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John J Stegeman - One of the best experts on this subject based on the ideXlab platform.
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molecular adaptation to high pressure in Cytochrome P450 1A and aryl hydrocarbon receptor systems of the deep sea fish coryphaenoides armatus
2018Co-Authors: Benjamin Lemaire, John J Stegeman, Jared V Goldstone, Mark E Hahn, Sibel I Karchner, David C Lamb, Jeffrey C Drazen, Jeanfrancois ReesAbstract:Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (Cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1Atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone".
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uncoupling of Cytochrome P450 1A and stimulation of reactive oxygen species production by co planar polychlorinated biphenyl congeners
2006Co-Authors: Jennifer J Schlezinger, William D J Struntz, Jared V Goldstone, John J StegemanAbstract:Abstract The non-ortho-polychlorinated biphenyl (PCB) congener 3,3′4,4′-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) Cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non-ortho-3,3′,4,4′,5′-pentachlorobiphenyl (PCB 126) and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), mono-ortho-2,3,3′,4,4′-pentachlorobiphenyl (PCB 105) and di-ortho-2,2′,5,5′-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non-ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined KInact values for inactivation were 0.14 and 0.08 μM, respectively, for PCB 126 and PCB 169, similar to the 0.05 μM for PCB 77 previously reported. The mono-ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di-ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non-ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.
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humic substances and crude oil induce Cytochrome P450 1A expression in the amazonian fish species colossoma macropomum tambaqui
2006Co-Authors: Aline Y O Matsuo, Bruce R Woodin, Christopher M Reddy, And Adalberto L Val, John J StegemanAbstract:Cytochrome P450 1A (CYP1A) induction is used widely as a biomarker of exposure to pollutants, such as petroleum hydrocarbons, yet CYP1A inducibility has been characterized in few tropical fish. Using Western blot analysis, catalytic assay, and immunohistochemistry, we evaluated CYP1A induction in an Amazonian fish (tambaqui; Colossoma macropomum) acclimated to humic substances (HS) and acutely exposed to crude oil. HS are ubiquitous in Amazonian waters, and they are known to affect the bioavailability of pollutants. CYP1A activity was also measured in fish exposed for 10 days to a range of concentrations of HS from both natural and commercial sources. Crude oil induced CYP1A expression in tambaqui, as expected. Exposure to both HS and crude oil resulted in greater levels of CYP1A expression relative to that in fish exposed to petroleum alone. Interestingly, CYP1A induction was also observed in fish exposed to HS alone. Induction by HS was concentration-dependent, and activity was higher in fish exposed to HS from the commercial source than in fish exposed to the HS from the natural source. The use of CYP1A as a biomarker of exposure to pollutants such as petroleum hydrocarbons in fish living in environments rich in humic substances should be considered with caution given that HS themselves induce CYP1A expression. Our results suggest that there may be as yet unknown CYP1A inducing components (aryl hydrocarbon receptor agonists) in humic substances.
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induction of Cytochrome P450 1A is required for circulation failure and edema by 2 3 7 8 tetrachlorodibenzo p dioxin in zebrafish
2003Co-Authors: Hiroki Teraoka, John J Stegeman, Wu Dong, Yoshikazu Tsujimoto, Hiroyuki Iwasa, Daiji Endoh, Naoto Ueno, Richard E Peterson, Takeo HiragaAbstract:The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of Cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.
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expression of p glycoprotein and Cytochrome P450 1A in intertidal fish anoplarchus purpurescens exposed to environmental contaminants
2002Co-Authors: Shannon Mala Bard, Bruce R Woodin, John J StegemanAbstract:Abstract Whether P-glycoproteins (P-gps) like those which confer multidrug resistance in tumor cell lines are important in adaptation to chemicals in natural populations of vertebrates exposed to contaminant mixtures is the focus of this study. P-gp expression was examined in the intertidal fish high cockscomb blenny (Anoplarchus purpurescens) exposed to crude oil or pulp mill effluent. The relationship between P-gp expression and Cytochrome P450 1A (CYP1A) induction also was investigated. Immunohistochemical (IHC) analysis revealed that levels of P-gp expression in the bile canaliculi were three- to five-fold greater in oil exposed fish than in control fish. Levels of P-gp expression were highly correlated with hepatic CYP1A levels previously measured in these fish. In fish from sites near pulp mills, P-gp expression in freshly caught fish did not correlate with proximity to pulp mills. However, hepatic P-gp expression levels in freshly caught fish were 14-fold higher than in fish from those sites that were depurated in clean water for 6 weeks. CYP1A levels were also elevated in liver of freshly caught as compared with depurated fish. Expression of neither CYP1A nor P-gp was elevated in depurated fish exposed to sediment and food from within the original pulp mill effluent stream. Depurated fish, which were injected with the aryl hydrocarbon receptor (AHR) agonist s-naphthoflavone (BNF) showed an expected induction of CYP1A but no induction of P-gp. These results suggest that in blennies, unlike CYP1A, P-gp expression is not regulated by the AHR pathway; although P-gp and CYP1A both may be induced by some compounds in petroleum and unidentified xenobiotics at field sites. While our data indicate that CYP1A and P-gp are not coordinately regulated, these proteins may play complementary roles in cellular detoxification. Thus the elevation of P-gp activity may be an important mechanism of multixenobiotic resistance for organisms, such as intertidal fish, which are commonly exposed to anthropogenic contaminants and naturally occurring toxins.
J P Boon - One of the best experts on this subject based on the ideXlab platform.
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temporal induction pattern of hepatic Cytochrome P450 1A in thermally acclimated dab limanda limanda treated with 3 3 4 4 tetrachlorobiphenyl cb77
1996Co-Authors: H M Sleiderink, J P BoonAbstract:Mature male dab (Limanda limanda) acclimated at 10° and 16°C were orally administered a single dose of 0.5 mg/kg 3,3′,4,4′-tetrachlorobiphenyl (CB77). At both temperatures, levels of Cytochrome P450 1A (CYP1A) protein and 7-ethoxyresorufin O-deethylase (EROD) activity showed a two to six fold induction 40 days after CB77 treatment compared to control groups. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed at 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both EROD activity and CYP1A protein was observed and maximum responses were measured 40 days after treatment. Absolute EROD activity and CYPIA protein levels of fish from both temperatures were equally high at 40 days after treatment. Since in the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, the magnitude of induction was higher in the warm acclimated ones. The highest concentrations of CB77 in muscle of fish from both temperatures were found at 5 and 10 days after treatment. The liver somatic index (LSI) showed 1.5 fold significantly higher values for the fish acclimated at 10°C.
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Cytochrome P450 1A response in north sea dab limanda limanda from offshore and coastal sites
1995Co-Authors: H M Sleiderink, J P BoonAbstract:Abstract Dab ( Limanda limanda ) were collected at four stations in the southern North Sea in November 1993. Highest muscle polychlorinated biphenyl (PCB) concentrations were found at a station near the Dutch coast. CB153 was the dominant congener at all stations. Since the concentrations of two mono- ortho Cl-substituted congeners covaried strongly with CB153, this congener appears to be a good marker for general differences in PCB concentrations. Mean CB153 concentrations ranged from 50–160 ng g −1 lipid. The highest Cytochrome P450 1A (CYP1A) levels were also found at the coastal station. The extent of induction, measured catalytically (7-ethoxyresorufin O -deethylase, EROD) and immunochemically (CYP1A ELISA), between the less contaminated offshore sites and the coastal site ranged up to five-fold. Both parameters were well correlated with muscle tissue PCB concentrations. The data indicate that the CYP1A induction response in dab populations along the Dutch coast is strong enough to separate the Dutch coastal area from more pristine offshore areas of the North Sea. Compared to results of previous studies in other seasons the present sampling period offered the best conditions to investigate the correlation between environmental contamination with PCBs and related compounds, since the variations in natural factors that also interfere, such as water temperature and condition of the fish, were very low.
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sensitivity of Cytochrome P450 1A induction in dab limanda limanda of different age and sex as a biomarker for environmental contaminants in the southern north sea
1995Co-Authors: H M Sleiderink, I Oostingh, Anders Goksoyr, J P BoonAbstract:The sensitivity of Cytochrome P450 1A (CYP1A) induction as a biomarker for environmental contaminants in the flatfish dab (Limanda limanda) was evaluated by studying fish of different age and sex from the southern North Sea. Mature and juvenile dab from both sexes were collected in autumn and winter during two surveys from four different stations with varying levels of polychlorinated biphenyls (PCBs) contamination in the southern North Sea.
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influence of temperature on Cytochrome P450 1A in dab limanda limanda from the southern north sea results from field surveys and a laboratory study
1995Co-Authors: H M Sleiderink, Jonny Beyer, Jan M Everaarts, J P BoonAbstract:Abstract Mature specimens of male dab were collected from several stations in the southern North Sea, and the Cytochrome P450 1A dependent 7-ethoxyresorufin O-deethylase (EROD) activity was measured with a microplate reader directly onboard the research vessel. During both surveys the highest levels of EROD activity were found at relatively clean off-shore stations with low bottom water temperatures due to stratification. Considerably lower EROD values were found along the more contaminated Dutch coast at stations with relatively high bottom water temperatures. The inverse relationship between water temperature and EROD activity was confirmed in a laboratory study, where dabs were acclimated to 8, 12 and 16 °C during a period of four weeks.
H M Sleiderink - One of the best experts on this subject based on the ideXlab platform.
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temporal induction pattern of hepatic Cytochrome P450 1A in thermally acclimated dab limanda limanda treated with 3 3 4 4 tetrachlorobiphenyl cb77
1996Co-Authors: H M Sleiderink, J P BoonAbstract:Mature male dab (Limanda limanda) acclimated at 10° and 16°C were orally administered a single dose of 0.5 mg/kg 3,3′,4,4′-tetrachlorobiphenyl (CB77). At both temperatures, levels of Cytochrome P450 1A (CYP1A) protein and 7-ethoxyresorufin O-deethylase (EROD) activity showed a two to six fold induction 40 days after CB77 treatment compared to control groups. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed at 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both EROD activity and CYP1A protein was observed and maximum responses were measured 40 days after treatment. Absolute EROD activity and CYPIA protein levels of fish from both temperatures were equally high at 40 days after treatment. Since in the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, the magnitude of induction was higher in the warm acclimated ones. The highest concentrations of CB77 in muscle of fish from both temperatures were found at 5 and 10 days after treatment. The liver somatic index (LSI) showed 1.5 fold significantly higher values for the fish acclimated at 10°C.
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Cytochrome P450 1A response in north sea dab limanda limanda from offshore and coastal sites
1995Co-Authors: H M Sleiderink, J P BoonAbstract:Abstract Dab ( Limanda limanda ) were collected at four stations in the southern North Sea in November 1993. Highest muscle polychlorinated biphenyl (PCB) concentrations were found at a station near the Dutch coast. CB153 was the dominant congener at all stations. Since the concentrations of two mono- ortho Cl-substituted congeners covaried strongly with CB153, this congener appears to be a good marker for general differences in PCB concentrations. Mean CB153 concentrations ranged from 50–160 ng g −1 lipid. The highest Cytochrome P450 1A (CYP1A) levels were also found at the coastal station. The extent of induction, measured catalytically (7-ethoxyresorufin O -deethylase, EROD) and immunochemically (CYP1A ELISA), between the less contaminated offshore sites and the coastal site ranged up to five-fold. Both parameters were well correlated with muscle tissue PCB concentrations. The data indicate that the CYP1A induction response in dab populations along the Dutch coast is strong enough to separate the Dutch coastal area from more pristine offshore areas of the North Sea. Compared to results of previous studies in other seasons the present sampling period offered the best conditions to investigate the correlation between environmental contamination with PCBs and related compounds, since the variations in natural factors that also interfere, such as water temperature and condition of the fish, were very low.
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sensitivity of Cytochrome P450 1A induction in dab limanda limanda of different age and sex as a biomarker for environmental contaminants in the southern north sea
1995Co-Authors: H M Sleiderink, I Oostingh, Anders Goksoyr, J P BoonAbstract:The sensitivity of Cytochrome P450 1A (CYP1A) induction as a biomarker for environmental contaminants in the flatfish dab (Limanda limanda) was evaluated by studying fish of different age and sex from the southern North Sea. Mature and juvenile dab from both sexes were collected in autumn and winter during two surveys from four different stations with varying levels of polychlorinated biphenyls (PCBs) contamination in the southern North Sea.
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influence of temperature on Cytochrome P450 1A in dab limanda limanda from the southern north sea results from field surveys and a laboratory study
1995Co-Authors: H M Sleiderink, Jonny Beyer, Jan M Everaarts, J P BoonAbstract:Abstract Mature specimens of male dab were collected from several stations in the southern North Sea, and the Cytochrome P450 1A dependent 7-ethoxyresorufin O-deethylase (EROD) activity was measured with a microplate reader directly onboard the research vessel. During both surveys the highest levels of EROD activity were found at relatively clean off-shore stations with low bottom water temperatures due to stratification. Considerably lower EROD values were found along the more contaminated Dutch coast at stations with relatively high bottom water temperatures. The inverse relationship between water temperature and EROD activity was confirmed in a laboratory study, where dabs were acclimated to 8, 12 and 16 °C during a period of four weeks.
Jennifer J Schlezinger - One of the best experts on this subject based on the ideXlab platform.
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uncoupling of Cytochrome P450 1A and stimulation of reactive oxygen species production by co planar polychlorinated biphenyl congeners
2006Co-Authors: Jennifer J Schlezinger, William D J Struntz, Jared V Goldstone, John J StegemanAbstract:Abstract The non-ortho-polychlorinated biphenyl (PCB) congener 3,3′4,4′-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) Cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non-ortho-3,3′,4,4′,5′-pentachlorobiphenyl (PCB 126) and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), mono-ortho-2,3,3′,4,4′-pentachlorobiphenyl (PCB 105) and di-ortho-2,2′,5,5′-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non-ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined KInact values for inactivation were 0.14 and 0.08 μM, respectively, for PCB 126 and PCB 169, similar to the 0.05 μM for PCB 77 previously reported. The mono-ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di-ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non-ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.
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induction and suppression of Cytochrome P450 1A by 3 3 4 4 5 pentachlorobiphenyl and its relationship to oxidative stress in the marine fish scup stenotomus chrysops
2001Co-Authors: Jennifer J Schlezinger, John J StegemanAbstract:The planar polychlorinated biphenyl (PCB) 3,3′,4,4′-tetrachlorobiphenyl (TCB) causes dose-dependent induction and post-transcriptional suppression of hepatic Cytochrome P450 1A (CYP1A) in the marine teleost scup (Stenotomus chrysops). That suppression is linked to inhibition and oxidative inactivation of CYP1A by TCB. Other planar PCBs, including 3,3′,4,4′,5-pentachlorobiphenyl (PeCB), inactivate scup CYP1A in vitro leading us to hypothesize that PeCB also will suppress CYP1A in vivo. We examined induction and suppression of CYP1A by PeCB in scup, as related to oxidative stress. PeCB at a low dose (0.01 mg/kg) induced hepatic microsomal spectral P450 and CYP1A protein and catalytic activities (ethoxyresorufin o-deethylase (EROD) and methoxyresorufin o-demethylase (MROD)) over an 18 day period. A high dose (1 mg PeCB/kg) only minimally induced hepatic spectral P450 and CYP1A content, and EROD and MROD rates remained at control levels at all sampling times, while CYP1A mRNA expression was induced strongly (up to 35-fold) at both doses. High dose PeCB had minimal effects on content of P450A (a CYP3A protein), P450B (a CYP2B-like protein) and Cytochrome b5 in scup liver, suggesting that the suppression was specific for CYP1A. High dose PeCB suppressed EROD but not CYP1A protein in the kidney but did not strongly suppress either CYP1A or EROD in the heart or gill. PeCB stimulated ROS production (oxidation of dihydroethidium) by liver microsomes from the low dose but not the high dose fish, and the rate of PeCB-stimulated ROS production was correlated with EROD activity (r2=0.641, P<0.0005). Oxidative stress, indicated by increased levels of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase activities, was stimulated in the liver by low dose but not high dose PeCB. The results support a hypothesis that many PHAH can inactivate teleost CYP1A in vivo, and that CYP1A is a source of ROS. However, there appears to be a complex balance between the effects of PeCB on the levels of active CYP1A, ROS release and oxidative stress.
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Cytochrome P450 1A expression in midwater fishes potential effects of chemical contaminants in remote oceanic zones
2001Co-Authors: John J Stegeman, Jennifer J Schlezinger, James E Craddock, Donald E TillittAbstract:Cytochrome P450 1A (CYP1A) induction is a robust marker for exposure to polynuclear aromatic hydrocarbons and planar halogenated aromatic hydrocarbons that are aryl hydrocarbon receptor agonists. We examined CYP1A expression in mesopelagic fishes from the western North Atlantic. Individuals in 22 species were obtained from slope water and the Sargasso Sea in 1977, 1978, and 1993. Aryl hydrocarbon hydroxylase (AHH), a CYP1A activity, was detected in liver from all species in 1977/78. In some, including Gonostoma elongatum, AHH was inhibited by the CYP1A inhibitor alpha-naphthoflavone. CYP1A-dependent ethoxyresorufin O-deethylase (EROD) was detected in liver microsomes of all species in 1993; rates were highest in G. elongatum and Argyropelecus aculeatus. Immunoblot analysis with the CYP1A-specific monoclonal antibody 1-12-3 detected a single microsomal protein band in most 1993 samples; the highest content was in G. elongatum. Immunohistochemical analysis showed CYP1A staining in gill, heart, kidney, and/or liver of several species. Extracts of the 1993 G. elongatum and A. aculeatus, when applied to fish hepatoma cells (PLHC-1) in culture, elicited a significant induction of EROD in those cells. The capacity of the extracts to induce CYP1A correlated with the content of PCBs measured in the same fish (2-4.6 ng/g total body weight). Mesopelagic fish in the western North Atlantic, which experience no direct exposure to surface waters or sediments, are exposed chronically to inducers of CYP1A at levels that appear to be biochemically active in those fish.
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induction of Cytochrome P450 1A in the american eel by model halogenated and non halogenated aryl hydrocarbon receptor agonists
2000Co-Authors: Jennifer J Schlezinger, John J StegemanAbstract:Eels (Anguilla sp.) have phylogenetic, life history, and morphological characteristics which distinguish them from many other species that have been examined for Cytochrome P450 1A (CYP1A) induction. Members of the family Anguillidae often occur in regions of the coastal environment that are heavily impacted by chemical contamination. Although eels have been suggested to be a useful species for biomonitoring, the sensitivity with which eel CYP1A is induced by aryl hydrocarbon receptor (AHR) agonists is not known. We investigated the dose-dependent induction of hepatic CYP1A in the American eel (Anguilla rostrata). Eels from an uncontaminated site were injected intra-peritoneally with the model AHR agonists s-naphthoflavone (BNF), benzo[a]pyrene (B[a]P) or 3,3',4,4-tetrachlorobiphenyl (TCB) at increasing doses (BNF at 0.1, 1, 10 and 100 mg/kg, B[a]P at 0.1, 1 and 10 mg/kg, and TCB at 0.1, 1, 10 and 20 mg/kg). All three compounds produced dose-dependent induction of CYP1A content and catalytic activity. An estimated ED(50) for induction of liver microsomal EROD activity by TCB was approximately 5 mg/kg, indicating only moderate sensitivity. At comparable doses of 1 and 10 mg/kg (or 3-4 and 30-40 µmol/kg), BNF and B[a]P had 2-3-fold greater effect than TCB in eliciting hepatic CYP1A induction. Injection of radiolabeled B[a]P and TCB resulted in similar dose-dependent concentrations of these compounds in eel liver, and the hepatic inducer concentrations and CYP1A levels were correlated positively. Eels collected from New Bedford Harbor (NBH), a Superfund site highly contaminated by polycyclic aromatic hydrocarbons and polychlorinated biphenyls, had levels of microsomal CYP1A protein and EROD activity that were equivalent to the highest levels induced experimentally. Eels from less contaminated sites had correspondingly less CYP1A expression. The responses to B[a]P or BNF as compared to TCB suggest a lower efficacy and/or potency for CYP1A induction by TCB which could involve differences in the mechanisms of responses to these compounds in eels. However, the moderate sensitivity and the CYP1A induction in NBH eels support suggestions that eels may be useful in monitoring more contaminated regions.
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3 3 4 4 tetrachlorobiphenyl oxidation in fish bird and reptile species relationship to Cytochrome P450 1A inactivation and reactive oxygen production
2000Co-Authors: Jennifer J Schlezinger, Jennifer M Keller, Lori A Verbrugge, John J StegemanAbstract:Previously we showed that the polychlorinated biphenyl 3,3′,4,4′-tetrachlorobiphenyl (TCB) caused a release of reactive oxygen species (ROS) from Cytochrome P450 1A (CYP1A) of the fish scup (Stenotomus chrysops), and from rat and human CYP1A1. This was linked to a TCB- and NADPH-dependent oxidative inactivation of the enzyme, which in scup and rat was inversely related to the rates of TCB oxidation. We examined the relationship between rates of TCB oxidation, CYP1A inactivation and ROS production in liver microsomes from additional vertebrate species, including skate (Raja erinacea), eel (Anguilla rostrata), killifish (Fundulus heteroclitus), winter flounder (Pleuronectes americanus), chicken (Gallus domesticus), cormorant (Phalacrocorax auritus), gull (Larus argentatus), and turtle (Chrysemys pictapicta). TCB oxidation rates were induced in all fish and birds treated with aryl hydrocarbon receptor agonists. Induced rates of TCB oxidation were <1 pmol/min/mg microsomal protein in all fish, and 6–14 pmol/min/mg in the birds. In all species but one, TCB oxidation rates correlated positively with EROD rates, indicating likely involvement of CYP1A in TCB oxidation. Incubation of liver microsomes of most species with TCB+NADPH resulted in an immediate (TCB-dependent) inhibition of EROD, and a progressive loss of EROD capacity, indicating an oxidative inactivation of CYP1A like that in scup. NADPH stimulated production of ROS (H2O2 and/or O2−) by liver microsomes, slightly in some species (eel) and greatly in others (chicken, turtle). Among the birds and the fish, NADPH-stimulated ROS production correlated positively with EROD activity. TCB caused a significant stimulation of ROS production by liver microsomes of flounder, killifish, cormorant and gull, as well as scup. The stimulation of CYP1A inactivation and ROS generation indicates an uncoupling of CYP1A by TCB in many species, and when compared between species, the rates of CYP1A inactivation correlated inversely with rates of TCB oxidation. Some feature(s) of binding/active site topology may hinder TCB oxidation, enhancing the likelihood for attack of an oxidizing species in the active site.
Jaeseong Lee - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and β naphthoflavone induced expression of a Cytochrome P450 1A cyp1A gene from an anadromous river pufferfish takifugu obscurus
2008Co-Authors: Jinhyoung Kim, Jaeseong Lee, Sheikh Raisuddin, Kyungnam HanAbstract:In recent years, there has been a decline in the wild populations of river pufferfish, Takifugu obscurus. Besides overexploitation for commercial purposes, environmental pollution is believed to have contributed to its decline. However, almost no information exists about genes involved in metabolism of xenobiotics by this species. Nevertheless, there is interest in fugu fishes, since they possess the smallest genome among vertebrates. We cloned and characterized the full-length cDNA sequence of a Cytochrome P450 1A (CYP1A) gene from T. obscurus. Phylogenic relationship of T. obscurus CYP1A was also compared to other fish species. The tissue distribution and time-dependant induction of CYP1A mRNA were studied by real-time PCR in T. obscurus exposed to an aryl hydrocarbon receptor (Ahr) agonist, beta-naphthoflavone (BNF). The greatest basal expression in livers of control as well as BNF-treated individuals. However, brain, gill, gonad, intestine, and kidney also expressed CYP1A. Muscles expressed the least CYP1A. The results of the time-course study revealed induction in brain and gills after 6h and at 12h in most tissues. Except for gills, all other organs retained induced expression of CYP1A mRNA up to 96h.
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cdna cloning and expression of a Cytochrome P450 1A cyp1A gene from the hermaphroditic fish rivulus marmoratus
2005Co-Authors: Youngmi Lee, Timothy Williams, Sangoun Jung, Jaeseong LeeAbstract:Abstract We previously described the genomic structure of the Cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus [Kim, I.-C., Kim, Y.J., Yoon, Y.-D, Kawamura, S., Lee, Y.-S., Lee, J.-S., 2004a. Cloning of Cytochrome P450 1A (CYP1A) genes from the hermaphroditic fish Rivulus marmoratus and the Japanese medaka Oryzias latipes. Mar. Environ. Res. 58, 125–129]. To further characterize R. marmoratus CYP1A, we cloned the cDNA sequence of a CYP1A gene from this species and also expressed its recombinant protein in an E. coli system. We exposed R. marmoratus to 4-nonylphenol, and found a small induction of CYP1A mRNA in the treated animals. In this paper, we discuss the characteristics of R. marmoratus CYP1A gene as well as its potential use in a biomonitoring assay.
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cloning of Cytochrome P450 1A cyp1A genes from the hermaphrodite fish rivulus marmoratus and the japanese medaka oryzias latipes
2004Co-Authors: Ilchan Kim, Young Ja Kim, Yongdal Yoon, Shoji Kawamura, Yongsung Lee, Jaeseong LeeAbstract:To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the Cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.
