Article

Phenanthrene and fluorene-mediated early development toxicity in zebrafish

Authors:
If you want to read the PDF, try requesting it from the authors.

Abstract

The early developmental toxicity in zebrafish exposed by Phenanthrene (Phe) and Fluorene (Fl) was investigated by determining mortality and morphological malformation endpoints, such as no inflation of swimming bladder (no ISB), axial malformations (AM, crooked/clubbed), pericardial edema (PE) and yolk sac edema (YSE). The lethal concentration to cause 50% mortality (LC50) in embryos was 78.632 μmol L-1 for Phe and 236.930 μmol L -1 for Fl, respectively. Embryos exposed to 40 μmol L-1 Phe exhibited a reduction of blood stream speed and sharply increasing of axial malformation rate. When exposed to 80 μmol L-1 Phe, the significant (at p<0.05) increase of PE or YSE index was observed. Embryos exposed to Fl had significant axial malformations, but didn't cause significant PE, YSE and speed reduction of blood stream as those exposed to Phe. The joint effect of Phe and Fl was antagonistic. The 42-h and 114-h Phe-exposure results showed simi ar mortality and malformation rates. All of the toxic endpoints showed the significant differences (p<0.05) between 18-h and 114-h exposure, suggesting that 42-h exposure produced very serious and irreversible toxicological effects, but that 18-h Phe-exposure only generated slight, reversible toxic effects. At low concentrations (3.12-12.5 μmol L -1) of Phe producing no obviously morphological abnormality or mortality, the locomotion analysis on the larval zebrafish showed that Phe-exposed groups have significantly (p<0.05) higher speed movements (>15 mm/s) than control groups.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... This particular observation is believed to be compound-specific, or only induced by PAHs and PAH-containing PS. These findings are in line with what has been reported in the literature, where the exposure to individual and mixtures of PAHs cause similar developmental effects (Goodale et al., 2013;Gu et al., 2010;Huang et al., 2012;Zhang et al., 2012;Wincent et al., 2015;Geier et al., 2018). For example, studies by Incardona et al. (2004;2006; showed that some 3-ring (fluorene, phenanthrene, dibenzothiophene), 4-ring (pyrene, benz[a]anthracene), and 5-ring PAHs (benzo[a]pyrene, benzo[k]fluoranthene), and mixtures of PAHs cause severe pericardial and yolk sac edemas, dorsal curvature of the trunk and tail, and growth retardation in the exposed-zebrafish embryo at 48-96 hpf. ...
... As a result, for the PS samples tested, the EC50 for teratogenic endpoints could not be derived and thus also the TI could not be established. These observed effects are in line with those of previous studies where the prominent developmental aberration upon exposure to PAHs and PAHmixture are limited to pericardial edema, yolk sac edema, and cumulative mortality (Goodale et al., 2013;Gu et al., 2010;Huang et al., 2012;Zhang et al., 2012;Wincent et al., 2015;Geier et al., 2018). In view of this, one may argue on the suitability of the ZET performed in the present study to evaluate possible PDT potency of PAHs-containing PS, also how do the morphological effects relate to mortality upon exposure to these substances. ...
... Together these results show that the fume condensate extracts of both bitumen and oxidized asphalt do not cause any in vitro developmental toxicity, in agreement with their test results observed in vivo. Moreover, exposed zebrafish embryos also developed pericardial and/or yolk sac edema, and such effects are common manifestations/profiles of zebrafish embryos exposed to individual PAHs or PAH-containing substances in the ZET, which are believed to be mediated via the AhR (Goodale et al., 2013;Gu et al., 2010;Huang et al., 2012;Incardona et al., 2004;Wincent et al., 2015;Geier et al., 2018). Hence, it is not surprising to see those edema effects as both test materials showed transient AhR induction in the AhR CALUX assay applied. ...
Article
The potential developmental toxicity and mode-of-action of fume condensate extracts of bitumen and oxidized asphalt were evaluated in the aryl hydrocarbon receptor (AhR) CALUX assay, the zebrafish embryotoxicity test (ZET), and the mouse embryonic stem cell test (mEST). In the AhR CALUX assay, both fume condensate extracts showed a concentration-dependent AhR induction following 6-h of exposure, but this activity was substantially reduced after 24-h, indicating a transient AhR activation. The main effects observed in the ZET were early embryonic lethality that occurred mostly in the 24 h-post-fertilization (hpf). This typically reflects non-specific toxicity rather than in vitro developmental toxicity of the fume condensate extracts tested since this effect was not seen as a result of the whole cumulative exposure period in the ZET (up to 96 hpf). No malformations were seen in any zebrafish embryo exposed to these fume condensate extracts, although some developed pericardial and/or yolk-sac edemas. Furthermore, both fume condensate extracts tested negative in the mEST. In conclusion, the results show that fume condensate extracts of bitumen and oxidized asphalt do not induce any in vitro developmental toxicity, which is in line with the results observed in the in vivo prenatal developmental toxicity studies performed with the same materials.
... This is most likely due to the induction of similar metabolic pathways, such as cytochrome P450-induced oxidation, as the systemic toxicity of these PAHs differs vastly. In fish, exposure to PAHs can induce developmental effects such as skeleton malformations, eye and cardiac defects (Incardona et al., 2006;Gu et al., 2010;Zhang et al., 2012;Huang et al., 2013). At a molecular level, transcriptomic analysis shows that exposure to specific PAHs may alter the gene expression profile of the organisms. ...
Article
Aromatic hydrocarbons (AH) are known to impair fish early life stages (ELS). However, poorly defined exposures often confound ELS test interpretation. Passive dosing (PD) overcomes these challenges by delivering consistent, controlled exposures. The objectives of this study were to: apply PD to obtain 5 d acute embryo lethality and developmental and 30 d chronic embryo-larval survival and growth effects data using zebrafish with different AHs; analyze study and literature toxicity data using target lipid (TLM) and chemical activity (CA) models; extend PD to a mixture and test the assumption of AH additivity. PD maintained targeted exposures over a six order of magnitude range in concentrations. AH toxicity increased with Log Kow up to pyrene (5.2). Pericardial edema was the most sensitive sub-lethal effect that often preceded embryo mortality although some AHs did not produce developmental effects at concentrations causing mortality. Cumulative embryo-larval mortality was more sensitive than larval growth with acute to chronic ratios < 10. More hydrophobic AHs did not exhibit toxicity at aqueous saturation. The relationship and utility of the TLM/CA models for characterizing fish ELS toxicity is discussed. Application of these models indicated concentration addition provided a conservative basis for predicting ELS effects for the mixture investigated.
Article
Phenanthrene (PHE) is among the most abundant and ubiquitous polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment as a result of human activities.this study, juvenile yellow catfish (Pelteobagrus fulvidraco) was exposed to 0.05(μ/L and 100 μ/L of waterborne PHE during 14 days. Hepatic enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were quantified. The ratio of specific antioxidant enzyme activity from treatments to the controls was expressed as an index, the relative enzymatic activity (REA). Hepatic SOD exposed to 0.05 μ/L PHE was induced significantly (P < 0.05) at day 0.25, 1, 3 and 14 except at day 7. Whereas, the SOD of 100 μg/L PHE treatment declined significantly at day 0.25 (P < 0.05), and recovered to the control level at day 1 or day 3 following increased at day 7 or day 14. Hepatic CAT of both treatments were declined with the exposure duration before day 14, and gradually increased at day 14. Hepatic GPx exposed to 0.05 and 100 μg/L PHE significant induced to the highest level at day 0.25 and day 3 respectively, and they tended to recover at day 14. Overall, the results indicate that yellow catfish is susceptible to the waterborne PHE that even in 0.05 νg/L could affect the antioxidant defense systems in liver, and demonstrate a higher adaptive competence expressed as hepatic antioxidant enzyme activation. So we considered that the hepatic antioxidant enzyme of yellow catfish has potential to be a sensitive biomarker of PHE exposure.
Article
Full-text available
Fish embryos exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) from petrogenic sources show a characteristic suite of abnormalities, including cardiac dysfunction, edema, spinal curvature, and reduction in the size of the jaw and other craniofacial structures. To elucidate the toxic mechanisms underlying these different defects, we exposed zebrafish (Danio rerio) embryos to seven non-alkylated PAHs, including five two- to four-ring compounds that are abundant in crude oil and two compounds less abundant in oil but informative for structure-activity relationships. We also analyzed two PAH mixtures that approximate the composition of crude oil at different stages of weathering. Exposure to the three-ring PAHs dibenzothiophene and phenanthrene alone was sufficient to induce the characteristic suite of defects, as was genetic ablation of cardiac function using a cardiac troponin T antisense morpholino oligonucleotide. The primary etiology of defects induced by dibenzothiophene or phenanthrene appears to be direct effects on cardiac conduction, which have secondary consequences for late stages of cardiac morphogenesis, kidney development, neural tube structure, and formation of the craniofacial skeleton. The relative toxicity of the different mixtures was directly proportional to the amount of phenanthrene, or the dibenzothiophene-phenanthrene total in the mixture. Pyrene, a four-ring PAH, induced a different syndrome of anemia, peripheral vascular defects, and neuronal cell death, similar to the effects previously described for potent aryl hydrocarbon receptor ligands. Therefore, different PAH compounds have distinct and specific effects on fish at early life history stages.
Article
Full-text available
Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.
Article
Full-text available
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. Traditionally, much of the research has focused on the carcinogenic potential of specific PAHs, such as benzo(a)pyrene, but recent studies using sensitive fish models have shown that exposure to PAHs alters normal fish development. Some PAHs can induce a teratogenic phenotype similar to that caused by planar halogenated aromatic hydrocarbons, such as dioxin. Consequently, mechanism of action is often equated between the two classes of compounds. Unlike dioxins, however, the developmental toxicity of PAH mixtures is not necessarily additive. This is likely related to their multiple mechanisms of toxicity and their rapid biotransformation by CYP1 enzymes to metabolites with a wide array of structures and potential toxicities. This has important implications for risk assessment and management as the current approach for complex mixtures of PAHs usually assumes concentration addition. In this review we discuss our current knowledge of teratogenicity caused by single PAH compounds and by mixtures and the importance of these latest findings for adequately assessing risk of PAHs to humans and wildlife. Throughout, we place particular emphasis on research on the early life stages of fish, which has proven to be a sensitive and rapid developmental model to elucidate effects of hydrocarbon mixtures.
Article
Results are presented of PAH measurements in Flanders and Brussels at different locations. Low flow sampling on filters is used to collect particulate phase PAH which are extracted with dichloromethane by ultrasonic or micro-Soxhlet extraction and analysed by means of HPLC with fluorescence detection or GC-MS in SIM-mode. The highest concentrations were found in winter periods exceeding those in the summer by a factor of 2.5 to 4.5 depending on the location. PAH concentration levels in Brussels are slightly higher than in the Flanders locations. On the absence of local occasional sources, annual averages calculated from 90 samples are within 10 % of the values calculated from all samples.
Article
This document presents an overview of the sources contributing to ambient polycyclic aromatic hydrocarbon (PAH) concentrations. The measurement and characterisation of these PAH and the preparation of inventories to allow source contribution is also discussed, along with the evidence and reasons for the measured decline in the ambient concentrations of PAH.
Article
Effects of naphthalene and phenanthrene on natural phytoplankton C-assimilation were examined and compared to effects of the same aromatics on the laboratory dgzPhaeodactylum tricornutum. Most of the concentrations of aromatics used depressed the photosynthesis of the algae. Marine phytoplankton was found more sensitive than freshwater phytoplankton and P. tricornutum was found intermediate. Freshwater phytoplankton collected in the spring and dominated by centric diatoms was found more sensitive to naphthalene than phytoplankton collected in the autumn and dominated by green algae. The species composition was believed to be responsible for this change of sensibility. The effects of naphthalene and phenanthrene on the light saturated oxygen evolution of P. tricornutum was found less than the effects on the light saturated C-assimilation of the same alga.
Article
Zebrafish has many advantages as a model of human pediatric research. Given the physical and ethical problems with performing experiments on human patients, biomedical research has focused on using model organisms to study biologic processes conserved between humans and lower vertebrates. The most common model organisms are small mammals, usually rats and mice. Although these models have significant advantages, they are also expensive to maintain, difficult to manipulate embryonically, and limited for large-scale genetic studies. The zebrafish model nicely complements these deficiencies in mammalian experimental models. The low cost, small size, and external development of zebrafish make it an excellent model for vertebrate development biology. Techniques for large-scale genome mutagenesis and gene mapping, transgenesis, protein overexpression or knockdown, cell transplantation and chimeric embryo analysis, and chemical screens have immeasurably increased the power of this model organism. It is now possible to rapidly determine the developmental function of a gene of interest in vivo, and then identify genetic and chemical modifiers of the processes involved. Discoveries made in zebrafish can be further validated in mammals. With novel technologies being regularly developed, the zebrafish is poised to significantly improve our understanding of vertebrate development under normal and pathologic conditions.
Article
Male Sprague-Dawley rats were treated with a single ip injection of physiological saline (3.0 ml/kg), dimethyl sulfoxide (DMSO, 3.0 ml/kg), phenanthrene (150 mg/kg), ozonized products of phenanthrene (150 mg/kg), pyrene (150 mg/kg), or ozonized products of pyrene (150 mg/kg). Phenanthrene, pyrene, and their ozonized products were dissolved in DMSO (50 mg/ml). Serum aspartate aminotransferase (AST) activity was increased significantly 24 hr after ip administration of DMSO when compared with physiological saline. Phenanthrene produced a significant elevation of serum AST and gamma-glutamyl transpeptidase (GGTP) levels related to physiological saline and DMSO-injected rats 24 hr after injection. However, GGTP levels for groups treated with DMSO or phenanthrene were not significantly increased when compared with saline groups 72 hr after injection. Ozonized products of phenanthrene produced a significant elevation of serum AST, alanine aminotransferase (ALT), GGTP, and bilirubin levels when compared with groups treated with physiological saline, DMSO, and phenanthrene 24 or 72 hr after injections. The ozonized products of phenanthrene also produced significant elevation of serum creatinine levels compared with physiological saline, DMSO, and phenanthrene groups at 24 hr after treatment and of blood urea nitrogen (BUN) levels at 24 and 72 hr. Although pyrene caused a small but significant increase in the serum AST and bilirubin levels 24 hr after treatment, no significant change in the serum AST, ALT, GGTP, BUN, and creatine levels were observed with the ozonized products of pyrene at 24 or 72 hr. This study demonstrates significant alterations in serum chemistry induced by reaction products of ozone with phenanthrene. No such effect was observed when the products of pyrene ozonation were administered. Although the ozonation products of pyrene were not toxic under the conditions of this study, phenanthrene products were more hepatotoxic than was phenanthrene itself. Nephrotoxicity was also an apparent effect of ozonized phenanthrene. Since ozone-polycyclic aromatic hydrocarbon (PAH) reactions may occur in the atmosphere, these reactions might produce compounds that are more toxic than either ozone or the PAH alone.
Article
Structure-toxicity relationships were investigated for six organic contaminants, representative of three chemical classes, likely to be found in coal conversion process waters and effluents. Using embryo-larval stages of the rainbow trout (Salmo gairdneri) and largemouth bass (Micropterus salmoides), continuous-flow toxicity tests were performed on hydroxylated aromatic hydrocarbons (phenol, beta-naphthol), azaarenes (quinoline, acridine), and polycyclic aromatic hydrocarbons (naphthalene, phenanthrene). Exposure was initiated at fertilization and maintained through 4 days post-hatching. Median lethal concentrations (LC50), based on combined frequencies of embryo-larval mortality and teratogenesis, were used to rank the toxicity of the compounds to each fish species. With the trout, the order of decreasing toxicity was phenanthrene (0.04 mg/L), beta-naphthol (0.07 mg/L), naphthalene (0.11 mg/L), phenol (0.15 mg/L), acridine (0.32 mg/L) and quinoline (11.0 mg/L). The toxicological ranking with the bass was phenanthrene (0.18 mg/L), naphthalene (0.51 mg/L), acridine (1.02 mg/L), beta-naphthol (1.77 mg/L), phenol (2.80 mg/L) and quinoline (7.50 mg/L). For each class of compounds, the chemical with the greater number of aromatic rings always exerted the greater toxicity. In tests with both fish species, beta-naphthol (two rings) was about twice as toxic as phenol (one ring), and phenanthrene (three rings) was nearly three times more toxic than naphthalene (two rings). Acridine (three rings) was seven times more toxic to bass and 34 times more toxic to trout than was quinoline (two rings). This relationship between ring number and toxicity was in excellent agreement with results from acute tests on the same compounds. Furthermore, a close correlation existed between toxicity and n-octanol:water partition coefficients within each class of compounds.
Article
The toxicity of two amines, nonylamine and decylamine, which have a narcotic effect on organisms, and ethylparathion (enzymatic inhibitor) on Daphnia magna has been studied when acting singly and in joint toxicity tests. This was done on the basis of an isobologram method where curves of constant response, i.e., isoboles are plotted vs. the concentrations of the two toxicants. The concentrations that immobilized 50, 10, and 0% of the test population in 48 h (IC50-48h, IC10-48h, and IC0-48h) were calculated. It is shown that nonylamine-decylamine mixtures follow a near concentration addition model of joint toxicity, while ethylparathion-decylamine mixtures follow a less than additive or near independent action model. The study was performed using three toxicity indices: additive index, sum of toxic units, and similarity parameter lambda. An analysis of the similarity parameter lambda is done to evaluate whether it has a constant value for IC50-48h, IC10-48h, and IC0-48h. The results suggest that it is quite probable that a constant value of lambda can be used to characterize all response levels, as the isoboles corresponding to the average value of the similarity parameter fit within the 95% confidence intervals of toxicant concentrations at all response levels. The average value of lambda is about 0.80 for the nonylamine-decylamine test and 0.30 for the ethylparathion-decylamine test.
Article
The zebrafish embryo has become an important vertebrate model for assessing drug effects. It is well suited for studies in genetics, embryology, development, and cell biology. Zebrafish embryos exhibit unique characteristics, including ease of maintenance and drug administration, short reproductive cycle, and transparency that permits visual assessment of developing cells and organs. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large-scale drug screening. Here we describe the use of zebrafish bioassays for assessing toxicity, angiogenesis, and apoptosis. Using 18 chemicals, we demonstrated that toxic response, teratogenic effects, and LC(50) in zebrafish are comparable to results in mice. The effects of compounds on various organs, including the heart, brain, intestine, pancreas, cartilage, liver, and kidney, were observed in the transparent animals without complicated processing, demonstrating the efficiency of toxicity assays using zebrafish embryos. Using endogenous alkaline phosphatase staining and a whole-animal enzyme assay, we demonstrated that SU5416 and flavopiridol, compounds shown to have antiangiogenic effects in mammals, inhibit blood vessel growth in zebrafish, and this bioassay is suitable for high-throughput screening using a 96-well microplate reader. We also demonstrated that in vivo acridine orange staining can be used to visualize apoptotic events in embryos treated with brefeldin A, neomycin, or caspase inhibitors. After in vivo staining, acridine orange can be extracted and quantitated using a fluorescence microplate reader, providing a screening system for agents that modulate apoptosis.
Article
Zebrafish combine the relevance of a vertebrate with the scalability of an invertebrate. They can live in 96-well plate format and readily absorb chemicals from the water. These features have stimulated the use of zebrafish by medical researchers to model human disease and then assess the action of compounds in a whole organism. Examples of the power of this system have been illustrated with the cloning of zebrafish human ether-a-go-go-related gene (HERG), which shows near 100% homology in key domains, and the associated ability to identify drugs that prolong the QT interval both rapidly and with tiny amounts (micrograms) of compound.