Is Cadmium Toxicity Tissue-Specific? Toxicogenomics Studies Reveal Common and Specific Pathways in Pulmonary, Hepatic, and Neuronal Cell Models.

Several harmful modifications in different tissues-organs, leading to relevant diseases (e.g., liver and lung diseases, neurodegeneration) are reported after exposure to cadmium (Cd), a wide environmental contaminant. This arises the question whether any common molecular signatures and/or Cd-induced modifications might represent the building block in initiating or contributing to address the cells towards different pathological conditions. To unravel possible mechanisms of Cd tissue-specificity, we have analyzed transcriptomics data from cell models representative of three major Cd targets: pulmonary (A549), hepatic (HepG2), and neuronal (SH-SY-5Y) cells. Further, we compared common features to identify any non-specific molecular signatures. The functional analysis of dysregulated genes (gene ontology and KEGG) shows GO terms related to metabolic processes significantly enriched only in HepG2 cells. GO terms in common in the three cell models are related to metal ions stress response and detoxification processes. Results from KEGG analysis show that only one specific pathway is dysregulated in a significant way in all cell models: the mineral absorption pathway. Our data clearly indicate how the molecular mimicry of Cd and its ability to cause a general metal ions dyshomeostasis represent the initial common feature leading to different molecular signatures and alterations, possibly responsible for different pathological conditions.

Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria.

In this paper, we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 µM CdCl2 for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two foci was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of the Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

Cadmium promotes glycolysis upregulation and glutamine dependency in human neuronal cells.

Cadmium is a widespread pollutant, which easily accumulates inside the human body with an estimated half-life of 25-30 years. Many data strongly suggest that it may play a role in the pathogenesis of neurodegenerative diseases. In this paper we investigated cadmium effect on human SH-SY5Y neuroblastoma cells metabolism. Results showed that, although SH-SY5Y cells already showed hyperactivated glycolysis, cadmium further increased basal glycolytic rate. Both glycolytic capacity and reserve were also increased following cadmium administration, endowing the cells with a higher compensatory glycolysis when oxidative phosphorylation was inhibited. Cadmium administration also led to an increase in glycolytic ATP production rate, paralleled by a decrease in ATP production by oxidative phosphorylation, due to an impairment of mitochondrial respiration. Moreover, following cadmium administration, mitochondria increased their dependency on glutamine, while decreasing lipids oxidation. On the whole, our data show that cadmium exacerbates the Warburg effect and promotes the use of glutamine as a substrate for lipid biosynthesis. Although increased glutamine consumption leads to an increase in glutathione level, this cannot efficiently counteract cadmium-induced oxidative stress, leading to membrane lipid peroxidation. Oxidative stress represents a serious threat for neuronal cells and our data confirm glutathione as a key defense mechanism.

In vitro and bioinformatics mechanistic-based approach for cadmium carcinogenicity understanding.

Cadmium is a toxic metal able to enter the cells through channels and transport pathways dedicated to essential ions, leading, among others, to the dysregulation of divalent ions homeostasis. Despite its recognized human carcinogenicity, the mechanisms are still under investigation. A powerful tool for mechanistic studies of carcinogenesis is the Cell Transformation Assay (CTA). We have isolated and characterized by whole genome microarray and bioinformatics analysis of differentially expressed genes (DEGs) cadmium-transformed cells from different foci (F1, F2, and F3) at the end of CTA (6 weeks). The systematic analysis of up- and down-regulated transcripts and the comparison of DEGs in transformed cells evidence different functional targets and the complex picture of cadmium-induced transformation. Only 34 in common DEGs are found in cells from all foci, and among these, only 4 genes are jointly up-regulated (Ccl2, Ccl5, IL6 and Spp1), all responsible for cytokines/chemokines coding. Most in common DEGs are down-regulated, suggesting that the switching-off of specific functions plays a major role in this process. In addition, the comparison of dysregulated pathways immediately after cadmium treatment with those in transformed cells provides a valuable means to the comprehension of the overall process.

Toxicogenomics applied to in vitro Cell Transformation Assay reveals mechanisms of early response to cadmium.

Cadmium is a well recognized carcinogen, primarily released into the environment by anthropogenic activities. In the effort to understand the early events responsible for cadmium carcinogenesis, we have used an in vitro biological system (the Cell Transformation Assay, CTA), that has been shown to closely model some key stages of the conversion of normal cells into malignant ones. Cadmium-triggered early responses in CTA were analysed through microarray-based toxicogenomics. Metallothioneins represent the earliest cell response, together with Slc30a1 encoding for a ZnT-1 zinc exporter. Other genes were found to be up-regulated in the first 24 h following Cd administration: phospatidylinositol-4-phospate 5-kinase alpha (Pip5k1a), glutathione S-transferase (Gstα 1-3), Gdf15 and aldolase. However, after the exposure, a number of genes expressing zinc proteins were found to be down-regulated, among which were many olfactory receptors (ORs) coding genes. Cd administration also promoted massive Zn release inside the cell that could be related to moonlighting activities of regulated genes (proteins). On the whole our data suggest that, despite the early involvement of defence mechanisms (metallothionein and GST), Cd-triggered Zn release, as well as Cd interference with different proteins, may lead to gene expression alterations which later induce metabolic changes, directing the cells towards uncontrolled growth.

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells.

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.

Impact of Cadmium on Intracellular Zinc Levels in HepG2 Cells: Quantitative Evaluations and Molecular Effects.

Cadmium is classified as a human carcinogen, and its disturbance in zinc homeostasis has been well established. However, its extent as well as molecular mechanisms involved in cadmium carcinogenesis has yet to be fully clarified. To this end, we used the zinc specific probe Zinquin to visualize and to quantitatively evaluate changes in the concentration of labile zinc, in an in vitro model of human hepatic cells (HepG2) exposed to cadmium. A very large increase (+93%) of intracellular labile zinc, displaced by cadmium from the zinc proteome, was measured when HepG2 were exposed to 10 µM cadmium for 24 hrs. Microarray expression profiling showed that in cells, featuring an increase of labile zinc after cadmium exposure, one of the top regulated genes is Snail1 (+3.6), which is included in the adherens junction pathway and linked to cancer. In the same pathway MET, TGF-ßR, and two members of the Rho-family GTPase, Rac, and cdc42 all implicated in the loss of adherence features and acquisition of migratory and cancer properties were regulated, as well. The microRNAs analysis showed a downregulation of miR-34a and miR-200a, both implicated in the epithelial-mesenchymal transition. These microRNAs results support the role played by zinc in affecting gene expression at the posttranscriptional level.

Modulation of different stress pathways after styrene and styrene-7,8-oxide exposure in HepG2 cell line and normal human hepatocytes.

Styrene is one of the most important monomers produced worldwide. IARC classified styrene as a possible carcinogen to humans (group 2B). Styrene-7,8-oxide (SO) is the main reactive metabolite of styrene, and it is found to be genotoxic in several in vitro test systems. Styrene and styrene-7,8-oxide (SO) toxicity to HepG2 cells was investigated by evaluating end-points such as heat shock proteins (Hsps), metallothioneins (MT), apoptosis-related proteins, accumulation of styrene within the cells and expression of two isoforms of cytochrome P450. The potential activity of styrene and styrene-7,8-oxide in modulating gene expression was also investigated. The results showed induction of Hsp70, metallothioneins, BclX(S/L) and c-myc expression and a decrease in Bax expression in HepG2 after treatments, confirming that these compounds activated protective mechanisms. Moreover, up-regulation of TGFbeta2 and TGFbetaRIII in HepG2 cells was found after exposure to styrene, while in human primary hepatocytes these genes were down-regulated after both treatments. Finally, it was found that styrene and SO treatments did not induce CYP1A2 and CYP2E1 protein expression. In conclusion, both compounds caused toxic stress in HepG2 cells, with SO being more toxic; in the meantime, a different effect of the two compounds in HepG2 cells and primary human hepatocytes was observed regarding their activity in gene modulation.