Oxidative stress induces the endoplasmic reticulum stress and facilitates inclusion formation in cultured cells

J Hepatol. 2007 Jul;47(1):93-102. doi: 10.1016/j.jhep.2007.01.039. Epub 2007 Mar 8.


Background/aims: The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell.

Methods: We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses.

Results: Generation of H(2)O(2) was detected in glucose oxidase (GO)-treated cells by 2',7'-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N-acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers.

Conclusions: The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cysteine Proteinase Inhibitors / pharmacology
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum / ultrastructure
  • Fluoresceins / pharmacology
  • Glucose Oxidase / pharmacology
  • Humans
  • Hydrogen Peroxide / metabolism
  • Inclusion Bodies / chemistry
  • Inclusion Bodies / metabolism*
  • Inclusion Bodies / ultrastructure
  • Keratin-18 / analysis
  • Keratin-8 / analysis
  • Leupeptins / pharmacology
  • Liver Diseases / metabolism*
  • Liver Diseases / pathology
  • Oxidative Stress*
  • Phenylbutyrates / metabolism
  • Proteasome Inhibitors
  • Tumor Cells, Cultured


  • 2',7'-dichlorodihydrofluorescein diacetate
  • Cysteine Proteinase Inhibitors
  • Fluoresceins
  • Keratin-18
  • Keratin-8
  • Leupeptins
  • Phenylbutyrates
  • Proteasome Inhibitors
  • acetylleucyl-leucyl-norleucinal
  • 4-phenylbutyric acid
  • Hydrogen Peroxide
  • Glucose Oxidase