PHASE II DETOXIFICATION ENZYMES

X. PHASE II DETOXIFICATION ENZYMES

A. NQO1 Induction by Isothiocyanates The ability of synthetic and plant-derived isothiocyanates to induce cystolic

quinone reductase NQO1[NADP(H):quinone reductase oxidoreductase], an FAD-containing flavoprotein, has been widely addressed. NQO1exists as a homodimmer, comprised of two homo-monomeric subunits of molecular weight 32 kDa with two identical catalytic sites providing the necessary pathway to detoxify quinones to hydroquinones using NADH and NADPH as reductants (reviewed in Ref. 129). The two-electron-mediated reduction by NQO1promotes the glucuronidation of hydroquinones that aid in their excretion (130). In addition, NQO1 also shows reductive activity toward quinone-imines and azo and nitrocompounds among others (reviewed in Ref. 131). With high localization within the liver of mammals NQO1 functions by competing with Phase I enzymes for quinone substrates. Indeed, the interac- tion of both NQO1and CYP450 isoenzymes can potentially reduce the amount of free radicals produced during normal metabolic processes. BITC, PEITC, and sulforaphane, but not their respective nitriles, and several cru- ciferous plant extracts containing ITCs have all been demonstrated to induce NQO1both in vitro and in vivo. Prochaska and Santamaria developed a rapid microtiter plate assay to assess the in vitro induction of NQO1in the murine hepa1c1c7 cell line (132). Of the hundreds of compounds tested, including ITCs, diphenols, and quinones, all appear to show several common features including their ability to react with sulfhydral groups and many are substrates for GSTs (reviewed in Refs. 133,134). Indeed, the in vitro induction of NQO1 has previously been used to identify the major Phase II enzyme inducers in broccoli and watercress (135,136). Furthermore, the assay has enabled the

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investigation of NQO1induction and structure function activities of a wide range of dietary constituent, including the potent N-methylsulfinylalkyl iso- thiocyanates (137,138). Results from in vivo assays have also demonstrated the induction of NQO1in several animal models. Kore et al. observed a significant increase in the NQO1and GST activities in intestinal tissues derived from Fischer F344 rats exposed to iberin, an ITC present in broccoli (139). Likewise, Zhang et al. demonstrated the potent induction of NQO1 and GST in rodents exposed to sulforaphane (140). However, as yet no in vivo research has shown that the induction of NQO1by dietary ITCs can alter the metabolism of, or indeed prevent, cellular damage by toxins, although current data are highly persuasive.

B. GST Induction by Isothiocyanates The second class of detoxification enzymes, the glutathione-S-transferases

(GSTs), and their functions in ITC metabolism have been of intense interest. GSTs function in detoxification of a wide range of electophilic species including aflatoxin-2,3-epoxide, tobacco carcinogens derived from benzo[a]- pyrene, and numerous pharmaceutical agents including acetaminophen. Distributed throughout the major organs and tissue such as the kidneys, lungs, intestine, skin, and the liver, GSTs catalyze the conjugation of glutathione (GSH), a tripeptide (g-glutamyl-cysteinyl-glycine) with electo- philic species, a process recognized as being the first step in the mercapturic acid pathway. Several ITCs have been demonstrated as efficient substrates for

human GSTs, such as GST M1 -1and P1 -1(1 41 ,1 42). However, more important from a chemopreventive aspect is the ability of ITCs to induce GSTs at the transcriptional and protein level. Rodents fed ITC-enriched diets often show elevated increases in GST activity in the liver, colon, pancreas, esophagus, and stomach. Furthermore, these increases are often associated with elevated levels of the cellular antioxidant GSH and several other pro- tective enzymes such as NQO1and g-glutamylcysteinesynthase (gGCS) (143,144).