©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
1-Chloro-2,4-dinitrobenzene Is an Irreversible Inhibitor of Human Thioredoxin Reductase
LOSS OF THIOREDOXIN DISULFIDE REDUCTASE ACTIVITY IS ACCOMPANIED BY A LARGE INCREASE IN NADPH OXIDASE ACTIVITY (*)

(Received for publication, November 29, 1994)

Elias S. J. Arnér Mikael Björnstedt Arne Holmgren (§)

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human thioredoxin reductase is a dimeric enzyme that catalyzes reduction of the disulfide in oxidized thioredoxin by a mechanism involving transfer of electrons from NADPH via FAD to a redox-active disulfide. 1-Chloro-2,4-dinitrobenzene (DNCB) is an alkylating agent used for depleting intracellular GSH and also showing distinct immunomodulatory properties. We have discovered that low concentrations of DNCB completely inactivated human or bovine thioredoxin reductase, with a second order rate constant in excess of 200 M s, which is almost 10,000-fold faster than alkylation of GSH. Total inactivation of 50 nM reduced thioredoxin reductase was obtained by 100 µM DNCB after 5 min of incubation at 20 °C also in the presence of 1 mM GSH. The inhibition occurred with enzyme only in the presence of NADPH and persisted after removal of DNCB, suggesting alkylation of the active site nascent thiols as the mechanism of inactivation. Thioredoxin reductase modified by DNCB lacked reducing activity with oxidized thioredoxin, 5,5`-dithiobis-(2-nitrobenzoic acid), or sodium selenite. However, the DNCB-modified enzyme oxidized NADPH at a rate of 4.7 nmol/min/nmol of enzyme in the presence of atmospheric oxygen. This activity was not dependent on the presence of DNCB in solution and constituted a 34-fold increase of the inherent low NADPH oxidase activity of the native enzyme. DNCB is a specific inhibitor of mammalian thioredoxin reductase, which reacted 100-fold faster than glutathione reductase. The inactivation of the disulfide reducing activity of thioredoxin reductase and thioredoxin with a concomitant large increase of the NADPH oxidase activity producing reactive oxygen intermediates may mediate effects of DNCB on cells in vivo.


INTRODUCTION

The thioredoxin (Trx) (^1)system (Trx and TR with NADPH) serves as a hydrogen donor system for ribonucleotide reductase and has general protein disulfide reductase activity(1, 2, 3) . TR catalyzes the reduction of oxidized Trx (Trx-S(2)) by NADPH, and reduced Trx (Trx-(SH)(2)) is a powerful protein disulfide reductase ( and ).

TR from Escherichia coli is a dimeric enzyme with subunits of 35 kDa that has been extensively characterized(1) . The enzyme mechanism involves the transfer of reducing equivalents from NADPH to a redox active disulfide (CATC) in the active site, via a FAD prosthetic group. A crystal structure at 2-Å resolution has demonstrated that the active site disulfide is located in a buried position in the NADPH domain(4) . Results also strongly suggest that upon generation of a dithiol in TR, the enzyme undergoes a large conformational change to create a binding site for Trx-S(2) and reduction by thiol-disulfide exchange(4) . TR from mammalian cells has been purified to homogeneity and characterized from calf liver and thymus(5) , rat liver(6) , and human placenta(7) . All mammalian enzymes are dimeric but have a higher native molecular weight (M(r) 116,000) compared with the E. coli enzyme (M(r) 70,000). Unlike the E. coli TR, the mammalian enzymes have a broad substrate specificity reducing thioredoxins also from distant species as well as DTNB(5) , SeO(3)(8) , selenodiglutathione(9) , vitamin K(10) , and alloxan(11) , indicating a more open active site structure.

Recent reports show that extracellular Trx plays several roles in cell regulation. Thus, human Trx is identical to an adult T-cell leukemia-derived factor from human T cell lymphotropic virus I-infected cells, which up-regulates the receptor for interleukin-2(12) , and a factor promoting the growth of Epstein-Barr virus-immortalized B-lymphocytes, which synergizes with several other lymphokines(13) . Trx expression is induced in activated lymphocytes, and Trx is secreted in a hormone-like manner in connection with its immunostimulatory effects(12, 14, 15, 16) . Trx also up-regulates interleukin-6 in Epstein-Barr virus-infected B cells(15) , affects lymphocyte rosetting as part of an early pregnancy factor(16) , and regulates the activation of the transcription factor NF-kappaB intracellularly by redox control (17, 18, 19) . These effects of Trx are related to the oxidation state and the activity of TR.

DNCB is an electrophilic compound alkylating SH groups used as a substrate in assays to determine glutathione S-transferase, which is involved in elimination of DNCB in vivo(20) . DNCB has also been used in cell culture experiments as a GSH-depleting agent. Furthermore, DNCB has an established use as an immunomodulatory agent to provoke delayed-type hypersensitivity(21) . Although proposed to function as a hapten, the mechanism of DNCB immunomodulation is not clear(22) . Recently, DNCB was also proposed as an immunostimulatory agent in AIDS treatment(23) .

Based on the reports described above in combination with the immunomodulatory and the chemical nature of DNCB, we determined to investigate possible interactions with the human Trx system.


MATERIALS AND METHODS

DNCB was from Sigma, and stock solutions were made in 99.5% ethanol. TR was purified from human placenta and calf thymus as described(6) . The enzyme concentration was determined from A measurements or by determining the activity with 5 mM DTNB, assuming that 1200 A units/ml corresponded to 1 mg of protein/ml and an M(r) of 120,000 as described(6) . Human Trx was a recombinant preparation(24) . Glutathione reductase was prepared from rat liver(25) .

Enzyme Activity Measurements

All experiments were performed in 0.5 ml of 50 mM Tris-Cl, 1 mM EDTA, pH 7.5 at 20 °C using a Zeiss PM Q3 or a Hitachi spectrophotometer with semimicro-quartz cuvettes. Details of activity determination using insulin reduction, DTNB reduction, or selenite reduction have been given in previous papers(5, 8, 26) . Oxidation of NADPH was followed at 340 nm, and a molar extinction coefficient of 6200 M cm was used in calculations. The activity of glutathione reductase was determined by using 1 mM GSSG as described by Carlberg and Mannervik (25) .

Anaerobic Experiments

The method previously described was used, utilizing argon equilibration in special cuvettes covered by rubber septa(9) .

Isolation of TR Modified by DNCB

Two samples each contained 0.36 µM calf thymus TR and 200 µM NADPH in 0.50 ml of Tris-Cl, 1 mM EDTA, pH 7.5 containing 1 mg/ml bovine serum albumin. To one was added 10 µl of 5 mM DNCB in ethanol (100 µM) and to the other 10 µl of ethanol. After incubation for 20 min at 20 °C each sample was applied to a Sephadex G-25 column (NAP-5, Pharmacia Biotech Inc.), equilibrated with 50 mM Tris-Cl, 1 mM EDTA, pH 7.5, and the enzyme was collected in 1.00 ml of buffer. The modified TR free from DNCB and the control enzyme were used to determine NADPH oxidase activity.

Rate of Reaction between GSH and DNCB

This was measured spectrophotometrically using 1 mM GSH and varying concentrations of DNCB (1-0.01 mM) in 50 mM Tris-Cl, 1 mM EDTA, pH 7.5 at 20 °C. The adduct between GSH and DNCB has an absorbance at 340 nm with the extinction coefficient 9600 M cm(20) .


RESULTS

Inhibition of Human TR by DNCB

Initial experiments showed that preincubation of human Trx and human TR with NADPH followed by incubation with DNCB blocked the activity of the Trx system as a protein disulfide reductase using insulin as a substrate ( and ). As shown in Fig. 1, 50 µM DNCB completely inhibited the activity of 50 nM TR and 1 µM Trx after 15 min of preincubation at 20 °C. Since both TR and Trx will be in the reduced or dithiol form in the presence of NADPH either could be inhibited by DNCB. However, full disulfide reductase activity with insulin was obtained by addition of 50 nM fresh TR. This demonstrated that TR was the target of inhibition. The inhibition of calf thymus TR by DNCB was identical to that with the human enzyme showing that this was a property of mammalian thioredoxin reductases.


Figure 1: Effects of DNCB on the NADPH-dependent reduction of insulin disulfide by thioredoxin reductase and thioredoxin. Human TR (0.05 µM) was preincubated with 200 µM NADPH and 1 µM Trx at room temperature for 10 min, whereafter 50 µM DNCB was added and the preincubation was continued for 15 min. Then 50 µl of insulin (10 mg/ml) was added, and the absorbance at 340 nm was followed. After 6.5 min, fresh TR (0.05 µM) was added, as indicated.



Incubation of 25 nM TR with NADPH and 100 µM DNCB for 10 min at 20 °C also destroyed all the activity of TR (>98%) with either 5 mM DTNB or 20 µM selenite. These small molecules are reduced by TR (5, 6, 9) via the active site dithiol. Selenite (SeO(3)) was used in further experiments since it lacks thiol groups after reduction.

Preincubation of 50 nM TR with DNCB for 15 min in the absence of NADPH followed by addition of 200 µM NADPH and 20 µM SeO(3) resulted in no inhibition. This demonstrated that only the dithiol form of TR was susceptible to inactivation by DNCB.

The inhibition of TR activity by DNCB was dependent both on the concentration of the inhibitor and the preincubation time. Removal of DNCB from the enzyme using Sephadex G-25 chromatography showed that the enzyme had lost activity (Fig. 2) and strongly suggested alkylation of one or both of the active site thiols as the mechanism of inactivation.


Figure 2: Reduction of selenite by thioredoxin reductase after incubation with DNCB followed by removal of the inhibitor by gel filtration. Calf thymus TR (0.36 µM) was preincubated with either 500 µM DNCB (bullet) or with only ethanol as control () at 20 °C for 15 min in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mg/ml bovine serum albumin, and 250 µM NADPH. The enzyme in the preincubation mixtures was then isolated using Sephadex G-25 columns. Then 250 µM NADPH and 20 µM SeO(3) was added to the enzyme solutions, and NADPH consumption was followed by the decrease in absorbance at 340 nm, in each case against a blank with only NADPH.



Rate of Inactivation of TR by DNCB

Inhibition of 50 nM TR by DNCB was measured in the presence of 1 mM GSH. As shown in Fig. 3, 10 µM DNCB resulted in 65% inhibition and 100 µM DNCB in 100% inhibition after 5 min of incubation. Assuming a second order reaction a k(2) of 200 M s was obtained. Separate determinations of the rate of the reaction between GSH and DNCB under the same conditions (pH 7.5 and 20 °C) gave a value of 0.03 M s. This shows that the reactivity of TR with DNCB is almost 4 orders of magnitude faster than that of GSH. A comparison with rat liver glutathione reductase (0.015 µM enzyme in 50 mM Tris-Cl, 1 mM EDTA, pH 7.5 incubated with 200 µM NADPH at 20 °C) showed that a 100-fold higher concentration of DNCB (1 mM) without GSH had to be used to give 65% inhibition during 5 min, demonstrating a high preference for TR.


Figure 3: Inhibition of the activity of TR by varying concentrations of DNCB in the presence of 1 mM GSH. Human TR (0.05 µM) was incubated with 200 µM NADPH and different concentrations of DNCB in the presence of 1 mM GSH in both reference and sample cuvettes for 5 min at 20 °C. Then 20 µM SeO(3) was added to the sample cuvette, and TR activity was measured as the initial decrease in absorbance at 340 nm.



Induction of NADPH Oxidase Activity of TR by DNCB

When inactivation of TR by DNCB in the presence of NADPH was performed in a cuvette we observed continuous oxidation of NADPH at 340 nm. To analyze this, the experiment was repeated under anaerobic conditions, and no decrease in A was noted (Fig. 4). However, by admission of air NADPH consumption was initiated (Fig. 4). The rate of NADPH oxidation was 4.7 nmol/min/nmol of enzyme. To determine if the NADPH oxidase activity was a property of the inactivated enzyme or dependent on the presence of DNCB, the experiment described in Table 1was performed. This showed that the NADPH oxidase activity was a property of the inactivated enzyme and independent of DNCB in solution. The low NADPH oxidase activity of native TR was calculated to be increased about 34-fold after DNCB inactivation. In fact, the slight NADPH oxidation seen with SeO(3) (Fig. 2) for the DNCB-inactivated enzyme was independent of the addition of SeO(3).


Figure 4: NADPH oxidation activity of thioredoxin reductase under anaerobic and aerobic conditions after inactivation by DNCB. Human TR (0.3 µM), 200 µM NADPH, and 25 µM DNCB (substituted by ethanol in the reference cuvette) was incubated under anaerobic conditions for 60 min, and the absorbance was followed at 340 nm. Then air was admitted with mixing in both the reference and sample cuvettes, and the absorbance at 340 nm was recorded.






DISCUSSION

In this paper we have shown that DNCB is an efficient and specific irreversible inhibitor of TR from human placenta or calf thymus. Inhibition only occurred after reduction of the active site disulfide in the enzyme by NADPH, strongly suggesting alkylation of one or both nascent thiols in the enzyme. The inhibition was a fast process; compared with alkylation of GSH it was almost 10^4 times faster, and glutathione reductase was 100-fold less sensitive than TR. These results clearly suggest that a prime target for DNCB in vivo will be TR. The concentration range of DNCB in this study was about 100-fold lower than that used in clinical applications of the agent. Furthermore, since previous work has shown that TR is localized at the plasma membrane in many cells (27, 28, 29, 30) the enzyme can be envisioned to be easily accessible to the membrane-permeable DNCB upon topical application.

Inactivation of TR by DNCB completely inhibited the reduction of Trx but increased the NADPH oxidase activity, independent of free DNCB in solution. Thus, the disulfide reducing activity of Trx-(SH)(2) required for deoxyribonucleotide and DNA synthesis and its general disulfide reduction in the cell will be lost. In addition, Trx has several newly discovered roles in cell regulation of receptors or transcription factors by redox reactions that may be affected. The NADPH oxidase activity of the DNCB-inactivated enzyme will produce reactive oxygen intermediates, which either are causing oxidative stress or operate as mediators of growth (18) in cell signaling. Since the DNCB-inactivated enzyme showed the NADPH oxidase activity independent of the presence of DNCB in solution, the production of reactive oxygen intermediates will be effective and long lasting even after clearance of DNCB from the cell by GSH and glutathione S-transferase.

In cell culture experiments, DNCB is often used as a GSH-depleting agent in order to study processes dependent on intracellular GSH levels (31) . Obviously inactivation of TR with loss of the disulfide reducing activity of Trx is a previously unknown effect of DNCB. This may be of much greater importance than the effect on GSH levels, which obviously must be dependent on the highly variable presence of GSH S-transferase activity since we have shown that a direct reaction between GSH and DNCB is negligible. As an example the induction of leukotriene synthesis in lymphoblastoid B-cells by DNCB was not correlated to lowering of GSH(32) . A possible hypothesis is production of reactive oxygen intermediates by DNCB-modified TR since peroxides will activate lipoxygenases(33) . Further studies on the effects of DNCB on TR will hopefully help to explain the immunomodulatory effects of DNCB.


FOOTNOTES

*
This study was supported by Grant 961 from the Swedish Cancer Society, Project 13X-3529 from the Swedish Medical Research Council, the Inga-Britt and Arne Lundbergs Stiftelse, the Knut and Alice Wallenbergs Stiftelse, Stiftelsen Sigurd och Elsa Goljes minne, and the Karolinska Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-8-728 7686; Fax: 46-8-728 4716.

(^1)
The abbreviations used are: Trx, thioredoxin; TR, thioredoxin reductase; Trx-S(2), oxidized Trx; Trx-(SH)(2), reduced Trx; DNCB, 1-chloro-2,4-dinitrobenzene; DTNB, 5,5`-dithiobis-(2-nitrobenzoic acid).


ACKNOWLEDGEMENTS

We are grateful to Dr. Sushil Kumar and Lena Hernberg for preparations of human placenta and calf thymus thioredoxin reductase and Joakim Rindå and Jonas Nordberg for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.