Identification of 3-MI-derived N-centered radicals obtained from incubation of 3-MI with microsomal-NADPH system by EPR-HPLC spin trapping.
3-Methylindole (3-MI) is a metabolite of tryptophan that causes acute pulmonary edema and emphysema in ruminants when administered orally or intravenously. Electron paramagnetic resonance (EPR) spin-trapping techniques have been used to investigate the in vitro and in vivo formation of free radicals during 3-MI metabolism by goat lung. Utilizing C-phenyl-N-tert-butyl nitrone (PBN), a nitrogen-centered free radical has been detected from 3-MI in goat lung microsomal incubation. The EPR spectrum of the spin adduct is identical to that observed when 3-MI is irradiated with ultraviolet light. The formation of a nitrogen-centered 3-MI free radical is followed by the appearance of a carbon-centered radical in microsomal preparations. The objective of the present study is to prove that the nitrogen-centered radical generated from the 3-MI incubation system is a 3-MI radical utilizing [14C]-3 MI and the EPR-HPLC technique. The HPLC chromatogram includes three peaks that give EPR signals. These peaks are assigned to nitrogen-, oxygen- and carbon-centered radical adducts. The polarity of the three peaks follows the order: carbon-centered radical adduct > oxygen-centered radical adduct > nitrogen-centered radical adduct. The last has a polarity that is weaker than 3-MI. Only the nitrogen-centered peak and the 3-MI peak possessed radioactivity. The retention time of the nitrogen centered radical is the same as the spin adduct generated by 3-MI irradiation with ultraviolet light. These results demonstrate that the nitrogen-centered radical is a 3-MI-PBN spin adduct, and supports the hypothesis that 3-MI-induced lung damage results from activation of 3-MI to a free radical. Also, in this study the stability of the radical spin adducts and the best conditions to produce the radicals in the incubation system was investigated.
Chen
G
G
Department of Nutritional Sciences, University of Guelph, Ontario, Canada.
Janzen
E G
EG
Bray
T M
TM
D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D012756:Sheep; D012862:Skatole / Q000378:metabolism*
D005609:Free Radicals; D009249:NADP; D012862:Skatole