Comparative Mechanisms of Benzo[a]pyrene Metabolism and DNA Repair in Two Species of Ictalurid CatfishEPA Grant Number: R827101
Title: Comparative Mechanisms of Benzo[a]pyrene Metabolism and DNA Repair in Two Species of Ictalurid Catfish
Investigators: Di Giulio, Richard T. , MacLean, Elizabeth , Meyer, Joel , Willett, Kristine L.
Current Investigators: Di Giulio, Richard T. , Lienesch, Laila , Rau, Michelle , Willett, Kristine L.
Institution: Duke University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1998 through September 30, 2001 (Extended to September 30, 2003)
Project Amount: $314,023
RFA: Exploratory Research - Environmental Biology (1998) RFA Text | Recipients Lists
Research Category: Health , Ecosystems , Biology/Life Sciences
Hypothesis 1. The channel catfish more rapidly eliminates benzo[a]pyrene (BaP) than the more hepatic neoplasia-sensitive 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., diols, phenols and epoxides) 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.
Phase II metabolism of BaP will be addressed in vitro and in vivo. Subcellular fractions (microsomes and cytosol as appropriate) will be used to determine relative activities of enzymes (EH, GST, UDPGT and ST) towards standard substrates and towards selected phase I metabolites of BaP. Primary hepatocytes of both species will be used to compare the integrated metabolism of BaP in these animals. Studies of bile metabolites of BaP from fish exposed in vivo will be employed to corroborate results from in vitro experiments. Nucleotide excision repair (NER), the dominant mechanism of bulky adduct repair in eukaryotes, will be addressed by comparing the abilities of cell free extracts from heptocytes of these two species to repair BaP metabolites adducted to plasmid DNA.
Previous work in our laboratory has yielded the counter-intuitive result that the cancer-sensitive bullhead has significantly less capacity to activate BaP to genotoxic metabolites, via CYP1A metabolism, than the resistant channel catfish. The project proposed herein will determine if differences in subsequent detoxification-oriented metabolic pathways and/or DNA repair can explain the marked differential susceptibility in these closely-related fishes. The results of this project will contribute significantly to our understanding of biological phenomena that underlie differential chemical sensitivity that is important to understanding why certain species, or individuals within a species (including humans) are relatively susceptible to toxic effects. As our approaches to risk assessment and environmental protection become more sophisticated, it is becoming increasingly evident that efforts should be directed towards sensitive components of ecosystems and populations, not the "mean."