5.1 Introduction: Epidemiology and Animal Carcinogenicity

Cadmium is a natural element of the earth’s crust, but its distribution in the environment and thus human exposure is greatly influenced by industrial use and agriculture. Significant exposure occurs via inhalation at the workplace. With respect to the general population, food and tobacco smoking are the main exposure sources. Mainly based on sufficient evidence for an increased relative risk of lung cancer in workers occupationally exposed to cadmium, in 1993 and 2009, cadmium and its inorganic compounds were classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (IARC Group 1) [2,3,4] and 2004 as carcinogens group 1 (carcinogenic to humans) by the German MAK Commission [5]. Cohorts in United Kingdom, Sweden and United States have been investigated, including extensive follow-up studies [6,7,8,9], revealing elevated risks of lung cancer in most cases. Some major constraints are, however, the small number of long-term, highly exposed workers and the lack of historical data on exposure to cadmium in some studies. Furthermore, confounding factors are cigarette smoke and simultaneous exposure to nickel and arsenic [4, 5]. Supportive evidence from environmental inhalative exposure provided a study in Belgium with subjects living near three smelters when compared to subjects not exposed to elevated levels of cadmium, investigated from 1985 until 2004. Based on urinary cadmium excretion and cadmium in garden soil as exposure indicators, elevated lung cancer risks were observed in the high-exposure group [10]. With respect to other cancer sites, especially the kidney may be of elevated risk due to high and persistent cadmium accumulation, going along with nephrotoxicity close to environmental exposure levels. No elevated risks for renal cancer due to cadmium exposure were observed in a Swedish or in a British cohort study [7, 9]. Nevertheless, case control studies elucidating the relative risk of kidney cancer due to occupational cadmium exposure, which have been conducted in the United States, in Finland, in Germany and in Canada and which estimated cadmium exposure via job-exposure-matrices (JEM), observed higher incidences of renal cancer upon cadmium exposure at the workplace [11,12,13,14]. Altogether, the German MAK Commission concluded that an increased relative risk of renal cancer has to be assumed [5] and also IARC stated a positive association with respect to renal and prostate cancer [4]. Finally, human cadmium exposure may also be associated with female breast and endometrial cancer, even though these tumor locations are not definitively established [15, 16]. Regarding data from experimental animals, cadmium did not induce lung tumors in the hamster [17] but at remarkably low concentrations in rats. In the latter species in long-term inhalation studies, several cadmium compounds (CdCl2, CdSO4, CdS, and CdO) caused lung cancer (mainly adenocarcinomas) [18, 19]. The lowest concentration inducing primary lung carcinoma in rats (15 versus 0% in controls) was 12.5 μg Cd/m3, even though under an unusual exposure regimen (23 h/day, 7 days per week for 18 months exposure to CdCl2 aerosols) [18]. In a later study, no lung tumors were induced when the rats were exposed continuously for 18 months to CdO fumes at a concentration of 10 μg Cd/m3, whereas 21% of the animals developed tumors when exposed to 30 μg Cd/m3 [19]. Considering oral exposure, adequately conducted studies revealed increased incidences of large granular lymphocytes, leukemia, prostate, and testis tumors in Wistar rats (summarized in [2, 5, 20]).

5.2 Direct and Indirect Genotoxicity

5.2.1 DNA Damage, Mutagenicity, and Clastogenicity

Cadmium does not cause DNA damage in cell extracts or in isolated DNA [21]. Furthermore, cadmium is not mutagenic in classical short-term test systems. Thus, in most bacterial assays, water-soluble cadmium compounds were not mutagenic, and in standard mammalian mutagenicity tests, the induction of point mutations was usually weak and/or restricted to comparatively high concentrations. In contrast, pronounced co-mutagenic effects in combination with DNA alkylating agents and with UVC radiation were observed both in bacteria and in mammalian cells, pointing towards an interaction with the cellular response to DNA damage (see below). In contrast to the missing mutagenicity, in mammalian cells cadmium compounds provoked clastogenic effects such as chromosomal aberrations and micronuclei [2, 5, 22,23,24]. This was also demonstrated by the pronounced positive effects of cadmium chloride in a modified mammalian test system capable of detecting large multi-locus deletions [25]. The clastogenicity is moreover evident in vivo in exposed rodents, while evidence for chromosomal damage in cadmium-exposed humans via environmental or workplace exposure is equivocal, partly due to simultaneous exposure to other metal compounds [2, 5, 26]. Recently, a reanalysis of the relationship between cadmium exposure and micronuclei in lymphocytes of exposed workers was performed, based on five studies published so far. While two studies with high exposure showed significant increases in micronuclei, three studies gave negative results, which may be due to lower exposure levels [27].

5.2.2 Oxidative Stress

Reactive oxygen species (ROS) such as superoxide anions (O2−) are continuously generated not only as by-products of mitochondrial respiration, due to incomplete reduction of oxygen to H2O but also formed in large quantities during the immune defense against invading pathogens in phagocytes via NADPH oxidase. Also, hydrogen peroxide (H2O2) is produced in specific cellular compartments and acts as a “second messenger” to regulate many important biological processes (for review see [28]). To enable the use of oxygen for energy production and yet to minimize oxygen-derived toxicity, a complex antioxidant network has evolved, including the scavenging of reactive species by glutathione and vitamins, the enzymatic conversion of highly reactive oxygen species to less harmful ones by superoxide dismutase, catalase, and glutathione peroxidase, and finally the repair or elimination of damaged macromolecules. However, even under normal cellular conditions, protection is not complete and for example, a measurable amount of oxidatively induced DNA damage exists in mammalian cells. Oxidative stress occurs if the equilibrium between the generation of ROS and the efficiency of their detoxification is disrupted [29]. Elevated levels of ROS have been implicated in the initiation as well as in later steps of carcinogenicity. Especially transition metal ions play an important role in the induction of oxidatively induced DNA damage. While neither superoxide anions nor hydrogen peroxide are able to react with DNA directly, in the presence of transition metals like iron, copper, cobalt, or nickel, they are converted into highly reactive hydroxyl radicals by Fenton-type reactions. DNA damage induced by ROS includes a range of lesions like DNA base modifications, sugar lesions, DNA single- and double-strand breaks, DNA–protein crosslinks, and abasic sites (for review see [30]). Among these, several oxidatively induced DNA base modifications such as 8-oxoG have miscoding and thus premutagenic properties and therefore may act as initiators in carcinogenesis [31]. In contrast, cadmium ions are not able to participate in redox reactions under physiological conditions, yet, oxidative stress and the interference with cellular redox regulation may be of high relevance in cadmium-induced carcinogenicity. Increased levels of ROS due to cadmium exposure have been observed both in vitro and in vivo [32]. Thus, different cadmium compounds have been shown to induce DNA strand breaks and oxidatively induced DNA base modifications in mammalian cells, but effects were usually small and/or restricted to comparatively high concentrations (e.g., [33, 34]). Similarly, the induction of DNA strand breaks and chromosomal aberrations by cadmium in mammalian cells was suppressed by antioxidants and antioxidant enzymes, indicating the involvement of ROS [35,36,37]. A pronounced oxidative stress response was also observed on the transcriptional level after exposure to BEAS-2B and A549 cells [38]. Even though cadmium ions themselves are not redox-active, several indirect effects may account for these observations, namely the release of Fenton-reactive metal ions from metallothioneins [39], the disturbance of the mitochondrial respiratory chain [40] and the inhibition of antioxidant enzymes, such as catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase [29]. Regarding oxidative DNA damage and resulting clastogenicity in cells and in vivo, an impaired DNA repair appears to be of major relevance (see below). Thus, ROS may be involved in cadmium-induced genotoxicity, but—perhaps more important—also in later steps of cadmium-induced carcinogenicity. With respect to the latter, moderately elevated levels of ROS have been implicated in later steps of tumor formation, such as cell proliferation due to mitotic stimuli and the activation of redox-sensitive transcription factors (see below). Furthermore, due to its reactivity towards thiol groups, cadmium may interfere directly with redox-controlled signaling pathways [37, 41, 42].

5.3 Interactions with DNA Repair and Tumor Suppressor Functions

Maintenance of genetic information is essential for basically all cellular processes and for the prevention of tumor development. However, many environmental agents as well as food mutagens have been identified which compromise genetic stability by inducing different types of DNA lesions. They include ionizing radiation, UV radiation, alkylating agents, polycyclic aromatic hydrocarbons as well as heterocyclic aromatic amines. Furthermore, the DNA is also damaged by endogenous processes, such as ROS generation due to leakage of the electron transport chain in cellular respiration [37, 43]. DNA damage interferes with DNA transcription and replication; potential consequences are cell cycle arrest, programmed cell death, mutagenesis, genomic instability, and cancer. To maintain the integrity of the genome, a complex DNA damage response network has evolved, consisting of DNA repair systems, cell cycle control, and apoptosis in case of heavily damaged DNA [44,45,46]. Cadmium has been shown to impair almost all major DNA repair pathways. Convincing evidence is available for its interference with nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR), with some information also on its impact on DNA double-strand break repair; frequently, effects were observed at comparatively low, noncytotoxic concentrations (reviewed in [47,48,49,50]). Since DNA repair systems are not only required for the repair of DNA damage induced by environmental, workplace, and food mutagens but also for the elimination of DNA lesions due to endogenous processes and to keep replication errors low, the disturbance of DNA repair processes may explain co-mutagenic effects in combination with UVC radiation, benzo[a]pyrene and alkylating agents on one side but may also lead to genomic instability and thus contribute to cadmium-induced carcinogenicity on the other side (for reviews see [22, 23, 49]).

5.3.1 Nucleotide Excision Repair (NER)

NER is the most versatile repair system involved in the removal of structurally unrelated bulky base adducts which cause significant helical distortions. It can be subdivided into global genome repair (GG-NER) and, as a sub-pathway, transcription-coupled nucleotide excision repair (TC-NER), which removes preferentially transcription-blocking bulky DNA lesions. At least 30 different proteins and enzymes are required in mammalian cells, including those which are defective in patients suffering from the DNA repair disorder Xeroderma Pigmentosum (XP) complementation groups A through G. The most crucial step is the damage recognition, followed by the incision at both sides of the lesion and the repair polymerization leading to the displacement of the damaged oligonucleotide; repair is completed by the ligation of the repair patch (for reviews see [44,45,46, 51]). Cadmium has been shown to inhibit GG-NER in several studies and with respect to different DNA damaging agents. Thus, it interfered with the removal of benzo[a]pyrene- and UVC-induced DNA lesions in cultured mammalian cells [34, 52, 53]. As one underlying mechanism, an interaction with zinc-binding proteins has been identified. They comprise a family of proteins where zinc is complexed to four cysteine and/or histidine residues, folding a protein domain mostly involved in DNA-protein- or protein–protein interactions [54]. First discovered in transcription factors, similar structures have been identified in DNA repair proteins and tumor suppressor proteins like p53. Examples for DNA repair proteins with zinc-binding structures include the bacterial formamidopyrimidine-DNA glycosylase (Fpg) involved in the removal of oxidative DNA base modifications and the mammalian Xeroderma Pigmentosum group A protein (XPA) essential for the formation of the DNA damage recognition complex during NER (reviewed in [55, 56]). In subcellular test systems, cadmium diminished DNA binding of XPA to an UVC-irradiated oligonucleotide [57, 58]. One molecular mechanism related to the inactivation of zinc-binding proteins appears to involve the displacement of zinc by cadmium, as evident from the reversal of cadmium-induced protein inactivation by excess of zinc as well as from structural investigations of XPA or a peptide resembling the zinc-binding domain of XPA [57,58,59,60]. Detailed studies in cadmium-treated A549 cells revealed an impaired assembly/disassembly of the DNA damage recognition proteins XPC and XPA at the repair complex after UVC irradiation [34].

5.3.2 Base Excision Repair (BER)

In contrast to the damage recognition complex in NER that detects a rather broad spectrum of DNA lesions, BER is initiated by glycosylases, which act specifically on one or few substrates. BER is mainly responsible for the removal of different types of endogenous DNA damage, including oxidative DNA base modifications like 8-oxoguanine (8-oxoG). This process generates abasic (AP) sites, which are further processed in a multistep process with slight differences depending on the type of damage [43,44,45, 51]. Regarding the impact of cadmium on this repair pathway, low concentrations of cadmium inhibited the activity of the bacterial Fpg [61] and disturbed the repair of oxidatively induced DNA base damage induced by visible light as well as DNA alkylation damage in mammalian cells [33, 62]. When compared with the induction of DNA base modifications such as 8-oxoG, inhibitory effects on the repair of this lesion were observed at much lower cadmium concentrations. This has been observed by direct comparison in HeLa cells: While the induction of DNA strand breaks by cadmium was restricted to 10 µM and higher, the removal of oxidatively induced DNA base modifications by visible light and recognized by the bacterial Fpg was inhibited starting at 0.5 µM cadmium, yielding complete inhibition at 5 µM, a noncytotoxic concentration in this test system [33]. With respect to isolated DNA repair enzymes, an inhibition of the murine 8-oxoguanine DNA glycosylase 1 (mOgg1), an enzyme responsible for recognition and excision of the premutagenic 8-oxoG during BER, as well as of 8-oxodG 5’triphosphate pyrophosphohydrolase (8-oxo-dGTPase), required for the removal of 8-oxo-dG from the deoxynucleotide pool, by cadmium have been described [63, 64]. Also, cadmium has been shown to inhibit the activity of the human 8-oxoguanine glycosylase (hOGG1) in mammalian cells [65,66,67]. Different mechanisms may be responsible, based on different experimental results, including the inactivation of the enzyme as such [66] or the diminished DNA binding of the zinc finger containing transcription factor SP1 to the OGG1 promotor [68], presumably due to the displacement of zinc by cadmium [69]. Inhibition of the repair of oxidatively induced DNA damage is also evident in vivo: When investigating, for example, the impact of cadmium on rat testis, a target organ for cadmium carcinogenesis, a gradual decrease in testicular 8-oxo-dGTPase activity was observed, accompanied with progressive increase of 8-oxo-dG levels in testicular DNA [70]. Therefore, increases in oxidatively induced DNA damage in vivo may at least in part be due to the repair inhibition and accumulation of endogenously induced DNA lesions. One other enzyme involved in DNA damage signaling, apoptosis and BER is poly(ADP-ribose) polymerase 1 (PARP-1). It contains three zinc fingers in its DNA binding domain involved in the recognition of DNA breaks and the subsequent synthesis of poly(ADP-ribose) [71, 72]. In HeLa cells, H2O2-induced PARP activity was decreased by cadmium chloride [73].

5.3.3 Mismatch Repair (MMR)

One other DNA repair system of particular relevance for maintaining genomic stability is MMR. This evolutionary conserved pathway is responsible for the repair of mismatched normal bases after DNA replication, contributing significantly to the extraordinary fidelity of DNA replication. Cells deficient in MMR exert a “mutator phenotype”, in which the rate of spontaneous mutations is greatly elevated. Also, microsatellite instability (MSI) is a hallmark of MMR deficiency. Defects in MMR are associated with an increased risk of different types of cancer, including hereditary human colorectal cancer. The MMR system also plays a key role in cell killing in response to alkylating agents, and MMR deficient cells are about 100 times more resistant to the cytotoxicity of alkylating agents [74,75,76,77]. Finally, MMR also participates in the DNA damage response (DDR) system by activating ATM and ATR, which regulate cell cycle control and apoptosis upon elevated levels of DNA damage [78]. With respect to cadmium, exposure towards low concentrations resulted in pronounced hypermutability in yeast. Furthermore, in extracts of human cells, cadmium inhibited at least one step leading to mismatch repair [79]. Since then, different studies demonstrated the interference by cadmium with proteins involved in the initial step of MMR, i.e., damage recognition by MSH2-MSH6 and MSH2-MSH3. Also, the induction of MSI was demonstrated in mice testis [80]. As underlying mechanisms, cadmium affected ATP binding and hydrolysis of MMR enzymes, reducing their DNA binding activity and their ability to discriminate between mismatched and matched DNA base pairing in isolated systems and in mammalian cells in culture [48, 81, 82]. Furthermore, the induction of MSI concomitantly with elevated levels of oxidatively induced DNA damage has recently been shown in human colorectal cancer cells and in zebrafish at sublethal cadmium concentrations; interestingly, in both systems, N-acetyl-L-cysteine (NAC) suppressed cadmium-induced MSI, thus linking MMR inhibition to oxidative stress [83].

5.3.4 DNA Double-Strand Break Repair (DSB)

Only little is known about the impact of cadmium on DNA double-strand break repair. Current evidence suggests that cadmium leads to the formation of DSB and inhibits their repair via nonhomologous end-joining (NHEJ) via interaction with the DNA-PK protein, leading to an over-activation of the MRE11-dependent repair pathway which in turn may favor more misrepair [84]. The delay of NHEJ upon γ-irradiation was confirmed in another study; here, the authors demonstrated an altered phosphorylation of DNA-PKcs as well as reduced expression of XRCC4 and Ligase IV [85]. Also, a significant downregulation of genes coding for ATM and BRCA1 associated with DSB repair was observed via high-throughput RT-qPCR [38]. Finally, when assessing the impact of several metal compounds on DNA double-strand break repair outcomes, exposure towards cadmium was shown to increase mutagenic, nonallelic recombination [86].

5.3.5 P53 Tumor Suppressor Functions

Besides DNA repair systems, further DNA damage responses are activated upon genotoxic stress in mammalian cells. They include cell cycle control mechanisms, increasing the time for DNA repair, as well as apoptosis eliminating heavily damaged cells. The DNA damage response is strictly coordinated, for example by the tumor suppressor protein p53. P53 regulates cell cycle control and apoptosis by several coordinated pathways and thus exerts pronounced impact on the processing of DNA damage and on genomic stability [87]. Cadmium has been shown to interfere with structure and function of p53, but opposite effects have been reported. In some studies, a stabilization of p53 through phosphorylation followed by the induction of the p53-mediated stress response was observed [88,89,90]; others demonstrated an inactivation of p53 via structural changes [34, 91]. P53 contains a zinc-binding structure in its DNA binding domain, essential for its tumor suppressor functions and rendering the protein redox-sensitive. Exposure of either the isolated p53 protein or human breast cancer MCF7 cells to cadmium resulted in the disruption of the zinc-binding structure, yielding a so-called “mutant” conformation; consequences were the inhibition of DNA binding and the inhibition of the activation of p53 target genes including p21. Furthermore, suppression of the p53-mediated cell cycle arrest in response to DNA damage induced by γ-irradiation was observed [91]. Similar effects were demonstrated in A549 human lung tumor cells, where CdO and CdCl2 induced structural alterations of the zinc-binding domain of p53, followed by diminished induction of the p53 regulated nucleotide excision repair gene XPC and diminished removal of UVC and benzo[a]pyrene induced DNA damage [34]. Thus, it appears that cadmium disrupts the zinc-binding structure of p53; whether or not this is due to the displacement of zinc is currently not clear.

5.4 Impact on Gene Expression Related to Genomic Instability and Deregulation of Cell Proliferation

Cadmium interacts with the expression of a large number of genes, including stress response genes and immediate early response genes. Major stress response genes induced by cadmium are those involved in the synthesis of metallothionein (MT), those encoding heat shock proteins, glutathione (GSH) synthesis and homeostasis and those mediating the oxidative stress response [23, 24, 42, 92,93,94]. Recently, the impact of cadmium was investigated via a high-throughput RT-qPCR test system specifically designed for the parallel and quantitative analysis of 95 selected genes crucial for genomic stability, including stress response as well as DNA repair, cell cycle control, apoptosis, and mitotic signaling. Gene expression analyses in cadmium-treated adenocarcinoma A549 and epithelial bronchial BEAS-2B cells revealed distinct dose- and time-dependent and also cell-type specific gene expression patterns, including the induction of genes coding for metallothioneins, the oxidative stress response, cell cycle control, mitotic signaling, and apoptosis. Interestingly, while genes coding for the DNA damage response were induced, distinct DNA repair genes were downregulated at the transcriptional level. Thus, this approach provided a comprehensive overview on the interaction by cadmium with distinct signaling pathways, also reflecting molecular modes of action in cadmium-induced carcinogenicity on the functional level, such as inhibition of DNA repair and tumor suppressor reactions [38]. On the molecular level, specific interactions with transcriptions factors, an interference with cellular redox regulation [42] as well as epigenetic alterations appear to be most relevant. With respect to transcription factors, cadmium exposure may lead to their activation or inactivation, depending on the actual transcription factor under investigation. Thus, c-fos and c-jun are overexpressed in cadmium-transformed cells; they constitute the AP-1 transcription factor, activating several genes involved in cell growth and division. Other transcription factors activated by cadmium are NF-κB and Nrf2. On the other hand, a suppression of the transcription factor SP1 has been observed in cadmium-treated cells (reviewed in [93, 94]). In support of a role for a redox-mediated mechanism in cadmium-induced activation of the MAPK-pathway, cadmium was shown to inhibit serine/threonine phosphatases 2A (PP2A) and 5 (PP5), leading to the activation of Erk1/2 and JNK. This effect was inhibited by N-acetyl-L-cysteine (NAC), and resembled the effects induced by hydrogen peroxide; therefore, the authors interpreted this effect being due to the enhanced generation of ROS [95, 96]. Nevertheless, since these phosphatases contain critical cysteine residues [97], the impact of cadmium may also be explained by a direct reaction with these cysteines. One other example is the cadmium-induced activation of Nrf2 [98, 99]. The Kelch-like ECH-associated protein (Keap1) serves as negative regulator of Nrf2. It contains critical cysteine residues as redox sensors; their oxidation results in the release of Nrf2 from the Keap1/cullin-3 E3-ubiquitin ligase (cul3) complex, preventing Nrf2 degradation and allowing for Nrf2 nuclear translocation. Additionally, Nrf2 contains a conserved cysteine located in the DNA binding domain (Cys-514) redox-regulated by Ref-1. After treatment of mouse embryo fibroblasts (MEF) with cadmium, Nrf2-Keap1 were stabilized in the cytoplasm and translocated to the nucleus, where the components dissociated. This was followed by the induction of the ARE-dependent expression of HO-1 in MEF cells and in a respective reporter gene assay [98].

In addition to directly stimulating mitogenic signals, cadmium also inhibits negative controls of cell proliferation, for example, by inactivation of p53 (see above) [91]. Furthermore, long-term treatment of prostate epithelial cells resulted in cadmium-induced malignant transformation; transformed cells exerted an acquired resistance to apoptosis, which appeared to be linked to an increase in the antiapoptotic action of Bcl-2 that perturbs the JNK signal transduction pathway [100]. One other aspect related to cadmium-induced alterations in gene expression consists in epigenetic changes. Three levels of interaction appear to be relevant, namely interference with DNA methylation patterns, histone modifications and miRNAs, all of which may be affected by cadmium in a time- and cell-type specific manner (for review see [101]). The mechanism in cadmium-induced epigenetic alterations has been further investigated in a rat liver epithelial cell line as a model for cadmium-induced malignant transformation. Cadmium provoked the suppression of ApoE, a key factor of cell invasion during malignant transformation via 5-aza-2’-deoxycytidine-sensitive hypermethylation of the regulatory region of ApoE, together with the suppression of liver X receptor α (LXRα), a transcriptional regulator for ApoE [102].

5.5 Mechanistic Considerations

Cadmium appears to be involved in tumor initiation as well as in later steps of tumor development. Regarding genotoxicity, direct interactions of cadmium ions with DNA are of little importance, as evident also by the lack of relevant mutagenicity in classical bacterial and mammalian test systems. However, indirect genotoxicity leads to clastogenicity and elevated levels of oxidative DNA damage; in this context, interactions with proteins are of high significance. Especially the DNA repair inhibitions but also altered cell proliferation and/or diminished cell cycle control have frequently been observed at low, noncytotoxic concentrations of cadmium, pointing towards particular sensitive targets of cadmium ions. Relevant mechanisms include elevated levels of ROS, interactions with homeostasis and cellular functions of essential metal ions like zinc, calcium, and iron and the interference with cellular redox regulation.

Current evidence suggests that Cd2+ is the ultimate damaging species since water-soluble and particulate water-insoluble cadmium compounds exert similar effects in experimental cell culture systems and in experimental animals. Thus, both cadmium chloride and largely water-insoluble cadmium oxide induced oxidative DNA lesions and inhibited the removal of benzo[a]pyren-induced DNA lesions. Furthermore, cadmium-induced conformational changes of p53 were comparable when applying cadmium chloride or cadmium oxide. Repair inhibitory effects were strongly correlated with cadmium levels in the nuclei, indicating the bioavailability of both compounds [34]. While water-soluble cadmium compounds are taken up via ion channels [103], particulate cadmium compounds may be taken up by phagocytosis and, due to the low pH, may dissolve gradually in lysosomes, yielding high concentrations of cadmium ions in the cytoplasm and in the nucleus, as described in detail for nickel compounds [104, 105]. This assumption is also supported by inhalation studies where water soluble cadmium sulfate, poorly water-soluble cadmium oxide and cadmium sulfide pigment with intermediate water solubility induced lung tumors in rats [106].

Since cadmium ions exert high affinity towards SH groups, potential targets are zinc-binding structures frequently found in transcription factors as well as in DNA repair and tumor suppressor proteins [55, 56]. As indicated above, one molecular mechanism related to the inactivation of zinc-binding proteins appears to involve the competition between zinc and cadmium. Compared to Zn2+, the radius of Cd2+ ion is larger, but still, cadmium ions can substitute for zinc ions in many enzymes and transcription factors [24, 107]. Considering the example of the nucleotide excision repair protein XPA or a peptide resembling the zinc-binding domain of XPA, binding constants for cadmium were about 1000-fold higher as compared to zinc. Replacement of zinc by cadmium yielded only minor structural alterations [59, 60], but provoked a pronounced disturbance of XPA within the assembly and disassembly of the nucleotide excision repair complex [34]. In addition to direct interactions with DNA repair proteins, cadmium may disturb DNA repair processes via interaction with zinc-containing transcription factors. Thus, human OGG1 (hOGG1), a glycosylase responsible for recognition and excision of the premutagenic 8-oxoG during BER in mammalian cells, was inhibited by cadmium [108]. Even though hOGG1 contains no zinc-binding motif itself, its inhibition was shown to be due to diminished DNA binding of the zinc finger containing transcription factor SP1 to the OGG1 promotor [68], presumably due to the displacement of zinc by cadmium [69]. Also, a downregulation of DNA repair genes like XPC has been observed recently in cultured cells [34] and in vivo in mouse testes [109], which may be due to a disturbed transcriptional activity of p53. However, whether or not the inactivation of the respective zinc-binding repair proteins are mediated via displacement of zinc by cadmium or whether interactions with other protein structures, such as critical thiols outside the zinc-binding structure, are relevant for the observed inhibitions has to be further elucidated. Furthermore, systematic investigations on the relevance of these mechanisms for in vivo situations are still missing. Multiple mechanisms appear to be involved in cadmium-induced alterations of gene expression. With respect to the induction of metallothionein, cadmium ions bind directly to the transcription factor MTF1 [110]. In some other cases, specific interactions have been identified. With respect to epigenetic effects, cadmium inhibited DNA-(cytosine-5) methyltransferase and lead to diminished DNA methylation during cadmium-induced cellular transformation, provoking augmented expression of cellular proto-oncogenes [111]. One example for a direct competition with calcium concerns the cadherin-mediated cell–cell adhesion system; here, cadmium specifically displaced calcium from the protein E-cadherin and impaired the cell–cell adhesion in kidney epithelial cells [112, 113]. A fast transient increase in levels of second messengers like Ca2+ and inositol-1,4,5-trisphosphate by low concentrations of cadmium may be due to its binding to G-protein coupled receptors in the plasma membrane; however, cadmium affects also intracellular signaling mediated by mitogen-activated protein kinases (MAPK) as well as cAMP-dependent and calmodulin-dependent pathways (see Chap. 3). Even though zinc-binding structures are involved in many of these pathways, at present, it is unclear whether a direct replacement of zinc is the underlying mechanism [24, 94, 114]. One hypothesis integrating many mechanistic observations consists in the interference by cadmium with the cellular redox regulation [42] (see also Chap. 3). Thus, diverse signaling pathways have been identified to be redox-regulated via reversible oxidation and reduction of thiol groups [115,116,117]. Cadmium has been shown to induce several redox-regulated signal transduction pathways, such as NF-κB and Nrf2, but also mitotic signaling, which may be due to the increased formation of ROS or to direct interaction with redox-sensitive cysteines in signal transduction proteins. In most cases, the molecular interactions have not been fully explored experimentally, but are subject of current research activities.

5.6 Concluding Remarks and Perspectives

In summary, cadmium-induced carcinogenicity is likely based on multiple distinct mechanisms. As opposed to direct DNA damage, interactions with proteins appear to be more relevant for carcinogenicity, and several targets have been identified such as antioxidative defense systems, DNA repair processes as well as tumor suppressor and signal transduction proteins. All these features taken alone could contribute to carcinogenicity, but most likely their combination seems to be of particular importance. Thus, long-term exposure to low concentrations of cadmium leads to adapted cells exerting increased cadmium accumulation, increased proliferation, diminished DNA repair, and cell cycle control as well as resistance to apoptosis. The outcome is a severe decrease in genomic stability, which may play an important role in cadmium-induced tumor initiation and progression (summarized in Fig. 5.1).

Fig. 5.1
figure 1

Integrating concept of cadmium-induced carcinogenicity

Full size image

One important question concerns specific mechanisms explaining the organ-specificity of cadmium-induced carcinogenicity. After inhalative exposure, the lung is a plausible target organ, but other organs like kidney, prostate, breast, and endometrium may be affected as well. Since tumors in prostate, breast, and endometrium are frequently hormone-dependent one aspect addressed by several groups concerns a potential impact of cadmium on steroid hormone-dependent signaling [118]. Nevertheless, respective experimental evidence is contradictory and needs to be further explored [49]. One other key issue in cadmium-induced carcinogenicity appears to be adaptation and the role of MT. Cadmium induces several genes for cadmium and ROS tolerance such as those coding for MT, GSH synthesis and function, catalase and superoxide dismutase. Hence, a condition for prolonged cell survival in the presence of cadmium is established, which may be beneficial in terms of protection from acute cadmium toxicity, also evident from comparative studies with MT-transgenic and MT-null mice [119]. However, adaptation may be a double-edged sword, since increased MT contents lead not only to cadmium accumulation and long half-lives but also to reduced DNA repair activities as well as suppressed apoptosis [92, 120].

Considering recent reports on cadmium-related carcinogenicity in different target organs under low exposure conditions, future research will have to focus on the relevance of the respective mechanisms in experimental animals and in exposed humans. Nevertheless, even though Cd blood levels are in the nanomolar concentration range and thus considerably lower as compared to low micromolar concentrations applied in most in vitro systems described above, it needs to be considered that far higher cadmium concentrations are observed for example in cadmium-exposed workers, reaching up to millimolar concentrations in the kidney cortex and high micromolar concentrations in the liver [121]. Also, indications for repair inhibition upon occupational cadmium exposure have been reported [122].