acetaminophen-induced liver injury...matthew sharpe, icu, kumc and ku hospital sean kumer, tim...
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Acetaminophen-induced Liver Injury: Translating Toxicity Mechanisms in Animals to
Humans
Hartmut Jaeschke, PhD, ATSProfessor and Chair
Dept. of Pharmacology, Toxicology & TherapeuticsSchool of Medicine
University of Kansas Medical CenterKansas City, KS
USA
Overview
• Clinical Relevance• Intracellular Signaling Mechanisms of Cell
Death in Mice• Mechanisms of Toxicity in Human
Hepatocytes• Mechanisms of Toxicity in Humans• Sterile Inflammation in Humans
Acetaminophen (Paracetamol)Hepatotoxicity
Clinical Relevance
Most consumed pain medication in the world: 35 billion doses/year sold
50-60,000 hospitalizations and 300-500 deaths/year in the US!
Largest cause of Acute Liver Failure in the US! (46%)
Overview
• Clinical Relevance• Intracellular Signaling Mechanisms of Cell
Death in Mice• Mechanisms of Toxicity in Human
Hepatocytes• Mechanisms of Toxicity in Humans• Sterile Inflammation in Humans
Mechanisms of APAP Bioactivation
NAPQI-Protein adduct
Oxidative stress
Superoxide
Nitric Oxide
Peroxynitrite
Hydrogen peroxide
SOD2 Nitrated SOD2
GSH supplementation
JNK P
JNK
Oxidative stress
Nitrosativestress
AIF Endonuclease G
Nucleus
DNA Fragmentation
Loss of membrane potential
ATP production
MPTInduction
H2O2
MitophagyMitochondrial fusion
and fission
Mkp-1
APAPNAPQI
APAP-GlucuronideAPAP-Sulfate
APAP-GSHCYP2E1GSH
Autophagy
Trx-SH Trx-S-S-Trx+
ASK-1ASK-1
MKK4
P
P
PASK-1
H2O2
MLK3MLK3
JNK
PSab
ETC
CYP1A2 CYP3A4
Mitochondrial Biogenesis
Mechanisms of APAP Hepatotoxicity in HumansHuman Hepatoma Cell Line HepaRG
McGill et al., Hepatology 53: 974-82, 2011
Time (h)0 12 24 36 48
LDH
(% R
elea
sed)
0
10
20
30
40
50
60
70
Time (h)0 6 12 18 24
Red
/Gre
en (
% o
f Con
trol)
0
20
40
60
80
100
120
AP
AP
-pro
t A
dduc
t (ng
/mg
prot
)
0
20
40
60
80
100
Mechanism of APAP Toxicity in HepaRG Cells
Time (h)0 6 12 18 24
GSH
(% o
f Con
trol)
0
20
40
60
80
100
120
*
***
0 1 3 6 12 24
Time (h)
*
*
*
* *GSH APAP-Cys
JC-1 LDH
*
***
McGill et al., Hepatology 53:974-82, 2011
Mechanism of APAP Toxicity in HepaRG Cells
Ctrl 20 mM APAP 6 h
MitoSOX Red: Mitochondrial Superoxide
McGill et al., Hepatology 53:974-82, 2011
Mechanism of APAP Toxicity in HepaRG Cells:Apoptosis
Cas
pase
3 A
ctiv
ity
(RF
U/m
in/m
g pr
otei
n)
0
2
4
6
8
10
12
Ctrl APAP G/TNF V
G/TNF CI
*
#
Caspase-3 Activity
LDH
Act
ivity
(% R
elea
sed)
0
5
10
15
20
25
30
Ctrl APAP V
APAP CI
**
LDH Release
McGill et al., Hepatology 53:974-82, 2011
Mechanism of APAP Toxicity in HepaRG Cells: Necrosis
APAP - 20mM – 24 hVehicle – 24 h
PI Staining
APAP-induced Nuclear Endonuclease G Translocation in HepaRG Cells
EndoG MergeDAPI
Ctl
APAP 24h
APAP, 400x, zoom
APAP-induced DNA Fragmentation in HepaRG Cells: TUNEL Assay
20 mM APAP, 24 h
Mechanisms of APAP Hepatotoxicity in Humans
Primary Human HepatocytesXie et al., Toxicol Appl Pharmacol 279: 266-74, 2014
Time (h)0 5 10 15 20 25 30
Glu
tath
ione
(% o
f con
trol)
0
20
40
60
80
100
120
**
* * *
Time (h)0 5 10 15 20 25 30
Red
/Gre
en R
atio
(% o
f con
trol)
0
20
40
60
80
100
120
*
**
0 2 4 6 8 10 12 14 16
Tota
l APA
P-C
YS
(nm
ol/m
g pr
otei
n)
0.0
0.5
1.0
1.5
2.0
2.5
Time (h)
*
**
*GSH Adducts
Time (h)0 10 20 30 40 50
ALT
(% o
f tot
al A
LT)
0
20
40
60
80
100Ctrl 5mM APAP10mM APAP
** *ALTJC-1
Mechanisms of APAP Hepatotoxicity in Primary Human Hepatocytes
Xie et al., Toxicol Appl Pharmacol 279: 266-74, 2014
Xie et al., Toxicol Appl Pharmacol 279: 266-74, 2014
APAPTime (h) 3
P-JNK
Total JNK
6 15 24 36 48054 kDa46 kDa
54 kDa46 kDa Cytosol
Mitochondria
P-JNK
Total JNK
P-JNK
Total JNK
APAPTime (h) 6 150
Cotreatment
Time (h)
ALT
(% o
f tot
al A
LT)
0
20
40
60
80
100
APAPAPAP+VehAPAP+JNK inh
24 48
Post-treatment
0
20
40
60
80
100
APAPAPAP+VehAPAP+JNK inh
*#
*#
APAP-induced JNK Activation in Human Hepatocytes
Yan et al., Toxicol Sci 117: 515-23, 2010.
Oxidant Stress in Cell Culture (Mouse Hepatocytes)MitoSox Red:
Mitochondrial Superoxide
Mechanisms of APAP Hepatotoxicity in Humans
Evaluation of mechanistic biomarkers in plasma of overdose
patients
APAP in Humans: Mitochondrial Biomarkers
Hepatic sinusoidal space
Mitochondrial Markers of APAP Toxicity in Humans: mtDNA
Pla
sma
mtD
NA
(ng/
mL)
0
50
100
150
200
ALT (U
/L)
0
1000
2000
3000
4000
5000
6000
7000
8000
1 2 3 4 5 6
Day in Hospital
Pla
sma
mtD
NA
(ng
/mL)
0
10
20
30
40
50
60Cyt C Ox NADH Deh
w/severeinjury
w/osevereinjury
Cyt C OxNADH Deh
V
**
ALT
mtDNA
McGill et al., J Clin Invest, 122:1574-83, 2012
Time (days) 0 2 4 6 8 10 12 14
DN
A Fr
agm
enta
tion
(%)
0
200
400
600
800
1000
1200
1400
1600
Plasma DNA
Marker of APAP Toxicity: Nuclear DNA Fragmentation
*D
DNA
Frag
(% o
f V)
0
1000
2000
3000
4000
5000
6000 *
w/Injury
w/oInjury
V
McGill et al., J Clin Invest, 122:1574-83, 2012
0 2 4 6 8 100
200
400
600
800
1000
0
2000
4000
6000
8000
10000
12000
K18 (U
/l): Caspase cleaved ---Total
ALT
Activ
ity (U
/l) K18
ccK18
Antoine, … Jaeschke, … Park - J Hepatol. 56: 1070-9, 2012.
0 1 2 3 4 50
500
1000
1500
2000
2500
0
2000
4000
6000
8000
10000
K18 (U
/l): Caspase cleaved ---Total
ALT
Activ
ity (U
/l)
K18
ccK18
Acetaminophen – Apoptosis or Necrosis?
K18 – full length cytokeratin-18 (M65)ccK18 – caspase-cleaved cytokeratin-18 (M30)
ALT ALT
Human Pathophysiology is Equivalent to Mechanisms in Mice
• GSH depletion with protein binding• Mitochondrial dysfunction and damage• Nuclear DNA damage• Cell necrosis with cell contents release
McGill et al., J Clin Invest, 122:1574-83, 2012
APAPCytochrome P450
NAPQI
ASK1 activation
Superoxide + Nitric oxide
Peroxynitrite
Thioredoxin oxidationThioredoxin
ASK1
Mitochondrial Electron Transport
Chain
Mitochondrial oxidative/nitrosative
stress
Thioredoxin oxidation
MLK3 activation
MKK4 Phosphorylation
JNK P-JNK
Mitochondrial oxidative
stress
Src
Mitochondrial Electron Transport
Chain
Superoxide
Bax
Bax
GSK-3β
GSK-3β
AIFEndo G
SmacCytochrome c
DNA fragmentation
Cell Necrosis
Glutathione depletion
RIP3/RIP1
Ramachandran and Jaeschke, Gene Expression 18: 19-30, 2018
Overview
• Clinical and Toxicological Relevance• Intracellular Signaling Mechanisms of Cell
Death in Mice• Mechanisms of Toxicity in Human
Hepatocytes• Mechanisms of Toxicity in Humans• Sterile Inflammation in Humans
Proposed Schematic of APAP-induced Inflammatory Liver Injury
Woolbright & Jaeschke, J Hepatol 66: 836-848, 2017
Pro-inflammatory Cytokine Formation in APAP Overdose Patients
0
200
400
600
800
1000 CtrlNLTNSSV
0
200
400
600
800
1000 CtrlNLTNSSV
D0 D1 D2 D3 D4 D5 D6NLTCD0 D1 D2 D3 D4 D5 D6NLTC
IL-1β TNF-α
Plas
ma
Leve
ls (p
g/m
l)
Plas
ma
Leve
ls (p
g/m
l)
Proposed Schematic of APAP-induced Inflammatory Liver Injury
Woolbright & Jaeschke, J Hepatol 66: 836-848, 2017
Time (days)0 2 4 6 8 10
Mea
n F
luor
esce
nce
Inte
nsity
0
200
400
600
800
1000Phagocytosis - E.coli
Neutrophil Activation in an APAP Overdose Patient
Time (days)0 2 4 6 8 10
Enz
yme
Act
ivity
(U
/L)
0
1000
2000
3000
4000
5000ALT GLDH
Time (days)0 2 4 6 8 10
Mea
n F
luor
esce
nce
Inte
nsity
0
500
1000
1500
2000
2500
3000
ROS - PMA
Williams et al., Toxicol Appl Pharmacol 275: 122-33, 2014.
Williams et al., Toxicol Appl Pharmacol 275: 122-33, 2014.
Neutrophil Activation during Recovery after APAP-induced Liver Injury in Patients
Circulating Levels of CCL2 (MCP-1) in APAP Overdose PatientsM
CP-
1 (p
g/m
L)
0
2000
4000
6000
8000
10000CtrlNLTNSSV
D0 D1 D2 D3 D4 D5 D6NLTC
* #* # *#†
* #
* #
* #
* #
* #* #* #
Non-Survivors
Survivors
Kupffer Cell
Acetaminophen Overdose
HMGB1
TLR4
Necrotic Hepatocytes
TNF-α, IL-1α/β
NF-κB
IL-8
ICAM-1
Hepatocytes
Neutrophils
Cell injury
TLR9 DNA Fragments mtDNA HMGB1
RAGE
Removal of necrotic cell debris
Host defense function
MCP-1Monocyte-derived Macrophages
M2
Necrotic Cell Death
Release of DAMPs
Innate Immune Cell Activation
Tissue Repair
Acknowledgement
Dave Williams*
Mitchell McGill*
Yuchao Xie*
Ben Woolbright*
Anup Ramachandran Mary Lynn Bajt
Chieko Saito*
Kuo Du*
Luqi DuanJames Weemhoff* Jeph Akakpo Nga Nguyen
Acknowledgement: Collaborators
Funding: NIH R01 DK070195, R01 DK102142, Training Grant (T32 ES007079, KUMC Liver Center Pilot, CTSA Pilot Grants (KUMC), COBRE (GM103549)
CollaboratorsJohn Lemasters, MUSC, Charleston, SCWen-Xing Ding, KUMC, Kansas City, KSKevin Park and colleagues, Liverpool, UK
Clinical Collaborators:Matthew Sharpe, ICU, KUMC and KU HospitalSean Kumer, Tim Schmitt, Surgery, KUMC and KU HospitalSteven Curry, U Arizona and Banner Health, Phoenix, AZ
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