Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide.

B G Taffe,N Takahashi,T W Kensler,R P Mason

doi:10.1016/s0021-9258(18)45328-8

Abstract

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.

Highlights

The organic hydroperoxides tert-butyl hydroperox- rarely yields malignant tumors.The second stage, promotion, ide and cumene hydroperoxidaere tumor promoters in serves to express the latent altered phenotyofptehe initiated the skinof SENCAR mice, and this activiitsypresumed cell through selection andclonalexpansion [2]
1-pyrrolineN-oxide or 2-methyl-2-nitrosopropane) originallydefined in mouse skin,multistage carcinogenesis and either tert-butyl hydroperoxidoer cumene hydro- models have been developed for many epithelial tissues inperoxide resulted in the generation and detection of cluding liver, lung,bladder, mammarygland, pancreas, esophradical adducts of these spin traps. tert-Butyl alkoxyl agus, stomach, andcolon, as well as cells in culture[3].These and alkyl radical adductosf 5,5-dimethyl-l-pyrroline models provide strong presumptive evidence for the role of
The spectrum generated on incubationof the keratinocytes with cumene hydroperoxide in the presence of the spin trap the experiments involving tNB, the spin trap was stirred in PBS overnight (1 mg/ml)in the dark

Summary

Free Radical Generation inMouse Keratinocytes

0.2 ml of PBS, DMPO (90 mM), and where indicated, tert-butyl hydroperoxide (30 mM). The spectrum generated on incubationof the keratinocytes with cumene hydroperoxide in the presence of the spin trap the experiments involving tNB, the spin trap was stirred in PBS overnight (1 mg/ml)in the dark. Omission of cumene hydroperoxide from this incubation (Fig. 3 F ) or heat inactivation of the cells prior to incubation withDMPO and cumene hydroperoxide(Fig. 3 E ) resulted,respectively,in the absence or significant diminutionof the spectrumgenerated. These data (30X with homogenizer clearance = 0.0005 inch), followed by centrifugation at 3,000 X g for 15 min. No alkoxylradical adduct of cumene hydroperoxide was detected in thissystem

RESULTS

Free Radical Generation in Mouse Keratinocytes

DISCUSSION

Ethyl Hydrogen