Mutagenesis vol.17 no.5 pp.
411–417, 2002
Cytogenetic and oxidative damage induced in human lymphocytes
by platinum, rhodium and palladium compounds
Lucia Migliore3, Giada Frenzilli1, Claudia Nesti,
Salvador Fortaner2 and Enrico Sabbioni2
Dipartimento di Scienze dell’Uomo e dell’Ambiente and 1Dipartimento di
Morfologia Umana e Biologia Applicata, University of Pisa, via S.
Giuseppe 22, 56126 Pisa, Italy and 2European Commission, JRC-Ispra,
Institute for Health and Consumer Protection (IHCP), ECVAM Unit,
21020-Ispra, Varese, Italy
This study of soluble compounds of platinum, palladium
and rhodium investigated the genotoxic properties of
(NH4)2PtCl4, PtCl2, PtCl4, (NH4)2PdCl4, PdCl2 and RhCl3
using the human lymphocyte micronucleus (MN) assay
coupled with fluorescence in situ hybridization (FISH).
A pancentromeric DNA probe was used to detect both
centromere-positive micronuclei (C⫹ MN) as well as centro-
mere-negative micronuclei (C– MN). A modified alkaline
single cell gel electrophoresis (SCGE) assay was used to
evaluate the possible role of oxidative damage in genotoxic-
ity of the Pt, Pd and Rh compounds tested. Two enzymes,
endonuclease III and formamidopyrimidine glycosylase,
were used to recognize and subsequently cut oxidized
pyrimidines and purines, respectively. A significant induc-
tion of MN by Pt and Rh compounds was observed
compared with controls, while (NH4)2PdCl4 and PdCl2
displayed weak significant MN induction. The FISH tech-
nique revealed no significant difference in the frequency
of C⫹ MN and C– MN for all compounds tested. These
findings suggest that MN induction is due both to a
clastogenic and an aneuploidogenic mechanism. SCGE
detected an increase in the level of DNA oxidative damage
for the Rh compound and for Pt(IV) which was also
capable of inducing an increase in primary DNA damage
at all the tested doses. This work highlights the stronger
genotoxicity, likely mediated by oxidative damage induc-
tion, of Pt and Rh compounds compared with Pd salts.
Introduction
This study is a part of a project aimed to screen metal
compounds for genotoxicity by micronucleus (MN) assay. In
particular, the aims of this investigation were: (i) to assess the
genotoxic properties of some Pt, Pd and Rh metal salts using
the human lymphocyte micronucleus assay (HLMA) and
fluorescence in situ hybridization (FISH) technique; (ii) to test
the possible role of oxidative damage in genotoxicity of Pt,
Pd and Rh salts as the toxicity of some metals is also due to
oxidative damage induction by production of O2 reactive
species (Stohs and Bagchi, 1995).
The FISH technique has been applied to those compounds
that gave clearly positive results with the HLMA, in order to
detect both centromere-negative micronuclei (C– MN) due to
chromosome breakage as well as centromere-positive
micronuclei (C⫹ MN) due to malsegregation phenomena. This
approach has already been employed to study the cytogenetic
3To whom correspondence should be addressed. Tel: ⫹39 050 836223; Fax: ⫹39 050 551290; Email: l.migliore@geog.unipi.it
© UK Environmental Mutagen Society/Oxford University Press 2002
damage induced by some Al, Cd, Hg, Sb, Te and Tl compounds
in human lymphocytes (Migliore et al., 1999). To study the
oxidative damage, a modified alkaline version of the SCGE,
optimized by Collins et al. (1995, 1997, 1998), using enzymes
able to recognize and cut oxidized pyrimidines and purines,
respectively, was applied.
There is a growing interest in the assessment of health risks
posed by increased human exposure to some platinum group
metals (PGM), namely platinum, rhodium and palladium. In
spite of their low natural occurrence in the Earth’s crust (0.05,
0.001 and 0.0004 p.p.m. for Pt, Pd and Rh, respectively)
(König and Schuster, 1994) these metals have important
applications in high technology processes related to their
catalytic properties and resistance to oxidation and corrosion
(König and Schuster, 1994).
Of environmental significance is the fact that these metals
are the three active components of automotive catalytic con-
verters. In this context, recent work has shown the presence
of 317 ng/g Pt and 74 ng/g Rh in road dust (Gómez et al.,
2001), confirming that these metals can be released into
the environment, representing ‘new’ potential environmental
pollutants (Pietra et al., 1994). In addition, there is also
growing interest in assessing exposure to PGM from an
occupational and a medical angle.
Pure PGM and their alloys are extensively used as materials
for melting tubes, in laboratory instruments, in jewellery and
in spinning jets in synthetic fibre production. In medicine,
while Pt and Pd are components of precious and semi-precious
alloys used in dental restoration (Wataha and Hanks, 1996),
Pt compounds are used as chemotherapeutic drugs as a part
of standard treatments.
Despite their increasing use, the health effects of Pt, Pd and
Rh remain little known. No information is available to date
on long-term low level exposure to Pt or its compounds, thus
preventing prediction of the environmental and health impacts
of Pt emitted from mobile sources (Pietra et al., 1994).
The well-known effects of Pt and its compounds are those
due to chronic industrial exposure to soluble salts of Pt,
which can lead to sensitization, with further induction of
conjunctivitis, asthma and contact dermatitis (platinosis). In
contrast, Pt has been reported as being non-toxic and non-
allergenic in its metallic state (Renner and Schmuckler, 1991).
Knowledge concerning the genotoxicity of Pt, Pd and Rh
compounds is fairly good for Pt, mainly in relation to Pt
complex compounds used as antitumor agents (IARC, 1987),
while little or no information is available for Pd and Rh
compounds. Inorganic Pt compounds but not Pt salts are
mutagenic in bacteria, Drosophila melanogaster and mam-
malian cells (Richardson and Gangotti, 1994). Mutations and
cell death can be introduced by binding of Pt to DNA,
which prevents cell replication (Choudhury et al., 2000). The
genotoxicity of Pd salts seems to be low in bacteria and
mammalian cells (Gebel et al., 1997).
411
L.Migliore et al.
In spite of this evidence, scientific information on the
mutagenicity and carcinogenic potential of PGM in mammals
is poor, making the assessment of health risk arising from
exposure to PGM compounds impossible.
Materials and methods
Chemicals
Metal salts used were (NH4)2PtCl4, PtCl2, PtCl4, (NH4)2PdCl4, PdCl2 and
RhCl3 (Alfachem, Cologno Monzese, Italy). Unless otherwise indicated, all
other chemicals and reagents were obtained from Sigma (St Louis, MO).
In order to assess metal contamination due to the addition of Pt, Pd or Rh
compounds to culture medium, 33 metal impurities were determined in the
10–2 M mother solutions of such compounds by inductively coupled plasma
mass spectrometry (model ELAN 6000; Perkin-Elmer/Sciex, Tornhill, Canada)
or graphite furnace atomic absorption spectroscopy (model SIMAA 6000;
Perkin-Elmer, Norwalk, CT) (Minoia et al., 1990).
The analysis of 33 metal impurities in the Pt, Pd and Rh solutions used in
our assays showed that 22 elements (Al, Au, Ba, Bi, Cd, Ce, Co, Cs, Cu, Ge,
Ir, La, Nb, Pb, Pd, Rh, Se, Sn, Sr, Th, U and W) were in concentrations
ranging from 1.3 (Al) to 0.0007 µg/l (Cs). Another 11 elements (Ag, As, Be,
Ga, Hg, Mn, Mo, Sb, Te, Tl and Zn) were below the detection limit, in any
case ⬍1 µg/l (results not shown).
Metal salts were dissolved in bi-distilled water at a concentration of 10–3
M, aliquoted and stored at 4°C until cell treatment. In the MN assay mitomycin
C (MMC) was used as a positive standard clastogenic mutagen at a dose of
0.51 µM, while for FISH analysis we also employed griseofulvin (GF) at a
dose of 43 µM, because of its known aneuploidogenic activity. In both cases
water was used as the negative control.
For each metal compound tested three doses at which a significant increase
in MN was obtained were selected for the Comet assay. Hydrogen peroxide
was used as a positive control for the Comet assay at doses of 50, 100, 150
and 200 µM.
Cell culture and chemical treatment
Cytogenetic analyses were carried out on whole blood obtained from a
young, healthy, non-smoking, male donor. Lymphocytes were stimulated with
phytohemagglutinin (PHA) and cultured in a standard fashion for 72 h for
the MN assay. Briefly, heparinized peripheral blood samples were cultured in
duplicate at 37°C for up to 72 h in RPMI 1640 (Gibco BRL, Milan, Italy)
supplemented with 20% fetal bovine serum (Gibco BRL), 1.5% PHA (Gibco
BRL) and 1% penicillin/streptomycin (Gibco BRL). Each culture was per-
formed in duplicate. Treatments with metals were performed 24 h after PHA
stimulation. For each metal salt a wider range of doses was exploited, starting
from an ineffective dose to a toxic one. Final experiments comprised at least
three doses, with the highest concentration showing a significant reduction in
the proportion of binucleate cells in the cultures. To block the cytokinesis of
interphase cells, cytochalasin B (final concentration 6 µg/ml) was added at
44 h to all tubes.
Binucleated lymphocytes were harvested after 72 h culture; the treatment
time was 48 h. Cells were treated with 0.075 M KCl to lyse erythrocytes,
prefixed in methanol, washed twice with fixative (methanol:acetic acid 5:1)
and then dropped onto clean glass slides. The air-dried slides were stained in
4% Giemsa solution or hybridized within 1 week of preparation.
FISH analysis
For FISH analysis a digoxigenin-labeled α-satellite DNA probe specific for
the centromeres of all human chromosomes (Oncor) was used. Prewarmed
slides were denatured in 70% formamide, 2⫻ SSC (saline sodium citrate
buffer), pH 7.0, at 70°C for 2 min, followed by dehydration via an ethanol
series. After heating at 70°C for 5 min, probes were placed on the denatured
slide and incubated overnight at 37°C in a moist chamber. Post-hybridization
washes were performed, first in a solution of 2⫻ SSC, pH 7.0, at 72°C for
5 min and then in 4⫻ SSC, 0.05% Tween 20 (SSCW) for 5 min at room
temperature. To minimize the background, slides were preincubated for 10 min
at 37°C in 4⫻ SSC, with 5% non-fat dry milk as immunological buffer (IB).
For detection of the digoxigenin-labeled probe, anti-digoxigenin (Boehringer,
Italy), TRITC-conjugated anti-mouse and TRITC-conjugated anti-rabbit anti-
bodies were diluted in IB and alternately incubated for 30 min at 37°C. Each
incubation step was followed by three 2 min washes in SSCW at 37°C.
After dehydrating through an ethanol series, slides were counterstained with
0.5 µg/ml DAPI dissolved in glycerol/DABCO antifade solution.
Slide scoring and statistical analysis
Giemsa stained slides were scored blind for MN analysis under an optical
microscope (final magnification 400⫻). The criteria for MN acceptance listed
by Fenech (1993) were followed. The results are expressed as the average
412
number of micronucleated cells ⫾ SD from two observations of 1000 cells
on two different slides from two culture tubes (according to Migliore et al.,
1999). MN frequency was expressed as the number of micronucleated
binucleate cells (MNBN, containing one or more MN) per 1000 cells. The
ratio of percent binucleate to mononucleate cells was used as a parameter of
cell proliferation in culture. For FISH analysis, preparations were analyzed
on a fluorescence microscope equipped with a triple bandpass filter for
simultaneous visualization of TRITC, FITC and DAPI fluorescence. A
sufficient number of lymphocytes were scored in order to record 50 MN for
each experimental point. MN were analyzed for the presence of the fluorescent
signal by considering a TRITC-labeled MN as centromere-positive MN
(C⫹ MN) and a non-labeled MN as centromere-negative MN (C– MN). Data
were analyzed by Fisher’s exact test to determine the significant difference
between each treatment and the control for MNBN. Fisher’s exact test was
also used to analyze the results obtained with the FISH technique: percentage
of C⫹ MN (or C– MN) in treated cultures versus percentage of C⫹ MN
(or C– MN) in control cultures.
Comet assay (SCGE)
Human leukocytes were isolated using the procedure described by Green et al.
(1992). Whole blood (0.5 ml) was centrifuged twice in lysing buffer (Na4Cl,
KHCO3, Na2EDTA) and resuspended in RPMI 1640 medium. The SCGE
assay was performed basically according to Singh et al. (1988), with some
modifications (Klaude et al., 1996).
Following their isolation, the cells were mixed with 0.4% trypan blue
solution and, after 15 min, they were counted and checked for viability.
Leukocytes resuspended in RPMI 1640 were treated with the test substances
for 2 h at 37°C immediately before the start of the assay, as suggested by
Hartmann and Speit (1994). The 2 h treatment makes sure that DNA primary
damage cannot be hidden by excision repair, as human leukocytes require 4 h
to repair strand breaks (Collins et al., 1995).
One day prior to the analyses, 1% normal melting point agarose was spread
on conventional slides and left to dry. On the following day, an 85 µl layer
of 0.5% low melting point agarose (LMA) together with 3⫻105 cells (10 µl
cell suspension ⫹ 75 µl LMA) were added to the slide surface. Finally, the
slide was covered with a third layer of 85 µl of LMA. Slides were immersed
in ice-cold freshly prepared lysing solution (2.5 M NaCl, 100 mM Na2EDTA,
10 mM Tris–HCl, 1% Triton X-100 and 10% DMSO, pH 10) to lyse the cells
and allow DNA unfolding. After at least 1 h at 4°C in the dark, slides were
placed on a horizontal electrophoresis unit. The unit was filled with fresh
buffer (1 mM Na2EDTA, 300 mM NaOH, pH ⬎ 13) to cover the slides. The
slides were allowed to set in the high pH buffer for 20 min to allow DNA
unwinding and expression of alkali-labile sites. Electrophoresis was conducted
for 20 min at 25 V (300 mA). Slides were then gently washed in neutralization
buffer (0.4 M Tris–HCl, pH 7.5) to remove alkali and stained with 100 µl
ethidium bromide (2 µg/ml). All steps described above were conducted under
yellow light to prevent additional DNA damage.
Low and normal melting point agarose, Triton X-100, Na2EDTA, Tris–
HCl, PBS and ethidium bromide were obtained from Sigma (St Louis, MO).
Enzyme preparation and treatment
In this study endonuclease III (endo III) was used to detect oxidized
pyrimidines, and formamidopyrimidine glycosylase (fpg) to detect damaged
purines, including 8-oxoguanine. The enzymes were kindly provided by
Dr A.R. Collins (Aberdeen, UK).
Enzymes were diluted (2 µl of enzyme in 2 ml) and stored at –80°C in
enzyme buffer (EB) (Na2 EDTA, 0.1 M KCl, 0.2 mg/ml BSA, 40 mM HEPES,
pH 8). For fpg, 2 µl of enzyme were diluted in 200 µl of buffer containing
10% glycerol. Aliquots of 50 µl of enzyme solution (or buffer alone, as a
control) were placed on gels and covered with 22⫻22 mm coverslips. Slides
were put into a moist box, in order to prevent desiccation, and incubated at
37°C for 45 (endo III) or 30 min (fpg). The enzymes recognize and cut
oxidized bases, converting these lesions into DNA single-strand breaks
(SSBs) and producing fragments which migrate towards the anode during
electrophoresis, making up the tail of the comet.
DNA migration assessment and statistical analysis
Images of 100 randomly selected cells (50 cells from each of the two replicate
slides) were analyzed from each sample under a fluorescence microscope
(200⫻) using a calibration scale considering two variables: nucleus diameter
and comet length, which included the nucleus diameter plus tail length. Two
independent cultures were performed per experimental point, for a total of
100 cells, and the mean was calculated.
The effects of dose, culture and experiment were evaluated by multifactor
analysis of variance (MANOVA). The multiple range test was performed
(P ⬍ 0.05) in order to detect differences in DNA migration among doses.
One hundred cells per point are shown as box and whisker plots, where
the y-axis displays the range of DNA migration values. Each box encloses

Fig. 1. Results of human lymphocyte micronucleus assay for some Pt, Pd and Rh salts.
50% of the data, with the median value of the variable displayed as a line.
The top and bottom of the plot mark the ⫾25% limits of the variable. The
lines extending from the top and bottom of each box mark the minimum and
the maximum values falling within an acceptable range. Any value outside
this range is displayed as an individual point. Genotoxicity was assessed in
the sub-toxic range (relative viability ⬎80%) to prevent potential artifacts due
to indirect toxicity-induced DNA damage.
Results
Table I and Figure 1 shows the results for MN frequency after
exposure of cells to different concentrations of Pt, Pd and Rh
compounds. A significant induction of MN for each Pt com-
pound and for the Rh salt as compared with the control
(Fisher’s exact test) was observed. In particular, PtCl4 shows
a significant (P ⬍ 0.001) induction of MN at a dose of 25 µM,
while (NH4)2PtCl4 and PtCl2 are weaker genotoxic inducers,
producing about the same effect on MN frequency at doses of
100 and 250 µM, respectively. Regarding RhCl3, a statistically
significant (P ⬍ 0.001) increase in MN frequency was observed
above a dose of 100 µM, not accompanied by a significant
decrease in binucleated cell percentage. The two Pd salts
showed quite weak but significant MN induction only at high
doses [500 µM for (NH4)2PdCl4, and 100, 200 and 300 µM
for PdCl2]. Data concerning FISH, applied only for those
compounds clearly positive in the MN assay and at the doses
that most significantly increased MN frequency, do not show
any significant difference in the frequency of C⫹ and C– MN
for all the compounds tested (Table II), even though for
Damage induced by platinum, rhodium and palladium
(NH4)2PtCl4, RhCl3 and PdCl2 a slight prevalence of C– MN
was observed.
Data concerning SCGE show oxidative DNA damage for
PtCl4 and RhCl3. All other metal compounds tested were
unable to induce this effect (Table III). A significant increase
in the amount of DNA migration after treating human leuko-
cytes with PtCl4, above a dose of 25 µM, was observed
(Figure 2A). A parallel statistically significant increase in
oxidized purines and pyrimidines was found at the same doses
(Figure 2B and C), indicating that the genotoxic potential of
PtCl4 acts via an oxidative mechanism under our experimental
conditions. All other Pt salts and PdCl2 gave negative or
inconclusive results. RhCl3 was ineffective in inducing any
increase in DNA migration at the doses tested. In contrast, a
statistically significant decrease was obtained at doses of
100 and 250 µM (Figure 3A). RhCl3 induced a statistically
significant increase in oxidized bases, both pyrimidines and
purines, at all exposure doses (Figure 3B and C).
A dose–effect response above the dose of 50 µM and an
increase in oxidized pyrimidines and purines were found for
hydrogen peroxide, a positive control known as an inducer of
oxidative damage.
Discussion
The data presented here provide evidence that the compounds
of Pt, Pd and Rh tested are able to induce significant cytogenetic
damage. Furthermore, the lack of any significant difference in
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Table I. Effects of Pt, Pd and Rh salts on MN induction in human Table II. Characterization of MN after hybridization with a pancentromeric
lymphocytes DNA probe.

Dose (mM) BN scored MN BNa MN BN/1000 BN (%)a Compound Dose C⫹ MN C– MN P

(mean ⫾ SD) (µM) (%) (%) (Fisher’s exact test)

(NH4)2PtCl4 Control 52.0 48.0

Control 2000 11 5.5 ⫾ 0.7 33.6 PtCl4 25 60.0 40.0 n.s.
100 2000 35 17.5 ⫾ 2.1b 31.4 50 68.0 32.0 n.s.
125 2000 34 17.0 ⫾ 7.1b 34.7 PtCl2 125 70.0 30.0 n.s.
150 2000 27 13.5 ⫾ 2.1c 26.5 250 70.0 30.0 n.s.
175 2000 28 14.0 ⫾ 9.9c 32 (NH4)2PtCl4 100 44.0 56.0 n.s.
200 2000 39 19.5 ⫾ 2.1b 22.3 125 38.0 62.0 n.s.
PtCl4 RhCl3 500 40.0 60.0 n.s.
Control 2000 5 2.5 ⫾ 0.7 39.5 1000 38.0 62.0 n.s.
25 2000 31 15.5 ⫾ 13.4b 29.5 PdCl2 100 40.0 60.0 n.s.
50 1682 24 14.3 ⫾ 3.7b 19.5 200 48.0 52.0 n.s.
75 759 28 37.1 ⫾ 8.9b 15 Mitomycin C 0.51 18.0 82.0 0.001
100 Toxic Griseofulvin 43 76.0 24.0 0.01
PtCl2 n.s., not significant.
Control 2000 5 2.5 ⫾ 0.7 39.5
125 2000 14 7.0 ⫾ 4.2d 31
250 2000 23 11.5 ⫾ 0.7b 27
500 2000 15 7.5 ⫾ 0.7d 27.5 Table III. Effects of Pt, Pd and Rh salts on DNA migration in human white
1000 Toxic blood cells
(NH4)2PdCl4 Dose (mM) DNA migration (mm) DNA migration (mm) (mean ⫾ SD)
Control 2000 6 3.0 ⫾ 0.0 40 (mean ⫾ SD)
100 2000 6 3.7 ⫾ 3.8 39.5
125 2000 12 6.0 ⫾ 1.4 41 ⫹ endo III ⫹ fpg
250 2000 14 7.0 ⫾ 1.4 32
500 2000 17 8.5 ⫾ 0.7d 21 (NH4)2PtCl4
750 Toxic 0 6.24 ⫾ 0.85 46.14 ⫾ 1.26 31.32 ⫾ 3.17
100 3.18 ⫾ 1.11 44.54 ⫾ 3.00 27.16 ⫾ 2.46
PdCl2 150 10.58 ⫾ 1.58a 45.72 ⫾ 1.23 37.52 ⫾ 1.76a
Control 2020 14 6.9 ⫾ 1.3 37.6 200 3.98 ⫾ 0.78 42.2 ⫾ 1.77 35.18 ⫾ 1.33
50 2000 11 5.5 ⫾ 0.7 36.3 PtCl4
100 2000 29 14.5 ⫾ 3.5d 27.9 0 7.22 ⫾ 1.21 42.72 ⫾ 1.96 35.08 ⫾ 1.14
200 2000 28 14.0 ⫾ 4.2d 33.6 25 19.28 ⫾ 1.29a 50.84 ⫾ 4.21a 42.84 ⫾ 1.52a
300 2000 30 15.0 ⫾ 1.4d 40.6 50 16.06 ⫾ 1.15a 51.28 ⫾ 1.96a 47.5 ⫾ 1.84a
400 2000 19 9.5 ⫾ 2.1 29 75 23.34 ⫾ 2.55a 56.08 ⫾ 2.8a 44.78 ⫾ 1.97a
600 Toxic PtCl2
RhCl3 0 19.0 ⫾ 1.75 59.82 ⫾ 3.73 48.48 ⫾ 3.04
Control 2020 14 6.9 ⫾ 1.3 37.6 125 19.22 ⫾ 2.18 55.08 ⫾ 4.38 49.18 ⫾ 3.23
10 2000 21 10.5 ⫾ 0.7 34.7 250 20.74 ⫾ 2.72 56.58 ⫾ 3.31 52.14 ⫾ 2.96
100 2000 43 21.5 ⫾ 2.1b 33.3 500 22.32 ⫾ 2.08 58.18 ⫾ 5.02 52.04 ⫾ 2.82
250 2000 51 25.5 ⫾ 9.2b 27.1 PdCl2
500 2000 44 22.0 ⫾ 7.1b 27.9 0 19.28 ⫾ 2.58 55.72 ⫾ 1.49 44.72 ⫾ 1.94
1000 2000 47 23.5 ⫾ 2.1b 25 100 7.74 ⫾ 0.79 51.26 ⫾ 2.49 41.46 ⫾ 2.14
Positive control (mytomicin C) 200 16.42 ⫾ 2.28 51.96 ⫾ 1.85 46.52 ⫾ 1.86
0.51 2000 199 99.5 ⫾ 37.5b 21.7 300 22.72 ⫾ 2.68 50.88 ⫾ 2.06 43.74 ⫾ 2.33
RhCl3
aMN BN, no. of micronucleated binucleated cells; BN (%), binucleated 0 10.42 ⫾ 1.21 32.76 ⫾ 1.44 29.26 ⫾ 2.16
cells/total cells. 100 8.02 ⫾ 1.39 47.4 ⫾ 1.86a 35.28 ⫾ 1.30a
bP ⬍ 0.001 (Fisher’s exact test). 250 7.16 ⫾ 0.94 54.14 ⫾ 1.53a 44.80 ⫾ 2.77a
cP ⬍ 0.01 (Fisher’s exact test). 500 10.04 ⫾ 1.36 56.1 ⫾ 1.51a 41.82 ⫾ 1.69a
dP ⬍ 0.05 (Fisher’s exact test). H2O2
0 3.76 ⫾ 0.74 48.22 ⫾ 3.19 35.95 ⫾ 3.09
50 9.52 ⫾ 0.68a 47.68 ⫾ 2.04 42.12 ⫾ 2.74a
100 25.46 ⫾ 1.36a 55.62 ⫾ 2.01a 42.56 ⫾ 2.17a
150 28.74 ⫾ 1.43a 58.16 ⫾ 2.15a 44.62 ⫾ 2.49a
the percentages of C⫹ MN and C– MN compared with the 200 23.16 ⫾ 1.98a 60.28 ⫾ 3.16a 41.04 ⫾ 1.97a
control, as shown by FISH analysis, indicates that all the
compounds studied can act by both a clastogenic and an aP ⬍ 0.05 MANOVA, multiple range test.
aneuploidogenic mechanism.
The genotoxic potential of PGM salts tested was assessed and anionic PdCl42–) were tested. In addition, in order to
taking into account the aspect of speciation, because it is compare the genotoxicity results, the Pt and Pd salts analyzed
known that the toxicological effects of individual metals had the corresponding anion/cation.
depend on their chemical forms (Sabbioni et al., 1985). In All three Pt compounds tested [(NH4)2PtCl4, PtCl2 and
particular, it is known that the genotoxicity of Pt is determined PtCl4] were found to produce a significant increase in MN
by oxidation state, conformation and structure (Gebel et al., frequency compared with the control. PtCl4, which was found
1997). For this, Pt compounds with two different oxidation to induce forward mutation in V-79 cells at a rate ~7 times
states (⫹2 as anionic PtCl42– or cationic Pt2⫹ species and ⫹4 the control rate (Kanematsu et al., 1990), seems to exert a
as the cationic Pt4⫹ form) and Pd salts (⫹2 as cationic Pd2⫹ stronger action in terms of both cytotoxicity and MN induction
414

Fig. 2. Comet formation in human lymphocytes by PtCl4. (A) Cells treated with PtCl4 only. (B) cells treated with PtCl4 ⫹ endoIII. (C) Cells treated with
PtCl4 ⫹ fpg.
compared with those of the divalent Pt salts. In fact, even at
the dose of 25 µM PtCl4 induced an ~6-fold increase in MN.
These findings are in agreement with the data of Gebel et al.
(1997), who showed that PtCl4 induces higher frequencies of
MN in human lymphocytes when compared with PtCl2. This
was confirmed by SCGE, which gave negative results with
(NH4)2PtCl4 and PtCl2 while PtCl4 showed a significant
increase in DNA migration above a dose of 25 µM. (NH4)2PtCl4
showed a significant increase in DNA migration and oxidized
purines at the only dose of 150 µM, but we consider a positive
response the presence of at least two other doses able to induce
a statistical effect. We cannot exclude that other mechanisms
of action not detectable by SCGE, such as crosslink formation,
could take place at the tested doses of Pt2⫹ compounds. This
could lead to the formation of bulky DNA fragments unable
to migrate in the electrophoresis gel under the experimental
Damage induced by platinum, rhodium and palladium
conditions used (Tice et al., 1992; Hartman and Speit, 1994).
PtCl2 exhibited mutagenicity and high toxicity for strains TA98
and TA100 in the Ames test (Uno and Morita, 1993). In
contrast, Pt4⫹ is able to induce DNA damage via both a direct
action and an oxidative mechanism.
On the other hand, metal carcinogenicity is known to be
mediated by a variety of different mechanisms: direct muta-
genic attack on DNA is a possible primary mode of action, as
also is the induction of oxidative stress or cellular immunity,
inhibition of DNA metabolism and DNA repair and the
formation of DNA and/or protein crosslinks (Snow, 1992).
Concerning the Pd compounds studied, both of them appear
to be weak inducers of cytogenetic damage. However, PdCl2
is unable to give rise to any increase in DNA migration at the
doses tested. A decrease in DNA migration was observed at
100 µM in the absence of endo III (primary DNA damage),
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L.Migliore et al.
Fig. 3. Comet formation in human lymphocytes by RhCl3. (A) Cells treated with RhCl3 only. (B) Cells treated with RhCl3 ⫹ endoIII. (C) Cells treated with
RhCl3 ⫹ fpg.
as well as at doses of 100, 200 and 300 µM in the presence
of endo III. As already stated, some metals can produce
crosslinks (Snow, 1992), and mutagenic and/or carcinogenic
compounds characterized by a crosslinking action are known
to be poor inducers of DNA migration (Tice et al., 1992;
Hartman and Speit, 1994; Frenzilli et al., 2000). Some of
them, such as active aldehydes, are even able to induce a
reduction in DNA migration at high doses (Frenzilli et al.,
2000). It is thus possible that crosslink formation might be
partially responsible for the lack of an effect obtained with
PdCl2. The decreased DNA migration observed in the presence
of endoIII at all doses might also represent the result of an
enzyme–metal interaction that may interfere with the action
of the enzyme itself on spontaneously oxidized pyrimidines,
whose presence is demonstrated by control strand break levels.
Bunger et al. (1996) showed both a cyto- and a genotoxic
416
activity for some Pt, Rh and Pd metal salts. In particular, they
found that spontaneous mutation rates increased by a factor
of 3–20 when the four strains tested were exposed to Pt
complexes, as assayed by the Ames test. The Rh compounds
proved to be considerably less mutagenic, while Pd showed a
non-mutagenic potential, in agreement with Uno and Morita
(1993). The reason for the weak Pd genotoxicity might be that
inorganic Pd2⫹ complexes seem to be more labile than their
corresponding Pt2⫹ counterparts (Gebel et al., 1997).
The MN assay coupled with FISH analysis revealed that
RhCl3 can act as both a clastogen and aneuploidogen in the
dose range 100–1000 µM. In a recent study Sadiq et al. (2000)
showed that a Rh3⫹ complex induced chromosomal aberrations
of all types in cultured human lymphocytes, exerting its
clastogenic effects without the need for metabolic activation,
in a radiomimetic S phase-independent way. The mutagenicity

of RhCl3 was studied in V-79 cells. At 300 µM a 4-fold mutation
rate compared with the control was observed (Kanematsu et al.,
1990). The same compound administered in drinking water to
mice at concentrations of 5 mg/l led to the development of
lymphomas and leukemia (Schäfer et al., 1999).
The mutagenic potential of Pt and Rh complexes appears
to be based on a variety of mechanisms that damage DNA
(Bunger et al., 1996). Our current knowledge on mechanisms of
metal genotoxicity suggests that there is no unique mechanism
which could account for the genotoxic potential of all the
diverse metals and their compounds, although the two main
actions seem to be enhanced formation of reactive oxygen
species, resulting in lipid peroxidation and DNA damage, and
interference with DNA repair and/or DNA replication processes
(Hartwig, 1995).
The present work shows the capability of Pt, Pd and Rh
compounds to act by different mechanisms. In this context,
the aneuploidogenic effect was proved by FISH assay and the
clastogenic effect by both FISH and SCGE assays, while
possible crosslink formation was drawn from SCGE data.
Concerning the occurrence of oxidative damage, PtCl4
displays an increase in oxidized purines, associated with a
clastogenic effect that was revealed in the absence of enzymes,
as shown by SCGE. In the case of RhCl3, oxidative damage
seemed to be prevalent. However, the decrease in DNA
migration observed at doses of 100 and 250 µM in the absence
of enzymes suggests that a possible alkylating mechanism
could occur. We can hypothesize that at the basal level
oxidative damage is likely hidden by crosslink formation, but
it is highlighted by the use of endoIII and fpg. On the other
hand, Pd compounds exert only weak effects when measured
with the MN assay and no significant effect with the other
methods employed, so we cannot make conclusive statements
about their possible mechanism of action.
We can conclude that a better understanding of metal
genotoxicity requires the simultaneous use of a wide spectrum
of tests in order to obtain evidence of the different modes of
action following exposure to metal compounds.
Acknowledgements
The authors are grateful to Leonardo Cocchi and Mariacarla Iorio for their
excellent technical assistance. This work is a contribution to contract no.
15469-1999 11F1ED ISP ES.
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Received on January 30, 2002; accepted on May 20, 2002
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