Mary Jane Long

Carcinogenesis is blocked by an extraordinary variety of agents belonging to many different classes--e.g., phenolic antioxidants, azo dyes, polycyclic aromatics, flavonoids, coumarins, cinnamates, indoles, isothiocyanates, 1,2-dithiol-3-thiones, and thiocarbamates. The only known common property of these anticarcinogens is their ability to elevate in animal cells the activities of enzymes that inactivate the reactive electrophilic forms of carcinogens. Structure-activity studies on the induction of quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] and glutathione S-transferases have revealed that many anti-carcinogenic enzyme inducers contain a distinctive and hitherto unrecognized chemical feature (or acquire this feature after metabolism) that regulates the synthesis of these protective enzymes. The inducers are Michael reaction acceptors characterized by olefinic (or acetylenic) bonds that are rendered electrophilic (positively charged) by conjugation with electron-withdrawing substrates. The potency of inducers parallels their efficiency in Michael reactions. Many inducers are also substrates for glutathione S-transferases, which is further evidence for their electrophilicity. These generalizations have not only provided mechanistic insight into the perplexing question of how such seemingly unrelated anticarcinogens induce chemoprotective enzymes, but also have led to the prediction of the structures of inducers with potential chemoprotective activity.

Induction of detoxification enzymes is a major mechanism whereby a wide variety of chemical agents protect rodents against neoplastic, mutagenic, and other toxicities of carcinogens. The enzyme NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) can protect against the toxicities of quinones and is a useful marker for protective enzyme induction. Quinone reductase can be induced in murine Hepa 1c1c7 hepatoma cells and 3T3 embryo fibroblasts by compounds that are chemoprotectors in vivo, including some phenolic antioxidants, azo dyes, aromatic diamines, and aminophenols. Structurally dissimilar catechols (1,2-diphenols) and hydroquinones (1,4-diphenols) induce quinone reductase in these systems, but resorcinol (1,3-diphenol) and its substituted analogues are inactive. Furthermore, only aromatic 1,2- and 1,4-diamines and aminophenols are inducers, whereas the 1,3-diamines are completely inactive. These findings suggest that the functional capacity to form quinones or quinone-diimines, rather than the precise structure, is essential for inductive activity and that the generation of the signal for enzyme induction depends upon oxidation-reduction lability. The observations that some chemoprotective compounds (e.g., azo dyes, beta-naphthoflavone) induce both cytochromes P-450 and quinone reductase, whereas others (e.g., tert-butylhydroquinone) induce only quinone reductase, can be reconciled by the fact that inducers of the first type are metabolized by P-450 enzymes to form products that are functionally similar to compounds of the second type.

Exposure of murine hepatoma (Hepa 1c1c7) cells to a variety of chemical agents known to protect animals against the neoplastic, mutagenic, and other toxic effects of chemical carcinogens results in dose- and time-dependent inductions of NAD(P)H:quinone reductase (EC 1.6.99.2). This enzyme protects against quinone toxicity by promoting obligatory two-electron reductions that divert quinones from oxidative cycling or direct interactions with critical nucleophiles. Quinone reductase levels are stable in culture, are easily measured, and are useful markers for the inductive effects of chemoprotective agents. The Hepa 1c1c7 system responds to chemoprotective compounds such as phenolic antioxidants (e.g., BHA [3(2)-tert-butyl-4-hydroxyanisole], BHT (3,5-ditert-butyl-4-hydroxytoluene), and tert-butylhydroquinone), lipophilic azo dyes belonging to the 1,1'-azonaphthalene, Sudan I (1-phenylazo-2-naphthol), and Sudan III [1-(4-phenylazophenylazo)-2-naphthol] families, polycyclic aromatic hydrocarbons, coumarin and various other lactones, flavonoids, and certain sulfur compounds (e.g., benzylisothiocyanate, dithiolthiones, and dithiocarbamates), all of which are recognized enzyme inducers and chemoprotectors in vivo. Quinone reductase induction in Hepa 1c1c7 cells therefore provides a simple, versatile, and reliable system for the evaluation of the potency, kinetics, and mechanism of action of anticarcinogens.

NAD(P)H:Quinone oxidoreductase (QR) is a widely-distributed enzyme that promotes obligatory two-electron reductions of quinones and thereby protects cells against the cytotoxicity of quinones and their metabolic precursors. QR is induced by a wide variety of chemoprotectors in many animal tissues as well as in the Hepa 1c1c7 murine hepatoma cell line. Such inducers fall into two families: dual inducers (e.g. polycyclic aromatics, azo dyes, beta-naphthoflavone) that elevate QR as well as cytochrome P1-450, and selective inducers of QR (e.g. tert-butylhydroquinone and other redox-labile diphenols). Induction by the first family of inducers depends on binding to the Ah (Aryl hydrocarbon) receptor and the associated expression of a functional cytochrome P1-450 enzyme, whereas the induction by redox-labile diphenols does not appear to be receptor-mediated. In order to analyze the possible role of the cytochrome P1-450 system in the induction of QR, we examined this process in the Hepa 1c1c7 cells and in four mutants of this cell line that are defective in the induction or expression of functional cytochrome P1-450. tert-Butylhydroquinone was an effective inducer of QR in all of the cell lines, and this process does not, therefore, depend on a functional cytochrome P1-450 enzyme. In contrast, azo dyes and polycyclic aromatics induce QR in the parent cell line but not in the various types of cytochrome P1-450-defective mutants. We conclude that the Ah receptor and cytochrome P1-450 function are involved in the induction of QR by certain azo dyes and polycyclic aromatics, but not by phenolic antioxidants.