Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S -transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2 -deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2 -deficient mice. In parallel with the enzymatic changes, nrf2 -deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[ a ]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2 -deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2 -disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

The cancer chemopreventive actions of oltipraz (4-methyl-5-[2-pyrazinyl]-1,2-dithiole-3-thione) have been primarily associated with the induction of phase 2 detoxifying enzymes through transcriptional activation of the antioxidant response element (ARE) in the promoter regions of these genes. The transcription factor Nrf2 has been shown to bind to and activate AREs. Previously, we demonstrated that nrf2 -deficient mice had low basal expression of phase 2 enzymes and were substantially more susceptible to benzo[ a ]pyrene (B[ a ]P)-induced neoplasia of the forestomach than wild-type. Moreover, loss of Nrf2 abrogated the chemopreventive action of oltipraz, when administered 48 h before B[ a ]P, an interval allowing maximal induction of many phase 2 enzymes. Oltipraz also inhibits some cytochrome P450s involved in the bioactivation of B[ a ]P. In the present study we observed that oltipraz had no protective effect on tumor burden in the forestomach of nrf2 -deficient mice when administered 1 h before B[ a ]P, a timeline that selectively optimizes for possible inhibitory effects on cytochrome P450s. To evaluate the role of nrf2 genotype on B[ a ]P disposition, levels of B[ a ]P–DNA adducts were measured as tetrols released from DNA isolated from target (forestomach) and non-target tissues (liver) of wild-type and nrf2 -deficient mice treated with either vehicle or oltipraz 1 or 48 h before B[ a ]P. Levels of B[ a ]P–DNA adducts in forestomach were significantly higher in nrf2 -deficient compared with wild-type mice. Oltipraz treatment at 1 or 48 h before B[ a ]P had no protective effect on forestomach tetrol levels in nrf2 -deficient mice, whereas a significant reduction was observed in wild-type mice treated with oltipraz 48 h, but not 1 h, before carcinogen. Combining all treatments and genotypes, there was a strong correlation ( R² = 0.91) between levels of B[ a ]P–DNA adducts in forestomach and subsequent yield of tumors. In contrast to the results in forestomach, nrf2 genotype did not modify hepatic B[ a ]P–DNA adduct levels while both oltipraz treatments were protective, suggesting that Nrf2-independent mechanisms (e.g. P450 inhibition) for oltipraz can also occur in vivo in some tissues.

The incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp²⁰⁰). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS.