Mutation Research. 275 (1992) 3 i -39 ~3 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8734/92/$05.(~)
3!
MUTAG! 091)79
Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells J.J.P. Gille, C . G . M . van B e r k e l a n d H. J o e n j e Department of Human Genetics, Free Unicersity, Amsterdam (171e Netherlands) (Received 16 July 1991) (Accepted 9 October 1991)
Keywords: Chromosome aberrations; Sister-ehromatid exchanges; Hyperoxia; Desferrioxamine; o-Phenanthroline: Chinese hamster ovary cells
Summary The iron chelators o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary cells against the eytotoxic and genotoxic effects of normobaric hyperoxia. Desferrioxamine added at sub-toxic concentrations (up to 2.5 #M) over a period of several days had no protective effect on hyperoxia-induced clonogenic cell killing and growth inhibition. The clastogenic effect of hyperoxia was strongly potentiated by desferrioxamine, while the induction of sister-chromatid exchanges (SCEs) by hyperoxia was unaffected. Similarly, o-phenanthroline (up to (i.25 #M) had no protective effect on hyperoxia-induced cell killing, growth inhibition, and SCE induction, while also this compound potentiated the clastogenic effect of hyperoxia. These results do not support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, the results may still be consistent with a critical involvement of particular iron fraction(s) not susceptible to the chelators used. Furthermore, our results show that concentrations of iron chelators known to protect against short-term (up to 1 h) toxic exposure to oxidative stress become toxic themselves when applied chronically, i.e., in the order of days.
During normal aerobic metabolism, activated oxygen species are continuously formed intracellularly, due to partial reduction of molecular oxy-
Correspondence: Dr. H. Joenje, Department of Human Genetics, Free University, Van der Boechorststraat 7, 1081 BT Amsterdam (The Netherlands). Tel.: + 31-20-5482764. Fax: + 31-20-6462228.
gem The potentially deleterious effects of such species are counterbalanced by a complex antioxidant defense system, consisting of the enzymes catalase, superoxide dismutase and glutathione peroxidase as well as low molecular weight antioxidants (DiGuiseppi and Fridovich, 1984; Gille and Joenje, 1991). Oxidative stress denotes a condition in which the balance between free radical fluxes and the capacity of the antioxidant defense
32 system is disturbed to such an extent that cellular function has become impaired (Giile and Joenje, 1991; Si,.-s, 1985). Oxidative stress is thought to play an important role in the initiation and promotion phases of oncogenesis, and may significantly contribute to spontaneous genetic instability and aging tames, 1989; Cerutti, 1985; Joenje, 1989; Loeb, 1989). According to the free radical theory of aging, loss of cellular function during aging is a consequence of accumulating subcellular damage inflicted by activated oxygen species (Harman, 1981; Mehlhorn and Cole, 1985; ames, 1989). Oxidative stress can be studied in vitro by exposing cell cultures to hyperoxia, i.e., an atmosphere containing more than 20% 0 2 (Gille and Joenje, 1991). Hyperoxia is an attractive model system for oxidative stress, because under hyperoxic conditions activated oxygen species .are thought to be formed excessively at those intracellular sites where they are also formed in the normal situation (Jamieson, 1989). Hyperoxia-induced oxidative stress may also be a relevant model in aging research, it has been shown that the in vitro lifespan of human diploid fibroblasts can be modulated by the oxygen tension over the culture (Balin et al., 1984). Furthermore, densityarrested human glial cells, when cultured at elevated oxygen concentrations, accumulate lipofuscin-like material, which can be prevented by exogenous antioxidar~ts (Thaw ctal., 1984). Hence, hyperoxia-induced oxidative stress in cell cultures may be considered as a model for accelerated cellular aging resulting from excessive free radical production by normal metabolic pathways (Joenje et al., 1990). The most striking consequences of exposure of permanent cell lines to hyperoxia are inhibition of cell growth (Rueckert and Mueller, 1960), loss of clonogenic cell survival (Gille et al., 1989b; Schoonen et al., 1990a,b), inactivation of respiratory enzymes (Schoonen et al., 1990a,b) and depletion of ATP (Gille et al., 1989b; Schoonen et al., 1990a,b). Genotoxic consequences of exposure to hyperoxia are increased levels of chromosomal aberrations (Gille et al., 1989a) and sister. chromatid exchanges (Gille et al., 1989a), whereas only a small increase in DNA-strand breaks, as detected by the alkaline elution technique (Gille
et al., 1989b), and mutations at the hprt locus (Oiler, 1989) have been reported. Partial reduction of oxygen yields a number of different activated species: superoxide (O~-), hydrogen peroxide (HzO2), hydroxyl radical (OH), and singlet oxygen (IO 2) (DiGuiseppi and Fridovich, 1984; Gille and Joenje, 1991). HzO 2 itself seems to be relatively unreactive and the toxicity of O~- has long been debated (Fee, 1982; Forman and Haugaard, 1982; Halliwell, 1982), but it has now been established that 02- may exert deleterious effects in some specific cases (Fridovich, 1986). Most of the oxygen-induced cell damage, however, is thought to be mediated by "OH, an extremely reactive species, which can be formed during the reduction of H 202 by transition metals, particularly Fe 2+ (Haber and Weiss, 1934; Bilinski et al., 1985). With limiting Fe z+ concentration, Fe "~÷ reduction by O~- or other reducing agents is required for continued "OH formation. Iron is likely to be the most important transition metal in vivo, and the availability of iron will determine if and where "OH radical formation may occur. In biological systems most iron is bound to transport or storage proteins, transfertin and ferritin, respectively. Iron bound to these proteins is unable to catalyze 'OH radical formation (Halliwell and Outteridge, 1986). However, O;- can mobilize iron from ferritin, making it available to catalyze 'OH formation (Biemond et a!., 1984). Cellular iron may also occur bound to compounds such as ATP, GTP, and citrate. Such iron chelates are very efficient catalysts for "OH formation. The iron chelators o-phenanthroline and desferrioxamine are powerful inhibitors of the formation of 'OH (Gutteridge et al., 1979; Graf et al., 1984; Mello Filho and Meneghini, 1985) and have proved to be valuable probes to study the importance of iron-dependent oxygen radical reactions in vivo. For example, desferrioxamine was found to protect cultured hepatocytes against cell killing by H202 as well as menadione (Starke and Farber, 1985), while o-phenanthroline was found to afford protection against the cell killing and DNA-damaging effects of active oxygen species generated by xanthine oxidase plus acetaldehyde in mouse fihroblasts (Mello Filho and Meneghini, 1985)and against H202-induced
33 DNA-strand breaks (Mello Filho and Meneghini, 1984) and SCEs (Larramendy et al., 1987) in Chinese hamster cell cultures. In this study, o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary (CHO) cells against the cytotoxic and genotoxic effects of normobaric hyperoxia. Materials and methods
Chemicals o-Phenanthroline (1,10-phenanthroline monohydrate) was obtained from Merck (Darmstadt, Germany) and desferrioxamine (desferrioxamine methanesulfonic acid: Desferal TM) from CibaGeigy (Basle, Switzerland). Cuhure methods Cells were cultured in Ham's F10 medium (Flow Laboratories Ltd, Irvine, U.K.) supplemented with 1 mM L-glutamine and 10% (v/v) heat-inactivated (20 rain, 56°(2) fetal calf serum (Flow). Routinely, cells were washed with phosphate-buffered saline (PBS), trypsinized with a 0.05% trypsin-0.02% EDTA solution (Gibco Europe Ltd, Paisley, U.K.) in PBS, and seeded at 5-20 x 103 cells per em 2. Cell cultures were gassed with gas mixtures containing 2% CO 2 and various percentages of 02 balanced with N2. Gas mixtures were prepared with calibrated flow meters (Brooks Instruments, Veenendaal, The Netherlands) as described previously (Joenje et al., 1985). Cells were cultured in polystyrene culture flasks and placed in a Forma Scientific incubator in which the atmosphere was maintained at either air/2% CO 2 or 98% 0 2 / 2 % CO 2. HzO z exposure CHO cells were seeded at a density of 2 x 105 cells per 25-cm 2 culture flask and allowed to grow for 2 days. Cells were washed twice with 5 ml Medium 199 and exposed to H 2 0 2 (Perhydrol, Merck) for 1 h at 37°C in 5 ml in Medium 199 containing no Phenol Red (Gibco) and without fetal calf serum. Because pyruvate is known to interfere with H 202-induced cytotoxicity (Andrae
et al., 1985), H202 exposure was carried out in Medium 199, which lacks pyruvate.
Clonogenic cell survical Cells were trypsinized, counted in a Coulter counter, diluted to an appropriate cell concentration and seeded in triplicate in 100-ram petri dishes (Nunclon). After clonal development for 7-10 days, clones were fixed with methanol : acetic acid (3:1), stained with Giemsa and counted by eye. Chromosomal preparations After treatment with colchicine (0.75/zg/ml) for 1 h, chromosome preparations were made as described earlier (Gille et al., 1989a). Chromosomal aberrations Air-dried preparations from duplicate cultures were stained in 5% Giemsa for 5 rain and chromosomal aberrations were scored on coded slides in 100 metaphases, 50 metaphases from each culture. Chromatid gaps, which were recorded in only clearly evident cases, were distinguished from chromatid breaks by a lack of dislocation of the distal part of the chromatid, according to UKEMS recommendations (Scott et al., 1990). Differences in the fraction of damaged cells were tested for statistical significance using Fisher's exact test by calculating the mid-P value (Richardson et al., 1989). Sister.chromatid exchanges Cells were cultured in the presence of 8 / z g / m l 5'-bromodeoxyuridine (BrdU; Sigma Chemical Company, St. Louis, MO, U.S.A.) for 2 cell cycles. Since CHO cells grown under 98% 0 2 had longer cell cycle times, BrdU-labeling time was adjusted: 24 h for CHO cells grown under 20% 0 2 and 40 h for CHO cells grown under 98% 0 2. Chromosome preparations were made as described above. Differential staining was achieved by the fluorescence-plus-Giemsa technique as described by Perry and Wolff (1974). Sister-chromatid exchanges (SCEs) were counted on coded slides in 100 second-division metaphases from duplicate cultures.
34 100
Results
In order to investigate the role of iron-catalyzed oxygen radical reactions in the toxicity of hype:oxia, the sensitivity of CHO cells towards hyperoxia was studied in the presence of the iron-chelating compounds o-phenanthroline and desferrioxamine. In pilot experiments chelator concentrations were determined that were not or only slightly inhibitory to cell growth when present for 3 days under 20% 0 2. These concentrations were subsequently tested for their ability to protect against hyperoxia-induced growth inhibition, cell killing, chromosomal aberrations and SCEs. in cell growth experiments, CHO cells were cultured for 5 days under oxygen tensions ranging from 20 to 98% O 2 in the presence of various concentrations of iron chelator. As shown in Fig. 1 desferrioxamine appeared to be unable to afford significant protection against the growth .inhibiting effect of hyperoxia. A slight protective effect of 2.5 p.M desferrioxamine against 60% O z cannot be excluded on the basis of these data, but 10'01
8O
0
' 2O
~ 40
60 80 o x y g e n (%)
98
Fig. I, Growth of CHO cells under oxygen tensions ranging from 2iir~ to 98¢;- O z for 5 days in the presence of various concentrations of desl'errioxamine: o, control; o, 2.5 p,M; n 5 p,M: A. 10 ~tM. Cell growth is expressed as percenta~,e of growth under 20% O, in the absence (control) or presence o1' the indicated concentrations of chelator, Growth in the presence of 2.5, 5 and 10 p,M desferrioxamine at 20% O: was 102%, 91f~ and 58%, respectively,
8O
6O
40
20
20
4O
60 8O 9B oxygen (%) Fig. 2. Growth of CHO cells under oxygen tensions ranging from 20% to 98% O z for 5 days in the presence of various concentrations of o-phenanthroline: o, control; o, 0.05 ~M; D, 0.10 p,M; A, 0.25 p,M. Cell growth is expressed as percentage of growth under 20% 0 2 in the absence (control) or presence of the indicated concentrations of chelator. Growth in the presence of 0.05, 0.1 and 0.25 p,M o-phenanthroline at 21)% O z was %%, 92%, and 77%, respectively.
higher conce~trations desferrioxamine had a clear potentiating effect, o-Phenanthroline was also unable to protect against hyperoxia-induced growth inhibition, while the highest concentration o-phenanthroline (0.25/zM) again had a potentiating effect (Fig. 2). in cell survival experiments, CHO cells were grown for 3 days under 20% or 98% 02 in the presence of various chelator concentrations, after which colony-forming ability was determined. In the series of experiments with desferrioxamine, exposure to 98% 0 2 resulted in 17% cell survival. As shown in Fig. 3, desferrioxamine did not protect against clonogenic killing by 98% 0 2, but had, at concentrations higher than 2.5 p.M, a clear potentiating effect, In the series of experiments with o-phenanthroline, exposure to 98% 0 2 resulted in 5% cell survival, o-Phenanthroline had neither a protective nor a clear potentiating effect (Fig. 4). Desferrioxamine, at the same concentrations as tested under 98% O z, was also tested for protective capacity against short-term exposure to H 2 0 2 under normoxic conditions. CHO cells were grown in the presence of desfer-
35 TABLE 1 E F F E C T O F D E S F E R R I O X A M I N E ON H Y P E R O X I A - I N D U C E D C H R O M O S O M A L A B E R R A T I O N S % O?
Conc. (/zM)
% Damaged
Aberrations
cells
chromatid-type
20 20 20 20
0.5 2.5 5.0
10 11 8 8
98 98 98 98
0.5 2.5 5.0
49 38 Ns 88 ** NM
chromosome-type ci
total
csg
af
dic
Total number o f b r e a k evens
ctg
ctb
total
5 9 7 2
2 2
-
7 9 7 4
4 2 1 3
-
? I
4 4 1 5
11 13 8 9
23 19 66
95 55 382
9 2 106
127 76 554
8 5 6
5 24
-
13 5 30
140 81 584
ctg, chromatid gaps: ctb, chromatid breaks; ci, chromatid interchanges: csg, c h r o m o s o m e gaps; at', acentric chromosome fragments: dic, dicentric chromosomes. For chromatid interchanges, the number o f break events necessary for their formation is recorded: ctg and csg were counted as single, dic as two break events. Numbers indicate aberrations per 100 metaphases. NM, no metaphases found. ** Significantly different from control, P < 0.0Ol; Ns not significantly different (Fisher's exact test).
rioxamine for 2 days, washed and treated with H 202 for 1 h in the absence of desferrioxamine (see Materials and methods), after which clonogenic cell survival was determined. The H202 treatment used resulted in 35% cell survival in
100,
100
>
non-pretreated cells. Desferrioxamine pretreatment was found to protect CHO cells against the cell killing action of H202 in a dose-dependent manner (Fig~ 5), 15/.~M giving complete protection. This suggests that H 202-induced cell killing is mediated by desferrioxamine-chclatable iron.
i#
"X
10
o~ 10 >
t 1
i
0
5 desferrioxamine
i
10
!
15
( 10 -6 M)
Fig. 3. Clonogenic cell survival of C H O cells grown for 3 days in the presence of various concentrations of desferrioxamine. o , 20% O2: o, 98% O z. Data points are means (+_ SE) of 3 i n d e p e n d e n t experiments.
1
0.0
i
,
,
0.1
0.2
0.3
o-ohenantnrohne
( 10 -6 M)
Fig. 4. Clonogenic cell survival of C H O cells grown for 3 days m the presence of various concentrations of o-phenanthroline. o , 20% Oe: e. 98% 0 2 . Data points are means ( ± SE) of 3 independent experiments.
3~ 100
TABLE 3
80
EFFECT OF DESFERRIOXA MINE ON HYPEROXIA-1ND U C E D SCEs ~
60 % 0 2
Desferrioxamine (#M)
SCEs/cell
20 20 98 98
0 0.5 0 0.5
9.4±3.7 9.6±3.7 29.4±9.0 28.9±8.7
40 >
20
" Cells were gro,:.,.,z in the presence of v a ~ s . ~ o n c e n t r a t i o n s of desferrioxamine for 2 cell cycles. SCEs/cell are expressed as means ( + SD) scored in 100 metaphases.
10 0
i
,
i
5
10
15
desferrroxamine
( 1 0 -6 M )
Fig. 5. Clonogenic cell survival of CHO cells cultured for 2 days in the presence of various concentrations of desferrioxamine and treated with H 2 0 ., (0.5 raM) for 1 h in the absence of desferrioxamine in the medium. Data points are means ( :1: SE) of 3 independent experiments.
o-Phenanthroline and desferrioxamine were also tested for possible protection against the genotoxie action of hyperoxia. CHO cells, grown for 2 days under 20% and 98% O, in the presence or absence of iron chelators, were scored for chromosomal aberrations. Desferrioxamine at 0.5 /.tM seemed to have a protective effect against hyperoxia-induced clastogenesis, but the reduc-
tion in the percentage of damaged cells was not statistically significant. At higher concentrations, desferrioxamine potentiated the clastogenic effect of hyperoxia, mainly by enhancing the frequency of chromatid breaks (Table 1). oPhenanthroline appeared to have a similar potentiating effect on hyperoxia-induced clastogenesis (Table 2). In cells exposed to 98% 0 2, the SCE frequency was 3-fold increased. No significant effect of desferrioxamine (Table 3) or o-phenanthroline (Table 4) was observed on hyperoxia-induced SCEs. Cultures exposed to desferrioxamine concentrations higher than 0.5 p,M, or o-phenanthroline concentrations in excess of 0.25/~M, for 40 h under 98% O ! in the presence of bromodeoxyuridine, which is necessary for visualization
TABLE 2 E F F E C T O F o - P H E N A N T H R O L I N E ON H Y P E R O X I A - I N D U C E D C H R O M O S O M A L A B E R R A T I O N S t~ 0 2
Cunc. (/zM)
c~ Damaged cells
Aberrations
chromatid-type etg
20 20 20 20
0.05 0.10 0.25
6 6 9 10
98 98 t)8 98
0.05 0.10 0,25
47 61 * 62 * NM
ctb
chromosome-type ci
total
Total number
csg
af
dic
total
of break events
3 4 6 7
I 7 2 3
-
4 11 8 I(I
1 2 2
I
I
0 1 2 5
4 12 i0 15
14 31 47
97 212 192
2 3 -
113 249 239
I 2
-
I 3
0 3 8
113 252 247
Abbreviations are as in Table I. Numbers indicate aberrations per I0{) metaphases, NM, no metaphases found.
* Significanlly different from control. P < 0,05 (Fisher's exact test),
37 TABLE 4 EFFECT OF o-PHENANTHROLINE ON HYPEROXIAINDUCED SCEs ~ % O2
o-Phenanthroline (p.M)
20 20 20 20
0 0.05 0.10 0.25
98 98 98 98
0
24.1_+ 7.9
0.05 O.lO
24.7+ 7.9 31.1 -+ !0.4 27.6+ 8.9 *
0.25
SCEs/cell 9.5 _+ 9.1 +_ 8.7+ 9.4_+
3.4 3.1 3.4 3.7
,L Cells were grown in the presence of various concentrations of o-phenanthroline for 2 cell cycles. SCEs/cell are expressed as means ( + SD) scored in 100 metaphases. * Based on 45 metaphases.
of SCEs, did not yield sufficient numbers of mitotic cells for an analysis. This suggests that in the presence of bromodeoxyuridine, iron chelators cause a much sxronger potentiation of the antimitotic effect of hyperoxia than in the absence of bromodeoxyuridine. Discussion Our results show that the iron chelators ophenanthroline and desferrioxamine do not protect CHO cells against intoxication by normobaric hyperoxia, as tested by clonogenic cell survival, growth inhibition, chromosomal breakage and SCEs. These results may be explained in two ways: (1) hyperoxia-induced cell damage is not mediated by iron-catalyzed reactions, or (2) iron chelators in the eoncenm, aons used are not able to remove iron from specific cellular sites where "OH formation is critical for toxicity. Our results seem to be in contrast to those of others who found significant protection against exogenous H202. However, an important difference between hyperoxia and other sources of oxidative stress, such as H202, is that under the conditions used in this study, a toxic treatment with (normobaric) hyperoxia resulting in less than 10% cell survival takes about 3 days of continuous exposure, whereas an equitoxic treatment with H202 can readily be given in ! h. Due to the
slow action of normobaric hyperoxia, iron chelators to be tested must also be present during these relatively long exposure times. For ophenanthroline, 0.25 #M was the highest concentration without a measurable toxic effect on cell growth during 3 days exposure under normoxic conditions (data not shown). This concentration is 200-fold lower than that used by Larramendy et al. (1987) to demonstrate significant protection of Chinese hamster V79 cells against the SCE-inducing effect of H 2 0 2. Therefore, it is conceivable that the low o-phenanthroline concentrations used in our experiments are insufficient to deplete the intracellular iron pool that might be involved in the toxicity of hyperoxia. A similar reasoning may apply to desferrioxamine, as the highest concentration used in our experiments (15/zM) is still 1000-fold lower than that used by Starke and Farber (1985) to protect hepatocytes against the cytotoxic effect of H 2 0 2, as measured by trypan blue exclusion. However, CHO cells pretreated with 15 /zM desferrioxamine for 2 days were found to be completely resistant towards a H 2 0 2 dose that yields 35% cell survival in control cells. Thus low desferrioxamine concentrations used in our experiments do protect against H20:-induced but not against hyperoxiainduced cell killing. The above-mentioned results do not clearly support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, our results may still be consistent with the involvement of an iron fraction bound to critical targets (e.g., DNA), which might not be readily modified by the treatment conditions used. Finally, our results show that concentrations of iror~ chelators that have been successfully used by others to protect against short-term (up to 1 h) toxic c:~posure to oxidative stress, become toxic themselves when applied chronically, i.e., in the order of days, to cells under hyperoxic stress. Toxic effects of desferrioxamine have been reported previously. Desferrioxamine was found to inhibit the growth of human and murine hematopoietic progenitor cells. This effect was due to binding of ferric iron, since the effects of desferrioxamine could be effectively neutralized by the addition of equimolar concentrations of
38
iron (Nocka and Pelus, 1988). The growth inhibiting effect of desferrioxamine is probably the result of inhibition of ribonucleotide reductase, an enzyme that requires iron for its activity (Brown et al., 1969). Other toxicity mechanisms that have been proposed with respect to desferrioxamine are the generation of hydroxyl radicals (Klebanoff et al., 1989) or nitroxide free radicals (Morehouse et al., 1987). Furthermore, it is interesting to note that in rat liver, desferrioxamine was found to induce alterations in the subcellular distribution of iron, i.e., the iron content of mitochondria, microsomes and nuclei was enhanced following administration of desferrioxamine (Wahba et al., 1990). In addition, a significant increase in both lipid peroxidation and DNA single-strand breaks was observed. The most remarkable observation in this study is a strong clastogenicity of the combination desferrioxamine plus hyperoxia. In our study, desferrioxamine was not identified as a clastogen in cells exposed to 20% O2 nor was desferrioxamine reported to be clastogenic in human lymphocytes (Porfirio ct al., 19891. Therefore, we suppose that the strong clastogenicity of the combination desferrioxamine plus hyperoxia is due to a potentiating effect of desferrioxamine on the clastogenicity of hyperoxia. Potentiating interact!ons between desferrioxamine and oxklative stress have also been observed in cells exposed to cumene hydroperoxide (Poet et al., 19891 and in cells during hyperthermia treatment (Freeman et al., 19901. Therefore, the present iron chelators may not be suitable protectors in cases of chronic oxidative stress.
Acknowledgements The authors wish to thank Prof. J.F. Koster and Prof. H.G. van Eijk (Erasmus University, Rotterdam) for valuable advice. This study was supported by the Dutch Cancer Society (Nederlandse Kankerbestrijding), grant IKA-VU-87.10,
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Andrae, U., J. Singh and K. Ziegler-Skylakakis (1985) Pyrurate and related alpha-ketoacids protect mammalian cells in culture against hydrogen peroxide-induced cytotoxicity, Toxicol. Lett., 28, 93-98. Balin, A.K., A.F. Fischer, I. Leong and D.M. Carter (1984J Physiologic oxygen tensions modulate the lifespan of human cells, Age, 7, 142. Biemond. P., H,G. Van Eijk and J.F. Koster (1984) Iron mobilization from ferritin by superoxide derived from stimulated polymorphonuelear leukocytes. Possible mechanism in inflammation diseases, J. Clin. Invest., 73, 15761579. Bilinski. T., Z. Krawiec, A. Liczmanski and J. Litwinska (1985) Is hydroxyl radical generated by the Fenton reaction in rive?, Biochem. Biophys. Res. Commun., 130, 533-539. Brown, N.C., R. Eliasson, P. Reiehard and L. Thelander (1969) Spectrum and iron content of protein B2 from ribonucleoside reductase, Ear. J. Biochem., 9, 512-518. Cerutti, P.A. (1985) Prooxidant states and tumor promotion, Science. 227, 375-381, DiGuiseppi, J., and I. Fridovich (19841 The toxicity of molecular oxygen, CRC Crit. Re~. Toxicol., 12, 315-342. Fee, J.A. (1982) ls superoxide important in oxygen poisoning?, Trends Biochem. Sci., 7, 84-86. Forman, H.J., and N. Haugaard (19821 Superoxide toxicity, Trends Biochem. Sci., 7, 279. Freeman, M.L., D.R. Spitz and M.J. Meredith (19911) Does heat shock enhance oxidative stress? Studio,, with ferrous and ferric iron. Radiat. Res,, 124, 288-293. Fridovich, I. (19861 Biological effects of superoxide radical, Arch. Biochem. Biophys,, 247, I - I I, Gillu, JJ.P., and H. Joenje (1991) Biological consequences of oxygen toxicity. An introduction, in: C. Vigo.Pelfrey (Ed.), Membrane Lipid Oxidation, Vol, IlL CRC Press, Boca Raton, FL, pp, 1-32. Gille, JJ.P,, E, Mullaart, J. Vijg, A.L. Leyva, F. Arwert aad H. Joenje (1989a) Chromosomal instability in an oxygentolerant variant of Chinese hamster ovary cells, Mutation Res., 219, 17-28, Gille, J.J,P., C.G.M. van Berkel, E, Mullaart, J. Vijg and H. Joenje (1989b) Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells, Mutation Res,, 214, 89-96, Graf, E,, J.R, Mahoney, R,G, Bryant and J,W. Eaton (19841 Iron-catalyzed hydroxyl radical formation: stringent requirement for free iron coordination site, J, Biol, Chem., 259, 362(I-3624, Gutteridge, J.M,C,, R, Richmond and B. Halliwell (19791 Inhibition of the iron-catalyzed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine, Biochem, J,, 184, 469-472. Haber, F,, and J, Weiss (1934)The catalytic decomposition of hydrogen peroxide by iron salts, Prec. R, Soc, Lend. Ser. A, 147, 332-351, Halliwell, B. (19821 Superoxide and superoxide-dependent formation of hydroxyl radicals are important in oxygen toxicity, Trends Biochem, Sci., 7, 270-272,
39 Halliwell, B., and J.M.J. Gutteridgc (1986)Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts, Arch. Biochcm. Biophys., 246, 501-514. Harman, D. (1981) The aging process, Prec. Natl. Acad. Sci. (U.S.A.), 78, 7124-7128. Jamieson, D. (1989) Oxygen toxicity and reactive oxygen metabolitcs in mammals, Free Radical Biol. Med., 7, 87108. Joenje, H. (1989) Genetic toxicology of oxygen, Mutation Res., 219, 193-208. Joenje, H., J.J.P. Gille, A.B. Oostra and P. Van der Valk (1985) Some characteristics of hyperoxia-adapted HeLa cells. A tissue culture model for cellular oxygen tolerance, Lab. Invest., 52, 420-428. Jocnje, H., JJ.P. Gille, P. Pasman and W.G.E.J. Schoonen (1990) Oxygen poisoning as a model for accelerated cellular aging, in: C.F.A van Bezooijen, R. Ravid and A.AJ. Verhofstad, From Gene to Man - Gerontological Research in The Netherlands, Stichting Gerontologie en Geriatrie, Rijswijk, pp. 44-51. Klebanoff, S.J., A.M. Waltersdorph, B.R. Michel and H. Rosen (1989) Oxygen-based free radical generation by ferrous 'ions and deferoxamine, J. Biol. Chem., 264, 19765-19771. Larramendy, M., A.C. Mello Filho, E.A. Leme Martins and R. Meneghini (1987) Iron-mediated induction of sisterchromatid exchanges by hydrogen peroxide and superoxide anion, Mutation Res., 178, 57-63. Loeb, L.A. (1989) Endogenous carcinogenesis: molecular oncology into the twenty-first century, Cancer R,.,s., 49, 5489-5496. Mehlhern, R.J., and G. Cole (1985)The free radical theory of aging: a critical review, Adv. Free Radical Biol. Med., I, 165~223. Mello Filho, A.C., and R. Meneghini (1984) In rive fi)rmation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haher-Weiss reaction, Biochim. Biophys. Acre, 781, 5h-63. M¢llo Filho, A.C., and R. Meneghini (1985) Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species, Biochim. Biophys. Acta, 847, 82-89. Morehouse, K.M., W.D. Flitter and R.P. Mason (1987) The enzymatic oxidation of Desferal to a nitroxid¢ free radical, FEBS Letl., 222, 246-250. Nocka, K.H., and L.M. Pelus (1988)Cell cycle specific effects of desferrioxamine on human and murine hematopoietic progenitor cells, Cancer Res., 48, 3571-3575. Oiler, A.R. (1989) Mutational spectra of molecular oxygen and hydrogen peroxide in human B-cells: comparison to spontaneous mutation, PhD thesis. Massachusetts Institute of Technology, Cambridge, MA.
Perry, P., and S. Wolff (19741 New Giemsa method for the differential staining of sister chromatids, Nature. 251. 156-158. Poet, M., P.S. Rabinovitch and H. Hoehn (1989) Free radical mediated cytotoxicity of desferrioxamine, Free Radical Res. Commun., 6, 323-328. Porfirio, B., G. Ambrose, G. Gianella, G. lsacchi and B. Dallapiccola (1989) Partial correction of chromosome instability in Fanconi anemia by desferrioxamine, Hum. Genet., 83, 49-51. Richardson, C., D.A. Williams, G. Amphlett, B. Phillips, J.A. Allen and D.O. Chanter (1989) Analysis of data from in vitro cytogenetic assays, in: J. Kirkland (Ed.), Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, pp. 141-154. Rueckert, R.R., and G.C. Mueller (1960) Effect of oxygen tension on HeLa cell growth, Cancer Res., 2(I, 944-949. Sehoonen, W,G.E.J., A.H. Wanamarta, J.M. van der Klei-van Moorsel, C. Jacobs and H. Joenje (1990a) Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes. Mutation Res., 237, 173-181. Schoonen, W.G.E.J., A.H. Wanamarta, J.M. van dee Klei-van Moorsel, C. Jacobs and H. Joenje (1990b) Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia, J. Biol. Chem., 265, i l l 18-11124. Scott, D., N.D. Danford, BJ. Dean and D.J. Kirkland (1990) Metaphase chromosome aberration assays in vitro, in: D.J. Kirkland (Ed.), Basic Mutagenicity Tests, UKEMS Recommended Procedures, Cambridge University Press. Cambridge, pp. 62-86. Sies, H. (1985) Oxidative stress: introductory remarks, in: 11. Sies (Ed.), Oxidative Slress. Academic Press, London. pp. ]-8. Starke. P,E., and J.L. Farher (1985) Ferric iron and supcroxide ions are required fl)r the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals fl)rmed by :m iron-catalyzed IlaberWeiss reaction, J. Biol. Chem., 260, 10()99=1(11ll4. Thaw, H.H., V.P. Collins and U.T. Brunk (1984) Influence cff oxygen tension, pro,oxidants and antioxidants on the fi)rmarion of lipid peroxidation products (lipofuscin) in individual cultivated human glial cells, Mech. Ageing Dev., 24. 211-223. Wahba. Z.Z., W.J. Murray and S.J. Stohs (1990) Desferrioxamine-induced alterations in hepatic iron distribution, DNA damage :rod lipid pcroxidation in control and 2,3,7,8.tetrachlorodihenzo.p-dioxin-treated rats. J. Appl. Toxicol.. 10, 119-124.
3!
MUTAG! 091)79
Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells J.J.P. Gille, C . G . M . van B e r k e l a n d H. J o e n j e Department of Human Genetics, Free Unicersity, Amsterdam (171e Netherlands) (Received 16 July 1991) (Accepted 9 October 1991)
Keywords: Chromosome aberrations; Sister-ehromatid exchanges; Hyperoxia; Desferrioxamine; o-Phenanthroline: Chinese hamster ovary cells
Summary The iron chelators o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary cells against the eytotoxic and genotoxic effects of normobaric hyperoxia. Desferrioxamine added at sub-toxic concentrations (up to 2.5 #M) over a period of several days had no protective effect on hyperoxia-induced clonogenic cell killing and growth inhibition. The clastogenic effect of hyperoxia was strongly potentiated by desferrioxamine, while the induction of sister-chromatid exchanges (SCEs) by hyperoxia was unaffected. Similarly, o-phenanthroline (up to (i.25 #M) had no protective effect on hyperoxia-induced cell killing, growth inhibition, and SCE induction, while also this compound potentiated the clastogenic effect of hyperoxia. These results do not support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, the results may still be consistent with a critical involvement of particular iron fraction(s) not susceptible to the chelators used. Furthermore, our results show that concentrations of iron chelators known to protect against short-term (up to 1 h) toxic exposure to oxidative stress become toxic themselves when applied chronically, i.e., in the order of days.
During normal aerobic metabolism, activated oxygen species are continuously formed intracellularly, due to partial reduction of molecular oxy-
Correspondence: Dr. H. Joenje, Department of Human Genetics, Free University, Van der Boechorststraat 7, 1081 BT Amsterdam (The Netherlands). Tel.: + 31-20-5482764. Fax: + 31-20-6462228.
gem The potentially deleterious effects of such species are counterbalanced by a complex antioxidant defense system, consisting of the enzymes catalase, superoxide dismutase and glutathione peroxidase as well as low molecular weight antioxidants (DiGuiseppi and Fridovich, 1984; Gille and Joenje, 1991). Oxidative stress denotes a condition in which the balance between free radical fluxes and the capacity of the antioxidant defense
32 system is disturbed to such an extent that cellular function has become impaired (Giile and Joenje, 1991; Si,.-s, 1985). Oxidative stress is thought to play an important role in the initiation and promotion phases of oncogenesis, and may significantly contribute to spontaneous genetic instability and aging tames, 1989; Cerutti, 1985; Joenje, 1989; Loeb, 1989). According to the free radical theory of aging, loss of cellular function during aging is a consequence of accumulating subcellular damage inflicted by activated oxygen species (Harman, 1981; Mehlhorn and Cole, 1985; ames, 1989). Oxidative stress can be studied in vitro by exposing cell cultures to hyperoxia, i.e., an atmosphere containing more than 20% 0 2 (Gille and Joenje, 1991). Hyperoxia is an attractive model system for oxidative stress, because under hyperoxic conditions activated oxygen species .are thought to be formed excessively at those intracellular sites where they are also formed in the normal situation (Jamieson, 1989). Hyperoxia-induced oxidative stress may also be a relevant model in aging research, it has been shown that the in vitro lifespan of human diploid fibroblasts can be modulated by the oxygen tension over the culture (Balin et al., 1984). Furthermore, densityarrested human glial cells, when cultured at elevated oxygen concentrations, accumulate lipofuscin-like material, which can be prevented by exogenous antioxidar~ts (Thaw ctal., 1984). Hence, hyperoxia-induced oxidative stress in cell cultures may be considered as a model for accelerated cellular aging resulting from excessive free radical production by normal metabolic pathways (Joenje et al., 1990). The most striking consequences of exposure of permanent cell lines to hyperoxia are inhibition of cell growth (Rueckert and Mueller, 1960), loss of clonogenic cell survival (Gille et al., 1989b; Schoonen et al., 1990a,b), inactivation of respiratory enzymes (Schoonen et al., 1990a,b) and depletion of ATP (Gille et al., 1989b; Schoonen et al., 1990a,b). Genotoxic consequences of exposure to hyperoxia are increased levels of chromosomal aberrations (Gille et al., 1989a) and sister. chromatid exchanges (Gille et al., 1989a), whereas only a small increase in DNA-strand breaks, as detected by the alkaline elution technique (Gille
et al., 1989b), and mutations at the hprt locus (Oiler, 1989) have been reported. Partial reduction of oxygen yields a number of different activated species: superoxide (O~-), hydrogen peroxide (HzO2), hydroxyl radical (OH), and singlet oxygen (IO 2) (DiGuiseppi and Fridovich, 1984; Gille and Joenje, 1991). HzO 2 itself seems to be relatively unreactive and the toxicity of O~- has long been debated (Fee, 1982; Forman and Haugaard, 1982; Halliwell, 1982), but it has now been established that 02- may exert deleterious effects in some specific cases (Fridovich, 1986). Most of the oxygen-induced cell damage, however, is thought to be mediated by "OH, an extremely reactive species, which can be formed during the reduction of H 202 by transition metals, particularly Fe 2+ (Haber and Weiss, 1934; Bilinski et al., 1985). With limiting Fe z+ concentration, Fe "~÷ reduction by O~- or other reducing agents is required for continued "OH formation. Iron is likely to be the most important transition metal in vivo, and the availability of iron will determine if and where "OH radical formation may occur. In biological systems most iron is bound to transport or storage proteins, transfertin and ferritin, respectively. Iron bound to these proteins is unable to catalyze 'OH radical formation (Halliwell and Outteridge, 1986). However, O;- can mobilize iron from ferritin, making it available to catalyze 'OH formation (Biemond et a!., 1984). Cellular iron may also occur bound to compounds such as ATP, GTP, and citrate. Such iron chelates are very efficient catalysts for "OH formation. The iron chelators o-phenanthroline and desferrioxamine are powerful inhibitors of the formation of 'OH (Gutteridge et al., 1979; Graf et al., 1984; Mello Filho and Meneghini, 1985) and have proved to be valuable probes to study the importance of iron-dependent oxygen radical reactions in vivo. For example, desferrioxamine was found to protect cultured hepatocytes against cell killing by H202 as well as menadione (Starke and Farber, 1985), while o-phenanthroline was found to afford protection against the cell killing and DNA-damaging effects of active oxygen species generated by xanthine oxidase plus acetaldehyde in mouse fihroblasts (Mello Filho and Meneghini, 1985)and against H202-induced
33 DNA-strand breaks (Mello Filho and Meneghini, 1984) and SCEs (Larramendy et al., 1987) in Chinese hamster cell cultures. In this study, o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary (CHO) cells against the cytotoxic and genotoxic effects of normobaric hyperoxia. Materials and methods
Chemicals o-Phenanthroline (1,10-phenanthroline monohydrate) was obtained from Merck (Darmstadt, Germany) and desferrioxamine (desferrioxamine methanesulfonic acid: Desferal TM) from CibaGeigy (Basle, Switzerland). Cuhure methods Cells were cultured in Ham's F10 medium (Flow Laboratories Ltd, Irvine, U.K.) supplemented with 1 mM L-glutamine and 10% (v/v) heat-inactivated (20 rain, 56°(2) fetal calf serum (Flow). Routinely, cells were washed with phosphate-buffered saline (PBS), trypsinized with a 0.05% trypsin-0.02% EDTA solution (Gibco Europe Ltd, Paisley, U.K.) in PBS, and seeded at 5-20 x 103 cells per em 2. Cell cultures were gassed with gas mixtures containing 2% CO 2 and various percentages of 02 balanced with N2. Gas mixtures were prepared with calibrated flow meters (Brooks Instruments, Veenendaal, The Netherlands) as described previously (Joenje et al., 1985). Cells were cultured in polystyrene culture flasks and placed in a Forma Scientific incubator in which the atmosphere was maintained at either air/2% CO 2 or 98% 0 2 / 2 % CO 2. HzO z exposure CHO cells were seeded at a density of 2 x 105 cells per 25-cm 2 culture flask and allowed to grow for 2 days. Cells were washed twice with 5 ml Medium 199 and exposed to H 2 0 2 (Perhydrol, Merck) for 1 h at 37°C in 5 ml in Medium 199 containing no Phenol Red (Gibco) and without fetal calf serum. Because pyruvate is known to interfere with H 202-induced cytotoxicity (Andrae
et al., 1985), H202 exposure was carried out in Medium 199, which lacks pyruvate.
Clonogenic cell survical Cells were trypsinized, counted in a Coulter counter, diluted to an appropriate cell concentration and seeded in triplicate in 100-ram petri dishes (Nunclon). After clonal development for 7-10 days, clones were fixed with methanol : acetic acid (3:1), stained with Giemsa and counted by eye. Chromosomal preparations After treatment with colchicine (0.75/zg/ml) for 1 h, chromosome preparations were made as described earlier (Gille et al., 1989a). Chromosomal aberrations Air-dried preparations from duplicate cultures were stained in 5% Giemsa for 5 rain and chromosomal aberrations were scored on coded slides in 100 metaphases, 50 metaphases from each culture. Chromatid gaps, which were recorded in only clearly evident cases, were distinguished from chromatid breaks by a lack of dislocation of the distal part of the chromatid, according to UKEMS recommendations (Scott et al., 1990). Differences in the fraction of damaged cells were tested for statistical significance using Fisher's exact test by calculating the mid-P value (Richardson et al., 1989). Sister.chromatid exchanges Cells were cultured in the presence of 8 / z g / m l 5'-bromodeoxyuridine (BrdU; Sigma Chemical Company, St. Louis, MO, U.S.A.) for 2 cell cycles. Since CHO cells grown under 98% 0 2 had longer cell cycle times, BrdU-labeling time was adjusted: 24 h for CHO cells grown under 20% 0 2 and 40 h for CHO cells grown under 98% 0 2. Chromosome preparations were made as described above. Differential staining was achieved by the fluorescence-plus-Giemsa technique as described by Perry and Wolff (1974). Sister-chromatid exchanges (SCEs) were counted on coded slides in 100 second-division metaphases from duplicate cultures.
34 100
Results
In order to investigate the role of iron-catalyzed oxygen radical reactions in the toxicity of hype:oxia, the sensitivity of CHO cells towards hyperoxia was studied in the presence of the iron-chelating compounds o-phenanthroline and desferrioxamine. In pilot experiments chelator concentrations were determined that were not or only slightly inhibitory to cell growth when present for 3 days under 20% 0 2. These concentrations were subsequently tested for their ability to protect against hyperoxia-induced growth inhibition, cell killing, chromosomal aberrations and SCEs. in cell growth experiments, CHO cells were cultured for 5 days under oxygen tensions ranging from 20 to 98% O 2 in the presence of various concentrations of iron chelator. As shown in Fig. 1 desferrioxamine appeared to be unable to afford significant protection against the growth .inhibiting effect of hyperoxia. A slight protective effect of 2.5 p.M desferrioxamine against 60% O z cannot be excluded on the basis of these data, but 10'01
8O
0
' 2O
~ 40
60 80 o x y g e n (%)
98
Fig. I, Growth of CHO cells under oxygen tensions ranging from 2iir~ to 98¢;- O z for 5 days in the presence of various concentrations of desl'errioxamine: o, control; o, 2.5 p,M; n 5 p,M: A. 10 ~tM. Cell growth is expressed as percenta~,e of growth under 20% O, in the absence (control) or presence o1' the indicated concentrations of chelator, Growth in the presence of 2.5, 5 and 10 p,M desferrioxamine at 20% O: was 102%, 91f~ and 58%, respectively,
8O
6O
40
20
20
4O
60 8O 9B oxygen (%) Fig. 2. Growth of CHO cells under oxygen tensions ranging from 20% to 98% O z for 5 days in the presence of various concentrations of o-phenanthroline: o, control; o, 0.05 ~M; D, 0.10 p,M; A, 0.25 p,M. Cell growth is expressed as percentage of growth under 20% 0 2 in the absence (control) or presence of the indicated concentrations of chelator. Growth in the presence of 0.05, 0.1 and 0.25 p,M o-phenanthroline at 21)% O z was %%, 92%, and 77%, respectively.
higher conce~trations desferrioxamine had a clear potentiating effect, o-Phenanthroline was also unable to protect against hyperoxia-induced growth inhibition, while the highest concentration o-phenanthroline (0.25/zM) again had a potentiating effect (Fig. 2). in cell survival experiments, CHO cells were grown for 3 days under 20% or 98% 02 in the presence of various chelator concentrations, after which colony-forming ability was determined. In the series of experiments with desferrioxamine, exposure to 98% 0 2 resulted in 17% cell survival. As shown in Fig. 3, desferrioxamine did not protect against clonogenic killing by 98% 0 2, but had, at concentrations higher than 2.5 p.M, a clear potentiating effect, In the series of experiments with o-phenanthroline, exposure to 98% 0 2 resulted in 5% cell survival, o-Phenanthroline had neither a protective nor a clear potentiating effect (Fig. 4). Desferrioxamine, at the same concentrations as tested under 98% O z, was also tested for protective capacity against short-term exposure to H 2 0 2 under normoxic conditions. CHO cells were grown in the presence of desfer-
35 TABLE 1 E F F E C T O F D E S F E R R I O X A M I N E ON H Y P E R O X I A - I N D U C E D C H R O M O S O M A L A B E R R A T I O N S % O?
Conc. (/zM)
% Damaged
Aberrations
cells
chromatid-type
20 20 20 20
0.5 2.5 5.0
10 11 8 8
98 98 98 98
0.5 2.5 5.0
49 38 Ns 88 ** NM
chromosome-type ci
total
csg
af
dic
Total number o f b r e a k evens
ctg
ctb
total
5 9 7 2
2 2
-
7 9 7 4
4 2 1 3
-
? I
4 4 1 5
11 13 8 9
23 19 66
95 55 382
9 2 106
127 76 554
8 5 6
5 24
-
13 5 30
140 81 584
ctg, chromatid gaps: ctb, chromatid breaks; ci, chromatid interchanges: csg, c h r o m o s o m e gaps; at', acentric chromosome fragments: dic, dicentric chromosomes. For chromatid interchanges, the number o f break events necessary for their formation is recorded: ctg and csg were counted as single, dic as two break events. Numbers indicate aberrations per 100 metaphases. NM, no metaphases found. ** Significantly different from control, P < 0.0Ol; Ns not significantly different (Fisher's exact test).
rioxamine for 2 days, washed and treated with H 202 for 1 h in the absence of desferrioxamine (see Materials and methods), after which clonogenic cell survival was determined. The H202 treatment used resulted in 35% cell survival in
100,
100
>
non-pretreated cells. Desferrioxamine pretreatment was found to protect CHO cells against the cell killing action of H202 in a dose-dependent manner (Fig~ 5), 15/.~M giving complete protection. This suggests that H 202-induced cell killing is mediated by desferrioxamine-chclatable iron.
i#
"X
10
o~ 10 >
t 1
i
0
5 desferrioxamine
i
10
!
15
( 10 -6 M)
Fig. 3. Clonogenic cell survival of C H O cells grown for 3 days in the presence of various concentrations of desferrioxamine. o , 20% O2: o, 98% O z. Data points are means (+_ SE) of 3 i n d e p e n d e n t experiments.
1
0.0
i
,
,
0.1
0.2
0.3
o-ohenantnrohne
( 10 -6 M)
Fig. 4. Clonogenic cell survival of C H O cells grown for 3 days m the presence of various concentrations of o-phenanthroline. o , 20% Oe: e. 98% 0 2 . Data points are means ( ± SE) of 3 independent experiments.
3~ 100
TABLE 3
80
EFFECT OF DESFERRIOXA MINE ON HYPEROXIA-1ND U C E D SCEs ~
60 % 0 2
Desferrioxamine (#M)
SCEs/cell
20 20 98 98
0 0.5 0 0.5
9.4±3.7 9.6±3.7 29.4±9.0 28.9±8.7
40 >
20
" Cells were gro,:.,.,z in the presence of v a ~ s . ~ o n c e n t r a t i o n s of desferrioxamine for 2 cell cycles. SCEs/cell are expressed as means ( + SD) scored in 100 metaphases.
10 0
i
,
i
5
10
15
desferrroxamine
( 1 0 -6 M )
Fig. 5. Clonogenic cell survival of CHO cells cultured for 2 days in the presence of various concentrations of desferrioxamine and treated with H 2 0 ., (0.5 raM) for 1 h in the absence of desferrioxamine in the medium. Data points are means ( :1: SE) of 3 independent experiments.
o-Phenanthroline and desferrioxamine were also tested for possible protection against the genotoxie action of hyperoxia. CHO cells, grown for 2 days under 20% and 98% O, in the presence or absence of iron chelators, were scored for chromosomal aberrations. Desferrioxamine at 0.5 /.tM seemed to have a protective effect against hyperoxia-induced clastogenesis, but the reduc-
tion in the percentage of damaged cells was not statistically significant. At higher concentrations, desferrioxamine potentiated the clastogenic effect of hyperoxia, mainly by enhancing the frequency of chromatid breaks (Table 1). oPhenanthroline appeared to have a similar potentiating effect on hyperoxia-induced clastogenesis (Table 2). In cells exposed to 98% 0 2, the SCE frequency was 3-fold increased. No significant effect of desferrioxamine (Table 3) or o-phenanthroline (Table 4) was observed on hyperoxia-induced SCEs. Cultures exposed to desferrioxamine concentrations higher than 0.5 p,M, or o-phenanthroline concentrations in excess of 0.25/~M, for 40 h under 98% O ! in the presence of bromodeoxyuridine, which is necessary for visualization
TABLE 2 E F F E C T O F o - P H E N A N T H R O L I N E ON H Y P E R O X I A - I N D U C E D C H R O M O S O M A L A B E R R A T I O N S t~ 0 2
Cunc. (/zM)
c~ Damaged cells
Aberrations
chromatid-type etg
20 20 20 20
0.05 0.10 0.25
6 6 9 10
98 98 t)8 98
0.05 0.10 0,25
47 61 * 62 * NM
ctb
chromosome-type ci
total
Total number
csg
af
dic
total
of break events
3 4 6 7
I 7 2 3
-
4 11 8 I(I
1 2 2
I
I
0 1 2 5
4 12 i0 15
14 31 47
97 212 192
2 3 -
113 249 239
I 2
-
I 3
0 3 8
113 252 247
Abbreviations are as in Table I. Numbers indicate aberrations per I0{) metaphases, NM, no metaphases found.
* Significanlly different from control. P < 0,05 (Fisher's exact test),
37 TABLE 4 EFFECT OF o-PHENANTHROLINE ON HYPEROXIAINDUCED SCEs ~ % O2
o-Phenanthroline (p.M)
20 20 20 20
0 0.05 0.10 0.25
98 98 98 98
0
24.1_+ 7.9
0.05 O.lO
24.7+ 7.9 31.1 -+ !0.4 27.6+ 8.9 *
0.25
SCEs/cell 9.5 _+ 9.1 +_ 8.7+ 9.4_+
3.4 3.1 3.4 3.7
,L Cells were grown in the presence of various concentrations of o-phenanthroline for 2 cell cycles. SCEs/cell are expressed as means ( + SD) scored in 100 metaphases. * Based on 45 metaphases.
of SCEs, did not yield sufficient numbers of mitotic cells for an analysis. This suggests that in the presence of bromodeoxyuridine, iron chelators cause a much sxronger potentiation of the antimitotic effect of hyperoxia than in the absence of bromodeoxyuridine. Discussion Our results show that the iron chelators ophenanthroline and desferrioxamine do not protect CHO cells against intoxication by normobaric hyperoxia, as tested by clonogenic cell survival, growth inhibition, chromosomal breakage and SCEs. These results may be explained in two ways: (1) hyperoxia-induced cell damage is not mediated by iron-catalyzed reactions, or (2) iron chelators in the eoncenm, aons used are not able to remove iron from specific cellular sites where "OH formation is critical for toxicity. Our results seem to be in contrast to those of others who found significant protection against exogenous H202. However, an important difference between hyperoxia and other sources of oxidative stress, such as H202, is that under the conditions used in this study, a toxic treatment with (normobaric) hyperoxia resulting in less than 10% cell survival takes about 3 days of continuous exposure, whereas an equitoxic treatment with H202 can readily be given in ! h. Due to the
slow action of normobaric hyperoxia, iron chelators to be tested must also be present during these relatively long exposure times. For ophenanthroline, 0.25 #M was the highest concentration without a measurable toxic effect on cell growth during 3 days exposure under normoxic conditions (data not shown). This concentration is 200-fold lower than that used by Larramendy et al. (1987) to demonstrate significant protection of Chinese hamster V79 cells against the SCE-inducing effect of H 2 0 2. Therefore, it is conceivable that the low o-phenanthroline concentrations used in our experiments are insufficient to deplete the intracellular iron pool that might be involved in the toxicity of hyperoxia. A similar reasoning may apply to desferrioxamine, as the highest concentration used in our experiments (15/zM) is still 1000-fold lower than that used by Starke and Farber (1985) to protect hepatocytes against the cytotoxic effect of H 2 0 2, as measured by trypan blue exclusion. However, CHO cells pretreated with 15 /zM desferrioxamine for 2 days were found to be completely resistant towards a H 2 0 2 dose that yields 35% cell survival in control cells. Thus low desferrioxamine concentrations used in our experiments do protect against H20:-induced but not against hyperoxiainduced cell killing. The above-mentioned results do not clearly support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, our results may still be consistent with the involvement of an iron fraction bound to critical targets (e.g., DNA), which might not be readily modified by the treatment conditions used. Finally, our results show that concentrations of iror~ chelators that have been successfully used by others to protect against short-term (up to 1 h) toxic c:~posure to oxidative stress, become toxic themselves when applied chronically, i.e., in the order of days, to cells under hyperoxic stress. Toxic effects of desferrioxamine have been reported previously. Desferrioxamine was found to inhibit the growth of human and murine hematopoietic progenitor cells. This effect was due to binding of ferric iron, since the effects of desferrioxamine could be effectively neutralized by the addition of equimolar concentrations of
38
iron (Nocka and Pelus, 1988). The growth inhibiting effect of desferrioxamine is probably the result of inhibition of ribonucleotide reductase, an enzyme that requires iron for its activity (Brown et al., 1969). Other toxicity mechanisms that have been proposed with respect to desferrioxamine are the generation of hydroxyl radicals (Klebanoff et al., 1989) or nitroxide free radicals (Morehouse et al., 1987). Furthermore, it is interesting to note that in rat liver, desferrioxamine was found to induce alterations in the subcellular distribution of iron, i.e., the iron content of mitochondria, microsomes and nuclei was enhanced following administration of desferrioxamine (Wahba et al., 1990). In addition, a significant increase in both lipid peroxidation and DNA single-strand breaks was observed. The most remarkable observation in this study is a strong clastogenicity of the combination desferrioxamine plus hyperoxia. In our study, desferrioxamine was not identified as a clastogen in cells exposed to 20% O2 nor was desferrioxamine reported to be clastogenic in human lymphocytes (Porfirio ct al., 19891. Therefore, we suppose that the strong clastogenicity of the combination desferrioxamine plus hyperoxia is due to a potentiating effect of desferrioxamine on the clastogenicity of hyperoxia. Potentiating interact!ons between desferrioxamine and oxklative stress have also been observed in cells exposed to cumene hydroperoxide (Poet et al., 19891 and in cells during hyperthermia treatment (Freeman et al., 19901. Therefore, the present iron chelators may not be suitable protectors in cases of chronic oxidative stress.
Acknowledgements The authors wish to thank Prof. J.F. Koster and Prof. H.G. van Eijk (Erasmus University, Rotterdam) for valuable advice. This study was supported by the Dutch Cancer Society (Nederlandse Kankerbestrijding), grant IKA-VU-87.10,
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