Many industrial sites are polluted by complex mixtures of polycyclic aromatic compounds (PACs). Besides polycyclic aromatic hydrocarbons (PAHs), these mixtures often contain significant amounts of ...more polar PACs including oxygenated PAHs (oxy-PAHs). The effects of oxy-PAHs are, however, poorly known. Here we used zebrafish embryos to examine toxicities and transcriptional changes induced by PAC containing soil extracts from three different industrial sites: a gasworks (GAS), a former wood preservation site (WOOD), and a coke oven (COKE), and to PAH and oxy-PAH containing fractions of these. All extracts induced aryl hydrocarbon receptor (Ahr)-regulated mRNAs, malformations, and mortality. The WOOD extract was most toxic and the GAS extract least toxic. The extracts induced glutathione transferases and heat shock protein 70, suggesting that the toxicity also involved oxidative stress. With all extracts, Ahr2-knock-down reduced the toxicity, indicating a significant Ahr2-dependence on the effects. Ahr2-knock-down was most effective with the PAH fraction of the WOOD extract and with the oxy-PAH fraction of the COKE extract. Our results indicate that oxy-PAH containing mixtures can be as potent Ahr activators and developmental toxicants as PAHs. In addition to Ahr activating potency, the profile of cytochrome P4501 inhibitors may also determine the toxic potency of the extracts.
Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading ...to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.
Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.
Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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6-Formylindolo3,2-bcarbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed ...physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.
Zebrafish (
Danio rerio) is a common model species in fish toxicology, and the zebrafish gill is potentially useful in screening waterborne pollutants. We have previously developed a gill-based ...ethoxyresorufin-
O-deethylase (EROD) assay, and proposed gill EROD activity as a biomarker for exposure to waterborne aryl hydrocarbon receptor (AHR) agonists. In this study we modified the gill EROD assay for use in zebrafish. We used immunohistochemistry to localize CYP1A induction, and microautoradiography to localize irreversible binding of the prototypic polycyclic aromatic hydrocarbon, 7,12-dimethylbenzaanthracene (DMBA) in zebrafish gills. Gill filament and liver microsomal EROD activities were measured after waterborne exposure of zebrafish and rainbow trout to benzoapyrene (BaP) or β-naphthoflavone (βNF). The results showed considerably lower relative EROD induction by βNF (1
μM) in zebrafish than in rainbow trout, both in gills (13-fold versus 230-fold compared to control) and in liver (5-fold versus 320-fold compared to control). The induced hepatic EROD activity was similar in the two species, whereas the basal activity was considerably higher in zebrafish than in rainbow trout. In zebrafish gills, βNF enhanced DMBA adduct formation and CYP1A immunostaining. Ellipticine blocked DMBA adduct formation and EROD activity following βNF exposure but had no effect on CYP1A immunostaining. A notable finding was that the localization of DMBA adducts differed from that of CYP1A protein in βNF-induced fish; CYP1A immunoreactivity was evenly distributed in the gills whereas DMBA adduction was confined to the leading edges of the filaments and the gill rakers,
i.e., structures being highly exposed to DMBA-containing inhaled water. The results show that the modified method is suitable for determination of gill EROD activity in zebrafish, although rainbow trout seems more sensitive. They also imply that the sites of DMBA adduct formation in zebrafish gills are markedly influenced by kinetic factors.
Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. ...AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14–21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3–100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4–2.7nM, Lowest observed effect concentration LOEC: 0.3–1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.
Gao, K.; Tian, J.; Wu, Y.; Mei, P.; Zhang, Z.; Jönsson, M., and Brandt, I., 2021. Induction of cytochrome P4501 genes by various inducers in rainbow trout (Oncorhynchus mykiss). Journal of Coastal ...Research, 37(1), 143–148. Coconut Creek (Florida), ISSN 0749-0208. Juvenile rainbow trout is widely found in various aquatic systems, such as freshwater, coastal, and marine habitats all over the world. Expression patterns of the cytochrome P4501 (CYP1) genes exposed to various stressors in such fish could be informative and useful in biomonitoring. In the present study, the induction of CYP1 in rainbow trout by indigo, humic acid (HA), and benzo(a)pyrene (BaP) was studied through a 12-hour exposure experiment by using rainbow trout as an experimental object. First, 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1 expression levels in fish exposed to different concentrations of indigo were analyzed. The EROD activities in fish gills were greatly affected by the concentration of indigo (1, 18, 50, and 80 nM). The expression level of CYP1A1 and CYP1A3 in gills was higher compared with CYP1B1 and CYP1Cs. Second, the gill EROD activity and CYP1 mRNA expressions levels were compared when induced by indigo, HA, and BaP at the same concentration (1 nM). Results indicated that rainbow trout were sensitive to all three inducers. Among them, the HA affected CYP1A1 and CYP1A3 most obviously. CYP1A, as an important biomarker, was more sensitive than CYP1B and CYP1C to those inducers. The observed effects of the different CYP1 inducers could provide a useful tool for monitoring of AhR-active pollutants in the aquatic environment.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NMLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR22Zebrafish cytochrome P450 family ...1 genes/mRNAs and proteins are referred to as CYP1 and CYP1 according to Nelson et al. (1996). For other genes/mRNAs and proteins in zebrafish we have followed the approved guidelines for zebrafish, e.g., ahr2 and Ahr2 (https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Guidelines). The aryl hydrocarbon receptor is denoted AHR when not referring to a particular species.) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC50 values of 1.4 to 2.0nM for induction of the CYP1s, 3.7 and 5.1nM (or higher) for cox-2a and cox-2b induction, and 2.5nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos33Fish embryos that have hatched but are still dependent on yolk as a nutrition source are technically “eleutheroembryos”, but for simplicity we will refer to them generically as “embryos” for the remainder of this paper. Once independent feeding begins (days 6–7 post fertilization in zebrafish), the fish are then called larvae. failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.
► PCB126 caused cellular changes in the developing swim bladder. ► Swim bladder inflation was not related to expression of CYP1 or cox-2. ► Failure of swim bladder inflation is mediated via an Ahr2-dependent mechanism. ► PCB126-exposed zebrafish larvae showed upregulation of the oncogene myca.
The tryptophan derivative formylindolo3,2-bcarbazole (FICZ) binds with high ligand affinity to the aryl hydrocarbon receptor (AHR) and is readily degraded by AHR-regulated cytochrome P450 family 1 ...(CYP1) enzymes. Whether in vivo exposure to FICZ can result in toxic effects has not been examined and the main objective of this study was to determine if FICZ is embryotoxic in birds. We examined toxicity and CYP1 mRNA induction of FICZ in embryos from chicken (Gallus domesticus) and Japanese quail (Coturnix japonica) exposed to FICZ (2–200μgkg−1) by yolk and air sac injections. FICZ caused liver toxicity, embryo mortality, and CYP1A4 and CYP1A5 induction in both species with similar potency. This is in stark contrast to the very large difference in sensitivity of these species to halogenated AHR agonists. We also exposed chicken embryos to a low dose of FICZ (4μgkg−1) in combination with a CYP inhibitor, ketoconazole (KCZ). The mixture of FICZ and KCZ was lethal while FICZ alone had no effect at 4μgkg−1. Furthermore, mixed exposure to FICZ and KCZ caused stronger and more long-lasting hepatic CYP1A4 induction than exposure to each compound alone. These findings indicate reduced biotransformation of FICZ by co-treatment with KCZ as a cause for the enhanced effects although additive AHR activation is also possible. To conclude, FICZ is toxic to bird embryos and it seems reasonable that the toxicity by FICZ involves AHR activation. However, the molecular targets and biological events leading to hepatic damage and mortality are unknown.
Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental ...toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between β-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the β-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). This led us to investigate the AHR interaction with β-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of β-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a β-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6-formylindolo3,2-bcarbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryo-toxic, and we found that in the presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholino-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of β-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of β-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating β-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.
Cytochrome P450 1 (CYP1) genes are biomarkers for aryl hydrocarbon receptor (AHR) agonists and may be involved in some of their toxic effects. CYP1s other than the CYP1As are poorly studied in birds. ...Here we characterize avian CYP1B and CYP1C genes and the expression of the identified CYP1 genes and AHR1, comparing basal and induced levels in chicken and quail embryos.
We cloned cDNAs of chicken CYP1C1 and quail CYP1B1 and AHR1. CYP1Cs occur in several bird genomes, but we found no CYP1C gene in quail. The CYP1C genomic region is highly conserved among vertebrates. This region also shares some synteny with the CYP1B region, consistent with CYP1B and CYP1C genes deriving from duplication of a common ancestor gene. Real-time RT-PCR analyses revealed similar tissue distribution patterns for CYP1A4, CYP1A5, CYP1B1, and AHR1 mRNA in chicken and quail embryos, with the highest basal expression of the CYP1As in liver, and of CYP1B1 in eye, brain, and heart. Chicken CYP1C1 mRNA levels were appreciable in eye and heart but relatively low in other organs. Basal transcript levels of the CYP1As were higher in quail than in chicken, while CYP1B1 levels were similar in the two species. 3,3',4,5,5'-Pentachlorobiphenyl induced all CYP1s in chicken; in quail a 1000-fold higher dose induced the CYP1As, but not CYP1B1.
The apparent absence of CYP1C1 in quail, and weak expression and induction of CYP1C1 in chicken suggest that CYP1Cs have diminishing roles in tetrapods; similar tissue expression suggests that such roles may be met by CYP1B1. Tissue distribution of CYP1B and CYP1C transcripts in birds resembles that previously found in zebrafish, suggesting that these genes serve similar functions in diverse vertebrates. Determining CYP1 catalytic functions in different species should indicate the evolving roles of these duplicated genes in physiological and toxicological processes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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