217
Biochimica et Biophysica Acta, 543 (1978) 217--225 © Elsevier/North-Holland Biomedical Press
BBA 28667 IRON UP TAKE BY CHANG CELLS FROM T R A N S F E R R I N , N I T R I L O A C E T A T E AND C I T R A T E COMPLEXES T H E E F F E C T S OF I R O N - L O A D I N G AND C H E L A T I O N WITH DESFERRIOXAMINE
G.P. WHITE and A. JACOBS Department of Haematology, Welsh National School of Medicine, Cardiff (U.K.) (Received February 15th, 1978)
Summary Iron u p tak e by Chang liver cells in culture is a b o u t thirty times as great when ferric nitriloacetate is used as a d o n o r as when iron-transferrin is used. Iron uptake f r o m ferric citrate is no greater than from iron-transferrin. Most of the intracellular iron derived from transferrin is f o u n d in the supernatant after 2 0 0 0 0 X g centrifugation of the cell h o m o g e n a t e for 40 min: a b o u t half of this is in the form o f ferritin. Iron derived from ferric nitriloacetate is f o u n d largely in the m e m b r a n o u s pellet after centrifugation and very little of this is in the form o f ferritin. Iron in co r p o r ate d in cyt os ol ferritin is easily available for chelation by desferrioxamine and this process is facilitated by ascorbic acid. Membrane-bound iron is less available for chelation. This tissue culture model forms a convenient basis for the study of iron overlead and iron chelation.
Introduction Previous studies have shown t ha t Chang liver cells in culture take up iron f r o m transferrin [1,2]. This process is accompanied by ferritin synthesis and m o s t o f the iron i nc or por a t e d in the cells is f o u n d in the cell sap, either as ferritin or as a low molecular weight intermediate complex. A small p r o p o r t i o n of the iron is f o u n d in a m e m b r a n e - b o u n d form. Chang cells appear to offer a useful model o f intracellular iron metabolism and can be used to observe the mechanism and effectiveness of potentially therapeutic iron chelating drugs [2,3]. The present studies were carried o u t to examine the possibility of inducing iron overload in cultured cells as a m e t h o d of investigating iron t o x i c i t y and intracellular iron chelation. Iron u p t a k e f r om non-transferrin sources was measured because o f the high c o n c e n t r a t i o n required t o p r o d u c e iron overload and
218 because of the possible importance of non-transferrin iron [4] as a source of parenchymal loading in patients with transfusional siderosis. Materials and Methods
Chang cells. Chang cells (Flow Laboratories Ltd.) were cultured in glass bottles in minimum essential medium containing 10% foetal bovine serum. Iron loaded cells were obtained by adding 2 ml stock ferric nitrilotriacetate solution to 60 ml culture medium. The iron concentration in the basic incubation medium was 6 pmol/1, all of which is bound to bovine transferrin, and in the iron supplemented medium concentrations up to 178 pmol/1,were used. The cells were harvested after one week, washed three times with fresh medium and counted using a Coulter counter Model ZF. Ferric nitrilotriacetate. 5 mmol nitrilotriacetic acid were partially dissolved in 300 ml isotonic saline (154 mmol/1) with a consequent drop in pH to 2. Ferric a m m o n i u m citrate (2.5 mmol) was slowly added and after complete solubilisation of the ferric nitriloacetate complex the pH was adjusted to 7 with solid NaHCO3 and the volume made up to 500 ml. The final concentration of ferric iron was 1.5 mmol/1. [59Fe]ferric nitriloacetate. Nitriloacetate (0.1 mmol) was dissolved in 100 ml isotonic saline after the addition of 3 drops 1 M NaOH. A 10-ml aliquot was taken and adjusted to pH 2 with conc. HC1. To this solution 100 pl [59Fe]ferric citrate (10 pCi/pg Fe; Radiochemical Centre, A m e r s h a m ) w e r e added followed by 100 pl ferric a m m o n i u m citrate (28.6 mg in 2 ml saline) and the solution was then adjusted to pH 7 with solid NaHCO3. Addition of 100 pl [S9Fe]nitriloacetate solution to 1.2 ml cell suspension gave a final iron concentration of 40 pmol/1. To obtain lower concentrations of [S9Fe]ferric nitriloacetate the stock nitriloacetate and ferric a m m o n i u m citrate solutions were diluted before the preparation of the final [ S9Fe] nitriloacetate solution. [S9Fe]ferric citrate. 382 mg sodium citrate • 2 H20, 81 mg NaC1 and 15 pl ferric chloride solution {60%, w/v FeC13; Koch-Light Laboratories Ltd.) were dissolved in distilled water, adjusted to pH 7 with solid NaHCO3 and made up to 100 ml. 100 pl of [S9Fe]ferric citrate (10 ttCi/pg Fe; Radiochemical Centre, Amersham) were added to 4.9 ml of the above solution. Addition of 100 pl of this [59Fe]ferric citrate solution to 1.2 ml of cell suspension gave a final iron concentration of 50 pmol/1. [S9Fe]transferrin. Serum was obtained from iron-deficient subjects and labelled with S9Fe. Preliminary estimation of serum iron concentration and total iron binding capacity [5] was followed by the addition of a calculated a m o u n t of iron as ferric a m m o n i u m citrate labelled with [S9Fe]ferric citrate (Radiochemical Centre, Amersham) to produce 100% saturation. The serum was passed through an anion-exchange column (IRA 400) to remove u n b o u n d iron. Iron uptake by Chang cells. 1 • 106 Chang cells were incubated at 37°C for 22 h either in 1.2 ml minimum essential medium or in a mixture of 0.6 ml minimum essential medium and 0.6 ml of S9Fe-labelled or unlabelled serum at pH 7.2. In some experiments 100-pl aliquots of [S9Fe]nitriloacetate, [S9Fe]ferric citrate or m i n i m u m essential medium alone were added to the 1.2 ml of
219 cell suspension to give the required concentrations of iron in the incubation mixtures. Following incubation the cells were washed three times with 5 ml minimum essential medium at 4°C and then twice with saline (154 mmol/1)before sonication in 10 ml saline. A 1-ml aliquot of the resulting cell homogenate was removed for the measurement of total cell iron uptake. The remainder was centrifuged at 2 0 0 0 0 × g for 4 0 m i n at 4°C and the supernatant removed leaving a cell membrane pellet. An aliquot of the supernatant (1 ml) was removed for the measurement of SgFe activity. 100 pg of purified human spleen ferritin was added to 7 ml of the s u p e m a t a n t followed by 50 pl of anti-human ferritin serum [6] and then incubated at 37°C for 1 h. After leaving at 4°C overnight the precipitate was washed three times with saline (154 mmol/1) and dissolved in 1 ml 0.01 M HC1. The three 1-ml fractions (cells, supematant and ferritin) were counted in an Intertechnique liquid scintillation counter in 10 ml scintillation cocktail (toluene/Triton X-100/PPO/POPOP, 700 ml/350 ml/3.5 g/ 750 mg). The results were corrected for counting efficiency, isotope decay, dilution and sample size to give estimates of iron uptake in picomoles per million cells. Iron loss from cells. 2 • 106 Chang cells were incubated for 22 h under the conditions described above. After incubation the cells were washed 5 times with fresh minimum essential medium and then suspended in the same medium. Tubes were prepared containing 106 cells in 1.2 ml of cell suspension, the medium containing desferrioxamine (Ciba) at a final concentration of 1 mmol/1. The cells were incubated under the standard conditions for 6 h together with control cultures containing no desferrioxamine. The incubation was stopped by the addition of 5 ml minimum essential medium at 4°C and the cells were treated as described above. In some cases the medium contained 10 mmol/1 ascorbic acid. Total cell iron. Iron concentrations were measured in triplicate in whole cell homogenates, 2 0 0 0 0 × g pellets and cell supematants by flameless atomic absorption spectroscopy using a Varian Techtron atomic absorption spectrop h o t o m e t e r Model 110 and a Model 63 carbon rod atomiser. The specimens were sonicated immediately before analysis of 2-pl aliquots. Results Table I shows the iron uptake by Chang cells from media containing concentrations of iron mainly towards the upper end of the physiological range. When the iron is present as 100% saturated transferrin with a concentration of 58 pmol/1 incorporation is 35 pmol/106 cells after 22 h. Most of the labelled iron is found in the membrane-free supernatant, as in earlier experiments, and about half of this is in the form of ferritin. Iron uptake from 8% saturated transferrin is 10 pmol/106 cells, less than from 100% saturated transferrin. When iron is present as [SgFe]ferric nitriloacetate at a concentration of 100 pmol/1 incorporation is increased 30-fold compared with transferrin-iron uptake from the fully saturated protein and in this case most of the intracellular iron is membrane-bound. The smaller proportion of iron in the cell sap is still half in the form of ferritin. Addition of unsaturated transferrin to a medium containing
220 TABLE I IRON UPTAKE ACETATE
BY C H A N G
CELLS
FROM
[59Fe]TRANSFERIN
I r o n in m e d i u m
AND [59Fe]FERRIC
Labelled iron concentration (#mol/1)
I r o n u p t a k e b y cells ( p m o l / 1 0 6 cells) Whole cell
[59Fe]Transferrin (100% saturation) [ 59 F e ] T r a n s f e r r i n ( 8% s a t u r a t i o n ) Transferrin (100% saturation) + [59Fe]ferric nitrlloacetate T r a n s f e r r i n (8% s a t u r a t i o n ) + [ 5 9 F e ] f e r r i c n i t r i l o a e e t a t e T r a n s f e r r i n (8% s a t u r a t i o n ) + [ 5 9 F e ] f e r r i c n i t r i l o a c e t a t e [ S9 Fe] Transferrin (100% saturation) + nitriloacetate
58 5 100 100 50 58
NITRILO-
Supernatant
35 10 1047 512 48 33
Total
Ferritin
30 8 273 158 41 28
17 4 141 81 21 16
T A B L E II IRON UPTAKE BY CHANG CELLS FROM [59Fe]TRANSFERRIN Transferrin (% s a t u r a t i o n )
I r o n in m e d i u m (#tool/l)
I r o n u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
15 25 38 69 93
6.5 10.5 16.5 31.0 42.0
Supernatant
23.4 39.8 48.8 50.3 73.2
Total
Ferritin
20.5 29.8 36.7 40.4 57.4
13.3 17.6 22.1 28.0 39.8
T A B L E III IRON UPTAKE BY CHANG CELLS FROM INCREASING NITROLOACETATE I r o n in m e d i u m Lumol/1)
I r o n u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
0.1 0.5 1 5 10 25 50 100 250 500 1000 2500 5000
CONCENTRATIONS
2 5 16 17 19 12 12
6 33 94 232 368 900 656 428 684 846 624 464 420
Supernatant Total
Ferritin
1 6 21 66 110 172 314 578 2718 2364 2740 2120 2054
1 3 10 38 56 66 144 250 818 828 738 694 806
OF [59Fe]FERRIC
221
T A B L E IV U P T A K E OF I R O N INTO C H A N G CELLS WITH I N C R E A S I N G C O N C E N T R A T I O N S OF [ 5 9 F e ] F E R RIC C I T R A T E
Iron in m e d i u m (#mol/1)
Iron u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
0.5 1.0 5.0 10.0 25.0 50.0
5.2 11.6 28.1 31.8 30.4 28.2
Supernatant Total
Ferritin
5.2 4.4 7.6 9.4 10.1 8.2
2.1 3.9 6.8 7.3 7.1 6.9
[ SVFe] ferric nitriloacetate results in reduction of iron uptake from this complex presumably because transferrin binding occurs in the medium. When the amount of unsaturated transferrin present is adequate to bind all the iron complexed to nitriloacetate then cell uptake is quantitatively similar to uptake directly from transferrin and in this case the intracellular distribution of iron is similar to that found when transferrin is the donor. The presence of nitriloacetate in the incubation medium does not affect iron uptake from transferrin. In order to define the difference in iron uptake when transferrin, nitriloacetate and citrate are used as donors a wide range of concentrations were studied and the results are shown in Tables II--IV. While there are minor differences between the uptake of iron by different cultures under the same conditions, uptake from ferric nitriloacetate is consistently greater than uptake from transferrin. At concentrations from 0.1 to 5000 pmol/1 intracellular iron derived from ferric nitriloacetate is predominantly membrane-bound while throughout the physiological range of concentrations iron derived from transferrin is found predominantly in the cell sap. Total uptake from ferric citrate is quantitatively comparable to that from transferrin though in this case also less of the iron is found in the cell sap. Maximum uptake from nitriloacetate TABLE V T H E E F F E C T O F C H A N G C E L L I R O N C O N T E N T ON I R O N U P T A K E F R O M [ 5 9 F e ] F E R R I C N I T R I LOACETATE 0.5 p m o l [ 5 9 F e ] ferric nitriloacetate per 1.
Iron c o n t e n t o f cells ( p m o l / 1 0 6 cells)
Iron u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
3 5 10 12 22
928 392 392 890 920 250
33.2 26.4 17.6 18.2 21.4 17.1
Supernatant Total
Ferritin
8.0 6.7 3.8 4.3 3.7 3.6
4.1 3.3 2.2 2.5 2.3 2.1
222 occurs from an iron c o n c e n t r a t i o n in the medium of 250 #mol/1 while maxim u m u p tak e from the citrate occurs at 5 pmol/1 concentration. When Chang cells were iron-loaded by incubation in media containing increasing co n cen tr a t i ons of ferric nitriloacetate their iron c o n t e n t after one week was p r o p o r t i o n a l to the c o n c e n t r a t i o n in the medium rising from a mean basal c o n t e n t o f 9 2 8 - - 2 2 2 5 0 pmol/106 cells (Table V). Iron uptake by these loaded cells was measured by incubating them in medium containing [SgFe]ferric nitriloacetate. A 5-fold increase in cell iron c o n t e n t results in a 50% re d u c tio n of iron upt a ke from 0.5 pmol/1 [59Fe]ferric nitriloacetate. Greater cellular iron loading did n o t appear to influence uptake (Table V). When 40 pmol/1 [SgFe]ferric nitriloacetate was used iron uptake was a b o u t 3000 pm ol / 106 cells and was n o t influenced by the iron c o n t e n t of the cells. When normal Chang cells were labelled by 22-h incubation with [SgFe]transferrin a f u r th e r 6 h incubation in medium containing 1 mmol/1 desferrioxamine resulted in 28% loss of total cell activity. This was largely from the soluble p o r t i o n of the cell h o m o g e n a t e and most of this was due to the mobilisation o f labelled iron from ferritin. Ferritin labelling was reduced by 42%. When cells were labelled with 40 pmol/1 [S9Fe]ferric nitriloacetate subsequent incubation with desferrioxamine was followed by 41% reduction of ferritin S9Fe activity b u t in this case only a very small p r o p o r t i o n of the cellular S9Fe was contained in this form. Membrane-bound S9Fe which accounts for 88% of the total a m o u n t taken up by the cells was reduced only by 10% on incubation with desferrioxamine. The ef f ect o f ascorbic acid on iron mobilisation is shown in Table VI. Chang cells were iron loaded by incubation for two weeks in a medium containing 536 pmol/1 iron as ferric nitriloacetate. At the end of this period they were reincubated in basal m e di um u n s u p p l e m e n t e d with iron and in medium containing desferrioxamine (1 mmol/1) with and w i t h o u t ascorbic acid (10 mmol/1). The medium was changed every 48 h. The fall in cellular iron c o n t e n t with time is related to rate o f proliferation and as this varies somewhat between cultures the
TABLE VI IRON CONTENT OF IRON LOADED CELLS FOLLOWING SUBCULTURE ING DESFERRIOXAMINE (1 r e t o o l / l ) A N D A S C O R B I C A C I D ( 1 0 m m o l f l )
INTO MEDIA CONTAIN-
T i m e a f t e r s u b c u l t u r e (h) 0
24
48
96
192
1331
1087 749 890 596
768 693 830 476
525 447 795 291
360 285 532 195
3.8 2.6 2.8 1.6
4.2 2.3 2.5 1.4
3.6 1.4 2.2 0.8
3.5 1.1 1.9 0.6
I r o n c o n t e n t of cells (fg per cell) Control Desferrioxamine Ascorbic acid Desferrioxamine plus ascorbic acid Iron content of total cell population Control Desferrioxamine Ascorbic acid Desferrioxamine plus ascorbic acid
---(pg per culture) 4.2 --
223 results are expressed on both a cellular basis and as iron c o n t e n t of the whole culture (Table VI). At all times the cells incubated with desferrioxamine contained less iron than the control cells. When ascorbic acid was also present in the medium the loss of iron from the cultures was greater than that induced by desferrioxamine alone. Discussion All cells normally obtain iron from extracellular transferrin and most of this is either directed to the synthesis of essential iron enzymes or is incorporated into ferritin where it functions as an intracellular iron store. In the case of certain specialised cells such as those in the intestinal epithelium or the macrophage system iron may enter the cell from dietary sources in the iron metabolism of different types of cell but relatively little detailed evidence is available regarding the intracellular pathways involved in these processes. Cultured Chang liver cells have proved a valuable model for the study of intracellular iron metabolism [2] and work carried out using this system has provided information about iron uptake from transferrin, ferritin synthesis and the low molecular weight transit iron pool within the cell [7]. This experimental model has also been of value for the in vitro assessment of potential iron chelating drugs [3,8]. Jandl et al. [9] showed that iron uptake from transferrin by liver slices increased when the transferrin saturation was greater than 60% though even at 100% saturation uptake was only equivalent to the a m o u n t taken up by reticulocytes at 12% saturation. Bothwell and Finch [10] pointed out that the normal saturation of transferrin, 30--60%, ensures an adequate supply of iron to erythroid cells while preventing overload in other tissues. Those clinical situations where plasma iron concentrations are increased together with an increase in transferrin saturation are associated with parenchymal depositions of iron, especially in the liver. Chang cells also show an increase in iron uptake when the transferrin saturation in the culture medium is increased [1] but this is limited in a m o u n t even at 100% saturation. Prolonged culture in medium containing 100% saturated transferrin does not lead to cellular iron loading [13]. Hepatic iron overload in patients whose source of loading is either from multiple transfusions or from dietary abnormalities is invariably associated with a pathologically high transferrin saturation but there is no direct evidence that high saturation itself leads directly to tissue overload. Hershko and Rachmilewitz [4] suggest t h a t in patients with iron overload non-transferrin iron may exist independently in the plasma. This may be attached to albumin and is easily removed by chelating agents. The possibility that non-transferrin iron may be responsible for hepatic iron loading is supported by observations in patients with congenital atransferrinaemia where failure of iron supply to erythroid cells is accompanied by a severe degree of iron deposition in the liver and at other sites [11]. Awai et al. [12] have shown that hepatic siderosis can be induced in vivo in rats by the administration of ferric nitriloacetate but it is not certain whether prior binding to transferrin occurred in this case. In the present study cellular uptake of ferric iron from the nitriloacetate
224 complex was about 30-times greater than from transferrin though when unsaturated transferrin was also present in the medium this effectively inhibited uptake from nitriloacetate. This greatly increased uptake was found whether or not transferrin was present in the medium and does n o t appear to be related to specific binding sites. The a m o u n t of iron incorporated in the cells increases with the concentration of ferric nitriloacetate in the medium up to 250/amol/1. Above this concentration iron uptake did not exceed 20000 pmol per million cells during the 22-h incubation. There appears to be little control over the rate of loading and the sequential measurement of cell iron content during 100 and 92 h of culture suggests that cell division with the subsequent halving of iron c o n t e n t is the major factor in limiting iron accumulation. Cellular incorporation of iron from the nitriloacetate complex in the absence of transferrin implies the presence of an alternative mechanism to that involving transferrin receptor sites. Penetration of the membrane may be facilitated by the negative charge of the complex. When Chang cells are incubated with [S9Fe] transferrin most of the cellular S9Fe is later found in the cytosol, largely in the form of ferritin but with a significant proportion in a diffusable form [ 1 ]. In contrast to this, after incubation with [ S9Fe] ferric nitriloacetate, most of the isotope appears in the membrane pellet after cell disruption. Electron microscope studies of these cells after iron loading show an accumulation of ferritin and haemosiderin in lysosomes [ 13 ]. It is known that the production of large amounts of iron-rich ferritin is followed by lysosomal uptake of the molecules and their iron-rich ferritin is followed by lysosomal uptake of the molecules and their subsequent degradation. It might be assumed that in the present experiments this process is accelerated due to the high level of uptake occurring from ferric nitriloacetate. Although a lower proportion of total iron remains in the cytosol in these cells the proportion of soluble iron in the form of ferritin is similar to that in iron-transferrin loaded cells. However, even when the concentration of ferric nitriloacetate in the medium is low and the subsequent iron uptake comparable to that from transferrin the abnormal distribution between cytosol and membranes remains. Although we have no evidence for the intracellular polymerisation and precipitation of ferric iron derived from the nitriloacetate complex this is a theoretical possibility and subsequent lysosomal incorporation might be expected. It is difficult however to see why this should happen as may additional potential ligands are present which could inhibit the process and we have no evidence that polymerisation occurs in the incubation medium itself. Iron uptake from ferric citrate is quantitatively similar to that from irontransferrin, though the intracellular distribution is similar to that derived from ferric nitriloacetate. The ferri nitriloacetate complex is perhaps more comparable than the citrate to the non-specific iron found in thalassaemic sera as the citrate has a strong tendency to polymerisation which might well affect its availability to cell uptake. The chelation experiments with desferrioxamine show that the iron incorporated in cytosol ferritin is equally available for mobilisation whether the cells have been initially labelled with iron from transferrin or nitriloacetate. In the latter case, however, most of the intracellular iron is membrane-bound and less available to the chelator. Ascorbic acid results in greater iron mobili-
225
sation than occurs with desferrioxamine alone. We may conclude that is is possible to produce a cell culture model of iron loading using ferric nitriloacetate as a donor. This type of loading may be analogous to the condition of iron-loaded patients whose plasma contains nontransferrin iron [4] though such a mechanism has never been demonstrated in vivo. The pattern of intracellular iron deposition is relevant to the pathological state in human subjects suffering from transfusional siderosis and the present experiments form a basis for studies of the effects of iron overload and the influence of potential iron chelating drugs. References 1 2 3 4 5 6 7 8 9 10 11
12 13
Bailey-Wood, R., White, G.P. and Jacobs, A. (1975) Brit. J. Exp. Pathol. 56, 358--362 White, G . P , Bailey-Wood, R., Jacobs, A. (1976) Clln. Sci. Mol. Med. 50, 145--152 White, G.P., Jaeobs, A., Grady, R.W. and Cerarai, A. (1976) Brit. J. Haematol. 33, 487--495 Hershko, C. and Rachrnilewitz, E.A. (1976) in P r o t e i n s o f I r o n S t o r a g e and Transport in B i o c h e m i s t r y and Medicine (Crichton, R.R., ed.), pp. 427--432, North-Holland, Oxford Young, D.S. and Hicks, J.M. (1965) J. Clln. Pathol. 18, 98--102 Worwood, M., Dawkins, S., Wagstaff, M. and Jacobs, A. (1976) Binchem. J. 157, 97--103 Jacobs, A. (1977) Blood 5 0 , 4 3 3 - - 4 3 9 White~ G.P., Jacobs, A.~ Grady, R.W. and Cerami, A. (1976) Blood 48, 923---929 Jandl, J.H., Inman , J.K., Simmons, R.L. and Allen, D.W. (1959) J. Clin. Invest. 3 8 , 1 6 1 - - 1 8 5 Bothwell, T.H. and Finch, C.A. (1962) Iron metabolism, p. 167, Churchill, L o n d o n Heilmeyer, L , Keller, W., Vivell, O., Keiderllng, W., Betke, K., Wohler, F. and Schultze, H.G. (1961) D e u t s c h e Med. Wochenscbx. 86, 1745--1751 Awai, M., Narasaki, M., Seno, S. (1977) in P r o t e i n s o f Iron Metabolism (Brown, E.B., ed.), pp. 411-414, Grune and Stratton, L o n d o n Jacobs, A., Hoy, T., Humphreys, J. and Perera, P. (1978) Br. J. Exp. Pathol., in t h e press
Biochimica et Biophysica Acta, 543 (1978) 217--225 © Elsevier/North-Holland Biomedical Press
BBA 28667 IRON UP TAKE BY CHANG CELLS FROM T R A N S F E R R I N , N I T R I L O A C E T A T E AND C I T R A T E COMPLEXES T H E E F F E C T S OF I R O N - L O A D I N G AND C H E L A T I O N WITH DESFERRIOXAMINE
G.P. WHITE and A. JACOBS Department of Haematology, Welsh National School of Medicine, Cardiff (U.K.) (Received February 15th, 1978)
Summary Iron u p tak e by Chang liver cells in culture is a b o u t thirty times as great when ferric nitriloacetate is used as a d o n o r as when iron-transferrin is used. Iron uptake f r o m ferric citrate is no greater than from iron-transferrin. Most of the intracellular iron derived from transferrin is f o u n d in the supernatant after 2 0 0 0 0 X g centrifugation of the cell h o m o g e n a t e for 40 min: a b o u t half of this is in the form o f ferritin. Iron derived from ferric nitriloacetate is f o u n d largely in the m e m b r a n o u s pellet after centrifugation and very little of this is in the form o f ferritin. Iron in co r p o r ate d in cyt os ol ferritin is easily available for chelation by desferrioxamine and this process is facilitated by ascorbic acid. Membrane-bound iron is less available for chelation. This tissue culture model forms a convenient basis for the study of iron overlead and iron chelation.
Introduction Previous studies have shown t ha t Chang liver cells in culture take up iron f r o m transferrin [1,2]. This process is accompanied by ferritin synthesis and m o s t o f the iron i nc or por a t e d in the cells is f o u n d in the cell sap, either as ferritin or as a low molecular weight intermediate complex. A small p r o p o r t i o n of the iron is f o u n d in a m e m b r a n e - b o u n d form. Chang cells appear to offer a useful model o f intracellular iron metabolism and can be used to observe the mechanism and effectiveness of potentially therapeutic iron chelating drugs [2,3]. The present studies were carried o u t to examine the possibility of inducing iron overload in cultured cells as a m e t h o d of investigating iron t o x i c i t y and intracellular iron chelation. Iron u p t a k e f r om non-transferrin sources was measured because o f the high c o n c e n t r a t i o n required t o p r o d u c e iron overload and
218 because of the possible importance of non-transferrin iron [4] as a source of parenchymal loading in patients with transfusional siderosis. Materials and Methods
Chang cells. Chang cells (Flow Laboratories Ltd.) were cultured in glass bottles in minimum essential medium containing 10% foetal bovine serum. Iron loaded cells were obtained by adding 2 ml stock ferric nitrilotriacetate solution to 60 ml culture medium. The iron concentration in the basic incubation medium was 6 pmol/1, all of which is bound to bovine transferrin, and in the iron supplemented medium concentrations up to 178 pmol/1,were used. The cells were harvested after one week, washed three times with fresh medium and counted using a Coulter counter Model ZF. Ferric nitrilotriacetate. 5 mmol nitrilotriacetic acid were partially dissolved in 300 ml isotonic saline (154 mmol/1) with a consequent drop in pH to 2. Ferric a m m o n i u m citrate (2.5 mmol) was slowly added and after complete solubilisation of the ferric nitriloacetate complex the pH was adjusted to 7 with solid NaHCO3 and the volume made up to 500 ml. The final concentration of ferric iron was 1.5 mmol/1. [59Fe]ferric nitriloacetate. Nitriloacetate (0.1 mmol) was dissolved in 100 ml isotonic saline after the addition of 3 drops 1 M NaOH. A 10-ml aliquot was taken and adjusted to pH 2 with conc. HC1. To this solution 100 pl [59Fe]ferric citrate (10 pCi/pg Fe; Radiochemical Centre, A m e r s h a m ) w e r e added followed by 100 pl ferric a m m o n i u m citrate (28.6 mg in 2 ml saline) and the solution was then adjusted to pH 7 with solid NaHCO3. Addition of 100 pl [S9Fe]nitriloacetate solution to 1.2 ml cell suspension gave a final iron concentration of 40 pmol/1. To obtain lower concentrations of [S9Fe]ferric nitriloacetate the stock nitriloacetate and ferric a m m o n i u m citrate solutions were diluted before the preparation of the final [ S9Fe] nitriloacetate solution. [S9Fe]ferric citrate. 382 mg sodium citrate • 2 H20, 81 mg NaC1 and 15 pl ferric chloride solution {60%, w/v FeC13; Koch-Light Laboratories Ltd.) were dissolved in distilled water, adjusted to pH 7 with solid NaHCO3 and made up to 100 ml. 100 pl of [S9Fe]ferric citrate (10 ttCi/pg Fe; Radiochemical Centre, Amersham) were added to 4.9 ml of the above solution. Addition of 100 pl of this [59Fe]ferric citrate solution to 1.2 ml of cell suspension gave a final iron concentration of 50 pmol/1. [S9Fe]transferrin. Serum was obtained from iron-deficient subjects and labelled with S9Fe. Preliminary estimation of serum iron concentration and total iron binding capacity [5] was followed by the addition of a calculated a m o u n t of iron as ferric a m m o n i u m citrate labelled with [S9Fe]ferric citrate (Radiochemical Centre, Amersham) to produce 100% saturation. The serum was passed through an anion-exchange column (IRA 400) to remove u n b o u n d iron. Iron uptake by Chang cells. 1 • 106 Chang cells were incubated at 37°C for 22 h either in 1.2 ml minimum essential medium or in a mixture of 0.6 ml minimum essential medium and 0.6 ml of S9Fe-labelled or unlabelled serum at pH 7.2. In some experiments 100-pl aliquots of [S9Fe]nitriloacetate, [S9Fe]ferric citrate or m i n i m u m essential medium alone were added to the 1.2 ml of
219 cell suspension to give the required concentrations of iron in the incubation mixtures. Following incubation the cells were washed three times with 5 ml minimum essential medium at 4°C and then twice with saline (154 mmol/1)before sonication in 10 ml saline. A 1-ml aliquot of the resulting cell homogenate was removed for the measurement of total cell iron uptake. The remainder was centrifuged at 2 0 0 0 0 × g for 4 0 m i n at 4°C and the supernatant removed leaving a cell membrane pellet. An aliquot of the supernatant (1 ml) was removed for the measurement of SgFe activity. 100 pg of purified human spleen ferritin was added to 7 ml of the s u p e m a t a n t followed by 50 pl of anti-human ferritin serum [6] and then incubated at 37°C for 1 h. After leaving at 4°C overnight the precipitate was washed three times with saline (154 mmol/1) and dissolved in 1 ml 0.01 M HC1. The three 1-ml fractions (cells, supematant and ferritin) were counted in an Intertechnique liquid scintillation counter in 10 ml scintillation cocktail (toluene/Triton X-100/PPO/POPOP, 700 ml/350 ml/3.5 g/ 750 mg). The results were corrected for counting efficiency, isotope decay, dilution and sample size to give estimates of iron uptake in picomoles per million cells. Iron loss from cells. 2 • 106 Chang cells were incubated for 22 h under the conditions described above. After incubation the cells were washed 5 times with fresh minimum essential medium and then suspended in the same medium. Tubes were prepared containing 106 cells in 1.2 ml of cell suspension, the medium containing desferrioxamine (Ciba) at a final concentration of 1 mmol/1. The cells were incubated under the standard conditions for 6 h together with control cultures containing no desferrioxamine. The incubation was stopped by the addition of 5 ml minimum essential medium at 4°C and the cells were treated as described above. In some cases the medium contained 10 mmol/1 ascorbic acid. Total cell iron. Iron concentrations were measured in triplicate in whole cell homogenates, 2 0 0 0 0 × g pellets and cell supematants by flameless atomic absorption spectroscopy using a Varian Techtron atomic absorption spectrop h o t o m e t e r Model 110 and a Model 63 carbon rod atomiser. The specimens were sonicated immediately before analysis of 2-pl aliquots. Results Table I shows the iron uptake by Chang cells from media containing concentrations of iron mainly towards the upper end of the physiological range. When the iron is present as 100% saturated transferrin with a concentration of 58 pmol/1 incorporation is 35 pmol/106 cells after 22 h. Most of the labelled iron is found in the membrane-free supernatant, as in earlier experiments, and about half of this is in the form of ferritin. Iron uptake from 8% saturated transferrin is 10 pmol/106 cells, less than from 100% saturated transferrin. When iron is present as [SgFe]ferric nitriloacetate at a concentration of 100 pmol/1 incorporation is increased 30-fold compared with transferrin-iron uptake from the fully saturated protein and in this case most of the intracellular iron is membrane-bound. The smaller proportion of iron in the cell sap is still half in the form of ferritin. Addition of unsaturated transferrin to a medium containing
220 TABLE I IRON UPTAKE ACETATE
BY C H A N G
CELLS
FROM
[59Fe]TRANSFERIN
I r o n in m e d i u m
AND [59Fe]FERRIC
Labelled iron concentration (#mol/1)
I r o n u p t a k e b y cells ( p m o l / 1 0 6 cells) Whole cell
[59Fe]Transferrin (100% saturation) [ 59 F e ] T r a n s f e r r i n ( 8% s a t u r a t i o n ) Transferrin (100% saturation) + [59Fe]ferric nitrlloacetate T r a n s f e r r i n (8% s a t u r a t i o n ) + [ 5 9 F e ] f e r r i c n i t r i l o a e e t a t e T r a n s f e r r i n (8% s a t u r a t i o n ) + [ 5 9 F e ] f e r r i c n i t r i l o a c e t a t e [ S9 Fe] Transferrin (100% saturation) + nitriloacetate
58 5 100 100 50 58
NITRILO-
Supernatant
35 10 1047 512 48 33
Total
Ferritin
30 8 273 158 41 28
17 4 141 81 21 16
T A B L E II IRON UPTAKE BY CHANG CELLS FROM [59Fe]TRANSFERRIN Transferrin (% s a t u r a t i o n )
I r o n in m e d i u m (#tool/l)
I r o n u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
15 25 38 69 93
6.5 10.5 16.5 31.0 42.0
Supernatant
23.4 39.8 48.8 50.3 73.2
Total
Ferritin
20.5 29.8 36.7 40.4 57.4
13.3 17.6 22.1 28.0 39.8
T A B L E III IRON UPTAKE BY CHANG CELLS FROM INCREASING NITROLOACETATE I r o n in m e d i u m Lumol/1)
I r o n u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
0.1 0.5 1 5 10 25 50 100 250 500 1000 2500 5000
CONCENTRATIONS
2 5 16 17 19 12 12
6 33 94 232 368 900 656 428 684 846 624 464 420
Supernatant Total
Ferritin
1 6 21 66 110 172 314 578 2718 2364 2740 2120 2054
1 3 10 38 56 66 144 250 818 828 738 694 806
OF [59Fe]FERRIC
221
T A B L E IV U P T A K E OF I R O N INTO C H A N G CELLS WITH I N C R E A S I N G C O N C E N T R A T I O N S OF [ 5 9 F e ] F E R RIC C I T R A T E
Iron in m e d i u m (#mol/1)
Iron u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
0.5 1.0 5.0 10.0 25.0 50.0
5.2 11.6 28.1 31.8 30.4 28.2
Supernatant Total
Ferritin
5.2 4.4 7.6 9.4 10.1 8.2
2.1 3.9 6.8 7.3 7.1 6.9
[ SVFe] ferric nitriloacetate results in reduction of iron uptake from this complex presumably because transferrin binding occurs in the medium. When the amount of unsaturated transferrin present is adequate to bind all the iron complexed to nitriloacetate then cell uptake is quantitatively similar to uptake directly from transferrin and in this case the intracellular distribution of iron is similar to that found when transferrin is the donor. The presence of nitriloacetate in the incubation medium does not affect iron uptake from transferrin. In order to define the difference in iron uptake when transferrin, nitriloacetate and citrate are used as donors a wide range of concentrations were studied and the results are shown in Tables II--IV. While there are minor differences between the uptake of iron by different cultures under the same conditions, uptake from ferric nitriloacetate is consistently greater than uptake from transferrin. At concentrations from 0.1 to 5000 pmol/1 intracellular iron derived from ferric nitriloacetate is predominantly membrane-bound while throughout the physiological range of concentrations iron derived from transferrin is found predominantly in the cell sap. Total uptake from ferric citrate is quantitatively comparable to that from transferrin though in this case also less of the iron is found in the cell sap. Maximum uptake from nitriloacetate TABLE V T H E E F F E C T O F C H A N G C E L L I R O N C O N T E N T ON I R O N U P T A K E F R O M [ 5 9 F e ] F E R R I C N I T R I LOACETATE 0.5 p m o l [ 5 9 F e ] ferric nitriloacetate per 1.
Iron c o n t e n t o f cells ( p m o l / 1 0 6 cells)
Iron u p t a k e b y cells ( p m o l / 1 0 6 cells) W h o l e cell
3 5 10 12 22
928 392 392 890 920 250
33.2 26.4 17.6 18.2 21.4 17.1
Supernatant Total
Ferritin
8.0 6.7 3.8 4.3 3.7 3.6
4.1 3.3 2.2 2.5 2.3 2.1
222 occurs from an iron c o n c e n t r a t i o n in the medium of 250 #mol/1 while maxim u m u p tak e from the citrate occurs at 5 pmol/1 concentration. When Chang cells were iron-loaded by incubation in media containing increasing co n cen tr a t i ons of ferric nitriloacetate their iron c o n t e n t after one week was p r o p o r t i o n a l to the c o n c e n t r a t i o n in the medium rising from a mean basal c o n t e n t o f 9 2 8 - - 2 2 2 5 0 pmol/106 cells (Table V). Iron uptake by these loaded cells was measured by incubating them in medium containing [SgFe]ferric nitriloacetate. A 5-fold increase in cell iron c o n t e n t results in a 50% re d u c tio n of iron upt a ke from 0.5 pmol/1 [59Fe]ferric nitriloacetate. Greater cellular iron loading did n o t appear to influence uptake (Table V). When 40 pmol/1 [SgFe]ferric nitriloacetate was used iron uptake was a b o u t 3000 pm ol / 106 cells and was n o t influenced by the iron c o n t e n t of the cells. When normal Chang cells were labelled by 22-h incubation with [SgFe]transferrin a f u r th e r 6 h incubation in medium containing 1 mmol/1 desferrioxamine resulted in 28% loss of total cell activity. This was largely from the soluble p o r t i o n of the cell h o m o g e n a t e and most of this was due to the mobilisation o f labelled iron from ferritin. Ferritin labelling was reduced by 42%. When cells were labelled with 40 pmol/1 [S9Fe]ferric nitriloacetate subsequent incubation with desferrioxamine was followed by 41% reduction of ferritin S9Fe activity b u t in this case only a very small p r o p o r t i o n of the cellular S9Fe was contained in this form. Membrane-bound S9Fe which accounts for 88% of the total a m o u n t taken up by the cells was reduced only by 10% on incubation with desferrioxamine. The ef f ect o f ascorbic acid on iron mobilisation is shown in Table VI. Chang cells were iron loaded by incubation for two weeks in a medium containing 536 pmol/1 iron as ferric nitriloacetate. At the end of this period they were reincubated in basal m e di um u n s u p p l e m e n t e d with iron and in medium containing desferrioxamine (1 mmol/1) with and w i t h o u t ascorbic acid (10 mmol/1). The medium was changed every 48 h. The fall in cellular iron c o n t e n t with time is related to rate o f proliferation and as this varies somewhat between cultures the
TABLE VI IRON CONTENT OF IRON LOADED CELLS FOLLOWING SUBCULTURE ING DESFERRIOXAMINE (1 r e t o o l / l ) A N D A S C O R B I C A C I D ( 1 0 m m o l f l )
INTO MEDIA CONTAIN-
T i m e a f t e r s u b c u l t u r e (h) 0
24
48
96
192
1331
1087 749 890 596
768 693 830 476
525 447 795 291
360 285 532 195
3.8 2.6 2.8 1.6
4.2 2.3 2.5 1.4
3.6 1.4 2.2 0.8
3.5 1.1 1.9 0.6
I r o n c o n t e n t of cells (fg per cell) Control Desferrioxamine Ascorbic acid Desferrioxamine plus ascorbic acid Iron content of total cell population Control Desferrioxamine Ascorbic acid Desferrioxamine plus ascorbic acid
---(pg per culture) 4.2 --
223 results are expressed on both a cellular basis and as iron c o n t e n t of the whole culture (Table VI). At all times the cells incubated with desferrioxamine contained less iron than the control cells. When ascorbic acid was also present in the medium the loss of iron from the cultures was greater than that induced by desferrioxamine alone. Discussion All cells normally obtain iron from extracellular transferrin and most of this is either directed to the synthesis of essential iron enzymes or is incorporated into ferritin where it functions as an intracellular iron store. In the case of certain specialised cells such as those in the intestinal epithelium or the macrophage system iron may enter the cell from dietary sources in the iron metabolism of different types of cell but relatively little detailed evidence is available regarding the intracellular pathways involved in these processes. Cultured Chang liver cells have proved a valuable model for the study of intracellular iron metabolism [2] and work carried out using this system has provided information about iron uptake from transferrin, ferritin synthesis and the low molecular weight transit iron pool within the cell [7]. This experimental model has also been of value for the in vitro assessment of potential iron chelating drugs [3,8]. Jandl et al. [9] showed that iron uptake from transferrin by liver slices increased when the transferrin saturation was greater than 60% though even at 100% saturation uptake was only equivalent to the a m o u n t taken up by reticulocytes at 12% saturation. Bothwell and Finch [10] pointed out that the normal saturation of transferrin, 30--60%, ensures an adequate supply of iron to erythroid cells while preventing overload in other tissues. Those clinical situations where plasma iron concentrations are increased together with an increase in transferrin saturation are associated with parenchymal depositions of iron, especially in the liver. Chang cells also show an increase in iron uptake when the transferrin saturation in the culture medium is increased [1] but this is limited in a m o u n t even at 100% saturation. Prolonged culture in medium containing 100% saturated transferrin does not lead to cellular iron loading [13]. Hepatic iron overload in patients whose source of loading is either from multiple transfusions or from dietary abnormalities is invariably associated with a pathologically high transferrin saturation but there is no direct evidence that high saturation itself leads directly to tissue overload. Hershko and Rachmilewitz [4] suggest t h a t in patients with iron overload non-transferrin iron may exist independently in the plasma. This may be attached to albumin and is easily removed by chelating agents. The possibility that non-transferrin iron may be responsible for hepatic iron loading is supported by observations in patients with congenital atransferrinaemia where failure of iron supply to erythroid cells is accompanied by a severe degree of iron deposition in the liver and at other sites [11]. Awai et al. [12] have shown that hepatic siderosis can be induced in vivo in rats by the administration of ferric nitriloacetate but it is not certain whether prior binding to transferrin occurred in this case. In the present study cellular uptake of ferric iron from the nitriloacetate
224 complex was about 30-times greater than from transferrin though when unsaturated transferrin was also present in the medium this effectively inhibited uptake from nitriloacetate. This greatly increased uptake was found whether or not transferrin was present in the medium and does n o t appear to be related to specific binding sites. The a m o u n t of iron incorporated in the cells increases with the concentration of ferric nitriloacetate in the medium up to 250/amol/1. Above this concentration iron uptake did not exceed 20000 pmol per million cells during the 22-h incubation. There appears to be little control over the rate of loading and the sequential measurement of cell iron content during 100 and 92 h of culture suggests that cell division with the subsequent halving of iron c o n t e n t is the major factor in limiting iron accumulation. Cellular incorporation of iron from the nitriloacetate complex in the absence of transferrin implies the presence of an alternative mechanism to that involving transferrin receptor sites. Penetration of the membrane may be facilitated by the negative charge of the complex. When Chang cells are incubated with [S9Fe] transferrin most of the cellular S9Fe is later found in the cytosol, largely in the form of ferritin but with a significant proportion in a diffusable form [ 1 ]. In contrast to this, after incubation with [ S9Fe] ferric nitriloacetate, most of the isotope appears in the membrane pellet after cell disruption. Electron microscope studies of these cells after iron loading show an accumulation of ferritin and haemosiderin in lysosomes [ 13 ]. It is known that the production of large amounts of iron-rich ferritin is followed by lysosomal uptake of the molecules and their iron-rich ferritin is followed by lysosomal uptake of the molecules and their subsequent degradation. It might be assumed that in the present experiments this process is accelerated due to the high level of uptake occurring from ferric nitriloacetate. Although a lower proportion of total iron remains in the cytosol in these cells the proportion of soluble iron in the form of ferritin is similar to that in iron-transferrin loaded cells. However, even when the concentration of ferric nitriloacetate in the medium is low and the subsequent iron uptake comparable to that from transferrin the abnormal distribution between cytosol and membranes remains. Although we have no evidence for the intracellular polymerisation and precipitation of ferric iron derived from the nitriloacetate complex this is a theoretical possibility and subsequent lysosomal incorporation might be expected. It is difficult however to see why this should happen as may additional potential ligands are present which could inhibit the process and we have no evidence that polymerisation occurs in the incubation medium itself. Iron uptake from ferric citrate is quantitatively similar to that from irontransferrin, though the intracellular distribution is similar to that derived from ferric nitriloacetate. The ferri nitriloacetate complex is perhaps more comparable than the citrate to the non-specific iron found in thalassaemic sera as the citrate has a strong tendency to polymerisation which might well affect its availability to cell uptake. The chelation experiments with desferrioxamine show that the iron incorporated in cytosol ferritin is equally available for mobilisation whether the cells have been initially labelled with iron from transferrin or nitriloacetate. In the latter case, however, most of the intracellular iron is membrane-bound and less available to the chelator. Ascorbic acid results in greater iron mobili-
225
sation than occurs with desferrioxamine alone. We may conclude that is is possible to produce a cell culture model of iron loading using ferric nitriloacetate as a donor. This type of loading may be analogous to the condition of iron-loaded patients whose plasma contains nontransferrin iron [4] though such a mechanism has never been demonstrated in vivo. The pattern of intracellular iron deposition is relevant to the pathological state in human subjects suffering from transfusional siderosis and the present experiments form a basis for studies of the effects of iron overload and the influence of potential iron chelating drugs. References 1 2 3 4 5 6 7 8 9 10 11
12 13
Bailey-Wood, R., White, G.P. and Jacobs, A. (1975) Brit. J. Exp. Pathol. 56, 358--362 White, G . P , Bailey-Wood, R., Jacobs, A. (1976) Clln. Sci. Mol. Med. 50, 145--152 White, G.P., Jaeobs, A., Grady, R.W. and Cerarai, A. (1976) Brit. J. Haematol. 33, 487--495 Hershko, C. and Rachrnilewitz, E.A. (1976) in P r o t e i n s o f I r o n S t o r a g e and Transport in B i o c h e m i s t r y and Medicine (Crichton, R.R., ed.), pp. 427--432, North-Holland, Oxford Young, D.S. and Hicks, J.M. (1965) J. Clln. Pathol. 18, 98--102 Worwood, M., Dawkins, S., Wagstaff, M. and Jacobs, A. (1976) Binchem. J. 157, 97--103 Jacobs, A. (1977) Blood 5 0 , 4 3 3 - - 4 3 9 White~ G.P., Jacobs, A.~ Grady, R.W. and Cerami, A. (1976) Blood 48, 923---929 Jandl, J.H., Inman , J.K., Simmons, R.L. and Allen, D.W. (1959) J. Clin. Invest. 3 8 , 1 6 1 - - 1 8 5 Bothwell, T.H. and Finch, C.A. (1962) Iron metabolism, p. 167, Churchill, L o n d o n Heilmeyer, L , Keller, W., Vivell, O., Keiderllng, W., Betke, K., Wohler, F. and Schultze, H.G. (1961) D e u t s c h e Med. Wochenscbx. 86, 1745--1751 Awai, M., Narasaki, M., Seno, S. (1977) in P r o t e i n s o f Iron Metabolism (Brown, E.B., ed.), pp. 411-414, Grune and Stratton, L o n d o n Jacobs, A., Hoy, T., Humphreys, J. and Perera, P. (1978) Br. J. Exp. Pathol., in t h e press
