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


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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.
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Arsenic and Its Derivatives as
Potent Environmental
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
Environmental Health Perspectives
VOLUME 113 |NUMBER 6 |June 2005
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@
The authors declare they have no competing
financial interests.
Received 4 October 2004; accepted 9 February
Intracellular Calcium Disturbances Induced by Arsenic and Its Methylated
Derivatives in Relation to Genomic Damage and Apoptosis Induction
Ana-Maria Florea,
Ebenezer N. Yamoah,
and Elke Dopp
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 [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.
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
). 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
appeared to pro-
tect cells against the induction of apoptosis.
However, treatment of U937 cells with As
also caused the Ca
-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
-induced apoptosis and
mitotic arrest was recently shown by Cai et al.
(2003), the latter being one of the common
mechanism for As
-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
Florea et al.
VOLUME 113 |NUMBER 6 |June 2005
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.
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
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
-induced apoptosis. Arsenic trioxide
increased the cellular content of ROSs, espe-
cially hydrogen peroxide, and the antioxidant
-cysteine. Furthermore, incubation of
the cells with catalase resulted in significant
suppression of As
-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
, 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
(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
is related to the
sensitivity of human cells to As
indicating that a critical intracellular Ca
nal transduction pathway could be involved
in As
-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
) was measured over the
course of apoptosis induction after exposure
with As
in esophageal carcinoma cells
(SHEEC1). SHEEC1 cells were exposed to
(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
level increased immediately after adding
, 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
increased Ca
and NO levels, the apoptotic
signal messengers initiate the mitochondria-
dependent apoptotic pathway.
In previous experiments (Florea 2004) we
assessed inorganic As
(III) and As
(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
concentration due to an efflux of Ca
the cell. Elevation of pH, however, increases
either in the presence or absence of exter-
nal Ca
(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
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
Arsenic-induced intracellular calcium changes
Environmental Health Perspectives
VOLUME 113 |NUMBER 6 |June 2005
Figure 4. Micronucleus formation in CHO cells after treatment of cells with (
) Asi(V), MMA(V), or DMA(V)
and (
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.
0.01, and **
0.001, Student
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
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
homeostatic control but can also be
positively or negatively controlled by changes
in Ca
distribution within intracellular com-
partments. It was shown that even non-
disruptive changes in Ca
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
import through the plasma
membrane occurs largely by receptor-operated
and voltage-sensitive channels. Once inside the
cell, Ca
can either interact with Ca
proteins or become sequestered to the endo-
plasmic reticulum (ER) or mitochondria,
reaching millimolar levels. Ca
levels in the
ER are regulated by Ca
-ATPase pumps, inos-
itol 1,4,5-trisphosphate (IP3) receptors, ryan-
odine receptors, and Ca
-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
inside the cell
is probably because mitochondria are able to
modulate the amplitude and shape of Ca
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
naling, the nuclear tubules provide a fast trans-
port system to direct the release of IP3 and
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
overloading, mitochondria can
take up a considerable amount of Ca
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
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
) may be regulated independently of
that of cytosolic Ca
. IP3 and cyclic ADP-
ribose are the major factors responsible for Ca
release into the nucleus from the perinuclear
space. [Ca
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
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.
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
Florea et al.
VOLUME 113 |NUMBER 6 |June 2005
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). (
) Control. (
) Control and
application of HEPES buffer (indicated by arrow). (
) Control after 1 min. (
) 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).
Relative intensity
197898173655749413325179 197898173655749413325179
185787164575043362922158 92 99 97898173655749413325179
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
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
increases immediately after adding As
The initiation of the mitochondria-dependent
apoptotic pathway was suggested (Iwama et al.
2001; Miller et al. 2002).
A precise cellular Ca
-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
Abernathy CO, Liu YP, Longfellow D, Aposhian HV, Beck B,
Fowler B, et al. 1999. Arsenic: health effects, mechanisms
of actions, and research issues. Environ Health Perspect
Akao Y, Yamada H, Nakagawa Y. 2000. Arsenic-induced apop-
tosis in malignant cells in vitro. Leuk Lymphoma 37:53–63.
Andrew AS, Karagas MR, Hamilton JW. 2003. Decreased DNA
repair gene expression among individuals exposed to
arsenic in United States drinking water. Int J Cancer
Babcock DF, Herrington J, Goodwin PC, Park YB, Hille B. 1997.
Mitochondrial participation in the intracellular Ca2+ network.
J Cell Biol 136:833–844.
Bucki R, Gorski J. 2001. Current views on function and regulation
of Ca2+ levels in cell nuclei. Postepy Hig Med Dosw
Bugreev DV, Mazin AV. 2004. Ca2+ activates human homolo-
gous recombination protein Rad51 by modulating its
ATPase activity. Proc Natl Acad Sci USA 101:9988–9993.
Buja LM, Eigenbrodt ML, Eigenbrodt EH. 1993. Apoptosis and
necrosis. Basic types and mechanisms of cell death. Arch
Pathol Lab Med 117:1208–1214.
Cabado AG, Alfonso A, Vieytes MR, Gonzalez M, Botana MA,
Botana LM. 2000. Crosstalk between cytosolic pH and
intracellular calcium in human lymphocytes: effect of
4-aminopyridin, ammoniun chloride and ionomycin. Cell
Signal 12:573–581.
Cai X, Yu Y, Huang Y, Zhang L, Jia PM, Zhao Q, et al. 2003. Arsenic
trioxide-induced mitotic arrest and apoptosis in acute
promyelocytic leukemia cells. Leukemia 17:1333–1337.
Challenger F. 1945. Biological methylation. Chem Rev
Cheng HY, Li P, David M, Smithgall TE, Feng L, Lieberman MW.
2004. Arsenic inhibition of the JAK-STAT pathway.
Oncogene 23:3603–3612.
Cohen SM, Arnold LL, Uzvolgyi E, Cano M, St John M,
Yamamoto S, et al. 2002. Possible role of dimethylarsinous
acid in dimethylarsinic acid-induced urothelial toxicity and
regeneration in the rat. Chem Res Toxicol 15:1150–1157.
Dopp E, Hartmann LM, Florea AM, Rettenmeier AW, Hirner AV.
2004a. Environmental distribution, analysis and toxicity of
organometal(loid) compounds. Crit Rev Toxicol 34:1–33.
Dopp E, Hartmann LM, Florea AM, von Recklinghausen U,
Pieper R, Shokouhi B, et al. 2004b. Uptake of inorganic and
organic derivatives of arsenic associated with induced
cytotoxic and genotoxic effects in Chinese hamster ovary
(CHO) cells. Toxicol Appl Pharmacol 201:156–165.
Dopp E, Muller J, Hahnel C, Schiffmann D. 1999. Induction of
genotoxic effects and modulation of the intracellular cal-
cium level in syrian hamster embryo (SHE) fibroblasts
caused by ochratoxin A. Food Chem Toxicol 37:713–721.
Duzkale H, Jilani I, Orsolic N, Zingaro RA, Golemovic M, Giles FJ,
et al. 2003. In vitro activity of dimethylarsinic acid against
human leukemia and multiple myeloma cell lines. Cancer
Chemother Pharmacol 51:427–432.
Elbekai RH, El-Kadi AO. 2004. Modulation of aryl hydrocarbon
receptor-regulated gene expression by arsenite, cadmium,
and chromium. Toxicology 202:249–269.
Florea AM. 2004. Toxicity of Alkylated Derivatives of Arsenic,
Antimony and Tin in Vitro: Cytotoxicity, Genotoxic Effects,
Cellular Uptake, Cytotoxicity, Genotoxic Effects, Perturba-
tion of Ca2+ Homeostasis, and Cell Death. Aachen,
Florea AM, Dopp E, Obe G, Rettenmeier AW. 2004. Genotoxicity
Arsenic-induced intracellular calcium changes
Environmental Health Perspectives
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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). (
i(III); (
i(V); (
) MMA(V);
) DMA(V); (
) TMAO. Note the drop in the fluo-
rescence signal immediately after application
(Florea 2004).
of organometallic species. In: Organic Metal and Metalloid
Species in the Environment: Analysis, Distribution,
Processes and Toxicological Evaluation (Hirner AV,
Emons H, eds). Heidelberg:Springer-Verlag, 205–216.
Gafter U, Malachi T, Ori Y, Breitbart H. 1997. The role of calcium
in human lymphocyte DNA repair ability. J Lab Clin Med
Gebel TW. 2001. Genotoxicity of arsenical compounds. Int J
Hyg Environ Health 203:249–262.
Gurr JR, Yih LH, Samikkannu T, Bau DT, Lin SY, Jan KY. 2003.
Nitric oxide production by arsenite. Mutat Res 533:173–182.
Hartwig A. 1998. Carcinogenicity of metal compounds: possible
role of DNA repair inhibition. Toxicol Lett 102–103:235–239.
Hartwig A, Blessing H, Schwerdtle T, Walter I. 2003. Modulation
of DNA repair processes by arsenic and selenium com-
pounds. Toxicology 193:161–169.
Hughes MF. 2002. Arsenic toxicity and potential mechanisms of
action. Toxicol Lett 133:1–16.
Iwama K, Nakajo S, Aiuchi T, Nakaya K. 2001. Apoptosis induced
by arsenic trioxide in leukemia U937 cells is dependent on
activation of p38, inactivation of ERK and the Ca2+-depen-
dent production of superoxide. Int J Cancer 92:518–526.
Jia G, Sone H, Nishimura N, Satoh M, Tohyama C. 2004.
Metallothionein (I/II) suppresses genotoxicity caused by
dimethylarsinic acid. Int J Oncol 25:325–333.
Jimi S, Uchiyama M, Takaki A, Suzumiya J, Hara S. 2004.
Mechanisms of cell death induced by cadmium and
arsenic. Ann NY Acad Sci 1011:325–331.
Kauppinen RA, Taipale HT, Komulainen H. 1989. Interrelation-
ships between glucose metabolism, energy state, and the
cytosolic free calcium concentration in cortical synapto-
somes from the guinea pig. J Neurochem 53:766–771.
Kitchin KT. 2001. Recent advances in carcinogenesis: modes of
action, animal model systems, and methylated arsenic
metabolites. Toxicol Appl Pharmacol 172:249–261.
Kitchin KT, Ahmad S. 2003. Oxidative stress as a possible mode
of action for arsenic carcinogenesis. Toxicol Lett 137:3–13.
Lau AT, He QY, Chiu JF. 2004. A proteome analysis of the arsen-
ite response in cultured lung cells: evidence for in vitro
oxidative stress-induced apoptosis. Biochem J 382:641–650.
Li W, Llopis J, Whitney M, Zlokarnik G, Tsien RY. 1998. Cell-per-
meant caged InsP3 ester shows that Ca2+ spike frequency
can optimize gene expression. Nature 392:936–941.
Liu YC, Huang H. 1996. Lowering extracellular calcium content
protects cells from arsenite-induced killing and micro-
nuclei formation. Mutagenesis 11:75–78.
Liu YC, Huang H. 1997. Involvement of calcium-dependent pro-
tein kinase C in arsenite-induced genotoxicity in Chinese
hamster ovary cells. J Cell Biochem 64:423–433.
Lui PP, Chan FL, Suen YK, Kwok TT, Kong SK. 2003. The nucleus
of HeLa cells contains tubular structures for Ca2+ signaling
with the involvement of mitochondria. Biochem Biophys
Res Commun 308:826–833.
Ma DC, Sun YH, Chang KZ, Ma XF, Huang SL, Bai YH, et al. 1998.
Selective induction of apoptosis of NB4 cells from G2+M
phase by sodium arsenite at lower doses. Eur J Haematol
Miller WH Jr, Schipper HM, Lee JS, Singer J, Waxman S. 2002.
Mechanisms of action of arsenic trioxide. Cancer Res
Murphy MJ, Lyon LW, Taylor JW. 1981. Subacute arsenic neuro-
pathy: clinical and electrophysiological observations.
J Neurol Neurosurg Psychiatry 44:896–900.
Namgung U, Xia Z. 2001. Arsenic induces apoptosis in rat cere-
bellar neurons via activation of JNK3 and p38 MAP
kinases. Toxicol Appl Pharmacol 174:130–138.
National Research Council. 1999. Arsenic in Drinking Water.
Washington, DC:National Academy Press.
National Research Council. 2001. Arsenic in Drinking Water:
2001 Update. Washington, DC:National Academy Press.
Nieminen AL. 2003. Apoptosis and necrosis in health and disease:
role of mitochondria. Int Rev Cytol 224:29–55.
Ochi T, Nakajima F, Sakurai T, Kaise T, Oya-Ohta Y. 1996.
Dimethylarsinic acid causes apoptosis in HL-60 cells via
interaction with glutathione. Arch Toxicol 70:815–821.
Orrenius S, Zhivotovsky B, Nicotera P. 2003. Regulation of cell
death: the calcium-apoptosis link. Nat Rev Mol Cell Biol
Paschen W. 2003. Mechanisms of neuronal cell death: diverse
roles of calcium in the various subcellular compartments.
Cell Calcium 34:305–310.
Patrick L. 2003. Toxic metals and antioxidants: part II. The role
of antioxidants in arsenic and cadmium toxicity. Altern
Med Rev 8:106–128.
Qian Y, Castranova V, Shi XJ. 2003. New perspectives in
arsenic-induced cell signal transduction. Inorg Biochem
Rojewski MT, Korper S, Thiel E, Schrezenmeier H. 2004.
Arsenic trioxide-induced apoptosis is independent of CD95
in lymphatic cell lines. Oncol Rep 11:509–513.
Rossman TG, Uddin AN, Burns FJ. 2004. Evidence that arsenite
acts as a cocarcinogen in skin cancer. Toxicol Appl
Pharmacol 198:394–404.
Sakurai T, Ochiai M, Kojima C, Ohta T, Sakurai MH, Takada NO,
et al. 2004. Role of glutathione in dimethylarsinic acid-
induced apoptosis. Toxicol Appl Pharmacol 198:354–365.
Schwerdtle T, Walter I, Mackiw I, Hartwig A. 2003. Induction of
oxidative DNA damage by arsenite and its trivalent and
pentavalent methylated metabolites in cultured human
cells and isolated DNA. Carcinogenesis 24:967–974.
Shen ZY, Shen J, Cai WJ, Hong C, Zheng MH. 2000. The altera-
tion of mitochondria is an early event of arsenic trioxide
induced apoptosis in esophageal carcinoma cells. Int J
Mol Med 5:155–158.
Shen ZY, Shen WY, Chen MH, Shen J, Cai WJ, Yi Z. 2002a. Nitric
oxide and calcium ions in apoptotic esophageal carcinoma
cells induced by arsenite. World J Gastroenterol 8:40–43.
Shen ZY, Shen WY, Chen MH, Shen J, Cai WJ, Zeng Y. 2002b.
Mitochondria, calcium and nitric oxide in the apoptotic
pathway of esophageal carcinoma cells induced by
As2O3. Int J Mol Med 9:385–390.
Sordo M, Herrera LA, Ostrosky-Wegman P, Rojas E. 2001.
Cytotoxic and genotoxic effects of As, MMA, and DMA on
leukocytes and stimulated human lymphocytes. Teratog
Carcinog Mutagen 21:249–260.
Tchounwou PB, Wilson B, Ishaque A. 1999. Important considera-
tions in the development of public health advisories for
arsenic and arsenic-containing compounds in drinking
water. Rev Environ Health 14:211–229.
Woo SH, Park IC, Park MJ, Lee HC, Lee SJ, Chun YJ, et al. 2002.
Arsenic trioxide induces apoptosis through a reactive oxy-
gen species-dependent pathway and loss of mitochondrial
membrane potential in HeLa cells. Int J Oncol 21:57–63.
Wu MM, Chiou HY, Ho IC, Chen CJ, Lee TC. 2003. Gene expres-
sion of inflammatory molecules in circulating lymphocytes
from arsenic-exposed human subjects. Environ Health
Perspect 111:1429–1438.
Xie Y, Trouba KJ, Liu J, Waalkes MP, Germolec DR. 2004.
Biokinetics and subchronic toxic effects of oral arsenite,
arsenate, monomethylarsonic acid, and dimethylarsinic
acid in v-Ha-
transgenic (Tg.AC) mice. Environ Health
Perspect 112:1255–1263.
Xu N, Luo KQ, Chang DC. 2003. Ca2+ signal blockers can inhibit
M/A transition in mammalian cells by interfering with the
spindle checkpoint. Biochem Biophys Res Commun
Yang C, Frenkel K. 2002. Arsenic-mediated cellular signal trans-
duction, transcription factor activation, and aberrant gene
expression: implications in carcinogenesis. J Environ
Pathol Toxicol Oncol 21:331–342.
Zhang TC, Cao EH, Li JF, Ma W, Qin JF. 1999. Induction of apop-
tosis and inhibition of human gastric cancer MGC-803 cell
growth by arsenic trioxide. Eur J Cancer 35:1258–1263.
Zhu XH, Shen YL, Jing YK, Cai X, Jia PM, Huang Y, et al. 1999.
Apoptosis and growth inhibition in malignant lymphocytes
after treatment with arsenic trioxide at clinically achiev-
able concentrations. J Natl Cancer Inst 91:772–778.
Florea et al.
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Environmental Health Perspectives
... Heavy metals appear to be implicated in gene expression modifications as well as related epigenetic changes (Arita & Costa 2009, Larson et al. 2010, Salnikow & Zhitkovich 2008. Disrupted regulation of calcium ions in the nucleus can cause cellular toxicity which is an established mechanism of triggering cell apoptosis (Florea et al. 2005). Other As-involved epigenetic modifications have been discovered, including intracellular glutathione-reactive oxidation, and increased expression of proteins (such as the alpha-B-crystallin, heat-shock antioxidative stress proteins, and ferritin light chain) as well as enzymes (such as heme oxygenase-1, reductase, aldose), while the glyceraldehyde-3-phosphate dehydrogenase is downregulated, and the extracellular signal-regulated kinases (ERK)-1 and ERK2 are inactivated. ...
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Untainted environment promotes health, but the last few decades experienced steep upsurge in environmental contaminants posing detrimental physiological impact. The responsible factors mainly include the exponential growth of human population, havoc rise in industrialization, poorly planned urbanization, and slapdash environment management. Environmental degradation can increase the likelihood of human exposure to heavy metals, resulting in health consequences such as reproductive problems. As a result, research into metal-induced causes of reproductive impairment at the genetic, epigenetic, and biochemical levels must be strengthened further. These metals impact upon the female reproduction at all strata of its regulation and functions, be it development, maturation, or endocrine functions, and are linked to an increase in the causes of infertility in women. Chronic exposures to the heavy metals may lead to breast cancer, endometriosis, endometrial cancer, menstrual disorders, and spontaneous abortions, as well as pre-term deliveries, stillbirths. For example, endometriosis, endometrial cancer, and spontaneous abortions are all caused by the metalloestrogen cadmium (Cd); lead (Pb) levels over a certain threshold can cause spontaneous abortion and have a teratogenic impact; toxic amounts of mercury (Hg) have an influence on the menstrual cycle, which can lead to infertility. Impact of environmental exposure to heavy metals on female fertility is therefore a well-known fact. Thus, the underlying mechanisms must be explained and periodically updated, given the growing evidence on the influence of increasing environmental heavy metal load on female fertility. The purpose of this review is to give a concise overview of how heavy metal affects female reproductive health.
... Cadmium also induces caspase-independent PCD via endonucleases or calpain activity [136,137]. Arsenic induces apoptosis by promoting ROS generation, caspase-3 activation, calcium balance disorders or the depletion of intracellular glutathione, supported by the involvement of mitochondria with Cyt c release [138,139]. The transforming growth factor beta pathway is involved in the apoptosis process of liver cells under Cr stress [140]. ...
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Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.
... There is increasing evidence that the inactivation of the Ca 2+ pump is accompanied by disturbed ER function (Doutheil et al., 2000). Our result is supported by previously reported that decreased Ca 2+ -ATPase activity results in a decrease in the uptake of Ca 2+ to the ER and pump-out toward extracellular, resulting an increase in the accumulation of Ca 2+ (Florea et al., 2005), thus causing dysfunction of the ER and triggers the UPR (Giorgi et al., 2015). As three signaling branches of UPR, PERK, IRE1 and ATF6 are evolved critically to orchestrate ERS. ...
Arsenic (As) occurs naturally and concentrations in water bodies can reach high levels, leading to accumulation in vital organs like the spleen. Being an important organ in immune response and blood development processes, toxic effects of As on the spleen could compromise immunity and cause associated disorders in affected individuals. Splenic detoxification is key to improving the chances of survival but relatively little is known about the mechanisms involved. Essential trace elements like zinc have shown immune-modulatory effects humans and livestock. This study aimed to investigate the mechanisms involved in As-induced splenic toxicity in the common carp (Cyprinus carpio), and the protective effects of zinc (Zn). Our findings suggest that environmental exposure to As caused severe histological injuries and Ca²⁺ accumulation in the spleen of common carp. Additionally, transcriptional and translational profiles of endoplasmic reticulum stress, apoptosis and autophagy-related genes of the spleen showed upward trends under As toxicity. Treatment with Zn appears to offer protection against As-induced splenic injury in common carp and the pathologic changes above were alleviated. Our results provide additional insight into the mechanism of As toxicity in common carp while elucidating the role of Zn, a natural immune-modulator, as a potential antidote against As poisoning.
... Florea et al. assessed inorganic iAs III and iAs V , as well as MMA V , DMA V , and TMAO V for early disturbances in calcium homeostasis in HeLa S3 cells within the first few seconds after application [150]. A drop in the fluorescence signal of the dye was recorded by confocal laser scanning microscopy. ...
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This title is not available to purchase from Royal Society of Chemistry. Please visit for title information. This volume, closely related to MILS-6, deals mainly with metal(loid)-alkyl derivatives but also with the rarer aryl compounds. Most of these (commonly toxic) compounds are formed in the environment by microorganisms, but some anthropogenic input occurs as well. MILS-7, providing a most up-to-date view, is of special relevance for researchers in analytical and bioinorganic chemistry, enzymology, environmental chemistry, physiology, toxicology, and related medical fields.
... As Ca 2+ is one of the crucial secondary messengers associated with phosphoinositol system, [Ca 2+ ] disturbance may affect both the molecular functions and the viability of the cell. Elevated intracellular [Ca 2+ ] modulates apoptosis and protein degradation, compromises mitochondrial function, and interferes with cell membrane integrity (Florea et al., 2005;Mutryn et al., 2015;Petracci et al., 2015). This could explain the extensive muscle protein degradation observed in WS samples collected from Cobb 500 broilers in the previous report of Vignale et al. (2017). ...
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Development of the white striping (WS) abnormality adversely impacts overall quality of broiler breast meat. Its etiology remains unclear. This study aimed at exploring transcriptional profiles of broiler skeletal muscles exhibiting different WS severity to elucidate molecular mechanisms underlying the development and progression of WS. Total RNA was isolated from pectoralis major of male 7-week-old Ross 308 broilers. The samples were classified as mild (n = 6), moderate (n = 6), or severe (n = 4), based on number and thickness of the white striations on the meat surface. The transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique. Gene expression patterns of each WS severity level were compared against each other; hence, there were three comparisons: moderate vs. mild (C1), severe vs. moderate (C2), and severe vs. mild (C3). Differentially expressed genes (DEGs) were identified using the combined criteria of false discovery rate ≤ 0.05 and absolute fold change ≥1.2. Differential expression of 91, 136, and 294 transcripts were identified in C1, C2, and C3, respectively. There were no DEGs in common among the three comparisons. Based on pathway analysis, the enriched pathways of C1 were related with impaired homeostasis of macronutrients and small biochemical molecules with disrupted Ca2+-related pathways. Decreased abundance of the period circadian regulator suggested the shifted circadian phase when moderate WS developed. The enriched pathways uniquely obtained in C2 were RNA degradation, Ras signaling, cellular senescence, axon guidance, and salivary secretion. The DEGs identified in those pathways might play crucial roles in regulating cellular ion balances and cell-cycle arrest. In C3, the pathways responsible for phosphatidylinositol 3-kinase-Akt signaling, p53 activation, apoptosis, and hypoxia-induced processes were modified. Additionally, pathways associated with a variety of diseases with the DEGs involved in regulation of [Ca2+], collagen formation, microtubule-based motor, and immune response were identified. Eight pathways were common to all three comparisons (i.e., calcium signaling, Ras-associated protein 1 signaling, ubiquitin-mediated proteolysis, vascular smooth muscle contraction, oxytocin signaling, and pathway in cancer). The current findings support the role of intracellular ion imbalance, particularly Ca2+, oxidative stress, and impaired programmed cell death on WS progression.
Over the past few decades, clinical trials conducted worldwide have demonstrated the efficacy of arsenic trioxide (ATO) in the treatment of relapsed acute promyelocytic leukemia (APL). Currently, ATO has become the frontline treatments for patients with APL. However, its therapeutic applicability is severely constrained by ATO-induced cardiac side effects. Any cardioprotective agents that can ameliorate the cardiac side effects and allow exploiting the full therapeutic potential of ATO, undoubtedly gain significant attention. The knowledge and use of natural products for evidence-based therapy have grown rapidly in recent years. Here we discussed the potential mechanism of ATO-induced cardiac side effects and reviewed the studies on cardiac side effects as well as the research history of ATO in the treatment of APL. Then, We summarized the protective effects and underlying mechanisms of natural products in the treatment of ATO-induced cardiac side effects. Based on the efficacy and safety of the natural product, it has a promising future in the development of cardioprotective agents against ATO-induced cardiac side effects.
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It can be challenging to deliver drugs to cancer cells in a targeted manner at an effective dose. Polymeric nanoparticles (NPs) are promising drug delivery systems that can be targeted to cancer cells using redox responsive elements. More specifically, intracellular and extracellular levels of the antioxidant glutathione (GSH) are elevated in cancer cells and therefore the use of NPs with a cleavable GSH-responsive element allowing these NPs to target cancer cells and trigger the release of their cargo (e.g. anticancer drugs). The aim of this study was to assess the hepatotoxicity of polymeric NP delivery systems with and without a redox sensitive element. Copolymer poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) NPs with (RR-NPs) and without (nRR-NPs) a redox responsive dithiylethanoate ester linker were synthesised and their toxicity assessed in vitro. As the liver is a primary site of NP accumulation, the C3A hepatocyte cell line was used to assess NP toxicity in vitro via investigation of cytotoxicity, cytokine production, genotoxicity, intracellular reactive oxygen species (ROS) production, intracellular calcium concentration, and hepatocyte function (albumin and urea production). The cellular uptake of NPs was also assessed as this may influence the cellular dose and, therefore, the cellular response. Both NPs had no detrimental impact on cell viability. However, both NPs stimulated an increase in cytokine (IL-1ra) and ROS production and decreased hepatocyte function, with the greatest effect observed for nRR-NPs. Only nRR-NPs caused DNA damage. Cells internalised both NPs and caused a (sub-lethal) increase in intracellular calcium levels. Therefore, whilst the NPs did not have a negative impact on cell viability, the NPs were able to elicit sub-lethal toxicity. By using a battery of tests we were able to demonstrate that RR-NPs may be less toxic than nRR-NPs. Our findings can therefore feed into the development of safer and more effective nanomedicines and into the design of testing strategies to assess polymeric NP safety based on knowledge of their mechanism of toxicity.
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Epidemiological studies demonstrate that arsenic exposure is associated with cognitive dysfunction. Experimental arsenic exposure models showed learning and memory deficits and molecular changes resembling the functional and pathologic neurodegeneration features. The present work focuses on hippocampal pathological changes in Wistar rats induced by continuous arsenic exposure from in utero up to 12 months of age, evaluated by magnetic resonance imaging along with immunohistochemistry. Diffusion-weighted images revealed age-related lower fractional anisotropy and higher radial-axial and mean diffusivity at 6 and 12 months, indicating that arsenic exposure leads to hippocampal demyelination. These structural alterations were paralleled by immunohistochemical changes that showed a significant loss of myelin basic protein in CA1 and CA3 regions accompanied by increased glial fibrillary acidic protein expression at all time-points studied. Concomitantly, arsenic exposure induced an altered morphology of astrocytes at all studied ages, whereas increased synaptogenesis was only observed at two months of age. These results suggest that environmental arsenic exposure is linked to impaired hippocampal connectivity and perhaps early glial senescence, which together might resemble a premature aging phenomenon leading to cognitive deficits.
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A new group of arsenic(III) complexes with bidentate S,S-donor ligands, 1,2-benzenedithiol (Ph(SH)2) and toluene-3,4-dithiol (MePh(SH)2), were synthesized. The use of arsenic(III) iodide and bromide promoted the formation of neutral complexes (1–4) with the general formula AsX(LS2) (X = I or Br, L = MePh or Ph). The crystal structures of these compounds were determined using single-crystal X-ray diffraction (scXRD). Unlike other arsenic(III) complexes, AsBr(PhS2) complex (2) was found to crystallize with a rare 13 molecules in the asymmetric unit. The compounds were also characterized by conventional physico-chemical techniques (Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), elemental analysis (EA) and electrospray ionization-mass spectrometry (ESI-MS)). The results from structural and spectroscopic studies were supported by DFT calculations using the B3LYP/LANL2DZ and (or) 6-31+G(d,p) approaches. The cytotoxicity of these complexes was estimated for human acute promyelocytic leukemia cell line (NB4). They exhibited remarkable cytotoxicities after 48 h of treatment with IC50 equal to about 10 µM and 40 µM for complexes with 1,2-benzenedithiolato and toluene-3,4-dithiolato ligand, respectively. Their toxicity was lower than that of commonly used chemotherapeutic As2O3 (IC50 = 1.4 µM).
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The modification of metals and metalloids by formation of volatile metal hydrides and alkylated species (volatile and non-volatile) has a major impact on the processing of these elements in the environment. In general, the formation of such species increases the mobility of the respective element and can result in its accumulation in biological systems (Craig and Glockling 1988).
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Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.
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Recent advances in our knowledge of arsenic carcinogenesis include the development of rat or mouse models for all human organs in which inorganic arsenic is known to cause cancer-skin, lung, urinary bladder, liver, and kidney. Tumors can be produced from either promotion of carcinogenesis protocols (mouse skin and lungs, rat bladder, kidney, liver, and thyroid) or from complete carcinogenesis protocols (rat bladder and mouse lung). Experiments with p53(+/-) and K6/ODC transgenic mice administered dimethylarsinic acid or arsenite have shown some degree of carcinogenic, cocarcinogenic, or promotional activity in skin or bladder. At present, with the possible exception of skin, the arsenic carcinogenesis models in wild-type animals are more highly developed than in transgenic mice. Recent advances in arsenic metabolism have suggested that methylation of inorganic arsenic may be a toxification, rather than a detoxification, pathway and that trivalent methylated arsenic metabolites, particularly monomethylarsonous acid and dimethylarsinous acid, have a great deal of biological activity. Accumulating evidence indicates that these trivalent, methylated, and relatively less ionizable arsenic metabolites may be unusually capable of interacting with cellular targets such as proteins and even DNA. In risk assessment of environmental arsenic, it is important to know and to utilize both the mode of carcinogenic action and the shape of the dose-response curve at low environmental arsenic concentrations. Although much progress has been recently made in the area of arsenic's possible mode(s) of carcinogenic action, a scientific concensus has not yet been reached. In this review, nine different possible modes of action of arsenic carcinogenesis are presented and discussed-induced chromosomal abnormalities, oxidative stress, altered DNA repair, altered DNA methylation patterns, altered growth factors, enhanced cell proliferation, promotion/progression, gene amplification, and suppression of p53.
Even though a well-known human carcinogen the underlying mechanisms of arsenic carcinogenicity are still not fully understood. For arsenite, proposed mechanisms are the interference with DNA repair processes and an increase in reactive oxygen species. Even less is known about the genotoxic potentials of its methylated metabolites monomethylarsonous [MMA(III)] and dimethylarsinous [DMA(III)] acid, monomethylarsonic [MMA(V)] and dimethylarsinic [DMA(V)] acid. Within the present study we compared the induction of oxidative DNA damage by arsenite and its methylated metabolites in cultured human cells and in isolated PM2 DNA, by frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg). Only DMA(III) (>10 μM) generated DNA strand breaks in isolated PM2 DNA. In HeLa S3 cells, short-term incubations (0.5-3 h) with doses as low as 10 nM arsenite induced high frequencies of Fpg-sensitive sites, whereas the induction of oxidative DNA damage after 18 h incubation was rather low. With respect to the methylated metabolites, both trivalent and pentavalent metabolites showed a pronounced induction of Fpg-sensitive sites in the nanomolar or micromolar concentration range, respectively, which was present after both short-term and long-term incubations. Furthermore MMA(III) and DMA(V) generated DNA strand breaks in a concentration-dependent manner. Taken together our results show that very low physiologically relevant doses of arsenite and the methylated metabolites induce high levels of oxidative DNA damage in cultured human cells. Thus, biomethylation of inorganic arsenic may be involved in inorganic arsenic-induced genotoxicity/carcinogenicity.