ARTICLE IN PRESS
Toxicology in Vitro xxx (2004) xxx–xxx
www.elsevier.com/locate/toxinvit
Oxidative activation of the human carcinogen chromate by
arsenite: A model for synergistic metal activation leading to
oxidative DNA damage
Kent D. Sugden *, Kevin M. Rigby, Brooke D. Martin
Department of Chemistry, The University of Montana, 32 Campus Drive, Missoula, MT 59812, USA
Received 22 January 2004; accepted 1 March 2004
Abstract
Human exposure to toxic metals and metalloids in the environment seldom occurs from a single pure compound. Most environmental exposure profiles are heterogeneous with co-exposure occurring coincident with multiple toxic metal species. This coexposure to metals and metalloids in complex mixtures can result in a synergistic, additive or even depletive toxic response. The
complexity of interactions presented by metal mixtures presents a need for convenient and sensitive methods to determine potential
toxic responses from such co-exposure. We have studied the reaction between the two commonly associated toxic metals of
chromate, Cr(VI), and arsenite, As(III), with regards to the ability of As(III) to reductively activate Cr(VI) to generate oxidative
stress and DNA damage. Using a DCF-based fluorescent dye assay we have demonstrated that the redox reaction between As(III)
and Cr(VI) yields high valent intermediates of chromium, Cr(V), that are highly oxidizing. This induction of oxidizing potential was
dose dependent and did not occur with As(III) or Cr(VI) alone or, with the other major oxidation state of arsenic, arsenate, As(V).
The mechanism of oxidation of DCFH to the fluorescent species, DCF, in this reaction was through a direct, metal-based oxidation
since addition of radical scavengers did not significantly decrease oxidation of the dye in this system. The addition of a ligand that
stabilizes the high valent Cr(V) oxidation state, 2-ethyl-2-hydroxybutyric acid (EHBA), to the chromate and arsenite mixture resulted in an enhancement of DCF fluorescence. The DCF fluorescence observed with the Cr(VI) and As(III) mixture was also found
to correlate with oxidative DNA damage as measured by a plasmid nicking assay. These data show how metal–metal interactions in
environmental mixtures could result in the synergistic induction of oxidative stress and DNA damage. Further, these data demonstrate the utility of the DCF fluorescence assay as a sensitive method for screening synergistic redox interactions in metal mixtures.
2004 Published by Elsevier Ltd.
Keywords: Chromium; Arsenic; Oxidation; DNA damage
1. Introduction
Occupational exposure of workers to chromium in
the chromate-producing, chrome-plating, and welding
industries is often associated with the simultaneous
exposure to other toxic metals such as arsenic, cadmium,
copper, lead, and nickel (Dawson et al., 1991; Nygren
et al., 1992; IARC Monographs, 1990). Exposure to
chromium in combination with arsenic and copper also
Abbreviations: DCF, dichlorofluorescein; DCFH, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; EHBA, 2-ethyl-2-hydroxybutyric acid; EPR, electron paramagnetic resonance spectroscopy
*
Corresponding author. Tel.: +1-406-243-4193; fax: +1-406-2434227.
E-mail address: [email protected] (K.D. Sugden).
0887-2333/$ - see front matter 2004 Published by Elsevier Ltd.
doi:10.1016/j.tiv.2004.03.001
occurs in the wood treatment industry where these
chemical mixtures have been used to impart resistance to
insect and fungal damage (Dawson et al., 1991; Nygren
et al., 1992). Environmental exposure of humans to
chromium is similarly heterogeneous with simultaneous
mixed-metal exposure arising from a number of industrial waste-streams (Colinet et al., 1983; Rinaldo-Lee
et al., 1984). While a significant amount of research has
focused on the toxicity and carcinogenicity of both
chromium and other metals as pure compounds, little is
known of the potential synergistic impact that metal
mixtures may have on biological systems. In part, this
lack of focus on metal mixtures is due to the complexity
that is presented in determining the mechanisms of
toxicity and the biological endpoints for such mixtures.
As such, convenient and sensitive methods that have the
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ability to identify potential synergistic metal interactions
would be beneficial in assessing the impact of these
mixtures in biological systems.
The current model for chromate genotoxicity and
carcinogenicity in humans proposes that reduction to
lower oxidation states is essential for activation of this
metal towards DNA damage (Connett and Wetterhahn,
1983; Miller and Costa, 1988). This reduction process
can occur through interaction with endogenous intraand extra-cellular reductants or with exogenous xenobiotics (Stearns and Wetterhahn, 1994; Connett and
Wetterhahn, 1985; Wiegand et al., 1984; Jennette, 1982;
Kawanishi et al., 1986). The interaction of Cr(VI) with
intracellular reductants has been well documented but
fewer studies have focused on the role that exogenous
reductants, and particularly other metals and metalloids, may have on activation of Cr(VI). The toxic
byproducts generated upon Cr(VI) reduction are
dependent upon the reductant and several candidates
for the ultimate genotoxic species formed in these
reactions are the transient high valent chromium oxidation states of Cr(V) and Cr(IV) as well as reactive
oxygen species, (ROS) (for a review see: Sugden and
Stearns, 2000; Aiyar et al., 1989). These products of
Cr(VI) reduction have all shown the ability to oxidize
biomolecules and the generation of oxidant stress in
cellular systems by these species is considered to be at
least partly responsible for this metals toxicity and carcinogenicity (Sugden and Stearns, 2000; Cantoni and
Costa, 1984).
Herein we report the synergistic reductive activation
of Cr(VI) by the metalloid arsenite (As(III)). As(III), as
well as the other oxidation state of this metalloid,
arsenate (As(V)) are common environmental co-pollutants with chromium (Dawson et al., 1991; Nygren
et al., 1992). Arsenic is also a constituent of cigarette
smoke and epidemiological studies have shown a link
between smoking and increased lung cancers in chromium-exposed populations (Hill and Faning, 1948;
Enterline and March, 1982). While arsenic itself has
been implicated as a human carcinogen, studies with
experimental animals have proven inconclusive and
have resulted in the suggestion that arsenic is not a
primary carcinogen but likely acts as a co-carcinogen
(Rossman, 1981).
The aim of this study was to evaluate the interaction
between Cr(VI) and As(III) for the formation of high
valent species of chromium and/or ROS that are capable
of oxidizing biological macromolecules. The assay that
we have used was based on the 20 70 -dichlorofluorescein
(DCFH) fluorophore which when converted to 20 ,70 -dichlorofluorescein (DCF) by an oxidant shows strong
fluorescence. Fluorogenic probes have been used in vitro
and in cellular assays to provide a convenient and sensitive method to detect oxidants (Cathcart et al., 1983).
We have shown, using this in vitro DCF fluorescence
assay, that a synergistic reaction between Cr(VI) and
As(III) exists that can result in a significant oxidizing
potential. This oxidation reaction was dose-dependent
and resulted from a direct oxidation between the fluorescent dye and the high valent chromium intermediates
as shown by the lack of radical scavengers to significantly reduce the level of oxidative fluorescence. The
influence of ligation of the high valent chromium oxidation states formed in this reduction reaction was
observed to significantly enhance the levels of DCFH
oxidation. Together, these results show that the redox
interaction of metals and metalloids could act to synergistically induce oxidative damage to cellular systems.
2. Materials and methods
2.1. DCF assay
The oxidant sensitive dye, 20 70 -dichlorofluorescein
diacetate (DCFH-DA) was obtained from Molecular
Probes Inc, (Eugene, OR). The DCFH-DA must be deesterified for in vitro studies to generate the oxidation
substrate 20 70 -dichlorofluorescein (DCFH). The deesterification reaction was performed by the method of
Cathcart et al. (1983) by mixing 125 ll of a 1.5 mM
DCFH-DA solution in EtOH with 0.5 ml of 0.01 N
NaOH for 30 min at room temperature in the dark. The
mixture was neutralized with 2.5 ml of 20 mM sodium
phosphate buffer (pH 7.0) to give a final concentration
of 60 lM of the activated DCFH dye stock solution.
Reactions were carried out in 96 well CoStar plates
using 10–30 lM dye stock solution and varying concentrations of Cr(VI) and arsenic as As(III) or As(V) as
given in the figure legends. Reactions with radical
scavengers were carried out using 100 mM of DMSO or
EtOH. Ligand effects were assessed in these same systems by the addition of 2-ethyl-2-hydroxybutyric acid
(EHBA).
2.2. Electron paramagnetic resonance (EPR or ESR)
EPR spectra were recorded using a Bruker ESP-300
spectrometer. The spectral parameters were 100 kHz
field modulation, 1.0 Gauss modulation amplitude, 5.12
ms time constant, 9.768–9.772 microwave frequency,
1 · 105 receiver gain, 2 mW microwave power, and
3380–3580 Gauss sweep width with a 21 s scan time.
Typical reactions were carried out on 2.0 ml volumes at
RT in 20 mM phosphate buffer (pH 7.0). Reactions to
visualize the formation of Cr(V) were carried out using
125 lM of Cr(VI) and excess (1.25 mM) As(III) and 1.0
mM of the EHBA ligand. For observation of 53 Cr
hyperfine splitting, the concentrations of Cr(VI) and
As(III) were increased to 2-fold and the EHBA con-
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The generation of oxidative DNA damage from the
reaction between Cr(VI) and As(III) was determined
using the plasmid relaxation assay. This assay uses the
formation of nicked circular DNA from supercoiled
DNA as a method to detect oxidative damage. The
plasmid DNA, pUC19, used in this study was purchased
from New England Biolabs. Reactions were carried out
using 1 lg of pUC19 DNA and various concentrations
of Cr(VI) and As(III) in 20 mM phosphate buffer (pH
7.0). Reactions were incubated at RT for one hour prior
to addition of 1 ll of gel loading buffer. The reactions
were loaded on a 1% agarose gel in 1X Tris-BorateEDTA (TBE) running buffer for 1 h at 90 V and the
resulting DNA bands were visualized with ethidium
bromide staining.
3. Results
3.1. Induction of DCF fluorescence by mixtures of Cr(VI)
and As(III)
We have used the sensitivity of the DCF fluorescence
assay towards oxidation by metals and ROS as a quick
and convenient screening method for assessing the
generation of an oxidative potential in mixtures of
Cr(VI) and As(III). Fig. 1 shows the synergistic response
for the time dependent oxidation of DCFH to the
fluorescent DCF form with a mixture of Cr(VI) and
As(III). Reactions were carried out at a 1:1 ratio with a
concentration of 125 lM of each metal in 20 mM
phosphate buffer (pH 7.0). The combination of Cr(VI)
and As(III) showed a dramatic increase in DCF fluorescence that occurs from As(III) reduction of Cr(VI) to
form the highly oxidizing chromium oxidation states of
Cr(V) and/or Cr(IV). As(V) is the fully oxidized form of
this metalloid and was unable to reduce Cr(VI) as
demonstrated by the lack of any DCF fluorescence in
this reaction. None of the metals, when treated with
DCFH separately, showed the ability to induce an oxidative potential as measured by the lack of DCF fluorescence. These data demonstrate that As(III), under
physiologically relevant conditions (aqueous neutral
pH), can reduce Cr(VI) and cause an oxidative response
as measured using the DCF assay.
Fluorescence Intensity
2.3. Plasmid nicking assay
50000
Cr(VI)
As(III)
As(V)
Cr(VI) + As(III)
Cr(VI) + As(V)
40000
30000
20000
10000
0
0
10
20
30
40
50
60
70
Time (min)
Fig. 1. Formation of the fluorescent form of the oxidant-sensitive dye,
DCF, over time from the reductive activation of 125 lM Cr(VI) with
125 lM As(III). Each data point represents the mean ± SD (N ¼ 8).
3.2. Concentration dependence of DCFH oxidation by
Cr(VI) and As(III)
Cr(VI) is taken up unidirectionally into cells and can
result in accumulation of this metal at concentrations of
1 mM or greater intracellularly from a 10 lM media
concentration (Martin et al., 1998). However, since a
steady-state level of reduction of Cr(VI) would be expected in cellular systems, the relevance of the in vitro
DCFH oxidation assay is dependent upon whether
detection levels are within extracellular Cr(VI) concentrations normally used for toxic and subtoxic studies in
cell systems (normally 10–100 lM). Fig. 2 shows the
40000
125 µM Cr(VI) + As(III)
35000
Fluorescence Intensity
centration was increased to 5 mM. Measurements were
made with 100 ll volumes of the reaction mixture drawn
into a capillary tube sealed on one end with Dow–
Corning high vacuum grease and placed in a quartz
EPR tube. The g-values were determined with respect
to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH, g ¼
2:0036).
3
50 µM Cr(VI) + As(III)
25 µM Cr(VI) + As(III)
10 µM Cr(VI) + As(III)
Cr(VI) control
DCF dye control
30000
25000
20000
15000
10000
5000
0
0
50
100
Time (min)
150
200
Fig. 2. The dose-dependence for Cr(VI) reductive activation (10–125
lM) with 125 lM As(III) for the formation of the fluorescent DCF
species. Each data point represents the mean ± SD (N ¼ 8).
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concentration dependence of DCFH oxidation with 10–
125 lM of Cr(VI) when the concentration of As(III) is
held constant at 125 lM. A similar concentration
dependence of DCFH oxidation was observed by varying the As(III) concentration, 10–125 lM, while holding
a constant Cr(VI) concentration, 125 lM, (data not
shown). The DCFH oxidation at relevant concentrations of Cr(VI) suggests that exposure to mixtures of
Cr(VI) and As(III) may induce a synergistic oxidant
insult in cellular systems.
3.3. Effects of radical scavengers on DCFH oxidation in
the Cr(VI) and As(III) system
Radical quenching studies with small molecular
weight substrates are frequently used to determine the
formation of diffusible reactive oxygen species (ROS).
Several radical quenchers such as dimethyl sulfoxide
(DMSO) and ethanol (EtOH) are commonly used for
quenching studies of these diffusible radical species. A
fundamental question in Cr(VI) reductive activation and
DNA damage is the nature of the species responsible for
the oxidation of DNA. Previously, we have shown that
synthetic Cr(V) species can oxidize both DNA and
DCFH directly (Martin et al., 1998; Sugden and Wetterhahn, 1996; Sugden and Wetterhahn, 1997; Sugden,
1999). Fig. 3 shows the reaction of Cr(VI) and As(III) in
the presence of 100 mM of the radical scavengers
DMSO and EtOH. Cr(VI) concentrations in this reaction were 50 lM and DCFH concentrations were 10 lM
respectively. The 2000-fold excess of radical scavengers
in relation to Cr(VI) and 10,000-fold excess with respect
to dye showed little effect on the induction of fluorescence in the reaction of Cr(VI) and As(III). These data
strongly suggested that dye oxidation was derived from
a direct oxidation with the +5 and/or +4 oxidation states
of chromium and not from the generation of ROS. The
small changes in fluorescence that were observed in these
reactions are likely due to a quenching effect of the
DMSO and EtOH on the activated dye or a change in
the stability of the high valent chromium due to polarity
changes from addition of organic solvents.
3.4. The effect of chelation on DCFH oxidation by
Cr(VI) and As(III)
Chelation of chromium, both intracellularly and
extracellularly, has the potential to effect the toxicity of
this metal by stabilizing reactive oxidation states of this
metal. The +5 oxidation state of chromium can be
chelated by a number of common intracellular ligands
but the impact of this chelation on toxicity is unknown.
The potential of chelation to stabilize hypervalent
chromium complexes formed in the reaction between
Cr(VI) and As(III) was investigated using the bidentate
ligand, 2-ethyl-2-hydroxybutyric acid (EHBA). The
EHBA ligand has been shown previously to stabilize the
+5 and +4 oxidation states of chromium (Krumpolc and
Rocek, 1979; Codd et al., 1997; Bose et al., 1992). Fig. 4
shows the effect of EHBA chelation on DCFH oxidation
using 10, 25, and 50 lM of Cr(VI) with excess (125 lM)
As(III). The presence of the EHBA ligand in the reaction mixture gave a dramatic, and dose-dependent increase in DCFH oxidation over the unchelated Cr(VI)
and As(III) reactions. In fact, in the presence of the
EHBA ligand, Cr(VI) showed a greater ability to oxidize
DCFH at its lowest concentration, 10 lM, than the
unchelated reaction showed at the highest concentra-
40000
32000
Cr(VI) + As(III) + EtOH
24000
Cr(VI) Control
Cr(VI) + DMSO Control
Cr(VI) + EtOH control
60000
Fluorescence Intensity
Fluorescence Intensity
Cr(VI) + As(III)
Cr(VI) + As(III) + DMSO
16000
10 µM Cr(VI)
25 µM Cr(VI)
50 µM Cr(VI)
Cr(VI) control
dye control
10 µM Cr(VI) + ehba
25 µM Cr(VI) + ehba
50 µM Cr(VI) + ehba
50000
40000
30000
20000
8000
10000
0
0
50
100
150
200
Time (min)
Fig. 3. The effect of 100 mM radical scavengers, DMSO and EtOH, on
the formation of the fluorescent dye, DCF, in the reaction between 125
lM Cr(VI) and 125 lM As(III). Each data point represents the
mean ± SD (N ¼ 8).
0
0
50
100
Time (min)
150
200
Fig. 4. The dose-dependence for Cr(VI) activation (10–50 lM) by 125
lM As(III) in the presence and absence of 1 mM Cr(V) stabilizing
ligand, EHBA. Each data point represents the mean ± SD (N ¼ 8).
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K.D. Sugden et al. / Toxicology in Vitro xxx (2004) xxx–xxx
5
tion, 50 lM. These data clearly demonstrate that a
chelating ligand can act to enhance the oxidative reactivity of the corresponding high valent chromium species
formed in this reaction.
3.5. EPR identification of Cr(V) through chelation
Identification of the reactive intermediates formed
in the reaction of Cr(VI) and As(III) is key to understand the induction of oxidative DNA damage and
ultimately the toxicity of these mixtures. Fig. 5(B) shows
that the reaction of Cr(VI) and As(III), when in the
presence of a stabilizing ligand such as EHBA, resulted
in the formation of an EPR detectable signal. The EPR
signal observed in this reaction had a g-value of 1.980
which was characteristic of the Cr(V)-EHBA complex
(Krumpolc and Rocek, 1979). With increased concentrations of Cr(VI) and As(III) (Fig. 5(A)) the 53 Cr
hyperfine splitting (18.3 Gauss) can be observed from
the 9.5% abundance of this I ¼ 3=2 isotope. The lack of
an observable EPR signal in the reaction between
Cr(VI) and As(III) without chelation by EHBA (Fig.
5(C)) suggested that Cr(V) levels do not accumulate to
sufficient levels to be observed by EPR in the absence of
ligand stabilization. This result, when correlated with
the increased oxidative activity of Cr(VI) towards
DCFH, suggests that metal ligation may be an impor-
A
B
C
3460
3480
3500
3520
3540
3560
3580
Fig. 6. Plasmid nicking assay in the reaction of Cr(VI) with As(III).
Lane 1: Control (no metal), Lane 2: 50 lM Cr(VI) + 10 lM As(III),
Lane 3: 50 lM Cr(VI) + 50 lM As(III), Lane 4: 50 lM Cr(VI) + 125
lM As(III), Lane 5: 50 lM Cr(VI) + 1.25 mM As(III), Lane 6: 50 lM
As(III) + 10 lM Cr(VI), Lane 7: 50 lM As(III) + 50 lM Cr(VI), Lane
8: 50 lM As(III) + 125 lM Cr(VI), Lane 9: 50 lM As(III) + 1.25 mM
Cr(VI), Lane 10: 1.25 mM Cr(VI) only, Lane 11: 1.25 mM As(III)
only, Lane 12: control (no metal).
tant mechanism in stabilizing high valent chromium in
the cellular milieu to afford a greater oxidation of biomolecules such as DNA.
3.6. Correlation of Cr(VI) and As(III) reactions with
DNA damage
A plasmid nicking assay was utilized to determine
whether the DCF fluorescence observed in the Cr(VI)
and As(III) reactions correlated with the ability to induce oxidative DNA damage. The plasmid nicking assay
is a sensitive assay that measures the induction of frank
strand breaks on DNA resulting in unwinding of supercoiled plasmid DNA, Form I, to the nicked circular
form of plasmid DNA, Form II. Fig. 6 shows a dosedependent nicking of the pUC19 plasmid in the reaction
of Cr(VI) with As(III). Nicking was enhanced with
increasing As(III), (Fig. 6; lanes 2–5) to a greater extent
than with increasing Cr(VI) (Fig. 6; lanes 6–9). This
result suggested that the reduction by As(III) was the
rate limiting step in the oxidation of the plasmid DNA.
Overall, the plasmid nicking induced by the reaction of
Cr(VI) with As(III) was consistent with the oxidative
response observed by DCF fluorescence.
Gauss
Fig. 5. The electron paramagnetic resonance, EPR, spectrum of the
high valent chromium intermediate, Cr(V)-EHBA, formed during the
reduction of Cr(VI) by As(III), (A) 53 Cr hyperfine showing the 18.3
Gauss splitting characteristic of the Cr(V)-EHBA complex formed
from As(III) reduction of 250 lM Cr(VI) in the presence of 5 mM
EHBA ligand; (B) EPR spectra of the Cr(V)-EHBA complex formed
from 1.25 mM As(III) reduction of 125 lM Cr(VI) in the presence
of excess, 1 mM, EHBA ligand; (C) Reaction of 250 lM Cr(VI) and
5 mM As(III) without the presence of stabilizing ligands.
4. Discussion
The interaction of metals and metalloids in mixtures
can give rise to a complex set of redox reactions and
toxic responses. Electron transfer reactions between
different redox metal centers can form more-or-less toxic
compounds depending on the metal oxidation state, the
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1998; Witmer et al., 1994; Kim and Yurkow, 1996). A
number of these studies (Witmer et al., 1994; Kim and
Yurkow, 1996) have suggested that intracellular reduction of Cr(VI) results in the formation of oxygen-based
radicals. Previously, we have shown that DCFH can be
oxidized in vitro and in cellular systems by the formation of Cr(V) as well as by ROS (Martin et al., 1998).
Since DCFH is sensitive to both direct metal oxidation
and ROS, we have used radical quenching compounds
in our reactions to show that the mechanism was via a
direct oxidation route suggesting that the oxidant
responsible for DCFH oxidation was a Cr(V) or Cr(IV)
species. We propose a direct metal oxidizing mechanism
for DCFH oxidation by Cr(V) as outlined in Scheme 1
although a single electron transfer mechanism with
Cr(IV) cannot be ruled out.
The redox reaction of Cr(VI) and As(III) resulted in
the formation of high valent chromium species of Cr(V)
and Cr(IV) as shown by EPR. Presumably, a concomitant formation of the +5 oxidation state of arsenic,
As(V) would also occur. As(III) has previously been
used as a two electron reductant of Cr(VI) under
highly acidic conditions for synthetic purposes. To our
knowledge, no reactions have been carried out under
physiologically relevant conditions to ascertain the impact that this reduction may have on Cr(VI) activation.
The final stable oxidation state of chromium following
reduction by As(III) in aqueous solution is Cr(III). The
impact of a strict two electron reductant, such as
As(III), on the reduction of Cr(VI) necessitates the
formation of a transient, and highly oxidizing, Cr(IV)
species. Cr(IV) can disproportionate to form the reactive
Cr(V) species as well as the oxidatively inert Cr(III)
availability of stabilizing ligands, and localization within
the cell. A measurable endpoint for determination of
toxic potential in these reactions is the formation of
highly oxidizing species in the form of high valent metal
complexes or oxygen radicals generated through
Fenton-like mechanisms. Both high valent metals and
radicals have been shown to directly damage DNA
through oxidative mechanisms (Pratviel et al., 1995;
Burrows and Muller, 1998). The ability to detect a
synergistic metal–metal interaction in regard to oxidant
formation may be a critical factor in assessing the toxicity of metal mixtures. We have chosen to investigate
the redox reaction between mixtures of Cr(VI) and
As(III) as these two compounds are often associated in
occupational and environmental exposure profiles. The
factors believed to influence the toxicity of metal and
metalloid mixtures, and the approach for the critical
understanding of mixtures are; (1) analysis of redox
reactions leading to the formation of more toxic oxidation states of the interacting species within the mixture, (2) the effects of ligand chelation on the
stabilization of toxic oxidation states that generate enhanced oxidizing profiles, (3) identification of the species
within the mixture that are responsible for the oxidative
potential as a precursor to mechanistic studies and, (4)
determination of the potential for the interaction of
these oxidants with biological macromolecules.
The redox reaction between Cr(VI) and As(III) has
demonstrated the ability to oxidize DCFH to its fluorescent form, DCF in vitro. This oxidant-sensitive
fluorescent dye has been used in a number of cellular
studies to show the induction of intracellular oxidant
stress with exposure to Cr(VI) exposure (Martin et al.,
O
L
HO
OH
Cl
H
Cl
+
L
L
O
Cr
V
O
HO
L
L
Cl
Cl
H
O
O
O
H
2O
DCFH
OH
L
III
L
Cr
L
L
+
L
L
O
HO
O
Cl
Cl
OH2
O
O
DCF
Scheme 1.
L
Cr
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K.D. Sugden et al. / Toxicology in Vitro xxx (2004) xxx–xxx
species (Codd et al., 1997; Stearns and Wetterhahn,
1994). A putative set of reactions between Cr(VI) and
As(III) which account for the generation Cr(V), Cr(IV)
and the final stable Cr(III) species in this system is
summarized in Eqs. (1)–(4) below.
CrðVIÞ þ AsðIIIÞ ! CrðIVÞ þ AsðVÞ
ð1Þ
2CrðIVÞ ! CrðVÞ þ CrðIIIÞ
ð2Þ
CrðIVÞ þ CrðVIÞ ! 2CrðVÞ
ð3Þ
CrðVÞ þ AsðIIIÞ ! CrðIIIÞ þ AsðVÞ
ð4Þ
During the reduction process of Cr(VI) by As(III),
chelation of Cr(V) was shown to stabilize this oxidation
state and enhance DCF fluorescence. This result suggests that stabilization of high valent oxidizing intermediates of chromium may be important in the toxic
mechanism of activity. A cell contains a number of
potential chelating ligands that are analogous to the
EHBA ligand and may serve to enhance the toxicity of
Cr(VI) in these reactions by stabilizing toxic oxidation
states of this metal.
We have further established a correlation between the
oxidation of DCFH and a corresponding oxidation of
DNA, using the plasmid nicking assay. Our data support that of Farrell et al. (1989) who have shown previously that Cr(V) complexes have the capability of
nicking plasmid DNA directly without the concomitant
formation of reactive oxygen species.
It has been well established that Cr(VI) is not the
oxidation state that generates DNA damage leading to
carcinogenesis, but from other intermediates formed
upon its reduction (Connett and Wetterhahn, 1983).
Thus, any species capable of reducing Cr(VI) has the
potential to exacerbate this metals toxicity. It should be
noted that reduction of Cr(VI) extracellularly could result in a lowering of toxicity due to the formation of the
less toxic, and less cell permeable, Cr(III) complex.
However, it has also been shown that Cr(V), much like
Cr(VI), can be readily transported across the cell
membrane to induce cellular toxicity (Dillon et al.,
2000). Reductive activation of Cr(VI) can also occur
with thiols such as glutathione which is present in millimolar concentrations in cells (Meister and Anderson,
1983). This competition for Cr(VI) reduction between
high concentrations of glutathione versus the relatively
low concentrations of As(III) in cellular systems may
relegate the As(III) reduction mechanism of chromate to
a predominantly extracellular phenomenon. A further
confounding factor in the interpretation of these in vitro
results to intracellular mechanisms of toxicity is the
presence of the As(III) reductant. The toxicity of As(III)
is well established and treatment of cells with As(III) has
been shown to cause oxidant stress through ROS formation (Wang and Huang, 1994). However, the mechanism of ROS formation is thought to be through the
interaction with critical thiols in the electron transport
7
system (Petrick et al., 2001) and not through a direct
interaction with biomolecules as seen for Cr(VI). As(III)
has also been shown to cause a lowering of DNA repair
capacity in cells (Hartwig, 1998) which could serve to
further exacerbate the oxidant stress derived from the
redox reaction with Cr(VI).
In conclusion, these data demonstrate that human
exposure to mixtures of Cr(VI) and As(III) have the
potential to act synergistically to produce oxidative
DNA damage. These data also serve to demonstrate the
utility of a simple and convenient DCFH oxidation assay as a screening mechanism to identify chemical
mixtures that have the potential to generate oxidative
damage to biomolecules.
Acknowledgements
Funding for this study was provided by the National
Institute of Environmental Health Sciences Grant
(ES10437) to KDS. KMR was supported through
an NSF-EPSCoR undergraduate fellowship and the
McNair Scholarship Foundation.
References
Aiyar, J., Borges, K.M., Floyd, R.A., Wetterhahn, K.E., 1989. Role of
chromium(V), glutathione thiyl radical and hydroxyl radical
intermediates in chromium(VI)-induced DNA damage. Toxicology
and Environmental Chemistry 22, 135–148.
Bose, R.N., Moghaddas, S., Gelerinter, E., 1992. Long-lived
chromium(IV) and chromium(V) metabolites in the chromium(VI)-glutathione reaction: NMR, ESR, HPLC and kinetic
characterization. Inorganic Chemistry 31, 1987–1994.
Burrows, C.J., Muller, J.G., 1998. Oxidative nucleobase modifications
leading to strand scission. Chemistry Reviews 98, 1109–1151.
Cantoni, O., Costa, M., 1984. Analysis of the induction of alkali
sensitive sites in the DNA by chromate and other agents that
induce single strand breaks. Carcinogenesis 5, 1207–1209.
Cathcart, R., Schwiers, E., Ames, B.N., 1983. Detection of picomole
levels of hydroperoxides using a fluorescent dichlorofluorescein
assay. Analytical Biochemistry 134, 111–116.
Codd, R., Lay, P.A., Levina, A., 1997. Stability and ligand exchange
reactions of chromium(IV) carboxylato complexes in aqueous
solutions. Inorganic Chemistry 36, 5440–5448.
Colinet, E., Griepnik, B., Muntau, H., 1983. The certification of the
contents (mass fraction) of cadmium, cobalt, copper, manganese,
mercury, nickel, lead and zinc in a sewage sludge of domestic
origin, Report no. 8836 EN, Commission of the European
Communities, Bureau of Community Reference, Luxembourg.
Connett, P.H., Wetterhahn, K.E., 1983. Metabolism of the carcinogen
chromate by cellular constituents. Structure Bonding 54, 93–124.
Connett, P.H., Wetterhahn, K.E., 1985. In vitro reaction of the
carcinogen chromate with cellular thiols and carboxylic acids.
Journal of the American Chemical Society 107, 4282–4288.
Dawson, B.S.W., Parker, G.F., Cowan, F.J., Hong, S.O., 1991.
Interlaboratory determination of copper, chromium and arsenic in
timber treated with preservative. Analyst 116, 339–346.
Dillon, C.T., Lay, P.A., Bonin, A.M., Cholewa, M., Legge, G.J., 2000.
Permeability, cytotoxicity and genotoxicity of Cr(III) complexes
ARTICLE IN PRESS
8
K.D. Sugden et al. / Toxicology in Vitro xxx (2004) xxx–xxx
and some Cr(V) analogues in V79 Chinese hamster lung cells.
Chemical Research in Toxicology 13, 742–748.
Enterline, P.E., March, G.H., 1982. Cancer among workers exposed to
arsenic and other substances in a smelter. American Journal of
Epidemiology 116, 895–911.
Farrell, R.P., Judd, R.J., Lay, P.A., Dixon, N.E., Baker, R.S.U.,
Bonin, A.M., 1989. Chromium(V)-induced cleavage of DNA: Are
chromium(V) complexes the active carcinogens in chromium(VI)induced cancers? Chemical Research in Toxicology 2, 227–229.
Hartwig, A., 1998. Carcinogenicity of metal compounds: possible role
of DNA repair inhibition. Toxicology Letters 102–103, 235–239.
Hill, A.B., Faning, E.L., 1948. Studies on the incidence of cancer in a
factory handling organic compounds or arsenic. I. Mortality
experience in the factory. British Journal of Industrial Medicine 5,
1–8.
IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans: Chromium, Nickel and Welding, vol. 49, International
Agency for Research on Cancer, Lyons, France, 1990.
Jennette, K.W., 1982. Microsomal reduction of the carcinogen
chromate produces chromium(V). Journal of the American Chemical Society 104, 874–875.
Kawanishi, S., Inoue, S., Sano, S., 1986. Mechanism of DNA cleavage
induced by sodium chromate(VI) in the presence of hydrogen
peroxide. Journal of Biological Chemistry 261, 5952–5958.
Kim, G., Yurkow, E.J., 1996. Chromium induces a persistent
activation of mitogen-activated protein kinases by a redox-sensitive
mechanism in H4 rat hepatoma cells. Cancer Research 56, 2045–
2051.
Krumpolc, M., Rocek, J., 1979. Synthesis of stable chromium(V)
complexes of tertiary hydroxy acids. Journal of the American
Chemical Society 101, 3206–3209.
Martin, B.D., Schoenhard, J.A., Sugden, K.D., 1998. Hypervalent
chromium mimics reactive oxygen species as measured by the
oxidant-sensitive dyes 20 ,70 -dichlorofluorescein and dihydrorhodamine. Chemical Research in Toxicology 11, 1402–1410.
Meister, A., Anderson, M.E., 1983. Glutathione. Annual Reviews of
Biochemistry 52, 711–760.
Miller III, C.A., Costa, M., 1988. Characterization of DNA-protein
complexes induced in intact cells by the carcinogen chromate.
Molecular Carcinogenesis 1, 125–133.
Nygren, O., Niesson, C.-A., Lindahi, R., 1992. Occupational exposure
to chromium, copper and arsenic during work with impregnated
wood in joinery shops. Annals of Occupational Hygiene 36, 509–
517.
Petrick, J.S., Jagadish, B., Mash, E.A., Aposhian, H.V., 2001.
Monomethylarsonous acid (MMA(III)) and arsenite: LD(50) in
hamsters and in vitro inhibition of pyruvate dehydrogenase.
Chemical Research in Toxicology 14, 651–656.
Pratviel, G., Bernadou, J., Meunier, B., 1995. Carbon–hydrogen bonds
of sugar units as targets for chemical nucleases and drugs.
Angewandt Chemie International Edition English 34, 746–769.
Rinaldo-Lee, M.B., Hagarman, J.A., Diefendorf, A.F., 1984. Siting of
a metal industry landfill on abandoned soda ash waste beds. In:
Jackson, L.P., Rohlik, A.R., Conway, R.A. (Eds.), Hazardous and
Industrial Waste Management and Testing. ASTM STP 851.
American Society for Testing and Materials, Philadelphia, pp. 61–
80.
Rossman, T.G., 1981. Enhancement of UV-mutagenesis by low
concentrations of arsenite in E. coli. Mutation Research 91, 207–
212.
Stearns, D.M., Wetterhahn, K.E., 1994. Reaction of chromium(VI)
with ascorbate produces chromium(V), chromium(IV) and carbonbased radicals. Chemical Research in Toxicology 7, 219–230.
Sugden, K.D., 1999. Formation of modified cleavage termini from the
reaction of chromium(V) with DNA. Journal of Bioinorganic
Chemistry 77, 177–183.
Sugden, K.D., Stearns, D.M., 2000. The role of chromium(V) in the
mechanism of chromate-induced oxidative DNA damage and
cancer. Journal of Environmental Pathology, Toxicology and
Oncology 19, 215–230.
Sugden, K.D., Wetterhahn, K.E., 1996. Identification of the oxidized
products formed upon reaction of chromium(V) with thymidine
nucleotides. Journal of the American Chemical Society 118, 10811–
10818.
Sugden, K.D., Wetterhahn, K.E., 1997. Direct- and hydrogen peroxide
induced-chromium(V) oxidation of deoxyribose in single-stranded
and double-stranded calf thymus DNA. Chemical Research in
Toxicology 10, 1397–1406.
Wang, T., Huang, H., 1994. Active oxygen species are involved in the
induction of micronuclei by arsenite in XRS-5 cells. Mutagenesis 9,
253–257.
Wiegand, H.J., Ottenwalder, H., Bolt, H.M., 1984. The reduction of
chromium(VI) to chromium(III) by glutathione: an intracellular
redox pathway in the metabolism of the carcinogen chromate.
Toxicology 33, 341–348.
Witmer, C., Faria, E., Park, H.-Y., Sadrieh, N., Yurkow, E.,
O’Connell, S., Sirak, A., Schleyer, H., 1994. In vivo effects of
chromium. Environmental Health Perspectives 102, 169–176.