Metabolism of the skin
tumor promoter butylated hydroxy-
toluene hydroperoxide (2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-
cyclohexadienone;
BHTOOH) to reactive intermediates is required for
tumor promotion by this compound. In particular, an electrophilic
quinone methide is known to mediate both in vivo
tumor promotion as well as in vitro cytotoxicity by
BHTOOH. In the present study, the role of this reactive intermediate in the induction of
ornithine decarboxylase (ODC), a gene strongly associated with
tumor promotion, was investigated in cultured keratinocytes.
BHTOOH stimulates a time-dependent increase in ODC
enzyme activity, paralleled by ODC
mRNA induction, suggesting transcriptional regulation of ODC by
BHTOOH. Depletion of intracellular
glutathione caused a 5-fold potentiation of keratinocyte sensitivity to
BHTOOH. Concordantly, ODC induction by
BHTOOH could be completely inhibited by soluble
thiol compounds. These results suggest that ODC induction is mediated by a
thiol-reactive metabolite of
BHTOOH. The
iron-specific
chelator desferal blocked ODC induction by
BHTOOH, indicating that formation of this intermediate is
iron-dependent. Substitution of the 4-methyl group of
BHTOOH with alkyl groups of incrementally larger size is known to reduce accordingly
quinone methide production; comparative study of these
BHTOOH analogs demonstrated a corresponding loss of potency for ODC induction, indicating that
BHT-quinone methide mediates the in vitro induction of ODC by
BHTOOH. Finally,
kinase inhibitor studies suggested a role for
protein kinase C in the induction of ODC by
BHTOOH. Taken together, these results provide insight into the cellular mechanisms through which the reactive electrophile
BHT-quinone methide can mediate alterations in gene expression, such as occur in
tumor promotion in vivo.