Free radical production during metabolism of organic hydroperoxides by normal human keratinocytes.
Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.
Iannone
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Institute of General Pathology, University of Modena, Italy.
Marconi
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A
Zambruno
G
G
Giannetti
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A
Vannini
V
V
Tomasi
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A
D001555:Benzene Derivatives / Q000378:metabolism; D001585:Benzoyl Peroxide / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012016:Reference Values; D020122:tert-Butylhydroperoxide
D001555:Benzene Derivatives; D005609:Free Radicals; D010545:Peroxides; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; C007164:cumene hydroperoxide; D001585:Benzoyl Peroxide