Toxic heavy metals including cadmium and nickel, which are common contaminants in occupational and environmental areas, may contribute to harmful effects on humans and other organisms. Unsafe persistent exposure to such metals is mediated through oral intake of contaminated water and food, inhalation of polluted air, or dermal contact from manufacturing processes and environmental contamination. Indeed, deleterious health effects caused by cadmium and nickel are similar, but the underlying mechanisms of toxicity and biological/toxicological signaling pathways are individually different (1–3).

Cadmium is a natural constituent of ocean water and the earth’s crust and is commonly associated with zinc, lead and copper ores (4). Due to its physicochemical properties, it is utilized in a variety of ways, such as in batteries, pigment, coating, plating, plastic stabilizers, non-ferrous alloys and photovoltaic devices. Ingestion of contaminated food and water and inhalation by active and passive smoking are considered the predominant routes for cadmium exposure (5). Cadmium is classified as carcinogenic to humans (group 1) by the World Health Organization’s International Agency for Research on Cancer, as it acts as an epigenetic or indirect genotoxic element. The molecular mechanisms of cadmium carcinogenicity have been recently reviewed (6–8) and range from interference with thiol-containing proteins and consequent induction of oxidative stress to genomic expression changes triggering cell cycle arrest, apoptosis, differentiation and immortalization. Strong data from epidemiological studies on occupational populations related to cadmium carcinogenicity showed that lung cancer occurs by breathing the metal (9). Cadmium affects the transcription of several genes; it induces protective genes, including those coding for metallothioneins with a cadmium-chelating property (10,11), heat shock proteins (HSPs) with roles in protein renaturation (12,13) and heme-oxygenase I with an anti-oxidative role (14). Notably, cadmium may induce proto-oncogenes (15,16) which can contribute to carcinogenesis. Its effects on the sex hormone receptor genes (17) also suggest endocrine-disrupting activity. However, these effects of cadmium on gene expression are only a fraction of the effects, and the unidentified effects on gene functioning in toxicity and protection against cadmium must be clarified.

Nickel, a non-essential metal, occurs naturally in soils and volcanic dust. It also has been categorized as a carcinogenic heavy metal by the World Health Organization’s International Agency for Research on Cancer. It combines with other metals to form alloys that are widely used to produce coins, jewelry and stainless steel. Nickel compounds are also applied to refining, electroplating, welding, color pigmentation in ceramics, the production of batteries, medical devices and carbon particles (18). Although several animal epidemiological and cell culture studies have documented that nickel compounds are carcinogenic; the molecular mechanisms of nickel carcinogenicity have not been investigated (19–22). The mutagenic activity of nickel compounds to mammalian cells in vitro in the Salmonella mutation assay was found to be low, indicating that nickel-mediated mutagenic activity is not the definite mechanism underlying nickel carcinogenicity (23,24). A number of studies have implicated that aberrant modification of structural chromatin and epigenetic alterations might be the predominant contributable factors to nickel carcinogenesis.

Both cadmium and nickel have adverse effects on human health by inducing oxidative stress, inhibiting DNA repair proteins, and dysregulating signal transduction, thereby leading to aberrant cell proliferation and differentiation (3). Furthermore, they have also been associated with an increased risk for diseases, including cancer, following chronic low-dose exposure (25,26). However, activation of these pathways still remains to be elucidated. In the present study, we comprehensively explored molecular candidates and the mechanisms of toxicity, both common and distinct, for nickel and cadmium using integrative toxicogenomic and bioinformatic tools. We compared the effects of cadmium to those of nickel using comparative genomic hybridization (CGH) array, gene expression microarray and functional proteomics-based technologies in p53-proficient human colon carcinoma RKO cells. The cells were subchronically exposed to low levels of cadmium chloride or nickel acetate, based on a survival rate of at least 80% after a 24-h treatment. We also identified both common and distinct mechanisms of toxicity underlying the individual metals. Similarities and differences in gene expression profiles, respective pathways and biological processes following low-level exposure to each metal are also discussed.

The mechanisms of toxicity for low-dose exposure to cadmium and nickel are unclear. Therefore, we comprehensively explored molecular candidates and the mechanisms of toxicity, both common and distinct, for both metals using integrative toxicogenomic and bioinformatic tools. Similarities and differences in gene subsets, pathways and biological processes in response to each metal are further discussed.

Using a high-resolution CGH array, we found no significant difference in recurrent CNV following continuous low-level exposure to both metals tested when comparing the tenth passage of the culture to the first. Less than 700 gene entities with altered expression at the mRNA and protein levels were identified by gene expression microarray and functional proteomic analysis after a 24-h exposure to either metal individually. This small subset of genes exhibiting a very slight to mild extent of metal-induced profiling change in terms of gene structure might be attributable to the low-level metal exposure of the cells. A number of studies have reported an association between DNA copy number variation and predisposition to several genetic diseases, including cancers, neurodegenerative disorders and obesity (29,30). These findings suggest that the significant contribution of a particular CNV to pathogenesis is involved in an altered dose of genes within the chromosomal region affected. Furthermore, cadmium and nickel induce carcinogenesis in diverse experimental models in vitro and in vivo. However, we found no significant alteration in recurrent CNV following low-level exposure to the metals after ten passages, suggesting that such a metal treatment has an undetectable impact on DNA structural variations based on aberrant copy number. However, exposure duration will be prolonged with such low doses in future research, as ten passages may have been insufficient for CNV to occur in p53-proficient carcinoma cells.

Alterations in gene and protein expression levels were analyzed in RKO cells following a 24-h low-dose exposure to either metals using gene expression microarray and functional proteomics, respectively. Nineteen transcripts and 69 protein entities were altered in the cadmium-exposed cells, whereas 629 transcripts and 69 protein entities were differentially expressed in nickel-exposed cells. Among them, 22 protein entities, including several members of the heat shock protein (HSP) family, were commonly altered in the cells treated with either cadmium or nickel. These proteins might be considered valuable biomarkers for cadmium and nickel exposure even at the low levels used. These entities are classified according to their functions, such as ligands (i.e., UTS2), stress proteins (i.e., ACTB, AKR1C2, DCD, HSPA1A, HSPA2, HSP90AA1, HSP90B1, KRT18 and MYH7), kinases and phosphatases (i.e., PGK1 and PPP1R13B, respectively), transcription factors (i.e., TWIST1), and other enzymes (i.e., AKR1C2, IDH1, P4HB and RNF128). Our data suggest that the molecular candidates identified here, including many regulatory factors and stress proteins, are potential metal carcinogenicity markers, which were consistently observed in previous studies (3). HSPs are stress-inducible proteins that fold and unfold other proteins and represent a suite of highly conserved and broadly distributed proteins in nature (31). HSP expression is regulated by temperature and other stressors (32). Recent studies have shown that diverse HSPs are activated by heavy metals including cadmium, copper, lead and zinc in cell lines, shrimp and the sea star (31,33,34). Although our data showed downregulation of HSPs, they can be suggested as potential carcinogenesis-related genes in response to low-dose cadmium or nickel. Six genes, including AFAP1, EIF3F, FASN, HSPA1A, JUN and TRIP10 were distinct in nickel-exposed cells. Interestingly, EIF3F increased significantly in response to low-level nickel exposure. EIF3F is a protein complex with at least 13 non-identical subunits (35). The functions of the individual subunits have not been clarified; however, a recent study demonstrated that EIF3F is involved in apoptotic signaling (36). The EIF3F gene is also differentially expressed by volatile organic compounds (37); however, no reports have found a nickel-modulated signaling network involving EIF3F. Thus, these data are the first results indicating that the EIF3F gene is dysregulated by subchronic exposure to low-dose nickel and also suggest that this gene might be involved in apoptotic signaling. The FASN gene product is a key enzyme for palmitate biosynthesis into long-chain saturated fatty acids in the presence of the reduced form of nicotinamide adenine dinucleotide phosphate (38,39). Additionally, this protein fuses with estrogen receptor α in some cancer cell lines. Another study demonstrated alterations in rat liver lipid metabolism induced by nickel deficiency (40). Thus, nickel has detrimental impacts on lipid metabolism involving FASN. JUN, a c-jun oncoprotein, is an essential component of the activator protein (AP) transcription complex. Upon heterodimerization with other Jun/Fos members, JUN forms an active AP-1 complex that regulates expression of many target genes harboring AP-1-binding DNA elements within their promoters. These AP-1 target genes are involved in various crucial cellular functions such as cell cycle progression, migration, proliferation and apoptosis. JUN is activated upon phosphorylation by c-jun-N-terminal kinase, which responds to diverse biological stress signals (41). Interestingly, JUN both induces and inhibits cellular apoptosis (42–44).

A number of reports have demonstrated that oxidative stress, interference with cell proliferation, dysregulation of oncogenes or tumor-suppressor genes, and an impaired DNA repair system are predominant mechanisms driven by carcinogenic metal compounds (3,45,46). We found that ACTB, HSP90AA1, HSPA5 and HSPA8 acted as key components of pathways associated with proliferation, apoptosis and neoplastic processes in response to cadmium and nickel (Fig. 1). The metals also interacted with important components in multiple signaling pathways crucial for cell growth, proliferation, survival, cell cycle, drug resistance, the stress response and carcinogenesis, including AKT1, BAG1, BAX, CDK1, CFTR, HSF1, TP53, MAPK3, MAPK8, STAT3 and TNF (Fig. 1). Herein, we showed that both cadmium and nickel influenced key components in the AKT1-mediated pathway, similar to a recent study showing that cadmium-induced reactive oxygen species activate the Akt/mTOR pathway by activating the positive regulator PI3K, resulting in neuronal apoptosis (47). These findings suggest that particular stress-inducible proteins, protein kinases, or transcription factors may be prominent biomarkers of subchronic exposure to cadmium or nickel and may be used to develop preventive approaches for associated disorders and/or diseases; however, the underlying mechanisms of toxicity must be identified. Furthermore, targeting regulatory component crosstalk among the mitogen-activated protein kinase pathways representing a cascade of sequential phosphorylation events might offer new opportunities to develop novel anticancer agents designed to be target-specific chemotherapeutic drugs. In addition to common responses, we also examined the mechanisms of toxicity that were unique to each metal compound. Our pathway analysis results of cadmium-treated cells found that the caspase (CASP)-associated pathway was a mechanism unique to cadmium involving CASP3, CASP7 and CASP9 (Fig. 2). Gene expression changes in nickel-exposed cells were associated with a hypoxic response. Our data showed that HSP90AA1, one of the main modulators, directly interacted with hypoxia inducible factor-1α (HIF-1α) (Fig. 3). HIF-1α is a transcription factor that induces the transcription of genes involved in glycolysis, glucose transport, apoptosis and other cellular processes as a result of a change in intracellular oxygen concentration (45). PGK1, encoding phosphoglycerate kinase 1, was found to be one of the HIF-1α targets to be dysregulated in nickel-exposed cells, suggesting that nickel induces a hypoxic-like response through HIF-1α, which was consistent with the results of a comparative genomic expression analysis in rat liver-derived cells exposed to nickel, cadmium, or chromium (46). Dysregulation of HIF-1α interferes with cellular energy metabolism, such as glycolysis, causing cells to shift toward non-oxidative forms of ATP production and altering production of glycolytic enzymes and glucose transporters (48). HSP90AA1 directly interacted with the androgen receptor (AR) based on the nickel-responsive pathway analysis. An abundance of evidence has revealed that the AR forms a heterodimer complex with Hsp90, resulting in a stable, unbound AR (49). Growth of a normal and neoplastic prostate is mediated by the AR, a ligand-dependent transcription factor activated by high affinity androgen binding. The AR is highly expressed in recurrent prostate cancer cells. Here, HIF-1α interacted with the AR in response to subchronic nickel exposure (Fig. 3). Interestingly, we also found that HSP90AA1 was involved in the BCL2-related apoptotic pathway uniquely in response to nickel exposure (Fig. 3), whereas it interacted with several CASP genes functioning in CASP-associated apoptotic signaling unique to cadmium exposure alone (Fig. 2). These data suggest that interference with the apoptotic process was likely common to both metals, but that the responsive interaction network with molecular targets was distinct in response to each metal. These findings support that HSP90 contributes to the pathogenesis of chronic diseases, including cancers, rheumatoid and arthritis, particularly through apoptosis (50,51). Targeting HSP90 has emerged as a potential avenue for therapeutic intervention. HSP90 is a molecular chaperone required for the post-translational stability and function of numerous key signal transduction proteins, termed ‘client’ proteins (52,53). A number of these client proteins have been causally implicated in the pathogenesis of prostate cancer, including AR, HER2, AKT and RAF1 (54–56). The interaction of these proteins with HSP90 regulates their half-life. We revealed from our pathway analysis that HSP90AA1 directly interacted with Src encoding Src protein-tyrosine kinase. Src is a member of a large family of structurally related kinases, many of which are expressed in highly differentiated cell types (57,58). Src and its family members play a key regulatory role transducing signals from cell surface receptors and coupling receptors with the cytoplasmic signaling machinery involved in several cellular processes, such as cell growth, differentiation, migration, proliferation, survival and specialized cell signaling. Additionally, JUN and FASN were shown here to be main modulators in unique nickel-responsive signaling pathways (Fig. 3) and their products have been implicated in tumor initiation and development of various tissues, as evidenced from cellular and/or epidemiological studies (59–61). JUN has a regulatory role in HIF-1α function through these interactions. Stabilizing HIF-1α is dependent on c-Jun domains for DNA binding and heterodimerization (62). Indeed, HIF-1α is activated by interacting with c-Jun, allowing increased expression of vascular endothelial growth factor, which is a signal protein involved in stimulating vasculogenesis and angiogenesis (63). JUN was regulated by interaction with the AR. Intriguingly, JUN and FASN were also governed by interacting with AKT1, INS and LEP in nickel-responsive signaling pathways. We also found that JUN was controlled by an interaction with APEX1, a redox-sensitive repair protein that plays an essential role in the base excision repair (BER) system. Apart from APEX1, we found that many other proteins were related to the DNA damage response and DNA repair particularly in the BER pathway; FEN1, PARP1, PCNA and POLB were uniquely responsible for the nickel-associated network. JUN interacted with MITF, whose product is called microphthalmia-associated transcription factor, which has a key role in the development, survival and function of particular cell types, such as melanocytes, retinal pigment epithelial cells and osteoclasts (64). FASN was found to regulate ERBB2 or HER2 expression and vice versa. Previous studies have consistently reported FASN-mediated regulation of ERBB2 expression and a suppressive effect on ERBB2 overexpression by inhibiting FASN-encoded fatty acid synthase in cancer cells (65). Other evidence supports that ERBB2, previously identified in preneoplastic breast lesions, upregulates FASN expression (66). Furthermore, FASN overexpression increases ERBB1 or epidermal growth factor receptor (EGFR) and ERBB2 protein expression levels as well as tyrosine phosphorylation (67). Treatment of EGFR and ERBB2 with kinase inhibitors suggests that both EGFR and ERBB2 are activated by regulating EGF-mediated FASN expression (68). Interestingly, nickel seemed to uniquely perturb the cell differentiation process associated with expression and function of both common (HSP90AA1, HSP90B1, HSPA5, NUDC and TWIST1) and distinct (JUN) components (Fig. 3). This result suggests that although a similar subset of genes was altered upon exposure to cadmium and nickel individually, they might contribute to unique network interactions and cellular processes (Figs. 1–3). This might be attributable to the combined effects of the expression patterns and regulation of the components involved in different signaling pathways. Taken together, our results identified valuable biomarkers and distinctive signaling networks in response to subchronic low-dose exposure of carcinogenic metals cadmium and nickel.