carcinogenic metal compounds recent insight into molecular and cellular mechanisms

Arch Toxicol (2008) 82:493512DOI 10.1007/s00204-008-0313-yREVIEW ARTICLE

Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms

Detmar Beyersmann Andrea Hartwig

Received: 15 April 2008 / Accepted: 30 April 2008 / Published online: 22 May 2008 Springer-Verlag 2008

Abstract Mechanisms of carcinogenicity are discussed formetals and their compounds, classiWed as carcinogenic tohumans or considered to be carcinogenic to humans: arsenic,antimony, beryllium, cadmium, chromium, cobalt, lead,nickel and vanadium. Physicochemical properties governuptake, intracellular distribution and binding of metal com-pounds. Interactions with proteins (e.g., with zinc Wngerstructures) appear to be more relevant for metal carcinoge-nicity than binding to DNA. In general, metal genotoxicity iscaused by indirect mechanisms. In spite of diverse physico-chemical properties of metal compounds, three predominantmechanisms emerge: (1) interference with cellular redox reg-ulation and induction of oxidative stress, which may causeoxidative DNA damage or trigger signaling cascades leadingto stimulation of cell growth; (2) inhibition of major DNArepair systems resulting in genomic instability and accumula-tion of critical mutations; (3) deregulation of cell prolifera-tion by induction of signaling pathways or inactivation ofgrowth controls such as tumor suppressor genes. In addition,speciWc metal compounds exhibit unique mechanisms suchas interruption of cellcell adhesion by cadmium, directDNA binding of trivalent chromium, and interaction of vana-date with phosphate binding sites of protein phosphatases.

Keywords Carcinogenic metals Mechanisms Genotoxicity Oxidative stress DNA repair Deregulation of cell proliferation

Introduction

A decisive step in the development of human technology andculture was the discovery of metals below the surface of ourplanet, their excavation, extraction and use as tools to fulWllhuman needs. Nowadays, we recognize that the wastes ofmetals are distributed over the soils and waters of the earthssurface and exert detrimental eVects on life in the environmentand human health. Unlike organic waste, metals and theircompounds are not degraded by living organisms and mayaccumulate up to harmful levels. Metals are small entitieswhen compared to organic materials and their reactions withliving matter are seemingly simple to evaluate. However, thepicture emerging today shows a very complex pattern of metalinteractions with cellular macromolecules, metabolic and sig-nal transduction pathways and genetic processes. Some metalcompounds even undergo metabolic transformation, such asreduction to lower oxidation state or alkylation. Hence, thetoxicological assessment of metal eVects is by no means sim-ple, which is especially true for mechanisms of metal carcino-genicity. Even for single metal species, the hitherto revealedmechanisms involved in their carcinogenic action are multi-ple. A special feature of metal biology is the fact that evenmetals that are essential for the sustainment of life (such asiron and copper) may become toxic depending on the oxida-tion state, complex form, dose and mode of exposure. In thisreview, Wrst we depict the possible common mechanisms ofmetal carcinogenicity without neglecting the speciWc diVer-ences and then discuss the peculiarities of speciWc metals.

Overview over classiWcation as carcinogens

Carcinogens are classiWed by both scientiWc committeesand regulatory agencies. The following assignments refer to

D. Beyersmann (&)Biochemistry, Department of Biology and Chemistry, University of Bremen, 28334 Bremen, Germanye-mail: [email protected]

A. HartwigInstitute of Food Technology and Food Chemistry, Technical University of Berlin, 13355 Berlin, Germanye-mail: [email protected]

494 Arch Toxicol (2008) 82:493512evaluations of scientiWc commissions only, since legal clas-siWcations depend on national policies. Table 1 summarizesthe classiWcation of some metals and metalloids by theInternational Agency for Research on Cancer (IARC) andthe German Commission for the Investigation of HealthHazards of Chemical Compounds in the Work Area (MAKcommission). The IARC classiWes the carcinogens accord-ing to carcinogenic hazard as carcinogenic to humans(Group 1), probably carcinogenic to humans (Group 2A),possibly carcinogenic to humans (Group 2B) or not classiW-able as to its carcinogenicity to humans. The GermanMAK-commission also has classiWed several metals eitheras carcinogenic to humans (category 1), as considered to becarcinogenic to man based on long-term animal studies(category 2) or as giving concern to cause cancer, but evi-dence is not suYcient for classiWcation in group 1 or 2 (cat-egory 3B) (DFG 2007a).

Physicochemical aspects

Metals may be carcinogenic in the form of free ions, metalcomplexes, or particles of metals and poorly soluble com-pounds. The toxicity of metals and their compounds is gov-erned by their physicochemical properties. Regarding metal

ions, oxidation state, charge and ionic radii are crucial. Withmetal complexes, the coordination number, the geometry andthe type of ligands (e.g., their lipophilicity) are important fortoxic interactions. Regarding metals and their poorly solublecompounds, particle size and crystal structure are important.Not only toxic metal cations, but also essential transitionmetal ions bind to biological ligands of opposite charge, suchas acid amino acid side chains of proteins and phosphategroups of nucleotides and nucleic acids, and form complexeswith oxygen, sulfur and nitrogen groups of proteins, nucleicacids and other biomolecules. If toxic metal ions have similarphysiochemical properties such as charge and size as those ofessential ions, they may compete for the biological bindingsites of the latter and cause structural perturbations resultingin aberrant function of biochemical macromolecules (Beyers-mann 1995; Nieboer et al. 1999; Hartwig 2001). Some exam-ples are discussed in more detail here.

Be2+ carries the same charge as Mg2+ and competes forMg2+ in biochemical binding sites. However, its ionic radiusin hexacoordination (0.45 ) is much smaller than that ofMg2+ (0.72 ), hence it polarizes ligands more strongly, andits bonds with proteins are more stable than those of Mg2+.

Cd2+ ions have ionic radii very similar to those of Ca2+(in hexacoordination 0.95 and 1.00 , respectively, in octa-coordination 1.10 and 1.12 , respectively). Although the

Table 1 ClassiWcation of met-als and/or their compounds as carcinogenic)

Substances IARC carcinogen group

MAK carcinogen category

Antimony and its compounds 2 (except SbH3)Antimony trioxide (Sb2O3) 2B 2Antimony trisulWde (Sb2S3) 3 2Arsenic and its compounds 1 1Beryllium and its compounds 1 1Cadmium and its compounds 1 1Chromium metal 3 Chromium(VI) compounds 1 2 (except ZnCrO4: cat. 1)Chromium(III) compounds 3 Cobalt and its compounds 2B 2Cobalt with tungsten carbide

(hard metal)2A 1

Gallium arsenide 1 Indium phosphide 2A 2Lead metal 2Lead compounds 2A 2Mercury and its compounds 2B 3BNickel metal 2B 1Nickel compounds 1 1Rhodium 3BSelenium and its compounds 3 3BVanadium and its compounds 2Vanadium pentoxide (V2O5) 2B 2

Origin: International Agency for Research on Cancer (IARC) Monographs, MAK (German Commission for the Investiga-tion of Health Hazards of Chem-ical Compounds in the Work Area). (, not classiWed)123

Arch Toxicol (2008) 82:493512 495preferred ligand of Ca2+ is oxygen, whereas Cd2+ preferssulfur, Cd2+ also accepts oxygen and is able to substituteCa2+ in protein binding sites. Cd2+ interferes with the func-tions of numerous Ca2+-transport and Ca2+-dependent sig-naling proteins. In some cases substitution of Ca2+ by Cd2+may even yield the normal protein function such as in theprotein calmodulin where substitution of Ca2+ by Cd2+ stillconserves 90% activity with cyclic phosphodiesterase(Cheung 1984). Cd2+ ions have an analogous electron con-Wguration with Zn2+ (4d10 vs. 3d10). Despite its larger radius(0.95 vs. 0.74 ), Cd2+ can often substitute for Zn2+ in zincenzymes and transcription factors and disturb or abolish thebiochemical functions of such proteins.

Pb2+ has ionic radii of 1.19 in hexacoordination and1.29 in octacoordination. These are suYciently close tothat of Ca2+, and Pb2+ interferes with many types of Ca2+-regulated physiological processes, especially the Ca2+-sig-naling system.

Ni2+ ions have nearly the same radius as Mg2+ ions (0.69and 0.66 , respectively) and similar ligand preferences, thatis for oxygen and nitrogen. Ni2+ ions can interfere with Mg2+functions in enzymes of nucleic acid synthesis and repair.

Co2+ ions have the same charge and size as Zn2+ ions(both 0.74 A). Both Co2+ and Zn2+ ions bind to the sametypes of ligands, that is, oxygen, nitrogen and sulfur. In sev-eral instances, the substitution of Zn2+ by Co2+ in zincenzymes results in proteins with modiWed catalytic activi-ties. A prominent example is the eVect of the carcinogenicmetal ions Cd2+, Co2+ and Ni2+ on structure and function ofzinc Wnger domains of several transcription factors andenzymes (Hartwig 2001; Kopera et al. 2004).

The toxicity of metals and their compounds largelydepends on their bioavailability, i.e. the mechanisms ofuptake through cell membranes, intracellular distributionand binding to cellular macromolecules (Fig. 1). Theanionic compounds of chromium and vanadium smoothlypenetrate into cells via the general anion channel of theplasma membrane. Hence, anionic chromium(VI) is readilytaken up into cells and, however, as soon as it is reduced to

chromium(III) by intracellular reductants, the metal istrapped and accumulates (Connett and Wetterhahn 1983).Several toxic metals occuring as divalent cations can passthrough plasma membranes via cation transporters. In con-trast, cellular plasma membranes are impermeable to thetrivalent Cr3+ ion. At variance, sparingly soluble metalcompounds may be taken up by phagocytosis, which maylead to considerable intracellular accumulation of thesemetals after gradual dissolution in the lysosomes.

General mechanisms of metal genotoxicity and carcinogenicity

The interactions of diverse metal carcinogens with livingmatter are complex, and at Wrst sight it seems daring toassume that there were common mechanisms of action.However, a closer look reveals three major mechanismswhich apply for the majority of carcinogenic metal com-pounds besides some distinct reactions of speciWc metalcompounds, namely oxidative stress, DNA repair modula-tion and disturbances of signal transduction pathways.

Induction of oxidative stress

The induction of oxidative stress is an attractive hypothesisto explain mutagenic and carcinogenic eVects of metals. Ionsof the carcinogenic metals, such asantimony, arsenic, chro-mium, cobalt, nickel and vanadium, are capable of perform-ing redox reactions in biological systems. They have beenshown to induce the formation of reactive oxygen and nitro-gen species in vivo and in vitro in mammalian cells. Fre-quently the formation of hydroxyl radicals, most probablyby Fenton- and HaberWeiss-type reactions, has beendetected. These radicals are known to cause oxidative dam-age to lipids, proteins and DNA (Fig. 2). Although the ionsof the carcinogenic metal cadmium are not capable of exert-ing redox reactions in biological systems, they have been

Fig. 1 Cellular uptake, intracellular distribution and binding of solu-ble and particulate metal compounds

Men+

Plasma Membrane

Nucleus

Lysosome pH 4.5

Phagocytosis

ParticulateMetal Compound

SolubleMetal Compound

Men+

Men+Ion Channel

Protein

Men+

DNA

Men+

Fig. 2 Metal ions and oxidative stress (modiWed from Hartwig 2007)

OxidativePhosphorylation

Activated Phagocytes Environmental factors

O21

Super Oxide Dismutase

Cr(V), Fe(II), Co(II), Ni(II), Cu(II)

OH

Catalase

Peroxidase

H2O + O2

H2O

Lipid Peroxidation Oxidative Protein Damage DNA Damage

H2O2123

496 Arch Toxicol (2008) 82:493512found to generate oxidative stress too. The reason for thisproperty of cadmium seems to be the inhibition of antioxida-tive enzymes in vitro and in vivo. Cadmium has been shownto inhibit catalase, superoxide dismutase, glutathione reduc-tase, and glutathione peroxidase (see below). In addition tothe metals classiWed as carcinogens, iron and copper are alsoeVective catalysts for Fenton and Fenton-type reactions.Nevertheless, in living systems iron and copper are tightlyregulated with respect to uptake, transport, storage, mobili-zation, transfer to target molecules and excretion, ensuringthat increased deliberation of free ions is restricted to condi-tions of extreme overload, genetic defects in metal homeo-stasis and/or metabolic stress. Besides generating DNAdamage directly, reactive oxygen species at low concentra-tions function as mitogenic signals and activate redox-sensi-tive transcription factors (Genestra 2007). Hence, oxidativestress may not only initiate tumor development by mutagen-esis but also deregulate cell growth and promote tumorgrowth depending on extent and time of interference.

One major objection against the oxidative-stress-hypoth-esis of metal carcinogenicity is the discrepancy between thecomparatively high, often cytotoxic doses of metal com-pounds that are required to evoke the formation of reactiveoxygen species and/or measurable increase in damage tocellular macromolecules and the often very low doses ofmetals that induce tumors. Hence, it seems that metal-induced oxidative stress is not the sole cause for metal car-cinogenesis but still contributes to the development ofmalignancy in a potentiating manner.

Interference with DNA repair

With the exception of chromium(VI), carcinogenic metalsare only weak mutagens in mammalian cells and often inac-

tive in bacterial assays. Since mutagenicity in bacterialassays is an indicator of reactivity of a substance withDNA, metals are thought to exert genotoxicity by indirectmechanisms. Carcinogenic metal compounds often arecomutagenic, that is, they enhance the mutagenicity ofother genotoxic agents. Indeed, many carcinogenic metalcompounds at low concentrations have been identiWed asinhibitors of the repair of DNA damage that is caused eitherby other xenobiotics or by endogeneous factors (Hartwig2007). The four main, partly overlapping DNA repair path-ways operating in mammalian cells are base excision, mis-match, nucleotide excision and recombinational repair.Figure 3 gives an overview over sources of DNA damageand the four major repair pathways involved in the removalof the respective DNA lesions. Inherited or acquired deW-ciencies in such pathways can contribute to the onset ofmalignant growth. DNA repair mechanisms are frequenttargets for interference by toxic metals as discussed indetail for the individual metals below. Inhibition of repairand persistent DNA damage results in genomic instability,which may become especially deleterious under conditionsof accelerated cell proliferation and/or impaired apoptosis.

Deregulation of cell proliferation

Tumor development is characterized by a deregulation ofcell growth and diVerentiation. Carcinogenic metal com-pounds may alter cell growth by several distinct mecha-nisms, either aVecting the expression of growth stimulatingfactors or inactivating growth control mechanisms. Withrespect to the former, some metal ions are found to activatemitogenic signaling pathways and induce the expression ofcellular proto-oncogenes. Furthermore, epigenetic mecha-nisms, such as hypo- or hyper-methylation of DNA or dis-

Fig. 3 Sources of DNA damage and major repair pathways Ionizing Radiation

Reactive OxygenSpecies,

Alkylating Agents

UV Irradiation,PolycyclicAromatic

Hydrocarbons

ReplicationErrors

Ionizing RadiationAntitumor Agents,

e.g. Cisplatin,Mitomycin C

Abasic Sites,Base Modification,

Single Strand Breaks

(6-4)-Photoproducts,

Pyridine-Dimers,Bulky Adducts

A-G Mismatch,T-C Mismatch

InterstrandCrosslinks,

Double Strand Breaks

Base ExcisionRepair

NucleotideExcision Repair

Base MismatchRepair

RecombinationalRepair123

Arch Toxicol (2008) 82:493512 497turbed histone acetylation, may contribute to modiWedpatterns of gene expression. Changes in gene regulation areobserved prior to manifestation of tumors. Initially, they arenot Wxed by mutation, and the agent must be present for anextended time period to cause persistent modiWcations,which can be genetically Wxed during tumor development.Concerning the interference with cellular growth control,some metal carcinogens have been shown to inactivate thetumor suppressor proteins p53 and/or downregulate theexpression of tumor suppressor genes Fhit, p16, p53 and ofsenescence genes. Finally, metal ions may deregulate cellproliferation by inactivating apoptotic processes resultingin adaptation to the cytotoxicity of the metal.

Mechanisms of action of speciWc metals

Arsenic

Arsenic is a well-documented human carcinogen. Numer-ous epidemiological studies have shown that arsenic cancause diVerent types of cancer via exposure to contami-nated drinking water and/or ambient air (reviewed by Yos-hida et al. 2004). In humans and many other mammals,inorganic arsenic prevalent in drinking water as arsenite orarsenate is metabolised in the liver through successive oxi-dative methylation and reduction steps to its trivalent andpentavalent mono- and di-methylated metabolites (Fig. 4;reviewed by Aposhian and Aposhian 2006).

While previously methylation has been considered as adetoxiWcation process, recent studies have shown that incontrast to the pentavalent methylated species, the trivalentmethylated metabolites monomethylarsonous [MMA(III)]and dimethylarsinous [DMA(III)] acid in various test sys-tems are at least as or even more genotoxic when comparedto inorganic arsenic (Styblo et al. 2000; Schwerdtle et al.2003a; Kligerman et al. 2003). Therefore, they may con-tribute to inorganic arsenic-induced carcinogenicity. Sev-eral modes of action have been proposed to be involved in

arsenic carcinogenicity, including the induction of oxida-tive stress, diminished DNA repair, altered DNA methyla-tion patterns, enhanced cell proliferation and suppression ofp53 (reviewed by Schoen et al. 2004). However, it is notentirely clear which mechanisms prevail in the carcinoge-nicity of arsenic.

Genotoxic eVects

Arsenite does not induce point mutations in bacterial ormammalian test systems. However, it increases the mutage-nicity of other DNA damaging agents, such as UVC radia-tion (Rossman et al.1986), which may be explained byinterference with DNA repair processes (see below). Incontrast, the induction of micronuclei, chromosomal aber-rations, DNA strand breaks and oxidative DNA base dam-age is well documented and has been observed atcomparatively low concentrations in cultured mammaliancell lines such as V79, CHO, A549, and in human periphe-ral lymphocytes (Schoen et al. 2004). With respect to theinorganic species, arsenate (with pentavalent As) and arse-nite (with trivalent As), similar genotoxic eVects have beenobserved, albeit at about tenfold higher concentrations ofarsenate as compared to arsenite. Regarding the methylatedspecies, MMA(III) and DMA(III) are genotoxic at lowerconcentrations than arsenite at all endpoints, while geno-toxic eVects of MMA(V) and DMA(V) are either absent orrestricted to much higher concentrations (Styblo et al.2000; Schwerdtle et al. 2003a; Kligerman et al. 2003).Micronuclei and chromosomal aberrations have also beenobserved in mice after oral exposure to comparatively lowconcentrations of arsenite. Furthermore, chromosomalaberrations and micronuclei were found in peripheral lym-phocytes, buccal and bladder cells of humans exposedtowards elevated concentrations of arsenite via drinkingwater (Schoen et al. 2004). Underlying mechanisms may bethe induction of oxidative stress, inhibition of DNA repairsystems and changes in DNA methylation patterns, all ofwhich may lead to genomic instability.


Several lines of evidence suggest the involvement of oxida-tive stress mediated by increased levels of reactive oxygenspecies in arsenic-induced genotoxicity (reviewed by Shiet al. 2004). Thus, arsenite increases the generation ofsuperoxide anions (O2) and hydrogen peroxide (H2O2) indiverse cellular systems, while the modulation of nitricoxid (NO) production appears to be restricted to higherconcentrations. With respect to genotoxicity, the applica-tion of radical scavengers revealed the involvement of arse-nite-induced ROS in the induction of lipid peroxidation aswell as in DNA damage. Furthermore, the ROS can activate

Fig. 4 Proposed mammalian arsenic metabolism. Note that the exactreaction sequence and enzymes involved are still debated (For detailssee Aposhian and Aposhian 2006)

Arsenate (V) Arsenite (III)Monomethyl-arsonic Acid[ MMA (V) ]

Reductase

Methyltransferase

Reductase

Monomethyl-arsonous Acid[ MMA (III) ]

Dimethyl-arsinic Acid[ DMA (V) ]

Dimethyl-arsinous Acid[ DMA (III) ] Reductase

Methyltransferase123

498 Arch Toxicol (2008) 82:493512signal cascades involving mitogen-activated proteinkinases (MAPKs) and transcription factors AP-1 and NFB(Leonard et al. 2004). Several origins of elevated levels ofROS are possible and have been suggested. They includeinteractions with the respiratory chain, the generation ofROS during oxidation of trivalent to pentavalent species asevident in liver metabolism as well as the release of ironfrom ferritin by trivalent arsenic species. Furthermore,interactions of arsenic with cellular protection mechanismsagainst ROS, in particular a decrease in glutathione levelsand the disturbance of DNA repair systems, contribute toincreased levels of oxidative damage in cells (reviewed byShi et al. 2004).

DNA repair inhibition

Arsenite as well as MMA(III) and DMA(III) have beenshown to inhibit the repair of UVC- and benzo[a]pyrene-diolepoxide (BPDE)-induced DNA damage in the lowmicromolar, non-cytotoxic concentration range (see forexample, Schwerdtle et al. 2003b). As one explanation, adecrease of poly(ADP-ribosyl)ation in HeLa S3 cells afterincubation with nanomolar concentrations of arsenite,MMA(III) or DMA(III) was observed (Hartwig et al. 2003;Walter et al. 2007), a reaction involved in DNA damagesignaling and the recruitment of DNA repair proteins to thesite of DNA damage. A possible mechanism of arsenic tox-icity may lie in its ability to react with thiols, for example inzinc binding structures prevalent in many transcription fac-tors, DNA repair and cell cycle control proteins. Recentstudies applying a 37 amino acid thiol-containing zincWnger peptide of XPA (XPAzf), a critical component of thenucleotide excision repair (NER) complex, where zinc iscomplexed to four cysteines, revealed diVerential eVects ofarsenite and MMA(III). Interestingly, reaction of arsenitewith the apopeptide resulted in thiol oxidation of two orfour cysteine residues, producing one or two disulWdebonds, respectively. In contrast, reaction of MMA(III) withXPAzf produced complexes containing two MMAs, or oneMMA with or without oxidation of the remaining two cys-teines. Thus, zinc-binding structures may be sensitive tar-gets for arsenicals, even though the actual species involvedin the speciWc interactions diVers (Piatek et al. 2008).


Accumulating evidence from cell culture studies, experi-mental animals and also from arsenic-exposed humans sug-gests that arsenic alters the DNA methylation pattern,thereby aVecting the expression of oncogenes and tumorsuppressor genes. Interestingly, both hypo- and hyper-methylation have been observed. For example, increasedcytosine methylation in the p53 promotor was detected in

A549 cells, and hypermethylation with the consequence ofdiminished gene expression of tumor suppressor genes suchas p16INK4a and RASSF1A were found in arsenic-exposed A/J mice (Cui et al. 2006). With respect tohumans, a dose-dependent hypermethylation of the p53gene was observed in blood samples of arsenic-exposedskin cancer patients in West Bengal (Chanda et al. 2006).The underlying mechanisms are still unclear. Whilehypomethylation may be due to inhibition of DNA-(cyto-sine-5) methyltransferase as in the instance of cadmium(Takiguchi et al. 2003) or the depletion of S-adenosylme-thionine, a common cofactor in DNA methylation andarsenic methylation, hypermethylation is not easily under-stood and further studies are required to resolve this ques-tion.

Antimony

Compared with arsenic, much less is known about the car-cinogenicity and the underlying mechanisms of action ofantimony. Epidemiological studies indicate a carcinogeniceVect of antimony and antimony compounds on the humanlung, but co-exposure to confounding substances does notallow a Wnal conclusion. In animal experiments, inhalationof antimony trioxide and of antimony trisulWde causedincreased incidences of lung tumors. Metallic antimony hasbeen classiWed as carcinogenic, too, because after inhala-tion exposure to metallic antimony the element has beendetected in human body Xuids, and metallic antimonyshowed the same toxicity proWle as antimony trioxide inrats (DFG 2007b).

Genotoxic eVects

The genotoxicity of antimony has been reviewed recently(DFG 2007b). In most bacterial mutation assays, inorganicantimony compounds were inactive. In mammalian cells,trivalent antimony compounds caused DNA strand breaks,enhanced chromosome aberrations and micronuclei, butgene mutations were not detected.

In animals, administration of antimony compoundsresulted in clastogenic eVects in some but not in all studies.Also, there are indications of an in vivo genotoxic potentialof inorganic antimony compounds in humans. Exposure ofworkers to antimony trioxide resulted in oxidative DNAdamage in whole blood.

Putative mechanisms of carcinogenesis

In aqueous solution, both trivalent and pentavalent anti-mony are stable and both forms may be mutually intercon-verted under physiological conditions. In reductivebiological milieus, pentavalent antimony is reduced to the123

Arch Toxicol (2008) 82:493512 499trivalent form, which is the stable form in physiologicalmedia (Reglinski 1998). Trivalent antimony reacts withsulfhydryl groups of proteins and thereby acts as anenzyme inhibitor (Gebel et al. 1997). There exist no con-clusive clues to the mechanism of carcinogenesis by anti-mony. A direct eVect on DNA is unlikely, since antimonycompounds were not mutagenic in most bacterial mutationtests. The clastogenic eVects of antimony trichloride inmammalian cells were not induced by DNA-protein cross-links, and the induction of micronuclei by antimony can-not be explained by oxidative stress (SchaumlVel andGebel 1998). As with many other carcinogenic metals,inhibition of DNA repair might contribute to the tumori-genic activity of antimony, but there are no published dataavailable yet. Furthermore, at present it is not clearwhether or not antimony is methylated to the same extentas arsenic. To sum up, it is evident that the mechanismsunderlying the carcinogenicity of antimony are stillobscure.

Beryllium

Occupational exposure to beryllium and beryllium com-pounds is associated with increased lung cancer mortality,and inhalation of beryllium metal, beryllium oxide andberyllium salts caused lung tumors in rats and rhesus mon-keys (IARC 1993; DFG 2003). The Be2+ ion carries thesame charge as Mg2+ and competes for Mg2+ in biochemi-cal binding sites such as the phosphate groups of nucleo-tides and nucleic acids. Like cadmium, beryllium does notparticipate in redox reactions under physiological condi-tions.

Genotoxic eVects

The genotoxicity of beryllium has been reviewed (DFG2003; Gordon and Browser 2003). In an acellular enzy-matic assay, a very high concentration of BeCl2 (10 mM)interfered with the Wdelity of DNA synthesis and causedincorporation of mispaired nucleotides (Zakour and Glick-man 1984). In bacterial assays, beryllium salts were notmutagenic, whereas in the majority of investigations withmammalian cells, beryllium salts induced sister chromatidexchanges, chromosomal aberrations and gene mutations(Gordon and Browser 2003). In rats exposed to berylliumsulfate, no increased number of micronuclei was observed,and in a group of workers exposed to beryllium oxide noenhanced frequencies of sister-chromatid exchanges andmicronuclei were found. In BALB 3/T3 cells, berylliumcaused a downregulation of genes involved in DNA repair(MCM4, MCM5, Rad23 and DNA ligase I) (Joseph et al.2001). Possibly, beryllium impairs the DNA repair capacityin mammalians.


A further mechanism related to the carcinogenicity ofberyllium may be the deregulation of cell proliferation.Like other carcinogenic metal compounds, beryllium acti-vates mitogenic signalling pathways. BeF2 inducedincreased phosphorylation of mitogenic protein kinases(MEK1, ERK1, p38, MAPK and JNK) and transcriptionfactors (NFkB and CREB) in human macrophages (Misraet al. 2002). Furthermore, Be(II) induced the expression ofcellular proto-oncogenes in vitro. In a study with BALB 3/T3 cells, beryllium activated the expression of K-ras and c-jun but not of c-myc, c-fos, c-sis or p53 genes (Keshevaet al. 2001). In another study with the same cell line, beryl-lium upregulated c-fos, c-jun, c-myc and c-ras, whereasseveral genes involved in DNA repair (MCM4, MCM5,Rad23 and DNA ligase I) were downregulated (Josephet al. 2001a). Both upregulation of mitotic signals anddownregulation of DNA repair functions are thought tocooperate to induce an unbalanced error-prone cell prolifer-ation. There is also evidence for epigenetic eVects in beryl-lium carcinogenesis. In lung tumors of rats induced byexposure to beryllium metal particles, there was hyperme-thylation of DNA in the tumor suppressor gene INK4, asso-ciated with a reduced synthesis of the gene productp16INK4a (Belinsky et al. 2002). Because this protein isinvolved in cell cycle arrest at the G1-S-phase transition, itsloss may contribute to tumor progression.

Cadmium

Human cadmium exposure is associated with cancers of thelung and the kidney (IARC 1993; DFG 2006a). In animals,cadmium induces carcinomas of the lung after inhalationand cancers of the prostate after ingestion or injection(Waalkes 2003). Physicochemical properties of the Cd2+ion may serve as clues to the interpretation of biologicaleVects: The Cd2+ ion easily substitutes for the calcium ionin biological systems, because it carries the same chargeand has a similar radius. Compared to the zinc ion, theradius of the Cd2+ ion is larger, but still Cd2+ ions can sub-stitute for Zn2+ ions in many enzymes and transcription fac-tors.

Genotoxic eVects

In rodent experiments, cadmium salts caused increased fre-quencies of micronuclei and chromosomal aberrations. Invitro, in mammalian cells cadmium compounds causedDNA strand breaks, gene mutations and chromosomal aber-rations, whereas in most bacterial assays soluble cadmiumcompounds were not mutagenic (Waalkes 2003; DFG2006a). Since cadmium salts do not cause DNA damage in123

500 Arch Toxicol (2008) 82:493512cell extracts or with isolated DNA (Valverde and Rojas2001), the genotoxicity of cadmium has to be explained byindirect mechanisms. Three frequently discussed mecha-nisms are (1) the generation of reactive oxygen species, (2)inhibition of DNA repair enzymes, and (3) deregulation ofcell proliferation.


Although cadmium(II) unlike many other carcinogenicmetal compounds, is not able to participate in redox reac-tions under physiological conditions, oxidative stressappears to be a relevant mechanism of cadmium-inducedgenotoxicity. Cadmium has been shown to induce the for-mation of reactive oxygen species, both in vitro and in vivo.Cadmium sulWde induced H2O2 formation in human poly-morphonuclear leukocytes, and cadmium chlorideenhanced the production of superoxide in rat and humanphagocytes (Sugiyama 1994). Accordingly, the inductionof DNA strand breaks and chromosomal aberrations bycadmium in mammalian cells was suppressed by antioxi-dants and antioxidative enzymes (Ochi et al. 1987; Stohset al. 2001; Valko et al. 2006). The induction of oxidativestress by cadmium is interpreted by its inhibitory eVects onantioxidant enzymes such as catalase, superoxide dismu-tase, glutathione reductase, and glutathione peroxidase(Stohs et al. 2001; Valko et al. 2006). In addition to theirprobable role in genotoxicity, reactive oxygen species mayfunction as mitogenic signals (see below).

Inhibition of DNA Repair

Cadmium is comutagenic and augments the mutagenicityof UV radiaton, alkylation and oxidation in mammaliancells. These eVects are explained by the observation thatcadmium inhibits several types of DNA repair, that is, baseexcision, nucleotide excision, mismatch repair and theelimination of the premutagenic DNA precursor 8-oxo-dGTP (reviewed by Hartwig and Schwerdtle 2002).Regarding base excision repair, low concentrations of cad-mium which did not generate oxidative damage as such,inhibited the repair of oxidative DNA damage in mamma-lian cells (Dally and Hartwig 1997; Fatur et al. 2003). Withrespect to nucleotide excision repair, cadmium interferedwith the removal of thymine dimers after UV irradiation byinhibiting the Wrst step of this repair pathway, that is, theincision at the DNA lesion (Hartwig and Schwerdtle 2002;Fatur et al. 2003). Furthermore, chronic exposure of yeastto very low cadmium concentrations resulted in hypermuta-bility, and in human cell extracts cadmium was shown toinhibit DNA mismatch repair (Jin et al. 2003). Addition-ally, cadmium disturbed the removal of 8-oxo-dGTP fromthe nucleotide pool by inhibiting the 8-oxo-dGTPases of

bacterial and human origin (Bialkowski and Kasprzak1998). A molecular mechanism related to the inactivationof DNA repair proteins involves the displacement of zincfrom zinc Wnger structures in the DNA repair proteins suchas from the XPA protein, which is required for nucleotideexcision repair, and from the Fpg protein, which is involvedin base excision repair in E. coli (Asmuss et al. 2000). Also,human OGG1 (hOGG1), a glycosylase responsible for rec-ognition and excision of the premutagenic 8-oxodG duringbase excision repair in mammalian cells, was inhibited bycadmium (Potts et al. 2003). Even though hOGG1 containsno zinc binding motif itself, its inhibition was shown to bedue to diminished DNA binding of the zinc Wnger contain-ing transcription factor SP1 (Youn et al. 2005). Finally,cadmium induces a conformational shift in the zinc bindingdomain of the tumor suppressor protein p53 (see below). Inaddition to inhibiting repair proteins directly, cadmiumdownregulates genes involved in DNA repair in vivo (Zhouet al. 2004).

The impairment of DNA repair by cadmium may beespecially deleterious in cadmium-adapted cells. Cadmiuminduces several genes for cadmium and ROS tolerance suchas those coding for metallothionein, glutathione synthesisand function, catalase and superoxide dismutase (Stohset al. 2001). Hence, a condition for prolonged cell survivalin the presence of cadmium is established (Chubatsu et al.1992). Taking into account the impairment of DNA repairby cadmium, tolerance to cadmium toxicity concurrentlymay constitute an extended chance for the induction of fur-ther critical mutations (Achanzar et al. 2002).


Cadmium interacts with a multitude of cellular signal trans-duction pathways, many of them associated with mitogenicsignaling. Submicromolar concentrations of cadmium stim-ulated DNA synthesis and proliferation of rat myoblastcells (Zglinicki et al. 1992) and of rat macrophages (Misraet al. 2002). In various cell types in vitro, cadmium evokesreceptor-mediated release of the second messengers inosi-tol-1,4,5-trisphosphate and calcium, it activates variousmitogenic protein kinases, transcription and translation fac-tors, and it induces the expression of cellular proto-onco-genes c-fos, c-myc and c-jun (reviewed by Waisberg et al.2003). However, it should be noted that the activation ofmitogen-activated protein kinases is not a suYcient condi-tion for enhanced cell proliferation, since persistent low-dose exposure of cells to cadmium has been shown to resultnot only in sustained activation of protein kinase ERK butalso to caspase activation and apoptosis (Martin et al.2006). In addition to directly stimulating mitogenic signals,cadmium also inhibits negative controls of cell prolifera-tion. It inactivates the tumor suppressor protein p53 and123

Arch Toxicol (2008) 82:493512 501inhibits the p53 response to damaged DNA (Meplan et al.1999).

Recently it was reported that cadmium modulates steroidhormone-dependent signaling in ovaries in rats, in a breastcancer cell line and in cadmium-transformed prostate epi-thelial cells (Benbrahim-Tallaa et al. 2007; Brama et al.2007). Nevertheless, in in-vitro estrogenicity assays basedon estrogen receptor activity, no eVect of cadmium wasdetected (Silva et al. 2006). It remains an open questionwhether cadmium might promote tumor growth by anestrogen-mediated mechanism.

In addition to eVects on genes and genetic stability, cad-mium also exerts epigenetic eVects which may contribute totumor development. It inhibited DNA-(cytosine-5) methyl-transferase and diminished DNA methylation during cad-mium-induced cellular transformation (Takiguchi et al.2003). Decreased DNA methylation is thought to have atumor promoting eVect, since it was associated with aug-mented expression of cellular proto-oncogenes.

A unique mechanism by which cadmium deregulatescell proliferation is the disruption of the cadherin-mediatedcellcell adhesion system and of cellcell communication(Fig. 5). Cadmium speciWcally displaced calcium from theprotein E-cadherin (Prozialeck and Lamar 1999) andimpaired the cellcell adhesion in kidney epithelial cells(Prozialeck et al. 2003). In conclusion, it is evident thatcadmium interferes with cellular controls of proliferation inseveral ways, which all can contribute to the observedderegulation of cell growth by this metal. However, it is notyet possible to assess the relative contributions of these var-ious mechanisms.

Chromium

Chromium occurs in various oxidation states. In technol-ogy, the prevalent materials are the chromates with hexava-lent chromium, the chromic compounds with trivalentchromium, and metallic chromium. Chromates are the tech-

nical intermediates in the manufacturing of Cr(III) com-pounds and metallic chromium. Therefore, Cr(III)compounds which have not been puriWed completely, stillcontain traces of hexavalent chromium; a fact that hascaused erraneous Wndings of genotoxicity with contami-nated Cr(III) compounds. Chromium traces are essential forhuman and animal nutrition and are taken up as complexesof Cr(III) with amino acids (Vincent 2000). Exposure tovarious chromium(VI) compounds has been consistentlyassociated with incidences of respiratory cancers in humansand experimental animals. At variance, there is no evidencefor a carcinogenic action of trivalent chromium compounds(IARC 1990; ATSDR 2000) and also the genotoxicity ofCr(VI) is much more pronounced than that of Cr(III).Hence, genotoxic eVects of Cr(VI) and Cr(III) are discussedseparately below.

Genotoxic eVects of chromium(VI)

For chromium, the oxidation state is most important for itsbiochemical activity. Chromium(VI) compounds have beenshown to exert genotoxicity both in vivo and in vitro. Lym-phocytes of workers exposed to dusts of chromium(VI)compounds showed elevated frequencies of DNA strandbreaks (Gambelunghe et al 2003), sister-chromatidexchanges (Wu et al. 2001) and micronuclei (Vaglenovet al. 1999; Benova et al. 2002). After intratracheal instilla-tion in rats, chromate(VI) induced DNA strand breaks inlymphocytes (Gao et al. 1992). After intraperitoneal injec-tion but not oral administration of chromate(VI) to mice,micronuclei were induced in bone marrow (De Flora et al.2006). Intraperitoneal injection of chromate(VI) induceddominant lethal mutations in rats (Bataineh et al. 1997). Invitro, soluble chromium(VI) compounds are mutagenic inmammalian and bacterial test systems (De Flora et al.1990). The mutagenicity of chromium(VI) in bacteria isexceptional because other carcinogenic metals are notgenotoxic or produce equivocal results in bacterial tests.DiVerent from chromate(VI), chromium(III) compoundsdid not induce genotoxic eVects in the majority of studieswith intact cells (discussed below).

Genotoxic eVects of chromium(III)

Cr(III) compounds have not been identiWed as carcinogenicand their genotoxicity is questionable. Unlike the chromateanion, the Cr3+ cation is very poorly taken up by cells.There is limited evidence for a possible genotoxicity ofCr(III) in vitro but not in vivo. After uptake of chro-mate(VI) and its intracellular reduction to Cr(III), the latterforms potentially genotoxic complexes with DNA. ThisWnding implies that Cr(III) can be genotoxic, if it over-comes the barrier of the plasma membrane (Fig. 6). This

Fig. 5 Interference of cadmium with the cadherin cellcell adhesionsystem (modiWed from Prozialeck 2003)

123

502 Arch Toxicol (2008) 82:493512can be achieved in various ways. Insoluble particles ofCr2O3 may be taken up by phagocytosis, and subsequentlybe solubilized in lysosomes to release Cr3+ ions. These cat-ions bind to cellular macromolecules including DNA.Indeed, there is some evidence for a low genotoxicity ofCr2O3 although results from diVerent laboratories are con-troversial (reviewed by De Flora et al. 1990). In intestinalepithelial cells, Cr(III) can be taken up via the transferrinuptake mechanism. Another mode of Cr(III) uptakeinvolves synthetic Cr(III) complexes with hydrophobicligands, which facilitate the permeation of chromiumthrough plasma membranes. Thus, complexes of Cr(III)with 1,10-phenanthroline or 2,2-bipyridine (Warren et al.1981) or with picolinic acid (Stearns et al. 2002) are takenup by cells and induce gene mutations. It has been sug-gested that chromium(III) might cause DNA damage if itwere reoxidised by intracellular hydrogen peroxide to pro-duce deleterious intermediates. However, this reaction hasbeen observed in cell-free systems only and with high con-centrations of hydrogen peroxide (Tsou et al. 1996).

Role of chromium-DNA adducts in the genotoxicity of Cr(III) and Cr(VI)

According to the uptake-reduction model developed byWetterhahn (Connett and Wetterhahn 1983), Cr(VI) as thechromate anion travels easily through anion channels of theplasma membrane and is reduced by intracellular electrondonors in three one-electron steps via chromium(V) andchromium(IV) to the stable form of chromium(III), whichis accumulated in cells and bound to biochemical macro-molecules (Fig. 7). The reduction of Cr(VI) keeps the intra-cellular concentration of chromate low and facilitateschromium(III) accumulation within cells. This model hasbeen conWrmed by various investigations with mammaliancells and in vivo. After treatment of diVerent cell lines withchromium(VI), the intracellular formation of chromium(V)and chromium(III) has been demonstrated by EPR spec-

troscopy (Arslan et al. 1987) and X-ray absorption spec-troscopy (Dillon et al. 1997). Cr(V) was also detected byEPR spectroscopy in living mice after intraveneous injec-tion of chromium(VI) (Liu et al. 1994). Whereas theanionic chromate is unable to react with DNA directly,chromium(III) forms stable complexes with DNA in chro-mate-treated cells (Fornace et al. 1981; Miller and Costa1988; Salnikow et al. 1992). Transfection of a bacterio-phage DNA treated with Cr(III) into E. coli cells lead to adose-dependent increase in mutation frequency (Snow1991). Also, after transfection of plasmids with ternaryamino acid-Cr(III)-DNA adducts into human Wbroblasts invitro, mutations were observed which predominantly weresingle base substitutions at G:C base pairs (Voitkun et al.1998). Hence, the formation of Cr-DNA adducts is dis-cussed as a relevant mechanism for chromium(VI) geno-toxicity (Zhitkovich 2005).


In the process of reduction of chromium(VI) to chro-mium(III) by cellular reductants, such as ascorbate or gluta-thione, potentially toxic intermediates such as oxygen andsulfur radicals are generated. In a cell-free assay, chro-mate(VI) reacted with glutathione to yield chromium(V)and thiyl radicals (Wetterhahn et al. 1989), whereas thereduction of Cr(VI) with ascorbate resulted in hydroxylradical formation (Shi et al. 1994). Furthermore, chro-mium(V) was detected as an intermediate during chro-mium(VI) reduction in experimental animals (Liu et al.1994). Pentavalent chromium reacted with isolated DNA toproduce 8-hydroxydeoxyguanosine, whereas hexavalentchromium performed this reaction only in the presence ofthe reductant glutathione (Faux et al. 1992). In culturedmammalian cells, chromate(VI) induced superoxide andnitric oxide production (Hassoun and Stohs 1995), whereastreatment of cells with chromium(VI) in the presence of

Fig. 6 Cellular uptake and reduction of chromium compounds

No entry!

CrO42

Cr2O3

[Cr(III)Ln]

Cr3+Ions

Cr(VI)O42

Cr(IV)

Cr(V)

Hydrophobiccomplex[Cr(III)Ln]

ParticulateCr2O3

Cr(III)-Transferrin

complex

Cr(III)-Transferrin

Endocytosisand

LysosomalSolubilisation

Cr(III) bound to biochemical ligands

AnionChannel

Fig. 7 Intracellular chromium metabolism, generation of oxidativestress and eVects on DNA

Cr(V)

X

Cr(IV)

Cr(VI) DNA

ReactiveOxygenSpecies

Cr(III)

e

Cr(III) - DNA

Oxidative Damageto Lipids, Proteins and DNA

e

e

Cellu

larR

educ

tant

s123

Arch Toxicol (2008) 82:493512 503glutathione reductase generated hydroxyl radicals. How-ever, the required concentration of chromate was in thecytotoxic range (2 mM) (Ye et al. 1995). Regarding geno-toxicity, the relative contributions of chromate-generatedoxidative stress on the one hand and Cr(III)-DNA adductson the other hand are still debated.


Besides directly causing DNA damage and mutations, chro-mium (VI) has been shown to activate various mitogen-acti-vated protein (MAP) kinases via formation of reactiveoxygen species. In a rat hepatome cell line, low doses ofCr(VI) activated extracellular signal-regulated kinases ERK-1 and ERK-2 in a persistent way (Kim and Yurkow 1996)and in human lung carcinoma cells, Cr(VI) activated threeMAP kinases, c-jun N-terminal kinase JNK and p38 (Chu-ang and Yang 2001). In addition to activation of mitogenicprotein kinases, chromium(VI) induced phosphorylation ofmitogenic transcription factors. Nuclear factor B (NF-B)was activated in a human lymphocyte culture (Ye et al.1995), and activating transcription factor 2 (ATF-2) and theoncogenic transcription factor c-Jun were activated inhuman bronchial epithelial cells (Samet et al. 1998). Sincethese protein kinases and transcription factors are known tobe involved in both inXammation and tumor growth, theiractivation constitutes a non-genotoxic mechanism of chro-mate(VI) carcinogenicity in addition to direct mutagenesis.

Cobalt

Inorganic cobalt compounds, both soluble and particulateforms, caused lung tumors in animal experiments, whereasthe epidemiological Wndings of increased lung cancer inci-dence of cobalt-exposed workers are regarded as not con-clusive because of co-exposure to other carcinogenicsubstances (IARC 1991, 2006a; DFG 2007c). At variance,workers exposed to cobalt in hard metals containing tung-sten carbide experienced a signiWcant increase in lung can-cer (DFG 2007d).

Genotoxicity

The genotoxicity of cobalt and cobalt compounds has beenreviewed (Beyersmann and Hartwig 1992; DFG 2007c).After intratracheal instillation in rodents, cobalt(II) chlorideinduced aneuploidies, micronuclei and chromosome aberra-tions in the bone marrow. In an inhalation carcinogenicitystudy with cobalt sulfate, mutations in the K-ras oncogenewere observed in lung tumor tissues of exposed mice (NTP1998). Soluble cobalt(II) salts induced DNA strand breaks,gene mutations, sister chromatid exchanges and micronu-clei in mammalian cells in vitro but were mostly not geno-

toxic in bacterial assays. The underlying mechanismsinclude direct mutagenicity by oxidative reactions andinterference with DNA repair processes (see below). Withrespect to metallic cobalt, cobalt dust caused DNA singlestrand breaks and micronuclei in mammalian cells in vitro,and these eVects of cobalt were considerably enhancedwhen cobalt was combined with tungsten carbide as it isencountered in hard metals (De Boeck et al. 2003).


Cobalt ions are able to induce the formation of reactive oxy-gen species (ROS) in vivo and in vitro. Cobalt(II) catalyzesthe generation of hydroxyl radicals from hydrogen peroxidein a Fenton type reaction. After intraperitoneal injection inrats, cobalt(II) evoked the formation of oxidative DNA basedamage in kidney, liver and lung (Kasprzak et al. 1994). Theanalysis of mutations in tumor tissues in a carcinogenicitystudy with cobalt sulfate in mice revealed that Wve of ninemutations were G-T transversions in codon 12 of the K-rasoncogene (Asmuss et al. 2000; National Toxicology Program1998). The authors interpret this eVect as evidence thatcobalt(II) causes oxidative DNA damage. A special mecha-nism was delineated for the increased genotoxicity of cobaltin combination with tungsten carbide as it is used in hardmetal dust. When metallic cobalt was combined with tungstencarbide in an acellular system, reactive oxygen species weregenerated (Lison et al. 1995). These authors conclude thattungsten carbide (WC) catalyzes the transfer of electrons fromcobalt to oxygen to yield superoxide as depicted in Fig. 8.

Inhibition of DNA repair

The genotoxicity of other mutagenic agents was augmentedby soluble cobalt salts (Beyersmann and Hartwig 1992) andcobalt metal dust (De Boeck et al. 1998). In human Wbro-

Fig. 8 Proposed mechanism of reactive oxygen species (ROS) forma-tion by transfer of electrons from cobalt metal to molecular oxygen ascatalyzed by tungsten carbide (modiWed from Lison et al. 1995)

O2

ROS

WC

eCo

Co2+

Co

Co Co2+ + 2 e O2 + e ROS123

504 Arch Toxicol (2008) 82:493512blasts, cobalt(II) inhibited the nucleotide excision repair ofDNA damage caused by UV-C radiation. Both the incisionand polymerisation steps were inhibited (Kasten et al.1997). In particular, cobalt inhibited the Xeroderma pig-mentosum group A (XPA) protein, a zinc Wnger proteininvolved in nucleotide excision repair (Asmuss et al. 2000)where it substituted for the zinc ion (Kopera et al. 2004).The comutagenicity of cobalt observed in vitro correspondsto its cocarcinogenic eVect in an animal study, wherecobalt(II) oxide enhanced the carcinogenicity ofbenzo[a]pyrene (SteinhoV and Mohr 1991).

Upregulation of hypoxia-inducible factor HIF-1

Cobalt(II) is known to evoke a hypoxia-like state in vivoand in vitro even in the presence of normal molecular oxy-gen pressure. The underlying mechanism involves the sta-bilization of hypoxia-inducible factor HIF-1, whichnormally is degraded when suYcient oxygen is present. Inthe hypoxic state, HIF-1 acts as a subunit of a transcrip-tion factor inducing the expression of genes controllingerythropoietin synthesis, glucose uptake, glycolytic enzymeactivities, blood vessel formation (angiogenesis) and otherprocesses allowing cell survival at low oxygen pressure.Hypoxia is a common feature of tumor tissues, and thegrowth of tumors beneWts from HIF-1 activation, whichleads to enhanced glycolytic and angiogenetic activities.For a more detailed discussion of this area, readers arereferred to the review of Maxwell and Salnikow (2004)who also discuss similar eVects of nickel(II).

Lead

The toxicity of lead and its compounds is well known formany centuries, with anemia and developmental distur-bances being most prominent. Nevertheless, during the lastyears potential carcinogenic eVects came into focus, leadingto the classiWcation of inorganic lead compounds as Proba-bly carcinogenic to humans (Group 2A) by IARC andGroup 2 by the German MAK Commission (considered tobe carcinogenic to man based on long-term animal studies).These classiWcations were mainly based on animal experi-ments, where increased tumor incidences were observed inmultiple organs, including kidney and brain. Nevertheless,the exact mechanisms are still unclear, but as with mostother metals and there compounds, indirect mechanisms likethe induction of oxidative stress and the interaction withDNA repair processes appear to be relevant.

Genotoxic eVects

Genotoxic eVects of lead compounds are well documentedin in-vitro systems, experimental animals and in lead-

exposed humans (summarized in IARC 2006b). Equivocalresults have been published with respect to the mutagenic-ity of water soluble lead compounds in mammalian cells inculture; in most classical test systems, eVects were ratherweak and/or restricted to toxic doses. Nevertheless, whenapplying mammalian AS52 cells carrying a single copy ofan E. coli gpt gene, which are suited for the detection ofsmall and large deletions, lead chloride induced mutationsin a dose-dependent manner, starting at the non-cytotoxicconcentration of 0.1 M (Ariza and Williams 1996; Arizaet al. 1998; Ariza and Williams 1999). High mutant fre-quencies and mutation spectra similar to those induced byreactive oxygen species were also observed in a diVerentstudy in CHO K1 cells (Yang et al. 1996). Furthermore,two studies revealed an increase in mutation frequency incombination with UVC irradiation and MNNG, indicativeof the disturbance of DNA repair processes (see below). Incontrast to the equivocal results of gene mutation studies,chromosomal damage and micronuclei have been observedconsistently in mammalian cells in culture, in experimentalanimals and in several cases also in lead-exposed humans;however, with respect to population-based studies, con-founding exposures cannot be ruled out (reviewed in IARC2006b). At low concentrations realistic for human expo-sure, two mechanisms may underlie lead-induced genotoxi-city, namely a disruption of the pro-oxidant/anti-oxidantbalance and an interference with DNA repair systems.


At diVerent experimental levels, there are strong indicationsfor the involvement of ROS in lead-induced genotoxicity.Proposed molecular mechanisms include enhanced lipidperoxidation, inhibition of antioxidant defense systems,catalysis of Fenton-type reactions and, interestingly, alsothe long-known inhibition of aminolevulinic acid dehydra-tase. The latter reaction leads to the accumulation of theheme precursor aminolevulinic acid, with the subsequentgeneration or ROS and oxidative DNA damage (reviewedin IARC 2006b).

DNA repair inhibition

A further mechanism, which has been quite well docu-mented during the last years, is the interaction of lead withtwo major DNA repair systems, that is nucleotide excisionrepair and base excision repair, and comutagenic eVectshave been observed in combination with UVC radiationand MNNG (reviewed in IARC 2006b). As one moleculartarget with respect to base excision repair, lead has beenshown to inhibit the apurinic/apyrimidinic endonuclease(APE1) in the low micromolar concentration range both inan isolated enzymic test system and in cultured AA8 cells,123

Arch Toxicol (2008) 82:493512 505leading to an accumulation of apurinic sites in DNA and anincrease in MMS-induced mutagenicity (McNeill et al.2007). Furthermore, lead interferes with the repair of DNAdouble strand breaks via interaction with the stress responsepathway induced by ATM (a phosphoinositol-3-kinaserelated kinase) (Gastaldo et al. 2007). Due to its high aYn-ity for sulfhydryl groups, one mechanism for lead interac-tion with proteins could be the displacement of zinc fromzinc binding structures. In support of this assumption, incell-free systems lead has been shown to reduce DNA bind-ing of transcription factors TFIIIA and Sp1 (Hanas et al.1999; Huang et al. 2004). However, no impact was seen onthe zinc-containing DNA repair proteins Fpg or XPA(Asmuss et al. 2000). Thus, zinc binding proteins cannot beconsidered as general target, but interactions depend on theactual protein.


Low concentrations of lead have been shown to stimulatecell growth (reviewed in IARC 2006b). A probable mecha-nism consists of the mobilization of free intracellular Ca2+and the activation of protein kinase C (PKC) by lead, whichtriggers a signal transduction cascade Wnally leading to thestimulation of DNA synthesis. In animals, lead signiWcantlyincreases proliferative lesions in the kidney below cyto-toxic concentrations, indicating that genotoxicity and accel-erated growth stimuli may act in concert in lead-inducedcarcinogenicity.

Nickel

Inorganic, both soluble and particulate nickel compoundswere associated with lung tumors in exposed workers (Doll1990). In experimental animals, inhalation of particulatenickel(II) compounds but not nickel(II) sulfate caused lungtumors in rats and mice (Dunnick et al. 1995). The absenceof carcinogenic eVects of nickel sulfate in experimental ani-mals may be attributed to the relatively low maximum tol-erated dose when compared with human exposure. Atvariance, insoluble particulate nickel oxides and sulWdesenter cells by phagocytosis, accumulate within cells to highconcentrations and release nickel ions after gradual dissolu-tion in lysosomes (Fig. 1) (Costa and Mollenhauer 1980).

Genotoxic eVects

The genotoxicity of nickel and its compounds has beenreviewed recently (DFG 2006b). Workers exposed to solu-ble nickel compounds or to poorly soluble sulWdic andoxidic nickel exhibited an elevated incidence of metaphaseswith gaps, but no signiWcant increase in sister-chromatidexchanges in lymphocytes. In animal experiments, intra-

peritoneal injection of soluble nickel salts caused chromo-some aberrations or micronuclei in some but not in allstudies. In mammalian cells, nickel(II) ions evoked chro-mosome aberrations, sister chromatid exchange, DNAbreaks and DNA-protein cross links, but only at millimolarcytotoxic concentrations. Furthermore, soluble nickel(II)salts were only weakly mutagenic in mammalian cells andinactive in almost all bacterial mutagenicity tests. Threemajor mechanisms are discussed for the genotoxic eVectsof nickel: generation of reactive oxgen species, interferencewith DNA repair processes, and epigenetic mechanismsinducing enhanced cell proliferation. In all mechanistic pro-posals, nickel ions are regarded as the ultimately genotoxicform of nickel and inorganic nickel compounds.


Like many other carcinogenic metals, nickel compoundsare able to induce the formation of reactive oxygen species.Nickel ions can catalyze the generation of hydroxyl radicalsfrom hydrogen peroxide in a Fenton type reaction. Accord-ingly, in the presence of hydrogen peroxide, nickel(II) ionsproduce oxidative DNA damage in isolated DNA and chro-matin (Kasprzak and Hernandez 1989; Lloyd and Phillips1999). In living cells, the contribution of oxidative mecha-nisms to the genotoxicity of nickel seems to depend onnickel speciation and cell type. While NiCl2 in HeLa cellscaused oxidative DNA damage only at elevated cytotoxicdoses (Dally and Hartwig 1997), soluble nickel carbonatehydroxide induced sister-chromatid exchanges involvingthe production of reactive oxygen species in human lym-phocytes at lower concentrations (MBemba-Meka et al.2007). Furthermore, the redox activity of nickel(II) maychange considerably if it is complexed to certain aminoacid sequences as demonstrated in subcellular systems forhistone binding (Bal et al. 2000).

Inhibition of DNA repair

Nickel is a distinct comutagen and it interferes with variousDNA repair pathways. Nickel ions enhanced the the muta-genicity of methyl methanesulfonate in E. coli and theinduction of mutations and sister chromatid exchanges byUV radiation in hamster cells. These comutagenic eVectsare explained by the inhibition of all major types of DNArepair processes. DNA excision repair, repair of O6-alkyl-guanine and repair of oxidative DNA damage were inhib-ited at subtoxic concentrations of nickel(II) chloride, whichwere not yet mutagenic themselves (Dally and Hartwig1997; Hartwig et al. 1994; Iwitzki et al. 1998; Kruegeret al. 1999). Recently, in human bronchial epithelial cellstransformed by nickel sulWde, silencing of the O6-methyl-guanine-DNA methyltransferase gene was observed (Ji123

506 Arch Toxicol (2008) 82:493512et al. 2008). Furthermore, the degradation of the promuta-genic DNA precursor 8-oxo-dGTP by a speciWc GTPase isalso inhibited by nickel(II) (Porter et al. 1997). The comu-tagenic properties of nickel ions are also reXected by epide-miological results. Occupational exposure to readilysoluble nickel salts led to lung tumors only at relativelyhigh exposure levels, but it increased the tumor incidenceafter simultaneous exposure to either poorly soluble nickelcompounds (Doll 1990) or tobacco smoke (Andersen et al.1996).


In addition to its genotoxic activity, nickel deregulates nor-mal growth control by several epigenetic mechanisms(reviewed by Salnikow and Zhitkovich 2008). In culturedmammalian cells, nickel chloride caused increased methyl-ation of cytosine bases and decreased expression of tumorsuppressor genes resulting in accelerated cell proliferation.Also in nickel-induced tumors, DNA hypermethylation wasobserved together with reduced expression of tumor sup-pressor genes p16 and Fhit. As a second epigenetic mecha-nism, nickel chloride inhibits acetylation of several histonesfollowed by chromatin condensation in eukaryotic cells,probably by binding of nickel ions to histone proteins.Since histone acetylation aids the access of transcriptionfactors to DNA, inhibition of histone acetylation is believedto contribute to the observed silencing of telomeric markergenes. As a third mechanism, the activation of hypoxic sig-naling is suggested. Nickel ions are strong inducers of thehypoxia-inducible factor HIF-1 and HIF-dependent tran-scription. Mimicking of the hypoxic state may provide themetabolic condition for the selection of transformed cellsthat have altered energy metabolism, changed growth con-trol and resistance to apoptosis (reviewed by Maxwell andSalnikow 2004).

Vanadium

Vanadum occurs in the oxidation states 0, +2, +3, +4, and+5. In the presence of oxygen, pentavalent vanadium is thestable state, whereas in biological media both the vanadateanion H2VO4 with pentavalent vanadium and the vanadylcation VO2+ with tetravalent vandium are stable and mutu-ally interconverted easily. Divanadium(V) pentoxideinduced lung tumors in mice and rats (NTP 2002).

Genotoxicity

The genotoxicity of vanadium compounds has beenreviewed recently (IARC 2006a; DFG 2006c). In animalexperiments, vanadium(V) and vanadium(IV) compoundsinduced micronuclei, vanadium(V) compounds caused

chromosomal aberrations and aneuploidy in bone marrowcells. Both vanadium(IV) and vanadium(V) compoundswere positive in dominant lethal tests. In human cells invitro, vanadium(V) compounds induced DNA strandbreaks. In mammalian cells, vanadium(III), vanadium(IV)and vanadium(V) compounds caused the formation of chro-mosome aberrations and vanadium(IV) and vanadium(V)induced aneuploidies in mammalian cells. Similar to mostcarcinogenic metal compounds, vanadate(V) exhibited noconsistent results in bacterial mutagenicity assays. Thegenotoxicity of vanadium compounds is interpreted bymechanisms of induction of oxidative stress, inhibition ofDNA repair and interference with the activity of proteinphosphatases and kinases. The observed induction of aneu-ploidy by vanadate(V) is interpreted by the inhibition ofspindle formation and the disruption of microtubule assem-bly (Ramirez et al. 1997; Mailhes et al. 2003). Vanadate(V)inhibits relative speciWcally the activity of protein-tyrosinephosphatases (Stankiewicz et al. 1995). Because theseenzymes regulate the aggregation of the meiotic spindleand the spindle checkpoint during meiosis, the inhibition ofprotein-tyrosine phophatases may contribute to the geno-toxicity of vanadium(V).


The genotoxicity of vanadium(V) can be attributed to oxi-dative mechanisms. In a cell-free system containing ratliver microsomes, vanadate(V) was reduced by NADH tovanadium(IV) and generated hydroxyl radicals as detectedby ESR spectroscopy (Shi and Dalal 1992). Vanadate(V)reacted with thiols to produce vanadium(IV) and thiyl radi-cals (Shi et al. 1990). Vanadyl(IV) sulfate catalyzed thereaction of 2-deoxyguanosin with molecular oxygen toform 8-hydroxydeoxyguanosin, and it caused strand breaksin isolated plasmid DNA (Shi et al. 1996).

Interference with DNA repair

In addition to its own genotoxicity, vanadium(V) mayenhance the eVects of other genotoxic agents. In humanWbroblasts, a low concentration of vanadate(V) (1 M)impaired the repair of DNA damage caused by UV irradia-tion or by bleomycin (Ivancsits et al. 2002).


Inhibition of protein tyrosine phosphatases by vanadate(V)is thought to enhance mitogenic signalling, because inhibi-tion of dephosphorylation stabilizes active phosphorylatedproteins. In mammalian cells, vanadyl(IV)sulfate activatedphosphatidylinositol-3 kinase, and vanadyl(IV)sulfate andvanadate(V) stimulated mitogen-activated protein kinases123

Arch Toxicol (2008) 82:493512 507ERK-1 and ERK-2 (Pandey et al. 1999; Wang and Bonner2000). In mouse epidermis cells, vanadate(V) activatedprotein kinase B (Akt kinase) and stimulated the entry ofcells into the S-phase (Zhang et al. 2004). A further mecha-nism stimulating proliferation is the activation of severaltranscription factors by oxidative mechanisms. In a murinemacrophage cell line, vanadate(V) induced the activation ofTNF (tumor necrosis factor ) (Ye et al. 1999), and inmurine epidermis cells vanadate(V) activated the transcrip-tion factor AP-1 (activator protein 1) (Ding et al. 1999). Onthe level of gene exporession, vanadate(V) activated theproliferin gene and induced morphological cell transforma-tion of murine Wbroblasts (Parfett and Pilon 1995). Thesestimulatory eVects of vanadium compounds on mitogenicsignalling enzymes, transcription factors and gene expres-sion are thought to promote cell transformation and malig-nant growth by carcinogenic vanadium compounds.

Conclusions

Carcinogenic metals are widely distributed over the peri-odic table of the elements. They occur in eight diVerentgroups from clear-cut metals to metalloids, from hard met-als like beryllium, which form ionic compounds only, tosoft metals like lead which are able to form covalent bonds.In spite of the wide range of physicochemical properties,some common mechanisms of carcinogenesis emergewhich can be regarded as typical for metal carcinogens ingeneral. They include the induction of oxidative stress,inhibition of DNA repair, activation of mitogenic signal-ling, and epigenetic modiWcation of gene expression, whichmay even be based on the same or similar molecular inter-

actions. Figure 9 gives an overview over these genetic andepigenetic mechanisms ultimately concurring in the dereg-ulation of cell growth and development of tumors. Never-theless, each metal and also each metal species exertcharacteristic interactions, and even though similar cellularpathways are aVected, the underlying mechanisms are quitediVerent.

One decisive factor in metal carcinogenesis is the bio-availability of diVerent metal species, and an important bar-rier is the cell membrane. Depending largely on the actualspecies present in physiological environments, metals canenter the cell via anion channels or cation transporters. Poorwater soluble particulate metal species may be endocytosedand are gradually dissolved in the acidic environment of thelysosomes, where respective metal ions are deliberated anddistributed within the cytoplasm and also the nucleus. Thepotential impact of the membrane passage is most evidentin case of chromium compounds: while the human and ani-mal carcinogen chromium(VI) is readily taken up via theanion transporter due to its similarity to sulfate, the cellmembrane is nearly impermeable for chromium(III), forwhich no carcinogenicity has been observed so far.

Once inside the cell, in most cases, the DNA appears notto be the primary binding site for carcinogenic metal ions.Even though due to their cationic character, in principlethey can form adducts with DNA bases as shown in isolatedsystems, interactions with proteins appear to be preferred inintact cells. One important exception is chromium(VI):after its intracellular reduction to chromium(III), it bindsreadily to DNA forming DNA-protein and DNADNAcrosslinks.

Nevertheless, in spite of the missing DNA binding, theinduction of oxidative DNA damage has been observed for

Fig. 9 Major mechanisms in metal carcinogenicity. Not shown are unique mechanisms found with speciWc metal com-pounds such as chromium-DNA adduct formation, cadmium interference with cellcell adhe-sion or vanadate inhibition of protein phosphatases

Inhibition of DNA Repair

Inhibition of AntioxidantDefences

Activation of Mitotic Signalling

Modulation ofGene Expression

DecreasedGenomic Stabilty

Oxidative Stress

Induction of Protooncogenes

Inactivation of TumorSuppressorGenes

Accumulationof CriticalMutations

Deregulation of Cell Proliferation

MetalCompound

TumorDevelopment123

508 Arch Toxicol (2008) 82:493512most metals, and common mechanisms include the interfer-ence with the cellular defense system against reactive oxy-gen species, including DNA repair systems, and/or thecatalysis of Fenton-type reactions where endogenouslyformed ROS like hydrogen peroxide are converted into thefar more reactive hydroxyl radicals. Furthermore, ROS maybe generated also in the course of intracellular reduction ofmetals, as is the case of chromium(VI) reduction to chro-mium(III) with instable chromium(V) and chromium(IV)intermediates, and also redox reactions occurring for exam-ple in the course of methylation of arsenite within meta-bolic competent cells. In many cases, the relevance ofoxidative DNA damage for metal carcinogenesis remainsquestionable, since in experimental systems, frequently butnot in all cases, comparatively high concentrations arerequired to yield signiWcant increases of the endogenouslevel of oxidative DNA damage. However, oxidative modi-Wcations may also play a role in the interaction with pro-teins, as outlined below.

For most metal compounds, interactions with proteinsappear to be more relevant for carcinogenicity as comparedto direct DNA damage, and several targets have been iden-tiWed, such as DNA repair, tumor suppressor and signaltransduction proteins. Even though diVerent metal com-pounds exert diVerent eVects on protein functions, commonmechanisms include the displacement of essential metalions and/or the oxidation of critical amino acids leadingalso to altered redox regulation, perhaps best investigatedfor DNA repair proteins. Since metal ions can bind in prin-ciple to many electron rich centers in proteins, this raisesthe question whether there are particularly metal-sensitiveprotein structures. During the last years, so-called zincWnger proteins have been identiWed as potential moleculartargets for toxic metal compounds. They represent a familyof proteins where zinc is complexed through four invariantcysteine and/or histidine residues forming a zinc Wngerdomain, which is mostly involved not only in DNA bindingbut also in proteinprotein interactions (Mackay and Cross-ley, 1998). Besides transcription factors, several proteinsinvolved in DNA damage signaling and repair belong tothis family, and also the tumor suppressor protein p53 has azinc binding structure in its DNA binding domain, essentialfor its transcriptional activity. For several zinc Wnger pro-teins, molecular interactions with toxic metal ions havebeen elucidated in detail. Thus, cadmium can substitute forzinc in the zinc Wnger domain of the nucleotide excisionrepair protein XPA, leading to structural distortions whichdisturb its correct function within the nucleotide excisionrepair complex. In contrast, nickel can substitute for zinc inthe XPA protein and increase its sensitivity towards oxidiz-ing agents. Perhaps most relevant are the results in the caseof arsenite and its trivalent methylated metabolites. Whileall of them inhibit the poly(ADP-ribosyl)ation, mediated

predominantly by the zinc Wnger protein PARP-1 atextremely low concentrations, detailed molecular studieswith the zinc Wnger structure of XPA revealed an oxidationof the zinc complexing thiol groups for arsenite, while incase of MMA(III) the binding to the thiol group andthereby the replacement of zinc was observed. Finally, withrespect to cadmium, an unfolding of the zinc bindingdomain of p53 was observed, leading to a complete loss oftumor suppressor functions. Thus, there is accumulatingevidence for zinc binding structures being very sensitivetargets for toxic metal compounds, but whether or not therespective proteins are indeed inhibited and if so by whichmechanism depends on the speciWc interaction of the metalion with the respective protein. Decisive factors appear tobe not only physicochemical properties but also assessibil-ity and the microenvironment within the protein underinvestigation.

Altogether, the inhibition of DNA repair processes andthe interference with cell growth, cell cycle control andtumor suppressor functions appears to be more evident forcarcinogenic metal compounds as opposed to direct muta-genicity. Nevertheless, the outcome, that is the decrease ingenomic stability, is very similar or even more severe.Since DNA repair systems not only provide pronouncedprotection towards environmental mutagens, but alsotowards endogenous DNA damage occurring permanently,for example due to oxygen metabolism their disturbanceresults in an increase in mutations and carcinogenesis. Thisis evident, for example, in the high tumor frequency inpatients with the DNA repair disorder Xeroderma pigmen-tosum. Nevertheless, other mechanisms contribute as well,such as epigenetic alterations of gene expression and altera-tions in signal transduction pathways leading, for example,to growth stimulation or deregulated apoptosis; the applica-tion of new techniques like genomics and proteomics willprovide much more information in the near future. Also,these common mechanisms do not exclude the existence ofunique interactions of speciWc metal species, such as thebinding of vanadate to phosphate binding sites. Consideringrisk assessment, future research will have to focus on therelevance of the respective mechanisms in experimentalanimals and exposed humans, especially with respect toeVective concentrations.

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