title: Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells.
abstract: Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.
authors.affiliation: Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania. ncenas@bchi.lt
authors.forename: Narimantas
authors.initials: N
authors.lastname: Cenas
authors.forename: Stefanie
authors.initials: S
authors.lastname: Prast
authors.forename: Henrikas
authors.initials: H
authors.lastname: Nivinskas
authors.forename: Jonas
authors.initials: J
authors.lastname: Sarlauskas
authors.forename: Elias S J
authors.initials: ES
authors.lastname: Arnér
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001665:Binding Sites; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004136:Dinitrobenzenes / Q000737:chemistry / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D009285:Naphthoquinones / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D017672:Nitrogen Compounds / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004136:Dinitrobenzenes; D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D017672:Nitrogen Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D013439:Sulfhydryl Reagents; D013481:Superoxides; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; C005134:juglone