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Originally published In Press as doi:10.1074/jbc.M307938200 on November 7, 2003

J. Biol. Chem., Vol. 279, Issue 6, 4490-4497, February 6, 2004
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Mitochondrial Expression of the Human Equilibrative Nucleoside Transporter 1 (hENT1) Results in Enhanced Mitochondrial Toxicity of Antiviral Drugs*

Yurong Lai{ddagger}, Chung-Ming Tse§, and Jashvant D. Unadkat{ddagger}

From the {ddagger}Department of Pharmaceutics, University of Washington, Seattle, Washington 98195 and the §Department of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, October 13, 2003 , and in revised form, October 13, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [14C]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nM nitrobenzylthioinosine. Moreover, FIAU (50 µM) produced significant mitochondrial toxicity (~70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fialuridine (FIAU),1 a uridine analog, was developed for the treatment of hepatitis B. In a phase II trial, administration of this antiviral nucleoside to patients with hepatitis B resulted in severe multisystem toxicity because of widespread mitochondrial damage including hepatotoxicity, pancreatitis, neuropathy, or myopathy. Of the seven patients exhibiting severe hepatotoxicity, five died and two survived after liver transplantation (1). Although the exact mechanism of mitochondrial toxicity of FIAU has not been determined (2), all of the evidence points toward inhibition of mitochondrial DNA polymerase {gamma} by the phosphorylated metabolites of FIAU that accumulate within the mitochondrial compartment (3, 4). Other antiviral nucleoside drugs also cause mitochondrial toxicity such as the anti-HIV dideoxynucleosides (e.g. DDI) (5) and the anticancer drugs (e.g. fludarabine) (6). The mitochondrial toxicity of the anti-HIV dideoxynucleosides is milder and occurs less frequently (7). Nevertheless, the mechanism of mitochondrial toxicity of these drugs is thought to be similar to that of FIAU.

To produce mitochondrial toxicity, these nucleoside drugs must first be phosphorylated either in the cytosol or the mitochondria. For example, the mitochondria-specific thymidine kinase (TK2) efficiently phosphorylates pyrimidine nucleosides including FIAU, gemcitabine, and AraC (8–10). If phosphorylated in the cytosol, the hydrophilic nucleotides may be transported across the mitochondrial membrane by either the ADP/ATP translocator (11) or as yet unidentified transporters (12). Alternatively, the nucleosides, which are mostly hydrophilic, must be transported across the highly impermeable mitochondrial membrane and then be phosphorylated to the nucleotides in the mitochondrial compartment (13–15). Here we report studies that demonstrate for the first time that one of these nucleoside transporters is the human equilibrative nucleoside transporter 1 (hENT1). By using confocal microscopy, our studies show that hENT1, fused to the yellow fluorescent protein (YFP; hENT1-YFP), is localized to the mitochondrial compartment in MDCK cells expressing hENT1-YFP. Mitochondria isolated from these cells demonstrate hENT1 activity and efficiently transport labeled uridine or FIAU. Moreover, we show that the mitochondrial expression of hENT1-YFP in MDCK cells results in enhanced mitochondrial toxicity of FIAU even when hENT1-YFP activity in the plasma membrane is blocked by a low nanomolar concentration of NBMPR, a potent hENT1 inhibitor. These studies demonstrate for the first time that the intracellular localization of hENT1 has important consequences for intracellular disposition and mitochondrial toxicity of nucleoside drugs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Gene Construction and Stable Expression of Nucleoside Transporters in MDCK Cells
hENT1 or hCNT1 genes were subcloned into yellow fluorescence vector pEYFP-C1 (Clontech, Palo Alto, CA). The resulting constructs, hENT1-YFP or hCNT1-YFP, were transfected into the MDCK cell line to produce a stable cell line as described previously (16). Cells were routinely maintained in minimum Eagle's medium with L-glutamine containing 10% fetal bovine serum, 100 units of penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 °C in 95% air, 5% CO2 with 95% humidity.

Mitochondria Isolation
Mitochondria were isolated from human liver or cells using the mitochondria isolation kit (Sigma). Human liver tissue was procured, prepared, and stored as described previously (17, 18). Liver tissue (100 mg) or 1–5 x 108 cells (pooled from 8 to 10 dishes) were homogenized on ice with 2 ml of the manufacturer's extraction buffer using a Teflon-glass homogenizer. As recommended by the vendor, to obtain a more purified "heavy" mitochondrial fraction, the low and high speed centrifugation steps listed in the kit were modified to three rounds of 1,000 and 3,500 x g, respectively. This modification reduced the contamination from lysosomes and peroxisomes.

JC-1 Uptake
The integrity of the inner mitochondrial membrane was determined by measuring the potential gradient across the mitochondrial membrane using the fluorescent stain, JC-1, as per the manufacturer's instructions (Sigma) (19).

Western Blot Analysis
Stable expressing cells or their mitochondria were suspended and fine-needle homogenized in cell lysis buffer containing 1% Nonidet P-40 (Sigma) and 20 µg/ml phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science) in 50 mM Tris-HCl, pH 7.4. Liver tissue (100 mg) was homogenized with lysis buffer using a Teflon-glass homogenizer and then sonicated on ice three times for 15 s separated by 30-s intervals. The cell debris was pelleted by centrifugation at 13,000 x g for 5 min. An aliquot (15 µl) of the resulting supernatant (10 µg protein) was boiled for 5 min with an equal volume of Laemmli buffer (Bio-Rad). The boiled samples were then run on 4–15% gradient SDS gel (Bio-Rad) and electrophoretically transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was washed and incubated with an anti-green fluorescent protein monoclonal antibody (for hENT1-YFP-expressing cell, JL-8 Clontech), or hENT1 antibody (for human liver) (20) or a Na+-K+-ATPase antibody (Sigma). Bound antibody was detected with horseradish peroxidaseconjugated rabbit anti-mouse IgG antibody and visualized by the ECL kit (Amersham Biosciences).

Visualization of hCNT1-YFP, hENT1-YFP, and Intracellular Organelles
MitroTracker Red 580, Golgi complex dye (N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine ceramide), LysoTracker, and 4',6'-diamidino-2-phenylindole, dihydrochloride (DAPI) were purchased from Molecular Probes, Inc. (Eugene, OR). The undifferentiated cells were grown on Lab-Tek Borosilicated Coverglass Chambers (Nalge Nunc International Corp., Naperville, IL) for 2 days. All cells were stained with DAPI (nuclei) and one of the intracellular organelle stains. Golgi complexes were stained by incubating with N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diazas-indacene-3-yl)phenoxy)acetyl)sphingosine ceramide (5 µM) for 30 min at 4 °C. Mitochondria and lysosomes were stained by incubating with MitoTracker-580 (100 nM) for 45 min or LysoTracker (300 nM) for 30 min at 37 °C, respectively. After incubation, the cells were rinsed several times with Hanks' balanced salt solution/HEPES containing 5 µM BSA to remove the excess dye. Endosomes were identified by immunolocalization. Briefly, the fixed cells were pretreated with 2% Triton X-100 for 15 min and blocked by 10% normal horse serum for 2 h. Then the cells were incubated overnight at 4 °C with 1:50 mouse monoclonal early endosome antigen 1 (Transduction Laboratories, Lexington KY). The cells were washed with phosphate-buffered saline and reacted with 1:250 anti-mouse IgG conjugated with Alexa-560 (Molecular Probes, Eugene, OR) at 37 °C for 2 h. Finally, the cell chromosomes were stained with DAPI (300 nM) for 5 min. The cells were observed and photographed by Leica confocal microscope equipped with a 2-photon and a krypton/argon laser as the light source. The DAPI-stained nuclei images were captured by excitation at 360 nm and emission at 440–460 nm. The YFP images were captured by excitation at 488 nm and emission at 520–540 nm. The lysosome, endosome, mitochondria, and Golgi complex stains were imaged by excitation at 580 nm and emission at 600–640 nm.

Nucleoside Transport and Inhibition Assays
All transport experiments were carried out in triplicate in sodium-containing transport buffer (Tris-HCl 20 mM, K2HPO4 3 mM, MgCl2·6H2O 1 mM, CaCl2 2 mM, glucose 5 mM, NaCl 130 mM, pH 7.4) or sodium-free transport buffer in which NaCl was replaced by 130 mM N-methyl-D-glucamine, pH 7.4. To determine transport into the mitochondria, the mitochondria (0.1 mg of protein) were incubated with 0.2 µM [3H]uridine (Moravek Chemicals) in a sodium-containing or sodium-free buffer, each containing 10 µM NBMPR (hCNT1 activity), or in a sodium-free buffer with or without 10 nM, 1 µM, or 10 µM NBMPR (hENT1/2 activity) (16). An equal concentration of Me2SO (a solvent used to dissolve NBMPR) was included in all experiments. After incubating at 37 °C for 10 min, nucleoside transport by the mitochondria was rapidly terminated by filtration followed by washing the filtered mitochondria three times with ice-cold Na+-free buffer containing 10 µM NBMPR. The mitochondria, retained on the filter, were solubilized with 0.5% Triton X-100 and then counted on a scintillation counter. To determine whether FIAU is transported by hCNT1 or hENT1, mitochondria from cells expressing these transporters were incubated for 10 min with 1 µCi of [14C]FIAU (8 µM) as described above.

Transport of labeled uridine or FIAU into MDCK cells (expressing empty vector (mock), hENT1-YFP, or hCNT1-YFP), was determined as described before (16). To determine the inhibition of hENT1-mediated [3H]uridine transport into MDCK cells by FIAU or raluridine, MDCK cells were incubated for 10 min with [3H]uridine (2 µCi/ml, 1 µM) in the presence of FIAU or raluridine (50 or 500 µM) in a sodium-free transporter buffer.

Mitochondrial Toxicity Assays
MTT Assay—103 cells were grown on 48-well cell culture plates. During the logarithmic phase of cell growth, FIAU or raluridine (0, 50, 100, 200, or 500 µM) was added to the cell culture medium for 4 days. Then the cells were washed and incubated for 4 h with MTT (5 mg/ml) dissolved in a Hanks' balanced salt solution. Then 0.2 ml of lysis buffer (isopropyl alcohol containing 10% Triton X-100 and 0.1 N HCl) was added to dissolve the formazan formed. 100 µl of the resulting formazan solution was transferred into a 96-well plate, and the absorbance was measured at 570 nm in a microplate spectrophotometer. Mitochondrial toxicity was evaluated as the formazan formed by cells incubated with FIAU or raluridine compared with that by control cells.

Lactate Dehydrogenase (LDH) Release Assay—The cell culture medium was collected at regular intervals in the above MTT assay. Cell debris in the medium was removed by centrifugation at 250 x g for 5 min. LDH released into the culture medium was measured by determining the absorbance at 490 nm using a NAD-linked assay kit according to the manufacturer's instructions (Sigma).

DNA Synthesis in Isolated Mitochondria
Freshly prepared mitochondria (~1 mg/ml) were incubated at 37 °C in a medium with or without FIAU (50 µM) and containing mitochondrial energy requirements and DNA synthesis components as described before (21). [3H]dTTP (2 µCi/ml, 79.6 Ci/mmol) was used as nucleic acid precursor. After 3 h of incubation, the mitochondria were precipitated by ice-cold 10% trichloroacetic acid, filtered, and then washed three times with trichloroacetic acid. The acid-precipitable radioactivity was quantified by a liquid scintillation counter.

Data Analysis
Data are expressed as mean ± S.D. of triplicate experiments. Data shown are representative of a minimum of two experiments carried out on different days on different batches of cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of hENT1-YFP in Undifferentiated MDCK Cells—Previously, when we simultaneously expressed hENT1 and hCNT1 in undifferentiated MDCK cells, we noted that hENT1-YFP (but not hCNT1-CFP) was found on the plasma membrane as well as discrete clusters within the cells (16). Others have also reported that hENT1 is also found intracellularly (22). To determine the localization of these observed hENT1-YFP clusters, we expressed hENT1-YFP or hCNT1-YFP in MDCK cells. These stable expressing cells, stained with organelle-selective dyes or antibodies, were examined using the confocal microscope. As expected, besides plasma membrane expression, hENT1-YFP was localized intracellularly (Fig. 1), whereas hCNT1-YFP was not (data not shown). By using the organelle-selective markers, we found that the intracellular localization of hENT1-YFP, both in the x-y and the z plane, overlapped with the mitochondria (Fig. 1, D and H) but not with the nuclear envelope, early endosomes (Fig. 1, A and E), Golgi complex (Fig. 1, B and F), or lysosomes (Fig. 1, C and G).



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  		FIG. 1.
As determined by organelle-selective dyes and confocal microscopy, the intracellular localization of hENT1-YFP in MDCK cells overlaps with the mitochondria (D and H) but does not overlap with the nuclear envelope (purple stain), endosomes (A and E), endoplasmic reticulum, and the Golgi apparatus (B and F) or lysosomes (C and G) as viewed in both the x-y and z plane.

 
Western Blotting of Mitochondria hENT1-YFP—To confirm mitochondrial localization of hENT1, mitochondria isolated from hENT1-YFP-expressing cells were examined for the presence of hENT1 by Western blotting. To do so, we first confirmed the lack of contamination of the mitochondrial fraction, by plasma membrane, by the minimal presence of Na+-K+-ATPase (Fig. 2A). Na+-K+-ATPase was present in abundance in the cell homogenates of both hENT1-YFP- and empty vector-expressing MDCK cells (Fig. 2A). In contrast, the mitochondrial fraction isolated from these cells showed minimal presence of this plasma membrane marker (Fig. 2A). When the mitochondrial fraction was separated by SDS-PAGE, hENT1-YFP (~67 kDa, Fig. 2B) was present at comparable levels in the mitochondrial fraction and the homogenate from MDCK cells expressing hENT1-YFP, but not in cell homogenate or the mitochondrial fraction of mock cells (Fig. 2B). To confirm that hENT1 is physiologically expressed in mitochondria of human livers, we determined its presence there by Western blotting. As expected, hENT1 (~40 kDa, Fig. 2C) was expressed in mitochondria isolated from human livers.



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  		FIG. 2.
Western blot analysis of mitochondrial fraction or cell homogenate (10 µg) of human liver, hENT1-YFP-expressing, or mock cells was conducted using either a Na+-K+-ATPase antibody (A), anti-green fluorescent protein antibody (to detect hENT1-YFP) (B), or anti-hENT1 antibody (to detect endogenous hENT1 in human liver) (C). A, Na+-K+-ATPase (~110 kDa) is present in cell homogenates (lanes 3 and 4) but minimally in the mitochondrial fraction (lanes 1 and 2) of both hENT1-YFP-expressing (lanes 1 and 3) and mock cells (lanes 2 and 4). B, hENT1-YFP (~67 kDa) was present in the mitochondrial fraction (lane 3) and the cell homogenate (lane 5) of MDCK cells expressing hENT1-YFP but not in cell homogenate (lane 2) or the mitochondrial fraction of mock cells (lane 4). YFP (~28 kDa) was detected in cell homogenate and mitochondrial fraction of mock cells (lanes 2 and 4) but not in MDCK cells (lane 1) not transfected with the empty vector. C, hENT1 (~40 kDa) was detected both in human liver homogenate (lane 1) and the mitochondrial fraction (lane 2).

 
Integrity of the Isolated Mitochondria—To confirm the integrity of the isolated mitochondria, we measured the mitochondria membrane potential, using the mitochondria membrane potentially sensitive lipophilic cationic dye JC-1, as described before (19, 23). An ~30-fold accumulation of JC-1 (fluorescent units) in the isolated mitochondrial fraction, over that obtained with the cell homogenate, indicated that the isolated mitochondrial fraction was enriched in intact mitochondria.

[3H]Uridine Transport and Inhibition—To determine whether hENT1-YFP expressed in the mitochondria was functional, we measured the transport of [3H]uridine over 10 min into the intact mitochondria in the presence or absence of the hENT1 inhibitor, NBMPR (10 µM). Preliminary studies showed that the uptake of [3H]uridine into the mitochondria was linear over 10 min. Mitochondria from hENT1-YFP-expressing cells showed about 4-fold greater hENT1 activity when compared with those isolated from hCNT1 or mock cells. The latter two cell types demonstrated some endogenous hENT-like transport activity that was inhibited by NBMPR (10 µM) (Fig. 3A). Further characterization of this endogenous hENT-like activity showed that it was also completely inhibited by 10 nM NBMPR indicating that it was an hENT1-like transporter (Fig. 3B). Previously, we have also shown that MDCK cells exhibit low level endogenous hENT-like activity (16). Moreover, the mitochondria isolated from human liver tissue also exhibited hENT-like [3H]uridine transport (Fig. 3C). This transport was sensitive to NBMPR at as low a concentration as 10 nM (Fig. 3C). Because hENT1 is completely inhibited by 10 nM NBMPR, the differences in transport of [3H]uridine in the presence of 10 nM and 10 µM NBMPR likely represents endogenous hENT2 activity. In contrast, mitochondria isolated from hCNT1 or mock-expressing cells showed no sodium-dependent transport activity (data not shown).



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  		FIG. 3.
A, mitochondria isolated from MDCK cells expressing hENT1-YFP demonstrate hENT1-mediated (NBMPR-sensitive) [3H]uridine (0.2 µM) transport. Mitochondria isolated from mock cells or those expressing hCNT1-YFP demonstrate low hENT-like endogenous transporter activity. B, [3H]uridine (2 µCi/ml, 1 µM) uptake into MDCK cells expressing hENT1-YFP, hCNT1-YFP or empty vector (mock) was completely inhibited by 10 nM NBMPR, indicating that MDCK cells express hENT1-like transporter. C, mitochondria isolated from human liver exhibit endogenous hENT1 (sensitive to 10 nM NBMPR) and hENT2 (sensitive to 10 µM NBMPR) transport of [3H]uridine (0.2 µM). Data are mean ± S.D. of triplicate experiments. *, p < 0.05 compared with the [3H]uridine uptake in the presence of NBMPR.

 
To ascertain if FIAU or raluridine are potential substrates of hENT1, we measured the ability of these two nucleoside analogs to inhibit the hENT1-mediated transport of [3H]uridine (Fig. 4A). FIAU was found to be a potent inhibitor of [3H]uridine transport (Fig. 4B). In contrast, raluridine is only a modest inhibitor even at the high concentration of 500 µM (Fig. 4B). These data suggest that, unlike FIAU, raluridine is unlikely to be a high affinity substrate of hENT1.



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  		FIG. 4.
Chemical structures of FIAU and raluridine (A) and the inhibition (B) by these two nucleosides of [3H]uridine (2 µCi/ml, 1 µM) transport into hENT1-YFP-expressing MDCK cells. Data are mean ± S.D. of the percentage of the [3H]uridine uptake observed in the absence of the inhibitors. *, p < 0.05 compared with [3H]uridine uptake observed in the absence of the inhibitors.

 
[14C]FIAU Transport by MDCK Cells and Their Mitochondria—To confirm that FIAU, a potent mitochondrial toxin, is indeed a substrate of hENT1, we measured the transport of [14C]FIAU (1 µCi, 8 µM) into cells expressing hENT1-YFP, hCNT1-YFP, or mock cells. [14C]FIAU is an excellent substrate of hENT1 but not of hCNT1 as shown by the significant inhibition of FIAU transport by NBMPR (Fig. 5A) but not by the absence of sodium (data not shown). As expected, [14C]FIAU transport into mitochondria of hENT1-YFP-expressing cells was significantly greater than that into mitochondria of mock cells (Fig. 5B). To allow comparison of these data with uridine uptake data (Fig. 3), the values have been adjusted to reflect the uptake that would be observed at 0.2 µM FIAU.



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  		FIG. 5.
[14C]FIAU uptake into cells (over 15 min) or mitochondria (over 10 min) expressing hENT1-YFP. A, [14C]FIAU uptake (8 µM) into hENT1-YFP-expressing MDCK cells shows that it is an excellent substrate of hENT1. B, as expected, hENT1-mediated [14C]FIAU uptake into mitochondria of hENT1-YFP-expressing cells was significantly greater than that into mitochondria of mock cells. To allow comparison with [3H]uridine uptake data shown in Fig. 3, the uptake data of FIAU are adjusted to values that would be observed if the FIAU concentration was 0.2 µM. *, p < 0.01 compared with the uptake observed in the presence of NBMPR.

 
Mitochondrial Toxicity of FIAU and Raluridine as Measured by the MTT and LDH Assay—Because we found that FIAU is an excellent substrate of hENT1, we compared its mitochondrial toxicity with that of raluridine, in hENT1-YFP-expressing cells. As measured by the MTT and the LDH release assay, FIAU, but not raluridine, produced significant mitochondrial toxicity when incubated with hENT1-YFP-expressing cells (Fig. 6, A and B). This observed mitochondrial toxicity of FIAU was greater in hENT1-YFP-expressing cells when compared with that in mock cells (Fig. 7). Mock cells exhibited some FIAU mitochondrial toxicity, most likely due to the transport of FIAU into the MDCK cells by the endogenous canine hENT-like transporter. To eliminate the presence of hENT1 on the plasma membrane from contributing to the enhanced mitochondrial toxicity of FIAU, we measured the toxicity of FIAU (plus tracer [14C]FIAU) when hENT1 activity on the plasma membrane was inhibited by low nanomolar (10 nM) concentrations of NBMPR. hENT1-YFP-expressing cells incubated with 50 µM FIAU showed mitochondrial toxicity that was significantly greater than that of mock cells, even though plasma membrane hENT1 activity was inhibited by 10 nM NBMPR (Fig. 7, A and B). We confirmed the latter by measuring the intracellular radioactivity contents of [14C]FIAU at 96 h. At this time, the intracellular [14C]FIAU content was similar in both hENT1-YFP- and mock cells (7.12 ± 1.54 versus 8.29 ± 2.14 pmol/mg protein/96 h).



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  		FIG. 6.
As determined by MTT (A) or LDH release assay (B) over 96 h, FIAU is significantly more toxic than raluridine to hENT1-YFP expressing cells. Data are expressed as mean ± S.D. as percent of values observed in the absence of drugs.

 



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  		FIG. 7.
In the absence of NBMPR, as determined by MTT (A)or LDH release (B) assay over 96 h, FIAU is a mitochondrial toxin toward hENT1-YFP-expressing cells and mock cells. In contrast, when NBMPR (10 nM) is used to block hENT1-YFP activity on the plasma membrane, FIAU is a mitochondrial toxin toward only hENT1-YFP-expressing cells but not toward mock cells. Data are expressed as percent of values observed in the absence of drugs. *, p < 0.05 compared with the values observed in the absence of drugs.

 
DNA Synthesis in Isolated Mitochondria Treated with FIAU—FIAU produces its mitochondrial toxicity by inhibiting synthesis of mitochondrial DNA (8, 24). To evaluate directly the contribution of hENT1 toward mitochondrial toxicity of FIAU, we examined the synthesis of DNA in freshly isolated mitochondria by measuring [3H]dTTP incorporation into mitochondrial DNA in the presence and absence of FIAU. Incubation of mitochondria with FIAU (50 µM, 3 h) decreased [3H]dTTP incorporation into mitochondrial DNA of hENT1-YFP-expressing cells to a much greater extent than in mock cells (Fig. 8). When the hENT1-mediated transport of FIAU into the mitochondria was blocked by 10 nM NBMPR, inhibition of mitochondrial DNA synthesis by FIAU was abrogated in both hENT1-YFP-expressing and mock cells (Fig. 8). Because mock cells demonstrate the same endogenous equilibrative nucleoside transporter-like activity, FIAU also moderately inhibited mitochondrial DNA synthesis in mitochondria isolated from these cells.



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  		FIG. 8.
Effect of FIAU on mitochondrial DNA synthesis. As measured by [3H]dTTP incorporation (over 3 h) into isolated mitochondria, FIAU (50 µM) inhibited DNA synthesis in mitochondria isolated from hENT1-YFP-expressing cells to a greater extent than in mitochondria isolated from MOCK cells. This inhibition of mitochondrial DNA synthesis was abrogated by blocking hENT1 activity (by 10 nM NBMPR) on the mitochondrial membrane. Data are expressed as percent of values observed in the absence of drugs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Many antiviral nucleoside analogs are known to cause mitochondrial toxicity resulting in a variety of adverse effects such as hepatotoxicity (FIAU), myopathy (AZT), peripheral neuropathy (d4T, DDI, lamivudine (2',3'-dideoxy-3'-thiacytidine)), pancreatitis (DDI), and lactic acidemia (DDI, d4T, AZT) (7, 25–27). In some cases, for example FIAU, this mitochondrial toxicity has resulted in death. Although the exact mechanism of this toxicity has not been elucidated, inhibition of mitochondrial DNA polymerase {gamma} and incorporation into mtDNA causing premature chain termination are widely accepted as the most likely mechanisms of the mitochondrial toxicity of nucleoside drugs (7, 28). Mitochondrial DNA polymerase {gamma}, which has poor proofreading capacity, is responsible for mitochondrial DNA replication. Inhibition of this enzyme by the triphosphate metabolite of the nucleoside drugs results in depletion of mitochondrial DNA, a signature of the mitochondrial toxicity of FIAU (29, 30).

To produce their mitochondrial toxicity, the nucleoside drugs must first be transported into the cytosol or diffused into the cells. Except for the few moderately lipophilic nucleoside drugs such as AZT, other nucleoside drugs that cause mitochondrial toxicity are too hydrophilic to diffuse into the cells in appreciable quantity (e.g. FIAU and gemcitabine). Thus, it is very likely that they are transported into the cells by the nucleoside transporters. Although gemcitabine, DDI, ddC, and AZT are substrates of hENT1 or hENT2, it is not known if FIAU is a substrate of nucleoside transporters (31, 32). In the cytosol, these drugs are phosphorylated by a variety of enzymes, depending on the nucleosides. For example, the thymidine and uridine analogs (e.g. AZT, FIAU, and d4T) are phosphorylated by the cytosolic thymidine kinase (TK1) (10, 33). The cytidine analogs (e.g. ddC, gemcitabine) are metabolized by the cytosolic deoxycytidine kinase (dCK) (34). Many phosphorylating enzymes are also present in the mitochondria, and some of these are unique to the mitochondria such as the mitochondria-specific thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK). TK2 can phosphorylate FIAU, gemcitabine, and AraC (35), whereas dGK phosphorylates purine deoxyribonucleosides (e.g. AraC) (36). TK2 is considered to be the rate-limited enzyme for FIAU phosphorylation (10, 37).

Consequently, antiviral nucleoside triphosphates present in the mitochondria can be derived from two sources. In the first, the nucleosides may diffuse into or be transported into the mitochondria where, if the necessary enzymes are present, they will be phosphorylated (e.g. phosphorylation of FIAU, AZT, and d4T) to the triphosphates to produce their mitochondrial toxicity. Alternatively, where the phosphorylating enzymes are present only in the cytosol (e.g. deoxycytidine kinase), the antiviral nucleotides may be transported into the mitochondria (e.g. dCTP) (11). There is evidence to suggest that the entry of nucleosides into the mitochondria is a rate-limiting step in the mitochondrial toxicity of some nucleosides. For example, overexpression of TK2 in a bacterial system increases the toxicity of many pyrimidines including FIAU, AZT, and gemcitabine (35). Likewise, overexpression of the mitochondrial dGK increases the cytotoxicity of purine nucleoside drugs, such as cladribine (2-chloro-2'-deoxyadenosine), to cancer cell lines (36). These data clearly demonstrate that some nucleosides traverse the highly impermeable mitochondrial membrane and are phosphorylated in the mitochondria to produce their toxicity there. Since, like FIAU, a majority of the nucleoside drugs is hydrophilic, it is unlikely that these nucleosides readily diffuse across the mitochondrial membrane. Therefore, we have hypothesized that nucleoside transporters must be expressed on the mitochondrial membrane to provide them access to the mitochondrial compartment. In this paper, we present evidence that one of these nucleoside transporters is hENT1, and the expression of this transporter in the mitochondrial membrane is a significant determinant of the mitochondrial toxicity of FIAU.

By using confocal microscopy and organelle-selective dyes, we found that, besides the plasma membrane, hENT1-YFP is localized to the mitochondria in MDCK cells expressing hENT1-YFP. However, hENT1-YFP was not localized to the Golgi apparatus, nuclear envelope, endosomes, or lysosomes (Fig. 1). This mitochondrial localization of hENT1-YFP is consistent with reports of nucleoside transport activity that can be inhibited by NBMPR in mitochondria isolated from the rat testes (14, 15). Interestingly, hENT-like transporters have been reported to be present intracellularly on the nuclear envelope or in lysosomes in human cell lines (38). The nucleoside transporter activity in the lysosomes demonstrates an atypically low affinity for nucleosides and NBMPR (in the millimolar range), making it unlikely that it is either hENT1 or hENT2. This transporter may well be hENT3, which is thought to be localized intracellularly (32).

The mitochondrial localization of hENT1 was functionally confirmed by measuring the transport of labeled uridine into intact mitochondria isolated from hENT1-YFP-expressing MDCK cells (Fig. 3). This transport activity was not due to contamination by the plasma membrane as demonstrated by the lack of Na+-K+-ATPase in the mitochondrial fraction and the fact that the plasma membrane would have to be prepared as membrane vesicles to demonstrate any transport activity (Fig. 2). hENT1-YFP, expressed in the mitochondrial membrane or the plasma membrane, also efficiently transported the mitochondrial toxin FIAU (Fig. 5). These data demonstrate that expression of hENT1-YFP on the mitochondrial membrane allows FIAU access to the mitochondrial compartment where, after phosphorylation, its triphosphate can produce its mitochondrial toxicity.

The efficient transport of FIAU into mitochondria by hENT1-YFP, comparable with that of uridine (when adjusted for the concentration), indicates that FIAU is a high affinity ligand of hENT1. This is not surprising as FIAU has the 3'-OH group that we and others have shown is necessary for a nucleoside to be a high affinity ligand of hENT1 (Fig. 4A) (39, 40). Interestingly, a closely related compound, raluridine, which is a potent inhibitor of DNA polymerase {gamma} (41), is not a mitochondrial toxin. Like FIAU, raluridine is phosphorylated by thymidine kinase (42). As raluridine lacks the 3'-OH group, we hypothesized that raluridine is unlikely to be a substrate of hENT1 (Fig. 4A). Unfortunately, as raluridine is not available in the radiolabeled form, this hypothesis cannot be tested directly. Therefore, we sought to determine whether it is a potent inhibitor of hENT1. At 50 and 500 µM, raluridine was a much weaker inhibitor of [3H]uridine transport into hENT1-YFP-expressing MDCK cells than FIAU (Fig. 4). These data suggest that raluridine is not a high affinity ligand of hENT1 and, therefore, is unlikely to be a high affinity substrate of hENT1. Based on these data, we hypothesized and have subsequently shown that FIAU would be a more potent mitochondrial toxin than raluridine when exposed to hENT1-YFP-expressing MDCK cells. Consistent with previous in vitro and in vivo data (43), raluridine produced no mitochondrial toxicity in hENT1-YFP-expressing cells, as measured by the MTT and LDH release assay, even at the high concentration of 500 µM (Fig. 6). In contrast, FIAU was a significant mitochondrial toxin to these cells at a concentration as low as 50 µM (Fig. 6). These data support our hypothesis that, although raluridine is a potent inhibitor of DNA polymerase {gamma}, it is not a significant mitochondrial toxin because of its lack of access to the cytosolic and/or mitochondrial compartment.

As indicated earlier, the enhanced mitochondrial toxicity of FIAU to hENT1-YFP-expressing cells could be due to increased access of the drug to the cytosolic or the mitochondrial compartment. To distinguish between these two possibilities, we sought to block hENT1-YFP activity only on the plasma membrane of hENT1-YFP-expressing cells. To do so, we took advantage of the potent inhibition of hENT1 at a low nanomolar (10 nM) NBMPR concentration. We hypothesized that this concentration of NBMPR would not result in appreciable inhibition of the mitochondrial hENT1 activity as NBMPR is not transported by hENT1, and, therefore, its intracellular concentration would be much lower than 10 nM. Thus, in the presence of NBMPR, we expected FIAU to diffuse into the cells in small quantity and be transported into the mitochondria by hENT1 expressed there. Our data show that hENT1-YFP-expressing and mock cells, incubated in the absence of NBMPR but with 50 µM FIAU for 4 days, resulted in hENT1-YFP-expressing cells experiencing significantly greater mitochondrial toxicity than the mock cells (Fig. 2). The mock cells experienced some toxicity of FIAU as they exhibit low level equilibrative nucleoside transporter-type activity that is capable of transporting FIAU. In the presence of 10 nM NBMPR, when plasma membrane hENT1-YFP activity was expected to be completely inhibited, mock cells incubated with 50 µM FIAU showed no significant mitochondrial toxicity, whereas those expressing hENT1-YFP showed reduced toxicity but greater than that of mock cells (Fig. 7). We confirmed that the plasma membrane activity was completely inhibited by 10 nM NBMPR by including [14C]FIAU and measuring the intracellular radioactivity contents. The intracellular [14C]FIAU content was similar in both hENT1-YFP and mock cells. These data indicate that, even when plasma membrane hENT1 was blocked, the FIAU that diffused into the cells was transported into the mitochondria by the hENT1 expressed there to produce its mitochondrial toxicity. This toxicity was, of course, reduced as the net flux of FIAU into the cytosolic compartment was considerably reduced by NBMPR. To confirm these findings, we directly evaluated the contribution of hENT1 toward the mitochondrial toxicity of FIAU. We determined the effect of FIAU on the synthesis of mitochondrial DNA by incubating FIAU with mitochondria isolated from hENT1-YFP-expressing cells. Inhibition of mitochondrial DNA synthesis by FIAU was greater for mitochondria isolated from hENT1-YFP-expressing cells when compared with those isolated from mock cells. This enhanced mitochondrial toxicity was abrogated when the hENT1 activity on the mitochondrial membrane was blocked by 10 nM NBMPR. These data conclusively show that the expression of hENT1 on the mitochondrial membrane is a significant determinant of the mitochondrial toxicity of FIAU.

Although hENT1 expression on the mitochondrial membrane can explain the mitochondrial toxicity of nucleoside drugs that are efficiently transported by hENT1 (e.g. FIAU and gemcitabine), it cannot completely explain the mitochondrial toxicity of the dideoxynucleosides (e.g. the anti-HIV AZT, D4T, or DDI), as they are low affinity substrates of hENT1 (39, 44). However, these drugs are more efficiently transported by h/rENT2 (44). Therefore, we speculate that they either diffuse into the mitochondria (e.g. AZT) or that they are transported into the mitochondria by hENT1 plus hENT2. Implicit in this speculation is the hypothesis that hENT2 is also expressed in the mitochondrial membrane. Although this hypothesis has yet to be thoroughly tested, it is supported by two observations. In the first observation, we observed hENT2-mediated transport of [3H]uridine into mitochondria isolated from human livers (Fig 3C). Second, the muscle, a tissue where hENT2 is highly expressed, is a target of mitochondrial toxicity of dideoxynucleosides (7). Indeed, all tissues (liver, muscle, and pancreas) that are energy-demanding (i.e. are significant consumers of ATP) are targets of mitochondrial toxicity of nucleoside drugs. It makes eminent physiological sense that the high ATP need of these tissues would be met by higher expression of nucleoside transporters on their mitochondrial membrane, thereby making them targets of dideoxynucleoside mitochondrial toxicity.

In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM54447. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98195. Tel.: 206-543-9434; Fax: 206-543-3204; E-mail: jash{at}u.washington.edu.

1 The abbreviations and trivial names used are: FIAU (fialuridine), 1-(2-deoxy-2-fluoro-{beta}-D-arabinofuranosyl)-5-iodouracil; AraC, arabinosyl cytosine; AZT (zidovudine), 3'-deoxy-3'-azidothymidine; d4T (stavudine), 2',3'-didehydro-2',3'-dideoxythymidine; ddC (zalcitabine), 2',3'-dideoxycytidine; DDI (didanosine), 2',3'-dideoxyinosine; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanine iodide; MTT, 3-4(dimethylthiazol-2,5-diphenyl)tetrazolium bromide; NBMPR, nitrobenzylthioinosine; raluridine (935U83), 5-chloro-2',3'-dideoxy-3'-fluorouridine; hCNT1, human concentrative nucleoside transporter 1; hENT1, human equilibrative nucleoside transporter 1; MDCK, Madin-Darby canine kidney; DAPI, 4,6-diamidino-2-phenylindole; YFP, yellow fluorescent fusion protein; HIV, human immunodeficiency disease; LDH, lactate dehydrogenase; dGK, deoxyguanosine kinase; TK2, thymidine kinase 2. Back


   ACKNOWLEDGMENTS
 
We thank GlaxoSmithKline for their gift of raluridine (935U83) and Drs. Aimee Bakken, Dhruba Sengupta, Eun-Woo Lee, and Chris Endres for helpful discussions and assistance.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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