[CANCER RESEARCH 41, 1628-1636, May 1981]
0008-5472/81/0041-0000302.00
Chromosome-damaging Activity of Ferritin and Its Relation to Chelation
and Reduction of Iron1
Robert F. Whiting,2 Lan Wei, and Hans F. Stich3
Environmental Carcinogenesis Unit. British Columbia Cancer Research Centre, and Department
British Columbia. Canada V6T 1W5
ABSTRACT
Ferritin from horse spleen was found to cause severe chro
mosome aberrations in cultured Chinese hamster ovary cells.
Ferritin at 15 to 170 ji¿g/mlwas clastogenic and at higher
doses was cytotoxic. At comparable concentrations of protein
or iron, neither apoferritin nor complexed iron was clastogenic.
Sulfhydryl compounds glutathione and cysteine reduced the
cytotoxic and clastogenic activities of ferritin. Physiological
concentrations of glutathione may normally be sufficient to
protect cells from damage. The reducing agent ascorbate had
little protective effect. Chelating agents varied in their inhibitory
activity: ethylenediaminetetraacetic
acid (hexadentate) > nitrilotriacetic acid (tetradentate) > salicylate (bidentate). 2,2'-Bipyridyl enhanced the chromosome-damaging
action of ferritin
while histidine did not markedly alter the frequencies of aber
rations. Catalase and Superoxide dismutase showed no inhibi
tory activity. The mechanism of DNA damage may involve
reduction of Fe(lll) in the ferritin core to Fe(ll), followed by
reoxidation of Fe(ll) with possible formation of free radicals.
INTRODUCTION
Ferritin is a major iron storage protein and is found widely
distributed in animals, plants, and fungi (6, 9, 21). In humans,
about 25% of the total body iron is contained in ferritin, stored
mainly in liver, spleen, and bone marrow (6, 9, 21). During
tumorigenesis, the amount of ferritin in tissues and serum may
significantly increase, and ferritin with different electrophoretic
mobilities was formed (23, 24). The ferritin molecule consists
of a central iron core surrounded by a spherical protein shell
called apoferritin. The iron core is a complex polymer of Fe(lll)
in the form of hydrous ferric oxide.-phosphate (6, 9,21). Ferritin
has been reconstructed from apoferritin, Fe(ll), and an oxidant
(2, 3, 10, 16, 29). Conversely, the reduction of Fe(lll) to Fe(ll)
is involved in the release of iron from ferritin (4,5,11,14,
20,
22, 25, 27, 30).
Ferritin (Fe3*)
Reduction
(mobilization)
Apoferritin
+ Fe2
Oxidation
(reconstitution)
Iron compounds have a variety of adverse biological effects.
lron:carbohydrate complexes are carcinogenic (12), and fer' This work was supported by the National Cancer Institute of Canada and the
Natural Sciences and Engineering Research Council of Canada.
2 Present address: Canadian Center for Occupational Health and Safety,
Health Sciences Center, 3N25, 1200 Main St. W., Hamilton, Ontario, Canada
L8N 325.
3 To whom requests for reprints should be addressed.
Received November 7, 1980; accepted January 22, 1981.
1628
of Medical Genetics. University of British Columbia. Vancouver,
rous sulfate is mutagenic in bacteria (1). In addition, low levels
of ferritin (0.25 to 5.0 jug/ml culture medium) were found to
suppress mitogen (phytohemagglutinin or concanavalin A)-induced blastogenesis in human peripheral blood lymphocytes
(19).
We have been investigating the mutagenic and comutagenic
activity of iron compounds (28, 31, 32). The interaction of iron
complexes with sodium ascorbate caused high frequencies of
chromosome aberrations in CHO4 cells (28). Ferritin was of
interest as a major physiological form of iron. In this report, we
describe (a) the induction of chromosome damage by ferritin in
cultured mammalian cells, (fa) the effect of chelating and re
ducing agents on the clastogenic activity of ferritin, and (c) a
comparison of the chromosome-damaging
activity of ferritin
with that of complexes of Fe(ll) and Fe(lll).
MATERIALS
AND METHODS
Cell Cultures. CHO cells were grown in MEM (Grand Island
Biological Co., Grand Island, N. Y.) supplemented with 15%
fetal calf serum, antibiotics (streptomycin sulfate, 29.6 ¿ig/ml;
penicillin G, 204 /ig/ml; kanamycin, 100 ¿tg/ml; and Fungizone, 2.5 /tg/ml), and 7.5% sodium bicarbonate (10 ml/800
ml medium). The stock cultures were maintained in 250-ml
plastic culture flasks (Falcon Plastics, div. Becton, Dickinson
and Co., Cockeysville, Md.) at 37° in a water-saturated CO2
incubator.
For each chromosome aberration experiment, approximately
140,000 CHO cells were seeded on 22-sq mm coverslips in
3.5-cm plastic dishes (Falcon) and kept in MEM with 15% fetal
calf serum at 37°for 2 days. Experiments were begun when
cells were 60 to 70% confluent.
Chemicals. The following chemicals were purchased from
Sigma Chemical Co., St. Louis, Mo.: ferritin [from horse spleen
as sterile solutions in 0.15 M NaCI, different lots containing 84
to 100 mg of ferritin per ml, and 144 to 188 /ig of iron per mg
of ferritin (determined by atomic absorption by Sigma)]; apo
ferritin (from horse spleen as a sterile solution in 0.15 M NaCI,
containing 50 mg of protein per ml); glutathione (reduced form,
98 to 100%); cysteine (HCI hydrate); histidine (free base); NTA
(disodium salt, 99.5%); catalase (from bovine liver, with activity
of 10,000 to 25,000 Sigma units per mg of protein); Superoxide
dismutase (from bovine blood, with approximately 3,000 units
per mg of protein); FMN (sodium salt, 95 to 97%); /S-NADH
(disodium salt, 98%); ascorbic acid (sodium salt); salicylic acid;
ferrous sulfate (heptahydrate, 99%); ferric chloride (hexahydrate); EDTA (disodium salt, dihydrate, 99%); and 2,2'-bipyridyl.
Iron Complexes. Complexes of Fe(ll) and Fe(lll) were freshly
4 The abbreviations
used are: CHO, Chinese hamster ovary; MEM. Eagle's
minimal essential medium; NTA, nitrilotriacetic
acid; FMN. flavin mononucleotide.
CANCER
RESEARCH
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Chromosome-damaging
prepared and diluted just before addition to the cultured cells.
Stock solutions of FeSO4-7H20 and FeCI3-6H2O were pre
pared in double-distilled H2O at concentrations of 0.1 M. Like
wise, stock solutions of complexing agents were prepared in
H2O at the following concentrations: glycine and cysteine, 0.5
M; histidine, NTA, and EDTA, 0.2 M. The stock solutions were
combined to give molar ratios of chelating agent to iron of 10:
1 (glycine, histidine, and cysteine) or 2:1 (NTA and EDTA). The
complexes were diluted as required into 2.5% MEM (MEM with
2.5% fetal calf serum) before addition to cells.
Treatment of Cells with Chemicals. All chemicals were
freshly prepared immediately before addition to cells. Catalase,
Superoxide dismutase, or FMN was dissolved in ice-cold 2.5%
MEM. All other chemicals were dissolved and diluted in 2.5%
MEM at room temperature.
Equal volumes (0.8 ml) of ferritin solutions were mixed with
the reducing or chelating agents directly on the cultured cells.
The order of addition was (a) the reducing agent, the chelating
agent, or the FMN:NADH system and (fa)ferritin. The cells were
incubated with the mixture for 3 hr at 37°,washed with MEM,
and incubated in 2 ml of 15% MEM for an additional 20 hr.
Analysis of Chromosome Aberrations. Cells were harvested
20 hr after the completion of chemical treatment. At 4 hr prior
to harvesting, colchicine was added (final concentration, 10
/¿g/ml). Cells were then treated with hypotonie solution (1%
sodium citrate) for 20 min, fixed in methanol:acetic acid (3:1,
v/v) for 30 min, and air dried. The slides were stained with 2%
orcein (in 50% acetic acid:water), dehydrated, and mounted.
For each sample, 50 to 300 metaphase cells were analyzed
for chromosome- and chromatid-type breaks and exchanges;
gaps were not scored. Whenever possible, at least 100 meta
phase cells were scored.
Chromosome aberration data in the tables are presented so
as to provide 2 major measures of damage. The first entry is
the frequency (percentage) of metaphase cells containing at
least one break or exchange. The second entry (in parenthe
ses) gives the average number of exchanges per cell, ex
pressed as percentage.
RESULTS
Ferritin and Apoferritin. The chromosome-damaging activity
of ferritin and its corresponding iron-free protein shell, apoferritin, were examined. Ferritin (3 to 2760 jug/ml) or apoferritin
(2 to 2240 ¿ig/ml) in culture medium was incubated for 3 hr
with CHO cells. The frequencies (percentage) of metaphase
cells with chromosome aberrations and the average number of
chromosome- or chromatid-type exchanges per cell are shown
in Table 1.
Extensive chromosome aberrations were induced in CHO
cells by ferritin at concentrations of 27 to 138 jug/ml. The
frequency of cells containing aberrations reached 60%, while
the number of exchanges per metaphase was also very high
(up to 87%). Higher concentrations of ferritin (276 to 2760 jug/
ml) inhibited mitosis and were cytocidal. Low concentrations of
ferritin (3 and 8 /¿g/ml) caused no detectable chromosome
damage (cf. Fig. 1).
In contrast, apoferritin did not induce chromosome aberra
tions over a wide range of concentrations, i.e., 2 to 1120 /¿g/
ml which is equivalent to the protein concentrations in 3 to
1380 /ig of ferritin per ml. A marginal increase in aberrations
MAY 1981
Activity of Ferritin and Iron Compounds
Table 1
Frequency of chromosome
aberrations
in CHO cells treated with ferritin and
apoferritinFerritin3Concentration(/ig/ml)27601380552276138552783,o'ChromosomeaberrationsTo
(86.7)"33.3
(0.0)0.7
(46.7)14.8
(13.8)0.8
(0.0)0.5(0.0)0.5(0.0)0.7(0.0)
(0.0)0.7
(0.0)0.5
(0.0)Apoferritin*1Concentration(Mg/ml)22401120448224112452272
Ferritin and apoferritin were used at concentrations
which gave equal
concentrations of protein, e.g., 2760 /ig of ferritin per ml contained 2240 fig of
protein per ml, since this lot of ferritin contained 18.8% iron and 81.2% protein.
CHO cells were treated for 3 hr with ferritin or apoferritin in MEM plus 2.5%
serum. Following a recovery period of 20 hr in MEM plus 15% serum, cells were
harvested.
6 Fifty to 300 metaphase cells were analyzed for chromosome- or chromatidtype exchanges and breaks. Whenever possible, at least 100 metaphase cells
were scored.
c Toxic, no detectable mitosis and variable cell loss; Ml, mitotic inhibition, less
than one metaphase cell per 5000 cells.
d Percentage of metaphase cells with at least one chromosome aberration.
" Numbers in parentheses, frequency (percentage) of chromosome- or chro
matid-type exchanges per metaphase cell.
' Controls CHO cells exposed to MEM only, not treated with chemicals.
(to 6.5%) was observed in cells treated with the highest con
centration of apoferritin (2240 /ig/ml, equivalent to the protein
concentrations in 2760 ^g of ferritin per ml).
These results clearly indicate that the chromosome-damag
ing activity of ferritin is not associated with apoferritin, the
spherical protein shell of ferritin.
Ferritin and Iron Complexes. In Table 2, the chromosomedamaging activity of ferritin and various complexes of Fe(ll)
and Fe(lll) is compared. The chromosome aberrations induced
by ferritin are expressed in relation to the iron content of the
ferritin and of the iron complexes (the calculation was based
on ferritin containing 18.8% iron). High concentration of ferritin
(298 to 1490 /¿g/ml, equivalent to 10~3 to 5 x 10~3 M iron)
was cytotoxic. Ferritin at lower concentrations (30 to 149
ml, equivalent to 10~4 to 5 x 10~" M iron) caused high
frequencies of breaks and exchanges (Figs. 2 and 4), while
ferritin at 3 /ug/ml was not detectably clastogenic.
In comparison to the severe clastogenic effects of ferritin,
iron complexes caused much less chromosome damage and
were less toxic. Complexes of Fe(ll) and Fe(lll) were prepared
with glycine, NTA, histidine, EDTA, and cysteine. At iron con
centrations up to 5 x 10~4 M, none of the complexes caused
increased frequencies of chromosome aberrations above those
in nontreated control cells (cf. 69% aberrations with ferritin at
5 x 10~4 M iron). Moreover, the cells treated with glycine or
histidine complexes of
amino acid molar ratio]
tions. Fe(lll):histidine at
5 x 10~3 M caused cell
iron [as Fe(ll) or Fe(lll) at 1:10 iron:
showed background levels of aberra
3 to 5 x 10~3 M and Fe(ll):histidine at
loss or mitotic inhibition.
Chromosome aberrations were detected only in cells treated
with HIM concentrations of ferrous sulfate or iron complexes of
NTA, EDTA, or cysteine. The NTA:iron or EDTA:iron molar ratio
was 2:1 and the cysteine:iron molar ratio was 10:1. An in
creased frequency of chromatid breaks (5.6%) was induced by
1629
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R. F. Whiting et al.
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CANCER
RESEARCH
VOL.
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41
Chromosome-damaging
Activity of Ferritin and Iron Compounds
ferrous sulfate at 5 x 10 3 M. Both breaks and exchanges (up
to 20%) were observed in cells exposed to ferrous:NTA com
plexes at 3 and 2 x 10~3 M, while a very low frequency of
aberrations (1%) was seen in cells treated with ferric:NTA
complexes. EDTA complexes of both Fe(ll) and Fe(lll) were
cytocidal and cytostatic at 5 and 3 x 10~3 M. Chromosome
Table 4
Effect of ascorbate on chromosome
aberrations
Treatment conditions and data presentation
induced by ferritin
are as described
FerritintrationascorbateFerritin
in Table 1.
plus
aloneToxicToxicMlMl38.7(58.1)18.6(21.4)2.9
(fig/ml)180072036018072361640Ferritin
aberrations (exchanges and breaks) were detected in cells
exposed to 2 x 10~3 M Fe(ll):EDTA or Fe(lll):EDTA complexes
(Fig. 3). Ferrous:EDTA induced a higher frequency of aberra
tions than did ferric:EDTA (12.1 versus 2.7%). EDTA alone at
concentrations above 10~2 M was found to cause cell loss
without cytostatic activity.
Among the ligands examined, cysteine has dual properties
as both a complexing and a reducing agent. Both chromosome
breaks and exchanges were detected in cells treated with
cysteine complexes of ferrous or ferric iron. The autooxidation
of cysteine yielded insoluble cystine crystals which were de
tected in samples where cysteine concentrations exceeded 2
x 1CT3 M.
During the 3-hr incubation period, precipitation occurred
with some iron complexes. Histidine is a weak ligand for ferric
iron, and precipitation of a ferric:histidine complex was ob
served over a wide range of iron concentrations (10~4 to 5 x
10~3 M). As well, complexes of iron with NTA and glycine
formed precipitates at iron concentrations higher than 10~3
M.
Among the complexing agents which have higher binding
constants for Fe(lll), such as EDTA and NTA, higher levels of
chromosomal damage (breaks and/or exchanges) were de
tected in the ferrous complexes than in the ferric ones. Con
versely, histidine, which preferentially binds to Fe(ll), was more
cytotoxic as a ferric complex than as a ferrous complex.
Results in Tables 1 and 2 showed that neither the protein
shell of ferritin nor iron complexes caused chromosome dam
age at comparable protein and iron concentrations to those in
ferritin.
Effect of Sulfhydryl Compounds, Glutathione and Cys
teine. Both cysteine and glutathione were effective in reducing
the chromosome-damaging
capacity of ferritin (Table 3). At
10~2 M, either sulfhydryl compound completely abolished the
cytocidal as well as the cytostatic action of high concentrations
of ferritin (333 to 3330 /¿g/ml). In addition, glutathione and
Table 3
Effects of sulfhydryl compounds (glutathione or cysteine) on chromosome
aberrations induced by ferritin
Treatment conditions and data presentation are as described in Table 1.
plusGlutathioneFerritin
Ferritin
concentra
tion(jig/ml)3330167066733316767331030Ferritin
aloneToxicToxicToxicM\78.1
(0.0)0.8(0.0)0.5
(0.0)1.4(0.0)0.9
(25.2)12.4(17.8)1
(0.5)0.9
.6
(4.0)1.0
(2.8)0.9
(0.9)0.9
(0.9)1.1
(0.0)Ml"
(0.0)0.8
(0.8)b10~3MToxicToxicMlMl31.6(47.4)13.8(20.1)3.6
(1.0)°10~2MToxicToxicMlMlMlMlM
(0.0)a10~*MToxicToxicMlMl33.3
Controls, CHO cells exposed to MEM only, not treated with chemicals.
6 Cells treated with 10"' M ascorbate alone.
c Cells treated with 10~3 M ascorbate alone.
d Cells treated with 10 2 M ascorbate alone.
cysteine effectively reduced the frequencies of chromosome
aberrations induced by lower concentrations of ferritin (33 to
167 jug/ml) to those in the nontreated cells.
A lower concentration of glutathione (10~3 M) provided sub
stantial protection from the clastogenic and cytotoxic activities
of ferritin. In the presence of 10~3 M glutathione, the frequen
cies of chromosome aberrations induced by ferritin at 33 to
167 ¿ig/mlwere reduced to background levels. The cytostatic
action of ferritin at 333 /¿g/mlwas inhibited. In addition, 10~3
M glutathione partially protected cells treated with high con
centrations of ferritin (667 to 3330 /¿g/ml);the cytocidal effect
of ferritin alone was lessened to mitotic inhibition.
Effect of an Enediol Reducing Agent, Ascorbate. In con
trast to the effects of sulfhydryl reducing agents, ascorbate did
not reduce the chromosome-damaging activity of ferritin (Table
4). Addition of ascorbate at 10~3 or 10"4 M with ferritin to cells
did not alter the cytocidal, cytostatic, or the clastogenic activ
ities of ferritin. Cells exposed to a higher concentration of
ascorbate (10~2 M) alone or to a mixture of ferritin (16 to 360
jug/ml) and 10~2 M ascorbate showed mitotic inhibition. How
ever, cells treated with a low dose of ferritin (4 jug/ml) and 10"2
M ascorbate exhibited normal mitotic rate and a background
level of chromosome aberrations. This effect is similar to the
reduced DNA damage observed in cells treated with 10~5 M
iron:EDTA (equivalent to 3 fig of ferritin per ml) and 10~2 M
ascorbate (28).
Effect of Nitrogen and Oxygen Ligands, EDTA, NTA, and
Salicylate. Three chelating agents (EDTA, NTA, and salicylate)
were examined for their effect on the induction of chromosome
aberrations by ferritin (Table 5). EDTA was most effective in
reducing the DNA-damaging action of ferritin. In the presence
of 10~3 M EDTA (Table 5, Column D), the clastogenic, cyto
static, and cytocidal effects of ferritin (15 to 750 jug/ml) were
essentially cancelled. In comparison, 10~3 M NTA (Table 5,
Column C) was less effective. It abolished the clastogenic
(0.0)0.7
(0.0)0.5
(93.8)36.0(51.7)13.4(11.8)1
(0.0)0.6(0.0)0.0
and
(0.0)0.8
the cytostatic effect of ferritin at 15 to 188 jug/ml. However,
(0.0)0.7(0.0)0.7(0.0)0.8
(0.0)0.6
higher concentrations of ferritin (375 to 1880 mg/ml) remained
(0.0)0.7
cytotoxic. Salicylate at 10~3 M did not reduce either the cyto
(0.0)0.7
(0.0)0.9
(0.0)0.5(0.0)0.5
(0.0)0.8
(0.8)0.9
.0
(0.0)0.8
(0.0)0.8(0.0)
(0.0)
(0.0)
(0.0)
0.8
(0.0)a10~2M0.5 0.8 (0.0)°10~3MMlMlMl0.9
0.5(0.0)cCysteine10~2M0.8
1.9(1.9)
a Controls, CHO cells exposed to MEM only, not treated with chemicals.
Cells treated with 10~2 M glutathione alone.
0 Cells treated with 10~3 M glutathione alone.
d Cells treated with 10~2 M cysteine alone.
toxic or the clastogenic activity of ferritin (Table 5, Column B).
An increase in the concentration of chelating agents to 10~2
M produced a variety of responses. EDTA itself caused a loss
of cells from the coverslips. NTA at 10~2 M (Table 5, Column F)
showed a protective activity comparable to that of 10~3 M
EDTA; the cytocidal,
cytostatic,
and clastogenic
effects
MAY 1981
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of
1631
R. F. Whiting et al.
Table 5
Effects of EDTA, NTA, and salicylate on chromosome aberrations induced by ferritin
Treatment conditions and data presentation are as described in Table 1.
Chromosome
aberrationsFerritin
agentsFerritin
concen
tration
alone
(/ig/ml)1880 (A)Toxic
plus chelating
(B)Toxic
(C)Toxic
(D)2.1
(E)Toxic
(F)1
(2.1)
.8 (0.0)
750
Toxic
Toxic
Ml
0.5(0.0)
Toxic
1.3 (0.0)
375
Ml
Ml
19.4(11.3)
0.6 (0.0)
Ml
1.1 (0.0)
188
Ml
Ml
1.4 (0.0)
0.0(0.0)
Ml
0.5(0.0)
38.2 (56.8)
75
38.3 (42.6)
0.8 (0.0)
0.5 (0.0)
32.6 (52.8)
0.6 (0.0)
12.1 (10.8)
14.7(13.0)
30
0.9 (0.0)
0.6 (0.0)
19.5(23.0)
0.5 (0.0)
3.2 (2.7)
7.6(10.8)
0.9 (0.0)
15
0.5(0.0)
12.9(18.6)
0.7 (0.0)
0.8 (0.0)
1.0 (0.0)
3
0.8 (0.0)
0.5(0.0)
0.8 (0.0)
0.5(0.0)
0.6 (0.0)"Sallcylate
0.8 (0.0)°EDTA
0.6 (0.0)dio-2Salicylate
0.5
(0.0)fiMNTA
0.5
(0.0)c
0Ferritin
0.8 (0.0)1Q-3MNTA
Control, CHO cells exposed to MEM only, not treated with chemicals.
6 Salicylate controls, cells treated with 10~3 M or 10~2 M salicylate.
c NTA controls, cells treated with 10 3 M or 10~2 M NTA.
d EDTA controls, cells treated with 10~3 M EDTA.
ferritin (15 to 1880 /¿g/ml)were essentially abolished. Finally,
10~2 M salicylate (Table 5, Column E) did not inhibit the
cytotoxic or clastogenic effects of ferritin. Salicylate may even
have a slight enhancing effect on the chromosome-damaging
action of ferritin at 15 and 30 /¿g/ml.
Effect of Heterocyclic Nitrogen Ligands, Histidine and
2,2'-Bipyridyl. The 2 nitrogen ligands histidine and bipyridyl
affected the chromosome-damaging action of ferritin differently
(Table 6). Histidine at 10~3 M or 10~2 M (Table 6) did not
appreciably change the cytocidal and cytostatic effect of fer
ritin. Little alteration of the clastogenic activity of ferritin was
detected with 10~3 M histidine. On the other hand, 10~2 M
histidine reduced the chromosome aberrations induced by
ferritin (35 jug/ml) from 13% to control levels.
In contrast, 2,2'-bipyridyl, which has a higher binding con
stant for ferrous iron than does histidine, increased the DNAdamaging action of ferritin (Table 6). For example, addition of
10~3 M bipyridyl with ferritin (19 /ig/ml) to cells resulted in an
increase of damage from 10.7% aberrations to mitotic inhibi
tion. Ferritin at low concentration (5 /¿g/ml) was not clasto
genic, and addition of bipyridyl caused an increase in the
aberrations to 33.5% with 50% chromosome- and chromatidtype exchanges. 2,2'-Bipyridyl alone at 10~3 M also induced
low levels of chromosome aberrations (6%) which were re
duced by low concentrations of ferritin (1 and 2 /¿g/ml).
Effect of Catalase and Superoxide Dismutase. Catalase
and Superoxide dismutase had little or no effect on the fre
quency of chromosome aberrations induced by ferritin (Table
7). Neither catalase (100 /¿g/ml) nor Superoxide dismutase
(100 fig/ml) detectably reduced (a) the toxic or cytostatic
effects of high concentrations of ferritin (333 to 1670 fig/ml)
or (b) the frequency of aberrations caused by ferritin at 167
and 67 /^g/ml. There may be a marginal protective effect of
catalase and Superoxide dismutase in cells treated with ferritin
at 33 fig/ml.
DISCUSSION
The data presented in this report demonstrate that exposure
of mammalian cells to ferritin caused severe chromosomal
damage. This raises questions of (a) the nature of the active
1632
Effect of histidine and 2,2'-bipyridyl
Table 6
on chromosome aberrations induced by
ferritin
Treatment conditions and data presentation
Ferritin
concen
tration
(ng/ml)Histidine
are as described in Table 1.
agentFerritin
aloneToxicToxicMl52.9
plus chelating
MToxicToxicMl61.5(69.2)33.8(47.9
500700350150703515302,2'-Bipyridyl
1
(58.8)38.7(58.1)13.0(11.2)2.9
(73.3)47.5(45.6)15.6(11.1)0.7
(0.9)0.5
(2.9)0.6
(0.0)0.7
(0.0)0.8
(0.0)0.5
(0.0)0.6
(0.0)0.5
(0.0)aMl44.5(83.2)23.2(31.7)10.7
(0.0)"ToxicToxicToxicMl66.7
(0.0)"
190954719105210Ferritin
(7.1)2.4
(3.2)0.0
(95.0)33.5(50.7)3.5
(0.0)0.5
(0.0)0.6
(3.5)1.6
(6.5)6.2(14.1)c10-*
(0.0)0.5
(0.0)a10~3MToxicToxicMl60.8
" Controls, CHO cells exposed to MEM only, not treated with chemicals.
^ Histidine controls, cells treated with 10~3 M or 10~2 M histidine.
: 2,2'-Bipyridyl controls, cells treated with 10 3 M 2.2'-bipyridyl.
component in ferritin which causes the damage and (b) the
mechanism of the reaction which leads to chromosome abnor
malities. As a partial answer to the first question, we found that
neither apoferritin (Table 1) nor complexes of iron [as Fe(ll) or
Fe(lll) (Table 2)] individually could account for the high cyto
toxic and clastogenic activity of ferritin. Thus, the intact ferritin
molecule appears to be required for DMA damage.
To examine the possible mechanism of mutagenic action of
ferritin, we have determined the effect of chemicals which
modify the physiological activities of ferritin. Chromosome dam
age could arise during mobilization of iron, during oxidationreduction of ferritin iron, or by a catalytic action of ferritin.
If mobilization of iron is important in the mechanism of
damage, then agents which affect mobilization should also
affect chromosome damage. Mobilization of ferritin iron is
enhanced by reducing agents. Physiological reducing agents
CANCER
RESEARCH
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Chromosome-damaging
Table 7
Effect of caÃ-a/aseand Superoxide disimilase on chromosome
induced by ferritin
Treatment conditions
and data presentation
aberrations
are as described in Table 1.
plusFerritin
Ferrltln
concentra
tion
Oig/ml)1670
aloneToxic
OOOnQ/ml)Toxic
dismutase
(100fig/ml>Toxic
Toxic
Toxic
Toxic
667
Ml
Ml
Ml
333
78.1 (93.8)
71.4(109.5)
167
82.6(100.5)
67
36.0(51.7)
31.4 (36.1)
36.4 (37.7)
13.4(11.8)
6.6
(1.6)
10.7
(5.8)
33
10
1.0 (0.8)
1.1
(0.0)
1.8
(0.6)
0.9 (0.0)
0.9
(0.0)
3
0.8
(0.0)
0.8 (0.0)aCatalase 0.8
(0.0)"Superoxide 1.0
<0.0)c
0Ferritin
a Control, CHO cells exposed to MEM only, not treated with chemicals.
Catalase control, cells treated with catalase (100 nig/ml) alone.
c Superoxide dismutase control, cells treated with Superoxide dismutase (100
fig/ml)
alone.
such as ascorbate, cysteine, glutathione, and reduced riboflavins released iron from ferritin (4, 5, 14, 25). The most efficient
ones were the dihydroflavin nucleotides. Relative to dihydroflavin mononucleotide, other reducing agents mobilized less
then 20% as much ferritin iron at up to 1% of the rate (25). The
rate of iron release was dependent on (a) the concentration
and the oxidation-reduction
potential of the reductant (4, 14,
25, 27), (o) the rate of diffusion of reductant through the 6
channels of apoferritin shell to the iron core (14), and (c) the
initial iron content and the crystallite surface of the iron micelles
of ferritin (5, 27).
Our results with reducing agents (Tables 3 and 4) do not
indicate that the mobilization of ferritin iron by reduction is a
critical step in DMA damage. This conclusion was derived from
the observation that (a) ascorbate had no significant effect on
the clastogenic activity of ferritin (Table 4), (b) glutathione and
cysteine had protective effects (Table 3), and (c) nontoxic
doses of reduced flavin (Table 8, 10~" M NADH plus 10~4 M
FMN) did not alter the frequencies of chromosome aberrations
induced by ferritin (data not shown). However, these are all
relatively weak mobilizing systems. Thus, mobilization may be
involved in the mechanism of damage but not be a rate-deter
mining step. As well, there may be mobilization of iron by
cellular reducing agents.
Next, we investigated the role of chelation in chromosome
damage by ferritin. In the absence of reducing agents, mobili
zation of ferritin iron is facilitated by chelating agents such as
EDTA, NTA, and bipyridyl (5, 22). Relative to dihydroflavin
mononucleotide, the rate and extent of mobilization are low.
We observed that chelating agents strongly affected the chro
mosome-damaging action of ferritin. However, their effect does
not appear to depend on the mobilization of ferritin iron by
chelation. NTA is a stronger agent for mobilization than is EDTA
(22), yet NTA showed a weaker protective effect than did EDTA
(Table 5).
The actions of chelating agents could be correlated with 3
properties. First, the effects appeared to be related to their
formation constants for Fe(ll) and Fe(lll). Chelating agents such
as EDTA and NTA, which have higher formation constants with
Fe(lll) than Fe(ll), reduced the clastogenic action of ferritin
(Table 5). On the other hand, bipyridyl, which preferentially
stabilizes Fe(ll), enhanced DNA damage (Table 6).
Activity of Ferritin and Iron Compounds
The second aspect is the effect of chelating agents to cata
lyze oxidation-reduction
of iron. EDTA and NTA enhance the
oxidation of Fe(ll), while bipyridyl can slowly reduce Fe(lll) (3).
DNA damage may be related to the amount of Fe(ll) present in
or near ferritin, and this would be decreased by EDTA and NTA
and increased by bipyridyl.
A third relationship is that between the protective effect and
the "denticity"
of the chelating agents. EDTA, NTA, and salicylate have similar effective stability constants for Fe(lll), log
Keff = 8.0 to 8.2 (17). Their ability to suppress the chromo
some-damaging action of ferritin is correlated with the denticity
(number of ligand-forming groups) of these compounds. EDTA
(hexadentate) was the most effective inhibitor followed by the
tetradentate NTA. Salicylate (bidentate) did not show inhibition.
The protective effect may be related to the rate of ligand
exchange. Further research is required to identify which of
these factors (relative stability constants for Fe(ll) and Fe(lll),
rate of oxidation-reduction,
and denticity) are most important
in affecting the mutagenicity of ferritin.
We observed strong inhibitory activities of glutathione and
cysteine on DNA damage caused by ferritin (Table 3). Sulfhydryl compounds glutathione and cysteine are very reactive
towards electrophilic species, including many free radicals
(15). They are able to trap not only reactive hydroxyl radicals
but also other less reactive radicals such as those found in
damaged lipids, proteins, and DNA. The observed protective
activity of the thiols appears to reflect more their radical-trap
ping activity than their reducing or chelating properties. Other
reducing agents such as ascorbate, which lacks such strong
trapping properties, did not exhibit similar inhibition (Table 4).
In addition, other ligands such as histidine and 2,2'-bipyridyl
(Table 6), which are like thiols in preferentially stabilizing iron
in ferrous state, either showed weak protection or enhanced
the clastogenic action of ferritin (Table 6).
The protective effect of cysteine against the clastogenic
activity of ferritin is in contrast to the chromosome-damaging
action of ironrcysteine mixtures (Table 2). Free iron catalyzes
the oxidation of cysteine (13), and an oxidation product of the
reaction may cause the DNA damage. Bound iron in the ferritin
core does not appear to have this catalytic activity towards
cysteine. Glutathione is different from cysteine in that free iron
does not catalyze its oxidation (13), and iron:glutathione mix
tures did not cause chromosome aberrations.5
Ascorbate also interacts differently with ferritin than with free
iron. Ascorbate had no significant effect on the clastogenic
action of ferritin (Table 4). In contrast, ascorbate reacted with
iron:EDTA complexes to cause severe chromosome aberra
tions (28).
Reduced oxygen species such as Superoxide radicals (O2~),
hydrogen peroxide (H2O2), and hydroxyl radicals (-OH) are
formed during the oxidation-reduction
of iron compounds (7,
8, 18) and may be responsible for the toxic and clastogenic
actions of ferritin. To test the possible involvement of superoxide radicals and hydrogen peroxide, we examined the effect
of Superoxide dismutase and catalase. Neither Superoxide
dismutase nor catalase influenced the chromosome-damaging
activity of ferritin (Table 7). This indicates that either there was
no appreciable accumulation of these species or the species
were not substantially involved in the mechanism of damage.
¡R. F. Whiting, L. Wei. and H. F. Stich, unpublished data.
MAY 1981
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1633
R. F. Whiting et al.
Moreover, Superoxide radicals and H2O2 were not detected
during deposition of ferritin iron (3, 29). It is possible that low
steady-state amounts of these or related species are present,
particularly inside the ferritin shell which is inaccessible to
enzymes.
A possible mechanism of DNA damage by ferritin is the
formation of hydroxyl radicals (-OH) during reduction and
reoxidation of ferritin iron. These latter processes occur contin
uously at the catalytic sites (Reactions A and B) (3, 14, 29). It
has been proposed (3) that ferriciperoxy species are formed
during oxidation-deposition
of iron in ferritin (Reaction C).
These peroxy species could react with Fe(ll) or other reductants to produce hydroxyl radicals in a Fenton-type reaction
(Reaction D) (7, 8, 18).
Fed!!),™,«,
+ reductant ?±Feilend
FedObound
+*
+ oxidant
FedOfree
(B)
nd + O2 «=!
Fe(ll).. .O2.. .Fe(ll) ;=»
Fe(lll)—O—O—Fe(lll)
Fe(lll)—O—O—Fedii) + Fe(ll) + 2H2O ^
(A)
normal human serum (0.01 to 0.1 /¿g/ml)and tissues (up to 5
/¿g/ml)are below the levels causing chromosome damage in
cells (6, 9, 21). However, in cancer and other pathological
states, serum ferritin concentration can reach 12 jug/ml (21,
23), which is near the lower limit of clastogenic concentration
of ferritin. In addition, an in vitro study showed that ferritin
concentration as low as 0.25 /¿g/mlcaused a marked suppres
sion of phytohemagglutinin and concanavalin A-induced blastogenesis in human lymphocytes (19). Furthermore, kinetic
studies of ferritin mobilization show that the rate of iron release
is dependent on the age and the fullness of the iron core.
Newly deposited iron is released faster (9). The maximum rate
was found in ferritin molecules which were less than one-third
full (1200 iron atoms per ferritin molecule) (5, 14). This may
imply that ferritins from different sources or with different iron
contents would have varied clastogenic activity. Moreover,
normal physiological levels of glutathione (10~4 to 5 x 10~3
3Fe(lll)-OH
+ •
OH
(C)
M) (15) are likely to be sufficient in suppressing
or clastogenic activity of ferritin.
any cytotoxic
(D)
ACKNOWLEDGMENTS
However, •OHis very short lived, and free -OH would be
scavenged by salicylate [salicylate had no protective effect
(Table 5)]. Thus, only secondary radicals or other reactive
species may escape from the ferritin molecule to react with
DNA. It may be these species which are trapped by glutathione
and cysteine.
Chemical studies have demonstrated that mobilization of
ferritin iron by dihydroriboflavin nucleotides(dihydroflavin
mononucleotide and dihydroflavin adenine dinucleotide) is rapid
and quantitative (4, 14, 25). Our preliminary studies of the
NADH:FMN system in CHO cells showed that, at millimolar
levels, NADH:FMN was cytocidal (1CT3 M NADH:1CT3 M FMN),
cytostatic (1CT3 M NADH:10~4 M FMN), and clastogenic (1CT4
M NADH:10'3 M FMN) to CHO cells (Table 8). No chromosome
The authors gratefully acknowledge
Wood.
the technical assistance of Grace Cecena-
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1635
R. F. Whiting et al.
Fig. 1. Metaphase plate of a CHO cell exposed to MEM only, not treated with chemicals.
Fig. 2. Metaphase plate of a CHO cell exposed to ferritin (30 /ig/ml) for 3 hr and harvested 20 hr posttreatment, showing one chromatid-type
Fig. 3. Metaphase plate of a CHO cell exposed to Fe(ll):EDTA (2 x 10 3 M), showing chromatid-type interchanges and breaks.
Fig. 4. Metaphase plate of a CHO cell exposed to ferritin (60 /ig/ml),
1636
interchange.
showing multiple exchanges and breaks.
CANCER
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41
Chromosome-damaging Activity of Ferritin and Its Relation to
Chelation and Reduction of Iron
Robert F. Whiting, Lan Wei and Hans F. Stich
Cancer Res 1981;41:1628-1636.
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