Heme Adducts from NitroPAH: Characterization and Mechanisms of FormationEPA Grant Number: R825346
Title: Heme Adducts from NitroPAH: Characterization and Mechanisms of Formation
Investigators: Ball, Louise M.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Hahn, Intaek
Project Period: November 18, 1996 through September 17, 1999
Project Amount: $390,896
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Ecosystems
Interactions of xenobiotics with cellular macromolecules such as DNA and proteins produce covalently-bound adducts that may serve as exposure markers. The environmental genotoxin 1-nitropyrene, widespread as a result of combustion processes, has been reported in laboratory animals (El-Bayoumy et al., 1994a) to bind stably to the heme moiety, as opposed to the protein, of hemoglobin, with linear dose-dependency over a dose range from 0.1 to 1000 ?g/kg. This finding suggests the existence of a novel class of adducts, heme adducts, which could potentially constitute useful markers for assessment of exposure. Since no chemical structure of a polycyclic aromatic hydrocarbon-heme adduct has yet been established, and mechanisms of formation and biological consequences of such adducts are as yet unknown, the objectives of this proposal are to characterize the chemical structure of the 1-nitropyrene-derived heme adduct, investigate its mechanism of formation, and determine the generalizability of heme adduct formation to other nitroPAH and arylamines, by means of the specific aims outlined below. The information gained will be of fundamental interest, and also of value in establishing the usefulness of heme adducts as monitors of exposure.
(1) Synthesis of model heme adducts, to establish regioselectivity of binding and the nature of the nitropyrene-derived moiety. Active intermediates known to arise from metabolism of 1-nitropyrene will be allowed to react with a model synthetic heme, octaethylporphyrin, to establish the position of substitution on the porphyrin moiety and the nature of the nitroarene-derived species capable of binding to heme. Products will be isolated then characterized chemically and spectroscopically.
(2) Generation of heme adducts by the heme protein hemoglobin. The capacity of the catalytically-active heme-containing protein hemoglobin to generate adducts of 1-nitropyrene with protoporphyrin IX itself will be investigated, along with adducts arising from reaction with the major known active metabolites of 1-nitropyrene, in order to distinguish between reaction of circulating active species and activation in situ by the heme protein itself, and thus gain insight into possible mechanisms of formation in vivo.
(3) Characterization of heme adducts formed in vivo. Characterization of in vivo adducts of 1-nitropyrene will be pursued by comparison of spectroscopic and chromatographic behavior with that of the adducts generated synthetically and in the protein model system.
(4) Formation of heme adducts from related nitroPAH. The generality of heme adduction by nitroPAH will be explored by investigation of in vivo heme binding and of interactions of the heme protein model system with other nitroarenes which are also ubiquitous environmental contaminants having important implications for human health.