Generation of reactive intermediates from the tumor promoter butylated hydroxytoluene hydroperoxide in isolated murine keratinocytes or by hematin

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Abstract
BHTOOH (2, 6-di-tert-butyl-4-hydroperoxyl-4-methyl-2, 5-cyclohexadienone), a metabolite of the food antioxidant BHT (2, 6-di-tert-butyl-4-methylphenol), has previously been shown to function as a tumor promoter in mouse skin. The metabolism of BHTOOH was examined to assess the role of reactive intermediates in mediating tumor promotion in this tissue. Free radical metabolites of BHTOOH were characterized in either isolated neonatal mouse keratinocytes or a cell-free hematin system using electron paramagnetic resonance (EPR) spectroscopy while non-radical, EPR silent products were characterized using HPLC separation coupled with UV or mass spectral detection. Incubation of BHTOOH with keratinocytes or hematin resulted in the generation of the BHT-phenoxyl radical detectable by EPR spectroscopy. Formation of the BHT-phenoxyl radical was prevented by heat inactivation of the cells prior to exposure to BHTOOH. Only one non-radical metabolite of BHTOOH was detected in keratinocytes: BHT-quinol (2, 6-di-tert-butyl-4-methyl-4-hydroxyl-2,5-cyclohexadianone), while incubation of BHTOOH with hematin produced several metabolites: oxacyclopen-tenone (2, 5-di-tert-butyl-5-(2'-oxopropyl)-4-oxa-2-cyclopen-tenone), BHT-quinone (2, 6-di-tert-butyl-p-benzoquinone), BHT, BHT-stilbenequinone (3, 5,3',5'-tetra-tert-butylstilbene-4, 4'-quinone), and BHT-quinone methide (2, 6-di-tert-butyl-4-methylene-2, 5-cyclohexadienone). Thus, radical as well as non-radical reactive intermediates can be formed during metabolism of BHTOOH. Several of the stable metabolites of BHTOOH were evaluated for possible promoter activity in a short-term bioassay, induction of ornithine decarboxylase (ODC) activity in mouse skin. In contrast to the action of BHTOOH, topical application of equimolar doses of BHT-quinol, BHT-quinone, BHT-stilbenequinone, as well as BHT itself, did not induce epidermal ODC activity. Thus, reactive metabolites of BHTOOH such as the BHT-phenoxyl radical or BHT-quinone methide may be involved in the molecular mechanisms of action of this hydroperoxide tumor promoter.