Figure 2.

LG-lysine adducts are found in cells and tissue, dependent on COX-2 activity. RAW264.7 mouse macrophage (A) and A549 human lung carcinoma (C) cells were stimulated to express COX-2 and then given 20 μM arachidonic acid (AA) or vehicle. A subgroup of cells was preincubated 45 min with 50 μM indomethacin. As a measure of COX activity, PGE₂ was determined by GC–MS from cell media prior to lysis (B and D). Nuclei were isolated, and histones were extracted and digested to individual amino acids prior to LC–ESI/MS/MS analysis. *p < 0.05; ***p < 0.001 by ANOVA followed by Tukey’s post-test (n ≥ 5). (E). Histones were extracted from nuclei of rat liver and analyzed as above for LG-lactam adduct. COX-2 protein was analyzed by Western blotting and plotted against lactam adduct levels. Each point corresponds to one liver, and shown is the line of regression (r² = 0.7237). Pearson r = 0.8507; two-tailed p = 0.0152. (F) LC–MS chromatograph of histones isolated from a rat liver with relatively high COX-2 expression (COX-2 band intensity of 117 arbitrary units).