Capture of Electrophilic Quinones in the Extracellular Space: Evidence for a Phase Zero Reaction

Abstract

Electrophilic quinones are produced during the combustion of gasoline in the atmosphere. Although these reactive species covalently bind to protein-based nucleophiles in cells, resulting in the formation of protein adducts involved in the modulation of redox signaling pathways and cytotoxicity, the extracellular regulation of quinones is not understood. In this study, incubation of 1,2-naphthoquinone (1,2-NQ) with the low-molecular-weight fraction of mouse plasma resulted in the consumption of cysteine (CysSH) in the plasma in a concentration-dependent manner. Covalent modification of albumin was markedly repressed by the addition of either the low-molecular-weight fraction of mouse plasma or CysSH, suggesting that CysSH protects by forming a conjugate with 1,2-NQ. Similar phenomena also occurred for other atmospheric quinones 1,4-NQ and 1,4-benzoquinone (1,4-BQ). The addition of cystine to a culture medium without amino acids enhanced the release of CysSH from A431 cells and blocked 1,2-NQmediated arylation of intracellular proteins, suggesting that 1,2-NQ interacts with extracellular CysSH. Liquid chromatography− tandem mass spectrometry analysis revealed that 1,2-NQ and 1,4-BQ undergoes nucleophilic attack by CysSH, yielding a 1,2-NQH 2 −SCys adduct and 1,4-BQH 2 −SCys adduct, respectively. Unlike 1,2-NQ and 1,4-BQ, the authentic 1,2-NQH 2 −SCys adduct and 1,4-BQH 2 −SCys adduct had little effect on the covalent modification of cellular proteins and viability of A431 cells. These results suggest that electrophilic quinones are readily trapped by CysSH released from A431 cells, forming less-toxic CysSH adducts and thereby repressing covalent modification of cellular proteins. These findings provide evidence for the existence of a "phase zero" reaction of electrophiles prior to their uptake by cells.