Identification and quantification of in vitro adduct formation between protein reactive xenobiotics and a lysine-containing model peptide

• Published in: Environmental toxicology Vol. 18; no. 1; pp. 29 - 36
• Main Authors: Reichardt, Peter, Schreiber, André, Wichmann, Gunnar, Metzner, Gerhard, Efer, Juergen, Raabe, Ferdinand
• Format: Journal Article
• Language: English
• Published: New York Wiley Subscription Services, Inc., A Wiley Company 2003; Wiley
• Subjects: adducts; Aldehydes - chemistry; Aldehydes - metabolism; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Chromatography, High Pressure Liquid; Environmental pollutants toxicology; Fundamental and applied biological sciences. Psychology; General aspects; immunotoxic effects; lysine; Lysine - analysis; Lysine - chemistry; Lysine - metabolism; Mass Spectrometry; Medical sciences; Microbiology; Models, Chemical; Molecular Structure; Peptide Fragments - chemistry; Peptide Fragments - metabolism; peptides; proteins; Toxicology; Tyrosine - analysis; Tyrosine - chemistry; Tyrosine - metabolism; xenobiotics; Xenobiotics - chemistry; Xenobiotics - metabolism; Peptides; Toxicity; xenobiotics; Immunity; In vitro; Protein; Pollutant; Lysine; adducts; proteins; Environment; immunotoxic effects; Models; Mass spectrometry; Molecular adduct; Xenobiotic
• ISSN: 1520-4081; 1522-7278
• DOI: 10.1002/tox.10097

Formation of in vitro adducts between different classes of xenobiotics and the lysine‐containing peptide Lys‐Tyr was monitored by high‐performance liquid chromatography and electrospray ionization mass spectrometry. The molecular structures of the main resulting products could be sensitively analyzed by mass spectrometry (flow injection analysis), enabling the detection of characteristic binding formations. Aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde were shown to form stable linkages to lysine amino groups via Schiff bases. Other electrophilic substances (e.g., toluene‐2,4‐diisocyanate, 2,4‐dinitro‐1‐fluorobenzene, 2,4,6‐trinitrobenzene sulfonic acid, dansyl chloride, and phthalic acid anhydride) also formed covalent adducts with lysine residues. The reactivity of the compounds was quantified by measuring the amount of peptide that remained unchanged after incubation for a certain period with the xenobiotic. Although reactivity levels within this group of aldehydes varied only to a small extent, as would be expected, extreme differences were seen among the structurally heterogeneous group of nonaldehyde xenobiotics. These results support the hypothesis that simple chemical reactions may lead to the adduction of nucleophilic macromolecules such as peptides or proteins. Such reactions, in particular, Schiff base formation of aldehydes, have previously been shown to be capable of specifically interfering with costimulatory signaling on T cells. Our results suggest that electrophilic xenobiotics of other classes may also inherit the capacity to exert similar effects. Forming covalent linkage to peptides may represent a possible molecular mechanism of electrophilic xenobiotics in vivo, yielding immunotoxic effects. The model utilized in this study is appropriate for monitoring the adduction of xenobiotics to basic peptides and for analyzing the resulting molecular structures. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 29–36, 2003.