Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

Abstract

NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O₂^.− and H₂O₂. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O₂ derivatives, activated the efflux of radioiodide but not of its analog ^99mTcO₄⁻ nor of the K⁺ cation mimic ⁸⁶Rb⁺ in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

Highlights

• •Diphenyleneiodonium (DPI) inhibits H₂O₂ generation by thyroid DUOXes in the μM range.
• •DPI activates iodide efflux from thyroid, thyroid-derived and unrelated cells in the μM range.
• •DPI does not activate the efflux of the iodide analog^99mTcO₄⁻ or of ⁸⁶Rb⁺ in the same cells.
• •DPI thus behaves as a specific ionophore for iodide.

1. Introduction

Seven specific O₂^.− and H₂O₂ generating enzymes have been demonstrated, the NADPH oxidases, NOXes and dual oxidases DUOXes. In many cell types, they generate both intracellular and extracellular O₂^.− and H₂O₂. They share many properties, including enzymatic mechanisms, but differ in terms of function, tissue distribution and control [1–3].

Diphenyleneiodonium (DPI) inhibits the activity of these flavoenzymes. It is often considered as a specific inhibitor. Inhibition of any metabolic step by DPI is therefore often attributed to this effect [4–9]. Although caution has been advised against the use of inhibition by DPI as a specific biomarker of NOX and DUOX actions [4], such arguments are still commonly used [10].

In this article we demonstrate another action of DPI which raises new interesting questions. In the course of our study of iodide metabolism in thyroid cells we observed that it increased the release of ¹²⁵I iodide but not ^99mTcO₄⁻, thus behaving as a specific carrier of iodide.

This shows an effect of DPI other than the inhibition of H₂O₂ generation. It presents an interesting chemical property of this iodinated derivative and may suggest a therapeutic application.

2. Materials and methods

2.1. Reagents

¹²⁵I as NaI (∼17Ci(629GBq)/mg) and ⁸⁶Rb⁺ (19.3 mCi(714.1 MBq)/mg) were purchased from Perkin Elmer Life and Analytical Science (Zaventem, Belgium), ^99mTc as Na Pertechnetate (^99mTcO₄⁻) was provided to us by the Medical School Hospital. It was prepared daily, carrier free, from a ^99mTc generator (Ultra TechneKow, Mallinckrodt Nuclear Medicine). We used around 1 μCi tracer per dish.

Homovanillic acid (HVA), horse radish peroxidase type II (HRP), methylmercaptoimidazole (MMI) and diphenyleneiodonium chloride (DPI) were provided by Sigma–Aldrich BVBA (Diegem, Belgium). Sodium iodide and sodium perchlorate were purchased from Merck KGaA (Darmstadt, Germany).

2.2. Tissue

Pig thyroids were collected at a local slaughterhouse. The fresh tissue was sliced at room temperature with a Stadie-Riggs microtome (Arthur Thomas, Philadelphia, PA, USA). Slices (0.3 mm thick, 45–60 mg wet weight) were incubated in 2 ml incubation medium.

2.3. Cell culture

PCCl3 cells, a rat thyroid cell line, were grown in Coon's modified Ham's F12 medium supplemented with 5% decomplemented fetal bovine serum, bovine TSH (1 mU/ml) transferrin (5 μg/ml), insulin (1 μg/ml), penicillin (100 IU/ml), streptomycin (100 μg/ml) and fungizone (2.5 μg/ml). COS–NIS cells are COS-7 cells transfected with the wild type human sodium/iodide symporter (NIS) cDNA. Stable cell lines were selected by resistance to geneticin [11]. They were cultured in DMEM supplemented with 10% fetal bovine serum, Na pyruvate (1 mM), penicillin (100 IU/ml), streptomycin (100 μg/ml) and fungizone (2.5 μg/ml).

2.4. Incubation

The slices and cells were incubated in Krebs–Ringer Hepes medium (NaCl 124 mM; KCl 5 mM; MgSO₄ 1.25 mM; CaCl₂ 1.45 mM; Hepes Buffer 25 mM) supplemented with glucose 8 mM and BSA (bovine serum albumin) 0.5 mg/ml [12].

2.5. Measurement of the H₂O₂ production

Thyroid slices were preincubated 1 h in KRH medium and then transferred to fresh medium containing 0.1 mg/ml HRP, 440 μM HVA and the tested agonists for 90 min. When H₂O₂ is produced by the tissue, the HVA is oxidized into a fluorescent dimer by the peroxidase. The fluorescence of the medium was measured at 315 and 425 nm, excitation and emission wavelengths respectively [13]. Generation of H₂O₂ was expressed as ng H₂O₂ produced by 100 mg of tissue, or ng H₂O₂ per dish in the case of cells, during the length of the incubation.

2.6. Measurement of iodide uptake

The thyroid slices were preincubated 1 h in KRH supplemented with 0.1 mM MMI to block organification of iodide, then incubated for 1 h with fresh KRH medium supplemented with ¹²⁵I⁻ (1 μCi/ml), 10⁻⁷ M KI, 0.1 mM MMI and the tested agents, washed and counted. The uptake was evaluated by the ratio: counts per min ¹²⁵I⁻/100 mg tissue to counts per min ¹²⁵I⁻/100 μl medium [14]. Uptake of investigated cells in culture was divided by the uptake of cells incubated in the presence of NaClO₄ 1 mM to obtain concentration ratios (cell to medium C/M).

2.7. Measurement of tracer efflux from cells

300,000 cells were seeded in 35-mm-diameter dishes and grown 24 h in the case of COS–NIS cells or 48 h in the case of PCCl₃ cells, in their respective culture medium. The cells were then rinsed and incubated under slight agitation in KRH medium supplemented with KI 10⁻⁷ M and 1 μCi/ml ¹²⁵I⁻, or 1 μCi/ml ^99mTcO₄⁻, or 1 μCi/ml ⁸⁶Rb⁺. As no iodination takes place in COS or PCCL3 cells, MMI was not used routinely in these experiments. For ⁸⁶Rb⁺ uptake, the K⁺ concentration in the medium was 0.5 mM instead of 5 mM.

After 45 min uptake with ¹²⁵I⁻ or ^99mTcO₄⁻ and 2 h with ⁸⁶Rb⁺, the cells were rinsed twice with KRH and incubated with fresh medium, which was supplemented with KI (10⁻⁷ M) and ClO⁻₄ (1 mM) in the case of ¹²⁵I⁻ or ^99mTcO₄⁻ efflux, to avoid tracer re-circulation. DPI (10 μM) is added at the start of this second incubation. Fifty microliter from 1 ml medium was withdrawn at the mentioned times and counted. At the end of incubation, the cells were rinsed twice with KRH medium kept on ice, then dissolved in 1 M NaOH and counted. The efflux was expressed in percent of the total radioactivity taken up by the cells (cells + medium + serial aliquots).

3. Results

DPI, in the same concentration range, inhibits both H₂O₂ generation and radioiodide uptake in pig thyroid slices (Fig. 1).

Fig. 1.

Effect of DPI on iodide uptake level at equilibrium (Empty columns) and H₂O₂ production (Filled columns) in pig thyroid slices. The incubation lasted 90 min. The results are the mean of 2 pig thyroids. The uptake control value, in absence of DPI, were 23.6 ± 4 and 18.5 ± 3 (mean of duplicates) in pig 1 and 2 respectively. The H₂O₂ control value, in absence of DPI, were 421 ± 12 and 482 ± 18 (mean of duplicates) ng H₂O₂/100 mg wet weight tissue for pig 1 and 2 respectively.

The effect of DPI on iodide uptake level at equilibrium results from the balance between the continuous uptake by NIS and the release. The inhibition of iodide uptake by DPI takes place at a lower concentration (IC50 1–4 μM) than the effect of competing iodide (IC50 40 μM) (Fig. 2). Moreover the C–I bond of the iodine to the benzene ring of DPI is not labile [9]. The inhibition therefore is not due to iodide release from DPI.

Fig. 2.

Comparison between the effect of the same range of DPI (Empty columns) and iodide (Filled columns) concentrations on iodide uptake value at equilibrium in pig thyroid slices. The incubation with DPI or KI lasted 1 h. The results presented are the mean of 2 pig thyroids. The uptake control value were 12.05 ± 1.4 and 7.1 ± 0.1 (means of duplicates) in pig 1 and 2 respectively. (Ratio of tissue vs medium radioactivity)

For the release experiments (Fig. 3, 4, 5), DPI is added after the labeling of the cells and the release is measured immediately after; it is, in effect, quasi instantaneous. The effect of DPI does not correspond to a general membrane permeabilization as preloaded ⁸⁶Rb⁺, used as analog of K⁺, is not discharged from PCCl3 nor COS–NIS cells (Fig. 3).

Fig. 3.

Comparison of DPI effect on ¹²⁵I⁻ and ⁸⁶Rb⁺ efflux from COS–NIS preloaded cells. The efflux of the tracer from the cell to the medium is expressed as percentage of total uptake by the cells. The ¹²⁵I⁻ uptake at equilibrium was 24.6 in this experiment. (Ratio of control vs NaClO₄ treated cells). Empty circles: control. Filled square: DPI 10 μM.

Moreover, DPI is a carrier more specific than NIS, the transporter of iodide (Na⁺/I⁻ symporter) as it does not release ^99mTcO₄⁻ from prelabeled cells. Thus, contrary to the NIS [15], DPI does not recognize ^99mTcO₄⁻ as radioiodide (Fig. 4).

Fig. 4.

Comparison of DPI effect on ¹²⁵I⁻ and ^99mTcO₄⁻ efflux from PCCl3 and COS–NIS preloaded cells. DPI was added at the beginning of the efflux. The uptake values at equilibrium for ¹²⁵I⁻ were 24 ± 1 and 24 ± 0.05 in PCCl₃ and COS–NIS cells respectively. The uptake values at equilibrium for ^99mTcO₄⁻ were 216 ± 6 and 228 ± 9 in PCCl₃ and COS–NIS cells respectively (mean of duplicates). The results of ¹²⁵I⁻ and ^99mTcO₄⁻ efflux are expressed as percentage of total uptake by the cells. Empty circles: control. Filled square: DPI 10 μM.

Spontaneous iodide release is much decreased when the temperature is lowered from 37 to 22 °C and 15 °C (Fig. 5). However, the relative effect of DPI is as important at the three temperatures.

Fig. 5.

Effect of temperature on DPI action on ¹²⁵I⁻ efflux from PCCl3 preloaded cells. The cells were loaded with ¹²⁵I⁻ at 37 °C for 45 min, then quickly rinsed with 2 × 1 ml KRH at 37 °C or 22 °C or 15 °C. One milliliter of KRH at 37 °C (□) or 22 °C (Δ) or 15 °C (○) was then added and efflux measured at those temperatures. The arrows bind the basal and DPI stimulated release curves at each temperature. Empty symbols: basal release. Filled symbols: DPI stimulated release.

To check the reversible or permanent character of DPI effect on iodide uptake (through its effect on efflux) and on H₂O₂ production we designed a protocol made of 3 sequential incubations called A, B and C. The measurement of iodide uptake (in the presence of ¹²⁵I and 10⁻⁷ M KI, for 45 min) and of H₂O₂ production (in the presence of 440 μM HVA and 100 μg/ml Peroxidase II for 90 min), took place in the last incubation C. Incubation A and B lasted 45 min each for either parameter. No DPI was added in any of these incubations, for the control. To test if DPI effect was short or long lasting we added it at 3 different times: In A only, or in B only, or in C only. The results presented in Fig. 6 show that the effect of DPI on iodide uptake is strongest when added in C, but fade away if it is added in B only and more so if added in A only, which suggests that the DPI involved in iodide release is rapidly washed in the absence of DPI.

Fig. 6.

Study of the reversibility of DPI effect on H₂O₂ production or iodide uptake (C/M) in PCCl3 cells. The experimental protocol is made up of 3 sequential incubations called A, B or C. ¹²⁵I⁻ for uptake (C/M) measurements or HVA and peroxydase II for H₂O₂ measurements were added in incubation C only, which lasted 45 min for ¹²⁵I⁻ uptake and 90 min for H₂O₂ production. Incubation A and B lasted 45 min each. DPI 10 μM was added either in A alone or in B alone, or in C alone. Empty columns: control. Filled columns: DPI 10 μM.

The effect of DPI on H₂O₂ is much more persistent. Indeed, if added previously, even in the first incubation A only, it works as well. This suggests a more stable binding of the DPI involved in this inhibition.

4. Discussion

We have shown that DPI, at concentrations of the same order as those that inhibit H₂O₂ generation by thyroid cells, also causes an immediate specific release of iodide from thyroid and non-thyroid cells.

The effect of NaClO₄ or NaSCN which only compete with the uptake of iodide and decrease by this mechanism the cell/medium equilibrium level [15] is relatively slow. The immediate effect of DPI on iodide efflux carried out in the presence of perchlorate is therefore not due to an inhibitory role on iodide uptake by NIS. It does not seem to require the specific iodide channel(s) involved in the efflux of iodide at the apical membrane of the thyrocyte [16,17], as it is reproduced in COS–NIS cells i.e. in non-hyroid cells.

Besides, an effect on NIS would similarly affect the efflux of ^99mTcO₄⁻ [15,18]. The absence of effect on ^99mTcO₄⁻ or ⁸⁶Rb⁺ release, excludes a general permeabilization of the cells by DPI.

Other unspecific effects of DPI have been observed but at higher concentrations than those effective on NOXes. For example, DPI (10 μM) also directly inhibits cholinesterase activity and to a lesser extent an internal Ca⁺⁺ pump [19]. The effect of DPI on iodide efflux is not due to the inhibition of H₂O₂ generation as it occurs in COS–NIS cells without DUOX and is, contrary to the effect on DUOX, relieved by incubation without the drug.

We have no explanation for this effect on iodide. Certainly because the covalent link in DPI between iodine and the other part of the DPI molecule is not labile, there is no dynamic exchange with medium iodide. The molecule is stable under stringent conditions [9].

Moreover, higher concentrations of iodide than DPI are needed to reach the same inhibition of C/M.

We can therefore only speculate on the mechanism of the DPI effect. A first explanation would be the rapid activation of an anion channel in the cell [20]. However DPI also acts on COS–NIS cells [11] which do not express any iodide specific channel, especially a channel distinguishing iodide and ^99mTcO₄⁻. A second explanation could be that DPI behaves as a membrane ionophore specific for iodide. Catalysis of an anion/hydroxyl ion exchange across mitochondrial membrane by DPI has been reported [21]. The fact that depressing membranes fluidity by decreasing the temperature of the cells slows down both basal and the increased efflux, does not distinguish the hypothesis of DPI acting as a shuttle for iodide from the concept that it acts as an intramembrane channel as is the case for valinomycin and K⁺ [22,23]. An ionic type of carrier mechanism would be similar to the mitochondrial protein carrier mechanism which is believed to account for the uncoupling effect on mitochondrial oxidative phosphorylation by dinitrophenol. Both hypotheses of channel and shuttle, would suggest a specific affinity of DPI molecule for iodide.

Nevertheless the present data confirm that DPI cannot be considered as a specific inhibitor, of NOXes and DUOXes. They suggest that DPI or an analog with similar properties could therefore be considered as drugs for iodine or amiodarone induced hyperthyroidism as they would combine in one molecule the inhibition of thyroid hormone synthesis and iodide uptake (in fact retention) as a combination of antithyroperoxidase drugs and perchlorate.