Chronic treatment of BALB and GRS mice with
BHT (2,6-di-tert-butyl-4-methylphenol) following a single
urethane injection increases lung
tumor multiplicity, but this does not occur in CXB4 mice. Previous data suggest that promotion requires the conversion of
BHT to a tert-butyl-hydroxylated metabolite (
BHTOH) in lung and the subsequent oxidation of this species to an electrophilic
quinone methide. To obtain additional evidence for the importance of
quinone methide formation, structural analogs that form less reactive
quinone methides were tested and found to lack promoting activity in
BHT-responsive mice. The possibility that promotion-unresponsive strains are unable to form
BHTOH was tested by substituting this compound for
BHT in the promotion protocol using CXB4 mice. No promotion occurred, and in-vitro work demonstrated that CXB4 mice are, in fact, capable of producing
BHTOH and its
quinone methide, albeit in smaller quantities. Incubations with BALB lung microsomes and radiolabeled substrates confirmed that more covalent binding to
protein occurs with
BHTOH than with
BHT and, in addition,
BHTOH quinone methide is considerably more toxic to mouse lung epithelial cells than
BHT quinone methide. These data are consistent with the hypothesis that a two-step oxidation process, i.e. hydroxylation and
quinone methide formation, is required for the promotion of mouse lung
tumors by
BHT.