deformities were also observed in the cranial and axial skeleton during the adult phase, and failures in reproduction, with a clear decrease in egg production and feminization of the population, where fish with distinctly feminine aspects carried testes instead of ovaries [124]. ...
... Kidney malformation (curved and cystic pronephric tube, pronephric duct, and cystic and atrophic glomerulus). The effects were dose-dependent [112] Aristolochic acid (AA) 0.5 μM Acute injury (led to a loss of function in the glomerular filtration barrier) [84] Endocrine system disruptions Water samples from a wastewater dominated stream Complex mixture of Endocrine Active Compounds (EAC) Hormonal imbalances affecting testicular growth, reproduction, inducing feminization of male fish, and consequent reduction of population [120] Sulfentrazone 0.0100; 0.0400; 0.400 mg/L Influenced steroid biosynthesis [122] 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) 50 pg/ml Feminization of the population (appearance of fish with clearly female bodies, yet carrying testes in place of ovaries) [124] Bisphenol A 0.01; 0.1; 1 mg/L Increased egg production, and reduced rate of fertilization. ...
Tel.: +55-19-997866505 (M.A.M.M.). Abstract: Anthropogenic interventions have had a compromising effect on environmental health, intensifying the degradation of ecosystems, and the quantity of chemical pollutants released into nature. Therefore, research areas within the scope of environmental assessments and monitoring such as ecotoxicology have contributed to the determination of the toxic potential of contaminants. A small cyprinid known as the zebrafish (Danio rerio), the use of which has exponentially grown, is an alternative vertebrate model for scientific research, mainly in the assessment of environmental risks. The species exhibits several advantages for breeding in a laboratory, in addition to presenting multi-biomarkers of environmental toxicity. Thus, this review aims to present the main characteristics and advantages of working with this species, as well as show studies related to ecotoxicology involving biomarkers of toxicity in zebrafish. The results show a progressive trend towards employing the species in environmental risk analyses, it is an increasingly recommended species in the assessment of the toxicity level of a range of chemical pollutants. The development of future technologies must contribute to scientific advancement, rendering the potential application of this model organism an even more widespread one, which will certainly help in bridging knowledge gaps in various areas of study. HIGHLIGHTS • Biomarkers of toxicity in zebrafish. • Toxicological screening with the embryonic, larval, and adult stages of zebrafish. • Scientific advancements in the assessment of the effects of environmental contaminants. Braga, A.P.A.; et al. 2
... Moreover, the most affected vertebral region in the common carp was the caudal one, whereas most of the anomalies affected the pre-haemal region in the other examined species. Except for the numerous laboratory experiments carried out to assess the effects of experimental diets (e.g., [46,47]) or toxic compounds (e.g., [48,49]) on zebrafish skeletogenesis, limited information on the occurrence of skeletal anomalies in juvenile cyprinids is available. In a comparative study on juvenile zebrafish, a higher percentage (93.4%) of reared individuals have been reported to be affected by skeletal anomalies compared with wild-caught fish (87.2%) [29]. ...
Cyprinids include some of the most widely farmed freshwater species. The aim of this study was to assess the incidence of skeletal malformations in carp species reared in semi-intensive systems in Albania: common carp (Cyprinus carpio), silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella) and bighead carp (Hypophthalmichthys nobilis). The common carp fingerlings had a mean total length of 28.9 ± 5.0 mm; the frequencies of individuals with at least one anomaly and at least one severe anomaly were 79.2% and 43.4%, respectively. The silver carp juveniles had a mean total length of 21.6 ± 2.1 mm; the frequencies of individuals with at least one anomaly and one severe anomaly were 93.1% and 57.5%, respectively. The grass carp fry had a mean total length of 33.5 ± 2.6 mm; all the analyzed specimens showed almost one anomaly and 86.4% showed at least one severe anomaly. The bighead carp juveniles had a mean total length of 34.4 ± 5.7 mm; the frequencies of individuals with at least one anomaly and at least one severe anomaly were 95.0% and 62.5%, respectively. The development of a more suitable feeding protocol for herbivorous species and the setting up of more efficient broodstock management protocols are suggested to reduce the high incidence of skeletal malformations.
... 8 Abnormalities caused by heavy metals, petroleum hydrocarbons and chlorinated hydrocarbons include amorphous embryonic tissue that usually results in early embryonic mortality and tissue necrosis. 8 Exposure to tetrachlorodibenzo-p-dioxin caused cranial and axial skeleton deformities, 161 and increase the frequency of deformities in newly hatched larvae. 80 Furthermore, exposure to these metals during the early development phase may adversely affect the recruitment process in nature. ...
Fish deformities are detrimental to aquaculture and conservation initiatives; it is also an ethical problem and causes substantial financial losses to the aquaculture industry. The exact mechanism of deformities is still a mystery. Several factors cause deformities in fish; it seems that the deformations are inevitable but can be minimised by adopting good aquaculture practices such as proper broodstock husbandry, standard breeding and larval rearing protocol, management of water quality parameters, and providing nutritionally balanced feed, especially to the broodstock and developing larvae. In addition, it can be minimised by early detection of deformities, especially at the embryonic and larval stages. Monitoring of stocking materials should be an essential part of responsible aquaculture and conservation programmes; stocking materials should be free from any types of deformities as it affects not only aquaculture production, profitability and success of conservation programmes but also the reoccurrence of deformities in nature. The mechanism behind deformities and the development of low-cost, robust methods/tools for detecting deformities in fish remain a challenge. Still, the opportunity exists to develop a robust marker that can predict deformities even at early embryonic and larval stages.
... According to one study, malformations and increased mortality were identified in the subsequent offspring of zebrafish fish during an embryogenesis experiment. Deformities were also observed in the cranial and axial skeleton during the adult phase, and failures in reproduction, with a clear decrease in egg production and feminization of the population, where fish with distinctly feminine aspects carried testes instead of ovaries [124]. ...
... Kidney malformation (curved and cystic pronephric tube, pronephric duct, and cystic and atrophic glomerulus). The effects were dose-dependent [112] Aristolochic acid (AA) 0.5 μM Acute injury (led to a loss of function in the glomerular filtration barrier) [84] Endocrine system disruptions Water samples from a wastewater dominated stream Complex mixture of Endocrine Active Compounds (EAC) Hormonal imbalances affecting testicular growth, reproduction, inducing feminization of male fish, and consequent reduction of population [120] Sulfentrazone 0.0100; 0.0400; 0.400 mg/L Influenced steroid biosynthesis [122] 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) 50 pg/ml Feminization of the population (appearance of fish with clearly female bodies, yet carrying testes in place of ovaries) [124] Bisphenol A 0.01; 0.1; 1 mg/L Increased egg production, and reduced rate of fertilization. ...
... The reason could be that the chorion surrounding the embryo acted as a protective barrier and inhibited the uptake of any chemical (Braunbeck et al., 2005;Tran et al., 2021b). A similar observation regarding chorion acting as a protective barrier against chemical uptake has been reported by Baker et al. (2013). In this regard, experiments were modified, and we exposed 5 days post-fertilized larvae for 15 days of treatments of various phthalates. ...
The plasticizer leaches from the microplastics are one of the significant concerns related to plastic pollution. These plasticizers are known to be endocrine disrupters; however, little is known about their long-term effect on the development of aquatic vertebrates. Hence, the present study has been conducted to provide a holistic understanding of the effect of the three most common plasticizers, dibutyl phthalate (DBP), diethyl phthalate (DEP), and di-ethylhexyl phthalate (DEHP) leaching out from the microplastics in zebrafish development. Zebrafish larvae were exposed to different phthalates at different concentrations. The phthalates have shown significantly higher mortality and morphological changes in the larva upon exposure compared to the control. A significant change in the genes related to cardiovascular development (krit1, fbn2b), dorsoventral axis development (chrd, smad5), tail formation (pkd2, wnt3a, wnt8a), and floorplate development (foxa2) were also observed under the effects of the phthalates in comparison to control.
... Studies have also demonstrated that exposure of fish embryos to heavy metals and POPs have adverse impacts on survival, growth rate, and quality of embryonic development, leading to larval abnormality, malformations, and increased mortality [40]. Moreover, heavy metals and POPs have been shown to induce morphological alterations and growth defects even at relatively low concentrations [37,41]. In a 2008 study [42], exposure during embryonic development of the common carp (Cyprinus carpio) to Cd, Pb, and Cu induced premature hatching, reduced hatchability, and impairment of embryonic development. ...
Although there is rising global concern over the environmental, ecological, and human health risks associated with the discharge of leachates from e-waste dumpsites into the aquatic ecosystems, little is known in this research area. Thus, for this study, we first defined the chemistry of the test leachate, followed by assessment of the leachate on the development of a model aquatic organism (Fundulus heteroclitus) used extensively as a bioassay organism in pollution studies. Chemical analyses revealed that levels of phosphate (20.03 mg/L), cadmium (Cd) (0.4 mg/L), lead (Pb) (0.2 mg/L), and chromium (Cr) (0.4 mg/L) were higher than the 2009 US EPA and the 2009 National Environmental Standards and Regulations Enforcement Agency (NESREA) permissible limits. Polycyclic aromatic hydrocarbon (PAH) burdens were dominated mainly by the high molecular weight congeners, specifically the ∑4rings (73 µg/L). Total polychlorinated biphenyls (PCB) levels ranged from 0.00 to 0.40 µg/L with the ∑deca PCBs reaching the highest concentration. For the biological studies, F. heteroclitus embryos (48-h post-fertilization) were divided randomly into groups and exposed to one of six e-waste leachate concentrations (10, 1, 0.1, 0.01, 0.001, 0.0001%). Significant differences (p ≤ 0.05) between treated and control groups were observed in standard and total length, and head size. Further analysis using Duncan’s post-hoc test of multiple comparison also revealed specific differences within and between specific treatment groups. We conclude that e-waste leachate arising from indiscriminate dumping into aquatic ecosystems in Nigeria contains mixtures of toxic constituents that can threaten ecosystem and public health.
... Interestingly, one study showed that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in juvenile zebrafish caused scoliosis in adult fish. Strikingly, scoliosis was also common in the next two generations (F1 and F2 offspring), indicating that TCDD exposure causes genetic defects in zebrafish spine development [155,156]. ...
Scoliosis is a common spinal deformity that considerably affects the physical and psychological health of patients. Studies have shown that genetic factors play an important role in scoliosis. However, its etiopathogenesis remain unclear, partially because of the genetic heterogeneity of scoliosis and the lack of appropriate model systems. Recently, the development of efficient gene editing methods and high-throughput sequencing technology has made it possible to explore the underlying pathological mechanisms of scoliosis. Owing to their susceptibility for developing scoliosis and high genetic homology with human, zebrafish are increasingly being used as a model for scoliosis in developmental biology, genetics, and clinical medicine. Here, we summarize the recent advances in scoliosis research on zebrafish and discuss the prospects of using zebrafish as a scoliosis model.
... In a rare human study measuring the influence of background levels of TCDD on sperm parameters, Mínguez-Alarcón et al. (11) took TCDD levels in peripubertal 8-to 9-year-old boys, measured semen parameters 10 years later, and found lowered sperm concentration, count, and motility associated with higher background TCDD levels. The link between early-life low-dose dioxin exposure and adult infertility has, in contrast, been well-studied in rodents and aquatic life (12)(13)(14)(15)(16). The NIH-approved model organism, the zebrafish (Danio rerio), has emerged as a useful tool to study these effects of exposure across the lifespan due to its quick generation time and genomic similarity to humans. ...
... By 7 wpf, gonocytes have been replaced by SPG (22), resulting in a landscape more similar to human postnatal testes. We have previously shown brief perinatal exposure to low levels of TCDD (1 hour, 50 pg/mL) at critical points of gonadal differentiation and maturation in zebrafish (21 and 49 dpf) led to decreased reproductive capacity and altered spermatogenesis as evidenced by changes in elicited fecundity (15) and altered cell populations of the seminiferous tubule (16). ...
... TCDD exposure during gonadal differentiation has previously been associated with reduced phenotypic spawning success in adult zebrafish (15). In the present study, multiple molecular pathways related to infertility were enhanced in response to exposure. ...
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical. Our previous work demonstrated the latent reproductive maladies of early-life TCDD exposure in zebrafish. Zebrafish acutely exposed to low, environmentally relevant levels of TCDD (50 pg/mL) during two windows of sexual differentiation in development (one hour of exposure at three and seven weeks post-fertilization) were later infertile, showed a reduction in sperm, and exhibited gene expression consistent with an altered microenvironment, even months after exposure. Due to the highly heterogeneous cell- type and -stage landscape of the testes, we hypothesized various cell types contribute markedly different profiles towards the pathology of TCDD exposure. To investigate the contributions of the diverse cell types in the adult zebrafish testes to TCDD-induced pathology, we utilized single-cell RNA-seq and the 10x Genomics platform. The method successfully captured every stage of testicular germ cell development. Testes of adult fish exposed during sexual differentiation to TCDD contained sharply decreased populations of late spermatocytes, spermatids and spermatozoa. Spermatogonia and early spermatocyte populations were, in contrast, enriched following exposure. Pathway analysis of differentially expressed genes supported previous findings that TCDD exposure resulted in male infertility, and suggested this outcome is due to apoptosis of spermatids and spermatozoa, even years after exposure cessation. Increased germ cell apoptosis was confirmed histologically. These results provide support for an environmental exposure explanation of idiopathic male infertility.
... One of the exogenous factors leading to the appearance of such abnormalities in different fish species is a short-term exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) within the embryonic development period [60][61][62][63]. Dong et al. [63] showed that formation of body curvature is accompanied by gene expression of COL2A1 in tail of medaka larvae. ...
... Exposure to this toxic agent may lead to a kink in the axial skeleton. To analyze polarization patterns in zebrafish with axial skeleton abnormalities, we prepared embryos according to protocol described in [61,63]. Polarization images illustrating the diversity of axial skeleton abnormalities are presented in Figure 5. AOLP and DOLP images of these species clearly demonstrate the difference in the muscle inhomogeneity map from healthy individuals. ...
Zebrafish is a well‐established animal model for developmental and disease studies. Its optical transparency at early developmental stages allows in vivo tissues visualization. Interaction of polarized light with these tissues provides information on their structure and properties. This approach is effective for muscle tissue analysis due to its birefringence. To enable real‐time Mueller‐matrix characterization of unanesthetized fish, we assembled a microscope for single‐shot Mueller‐matrix imaging. First, we performed a continuous observation of 48 species within the period of 2‐96 hpf and measured temporal dependencies of the polarization features in different tissues. These measurements show that hatching was accompanied by a sharp change in the angle and degree of linearly polarized light after interaction with muscles. Second, we analyzed 9 species with skeletal disorders and demonstrated that the spatial distribution of light depolarization features clearly indicated them. Obtained results demonstrated that real‐time Mueller‐matrix imaging is a powerful tool for label‐free monitoring zebrafish embryos. This article is protected by copyright. All rights reserved.
... However, understanding the sources of divergent information and their implications is critical. Our previous toxicological work with scRNA-seq in zebrafish (Danio rerio) testes revealed differential cell type population alterations in response to exposure to the classic environmental contaminant 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) (Haimbaugh et al., 2022), which is known to induce male fertility defects in both fish and humans (King Heiden et al., 2009;Baker et al., 2013;Baker et al., 2016;Eskenazi et al., 2018;Bruner-Tran et al., 2019). One marked difference was a loss of spermatids and spermatozoa; in these cells, pathways of apoptosis and sperm disorder were upregulated. ...
... TCDD (>99% purity) (Chemsyn, Concord, ON, Canada) was used as a 0.4 ng/ml stock solution in dimethyl sulfoxide (DMSO) Zebrafish were exposed as previously described (Henry et al., 1997;Baker et al., 2013). Briefly, fish were exposed at three wpf and again at seven wpf to water-borne TCDD (50 pg/ml) or vehicle (0.1% DMSO) for 1 h each time in small glass beakers with gentle rocking. ...
In this report, we compare the outcomes and limitations of two methods of transcriptomic inquiry on adult zebrafish testes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during sexual differentiation: conventional or bulk RNA-seq (bulk-seq) and single cell RNA sequencing (scRNA-seq) data. scRNA-seq has emerged as a valuable tool for uncovering cell type-specific transcriptome dynamics which exist in heterogeneous tissue. Our lab previously showed the toxicological value of the scRNA-seq pipeline to characterize the sequelae of TCDD exposure in testes, demonstrating that loss of spermatids and spermatozoa, but not other cell types, contributed to the pathology of infertility in adult male zebrafish exposed during sexual differentiation. To investigate the potential for technical artifacts in scRNA-seq such as cell dissociation effects and reduced transcriptome coverage, we compared bulk-sequenced and scRNA-seq-paired samples from control and TCDD-exposed samples to understand what is gained and lost in scRNA-seq vs bulk-seq, both transcriptomically and toxicologically. We hypothesized that the testes may be sensitive to tissue disruption as they contain multiple cell types under constant division and/or maturation, and that TCDD exposure may mediate the extent of sensitivity. Thus, we sought to understand the extent to which this dissociation impacts the toxicological value of data returned from scRNA-seq. We confirm that the required dissociation of individual cells from intact tissue has a significant impact on gene expression, affecting gene pathways with the potential to confound toxicogenomics studies on exposures if findings are not well-controlled and well-situated in context. Additionally, a common scRNA-seq method using cDNA amplified from the 3’ end of mRNA under-detects low-expressing transcripts including transcription factors. We confirm this, and show TCDD-related genes may be overlooked by scRNA-seq, however, this under-detection effect is not mediated by TCDD exposure. Even so, scRNA-seq generally extracted toxicologically relevant information better than the bulk-seq method in the present study. This report aims to inform future experimental design for transcriptomic investigation in the growing field of toxicogenomics by demonstrating the differential information extracted from sequencing cells—despite being from the same tissue and exposure scheme—is influenced by the specific protocol used, with implications for the interpretation of exposure-induced risk.
