Abstract
Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.
Highlights
A large body of evidence supports the concept that electrophiles, generated intracellularly from chemically stable compounds, play a key role in chemical-induced cytotoxicity [1,2]
These inconsistencies suggest that simple measures of reactive metabolite formation cannot, a priori, be used to exclude a prospective therapeutic agent from development and that better methods are needed to discriminate those reactive metabolites with real potential for tissue injury from those which are unlikely to cause deleterious effects [5]
The purpose of this study was to characterize the amino acid residues that react with these reactive metabolites, to determine whether these could be measured effectively in a very controlled environment where the pH and peptide/metabolite ratios could be controlled and to measure relative rates of adduction
Summary
A large body of evidence supports the concept that electrophiles, generated intracellularly from chemically stable compounds, play a key role in chemical-induced cytotoxicity [1,2]. Despite positive correlations between total levels of reactive metabolite binding and the extent of toxicity for many chemicals, there are notable exceptions where high levels of bound metabolite do not lead to detrimental effects in the cell [3,4]. Considerable progress has been made in enhancing the ease and speed of studying proteins adducted by reactive metabolites and, owing largely to Hanzlik’s work, a database is available which lists many of the cellular proteins adducted by reactive intermediates [6]. Hypotheses have emerged on which proteins may be important in the pathways leading to cell death, the identification of many of the critical proteins remains elusive
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