Grantee Research Project Results
1999 Progress Report: Comparative Mechanisms of Benzo[a]pyrene Metabolism and DNA Repair in Two Species of Ictalurid Catfish
EPA Grant Number: R827101Title: Comparative Mechanisms of Benzo[a]pyrene Metabolism and DNA Repair in Two Species of Ictalurid Catfish
Investigators: Di Giulio, Richard T. , Willett, Kristine L. , Lienesch, Laila , Rau, Michelle
Institution: Duke University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1998 through September 30, 2001 (Extended to September 30, 2003)
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $314,023
RFA: Exploratory Research - Environmental Biology (1998) RFA Text | Recipients Lists
Research Category: Human Health , Aquatic Ecosystems , Biology/Life Sciences
Objective:
The goal of this project is to elucidate underlying biochemical and molecular mechanisms that account for the greater sensitivity of the brown bullhead (Ameriurus nebulosus) to hydrocarbon-mediated liver cancer versus the related channel catfish (Ictalurus punctatus). Previous work in our laboratory has demonstrated that the channel catfish has about 10X greater activities of hepatic CYP1A and correspondingly greater metabolism of model carcinogenic hydrocarbons (such as benzo[a]pyrene, BaP) and enhanced formation of DNA adducts in vitro. These results are counter-intuitive, based upon the difference in sensitivity between the two species. However, following in vivo exposures to BaP, DNA adducts in liver were significantly greater and more persistent in the more sensitive bullhead. These results collectively underlie the hypotheses and objectives of this current project:Hypothesis 1. The channel catfish more rapidly eliminates BaP than the brown bullhead due to greater hepatic phase II metabolic activities towards BaP.
Objective 1a: To determine the hepatic activities of the major enzymes likely to be involved in the further metabolism of the BaP products (e.g., epoxides, dihydrodiols, and phenols) resulting from CYP1A-catalyzed oxidations in these species. These enzymes are: epoxide hydrolase (EH), glutathione S-transferases (GST), uridine diphosphate glucuronosyltransferases (UDPGT), and sulfotransferases (ST).
Objective 1b: To determine rates of BaP metabolite formation catalyzed by these enzymes in isolated hepatocytes from both species.
Objective 1c: To determine rates of biliary excretion of BaP metabolites by these species following in vivo exposures.
Hypothesis 2. The channel catfish more rapidly repairs BaP-DNA adducts in liver tissue than the brown bullhead.
Objective 2: To compare the activities of nucleotide excision repair systems in tissue extracts from both species targeted to plasmid BaP-DNA adducts.
Progress Summary:
Significant progress has been made with respect to hypothesis 1. A major in vivo experiment was conducted wherein livers and bile were collected from each species 6, 24, 72, and 168 hr after a single 10 mg/kg i.p. BaP exposure. Phase II enzyme activities were quantified in liver tissue, and BaP metabolites were quantified in bile samples. Key results included the following: Cytosolic 1-chloro-2,4?dinitrobenzene? glutathione-S-transferase (GST) and cis-stilbene oxide-microsomal EH activities were not significantly different in BaP-treated and control animals of either species. Channel catfish EH and GST activities were 1.2-fold higher than brown bullhead activities (p = 0.058 and p = 0.002, respectively). HPLC-APCI-MS of extracted bile and bile enzymatically digested to detect glucuronyl transferase (GT), GST, and ST conjugated metabolites indicated no species difference in elimination of total biliary metabolites. GT conjugates predominated; ST and GST conjugates were minimal. BaP-diones accounted for the majority of metabolites in both species. However, brown bullhead preferentially formed BaP-7,8-dihydrodiol, a precursor to the DNA reactive BaP-7,8-dihydrodiol-9,10-epoxide (BPDE), which may be linked to the increased PAH susceptibility in this species.Current work is focusing upon hypothesis 2. We are collaborating with Dr. Aziz Sancar (University of North Carolina, Chapel Hill) to optimize techniques developed in his laboratory to study nucleotide excision repair in these species. Preliminary results suggest limited capacities for repair in both species, relative to mammalian models.
Future Activities:
Future activities will include research along three lines: (1) continued work to elucidate nucleotide excision repair in the two species; (2) investigations into the relative roles of CYP1A and CYP1B1 in activating BaP to DNA-reactive metabolites in these species; and (3) investigations of the tumor promoter gene p53, including structural differences between the two species, and mutational and regulatory effects of BaP.Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 5 publications | 1 publications in selected types | All 1 journal articles |
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Type | Citation | ||
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Willett KL, Gardinali PR, Lienesch LA, Di Giulio RT. Comparative metabolism and excretion of benzo(a)pyrene in 2 species of ictalurid catfish. Toxicological Sciences 2000, Volume: 58, Number: 1 (NOV), Page: 68-76. |
R827101 (1999) |
not available |
Supplemental Keywords:
PAHs, cancer susceptibility, surface water, aquatic ecosystems., RFA, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, National Recommended Water Quality, Aquatic Ecosystem, Environmental Microbiology, Molecular Biology/Genetics, Biology, ictalurid catfish, bullhead catfish, channel catfish, microbiology, cellular biology, ecology, cellular physiology, water quality, DNA repair, BaP metabolismProgress 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.