Arch Toxicol

DOI 10.1007/s00204-015-1579-5

REVIEW ARTICLE

Redox‑ and non‑redox‑metal‑induced formation of free radicals

and their role in human disease
Marian Valko1,4 · Klaudia Jomova2 · Christopher J. Rhodes3 · Kamil Kuča4,5 ·
Kamil Musílek5,6

Received: 2 August 2015 / Accepted: 11 August 2015

© Springer-Verlag Berlin Heidelberg 2015

Abstract Transition metal ions are key elements of vari- arsenic (As) and cadmium (Cd), the primary route for their
ous biological processes ranging from oxygen formation to toxicity is depletion of glutathione and bonding to sulfhydryl
hypoxia sensing, and therefore, their homeostasis is main- groups of proteins. While arsenic is known to bind directly
tained within strict limits through tightly regulated mecha- to critical thiols, other mechanisms, involving formation of
nisms of uptake, storage and secretion. The breakdown of hydrogen peroxide under physiological conditions, have been
metal ion homeostasis can lead to an uncontrolled formation proposed. Redox-inert zinc (Zn) is the most abundant metal
of reactive oxygen species, ROS (via the Fenton reaction, in the brain and an essential component of numerous proteins
which produces hydroxyl radicals), and reactive nitrogen spe- involved in biological defense mechanisms against oxidative
cies, RNS, which may cause oxidative damage to biological stress. The depletion of zinc may enhance DNA damage by
macromolecules such as DNA, proteins and lipids. An imbal- impairing DNA repair mechanisms. Intoxication of an organ-
ance between the formation of free radicals and their elimina- ism by arsenic and cadmium may lead to metabolic distur-
tion by antioxidant defense systems is termed oxidative stress. bances of redox-active copper and iron, with the occurrence
Most vulnerable to free radical attack is the cell membrane of oxidative stress induced by the enhanced formation of ROS/
which may undergo enhanced lipid peroxidation, finally pro- RNS. Oxidative stress occurs when excessive formation of
ducing mutagenic and carcinogenic malondialdehyde and ROS overwhelms the antioxidant defense system, as is main-
4-hydroxynonenal and other exocyclic DNA adducts. While tained by antioxidants such as ascorbic acid, alpha-tocopherol,
redox-active iron (Fe) and copper (Cu) undergo redox-cycling glutathione (GSH), carotenoids, flavonoids and antioxidant
reactions, for a second group of redox-inactive metals such as enzymes which include SOD, catalase and glutathione per-
oxidase. This review summarizes current views regarding the
role of redox-active/inactive metal-induced formation of ROS,
* Marian Valko and modifications to biomolecules in human disease such as
[email protected] cancer, cardiovascular disease, metabolic disease, Alzheimer’s
1 disease, Parkinson’s disease, renal disease, blood disorders
Faculty of Chemical and Food Technology, Slovak University
of Technology, 812 37 Bratislava, Slovakia and other disease. The involvement of metals in DNA repair
2 mechanisms, tumor suppressor functions and interference
Department of Chemistry, Faculty of Natural Sciences,
Constantine the Philosopher University, 949 74 Nitra, with signal transduction pathways are also discussed.
Slovakia
3
Fresh Lands, P.O. Box 2074, Reading, Berkshire RG4 5ZQ, Keywords Metals · Toxicity · Oxidative stress · Human
UK disease · Reactive oxygen species
4
The Center for Basic and Applied Research, University
Hradec Kralove, Hradec Kralove, Czech Republic
5
Biomedical Research Center, University Hospital Hradec Introduction
Kralove, Hradec Kralove, Czech Republic
6
Department of Chemistry, Faculty of Science, University In comparison with the majority of organic molecules, met-
Hradec Kralove, Hradec Kralove, Czech Republic als or metal ions are rather small and simple species, and

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 Arch Toxicol

consequently, an understanding of their interactions with interact with physiologically relevant metabolites derived
large biological molecules might appear deceptively straight- from oxygen (molecular oxygen, superoxide anion radical,
forward. However, the currently emerging picture is that the hydroxyl radical, etc.), low molecular weight antioxidants
interactions of such species with bio-macromolecules and in (ascorbic acid, tocopherols, etc.) and other components
signal transduction pathways are actually rather complex in (Stohs and Bagchi 1995).
nature. Some metal ions may be transformed by processes In addition to kinetic criteria, the outcome of chemical
which include reduction/oxidation reactions, and lead to reactions between various substances is governed by ther-
the formation of reactive oxygen species (ROS), or reactive modynamic principles. The most important parameter to
nitrogen species (RNS) (Halliwell and Gutteridge 1990). As characterize the course of a chemical reaction is the half-
already noted, the cumulative formation of ROS and RNS is cell reduction potential. The overall redox potential for two
termed oxidative stress which may induce a cellular redox redox pairs is given by the electromotive force (ΔE) and
imbalance, and this may be linked with various disease states is calculated according to the equation (Jomova and Valko
of an organism (Valko et al. 2007). 2011a, b)
Metal toxicity is a rather complex phenomenon to evalu-
ate, as is especially true for mechanisms of metal-induced
�E = E2 (electron-acceptor) − E1 (electron-donor) (1)
carcinogenesis (Beyersmann and Hartwig 2008). Metal where E1 and E2 are the reduction potentials for both half-
carcinogenicity occurs via complex mechanisms which cell reactions.
involve oxidative damage, DNA repair, maintenance of Disruption of the homeostasis of redox-active metals
redox homeostasis and disturbance of signal transduction such as iron, copper, cobalt, chromium, nickel and other
pathways. metals renders them available as catalysts to form excess
An intriguing feature of metals in biological systems is of ROS and RNS. In addition, redox-active metals can
that some metals essential for health (such as copper and bind to phospholipids, so changing the integrity of the lipid
iron) may become toxic under certain circumstances. These bilayer and enhancing its susceptibility to lipid peroxida-
metal ions may compete with essential ions for high-affin- tion. Conversely, redox-inactive metals (cadmium, arsenic,
ity metal-binding sites, so causing structural modifications lead and other metals) mediate their oxidative stress effects
and disturbances in metal homeostasis (Kozlowski et al. via bonding to sulfhydryl groups of proteins, which results
2014; Valko et al. 2005). in depletion of glutathione (Koedrith and Seo 2011). Zinc
In contaminated areas, humans may be exposed to has a special role among metals in living organisms, since
redox-inert toxic elements such as arsenic and cadmium, it is a non-redox metal and can impact both on the immune
which are known to be toxic at low concentrations (Hartwig system and on various important neurological processes.
2013). For this group of metals, the primary mechanism for To avoid the excessive formation of ROS/RNS by redox
their toxicity and carcinogenicity is depletion of the power- metal imbalance, and consequent oxidative stress to an
ful cellular antioxidant glutathione, by bonding to protein organism, redox homeostasis is tightly regulated (Forman
sulfhydryl groups and other modes of action. et al. 2010). Indeed, many disease states are a result of dis-
The present article surveys all the aforementioned rupted homeostasis of metals (most frequently iron and
aspects of metals acting in living systems, including provid- copper) which catalyze and participate in the formation of
ing an overview of the role of redox-active copper and iron damaging free radicals.
and non-redox toxic metals arsenic and cadmium. Redox-
inert zinc is the most abundant trace metal in the brain and Radicals derived from oxygen
has various functions in health and in disease. Accordingly,
special attention is paid to the anti-inflammatory role of Mitochondria are known to play a key role in maintaining
zinc and its potential to suppress oxidative stress, while the the bioenergetic status of multiple basic cellular processes.
mechanism by which zinc may act against oxidative stress The formation of superoxide anion radical by mitochon-
and chronic inflammation is discussed. dria is dependent on the reaction between potential elec-
tron donors and molecular oxygen. The formation of the
superoxide radical anion within the mitochondrial matrix
Biochemistry of metal‑induced oxidative stress depends on the proton-motive force (Δp), the NADH/
NAD+ and CoQH2/CoQ (coenzyme Q) ratios and the local
The toxicity of redox-active transition metal ions in living O2 concentration (Murphy 2009)
systems is a very complex process. Transition metal ions
O2 + e− → O·− (2)
are integral parts of protein active sites and may undergo 2

redox-cycling reactions. Metal ions are present in bio- The superoxide radical anion further reduces Fe(III) or
logical systems in a variety of oxidation states and may Cu(II) ions (or another metal ions) according to reaction

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Arch Toxicol

Fe(III)/Cu(II) + O·−
2 → Fe(II)/Cu(I) + O2 (3) Fe(III)/Cu(II) + HO·2 + Fe(II)/Cu(I) + O2 + H+ (12)

Metal ions in reduced oxidation states are able to Redox metals, mainly iron and copper, can mediate lipid
enter catalytic decomposition of hydrogen peroxide peroxidation by the reductive cleavage of hydroperoxides
(ΔEΘ = +0.307 V) (ROOH) derived from membrane phospholipids
Fe(II)/Cu(I) + H2 O2 → Fe(III)/Cu(II) + HO· ROOH + Fe(II)/Cu(I) → RO· + OH− + Fe(III)/Cu(II)
+ OH− (Fenton reaction) (4) (13)
The superoxide radical anion is not only produced as a ROOH + Fe(III)/Cu(II) → RO·2 + H+ + Fe(II)/Cu(I)
by-product of mitochondrial respiration, but is formed in (14)
large quantities in phagocytes by NADPH oxidase, for the resulting in the formation of alkoxyl (RO·) and peroxyl
purpose of killing invading pathogens. Hence, the immune (RO·2) radicals. The mechanism of reactions (13) and (14)
system defends its host against microorganisms by produc- involves formation of Fe(II)–Fe(III) or Fe(II)–O2–Fe(III)
ing superoxide. Superoxide is also produced by a number complexes. Fe(II) oxidation is stimulated at increased pH
of other enzymes, for example, xanthine oxidase (Valko and, by the addition of Fe(III), reaching a maximum at a
et al. 2004). Fe(II)/Fe(III) ratio approaching 1:1 (Tadolini and Hakim
Hydrogen peroxide is produced in specific cellular 1996). At low concentrations of hydrogen peroxide, Fe(II)
compartments and acts as a second messenger molecule induces decomposition of lipid peroxides forming alkoxyl
in regulating various important biological processes. Met- and peroxyl radicals.
als in low oxidation states can catalyze decomposition of Cu(I) ions are effective catalysts for the Fenton reaction,
hydrogen peroxide via the Fenton reaction (Eq. 4) which while both Cu(II) and Cu(I) ions are effective species for
yields hydroxyl radicals (Lloyd et al. 1997). Hydroxyl radi- decomposing organic hydroperoxides to form peroxyl and
cals can subsequently react at diffusion-limited rates with alkoxyl radicals according to reactions (15–17) (Li et al.
various biomolecules, including lipids, proteins and DNA 2000a, b).
and cause serious damage to them. Following reaction (2),
Cu(I) + H2 O2 → [Cu(I)−H2 O2 ] → Cu(II) + HO· + OH−
the resulting superoxide radical anion can reduce hydrogen (15)
peroxide via the so-called Haber–Weiss reaction which can
be described according to reaction (5) (Liochev and Fri-
Cu(I) + ROOH → RO· + OH· + Cu(II) (16)
dovich 2002):
O·− ·
2 + H2 O2 → O2 + HO + OH
−
(5) Cu(II) + ROOH → RO·2 + H+ + Cu(I) (17)
SOD enzymes can scavenge superoxide radical anions by Based on the above discussion and various in vitro stud-
either the removal or addition of an electron, so resulting in ies, it has been shown that the mechanism of lipid peroxi-
the less damaging species, molecular dioxygen or hydrogen dation is a radical-dependent process with physiological
peroxide relevance. Lipid peroxidation results in the formation of
a wide variety of oxidation products, the most common
M(II)-SOD + O·−
2 → M(I)-SOD + O2 (6) of which are aldehydes. Among the most abundantly pro-
duced aldehydes are malondialdehyde (MDA), propanal,
M(I)-SOD + O·− +
2 + 2H → M(II)-SOD + H2 O2 (7) hexanal and 4-hydroxynonenal (4-HNE) (Marnett 2000).
where M is copper, manganese or iron. We may note that MDA appears to be the most mutagenic
The biologically acceptable level of hydrogen peroxide product of lipid peroxidation, whereas 4-HNE is the most
is controlled by the enzymes catalase and glutathione per- toxic (Esterbauer et al. 1991).
oxidase, both of which convert it to molecular dioxygen. The initiation reaction is mediated by formation of
hydroxyl radicals via the Fenton reaction, or by ferryl inter-
2H2 O2 → 2H2 O + O2 (8) mediates which are similarly powerful oxidants. Both spe-
The following reactions involving hydrogen peroxide cies are able to abstract hydrogen atoms from unsaturated
may also take place: fatty acids forming alkyl radicals (R·):
·
OH + H2 O2 → H2 O + HO·2 (9) HO· + RH → H2 O + R· (18)
· Studies of the lipid peroxidation process in brain tissue
OH + Fe(II) → Fe(III) + OH− (10) revealed that hydrogen abstraction occurs at the allylic
carbons in the ninth and tenth positions of the oleic acid
HO·2 + HO·2 → H2 O2 + O2 (11) chain (Hall et al. 2015). Secondary reactions then proceed

13
 Arch Toxicol

by hydrogen abstraction by RO· and ROO· at the tertiary the toxicity of nitric oxide is a consequence of its reactivity
carbon atoms: with superoxide radical anion to form peroxynitrite.
RO· + RH → ROH + R· (19)
Iron
R· + O2 → RO·2 (20)
The electronic structure of an iron atom predetermines its
RO·2 + RH → ROOH + R· (21) ability to carry out one-electron reactions, such that it is
The radicals R· and ROO· are key intermediates in the a key element in the formation and metabolism of ROS. In
ROS-mediated mechanism of lipid peroxidation. Thus, iron biological systems, iron is most frequently present in fer-
and copper ions promote peroxidation of lipids in phospho- rous [Fe(II)] and ferric [Fe(III)] oxidation states and to a
lipid liposomes by homolytic cleavage of hydrogen perox- lesser extent in the highly oxidizing ferryl [Fe(IV)] form.
ide via formation of hydroxyl radicals and by scission of As discussed above, iron is a redox-active metal which
ROOH to form alkoxyl and peroxyl radicals, in addition to can be involved in the various redox reactions that catalyze
the effect of altering the surface properties of liposomes. formation of ROS (Jungwirth et al. 2011). The standard
Nickel and cobalt ions have been shown to catalyze redox potentials of Fe(III) complex + e− → Fe(II) complex
metal-induced peroxidation; however, the mechanism is may vary in the range of −1 to +1 V depending on the struc-
not fully understood. Nickel exists in a variety of oxida- ture, strength of coordination and nature of ligands bound to
tion states ranging from −1 to +4 of which +2 is the most the iron atom. Thus, variation of the ligand coordinated to
prevalent form (Valko et al. 2005). Nickel(II) ions inter- iron allows a fine tuning of the various electron transfer reac-
fere with DNA repair and promote peroxidation of lipids, tions that are possible in a given system (Valko et al. 2005).
in addition to the formation of protein carbonyl moieties. Of the order of 65 % of iron in living organisms is bound
Ni(II) does not catalyze the lipid peroxidation process to the carrier protein hemoglobin, about 25 % is bound to the
when hydroperoxides are present alone, but it does so when iron-storage proteins ferritin and hemosiderin, and about 10 %
included in the Fenton system (Fe(II)/hydrogen peroxide) is an integral part of oxygen carrier proteins, myoglobin and
(Salnikow and Kasprzak 2005). Cobalt(II) ions are directly cytochromes (Winter et al. 2014). Only a very small amount
not involved in the peroxidation of lipids and exhibit only a of iron is present in the form of a redox-active iron pool, some-
negligible effect when incorporated into the Fenton system. times termed as the labile iron pool (LIP), which represents
iron bound to low-affinity intracellular low molecular weight
Radicals derived from nitrogen ligands. It has been estimated that less than 5 % of the total cell
iron is present in the redox-active form (50–100 μM) (Kakhlon
Nitric oxide (NO·) contains one unpaired electron on the and Cabantchik 2002; Wang and Pantopoulos 2011). The pres-
antibonding 2πy orbital and is, therefore, a radical. Nitric ence of a free redox-active iron pool in a cell may serve as a
oxide is formed in biological tissues by specific nitric oxide catalyst for the formation of free radicals via the Fenton reac-
synthases (NOSs). NOSs are a family of enzymes catalyz- tion (4) which may cause damage to cellular components.
ing formation of NO· from L-arginine. Nitric oxide is an Since iron availability is essential for the synthesis of many
important molecule playing significant roles in a variety proteins, including oxygen carriers, electron transport proteins
of physiological processes, such as blood pressure regu- and cytochromes, the iron balance has to be tightly regulated.
lation, neurotransmission, defense mechanisms, smooth In summary, iron is released into the plasma from intestinal
muscle relaxation and immune regulation. In an aqueous enterocytes or macrophages, where it is further scavenged by
environment, nitric oxide has a half-life of only several sec- transferrin which maintains Fe3+ in a redox-conserved state.
onds (Matsubara et al. 2015). In analogy with the defini- Scavenged iron is then delivered to and absorbed by tissues.
tion of oxidative stress, overproduction of radicals derived Received iron is transported into mitochondria for the synthe-
from nitrogen (RNS) is termed nitrosative stress. During sis of iron–sulfur or hem cores which represent the active sites
the inflammatory processes, the immune system produces of various metalloproteins. Excess of iron is stored and detoxi-
both the superoxide anion radical and nitric oxide. Under fied in cytosolic ferritin.
these conditions, these two radical species may react
together to from significant amounts of peroxynitrite anion Iron homeostasis
ONOO− (NO· + O·− −
2 → ONOO ), which is a very potent
oxidant capable of participating in several oxidative reac- Homeostasis of cellular iron is accomplished by the con-
tions causing DNA fragmentation and oxidation of lipids certed expression of various proteins that are involved in
(Prolo et al. 2015). The reaction of peroxynitrite formation iron uptake, export, storage and utilization (Santamaria
has one of the highest rate constants (~1010 M−1 s−1). Thus, et al. 2011).

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Arch Toxicol

Dietary free iron, on reduction from the ferric to the state) is critical for catalysis through ubiquitination and
ferrous state on the luminal surface of the proximal small degradation by the proteasome. The iron homeostasis is
intestine, is transported into the cell through an energeti- also achieved by a transmembrane protein ferroportin
cally active transport mechanism involving the divalent arising on the cell surfaces, which transport iron from the
metal transporter-1 (DMT-1). DMT-1 is a protein expressed intracellular to the extracellular space.
on the surface of the apical membrane of intestinal entero- The labile iron pool (LIP) or cytosolic free iron influ-
cytes in the initial part of the duodenum (Bou-Abdallah ences the RNA-binding capacity of IRP1 or induces IRP2
2010). DMT-1 is structurally not only specific to iron, but degradation (Bregen and Eckl 2015). The IRPs RNA-
it can also transport other metal ions such as zinc, copper, binding activity is also regulated by other exogenous and
cobalt, manganese, cadmium or lead. Following the uptake endogenous factors, such as oxidative stress, nitric oxide
of iron into the circulation, there are very limited pos- signaling and other factors. The cellular labile iron content
sibilities for its elimination from human body (except by can be quantified by a fluorimetric assay using metal-sensi-
menstruation). tive probes, such as calcein (CA) and the strong membrane-
The mechanism of iron transport, storage and use permeant iron chelator, for example, salicylaldehyde isoni-
involves the iron-responsive/regulatory elements/proteins cotinoyl hydrazine (Santamaria et al. 2011).
(IRE, IRP) (Anderson et al. 2012). IRE/IRP is a posttran-
scriptional regulatory circuit that controls iron homeosta- Iron‑induced oxidative stress
sis in types of cells, but they also maintain the overall bal-
ance of iron in an organism. Iron metabolism is regulated The alterations and damage that occurs at the cellular level,
in higher eukaryotes through interactions between two iron as a consequence of iron overload, seem to be principally
regulatory proteins, IRP-1 and IRP-2, with iron-responsive related to free radical-mediated damage of cell compo-
elements (IREs). IRPs contain four domains which are nents (Kell 2010; Fraga and Oteiza 2002). In fact, free
members of the aconitase family. The protein Fe–S clusters radical-induced damage to all key biological targets includ-
are structurally well separated. ing DNA, proteins and membranes has been accepted as a
IRP1 is a dual-functioning protein with either aconitase key mechanism and common denominator of many chronic
activity when cellular iron is abundant or RNA-binding diseases.
activity when cellular iron is in deficiency. Conversely, Many in vitro experiments confirmed the formation of
IRP2 functions exclusively as an RNA-binding protein, and hydroxyl radicals (·OH) which can be described by reac-
thus, it lacks aconitase activity. In the case of a low level tions (3) and (4). Moreover, the hydroxyl radical can be
of iron, both IRP1 and IRP2 function as RNA-binding generated under in vitro conditions when ferric ions are
proteins and bind to stem–loop structures (Anderson et al. reduced in the presence of reducing agents such as ascorbic
2012). These structures are termed iron-responsive ele- acid (Du et al. 2012; Valko et al. 2005)
ments (IREs) and are located in the 5′ or 3′ untranslated
Fe(III) + Asc− → Fe(II) + Asc·− (22)
regions (UTRs) of specific mRNAs that encode proteins
which are important in the metabolism of iron. The generation of hydroxyl radicals may also be cata-
The binding of IRP1/IRP2 to mRNA motifs, termed lyzed by ferric ions in the absence of a redox agent. This
iron-responsive elements (IREs), regulates the expression process can be considered as a special example of the Fen-
of proteins important for iron acquisition, including trans- ton reaction in which one electron from the hydroxide ion
ferrin receptor 1 (TfR-1) and divalent metal transporter 1 (originating from the self-ionization reaction of water) is
(DMT-1) and storage protein (ferritin) (Santamaria et al. transferred to the ferric ion [reducing it to Fe(II)] and the
2011). hydroxyl radical is formed (Eq. 23).
Under increased iron levels, IRP1 binds a 4Fe–4S
Fe(III) + OH− → Fe(II) + HO· (23)
cluster to become a functioning cytosolic aconitase
enzyme (Dupuy et al. 2006). Low concentrations of iron In addition, redox-active iron mediates the peroxidation of
or heme, formation of ROS/RNS, phosphorylation of lipids by cleavage of hydroperoxides and membrane phos-
the protein and other factors, all help the Fe–S cluster to pholipids (Eqs. 24, 25).
disassemble. While ROS/RNS-induced oxidative stress
ROOH + Fe(II) → RO· + OH− + Fe(III) (24)
is considered to provide a direct attack on the iron–sul-
fur cluster, phosphorylation is considered as an indirect
ROOH + Fe(III) → RO·2 + H+ + Fe(II) (25)
event that affects the IRP1 switch. Indeed, different types
of cluster perturbations are probable. IRP2 is stabilized As formed by the Fenton reaction, hydroxyl radicals
in response to hypoxia; however, the presence of Fe2+ can react at diffusion-limited rates with various biomol-
ions, oxygen and ascorbate (to maintain iron in a reduced ecules. As noted, the reactivity of iron in redox reactions is

13
 Arch Toxicol

strongly dependent on the ligand environment (Lloyd et al.

1997). [Fe(II) 3Fe(III)−4S]3+ → [3Fe(III)−4S]+ + Fe(II) (27)
The maximum coordination number of iron is six, and The released ferrous ions can catalyze the decomposi-
so hexadentate ligand–chelators can provide iron atoms tion of hydrogen peroxide to form hydroxyl radicals, via
with a coordination sphere around the metal center which the Fenton reaction. The Fenton reaction has in vivo rel-
is completely saturated. Consequently, a chelator mol- evance predominantly under state of an organisms over-
ecule that binds to all six coordination sites of the iron ion loaded by iron, such as conditions of hemochromatosis,
completely deactivates the catalytic activity of “free iron,” b-thalassemia and hemodialysis.
for example, desferrioxamine (Lovejoy and Richardson As already discussed, if iron is tightly coordinated, it
2003). can catalyze the formation of hydroxyl radicals. Cytoplasm
The coordination of biomolecules to an iron atom contains low molecular weight chelators that can bind iron
involves the d-orbitals of the latter. Oxygen is similarly and thus form a labile iron pool which consists of both fer-
able to bind to iron via electrons occupying antibonding rous and ferric ions. LIP is known to cross biological mem-
π* orbitals. Iron can simultaneously bind both oxygen branes. A controversy exists regarding an appropriate meth-
and biomolecules, to form a bridge between the two bond odology for the quantitative assessment of LIP, given the
types of substrate. The “flexibility” of iron to bind various generally accepted large range of LIP which is somewhere
ligands is reflected by the variation of its oxidation state but in between 0.2 and 230 μM for the same type of cell (Dar-
also by changes in its electronic spin properties and rela- bari et al. 2003).
tive redox potential. Coordinating ligands to iron allows the
physical properties of iron to be “fine tuned” in response to Iron, free radicals and human disease
the particular needs of the biomolecule in which it operates
(Toyokuni 1996). A number of studies and trials reported a positive correla-
As already discussed, superoxide radical anion is formed tion between the elevated body iron stores and the risk of
in living systems by several routes. Of itself, superox- the development of various ailments such as cardiovascular,
ide possesses only a moderate reactivity and cannot cause cancers, neurological and other diseases (Jomova and Valko
damage to DNA; however, it has been shown that under 2011a, b). As discussed above, it is the natural tendency for
in vitro conditions superoxide favors the Fenton reaction by an organism to minimize the presence of free iron, in view
reducing free ferric iron into ferrous iron leading to produc- of its ability to catalyze the formation of free radicals. In its
tion of hydroxyl radicals which can then be damaging to all reduced (ferrous) state, iron acts as a most efficient cata-
important biomolecules. lyst mainly via Fenton chemistry forming hydroxyl radi-
An in vivo study has been made which demonstrated an cals, while in its oxidized (ferric) state, iron is an integral
iron-mediated superoxide radical toxicity from an analy- element of various proteins and therefore is less actively
sis of the phenotype of sodA sodB E. coli mutants. It was involved in free radical reactions. As we have stressed, it is
shown that the excess of superoxide radical anion causes not only the oxidation state of the iron atom, but the effect
DNA damage that cannot be due to the direct effects of of ligand chelation from amino acid residues of proteins is
superoxide itself and the requirement of iron (Stolc et al. of importance since it can reduce the catalytic activity of
1996). iron, which counteracts formation of free radicals.
Rather, under stress conditions, when excess of super-
oxide radical is formed, it has capacity to release “free Iron overload diseases
iron” from iron-containing proteins such as ferritin (Touati
2000). When iron absorption exceeds the storage capacity of ferri-
The release of iron mediated by superoxide radical tin molecules, free iron may catalyze the formation of free
anion has been demonstrated for the enzymes containing radicals within the cells, resulting in damage to all impor-
[4Fe–4S] clusters, which contain two ferrous and two ferric tant biomolecules. Free iron can be deposited in various
ions. Superoxide oxidizes one ferrous ion into a ferric ion organs including the liver, pancreas, heart, joints and other
according to the reaction (Liochev and Fridovich 1994) tissues. The accumulated amount of iron in the human body
may reach as much as 20 g. After several years, patients
[2Fe(II) 2Fe(III)−4S]2+ + O·−
2 + 2H
+
develop specific symptoms (weakness, fatigue) followed by
→ [Fe(II)3Fe(III)−4S]3+ + H2 O2 (26) more serious conditions such as cirrhosis, liver cancer and
arthritis.
For which the rate constant is about 108–109 M−1 s−1. The As we have already stressed, excess iron is toxic to cells,
oxidized protein binds ferric ions more strongly, and so fer- mainly via iron-mediated formation of free radicals involv-
rous ions are released from the protein (Eq. 27) ing the Fenton reaction (Fig. 1).

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Arch Toxicol

Fig. 1  Link between iron-

induced formation of ROS and Cancer
human disease
Fenton and
Haber-Weiss
Reacons

High Body Reacve Oxidaon of Cardiovascular

Iron Stores Oxygen Species Biomolecules Disease

Neurodegenerave
Disorders

We note that various in vitro studies related to iron tox- is a hormone synthesized mainly in the liver which affects
icity used supraphysiologic concentrations of iron. Fur- dietary iron absorption across the gut mucosa (enterocytes).
thermore, iron toxicity linked with oxidative stress con- Hepcidin functions are based on a feedback mechanism
sequences to cause tissue damage has been difficult to that on one side allows sufficient iron to enter the plasma
demonstrate in animal models (Gammella et al. 2015). when the requirements for it are high and on the other to
The most common disease of iron overload is hemo- limit iron intake/release when iron stores are adequate
chromatosis, which may be of genetic or non-genetic ori- (Nakanishi et al. 2011).
gin. Genetic origin is linked with mutations of the HFE Hepcidin is linked with anemia which is characterized
gene, which encodes human hemochromatosis protein (also by a decreased serum level of iron and iron-binding capac-
known as HFE protein) (Adams and Barton 2010). ity via transferrin, and accumulation of iron in endothelial
However, the exact role of HFE protein in the pathogen- cells, showing impaired mobilization of iron from iron-
esis of hemochromatosis has not been fully elucidated. In storage sites (Pasricha et al. 2011). Ferroportin, a protein
fact, there are other factors affecting iron absorption, trans- responsible for iron transport from intracellular into extra-
port and storage, of which most important include hepci- cellular space has been found to be inhibited by hepcidin,
din, bone morphogenic protein 6, hemojuvelin, transferrin which binds to ferroportin and internalizes it within the
receptors and ferroportin (Gao et al. 2009). Clinical stud- cell.
ies involving iron depletion have been found to improve the
length and quality of life of patients with hemochromato- Iron and cardiovascular and metabolic diseases
sis. Thus, the iron depletion has been considered to be the
key action to prevent iron-mediated tissue injury. The prac- In a pioneering paper published by Sullivan more than
tice of phlebotomy, which among other affects stimulates 30 years ago, it was claimed that an increased level of car-
erythropoiesis, was used for several decades to reduce body diovascular disease in men and postmenopausal women is
iron stores in patients suffering from hemochromatosis related to increased iron deposits (Sullivan 1981). In con-
(Anderson and Shah 2013). The standard therapy involves trast, premenopausal women have lower iron deposits as a
a weekly removal of blood to bring the level of serum fer- result of menstrual bleeding. Sullivan’s hypothesis has been
ritin into the low reference range. The major benefit of this supported by (1) increased iron stores in postmenopausal
therapy is prevention of liver fibrosis (Adams and Barton women, which reached the same level as men, (2) myocar-
2010). Some patients reported suppression of various side dial failure of patients suffering of iron-storage disease and
effects of hemochromatosis, including a simultaneous (3) increased level of iron with age in men.
improvement in their control of diabetes mellitus. In addi- Since Sullivan formulated his hypothesis, more than
tion to phlebotomy, an alternative treatment based on the 55 epidemiological studies related to iron and cardio-
iron chelation therapy exists in clinical praxis. This type of vascular disease have been conducted, of which half are
treatment is hardly suitable for hemochromatosis patients, in support of it; however, about 35 % of studies found
mainly because of potential ligand toxicity, and a relative no evidence to support the hypothesis and the remain-
lack of documentation of benefits. Dietary restrictions and der gave results that were in disagreement with the idea
medications to reduce iron absorption are also important. (Munoz-Bravo et al. 2013; Baggott and Tamura 2015). In
The level of iron in the body is tightly linked with hep- the following paragraphs, we attempt to summarize the
cidin, a key regulator of the iron entry into the circulatory available data and their link with processes occurring at
system in humans. Hepcidin was discovered in 2000 and molecular level.

13
 Arch Toxicol

The iron–cardiovascular hypothesis is based on the fact An interesting correlation has been found between
that elevated low-density lipoprotein cholesterol (LDL- patients with chronic kidney disease and atherosclerosis.
C) is associated with cardiovascular disease and oxidative Patients with kidney disease exhibit high serum hepcidin
modifications to LDL-C alter the structure and function of levels, favouring iron sequestration in several cell types
endothelial cells which in turn leads to the development of leading thus to iron-related complications (Nakanishi et al.
atherosclerotic plaques (Holvoet and Collen 1994). Ather- 2011). Serum hepcidin is closely linked with serum ferritin
osclerosis leads to hardening of the arteries causing heart in healthy subjects and in chronic kidney disease patients.
attacks, strokes and peripheral vascular disease. The expression of the iron exporter, ferroportin, limits the
The link between iron and atherosclerosis may be sub- growth of intracellular bacteria by depleting cytosolic iron.
stantiated by the following mechanism. Iron is released An up-regulation of hepcidin which may interact with fer-
from transferrin, ferritin and senescent erythrocytes. roportin could suppress ferroportin functioning and thus
Release of iron from transferrin involves protonation of promote bacterial growth. Thus, it may be hypothesized
the carbonate anion, loosening the metal–protein bond that hepcidin, following interaction with ferroportin, might
(Bacon and Tavill 1984). Uptake of transferrin and release prevent iron efflux from macrophages or other cells in the
of iron occur in the endosome of all cells, and the daily arterial wall and thus accelerate atherosclerosis. Thus, the
amount of exchanged iron is about 0.03 g. The endoso- role of hepcidin in chronic kidney disease and atherosclero-
mal iron is poorly ligated and so capable of catalyzing sis deserves further attention (Nakanishi et al. 2010).
the formation of ROS, increasing thus the oxidative status Cardiac metabolism is affected not only by iron overload
which in turn causes oxidation of LDL-C. Experiments but also by iron deficiency. Iron deficiency induces abnor-
performed using animal models revealed an increased mal muscle metabolism and function and leads to increased
amount of iron in atherosclerotic lesions. Thus, iron-cat- lactic acidosis (Guertl et al. 2000). Iron deficiency may
alyzed formation of ROS via Fenton chemistry may play impair enzyme functions (e.g., cytochrome) in the skeletal
a role in the mechanism of atherosclerosis development muscle cells. Contrary, iron overload results in parenchy-
(Yuan and Li 2003). mal damage to the heart and other organs.
Atherosclerosis is a condition in which plaque builds up In conclusion, it is clear that iron overload is associated
on the insides of the arteries casing them to “harden.” Ath- with a number of disease states of an organism, including
erosclerotic plaques are composed of cholesterol, various myocardial disease and enhanced susceptibility to reperfu-
cellular waste products, calcium and fibrin. Hypercholes- sion damage. However, an exact role for the iron overload
terolemia appears to be a major factor in the development state in atherosclerosis requires further clarification. The
and progression of atherosclerosis (Delporte et al. 2013). main difficulty in linking iron status with various disorders
Elevated LDL is linked with an increased risk of cardiovas- is how the exact level of iron is measured. Iron is most fre-
cular disease, and oxidatively modified low-density lipo- quently determined in plasma or serum, of which plasma
protein (Oxid-LDL) has been considered to be involved in measurements have been considered to be directly relevant
several steps of atherogenesis (Hansson 2001). Oxid-LDL to the risk of development of arterial disease. Further inves-
alters the structure and function of endothelial cells and tigations including fundamental research and epidemio-
stimulates the synthesis of autoantibodies that are involved logic trials are needed to clarify the link between iron and
in atherogenesis, and oxid-LDL is taken up into foam cells risk of cardiovascular disease. Of special practical interest
via a scavenger receptor. Oxidative stress mediated by free are dietary recommendations as well as a clarification of
radicals induces oxid-LDL. In vitro studies employing iron some open questions regarding food fortification with iron.
and copper have shown their ability to catalyze the oxi- Metabolic syndrome involves a combination of obesity,
dation of lipids and proteins, in addition to LDL. These insulin resistance, high blood pressure and elevated triglyc-
results indicate the possibility of transition metal-mediated erides and is considered as a clinical condition associated
oxidative stress under in vivo conditions which may thus with high risk of diabetes and cardiovascular disease, as
promote atherogenesis. This makes a direct link between discussed above (Roberts and Sindhu 2009). As we have
redox-active iron or copper and risk of cardiovascular dis- noted, absorption of iron by enterocytes in the duodenum
ease feasible. is tightly regulated. Excess of iron is accumulated in the
In conclusion, lipoproteins oxidized by redox-active liver and pancreatic β-cells (Leiva et al. 2013). Increased
metals are key factors in the initiation and progression of iron content may increase the risk of type 2 diabetes via
atherosclerosis. The progression starts from the early-stage several mechanisms. In the liver, elevated iron may trig-
conversion of monocytes/macrophages into lipid-laden ger insulin resistance through the reduced capacity of the
foam cells and fatty streaks to the late-stage development of liver to extract insulin, which in turn results in impaired
coronary artery stenosis, plaque instability, plaque rupture, control of production of hepatic glucose. Excess of iron in
coronary thrombosis and finally myocardial infarction. adipocytes may worsen insulin functioning and interfere

13
Arch Toxicol

with the uptake of glucose. Muscles are also affected by is 8-hydroxy-2′-deoxyguanosine (8-OHdG) or 8-oxo-
the increased iron deposits via enhanced oxidation of fatty 7,8-dihydro-2′-deoxyguanosine (8-oxodG) (Dizdaroglu
acids which thus influence uptake of glucose. Iron has et al. 2002). The occurence of 8-OH-G was first reported
been also found to affect insulin secretion and activation of in human urine (Shigenaga et al. 1989). Various studies
apoptosis in pancreatic β-cells. confirmed that the level of urinary 8-OH-G is a good bio-
marker for risk assessment of various degenerative disor-
Iron and cancer ders and cancers. This biomarker has been used to monitor
the DNA damage in humans following exposure to carcino-
The link between cancer and elevated iron levels is more genic agents, such as asbestos fibers, heavy metals, tobacco
straightforward than that between iron and cardiovascular and polycyclic aromatic hydrocarbons as well as a risk fac-
and metabolic diseases, discussed above. tor for many diseases including cancer. The formation of
Reactive species such as free radicals modify the most 8-hydroxy-guanine adduct is shown in Fig. 2, and a selec-
important biomolecules including DNA. Modification of tion of free radical adducts with DNA bases is shown in
biological material represents the primary step (lesion) Fig. 3.
resulting in mutagenesis, carcinogenesis and aging. Vari- While progress in qualitative analysis of free radical
ous cancer tissues in fact revealed signs of DNA damage adducts with DNA bases has been relatively straightfor-
(Marnett 2000). The most representative damage caused ward, quantitative analysis has been the subject of meth-
by formation of free radicals catalyzed by iron ions is odological problems (Dizdaroglu et al. 2002). The most
substantiated by the occurence of a variety of DNA oxi- frequently used method for the quantitative analysis of
dation products. The hydroxyl radicals produced by an radical adducts with DNA is high-performance liquid chro-
iron-catalyzed Fenton reaction are able to add to dou- matography (HPLC) with electrochemical detection (EC),
ble bonds of DNA bases to form oxidative DNA adducts gas chromatography–mass spectrometry (GC–MS) and
(Valavanidis et al. 2009). To date, more than 100 oxida- HPLC–tandem mass spectrometry. To resolve the problems
tive DNA products have been identified, of which the most involved in unification of isolation and purification proce-
predominant adduct of guanine with hydroxyl radicals dures in oxidative DNA damage, the European Standards
Committee on Oxidative DNA Damage was established in
1997 (Collins 2002).
The main aim of the Committee is to resolve the prob-
O O lems associated with the measurement of background lev-
N N
els of oxidative DNA damage in human cells. A principal
HN HN
+ OH OH problem was DNA oxidation prior to instrumental analysis.
H2N N N H2 N N N To eliminate any artefacts, the Committee has suggested
H H
the distribution to associated Laboratories of standard sam-
guanine (G) 8-hydroxyguanine ples of pig liver and HeLa cells for reference analysis. In
(8-OH-G) spite of the progress to quantitatively analyze the level of
oxidative damage, there are still discrepancies between dif-
Fig. 2  Reaction of DNA base guanine with hydroxyl radical ferent methods and different laboratories. Similar problems

Fig. 3  Adducts of free radicals O NH2 NH2

with DNA bases
N N N
HN N N
OH H OH
H2N N N N H N N
H HO N H
H
8-hydroxyguanine 2-hydroxyadenine 8-hydroxyadenine

O O NH2
CH3
HN CH2OH OH
OH HN N
H
O N H O N H O N OH
H H H

5-hydroxy-6-hydroxo- 5-(hydroxymethyl)- 5,6-dihydroxy-

thymine uracil cytosine

13
 Arch Toxicol

Fig. 4  Iron and lipid peroxida- .−

tion process NAD(P)H oxidases
O2
Fe2+
.− SOD
xanthine oxidase O2 H2O2 Fe3+
O2
hypoxanthine
xanthine
xanthine
uric acid DNA
damage
mitochondria .OH
LOOH
Fe3+
LH
Fe2+
Fe2+
Fe3+
. O2
. Lipid peroxidation
Lipid peroxidation process LOO . L
H
LO process
.
C5H11 R

.
cyclisation OO

.
H
O
cyclisation
cyclisation
O O H O H
O2 /RH
malondialdehyde β-hydroxyacrolein
O
C5H11 . R
(MDA) O
OOH
H H

O H HO H
O
OH
O O Cytosine
Guanine O CH
Adenine
O O
H H 4-hydroxynonenal
N H
N - 2H2O H
H H
H N
N N N N H
deoxyR N N
N
M1G
N N N O
deoxyR
M1A deoxyR M1C

have been monitored involving the quantitative analysis of in a variety of pathways and has been considered as causal
the level of free radicals in studied samples using electron agent in numerous diseases including various cancers,
spin resonance spectroscopy. neurodegenerative disease and diabetes (Koh et al. 2000).
The lipid peroxidation process is linked with the cata- Thus, the role of iron in lipid peroxidation process has seri-
lytic action of iron and formation of peroxyl radicals ous health consequences.
(Fig. 4) (Spiteller 2006). Lipid hydroperoxides can react The risk of developing cancer is linked with a number
relatively rapidly with ferrous ions to form lipid alkoxyl of environmental factors, including radiation, pollution and
radicals (RO·) or more slowly with ferric ions to form occupational exposures to a variety of substances. It is known
peroxyl radicals (ROO·). Alkoxyl radicals derived from that occupational exposure to asbestos causes cancer, offi-
arachidonic acid undergo a cyclization reaction to form a cially termed as malignant mesothelioma (Toyokuni 2009).
six-membered ring hydroperoxide which may subsequently It is noteworthy that asbestos contains about 30 % of iron
undergo further reactions to form 4-hydroxynonenal. (Stayner et al. 1996). The mutagenic and carcinogenic prop-
Peroxyl radicals containing an unpaired electron in the erties of asbestos have been related to the catalytic properties
internal position of the fatty acid chain can react by cycli- of iron to decompose hydrogen peroxide leading to formation
zation to produce a cyclic peroxide moiety adjacent to a of hydroxyl radicals. There are three proposed mechanistic
carbon-centered radical. This radical can undergo a second routes to asbestos-induced carcinogenesis (Toyokuni 2009).
cyclization to form a bicyclic peroxide which after cou- The first of these is termed the “oxidative stress hypothesis”
pling to dioxygen and reduction yields a molecule that is and is based on the fact that phagocytic cells absorb asbestos
structurally analogous to the endoperoxide. An endoperox- fibers and produce damaging free radicals. Epidemiological
ide is an intermediate product in the formation of malondi- studies indicate that asbestos fibers with a higher iron content
aldehyde which can react with DNA bases to form adducts. are more carcinogenic due to the catalytic properties of iron
Malondialdehyde is a potentially mutagenic substance, to produce hydroxyl radicals via Fenton reaction. The second
and 4-hydroxynonenal is a key molecule in cell signaling hypothesis is based on the “chromosome tangling theory”

13
Arch Toxicol

which rests upon the assumption that asbestos fibers damage ._ ._

chromosomes during the course of the cell division. The final O2 _ O2 O2 _ O2
e . _
e
hypothesis is based on the preferential adsorption of many KH2 KSQ KQ
specific proteins and carcinogenic chemicals, including ciga- OH O
. O
rette smoke components, on asbestos fibers which may fur- _
_ _
_
e 2H+ _ e
ther promote the process of carcinogenesis. From the above, _ _
+ e + 2H+ + e
it is clear that the molecular mechanism of asbestos-induced OH O_ O
carcinogenesis is a complex process. It is generally believed hydroquinone semiquinone quinone
that catalytic iron plays a significant role in the process of
carcinogenesis. Measurements of iron content in asbestos Fig. 5  Redox cycling of K vitamins
in industrial plants employing asbestos workers can provide
a useful prophylaxis against the development of asbestos-
induced cancers. However, further work to elucidate molecu- The consequence of O·− ·
2 and HO formation may reflect
lar mechanism of asbestos carcinogenesis is necessary. the fact that these highly reactive species can initiate DNA
The gastrointestinal tract, in particular the colon, is a site damage and hence act as initiators and promoters of colo-
where various signs of enhanced oxidative stress have been rectal cancer. The formation of hydroxyl radicals in the
observed (Halliwell et al. 2000). Models describing the for- colon is supported by experiments with 1/10,000 diluted
mation of free radicals in the colon may correlate with the feces in which hydroxyl radicals have been detected using
high incidence of cancer in the colon and rectum compared DMSO as a molecular probe (Babbs 1990). Based on the
with other regions of the gastrointestinal tract. Epidemiologi- above discussion, we can conclude that a high intake of red
cal trials and model studies support the hypothesis that intra- meat increases stool iron and bile pigments can promote
colonic formation of free radicals linked with the consump- Fenton chemistry and the formation of damaging hydroxyl
tion of red meat containing high levels of iron, and diets low radicals, thus supporting the notion of oxidative stress-
in fiber correlate well with a high incidence of cancers of induced colorectal cancer.
colon and rectum. Red meat increases stool iron, and bile pig- Environmental toxicology is concerned with the circu-
ments (resulting from diets rich in animal fat) may promote lation of toxic substances and degradation products in the
free radical formation via Fenton reactions at the mucosal/ human environment, for example, the toxic chemicals used
luminal interface. We have proposed a role for vitamins K in in households. Nitrilotriacetic acid (NTA) is an important
the formation of free radicals in the colon (Valko et al. 2001). chelating agent with many industrial applications. NTA
Endogenous menaquinones (vitamin K2) are synthe- can sequester metal ions to form water-soluble complexes
sized primarily by the bile acid-tolerant Bacteroides fra- (Gosriwatana et al. 1999). Since NTA can chelate cal-
gilis strains and are present in relatively high concentra- cium and magnesium ions, it is used in household laundry
tions in the human gastrointestinal tract. A diet high in fat detergents as a “builder” to replace phosphates, the use of
increases bile secretion and production of the co-mutagenic which is legislatively restricted since they may give rise to
and cocarcinogenic secondary bile acids by the colon eutrophication, algal blooms and “dead zones” when they
microflora. We have proposed that bile-tolerant Bacteroides enter the environmental water systems. Interestingly, the
strains synthesize menaquinones and that vitamin K1 is a iron–NTA complex has been found to induce renal carcino-
cofactor in the dehydrogenation of bile acid substrates to genesis and acts as an efficient catalyst for the decomposi-
yield reduced vitamins K (KH2). Reduced vitamins can tion of hydrogen peroxide via the Fenton reaction. Regard-
be transported into the mature colonocytes in the form of less of the number of saturated binding sites present, this
mixed micelles to trigger formation of the superoxide radi- complex works as effectively as free iron at pH ~7 under
cal anion (Fig. 5) and further redox-cycling reactions form- in vitro conditions.
ing a variety of free radicals including hydroxyl radicals
(Valko et al. 2001). Iron and neurodegenerative disease
KSQ·− + O2 → KSQ + O·− (28)
2 Disruption in homeostasis of iron, copper and zinc has been
associated with neurological disorders such as Alzheimer’s
O·−
2 + Fe(III) → O2 + Fe(II) (29) disease (AD) and Parkinson’s disease (PD) (Bush 2003;
Bonda et al. 2010; Montes et al. 2014).
KSQ·− + Fe(III) → KSQ + Fe(II) (30) A histopathological hallmark of AD represents deposits
called amyloid plaques which are found postmortem in the
2O·− +
2 + 2H → H2 O2 + O2 (31) brains of patients. The main component of amyloid plaques
− ·
Fe(II) + H2 O2 → Fe(III) + OH + HO (32) is a peptide of 36–43 amino acids called amyloid-β peptide

13
 Arch Toxicol

(Aβ). Aβ has been shown to play an important role in the In the development process of highly potent tacrine ana-
pathology of AD via formation of ROS (Jomova and Valko logues, heterodimers bearing tacrine and 7-methoxytacrine
2012). The mechanism of free radical formation mediated congeners in single molecule were synthesized, compu-
by Ab involves the participation of redox metals, mainly tationally designed and tested for their ability to inhibit
iron and copper and non-redox metal zinc. AChE (Fig. 6) (unpublished results). The length of the
Significant progress in the precise quantification of linker in new 7-MEOTA-tacrine thioureas and 7-MEOTA-
redox-active metals such as iron, copper and non-redox tacrine urease was optimized to achieve the simultaneous
metal zinc in Alzheimer’s tissue has been made by apply- interaction between the heterodimers and both the catalytic
ing three unique physical techniques: scanning transmis- and peripheral sites of the enzyme AChE.
sion ion microscopy, Rutherford backscattering spectrom- The binding mode of bis(7)-tacrine (magenta carbon
etry and particle-induced X-ray emission in conjunction atoms) and highlighted one of the synthesized 7-MEOTA-
with a high-energy (MeV) proton microprobe. The results tacrine derivatives (green carbon atoms) in the AChE active
obtained revealed an increased concentration of metals in site is shown in Fig. 7.
the amyloid plaques of Alzheimer’s tissues compared with The compounds were found to exhibit moderate radi-
the surrounding tissues (Rajendran et al. 2009). The level of cal scavenging activity and strong metal-chelating ability,
iron in amyloid plaques was determined to be nearly dou- which indicate their potential ability to suppress the oxida-
ble, while those of copper and zinc were found to be nearly tive stress component that had been identified in AD tis-
triple that in the surrounding tissue. These data document sues. At present, our aim is to prepare a series of heterodi-
the catalytic role of transition metal ions in the formation mers bearing 7-methoxytacrine linked with molecules with
of oxidized species which in turn contribute to the occur- strong antioxidant potential such as lipoic acid and other
rence of various signs of oxidative stress in brain tissues. biological molecules (e.g., flavonoids) possessing strong
The major signs of an oxidative stress component in AD antioxidant potential. Thus, we believe that bifunctional
brains include increased lipid peroxidation which was sub- compounds bearing not only acetylcholinesterase inhibi-
stantiated by the enhanced abundance of 4-hydroxynonenal tory activity but also oxidative stress-reducing potential
(Zhu et al. 2012). In addition, increased levels of oxidized represent a new strategy to combat this threatening disease.
DNA and proteins, diminished energy metabolism, inhib- Similarly to Alzheimer’s disease, Parkinson’s dis-
ited synthesis of cytochrome c oxidase, increased levels of ease (PD) also exhibits evidence for an oxidative stress
malondialdehyde and observation of advanced glycation component. The earliest features of the development of
end products (AGEs) have all been observed (Varadarajan
et al. 2000).
The therapy is still in the early stages of development,
and various hypotheses involving cholinergic, β-amyloidal,
oxidative and other are being investigated with the aim to
understand the pathophysiology of the disease and on this
basis to optimize an effective treatment.
Acetylcholinesterase (AChE) is considered to be prob-
ably the most viable therapeutic target for improving the
cognitive status of patients suffering from AD (Patocka
et al. 2008). Inhibition of AChE was considered to be
achievable as a therapeutic target because of the proven
efficacy of inhibition of peripheral AChE as a treatment for
myasthenia gravis (MG), endorsing the feasibility proving
of the approach.
More than two decades ago, tacrine was the first
approved drug for the treatment of ADt. Tacrine has been
reported to be a multitargeted drug; however, it was with-
drawn from the market, mainly on the basis of its hepa-
totoxicity and gastrointestinal effects. Later, the tacrine
derivative, 7-methoxytacrine (7-MEOTA), was introduced,
and probably due to its different metabolic behavior, it did
not exhibit the serious side effects. Since then, many drugs
based on the 7-methoxytacrine moiety (Mehta et al. 2012) Fig. 6  Structures of 7-methoxytacrine thioureas (upper structure)
have been synthesized and tested. and 7-methoxytacrine ureas (bottom structure)

13
Arch Toxicol

Fig. 7  Binding mode of bis(7)-

tacrine (magenta carbon atoms)
and highlighted 7-MEOTA-
tacrine derivative (green carbon
atoms) in the human AChE
active site. Selected residues
interacting with one of the drugs
are rendered with yellow carbon
atoms. Nonpolar hydrogens are
omitted to improve figure clar-
ity. The figure was created with
PyMol (color figure online)

Parkinson’s disease are substantiated by the rapid depletion the mitochondrial respiratory chain (Anderson et al. 2005).
of the main cellular antioxidant glutathione in the substan- The defect leads to the release of mitochondrial iron with
tia nigra (Jenner 2003). in turn may catalyze hydroxyl radical formation via Fenton
A decreased level of glutathione may influence mito- chemistry (Huang et al. 2009).
chondrial energy production most probably via disrupted
activity of mitochondrial complex I (Chinta and Andersen
2008). These and other signs signify that PD is linked with Copper
a relatively high level of oxidative stress, as is also evi-
denced by biochemical analysis of postmortem tissues of In biological systems, copper is present predominantly
the PD brain (Andersen 2004). The features of enhanced in oxidation states +1 [cuprous (I)] and +2 [cupric (II)].
oxidative stress correlated with the increased iron levels The trivalent form +3 occurs; however, from biological
reported from the studies of PD brains. Thus, increased point of view, it is relatively unimportant. Cu(II) contains
signs of oxidative stress and broken homeostasis of redox- one unpaired electron in its 3d-orbitals (nine electrons)
active metals contribute to neurodegeneration, typical for and so this form of copper is paramagnetic. In contrast, the
PD (Jomova et al. 2010). cuprous [Cu(I)] form has a completely filled set of d-orbit-
Along with the superoxide radical anion, iron from fer- als (10 electrons) so this form of copper is diamagnetic
ritin can be released by 6-hydroxydopamine, a neurotoxin (Gala et al. 2014).
implicated in PD. Released iron behaves as “free iron labile In living systems, majority of copper is bound to bio-
pool” and can catalyze the formation of hydroxyl radicals logical macromolecules. Cuprous [Cu(I)] copper is pre-
via Fenton reaction. dominantly present in the reducing intracellular environ-
Although less common, the ailments, Huntington’s dis- ment, while the cupric [Cu(II)] oxidation state is typical
ease and Friedreich’s ataxia are neurological disorders for for oxidizing extracellular space. Cu+ ions are unstable in
which typical signs of oxidative stress are also present. It aqueous environments and undergo oxidation to Cu2+. In
has been shown that huntingtin (HTT) inclusion bodies are the oxygenated system, formation of the superoxide radical
targets of oxidative stress containing large amounts of oxi- anion occurs (Eq. 33).
dized proteins.
Cu(I) + O2 → Cu(II) + O·− (33)
Friedreich’s ataxia is associated with impaired mus- 2

cle coordination (ataxia). The critical factor appears to be The standard reduction potential of the Cu(II)/Cu(I) cou-
mutation in a gene labeled FXN that codes for frataxin, ple is 0.153 V. The ability to adopt two distinct redox
which is located on chromosome 9. Frataxin is a mitochon- states makes copper a very important element as an inte-
drial iron chaperone protein (Atkinson and Winge 2009; gral part of metalloenzymes and also in redox reactions
Babady et al. 2007) and plays a key role in the insertion of and important biological functions including cell signaling.
ferrous ions during the assembly of iron–sulfur clusters in Redox properties of copper are very important in processes

13
 Arch Toxicol

resulting in electron transfer within biological molecules. plasma proteins is formed by the histidine residues. The
Copper is involved as a cofactor of various proteins in bio- latter probably also participate in the copper reduction pro-
logical processes such as photosynthesis, respiration, iron cess. At the C-terminal intracellular site of Ctr-1, there are
metabolism, neurological function and the interception and cysteine clusters participating in tight binding of cuprous
scavenging of damaging free radicals (Kozlowski et al. ions. Cupric ions delivered inside the cells are handled by
2014). copper chaperones which maintain the transportation to
Copper occurs most often as coordinated by imidazole subcellular compartments for subsequent incorporation in
nitrogen atoms, or nitrogen atoms that originate from pep- copper enzymes. Besides copper chaperones, copper is also
tide bonds and amino groups, or oxygen atoms from car- transported to specific cellular destinations within the cyto-
boxylate groups or hydroxyl groups and sulfur atoms from plasm by metallothioneins which protect cells from copper
the amino acids methionine and cysteine. toxicity.
Copper in blood is bound by serum albumin and cerulo-
Copper homeostasis plasmin. Ceruloplasmin is responsible for transport of the
majority of copper, while serum albumin also binds zinc,
Copper homeostasis is the regulation of the body’s copper calcium and nickel ions. An exact description of the high-
content. Homeostatic regulation involves copper absorp- affinity binding site for copper in human serum albumin
tion and excretion (Tegoni et al. 2014). Among other fac- has been made using results from EPR spectroscopy (Valko
tors, metal ion homeostasis is achieved by the metallochap- et al. 1999). Ascorbic acid may reduce bound cupric ions
erones, which are proteins that safely deliver metal ions on serum albumin, which are easily reoxidized in the pres-
to the appropriate protein receptors. In the course of the ence of molecular oxygen.
investigation of copper chaperone for the superoxide dis- Small amounts of copper (less than 5 %), known as “free
mutase gene, Rae et al. (1999) made the outstanding dis- copper pools” (discussed above), are copper ions either
covery that the upper limit of so-called free pools of copper loosely bound to ceruloplasmin or as present in the form of
was far less than one single atom per cell. This finding is small molecular weight chelates.
of importance because it had been commonly believed that
metal ions were in equilibrium with metalloproteins. These Copper‑induced oxidative stress
results indicate that cells possess significant capacity for
copper chelation via mechanisms that preventing copper In interpreting copper-induced cellular toxicity, it is
from being randomly available. This finding has important assumed that copper ions are able to participate in the for-
consequences regarding free radical formation by the Fen- mation of free radicals. Cupric ions in the presence of bio-
ton reaction. logical reductants such as ascorbic acid or glutathione can
Beacuse of the free radical-induced toxicity mediated by be reduced to cuprous species capable of catalytic decom-
the circulation of free copper ions, living organisms have position of hydrogen peroxide (Prousek 2007)
evolved a sophisticated regulatory machinery for copper
Cu(I) + H2 O2 → Cu(II) + · OH + OH− (34)
adsorption, distribution, utilization and excretion, mediated
by a variety of specific proteins capable of tightly binding Hydroxyl radicals are very reactive, with a lifetime of
copper ions (Banci et al. 2010). several nanoseconds. They can react in the proximity of
Eucaryotic organisms have developed sophisticated various biological molecules through the mechanisms of
systems for copper delivery to all specific cellular targets. hydrogen abstraction from unsaturated fatty acids. Thus,
Copper from the diet is absorbed in the small intestine by the copper-induced formation of free radicals induces DNA
epithelial cells (Zhao et al. 2003). Excess copper levels are damage and oxidation of bases.
stored in the liver or excreted in bile. Translocation of cop- The role of copper in low-density lipoprotein (LDL) oxi-
per across the cellular membrane is carried out by a small dation was studied. Oxidation of LDL has various health
transmembrane high-affinity transport protein of copper consequences including occurence of atherogenesis. In
(hCtr-1), expressed mainly in the kidney or liver. There is addition to oxidation of LDL, high-density lipoproteins
strong evidence that cuprous (Cu+) ions rather than cupric (HDL) are also susceptible to oxidation. Studies of the oxi-
ions (Cu2+) are bound to the Ctr-1 transporter. This is in dation of HDL are of great importance since oxidized HDL
agreement with a current view that cupric ions are first directly affects cardioprotective features. It appears that
reduced to cuprous species by a variety of small molecu- HDL is even more susceptible to copper-induced oxida-
lar weight reductants (vitamins, amino acids, antioxidants tive damage than LDL due to the effect of the α-tocopherol
such flavonoids). Ctr-1 is able to bind copper at both N- radical which is formed during the course of the reduction
and C-terminus sites (Tegoni et al. 2014). An N-terminal of cupric ions to cuprous species by α-tocopherol (Raveh
extracellular site capable of binding copper ions from et al. 2000). In accord with the above discussion, the

13
Arch Toxicol

enzyme ceruloplasmin may be involved in LDL serving as stage involves the typical formation of an identifiable focal
a source of free copper (Mukhopadhyay and Fox 1998). lesion. This is a reversible process and requires permanent
Copper is an integral part of many enzymes, including activity of tumor promotion stimulus to proceed further
Cu, Zn-SOD. Overexpression of Cu, Zn-SOD may lead to (Loft and Poulsen 1996). Tumor promoters are known to
occurence of oxidative damage. The mechanism by which have inhibitory effects on antioxidant defense systems
SOD induces oxidative damage was investigated. SOD including antioxidant enzymes. A high level of oxidative
enzymes (Cu, Zn-SOD, Mn-SOD, Ni-SOD) in the presence stress suppresses cell proliferation by inducing apoptosis
of hydrogen peroxide cause DNA damage (Barbouti et al. or necrosis. On the other hand, a low/intermediate level
2001). Hydrogen peroxide caused copper-dependent DNA of oxidative stress may stimulate the cell division in the
damage with a dominant abundance at the 5-guanine of promotion stage and therefore in fact stimulate the tumor
poly G sequences which was enhanced following addition growth (Dreher and Junod 1996). ROS formation during
of SODs. It may be thus deduced that overexpressed SODs this stage of the carcinogenesis process is responsible for
represent a potential carcinogenic risk. free radical-promoted tumor growth. Progression is the
final stage of the overall mechanism of carcinogenesis.
Copper and cancer This final stage is irreversible and is characterized by bio-
chemical changes during the transformation of cells from a
As discussed above, excess of copper is a potent oxidative preneoplastic to a neoplastic state. Genetic instability and
risk, as is evidenced by the generation of free radicals. The disruption of chromosome integrity are typical features of
role of copper in etiology and growth of tumors has been this stage. The critical point regarding oxidative stress is its
studied, and the results have shown that the levels of copper level in cancer cells sufficient to maintain cell proliferation;
in various types of tumors are significantly elevated com- however, such a level is insufficient to cause apoptosis or
pared with healthy subjects. Interestingly, levels of ceru- necrosis (Hanahan and Weinberg 2011).
loplasmin have also been shown to be elevated in various Signs of oxidative stress in the circulation have been
types of cancers such as those of the colon, breast and lym- reported in ovarian cancer patients (Senthil et al. 2004).
phomas. Ceruloplasmin seems to be an angiogenic stimula- The levels of enzymatic enzymes SOD and catalase as well
tor issued for the formation of new blood vessels (Gupte as vitamins C and E were found to be significantly lowered.
and Mumper 2009). In addition, it has also been reported that thiobarbituric
For healthy cells, a typical redox equilibrium is achieved acid-reactive substances (TBARS) (a sensitive marker of
by a balanced formation and elimination of ROS and RNS. oxidative stress) are increased (nanomoles malondialde-
The direct proof of oxidative stress is based on the detec- hyde in plasma) in plasma as compared to control samples.
tion of free radicals by a variety of techniques; indirect A disturbed oxidant/antioxidant ratio in the circulation
proof is based on the detection and quantification of vari- has been found in patients with benign and malignant pros-
ous products following modification of biological material tate cancers. SOD and glutathione peroxidase have been
by ROS. An accompanying feature typical for cancer cells found to be significantly lowered than in control subjects. A
is a reduced level of cellular antioxidants: both antioxidant slight decrease in the level of catalase has also been noted.
enzymes and low molecular weight antioxidants. Similar findings reporting decreased levels of antioxidant
Cancer development is a complex multistage process, enzymes (SOD and catalase) have also been reported also
characterized by the cumulative actions of multiple events in patients suffering from stomach cancer.
occurring in a single cell (Valko et al. 2006). Such pro- In most of the cancer patients, changes have been noted
cesses can be described by three stages: initiation, pro- in glutathione status and antioxidant systems in blood. The
motion and progression. Free radicals participate in all GSH/GSSG ratio is significantly reduced, mainly due to an
three stages of carcinogenesis. Initiation involves an ini- increase in oxidized GSSG: The latter has been attributed
tial non-lethal DNA mutation. Oxidative DNA damage to an increased formation of hydrogen peroxide by tumors
may occur via reactions of free radicals such as hydroxyl (Navarro et al. 1999).
radicals. Several studies reported a direct link between size Oxidative stress markers have been increased not only
of tumors and amount of oxidized DNA adducts such as in case of solid tumors but also in specific malignancies
8-OH-Gua. The process of cancer initiation is accompa- such as various forms of leukemia, including acute lym-
nied by calcium-induced oxidative stress evidenced by an phocytic leukemia, acute non-lymphocytic leukemia and
increase in intracellular free calcium, arising from an influx chronic myeloid leukemia. Redox-active trace metals, pre-
of extracellular Ca(II). dominantly copper, and superoxide radical anion have been
The promotion stage of carcinogenesis is character- found to be significantly increased in leukemia patients
ized by the process of induction of cell proliferation and/ (Devi et al. 2000). Interestingly, levels of SOD were ele-
or inhibition of programmed cell death (apoptosis). This vated threefold and levels of glutathione peroxidase by

13
 Arch Toxicol

a factor of four, as compared to normal levels. A slight N-terminally complexed Cu(II) is reduced by electrons
increase in the levels of hydrogen peroxide has been noted originating from the C-terminal methionine (Met35) resi-
in leukemia patients. An increase in the formation of free dues according to reaction (Pogocki 2003)
radicals by leukemia leukocytes led to a stimulation of the
MetS + Aβ-Cu(II) ↔ MetS·+ + Aβ-Cu(I) (39)
expression of antioxidant enzyme activities.
The levels of antioxidant enzymes and lipid peroxida- leaving behind the methionine radical of Met35 (MetS·+)
tion in different stages of the disease progression have been and forming cuprous ions from cupric species. From a ther-
investigated in breast cancer patients. It was found that modynamic point of view, based on an inspection of the
lipid peroxidation and free radical production (superoxide reduction potentials of the Cu(II)/Cu(I) and Met/MetS·+
radical and hydrogen peroxide) in the plasma were signifi- couples, the above reaction is rather unfavorable; how-
cantly increased at all stages of breast cancer (Sener et al. ever, electron transfer between MetS and Aβ-Cu(II) may be
2007). accelerated by the subsequent exergonic reaction of depro-
tonation of MetS·+, leaving behind the 4-methylbenzyl
Copper and neurodegenerative disease radical, making the reaction thus viable in vivo (Pogocki
2003). The sulfide radical MetS·+ may efficiently react with
The majority of studies devoted to Alzheimer’s disease superoxide radical anion. The occurence of this reaction is
have concerned amyloid beta (Aβ) (Ow and Dunstan 2014), confirmed by the formation of Met-sulfoxide (MetO) which
which is a main constituent of amyloid plaques occurring in has been isolated from AD senile plagues
the brains of Alzheimer’s patients. Formation of amyloid-β
Met
(Aβ) associated with increased oxidative stress and neuro- MetS·+ + O·−
2 −→2MetO (40)
toxicity is proposed to represent a major event in the devel-
opment of Alzheimer’s disease. Generally, the amino acid methionine is related to the
As already noted above, besides the amyloid-β hypoth- pathogenesis of Alzheimer’s disease, since this amino acid
esis, Alzheimer’s disease is characterized by a disrupted is prone to oxidation under in vivo conditions. It has been
metabolism of redox-active metals (iron, copper) and non- proposed that Met35 oxidation to Met-sulphoxide reduced
redox metal (zinc) (Cuajungco et al. 2005). Copper binds to toxic and pro-apoptotic effects of the amyloid beta protein
amyloid-β via the following amino acid residues: histidine fragment on isolated mitochondria (Pogocki 2003).
(His13, His14, His6) and tyrosine (Tyr10). Amyloid beta As a direct result of disturbed metabolism of redox
also binds zinc(II) and iron(III). metals (copper, iron), a variety of oxidative products have
Interaction of copper ions with amyloid beta promotes been quantified in the brains of Alzheimer’s patients.
its neurotoxicity, according to in vitro studies which con- These include heme oxygenase-1 (HO-1), 8-hydroxy-
firm the reduction of Cu(II) to Cu(I) and the formation of guanine, oxidative-modified proteins, lipids and nucleic
hydrogen peroxide (Cuajungco et al. 2000). acids. The concentration of lipid hydroperoxides has been
The protective role of antioxidants in Alzheimer’s dis- found to be increased in the brain tissue from Alzheimer’s
ease has been extensively studied. The results show that patients as compared to control experiments. A process
amyloid-β is able to stimulate Cu-catalyzed oxidation of of lipid peroxidation has been substantiated by various
ascorbate anion (AscH−) to ascorbyl radical (Asc·−) with markers, including 4-hydroxy-trans-2-nonenal (HNE),
the subsequent formation of reactive hydroxyl radicals 4-oxo-trans-2-nonenal (4-ONE), acrolein and 4-oxo-
according to the following reactions (Dikalov et al. 2004) trans-2-hexenal, all of which are well-recognized neuro-
toxic agents.
Aβ - Cu(II) + AscH− ↔ Aβ-Cu(I) + Asc·− + H+ (35) Ascorbic acid levels in plasma and in cerebrospinal flu-
ids in patients with AD have been found to be lowered in
Aβ-Cu(II) + Asc·− ↔ Aβ-Cu(I) + Asc (36) comparison with control patients (Sultana et al. 2013). This
may suggest suppressed regeneration of the α-tocopherol
Aβ-Cu(I) + H2 O2 → Aβ-Cu(II) + · OH + OH− (Fenton) (37) radical of vitamin E back to α-tocopherol. The combination
of both ascorbate and tocopherol resulted in an increase
Aβ-Cu(I) + O2 ↔ Aβ-Cu(II) + O·− (38)
2 in both these antioxidants in plasma and cerebrospinal
The superoxide radical anion is a by-product of this reac- fluids, making them thus less susceptible to oxidation
tion scheme. under in vitro conditions. However, supplementation of
Electron paramagnetic resonance (EPR) spectros- α-tocopherols alone to AD patients did not show protection
copy has been employed to show that the N-terminal resi- against in vivo oxidation. This study confirms the impor-
dues of His 13, His14, His6 and Tyr10 are involved in the tance of synergism between vitamins E and C in patients
binding of Cu with amyloid-β. It has been proposed that with Alzheimer’s disease.

13
Arch Toxicol

Various trials confirmed that flavonoids are beneficial to Arsenic

patients with Alzheimer’s disease (Solanki et al. 2015). Neu-
rotoxicity induced by amyloid-β, whose deposition in the Arsenic is the 33rd element of periodic table, and its prop-
brain resulted in neuronal loss in brain, has been shown to be erties can be described as a brittle steel-gray solid display-
attenuated in the presence of epigallocatechin gallate. Epigal- ing some features of both a metal and a non-metal; how-
locatechin gallate (present in green tea extracts) is currently ever, most frequently it is referred to as a metal and from a
under investigation as a prospective chemoprotective agent. toxicological point of view as a heavy metal (Mandal and
In addition, catechins are of interest due to their ability to Suzuki 2002). Arsenic ranks twentieth in abundance of
directly scavenge free radicals and excerpt indirect antioxi- all elements present in the Earth’s crust and fourteenth in
dant effects via activation of transcription factors and antioxi- seawater.
dant enzymes, modulating thus the cellular redox state. The most common oxidation states of arsenic are +5,
+3 and −3 forming either inorganic or organic com-
Copper and metabolic disease pounds. Combined with oxygen, sulfur or chlorine, arsenic
is able to form inorganic species, while combination with
Perturbations in the balance of transition metal ions such as hydrogen or carbon leads to formation of organo-arsenic
copper and zinc may shift redox equilibria in physiological compounds.
milieu leading to oxidative stress and finally to the develop- Arsenic compounds represent a serious threat to human
ment of diabetes and diabetic complications. health since they lack color or smell, and hence, their pres-
Increased concentrations of copper ions in the plasma ence in the food chain, in drinking water or in air is not
of diabetic patients have been related to hypertension and immediately obvious. Various cosmetic products including
retinopathy (Kang et al. 2000). The level of ceruloplasmin eye shadows frequently contain toxic elements including
has been found to be increased in both type 1 and type 2 arsenic (Borowska and Brzoska 2015). Due to the rela-
diabetic patients (Uriu-Adams and Keen 2005). A disturbed tively good water solubility, arsenic particles may penetrate
metabolism of copper may interfere with increased levels through the skin via intercellular microroute and thus enter
of glycated proteins which in turn may contribute to the into the body. Since arsenic that has entered the circulatory
progression of pathologies related to diabetes. Glycated system represents a potential risk of carcinogenesis, cos-
proteins have an increased affinity to bind transition metal metic products should contain no more than 5 ppm of the
ions, including copper. Copper bound to proteins can still element.
act catalytically in the formation of ROS and thus provide Contamination of groundwater by arsenic (and also by
stable active sites for producing reactive species that may other toxic metals) may affect the health status of people
lead to occurence of oxidative stress in diabetes (McCarty living in various regions. In India and in Bangladesh, there
and DiNicolantonio 2015). are more 45 million people living in areas where they are
Copper has been found to be involved in the etiology of exposed to arsenic in their drinking water (Chowdhury
atherosclerosis (Grammer et al. 2014). Increased levels of et al. 2000).
homocysteine also represent a risk factor for atherosclerosis.
Copper may interact with homocysteine and consequently Arsenic toxicity
produce free radicals which in turn may lead to the forma-
tion of oxidized LDL as is found in atherosclerotic plaques. Arsenic enters the body predominantly in the form of tri-
In addition, an association between elevated copper and valent inorganic arsenic As(III) via diffusion (Cohen et al.
ceruloplasmin levels with atherosclerotic disease has been 2006). Pentavalent inorganic arsenic is able to penetrate
noted (Wei et al. 2012), suggesting that the ceruloplasmin through cell membranes by an energy-dependent transport
may be involved in the pathology of atherosclerosis. The mechanism. Pentavalent arsenic is immediately reduced by
mechanism explaining this link is based on the proposal biological reductants to trivalent arsenic.
that one of the copper ions of ceruloplasmin may interact Inorganic arsenic is represented by arsenite [As(III)]
with LDL which is a component of atherosclerotic plaques and arsenate [As(V)], both of which can be methylated or
and thus may promote free radical damage. dimethylated to form monomethylarsonic acid [MMA(V)],
Increased copper levels have also been found in response monomethyl arsonous acid [MMA(III)], or dimethylars-
to inflammation, chronic diseases such as arthritis. The inic acid [DMA(V)] and dimethylarsinous acid [DMA(III)]
increase in copper levels in inflammation is related to the (Fig. 8) (Jomova et al. 2011).
increase in ceruloplasmin, a recognized process in arthritis As we have already mentioned, the metabolism of inor-
in order to neutralize ROS, mainly superoxide anion radical ganic arsenic involves a two-electron reduction of penta-
(Sokolov et al. 2015). valent arsenic to trivalent arsenic, mediated by biological

13
 Arch Toxicol

Arsenate (V) Dimethylarsinous acid Dimethylarsinic acid

DMA(III) DMA(V)
OH CH3 CH3
V V
O As OH III O As CH3
_ As _
O HO CH3 O

GSH As-
Methyltransferase

2e
OH OH
OH
As-Methyltransferase V III
III O As CH3 As CH3
As
_
HO OH O OH
Arsenite(III) Methylarsonic acid Methylarsonous acid
MMA(V) MMA(III)

Fig. 8  Various pathways of the metabolism of inorganic arsenic

reducing agents, the most efficient of which is glutathione. the mitochondrial membrane potential. The mitochondrial
The reduction is followed by oxidative methylation to form alterations in turn lead to random formation of the superox-
pentavalent organic arsenic (Hughes 2002). ide radical anion which is a primary radical from which all
Trivalent inorganic arsenic is known to inhibit pyruvate other damaging ROS can be formed. The process of radical
dehydrogenase by binding to the sulfhydryl groups of dihy- formation is accompanied by depletion of prominent cel-
drolipoamide (Bergquist et al. 2009). In addition, trivalent lular antioxidant, glutathione (Flora 2011).
arsenic inhibits various other enzymes through binding to The most damaging radicals formed in the process of
sulfhydryl groups, glucose uptake into cells, oxidation of arsenic-induced oxidative stress include hydroxyl radicals,
fatty acids and production of glutathione, which is a major peroxyl radicals, singlet oxygen, hydrogen peroxide and
player in the protection against oxidative stress (Miller other species. The detailed mechanism behind the forma-
et al. 2002). The toxicity of pentavalent inorganic arsenic tion of all these ROS has yet to be fully identified; however,
can be explained by its reduction to trivalent arsenic. More some studies proposed the formation of intermediate arsine
specifically, pentavalent arsenic emulates inorganic phos- species.
phate and replaces phosphate in glycolytic and cellular Formation of intermediate arsine species may lead
respiration pathways resulting in uncoupling of oxidative to excessive formation of free radicals (Yamanaka et al.
phosphorylation (Hughes 2002). 1997). Methylated arsenic species have been shown to
The mechanism of action of arsenicals is complicated release iron from ferritin which may catalyze formation
and depends on various factors, which include physico- of free radicals including hydroxyl radicals (Ahmad et al.
chemical properties, such as the valence state, extent of 2000). Free radicals of oxygen origin were found to be
methylation and electrostatic and van der Walls interac- generated in the course of oxidation of arsenite to arsenate
tions to active sites of biomacromolecules. Further factors (Del Razo et al. 2001).
include pharmacokinetic features such as rate of intake, Experimental results confirmed formation of superox-
distribution, metabolism and excretion. Among the cur- ide radical anions and hydrogen peroxide following arsenic
rently accepted mechanisms of arsenic toxicity is oxidative/ exposure in some cell lines such as human vascular smooth
nitrosative stress. muscle cells (Lynn et al. 2000) and vascular endothelial
cells (Barchowsky et al. 1999), whereas other cell lines
Arsenic and oxidative stress revealed formation of hydrogen peroxide (Wang et al.
1996).
Various studies of arsenic toxicity have confirmed that ROS Indirect proof of arsenic-induced formation of free radi-
and RNS are formed in the course of inorganic arsenic cals is substantiated by the alterations in functioning of the
metabolism in cells (Shi et al. 2004). Arsenic causes struc- antioxidant enzymes such as catalase and superoxide dis-
tural changes in mitochondria resulting in a decrease in mutase (Wang and Huang 1994).

13
Arch Toxicol

An interesting mechanism of hydrogen peroxide forma- Arsenic interferes with signal transduction pathways via
tion involving the oxidation of As(III) to As(V) was pre- ROS directly inducing transcription factors (NF-kappaB,
sented (Valko et al. 2005) AP-1). NF-kappaB plays an important role in cell prolif-
eration and cell–cell interactions. The effects of arsenic-
H3 AsO3 + H2 O + O2 → H3 AsO4 induced formation or ROS on NF-kappaB are both dose
+ H 2 O2 (�r G� = −41 kcal/mol) (41) and cell type dependent. Low levels of arsenic (up to
10 μM) cause activation of NF-kappaB, while high doses
The above reaction is exergonic with an estimated standard of arsenic inhibit NF-kappaB via IKK.
Gibbs free reaction energy of –40.82 kcal/mol (−172 kJ/ Although low levels of arsenic-induced NF-kappaB
mol). Hydrogen peroxide may further be decomposed by increase keratinocyte proliferation, high levels of arsenic
traces of redox-active metals to hydroxyl radicals by means induce caspase activity of AP-1 expression (Lemarie et al.
of the Fenton reaction (see above). 2006). Arsenic-induced activation of NF-kappaB appears
Other than the formation of oxygen-derived free radi- to be independent of I-kappaB phosphorylation and subse-
cals, arsenic exposure can initiate the formation of reac- quent degradation.
tive nitrogen species (RNS). In this respect, several con- AP-1 is a heterodimeric protein composed of proteins
tradictory results regarding the formation of nitric oxide belonging to the fos and jun families (Leonard et al. 2004).
were reported. One study concluded that arsenite was Similarly to NF-kappaB, the activity of AP-1 changes with
able to inhibit inducible NO synthase gene expression in the level and exposure to arsenic (Drobna et al. 2003).
rats (Kodavanti et al. 1998). Another study concluded that Short-term exposure to arsenic leads to enhanced bind-
arsenic was not able to induce increase in formation of ing to DNA via c-fos and c-jun, while chronic exposure
nitric oxide in hepatocytes; however, arsenic was found to decreases DNA-binding efficiency of AP-1 making cells
inhibit inducible NO synthase gene expression in cytokine more tolerant to arsenic (Hu et al. 2002).
(Hughes 2002; Flora et al. 2008). While As(III) activates p53 significantly, MMA has no
effect on p53. Activation of p53 triggers apoptosis via DNA
Arsenic and signaling pathways and enzyme activity damage. This observation can be used for the development
of chemotherapeutic agents against cancer (Zhu et al. 2002).
Arsenic is known to modulate signal transduction pathways Arsenic was also found to interfere with a cell cycle
(Flora 2011). While arsenic toxicity depends on the exact which is regulated by a group of proteins called cyclins and
type of species and target cells, the common denominator cyclin-dependent kinases (CDKs). An important role of p53
in these processes is formation of free radicals. Since free on arsenic-induced cell cycle arrest was confirmed (Filip-
radicals are critical in the modulation of signal transduction pova and Duerksen-Hughes 2003); however, the results are
pathways, we now briefly discuss the mechanism of oxida- not fully convincing. Arsenic induces cell cycle arrest, pre-
tive stress-induced modulation of signaling pathways by dominantly at the G1 or G2-M phase (Park et al. 2000).
arsenic species. The apoptotic potential of arsenic was also the subject of
The main pathways affected by arsenic involve mitogen- various studies (Gupta et al. 2002). Arsenic mediates cell
activated protein kinases (MAPKs), transcription factors death not only in acute promyelocytic leukemia cells (APL)
(NF-kappaB and AP-1) and the tyrosine phosphorylation but also in myeloma cells, chronic myeloid leukemia cells
system. The tyrosine phosphorylation system is mediated and esophageal solid tumor cells. In addition, arsenic was
by a complex interplay of two major types of protein tyros- found to mediate cell death in ovarian and prostate cells
ine kinases: receptor tyrosine kinases (RTKs) and non- (Cheng et al. 2010). Glutathione was found to have an
receptor tyrosine kinases (NTKs) (Jomova et al. 2011). impact on apoptosis. Cells exposed to arsenic and insuffi-
Receptor tyrosine kinases include growth factor recep- cient in GSH synthesis exhibit reduced levels of Akt and
tors such as epidermal growth factor receptor (EGFR), c-fos and underwent apoptosis via ubiquitin–proteasome-
platelet-derived growth factor receptor and vascular mediated degradation. Cells under insufficient glutathione
endothelial growth (VEGF). Arsenic has been shown to conditions treated with arsenic show down-regulated heat-
up-regulate total cellular tyrosine phosphorylation with the shock proteins (Hsp90) at both transcriptional and post-
central target being phosphorylation of EGFR. Although transcriptional levels via ATM kinase and p-53-dependent
convincing evidence is still being sought, the direct inter- apoptosis (Habib 2009).
action of arsenic with EGFR involving sulfhydryl groups Low concentrations of arsenic were found to induce
(-SH) was proposed. In addition, an indirect mechanism apoptosis in human proximal tubule cell lines via a mito-
involving free radicals may cause structural changes to pro- chondrial-dependent pathway (Peraza et al. 2006). In
tein (Wu et al. 1999). conclusion, mitochondria-driven apoptosis appears to be

13
 Arch Toxicol

probable mechanism for arsenic-induced and free radical- Arsenic negatively affected the antioxidant defense sys-
mediated cell death. tem which was substantiated by the increased lipid per-
Arsenic significantly affects antioxidant enzymes oxidation and hepatic fibrosis (Santra et al. 2000). Chronic
including catalase, superoxide dismutase, glutathione per- arsenic exposure in some Indian regions led to observation
oxidase, glutathione S-transferase and glutathione per- of elevated hepatic enzymes, jaundice and hepatic fibrosis
oxidase. While short time exposure of low concentrations that cause portal hypertension but do not progress to cirrho-
of arsenic leads to increase in activity of these enzymes, sis (Waalkes et al. 2007).
long-term exposure resulted in a decline in the activity of ROS formation negatively altered the GSH/GSSG ratio
all these enzymes. In addition to those enzymes just men- and antioxidant enzymes and increased lipid peroxidation
tioned, arsenic is known to interfere with the activity of in hepatic tissues. In the course of arsenic metabolism,
thioredoxin reductase, NADPH oxidase and heme oxyge- conversion of As3+ to As5+ species under physiological
nase reductase (Shi et al. 2004). conditions leads to ROS formation. Injury to hepatocyte
NADPH oxidase appears to be an important target for mitochondria suggests mitochondria to be one of the prime
arsenic toxicity. Arsenic is known to induce up-regulation, targets of arsenic induced (Flora 2011).
and phosphorylation as well as membrane translocation of The kidney is another key target organ of arsenic toxic-
key subunits of NADPH oxidase (Cooper et al. 2009). Up- ity. The kidney and liver accumulate the highest amount of
regulation of NADPH oxidase is linked to DNA damage in arsenic following acute arsenic exposure. Similarly to the
vascular smooth muscle cells. Mutations of enzyme subu- mechanism which occurs in the liver, arsenic induces oxi-
nits due to the interaction with arsenic result in suppressed dative stress in renal microsomes, damages cellular lipids
immunity and development of congenital disease such as and proteins and finally leads in decline in the level of anti-
chronic granulomatous disease. oxidant enzymes (Ramanathan et al. 2003). Arsenic is able
Arsenic interacts with the molybdenum center of xan- to cause acute renal failure and chronic renal insufficiency.
thine oxidase which in turn leads to alterations in oxida- Chronic renal insufficiency is further linked with hyperten-
tion–reduction potentials which finally affects the EPR sion. Arsenic-induced oxidative stress may contribute to
spectrum of the enzyme. In contrast, the iron–sulfur center DNA damage that leads to mutagenesis and carcinogenesis.
of the enzyme is not affected by arsenic interaction; how- Arsenic poisoning further impacts on cardiovascular dis-
ever, the midpoint potential of the flavin adenine dinucleo- eases such as hypertension, peripheral arterial disease, ath-
tide was lowered (Boer et al. 2004). Generally, the arsenic– erosclerosis, coronary heart disease and stroke (States et al.
protein binding was linked with carcinogenesis. Arsenic 2009).
binds preferentially to sulfhydryl groups and competitively A positive link between arsenic exposure and ischemia
replaces zinc leading to inhibition of zinc-finger-dependent and carotid atherosclerosis was found (Chen et al. 1996).
enzymes. Zinc(II)-finger motifs are protein domains con- Arsenic-induced initiation of atherosclerosis can be char-
taining coordinated zinc(II), which binds at the cysteine acterized by the following facts. Arsenic is an oxidative
binding site. Arsenic–zinc finger interactions were associ- element in the cellular endothelium leading to activation
ated with several effects, especially carcinogenesis. Arse- of inflammatory mediators, and a reduced bioavailability
nic was also found to induce zinc(II)-finger inhibition most of nitric oxide contributing to activation of NF-kappaB
probably via oxidative stress. Since glutathione participates which in turn stimulates inflammatory processes. In addi-
in zinc homeostasis, arsenic-induced glutathione depletion tion, arsenic increases the expression of cyclooxygenase-2
indirectly affects zinc(II)-containing proteins (Witkiewicz- through formation of peroxynitrite and activation of NF-
Kucharczyk and Bal 2006). kappaB (Bunderson et al. 2004).
As we have already noted, arsenic was reported to
Arsenic and human disease induce hypertension. In several studies, a dose-dependent
relationship between arsenic intake and hypertension was
Arsenic-induced toxicity may involve alterations in the reported (Tseng et al. 2005). The underlying mechanism
integrity of the cellular genetic material by free radicals. for arsenic-induced hypertension is similar to that proposed
The primary site of arsenic metabolism is the liver, which for atherosclerosis and involves vascular redox signal-
is subject to interaction with various arsenic species, such ing. In addition, suppressed bioavailability of nitric oxide
that a link between arsenic exposure and oxidative stress- contributes to arsenic-triggered hypertension (Zalba et al.
induced hepatotoxicity was noted. MMAIII exhibits tight 2000).
binding with hepatocytes, followed by arsenite, DMAIII, The effect of arsenic on type 2 diabetes is questionable.
arsenate, MMAV and DMAV. Free radical-induced oxida- Arsenic was able to induce symptoms which mimicked
tive stress by arsenic can cause damage to cells via activa- type 2 diabetes. The metabolism of glucose leads to the for-
tion of signaling pathways (Cohen et al. 2006). mation of free radicals and represents an important signal

13
Arch Toxicol

for insulin secretion (Diaz-Villasenor et al. 2008). A low trioxide is used to treat a specific type of acute promyelo-
level of arsenic prevents proper β-cell functioning by acti- cytic leukemia (APL), albeit under special conditions. In
vating the adaptive response to oxidative stress. Subchronic the case of anticancer drugs, the cytotoxic action of arse-
low level of arsenic suppresses oscillations of free calcium nic proceeds via necrosis or apoptosis, whereas the basis
which is important for glucose-stimulated insulin secretion for the carcinogenic effect of arsenic is an uncontrolled
(Woods et al. 2009). proliferation.
The molecular mechanism of arsenic-induced neuro- Arsenic exhibits several mechanisms toward the exposed
logical defects is insufficiently established. Neurologi- cell, which may include oxidative stress via free radical
cal disorders related to arsenic exposure include memory formation, protein binding and DNA damage. A prooxidant
disturbances, cognitive impairments, visual defects and mechanism of arsenic action is generally accepted. Exten-
encephalopathies and neuropathies. Further symptoms sive formation of free radicals stimulates defense mecha-
include suppressed sensitivity, nausea and vomiting. nisms that suppress stress by terminating free radicals and
Histological results of samples taken from patients suf- repair the damaged biomolecules (Flora et al. 2007). While
fering of arsenic-induced neuropathy revealed fragmenta- a low level of arsenic activates certain signaling path-
tion and resorption of myelin and depleted myelinated fib- ways, a high level of arsenic inhibits the same pathways
ers (Rodriguez et al. 2003). In addition, in vitro and in vivo such as nuclear factor kappa B, angiogenesis and other
preclinical studies reported that 10 μM doses of arsenic mechanisms.
inhibit myelination and significantly reduce neurite growth Arsenic metabolites may form free radicals which inter-
in a dorsal root ganglia culture (Rodriguez et al. 2003). It act with proteins
was proposed that arsenic-induced demyelination may be
(CH3 )2 As + O2 → (CH3 )2 As· + O·− (42)
due to the decreased methylation of basic myelin protein at 2

the arginine residues (Zarazua et al. 2010). It appears that

(CH3 )2 As + O2 → (CH3 )2 AsOO· (43)
observed neurotoxicity results from arsenic interference
with other methylation pathways. Arsenic also induces Methylated arsenic is able to release iron from ferritin
electrophysiological changes which are in agreement with which may catalyze the formation of hydroxyl radicals by
histological changes showing demyelination and enhanced the decomposition of hydrogen peroxide via the Fenton
lipid peroxidation in peripheral nerves. reaction. Hydroxyl radicals are highly reactive toward the
The common denominator behind arsenic-induced heterocyclic DNA bases and the sugar moiety, at diffusion-
neurotoxicity is oxidative stress, which is supported by controlled rates. DNA modifications caused by arsenic-
in vitro and in vivo studies. Arsenic induces DNA damage induced free radical attack involve oxidation, methylation,
and apoptosis in neuronal cells which in turn may lead to depurination and deamination (Valko et al. 2007). The
increased intracellular calcium that triggers mitochondrial DNA repair mechanisms are inhibited by arsenic by alter-
stress and formation of free radicals (Florea et al. 2007). ing cellular redox state affecting signal transduction path-
Chronic arsenic exposure disturbs the cholinergic sys- ways or protein phosphorylation (Flora 2011).
tem, as evidenced by the synthesis, release and uptake of While DNA damage may cause tumor initiation, As-
acetylcholine. A decrease in the activity of acetylcholinest- induced ROS formation may support tumor promotion.
erase in various brain regions following arsenic exposure This may be achieved via secondary messengers modifying
was observed (Rodriguez et al. 2003). The distribution of signaling pathways, gene expression and transcription fac-
arsenic in the brain results in the redistribution of redox- tors. The As-triggered elevated levels of tyrosine phospho-
active copper and iron and redox-inactive zinc which is rylation are linked with abnormal cell signaling and abnor-
a probable mechanism for its neurotoxic effects (Rubio mal cellular growth and development of prostate, bladder,
et al. 2008). kidney, colon, skin and lung cancers (Kolibaba and Druker
Arsenic was closely linked with the incidence of can- 1997).
cer. Epidemiological trials concluded that arsenic may lead All these tissues are characterized by a high arsenic
to development of skin, bladder, liver and lung cancers turnover. Arsenic is deposited in the lung, liver and kerati-
(Meliker et al. 2010). The mechanisms of arsenic carcino- nized tissues such as hair, nails and skin. DMA is elimi-
genesis are as yet not fully understood. The environmental nated through the pulmonary tissue, where formation of
protection agency has classified arsenic as a group 1 carcin- free radicals may occur, mainly because of oxygen partial
ogen; however, safe limits of exposure were not specified pressure. The bladder lumen retains high concentrations
(Yoshida et al. 2004). There are several reasons for this, of pentavalent DMA and MMA and their trivalent species,
for example, the varying degree of toxicity and conflict- formed after reduction. Keratinized tissues such as skin
ing interpretations regarding a risk assessment model for are susceptible to damage because of high arsenic depo-
cancer incidence (Wang et al. 2002). Conversely, arsenic sition in these tissues. The most profound human arsenic

13
 Arch Toxicol

carcinogens include monomethylated and dimethylated tri- and other genes that play a role in apoptosis (Cannino et al.
valent arsenic species (Kitchin 2001). 2009). It appears that submicromolar concentrations of cad-
mium lead to delayed apoptosis, medium concentrations of
cadmium trigger apoptosis, and high concentrations of cad-
Cadmium mium (>60 μM) cause necrosis (Templeton and Liu 2010).
Cadmium interferes with cellular redox states (Cuypers
Cadmium (Cd) is a transition metal located in the d-block et al. 2010) which in turn leads to Cd-induced patholo-
of the periodic table. This element was very commonly gies. Cadmium is not a redox-active metal and therefore is
used as cathodic protection to prevent iron and steel from unable to form free radicals directly (Cuypers et al. 2010).
corroding too rapidly. Cadmium is also used in nickel–cad- Indirect mechanisms of Cd-induced oxidative stress involve
mium batteries. Cadmium is a toxic element and therefore the depletion of antioxidants, inhibition of antioxidant
should be handled with caution. Anthropogenic activities enzymes, displacement of redox-active metals and inhibi-
such as mining have altered the environment significantly, tion of electron transport chains in mitochondria (Cuypers
such that environmental contaminants including cadmium et al. 2010).
are widely distributed in air, water and soils, and therefore, The indirect mechanism underlying cadmium-induced
they will have an impact on human health (Hartwig 2012, oxidative stress is replacement of iron by cadmium, thereby
2013; Rani et al. 2014; Nair et al. 2013). increasing the content of free iron which may participate in
Cadmium compounds are highly soluble and therefore the catalytic decomposition of hydrogen peroxide, leading
are readily taken up by plants, rendering vegetables and to the formation of hydroxyl radicals. Hydroxyl radicals are
cereals as the main dietary source of cadmium. Cadmium reactive species able to induce the lipid peroxidation pro-
also occurs in tobacco, and so cadmium oxide formed dur- cess, finally producing malondialdehyde and hydroxynon-
ing smoking is deposited in the lungs or absorbed into the enal (Cuypers et al. 2010).
circulatory system. Cadmium interacts primarily with the highly abundant
The first experimental studies dealing with cadmium cellular antioxidant glutathione. Depletion of glutathione
toxicity were conducted in 1919; however, the chronic results in suppressed scavenging of cadmium which leads
effects of this metal on humans were recognized one or two to a disturbed cellular redox state.
decades later, when the first reports appeared of pulmo- Depletion of hepatic glutathione by diethyl maleate also
nary, bone and renal lesions caused by cadmium in indus- significantly enhances the cadmium-generated POBN radi-
trial workers (Bulmer et al. 1938; Friberg 1950). Cadmium cal adduct signals in the bile, suggesting that disruption of
is classified as a type I carcinogen by the International the cellular GSH system is a key mechanism for cadmium-
Agency for Cancer Research (Arroyo et al. 2012). induced oxidative stress in the liver (Liu et al. 2009).
Cadmium also affects the activity of various antioxidant
Cadmium toxicity and oxidative stress enzymes, for example, Cu, Zn-SOD, the activity of which
is changed following cadmium intoxication. The results
Administration of cadmium chloride caused a statistically show that the time of intoxication plays an important role
significant increase in lipid peroxidation and depletion of in protein inhibition. Cadmium-induced inhibition was also
the reduced form of glutathione in the liver of laboratory observed for glutathione peroxidase, glutathione reductase
animals, accompanied by elevated activity of alanine ami- and catalase (Casalino et al. 1997).
notransferase (Caisova and Eybl 1986). As mentioned above, cadmium interferes with the
It has been proposed that cellular cadmium uptake is mitochondrial electron transfer chain. The main target for
linked with dysregulation of transition metal ion homeosta- Cd inhibition is complex II and complex III, while weak
sis and some essential elements caused by their competi- inhibition was observed for complex I and complex IV.
tive displacement by cadmium. Cadmium taken up by the Cd interaction with complex III results in formation semi-
cells accumulates in the liver and binds to metallothioneins, ubiquinone which can form the superoxide anion radical
glutathione and other proteins (Thevenod 2009). Cadmium from molecular oxygen by electron transfer reaction.
binding to metallothionein can be considered as a detoxi- Cadmium significantly depletes selenium. Selenium
fying process which removes it from the cellular environ- interacts with cadmium, and the complex formed is elim-
ment, but by binding to thiol groups of metallothionein, inated from the body via the bile system. Thus, selenium
cadmium also participates in scavenging of free radicals deficiency results in the decreased synthesis of glutathione
that are formed as a result of cadmium-induced oxidative peroxidase which is one of the more important antioxi-
stress (Thevenod and Lee 2013; Hart et al. 2001). dants. As a result, increased signs of oxidative stress are
Cadmium is known to affect oxidative phosphorylation observed. An important role in the detoxification of cad-
in mitochondria and mitochondrial genes such as hsp60 mium is played by metallothionein; however, no effective

13
Arch Toxicol

therapy for cadmium poisoning has so far been adopted 2003). The remaining cells may become resistant to apop-
(Klaassen and Liu 1997). tosis. It has been reported that cells adapted to cadmium are
An important consequence of oxidative stress is the characterized by an enhanced resistance to apoptosis (Hart
phosphorylation of mitogen-activated protein kinases et al. 2001) which in turn leads to the accumulation of
(MAPK). Cadmium activates the MAPK pathways through mutations and neoplastic transformation. In fact, acquired
ROS formation (Qu et al. 2007; Chen et al. 2008). Thus, it resistance to apoptosis is a general feature of cancer (Hana-
may be concluded that while pathology behind Cd-induced han and Weinberg 2011).
toxicity is not completely known, the ability of cadmium to Cadmium induces apoptosis by interfering with the
induce oxidative stress is unequivocal. activity of stress-sensitive transcription factors (e.g., NF-
kappaB) responsible for expression of apoptotic genes
Cadmium and genotoxicity (Watkin et al. 2003). The link between cadmium and cal-
cium with respect to apoptosis was studied. It has been
Cadmium affects cell cycle progression, apoptosis, DNA proposed that cadmium enters into cells through voltage-
synthesis and other cellular activities (Aimola et al. 2012). dependent calcium channels and results in calcium release
Cadmium may also interfere with DNA repair causing from the endoplasmic reticulum. Released calcium induces
genomic instability associated with tumorigenesis (Wais- DNA fragmentation and apoptosis (Li et al. 2000a, b).
berg et al. 2003). Several different possible mechanisms of
action were proposed for cadmium, including the above- Cadmium and human disease
discussed oxidative stress, altered DNA methylation, acti-
vation of proto-oncogenes and disturbed gene expression Primary sites through which cadmium enters the body
(Beyersmann and Hechtenberg 1997). involve the gastrointestinal tract and lungs. Cadmium
Probably the most profound carcinogenic risk of cad- enters the lung via occupational exposure, smoking and
mium is linked with its ability to inhibit DNA damage dust inhalation (Hogervorst et al. 2007). Smoking is one of
repair mechanisms (Giaginis et al. 2006). Cadmium is a co- the major sources of cadmium intoxication. Cadmium con-
mutagen and synergistically increases the mutagenicity of tained in the fumes has been shown to cause acute respira-
UV radiation and oxidation of biomolecules. Examples of tory distress syndrome (Barbee and Prince 1999).
cadmium inhibition include base excision repair, nucleotide Cd is able to trigger apoptosis in lung epithelial cells
excision repair, elimination of 8-oxo-guanosine (one of the of laboratory animals, most probably via a free radical-
most common DNA lesions) and mismatch repair. A low induced mechanism. A free radical-induced mechanism
concentration of cadmium producing mild oxidative dam- was proposed on the basis of fourfold increase in the level
age was found to inhibit repair of oxidative DNA damage of oxidized glutathione GSSG following cadmium expo-
in mammalian cells (Dally and Hartwig 1997). sure (Nair et al. 2013).
The lesions on apurinic/apyrimidinic sites are common Experiments using laboratory animals revealed that cad-
in any cell and must be repaired to prevent mutagenesis. It mium is responsible for pulmonary oxidative stress, asso-
was shown that cadmium inhibits (Candéias et al. 2010) the ciated with permanent airway inflammation. Nrf2 controls
repair of uracil in DNA resulting both from mis-incorpo- more than 100 genes responsible for cellular antioxidant
ration and cytosine (C) deamination. Thus, the genotoxic defense. Recent results suggested that antioxidant tran-
consequence of cadmium exposure appears to involve the scription factor Nrf2 is involved in a variety of responses in
deregulation of normal cellular processes by disturbing various stages of lung injury caused by smoking (Tuder and
gene expression (repair of uracil) and enhancing rate of Petrache 2012). An increased incidence of lung cancer has
mutations (on apurinic/apyrimidinic sites) interfering with been observed in population-based studies of environmen-
the normal control of cell growth and division. tal exposure to cadmium (Nawrot et al. 2006). A positive
Deficiencies in repair systems can lead to malignant correlation was found between the incidence of lung cancer
growth. Genetic defects and genomic polymorphisms and the urinary cadmium level.
in genes controlling DNA repair are tightly linked with Renal dysfunction by cadmium exposure has been well
human cancers (Nohmi et al. 2005). Broken DNA repair documented in a study from cadmium-polluted areas in
mechanism and permanent DNA damage result in genomic Belgium (Jarup 2002). A positive correlation between tubu-
instability and/or imperfect apoptosis. lar dysfunction and cadmium excretion was found. Other
Apoptosis is observed in cadmium-treated cells and trials reported increased mortality of populations living in
therefore can be considered to have an anticancer func- cadmium-polluted areas (Iwata et al. 1992).
tion. Since only a small number of cells undergo apoptosis, All these studies suggest a positive correlation between
cadmium-triggered apoptosis might not be completely pro- chronic exposure to cadmium and nephrotoxicity. Early
tective against malignant transformation (Waisberg et al. stages of the disease are characterized by changes in

13
 Arch Toxicol

cell–cell adhesion and disrupted cellular signaling before Reproductive anomalies and impaired testicular func-
the occurence of necrosis/apoptosis (Prozialeck and tions can be associated with cadmium exposure. Cadmium
Edwards 2010). causes damage to vascular endothelium of testis. In addi-
Cadmium is transported to the liver and stimulates the tion, an impaired activity of antioxidant enzymes was
synthesis of metallothionein which reacts with cadmium observed.
and attenuates its toxic effects. Cadmium–metallothionein Cadmium negatively affects the hormonal activity of
is not toxic to most of the organs and represents a transfer prostate function in men (Sarkar et al. 2013). Cigarette
route for cadmium from the liver to the kidney (Klaassen smoke containing high levels of cadmium is linked with
and Liu 1997). smaller size of testes and decreased sperm volume (Juraso-
The acute hepatotoxicity is characterized by the direct vic et al. 2004).
effect of cadmium (initial injury) followed by the injury due Cadmium cannot penetrate the blood brain barrier
to inflammation. Initial injury involves cadmium binding to (BBB) of adults; however, it might diffuse across the
sulfhydryl groups of proteins in mitochondria. Saturation BBB with the help of a supporting carrier such as etha-
of protein thiol groups leads to the occurence of oxidative nol (Pal et al. 1993). Cadmium can penetrate the BBB
stress and mitochondrial dysfunction. Hepatocellular injury in newborns and during the developmental stage of an
caused by cadmium may involve ischemia and activation organism (Sinha et al. 2008). Industrial workers exposed
of Kupffer cells and multiple events including inflamma- to cadmium may develop amyotrophic lateral sclero-
tory and cytotoxic mediators (Rikans and Yamano 2000). sis as a consequence of increased oxidative stress and
Activated Kupffer cells can release various inflammatory decreased SOD activity in the brain. Cadmium injected
mediators (free radicals, nitric oxide and cytokines), some into laboratory animals led to formation of free radicals
of which have been recognized to stimulate the expression in the brain causing spatial-specific membrane altera-
of adhesion molecules that can initiate a cascade of cellu- tions that affected membrane fluidity and the intracellular
lar responses leading to inflammation and secondary liver concentration of calcium (Kumar et al. 1996). Cadmium
damage during Cd-induced hepatotoxicity (Yamano et al. results in a decrease in glutathione level (GSH/GSSG
2000). ratio) in the midbrain and hippocampus (Shukla et al.
Being a non-redox metal, cadmium-induced oxida- 1988). Cadmium also induced lipid peroxidation and
tive stress is achieved by depleting cellular glutathione. In protein carbonylation in mice that were fed with 4 mg of
addition, cadmium competes with various metals, such as cadmium chloride for three days (Sinha et al. 2008). An
zinc, copper and calcium (Martelli et al. 2006). Cd affects increased level of free radicals, reduced glutathione pool
enzyme activities, DNA repair system, cellular redox state and reduced activity of catalase, glutathione reductase,
and signal transduction. superoxide dismutase and other enzymes were observed.
Cadmium interferes with homeostasis of redox-active Interestingly, vitamin C and taurine showed a protec-
metal ions (iron, copper) and non-redox metal zinc. Cad- tive effect against cadmium-induced toxicity. Vitamin C
mium and zinc are both non-redox metals of the same reversed cadmium-induced apoptosis in cortical neurons
group, and both have stable divalent oxidation states. Sub- (Lopez et al. 2006).
stitution of copper and zinc by cadmium is regarded as the According to the International Agency for Research
key molecular mechanism of cadmium-induced hepatotox- on Cancer and by the US National Toxicology Program,
icity (Arroyo et al. 2012). cadmium is classified as a human carcinogen. The effect
Cadmium has various detrimental effects on mamma- of cadmium on lung cancers was confirmed by experi-
lian reproduction, mainly as an endocrine disrupting ele- mental and epidemiological trials (Waalkes 2003). A
ment. Cadmium is known to enhance or inhibit the biosyn- clear correlation between cadmium exposure and pros-
thesis of progesterone. Progesterone is a steroid hormone tate, kidney, breast, bladder and pancreas cancer was
involved in the pregnancy, embryogenesis and menstrual established (Amaral et al. 2012). A less convincing cor-
cycle of humans and other species (Chedrese et al. 2006). relation was found for the incidence of stomach and liver
Intoxication with cadmium and increased placental content cancers (Filipic 2012). The common denominator for
of cadmium negatively affected birth weight and premature cadmium-induced cancer is oxidation stress and inhibi-
birth. In mammalian cell cultures, cadmium was found to tion of the repair of DNA damage (Qu et al. 2005). In
induce the expression of estrogen genes, triggering activa- addition, Cd-induced formation of ROS which in turn
tion of cytoplasmic kinases. affects various signaling pathways plays an important
Cadmium tends to accumulate in embryos from the four-cell role in the development of malignancies. The process of
stage onward. Enhanced cadmium exposure inhibits cell pro- cadmium-induced carcinogenesis is based on alterations
gression to the blastocyst stage and can induce apoptosis and in redox state of the cell and the role of ROS in the neo-
breakdown in cell adhesion (Thompson and Bannigan 2008). plastic transformations.

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Arch Toxicol

Zinc Biological roles of zinc

Zinc is one of the most important trace elements found in There are three important roles played by zinc in a range of
all body tissues and secretions in relatively high concen- biological processes. They include catalytic, structural and
trations. The average amount of zinc in the adult body is regulatory functions.
approximately 1.5–2.5 g. About 85–90 % of total body Catalytic role of zinc is substantiated by the action of
zinc is found in skeletal muscle and bone, 8–10 % in the zinc-containing enzymes controlling many cellular pro-
skin and the liver and the rest of the zinc in other tissues cesses including DNA synthesis, brain development, nor-
(Chasapis et al. 2012). The level of free intracellular zinc mal growth, reproduction, membrane integrity, bone forma-
is as low as 0.5 nM as it was estimated from zinc specific tion and wound healing (Barceloux 1999).
19 Electronic structure and physicochemical properties
F NMR signal of a fluorinated metal probe (Benters et al.
1997). The most common and most stable oxidation state of zinc predetermine it as a key element in structural and
of zinc is +2 [Zn(II)]. Compounds containing zinc(I) are functional roles in various proteins involved in replication
rare and require bulky ligands to stabilize the low oxidation of DNA and other important biological processes (Mocche-
state. giani et al. 2000a, b). Zinc ions are hydrophilic and do not
In the early 1960s, Prasad and collaborators found that cross cell membranes by passive diffusion. In addition, zinc
low levels of zinc in blood plasma were related to a rare ions are involved in expression of genetic information, syn-
condition of dwarfism, susceptibility to infections and thesis and storage of peptide hormones (Tapiero and Tew
testicular retardation in a group of patients who were on 2003).
a diet poor in zinc (Prasad et al. 1963). Since this find- The regulatory role of zinc involves enzymatic activ-
ing, zinc is considered to be an essential component of ity and the stability of the proteins as an activator or as an
the human diet. Zinc is not accumulated in any particu- inhibitor ion. Zinc is known to modulate cellular signaling
lar storage organ, so regular intake of zinc-rich foods is pathways and to function as a neurotransmitter in the case
required. of zinc-containing neurons found almost exclusively in the
Symptoms of zinc deficiency include poor growth and forebrain (Beyersmann 2002).
development (see above), loss of appetite, dermatitis, Zinc participates in the regulation of cell proliferation
delayed wound healing, impaired reproduction and poor in several ways. The regulation of cell proliferation by
immune function (Bray and Bettger 1990). Severe zinc zinc can occur at different levels. These involve the direct
deficiency is rare and usually caused by genetic or acquired requirement of zinc for the activity of enzymes involved in
conditions (Roth and Kirchgessner 1994). DNA synthesis and signal regulations as well as the indi-
Zinc is important for cell membrane integrity, and it rect activity via the effect of zinc on hormonal regulation of
helps manage insulin action and has an essential role in cell division (Wong et al. 2007; Corniola et al. 2008). How-
the maintenance of the immune system. Zinc is an inte- ever, the role of zinc deficiency associated with suppressed
gral component of more than 70 different metalloen- cell proliferation is still a controversial issue requiring fur-
zymes that function in chemical reactions, metabolism, ther research and investigations (Ninh et al. 1998).
digestion, immune response and many other processes. Under physiological conditions, majority of intracellular
Zinc is known to modulate cellular signal transduction zinc is protein bound and its distribution is approximately
pathways and to function as a modulator of synaptic 50 % in the cytoplasm and 40 % in the nucleus (Vallee and
neurotransmission. Auld 1993). Zinc homeostasis is maintained by two pro-
Acute zinc toxicity is a rare condition, but has been tein families: (1) the zinc-importer family of proteins (ZIP)
reported. It has occurred as a result of food and beverage required for an efficient allocation of zinc to cytosol and (2)
intake contaminated with zinc from galvanized contain- zinc transporter family (ZnT) containing proteins removing
ers (Duncan et al. 1992). Intakes of 2 g or more of zinc zinc ions from the cytosol (Lichten and Cousins 2009).
salts can cause irritation of gastrointestinal tract. Symp- ZIP proteins contain eight potential transmembrane
toms of acute zinc toxicity include nausea, vomiting, domains and a similar membrane topology in which the N-
lethargy, diarrhea, muscle pain and fever. More com- and C-terminal ends of the protein are located at the outside
mon is chronic zinc toxicity (100–300 mg/day) which surface of the plasma membrane. ZIP proteins contain from
may disturb immune functions. Excess of zinc may also 309 to 476 amino acids; the length difference is mainly due
affect copper and iron status and lower plasma HDL to the length between transmembrane domains III and IV
concentrations. Zinc is excreted mostly through feces (the “variable region”). The variable region is rich in his-
(12–15 mg/day) and lesser amounts (0.5 mg/day) are tidine residues and contains a metal-binding domain that
eliminated in urine. is predicted to be cytoplasmic. A number of the C. elegans

13
 Arch Toxicol

ZIP proteins have potential metal-binding site located in humans has also been made (Prasad et al. 2007). These
positions other than the variable region. Transmembrane findings support the proposal that zinc deficiency may rep-
domain IV of the ZIP family of proteins is the most con- resent an important factor contributing to the progression of
served histidine portion of the ZIP family (Eng et al. 1998). cancer (Ho 2004).
In contrast, ZnT proteins regulate the extrusion of zinc Increased oxidative stress may emerge from declined
ions outside the cytoplasm. They belong to the cation diffu- activity of key antioxidant enzymes containing zinc, for
sion facilitator family, and majority of them share six mem- example, Cu, Zn-SOD. In addition, Zn–metallothionein
brane-spanning domains and a histidine-rich intracellular complex has recently been shown to possess antioxidant
loop (Devergnas et al. 2004). Seven homologous (ZnT1-7) activity (Maret 2003). Competition of zinc with redox-
zinc export proteins have been identified. ZnT-1 is the only active metals such as copper and iron may also play a role
transporter located in the plasma membrane that transports in oxidative stress-mediated damage to biomolecules. Zinc
zinc efflux from the cell. The remaining ZnT transport- may bind and protect sulfhydryl groups of proteins repre-
ers assist with zinc sequestration into vesicles called zin- senting thus another antioxidant mechanism of action of
cosomes (Haase and Maret 2003; Taylor et al. 2008). zinc.
Metallothioneins (MTs) are another group of metal- Zn deficiency resulting from long-term decreased intake
binding, cysteine-rich, low molecular weight proteins, of zinc has been revealed in both humans and laboratory
which belong to the family of intracellular metal-binding animals and is linked with increased markers of oxidative
proteins responsible for metal homeostasis (Chimienti damage, such as oxidation of DNA, protein oxidation and
et al. 2003). MTs have high affinity for copper and zinc. peroxidation of lipids (Jomova and Valko 2012).
They bind them and support their detoxification, thus mak- Recent results have shown that metallothioneins repre-
ing protection against oxidative stress and suppressing cell sent a link between zinc levels and redox state of the cell.
death by apoptotic pathways. MTs bind about one-fifth Increased oxidative stress causes fluctuations in redox state
of the intracellular zinc (Stefanidou et al. 2006) and play of the cell and results in release of zinc from metallothio-
a significant role in zinc absorption by competing with or nein as a consequence of altered sulfide/disulfide ratios
supplying zinc to a variety of transporter proteins (Vasak (Maret 2008).
and Hasler 2000). One molecule of metallothionein can Blood brain barrier (BBB) is highly specialized blood
bind seven zinc ions (Kelly et al. 1996). MTs are able to vessel system of CNS that serves to protect the brain by
scavenge hydroxyl radicals very efficiently providing thus excluding the toxic agents (Valko et al. 2005). It is known
protection against radical-induced oxidative stress (Sato that polyunsaturated fatty acids of BBB membranes are
1992). prone to ROS attack. Magnetic resonance imaging of zinc-
deficient laboratory animals has shown that zinc deficiency
Zinc and oxidative stress increases permeability of the BBB and induces oxidative
stress. These results concluded that under normal condi-
Several studies reported that zinc deficiency increases pro- tions antioxidant properties of Zn protect the BBB against
duction of free radicals (Fig. 9). Most of these studies have free radical-induced damage and attenuate the development
been performed using zinc-deficient cell cultures (Eide of neurological disorders.
2011). Increased formation of ROS and resulting damage
has been observed in animals fed low dose zinc. It has been Zinc and human disease
shown that zinc-deficient rats exhibited increased lipid
peroxidation in lung microsomes (Sullivan et al. 1980). As already discussed above, zinc is integral to various
These results were confirmed and extended to other types enzymes and transcription factors that regulate key cellular
of tissues (Bray et al. 1986). Extension of these results to functions such as DNA replication, repair of DNA damage,
cell cycle progression and apoptosis. Reduced availability
of zinc due to the zinc deficiency could result in a loss of
Enzymes containing activity of zinc-containing proteins involved in the mainte-
Zinc (Cu, Zn-SOD)
Metallothionein nance of DNA integrity and may contribute to the cancer
Zinc Free incidence (Dhawan and Chadha 2010).
radicals
Various dietary components including zinc have been
Fenton reaction -
hydroxyl radicals proposed to exhibit anticancer properties. The content of
NADPH oxidase activity zinc in serum and malignant tissues of patients suffering
from various types of cancer exhibits significant anomalies.
Fig. 9  Sources of free radicals in zinc-deficient cells (adapted from This points to involvement of zinc in cancer development
Eide 2011) (John et al. 2010).

13
Arch Toxicol

Zinc deficiency is known to up-regulate expression of during reperfusion led to full myocardial recovery and
the tumor suppressor protein (p53) but impair the DNA- twofold decrease in cardiac arrhythmias (Karagulova et al.
binding ability of p53, NF-kappaB, as well as AP-1 tran- 2007). Beneficial role of zinc homeostasis substantiated by
scription factors in rat glioma cells (Ho and Ames 2002). the higher levels of zinc transporter ZnT1 has been shown
Based on this and other studies, researchers suggested that in patients with atrial fibrillation (Beharier et al. 2007).
a decrease in content of cellular zinc causes DNA damage Increased expression of ZnT1 has been found in turn to
and impairs DNA repair mechanisms resulting in a loss of inhibit L-type calcium channel.
DNA integrity and potential cancer risk. Various key zinc metallopeptidases such as angiotensin-
Patients with breast (Schlag et al. 1978), gallblad- converting enzyme are suitable targets for the treatment of
der (Gupta et al. 2005), lung (Issell et al. 1981), colorec- a variety of cardiovascular disease. The knowledge of the
tal, head and neck (Buntzel et al. 2007) cancers have been structure–activity relationships of the metallopeptidases is
demonstrated to have reduced serum levels of zinc. While of great importance in the design of highly specific thera-
serum levels of zinc are lower in most of the cancers, peutic agents.
tumor tissues of lung and breast cancers revealed increased Aging is a continuous and complex biological process of
amounts of zinc (Margalioth et al. 1983). Up-regulated living systems accompanied with biochemical and physi-
cellular zinc importers found in many cancer tissues are ological changes. Aging is linked with the immunological
in agreement with increased zinc concentrations in most decline of an organism, including neuroendocrine func-
tumors. Exceptions to these results are prostate and skin tions and increase in apoptosis regulated by zinc deficiency.
cancer tumor cells indicating lowered zinc content with Both low level of zinc and high level of metallothioneins
respect to normal cells (Costello and Franklin 2006). represent risk factors for infection relapses in the elderly.
Chemoprevention of zinc includes the effects of zinc on Dysfunction of the elderly organism results in decreased
the inhibition of terminal oxidation, induction of mitochon- response to external harmful stimuli and the appearance
drial apoptogenesis and suppressed activity of NF-kappaB of age-related degenerative disease such as infections and
activity. Zinc plays an important role in the maintenance of cancer (Mocchegiani et al. 2000a, b). Zinc affects thyroid
DNA integrity in normal prostate cells by modulating both hormones receptors and cognitive functions (Darling et al.
DNA damage and repair proteins, most significantly p53 1998; Weiss et al. 2000). The supply of zinc is considered
(Yan et al. 2008). In addition, the role of zinc transporters necessary in aging which in turn leads to improved cogni-
as tumor suppressors in prostate cells has been observed tive functions, immune functions, stress response and age-
(Franklin et al. 2005). These results provide a basis for the related neurodegenerative disorders (Mocchegiani et al.
concept that restoration of zinc pools in malignant cells 2011).
could represent an efficient way in the treatment and pre- Diabetes greatly increases the risk of heart disease and
vention of cancer. stroke. The link between zinc and insulin has been known
Cardiovascular disease involves a number of conditions for decades. Zinc binding to insulin is important for the
affecting the function of the heart. Cells of cardiovascular crystallization of the zinc–hormone complex (Dodson
system interact with zinc. Endothelial cells rapidly take and Steiner 1998) and adequate amounts of stored insu-
up zinc ions, probably by albumin which contains a high- lin in pancreatic b-cells to maintain its sufficient release.
affinity binding site for zinc. Plasma zinc levels tend to A complex relationship between zinc and both type 1 and
decrease with age which in turn correlates with increased 2 diabetes has been proposed. Diabetes is characterized by
incidence of cardiovascular disease (Little et al. 2010). increased levels of oxidative markers and decreased intra-
Ischemic cardiomyopathy usually leading to heart failure cellular zinc and zinc-containing antioxidant enzymes.
has been subject of various studies taking into account Zinc transporter ZnT8 is involved in the control of insulin
the status of the trace elements (Shokrzadeh et al. 2009). secretion and may play a role in the development of type
The results of these studies showed a higher level of cop- 2 diabetes, making this transporter a promising therapeutic
per and lower level of zinc in patients with cardiomyopa- target for pharmacological intervention (Rutter 2010).
thy. Similar trend was found in patients with type 2 diabe- Considerable evidence exists that zinc plays important
tes and congestive heart failure (Shokrzadeh et al. 2009). roles in neurological disorders (Wang et al. 2010). Particu-
It has been suggested that heart failure is a syndrome with larly, the role of zinc in the etiology of Alzheimer’s disease
nutritional deficiency of magnesium, copper, selenium and is questionable. As mentioned in the paragraph devoted to
zinc (Witte et al. 2001). The cardioprotective role of zinc copper, the main feature of AD is a marked accumulation
has been confirmed in a rodent model of ischemia/reperfu- of amyloid-β (Aβ), one of the pathological hallmarks of
sion injury. The results have shown that depleted levels of AD. A large body of evidence including marked accumu-
zinc in ischemic/reperfusion with the subsequent adminis- lation of zinc found in AB plaques has demonstrated that
tration of zinc (in the form of zinc ionophore pyrithione) aberrant zinc homeostasis is involved in the pathogenesis of

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Fig. 10  Various sources of ROS, damage to biomolecules and incidence of chronic disease

AD (Atwood et al. 1999; Bush 2000). Aβ peptide has high- resulting in improvement of some clinical symptoms of the
affinity binding sites for zinc (and also for copper), and patients (Huster 2010).
zinc is the only metal able to precipitate Aβ. The abnormal In the context of Alzheimer’s disease, zinc has a clear
abundance of zinc in Alzheimer’s brain indicates that zinc relationship with copper. Protective role of zinc is substan-
binding to amyloid-β plays a role in the formation of amy- tiated by its competition with copper ions (or iron ions) to
loid plaques. Specific chelators of zinc, such as clioquinol, bind amyloid-β. Zinc bound to Aβ changes its conforma-
have been found to be effective in preventing formation of tional state to the extent that copper ions cannot bind to the
amyloid plaques (Bush 2002). designed binding sites. Preventing copper from interaction
The disturbed zinc homeostasis in Alzheimer’s brain is with amyloid-β may preclude the catalytic activity of Cu–
linked with the abnormal distribution and altered expres- amyloid-β complex to produce damaging hydroxyl radicals
sion of metallothionein, zinc transporters and divalent by means of Fenton reaction.
metal transporter 1 (DMT1), collectively known as zinc-
regulated metalloproteins. Metallothioneins protect neurons
against oxidative stress. It was found that extracts from AD Conclusions
tissues contained deficient amounts of MT-3 (Coyle et al.
2002). In addition, increased levels of DMT1 and ZnT1, Convincing experimental evidence exists that ROS and
3-7 proteins were found in the amyloid-β plaques of AD RNS may play a significant role in the regulation of a vari-
brains (Zhang et al. 2010). Interestingly, long-term intake ety of cellular processes. Formation of ROS/RNS is signifi-
of water containing high levels of zinc accelerated deposi- cantly determined by the action of redox-active and redox-
tion of amyloid-β. These data support the hypothesis that inactive metals.
zinc overload is tightly linked with the pathophysiology of Toxic metals are capable of interacting with proteins
Alzheimer’s disease (Wang et al. 2010). and DNA causing site-specific damage. The direct dam-
Wilson’s disease is a rare inherited disorder that causes age involves conformational changes to bio-macromole-
too much copper to accumulate in liver, brain and other cules due to the coordination of metals. The indirect dam-
organs leading to hepatic damage and neurological dis- age involves metal-mediated formation of ROS and RNS;
turbances. Pharmacological therapy is based on the use of redox-active transition metal compounds may catalyze
copper chelators and zinc compounds (zinc acetate) (Prasad Fenton-type reactions through which free radicals includ-
2009). The major role of zinc compounds is the induc- ing hydroxyl radicals can cause damage to lipids, proteins
tion of intestinal and hepatic synthesis of metallothioneins and DNA.

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A shift in the balance between oxidized and reduced bio- effective classes of antioxidants is thiol compounds, espe-
molecules within the cells, in favor of the former, is termed cially glutathione, which provide significant protection by
oxidative stress that has been recognized as a contributing trapping ROS and help to maintain the redox state of the cell.
factor in the toxicity of a large number of toxins includ- As discussed above, metal-induced oxidative stress is
ing toxic metals. The sensitive marker for characterization a common denominator of many diseases and results partly
of oxidative stress is the glutathione/glutathione disulfide from decreased antioxidant mechanisms (Fig. 10). The
redox couple mainly because of its high concentration and design of dual-functioning antioxidants possessing both
direct role(s) as an efficient cellular antioxidant. metal-chelating and ROS/RNS-scavenging properties is
Metal-induced formation of ROS/RNS has most sig- awaited.
nificantly been observed for iron and copper (both essen-
tial elements). Formation of the superoxide radical anion is Acknowledgments We thank The Slovak Grant Agency—
VEGA (#1/0765/14) and University Hradec Kralove (Long-Term
followed by its reaction with oxidized forms of metals and Development Plan) and Ministry of Health of the Czech Republic
subsequently by Fenton chemistry leading to formation of (FNHK00179906) for financial support.
hydroxyl radicals
O2 + e− → O·−
2 (44)
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