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