DT diaphorase

The requirement for reduction prior to DNA alkylation and crosslinking was first demonstrated by Iyer and Szybalski in 1964 [29], and can be induced both by chemical reducing agents such as sodium dithionite and thiols in vitro and by various reductive enzymes such as DT-diaphorase (NAD(P)H-quinone oxidoreduc-tase) in vitro and in vivo [47]. Much work to characterize the mechanism of reductive activation and alkylation has been carried out, principally by the Tomasz and Kohn groups, and Figure 11.1 illustrates a generally accepted pathway for mitomycin C [16, 48-50] based on these experiments, which is very similar to the mechanism originally proposed by Iyer and Szybalski [29]. 

The third study has employed 4,6-dinitrobenzofuroxan and as metabolic systems the one-electron reductants NADPHxytochrome P450 reductase and ferredoxin NADP(+) reductase and the two-electron reductants DT-diaphorase and Enterobacter cloacae nitroreductase [239]. The compound is activated either by DT-diaphorase or nitroreductase. 

The continuing interest in bioreductive alkylation is largely due to the clinical success of mitomycin C and the low reduction potentials observed in many tumors.9 The low reduction potentials favor the quinone to hydroquinone conversion necessary for bioreductive alkylation. Hypoxia due to low blood flow3 and/or the unusually high expression of the quinone two-electron reducing enzyme DT-diaphorase in some histological cancer types10-14 contribute to the tumor s tendency to reduce quinones. 

Selective bioreductive alkylation in high DT-diaphorase cancer types (melanoma renal and nonsmall-cell lung cancers)15 would exhibit maximal antitumor activity with minimal side effects. 

Belcourt, M.F. Hodnick, W. F. Rockwell, S. Sartorelli, A. C. Bioactivation of mitomycin antibiotics by aerobic and hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase. Biochem. Pharm. 1996, 51, 1669-1678. 

Gutierrez, P. L. The role of NAD(P)H oxidoreductase (DT-diaphorase) in the bioactivation of quinone-containing antitumor agents a review. Free Radio. Biol. Med. 2000, 29, 263-275. 

Rauth, A. M. Goldberg, Z. Misra, V. DT-diaphorase possible roles in cancer chemotherapy and carcinogenesis. Oncol. Res. 1997, 9, 339-349. 

Workman, P. Enzyme-directed bioreductive drug development revisted a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase. Oncol. Res. 1994, 6, 461 175. 

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

Suresh Kumar G, Lipman R, Cummings J, et al. Mitomycin C-DNA adducts generated by DT-diaphorase. Revised mechanism of the enzymatic reductive activation of mitomycin C. Biochemistry 1997 36(46) 14128—14136. 

Conversion of haloperidol to reduced haloperidol (a secondary alcohol) Glutathine dependent reduction of disulfiram to deithyldithiocarbamate Thioredoxin dependent of sulindac to sulindac sulfide DT diaphorase reduction of menadione to hydroquinone Conversion of pentabromoethane to tetra bromoethane (releasing free bromide ion)... 

Buffinton, G.D., Olhnger, K., Brunmark, A., and Cadenas, E., 1989, DT-diaphorase catalyzed reduction of 1,4-naphthoquinone derivatives and glutathione-conjugates. Biochem. J. 257 561-571... 

Riley, R.J. and Workman, P., 1992, DT-diaphorase and cancer chemotherapy. Biochem. Pharmacol. 43 1657-1669... 

Stoka, V., Turk, B., Schendd, S.L., Kim, T.-H., Cirman, T, Srripas, SJ., EUerby, L.M., Bredesen, L., Freeze, H, Abrahamson, M., Bromme, D., Krajewski, S., Reed, J.C., Yin, X.-M., Turk, V., and Salvesen G.S., 2001, Lysosomal Protease Pathways to Apoptosis. Cleavage of Bid, not pro-caspases, is the most likely route. J. Biol. Chem. 276 3149-3157 Srm, X. and Ross, D., 1996, Quinone-induced apoptosis in human colon adenocarcinoma ceUs via DT-diaphorase mediated bioactivation. Chem. Biol. Interact. 100 267-76 Taatjes, D.J. and Koch, T.H., 2001, Nuclear targeting and retention of anthracycUne antitumor dmgs in sensitive and resistant tumor ceUs. Curr. Med Chem. 8 15-29 Tarr, M. and van Helden, PT)., 1990, Inhibition of transcription by adriamycin is a... 

Rat brain DT-diaphorase enzyme hypothesized to be cytochrome P450 reductase (Kemp... 

Eight human brain tumors contained DT-diaphorase, NADH cytochrome b5 reductase and NADPH cytochrome P450 reductase as assessed by enzyme activity and WB (Rampling et al., 1994). 

This enzyme [EC 1.6.99.2] (also known as NAD(P)H quinone reductase, DT diaphorase, quinone reductase, azoreductase, phylloquinone reductase, and menadione reductase) catalyzes the reaction of NAD(P)H with an acceptor to produce NAD(P) and the reduced acceptor. This FAD-dependent enzyme is inhibited by dicoumarol. 

Fig. 2. Mechanisms causing resistance to antitumor treatment. ATM. ataxia telangiectasia gene, (Westphal et al., 1998 Xu and Baltimore, 1996), bcl-2/bax (Farrow and Brown, 1996, Zunino et al., 1997 Haq and Zanke, 1998), bcr/abl (McGahon et al., 1994), BCRP, breast cancer resistance protein (Doyle et d., 1998 Ross et al, 1999) bleomycin hydrolase (El-Deiry, 1997), BRCAl (Husain et al., 1998 Chen et al., 1998), BRCA2 (Chen et al., 1998 Chen et 1999), c-abl (White and Prives, 1999), c-jun (Sanchez-Perez and Perona, 1999), cytidine deaminase (El-Deiry, 1997), DNA poip, DNA polymerase p (Ochs et al., 1999), dihydrofolate reductase (Schimke, 1986), DT-diaphorase (Riley and Workman, 1992 Fitzsimmons et al., 1996 El-Deiry, 1997), EGR-1 (Ahmed et al., 1996), fos (Niimi et al., 1991), glucosylceramide synthase...

Reduction of the azo dye piontosil to produce the antibacterial drug sulfanilamide (Fig. 4.38) is a well-known example of azo reduction. This reaction is catalyzed by cytochromes P-450 and is also carried out by the reductases in the gut bacteria. The reduction of azo groups in food coloring dyes such as amaranth is catalyzed by several enzymes, including cytochromes P-450, NADPH cytochrome P-450 reductase, and DT-diaphorase, a cytosolic enzyme. 

An alternative route of reduction is catalyzed by the enzyme DT diaphorase. This is a two-electron reduction, which produces a hydro quinone. These tend to be less toxic than semiquinones, as they do not tend to undergo redox cycling. 

The one-electron reduction of a chemical to cytotoxic products is the basis of certain anticancer drugs. This is because tumor cells tend to be anaerobic, and so reduction is favored. However, anaerobic conditions can induce DT diaphorase, which carries out two-electron reduction reactions. For example, the anticancer drug tirapazamine is activated by a one-electron reduction catalyzed by NADPH cytochrome P-450 reductase in anaerobic conditions to a nitroxide radical, which is toxic to tumor cells. It is detoxified however, by DT diaphorase, to another product. 

Major reduction reactions are azo reduction and nitro reduction. The enzymes (reductases) are found in gut flora but also mammalian tissues. Reduction catalyzed by cytochrome P-450 can occur (e.g., dehalogenation). DT diaphorase carries out two-electron reductions. 

For electrophiles, the general mechanism of detoxication is conjugation with GSH. Quinones and hydroquinones are often reactive intermediates, and the enzyme DT diaphorase will carry out a two-electron reduction of these. 

DT diaphorase Dicoumarol-sensitive NADH/NADPH dye reductase, ductule small duct, dyspnea labored breathing. 

CDDs induce both phase I and phase II drug-metabolizing enzymes including AHH, EROD, ACOH, glucuronosyl transferase, glutathione S-transferase, and DT-diaphorase (Van den Berg et al. 1994). 

Belinsky M, Jaiswal AK. NAD(P)H quinone oxidoreductasel (DT-diaphorase) expression in normal and tumor tissues. Cancer Metastasis Rev 1993 12 103-117. 

Smitskamp-Wilms E, Giaccone G, Pinedo HM, van der Laan BF, Peters GJ. DT-diaphorase activity in normal and neoplastic human tissues an indicator for sensitivity to bioreductive agents . Br J Cancer 1995 72 917-921. 

Marin A, Lopez de Cerain A, Hamilton E, Lewis AD, Martinez-Penuela JM, Idoate MA, Bello J. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours. Br J Cancer 1997 76 923-929. 

Sharp SY, Kelland LR, Valenti MR, Brunton LA, Hobbs S, Workman P. Establishment of an isogenic human colon tumor model for NQOl gene expression application to investigate the role of DT-diaphorase in bio-reductive drug activation in vitro and in vivo. Mol Pharmacol 2000 58 1146-1155. 

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