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![Timeline showing key events during zebrafish development, from fertilization to juvenile stages (j.) Color bars indicate the developmental phases with gradients representing embryo-larval and larval-juvenile transitions. The period of embryonic development includes pre-hatching stage and eleutheroembryo stage, i.e., the stage between hatching and onset of exogenous feeding [5,10]. The period of organogenesis, i.e., development of brain, heart, liver, intestine and pronephros, coincides with embryonic development. Embryo-larval transition implies the period between the onset of exogenous feeding and complete yolk absorption. Larval-juvenile transition reflects the period of metamorphosis in which the larval morphology is transformed into that of a juvenile (e.g., metamorphosis of the pigment pattern and fin morphology) [43,64]. Developmental stages of the organogenesis period are represented as h post-fertilization (hpf). Older developmental stages are shown as d post-fertilization (dpf). Th: thyroid hormone. The timeline is adapted from Vergauwen et al. [54] and based on Chang et al. [65], Drummond et al. [66], Field et al. [15], Kimmel et al. [6], Li et al. [67], Ng et al. [16], Ober et al. [17], Parichy et al. [64], Strähle et al. [5] and Wilson et al. [43].](publication/329546261/figure/fig1/AS:702467226480641@1544492646937/Timeline-showing-key-events-during-zebrafish-development-from-fertilization-to-juvenile.png)
Timeline showing key events during zebrafish development, from fertilization to juvenile stages (j.) Color bars indicate the developmental phases with gradients representing embryo-larval and larval-juvenile transitions. The period of embryonic development includes pre-hatching stage and eleutheroembryo stage, i.e., the stage between hatching and onset of exogenous feeding [5,10]. The period of organogenesis, i.e., development of brain, heart, liver, intestine and pronephros, coincides with embryonic development. Embryo-larval transition implies the period between the onset of exogenous feeding and complete yolk absorption. Larval-juvenile transition reflects the period of metamorphosis in which the larval morphology is transformed into that of a juvenile (e.g., metamorphosis of the pigment pattern and fin morphology) [43,64]. Developmental stages of the organogenesis period are represented as h post-fertilization (hpf). Older developmental stages are shown as d post-fertilization (dpf). Th: thyroid hormone. The timeline is adapted from Vergauwen et al. [54] and based on Chang et al. [65], Drummond et al. [66], Field et al. [15], Kimmel et al. [6], Li et al. [67], Ng et al. [16], Ober et al. [17], Parichy et al. [64], Strähle et al. [5] and Wilson et al. [43].
Source publication
The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study w...
Contexts in source publication
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... in view of cost and time effectiveness, and within the framework of the three Rs-Replacement, Reduction, and Refinement- described by Russell and Burch [3], the zebrafish (Danio rerio) embryo has been proposed as an alternative non-rodent animal model for developmental toxicity studies. Indeed, the zebrafish embryo is not considered to be a test animal until it reaches the stage of independent feeding, i.e., at 120 h post-fertilization (hpf) (Figure 1) [4,5]. Moreover, zebrafish are characterized by a rapid and ex utero embryonic development and embryos may be used in medium-or high-throughput screening because of their small size [6]. ...
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... zebrafish liver and intestine, which are pivotal in the biotransformation of xenobiotics, become functional towards the end of the organogenesis period, i.e., around 96 hpf (Figure 1) [5,[15][16][17]. Since zebrafish embryos develop ex utero, they are directly exposed to the parent compound in developmental toxicity assays. ...
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... we extended the developmental stages beyond the period of organogenesis (120 hpf) in the present study, i.e., including 9 and 14 d post-fertilization (dpf). At 96-120 hpf, exogenous feeding starts ( Figure 1) and exposure to environmental compounds is expected to increase in a natural situation, which may activate the pregnane X receptor (PXR) or the aryl hydrocarbon receptor (AhR) that regulate CYP expression [41,42]. Hence, the onset of exogenous feeding may affect CYP activity in zebrafish larvae. ...
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... the onset of exogenous feeding may affect CYP activity in zebrafish larvae. Furthermore, larvae between 8 and 15 dpf often show increased mortality due to starvation in the period between complete yolk absorption and successful exogenous feeding (Figure 1), and this may affect CYP activity [5,43]. Besides an in vitro assessment, we also localized CYP-mediated biotransformation in intact zebrafish embryos and larvae, as organ-specific concentrations of the metabolite may be diluted when using microsomes prepared from whole organisms. ...
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... results of the CYP activity assays support the hypothesis that the intrinsic CYP-mediated biotransformation capacity in zebrafish embryos is immature during early development although differences in CYP isoforms do occur. More specifically, biotransformation of the non-specific CYP substrate BOMR was above the LLOQ in intact embryos from 74 hpf onwards, i.e., towards the end of zebrafish organogenesis ( Figure 1). Furthermore, these findings are in agreement with the present in vitro data that showed no BOMR biotransformation before 72 hpf in microsomes prepared from whole zebrafish embryo homogenates. ...
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... these findings are in agreement with the present in vitro data that showed no BOMR biotransformation before 72 hpf in microsomes prepared from whole zebrafish embryo homogenates. This onset of CYP activity at 72 hpf coincides with vascularization of the liver, development of the intestinal epithelium and opening of the mouth (Figure 1). By 96 hpf, the liver has reached its adult configuration and the intestine has developed into an open-ended tube, which is reflected in CYP activity in the respective organs of intact embryos at 98 hpf [6,[15][16][17]68]. ...
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... versus 7-Ethoxyresorufin BOMR and ER biotransformation showed similar activity in the digestive system, i.e., between 74 and 122 hpf. The detection of resorufin formation in the digestive system at 74 hpf coincides with opening of the mouth around 72 hpf (Figure 1) [6]. At this stage, oral ingestion of xenobiotics complements uptake of compounds by the skin. ...
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... BOMR and ER were also metabolized in the otic vesicle-the zebrafish counterpart of the mammalian inner ear-at 74, 98, 122 hpf and 9 dpf for BOMR and at 74 and 98 hpf for ER. These stages do not coincide with the development of the respective organ as the otic vesicle and its corresponding otoliths have already been developed around 19 hpf and 22 hpf, respectively (Figure 1) [6]. However, in mammals, RA, and thus indirectly CYP enzymes, are suggested to be essential in embryonic development as well as in postnatal maintenance of the mammalian inner ear [74]. ...
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... CYP1B1 transcripts peaked at 36 hpf, followed by a decline until 48 hpf after which mRNA levels started to level out for the remaining developmental time points. This peak, which was also detected by Goldstone et al. [22] at the same time point, coincides with the development of the eye cup and retina (Figure 1) [6,67]. Moreover, Yin and colleagues [78] already reported basal CYP1B1 mRNA expression in ocular cells of zebrafish at 24 hpf after which transcription levels peaked between 30 and 48 hpf. ...
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... the presence of CYP2K6 transcripts in adult zebrafish liver and ovary cannot explain the high transcript level at 14 hpf in the current study since these organs develop later. Yet, the early CYP2K6 mRNA peak coincides with the development of the brain neuromeres (Figure 1) around 16 hpf and with the onset of heart development around 16-19 hpf [6,80]. With regards to CYP1C1 and CYP1C2, Jönsson et al. [72] described an increase in basal mRNA levels from 8 to 96 hpf and from 8 to 72 hpf for the respective enzymes, which is similar to the present study. ...
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... for CYP1B1, all CYP transcripts that were investigated reached maximum expression levels during embryo-larval transition, i.e., between 4 and 7 dpf (Figure 1), which comprises the period between the onset of exogenous feeding and complete yolk absorption. Exogenous feeding implies increased exposure to environmental compounds, which may cause a slight induction of CYP mRNA expression due to PXR or AhR activation [41,42]. ...
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... reaching maximum mRNA levels during the embryo-larval transition, CYP1A, CYP1C1, CYP1C2, and CYP3A65 transcript levels remained stable throughout the larval period, whereas mRNA levels of CYP1B1, CYP2K6, and CYP3C1 fluctuated to some extent. The decline of transcript levels around 10 dpf that was observed for CYP1B1, CYP2K6, and CYP3C1 coincides with the period of increased mortality due to starvation (Figure 1). However, the correlation between both observations remains unclear. ...
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... UGT1A was first identified by Huang and Wu [50] and is expressed in liver and intestine and, to a lesser extent, in brain and testis of adult zebrafish [48]. In the current study, UGT1A1 transcripts reached maximum expression levels during embryo-larval transition (Figure 1) after which mRNA levels levelled off throughout the larval period. Since embryo-larval transition coincides with the onset of exogenous feeding and since UGT1A is supposed to be regulated through the AhR pathway [48], we assume that the increased exposure to environmental compounds induced UGT1A1 mRNA expression due to AhR activation. ...
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... in contrast to the current study, UGT1A mRNA levels at 24 hpf were higher than at 48 hpf [48]. Zebrafish SULT1ST1, which was first identified by Liu et al. [51], showed maximum transcript levels already around 72 hpf, which coincides with the first observation of thyroid hormone synthesis (Figure 1) [65]. Moreover, zebrafish SULT1ST1 enzymes are involved in the sulfonation of endogenous thyroid hormones [85], which might explain the maximum SULT1ST1 mRNA levels around 72 hpf. ...
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... only one biological replicate of ZLM was used in the current assay. The ZEM (Batch 1-3), which had already been used in a previous study [25] were included in the assay to assess CYP activity throughout zebrafish development. Positive and negative controls and ZEM were subjected to the same protein and substrate concentration as for ZLaM and ZM. ...
Citations
... In a recent review, Loerracher and Braunbeck [31] pointed out that-whenever looked at in detail-all CYP activities could be demonstrated in the zebrafish embryo so far. However, in general, CYP activities [32][33][34][35][36][37] are comparatively low in Danio rerio up to 72/96 hpf compared to later larval developmental stage or in juvenile fish. More specifically, other studies by Verbueken et al. [37] and Otte et al. [38] demonstrated albeit low, but in some cases even constitutive CYP1A activity from the very beginning of zebrafish embryonic development. ...
... However, in general, CYP activities [32][33][34][35][36][37] are comparatively low in Danio rerio up to 72/96 hpf compared to later larval developmental stage or in juvenile fish. More specifically, other studies by Verbueken et al. [37] and Otte et al. [38] demonstrated albeit low, but in some cases even constitutive CYP1A activity from the very beginning of zebrafish embryonic development. Another method to differentiate metabolism capacities is to study effects of individual pro-toxicants. ...
... Interestingly, after 48 h exposure in the FET, the addition of ewoS9R resulted in a significantly higher than the addition of pre-metabolization with the rat S9; likewise, the hill slopes were different, if compared to the results without metabolization. The stronger effects at 48 hpf might be due to transient increase of toxic metabolites at the beginning of the FET followed by time-dependent degradation during subsequent development [32,33,37]. ...
Background
The fish embryo acute toxicity (FET) test with the zebrafish (Danio rerio) was developed to assess the acute fish toxicity of chemicals or environmental samples as a replacement for the Acute Fish Test (AFT) with juvenile fish. However, the FET is not yet established in the regulatory context. One reason is the (postulated) difference between the biotransformation capacities of embryos and juvenile fish.
The present study was designed to develop a procedure for external metabolization of test substances prior to testing in the FET. The workflow allows simultaneous exposure of the embryos to the maternal substances and their potential metabolites throughout the entire exposure period. After a 2 h incubation of the samples at 37 °C with non-toxic concentrations of a rat liver S9 homogenate or an animal-free (ewoS9R) metabolization system, freshly fertilized zebrafish embryos are added and incubated up to 120 h post-fertilization at 26 °C. Five biotransformable model substances (allyl alcohol, benzo[a]pyrene (B[a]P), chlorpyrifos (CP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and bisphenol A (BPA)) were evaluated for embryotoxicity with and without external metabolization.
Results
Only for allyl alcohol, external metabolization with both rat S9 and ewoS9R resulted in significantly higher embryotoxicity than under non-premetabolized conditions and, thus, in a better correlation of FET and AFT data. For B[a]P, CP, TDCPP and BPA, there was no relevant difference between data derived from the FET (with and without pre-metabolization) and literature AFT data; even though the FET results with and without pre-metabolization differed significantly for BPA (with rat S9 and ewoS9R) and TDCPP (rat S9 only).
Conclusions
External pre-metabolization appears a promising add-on to the FET protocol to improve the correlation with AFT data of certain biotransformable substances and might help to strengthen the FET as an alternative to the AFT and finally to reduce or replace sentient animals used for acute fish toxicity data in the regulatory context.
Graphical Abstract
... Susceptibility to CAF (0,13,20,44,67, and 100 mg.L −1 ) considering locomotion behavior and biochemical parameters was compared in three different scenarios: exposure from 2 to 120 hpf (5 days exposure-5dE), exposure from 72 to 120 hpf (2dE), and exposure from 96 to 120 hpf (1dE). In the 5dE scenario, the embryos are surrounded by the chorion when exposure begins, therefore the chorion can operate as a protective barrier during this time (Qiang et al. 2020) decreasing the absorption of CAF by the embryo (Halbach et al. 2020), and within this period the cytochrome P450 activity (CYP1; involved in the oxidative metabolism (phase I) of xenobiotics) is detected (approximately between 7 and 26 hpf) (Verbueken et al. 2018). The 2dE scenario (from 72 to 120 hpf) is a post-hatching exposure period that coincides with the opening of the mouth, a potential additional portal of entry for CAF. ...
... The 2dE scenario (from 72 to 120 hpf) is a post-hatching exposure period that coincides with the opening of the mouth, a potential additional portal of entry for CAF. The 1dE scenario (exposure from 96 to 120 hpf) is a later exposure period when other CYP (CYP2ad2, 2k18, 2n13, 3a65, 3c2/3, and 3c4) transcripts increase suddenly, and a period closest to the total formation of the liver (i.e., 120 hpf) (Nawaji et al. 2020;Verbueken et al. 2018), and simultaneously present potential new drug targets (Pruvot et al. 2012). To assess the reversibility of potential CAF effects, after each exposure period, some fish were subjected to a 10 days depuration period in clean water (10dpE)-the predicted time for complete elimination of CAF in human newborns (Christian and Brent 2001). ...
... The presence of chorion has been considered as a protective barrier that provides security to the embryo limiting the entrance of chemicals (Halbach et al. 2020;Qiang et al. 2020). In this sense, it has been described that xenobiotic presence in zebrafish embryos is low before 72 hpf (Verbueken et al. 2018). For instance, Chen et al. (2008) reported that in zebrafish embryos exposed by immersion to 150 mg.L −1 of CAF for 2 days, only 172.5 ng of CAF entered the embryo. ...
The chorion acts as a protective barrier, restricting some chemical absorption into the embryo and the surrounding fluids. In this sense, larvae may only have direct contact with some chemicals after dechorionation. This study aimed to evaluate the effects of caffeine (CAF) (0, 13, 20, 44, 67, and 100 mg.L⁻¹) under different exposure scenarios (embryos with chorion or embryos/larvae already hatched) and rank the stage sensitivity. Thus, three scenarios were investigated: from 2 to 120 hours post fertilization (hpf) (5 days of exposure- 5dE), from 72 to 120 hpf (2dE), and from 96 to 120 hpf (1dE). Heart rate (48 hpf) and energy reserves (120 hpf) were measured in the 5dE scenario, and behavior and acetylcholinesterase (AChE) activity were evaluated at 120 hpf in all scenarios (5dE, 2dE, and 1dE). At 120 hpf, some of the fish was transferred to clean medium for a 10 days depuration period (10dPE). Behavior and AChE activity were assessed after this period. In the 5dE scenario, CAF increased heartbeat (13, 20, and 30 mg.L⁻¹) and reduced carbohydrates (67, and 100 mg.L⁻¹), while inhibiting AChE activity (100 mg.L⁻¹) in the 5dE, 2dE, and 1dE scenarios. CAF reduced the total distance moved in the 5dE (67, and 100 mg.L⁻¹), 2dE (20, 30, 44, 67, and 100 mg.L⁻¹), and 1dE fish (67, and 100 mg.L⁻¹) and increased erratic movements. Based on the lowest observed effect concentration (LOEC) for total distance moved (20 mg.L⁻¹) and higher inhibition of AChE activity (100 mg.L⁻¹) (65%), 2dE fish appear to be more sensitive to CAF. After 10dPE, a recovery in behavior was detected in all scenarios (5dE, 2dE, and 1dE). AChE activity remained inhibited in the 2dE scenario while increasing in the 1dE scenario. This study demonstrated that the presence of the chorion is an important factor for the analysis of CAF toxicity. After the loss of the chorion, organisms show greater sensitivity to CAF and can be used to evaluate the toxicity of various substances, including nanomaterials or chemicals with low capacity to cross the chorion. Therefore, the use of hatched embryos in toxicity tests is suggested, as they allow a shorter and less expensive exposure scenario that provides similar outcome as the conventional scenario.
... Phase II biotransformation enzymes in embryos, like glutathione S-transferase and sulfotransferase, help in the efflux process. They convert exogenous chemicals to different conjugates, which are then pumped out by MXR transporters (Cunha et al., 2016;Verbueken et al., 2018). ...
ATP-binding cassette (ABC) transporters are believed to protect aquatic organisms by pumping xenobiotics out, and recent investigation has suggested their involvement in the detoxification and efflux of nanoparticles (NPs), but their roles in fish embryos are poorly understood. In this regard, this paper summarizes the recent advances in research pertaining to the development of ABC transporter-mediated multi-xenobiotic resistance (MXR) mechanism in fish embryos and the potential interaction between ABC transporters and NPs. The paper focuses on: (1) Expression, function, and modulation mechanism of ABC proteins in fish embryos; (2) Potential interaction between ABC transporters and NPs in cell models and fish embryos. ABC transporters could be maternally transferred to fish embryos and thus play an important role in the detoxification of various chemical pollutants and NPs. There is a need to understand the specific mechanism to benefit the protection of aquatic resources.
... In aquatic habitats, organisms are frequently exposed to a complex combination of chemicals, including parent substances and their transformation products. These pollutants can harm biological diversity, organ function, J. Mori Krinal · G. Ad Viral · S. Kumar (✉) Verbueken et al. (2018) assessed cytochrome P450 activity in zebrafish embryos and larvae until 14 day post-fertilization (dpf) by using a nonspecific CYP substrate, (benzyloxy-methyl-resorufin (BOMR), CYP1-specific substrate like 7-ethoxyresorufin (ER) and transcript analysis of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine di-phosphate glucuronosyltransferase 1A1 (UGT1A1), sulfo-transferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter like abcb4 gene in zebrafish for 32 dpf. Using quantitative PCR, they reported a low to undetectable role of these proteins in the disposition of xenobiotics before 72 h post-fertilization. ...
Among the major concerns associated globally with aquaculture or fish farming, top-notch and prime one is the treatment and management of xenobiotics. Aquatic ecosystems are heavily and negatively impacted by these chemicals. Xenobiotics impact aquaculture industry in a number of ways, be it the decline in the production or remaining in the system for so long that it hampers treatment and management. Because of its presence for a large time in waterbodies, these compounds can build up in food chains via the consumption of aquaculture products. There are good reports where these chemicals build-up in human body leading to cancers. One of the main agendas of xenobiotic management is its degradation. Degradation involves bioremediation (bioattenuation, biostimulation, and bioaugmentation). Burkholderia, Bacillus, Pseudomonas, Sphingomonas, Kocuria, Chromohalobacter, and Achromobacter have been reported to play a critical role in the degradation of these compounds. Bioremediation process can be improved with advancements in molecular biological techniques like genome editing, which allows the modification of microbial strains with an increased capacity for digesting several xenobiotics simultaneously and/or at a quick rate the bioremediation process can be improved.KeywordsXenobioticDegradationBioremediationGenome editing
... Transcriptomic changes following 1 ppm benzene exposure were associated with detoxification processes, hematological development, and inflammation response. We detected upregulation of two genes from the cytochrome P450 (CYP) family commonly involved in the biotransformation of xenobiotics, cyp2k6 (orthologous to human cyp2w1) and cyp2n13 [35,36]. Many genes from the solute carrier (slc) family were also dysregulated, including downregulation of slc2a1a and slc9a5, as well as upregulation of slc23a3, slc27a2b, and slc26a3.2. ...
Urban environments are plagued by complex mixtures of anthropogenic volatile organic compounds (VOCs), such as mixtures of benzene, toluene, ethylene, and xylene (BTEX). Sources of BTEX that drive human exposure include vehicle exhaust, industrial emissions, off-gassing of building material, as well as oil spillage and leakage. Among the BTEX mixture, benzene is the most volatile compound and has been linked to numerous adverse health outcomes. However, few studies have focused on the effects of low-level benzene on exposure during early development, which is a susceptible window when hematological, immune, metabolic, and detoxification systems are immature. In this study, we used zebrafish to conduct a VOC exposure model and evaluated phenotypic and transcriptomic responses following 0.1 and 1 ppm benzene exposure during the first five days of embryogenesis (n = 740 per treatment). The benzene body burden was 2 mg/kg in 1 ppm-exposed larval zebrafish pools and under the detection limit in 0.1 ppm-exposed fish. No observable phenotypic changes were found in both larvae except for significant skeletal deformities in 0.1 ppm-exposed fish (p = 0.01) compared with unexposed fish. Based on transcriptomic responses, 1 ppm benzene dysregulated genes that were implicated with the development of hematological system, and the regulation of oxidative stress response, fatty acid metabolism, immune system, and inflammatory response, including apob, nfkbiaa, serpinf1, foxa1, cyp2k6, and cyp2n13 from the cytochrome P450 gene family. Key genes including pik3c2b, pltp, and chia.2 were differentially expressed in both 1 and 0.1 ppm exposures. However, fewer transcriptomic changes were induced by 0.1 ppm compared with 1 ppm. Future studies are needed to determine if these transcriptomic responses during embryogenesis have long-term consequences at levels equal to or lower than 1 ppm.
... Until now, the metabolic capacity of the zebrafish embryo has mainly been investigated by CYP-non-specific (benzyloxy-methyl-resorufin) [18,19] and -specific (e.g., 7ethoxyresorufin, for the CYP1 family; 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylation, for the CYP3A family; 7-methoxycoumarin-O-demethylasem for CYP2 family) [20-26] fluorescent probes. The activity of these CYP enzymes can also be assessed in the embryos/larvae homogenate microsomes, which are endoplasmic reticulum vesicles containing anchored CYP enzymes on their outer membranes. ...
... Recent in-vitro and in-vivo research in zebrafish embryos showed that the mRNA expression of most CYPs-families 1-3-reaches a plateau between 72 and 120 hpf and stays stable throughout larval development (up until 32 days post fertilization). Most importantly, it correlates with the onset of CYP (1-3) activity in developing zebrafish embryos [19]. On the other hand, the transcripts and/or the activity of these Phase I enzymes appeared to be low or undetectable before 72 hpf and only reach mature capacity by 120 hpf. ...
... In our study, the exposure window was extended to 120 hpf to investigate the potential formation of the metabolites over time in zebrafish embryos/larvae and observe the induction of additional teratogenic effects by the parent compound and their potential active metabolite(s) beyond 72 hpf. This time point, 72 hpf, appears to be a threshold for CYPs activity in zebrafish embryos [19]. In addition, in vitro metabolism of these compounds in adult zebrafish was also compared with human and other routinely used species in the in-vivo developmental toxicity studies, the rat, and the rabbit, to get a better view of potential species differences. ...
The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.
... The zebrafish CYP family 1 contains five CYP genes, i.e. CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 (GRCz11, see Table 1), which all differ in their developmental expression patterns (Goldstone et al. 2009(Goldstone et al. , 2010Jönsson et al. 2007a;Verbueken et al. 2018), tissue and organ distributions (Jönsson et al. 2007b), responses to xenobiotic inducers and inhibitors (Jönsson et al. 2007a) and catalytic activities towards xenobiotic and endogenous compounds Fig. 1 Numbers of CYP1-4 isoforms whose constitutive expression patterns have been determined throughout embryonic, juvenile and adult development of the zebrafish (Danio rerio). Illustrations by Karlotta Boßung ...
... This suggests that each may have distinct physiological functions and/or roles in xenobiotic biotransformation. For all genes of the CYP1 family, transcripts have been detected whenever studied, i.e. in zebrafish from at least 3 hpf onwards (Table 2; Goldstone et al. 2010;Jönsson et al. 2007a;Verbueken et al. 2018). CYP1A is the only member of the CYP family 1 for which transcripts have also been detected in unfertilized zebrafish oocytes, indicating a maternal transfer of CYP1A mRNA to the embryo (Goldstone et al. 2010;Verbueken et al. 2018). ...
... For all genes of the CYP1 family, transcripts have been detected whenever studied, i.e. in zebrafish from at least 3 hpf onwards (Table 2; Goldstone et al. 2010;Jönsson et al. 2007a;Verbueken et al. 2018). CYP1A is the only member of the CYP family 1 for which transcripts have also been detected in unfertilized zebrafish oocytes, indicating a maternal transfer of CYP1A mRNA to the embryo (Goldstone et al. 2010;Verbueken et al. 2018). The constitutive expression of CYP1A fluctuates during the first 48 hpf (Glisic et al. 2016;Goldstone et al. 2010) and considerably increases around hatching, reaching a first peak in late embryogenesis (96-120 hpf; Glisic et al. 2016;Jones et al. 2010;Verbueken et al. 2018). ...
Given the strong trend to implement zebrafish ( Danio rerio ) embryos as translational model not only in ecotoxicological, but also toxicological testing strategies, there is an increasing need for a better understanding of their capacity for xenobiotic biotransformation. With respect to the extrapolation of toxicological data from zebrafish embryos to other life stages or even other organisms, qualitative and quantitative differences in biotransformation pathways, above all in cytochrome P450-dependent (CYP) phase I biotransformation, may lead to over- or underestimation of the hazard and risk certain xenobiotic compounds may pose to later developmental stages or other species. This review provides a comprehensive state-of-the-art overview of the scientific knowledge on the development of the CYP1-4 families and corresponding phase I biotransformation and bioactivation capacities in zebrafish. A total of 68 publications dealing with spatiotemporal CYP mRNA expression patterns, activities towards mammalian CYP-probe substrates, bioactivation and detoxification activities, as well as metabolite profiling were analyzed and included in this review. The main results allow for the following conclusions: (1) Extensive work has been done to document mRNA expression of CYP isoforms from earliest embryonic stages of zebrafish, but juvenile and adult zebrafish have been largely neglected so far. (2) There is insufficient understanding of how sex- and developmental stage-related differences in expression levels of certain CYP isoforms may impact biotransformation and bioactivation capacities in the respective sexes and in different developmental stages of zebrafish. (3) Albeit qualitatively often identical, many studies revealed quantitative differences in metabolic activities of zebrafish embryos and later developmental stages. However, the actual relevance of age-related differences on the outcome of toxicological studies still needs to be clarified. (4) With respect to current remaining gaps, there is still an urgent need for further studies systematically assessing metabolic profiles and capacities of CYP isoforms in zebrafish. Given the increasing importance of Adverse Outcome Pathway (AOP) concepts, an improved understanding of CYP capacities appears essential for the interpretation and outcome of (eco)toxicological studies.
... Although the embryonic development of zebrafish has been well-characterized, current knowledge of the capacity of zebrafish early life-stages to metabolize xenobiotic substances, especially with regard to CYP-mediated metabolism, is still limited (for review, see Saad et al., 2016a). While the full complement of CYP genes has been demonstrated to be constitutively expressed as mRNA at least once during zebrafish embryogenesis (Bräunig et al., 2015;Glisic et al., 2016;Goldstone et al., 2010;Jönsson et al., 2007;Poon et al., 2017;Wang-Buhler et al., 2005;Wang et al., 2007), apart from CYP1A, i.e. 7-ethoxyresorufin-O-deethylase, evidence for their functional activity is still lacking almost completely (Otte et al., 2010;Saad et al., 2016b;Verbueken et al., 2018). Furthermore, the few studies that have been conducted to investigate the capability of zebrafish embryos to metabolize mammalian CYP-specific substrates (known from mammalian studies), have come to different and even conflicting results. ...
... On the one hand, they demonstrate a lack of CYP activity and/or negligible xenobiotic biotransformation during a major part of zebrafish embryogenesis Verbueken et al., 2017Verbueken et al., , 2018; on the other hand, some studies documented functional CYP-dependent biotransformation and bioactivation activities from earliest stages of development onward (Bräunig et al., 2015;Brox et al., 2016;Otte et al., 2010Otte et al., , 2017Verbueken et al., 2018;Weigt et al., 2010). Given the uncertainty about the capacities of zebrafish early life-stages to metabolize xenobiotic substances, as well as the associated discussion about the applicability domain of the zebrafish embryo as an alternative model for regulatory testing purposes (Sobanska et al., 2018), there is a need to better characterize the CYP-dependent metabolic capacities of zebrafish across different developmental stages. ...
... On the one hand, they demonstrate a lack of CYP activity and/or negligible xenobiotic biotransformation during a major part of zebrafish embryogenesis Verbueken et al., 2017Verbueken et al., , 2018; on the other hand, some studies documented functional CYP-dependent biotransformation and bioactivation activities from earliest stages of development onward (Bräunig et al., 2015;Brox et al., 2016;Otte et al., 2010Otte et al., , 2017Verbueken et al., 2018;Weigt et al., 2010). Given the uncertainty about the capacities of zebrafish early life-stages to metabolize xenobiotic substances, as well as the associated discussion about the applicability domain of the zebrafish embryo as an alternative model for regulatory testing purposes (Sobanska et al., 2018), there is a need to better characterize the CYP-dependent metabolic capacities of zebrafish across different developmental stages. ...
Zebrafish (Danio rerio) early life-stages are increasingly gaining attention as an alternative model in both human and environmental toxicology. Whereas there is amble knowledge about the transcription of various cytochrome P450 isoforms, the level of information about functional implications is still limited. This study investigated the development of CYP2-dependent 7-methoxycoumarin-O-demethylase (MCOD) activity throughout the early zebrafish development from 5 to 118 h post-fertilization (hpf) via confocal laser scanning microscopy. Results demonstrate that zebrafish embryos exhibit constitutive MCOD activity from as early as 5.5 hpf. Characteristic spatiotemporal patterns were documented with MCOD activities localized in several tissues and organs, namely the cardiovascular system, the brain, the digestive system, and the urinary tract. The study thereby contributes to a better understanding of the development and functional role of CYP enzymes in zebrafish early life-stages.
... At this stage of development, the embryo faces one of the most serious threats to its existence because it is exposed to environmental pollution caused by xenobiotics. At this time period, the full capacity of zebrafish xenobiotic metabolism should be reached; Verbueken et al. (2018) reported that the metabolism function should be completed at approximately 120 hpf. In our experiment, the mRNA amount at 144 hpf decreased at lower levels than at 96 hpf (except cyp3a65) without induction to venlafaxine. ...
... Regarding cyp1a and cyp3a65, Goldstone et al. (2010) referred that zebrafish cytochrome P450 could have the similar function as in mammal's xenobiotics biotransformation, while the P450 families 1-3 have the main part in drug metabolism (Saad et al. 2016). Moreover, as referred by Verbueken et al. (2018), the cyp1a gene is probably immature in the earlier ontogenesis thus its expression is probably not yet fully started at 24 hpf. However, at 144 hpf, the total mRNA amounts of cyp1a increased multiply than for the other genes as its expression was stimulated by higher venlafaxine exposure. ...
... Additionally, the higher the venlafaxine concentration and longer the time of exposure, the more of the disrupting of regulation by pxr was observed. According to Verbueken et al. (2018), the start of the exogenous nutrition at approximately 144 hpf most probably activates the pxr regulator. Based on our results, this regulation seems to be disrupted in L venlafaxine concentration already after 24 hpf, in higher concentration after 96 hpf. ...
The effect of venlafaxine, a pharmaceutical commonly found in aquatic environment, was analyzed on non-target organism, Danio rerio (Hamilton, 1822). D. rerio embryos were treated by two different concentrations of venlafaxine: either concentration relevant in aquatic environment (0.3 μg/L) or concentration that was two orders of magnitude higher (30 μg/L) for the evaluation of dose-dependent effect. Time-dependent effect was rated at 24, 96, and 144 h post-fertilization (hpf). For gene expression, genes representing one of the phases of xenobiotic biotransformation (0 to III) were selected. The results of this study showed that the effect of venlafaxine on the zebrafish embryos is the most evident at hatching (96 hpf). At this time, the results showed a downregulation of gene expression in each phase of biotransformation and in both tested concentrations. In contrast, an upregulation of most of the genes was observed 144 hpf for both tested venlafaxine concentrations. The study shows that venlafaxine can affect the gene expression of biotransformation enzymes in D. rerio embryos even in the environmentally relevant concentration and thus disrupt the process of biotransformation. Moreover, the pxr regulation of genes seems to be disrupted after venlafaxine exposure in dose- and time-dependent manner.
... Zebrafish possesses 94 CYP genes corresponding to mammals. However, some of cytochrome P450 genes (cyp1c, cyp2ae and cyp2x) have no orthologue in human (de Souza Anselmo et al., 2018;Goldstone et al., 2010;Verbueken et al., 2018). Out of the 94 identified CYP genes, 86 genes belong to CYP families 1-3, and are implicated in xenobiotic metabolism (Saad et al., 2016;Verbueken et al., 2018). ...
... However, some of cytochrome P450 genes (cyp1c, cyp2ae and cyp2x) have no orthologue in human (de Souza Anselmo et al., 2018;Goldstone et al., 2010;Verbueken et al., 2018). Out of the 94 identified CYP genes, 86 genes belong to CYP families 1-3, and are implicated in xenobiotic metabolism (Saad et al., 2016;Verbueken et al., 2018). The two most important CYP isoforms in human, CYP3A4 and CYP2E1, have at least one orthologue in zebrafish. ...
The rate of obesity and NAFLD prevalence is growing proportionately. Considering other etiological factors of NAFLD, exposure to environmental contaminants has been described, in recent years, as an essential cause of NAFLD development and progression. Among these toxicants, benzo[a]pyrene (B[a]P), a widely distributed environmental pollutant, is believed to contribute in NAFLD pathogenesis. Another well-known hepatotoxicant and contributor of fatty liver disease is ethanol. It has already been described by our team that B[a]P and ethanol, even at low doses, exert hepatotoxicity notably upon co-exposure, and can lead to NAFLD progression, if liver is already compromised with steatosis in both in vitro (HepaRG and WIF-B9) and in vivo (zebrafish larva) models. Furthermore, several mechanisms, responsible for this pathological progression to steatohepatitis-like state have also been described by the team using two in vitro models. However, in vivo mechanisms underlying steatosis progression in response to B[a]P/ethanol co-exposure are yet not elucidated. In this context, we have used high fat diet (HFD)-fed zebrafish larva model to assess NAFLD pathogenesis. Our team has recently demonstrated that, in this zebrafish larva model, prior steatosis can progress to steatohepatitis-like state following co-exposure to 43 mM ethanol with 25 nM B[a]P for 7 days. With this in vivo model, we observed two important key mechanisms involved in NAFLD progression i.e. membrane remodeling and mitochondrial iron accumulation, likely associated with AhR activation. In conclusion, we proposed that membrane remodeling could act as an initial signaling element to induce this mitochondrial iron accumulation, hence mitochondrial dysfunction leading to cell death. Taking into account our results, one might propose that an iron-associated cell death, possibly ferroptosis, would be principally responsible for the NAFLD progression following B[a]P/ethanol co-exposure.
