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Requirement of endogenous iron for cytotoxicity caused by hydrogen peroxide in Euglena gracilis
31arine Emironmental Research 3-111992~ 339-343
Requirement of Endogenous Iron for Cytotoxicity Caused by Hydrogen Peroxide in Euglena gracilis Kimette Radtke, Robert W. Byrnes, Pamela Kerrigan, William E. Antholine & David H. Petering Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, USA A BSTRA C T
h Ls" widely thought that redox-actice metals hi cells such as iron or copper eataO'ze the re~hlction o f hydrogen peroxide to toxic hydroxyl radicals or their equicalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intraeellular iron content can he mo~hdated by the concentration o f iron in the dt~[ined growth medium without eJfect on the proliferation rate of the cells. E. gracilis contains two large storage pools of eytosolic iron which can he monitored to assess celluhtr iron status. The toxieio' o f H , O , hz E. gracilis was im'erselv related to the amottnt of iron ht the extraeelluhtr tne~#um. ,4 t h~k,h lereLs" ¢~/ external iron. the metal carried out the Fenton reaction with H,_O, outside the cell. pro~hwing hv~h'oxvl ra~ficals as detected hi' electron-spin resonance spin-trapping experinwnts. This reaction rethwed the amount o f t t , 0 2 that couht d([]use into cells to cause toxieitv. When cells with d([]~,rent httracelluhtr iron content were phwed in iron-dt~[ieient media. the toxicitv o f tt,_ 0 z, measured h v inhibition o/ prol([eration, was directly rehtted to the concentration qfinternal iron. Using cells de[icient ht iron, th{s" oxi~hmt did not inhibit proliferation at low concentration hut was somewhat ~[]?ctiee at h~g,her concentrations. Although the iron chehtthtg agents, 1,lOphenanthrolhte and de,~ferrioxamine, also depressed cytosolic iron, they were cvtotoxic to E. gracilis a/td so could not he used unamhiguotcs O' to examine the role of intracelluhtr iron bt the toxic ~[]'ects ~)['h)'dro~en peroxide.
Cellular oxidative stress can result from the reduction of molecular oxygen to species such as the hydroxyl radical (reaction (1)): 0 2 ~ ,O;[a)
~-~H_,O_, (bl
~OH'+OH(c~
339
(1)
340
Kimette Radtke et al.
Superoxide ion and hydrogen peroxide may form as side-products of enzymatic reactions that use 02 as a substrate or can be generated through oxidation-reduction reactions of xenobiotic compounds in cells, t It is hypothesized that the reduction of H202 to hydroxide ion and hydroxyl radical is catalyzed by redox-active metal ions in the Fenton reaction." This might occur by the Haber-Weiss pathway, in which 0 2 + M ~+. H 2 0 2 + M ~n- 1~+
" O 2 + M I"-t~+
(2)
" O H - + O H " + M "+
(3)
To examine the role of intracellular iron in eqn (1), step (c), chelating agents, thought to be specific for iron, have been added to cells exposed to H202 to see if they inhibit oxidant damage) Possible target species may be small molecular weight complexes of iron with ligands such as ADP or citrate. Concern about this approach lies in the non-specificity of such compounds for iron and the possibility that they may interact with cells in ways unrelated to metal-binding, including radical scavenging, a's Because of this, the reqt, irement of iron for step (c) has been reexamined. In the present investigation three procedures for iron removal from Euglena gracilis were compared--direct depletion from the defined growth medium and chelation of iron by 1,10-phenanthroline (OP) or desferrioxamine (D). According to Sephadex G-75 chromatography of cytosol, followed by atomic absorption spectrophotometric analysis of fractions for iron and zinc, E. gracilis has two large pools of cytosolic iron. One is associated with components greater than 70 kDa in molecular weight and the other with species about I-5 kDa in molecular weight.'* There is also a smaller pool of iron bound to cytochrome, identified by spectrophotometry. All three were lost when cells were placed in growth medium made up without its normal complement of added FeSO4 (361tM), which only contained 10-15nM residual iron. Even under this constraint, the proliferation rate remained unchanged. According to Fig. 1, the amount of high and low M r species of Fe per 10s cells declined over time after cells were placed in the iron-deficient medium. A calculation showed that the metal in these pools was essentially diluted out as the cells repeatedly divided. The chelating agents, OP and D, also reduced each pool of cytosolic Fe when supplied in the normal growth medium at 36 and 101tM, respectively, although they were somewhat less effective than the iron-depleted growth medium in reducing cellular iron. In addition, OP substantially lowered cytosolic zinc. Notably, these chelators were present at only one-third the stoichiometric concentrations needed for complexation of all the iron in the medium. However, at such higher levels, these reagents significantly inhibited cell proliferation. Because of these complications, it was not
Hydrogen peroxide in Euglena gracilis
341
0o _1 _J ILl ¢,.9
%4 -J I-ILl
2
HIGH Mr F~ LOW Mr F o
~o
zo
30
40
50
6o
to
TIME (HOURS)
Fig. i. Kinetics of loss of pools of iron I'rom cytosol. High and low .~1, pools were quantitatcd by Scphadex G-75 chromatography followed by atomic absorption spectrophotometry. possible to interpret possible effects of UP or D on hydrogen peroxide toxicity in terms of changes in intracellular-iron availability. It was recognized that the growth medium containing 36 l~M FeSO,~ might, itself, participate in the formation of hydroxyl radicals from H20 z. This was confirmed by adding the spin-trapping agent, 5,5-dimethyl-pyrolline-Noxide (DMPO), to the medium in the absence orE. gracilis immediately after addition of 2 ILMH 20_,. The electron spin resonance spectrum of this mixture revealed the presence of the four-line free-radical spectrum indicative of the spin adduct of DMPO and hydroxyl radical. No radicals were detected in the iron-deficient medium incubated with H202.To the extent that this reaction occurs, extracellular H20 2 will be degraded before it diffuses into cells and reacts there to cause cell damage. Thus, H202 might be less toxic when presented to cells in the iron-normal than in the iron-deficient growth medium. Hydrogen peroxide was more toxic to cells suspended in the iron-depleted medium than it was to cells placed in the normal growth medium. In the latter experimental regime, over a 120h incubation period, 100/.LM HzO 2 slowed proliferation of control cells 40% when placed in the iron-deficient growth medium but had no effect on a parallel culture of cells placed in the iron-normal medium. Therefore, the effects of H202 on cell proliferation
342
Kimette Radtke et al.
m 100~ rr"
o=
95
m
90
-u
F-
<
85-
~ o rr
80-
(5
75-
...J
©
re I-z o o LL O
7065-
6055
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lb
16o
HYDROGEN PEROXIDE (,qM)
Fig. l. Dependenceof proliferation of cells on H.,Oz,concentration. Cells grov.n for two ~aecks in iron-normM136Itg Fe ml) or iron-delicicnt10' 14)-5 fig Fe roll media were placcci in iron-deficieilt media at time zero and exarnincd 8 h later. were examined in iron-normal and iron-deficient cells that had been placed in media containing 36 or 0"1-0-51~xl Fe just prior to the addition of the oxidant. Figure 2 summarizes the growth effects of H 2 0 , on E. ,,,racilis under various conditions. It can be seen that cells containing normal levels of iron had lower proliferation rates in the presence of H_,O2 than did cells previously grown under iron-deficient conditions. Thus, it is clear that intracellular iron is required to mediate the toxicity of hydrogen peroxide. At higher concentrations of the oxidant, some growth reduction was seen, even in the iron-deficient cells. Whether this means that higher levels of H,_O, have gained access to other pools of iron or that they encounter another catalytic metal such as copper is not known. ACKNOWLEDGEMENTS The authors were supported by NIH grant ES-04026 and American Cancer Society grant CH-466.
REFERENCES 1. Sies, H. in Oxidatire Stress, ed. H. Sies. Academic Press, New York, 1985. 2. Aust, S. D., Morehouse, L. A. & Thomas, C. E., J. Free Radicals Biol. Med., I, 2-25 (1985).
Hydrogen peroxide in Euglena gracilis
343
3. Mello Filho, A. C. & Meneghini, R. Biochim. Biophys. Acta, 847, 82-9 11985). 4. Lyman, S., Taylor, P., Lornitzo, F., Weir, A., Stone, D., Antholine, W. E. & Petering, D. H. Biochem. Pharmacol., 38, 4273-82 (1989). 5. Sinaceur, J., Ribiere, C.. Nordmann, J. & Nordmann, R. Biochem. Pharmacol.. 33, 1693-9 (1984).
Requirement of Endogenous Iron for Cytotoxicity Caused by Hydrogen Peroxide in Euglena gracilis Kimette Radtke, Robert W. Byrnes, Pamela Kerrigan, William E. Antholine & David H. Petering Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, USA A BSTRA C T
h Ls" widely thought that redox-actice metals hi cells such as iron or copper eataO'ze the re~hlction o f hydrogen peroxide to toxic hydroxyl radicals or their equicalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intraeellular iron content can he mo~hdated by the concentration o f iron in the dt~[ined growth medium without eJfect on the proliferation rate of the cells. E. gracilis contains two large storage pools of eytosolic iron which can he monitored to assess celluhtr iron status. The toxieio' o f H , O , hz E. gracilis was im'erselv related to the amottnt of iron ht the extraeelluhtr tne~#um. ,4 t h~k,h lereLs" ¢~/ external iron. the metal carried out the Fenton reaction with H,_O, outside the cell. pro~hwing hv~h'oxvl ra~ficals as detected hi' electron-spin resonance spin-trapping experinwnts. This reaction rethwed the amount o f t t , 0 2 that couht d([]use into cells to cause toxieitv. When cells with d([]~,rent httracelluhtr iron content were phwed in iron-dt~[ieient media. the toxicitv o f tt,_ 0 z, measured h v inhibition o/ prol([eration, was directly rehtted to the concentration qfinternal iron. Using cells de[icient ht iron, th{s" oxi~hmt did not inhibit proliferation at low concentration hut was somewhat ~[]?ctiee at h~g,her concentrations. Although the iron chehtthtg agents, 1,lOphenanthrolhte and de,~ferrioxamine, also depressed cytosolic iron, they were cvtotoxic to E. gracilis a/td so could not he used unamhiguotcs O' to examine the role of intracelluhtr iron bt the toxic ~[]'ects ~)['h)'dro~en peroxide.
Cellular oxidative stress can result from the reduction of molecular oxygen to species such as the hydroxyl radical (reaction (1)): 0 2 ~ ,O;[a)
~-~H_,O_, (bl
~OH'+OH(c~
339
(1)
340
Kimette Radtke et al.
Superoxide ion and hydrogen peroxide may form as side-products of enzymatic reactions that use 02 as a substrate or can be generated through oxidation-reduction reactions of xenobiotic compounds in cells, t It is hypothesized that the reduction of H202 to hydroxide ion and hydroxyl radical is catalyzed by redox-active metal ions in the Fenton reaction." This might occur by the Haber-Weiss pathway, in which 0 2 + M ~+. H 2 0 2 + M ~n- 1~+
" O 2 + M I"-t~+
(2)
" O H - + O H " + M "+
(3)
To examine the role of intracellular iron in eqn (1), step (c), chelating agents, thought to be specific for iron, have been added to cells exposed to H202 to see if they inhibit oxidant damage) Possible target species may be small molecular weight complexes of iron with ligands such as ADP or citrate. Concern about this approach lies in the non-specificity of such compounds for iron and the possibility that they may interact with cells in ways unrelated to metal-binding, including radical scavenging, a's Because of this, the reqt, irement of iron for step (c) has been reexamined. In the present investigation three procedures for iron removal from Euglena gracilis were compared--direct depletion from the defined growth medium and chelation of iron by 1,10-phenanthroline (OP) or desferrioxamine (D). According to Sephadex G-75 chromatography of cytosol, followed by atomic absorption spectrophotometric analysis of fractions for iron and zinc, E. gracilis has two large pools of cytosolic iron. One is associated with components greater than 70 kDa in molecular weight and the other with species about I-5 kDa in molecular weight.'* There is also a smaller pool of iron bound to cytochrome, identified by spectrophotometry. All three were lost when cells were placed in growth medium made up without its normal complement of added FeSO4 (361tM), which only contained 10-15nM residual iron. Even under this constraint, the proliferation rate remained unchanged. According to Fig. 1, the amount of high and low M r species of Fe per 10s cells declined over time after cells were placed in the iron-deficient medium. A calculation showed that the metal in these pools was essentially diluted out as the cells repeatedly divided. The chelating agents, OP and D, also reduced each pool of cytosolic Fe when supplied in the normal growth medium at 36 and 101tM, respectively, although they were somewhat less effective than the iron-depleted growth medium in reducing cellular iron. In addition, OP substantially lowered cytosolic zinc. Notably, these chelators were present at only one-third the stoichiometric concentrations needed for complexation of all the iron in the medium. However, at such higher levels, these reagents significantly inhibited cell proliferation. Because of these complications, it was not
Hydrogen peroxide in Euglena gracilis
341
0o _1 _J ILl ¢,.9
%4 -J I-ILl
2
HIGH Mr F~ LOW Mr F o
~o
zo
30
40
50
6o
to
TIME (HOURS)
Fig. i. Kinetics of loss of pools of iron I'rom cytosol. High and low .~1, pools were quantitatcd by Scphadex G-75 chromatography followed by atomic absorption spectrophotometry. possible to interpret possible effects of UP or D on hydrogen peroxide toxicity in terms of changes in intracellular-iron availability. It was recognized that the growth medium containing 36 l~M FeSO,~ might, itself, participate in the formation of hydroxyl radicals from H20 z. This was confirmed by adding the spin-trapping agent, 5,5-dimethyl-pyrolline-Noxide (DMPO), to the medium in the absence orE. gracilis immediately after addition of 2 ILMH 20_,. The electron spin resonance spectrum of this mixture revealed the presence of the four-line free-radical spectrum indicative of the spin adduct of DMPO and hydroxyl radical. No radicals were detected in the iron-deficient medium incubated with H202.To the extent that this reaction occurs, extracellular H20 2 will be degraded before it diffuses into cells and reacts there to cause cell damage. Thus, H202 might be less toxic when presented to cells in the iron-normal than in the iron-deficient growth medium. Hydrogen peroxide was more toxic to cells suspended in the iron-depleted medium than it was to cells placed in the normal growth medium. In the latter experimental regime, over a 120h incubation period, 100/.LM HzO 2 slowed proliferation of control cells 40% when placed in the iron-deficient growth medium but had no effect on a parallel culture of cells placed in the iron-normal medium. Therefore, the effects of H202 on cell proliferation
342
Kimette Radtke et al.
m 100~ rr"
o=
95
m
90
-u
F-
<
85-
~ o rr
80-
(5
75-
...J
©
re I-z o o LL O
7065-
6055
i
lb
16o
HYDROGEN PEROXIDE (,qM)
Fig. l. Dependenceof proliferation of cells on H.,Oz,concentration. Cells grov.n for two ~aecks in iron-normM136Itg Fe ml) or iron-delicicnt10' 14)-5 fig Fe roll media were placcci in iron-deficieilt media at time zero and exarnincd 8 h later. were examined in iron-normal and iron-deficient cells that had been placed in media containing 36 or 0"1-0-51~xl Fe just prior to the addition of the oxidant. Figure 2 summarizes the growth effects of H 2 0 , on E. ,,,racilis under various conditions. It can be seen that cells containing normal levels of iron had lower proliferation rates in the presence of H_,O2 than did cells previously grown under iron-deficient conditions. Thus, it is clear that intracellular iron is required to mediate the toxicity of hydrogen peroxide. At higher concentrations of the oxidant, some growth reduction was seen, even in the iron-deficient cells. Whether this means that higher levels of H,_O, have gained access to other pools of iron or that they encounter another catalytic metal such as copper is not known. ACKNOWLEDGEMENTS The authors were supported by NIH grant ES-04026 and American Cancer Society grant CH-466.
REFERENCES 1. Sies, H. in Oxidatire Stress, ed. H. Sies. Academic Press, New York, 1985. 2. Aust, S. D., Morehouse, L. A. & Thomas, C. E., J. Free Radicals Biol. Med., I, 2-25 (1985).
Hydrogen peroxide in Euglena gracilis
343
3. Mello Filho, A. C. & Meneghini, R. Biochim. Biophys. Acta, 847, 82-9 11985). 4. Lyman, S., Taylor, P., Lornitzo, F., Weir, A., Stone, D., Antholine, W. E. & Petering, D. H. Biochem. Pharmacol., 38, 4273-82 (1989). 5. Sinaceur, J., Ribiere, C.. Nordmann, J. & Nordmann, R. Biochem. Pharmacol.. 33, 1693-9 (1984).