N6-Formyllysine as a Biomarker of Formaldehyde Exposure: Formation and Loss of N6-Formyllysine in Nasal Epithelium in Long-Term, Low-Dose Inhalation Studies in Rats.

Exposure to both endogenous and exogenous formaldehyde has been established to be carcinogenic, likely by virtue of forming nucleic acid and proteins adducts such as N6-formyllysine. To better assess N6-formyllysine as a biomarker of formaldehyde exposure, we studied accumulation of N6-formyllysine adducts in tissues of rats exposed by inhalation to 2 ppm [13C2H2]-formaldehyde for 7, 14, 21, and 28 days (6 h/day) and investigated adduct loss over a 7-day postexposure period using liquid chromatography-coupled tandem mass spectrometry. Our results showed formation of exogenous adducts in nasal epithelium and to some extent in trachea but not in distant tissues of lung, bone marrow, or white blood cells, with a 2-fold increase over endogenous N6-formyllysine over a 3-week exposure period. Postexposure analyses indicated a biexponential decay of N6-formyllysine in proteins extracted from different cellular compartments, with half-lives of ∼25 and ∼182 h for the fast and slow phases, respectively, in cytoplasmic proteins. These results parallel the behavior of DNA adducts and DNA-protein cross-links, with protein adducts cleared faster than DNA-protein cross-links, and point to the potential utility of N6-formyllysine protein adducts as biomarkers of formaldehyde.

Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6)-formyllysine that is refractory to histone deacetylases.

Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6)-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6)-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6)-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6)-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4) lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4) lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6)-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6)-formyllysine, with use of [(13)C,(2)H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6)-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N(6)-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification.

Dosimetry of N6-formyllysine adducts following [¹³C²H2]-formaldehyde exposures in rats.

With formaldehyde as the major source of endogenous N6-formyllysine protein adducts, we quantified endogenous and exogenous N6-formyllysine in the nasal epithelium of rats exposed by inhalation to 0.7, 2, 5.8, and 9.1 ppm [¹³C²H2]-formaldehyde using liquid chromatography-coupled tandem mass spectrometry. Exogenous N6-formyllysine was detected in the nasal epithelium, with concentration-dependent formation in total as well as fractionated (cytoplasmic, membrane, nuclear) proteins, but was not detected in the lung, liver, or bone marrow. Endogenous adducts dominated at all exposure conditions, with a 6 h 9.1 ppm formaldehyde exposure resulting in one-third of the total load of N6-formyllysine being derived from exogenous sources. The results parallel previous studies of formaldehyde-induced DNA adducts.