Physiological and toxicological transcriptome changes in HepG2 cells exposed to copper

Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
Physiological Genomics (Impact Factor: 2.37). 07/2009; 38(3):386-401. DOI: 10.1152/physiolgenomics.00083.2009
Source: PubMed


Copper is an essential trace element; however, at supraphysiological levels, it can be extremely toxic. Microarray data from HepG2 cells exposed to 100, 200, 400, and 600 microM copper for 4, 8, 12 and 24 h were generated and analyzed. Principal components, K-means, and hierarchical clustering, interactome, and pathway mapping analyses indicated that these exposure conditions induce physiological and toxicological changes in the HepG2 transcriptome. As a general trend, when the level of toxicity increases, the number and diversity of affected genes, Gene Ontology categories, regulatory pathways, and complexity of interactomes increase. Physiological responses to copper include transition metal ion binding and responses to stress/stimulus, whereas toxicological responses include apoptosis, morphogenesis, and negative regulation of biomolecule metabolism. The global gene expression profile was overlaid onto biomolecular interaction networks and signal transduction cascades using pathway mapping and interactome identification. This analysis indicated that copper modulates signal transduction pathways associated with MAPK, NF-kappaB, death receptor, IGF-I, hypoxia, IL-10, IL-2, IL-6, EGF, Toll-like receptor, protein ubiquitination, xenobiotic metabolism, leukocyte extravasation, complement and coagulation, and sonic hedgehog signaling. These results provide insights into the global and molecular mechanisms regulating the physiological and toxicological responses to metal exposure.

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    • "Limited evidence for the role of GPX in organismal copper detoxification has been documented [5], [50]. The results in fig 3A show that GPX activity increased in a dose-dependent manner in C. elegans. "
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    ABSTRACT: Antioxidant enzymes play a major role in defending against oxidative damage by copper. However, few studies have been performed to determine which antioxidant enzymes respond to and are necessary for copper detoxification. In this study, we examined both the activities and mRNA levels of SOD, CAT, and GPX under excessive copper stress in Caenorhabditis elegans, which is a powerful model for toxicity studies. Then, taking advantage of the genetics of this model, we assessed the lethal concentration (LC50) values of copper for related mutant strains. The results showed that the SOD, CAT, and GPX activities were significantly greater in treated groups than in controls. The mRNA levels of sod-3, sod-5, ctl-1, ctl-2, and almost all gpx genes were also significantly greater in treated groups than in controls. Among tested mutants, the sod-5, ctl-1, gpx-3, gpx-4, and gpx-6 variants exhibited hypersensitivity to copper. The strains with SOD or CAT over expression were reduced sensitive to copper. Mutations in daf-2 and age-1, which are involved in the insulin/insulin-like growth factor-1 signaling pathway, result in reduced sensitivity to stress. Here, we showed that LC50 values for copper in daf-2 and age-1 mutants were significantly greater than in N2 worms. However, the LC50 values in daf-16;daf-2 and daf-16;age-1 mutants were significantly reduced than in daf-2 and age-1 mutants, implying that reduced copper sensitivity is influenced by DAF-16-related functioning. SOD, CAT, and GPX activities and the mRNA levels of the associated copper responsive genes were significantly increased in daf-2 and age-1 mutants compared to N2. Additionally, the activities of SOD, CAT, and GPX were greater in these mutants than in N2 when treated with copper. Our results not only support the theory that antioxidant enzymes play an important role in copper detoxification but also identify the response and the genes involved in these processes.
    PLoS ONE 09/2014; 9(9):e107685. DOI:10.1371/journal.pone.0107685 · 3.23 Impact Factor
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    • "MeHg hypomethylated the pyrimidinergic receptor P2Y, G-protein coupled, 6 (p2ry6) and CDC42 effector protein (Rho GTPase binding) 4 (cdc42ep4) promoters in F1 zebrafish and induced transcription of orthologous genes in other vertebrates (Robinson et al., 2011; Toyama et al., 2011), while somatostatin (sst), hypermethylated in F1, was transcriptionally repressed by copper, another heavy metal (Song et al., 2009). For TCDD, lecithin retinol acyltransferase (lrata) and THUMP domain containing 1 (thumpd1) promoters were hypomethylated in F0 and cadherin-related family member 1 (cdhr1) in F1 and these genes can be transcriptionally activated by TCDD (Hoegberg et al., 2003; Fracchiolla et al., 2011; Thornley et al., 2011). "
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    ABSTRACT: This study aimed to investigate whether dioxin (TCDD) and methylmercury (MeHg) pose a threat to offspring of fish exposed to elevated concentrations of these chemicals via epigenetic-based mechanisms. Adult female zebrafish were fed diets added either 20μg/kg 2,3,7,8 TCDD or 10mg/kg MeHg for 47days, or 10mg/kg 5-aza-2'-deoxycytidine (5-AZA), a hypomethylating agent, for 32days, and bred with unexposed males in clean water to produce F1 and F2 offspring. Global DNA methylation, promoter CpG island methylation and target gene transcription in liver of adult females and in 3days post fertilization (dpf) F1 and F2 embryos were determined with HPLC, a novel CpG island tiling array containing 54,933 different probes and RT-qPCR, respectively. The results showed that chemical treatment had no significant effect on global DNA methylation levels in F1 (MeHg and TCDD) and F2 (MeHg) embryos and only a limited number of genes were identified with altered methylation levels at their promoter regions. CYP1A1 transcription, an established marker of TCDD exposure, was elevated 27-fold in F1 embryos compared to the controls, matching the high levels of CYP1A1 expression observed in F0 TCDD-treated females. This suggests that maternal transfer of TCDD is a significant route of exposure for the F1 offspring. In conclusion, the selected doses of TCDD and MeHg, two chemicals often found in high concentrations in fish, appear to have only modest effects on DNA methylation in F1 (MeHg and TCDD) and F2 (MeHg) embryos of treated F0 females.
    Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 05/2014; 165. DOI:10.1016/j.cbpc.2014.05.004 · 2.30 Impact Factor
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    • "In the present study, Nrf1 or Nrf2 centered networks generated through IPA using HepG2 transcriptome [30] showed that the target genes of Nrf1 and Nrf2 included NAD(P)H:quinone reductase (NQO1), glutathione S-transferase A4 (GSTA4), glutathione peroxidase 1 (GPX1), glutamate-cysteine ligase, catalytic subunit (GCLC), heme oxygenase 1 (HMOX1) and thioredoxin reductase 1 (TXNRD1), which were well known to be regulated mainly by Nrf2-dependent mechanism [12] [31]. A couple of previous studies demonstrated that expressions of some of these genes were modulated by metals via Nrf2 in HepG2 cells [1] [14]. "
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    ABSTRACT: Recent evidences indicated Nrf2 is more potent than Nrf1 in the activation of antioxidant genes. However, the roles of Nrf proteins in the regulation of copper-responsive transcription have not been well addressed. We took the toxicogenomic approach and the present network and Gene Ontology analyses results showed that Nrf1 and Nrf2 are distinctively involved in copper-responsive transcriptional regulation in HepG2 transcriptome. Cells deficient in either Nrf1 or Nrf2 were more susceptible to copper exposure than wild type cells. Nrf1 and Nrf2 null cells were transfected with the luciferase reporters containing either ARE(s) or a combination of ARE(s) and MREs, and then treated with copper. In Nrf2-null (Nrf2(-/-)) cells, copper did not activate transcription of reporter genes, whereas Nrf1 deficiency did not affect copper-inducible activation. Ectopic expression of Nrf2 restored copper-inducible transcription in Nrf2(-/-) cells. However, the changes in the intrinsic mRNA levels of MT-1 in Nrf null cells following copper treatment showed that Nrf1 and Nrf2 equally contributed to MT-1 activation after 4h, while Nrf1involved more than Nrf2 following 24h exposure. These results suggest that while Nrf2 is crucial for MRE/ARE-mediated transcription in response to copper, Nrf1 may activate MT-1 expression by a mechanism different from that Nrf2 employs.
    Experimental Cell Research 01/2014; 322(1). DOI:10.1016/j.yexcr.2014.01.013 · 3.25 Impact Factor
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