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Control of cellular redox status and upregulation of quinone reductase NQO1 via Nrf2 activation by alpha-lipoic acid in human leukemia HL-60 cells
Abstract and Figures
alpha-Lipoic acid (LA) is a naturally-occurring micronutrient that has been actively investigated for the treatment and management of various chronic medical conditions including neurodegenerative diseases, diabetes and hepatic disorders. However, relatively few studies have examined the effects of LA as a chemopreventive agent, particularly in regard to its ability to modulate homeostasis of oxidoreductive state and to regulate detoxification enzymes such as quinone reductase NQO1 in LA-responsive cells. We tested the hypothesis that LA affects the intracellular redox status and induces NQO1 expression using the human promyelocytic HL-60 leukemia cells. We showed that treatment by LA maintains HL-60 cells in a relatively reduced state, supported by the dose/time-dependent increase in the activities of glutathione peroxidase and glutathione reductase and decrease in the activity of catalase. Moreover, LA significantly increased the activity and protein expression of NQO1. The induction of NQO1 was accompanied by the nuclear accumulation of transcription factor Nrf2, which was correlated with a decreased level of Nrf2 in the cytosol as well as the concomitant reduction in the expression of cytoplasmic repressor of Nrf2, Keap1.
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... Subsequently, cell suspensions were subjected to centrifugation at 13,000×g for 10 min at 4 °C. The resulting supernatant fractions were collected for the measurement of enzyme activities shown below [40]. ...
... NQO1 activity was measured by the modified method of Lind et al. [41]. Briefly, the reaction was started with the addition of menadione into the reaction mixture containing 33 mM potassium phosphate buffer (pH 7.4), 0.18 mM NADPH, 0.02 % BSA, 0.01 % Tween-20 and cell lysates [40]. The oxidation of NADPH was monitored spectrophotometrically at 340 nm, 25 °C for 2 min with and without 20 μM dicoumarol. ...
Background
The NAD(P)H: quinone oxidoreductase (NQO1) confers protection against semiquinones and also elicits oxidative stress. The C609T polymorphism of the NQO1 gene, designated NQO1*2, significantly reduces its enzymatic activity due to rapid degradation of protein. Since down regulation of NQO1 mRNA expression correlates with increased susceptibility for developing different types of cancers, we investigated the link between leukemia and the NQO1*2 genotype by mining a web-based microarray dataset, ONCOMINE. Phytochemicals prevent DNA damage through activation of phase II detoxification enzymes including NQO1. Whether NQO1 expression/activity in leukemia cells that carry the labile NQO1*2 genotype can be induced by broccoli-derived phytochemical sulforaphane (SFN) is currently unknown. Methods and ResultsThe ONCOMINE query showed that: (1) acute lymphoblastic leukemia and chronic myelogenous leukemia are associated with reduced NQO1 levels, and (2) under-expressed NQO1 was found in human HL-60 leukemia cell line containing the heterozygous NQO1*2 polymorphism. We examined induction of NQO1 activity/expression by SFN in HL-60 cells. A dose-dependent increase in NQO1 level/activity is accompanied by upregulation of the transcription factor, Nrf2, following 1–10 μM SFN treatment. Treatment with 25 µM SFN drastically reduced NQO1 levels, inhibited cell proliferation, caused sub-G1 cell arrest, and induced apoptosis, and a decrease in the levels of the transcription factor, nuclear factor-κB (NFκB). Conclusions
Up to 10 μM of SFN increases NQO1 expression and suppresses HL-60 cell proliferation whereas ≥ 25 μM of SFN induces apoptosis in HL-60 cells. Further, SFN treatment restores NQO1 activity/levels in HL-60 cells expressing the NQO1*2 genotype.
... Thus, we took a systems approach to administer participants with a supplement (NT) targeting multiple enzymes involved in NAD+ metabolism whilst also providing NAD + precursor (Fig. 1b). Table 1 lists the components of NT and specifies the basis (intended action) for including them in the supplement [38][39][40][41][42][43][44][45][46][47][48][49][50] . ...
Reversal or mitigation against an age-related decline in NAD ⁺ has likely benefits, and this premise has driven academic and commercial endeavour to develop dietary supplements that achieve this outcome. We used a systems-based approach to improve on current supplements by targeting multiple points in the NAD ⁺ salvage pathway. In a double-blind, randomised, crossover trial, the supplement – Nuchido TIME+® (NT) - increased NAD ⁺ concentration in whole blood. This was associated with an increase in SIRT1 and an increase in nicotinamide phosphoribosyltransferase (NAMPT) in peripheral blood mononucleocytes, lower concentrations of pro-inflammatory cytokines in plasma, including a reduction in interleukin 2 (IL2), a reduction in glycated serum protein and a shift in the glycosylation profile of immunoglobulin G (IgG) toward a younger biological age, all of which are likely to promote a healthier ageing trajectory.
... Compound 36 could significantly upregulate the activity and protein expression level of NQO1, which is accompanied by the Nrf2 nuclear accumulation in the human promyelocytic HL-60 leukemia cells. 183 Natural isothiocyanates are abundant in cruciferous vegetables. 184 For instance, as shown in the structure of sulforaphane (SFN, 37) in Figure 5C, THE central carbon atom of the isothiocyanate group is highly electrophilic, and it can react with sulfhydryl group in Keap1 to form a dithiocarbamate easily. ...
Elevated intracellular reactive oxygen species (ROS) and antioxidant defense systems have been recognized as one of the hallmarks of cancer cells. Compared with normal cells, cancer cells exhibit increased ROS to maintain their malignant phenotypes and are more dependent on the "redox adaptation" mechanism. Thus, there are two apparently contradictory but virtually complementary therapeutic strategies for the regulation of ROS to prevent or treat cancer. The first strategy, that is, chemoprevention, is to prevent or reduce intracellular ROS either by suppressing ROS production pathways or by employing antioxidants to enhance ROS clearance, which protects normal cells from malignant transformation and inhibits the early stage of tumorigenesis. The second strategy is the ROS-mediated anticancer therapy, which stimulates intracellular ROS to a toxicity threshold to activate ROS-induced cell death pathways. Therefore, targeting the regulation of intracellular ROS-related pathways by small-molecule candidates is considered to be a promising treatment for tumors. We herein first briefly introduce the source and regulation of ROS, and then focus on small molecules that regulate ROS-related pathways and show efficacy in cancer therapy from the perspective of pharmacophores. Finally, we discuss several challenges in developing cancer therapeutic agents based on ROS regulation and propose the direction of future development.
... To expand the application scope of the NAPA therapy to NQO1-low tumors, we explored the strategy of combined regimens to increase NQO1 expression that could facilitate NAPA cytotoxicity. NQO1 expression is mainly regulated by the transcription factor NRF2 [39][40][41][42]. We asked whether manipulation of NRF2 activation can lead to elevated NAPA efficacy. ...
Napabucasin (NAPA) is thought to be a potent cancer stemness inhibitor in different types of cancer cell lines. While it has shown promising activity in early phase clinical trials, two recent phase III NAPA clinical trials failed to meet the primary endpoint of overall survival. The reason for the failure is not clear, but a possible way to revive the clinical trial is to stratify patients with biomarkers that could predict NAPA response. Here, we report the identification of NAD(P)H dehydrogenase 1 (NQO1) as a major determinant of NAPA efficacy. A proteomic profiling of cancer cell lines revealed that NQO1 abundance is negatively correlated with IC50; in vitro assays showed that NAPA is a substrate for NQO1, which mediates the generation of ROS that leads to cell death. Furthermore, activation of an NQO1 transcription factor NRF2 by chemicals, including an FDA approved drug, can increase the NAPA cytotoxicity. Our findings suggest a potential use of NQO1 expression as a companion diagnostic test to identify patients in future NAPA trials and a combination strategy to expand the application of NAPA-based regimens for cancer therapy.
... Irrespective of their genetic characteristics, leukemia cells with high growth fractions generate an excess of ROS. In turn, leukemia cells have prominent antioxidant machinery to mitigate the effects of excess ROS and maintain cellular oxidative states compatible with cell survival [25,26]. Thus, mechanisms maintaining the oxidative state in leukemia cells may be exploitable targets to selectively eradicate neoplastic cells [7], as compared to normal cells. ...
Selective targeting of the oxidative state, which is a tightly balanced fundamental cellular property, is an attractive strategy for developing novel anti-leukemic chemotherapeutics with potential applications in the treatment of acute myeloid leukemia (AML), a molecularly heterogeneous disease. Dimeric naphthoquinones (BiQs) with the ability to undergo redox cycling and to generate reactive oxygen species (ROS) in cancer cells are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against AML cells. We evaluated the effect of two BiQ analogues and one monomeric naphthoquinone in AML cell lines and primary cells from patients. All compounds possess one halogen and one hydroxyl group on the quinone cores. Dimeric, but not monomeric, naphthoquinones demonstrated significant anti-AML activity in the cell lines and primary cells from patients with favorable therapeutic index compared to normal hematopoietic cells. BiQ-1 effectively inhibited clonogenicity and induced apoptosis as measured by Western blotting and Annexin V staining and mitochondrial membrane depolarization by flow cytometry. BiQ-1 significantly enhances intracellular ROS levels in AML cells and upregulates expression of key anti-oxidant protein, Nrf2. Notably, systemic exposure to BiQ-1 was well tolerated in mice. In conclusion, we propose that BiQ-induced therapeutic augmentation of ROS in AML cells with dysregulation of antioxidants kill leukemic cells while normal cells remain relatively intact. Further studies are warranted to better understand this class of potential chemotherapeutics.
... In the light of these findings, we verified that the Nrf2 pathway could be pharmacologically modulated in the kidneys of Nrf2 +/+ mice and was non-functional in Nrf2 − / − mice, before conducting transcriptomic and proteomic analyses, by determining the expression level of Nqo1 in kidney homogenates from both genotypes, 24 h after dosing with vehicle or CDDO-Me. 20 The level of Nqo1 protein (Figure 1a and b) and mRNA (Figure 1c) was significantly lower in the kidneys of vehicleexposed Nrf2 − / − mice, compared with those of Nrf2 +/+ counterparts, consistent with loss of Nrf2 function. Moreover, while the Nrf2 inducer CDDO-Me provoked the induction of Nqo1 24 h after administration to Nrf2 +/+ mice, this effect was abrogated in Nrf2 − / − mice (Figure 1a-c). ...
The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control. In the light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis, and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.Kidney International advance online publication, 30 September 2015; doi:10.1038/ki.2015.286.
Background: Visceral obesity and the metabolic syndrome that often accompanies it are robust and well-established risk factors for postmenopausal breast cancer. This increased risk is thought to be mediated, in large part, by increased aromatase expression in the breast. stromal adipocytes and in mammary epithelium; this results in a high local concentration of estrogen capable of supporting the cancerous transformation of estrogen receptor-positive (ER+) epithelial cells. Aromatase, a member of the cytochrome 450 family, is expressed in the endoplasmic reticulum. After menopause, it converts androgens produced within the adrenal cortex (androstenedione and testosterone) into estrogens (estrone and estradiol, respectively. Therefore, the main objective of this narrative article is to review dietary and nutraceutical remedies for the prevention of menopause. Breast cancer: leading strategies to suppress breast aromatase Methods: The research articles to carry out this work were focused based on many searches and reviews in the following databases: Google Scholar, MDPI, PubMed, ScienceDirect and using the following keywords and combined synonyms: (“nutraceuticals” or “supplements” or “aromatase inhibitors” or “aromatase” or "antioxidants”) AND (“breast cancer” or “vascular function” or “inflammation”). The keywords were also searched in the references of the original articles included in this study Conclusion: A number of nutraceutical, dietary, and lifestyle measures hold promise for breast cancer prevention and may be considered practical options by women seeking to minimize their risk. for this cancer, particularly as these measures appear to have few downsides and are likely to work in other ways to promote health.
Reversal or mitigation against an age-related decline in NAD ⁺ has likely benefits, and this premise has driven academic and commercial endeavour to develop dietary supplements that achieve this outcome. We used a novel systems-based approach to improve on current supplements by targeting multiple points in the NAD ⁺ salvage pathway. In a double-blind, randomised, crossover trial, the supplement – Nuchido TIME+ ® (NT) - increased NAD ⁺ concentration in whole blood. This was associated with an increase in SIRT1 and an increase in nicotinamide phosphoribosyltransferase (NAMPT) in peripheral blood mononucleocytes, lower concentrations of pro-inflammatory cytokines in plasma, including a reduction in interleukin 2 (IL2), a reduction in glycated serum protein and a shift in the glycosylation profile of immunoglobulin G (IgG) toward a younger biological age, all of which are likely to promote a healthier aging trajectory.
Nanotechnology has become a ubiquitous part of our everyday life. Besides the already-known nanoparticles (NPs), plenty of new nanomaterials are being synthesized every day. Here, we explain the mechanism of the zinc oxide nanoparticles (ZnONPs) cytotoxicity in a cellular model of acute lymphoblastic leukaemia (CCRF-CEM). To do so, we investigated both possible hypotheses about the ZnONPs mechanism of toxicity: a free zinc ions release and/or reactive oxygen species (ROS) generation. Presented here results show that:
A) EDTA protects the cells, whereas a common antioxidant agent (NAC) enhances the cytotoxic effect induced by ZnONPs,
B) ZnONPs release zinc ions in vitro,
C) ZnONPs upregulate the activity of antioxidative defence enzymes, which are Nrf2 transcription factor-dependent,
D) The upregulation of ROS generation is correlated with the upregulation of reductive stress markers,
E) ZnONPs treatment induces NADPH oxidases that cause excessive production of superoxide radicals in tested cellular model.
Our results support the hypothesis that the mechanism of ZnONPs cytotoxicity is based on the release of free zinc ions. Nevertheless, both previously quoted hypotheses incompletely described the mechanism of action of ZnONPs. In this paper, we show that the mechanism of cytotoxicity of ZnONPs is based on the induction of reductive stress in CCRF-CEM cells, which is caused by free zinc ions released from ZnONPs. Therefore, the increase of oxidative stress markers is most likely a secondary response of the cells towards the Zn2+. These results provide a crucial expansion of the zinc ion hypothesis and thus explain the biphasic cellular response of CCRF-CEM cells treated with ZnONPs
Nad(p)h:
Quinone Oxidoreductase 1 (NQO1) is an antioxidant enzyme that catalyzes the two-electron reduction of several different classes of quinone-like compounds (quinones, quinone imines, nitroaromatics, and azo dyes). One-electron reduction of quinone or quinone-like metabolites is considered to generate semiquinones to initiate redox cycling that is responsible for the generation of reactive oxygen species and oxidative stress and may contribute to the initiation of adverse drug reactions and adverse health effects. On the other hand, the two-electron reduction of quinoid compounds appears important for drug activation (bioreductive activation) via chemical rearrangement or autoxidation. Two-electron reduction decreases quinone levels and opportunities for the generation of reactive species that can deplete intracellular thiol pools. Also, studies have shown that induction or depletion (knockout) of NQO1 were associated with decreased or increased susceptibilities to oxidative stress, respectively. Moreover, another member of the quinone reductase family, NRH: Quinone Oxidoreductase 2 (NQO2), has a significant functional and structural similarity with NQO1. The activity of both antioxidant enzymes, NQO1 and NQO2, becomes critically important when other detoxification pathways are exhausted. Therefore, this article summarizes the interactions of NQO1 and NQO2 with different pharmacological agents, endogenous biochemicals, and environmental contaminants that would be useful in the development of therapeutic approaches to reduce the adverse drug reactions as well as protection against quinone-induced oxidative damage. Also, future directions and areas of further study for NQO1 and NQO2 are discussed.
α-lipoic acid (α-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal
poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, α-LA induces apoptosis
in different cancer cells types. However, whether α-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated
whether α-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that α-LA inhibits the
growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased
levels of the cyclin/CDKs inhibitors, p27Kip1 and p21Cip1. Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased
generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome
c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and
inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that α-LA induces
apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests
that it may prove useful in liver cancer therapy.
To determine glutathione peroxidase reliably, some factors of potential pitfall have to be considered, for example, enzymatic side reactions of substrates (especially when crude tissue samples are assayed), high and variable spontaneous reaction rates of substrates, and the peculiar kinetics of the enzyme itself. With the best documented example, the enzyme of bovine red blood cells, ping-pong kinetics with infinite limiting maximum velocities, and Michaelis constants have been established. This means that the generally recommended conditions for determination of enzyme activity––that is, “saturating” concentrations of all substrates, cannot possibly be fulfilled. In consequence, compromises are inevitable in the choice of substrate concentration for the assay and in the definition of the unit of activity. Fixed-time assay measuring H2O2 consumption and continuous monitoring of Glutathione disulfide (GSSG) formation are cited here. The main differences between the assay procedure described and those proposed by others are listed in the chapter. To compare the results obtained by different procedures, appropriate empirical converting factors are also given.
Many chemopreventive agents have been shown to modulate gene expression including induction of phase II detoxifying enzymes, such as glutathione S-transferases (GST) and quinone reductases (QR). Induction of phase II enzymes in general leads to protection of cells/tissues against exogenous and/or endogenous carcinogenic intermediates. The antioxidant or electrophile response element (ARE/EpRE) found at the 5′-flanking region of these phase II genes may play important role in mediating their induction by xenobiotics including chemopreventive agents. Members of the basic leucine zipper (bZIP) transcription factor, Nrf2 which heterodimerizes with Maf G/K, are found to bind to the ARE, and transcriptionally-activated ARE. Recently, we showed that the mitogen-activated protein kinases (MAPK) were activated by phase II gene inducers such as phenolic antioxidant butylated hydroxyanisol (BHA) and isothiocyanate sulforaphane (SUL), and involved in the transcription activation of ARE-mediated reporter gene. Transfection studies with wild-type and dominant negative mutants of Nrf2 and MAPK showed synergistic response during co-transfection as well as to phase II gene inducers. However, increasing the concentrations of these compounds such as BHA, the activities of cell death signaling molecules, caspases, were stimulated and resulted in apoptotic cell death. At these concentrations, BHA stimulated loss of mitochondrial membrane potential, cytochrome c release, and activation of caspase 3, 8 and 9 preceding apoptosis. Further increase in concentrations led to rapid cell necrosis. A model is proposed for BHA and SUL, in that at low concentrations, these potential chemopreventive agents may modulate MAPK pathway leading to transcription activation of Nrf2 and ARE with subsequent induction of cellular defensive enzymes including phase II detoxifying enzymes as well as other defensive genes, which may protect the cells against cellular injury, which is a homeostatic response. At higher concentrations, these agents may activate the caspase pathways, leading to apoptosis, a potential beneficial effect if occurs at preneoplastic/neoplastic tissues, but a potential cytotoxic response if occurs in normal tissues. On the other hand, some phenolic compounds such as resveratrol inhibits TPA- or UV-induced AP-1-mediated activity through the inhibition of c-Src non-receptor tyrosine kinase and MAPK pathways. It is possible that in proliferating or stimulated cells, these chemopreventive compounds may block proliferation by inhibiting these signaling kinases, whereas in non-proliferating or quiescent cells, some of these compounds may activate these signaling kinases leading to gene expression of cellular defensive enzymes such as phase II detoxifying enzymes. The studies of these and other signaling pathways may yield insights into the development of potential chemopreventive compounds.
The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides cptimal conditions for determinime the basic kinetic parameters of the enzyme.
The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates.
In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site.
The relation of the properties of glutathione reductase to cellular conditions is discussed.
DT-diaphorase is a highly active diaphorase in the soluble fraction of rat liver homogenates, which catalyzes the oxidation of nicotine adenine dinucleotide, reduced nicotine adenine dinucleotide hydrogenase (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH) at equal rates. This chapter discusses the purification, properties, and function of DT-diaphorase. Most of the present knowledge about DT-diaphorase has been obtained in studies of the cytosolic enzyme, which was first purified from both rat and beef liver 44 by employing conventional methods. The introduction of affinity chromatography for the purification of DTdiaphorase reduced the number of purification steps considerably. In the method presented in the chapter for the purification of DT-diaphorase, the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, which is a potent competitive inhibitor of the enzyme with respect to NAD(P)H. A gel with both a high affinity and a high binding capacity for DT-diaphorase is obtained by coupling dicoumarol through an azo linkage to divinyl sulfonate (DVS)-activated Sepharose 6B. In the assay, DT-diaphorase activity is measured routinely with NADH or NADPH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) or 2-methyl-l,4-naphthoquinone (menadione) as the electron acceptor. The standard assay system contains 50 mM Tris-HC1, pH 7.5, 0.08% Triton X-100, 0.5 mM NADH or NADPH, and 40 μM DCPIP or 10 μM menadione.
NF-kappa B transcription factor regulates a wide variety of cellular and viral genes including the human immunodeficiency virus type 1. Here, we demonstrate that dihydrolipoate/alpha-lipoate redox couple which is a cofactor for mitochondrial dehydrogenases reactions, influences the DNA binding activity of NF-kappa B. The elimination of dithiothreitol in the electrophoretic mobility shift assay protocol resulted in the inability to detect DNA binding activity of activated NF-kappa B. The DNA binding activity was restored by the addition of dihydrolipoate in the binding reaction mixture. Inhibition of NF-kappa B DNA binding activity by in vitro exposure to a sulfhydryl oxidizing agent, diamide was also blocked by dihydrolipoate. In contrast, the addition of the oxidized form, alpha-lipoate inhibited the NF-kappa B DNA binding activity. Coincidentally, preincubation of Jurkat cells with dihydrolipoate potentiated and alpha-lipoate inhibited the okadaic acid-induced NF-kappa B activation as detected by assessing its DNA binding activity. These results suggest the redox exchange between lipoate and NF-kappa B molecules. Furthermore, since the inhibition of AP-1 DNA binding activity by diamide was also blocked by dihydrolipoate, this natural reductant may participate in the redox regulation of transcription factors by enhancing the DNA-protein interactions.