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Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs
ABB Archives of Biochemistry and Biophysics 421 (2004) 1–9 www.elsevier.com/locate/yabbi
Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs Joan L. Buss,a Jiri Neuzil,b,1 and Prem Ponkac,* a
c
Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC, USA b Department of Pathology II, University Hospital, Linkoping, Sweden Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and Departments of Physiology and Medicine, Que., Canada H3T 1E2 Received 21 May 2003, and in revised form 15 September 2003
Abstract Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe3þ –chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe3þ complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe3þ complexes, and toxicity of the chelators increased with oxygen tension. Fe3þ complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)2 did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)2 in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe3þ complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Oxidative stress; Pyridoxal isonicotinoyl hydrazone (PIH) analogs; Iron chelators; Toxicity; K562 cells; Jurkat T lymphocytes
Iron (Fe) is involved in many essential biological functions such as oxygen transport, electron transfer, and DNA synthesis. However, the chemical properties of iron which allow this versatility also lead to the paradoxical situation that Fe acquisition by the organism is difficult. Under physiological conditions, Fe2þ is readily oxidized to Fe3þ , which is virtually insoluble in the absence of chelators. Moreover, unless bound to specific ligands, iron plays a key role in the formation of *
Corresponding author. Fax: +514-340-7502. E-mail address: [email protected] (P. Ponka). 1 Present address: Heart Foundation Research Center, School of Health Sciences, Griffith University, Southport, Queensland, Australia. 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.09.044
reactive oxygen species (ROS2), causing oxidative damage to vital cell structures [1]. To overcome the challenge posed by the safe acquisition, transport, and storage of Fe, specialized mechanisms and molecules
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Abbreviations used: BSA, bovine serum albumin; BSO, buthionine sulfoximine; DFO, desferrioxamine; EPA, eicosapentenoic acid; FBS, fetal bovine serum; GSH, glutathione; INH, isonicotinic acid hydrazide; MDA, malondialdehyde; MTS, 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; PIH, pyridoxal isonicotinoyl hydrazone; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid.
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have evolved to meet cellular and organismal iron requirements. b-Thalassemia, caused by decreased or absent synthesis of b-globin which causes ineffective erythropoiesis, reduces the lifespan of erythrocytes. Severe b-thalassemia requires chronic blood transfusion as a supply of functional erythrocytes, which results in secondary Fe overload due to the absence of a physiological mechanism for its excretion. Subcutaneous infusion of desferrioxamine (DFO), a Fe chelator, is the standard pharmacotherapy to prevent toxic accumulation of Fe in the liver and other organs. Since this treatment is costly and inconvenient, there is an ongoing search for an effective orally available Fe chelator. Pyridoxal isonicotinoyl hydrazone (PIH), the structure of which is shown in Fig. 1, is effective orally [2], causes Fe excretion in vivo at non-toxic doses [2,3], and mobilizes 59 Fe from cells in vitro [4–7]. The study of PIH analogs, therefore, is of value for the development of other orally available chelators. A recent study of the mechanism of action of PIH analogs demonstrated that the relationship between the lipophilicity and effectiveness of these chelators at mobilizing 59 Fe from cells is due to relatively high intracellular accumulation of the most lipophilic 59 Fe– chelator complexes [8]. The toxicity of PIH analogs, mediated by the induction of apoptosis [9], exhibits the same structure–activity relationship as observed for their 59 Fe mobilization [10], suggesting that the intra-
cellular levels of Fe–chelator complexes are, in part, responsible for the toxicity of the chelators [9,10]. Redox cycling of Fe is a well-known mechanism of initiation and propagation of oxidative stress [1]. Ferrous ion participates in the Fenton reaction: Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH
ð1Þ
and is regenerated by cellular reductants: Fe3þ þ e ! Fe2þ
ð2Þ
The contribution of oxidative stress to the overall mechanism of toxicity of PIH analogs and their Fe complexes is of interest, especially since some of these compounds have been shown to limit the participation of Fe in producing oxidative damage [11–15]. In this study, induction of oxidative stress by a series of PIH analogs is examined.
Materials and methods PIH analogs and their Fe3þ complexes PIH analogs were synthesized by the condensation of the corresponding aldehydes and hydrazides as described [16]. Fe complexes were formed by the addition of FeCl3 (Fisher, Fair Lawn, NJ), dissolved in sodium citrate, and chelator, dissolved in NaOH, in a 1:2 molar ratio, and incubation at room temperature for 1 h following neutralization [10]. Toxicity Jurkat T lymphocytes and K562 cells were cultured in phenol-red-free RPMI-1640 (Cellgro, Herndon, VA) supplemented with 10% FBS and 2 mM glutamine (Gibco, Burlington, Canada). Experiments were performed using log phase cultures. For 72 h toxicity experiments, Jurkat cells were seeded at 20,000 cells/well in 96-well plates, with a final volume of 200 lL. Cell viability was measured using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium) assay (Promega, Madison, WI) [10] in 96-well plates, with a final concentration of 0.14 mg/mL MTS. Plates were analyzed at 490 nm, and corrected for background absorbance at 630 nm, on an ELx 800 microplate reader (Bio-tek Instruments, Winooski, VT) and, when necessary, corrected for the absorbance of the Fe–chelator complexes. Effect of pO2 on toxicity of PIH analogs
Fig. 1. Structures of the PIH analogs. Pyridoxal isonicotinoyl hydrazone (PIH), Pyridoxal benzoyl hydrazone (PBH), salicylaldehyde isonicotinoyl hydrazone (SIH), salicylaldehyde benzoyl hydrazone (SBH), 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH), and 2-hydroxy-1-naphthaldehyde benzoyl hydrazone (NBH).
Cells were seeded at 0.5 million cells/mL in RPMI containing 10% fetal bovine serum (FBS) and incubated in a Haereus B 5060 EC/O2 incubator (8, 20, 40% O2 ) or a modular incubator chamber filled with 95% N2 and
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5% CO2 (0% O2 ). Phosphatidylserine externalization, a marker of apoptosis, was measured by the binding of annexin-V-FITC (PharMingen) [17]. Briefly, cells were washed in PBS, resuspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2 ), incubated for 20 min at room temperature with 2 lL annexin-V-FITC (PharMingen), and analyzed by flow cytometry using a FACScan instrument (Becton– Dickinson). Since control cells contained a small percentage of cells with high fluorescence, the gate was set on the control cells to encompass the approximately 95% of cells in the low-fluorescence peak. Cells with fluorescence above this range were considered apoptotic. EPA-loaded K562 cells The naturally occurring fatty acid, eicosapentenoic acid (EPA, Aldrich, Oakville, Canada), was dissolved in 0.1 M NaOH, stored at )20 °C for a maximum of four weeks, and diluted in RPMI/FBS and neutralized immediately prior to its use. For experiments using [EPA] > 200 lM, EPA was incubated with bovine serum albumin (BSA, Sigma, Oakland, Canada) dissolved in RPMI at a 4:1 ratio prior to dilution in RPMI/FBS to allow controlled delivery of EPA to the cells. For experiments using [EPA] of 200 lM or less, EPA was incubated directly in RPMI/FBS, since the concentration of BSA in the FBS results in an EPA:BSA ratio of at least 4:1. No toxicity was observed under any conditions of EPA loading, either by the MTS assay or by trypan blue exclusion. No difference in doubling time was observed between control cells and EPA-loaded cells. Nonesterified fatty acids in the medium were determined using a kit (Calbiochem, San Diego, CA). Spectrophotometric data were collected at 25 °C using a Cary 1 spectrophotometer. Lipid peroxidation assays Thiobarbituric acid reactive substances (TBARS) were determined by the addition of 200 lL of 50% trichloroacetic acid (TCA) and 235 lL of 0.67% thiobarbituric acid (TBA) dissolved in 1 M NaOH to a 500 lL sample, resulting in a pH between 2 and 3, which is necessary for optimum detection of malondialdehyde (MDA) [18]. After boiling for 15 min, the difference in absorbance between 532 and 700 nm was determined. The assay for MDA using 1-methyl-2-phenylindole (Aldrich) was essentially as described [19], adding 1950 lL of 0.12% 1-methyl-2-phenylindole in 25% methanol/acetonitrile and 780 lL concentrated HCl to a 900 lL sample, incubating at 45 °C for 90 min, and determining the difference in absorbance between 586 and 700 nm. Addition of PIH analogs or their Fe complexes to identical samples immediately prior to boiling them had no measurable effect on MDA detection, indicating
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that these species neither promote nor inhibit the degradation of lipid hydroperoxides to MDA, which may be present in the samples. GSH assay Glutathione (GSH, Sigma) was determined in 96-well plates at 405 nm [20]. Data were corrected by subtracting the background absorbance at 630 nm. Protein was precipitated from samples with 5% sulfosalicylic acid, which were then centrifuged. Equal volumes of the reagent, a 3:1 mixture of 1.2 M Na2 HPO4 and 0.2 mg/mL of 5,50 -dithiobis (2-nitrobenzoic acid, Sigma) in 1% sodium citrate, and the sample were added to the wells, mixed, and incubated for 5 min. Ascorbate oxidation assay Measurement of Fe-catalyzed ascorbic acid (Sigma) oxidation was essentially as described [21]. Experiments were performed in chelexed phosphate-buffered saline (PBS), prepared by overnight stirring of a 10% slurry of chelex-100 (Bio-Rad) in PBS. Aliquots of 5 lL of fresh stock solutions of 20 mM ascorbic acid were added to 995 lL samples. Pseudo-first-order rate constants, determined from initial rates of oxidation of ascorbic acid, were calculated from spectrophotometric data collected over 20 min at 265 nm. Following the entire course of the reaction demonstrated complete oxidation of ascorbate (100 lM) by the Fe complexes (25 lM), indicating that the complexes were catalytically, and not stoichiometrically, degrading the ascorbate. Ferrozine assay Reduction of ferric complexes of PIH analogs by 2 106 /mL K562 cells was determined in PBS over 4 h using 50 lL of 0.017 g/mL ferrozine (Sigma) and 950 lL sample. In one series of experiments, ferrozine was added after centrifugation of the cells to measure [Fe2þ ] present at the end of the incubation, and samples were incubated at 37 °C for 30 min. In a second series, ferrozine was added with the Fe–chelator complexes, to bind all Fe2þ formed during the 4 h incubation period. Data were analyzed spectrophotometrically by determining the difference in absorbance between 562 and 700 nm.
Results Toxicity of PIH analogs and their Fe3þ complexes; effect of GSH depletion The toxicity of PIH analogs, the structures of which are shown in Fig. 1, and their Fe3þ complexes toward
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Table 1 Effect of BSO on the toxicity of PIH analogs and their Fe3þ complexes toward Jurkat T lymphocytesa Fe3þ complexes
Chelators Control IC50 (lM) PIH PBH SIH SBH NIH NBH
45 6 20 4 5.5 1.4 0.76 0.07 1.0 0.16 1.2 0.4
Low GSH IC50 (lM) 41 4 14 2 1.5 0.6 0.41 0.02 0.69 0.11 0.56 0.1
Control IC50 (lM) b
>150 60 4 67 12 0.37 0.02 82 11 NDc
Low GSH IC50 (lM) 19 2 31 7 0.44 0.13 0.19 0.01 2.3 0.4 NDc
a
Cells were incubated for 72 h as described in Materials and methods. Data are averages standard deviations (n ¼ 2–13). Solubility limit of the complex in RPMI/FBS. c Not determined. b
Jurkat T lymphocytes was determined after 72 h incubation (Table 1). The structure–activity relationships describing the toxicity of PIH analogs in Jurkat cells were similar to those in K562 cells [10] and SK-N-MC cells [22], suggesting a common mechanism of action of these iron chelators among different cell types. Toxicity increased with the lipophilicity of the analogs, as has been observed for K562 cells [10]. With the exception of Fe(SBH)2 , the Fe complexes were significantly less toxic than the corresponding free chelators. Buthionine sulfoximine (BSO) depletes the important cellular antioxidant, GSH, by inhibiting c-glutamyl transpeptidase [23], an enzyme in the biosynthetic pathway of GSH. Jurkat T cells grow normally in the presence of 20 lM BSO, although their GSH levels are greatly reduced: control cells had 77 2 nmol GSH/106 cells, whereas cells treated for 6 and 72 h with BSO had 27 0.6 and 2.7 0.2 nmol GSH/106 cells, respectively. GSH-depleted Jurkat cells were significantly (p < 0:05) more sensitive to the Fe complexes of PIH analogs than control cells (Table 1). The extent of this effect ranged from approximately twofold for Fe(PBH)2 and
Fe(SBH)2 to 150-fold for Fe(SIH)2 . GSH-depleted Jurkat cells were also significantly (p < 0:05) more sensitive to the PIH analogs themselves than control cells (Table 1), although sensitization of Jurkat cells to the chelators by GSH depletion was much weaker than for the corresponding Fe complexes. When all determinations were compared, there was no significant difference between the PIH IC50 values for control and GSH-depleted cells (Table 1). However, a paired t test of IC50 values determined within single experiments was significant (p < 0:05), indicating that exacerbation of PIH toxicity by GSH depletion is weak, but reproducible. Effect of oxygen tension on toxicity of PIH analogs The effect of oxygen tension (pO2 ) on the induction of apoptosis by PIH, SIH, and NIH was assessed at 100, 20, and 2 lM, respectively, concentrations which caused significant toxicity under normal cell culture conditions (Table 1). The increase in phosphatidylserine externalization for all three analogs with respect to pO2 is shown in Fig. 2. At 0% pO2 , toxicity of PIH and SIH is nearly
Fig. 2. Enhancement of toxicity of PIH analogs by O2 . Jurkat T lymphocytes were treated with 100 lM PIH (A), 20 lM SIH (B), and 2 lM NIH (C) for 24 (d) or 48 (s) h in the presence of varying pO2 . Apoptosis was measured by FITC-annexin V binding. The percentages of dead control cells at the corresponding pO2 and incubation times were subtracted from the data. Symbols and error bars represent means and standard deviations, respectively, of data collected in triplicate.
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zero after a 24 h incubation. In contrast, at 0% pO2 , NIH causes induction of apoptosis in approximately 20 and 35% of the cells at 24 and 48 h, respectively, suggesting that NIH may also cause toxicity by non-oxidative mechanisms. At 20% pO2 , to which cultured cells are normally exposed, toxicity is maximal for all three analogs. Interference of PIH analogs with the TBARS assay Measurement of lipid peroxidation by detection of the lipid oxidation product, MDA, is a simple assay to further evaluate the role of oxidative stress in the toxicity of PIH analogs [18]. However, both PIH and Fe(PIH)2 inhibit the formation of the TBARS product, causing underestimation of the amount of MDA detected by this assay. Inhibition of MDA detection by Fe(PIH)2 , PIH, and isonicotinic acid hydrazide (INH), the hydrolysis product of PIH, was linear over the concentration range 0–50 lM; concentrations resulting in 10% inhibition were 34, 20, and 16 lM, respectively. Since INH had an inhibitory effect stronger than that of PIH, it is likely that hydrolysis of PIH [24,25] during the conjugation of MDA to TBA produces INH, which competes with TBA for MDA. Inhibition of TBARS formation by PIH to a greater extent than its antioxidant activity in other assays has been previously observed [13], likely due to the underestimation of MDA in the presence of PIH analogs. As a result of interference between PIH analogs in the TBARS assay, another method of detecting lipid peroxidation by measuring MDA formation was assessed, which uses a different chromophore, 1-methyl-2-phenylindole, a different solvent system, and a lower incubation temperature [19]. Since 90% of 5 lM MDA was detected by this assay in the presence of 200 lM PIH, the most readily hydrolyzed analog in the series [26], this method is suitable for measuring lipid peroxidation in the presence of PIH analogs.
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linear, reaching 25 nmol/106 cells by 16 h. EPA uptake during a 6 h incubation was saturable, reaching halfmaximal levels at 90 lM. There was no detectable increase in lipid peroxidation in EPA-loaded cells (Fig. 3), indicating that the antioxidant defenses of the cell are sufficient to repair any damage to the more susceptible lipids. In the presence of 50 lM Fe(PBH)2 , cell viability decreased and lipid peroxidation increased with [EPA] (Fig. 3), whereas Fe(PBH)2 did not oxidize EPA in a cell-free system (data not shown). To assess the relationship between oxidative stress and toxicity induced by Fe complexes of PIH analogs, K562 cells were incubated with 200 lM EPA for 6 h, as these conditions allow maximal detection of lipid peroxidation, with no EPA-induced toxicity (Fig. 3). Uptake of EPA under these conditions was 19 nmol/106 cells. Fe(PIH)2 was not toxic toward K562 cells, whether or not they were EPA-loaded (Fig. 4A), even at concentrations as high as 150 lM, the limit of its solubility in RPMI/FBS. In contrast, Fe(PBH)2 and Fe(SIH)2 were toxic toward EPA-loaded (Fig. 4A), but not control, cells. For these two complexes, cell viability and lipid peroxidation were inversely correlated (Figs. 4A and B). The lipophilic antioxidant, vitamin E, added at the same time as Fe(PBH)2 , completely prevented both effects (Figs. 4A and B). Since no toxicity was observed on incubation of EPAloaded cells with any PIH analogs for 24 h, a 48 h incubation period was used to assess the effects of the free chelators (Figs. 4C and D). PIH was not toxic toward K562 cells (48 h) at concentrations as high as 500 lM, whether or not they were loaded with EPA (Fig. 4C), and caused no lipid peroxidation (Fig. 4D). At concentrations
Relationship of lipid peroxidation and toxicity of PIH analogs and their Fe3þ complexes Simultaneous measurement of toxicity and lipid peroxidation in K562 cells (which produce undetectable levels of MDA in the lipid peroxidation assay) was achieved by loading cells with eicosapentenoic acid (EPA), a relatively rare, polyunsaturated fatty acid (PUFA). PUFA-loaded cells have frequently been used to facilitate detection of oxidative damage [27–31]. EPA was bound to BSA to allow its controlled delivery to the cells [32] and its uptake was determined by its depletion in the medium following incubation. In the presence of 200 lM EPA, the kinetics of its uptake by K562 cells were biphasic. Approximately 15 nmol/106 cells was taken up in the first hour, following which uptake was
Fig. 3. Toxicity and lipid peroxidation of EPA-loaded K562 cells by Fe(PBH)2 . Cells were incubated with EPA in RPMI/FBS for 6 h, washed, and incubated in RPMI/FBS with (filled symbols) or without (hollow symbols) 50 lM Fe(PBH)2 for 24 h. Cell viability (circles), measured by the MTS assay, and lipid peroxidation (triangles), measured using 1-methyl-2-phenylindole, represent means of triplicate samples and error bars represent standard deviations.
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A
B
C
D
Table 2 GSH levels and lipid peroxidation in EPA-loaded cellsa GSH (nmol/106 cells)
Lipid peroxidation (nmol/106 cells)
Cells + control/6 h Cells + EPA/6 h
83 1 71 2
NDb ND
Cells + control/30 h Cells + EPA/6 h + control/24 h Cells + EPA/6 h + 18 lM Fe(PBH)2 /24 h Cells + EPA/6 h + 32 lM Fe(PBH)2 /24 h Cells + EPA/6 h + 50 lM Fe(PBH)2 /24 h
72 0.9 67 0.3 55 1.1
ND 0.00 0.01 0.27 0.009
42 1.7
0.97 0.02
6.6 0.2
5.72 0.07
63 0.1 62 0.3 60 0.4
ND 0.00 0.002 0.37 0.007
Cells + control/54 h Cells + EPA/6 h + control/48 h Cells + EPA/6 h + 50 lM PBH/48 h
a Cells were incubated in RPMI/FBS (control) or 200 lM EPA for 6 h, washed, and incubated in RPMI/FBS (control), Fe(PBH)2 , or PBH as indicated. b Not determined.
Fig. 4. Toxicity and lipid peroxidation of EPA-loaded K562 cells by PIH analogs and their Fe complexes. Cells were incubated with 200 lM EPA for 6 h and washed. Toxicity (A) and lipid peroxidation (B) were measured after incubation for 24 h with Fe(PIH)2 (d), Fe(PBH)2 (.), and Fe(SIH)2 (j). The curves in (A) represent weighted, least-squares fits of the data to a three-parameter logistic equation; the IC50 values from these fits were 22 0.5 and 16 1 lM for Fe(PBH)2 and Fe(SIH)2 , respectively. The presence of 1 lM vitamin E during the 24 h incubation prevented both the toxicity and the lipid peroxidation caused by Fe(PBH)2 (r). Neither toxicity nor lipid peroxidation caused by the Fe complexes was detected in cells which were not EPA-loaded. Toxicity (C) and lipid peroxidation (D) were measured after incubation for 48 h with PIH (d), PBH (m), and SIH (j). Toxicity, but not lipid peroxidation, caused by SIH () was observed in cells, which were not EPA-loaded. Cell viability was measured by the MTS assay and lipid peroxidation was measured using 1-methyl-2phenylindole. Symbols and error bars represent means and standard deviations of triplicate measurements.
below 20 lM, PBH caused lipid peroxidation and was toxic toward EPA-loaded, but not control, cells (Figs. 4C and D). Above 20 lM, PBH reversed its own toxicity and lipid peroxidation. SIH was equally toxic toward EPAloaded and control cells (Fig. 4C), but caused no lipid peroxidation (Fig. 4D). GSH levels in EPA-loaded cells were correlated with lipid peroxidation (Table 2). Immediately following EPA loading, GSH levels were decreased from 83 1 to 71 2 nmol/106 cells. After 24 h, GSH levels of EPAtreated cells had partially recovered. Treatment with Fe(PBH)2 exacerbated the decrease in a concentrationdependent manner; incubation with 50 lM Fe(PBH)2
for 24 h caused GSH levels to decline to 6.6 0.2 nmol/ 106 cells, which was associated with significant lipid peroxidation, as observed in Fig. 4. After 48 h, GSH levels had completely recovered. Treatment with PBH for 48 h did not greatly affect GSH levels, but caused a small amount of lipid peroxidation. Of the compounds tested, Fe(PBH)2 , Fe(SIH)2 , and PBH caused concomitant toxicity and lipid peroxidation in EPA-loaded K562 cells (Fig. 4). Despite the differences in concentrations and incubation times at which these effects were assessed, the dependence of toxicity on lipid peroxidation was similar for all the above species (Fig. 5); lipid peroxidation of 3.3 nmol/106 cells corresponded to 50% toxicity. Hydrogen peroxide, which caused 50% toxicity in 24 h at a concentration of approximately 1.4 mM in both control and EPA-loaded cells, caused lower levels of lipid peroxidation than Fe(PBH)2 , Fe(SIH)2 , and PBH (Fig. 5). Vitamin E (a-tocopherol) completely prevented the lipid peroxidation, but only partially prevented the toxicity, caused by hydrogen peroxide (data not shown), likely because the lipophilic antioxidant, vitamin E, is unlikely to be found in the same cellular compartments. Reduction of Fe3þ complexes of PIH analogs by cells The Fe complexes of PIH analogs, at concentrations of 25 lM, oxidized ascorbate with pseudo-first-order kinetics. The rate constants varied, with no apparent structure–activity relationship, from 1.0 0.08 to 4.7 0.2 103 min1 for Fe(SBH)2 and Fe(NIH)2 , respectively, indicating that reduction of these complexes by physiological species is possible. Reduction of Fe3þ
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Table 3 Reduction of Fe complexes of PIH analogs by K562 cellsa
Fe(PIH)2 Fe(PBH)2 Fe(SIH)2 FeEDTA
Fe reduced ) ferrozineb (pmole/106 cells/h)
Fe reduced + ferrozinec (pmole/106 cells/h)
16 2 21 2 41 50 4
150 8 153 7 90 20 220 20
a Incubations of 2 106 cells per sample were for 4 h at 37 C in 1 mL PBS, in the presence of 10 lM Fe complex. Data are means of triplicate samples and errors are standard deviations. b Ferrozine was added after cells were centrifuged from the samples, as described in Materials and methods. c Ferrozine was added at the beginning of the 4 h incubation, as described in Materials and methods.
Fig. 5. Dose-dependence of chelator- and complex-mediated toxicity on lipid peroxidation. Fe(PIH)2 (d), Fe(PBH)2 (.), Fe(SIH)2 (j), and PBH (r) elicit the same relationship between toxicity and lipid peroxidation. The IC50 from the weighted, least-squares fit of these data to a three-parameter logistic equation (solid line) corresponds to 3.3 nmol MDA/106 cells. The IC50 for 24 h H2 O2 treatment (r), 1.4 mM, corresponds to 0.7 nmol MDA/106 cells.
complexes of PIH analogs by K562 cells was measured using the ferrozine assay (Table 3). When ferrozine was added to the supernatant of cells incubated for 4 h with the Fe complexes of PIH analogs, low levels of Fe2þ were detected. In the absence of cells, no Fe3þ reduction occurred with any of the complexes. Since it is likely that, under these conditions, some of the Fe2þ produced is re-oxidized to Fe3þ by dissolved O2 , these values represent minimum levels of Fe3þ reduction by cells. When ferrozine was included in the incubation medium with the Fe complexes, the amounts of Fe2þ detected were four- to 20-fold higher (Table 3). Since it is possible that, under these conditions, the presence of ferrozine, a Fe2þ chelator, shifted the Fe3þ –Fe2þ equilibrium toward Fe reduction, these values represent maximum amounts of Fe3þ reduction by cells. By comparison to EDTA, a complex well known to redox cycle under physiological conditions, it is apparent that Fe complexes of PIH analogs are less readily reduced by cells.
Discussion Redox cycling by Fe complexes of PIH analogs K562 cells reduce the Fe complexes of PIH analogs (Table 3), as does the ubiquitous cellular reductant, ascorbate. In contrast, cyclic voltammetry of Fe(SBH)2 [33] and Fe(NIH)2 [34] demonstrated the absence of a reversible redox potential in the biologically accessible range; from these data, it was concluded that reduction of these complexes by cells is not possible. It may be, however, that these experiments were confounded by the relatively slow kinetics of ligand exchange characteristic
of the Fe3þ complexes of these ligands. It has been demonstrated that Fe2þ forms a complex with PIH almost immediately [11], whereas complex formation of PIH with Fe3þ requires minutes or hours to achieve completion [8], depending on the concentrations of the two species. It is therefore likely that the complexes are not reduced quickly enough to allow the identification of a redox potential by this method. The capacity of these complexes to redox cycle in biological systems provides a compelling explanation for the lipid peroxidation, GSH depletion, and toxicity of PIH analogs and their Fe complexes in EPA-loaded K562 cells (Figs. 3–5, Table 2), as well as the sensitization of Jurkat cells to these species by high pO2 (Fig. 2) and GSH depletion (Table 1). However, it is also possible that dissociation of Fe from the chelators precedes its reduction, since both FePIH2 [35] and FeSIH2 [36] are known to donate Fe to cells in vitro. The donation of Fe to human T-lymphocytes by Fe(PIH)2 has been demonstrated to be non-specific, causing the accumulation of Fe in non-physiological pools [37]. Therefore, it is not possible, from the results presented here, to determine whether the oxidative stress induced by PIH analogs and their Fe complexes is due to redox cycling of the complexes themselves, or to donation of Fe to cellular ligands which redox cycle. It is also important to consider the possibility that mixed ligand complexes are formed, in which one of the two coordinating chelator molecules is replaced by a cellular ligand. The redox properties of such complexes, whether they exist, are unknown. A series of studies of PIH analogs [11,13,14] have demonstrated their capacity to inhibit redox cycling of Fe-EDTA in vitro. Likely, this is due to formation of Fe–chelator complexes, which have slower kinetics of reduction (i.e., Reaction 2) than Fe-EDTA. While PIH analogs are less redox active than Fe-EDTA (Table 3 and [11,13,14]), and therefore act as antioxidants in its presence, the conclusion that Fe complexes of PIH analogs cannot redox cycle [13–15] is not supported by the present results.
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Contribution of oxidative stress to the toxicity of PIH analogs GSH-depleted Jurkat cells are more sensitive to both PIH analogs and their Fe complexes (Table 1). Therefore GSH, which supplies reducing equivalents for enzymes involved in the antioxidant response such as GSH-S-transferase and GSH peroxidases, plays a role in protecting Jurkat cells against the toxicity of these compounds, implicating oxidative stress in their mechanisms of toxicity. The sensitization of Jurkat cells to PIH analogs by increased pO2 further supports the role of oxidative stress in the toxicity of this series of chelators. It has been proposed that, since exposure of cells to the free chelators results in the formation of intracellular Fe–chelator complexes, the complexes are, in part, responsible for the toxicity of the chelators [8–10]. The Fe complexes redox cycle (Table 3), causing oxidative stress (Table 2, Figs. 3–5), which contributes to their toxicity, and that of the free chelators. That the sensitization of Jurkat cells to the chelators by GSH depletion is weaker than the corresponding Fe complexes (Table 1) suggests that multiple mechanisms of toxicity, including Fe depletion, are also involved in the toxicity of PIH analogs. The concomitant lipid peroxidation and toxicity caused by Fe(PBH)2 (Fig. 4) and hydrogen peroxide (Fig. 5), and the reversal of these effects by vitamin E, demonstrate that oxidative stress is a causal event in the toxicity of these compounds. Because of the effectiveness of vitamin E in protection against the toxicity of Fe(PBH)2 , which is lipophilic [10,38], it is likely that the target of oxidative stress is localized to a membrane. However, it cannot be concluded that lipid peroxidation, and not some other, possibly more specific, form of oxidative stress, causes the toxicity. Lipid peroxidation may simply serve as a reporter for damage to other cellular structures. It is noteworthy that the lipophilic antioxidant, vitamin E, completely abolished the toxicity of Fe(PBH)2 in EPA-loaded K562 cells, while it only partially prevented the toxicity of H2 O2 , a polar oxidant. Thus, in addition to lipid peroxidation, H2 O2 likely causes oxidative damage to polar structures, which may not be well protected by vitamin E. Lipid peroxidation certainly does not represent all of the oxidative damage caused by H2 O2 , and may not do so for Fe(PBH)2 , either. Depletion of cellular GSH levels, while also reflecting oxidative stress (Table 2), similarly does not appear to be the specific cause of toxicity. Since treatment of K562 cells with 20 lM BSO for 72 h caused GSH levels to drop to 2.7 0.2 nmol/106 cells without evidence of toxicity, it is unlikely that GSH depletion per se (which reached a minimum level of 6.6 0.2 nmol/106 cells following Fe(PBH)2 treatment, Table 2) is the cause of the observed toxicity and lipid peroxidation by Fe(PBH)2 and Fe(SIH)2 .
While Fe(PBH)2 and Fe(SIH)2 caused toxicity and lipid peroxidation in EPA-loaded cells, Fe(PIH)2 did not. However, the toxicity of PIH and Fe(PIH)2 toward Jurkat cells was enhanced by GSH depletion (Table 1) and toxicity of PIH was reduced by low pO2 (Fig. 2). Furthermore, Fe(PIH)2 increased glutathione peroxidase mRNA levels in MEL cells [39], suggesting induction of oxidative stress. Therefore, the cellular effects of both PIH and its Fe complex are sensitive to the redox state of the cell, as are those of the other PIH analogs. Since Fe(PIH)2 can be reduced by ascorbate and cells (Table 3) as readily as Fe complexes of its analogs, its redox chemistry is unlikely to account for the insensitivity of Fe(PIH)2 to EPA loading. An explanation may be found in the lower lipophilicity of Fe(PIH)2 in comparison with the other Fe complexes [8,38], which results in its weaker association with the cell membrane [8], and possibly lower intracellular accumulation. Modulation of toxicity of PIH analogs by oxygen tension The oxygen tension at which cultured cells are routinely grown (20% O2 ) exacerbated the toxicity of PIH analogs (Fig. 2). This effect could be partly due to changes in the regulation of redox-sensitive proteins, but the simplest explanation is that reduced oxygen tension causes lower production of reactive oxygen species (ROS), which initiate Fe-catalyzed oxidative stress. Since initiation of oxidative stress by oxidation of Fe2þ by O2 may be a more significant pathway than by preexisting hydrogen peroxide [40], lower pO2 should directly limit the production of ROS. That many PIH analogs are safely administered to rats at high doses (0.2 mmol/kg, [2]) without toxicity, despite their toxicity in vitro, may be explained in part by the much lower pO2 in tissues (2–5%) than in the atmosphere in which cultured cells are grown. Certainly, other factors, including pharmacokinetics, metabolism, and hydrolysis, also contribute to the exposure of tissues to PIH analogs in vivo. Conclusions While the development of PIH analogs for use in treating secondary iron overload has so far included few studies in vivo, the small number of carefully chosen compounds selected for animal studies has been effective at doses which do not cause significant adverse effects [2]. The basis for their selection was their efficiency in causing 59 Fe release from a series of cell culture models [41], which reflect important targets of iron chelation in vivo. Since the completion of that study, it has been demonstrated that the vast differences in the efficacy of these chelators lie in their abilities to translocate the 59 Fe–chelator complexes out of the cell. Because the most effective analogs, which cause minimal intracellular
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accumulation of 59 Fe–chelator complexes [8,10] are, fortuitously, also the least toxic [10], the selection of analogs for in vivo study based on mobilization of 59 Fe in vitro appears to be sound. These data underscore the importance of evaluating the capacity to induce oxidative stress of Fe chelators with therapeutic potential, since Fe-overloaded patients receiving chelator therapy are already at risk for oxidative stress [1]. The relative contributions of Fe depletion and oxidative stress to the overall mechanism of toxicity of PIH analogs may be altered in Fe-overloaded tissues. It is possible that, in the presence of the same concentration of chelator, toxicity due to Fe depletion is lower, and toxicity due to accumulation of Fe–chelator complexes is higher, than in non-overloaded tissues. There is increasing interest in the use of some of the more toxic PIH analogs, particularly NIH, for the treatment of cancer. While there are some encouraging reports of their effectiveness in treating neuroblastoma cells in vitro [42], which are highly sensitive to Fe depletion, the mechanism of its action will require further study. NIH is among the most toxic PIH analogs in vitro [9,10,22], and NIH-treated, 59 Fe-labeled cells accumulate high levels of intracellular 59 FeNIH2 [9]. In addition to Fe depletion, the mechanism of toxicity of NIH includes an oxidative component (Table 1), which may contribute to its effectiveness in treating cancer, in which oxidative damage to DNA and other cell structures may be considered advantageous.
Acknowledgments This work was supported by grants from the Canadian Institutes for Health Research (P.P.) and University of Linkoping Grant #83081030 (J.N.), and postdoctoral fellowships from the National CooleyÕs Anemia Foundation and the Thalassemia Foundation of Canada (J.L.B.).
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[8] J.L. Buss, E. Arduini, P. Ponka, Biochem. Pharmacol. 64 (2002) 1689–1701. [9] J.L. Buss, J. Neuzil, N. Gellert, C. Weber, P. Ponka, Biochem. Pharmacol. 65 (2003) 161–172. [10] J.L. Buss, E. Arduini, K.C. Shephard, P. Ponka, Biochem. Pharmacol. 65 (2003) 349–360. [11] M. Hermes-Lima, N.C. Santos, J. Yan, M. Andrews, H.M. Schulman, P. Ponka, Biochim. Biophys. Acta 1426 (1999) 475– 482. [12] M. Hermes-Lima, E. Nagy, P. Ponka, H.M. Schulman, Free Radic. Biol. Med. 25 (1998) 875–880. [13] H.M. Schulman, M. Hermes-Lima, E.M. Wang, P. Ponka, Redox Rep. 1 (1995) 373–378. [14] M. Hermes-Lima, P. Ponka, H.M. Schulman, Biochim. Biophys. Acta 1523 (2000) 154–160. [15] N.C. Santos, R.F. Castilho, A.R. Meinicke, M. Hermes-Lima, Eur. J. Pharmacol. 428 (2001) 37–44. [16] J.T. Edward, M. Gauthier, F.L. Chubb, P. Ponka, J. Chem. Eng. Data 33 (1988) 538–540. [17] J. Neuzil, I. Svensson, T. Weber, C. Weber, U.T. Brunk, FEBS Lett. 445 (1999) 295–300. [18] R.P. Bird, H.H. Draper, Methods Enzymol. 105 (1984) 299–305. [19] E.K. Lund, S.J. Fairweather-Tait, S.G. Wharf, I.T. Johnson, J. Nutr. 131 (2001) 2928–2931. [20] M.E. Anderson, Methods Enzymol. 113 (1985) 548–555. [21] G.R. Buettner, Methods Enzymol. 186 (1990) 125–127. [22] D.R. Richardson, E.H. Tran, P. Ponka, Blood 86 (1995) 4295– 4306. [23] A. Meister, Methods Enzymol. 252 (1995) 26–30. [24] N.J. Lees-Gayed, M.A. Abou-Taleb, I.A. El-Bitash, M.F. Iskander, J. Chem. Soc. Perkin 2 (1992) 213–217. [25] D.R. Richardson, L.W. Vitolo, E. Baker, J. Webb, Biol. Met. 2 (1989) 69–76. [26] J.L. Buss, P. Ponka, Biochim. Biophys. Acta 1619 (2003) 177–186. [27] J. Naval, M.J. Martinez-Lorenzo, I. Marzo, P. Desportes, A. Pineiro, Biochem. J. 291 (1993) 841–845. [28] B.A. Wagner, G.R. Buettner, C.P. Burns, Arch. Biochem. Biophys. 334 (1996) 261–267. [29] N. Sugihara, T. Arakawa, M. Ohnishi, K. Furuno, Free Radic. Biol. Med. 27 (1999) 1313–1323. [30] S.Y. Qian, H.P. Wang, F.Q. Schafer, G.R. Buettner, Free Radic. Biol. Med. 29 (2000) 568–579. [31] F.Q. Schafer, S.Y. Qian, G.R. Buettner, Cell Mol. Biol. 46 (2000) 657–662. [32] N. Sugihara, Y. Tsuruta, Y. Date, K. Furuno, K. Kohashi, Toxicol. Appl. Pharmacol. 126 (1994) 124–128. [33] N.R. Sangeetha, S. Pal, J. Coord. Chem. 42 (1997) 157–163. [34] D.R. Richardson, P.V. Bernhardt, J. Biol. Inorg. Chem. 4 (1999) 266–273. [35] P. Ponka, H.M. Schulman, A. Wilczynska, Biochim. Biophys. Acta 718 (1982) 151–156. [36] L.M. Garrick, K. Gniecko, J.E. Hoke, A. al-Nakeeb, P. Ponka, M.D. Garrick, J. Cell. Physiol. 146 (1991) 460–465. [37] L.L. Koh, O.L. Kon, K.W. Loh, Y.C. Long, J.D. Ranford, A.L. Tan, Y.Y. Tjan, J. Inorg. Biochem. 72 (1998) 155–162. [38] J.T. Edward, P. Ponka, D.R. Richardson, Biometals 8 (1995) 209– 217. [39] O. Fuchs, Neoplasma 44 (1997) 184–191. [40] S.Y. Qian, G.R. Buettner, Free Radic. Biol. Med. 26 (1999) 1447– 1456. [41] P. Ponka, D. Richardson, E. Baker, H.M. Schulman, J.T. Edward, Biochim. Biophys. Acta 967 (1988) 122–129. [42] D.B. Lovejoy, D.R. Richardson, Curr. Med. Chem. 10 (2003) 1035–1049.
Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs Joan L. Buss,a Jiri Neuzil,b,1 and Prem Ponkac,* a
c
Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC, USA b Department of Pathology II, University Hospital, Linkoping, Sweden Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and Departments of Physiology and Medicine, Que., Canada H3T 1E2 Received 21 May 2003, and in revised form 15 September 2003
Abstract Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe3þ –chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe3þ complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe3þ complexes, and toxicity of the chelators increased with oxygen tension. Fe3þ complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)2 did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)2 in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe3þ complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Oxidative stress; Pyridoxal isonicotinoyl hydrazone (PIH) analogs; Iron chelators; Toxicity; K562 cells; Jurkat T lymphocytes
Iron (Fe) is involved in many essential biological functions such as oxygen transport, electron transfer, and DNA synthesis. However, the chemical properties of iron which allow this versatility also lead to the paradoxical situation that Fe acquisition by the organism is difficult. Under physiological conditions, Fe2þ is readily oxidized to Fe3þ , which is virtually insoluble in the absence of chelators. Moreover, unless bound to specific ligands, iron plays a key role in the formation of *
Corresponding author. Fax: +514-340-7502. E-mail address: [email protected] (P. Ponka). 1 Present address: Heart Foundation Research Center, School of Health Sciences, Griffith University, Southport, Queensland, Australia. 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.09.044
reactive oxygen species (ROS2), causing oxidative damage to vital cell structures [1]. To overcome the challenge posed by the safe acquisition, transport, and storage of Fe, specialized mechanisms and molecules
2
Abbreviations used: BSA, bovine serum albumin; BSO, buthionine sulfoximine; DFO, desferrioxamine; EPA, eicosapentenoic acid; FBS, fetal bovine serum; GSH, glutathione; INH, isonicotinic acid hydrazide; MDA, malondialdehyde; MTS, 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; PIH, pyridoxal isonicotinoyl hydrazone; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid.
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have evolved to meet cellular and organismal iron requirements. b-Thalassemia, caused by decreased or absent synthesis of b-globin which causes ineffective erythropoiesis, reduces the lifespan of erythrocytes. Severe b-thalassemia requires chronic blood transfusion as a supply of functional erythrocytes, which results in secondary Fe overload due to the absence of a physiological mechanism for its excretion. Subcutaneous infusion of desferrioxamine (DFO), a Fe chelator, is the standard pharmacotherapy to prevent toxic accumulation of Fe in the liver and other organs. Since this treatment is costly and inconvenient, there is an ongoing search for an effective orally available Fe chelator. Pyridoxal isonicotinoyl hydrazone (PIH), the structure of which is shown in Fig. 1, is effective orally [2], causes Fe excretion in vivo at non-toxic doses [2,3], and mobilizes 59 Fe from cells in vitro [4–7]. The study of PIH analogs, therefore, is of value for the development of other orally available chelators. A recent study of the mechanism of action of PIH analogs demonstrated that the relationship between the lipophilicity and effectiveness of these chelators at mobilizing 59 Fe from cells is due to relatively high intracellular accumulation of the most lipophilic 59 Fe– chelator complexes [8]. The toxicity of PIH analogs, mediated by the induction of apoptosis [9], exhibits the same structure–activity relationship as observed for their 59 Fe mobilization [10], suggesting that the intra-
cellular levels of Fe–chelator complexes are, in part, responsible for the toxicity of the chelators [9,10]. Redox cycling of Fe is a well-known mechanism of initiation and propagation of oxidative stress [1]. Ferrous ion participates in the Fenton reaction: Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH
ð1Þ
and is regenerated by cellular reductants: Fe3þ þ e ! Fe2þ
ð2Þ
The contribution of oxidative stress to the overall mechanism of toxicity of PIH analogs and their Fe complexes is of interest, especially since some of these compounds have been shown to limit the participation of Fe in producing oxidative damage [11–15]. In this study, induction of oxidative stress by a series of PIH analogs is examined.
Materials and methods PIH analogs and their Fe3þ complexes PIH analogs were synthesized by the condensation of the corresponding aldehydes and hydrazides as described [16]. Fe complexes were formed by the addition of FeCl3 (Fisher, Fair Lawn, NJ), dissolved in sodium citrate, and chelator, dissolved in NaOH, in a 1:2 molar ratio, and incubation at room temperature for 1 h following neutralization [10]. Toxicity Jurkat T lymphocytes and K562 cells were cultured in phenol-red-free RPMI-1640 (Cellgro, Herndon, VA) supplemented with 10% FBS and 2 mM glutamine (Gibco, Burlington, Canada). Experiments were performed using log phase cultures. For 72 h toxicity experiments, Jurkat cells were seeded at 20,000 cells/well in 96-well plates, with a final volume of 200 lL. Cell viability was measured using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium) assay (Promega, Madison, WI) [10] in 96-well plates, with a final concentration of 0.14 mg/mL MTS. Plates were analyzed at 490 nm, and corrected for background absorbance at 630 nm, on an ELx 800 microplate reader (Bio-tek Instruments, Winooski, VT) and, when necessary, corrected for the absorbance of the Fe–chelator complexes. Effect of pO2 on toxicity of PIH analogs
Fig. 1. Structures of the PIH analogs. Pyridoxal isonicotinoyl hydrazone (PIH), Pyridoxal benzoyl hydrazone (PBH), salicylaldehyde isonicotinoyl hydrazone (SIH), salicylaldehyde benzoyl hydrazone (SBH), 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH), and 2-hydroxy-1-naphthaldehyde benzoyl hydrazone (NBH).
Cells were seeded at 0.5 million cells/mL in RPMI containing 10% fetal bovine serum (FBS) and incubated in a Haereus B 5060 EC/O2 incubator (8, 20, 40% O2 ) or a modular incubator chamber filled with 95% N2 and
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5% CO2 (0% O2 ). Phosphatidylserine externalization, a marker of apoptosis, was measured by the binding of annexin-V-FITC (PharMingen) [17]. Briefly, cells were washed in PBS, resuspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2 ), incubated for 20 min at room temperature with 2 lL annexin-V-FITC (PharMingen), and analyzed by flow cytometry using a FACScan instrument (Becton– Dickinson). Since control cells contained a small percentage of cells with high fluorescence, the gate was set on the control cells to encompass the approximately 95% of cells in the low-fluorescence peak. Cells with fluorescence above this range were considered apoptotic. EPA-loaded K562 cells The naturally occurring fatty acid, eicosapentenoic acid (EPA, Aldrich, Oakville, Canada), was dissolved in 0.1 M NaOH, stored at )20 °C for a maximum of four weeks, and diluted in RPMI/FBS and neutralized immediately prior to its use. For experiments using [EPA] > 200 lM, EPA was incubated with bovine serum albumin (BSA, Sigma, Oakland, Canada) dissolved in RPMI at a 4:1 ratio prior to dilution in RPMI/FBS to allow controlled delivery of EPA to the cells. For experiments using [EPA] of 200 lM or less, EPA was incubated directly in RPMI/FBS, since the concentration of BSA in the FBS results in an EPA:BSA ratio of at least 4:1. No toxicity was observed under any conditions of EPA loading, either by the MTS assay or by trypan blue exclusion. No difference in doubling time was observed between control cells and EPA-loaded cells. Nonesterified fatty acids in the medium were determined using a kit (Calbiochem, San Diego, CA). Spectrophotometric data were collected at 25 °C using a Cary 1 spectrophotometer. Lipid peroxidation assays Thiobarbituric acid reactive substances (TBARS) were determined by the addition of 200 lL of 50% trichloroacetic acid (TCA) and 235 lL of 0.67% thiobarbituric acid (TBA) dissolved in 1 M NaOH to a 500 lL sample, resulting in a pH between 2 and 3, which is necessary for optimum detection of malondialdehyde (MDA) [18]. After boiling for 15 min, the difference in absorbance between 532 and 700 nm was determined. The assay for MDA using 1-methyl-2-phenylindole (Aldrich) was essentially as described [19], adding 1950 lL of 0.12% 1-methyl-2-phenylindole in 25% methanol/acetonitrile and 780 lL concentrated HCl to a 900 lL sample, incubating at 45 °C for 90 min, and determining the difference in absorbance between 586 and 700 nm. Addition of PIH analogs or their Fe complexes to identical samples immediately prior to boiling them had no measurable effect on MDA detection, indicating
3
that these species neither promote nor inhibit the degradation of lipid hydroperoxides to MDA, which may be present in the samples. GSH assay Glutathione (GSH, Sigma) was determined in 96-well plates at 405 nm [20]. Data were corrected by subtracting the background absorbance at 630 nm. Protein was precipitated from samples with 5% sulfosalicylic acid, which were then centrifuged. Equal volumes of the reagent, a 3:1 mixture of 1.2 M Na2 HPO4 and 0.2 mg/mL of 5,50 -dithiobis (2-nitrobenzoic acid, Sigma) in 1% sodium citrate, and the sample were added to the wells, mixed, and incubated for 5 min. Ascorbate oxidation assay Measurement of Fe-catalyzed ascorbic acid (Sigma) oxidation was essentially as described [21]. Experiments were performed in chelexed phosphate-buffered saline (PBS), prepared by overnight stirring of a 10% slurry of chelex-100 (Bio-Rad) in PBS. Aliquots of 5 lL of fresh stock solutions of 20 mM ascorbic acid were added to 995 lL samples. Pseudo-first-order rate constants, determined from initial rates of oxidation of ascorbic acid, were calculated from spectrophotometric data collected over 20 min at 265 nm. Following the entire course of the reaction demonstrated complete oxidation of ascorbate (100 lM) by the Fe complexes (25 lM), indicating that the complexes were catalytically, and not stoichiometrically, degrading the ascorbate. Ferrozine assay Reduction of ferric complexes of PIH analogs by 2 106 /mL K562 cells was determined in PBS over 4 h using 50 lL of 0.017 g/mL ferrozine (Sigma) and 950 lL sample. In one series of experiments, ferrozine was added after centrifugation of the cells to measure [Fe2þ ] present at the end of the incubation, and samples were incubated at 37 °C for 30 min. In a second series, ferrozine was added with the Fe–chelator complexes, to bind all Fe2þ formed during the 4 h incubation period. Data were analyzed spectrophotometrically by determining the difference in absorbance between 562 and 700 nm.
Results Toxicity of PIH analogs and their Fe3þ complexes; effect of GSH depletion The toxicity of PIH analogs, the structures of which are shown in Fig. 1, and their Fe3þ complexes toward
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Table 1 Effect of BSO on the toxicity of PIH analogs and their Fe3þ complexes toward Jurkat T lymphocytesa Fe3þ complexes
Chelators Control IC50 (lM) PIH PBH SIH SBH NIH NBH
45 6 20 4 5.5 1.4 0.76 0.07 1.0 0.16 1.2 0.4
Low GSH IC50 (lM) 41 4 14 2 1.5 0.6 0.41 0.02 0.69 0.11 0.56 0.1
Control IC50 (lM) b
>150 60 4 67 12 0.37 0.02 82 11 NDc
Low GSH IC50 (lM) 19 2 31 7 0.44 0.13 0.19 0.01 2.3 0.4 NDc
a
Cells were incubated for 72 h as described in Materials and methods. Data are averages standard deviations (n ¼ 2–13). Solubility limit of the complex in RPMI/FBS. c Not determined. b
Jurkat T lymphocytes was determined after 72 h incubation (Table 1). The structure–activity relationships describing the toxicity of PIH analogs in Jurkat cells were similar to those in K562 cells [10] and SK-N-MC cells [22], suggesting a common mechanism of action of these iron chelators among different cell types. Toxicity increased with the lipophilicity of the analogs, as has been observed for K562 cells [10]. With the exception of Fe(SBH)2 , the Fe complexes were significantly less toxic than the corresponding free chelators. Buthionine sulfoximine (BSO) depletes the important cellular antioxidant, GSH, by inhibiting c-glutamyl transpeptidase [23], an enzyme in the biosynthetic pathway of GSH. Jurkat T cells grow normally in the presence of 20 lM BSO, although their GSH levels are greatly reduced: control cells had 77 2 nmol GSH/106 cells, whereas cells treated for 6 and 72 h with BSO had 27 0.6 and 2.7 0.2 nmol GSH/106 cells, respectively. GSH-depleted Jurkat cells were significantly (p < 0:05) more sensitive to the Fe complexes of PIH analogs than control cells (Table 1). The extent of this effect ranged from approximately twofold for Fe(PBH)2 and
Fe(SBH)2 to 150-fold for Fe(SIH)2 . GSH-depleted Jurkat cells were also significantly (p < 0:05) more sensitive to the PIH analogs themselves than control cells (Table 1), although sensitization of Jurkat cells to the chelators by GSH depletion was much weaker than for the corresponding Fe complexes. When all determinations were compared, there was no significant difference between the PIH IC50 values for control and GSH-depleted cells (Table 1). However, a paired t test of IC50 values determined within single experiments was significant (p < 0:05), indicating that exacerbation of PIH toxicity by GSH depletion is weak, but reproducible. Effect of oxygen tension on toxicity of PIH analogs The effect of oxygen tension (pO2 ) on the induction of apoptosis by PIH, SIH, and NIH was assessed at 100, 20, and 2 lM, respectively, concentrations which caused significant toxicity under normal cell culture conditions (Table 1). The increase in phosphatidylserine externalization for all three analogs with respect to pO2 is shown in Fig. 2. At 0% pO2 , toxicity of PIH and SIH is nearly
Fig. 2. Enhancement of toxicity of PIH analogs by O2 . Jurkat T lymphocytes were treated with 100 lM PIH (A), 20 lM SIH (B), and 2 lM NIH (C) for 24 (d) or 48 (s) h in the presence of varying pO2 . Apoptosis was measured by FITC-annexin V binding. The percentages of dead control cells at the corresponding pO2 and incubation times were subtracted from the data. Symbols and error bars represent means and standard deviations, respectively, of data collected in triplicate.
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zero after a 24 h incubation. In contrast, at 0% pO2 , NIH causes induction of apoptosis in approximately 20 and 35% of the cells at 24 and 48 h, respectively, suggesting that NIH may also cause toxicity by non-oxidative mechanisms. At 20% pO2 , to which cultured cells are normally exposed, toxicity is maximal for all three analogs. Interference of PIH analogs with the TBARS assay Measurement of lipid peroxidation by detection of the lipid oxidation product, MDA, is a simple assay to further evaluate the role of oxidative stress in the toxicity of PIH analogs [18]. However, both PIH and Fe(PIH)2 inhibit the formation of the TBARS product, causing underestimation of the amount of MDA detected by this assay. Inhibition of MDA detection by Fe(PIH)2 , PIH, and isonicotinic acid hydrazide (INH), the hydrolysis product of PIH, was linear over the concentration range 0–50 lM; concentrations resulting in 10% inhibition were 34, 20, and 16 lM, respectively. Since INH had an inhibitory effect stronger than that of PIH, it is likely that hydrolysis of PIH [24,25] during the conjugation of MDA to TBA produces INH, which competes with TBA for MDA. Inhibition of TBARS formation by PIH to a greater extent than its antioxidant activity in other assays has been previously observed [13], likely due to the underestimation of MDA in the presence of PIH analogs. As a result of interference between PIH analogs in the TBARS assay, another method of detecting lipid peroxidation by measuring MDA formation was assessed, which uses a different chromophore, 1-methyl-2-phenylindole, a different solvent system, and a lower incubation temperature [19]. Since 90% of 5 lM MDA was detected by this assay in the presence of 200 lM PIH, the most readily hydrolyzed analog in the series [26], this method is suitable for measuring lipid peroxidation in the presence of PIH analogs.
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linear, reaching 25 nmol/106 cells by 16 h. EPA uptake during a 6 h incubation was saturable, reaching halfmaximal levels at 90 lM. There was no detectable increase in lipid peroxidation in EPA-loaded cells (Fig. 3), indicating that the antioxidant defenses of the cell are sufficient to repair any damage to the more susceptible lipids. In the presence of 50 lM Fe(PBH)2 , cell viability decreased and lipid peroxidation increased with [EPA] (Fig. 3), whereas Fe(PBH)2 did not oxidize EPA in a cell-free system (data not shown). To assess the relationship between oxidative stress and toxicity induced by Fe complexes of PIH analogs, K562 cells were incubated with 200 lM EPA for 6 h, as these conditions allow maximal detection of lipid peroxidation, with no EPA-induced toxicity (Fig. 3). Uptake of EPA under these conditions was 19 nmol/106 cells. Fe(PIH)2 was not toxic toward K562 cells, whether or not they were EPA-loaded (Fig. 4A), even at concentrations as high as 150 lM, the limit of its solubility in RPMI/FBS. In contrast, Fe(PBH)2 and Fe(SIH)2 were toxic toward EPA-loaded (Fig. 4A), but not control, cells. For these two complexes, cell viability and lipid peroxidation were inversely correlated (Figs. 4A and B). The lipophilic antioxidant, vitamin E, added at the same time as Fe(PBH)2 , completely prevented both effects (Figs. 4A and B). Since no toxicity was observed on incubation of EPAloaded cells with any PIH analogs for 24 h, a 48 h incubation period was used to assess the effects of the free chelators (Figs. 4C and D). PIH was not toxic toward K562 cells (48 h) at concentrations as high as 500 lM, whether or not they were loaded with EPA (Fig. 4C), and caused no lipid peroxidation (Fig. 4D). At concentrations
Relationship of lipid peroxidation and toxicity of PIH analogs and their Fe3þ complexes Simultaneous measurement of toxicity and lipid peroxidation in K562 cells (which produce undetectable levels of MDA in the lipid peroxidation assay) was achieved by loading cells with eicosapentenoic acid (EPA), a relatively rare, polyunsaturated fatty acid (PUFA). PUFA-loaded cells have frequently been used to facilitate detection of oxidative damage [27–31]. EPA was bound to BSA to allow its controlled delivery to the cells [32] and its uptake was determined by its depletion in the medium following incubation. In the presence of 200 lM EPA, the kinetics of its uptake by K562 cells were biphasic. Approximately 15 nmol/106 cells was taken up in the first hour, following which uptake was
Fig. 3. Toxicity and lipid peroxidation of EPA-loaded K562 cells by Fe(PBH)2 . Cells were incubated with EPA in RPMI/FBS for 6 h, washed, and incubated in RPMI/FBS with (filled symbols) or without (hollow symbols) 50 lM Fe(PBH)2 for 24 h. Cell viability (circles), measured by the MTS assay, and lipid peroxidation (triangles), measured using 1-methyl-2-phenylindole, represent means of triplicate samples and error bars represent standard deviations.
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A
B
C
D
Table 2 GSH levels and lipid peroxidation in EPA-loaded cellsa GSH (nmol/106 cells)
Lipid peroxidation (nmol/106 cells)
Cells + control/6 h Cells + EPA/6 h
83 1 71 2
NDb ND
Cells + control/30 h Cells + EPA/6 h + control/24 h Cells + EPA/6 h + 18 lM Fe(PBH)2 /24 h Cells + EPA/6 h + 32 lM Fe(PBH)2 /24 h Cells + EPA/6 h + 50 lM Fe(PBH)2 /24 h
72 0.9 67 0.3 55 1.1
ND 0.00 0.01 0.27 0.009
42 1.7
0.97 0.02
6.6 0.2
5.72 0.07
63 0.1 62 0.3 60 0.4
ND 0.00 0.002 0.37 0.007
Cells + control/54 h Cells + EPA/6 h + control/48 h Cells + EPA/6 h + 50 lM PBH/48 h
a Cells were incubated in RPMI/FBS (control) or 200 lM EPA for 6 h, washed, and incubated in RPMI/FBS (control), Fe(PBH)2 , or PBH as indicated. b Not determined.
Fig. 4. Toxicity and lipid peroxidation of EPA-loaded K562 cells by PIH analogs and their Fe complexes. Cells were incubated with 200 lM EPA for 6 h and washed. Toxicity (A) and lipid peroxidation (B) were measured after incubation for 24 h with Fe(PIH)2 (d), Fe(PBH)2 (.), and Fe(SIH)2 (j). The curves in (A) represent weighted, least-squares fits of the data to a three-parameter logistic equation; the IC50 values from these fits were 22 0.5 and 16 1 lM for Fe(PBH)2 and Fe(SIH)2 , respectively. The presence of 1 lM vitamin E during the 24 h incubation prevented both the toxicity and the lipid peroxidation caused by Fe(PBH)2 (r). Neither toxicity nor lipid peroxidation caused by the Fe complexes was detected in cells which were not EPA-loaded. Toxicity (C) and lipid peroxidation (D) were measured after incubation for 48 h with PIH (d), PBH (m), and SIH (j). Toxicity, but not lipid peroxidation, caused by SIH () was observed in cells, which were not EPA-loaded. Cell viability was measured by the MTS assay and lipid peroxidation was measured using 1-methyl-2phenylindole. Symbols and error bars represent means and standard deviations of triplicate measurements.
below 20 lM, PBH caused lipid peroxidation and was toxic toward EPA-loaded, but not control, cells (Figs. 4C and D). Above 20 lM, PBH reversed its own toxicity and lipid peroxidation. SIH was equally toxic toward EPAloaded and control cells (Fig. 4C), but caused no lipid peroxidation (Fig. 4D). GSH levels in EPA-loaded cells were correlated with lipid peroxidation (Table 2). Immediately following EPA loading, GSH levels were decreased from 83 1 to 71 2 nmol/106 cells. After 24 h, GSH levels of EPAtreated cells had partially recovered. Treatment with Fe(PBH)2 exacerbated the decrease in a concentrationdependent manner; incubation with 50 lM Fe(PBH)2
for 24 h caused GSH levels to decline to 6.6 0.2 nmol/ 106 cells, which was associated with significant lipid peroxidation, as observed in Fig. 4. After 48 h, GSH levels had completely recovered. Treatment with PBH for 48 h did not greatly affect GSH levels, but caused a small amount of lipid peroxidation. Of the compounds tested, Fe(PBH)2 , Fe(SIH)2 , and PBH caused concomitant toxicity and lipid peroxidation in EPA-loaded K562 cells (Fig. 4). Despite the differences in concentrations and incubation times at which these effects were assessed, the dependence of toxicity on lipid peroxidation was similar for all the above species (Fig. 5); lipid peroxidation of 3.3 nmol/106 cells corresponded to 50% toxicity. Hydrogen peroxide, which caused 50% toxicity in 24 h at a concentration of approximately 1.4 mM in both control and EPA-loaded cells, caused lower levels of lipid peroxidation than Fe(PBH)2 , Fe(SIH)2 , and PBH (Fig. 5). Vitamin E (a-tocopherol) completely prevented the lipid peroxidation, but only partially prevented the toxicity, caused by hydrogen peroxide (data not shown), likely because the lipophilic antioxidant, vitamin E, is unlikely to be found in the same cellular compartments. Reduction of Fe3þ complexes of PIH analogs by cells The Fe complexes of PIH analogs, at concentrations of 25 lM, oxidized ascorbate with pseudo-first-order kinetics. The rate constants varied, with no apparent structure–activity relationship, from 1.0 0.08 to 4.7 0.2 103 min1 for Fe(SBH)2 and Fe(NIH)2 , respectively, indicating that reduction of these complexes by physiological species is possible. Reduction of Fe3þ
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Table 3 Reduction of Fe complexes of PIH analogs by K562 cellsa
Fe(PIH)2 Fe(PBH)2 Fe(SIH)2 FeEDTA
Fe reduced ) ferrozineb (pmole/106 cells/h)
Fe reduced + ferrozinec (pmole/106 cells/h)
16 2 21 2 41 50 4
150 8 153 7 90 20 220 20
a Incubations of 2 106 cells per sample were for 4 h at 37 C in 1 mL PBS, in the presence of 10 lM Fe complex. Data are means of triplicate samples and errors are standard deviations. b Ferrozine was added after cells were centrifuged from the samples, as described in Materials and methods. c Ferrozine was added at the beginning of the 4 h incubation, as described in Materials and methods.
Fig. 5. Dose-dependence of chelator- and complex-mediated toxicity on lipid peroxidation. Fe(PIH)2 (d), Fe(PBH)2 (.), Fe(SIH)2 (j), and PBH (r) elicit the same relationship between toxicity and lipid peroxidation. The IC50 from the weighted, least-squares fit of these data to a three-parameter logistic equation (solid line) corresponds to 3.3 nmol MDA/106 cells. The IC50 for 24 h H2 O2 treatment (r), 1.4 mM, corresponds to 0.7 nmol MDA/106 cells.
complexes of PIH analogs by K562 cells was measured using the ferrozine assay (Table 3). When ferrozine was added to the supernatant of cells incubated for 4 h with the Fe complexes of PIH analogs, low levels of Fe2þ were detected. In the absence of cells, no Fe3þ reduction occurred with any of the complexes. Since it is likely that, under these conditions, some of the Fe2þ produced is re-oxidized to Fe3þ by dissolved O2 , these values represent minimum levels of Fe3þ reduction by cells. When ferrozine was included in the incubation medium with the Fe complexes, the amounts of Fe2þ detected were four- to 20-fold higher (Table 3). Since it is possible that, under these conditions, the presence of ferrozine, a Fe2þ chelator, shifted the Fe3þ –Fe2þ equilibrium toward Fe reduction, these values represent maximum amounts of Fe3þ reduction by cells. By comparison to EDTA, a complex well known to redox cycle under physiological conditions, it is apparent that Fe complexes of PIH analogs are less readily reduced by cells.
Discussion Redox cycling by Fe complexes of PIH analogs K562 cells reduce the Fe complexes of PIH analogs (Table 3), as does the ubiquitous cellular reductant, ascorbate. In contrast, cyclic voltammetry of Fe(SBH)2 [33] and Fe(NIH)2 [34] demonstrated the absence of a reversible redox potential in the biologically accessible range; from these data, it was concluded that reduction of these complexes by cells is not possible. It may be, however, that these experiments were confounded by the relatively slow kinetics of ligand exchange characteristic
of the Fe3þ complexes of these ligands. It has been demonstrated that Fe2þ forms a complex with PIH almost immediately [11], whereas complex formation of PIH with Fe3þ requires minutes or hours to achieve completion [8], depending on the concentrations of the two species. It is therefore likely that the complexes are not reduced quickly enough to allow the identification of a redox potential by this method. The capacity of these complexes to redox cycle in biological systems provides a compelling explanation for the lipid peroxidation, GSH depletion, and toxicity of PIH analogs and their Fe complexes in EPA-loaded K562 cells (Figs. 3–5, Table 2), as well as the sensitization of Jurkat cells to these species by high pO2 (Fig. 2) and GSH depletion (Table 1). However, it is also possible that dissociation of Fe from the chelators precedes its reduction, since both FePIH2 [35] and FeSIH2 [36] are known to donate Fe to cells in vitro. The donation of Fe to human T-lymphocytes by Fe(PIH)2 has been demonstrated to be non-specific, causing the accumulation of Fe in non-physiological pools [37]. Therefore, it is not possible, from the results presented here, to determine whether the oxidative stress induced by PIH analogs and their Fe complexes is due to redox cycling of the complexes themselves, or to donation of Fe to cellular ligands which redox cycle. It is also important to consider the possibility that mixed ligand complexes are formed, in which one of the two coordinating chelator molecules is replaced by a cellular ligand. The redox properties of such complexes, whether they exist, are unknown. A series of studies of PIH analogs [11,13,14] have demonstrated their capacity to inhibit redox cycling of Fe-EDTA in vitro. Likely, this is due to formation of Fe–chelator complexes, which have slower kinetics of reduction (i.e., Reaction 2) than Fe-EDTA. While PIH analogs are less redox active than Fe-EDTA (Table 3 and [11,13,14]), and therefore act as antioxidants in its presence, the conclusion that Fe complexes of PIH analogs cannot redox cycle [13–15] is not supported by the present results.
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Contribution of oxidative stress to the toxicity of PIH analogs GSH-depleted Jurkat cells are more sensitive to both PIH analogs and their Fe complexes (Table 1). Therefore GSH, which supplies reducing equivalents for enzymes involved in the antioxidant response such as GSH-S-transferase and GSH peroxidases, plays a role in protecting Jurkat cells against the toxicity of these compounds, implicating oxidative stress in their mechanisms of toxicity. The sensitization of Jurkat cells to PIH analogs by increased pO2 further supports the role of oxidative stress in the toxicity of this series of chelators. It has been proposed that, since exposure of cells to the free chelators results in the formation of intracellular Fe–chelator complexes, the complexes are, in part, responsible for the toxicity of the chelators [8–10]. The Fe complexes redox cycle (Table 3), causing oxidative stress (Table 2, Figs. 3–5), which contributes to their toxicity, and that of the free chelators. That the sensitization of Jurkat cells to the chelators by GSH depletion is weaker than the corresponding Fe complexes (Table 1) suggests that multiple mechanisms of toxicity, including Fe depletion, are also involved in the toxicity of PIH analogs. The concomitant lipid peroxidation and toxicity caused by Fe(PBH)2 (Fig. 4) and hydrogen peroxide (Fig. 5), and the reversal of these effects by vitamin E, demonstrate that oxidative stress is a causal event in the toxicity of these compounds. Because of the effectiveness of vitamin E in protection against the toxicity of Fe(PBH)2 , which is lipophilic [10,38], it is likely that the target of oxidative stress is localized to a membrane. However, it cannot be concluded that lipid peroxidation, and not some other, possibly more specific, form of oxidative stress, causes the toxicity. Lipid peroxidation may simply serve as a reporter for damage to other cellular structures. It is noteworthy that the lipophilic antioxidant, vitamin E, completely abolished the toxicity of Fe(PBH)2 in EPA-loaded K562 cells, while it only partially prevented the toxicity of H2 O2 , a polar oxidant. Thus, in addition to lipid peroxidation, H2 O2 likely causes oxidative damage to polar structures, which may not be well protected by vitamin E. Lipid peroxidation certainly does not represent all of the oxidative damage caused by H2 O2 , and may not do so for Fe(PBH)2 , either. Depletion of cellular GSH levels, while also reflecting oxidative stress (Table 2), similarly does not appear to be the specific cause of toxicity. Since treatment of K562 cells with 20 lM BSO for 72 h caused GSH levels to drop to 2.7 0.2 nmol/106 cells without evidence of toxicity, it is unlikely that GSH depletion per se (which reached a minimum level of 6.6 0.2 nmol/106 cells following Fe(PBH)2 treatment, Table 2) is the cause of the observed toxicity and lipid peroxidation by Fe(PBH)2 and Fe(SIH)2 .
While Fe(PBH)2 and Fe(SIH)2 caused toxicity and lipid peroxidation in EPA-loaded cells, Fe(PIH)2 did not. However, the toxicity of PIH and Fe(PIH)2 toward Jurkat cells was enhanced by GSH depletion (Table 1) and toxicity of PIH was reduced by low pO2 (Fig. 2). Furthermore, Fe(PIH)2 increased glutathione peroxidase mRNA levels in MEL cells [39], suggesting induction of oxidative stress. Therefore, the cellular effects of both PIH and its Fe complex are sensitive to the redox state of the cell, as are those of the other PIH analogs. Since Fe(PIH)2 can be reduced by ascorbate and cells (Table 3) as readily as Fe complexes of its analogs, its redox chemistry is unlikely to account for the insensitivity of Fe(PIH)2 to EPA loading. An explanation may be found in the lower lipophilicity of Fe(PIH)2 in comparison with the other Fe complexes [8,38], which results in its weaker association with the cell membrane [8], and possibly lower intracellular accumulation. Modulation of toxicity of PIH analogs by oxygen tension The oxygen tension at which cultured cells are routinely grown (20% O2 ) exacerbated the toxicity of PIH analogs (Fig. 2). This effect could be partly due to changes in the regulation of redox-sensitive proteins, but the simplest explanation is that reduced oxygen tension causes lower production of reactive oxygen species (ROS), which initiate Fe-catalyzed oxidative stress. Since initiation of oxidative stress by oxidation of Fe2þ by O2 may be a more significant pathway than by preexisting hydrogen peroxide [40], lower pO2 should directly limit the production of ROS. That many PIH analogs are safely administered to rats at high doses (0.2 mmol/kg, [2]) without toxicity, despite their toxicity in vitro, may be explained in part by the much lower pO2 in tissues (2–5%) than in the atmosphere in which cultured cells are grown. Certainly, other factors, including pharmacokinetics, metabolism, and hydrolysis, also contribute to the exposure of tissues to PIH analogs in vivo. Conclusions While the development of PIH analogs for use in treating secondary iron overload has so far included few studies in vivo, the small number of carefully chosen compounds selected for animal studies has been effective at doses which do not cause significant adverse effects [2]. The basis for their selection was their efficiency in causing 59 Fe release from a series of cell culture models [41], which reflect important targets of iron chelation in vivo. Since the completion of that study, it has been demonstrated that the vast differences in the efficacy of these chelators lie in their abilities to translocate the 59 Fe–chelator complexes out of the cell. Because the most effective analogs, which cause minimal intracellular
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accumulation of 59 Fe–chelator complexes [8,10] are, fortuitously, also the least toxic [10], the selection of analogs for in vivo study based on mobilization of 59 Fe in vitro appears to be sound. These data underscore the importance of evaluating the capacity to induce oxidative stress of Fe chelators with therapeutic potential, since Fe-overloaded patients receiving chelator therapy are already at risk for oxidative stress [1]. The relative contributions of Fe depletion and oxidative stress to the overall mechanism of toxicity of PIH analogs may be altered in Fe-overloaded tissues. It is possible that, in the presence of the same concentration of chelator, toxicity due to Fe depletion is lower, and toxicity due to accumulation of Fe–chelator complexes is higher, than in non-overloaded tissues. There is increasing interest in the use of some of the more toxic PIH analogs, particularly NIH, for the treatment of cancer. While there are some encouraging reports of their effectiveness in treating neuroblastoma cells in vitro [42], which are highly sensitive to Fe depletion, the mechanism of its action will require further study. NIH is among the most toxic PIH analogs in vitro [9,10,22], and NIH-treated, 59 Fe-labeled cells accumulate high levels of intracellular 59 FeNIH2 [9]. In addition to Fe depletion, the mechanism of toxicity of NIH includes an oxidative component (Table 1), which may contribute to its effectiveness in treating cancer, in which oxidative damage to DNA and other cell structures may be considered advantageous.
Acknowledgments This work was supported by grants from the Canadian Institutes for Health Research (P.P.) and University of Linkoping Grant #83081030 (J.N.), and postdoctoral fellowships from the National CooleyÕs Anemia Foundation and the Thalassemia Foundation of Canada (J.L.B.).
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