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www.elsevier.com/locate/jhep
Center for Liver, Digestive and Metabolic Diseases, University Medical Center Groningen, Groningen, P.O. 30.001, 9700 RB, The Netherlands
2
Department of Gastroenterology and Hepatology, Academic Medical Center Amsterdam, The Netherlands
Background/Aims: In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the
mechanisms are not completely elucidated. To elucidate the mechanisms of hepatocyte cell death induced by the ROS
superoxide anions and hydrogen peroxide, primary cultures of hepatocytes were exposed to the superoxide anion donor
menadione (1050 mmol/L) or H2O2 (15 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors,
superoxide dismutase (PEG-SOD) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9,
-6, -3 activation and cleaved PARP, and necrotic cell death by Sytox Green staining.
Results: (1) Menadione (50 mmol/L) induces JNK phosphorylation, caspase-9, -6, -3 activation, PARP cleavage and
apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50 mmol/L) induces the
phosphorylation of ERK1/2 and this attenuates cell death. (2) H2O2 increases necrotic cell death at high concentration
or when H2O2 detoxification is impaired. H2O2 does not activate MAPKs signalling. (3) PEG-SOD prevents ERK1/2-,
JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases
menadione-induced apoptosis. (4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death.
Conclusions: In normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on
JNK activity and totally abolished by superoxide scavengers.
q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Oxidative stress; Apoptosis; Necrosis; Signal transduction; Caspase; Glutathione.
1. Introduction
Received 17 February 2005; received in revised form 8 July 2005; accepted
21 July 2005; available online 12 September 2005
* Corresponding author. Tel.: C31 50 3611408; fax: C31 50 3619306.
E-mail address: l.conde.de.la.rosa@med.umcg.nl (L. Conde de la Rosa).
Abbreviations: Ad5LacZ, adenoviral b-galactosidase; Ad5IkBAA,
adenoviral dominant-negative IkB-a; 3-AT, 3-amino-1,2,4-triazole; BSO,
D,L-buthionine-(S,R)-sulfoximine; GPx, Glutathione Peroxidase; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; GSH, Glutathione; GSHMEE, Glutathione monoethylester; H2O2, hydrogen peroxide; HO.,
hydroxyl radicals; O.K
2 , superoxide anions; MAPK, mitogen-activated
protein kinase; MS, mercaptosuccinic acid; NO., Nitric oxide; PARP,
Poly(ADP-ribose) polymerase; PEG-SOD, polyethylene glycol-superoxide
dismutase; ROS, reactive oxygen species; SNAP, S-nitro-N-acetylpenicillamine; SOD, superoxide dismutase; TUDCA, Tauroursodeoxycholic
acid.
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jhep.2005.07.034
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3. Results
3.1. Superoxide anions, but not hydrogen peroxide,
induce apoptosis in primary rat hepatocytes
Fig. 2. (A) Menadione induces caspase-3 activity and PARP cleavage only at 50 mmol/L. Hepatocytes were incubated or not (controls) with different
concentrations of menadione (Men) for 9 h. (B) Caspase-3 activity and Western blot. Paraquat (50 mmol/L) and hypoxanthine (0.5 mmol/L)/xanthine
oxidase (20 mU/ml) (HX/XO) induce apoptosis, PARP and caspase-3 cleavage peaking at 4 and 9 h, respectively. (C) Menadione, but not hydrogen
peroxide, induces nuclear condensation (apoptosis indicator), as determined by acridine orange staining, only at 50 mmol/L. Hepatocytes were treated
with different concentrations of menadione (9 h) or H2O2 (14 mmol/L, 6 h). Original magnification: 1000!. (D) H2O2 does not induce caspase-3
activity in primary hepatocytes at concentrations up to 5 mmol/L.
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Fig. 3. Sytox Green nuclear staining of necrotic cells. (A) Menadione (Men, 50 mmol/L, 9 h) slightly increases necrotic cell death in primary
hepatocytes (magnification: 40!). (B) H2O2 induces necrotic cell death only at high concentrations. Hepatocytes were exposed to increasing
concentrations of H2O2 (15mmol/L, 6 h). Upper panel phase contrast and fluorescence; lower panel fluorescence only. Magnification: 40!. [This
figure appears in colour on the web.]
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Fig. 4. (A) Hydrogen peroxide (H2O2) slightly increases caspase-3 activity in primary hepatocytes when H2O2 detoxification is impaired. Cells were
exposed to H2O2 (1 mmol/L) for 6hrs in the presence of the catalase inhibitor 3-AT and/or GPx inhibitor MS. (B) H2O2 (1 mmol/L) induces massive
necrotic cell death in hepatocytes with impaired H2O2 detoxification, as determined by Sytox Green nuclear staining. Magnification: 40!. (C) The
catalase inhibitor 3-AT does not affect menadione-induced apoptosis (upper graph) or necrotic cell death (lower pictures). Upper panels phase
contrast and fluorescence; lower panels fluorescence only. [This figure appears in colour on the web.]
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Fig. 6. (A) Phosphorylation of MAP kinases. Menadione 50 mmol/L, but not menadione 25 mmol/L or hydrogen peroxide (H2O2), induces p-JNK
and p-ERK1/2 after 1 and 2 h, respectively; MAPK p38 was not phosphorylated by menadione or H2O2. (B) ERK activation attenuates
menadione-induced apoptosis. Western blot demonstrates functionality of U0126, used as ERK inhibitor. Inhibition of ERK phosphorylation
increases menadione-induced caspase-3 activity (P!0.05; statistically significant according to MannWhitney U-test). (C) Caspase-9, -6 and -3
activation and (D) Menadione-induced caspase-6 activity and apoptosis are dependent on JNK activity. Cells were exposed to menadione with
or without JNK inhibitor SP600125 (SP).
30000
25000
20000
15000
10000
5000
0
B
caspase-3 activity (AFU)
Menadione 50 M
Ad
I
BA
A+
Con
Ad
La
cZ
+
35000
25000
15000
5000
Control
CM
CM
+AdIBAA
Fig. 7. NF-kB-activation is not involved in menadione-induced caspase3 activation. (A) Menadione-induced caspase-3 activity (50 mmol/L,
9 h) is not changed by the inhibitor of NF-kB pathway Ad5IkBAA or
Ad5LacZ (virus control). (B) Functionality of the NF-kB inhibitor
Ad5IkBAA is demonstrated by increased caspase-3 activity after
exposure to cytokines (CM: m-TNF-a (20 ng/ml), h-Interleukin-1b
(10 ng/ml) and r-Interferon-g (10 ng/ml)).
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4. Discussion
In this study we investigated the role of ROS in
primary rat hepatocyte cell death. We demonstrate that the
superoxide anion donor menadione mainly induces
apoptosis and to a much lesser extent necrotic cell death
in primary hepatocytes. In other studies, generation of
superoxide in Sprague Dawley rats in vivo [30] and in
isolated perfused rat liver [31] causes limited toxicity and
only necrotic cell death. Several differences may explain
these contrasting results: (1) Different rat strains differ
with respect to the susceptibility to oxidant injury, (2) Site
of ROS formation: diquat was added to the perfusion
medium whereas in our study, superoxide-generators were
added directly to the culture medium, which may result in
different uptake and final intracellular concentration of the
ROS-donor in vivo and in vitro. (3) Mode of cell death: in
the previous in vivo studies, only LDH/AST/ALT leakage
was used as a determinant of cell death, however
apoptosis was not specifically analyzed. In our experiments, we observed that paraquat as well as HX/XO also
induced apoptosis.
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Fig. 9. Nitric oxide blocks caspase-3 activity induced by superoxide anions but increases necrotic cell death. Hepatocytes were incubated with
menadione (Men, 50 mmol/L, 9 h) with or without the nitric oxide donor SNAP. (A) Caspase-3 activity (apoptosis indicator). (B) Sytox Green nucleic
acid staining (necrotic cell death indicator). Original magnification: 40!. [This figure appears in colour on the web.]
Fig. 10. Superoxide dismutase prevents menadione-induced caspase-6 and -3 activity, processing of caspases and phosphorylation of JNK and ERK
pathways but does not increase necrotic cell death. PEG-SOD (100 U/ml) was added 30 min before menadione (Men, 50 mmol/L) and cells were
harvested 9 h after menadione addition. (A) Caspase-6 and -3 activity (apoptosis). (B) Sytox Green nucleic acid staining (necrotic cell death indicator).
(C) Western blot analysis for cleaved caspase-9, -6, and -3. (D) Nuclear condensation determined by acridine orange staining. Magnification: 1000!.
(E) Western blot analysis for phospho-JNK and phospho-ERK.
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Acknowledgements
This work is supported by grants from the Dutch
Digestive Diseases Foundation and the J.K. de Cock
Foundation Groningen. Part of this work was presented at
the annual meeting of the American Association for the
Study of Liver Diseases, Boston 2003.
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