Spin-trapping of free radicals formed during in vitro and in vivo metabolism of 3-methylindole.

S Kubow,E G Janzen,T M Bray

doi:10.1016/s0021-9258(17)43067-5

Abstract

Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.

Highlights

Utilizing thespintrap reactive electrophilic intermediates such as epoxides and free phenyl-t-butylnitrone,a nitrogen-centered free radi- radicals [7]
Stants identical to those carbon-centeredfree radicals Chemical compounds termedspin-trappingagents have trapped in in vitro incubationsof3MI.Thesedata recently been used to detect and identi$ short lived radicals demonstrate that microsomal metabolismof 3MI pro- produced in biochemical systems
The results obtained inthis study indicate that a 3MI free radical intermediate resulting from the microsomal metabolism of 3MI is trapped by PBN, and this nitroxyl adduct detected by ESR is dependent on the presence of NADPH, 02,and microsomes

Summary

THEJOURNALOF BIOLOGICACLHEMISTRY

0 1984 by The American Society of Biological Chemists, Inc. Vol,259, No h u e of April 10,pp. 4447-4451,1984 Prmted m U.S.A. Thtehe exact mechanism of 3MI toxicity is not known, a free concept that3MI causes lipid peroxidation in the lungradical produced from 3MI may be of significance.Free radical was supported bythe in vivo study in which a carbon- intermediates with short biological half-lives have been demcentered radical was spin-trapped by phenyl-t-butyl- onstrated to be of importance in many examples of tissue nitrone in lungs of intact goats infuswedith 3MI. This injury that require metabolic activation of a primary toxic carbon-centered radical had hyperfine splitting con- agent [11]. Oral or intravenous administration of 3MI has been shown to induce

MATERIALS ANDMETHODS

RESULTS

DISCUSSION