Grantee Research Project Results
Final Report: Catalytic Function of Expressed Teleost Cytochrome P4501A
EPA Grant Number: R827102Title: Catalytic Function of Expressed Teleost Cytochrome P4501A
Investigators: Stegeman, John J.
Institution: Woods Hole Oceanographic Institution
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 1995 through August 1, 1996
Project Amount: $251,946
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
The long term goal of these studies is to compare the functional properties and homologous relationships of CYP among vertebrates. Identifying relationships and catalytic functions of CYP is important to understanding species differences and similarities in susceptibility to drugs and chemical pollutants. Such studies across taxa may indicate how structure-function relationships and physiological roles of CYP have been conserved during evolution.Background And Significance - P450 Functions. Understanding species sensitivity to xenobiotics requires knowledge of the metabolism, activation and/or detoxication of those compounds by those species. Enzymes in the CYP superfamily are the dominant catalysts in the oxidative metabolism of organic foreign compounds including natural products, drugs and pollutants. CYP functions in liver and extrahepatic organs can determine the susceptibility of individuals, populations and species to the action of toxic foreign compounds that are substrates, and the validity of extrapolation between species. Expanding libraries of substrates linked to mammalian CYP are revealing common structural features of substrates preferred by those forms, defining their active sites. How well the substrate structure-activity relationships (S-SAR) being developed for mammalian CYP will apply to homologous CYP in other species groups is not yet known. The latitude for substrate binding is a feature of protein structure that can differ substantially between homologous enzymes from different species. CYP alleles differing by a single amino acid can have different activities. Conversely, the same activities in different species could be catalyzed by CYP that may or may not be closely related. Such possible differences create uncertainty in extrapolating the details of xenobiotic metabolism between species.
The Objectives of this study were to determine the functional properties of CYP1A from a fish species, and to compare these to properties of CYP1A in mammals. Knowledge of CYP1A activity is important to the study of environmental carcinogenesis in fish. Fish in several locations have been shown to have high prevalences of hepatic neoplasms. The project included studies on other species, identification of additional genes whose activity may bear on the function of CYP1A. and a continued effort to define the induction of CYP1A in the environment.
This molecular aspects of study represent a collaboration involving our lab and that of Dr. Johannes Doehmer, Technical University of Munich. Aims addressed were to: 1. Express CYP1A cDNA from the marine fish scup (Stenotomus chrysops) in V79 cells. 2. Establish that the expressed CYP1A was functional. 3. Determine the metabolite profiles of expressed, purified and microsomal CYP1A with various PAH and selected diagnostic substrates. 4. Compare these activities of scup CYP1A with expressed human and rat 1A1 and 1A2. 5. Compare activity of the expressed scup CYP1A with microsomal CYP1A.
Scup were selected in part because of extensive information on other properties, including the data on the catalytic function and regulation of CYP1A in this species.
Expression: Scup CYP1A was stably expressed in Chinese hamster V79 cells, by chromosomal integration of CYP1A cDNA under the control of the SV40 promoter, in collaboration with Dr. Johannes Doehmer, University of Munich, as originally proposed. Chromosomal integration was verified by Southern analysis using the homologous cDNA probe. Effective transcription and translation of the CYP1A was established by Northern analysis using the cDNA probe and by Western analysis using monoclonal antibody 1-12-3 to scup CYP1A. This new cell line, termed V79MZf1A1, expresses authentic and active scup CYP1A.
Verification: Catalytic activity of the expressed protein was established by analysis of EROD activity in microsomal fractions, and conditions supporting maximal activity defined. EROD activity was also demonstrated in intact V79MZf1A1 cells, cultured in 48 well plates. P450 reductase and epoxide hydrolase activities inherent in the V79 cells expressing the scup CYP1A were determined.
PAH Metabolism: The activity of the expressed scup CYP1A was established with PAH substrates of toxicological and environmental significance. Metabolites of PAH were generated by exposing live V79MZf1A1 cells to compounds at maximally tolerated doses for 48 hours, and extraction and analysis of the media by HPLC and GC/MS. Selectivity for (+) and (-) enantiomers of trans-7,8-dihydriol-BP was determined. Compounds examined included phenanthrene, benzo[c]phenanthrene, pyrene, chrysene, and benzo[a]pyrene and dibenzo[a,l]pyrene. Each PAH tested has been found to be extensively metabolized by the cells expressing scup CYP1A. Between 20 and 100% of the compound was metabolized in 48 hours, depending on the substrate.
Cytotoxicity: PAHs were tested for metabolic activation by scup CYP1A. Control V79 cells and cells expressing scup CYP1A were exposed to various doses of PAH and cytotoxicity measured by neutral red uptake. PAH and PAH-dihydrodiols were cytotoxic to the cells expressing scup CYP1A, but not to control cells. Dose-response for cytotoxicity was similar to that in V79 cells expressing human or rat CYP1A1, depending on the substrate.
Substrate Specificity: Selected PAH and other compounds have been examined over the years, using microsomal preparations, purified reconstituted CYP, and inhibition studies. Metabolism of BP by the expressed protein was identical to results obtained by these other methods. PAH and related compounds thus far indicated or proven substrates of scup CYP1A, based on total metabolism, cytotoxicity assays, metabolite assays, and studies with purified and microsomal CYP1A include: ethoxy- and, methoxyresorufin, a-naphthoflavone, ?-naphthoflavone, phenanthrene, benzo[c]phenanthrene, pyrene, chrysene, benzo[a]pyrene, (+)-7,8-diol-BP, (-)-7,8-diol-BP, dibenzo[a,l]pyrene, (+)-11,12-di-B[a,l]P, (-)-11,12-di-B[a,l]P, benzo[f]quinoline, dibenzo[a,h]acridine, 3,3',4,4'-tetrachlorobiphenyl.
Metabolite Profiles: Expressed scup CYP1A was determined to catalyze oxidation at the following positions with the indicated compounds: 1,2- and 3,4- of phenanthrene; 3,4- and 5,6- of benzo[c]phenanthrene; 1,2- and 3,4- of chrysene; 3-, the 7,8- and the 9,10- of benzo[a]pyrene. Little or no capability to oxidize K-regions was seen with any PAH. Combining the data from the expressed scup 1A and from in vitro studies, we are beginning to model the substrate binding and active sites of scup CYP1A, the first such analysis for a fish CYP.
Comparisons to human and rat CYP1A1 or 1A2 expressed in V79 cells: To summarize results, phenanthrene metabolism by scup CYP1A most resembled that of human 1A2. Chrysene metabolism by scup CYP1A resembled that of both human and rat 1A1, and was distinct from both human and rat 1A2. Benzo[a]pyrene metabolites formed by expressed, purified and microsomal scup CYP1A all indicate that scup CYP1A oxidizes BP at both the 7,8- and 9,10-positions more efficiently than do mammalian CYP1A1 forms. Scup 1A1 does not form K-region metabolites of BP, and in that respect is more like the expressed human 1A1. Thus, scup CYP1A has some catalytic properties resembling CYP1A1, some resembling CYP1A2, and some distinct. This is consistent with the possibility that teleost CYP1As represent a type ancestral to both 1A1 and 1A2.
The V79MZf1A1 line was constructed with scup CYP1A alone. It appears that this cell line has reductase limitation, and insufficient catalytic activity for proper evaluation of some slowly metabolized substrates. Thus, the results below were with microsomal systems. Activity with selected other substrates was examined, preparatory to analysis with the expressed scup CYP1A.
Caffeine: As proposed, scup liver microsomal metabolism of caffeine was examined, using both polyclonal and monoclonal antibodies to scup CYP1A to judge its contribution to metabolites. Substrate concentration were varied appropriately, and analysis was by HPLC. HPLC eluate was analyzed spectrally from 200 to 400 nm using an HP diode array detector with a continuous elution profile determined at 285 nm. Identities and quantities of metabolites were determined by comparing elution positions and spectra with authentic metabolite standards. Separated 14C labelled CA metabolites were dried under N2 and counted in Scintaverse II using a Beckman liquid scintillation counter. The results indicate that microsomal scup CYP1A forms the majority (95+ %) of the 1,7- and 3,7-dimethylxanthine. We did carry out studies with the V79MZf1A1 cell line, and got slight metabolism, too little to confidently identify the metabolites.
3,3',4,4'-Tetrachlorobiphenyl: We obtained the first direct information on the identity of fish CYPs involved in metabolism of this pHAH. Stringent care was required as maximal rates of metabolism were < 0.5 picomoles/min/mg, only 3-5-fold above background.
Arachidonic acid: We have characterized in detail the metabolism of AA by scup organs in vitro and in vivo, comparing male and female fish, BP-treated, TCDD-treated and control fish and examining antibody inhibition (in collaboration with Dr. D. Zeldin, NIEHS). Until these studies, AA metabolism by teleost CYP was unknown. Several microsomal P450s metabolized AA, apparently including CYP1A. Products were 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), the corresponding HETEs, and the w- and w-1-HETEs. Microsomal rates were only slightly induced by TCDD or B[a]P. Endogenous AA metabolism also was induced by TCDD or BP, and the effect was stronger than that measured in vitro, but the results in heart and kidney were not consistent with results in liver. Studies with expressed CYP1A are required to resolve the issue.
Reactive oxygen: Incubation of induced scup liver microsomes with TCB results in a time-, TCB- and NADPH- and oxygen-dependent loss of CYP1A catalytic activity (85% loss) and a loss of spectral P450. Rates of covalent binding of TCB were negligible, indicating that inactivation does not proceed via a TCB metabolite. Rather, the inactivation appears to result from formation of ROS in the active site. NADPH oxidation, hydrogen peroxide formation, and superoxide formation, measured by multiple methods, all were stimulated by TCB. Our studies _(59, 71)_ indicate that TCB strongly uncouples CYP1A, with resultant formation and release of ROS from CYP1A. Subsequently, BP and various PCB congeners were tested for the ability to stimulate ROS production by CYP1A in scup liver microsomes. Production of ROS during incubation of liver microsomes from untreated scup was not stimulated by any congener. Incubation of liver microsomes from BP-induced scup with NADPH and the non-ortho 3,3',4,4'-tetra- (CB-77), 3,3',4,4',5-penta- (CB-126), and 3,3',4,4',5,5'-hexa-chlorobiphenyl (CB-169) significantly increased O2-o production compared to that with NADPH alone, as measured by oxidation of HE to ethidium (Fig. 2). BP and the ortho-substituted CBs 2,2',5,5-tetra- (CB-52) and 2,3,3',4,4'-pentachlorobiphenyl (CB-105), did not alter ROS production from that obtained with acetone+NADPH. It is the non-ortho-PCBs that exert developmental toxicity; ROS release is a possible mechanism.
Summary/Accomplishments (Outputs/Outcomes):
The long term goal of these studies is to compare the functional properties and homologous relationships of CYP among vertebrates. Identifying relationships and catalytic functions of CYP is important to understanding species differences and similarities in susceptibility to drugs and chemical pollutants. Such studies across taxa may indicate how structure-function relationships and physiological roles of CYP have been conserved during evolution.Understanding species sensitivity to xenobiotics requires knowledge of the metabolism, activation and/or detoxication of those compounds by those species. Enzymes in the CYP superfamily _(1)_ are the dominant catalysts in the oxidative metabolism of organic foreign compounds including natural products, drugs and pollutants _(2-4)_. CYP functions in liver and extrahepatic organs can determine the susceptibility of individuals, populations and species to the action of toxic foreign compounds that are substrates, and the validity of extrapolation between species. Expanding libraries of substrates linked to mammalian CYP are revealing common structural features of substrates preferred by those forms, defining their active sites _(5)_ _(6)_. How well the substrate structure-activity relationships (S-SAR) being developed for mammalian CYP will apply to homologous CYP in other species groups is not yet known. The latitude for substrate binding is a feature of protein structure that can differ substantially between homologous enzymes from different species. CYP alleles differing by a single amino acid can have different activities _(7)_. Conversely, the same activities in different species could be catalyzed by CYP that may or may not be closely related. Such possible differences create uncertainty in extrapolating the details of xenobiotic metabolism between species. This study has examined the functional properties of a CYP1A from a fish species, and compared these to properties of CYP1A in mammals. The project included studies on other species, identification of additional genes whose activity may bear on the function of CYP1A. and a continued effort to define the induction of CYP1A in the environment.
This molecular aspects of study represent a collaboration involving our lab and that of Dr. Johannes Doehmer, Technical University of Munich, and involved expression of CYP1A cDNA from the marine fish scup (Stenotomus chrysops) in V79 cells, and the characterization of the new cell line. That line was used in combination with microsomal studies to characterize catalytic functions of the scup CYP1A. Scup were selected in part because of extensive information on other properties, including the data on the catalytic function and regulation of CYP1A in this species.
Scup CYP1A was stably expressed in Chinese hamster V79 cells, by chromosomal integration of CYP1A cDNA under the control of the SV40 promoter, in collaboration with Dr. Johannes Doehmer, University of Munich, as originally proposed. Chromosomal integration was verified by Southern analysis using the homologous cDNA probe. Effective transcription and translation of the CYP1A was established by Northern analysis using the cDNA probe and by Western analysis using monoclonal antibody 1-12-3 to scup CYP1A. This new cell line, termed V79MZf1A1, expresses authentic and active scup CYP1A. The cells line was examined for metabolism and cytotoxicity of PAH, and cytotoxicity. Studies on metabolite profiles, and substrate specificity. Comparisons to human and rat CYP1A1 or CYP1A2 expressed in V79 cells were accomplished. Cells and purified protein and microsomes were used to establish metabolic profiles with caffeine, 3,3',4,4'-tetrachlorobiphenyl, and arachidonic acid. Uncoupling of CYP1A by 3,3',4,4'-tetrachlorobiphenyl and the resultant release of reactive oxygen were established. Fourteen publications have resulted thus far from these studies.
Journal Articles on this Report : 14 Displayed | Download in RIS Format
Other project views: | All 18 publications | 17 publications in selected types | All 17 journal articles |
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Bainy ACD, Woodin BR, Stegeman JJ. Elevated levels of multiple cytochrome P450 forms in tilapia from Billings Reservoir-Sao Paulo, Brazil. Aquatic Toxicology 1999;44(4):289-305. |
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Bishop CA, Ng P, Pettit KE, Kennedy SW, Stegeman JJ, Norstrom RJ, Brooks RJ. Environmental contamination and developmental abnormalities in eggs and hatchlings of the common snapping turtle (Chelydra serpentina serpentina) from the Great Lakes—St Lawrence River basin (1989-1991). Environmental Pollution 1998;101(1):143-156. |
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Cantrell SM, Joy-Schlezinger J, Stegeman JJ, Tillitt DE, Hannink M. Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptotic cell death in the embryonic vasculature with embryotoxicity. Toxicology and Applied Pharmacology 1998;148(1):24-34. |
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Celander M, Weisbrod R, Stegeman JJ. Glucocorticoid potentiation of cytochrome P4501A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in porcine and human endothelial cells in culture. Biochemical and Biophysical Research Communications 1997;232(3):749-753. |
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Celander M, Stegeman JJ. Isolation of a cytochrome P450 3A cDNA sequence (CYP3A30) from the marine teleost Fundulus heteroclitus and phylogenetic analysis of CYP3A genes. Biochemical and Biophysical Research Communications 1997;236(2):306-312. |
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Doehmer J, Buters JTM, Luch A, Soballa V, Baird WM, Morrison H, Stegeman JJ, Townsend AJ, Greenlee WF, Glatt HR, Seidel A, Jacob J, Greim H. Molecular studies on the toxifying effects by genetically engineered cytochromes P450. Drug Metabolism Reviews 1999;31(2):423-435. |
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Guiney PD, Smolowitz RM, Peterson RE, Stegeman JJ. Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in vascular endothelium with toxicity in early life stages of lake trout. Toxicology and Applied Pharmacology 1997;143(2):256-273. |
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Jacob J, Raab G, Soballa VJ, Luch A, Grimmer G, Greim H, Doehmer J, Morrison HG, Stegeman JJ, Seidel A. Species-dependent metabolism of benzo[c]phenanthrene and dibenzo[a,1]pyrene by various CYP450 isoforms. Polycyclic Aromatic Compounds 1999;16(1-4):191-203. |
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Morrison HG, Weil EJ, Karchner SI, Sogin ML, Stegeman JJ. Molecular cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of CYP1A genes: update with new sequences. Comparative Biochemistry and Physiology C-Pharmacology, Toxicology, and Endocrinology 1998;121(1-3):231-240. |
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Schlezinger JJ, Parker C, Zeldin DC, Stegeman JJ. Arachidonic acid metabolism in the marine fish Stenotomus chrysops (Scup) and the effects of cytochrome P450 1A inducers. Archives of Biochemistry and Biophysics 1998;353(2):265-275. |
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Schlezinger JJ, White RD, Stegeman JJ. Oxidative inactivation of cytochrome P-450 1A (CYP1A) stimulated by 3,3',4,4'-tetrachlorobiphenyl: Production of reactive oxygen by vertebrate CYP1As. Molecular Pharmacology 1999;56(3):588-597. |
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Schlezinger JJ, Stegeman JJ. Induction of cytochrome P450 1A in the American eel by model and by model halogenated and non-halogenated aryl hydrocarbon receptor agonists. Aquatic Toxicology 2000;50(4):375-386. |
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White RD, Shea D, Stegeman JJ. Metabolism of the aryl hydrocarbon receptor agonist 3,3',4,4'-tetrachlorobiphenyl by the marine fish scup (Stenotomus chrysops) in vivo and in vitro. Drug Metabolism and Disposition 1997;25(5):564-572. |
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White RD, Shea D, Solow AR, Stegeman JJ. Induction and post-transcriptional suppression of hepatic cytochrome P450 1A1 by 3,3',4,4'-tetrachlorobiphenyl. Biochemical Pharmacology 1997;53(7):1029-1040. |
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Supplemental Keywords:
Health, Scientific Discipline, Ecology, Environmental Chemistry, Chemistry, Risk Assessments, Biology, diagnostic substrates, catalytic function, hydrocarbon, animal model, genetic analysis, genetic engineering, fish proteins, cytochrome P4501A, biomarker, genetic susceptibilityThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.