Intracellular Calcium Disturbances Induced by Arsenic and Its Methylated Derivatives in Relation to Genomic Damage and Apoptosis Induction

Abstract

Arsenic and its methylated derivatives are contaminants of air, water, and food and are known as toxicants and carcinogens. Arsenic compounds are also being used as cancer chemotherapeutic agents. In humans, inorganic arsenic is metabolically methylated to mono-, di-, and trimethylated forms. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In recent years, the correlation between arsenic exposure, cytotoxicity and genotoxicity, mutagenicity, and tumor promotion has been established, as well as the association of arsenic exposure with perturbation of physiologic processes, generation of reactive oxygen species, DNA damage, and apoptosis induction. Trivalent forms of arsenic have been found to induce apoptosis in several cellular systems with involvement of membrane-bound cell death receptors, activation of caspases, release of calcium stores, and changes of the intracellular glutathione level. It is well known that calcium ion deregulation plays a critical role in apoptotic cell death. A calcium increase in the nuclei might lead to toxic effects in the cell. In this review, we highlight the relationship between induced disturbances of calcium homeostasis, genomic damage, and apoptotic cell death caused by arsenic and its organic derivatives.

Full text

Research
Review
Arsenic and Its Derivatives as
Potent Environmental
Toxicants
Exposure to high levels of arsenic in drinking
water has been recognized for many decades
in some regions of the world, notably in
China, India, and some countries in Central
and South America. Millions of people are at
risk of cancer and other diseases because of
chronic arsenic exposure (National Research
Council 1999, 2001).
General adverse health effects that are
associated with human exposure to arsenicals
include cardiovascular diseases, developmental
abnormalities, neurologic and neurobehavioral
disorders, diabetes, hearing loss, fibrosis of
the liver and lung, hematologic disorders,
blackfoot disease, and cancers (Abernathy
et al. 1999; Sordo et al. 2001; Tchounwou
et al. 1999). In humans, arsenic is known to
cause cancer of the skin (in combination with
ultraviolet irradiation; Rossman et al. 2004)
and cancer of the lung, bladder, liver, and
kidney (Abernathy et al. 1999; Kitchin 2001;
Tchounwou et al. 1999). The principal pro-
posed mechanisms of arsenic carcinogenicity
are induction of chromosomal abnormalities,
promotion, and oxidative stress (Kitchin 2001;
Kitchin and Ahmad 2003). Also, chronic
exposure to arsenic has been found to cause
immunotoxicity and has been associated with
the suppression of hematopoiesis (anemia and
leukopenia; Cheng et al. 2004). In its inor-
ganic form, arsenic is known to be cytotoxic
and genotoxic in vivo and in vitro (for review,
see Dopp et al. 2004a).
Inorganic arsenic is methylated via gluta-
thione (GSH) conjugation to the pentavalent
species: monomethylarsonic acid [MMA(V)],
dimethylarsinic acid [DMA(V)], and tri-
methylarsenic oxide [TMAO(V)] (Kitchin
2001; Sordo et al. 2001). This process requires
the metabolic reduction of As(5
+
) to As(3
+
),
and in this way, trivalent monomethylarsonous
acid [MMA(III)], dimethylarsinous acid
[DMA(III)], and trimethylarsine [TMA(III)]
appear as metabolic products (Kitchin 2001;
Kitchin and Ahmad 2003; Sordo et al. 2001)
(Figure 1). Recent findings show that the tri-
valent methylated arsenic metabolites are
highly toxic; DMA(III) has been shown to
cause several genotoxic and/or clastogenic
effects such as single-strand breaks, formation
of apurinic/apyrimidinic sites, DNA and
oxidative base damages, DNA–protein cross-
links, chromosomal aberrations, and aneu-
ploidy (Dopp et al. 2004b; Schwerdtle et al.
2003; Sordo et al. 2001). The genotoxic effects
of arsenic and its methylated metabolites
in vivo and in vitro, as well as the carcinogenic
potencies of these substances, are discussed in
detail by Dopp et al. (2004a), Florea et al.
(2004), Patrick (2003), Hughes (2002), and
Gebel (2001).
The major mechanisms in which toxic
metallic entities may damage cells are direct
binding to cellular molecules, induction of
conformational changes, replacement of physi-
ologic metals from their binding sites (Qian
et al. 2003), or inhibition of DNA repair func-
tions (Hartwig 1998). Thus, they may act as
catalysts for the redox reactions that produce
reactive oxygen species (ROSs). ROSs are
capable of damaging a wide variety of cellular
macromolecules, including DNA, lipids, and
proteins. Finally, cellular signal transduction
can be altered (e.g., activation of transcription
factors, changes of gene expression); cell
growth, proliferation, and differentiation can
be promoted; and apoptosis leading to cell
death or cancer development can be induced
(Qian et al. 2003; Yang and Frenkel 2002).
In addition, Murphy et al. (1981) sug-
gested a neurotoxic potential of arsenic after
acute arsenic intoxication of human patients
that caused a polyneuropathy with prolonged
sensory and motor deficits. Namgung and
Xia (2001) have shown that primary cultures
of rat cerebellar neurons exposed to 5–15 µM
sodium arsenite and 1–5 mM DMA(V) had
reduced viability. These authors reported
nuclear fragmentation, DNA degradation,
and apoptosis induction in neuronal cells
treated with sodium arsenite or DMA(V).
They concluded that the neurotoxicity of
arsenite might be caused by an activation of
p38 and c-Jun N-terminal kinase 3 (JNK3)
mitogen-activated protein kinases (MAPKs),
which are involved in the apoptotic process.
The role of metallothionein (MT) in
modifying DMA(V) genotoxicity was recently
studied in MT-I/II null mice and in the cor-
responding wild-type mice by Jia et al.
(2004). In this study, increased formation of
8-hydroxy-2´-deoxyguanosine was found
together with elevated numbers of DNA
strand breaks. The observed levels were signif-
icantly higher in MT-I/II null mice than in
wild-type mice. Furthermore, the appearance
of apoptotic cells was significantly higher in
the urinary bladder epithelium of MT-I/II
null mice than in dose-matched wild-type
mice exposed to DMA(V) (Jia et al. 2004).
Genetic Damage and Apoptosis
Induction by Arsenic Compounds
Arsenite is widely used as a chemothera-
peutic agent for the treatment of several
human diseases. Arsenic trioxide has been used
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VOLUME 113 |NUMBER 6 |June 2005
659
Address correspondence to E. Dopp, University of
Duisburg-Essen, University Hospital Essen, Institute of
Hygiene and Occupational Medicine, Hufelandstraße
55, 45122 Essen, Germany. Telephone: 0201-723-
4578. Fax: 0201-723-4546. E-mail: elke.dopp@
uni-essen.de
The authors declare they have no competing
financial interests.
Received 4 October 2004; accepted 9 February
2005.
Intracellular Calcium Disturbances Induced by Arsenic and Its Methylated
Derivatives in Relation to Genomic Damage and Apoptosis Induction
Ana-Maria Florea,
1,2
Ebenezer N. Yamoah,
2
and Elke Dopp
1
1Institute of Hygiene and Occupational Medicine, University Hospital, Essen, Germany; 2Department of Otolaryngology, Center for
Neuroscience, University of California, Davis, California, USA
Arsenic and its methylated derivatives are contaminants of air, water, and food and are known as
toxicants and carcinogens. Arsenic compounds are also being used as cancer chemotherapeutic
agents. In humans, inorganic arsenic is metabolically methylated to mono-, di-, and trimethylated
forms. Recent findings suggest that the methylation reactions represent a toxification rather than a
detoxification pathway. In recent years, the correlation between arsenic exposure, cytotoxicity and
genotoxicity, mutagenicity, and tumor promotion has been established, as well as the association
of arsenic exposure with perturbation of physiologic processes, generation of reactive oxygen
species, DNA damage, and apoptosis induction. Trivalent forms of arsenic have been found to
induce apoptosis in several cellular systems with involvement of membrane-bound cell death
receptors, activation of caspases, release of calcium stores, and changes of the intracellular glu-
tathione level. It is well known that calcium ion deregulation plays a critical role in apoptotic cell
death. A calcium increase in the nuclei might lead to toxic effects in the cell. In this review, we
highlight the relationship between induced disturbances of calcium homeostasis, genomic damage,
and apoptotic cell death caused by arsenic and its organic derivatives. Key words: apoptosis,
arsenic, genomic damage, intracellular calcium. Environ Health Perspect 113:659–664 (2005).
doi:10.1289/ehp.7634 available via http://dx.doi.org/ [Online 10 February 2005]

as a mitochondria-targeting drug in acute pro-
myelocytic leukemia (Jimi et al. 2004; Lau et al.
2004; Miller et al. 2002; Rojewski et al. 2004;
Zhang et al. 1999). Thus, arsenite and arsenic
trioxide are cytotoxic (Jimi et al. 2004; Lau
et al. 2004) and are capable of triggering apop-
tosis (Akao et al. 2000; Cai et al. 2003; Iwama
et al. 2001; Shen et al. 2000; Zhang et al.
1999). Cellular targets of arsenic trioxide action
are presented in Figure 2. Arsenic facilitates
profound cellular alterations, including induc-
tion of apoptosis, inhibition of proliferation,
stimulation of differentiation, and inhibition of
angiogenesis via numerous pathways. The bio-
logic effects of arsenic (principally the trivalent
forms, arsenite and arsenic trioxide) may be
mediated by reactions with closely spaced cys-
teine residues on critical cell proteins.
The cytotoxic potential of arsenic trioxide
leads to decreased mitochondrial membrane
potential, fragmented DNA, and finally to
apoptotic cell death. Additionally, apoptosis
induced by arsenic is mediated by a mecha-
nism involving intracellular GSH-reactive
oxidation (Akao et al. 2000; Jimi et al. 2004;
Zhang et al. 1999).
At the molecular level of the cellular
response, arsenite is able to up-regulate or
down-regulate several proteins involved in dif-
ferent physiologic and pathologic pathways. In
rat lung epithelial cells treated with arsenite,
7 of 1,000 proteins changed expression levels
significantly. The up-regulated proteins were
mostly heat-shock proteins (HSPs) and anti-
oxidative stress proteins, including HSP70,
aldose reductase, heme oxygenase-1, HSP27,
ferritin light chain, and alphaB-crystallin. The
glycolytic enzyme, glyceraldehyde-3-phosphate
dehydrogenase, was down-regulated (Lau et al.
2004).
In addition, extracellular signal-regulated
kinases ERK1 and ERK2 were completely
inactivated, whereas p38 was found activated
in human leukemia U937 cells treated with
arsenic trioxide (As
2
O
3
). Experiments with
transfected cells that expressed constitutively
activated MAPK kinase MEK1 and a specific
inhibitor of p38 have shown that inactivation
of ERKs and activation of p38 might be associ-
ated with the induction of apoptosis by arsenic
trioxide (Iwama et al. 2001). In contrast to the
inactivation of ERKs and the activation of p38,
activation of JNK by As
2
O
3
appeared to pro-
tect cells against the induction of apoptosis.
However, treatment of U937 cells with As
2
O
3
also caused the Ca
2+
-dependent production of
superoxide, intracellular acidification, and a
decrease in the mitochondrial membrane
potential at the early stages of apoptosis. These
changes preceded the release of cytochrome c
from mitochondria and the activation of cas-
pase-3 (Figures 2 and 3) (Iwama et al. 2001;
Miller et al. 2002).
Arsenic trioxide induces apoptosis in vari-
ous cancer cells via complex mechanisms,
which seem to be cell type dependent (Cai
et al. 2003; Miller et al. 2002). Involvement of
caspase 3 and caspase 8 was shown together
with the down-regulation of Bcl-2 protein
(Akao et al. 2000; Miller et al. 2002). A tight
link between As
2
O
3
-induced apoptosis and
mitotic arrest was recently shown by Cai et al.
(2003), the latter being one of the common
mechanism for As
2
O
3
-induced apoptosis in
cancer cells. Arsenic can either enhance or
reduce nitric oxide (NO) production, depend-
ing on the type of cell, the species, and dose of
arsenical tested. The mechanisms of how
arsenic increases or decreases NO production
remain unclear (Gurr et al. 2003).
The Janus kinase (JAK)-signal transduc-
tion and activation of transcription (STAT)
pathway is an essential cascade for mediating
normal functions of different cytokines in the
development of the hematopoietic and
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Florea et al.
660
VOLUME 113 |NUMBER 6 |June 2005
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Environmental Health Perspectives
Figure 1. Challenger (1945) mechanism for arsenic biomethylation. R = reduction, OM = oxidative methyla-
tion. [Copyright 2004 from “Environmental Distribution, Analysis and Toxicity of Organometal(loid)
Compounds” by Dopp et al. (2004a). Reproduced by permission of Taylor & Francis Group, LLC.
(http://www.taylorandfrancis.com).]
Figure 2. Cellular targets of arsenic trioxide action, with multiple pathways in malignant cells resulting in
apoptosis or in the promotion of differentiation. Potential molecular targets for arsenic trioxide and arsenite
are shown in gray. Abbreviations: AP1, activator protein-1; Apaf, apoptotic protease-activating factor; CK2,
casein kinase; Co-A, coenzyme A; DAXX, death-associated protein; ER, estrogen receptor; FADH, flavin
adenine dinucleotide; PARP, poly-(ADP-ribose)-polymerase; PML, promyelocytic leukemia. Modified from
Miller et al. (2002) with permission from the American Association for Cancer Research.
Figure 3. Apoptosis induced by arsenic trioxide by
way of changes in mitochondrial membrane poten-
tial and increased H2O2in cells; this lowers the
mitochondrial membrane potential, leading to the
release of cytochrome c and the activation of the
caspase pathway. Abbreviations: ψM, mitochon-
drial inner transmembrane potential; Apaf-1, apop-
totic protease-activating factor 1; GPx, glutathione
peroxidase 1; GS, glutathione. Modified from Miller
et al. (2002) with permission from the American
Association for Cancer Research.

immune systems (Cheng et al. 2004). It has
been suggested that arsenic-induced MAPK
signal transduction leads to activation of tran-
scription factors such as activator protein-1
(AP-1) and nuclear factor-κB (NFκB), which
in turn alters gene expression (Yang and
Frenkel 2002). This might be associated with
the carcinogenicity of arsenic.
Ma et al. (1998) studied apoptosis in NB4
cells induced by sodium arsenite and arsenate
using flow cytometry and DNA gel elec-
trophoresis. The authors concluded that arsen-
ite and arsenate induced apoptosis in NB4
cells by two different mechanisms: at low
doses, arsenic might directly induce apoptosis
through regulation of the cell cycle check-
point, whereas at high doses it might directly
induce apoptosis, but in this case Bcl-2 might
not play an important role. Thus, the chemi-
cal valence of arsenic in a compound might be
related to the efficiency of arsenical-induced
apoptosis (Ma et al. 1998).
Woo et al. (2002) reported that HeLa cells
underwent apoptosis in response to As
2
O
3
,
accompanied by a decrease in mitochondrial
membrane potential and caspase-3 activation.
Overexpression of Bcl-2, however, prevented
the dissipation of mitochondrial membrane
potential, subsequently protecting the cells
from As
2
O
3
-induced apoptosis. Arsenic trioxide
increased the cellular content of ROSs, espe-
cially hydrogen peroxide, and the antioxidant
N-acetyl-
L
-cysteine. Furthermore, incubation of
the cells with catalase resulted in significant
suppression of As
2
O
3
-induced apoptosis. The
above results indicate that the induction of
apoptosis in HeLa cells by arsenic trioxide
include an early decrease in cellular mitochon-
drial membrane potential and an increase in
ROS content, predominantly H
2
O
2
, followed
by caspase-3 activation and DNA fragmenta-
tion (Miller et al. 2002; Woo et al. 2002).
For decades, arsenic has been considered a
nongenotoxic carcinogen because it is only
weakly active or, more often, completely inac-
tive in bacterial and mammalian cell mutation
assays. In recent studies, methylated metabo-
lites of inorganic arsenic have been extensively
investigated because of their high cytotoxic
and genotoxic potential. Trivalent dimethy-
lated arsenic, which can be produced by the
metabolic reduction of DMA, has attracted
considerable attention from the standpoint of
arsenic carcinogenesis. Several groups have
shown that DMA(III) is highly genotoxic
compared with the pentavalent species and
inorganic arsenic (e.g., Dopp et al. 2004b;
Schwerdtle et al. 2003) (Figure 4).
Ochi et al. (1996) studied the induction of
apoptosis caused by the methylated arsenic
species. These authors showed that DMA(V)
induces apoptosis in cultured human HL-60
cells at concentrations of 1–5 mM after an
incubation period of 18 hr. On the other
hand, Cohen et al. (2002) showed that in vivo
administration of DMA(V) results in cytotoxi-
city with necrosis, followed by regenerative
hyperplasia of the bladder epithelium.
DMA(V) exerted differential antiproliferative
and cytotoxic activity against leukemia and
multiple myeloma cells, with no significant
effect on normal progenitor cells (Duzkale
et al. 2003).
In comparison with the trivalent inorganic
arsenic form, therapeutic concentrations of
As
2
O
3
(1–2 µM) had dual effects on malignant
lymphocytes: a) inhibition of growth through
adenosine triphosphate (ATP) depletion and
prolongation of cell cycle time, and b) induc-
tion of apoptosis (Zhu et al. 1999).
Zhang et al. (1999) suggested that the
increase in intracellular Ca
2+
is related to the
sensitivity of human cells to As
2
O
3
exposure,
indicating that a critical intracellular Ca
2+
sig-
nal transduction pathway could be involved
in As
2
O
3
-mediated cell death.
The Toxicity of Arsenicals
Is Related to Calcium
Homeostasis Disturbances
In order to explore the early apoptotic signal
messengers and the apoptotic pathway, the
morphologic and functional changes of mito-
chondria were examined in a study by Shen
et al. (2002b). The content of NO and free
calcium ions (Ca
2+
) was measured over the
course of apoptosis induction after exposure
with As
2
O
3
in esophageal carcinoma cells
(SHEEC1). SHEEC1 cells were exposed to
As
2
O
3
(1, 3, and 5 µmol/L), and after 0, 2, 4,
8, 12, and 24 hr, the fluorescence intensity
(FI) of rhodamine 123 (Rho123)-labeled cells
was detected using a confocal laser scanning
microscope for evaluation of the mitochon-
drial membrane potential. After adding arsenic
trioxide, SHEEC1 cells showed characteristic
morphologic and functional changes of mito-
chondria such as hyperplasia, disruption, and
an accompanying decrease in transmembrane
potential (FI of Rho123 decreased). The Ca
2+
level increased immediately after adding
As
2
O
3
, and the NO concentration increased
in a step-wise manner up to 24 hr. At this
time the cells appeared to have an apoptotic
morphology. The results of Shen et al. (2002a,
2002b) suggest that by inducement of As
2
O
3
-
increased Ca
2+
and NO levels, the apoptotic
signal messengers initiate the mitochondria-
dependent apoptotic pathway.
In previous experiments (Florea 2004) we
assessed inorganic As
i
(III) and As
i
(V), as well as
MMA(V), DMA(V), and TMAO(V) (0.5
mM concentration) for early disturbances in
calcium homeostasis in HeLa S3 cells within
the first few seconds after application. If cal-
cium homeostasis was disturbed, a drop in the
fluorescence signal of the dye was recorded by
confocal laser scanning microscopy. The drop
was transient, and the signal returned rapidly
to the initial level within 20 sec (Figures 5
and 6). These calcium signals might occur as
active efflux from the cell to the exterior
(energy consuming) or as deregulation of other
ion transports. A mechanism via membrane
receptor activation or membrane damage can-
not be excluded (Florea 2004).
Recently, the original calcium hypothesis
has been modified, taking into account that
cell death is induced under experimental con-
ditions not only by a rise in cytoplasmatic cal-
cium but also when cytoplasmatic calcium
activity drops below physiologic levels
(Paschen 2003). Cellular stimulation can lead
to activation of different signal transduction
mechanisms, such as alterations of the cyto-
plasmatic levels of different ions. Cell alkaliza-
tion slightly decreases the intracellular Ca
2+
concentration due to an efflux of Ca
2+
from
the cell. Elevation of pH, however, increases
Ca
2+
either in the presence or absence of exter-
nal Ca
2+
(Cabado et al. 2000). In contrast to
these findings, Kauppinen et al. (1989)
reported a study involving cortical synapto-
somes in the guinea pig. Cytosolic calcium
drops were seen in this study in the absence of
Ca
2+
in the external solution and were related
to an increased glucose utilization (Kauppinen
et al. 1989). On the other hand, Buja et al.
(1993) suggested that the initial modifications
of cellular metabolism and calcium homeo-
stasis may activate major pathways leading to a
loss of membrane integrity by a) membrane
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|
Arsenic-induced intracellular calcium changes
Environmental Health Perspectives
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VOLUME 113 |NUMBER 6 |June 2005
661
Figure 4. Micronucleus formation in CHO cells after treatment of cells with (
A
) Asi(V), MMA(V), or DMA(V)
and (
B
)As
i(III), MMA(III), or DMA(III). The cells were incubated with the arsenic species for 1 hr. Two
thousand binucleated cells were evaluated for micronucleus induction in each case. Data from Dopp et al.
(2004b).
*
p
≤0.01, and **
p
≤0.001, Student
t
-test.
BA
140
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
Control 1 µM5 µM 10 µM 30 µM 100 µM 500 µM Control 1 µM5 µM 10 µM 30 µM 100 µM 500 µM
Concentrations Concentrations
No. of MN/1,000 cells
No. of MN/1,000 cells
**
**
**
**
*
Asi(V)
MMA(V)
DMA(V)
Asi(III)
MMA(III)
DMA(III)

phospholipid degradation, b) production of
amphipathic lipids, c) damage of the cyto-
skeleton, and d) generation of toxic oxygen
species and free radicals.
Cellular Mechanisms of
Intracellular Calcium Changes
in Relation to Genetic Damage
Regulation of intercellular and intracellular sig-
naling is fundamental for survival and death in
biologic organisms; the systems that control
ion movements across cell membranes are
essential for cell survival. A deregulation of
channels or pumps can cause events that lead
to cell death. Apoptosis can be caused by loss
of Ca
2+
homeostatic control but can also be
positively or negatively controlled by changes
in Ca
2+
distribution within intracellular com-
partments. It was shown that even non-
disruptive changes in Ca
2+
signaling could have
adverse effects, including alterations in cell pro-
liferation and differentiation, as well as in the
modulation of apoptosis (Orrenius et al. 2003).
Cellular Ca
2+
import through the plasma
membrane occurs largely by receptor-operated
and voltage-sensitive channels. Once inside the
cell, Ca
2+
can either interact with Ca
2+
-binding
proteins or become sequestered to the endo-
plasmic reticulum (ER) or mitochondria,
reaching millimolar levels. Ca
2+
levels in the
ER are regulated by Ca
2+
-ATPase pumps, inos-
itol 1,4,5-trisphosphate (IP3) receptors, ryan-
odine receptors, and Ca
2+
-binding proteins
(Orrenius et al. 2003). Thus, the mitochon-
drial permeability transition is involved in
apoptotic cell death, in that it releases pro-
apoptotic proteins from the mitochondria into
the cytosol where, with the aid of cellular ATP,
they complete the apoptotic cascade. The com-
plexity of the regulation of Ca
2+
inside the cell
is probably because mitochondria are able to
modulate the amplitude and shape of Ca
2+
sig-
nals (Babcock et al. 1997). However, mito-
chondria contribute to both apoptotic and
necrotic cell death (Nieminen 2003).
It was previously demonstrated by Lui
et al. (2003) that tubules, in a vertical or hori-
zontal orientation, extend deep inside the
nucleus of HeLa cells. These extensions,
together with the nuclear envelope and ER,
physically form a spatial network. For Ca
2+
sig-
naling, the nuclear tubules provide a fast trans-
port system to direct the release of IP3 and
Ca
2+
from the cytosol to the nucleus or vice
versa. The lumen of the nuclear tubules con-
tains many organelles, including mitochondria
that move in and out of the nuclear tubules.
To reduce Ca
2+
overloading, mitochondria can
take up a considerable amount of Ca
2+
inside
the nuclear tubules (Lui et al. 2003). Li et al.
(1998) described that oscillations in cytosolic
calcium at physiologic rates maximize gene
expression depending on IP3. Spikes of cyto-
solic calcium were able to stimulate gene
expression via the nuclear factor of activated
T cells (Li et al. 1998).
Another study by Liu and Huang (1996)
demonstrated that calcium ions are accumu-
lated in the nuclei of Chinese hamster ovary
(CHO)-K1 cells after arsenite treatment.
These observed effects were related to distur-
bances in intracellular calcium homeostasis
and with arsenite-induced cytotoxicity and
micronucleus formation. A modulation of the
calcium level within the nucleus might have
toxic effects leading to DNA damage and/or
inhibition of DNA repair function. Some
authors have shown that micronucleus forma-
tion (expressing DNA damage) as well as
induction of mitotic disturbances is strongly
correlated with disturbances of calcium
homeostasis (Dopp et al. 1999; Liu and
Huang 1996; Xu et al. 2003). The correlation
between DNA damage and calcium home-
ostasis disturbances was supported for As
i
(III)
when Liu and Huang (1997) observed an ele-
vation of intracellular calcium after arsenite
treatment. These authors showed that cal-
cium ions play an essential role in arsenite-
induced genotoxicity and concluded that
arsenite exposure perturbs intracellular calcium
homeostasis and activates protein kinase C
activity in a dose-dependent manner.
Bucki and Gorski (2001) even suggested
that the nucleoplasmic calcium concentrations
([Ca
2+
]
n
) may be regulated independently of
that of cytosolic Ca
2+
. IP3 and cyclic ADP-
ribose are the major factors responsible for Ca
2+
release into the nucleus from the perinuclear
space. [Ca
2+
]
n
is involved in the regulation of
many events in the nucleus, such as gene
expression, DNA replication, DNA repair,
chromatin fragmentation in apoptosis, and
modulation of an intranuclear contractile sys-
tem. The importance of a precise cellular Ca
2+
-
level regulation for an optimal DNA repair
process was mentioned already by Gafter et al.
(1997). Bugreev and Mazin (2004) showed
that the human Rad51 protein, which plays a
key role in homologous recombination and
DNA repair, is dependent upon the intra-
cellular calcium level. Arsenic and its methy-
lated derivatives are able to modulate DNA
repair processes (e.g., Andrew et al. 2003;
Hartwig 1998; Hartwig et al. 2003) and gene
expression (e.g., Elbekai and El-Kadi 2004; Wu
et al. 2003). A possible correlation between
inhibition of DNA repair function as well as
changed gene expression profiles caused by
arsenicals and disturbed intracellular calcium
homeostasis requires further investigations.
Conclusions
Epidemiologic evidence suggests that expo-
sure to inorganic arsenic causes cancer (e.g.,
National Research Council 1999). However,
the mechanism of arsenic carcinogenesis is
still unclear. A complicating factor receiving
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VOLUME 113 |NUMBER 6 |June 2005
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Environmental Health Perspectives
Figure 5. Intracellular calcium changes (relative intensity units measured by confocal laser scanning
microscopy) in HeLa S3 cells after application of HEPES buffer (negative control). (
A
) Control. (
B
) Control and
application of HEPES buffer (indicated by arrow). (
C
) Control after 1 min. (
D
) Control after 3 min. The incuba-
tion buffer did not modify the initial level of fluorescence intensity. No photo bleaching occurred. Data from
Florea (2004).
BA
DC
250
200
150
100
50
0
250
200
150
100
50
0
250
200
150
100
50
0
250
200
150
100
50
0
Relative intensity
197898173655749413325179 197898173655749413325179
185787164575043362922158 92 99 97898173655749413325179
1
Time (scan cycles)
Relative intensity
Relative intensity
Relative intensity
Time (scan cycles)
Time (scan cycles) Time (scan cycles)

increasing attention is that arsenic is bio-
methylated to form various metabolites.
Methylated arsenic species are able to induce
genomic damage as well as apoptosis in vivo
and in vitro. Most research has been done
with DMA(V) because it has neurotoxic
effects and induces bladder cancer in rats and
apoptosis in cultured human cells (Jia et al.
2004; Namgung and Zia 2001; Xie et al.
2004). The conjugation of DMA(V) with
cellular GSH appears to be of mechanistic sig-
nificance. More research is needed to deter-
mine the role of intracellular GSH and
methylation in the toxicity of arsenicals in
chronic arsenic poisoning or in cases where
arsenicals are used as chemotherapeutics.
Several investigations have shown that
DMA(V) exposure causes oxidative stress,
DNA damage, and specific induction of apop-
tosis in target organs of arsenic carcinogenesis
(Jia et al. 2004; Sakurai et al. 2004), which
may be attributable to the mechanism(s) of
arsenic-induced carcinogenesis in rodents.
Compared with the pentavalent methylated
arsenic species, the trivalent species are even
more reactive and cause calcium homeostasis
disturbances, oxidative stress, DNA damage,
and apoptosis to a higher extent. The involve-
ment of internal calcium stores, particularly
mitochondria, can be assumed. This specific
area requires further research. Also, more
research should focus on the cellular effects of
arsenic metabolites, which are generated inside
the cell and may cause cellular damage at
much lower concentrations than the inorganic
arsenic species.
Many studies in the literature describe the
effects of arsenite and arsenic trioxide on cellu-
lar targets, because these chemicals are or have
been used as chemotherapeutic agents for the
treatment of several human diseases. Apoptosis
induction caused by As
2
O
3
has been shown to
be related with changes of the intracellular cal-
cium concentration (e.g., Akao et al. 2000;
Cai et al. 2003). The intracellular Ca
2+
level
increases immediately after adding As
2
O
3
.
The initiation of the mitochondria-dependent
apoptotic pathway was suggested (Iwama et al.
2001; Miller et al. 2002).
A precise cellular Ca
2+
-level regulation is
also necessary for optimal DNA repair
processes, DNA replication, and gene expres-
sion. Arsenicals are able to modulate these
processes. A direct correlation between
genotoxic effects caused by arsenicals and dis-
turbances of intracellular calcium concentra-
tion is partially proven but requires further
investigations.
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BA
DC
140
120
100
80
60
40
20
0
185787164575043362922158 92 99
Time (scan cycles)
140
120
100
80
60
40
20
0
185787164575043362922158 92 99 185787164575043362922158 92 99
185787164575043362922158 92 99
E
185787164575043362922158 92 99
140
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80
60
40
20
0
140
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80
60
40
20
0
140
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60
40
20
0
Relative intensity
Time (scan cycles)
Time (scan cycles) Time (scan cycles)
Time (scan cycles)
Relative intensity
Relative intensity
Relative intensity
Relative intensity
Figure 6. Intracellular calcium changes (relative
intensity units measured by confocal laser scan-
ning microscopy) in HeLa S3 cells after application
of 0.5 mM of different arsenic species (indicated
by arrows). (
A
)As
i(III); (
B
)As
i(V); (
C
) MMA(V);
(
D
) DMA(V); (
E
) TMAO. Note the drop in the fluo-
rescence signal immediately after application
(Florea 2004).

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