The significance of blood and salivary oxidative stress markers and chemerin in gestational diabetes mellitus.

OBJECTIVE: Gestational diabetes mellitus (GDM) is a medical complication of pregnancy. The aim of this study was to evaluate the correlations between the salivary and blood levels of oxidative stress markers and an adipokine chemerin, which play a role in the pathogenesis of GDM. MATERIALS AND METHODS: Study groups (Control (n = 29), GDM (n = 22)) had been assessed clinically healthy oral hygiene, according to the age range between 25 and 40 years, BMI<30 kg/m2, who were non-smokers and who were not having systemic diseases. GDM was diagnosed using a 100 g OGTT. Saliva samples were collected without stimulation between 08.30 and 10.00 a.m.. Chemerin and TrxR levels were measured by ELISA. Malondialdehyde, sulfhydryl and NO levels were determined by spectrophotometric analysis. Statistical analysis were performed by Shapiro Wilk, Mann Whitney U, Student's t test. RESULTS: Blood pressure, BMI, and plasma chemerin, salivary chemerin, fasting glucose, LDL, triglyceride, CRP levels in GDM were not different when compared to Control. There were significant differences between Plasma TrxR and HDL levels. Also, significant differences between salivary TrxR and Malondialdehyde levels were observed in GDM. CONCLUSION: It was concluded that the optimal cut-off points for oxidative stress parameters and chemerin level can be used to distinguish between healthy pregnant and GDM.

A review of bioselenol-specific fluorescent probes: Synthesis, properties, and imaging applications.

Bioselenols are important substances for the maintenance of physiological balance and offer anticancer properties; however, their causal mechanisms and effectiveness have not been assessed. One way to explore their physiological functions is the in vivo detection of bioselenols at the molecular level, and one of the most efficient ways to do so is to use fluorescent probes. Various types of bioselenol-specific fluorescent probes have been synthesized and optimized using chemical simulations and by improving biothiol fluorescent probes. Here, we review recent advances in bioselenol-specific fluorescent probes for selenocysteine (Sec), thioredoxin reductase (TrxR), and hydrogen selenide (H2Se). In particular, the molecular design principles of different types of bioselenols, their corresponding sensing mechanisms, and imaging applications are summarized.

Inhibition of the Nrf2-TrxR Axis Sensitizes the Drug-Resistant Chronic Myelogenous Leukemia Cell Line K562/G01 to Imatinib Treatments.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

Carbon dots as analytical tools for sensing of thioredoxin reductase and screening of cancer cells.

Thioredoxin Reductase (TrxR) is a redox regulating enzyme which is predestined for the maintenance of redox homeostasis of mammalian cells. However, the elevated level of TrxR is associated with the progress of various types of tumors and therefore, this is a significant target for the detection of cancer cells. Herein, an easily engineered 'Turn ON' fluorescent sensor probe has been synthesized for the detection of TrxR and cell imaging using carbon dots. The emission intensity of fCDs on complexation with Cu2+ ions was drastically quenched. Subsequently, the addition of TrxR to the solution of the fCDs-Cu2+ complex leads to the cleavage of the disulfide bond of the fCDs, which acclaim the release of 3-mercaptopropionic acid. 3-Mercaptopropionic acid, being a strong bi-dentate chelating agent for Cu2+ ions, extracted Cu2+ from the coordination sphere of fCDs and restored the original fluorescence intensity of fCDs. Thus, the probe is operating with a simple process of "ON-OFF" emission switching due to Cu2+ and "OFF-ON" switching with TrxR. The probe has been successfully used for real-time application to monitor TrxR activities in the complex biological system. The fluorescence images of MCF-7 and HeLa cells after incubation with the fCDs-Cu2+ complex were recorded under a confocal laser scanning microscope (CLSM) as a function of time. Enhancement in the emission intensity of cancer cells after 2 h of treatment demonstrates the potential application of the sensor probe for the bioimaging of endogenous TrxR in living cells and screening of cancer cells. Such fluorescent probes will open the door for the development of promising clinical devices for the diagnosis of cancer cells.

Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR.

To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential ( EGSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and EGSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured in vitro (∼ -300 mV), the intracellular Trx exhibited the redox transition at EGSH between -250 and -200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of EGSH and the reduction by Trx reductase and other endogenous molecules.

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging.

The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD+/NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD+/NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.

Sodium selenate regulates the brain ionome in a transgenic mouse model of Alzheimer's disease.

Many studies have shown that imbalance of mineral metabolism may play an important role in Alzheimer's disease (AD) progression. It was recently reported that selenium could reverse memory deficits in AD mouse model. We carried out multi-time-point ionome analysis to investigate the interactions among 15 elements in the brain by using a triple-transgenic mouse model of AD with/without high-dose sodium selenate supplementation. Except selenium, the majority of significantly changed elements showed a reduced level after 6-month selenate supplementation, especially iron whose levels were completely reversed to normal state at almost all examined time points. We then built the elemental correlation network for each time point. Significant and specific elemental correlations and correlation changes were identified, implying a highly complex and dynamic crosstalk between selenium and other elements during long-term supplementation with selenate. Finally, we measured the activities of two important anti-oxidative selenoenzymes, glutathione peroxidase and thioredoxin reductase, and found that they were remarkably increased in the cerebrum of selenate-treated mice, suggesting that selenoenzyme-mediated protection against oxidative stress might also be involved in the therapeutic effect of selenate in AD. Overall, this study should contribute to our understanding of the mechanism related to the potential use of selenate in AD treatment.

Autophagy and redox status in carcinoma of an unknown primary.

AIM AND BACKGROUND: The purpose of the study was to investigate the role and clinical implications of autophagy and reactive oxygen species-related proteins in carcinoma of an unknown primary (CUP). METHODS AND STUDY DESIGN: Tissue microarray was constructed for a total of 77 CUP cases. Immunohistochemical stains conducted were as follows: autophagy-related beclin-1, LC3A, LC3B, and p62; redox-related catalase, thioredoxin reductase, glutathione S-transferase π, thioredoxin-interacting protein, and manganese superoxide dismutase. Immunohistochemical results were then related to their clinicopathologic parameters. RESULTS: The degree of LC3A expression showed a difference according to histologic subtype. In undifferentiated carcinoma, LC3A had the highest expression and adenocarcinoma had the lowest expression (P = 0.021). According to clinical subtype, there was a significant difference between LC3A and glutathione S-transferase π in expression. LC3A had the highest expression in single-organ types and the lowest in intermediate and carcinomatosis types (P = 0.003). Glutathione S-transferase π showed the highest expression in nodal-type tumors and the lowest in carcinomatosis types (P = 0.010). In univariate analysis, shorter overall survival was related to tumor glutathione S-transferase π negativity (P = 0.030). CONCLUSIONS: Different expression levels of the autophagy and reactive oxygen species-related proteins, LC3A and glutathione S-transferase π, were observed according to histologic and/or clinical subtype of carcinoma of an unknown primary.

A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates.

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in TR activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes; therefore, quantification of thioredoxin activity can be a valuable indicator of clinical conditions. Here we describe a new direct assay, termed the SC-TR assay, to determine the activity of TR based on the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as in older methods, the SC-TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC-TR assay can be used in a cuvette using traditional spectrophotometry or as a 96-well plate-based format using a plate reader. In addition, the SC-TR assay is compatible with the use of nonionic detergents, making it more versatile than other methods using cell lysates.

Organoselenium compounds modulate extracellular redox by induction of extracellular cysteine and cell surface thioredoxin reductase.

The effect of selenium compounds on extracellular redox modulating capacity was studied in murine macrophage RAW 264.7 cells and differentiated human THP-1 monocytes. The arylselenium compounds benzeneselenol (PhSeH), dibenzyl diselenide (DBDSe), diphenyl diselenide (DPDSe), and ebselen were capable of inducing extracellular cysteine accumulation via a cystine- and glucose-dependent process. Extracellular cysteine production was dose-dependently inhibited by glutamate, an inhibitor of cystine/glutamate antiporter (Xc(-) transporter), supporting the involvement of Xc(-) transporter for cystine uptake in the above process. These arylselenium compounds also induced cellular thioredoxin reductase (TrxR) expression, particularly at the exofacial surface of cells. TrxR1 knockdown using small interfering RNA attenuated TrxR increases and cysteine efflux induced in cells by DPDSe. Sodium selenite (Na2SeO3), selenomethionine (SeMet), seleno-l-cystine (SeCySS), and Se-methylselenocysteine (MeSeCys) did not have these effects on macrophages under the same treatment conditions. The effects of organoselenium compounds on extracellular redox may contribute to the known, but inadequately understood, biological effects of selenium compounds.

Nuclear expression of thioredoxin-1 in the invasion front is associated with outcome in patients with gallbladder carcinoma.

BACKGROUND: Multifunctional redox protein human thioredoxin (TRX-1) is reduced by thioredoxin reductase (TRX-R). The aim of the present study was to examine the distribution of TRX-1 and TRX-R expressions in gallbladder carcinoma (GBC) to clarify their usefulness as prognostic factors after surgical resection. METHODS: Immunohistochemical staining for TRX-1 and TRX-R was performed in GBC tissue from 38 patients who underwent surgical resection, and TRX-1/TRX-R localization in relation to outcome was examined. RESULTS: TRX-1 protein levels were significantly higher in GBC samples than in cholecystolithiasis samples (P = 0.0174). TRX-1 expression was observed in 100% (38/38) of tumour samples and in the nucleus in 76% (29/38), with nuclear expression in the invasion front observed in 45% (13/29). TRX-R expression was only detected in the cytoplasm of cancer cells and in the invasion front in 28 samples. In all of the samples, the depth of tumour invasion, lymph node metastasis, surgical margin, curability and nuclear expression of TRX-1 in the invasion front were significant prognostic factors by univariate analysis. In 27 selected patients who underwent curative resection, both TRX-1 nuclear expression and TRX-R cytoplasmic expression in the invasion front was a significantly prognostic factor. CONCLUSION: TRX-1 nuclear expression in the GBC invasion front is a significant prognostic marker. Patients with both TRX-1 nuclear expression and TRX-R cytoplasmic expression in the tumour invasion front should be observed carefully even if after curative resection.

Association of the time course of pulmonary arterial hypertension with changes in oxidative stress in the left ventricle.

1. This study investigates the time course of pulmonary arterial hypertension (PAH) due to monocrotaline (MCT) and its association with cardiac function and oxidative stress markers in the left ventricle (LV). 2. Male Wistar rats were divided into six groups: 7 days, 21 days, and 31 days for both control and MCT groups. Following echocardiographic analysis, the heart was removed. The LV was separated and homogenized to analyze oxidized-to-total glutathione ratio and thioredoxin reductase (TrxR) activity as well as hydrogen peroxide (H(2) O(2) ) and ascorbic acid levels. 3. There was significant (P < 0.01) cardiac and right ventricle (RV) hypertrophy and pulmonary congestion in the MCT 21 day and 31 day groups. Echocardiography showed a change in the flow wave of the pulmonary artery at 21 days after MCT treatment. There was an increase in the LV ejection time (P < 0.05) at 31 days after MCT. The LV H(2)O(2) concentration was increased (P < 0.05) in the MCT 21 day and MCT 31 day groups compared with controls. There was a reduction (P < 0.05) in the LV ascorbic acid concentration and an increase (P < 0.05) in TrxR activity in the MCT 31 day rats. 4. Our findings showed RV changes due to pulmonary hypertension at 21 days after MCT injection. There was a correlation between the degree of dysfunction and the morphometry of the heart chambers, along with impairment of the antioxidant/pro-oxidant balance in the LV 31 days after the beginning of the protocol. This study suggests that LV changes follow RV dysfunction subsequent to pulmonary hypertension.

Engineering of fluorescent reporters into redox domains to monitor electron transfers.

The rate of electron transfer through multicomponent redox systems is often monitored by following the absorbance change due to the oxidation of the upstream pyridine nucleotide electron donor (NADPH or NADH) that initiates the process. Such coupled assay systems are powerful, but because of problems regarding the rate-limiting step, they sometimes limit the kinetic information that can be obtained about individual components. For peroxiredoxins, such assays have led to widespread underestimates of their catalytic power. We show here how this problem can be addressed by a protein engineering strategy inspired by some bacterial and eukaryotic thioredoxins for which a significant fluorescence signal is generated during oxidation that provides a highly sensitive tool to directly measure electron transfers into and out of these domains. For the N-terminal domain of AhpF (a flavoprotein disulfide reductase) and Escherichia coli glutaredoxin 1, two cases not having such fluorescence signals, we have successfully added "sensor" tryptophan residues using the positions of tryptophan residues in thioredoxins as a guide. In another thioredoxin-fold redox protein, the bacterial peroxiredoxin AhpC, we used chemical modification to introduce a disulfide-bonded fluorophore. This modified AhpC still serves as an excellent substrate for the upstream AhpF electron donor but now generates a strong fluorescence signal during electron transfer. These tools have fundamentally changed our understanding of the catalytic power of peroxiredoxin systems and should also be widely applicable for improving quantitative assay capabilities in other electron transfer systems.

Alteration of thioredoxin reductase 1 levels in elucidating cancer etiology.

Thioredoxin reductase 1 (TR1) is a major antioxidant and redox regulator in mammalian cells and appears to function as a double-edged sword in that it has roles in preventing and promoting/sustaining cancer. TR1 is overexpressed in many cancer cells and targeting its removal often leads to a reversal in numerous malignant characteristics which has marked this selenoenzyme as a prime target for cancer therapy. Since alterations in TR1 activity may lead to a better understanding of the etiology of cancer and new avenues for providing better therapeutic procedures, we have described herein techniques for removing and reexpressing TR1 employing RNAi technology and for assessing the catalytic activity of this enzyme.

Transgenic mice overexpressing methionine sulfoxide reductase A: characterization of embryonic fibroblasts.

Methionine residues in protein can be oxidized by reactive oxygen species to generate methionine sulfoxide. Aerobic organisms have methionine sulfoxide reductases capable of reducing methionine sulfoxide back to methionine. Methionine sulfoxide reductase A acts on the S-epimer of methionine sulfoxide, and it is known that altering its cellular level by genetic ablation or overexpression has notable effects on resistance to oxidative stress and on life span in species from microorganisms to animals. In mammals, the enzyme is present in both the cytosol and the mitochondria, and this study was undertaken to assess the contribution of each subcellular compartment's reductase activity to resistance against oxidative stresses. Nontransgenic mouse embryonic fibroblasts lack methionine sulfoxide reductase A activity, providing a convenient cell type to determine the effects of expression of the enzyme in each compartment. We created transgenic mice with methionine sulfoxide reductase A targeted to the cytosol, mitochondria, or both and studied embryonic fibroblasts derived from each line. Unexpectedly, none of the transgenic cells gained resistance to a variety of oxidative stresses even though the expressed enzymes were catalytically active when assayed in vitro. Noting that activity in vivo requires thioredoxin and thioredoxin reductase, we determined the levels of these proteins in the fibroblasts and found that they were very low in both the nontransgenic and the transgenic cells. We conclude that overexpression of methionine sulfoxide reductase A did not confer resistance to oxidative stress because the cells lacked other proteins required to constitute a functional methionine sulfoxide reduction system.

Comparison of the protective effects of steamed and cooked broccolis on ischaemia-reperfusion-induced cardiac injury.

Recently, broccoli, a vegetable of the Brassica family, has been found to protect the myocardium from ischaemic reperfusion injury through the redox signalling of sulphoraphane, which is being formed from glucosinolate present in this vegetable. Since cooked broccoli loses most of its glucosinolate, we assumed that fresh broccoli could be a superior cardioprotective agent compared to cooked broccoli. To test this, two groups of rats were fed with fresh (steamed) broccoli or cooked broccoli for 30 d, while a third group was given vehicle only for the same period of time. After 30 d, all the rats were sacrificed, and the isolated working hearts were subjected to 30 min ischaemia followed by 2 h of reperfusion. Both cooked and steamed broccolis displayed significantly improved post-ischaemic ventricular function and reduced myocardial infarction and cardiomyocyte apoptosis compared to control, but steamed broccoli showed superior cardioprotective abilities compared with the cooked broccoli. Corroborating with these results, both cooked and steamed broccolis demonstrated significantly enhanced induction of the survival signalling proteins including Bcl2, Akt, extracellular signal-regulated kinase 1/2, haemoxygenase-1, NFE2 related factor 2, superoxide dismutase (SOD1) and SOD2 and down-regulation of the proteins (e.g. Bax, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase) of the death signalling pathway, steamed broccoli displaying superior results over its cooked counterpart. The expressions of proteins of the thioredoxin (Trx) superfamily including Trx1 and its precursor sulphoraphane, Trx2 and Trx reductase, were enhanced only in the steamed broccoli group. The results of the present study documented superior cardioprotective properties of the steamed broccoli over cooked broccoli because of the ability of fresh broccoli to perform redox signalling of Trx.

Oral administration of potassium dichromate inhibits brush border membrane enzymes and alters anti-oxidant status of rat intestine.

Potassium dichromate (K2Cr2O7) is a soluble hexavalent chromium compound that is widely used in several industries. In the present work the effect of administration of K2Cr2O7 on rat intestinal brush border membrane(BBM) enzymes and anti-oxidant system was studied. Rats were given a single oral dose of K2Cr2O7 (100 mg/kg bodyweight) and sacrificed 6, 12, 24, 48 and 96 h after the treatment.Control animals were not given K2Cr2O7. The administration of K2Cr2O7 resulted in a reversible decline in the specific activities of several BBM enzymes. The decrease in the activities of these enzymes was due to changes in the maximum velocity while their affinities for the substrates remained unchanged. Lipid peroxidation increased while total SH groups decreased in K2Cr2O7-treated rats as compared to controls indicating increased oxidative stress in the intestinal mucosa. The activities of superoxide dismutase and glutathione-S-transferase increased while those of catalase, glutathione reductase, thioredoxin reductase and glucose-6-phosphate dehydrogenase decreased. The maximum changes in all the parameters studied above were 24 h after administration of K2Cr2O7 after which recovery took place,in most cases almost to control values after 96 h. These results show that oral administration of K2Cr2O7 to decrease in the activities of BBM enzymes, increase in oxidative stress and alters the activities of anti-oxidant enzymes in rat intestine.

Aberrant expression of selenoproteins in the progression of colorectal cancer.

Since damage to DNA and other cellular molecules by reactive oxygen species ranks high as a major culprit in the onset and development of colorectal cancer, the aim of the present study is to clarify the role of antioxidant seleonoproteins including glutathione peroxidase (GPx), thioredoxin reductase (TXR) and selenoprotein P (SePP), and the effect of oxidative stress on the progression of colorectal cancer. Expression of 14 oxidative stress-related molecules in both tumorous and non-tumorous tissues in 41 patients was examined by immunohistochemistry and Western blot analysis. Expression levels of proteins modified by 4-hydroxy-2-nonenal (4-HNE), malonyldialdehyde (MDA) and 4-hydroxy-2-hexenal (4-HHE), and the positive rate of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in tumorous tissues were much higher than those in non-tumorous tissues. Glutathione (GSH) content in tumor tissues was much lower than that in non-tumorous tissues. Expression level of selenoproteins such as GPx-1, GPx-3, and SePP, which are rapidly degraded during selenium deprivation, was significantly decreased in tumorous tissues, whereas that of GPx-2, which is resistant to selenium deprivation, was increased. Expression of SePP was decreased at stage III and IV, compared to that of stage II. These data suggest that contrasting expression pattern of the antioxidant selenoproteins plays an important role in the progression of colorectal cancer.

Elemental selenium at nano size possesses lower toxicity without compromising the fundamental effect on selenoenzymes: comparison with selenomethionine in mice.

Glutathione peroxidase and thioredoxin reductase are major selenoenzymes through which selenium exerts powerful antioxidant effects. Selenium also elicits pro-oxidant effects at toxic levels. The antioxidant and pro-oxidant effects, or bioavailability and toxicity, of selenium depend on its chemical form. Selenomethionine is considered to be the most appropriate supplemental form due to its excellent bioavailability and lower toxicity compared to various selenium compounds. The present studies reveal that, compared with selenomethionine, elemental selenium at nano size (Nano-Se) possesses equal efficacy in increasing the activities of glutathione peroxidase and thioredoxin reductase but has much lower toxicity as indicated by median lethal dose, acute liver injury, and short-term toxicity. Our results suggest that Nano-Se can serve as an antioxidant with reduced risk of selenium toxicity.

Dual action of sulforaphane in the regulation of thioredoxin reductase and thioredoxin in human HepG2 and Caco-2 cells.

We have previously demonstrated that sulforaphane is a potent inducer for thioredoxin reductase in HepG2 and MCF-7 cells (Zhang et al. Carcinogenesis 2003, 24, 497-503; Wang et al. J. Agric. Food Chem. 2005, 53, 1417-1421). In this study, we have shown that sulforaphane is not only an inducer for thioredoxin reductase but also an inducer for its substrate, thioredoxin in HepG2, and undifferentiated Caco-2 cells. Sulforaphane acts at two levels in the regulation of thioredoxin reductase/thioredoxin system by the upregulation of the expression of both the enzyme and the substrate. In human hepatoma HepG2 cells, sulforaphane induced thioredoxin reductase mRNA and protein by 4- and 2-fold, respectively, whereas thioredoxin mRNA was induced 2.9-fold and thioredoxin protein was unchanged in whole cell extracts, but an increase in nuclear accumulation (1.8-fold) was observed. Moreover, the induction of thioredoxin reductase was found faster than that of thioredoxin. The effects of PI3K and MAPK kinase inhibitors, LY294002, PD98059, SP600125, and SB202190, have been investigated on the sulforaphane-induced expression of thioredoxin reductase and thioredoxin. PD98059 abrogates the sulforaphane-induced thioredoxin reductase at both mRNA and protein levels in HepG2 cells, although other inhibitors were found less effective. However, both PD98059 and LY294002 significantly decrease thioredoxin mRNA expression in HepG2 cells. None of the inhibitors tested were able to modulate the level of expression of either thioredoxin reductase mRNA or protein in Caco-2 cells suggesting that there are cell-specific responses to sulforaphane. In summary, the dietary isothiocyanate, sulforaphane, is important in the regulation of thioredoxin reductase/thioredoxin redox system in cells.