Grantee Research Project Results
1999 Progress 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 Period Covered by this Report: September 1, 1998 through August 1, 1999
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 this research is to establish the catalytic capabilities of the polynuclear and planar halogenated aromatic hydrocarbon (PAH and HAH)-inducible cytochrome P450 1A (CYP1A) proteins in non-mammalian vertebrates. The toxicological consequences of environmental induction of CYP1A, and the validity of extrapolation of these consequences between species, will remain uncertain until the substrate specificity with xenobiotic and endogenous compounds is adequately known for CYP1As in different taxonomic groups. Here we propose further studies to define the functions of scup CYP1A, in three broad areas?the specificity for xenobiotic and endogenous substrates, the molecular and cellular responses to slowly metabolized substrates, and testing a hypothesis that pHAH contribute to toxicity through oxidative stress due to uncoupling of CYP1A catalytic cycle.Progress Summary:
The previously prepared Chinese hamster V79 cell line was re-evaluated and found to have adequate reductase activity to address selected questions. While microsomes prepared from that cell line were not active, the intact cells could be used and activity was adequate. Attempts to express the fish CYP1A in the mouse hepatoma Hepa-1c1c7 mutant c37 line, that has an AhR, but is deficient in Cyp1a1 and 1a2, are envisioned.We continued to employ model compounds to determine the substrate specificity of teleost CYP1As, and to assess functional similarities to both CYP1A1 and CYP1A2, vis-a-vis the structural relationships among these proteins. Caffeine (CA; 1,3,7-trimethylxanthine) is N-demethylated by CYP1A2 in mammals, to paraxanthine (PX; N-3-demethylation) and theobromine (TB; N-1-demethylation). BNF-induced scup liver microsomes oxidized CA primarily to PX and TB. TP and TMU formation was not detected. Polyclonal and the 1A1-specific monoclonal (1-12-3) antibodies to scup CYP1A inhibited CA metabolism by 80-90 percent. Chinese hamster V79 cells stably expressing scup CYP1A metabolized CA to PX (major) and TB (minor). Teleost CYP1As metabolize caffeine with regiospecificity more similar to CYP1A2s than to CYP1A1s.
In addition to approaching the metabolic capabilities experimentally, we initiated a molecular modeling approach to assessing the substrate-structure activity relationships for scup CYP1A. Computational and experimental approaches can be combined to study the interactions of CYP molecules with xenobiotic and endogenous substrates. The structure of teleost (scup) CYP1A was assessed by homology modeling from the known crystal structures of bacterial CYP. We will address the hypothesis that teleost fish CYP1As have a more restricted access channel or active site than do mammalian 1A1s, a hypothesis based on findings made primarily with scup CYP1A.
Using baculovirus expressed human enzymes, we have determined that it is indeed human CYP1A1 and not CYP1A2 that is uncoupled and releases ROS. Unlike the results with scup and rat CYP1As, inactivation of human CYP1A1 and CYP1A2 occurred during incubation with NADPH alone, but was the inactivation of CYP1A1 was increased when TCB was added.
We determined that 3,3',4,4',5-pentachlorobiphenyl can suppress CYP1A in vivo, as occurs with 3,3',4,4'-TCB. Low dose PeCB induced hepatic microsomal P450 and CYP1A protein and catalytic activities over an 18-day period, while high dose PeCB did not induce hepatic P450 and CYP1A content, and catalytic activities remained at control levels. CYP1A mRNA expression was induced strongly at both doses. PeCB stimulated reactive oxygen species production by scup liver microsomes. Oxidative stress, (i.e., increased levels of catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase activities) was simulated in liver by low dose but not high dose PeCB.
To address whether the oxidative stress might have further consequences, we cloned a Rel homology domain from scup (the first fish Rel sequence) and evaluated the activation of NF-kB using gel shift assays. Nuclear proteins from liver, kidney, and heart of scup all showed specific binding to an NF-kB consensus sequence. NF-kB activation was elevated in the PeCB-treated fish. The data demonstrate the presence of Rel family proteins in fish and support a hypothesis that some aryl hydrocarbon receptor agonists can activate NF-kB in vivo.
We have cloned additional CYP1 gene family members from scup, relevant to the evaluation of CYP1A functions considered above. Two CYP1B genes, prov. CYP1B2 and CYP1B3 were cloned from scup liver.
Future Activities:
Studies will continue as proposed, addressing parts of all of the Aims. We will focus principally on the studies using transgenic cell lines expressing CYP1As in culture to assess ROS generation, the activation of NFkB and also DNA modification in those cells, and the metabolism of retinoids. These are the most relevant to the toxicological implications of the work.Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 18 publications | 17 publications in selected types | All 17 journal articles |
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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. |
R827102 (1999) R827102 (Final) |
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Iwata H, Stegeman JJ. In situ RT-PCR detection of CYP1A mRNA in pharyngeal epithelium and chondroid cells from chemically untreated fish: Involvement in vertebrate craniofacial skeletal development? Biochemical and Biophysical Research Communications 2000;271(1):130-137. |
R827102 (1999) |
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Schlezinger JJ, Blickarz CE, Mann KK, Doerre S, Stegeman JJ. Identification of NF-κB in the marine fish Stenotomus chrysops and examination of its activation by aryl hydrocarbon receptor agonists. Chemico-Biological Interactions 2000;126(2):137-157. |
R827102 (1999) |
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Schlezinger JJ, Stegeman JJ. 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). Aquatic Toxicology 2001;52(2):101-115. |
R827102 (1999) |
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Supplemental Keywords:
cytochrome P4501A, biology, toxicology, polynuclear aromatic hydrocarbons, halogenated aromatic hydrocarbons, molecular response, cellular response, metabolism., 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 susceptibilityProgress and Final Reports:
Original AbstractThe 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.