Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular ''sensor'' molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho-(but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the ␤-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies. Mammalian cells have developed remarkably efficient protective mechanisms against both acute and chronic toxicities of electrophiles and reactive oxygen species that are the major causes of malignancy. The two primary lines of defense are (i) a family of phase 2 enzymes that detoxify electrophiles and serve as indirect antioxidants; and (ii) glutathione (GSH), the most abundant cellular nonprotein thiol. Phase 2 enzyme activities and GSH levels do not normally operate at their maximal capacity, but they can be transcriptionally induced by a wide variety of natural and synthetic chemical agents, thereby achieving efficient protection against carcinogenesis (1-3). The family of inducible phase 2 proteins is enormously diverse, and each member plays a distinct role in cellular protection. In addition to inducing the ''classical'' phase 2 drug-metabolizing enzymes, such as glutathione S-transferases (GST) and UDP-glucuronosyltransferase (4, 5) that conjugate xenobiotics with endogenous ligands, this group of inducible proteins now includes NAD(P)H:quinone reductase (NQO1) (6), epoxide hydrolase (7), heme oxygenase 1 (8, 9), ferritin (9), ␥-glutamylcysteine synthetase (10-12), aflatoxin aldehyde reductase (13, 14), catalase and superoxide dismutase (12), dihydrodiol dehydrogenase (15), leukotriene B 4 dehydrogenase (16), and glutathione Sconjugate efflux pumps (see ref. 17 for a review).The genes for many of these proteins contain 5Ј-upstream antioxidant (electrophile) responsive elements (ARE͞EpRE; consensus sequence TGACNNNGC), which regulate both their basal and inducible expression. The identities of the transcription factors that interact with the ARE...

The implications of oxidative stress in the pathogenesis of many chronic human diseases has led to the widely accepted view that low molecular weight antioxidants could be beneficial and postpone or even prevent these diseases. Small molecules of either plant or synthetic origins, which contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron-withdrawing groups) protect against the toxicity of oxidants and electrophiles indirectly, i.e., by inducing phase 2 cytoprotective enzymes. Some of these molecules, e.g., flavonoid and curcuminoid analogues that have phenolic hydroxyl groups in addition to Michael acceptor centers, are also potent direct antioxidants, and may therefore be appropriately designated: bifunctional antioxidants. By use of spectroscopic methods we identified phenolic chalcone and bis(benzylidene)acetone analogues containing one or two Michael acceptor groups, respectively, as very efficient scavengers of two different types of radicals: (a) the nitrogen-centered 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS.+) radical cation, and (b) the oxygen-centered galvinoxyl (phenoxyl) radical. The most potent scavengers are those also bearing hydroxyl substituents on the aromatic ring(s) at the ortho-position(s). The initial reaction velocities are very rapid and concentration-dependent. In the human keratinocyte cell line HaCaT, the same compounds coordinately increase the intracellular levels of glutathione, glutathione reductase, and thioredoxin reductase. Thus, such bifunctional antioxidants could exert synergistic protective effects against oxidants and electrophiles which represent the principal biological hazards by: (i) scavenging hazardous oxidants directly and immediately; and (ii) inducing the phase 2 response to prevent and resolve the consequences of hazardous processes that are already in progress, i.e., acting indirectly, but with much more diverse and long-lasting effects.

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