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Gene expression profiling of liver cells after copper overload in vivo and in vitro reveals new copper-regulated genes

  • Patricia Muller
  • Harm van Bakel
  • Bart van de Sluis
  • Frank Holstege
  • Cisca Wijmenga
  • Leo W. J. Klomp
Original Paper

Abstract

Copper toxicity in the liver is mediated by free-radical generation, resulting in oxidative stress. To prevent toxic accumulation of copper, liver cells adapt to high copper levels. Here, we used microarray analysis to compare the adaptive responses on global gene expression in liver cells exposed to high copper levels in vitro and in vivo. In HepG2 cells we identified two clusters of upregulated genes over time, an “early” cluster that comprised metallothionein genes and a “late” cluster, highly enriched in genes involved in proteasomal degradation and in oxidative stress response. Concomitant with the “late” cluster, we detected a significant downregulation of several copper metabolism MURR1 domain (COMMD) genes that were recently implicated in copper metabolism and inhibition of nuclear transcription factor κB (NF-κB) signaling. As metal-induced oxidative stress increases NF-κB activity, our data suggest a role for reduced COMMD protein levels in prolonged activation of NF-κB, thus inducing cell survival. Mice exposed to a copper diet that highly exceeded normal daily intake accumulated only twofold more hepatic copper than control mice. Although a moderate, but significant upregulation of a set of 22 genes involved in immunity, iron and cholesterol metabolism was detected, these cannot account for direct mechanisms involved in copper excretion. In conclusion, we identified a novel set of genes that represent a delayed response to copper overload, thus providing insight into the adaptive transcriptional response to copper-induced oxidative stress.

Keywords

Copper Liver Microarray Gene expression HepG2 

List of abbreviations

BCS

Bathocuproine disulfonic acid

cDNA

Complementary DNA

COMMD

Copper metabolism MURR1 domain

cRNA

Complementary RNA

CYP

Cytochrome P450

FAAS

Flame atomic absorption spectrometry

HEK

Human embryonic kidney

HMOX1

Heme oxygenase 1

LEC

Long–Evans cinnamon

MAANOVA

Microarray analysis of variance

MIAME

Minimum information about a microarray experiment

mRNA

Messenger RNA

NF-κB

Nuclear transcription factor κB

qRT-PCR

Quantitative reverse transcriptase polymerase chain reaction

TNF

Tumor necrosis factor

Notes

Acknowledgements

We are grateful to S. Bos (Pharmaceutical Department, University Medical Centre Utrecht, The Netherlands) for the use and support of the flame atomic absorption spectrometer. This study was supported by grants from the WKZ Fund and the Dutch Organization for Scientific Research Zon-MW (40-00812-98-03106 and 901-04-219).

Supplementary material

775_2006_201_MOESM1_ESM.pdf (2 mb)
Supplementary Table S1. Overview of all the genes that were differentially expressed after at least one time point in copper-treated HepG2 cells (MAANOVA analysis). The common name, chip annotation, the Genbank identifiers, the GO database annotation, a short description and the fold induction after each time point are indicated for each gene. (PDF 1.98 Mb)

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Copyright information

© SBIC 2007

Authors and Affiliations

  • Patricia Muller
    • 1
    • 2
  • Harm van Bakel
    • 2
    • 3
  • Bart van de Sluis
    • 2
    • 4
  • Frank Holstege
    • 3
  • Cisca Wijmenga
    • 2
  • Leo W. J. Klomp
    • 1
    • 5
  1. 1.Laboratory for Metabolic and Endocrine DiseasesUniversity Medical Centre UtrechtThe Netherlands
  2. 2.Complex Genetics Section, DBG Department of Medical GeneticsUniversity Medical CentreUtrechtThe Netherlands
  3. 3.Genomics LabUniversity Medical CentreUtrechtThe Netherlands
  4. 4.Oncogenesis and Development SectionNHGRI, National Institutes of HealthBethesdaUSA
  5. 5.Laboratory of Metabolic and Endocrine DiseasesWilhelmina Children’s HospitalUtrechtThe Netherlands

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