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Cite this: DOI: 10.1039/c4mt00214h
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Iron-sensitive fluorescent probes: monitoring intracellular iron pools Yongmin Ma,a V. Abbateb and R. C. Hider*b Several iron-sensitive fluorophores have been investigated in a range of cell types in order to quantify iron(II) levels in the cytosol and the cytoplasm. Both iron(II) and iron(III) cause fluorescence quenching of these probes and changes in cytosolic iron levels can be monitored in a reproducible manner. However
Received 12th August 2014, Accepted 7th October 2014
the precise quantification of iron(II) in the cytosol is complicated by the uncertainty of the structure of
DOI: 10.1039/c4mt00214h
is essential for quantitative purposes. The lysosomal and mitochondrial iron pools have only been the
many of the quenched species that exist under in vivo conditions. Precise knowledge of these structures subject of relatively few studies at the time of writing. Calcein–AM has been widely adopted for the
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monitoring of changes in iron levels in a range different cell types.
1. Introduction: the chemical nature of intracellular iron pools There are a number of intracellular pools of labile iron, associated with the cytosol, mitochondrion, nucleus and lysosome.1 Whereas there is an increasing knowledge relating to the pools associated with the cytosol, mitochondrion and lysosome, there
is very little information relating to that of the nucleus.2 The labile iron pool in each of these organelles plays an essential role in supplying iron to metalloproteins. As non coordinated iron salts can catalyse the formation of toxic oxygen-containing radicals in aerobic organisms,3 the levels of this labile iron pool must be tightly controlled. Cells must be able to sense iron levels and regulate iron homeostasis in such a manner as to maintain non-toxic levels of this essential metal.2
a
College of Pharmaceutical Science, Zhejiang Chinese Medical University, 548 Binwen Road, Hangzhou, P. R. China 310053 b Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK. E-mail: [email protected]
Yong-Min Ma received his MSc degree in Organic Chemistry in 2001 from Zhejiang University (P. R. China) under the direction of Professor Yong-Min Zhang. He obtained his PhD degree in Medicinal Chemistry in 2005 at King’s College London under the guidance of Professor Robert C Hider. He continued to work as a research fellow in Prof. Hider’s group until 2013, when he was appointed associate professor at Yongmin Ma Zhejiang Chinese Medical University. His research interests involve the design of iron chelators for the treatment of iron overload diseases, including fluorescencelabeled 3-hydroxypyridin-4-ones and fluorinated 3-hydroxypyridin4-ones.
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Cytoplasmic iron pool The redox state of this iron pool has been demonstrated to be iron(II) on the basis of thermodynamic arguments,4 the use of
Dr Abbate completed his MSc in Pharmacy in 2004 at ‘‘Federico II’’ University of Naples, where he also joined the Department of Medicinal & Toxicological Chemistry for one year working on the synthesis of novel analgesic compounds. He then moved to the UK to undertake a PhD in Chemistry and Analytical Sciences at The Open University (awarded in 2009). In 2008 he was awarded a prestigious Maplethorpe Fellowship of the University of Vincenzo Abbate London for the promotion of teaching and research in the pharmaceutical area. Dr Abbate currently works at KCL with Professor Hider on a BBSRC-funded project aimed at developing novel iron-chelating probes for monitoring and adjusting excess labile iron pool in mitochondria.
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ionophores5 and the use of iron-sensitive fluorophores.6,7 The concentration of this iron pool falls in the range 2 10 7–5 10 6 M.8 The nature of the iron-binding ligands has been much debated.9–13 Recently glutathione (GSH) has been demonstrated to be the most probable candidate, forming a monodentate complex via the sulfur atom (1).8,14 GSH has a high cytosolic concentration in most eukaryote cells (2–8 mM).15,16
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delivered to the lysosome will be iron(III), there is likely to be a mixture of both iron(III) and iron(II) present, the solubility of iron(III) being enhanced by the acid pH (5.5–6.0) of the lysosome. This labile iron renders lysosomes susceptible towards oxidative stress and this in turn leads to autophagy, apoptosis or necrosis.20 A wide range of ligands are available for the coordination of iron in this pool. For iron(II) it is likely that cysteine and glutathione will dominate and for iron(III), citrate will dominate.21 Nucleus iron pool
Mitochondrial iron pool Mitochondria are major sites for heme and iron–sulfur cluster synthesis and consequently there is a constant iron influx into mitochondria. Iron(II) is believed to be the dominant redox state and as the GSH concentration is even higher than that of the cytosol, namely 10–12 mM,16 again FeIISG (1) is likely to be the dominant chemical form of the labile iron pool. Significantly FeIISG could be a direct precursor for iron–sulfur clusters.8 Lysosome iron pool Lysosomes play a major role in the breakdown of both ferritin17,18 and mitochondria.19 Ferritin turns over with a half life between 12 and 24 h in most cell types,17 thus the resulting iron pool is likely to be relatively high. As a large proportion of the iron
R. C. Hider is emeritus professor of medicinal chemistry at King’s College London, where he has worked since 1987. Prior to this he was a lecturer in biological chemistry at Essex University. He has worked with siderophore-based iron uptake processes in microorganisms, plant root absorption of iron and the absorption of different molecular forms of iron by mammalian cells. His work on membrane structure and transport R. C. Hider mechanisms has led to the development of novel oral iron chelators for the treatment of iron overload. His group has synthesized a range of iron chelators, all specifically designed to permeate cell membranes in both the iron-free and iron-complexed form. As a result, N-alkyl-3-hydroxypyridin-4-ones have been identified as possessing potential for clinical application. Deferiprone is now used worldwide for the treatment of iron overload.
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The boundary between the nucleus and the cytosol is a double membrane, termed the nuclear envelope which is perforated by nuclear pores. There is a rapid equilibrium of labile iron between these two pools, presumably via nuclear pores.22 Iron–sulfur cluster proteins play a key role inside the nucleus23 and ferritin has a regulatory function in the nucleus.24 Nuclear redox active labile iron is thought to be involved with DNA damage induced by H2O2 and other reactive oxygen species and so it is important to be able to monitor nuclear LIP levels, particularly if they are not identical to those of the cytosol.25
2. Probes that have been used to measure the labile iron pool (LIP) A small range of fluorescent molecules have been utilized to provide information on the intracellular labile iron pool (2, 3, 4 and 5; Table 1). Those containing the acetoxymethoxy group are limited to the cytosol, while the others monitor intracellular organelle iron levels together with those of the cytosol. The acetoxymethoxy group (AM) renders probes membrane permeable, and cytosolic esterases rapidly cleave the AM esters to hydrophilic carboxylate groups (eqn (i)), creating the highly charged iron probe in the cytosol. Due to the hydrophilic nature of the probe, it remains trapped within the cytosol.30 As indicated in Table 1 none of the listed probes are completely selective for iron, they all bind both iron(II) and iron(III) and a limited range of other divalent metals. However under physiological conditions, the cytosolic zinc(II) and copper(II) levels are extremely low8 and are unlikely to interfere with the detection of iron. With fura-2 (4), although binding with calcium(II) will occur in the cytosol, this leads to a shift in the peak excitation wavelength, whereas the binding with iron(II) leads to fluorescence quenching.28 Thus the binding of calcium(II) and iron(II) can be distinguished.
(i) There are many fluorescent probes which have been described,31 some selective for iron(II) others selective for iron(III). However the properties of most of these probes have not been carefully investigated in cell culture systems.
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Iron-sensitive fluorophores
Probe
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Structure
Ion selectivity Ref.
Calcein–AM (Calcein, 2)
FeII, FeIII 26 NiII, CuII, CoII
Phen Green SK (3)
FeII, FeIII CaII, ZnII
27
Fig. 1 LIP levels in a single human hepatoma HepG2 cell. Iron(II) ammonium sulphate (FAS, 20 mM) was added to half the cells. SIH (100 mM) was added after the indicated time interval.34
Fura-2AM (Fura-2, 4)
FeII, FeIII CuII, CaII
28
CP655 (5)
FeII, FeIII CuII
29
We discuss below the probes which have been investigated in some detail. Calcein–AM Calcein–AM (2) is the most widely adopted iron(II) fluorescent probe, it having been studied in reticulocytes, CHO cells, fibroblasts, hepatocytes and colon cells.32,33 Typical behavior is illustrated in Fig. 1 where a single human hepatoma HepG2 cell is monitored.34 The top trace is fluorescence of a control cell treated with calcein–AM and lower shows the influence of incubation with ferrous ammonium sulfate (20 mM). As iron enters the cytosol it quenches the calcein fluorescence. After approximately 12 min, 100 mM SIH (6) was added. This chelator rapidly gains entry into the cytosol where it out competes calcein for iron(II) and so the fluorescence rapidly increases. Chelators with a high affinity for iron(II), for instance SIH (6) and PIH (7), compete with calcein for iron(II) very rapidly (Fig. 2) whereas chelators with a high affinity for iron(III), for instance DFO (8) and CP41 (9) compete at different rates. DFO, by virtue of its extremely high affinity for iron(III) facilitates the rapid autoxidation of iron(II)35 and reacts rapidly just like SIH, whereas CP41, a simple hydroxypyridin-4-one reacts more slowly (Fig. 2). The reason for this difference is the rate of
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Fig. 2 Dequenching of calcein–iron complexes by chelators in the absence of cells. Calcein (40 nM) at pH 7.2 was pretreated with iron(II) ammonium sulphate for 45 min. Chelators (1 mM) were added (*). The arrows indicate addition of SIH (100 mM).32
autoxidation of the CP41iron(II) complex is slower than that of DFOiron(II) (eqn (ii)). Thus the driving force for iron(II) chelation by these two iron(III) selective ligands is the enhanced stability of the iron(III) complex, which results from autoxidation of the corresponding iron(II) complex. When the behavior of the same group of 4 compounds was investigated in the presence of erythroleukemia K562 cells containing calcein, the properties of SIH and PIH were found to be similar to that observed in free solution, but the behavior of CP41 and DFO were reversed (Fig. 3). Clearly within the time course of the experiment DFO does not enter the cell, whereas although the smaller CP41 does enter the cell rapidly, it takes longer than SIH to stabilise the calcein fluorescence; 8 minutes as compared to 2 minutes. This is a result of the necessity of autoxidation to occur with CP41,
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Fig. 3 Dequenching of calcein–iron complexes in K562 cells by extracellularly added chelators (100 mM) at zero time. Arrow indicates the addition of SIH (100 mM).
whereas PIH and SIH are each able to form a relatively stable complex with iron(II).
constant KCA = [FeIICA]/[FeII] + [CA] can be used to quantify LIP, where LIP = [FeII] + [FeIICA]. In principle, KCA, [FeIICA] and [CA] can be determined. Thus [CA] can be determined from the fluorescence of a single cell or a suspension of cells of known density and cell volume, an anticalcein antibody being used to remove extracellular fluorescence. [FeIICA] is measured from the change in fluorescence on addition of SIH. KCA is determined from normal physico-chemical measurements. Herein lies a difficulty with the application of calcein for the quantification of the labile iron pool (LIP), as the precise nature of the iron–calcein complex in the cytosol is uncertain. Initially it was assumed to be similar to a 1 : 1 EDTA-type complex and the affinity constant for FeIIEDTA was adopted for the calculations used to quantify LIP. However under cytosolic conditions rapid autoxidation of FeIIEDTA occurs, as a consequence the CoII EDTA conditional constant was adopted for these studies, as this could be reliably measured.34 However studies based on ESR and cyclic voltammetry indicate that under physiological conditions calcein also binds iron at a phenolic site on the molecule (10), leading to a preference for iron(III).36 This has an entirely different affinity constant for iron and there is the possibility of each calcein molecule binding two such iron(III) cations. Thus there is uncertainty associated with the nature of the physiologically relevant iron complex that acts as a sink, indeed a mixture of iron complexes probably form. Thus it is difficult to determine LIP values based on this type of calculation. Never-the-less LIP values have been reported for a range of cell types using this method (Table 2).32
(iii)
Table 2 LIP values obtained from the application of calcein in various cell types, following iron loading and chelator – mediated deprivationa
(ii) It is possible to determine the concentration of iron(II) in the cytosol, often termed LIP (labile iron pool) or [Fe]LIP, from studies centred on the use of SIH as indicated in Fig. 3. Thus based on the equilibrium depicted in eqn (iii), the equilibrium
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Cell
Treatment
LIP (mM)
K562 K562 U937 U937 U937 Hepatocytes Hepatocytes Hepatocytes
Control +DFO Control +DFO +FAC Control +DFO +FAC
0.113 0.058 0.176 0.071 0.412 0.203 0.046 0.923
a K562, human erythroleukemia cells; U937, human promonocyte cells; hepatocytes, human hepatoma (HEPG2) cells. DFO, desferrioxamine, 100 mM; FAC, ferric ammonium citrate (20 mM).32,34
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Fig. 4 Dequenching of calcein–iron complexes in K562 cells. Various chelators (100 mM) added at different time points, SIH (100 mM) marked by *. The chelators 102, 41, 165 and 117 are analogues of Deferiprone.37
Despite this limitation, calcein has been utilized extremely effectively to provide information on changes in LIP levels. Thus the rate of influx of iron chelators into cells can be directly compared (Fig. 4),37 the UV-induced production of LIP can be monitored (Fig. 5)38 and the influence of LIP on the erythrocyte stage of Plasmodium falciparum monitored during cell culture (Fig. 6).39 Phen green SK diacetate Phen green SK diacetate is metabolized by cytosol esterases to phen green SK (3) (eqn (iv)) and hence becomes trapped
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Fig. 6 LIP levels in various malaria parasite stages. The LIP increases as the parasite matures within the host red blood cell. The LIP of uninfected and different stage parasitized RBCs was determined by assessing the fluorescence of calcein-loaded cells with the addition of the iron chelator, deferiprone.39
within the cell, although a proportion of the probe also enters mitochondria, lysosomes and the nucleus.27 Leakage from cells is generally superior to that of calcein, the latter acting as a substrate for a xenobiotic exporter protein.27 Using techniques similar to those outlined under the section on calcein, phen green SK has been used to estimate cytosolic levels of LIP (Fig. 7). Iron enters the cell more quickly in the presence of 8-hydroxyquinoline (11) than iron(III) citrate. In both cases the LIP is scavenged in the presence of bipyridyl (12). From these measurements an estimate of the cytosolic LIP concentration can be made. Petrat and coworkers40 prefer to use an ‘ex situ’ calibration method in order to facilitate such measurements. This is because the ‘in situ’ calibration requires a free equilibration between intra- and extracellular LIP and the iron(III) 8-hydroxyquinoline complex accumulates intracellularly, therefore ruling out this calibration procedure. Free phen green SK at a concentration loaded in hepatocytes was used to perform the calibration in the ‘‘cytosolic’’ – like medium. The fluorescence of intracellular phen green SK in an excess of bipyridyl (5 mM) was set at 100% fluorescence and the autofluorescence from a parallel incubation of phen green SK unloaded control hepatocytes was set at 0%. Thus the percentage of fluorescence recovery in hepatocytes by the addition of excess bipyridyl could be calculated and compared with the values from ‘ex situ’ calibration curve to obtain a LIP concentration.27
Fig. 5 UVA induced production of LIP in skin fibroblasts. EC – epicatechin (30 mM).38
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(iv)
Fig. 8 Frequency distribution of the hepatocellular concentration of LIP. Cells were incubated with PG SK diacetate (20 mM) for 10 min. Bipyridyl (5 mM) was added and the resulting fluorescence noted.40
9.0 mM, with a large proportion of the cells possessing LIP in the range 0.8–2 mM (Fig. 8).40 Hydroxypyridinone CP655 A range of fluorescent hydroxypyridinones have been described41,42 and one, CP655 (5) has been selected to monitor LIP levels.29 The probe gains rapid entry into cells without the necessity of using AM or acetyl esters. However this ability to penetrate plasma membranes renders it also possible to enter lysosomes and mitochondria, thus providing a measure of cellular LIP as opposed to cytosolic LIP. A further limitation of this class of fluorescent probe is that they are not trapped intracellularly and will efflux, once the incubation media is changed.29 However changes in LIP levels induced by the addition of iron(III) citrate and a competing chelator CP94 (13), similar to those reported for calcein and phen green SK, can be monitored by changes in fluorescence (Fig. 9). We are currently synthesising hydroxyl-pyridinone fluorescent probes which are linked to AM moieties. 14 is an example of such a molecule. This class of molecule is predicted to have a similar distribution to calcein–AM but will have the advantage of possessing a well characterized affinity constant for iron(III).
Fig. 7 Effect of an increase in the cellular chelatable iron on the fluorescence of PG SK enclosed in rat hepatocytes. A, iron(III) chloride; B, iron(II) ammonium sulphate.27
When this method was applied to isolated rat hepatocytes, a wide range of values for LIP was obtained ranging from 0.2 to
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Fig. 9 Fluorescence of the hydroxypyridinone CP655 (6 mM) in primary hepatocytes, quenched by iron(III) (2 mM) and recovered in the presence of CP94, an analogue of deferiprone.42
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mobilize lysosomal labile iron by monitoring the iron pools of single cells by flow cytometry.48
3. Lysosomal targeting probes The endosomal/lysosomal compartment of cells contains a relatively high level of labile iron. This will not be registered with the use of calcein–AM, as once calcein is generated in the cytosol it lacks the ability to penetrate membranes.43 The lysosome contains the breakdown products of both mitochondria44 and ferritin17 (Fig. 10) and chelation of this iron pool protects against oxidative stress-induced cellular damage.45 The measurement of labile iron levels in the endosomal/ lysosomal compartment of hepatocytes is difficult because of its small size, approximately 1% of the cell volume.46 However the larger lysosomes present in liver endothelial cells are amenable to measurement. Using Phen Green SK and 1,10phenanthroline in combination, Petrat and coworkers46 determined a labile iron level for this intracellular compartment of 15.8 4.1 mM. The only probe specifically designed to target the lysosome is the hydroxypyridinone SF34 (15).47 This fluorescein-labelled probe is only distributed to the endosomal/lysosomal compartments as demonstrated by colocation studies in macrophages (Fig. 11). Unlike the smaller more lipophilic probe CP655 (5), SF34 does not permeate membranes rapidly and so can be trapped within the endosomal/liposomal compartment. It apparently directly enters this organelle system by endocytosis. This probe has been used to compare the ability of different chelators to
4. Mitochondrial targeting probes As mitochondria are the main utilisers of iron in the cell as well as being a major source of the superoxide anion, O2 , their iron homeostasis is critically important. Clearly there must be a tight coordination between the influx of iron and its incorporation into heme and iron–sulfur clusters. An increase in mitochondrial iron burden is likely to be linked to associated pathology, for instance with Friedreich’s ataxia where there is inefficient iron– sulfur cluster synthesis.49,50 The mitochondrial labile iron pool can be buffered to some extent by mitochondrial ferritin; but not in all tissues, for instance liver mitochondria lack mitochondrial ferritin.51 One widely adopted method of targeting molecules to the mitochondrial matrix is by attachment to delocalized lipophilic cations. By taking advantage of the substantial negative electrochemical potential maintained across the inner mitochondrial membrane (typically 200 mV), delocalised cations are able to cross the membrane and hence be accumulated with a distribution
Fig. 10 Endosome, autophagosome, lysosomal pathway.44
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Fig. 11 Representative confocal microscopy images of intracellular colocalization studies of SF34 (15) in bone marrow derived macrophages using dextran Texas Red labeled lysosomes. Colocalization studies were carried out in DFO (E, F) and iron dextran (I, J) pretreated cells as well as untreated cells (A, B), with 1 h of incubation with 75 mM (15). Findings are illustrated together with merged fluorescence data showing a high degree of lysosomal colocalization in yellow at a later stage of cellular probe processing (C, G, K). Fluorescence data are shown in comparison to the corresponding bright field images (D, H, L).47
associated with the Nernst potential, typically in excess of 1000 fold. Triphenyl phosphonium salts (16) are typical examples where the R group can be, for instance the anti-oxidants vitamin E or Trolox.52 Attachment to 18F-labelled moieties renders it possible to monitor mitochondria by PET/CT.53 The positive charge formally associated with the central phosphorus atom is delocalised over the three phenyl rings, resulting in a low electron density. This, together with the overall lipophilic character of the molecule, enables rapid penetration of membranes. This useful property of lipophilic cations has been utilised to target fluorescent iron sensors to the mitochondrion. Both the Petrat54 and Cabantchik55,56 groups have utilised a derivative of rhodamine (RPA, 17) to monitor mitochondrial iron levels. Rhodamine is a positively charged fluorescent molecule where the single positive charge is delocalised on both nitrogen atoms and over the plane of the tricyclic structure. Petrat and coworkers introduced RPA (17) for the monitoring of mitochondrial iron levels.54 By adopting methods developed for the cytosol and using PIH (7), estimates of the mitochondrial labile iron pool were made (Fig. 12).54 The values for mitochondria of different cells were found to be distributed over a wide concentration range, centred on 12 mM (Fig. 13).54 Cabantchik has also utilised RPA to monitor mitochondrial iron levels.55 A limitation of this probe is the difficulty in reversing the quenched signal under physiological conditions56 and thus the associated difficulty of unequivocally assigning the quenched signal to labile iron.
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Fig. 12 Time course of dequenching of mitochondrial RPA (0.2 mM) fluorescence in rat hepatocytes after the addition of PIH (2 mM).54
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5. Other iron-sensitive fluorescent probes In addition to the probes listed in Table 1, there have been a number of reports describing other iron sensitive probes which have not been widely adopted. Calcein blue (CALB) (19) has been investigated by Cabantchik;56 its use as an acetomethoxy ester precursor has been introduced for the measurement of cytosolic iron. A rhodamine-based iron-sensor (20) which, in contrast to most other iron probes, undergoes an increase in fluorescence in the presence of iron has been investigated under in vitro conditions.60 However the molecular weight of this probe may reduce its ability to penetrate cell membranes. In similar fashion an anthracene-based probe (21) has also been reported to increase in fluorescence intensity in the presence of iron(III), but not iron(II). Again the molecular size of this probe and its highly charged nature are likely to seriously influence its ability to penetrate cell membranes.61 A boron-based iron(III) probe (BOD-NHOH) (22) has been described which functions by iron(III) catalysed hydroxylamine oxidation.62 However the fluorescence profiles of this probe are unchanged in the presence of iron(II) and so it is unlikely to be able to register the cytosolic labile iron pool. Other iron sensitive probes are listed in general reviews on fluorescent sensors.31,63 Fig. 13 Frequency distribution of concentration of mitochondrial chelatable iron in single hepatocytes. RPA (0.2 mM); PIH (2 mM). Mitochondria (n = 50) from three hepatocytes were investigated.54
Largely as a result of the above limitation, a different approach has recently been introduced for monitoring the mitochondrial matrix labile iron pool. A series of peptides has been prepared, members of which are capable of being targeted to and accumulated in mitochondria. These peptides contain alternate hydrophobic and basic amino acids and typically contain between 4 to 6 residues.57,58 Using this general design, a series of small fluorescent peptides have been synthesised that are selectively accumulated by mitochondria.59 By incorporating an iron chelating moiety into the molecule (18) we are able to monitor the mitochondrial labile iron pool.
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6. Conclusions Methods for the quantification of intracellular labile iron pools are still under development. The cytosolic pool is the best characterized using the probes Calcein–AM and phen green SK, although quantification is difficult. The mitochondrial and lysosomal pools are less well characterized, but there is progress in this area. The nuclear LIP has yet to be investigated. None of the probes so far studied are ideal, but many lessons have been learned and the design of probes to specifically quantify the cytosol, mitochondria and lysosome are likely to be introduced over the next few years.
List of abbreviations AM CA CALB CHO DFO EC EDTA ESR FAC FAS HepG2 GSH LIP PET PIH RPA SIH
Acetoxymethoxy group Calcein Calcein blue Chinese hamster ovary cells Desferrioxamine Epicatechin Ethylenediaminetetraacetic acid Electron spin resonance Ferric ammonium citrate Ferrous ammonium sulphate Human hepatoma cells Glutathione Labile iron pool Positron emission tomography Pyridoxal isonicotinoyl hydrazone Rhodamineb-[(1,10-phenanthrolin-5yl)aminocarbonyl]benzyl ester Salicylaldehyde isonicotinoyl hydrazone
References 1 L. A. Ba, M. Doering, T. Burkholz and C. Jacob, Metallomics, 2009, 1, 292–311. 2 M. W. Hentze, M. U. Muckenthaler, B. Galy and C. Camaschella, Cell, 2010, 142, 24–38. 3 B. Halliwell and J. M. C. Gutteridge, Biochem. J., 1984, 219, 1–14. 4 R. J. P. Williams, FEBS Lett., 1982, 140, 3–10. 5 A. Egyed and P. Saltman, Biol. Trace Elem. Res., 1984, 6, 357–364. 6 W. Breuer, S. Epsztejn and Z. I. Cabantchik, J. Biol. Chem., 1995, 270, 24209–24215. 7 F. Petrat, D. Weisheit, M. Lensen, H. de Groot, R. Sustmann and U. Rauen, J. Biol. Chem., 2002, 362, 137–147. 8 R. C. Hider and X. Kong, Dalton Trans., 2013, 42, 3220–3229. 9 J. P. G. Miller and D. J. Perkins, Eur. J. Biochem., 1969, 10, 146–151. 10 J. Weaver and S. Pollack, Biochem. J., 1989, 261, 787–792. 11 N. Veiga, J. Torres, D. Mansell, S. Freeman, S. Dominguez, C. J. Barker, A. Diaz and C. Kremer, JBIC, J. Biol. Inorg. Chem., 2009, 14, 51–59.
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12 L. R. Devireddy, D. O. Hart, D. H. Goetz and M. R. A. Green, Cell, 2010, 141, 1006–1017. 13 C. G. D. Morley and A. Bezkorovainy, IRCS Med. Sci., 1983, 11, 1106–1107. 14 R. C. Hider and X. L. Kong, BioMetals, 2011, 24, 1179–1187. 15 T. Kondo, G. L. Dale and E. Beutler, Methods Enzymol., 1995, 252, 72–82. ¨ndel, J. Harris, V. Kolb-Bachofen, B. Ketterer 16 S. Soboll, S. Gru and H. Sies, Biochem. J., 1995, 311, 889–894. 17 T. Z. Kidane, E. Sauble and M. C. Linder, Am. J. Physiol.: Cell Physiol., 2006, 291, C445–C455. ¨ger, 18 N. Bresgen, H. Jaksch, H. Lacher, I. Ohlenschla K. Uchida and P. M. Eckl, Free Radical Biol. Med., 2010, 48, 1347–1357. 19 A. Terman, B. Gustafsson and U. T. Brunk, J. Pathol., 2007, 211, 134–143. 20 T. Kurz, A. Terman, B. Gustafsson and U. T. Brunk, Biochim. Biophys. Acta, 2008, 1780, 1291–1303. 21 A. M. N. Silva, X. L. Kong, M. C. Parkin, R. Cammack and R. C. Hider, Dalton Trans., 2009, 8616–8625. 22 H. Glickstein, R. Ben El, G. Link, W. Breuer, A. M. Konijn, C. Hershko, H. Nick and Z. I. Cabantchik, Blood, 2006, 208, 3195–3203. 23 R. Lill, R. Dutkiewicz, H. P. Elsasser, A. Hausmann, D. J. A. Netz, A. J. Pierik, O. Stehling, E. Urzica and U. Muhlenhott, Biochim. Biophys. Acta, 2006, 1763, 652–667. 24 K. J. Thompson, M. G. Fied, Z. Ye, P. Boyer and J. R. Connor, J. Cell Sci., 2002, 115, 2165–2177. 25 F. Petrat, H. de Groot, R. Sustmann and U. Rauen, Biol. Chem., 2002, 383, 489–502. 26 W. Breuer, S. Epsztejn, P. Millgram and I. Z. Cabantchik, Am. J. Physiol., 1995, 268, C1345–C1361. 27 F. Petrat, U. Rauen and H. de Groot, Hepatology, 1999, 29, 1171–1179. 28 G. J. Kress, K. E. Dineley and I. J. Reynolds, J. Neurosci., 2002, 22, 5848–5855. 29 Y. M. Ma, H. de Groot, Z. Liu, R. C. Hider and F. Petrat, Biochem. J., 2006, 395, 49–55. 30 M. Tenopoulou, T. Kurz, P. T. Doulias, D. Galaris and U. T. Brunk, Biochem. J., 2007, 403, 261–266. 31 K. P. Carter, A. M. Young and A. E. Palmer, Chem. Rev., 2014, 114, 4564–4601. 32 Z. I. Cabantchik, H. Glickstein, P. Milgram and W. Breuer, Anal. Biochem., 1996, 233, 221–227. 33 H. Glickstein, R. B. El, M. Shvartsman and Z. I. Cabantchik, Blood, 2005, 106, 3242–3250. 34 S. Epsztejn, O. Kakhlon, H. Glickstein, W. Breuer and Z. I. Cabantchik, Anal. Biochem., 1997, 248, 31–40. 35 D. C. Harris and P. Aisen, Biochim. Biophys. Acta, 1973, 329, 156–158. 36 F. Thomas, G. Serratrice, C. Beguin, E. StaintAman, J. L. `re, J. Biol. Chem., 1999, Pierre, M. Fontecave and J. P. Laulhe 274, 13375–13383. 37 G. Zanninelli, H. Glickstein, W. Breuer, P. Milgram, P. Brissot, R. C. Hider, A. M. Konijn, J. Lipman, A. Shanzer and Z. I. Cabantchik, Mol. Pharmacol., 1997, 51, 842–852.
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38 S. Basu-Modak, D. Ali, M. Gordon, T. Polte, A. Yiakouvaki, C. Pourzand, C. Rice-Evans and R. M. Tyrrell, Free Radical Biol. Med., 2006, 41, 1197–1204. 39 M. Clark, N. C. Fisher, R. Kasthvri and C. Cerami Hand, Br. J. Haematol., 2013, 161, 262–269. 40 F. Petrat, H. de Groot and U. Rauen, Arch. Biochem. Biophys., 2000, 376, 74–81. 41 Y. M. Ma, W. Luo, P. J. Quinn, Z. Liu and R. C. Hider, J. Med. Chem., 2004, 47, 6349–6362. 42 Y. M. Ma, W. Luo, M. Camplo, Z. Liu and R. C. Hider, Bioorg. Med. Chem. Lett., 2005, 15, 3450–3452. 43 M. Tenopoulou, T. Kurz, P. T. Doulias, D. Galaris and U. T. Brunk, Biochem. J., 2007, 403, 261–266. 44 T. Kurz, A. Terman, B. Gustafsson and U. T. Brunk, Biochim. Biophys. Acta, 2008, 1780, 1291–1303. 45 T. Kurz, B. Gustafsson and U. T. Brunk, FEBS J., 2006, 273, 3106–3117. 46 F. Petrat, H. de Groot and U. Rauen, Biochem. J., 2001, 356, 61–69. 47 S. Fakih, M. Podinovskaia, X. Kong, H. L. Collins, U. E. Schaible and R. C. Hider, J. Med. Chem., 2008, 51, 4539–4552. 48 S. Fakih, M. Podinovskaia, X. Kong, U. E. Schaible, H. L. Collins and R. C. Hider, J. Pharm. Sci., 2009, 98, 2212–2226. 49 I. Napier, P. Ponka and D. R. Richardson, Blood, 2005, 105, 1867–1874.
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50 W. Breuer, M. Shvartsman and Z. I. Cabantchik, Int. J. Biochem. Cell Biol., 2008, 40, 350–354. 51 A. Campanella, E. Rovelli, P. Santambrogio, A. Cazzi, F. Taroni and S. Levi, Hum. Mol. Genet., 2009, 8, 1–9. 52 M. C. Frantz and P. Wipf, Environ. Mol. Mutagen., 2010, 51, 462–475. 53 H. Yuan, H. Cho, H. H. Chen, M. Panagia, D. E. Sosnovik and L. Josephson, Chem. Commun., 2013, 49, 10361–10363. 54 F. Petrat, D. Weisheit, M. Lensen, H. de Groot, R. Sustmann and U. Rauen, Biochem. J., 2002, 362, 137–147. 55 M. Shvartsman, E. Fibach and Z. I. Cabantchik, Biochem. J., 2010, 429, 185–193. 56 Z. I. Cabantchik, Front. Pharmacol., 2014, 5, 1–11. 57 K. L. Horton, K. M. Steward, S. B. Fonseca, Q. Guo and S. O. Kelley, Chem. Biol., 2008, 15, 375–382. 58 L. F. Yousif, K. M. Stewart, K. L. Horton and S. O. Kelly, ChemBioChem, 2009, 10, 2081–2088. 59 V. Abbate, R. C. Hider, C. Pourzand and O. Reelfs, Am. J. Hematol., 2013, 88, E68. 60 Y. Xiang and A. Tong, Org. Lett., 2006, 8, 1549–1552. 61 L. Qiu, C. Zhu, H. Chen, M. Hu, W. He and Z. Guo, Chem. Commun., 2014, 50, 4631–4634. 62 R. Wang, F. Yu, P. Liu and L. Chen, Chem. Commun., 2012, 48, 5310–5312. 63 D. W. Domaille, E. L. Que and C. J. Chang, Nat. Chem. Biol., 2008, 4, 168–175.
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Cite this: DOI: 10.1039/c4mt00214h
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Iron-sensitive fluorescent probes: monitoring intracellular iron pools Yongmin Ma,a V. Abbateb and R. C. Hider*b Several iron-sensitive fluorophores have been investigated in a range of cell types in order to quantify iron(II) levels in the cytosol and the cytoplasm. Both iron(II) and iron(III) cause fluorescence quenching of these probes and changes in cytosolic iron levels can be monitored in a reproducible manner. However
Received 12th August 2014, Accepted 7th October 2014
the precise quantification of iron(II) in the cytosol is complicated by the uncertainty of the structure of
DOI: 10.1039/c4mt00214h
is essential for quantitative purposes. The lysosomal and mitochondrial iron pools have only been the
many of the quenched species that exist under in vivo conditions. Precise knowledge of these structures subject of relatively few studies at the time of writing. Calcein–AM has been widely adopted for the
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monitoring of changes in iron levels in a range different cell types.
1. Introduction: the chemical nature of intracellular iron pools There are a number of intracellular pools of labile iron, associated with the cytosol, mitochondrion, nucleus and lysosome.1 Whereas there is an increasing knowledge relating to the pools associated with the cytosol, mitochondrion and lysosome, there
is very little information relating to that of the nucleus.2 The labile iron pool in each of these organelles plays an essential role in supplying iron to metalloproteins. As non coordinated iron salts can catalyse the formation of toxic oxygen-containing radicals in aerobic organisms,3 the levels of this labile iron pool must be tightly controlled. Cells must be able to sense iron levels and regulate iron homeostasis in such a manner as to maintain non-toxic levels of this essential metal.2
a
College of Pharmaceutical Science, Zhejiang Chinese Medical University, 548 Binwen Road, Hangzhou, P. R. China 310053 b Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK. E-mail: [email protected]
Yong-Min Ma received his MSc degree in Organic Chemistry in 2001 from Zhejiang University (P. R. China) under the direction of Professor Yong-Min Zhang. He obtained his PhD degree in Medicinal Chemistry in 2005 at King’s College London under the guidance of Professor Robert C Hider. He continued to work as a research fellow in Prof. Hider’s group until 2013, when he was appointed associate professor at Yongmin Ma Zhejiang Chinese Medical University. His research interests involve the design of iron chelators for the treatment of iron overload diseases, including fluorescencelabeled 3-hydroxypyridin-4-ones and fluorinated 3-hydroxypyridin4-ones.
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Cytoplasmic iron pool The redox state of this iron pool has been demonstrated to be iron(II) on the basis of thermodynamic arguments,4 the use of
Dr Abbate completed his MSc in Pharmacy in 2004 at ‘‘Federico II’’ University of Naples, where he also joined the Department of Medicinal & Toxicological Chemistry for one year working on the synthesis of novel analgesic compounds. He then moved to the UK to undertake a PhD in Chemistry and Analytical Sciences at The Open University (awarded in 2009). In 2008 he was awarded a prestigious Maplethorpe Fellowship of the University of Vincenzo Abbate London for the promotion of teaching and research in the pharmaceutical area. Dr Abbate currently works at KCL with Professor Hider on a BBSRC-funded project aimed at developing novel iron-chelating probes for monitoring and adjusting excess labile iron pool in mitochondria.
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ionophores5 and the use of iron-sensitive fluorophores.6,7 The concentration of this iron pool falls in the range 2 10 7–5 10 6 M.8 The nature of the iron-binding ligands has been much debated.9–13 Recently glutathione (GSH) has been demonstrated to be the most probable candidate, forming a monodentate complex via the sulfur atom (1).8,14 GSH has a high cytosolic concentration in most eukaryote cells (2–8 mM).15,16
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delivered to the lysosome will be iron(III), there is likely to be a mixture of both iron(III) and iron(II) present, the solubility of iron(III) being enhanced by the acid pH (5.5–6.0) of the lysosome. This labile iron renders lysosomes susceptible towards oxidative stress and this in turn leads to autophagy, apoptosis or necrosis.20 A wide range of ligands are available for the coordination of iron in this pool. For iron(II) it is likely that cysteine and glutathione will dominate and for iron(III), citrate will dominate.21 Nucleus iron pool
Mitochondrial iron pool Mitochondria are major sites for heme and iron–sulfur cluster synthesis and consequently there is a constant iron influx into mitochondria. Iron(II) is believed to be the dominant redox state and as the GSH concentration is even higher than that of the cytosol, namely 10–12 mM,16 again FeIISG (1) is likely to be the dominant chemical form of the labile iron pool. Significantly FeIISG could be a direct precursor for iron–sulfur clusters.8 Lysosome iron pool Lysosomes play a major role in the breakdown of both ferritin17,18 and mitochondria.19 Ferritin turns over with a half life between 12 and 24 h in most cell types,17 thus the resulting iron pool is likely to be relatively high. As a large proportion of the iron
R. C. Hider is emeritus professor of medicinal chemistry at King’s College London, where he has worked since 1987. Prior to this he was a lecturer in biological chemistry at Essex University. He has worked with siderophore-based iron uptake processes in microorganisms, plant root absorption of iron and the absorption of different molecular forms of iron by mammalian cells. His work on membrane structure and transport R. C. Hider mechanisms has led to the development of novel oral iron chelators for the treatment of iron overload. His group has synthesized a range of iron chelators, all specifically designed to permeate cell membranes in both the iron-free and iron-complexed form. As a result, N-alkyl-3-hydroxypyridin-4-ones have been identified as possessing potential for clinical application. Deferiprone is now used worldwide for the treatment of iron overload.
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The boundary between the nucleus and the cytosol is a double membrane, termed the nuclear envelope which is perforated by nuclear pores. There is a rapid equilibrium of labile iron between these two pools, presumably via nuclear pores.22 Iron–sulfur cluster proteins play a key role inside the nucleus23 and ferritin has a regulatory function in the nucleus.24 Nuclear redox active labile iron is thought to be involved with DNA damage induced by H2O2 and other reactive oxygen species and so it is important to be able to monitor nuclear LIP levels, particularly if they are not identical to those of the cytosol.25
2. Probes that have been used to measure the labile iron pool (LIP) A small range of fluorescent molecules have been utilized to provide information on the intracellular labile iron pool (2, 3, 4 and 5; Table 1). Those containing the acetoxymethoxy group are limited to the cytosol, while the others monitor intracellular organelle iron levels together with those of the cytosol. The acetoxymethoxy group (AM) renders probes membrane permeable, and cytosolic esterases rapidly cleave the AM esters to hydrophilic carboxylate groups (eqn (i)), creating the highly charged iron probe in the cytosol. Due to the hydrophilic nature of the probe, it remains trapped within the cytosol.30 As indicated in Table 1 none of the listed probes are completely selective for iron, they all bind both iron(II) and iron(III) and a limited range of other divalent metals. However under physiological conditions, the cytosolic zinc(II) and copper(II) levels are extremely low8 and are unlikely to interfere with the detection of iron. With fura-2 (4), although binding with calcium(II) will occur in the cytosol, this leads to a shift in the peak excitation wavelength, whereas the binding with iron(II) leads to fluorescence quenching.28 Thus the binding of calcium(II) and iron(II) can be distinguished.
(i) There are many fluorescent probes which have been described,31 some selective for iron(II) others selective for iron(III). However the properties of most of these probes have not been carefully investigated in cell culture systems.
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Iron-sensitive fluorophores
Probe
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Structure
Ion selectivity Ref.
Calcein–AM (Calcein, 2)
FeII, FeIII 26 NiII, CuII, CoII
Phen Green SK (3)
FeII, FeIII CaII, ZnII
27
Fig. 1 LIP levels in a single human hepatoma HepG2 cell. Iron(II) ammonium sulphate (FAS, 20 mM) was added to half the cells. SIH (100 mM) was added after the indicated time interval.34
Fura-2AM (Fura-2, 4)
FeII, FeIII CuII, CaII
28
CP655 (5)
FeII, FeIII CuII
29
We discuss below the probes which have been investigated in some detail. Calcein–AM Calcein–AM (2) is the most widely adopted iron(II) fluorescent probe, it having been studied in reticulocytes, CHO cells, fibroblasts, hepatocytes and colon cells.32,33 Typical behavior is illustrated in Fig. 1 where a single human hepatoma HepG2 cell is monitored.34 The top trace is fluorescence of a control cell treated with calcein–AM and lower shows the influence of incubation with ferrous ammonium sulfate (20 mM). As iron enters the cytosol it quenches the calcein fluorescence. After approximately 12 min, 100 mM SIH (6) was added. This chelator rapidly gains entry into the cytosol where it out competes calcein for iron(II) and so the fluorescence rapidly increases. Chelators with a high affinity for iron(II), for instance SIH (6) and PIH (7), compete with calcein for iron(II) very rapidly (Fig. 2) whereas chelators with a high affinity for iron(III), for instance DFO (8) and CP41 (9) compete at different rates. DFO, by virtue of its extremely high affinity for iron(III) facilitates the rapid autoxidation of iron(II)35 and reacts rapidly just like SIH, whereas CP41, a simple hydroxypyridin-4-one reacts more slowly (Fig. 2). The reason for this difference is the rate of
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Fig. 2 Dequenching of calcein–iron complexes by chelators in the absence of cells. Calcein (40 nM) at pH 7.2 was pretreated with iron(II) ammonium sulphate for 45 min. Chelators (1 mM) were added (*). The arrows indicate addition of SIH (100 mM).32
autoxidation of the CP41iron(II) complex is slower than that of DFOiron(II) (eqn (ii)). Thus the driving force for iron(II) chelation by these two iron(III) selective ligands is the enhanced stability of the iron(III) complex, which results from autoxidation of the corresponding iron(II) complex. When the behavior of the same group of 4 compounds was investigated in the presence of erythroleukemia K562 cells containing calcein, the properties of SIH and PIH were found to be similar to that observed in free solution, but the behavior of CP41 and DFO were reversed (Fig. 3). Clearly within the time course of the experiment DFO does not enter the cell, whereas although the smaller CP41 does enter the cell rapidly, it takes longer than SIH to stabilise the calcein fluorescence; 8 minutes as compared to 2 minutes. This is a result of the necessity of autoxidation to occur with CP41,
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Fig. 3 Dequenching of calcein–iron complexes in K562 cells by extracellularly added chelators (100 mM) at zero time. Arrow indicates the addition of SIH (100 mM).
whereas PIH and SIH are each able to form a relatively stable complex with iron(II).
constant KCA = [FeIICA]/[FeII] + [CA] can be used to quantify LIP, where LIP = [FeII] + [FeIICA]. In principle, KCA, [FeIICA] and [CA] can be determined. Thus [CA] can be determined from the fluorescence of a single cell or a suspension of cells of known density and cell volume, an anticalcein antibody being used to remove extracellular fluorescence. [FeIICA] is measured from the change in fluorescence on addition of SIH. KCA is determined from normal physico-chemical measurements. Herein lies a difficulty with the application of calcein for the quantification of the labile iron pool (LIP), as the precise nature of the iron–calcein complex in the cytosol is uncertain. Initially it was assumed to be similar to a 1 : 1 EDTA-type complex and the affinity constant for FeIIEDTA was adopted for the calculations used to quantify LIP. However under cytosolic conditions rapid autoxidation of FeIIEDTA occurs, as a consequence the CoII EDTA conditional constant was adopted for these studies, as this could be reliably measured.34 However studies based on ESR and cyclic voltammetry indicate that under physiological conditions calcein also binds iron at a phenolic site on the molecule (10), leading to a preference for iron(III).36 This has an entirely different affinity constant for iron and there is the possibility of each calcein molecule binding two such iron(III) cations. Thus there is uncertainty associated with the nature of the physiologically relevant iron complex that acts as a sink, indeed a mixture of iron complexes probably form. Thus it is difficult to determine LIP values based on this type of calculation. Never-the-less LIP values have been reported for a range of cell types using this method (Table 2).32
(iii)
Table 2 LIP values obtained from the application of calcein in various cell types, following iron loading and chelator – mediated deprivationa
(ii) It is possible to determine the concentration of iron(II) in the cytosol, often termed LIP (labile iron pool) or [Fe]LIP, from studies centred on the use of SIH as indicated in Fig. 3. Thus based on the equilibrium depicted in eqn (iii), the equilibrium
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Cell
Treatment
LIP (mM)
K562 K562 U937 U937 U937 Hepatocytes Hepatocytes Hepatocytes
Control +DFO Control +DFO +FAC Control +DFO +FAC
0.113 0.058 0.176 0.071 0.412 0.203 0.046 0.923
a K562, human erythroleukemia cells; U937, human promonocyte cells; hepatocytes, human hepatoma (HEPG2) cells. DFO, desferrioxamine, 100 mM; FAC, ferric ammonium citrate (20 mM).32,34
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Fig. 4 Dequenching of calcein–iron complexes in K562 cells. Various chelators (100 mM) added at different time points, SIH (100 mM) marked by *. The chelators 102, 41, 165 and 117 are analogues of Deferiprone.37
Despite this limitation, calcein has been utilized extremely effectively to provide information on changes in LIP levels. Thus the rate of influx of iron chelators into cells can be directly compared (Fig. 4),37 the UV-induced production of LIP can be monitored (Fig. 5)38 and the influence of LIP on the erythrocyte stage of Plasmodium falciparum monitored during cell culture (Fig. 6).39 Phen green SK diacetate Phen green SK diacetate is metabolized by cytosol esterases to phen green SK (3) (eqn (iv)) and hence becomes trapped
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Fig. 6 LIP levels in various malaria parasite stages. The LIP increases as the parasite matures within the host red blood cell. The LIP of uninfected and different stage parasitized RBCs was determined by assessing the fluorescence of calcein-loaded cells with the addition of the iron chelator, deferiprone.39
within the cell, although a proportion of the probe also enters mitochondria, lysosomes and the nucleus.27 Leakage from cells is generally superior to that of calcein, the latter acting as a substrate for a xenobiotic exporter protein.27 Using techniques similar to those outlined under the section on calcein, phen green SK has been used to estimate cytosolic levels of LIP (Fig. 7). Iron enters the cell more quickly in the presence of 8-hydroxyquinoline (11) than iron(III) citrate. In both cases the LIP is scavenged in the presence of bipyridyl (12). From these measurements an estimate of the cytosolic LIP concentration can be made. Petrat and coworkers40 prefer to use an ‘ex situ’ calibration method in order to facilitate such measurements. This is because the ‘in situ’ calibration requires a free equilibration between intra- and extracellular LIP and the iron(III) 8-hydroxyquinoline complex accumulates intracellularly, therefore ruling out this calibration procedure. Free phen green SK at a concentration loaded in hepatocytes was used to perform the calibration in the ‘‘cytosolic’’ – like medium. The fluorescence of intracellular phen green SK in an excess of bipyridyl (5 mM) was set at 100% fluorescence and the autofluorescence from a parallel incubation of phen green SK unloaded control hepatocytes was set at 0%. Thus the percentage of fluorescence recovery in hepatocytes by the addition of excess bipyridyl could be calculated and compared with the values from ‘ex situ’ calibration curve to obtain a LIP concentration.27
Fig. 5 UVA induced production of LIP in skin fibroblasts. EC – epicatechin (30 mM).38
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(iv)
Fig. 8 Frequency distribution of the hepatocellular concentration of LIP. Cells were incubated with PG SK diacetate (20 mM) for 10 min. Bipyridyl (5 mM) was added and the resulting fluorescence noted.40
9.0 mM, with a large proportion of the cells possessing LIP in the range 0.8–2 mM (Fig. 8).40 Hydroxypyridinone CP655 A range of fluorescent hydroxypyridinones have been described41,42 and one, CP655 (5) has been selected to monitor LIP levels.29 The probe gains rapid entry into cells without the necessity of using AM or acetyl esters. However this ability to penetrate plasma membranes renders it also possible to enter lysosomes and mitochondria, thus providing a measure of cellular LIP as opposed to cytosolic LIP. A further limitation of this class of fluorescent probe is that they are not trapped intracellularly and will efflux, once the incubation media is changed.29 However changes in LIP levels induced by the addition of iron(III) citrate and a competing chelator CP94 (13), similar to those reported for calcein and phen green SK, can be monitored by changes in fluorescence (Fig. 9). We are currently synthesising hydroxyl-pyridinone fluorescent probes which are linked to AM moieties. 14 is an example of such a molecule. This class of molecule is predicted to have a similar distribution to calcein–AM but will have the advantage of possessing a well characterized affinity constant for iron(III).
Fig. 7 Effect of an increase in the cellular chelatable iron on the fluorescence of PG SK enclosed in rat hepatocytes. A, iron(III) chloride; B, iron(II) ammonium sulphate.27
When this method was applied to isolated rat hepatocytes, a wide range of values for LIP was obtained ranging from 0.2 to
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Fig. 9 Fluorescence of the hydroxypyridinone CP655 (6 mM) in primary hepatocytes, quenched by iron(III) (2 mM) and recovered in the presence of CP94, an analogue of deferiprone.42
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mobilize lysosomal labile iron by monitoring the iron pools of single cells by flow cytometry.48
3. Lysosomal targeting probes The endosomal/lysosomal compartment of cells contains a relatively high level of labile iron. This will not be registered with the use of calcein–AM, as once calcein is generated in the cytosol it lacks the ability to penetrate membranes.43 The lysosome contains the breakdown products of both mitochondria44 and ferritin17 (Fig. 10) and chelation of this iron pool protects against oxidative stress-induced cellular damage.45 The measurement of labile iron levels in the endosomal/ lysosomal compartment of hepatocytes is difficult because of its small size, approximately 1% of the cell volume.46 However the larger lysosomes present in liver endothelial cells are amenable to measurement. Using Phen Green SK and 1,10phenanthroline in combination, Petrat and coworkers46 determined a labile iron level for this intracellular compartment of 15.8 4.1 mM. The only probe specifically designed to target the lysosome is the hydroxypyridinone SF34 (15).47 This fluorescein-labelled probe is only distributed to the endosomal/lysosomal compartments as demonstrated by colocation studies in macrophages (Fig. 11). Unlike the smaller more lipophilic probe CP655 (5), SF34 does not permeate membranes rapidly and so can be trapped within the endosomal/liposomal compartment. It apparently directly enters this organelle system by endocytosis. This probe has been used to compare the ability of different chelators to
4. Mitochondrial targeting probes As mitochondria are the main utilisers of iron in the cell as well as being a major source of the superoxide anion, O2 , their iron homeostasis is critically important. Clearly there must be a tight coordination between the influx of iron and its incorporation into heme and iron–sulfur clusters. An increase in mitochondrial iron burden is likely to be linked to associated pathology, for instance with Friedreich’s ataxia where there is inefficient iron– sulfur cluster synthesis.49,50 The mitochondrial labile iron pool can be buffered to some extent by mitochondrial ferritin; but not in all tissues, for instance liver mitochondria lack mitochondrial ferritin.51 One widely adopted method of targeting molecules to the mitochondrial matrix is by attachment to delocalized lipophilic cations. By taking advantage of the substantial negative electrochemical potential maintained across the inner mitochondrial membrane (typically 200 mV), delocalised cations are able to cross the membrane and hence be accumulated with a distribution
Fig. 10 Endosome, autophagosome, lysosomal pathway.44
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Fig. 11 Representative confocal microscopy images of intracellular colocalization studies of SF34 (15) in bone marrow derived macrophages using dextran Texas Red labeled lysosomes. Colocalization studies were carried out in DFO (E, F) and iron dextran (I, J) pretreated cells as well as untreated cells (A, B), with 1 h of incubation with 75 mM (15). Findings are illustrated together with merged fluorescence data showing a high degree of lysosomal colocalization in yellow at a later stage of cellular probe processing (C, G, K). Fluorescence data are shown in comparison to the corresponding bright field images (D, H, L).47
associated with the Nernst potential, typically in excess of 1000 fold. Triphenyl phosphonium salts (16) are typical examples where the R group can be, for instance the anti-oxidants vitamin E or Trolox.52 Attachment to 18F-labelled moieties renders it possible to monitor mitochondria by PET/CT.53 The positive charge formally associated with the central phosphorus atom is delocalised over the three phenyl rings, resulting in a low electron density. This, together with the overall lipophilic character of the molecule, enables rapid penetration of membranes. This useful property of lipophilic cations has been utilised to target fluorescent iron sensors to the mitochondrion. Both the Petrat54 and Cabantchik55,56 groups have utilised a derivative of rhodamine (RPA, 17) to monitor mitochondrial iron levels. Rhodamine is a positively charged fluorescent molecule where the single positive charge is delocalised on both nitrogen atoms and over the plane of the tricyclic structure. Petrat and coworkers introduced RPA (17) for the monitoring of mitochondrial iron levels.54 By adopting methods developed for the cytosol and using PIH (7), estimates of the mitochondrial labile iron pool were made (Fig. 12).54 The values for mitochondria of different cells were found to be distributed over a wide concentration range, centred on 12 mM (Fig. 13).54 Cabantchik has also utilised RPA to monitor mitochondrial iron levels.55 A limitation of this probe is the difficulty in reversing the quenched signal under physiological conditions56 and thus the associated difficulty of unequivocally assigning the quenched signal to labile iron.
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Fig. 12 Time course of dequenching of mitochondrial RPA (0.2 mM) fluorescence in rat hepatocytes after the addition of PIH (2 mM).54
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5. Other iron-sensitive fluorescent probes In addition to the probes listed in Table 1, there have been a number of reports describing other iron sensitive probes which have not been widely adopted. Calcein blue (CALB) (19) has been investigated by Cabantchik;56 its use as an acetomethoxy ester precursor has been introduced for the measurement of cytosolic iron. A rhodamine-based iron-sensor (20) which, in contrast to most other iron probes, undergoes an increase in fluorescence in the presence of iron has been investigated under in vitro conditions.60 However the molecular weight of this probe may reduce its ability to penetrate cell membranes. In similar fashion an anthracene-based probe (21) has also been reported to increase in fluorescence intensity in the presence of iron(III), but not iron(II). Again the molecular size of this probe and its highly charged nature are likely to seriously influence its ability to penetrate cell membranes.61 A boron-based iron(III) probe (BOD-NHOH) (22) has been described which functions by iron(III) catalysed hydroxylamine oxidation.62 However the fluorescence profiles of this probe are unchanged in the presence of iron(II) and so it is unlikely to be able to register the cytosolic labile iron pool. Other iron sensitive probes are listed in general reviews on fluorescent sensors.31,63 Fig. 13 Frequency distribution of concentration of mitochondrial chelatable iron in single hepatocytes. RPA (0.2 mM); PIH (2 mM). Mitochondria (n = 50) from three hepatocytes were investigated.54
Largely as a result of the above limitation, a different approach has recently been introduced for monitoring the mitochondrial matrix labile iron pool. A series of peptides has been prepared, members of which are capable of being targeted to and accumulated in mitochondria. These peptides contain alternate hydrophobic and basic amino acids and typically contain between 4 to 6 residues.57,58 Using this general design, a series of small fluorescent peptides have been synthesised that are selectively accumulated by mitochondria.59 By incorporating an iron chelating moiety into the molecule (18) we are able to monitor the mitochondrial labile iron pool.
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6. Conclusions Methods for the quantification of intracellular labile iron pools are still under development. The cytosolic pool is the best characterized using the probes Calcein–AM and phen green SK, although quantification is difficult. The mitochondrial and lysosomal pools are less well characterized, but there is progress in this area. The nuclear LIP has yet to be investigated. None of the probes so far studied are ideal, but many lessons have been learned and the design of probes to specifically quantify the cytosol, mitochondria and lysosome are likely to be introduced over the next few years.
List of abbreviations AM CA CALB CHO DFO EC EDTA ESR FAC FAS HepG2 GSH LIP PET PIH RPA SIH
Acetoxymethoxy group Calcein Calcein blue Chinese hamster ovary cells Desferrioxamine Epicatechin Ethylenediaminetetraacetic acid Electron spin resonance Ferric ammonium citrate Ferrous ammonium sulphate Human hepatoma cells Glutathione Labile iron pool Positron emission tomography Pyridoxal isonicotinoyl hydrazone Rhodamineb-[(1,10-phenanthrolin-5yl)aminocarbonyl]benzyl ester Salicylaldehyde isonicotinoyl hydrazone
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