GENERATION OF NITRO AND SUPEROXIDE RADICAL-ANIONS FROM 2,4,6-TRINITROBENZENESULFONIC ACID BY RAT GASTROINTESTINAL CELLS

Reactive oxygen and nitrogen species have been implicated in the infla mmation of the gastrointestinal tract. The objective of this study was to investigate mechanisms of free radical formation from the colitis inducer 2,4,6-trinitrobenzene sulfonic acid (TNBS). We showed that TNB S was rapidly metabolized to TNBS nitro radical anion via metabolic re duction by flavinmononucleotide/NADPH, xanthine/xanthine oxidase as we ll as the rat small intestine and colon. TNBS nitro radical anion was directly detected with electron paramagnetic resonance (EPR) spectrosc opy. EPR spectra of TNBS nitro radical anion showed hyperfine coupling constants from the proximal nitrogen, two hydrogens and the two dista l nitrogens with respective magnitudes of a(N)(4) = 9.7 G; a(H)(3,5) = 3.2 G (2); and a(N)(2,6) = 0.25 G. EPR spin trapping using 5,5-dimeth yl-1-pyrroline N-oxide in aerobic incubations of isolated enterocytes (or colonocytes, or red blood cells) and TNBS, in the presence or abse nce of NADPH, produced radical adducts characteristic of superoxide an d hydroxyl radicals. Our EPR data showing generation of TNBS nitro and superoxide radical anions demonstrate that one-electron reduction of TNBS may be an initial step in the cascade of the in vivo inflammatory events in TNBS-induced colitis. (C) 1997 Elsevier Science B.V.