Role of the NRF2-mediated oxidative stress response and lysosomal dysfunction in drug-induced liver injury associated with mitochondrial damage

Roos, Noëmi Johanna. Role of the NRF2-mediated oxidative stress response and lysosomal dysfunction in drug-induced liver injury associated with mitochondrial damage. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/88502/

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Drug-induced liver injury is a rare, but potentially severe adverse drug reaction that is caused by
various mechanisms including mitochondrial dysfunction and oxidative stress. Highly expressed in
the liver, the transcription factor NRF2 stimulates the expression of phase II-detoxifying and antioxidant
genes in response to electrophilic and oxidative stress. In unstressed conditions, NRF2 activation
is suppressed by the cytosolic redox-sensitive protein KEAP1. As master regulator of the
oxidative and electrophilic stress response, NRF2 might protect against drug-induced liver injury
caused by mitochondrial damage and oxidative stress. To test this hypothesis, we assessed whether
the hepatotoxic drugs benzbromarone and lapatinib, which are both associated with mitochondrial
dysfunction and oxidative stress, activate the KEAP1-NRF2 pathway in HepG2 cells, a human hepatoma
cell line. Moreover, lapatinib has lysosomotropic properties, which have also been described
for the tyrosine kinase inhibitor imatinib. Similar to benzbromarone and lapatinib, imatinib caused
severe liver injury in patients. As lipophilic weak bases, lapatinib and imatinib accumulate in acidic
cellular compartments such as lysosomes. Thus, we assessed the effects of lapatinib and imatinib
on lysosomal functions and related processes such as mammalian target of rapamycin complex 1
activation, lysosomal biogenesis, and autophagy in HepG2 cells.
Benzbromarone is a uricosuric drug that was withdrawn from the drug market by its manufacturer
due to severe cases of liver toxicity. In our first project, benzbromarone (1-100 μM) lead to accumulation
of mitochondrial superoxide radicals and cellular reactive oxygen species in HepG2 cells.
The uricosuric drug caused oxidation of glutathione, the most prevalent antioxidant molecule in
hepatocytes, to glutathione disulfide. Glutathione disulfide levels increased in entire HepG2 cells
and especially in mitochondria. Moreover, benzbromarone increased the level of oxidized mitochondrial
antioxidant protein thioredoxin 2. These findings indicate that mainly mitochondria were exposed
to benzbromarone-induced oxidative stress and might have been the origin of ROS generation.
Furthermore, benzbromarone activated the KEAP1-NRF2 pathway in HepG2 cells, demonstrated
by nuclear accumulation of NRF2 and upregulation of several NRF2-regulated antioxidant
proteins. Downregulation of KEAP1, which led to NRF2 activation, protected HepG2 cells from
benzbromarone-induced ATP depletion and cell membrane permeabilization.
Approved for the treatment of human epidermal growth factor receptor 2-positive breast cancer,
lapatinib received a black box warning by the U.S. Food and Drug Administration due to severe
cases of hepatotoxicity in patients. We observed that lapatinib (2-20 μM) induced the generation of
mitochondrial superoxide and cellular hydrogen peroxide in HepG2 cells in our second project. In
this cellular system, lapatinib activated the KEAP1-NRF2 pathway at clinically relevant concentrations.
Consequently, lapatinib upregulated several prototypical NRF2-regulated antioxidant genes
and glutathione biosynthesis. As observed for benzbromarone, lapatinib increased the levels of glutathione
disulfide, confirming that lapatinib caused oxidative stress in HepG2 cells. Co-treatment
with the antioxidant N-acetylcysteine reduced the accumulation of NRF2 induced by lapatinib, indicating
that reactive oxygen species were involved in the activation. N-acetylcysteine co-treatment
also reduced the decrease in KEAP1 protein levels caused by lapatinib. Finally, lapatinib upregulated
mitochondria-specific antioxidant genes more strongly than their cytosolic counterparts.
As lysosomotropic drugs, lapatinib and imatinib increased the lysosomal volume, raised the lysosomal
pH, and showed signs of lysosomal membrane permeabilization in HepG2 cells in our third
project. Both drugs disturbed the proteolytic activity of lysosomes. Moreover, imatinib reduced the
activity of the mammalian target of rapamycin complex 1. This protein complex is activated on the
lysosomal surface and regulates essential metabolic pathways such as protein synthesis and autophagy.
Consequently, imatinib activated the transcription factor EB. This transcription factor is
the master regulator of lysosomal biogenesis as well as autophagy and a substrate of the mammalian
target of rapamycin complex 1. In response to imatinib, the transcription factor EB accumulated
in the nucleus and upregulated the expression of lysosomal as well as autophagic genes. Inactivation
of the mammalian target of rapamycin complex 1 and upregulation of autophagic genes together
with the increased expression of autophagic proteins implied that imatinib induced autophagy
in HepG2 cells. However, the concomitant lysosomal dysfunction caused by imatinib might impair
a complete autophagic flux.
In conclusion, the hepatotoxic drugs benzbromarone and lapatinib activated the NRF2 pathway in
HepG2 cells as an adaptive stress response. Activation of NRF2 protected against benzbromarone-
induced hepatotoxicity. Moreover, oxidative stress was partially involved in the activation of
NRF2 by lapatinib. Finally, we revealed that the impairment of lysosomal functions and related
pathways might contribute to lapatinib- and imatinib-induced liver toxicity.
Advisors:Krähenbühl, Stephan and Huwyler, Jörg and Simon, Hans-Uwe
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Clinical Pharmacology (Krähenbühl)
UniBasel Contributors:Roos, Noëmi Johanna and Krähenbühl, Stephan and Huwyler, Jörg
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14724
Thesis status:Complete
Number of Pages:V, 129
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss147242
edoc DOI:
Last Modified:14 Jun 2022 04:30
Deposited On:13 Jun 2022 10:14

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