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°C using 50 M NADPH. As a nitroreductase, TrxR cycled between four-and twoelectron-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-Nmethylnitramine, 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.

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

“…The Cys-59 thiolateand charge-transfer-containing active site is thereby instead available for other types of reactions, including one-(or two-) electron reduction of quinones such as juglone. This line of reasoning is supported by the known high capacity of native, Sec-derivatized as well as Sec-lacking truncated forms of TrxR in reduction of juglone directly through the N-terminal redox active motif (38,39,68). Hence, without resonance effects of Tyr-116 and therefore a lack of an efficient reductive half-reaction, the result would easily be the observed increased propensity for juglone reduction but diminished activity with Trx as substrate.…”

“…However, considering that juglone can be efficiently reduced by either the C-terminal selenolthiol motif or directly by the N-terminal FAD/dithiol motif of TrxR (38,39,68) and considering the plausible roles of Tyr-116 in the reductive and oxidative half-reactions, as described above, the effects of Tyr-116 mutations become explainable. Hence, we propose that the Tyr-116 substitutions diminish the possibility for the Cys-59 thiolate to attack Sec-498 of the selenenylsulfide, thereby mainly leaving the selenenylsulfide in the inactive oxidized state.…”

“…mutants of mouse TrxR2 (33)(34)(35)(36), human TrxR1 (37) (also as directly deposited PDB entry 2CFY), and the non-selenoprotein orthologue of Drosophila (34,35). These structures as well as additional modeling studies and several extensive biochemical analyses (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) have generated a generally accepted model for the major features of the catalytic mechanism of mammalian TrxRs. This model is summarized in Fig.…”

Modulation of Nitro-fatty Acid Signaling

“…In particular, OYE (EC 1.6.99.1) the archetypical member of the old yellow enzyme flavoprotein family, reacts with non-native, non-aromatic nitroalkenes such as nitrocyclohexene, nitrostyrene, and nitrovinylthiophene via NADPH-dependent mechanisms to generate the corresponding nitroalkane by the intermediate formation of a nitronate derivative (32). Finally, mammalian enzymes such as thioredoxin reductase, NAD(P)H:quinone oxidoreductases, NADPH: cytochrome P450 reductase, and ferredoxin:NADP ϩ reductase reduce nitroaromatic compounds either by one-or two-electron transfer to the nitro moiety, yielding the corresponding nitroso, hydroxylamine, or amine derivatives (57)(58)(59)(60)(61). The efficiency (k cat /K m ) of nitro reduction by these enzymes is typically lower than that reported herein for nitroalkene reduction by PtGR-1 (60,61), and none of these enzymes was identified in the nitroalkene reductase-purified fraction.…”

Role of cytochrome P450 reductase in nitrofurantoin-induced redox cycling and cytotoxicity

“…Several enzymes in mammalian cells that mediate one-electron reduction of nitroaromatic compounds to reactive electrophilic intermediates have been identified including cytochrome P450 reductase [14,26], NADH:lipoamide oxidoreductase [27], NAD(P)H:quinone oxidoreductase (NQO1) [28], thioredoxin reductase [29], nitric oxide synthase [30], xanthine oxidase [31], and ferredoxin: NADP + reductase [32]. Oxidation of reactive electrophilic intermediates back to the parent compound generates toxic ROI.…”