Derick Han PhD

USC Research Center for Liver

Diseases

Interplay of Signal Transduction and Mitochondria

in the Acetaminophen Model

Drug-induced liver injury (DILI)

Drug induced liver injury is major cause of:

- warnings

- drug withdrawals from the market

- failure during clinical trials

Withdrawn Not Approved in the U.S.

BenoxaprofenFlutamideIproniazidNefazodoneTienilic acidTroglitazoneBromfenac

AlpidemAmineptineAmodiaquineCincophenDihydralazineDilevalolEbrotidine

GlafenineIbufenacIsoxicamNiperotidinePerhexilinePirprofenTilbroquinol Ximelagatran

Warnings on labels

Acetaminophen (Tylenol)CarbamazepineClozapineDacarbazineDantroleneDiclofenacDisulfiramFelbamateHalothaneIsoniazidLeflunomide

KetoconazoleMethyldopaPyrazinamideRifampinTacrineTerbinafineTiclopidineTolcaponeValproic AcidZileutin

Adapted from Kaplowitz Nature Reviews: Drug Discovery (2005) 4, 489

Acetaminophen – the major cause of acute

liver failure in the United States

Acetaminophen 46%

Other Drugs 12%

Indeterminate 15%

Viral Hepatitis 11%

Autoimmune 5%

Miscellaneous 11%

Causes of Acute Liver Failure Percent

Adapted from Ostapowicz et al. Ann Intern Med (2002) 137, 947

Acetaminophen – model to study drug-

induced liver injury in animals

Acetaminophen primarily causes necrotic death in hepatocytes

Acetaminophen-induced liver injury is mediated by the

reactive metabolite, NAPQI

Cytochrome P450

(CYP2E1 isoform)

N

OH

O

CCH3

N

O

O

CCH3

GSH depletion

Covalent binding

Hepatocyte

Necrosis

+

Cys S NAPQI

GS NAPQI

APAP

NAPQI

Drug metabolite(excretion product)

Traditional model of hepatocyte necrosis-

induced by acetaminophen

Acetaminophen

“Overwhelming Injury Model”

Mitochondrial

permeability

transition

Plasma membrane

rupture

ALT

NAPQI formation

GSH depletion

Covalent binding

Role of signal transduction pathways in APAP-

induced liver injury

Protective effects of JNK inhibitor against APAP-induced liver injury

APAP (mg/kg)

Se

rum

ALT

(U

/L)

* **

APAP

JNK inhibitor + APAP

JNK inhibitor (SP600125; 10 mg/kg) was dissolved in a DMSO-PBS solution. APAP treated mice were given vehicle control

of DMSO-PBS.

Hanawa et al. JBC (2008) 283, 13565-77

Gunawan et al. Gastro (2006) 131, 165-78

C-Jun N-terminal protein kinase (JNK)

Stimulus

MAPKKK

MAPKK

MAPK

stress,

cytokines, ROS

MEKK,

MLK,

ASK1

MKK 4/7

JNK 1,2,3autophosphorylation

p

p

p

JNK belongs to the

mitogen-activated protein

kinase family

JNK is activated early during APAP-induced

liver

p-JNK 54

46

Cont 1 2 4 6

APAP (h)

JNK54

46

JNK activation in cytoplasm of liver JNK inhibitor prevents JNK activation (4 h following APAP treatment)

p-JNK

JNK

54

46

actin

Cont APAPAPAP+

JNK inh

54

46

Mice were treated with APAP 600 mg/kg. JNK inhibitor (SP600125; 10 mg/kg) was dissolved in a DMSO-PBS solution.

APAP treated mice were given vehicle control of DMSO-PBS.

Hanawa et al. JBC (2008) 283, 13565-77

JNK inhibitor does not affect GSH depletion

caused by NAPQI

GS

H (

nm

ol/m

g)

0

20

40

60

80

1 2 3 4 5 6

Mitochondria

APAP+JNK inh

control

APAP

0

20

40

60

80

control

APAP+JNK inh

APAPG

SH

(nm

ol/m

g)

1 2 3 4 5 6(h) (h)

Hanawa et al. JBC (2008) 283, 13565-77

Total Liver homogenate

Silencing JNK 1 and 2 using antisense protects

against APAP-induced liver injury

Control

ASO

Seru

m A

LT

(U

/L)

JNK1

ASO

JNK2

ASO

Control

2 x ASO

JNK 1+2

ASO

*

Mice were injected with 300mg/kg APAP. ASO = antisense. Hanawa et al. JBC (2008) 283, 13565-77

Acetaminophen-induced necrosis in liver is an

active process

Acetaminophen

“Overwhelming Injury Model”

Mitochondrial

permeability

transition

Plasma membrane

rupture

ALT

NAPQI formation

GSH depletion

Covalent binding

JNK activation

JNK inhibitor

An active process –

not a passive process

“programmed necrosis”

“Programmed necrosis” in primary hepatocytes

treated with H2O2 – role of PKC

0

20

40

60

80

100

0 100 200 300 400 500

H2O2 ( M)

Via

bili

ty (

)

*

*

*H2O2

PKC inhibitor + H2O2

H2O2 PKC inhibitor + H2O2

Sytox green stainingSaberi et al. AJP (2008) 295, C50-63

Delaying treatment of JNK inhibitor still protects

against APAP-induced liver injury

JNK

0

2000

4000

6000

8000

10000

12000

14000

0 2 4

Time (h) following APAP treatment when

JNK inhibitor was injected

Seru

m A

LT

(U

/L)

**

p-JNK

JNK

Cytoplasm

54

46

Cont APAP

APAP+

JNK inh

54

46

JNK levels 2 hours

following APAP treatment

Hanawa et al. JBC (2008) 283, 13565-77

Signal Transduction Pathways Involved in

APAP-induced Liver

Cont 1 2 4

p-AKT

AKT

APAP (h)

p-PKA

PKA

Actin

p-glycogen synthase

Cytosol

p-GSK-3β (Tyr 216)

GSK-3β

p-GSK-3β (Ser 9)

0.0

0.4

0.8

1.2

1.6

2.0

0 1 2 3 4

p-JNK54

46

Time (h)

Ph

osp

ho

ryla

tion

(a

.u.)

p-GSK(ser 9)

1 2 4

APAP (h)

Cont

Shinohara et al. JBC (2010) 285, 8244-55

Signaling pathways activated in liver following

APAP treatment

Mice were injected with 300mg/kg APAP.

glycogen synthase

GSK-3β

JNK

DMSO used to dissolve JNK inhibitor modulates

time course of JNK activation

Cytoplasm

JNK

C 1h 2h 4h 6h C 1h 2h 4h 6h

APAP 600DMSO (8.3%) +

APAP 600

p－JNK65

54

65

54

Actin

Cont 1 2 4

p-AKT

AKT

APAP (h)

p-PKA

PKA

Actin

p-glycogen synthase

Cytosol

p-GSK-3β (Tyr 216)

GSK-3β

p-GSK-3β (Ser 9)

0.0

0.4

0.8

1.2

1.6

2.0

0 1 2 3 4

p-JNK54

46

Time (h)

Ph

osp

ho

ryla

tion

(a

.u.)

p-GSK(ser 9)

1 2 4

APAP (h)

Cont

Shinohara et al. JBC (2010) 285, 8244-55

Signaling pathways activated in liver following

APAP treatment

Mice were injected with 300mg/kg APAP.

glycogen synthase

GSK-3β

JNK

GSK-3

p

GSK-3

GSK-3

p

inactive

enhanced activity

active

serine 9

tyrosine 216

GSK-3 regulates metabolic and apoptotic

pathways in cells

Akt

p

insulin

glycogen

synthase

p

glycogen

synthase

inactive

active

Wnt Wnt

p

degradation

Mcl-1 Mcl-1

p

degradation

0

4000

8000

12000

16000

Se

rum

ALT

(U

/L)

Cont ASO GSK-3 ASO

Actin

Cont ASO GSK-3 ASO

*0

5000

10000

15000

20000

0 1 2 4 8 24 48

Time (h)

Seru

m A

LT

(U

/L)

** *

Silencing GSK-3 significant reduces APAP-

induced liver injury

Shinohara et al. JBC (2010) 285, 8244-55

GSK-3β

Control

ASO = antisense.

Silencing Akt-2 or GSK-3 does not modulate

APAP-induced liver injury

0

4000

8000

12000

16000

20000

Se

rum

ALT

(U

/L)

Cont ASO Akt 2 ASO

Akt 2 Cont ASO Akt 2 ASO

Se

rum

ALT

(U

/L)

0

4000

8000

12000

16000

20000

Cont ASO GSK-3 ASO

GSK-3Cont ASO GSK-3 ASO

Shinohara et al. JBC (2010) 285, 8244-55

Interplay of Signal Transduction and

Mitochondria in the Acetaminophen Model

APAP-induced liver injury involves mitochondria

dysfunction

RCR – respiratory control ratio

(state III/state IV)

ATP levels in liver homogeneate

0

1

2

3

4

5

6

0 1 2 3 4 5 6

control

APAP+

JNK inh.

APAP

RC

R

time (h)

**

AT

P (

nm

ol/m

g)

control

APAP+

JNK inh.

APAP

**

time (h)

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6

Hanawa et al. JBC (2008) 283, 13565-77

JNK is activated by stresses including reactive

oxygen species

Stimulus

MAPKKK

MAPKK

MAPK

stress,

cytokines, ROS

MEKK,

MLK,

ASK1

MKK 4/7

JNK 1,2,3autophosphorylation

p

p

p

Mitochondria are the major sources of reactive

oxygen species in cells

I

cyt cInner membrane

I

II

III IV VUQ

NADH

e+

O2O2

.-

Outer membrane

Matrix

H2O2 H2O

GSH GSSG

GSH reductase

UQI.-

UQO.-

Mn-SODGSH

peroxidase

H2O2

O2

VDAC

O2

O2.-

O2.-

O2.-

Han et al. Mol Pharm (2003) 64, 1136-44

Han et al. JBC (2003) 278, 5557-63

Does increased mitochondrial H2O2 caused by GSH

depletion activate JNK during APAP-induced liver injury?

APAP

NAPQI

H2O2

CYP2E1

MitochondriaJNK

activation

JNK

?

PJNK

GSH

covalent binding

GSH depletion in mitochondria is associated with

increase H2O2 generation.

Han et al. Mol Pharm (2003) 64, 1136-44

H2O2 production increases from mitochondria

following APAP but not AMAP treatment

0

10

20

30

APAPControl AMAP

H2O

2(p

icom

ole

/min

/mg

) *

Mice were treated with APAP 400 mg/kg or AMAP 600 mg/kg. Liver mitochondria were isolated 1 hour following APAP

or AMAP treatment. Hanawa et al. JBC (2008) 283, 13565-77

AMAP, a non-hepatotoxic regioismer of APAP, does not

target mitochondria

CYP2E1 isoform

N

OH

O

CCH3

N

O

O

CCH3

GSH depletion

Covalent binding

N

OH

O

CCH3

N

O

O

CCH3

O

CYP2E1 isoform

APAP

NAPQI

AMAP

Reactive

metabolite

GSH depletion

Covalent binding GSH

Cytoplasm

diffusion

Mitochondria

Mitochondria

little

diffusion

Rashed et al. Drug Met Disp (1990) 18, 765-770

AMAP lowers cytoplasmic but not mitochondrial

GSH levels

0

10

20

30

40

50

60

0

0.5

1

1.5

2

2.5

3

Control AMAP

GS

H (

nm

ol/m

g)

GS

H (

nm

ol/m

g)

*

Control AMAP

Hanawa et al. JBC (2008) 283, 13565-77

Mice were treated with AMAP 600 mg/kg. Measurements were made 2 hour following AMAP treatment.

MitochondriaTotal Liver homogenate

JNK is activated by APAP treatment but not by

AMAP treatment

Control APAP AMAP

p-JNK

JNK

actin

54

46

54

46

Mice were treated with APAP 400 mg/kg or AMAP 600 mg/kg. Measurements were taken 2 hours following APAP or

AMAP treatment. Hanawa et al. JBC (2008) 283, 13565-77

H2O2 production increases from mitochondria

following APAP but not AMAP treatment

0

10

20

30

APAPControl AMAP

H2O

2(p

icom

ole

/min

/mg

) *

Mice were treated with APAP 400 mg/kg or AMAP 600 mg/kg. Liver mitochondria were isolated 1 hour following APAP

or AMAP treatment. Hanawa et al. JBC (2008) 283, 13565-77

Exogenous H2O2 or mitochondrial inhibitors that increase

mitochondrial H2O2 production activates JNK in primary cultured

hepatocytes

p-JNK

JNK

54

46

54

46

actin

Cont

H2O2 Rotenone Antimycin

300 400 2.5 5 5 10 M

JNK levels were measured following H2O2 (30 min), antimycin (60 min; complex III inhibitor), and rotenone (60 min;

complex I inhibitor) treatment to primary cultured hepatocytes.

Hanawa et al. JBC (2008) 283, 13565-77

JNK is activated by H2O2 released by mitochondria

during APAP-induced liver injury

APAP

NAPQI

H2O2

CYP2E1

MitochondriaJNK

activation

JNK

?

PJNK

GSH

covalent binding

JNK translocates to mitochondria during APAP-

induced liver injury

p-JNK

JNK

54

46

Cont 1 2 4 6

APAP (hrs)

COX Ⅳ

54

46

Time course of JNK translocation to

mitochondria following APAP treatment

Cont APAPAPAP+

JNK inh

JNK inhibitor prevents JNK

translocation to mitochondria(4 h following APAP treatment)

p-JNK

JNK

54

46

COX Ⅳ

54

46

Hanawa et al. JBC (2008) 283, 13565-77

APAP-induced liver injury involves mitochondria

dysfunction

RCR – respiratory control ratio

(state III/state IV)

ATP levels in liver homogeneate

0

1

2

3

4

5

6

0 1 2 3 4 5 6

control

APAP+

JNK inh.

APAP

RC

R

time (h)

**

AT

P (

nm

ol/m

g)

control

APAP+

JNK inh.

APAP

**

time (h)

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6

Hanawa et al. JBC (2008) 283, 13565-77

JNK translocation to mitochondria following activation

APAP

NAPQI

H2O2

CYP2E1

MitochondriaJNK

activation

JNK

?

PJNK

PJNK

respiration

GSH

covalent binding

ATP

Does JNK modulate mitochondria function?Exp: Addition of purified JNK1 or JNK2 to isolated mitochondria

Isolate mitochondria

1. Control

or

2. APAP +JNK inhibitor(Mitochondria with GSH

depleted and covalent binding)

+JNK1

+JNK1 or JNK2

+CyA

+JNK2RCR Measurements

(10 minutes in

glutamate/malate, ADP)

incubation

Western blot of JNK1 and JNK2

associated with mitochondria

following incubation

54JNK

COX

+ JNK 1

Control APAP +

JNK inhControl APAP +

JNK inh

+ JNK 2

46

Mitochondria GSH

APAP+JNK inh

control

0

40

80

GS

H (

nm

ol/m

g)

1 2 3 4 5 6 (h)

Hanawa et al. JBC (2008) 283, 13565-77

Addition of purified JNK to redox-modified isolated

mitochondria inhibits respiration

RC

R

0

1

2

3

4

5

6

Mitochondria

control

APAP+JNK inh.

APAP

+CsA +CsA

0

1

2

3

4

5

6

Mitochondria

•Addition of purified active JNK 1 or 2 to mitochondria isolated from mice treated with APAP plus JNK inhibitor

(mitochondria with severe GSH depletion, covalent binding, and respiration partially impaired) directly inhibited

mitochondrial respiration.

•Cyclosporin A blocked the inhibitor effect of JNK on mitochondria respiration suggesting that JNK induces MPT.

RC

R

APAP+JNK inh.

APAP

+ active JNK1 + active JNK2

Hanawa et al. JBC (2008) 283, 13565-77

Proposed model of the JNK–mitochondria signaling

loop important in mediating liver injury

APAP

NAPQI

H2O2

CYP2E1

MitochondriaJNK

activation

JNK

?

PJNK

PJNK

respiration

GSH

covalent binding

MPT ATP

Two hit hypothesis to mitochondria

1

2

Interaction of GSK-3 with Mitochondria in

the Acetaminophen Model

Cont 1 2 4

APAP (h)

p-GSK-3β (Tyr 216)

GSK-3β

p-GSK-3β (Ser 9)

COX

JNK

p-JNK54

46

54

46

Mitochondria

0

20

40

60

80

100

120

0 1 2 3 4

Tra

nslo

cation t

o

mitochondria (

a.u

.)

GSK-3 and JNK translocate to mitocondria

during APAP-induced liver injury

GSK-3β

JNK

Time (h)

Shinohara et al. JBC (2010) 285, 8244-55Mice were injected with 300mg/kg APAP.

Translocation of kinases to mitocondria is a

growing theme in many pathologies

Kinases that translocate to mitochondria

GSK-3 - during cardiac ischemia reperfusion - JCI (2004) 113, 1535

- J Mol Cell Card (2007) 42, 564

PKC ( ) - during cardiac ischemia reperfusion - Biochem Soc Trans (2007) 35, 1040

- Pharm Res (2007) 55, 523

JNK - ionizing radiation, oxidative stress - J Neurochem (2008) 104, 325

aging - FEBS lett (2009) 583, 1132

- JBC (2000) 275, 322

Akt - growth factor, stress - J Cell Biochem (2007) 102, 196

0

1

2

3

4

5

6

0 2 4 6 8

0

4

8

12

16

0 2 4 6 8

Time (h)

Sta

te III

re

sp

ira

tio

n(O

2nm

ol/m

in/m

g)

Time (h)

RC

R

A

**

* *

*

Shinohara et al. JBC (2010) 285, 8244-55

GSK-3β ASO

Control ASO

GSK-3β ASO

Control ASO

Silencing GSK-3 protects against mitochondria

dysfunction in liver caused by APAP

Respiratory Control RatioState III Respiration

Mice were injected with 300mg/kg APAP.

Mcl-1

Cont ASO (h) GSK3β ASO (h)

0 1 2 4 0 1 2 4

COX

Bax

Silencing GSK-3 inhibits Mcl-1 degradation in

mitochondria caused by APAP treatment

Shinohara et al. JBC (2010) 285, 8244-55

Anti-apoptotic bcl-2 family members

Mcl-1 - degradation - Shinohara et al. JBC (2010) 285, 8244

Bcl-xl - phosphorylation - Latchoumycandane et al. Hep (2007) 45, 412

- degradation - Hu et al. APJ (2008) 295, G24

Bcl-2 - phosphorylation - Latchoumycandane et al. Hep (2007) 45, 412

- degradation - Hu et al. APJ (2008) 295, G24

Pro-apoptotic bcl-2 family members

Bax - translocation to mitochondria - Gunawan et al. Gastro (2006) 131, 165

- Bajt et al. JPET (2008) 324, 8

Bak - no change - Hanawa et al. JBC (2008) 283, 13565-77

tBid - no change - Hanawa et al. JBC (2008) 283, 13565-77

Changes in pro- and anti-apoptotic bcl-2 family

members during acetaminophen-induced liver injury

Cont ASO (h) GSK-3β ASO (h)

0 1 2 4 0 1 2 48 8

p-JNK

JNK

54

46

0 1 2 4 8 0 1 2 4 8

time (h)time (h)

80

60

40

20

0

De

nsity (

AU

) p-JNK JNK

*

*

*

80

60

40

20

0

*

Silencing GSK-3 delays and blunts JNK activation and

translocation to mitochondria during acetaminophen-

induced liver injury

JNK translocation to mitochondria

APAP treatment to ASK-1 knockout mice induces an early ASK-1

independent JNK activation but inhibits a late ASK-1 dependent

JNK activation

Nakagawa et al. Gastro (2008) 135,

1311-1321

Cont ASO (h) GSK-3β ASO (h)

0 1 2 4 0 1 2 48 8

p-JNK

JNK

54

46

0 1 2 4 8 0 1 2 4 8

time (h)time (h)

80

60

40

20

0

De

nsity (

AU

) p-JNK JNK

*

*

*

80

60

40

20

0

*

Silencing GSK-3 delays and blunts JNK activation and

translocation to mitochondria during acetaminophen-

induced liver injury

JNK translocation to mitochondria

early

GSK-3 acts upstream of JNK during acetaminophen-

induced liver injury

ROS

ASK1

MKK 4/7

JNK 1,2,3

p

p

p

MLK,MEKK

p

GSK-3

ROS?

late

ASK1 knockout mice treated

with APAP had an early initial

ASK1 independent JNK

activation (~ 1.5 h), but no late

(> 3 h) JNK activation.

Nakagawa et al. Gastro (2008) 135,

1311-1321

GSK-3 activates MEKK1 or

MLK to activate JNK in several

cell lines

Mishra et al. JBC (2007) 282, 30393-405

Kim et al. JBC (2003) 278, 13995-4001

JNK and GSK-3 activation and interaction with

mitochondria during APAP-induced liver injury

NAPQIAPAP

Mitochondria

Mcl-1

GSH

ROS

GSK-3activation

src ?

Early - MLK, MEKK ?

JNKactivation

ASK-1activation

Later

1

2

3?

Role of signal transduction pathway in APAP-

induced liver injury depend on dose

Acetaminophen

“Overwhelming Injury Model” – occurs at extremely high lethal

doses of APAP

Mitochondrial

permeability

transition

Plasma membrane

rupture

ALT

NAPQI formation

GSH depletion

Covalent binding

JNK activation

Signal transduction pathways play a role in APAP-induced liver

injury – at high non-lethal doses of APAP

“Programmed necrosis” in primary hepatocytes

treated with H2O2 – role of PKC

0

20

40

60

80

100

0 100 200 300 400 500

H2O2 ( M)

Via

bili

ty (

)

*

*

*H2O2

PKC inhibitor + H2O2

H2O2 PKC inhibitor + H2O2

Sytox green stainingSaberi et al. AJP (2008) 295, C50-63

Is JNK a common factor in liver disease?

ASK1 knockout mice are not protected

from CCl4 hepatotoxicity

Gunawan et al. Gastro (2006) 131, 165-78

CCl4–induced liver injury

Hepatic ischemia reperfusion injury

JNK mediates hepatic ischemia reperfusion injury – Uehara et al. J Hepatol (2005)

42, 850-9.

Effect of JNK inhibitor (SP600125) on CCl4hepatotoxicity in mice.

Nakagawa et al. Gastro (2008) 135, 1311-1321

Is a GSK-3 a common factor in liver disease?

0

5000

10000

15000

20000

Cont ASO GSK-3b ASO

CCl4–induced liver injury (0.1 ml/kg)

Se

rum

ALT

(U

/L)

Summary – role of JNK and GSK-3 during APAP-

induced liver injury

NAPQIAPAP

Mitochondria

Mcl-1

GSH

ROS

GSK-3activation

src ?

Early - MLK, MEKK ?

JNKactivation

ASK-1activation

Later

1

2

3?

An active process –

not a passive process

“programmed necrosis”

Binding partners?

Acknowledgements

Maria Ybanez

Behnam Saberi

Naoko Hanawa

Mie Shinohara

William A. Gaarde

Neil Kaplowitz