Quantitation of N6-Formyl-lysine Adduct Following Aristolochic Acid Exposure in Cells and Rat Tissues by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method.

N⁶-Formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational N⁶-formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentration-dependent formation of FLys in cells exposed to formaldehyde and Fe²⁺-EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe²⁺-EDTA produced FLys at a frequency of 20.2 and 4.1 per 10⁴ lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed Escherichia coli cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time- and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic N⁶-formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of N⁶-formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.