The Keap1 Nrf2 Pathway Toxicology · The Keap1‐Nrf2 Pathway in Toxicology Curtis Klaassen...

The Keap1‐Nrf2 Pathway in Toxicology

Curtis KlaassenUniversity of Kansas Medical CenterUniversity of Kansas Medical Center

Cd tolerance from Cd pretreatment

Time after Cd pretreatment n Mortality (%)No pretreatment 10 100p2 hr 10 1004 hr 10 1006 hr 10 608 hr 10 5024 hr 10 02 day 10 04 day 10 08 day 10 016 day 10 0

Rats were pretreated with CdCl2 (2.0 mg Cd/kg, sc) and at indicated time challenged with a lethal dose of CdCl2 (4.0 mg/kg, iv), and mortality was recorded within 48 hrs after Cd challenge.

Goering and Klaassen JTEH 14:803, 1984

Cd pretreatment protected against acute Cd hepatotoxicityhepatotoxicity

1000000No pretreatment

Cd t t t

100000

/ml)

Cd pretreatment

* * *

10000

DH (S

units/

100

1000S

Control 2.0 3.0 4.0 5.0mg Cd/kg

Rats were pretreated with CdCl2 (2.0 mg Cd/kg, sc) and 24 hrs later were

Goering and Klaassen TAAP 74:308, 1984

p 2 ( g g, )challenged with hepatotoxic doses of CdCl2 (2.0-4.0 mg/kg, iv), and serum SDH was determined 10 hrs later. Data are Mean ± SE of 4-6 rats.

Cd pretreatment alters subcellular Cd distributionsubcellular Cd distribution

80 No pretreatment*

50

60

70n the liver

No pretreatment

Cd pretreatment

30

40

50

istribution in

** *

0

10

20

% Cd di

Nuclei Mitochondria Microsome Cytosol

Rats were pretreated with CdCl2 (2.0 mg Cd/kg, sc) and 24 hrs later were challenged with hepatotoxic doses of CdCl2 (3.5 mg Cd/kg, iv), and subcellular Cd di t ib ti d t i d 2 h l t D t M ± SE f 6 t


Cd distribution was determined 2 hrs later. Data are Mean ± SE of 6 rats.

Cd pretreatment alters cytosolic Cd distributiondistribution

3 5No pretreatment

2.5

3

3.5ver

Cd pretreatment

1

1.5

2

ug Cd/g liv

0

0.5

0.6 1.0 1.4 1.8 2.2 2.6Ve/Vo

Rats were pretreated with CdCl2 (2.0 mg Cd/kg, sc) and 24 hrs later were challenged with hepatotoxic doses of CdCl2 (3.5 mg Cd/kg, iv), and cytosolic Cd di t ib ti d t i d 2 h l t


distribution was determined 2 hrs later.

MT-null mice are highly susceptible to chronic Cd-induced nephrotoxicity

Liu……Klaassen ToxSci 46: 197, 1998

Wild-type control and MT-null mice were given CdCl2 (0.05-2.4 mg/kg, sc) daily for 6 weeks, and Blood urea nitrogen (BUN) and renal MT were determined. N =6-8 mice.

The relationship between human exposure to cadmium and renal injuryj y

Klaassen et al., Ann Rev Pharmacol Tixocol 39, 267, 1999

• In 1987, a post‐doctoral fellow, Jie Liu, came to my Lab with a “suitcase” of chemicals in Traditional Chinese Medicine that he thought would protect againstchemicals in Traditional Chinese Medicine that he thought would protect against liver injury.

OA Oleanolic acid

UA Ursolic acid

UV Uvaol

α‐H α‐Hederin

Jie Liu 87‐98

HD Hederagenin

GL Glycyrrhizin

18β‐GA 18 β‐Glycyrrhetinic acid

18 α‐GA 18 α‐Glycyrrhetinic acid

HAG 19 α‐Hydroxyl asiatic acid 29‐0‐ β‐glucoside

HA 19 α‐Hydroxyl asiatic acid

CCl42500

3000

ALT

(U/L

)

1000

1500

2000 ∗

∗

∗∗

∗

0

500

20000

25000

∗∗

SDH

(U/ m

l)

5000

10000

15000 ∗

∗∗

∗

∗

0

5000

ade)

2.0

∗

∗∗

NEC

RO

SIS

(gra

0.5

1.0

1.5

∗

∗∗∗

0.0CON Jgs Ngs Ful OA Swe Om DDB

y (%

)

30

40

Vehicle OA

SalineBrom obenzeneAcetam inophenCarbon tetThioacetam ideFurosem idePhalloidinColchicineCadm iumD-GalN/LPS

Mor

talit

y

0

10

20

ALT

(U/L

)

4000

6000

8000

10000

∗ ∗∗

SalineBrom obenzeneAcetam inophenCarbon tetThioacetam ideFurosem idePhalloidinColchicineCadm iumD-GalN/LPS0

2000

U/L

) 20000

25000

∗ ∗ ∗ ∗∗ ∗

SalineBrom obenzeneAcetam inophenCarbon tetThioacetam ideFurosem idePhalloidinColchicineCadm iumD-GalN/LPS

SDH

(kI

0

5000

10000

15000

∗∗ ∗ ∗

∗∗

∗ ∗ ∗

∗

Nec

rosi

s (g

rade

)

1

2

3

∗ ∗ ∗

∗ ∗

∗∗ ∗

∗

Saline

Bromobenzene

Acetaminophen

Carbon tet

Thioacetamide

Furosemide

Phalloidin

Colchicine

Cadmium

D-GalN/LPS0

Oleanolic acid

How?

We spent a considerable amount of time tying to determine how Oleanolic Acid protects against this largedetermine how Oleanolic Acid protects against this large panel of hepatotoxicants, unfortunately, with little success.

Anticarcinogenic enzyme inducers are of two types: (a) bifunctional inducers (e g

Precarcinogens activation

(a) bifunctional inducers (e.g., TCDD, polycyclic aromatics) that elevate both Phase II enzymes, NAD(P)H: quinone

d d doxidoreductase, and certain Phase I enzymes;

(b) monofunctional inducers (e.g., diphenols, ( g , p ,thiocarbamates, 1,2‐dithiol‐3‐thiones, beta‐naphthoflavone) that elevate i il Ph II

Prochaska and TalalayC R 1988

primarily Phase II enzymes.

Cancer prevention

Cancer Res 1988

Ph li ti id tPhenolic antioxidants Phenolic antioxidants

ARE GST‐Ya AREGST‐YaGST‐Pi

MafNrf2

ARE

Antioxidants

GST PiNqo1

Itoh et al., BBRC 1997.

MafNrf2

AREHo‐1 (Alam et al., JBC 1999)Gclc (Moinova et al., BBRC 1999)

Oxidative stress and activatorsactivators

Keap1

Keap1

CUL3

Nrf2

Nrf2

ARE

MafNrf2Anti‐oxidant and inflammation

Cell survival and proliferationDrug metabolismLipid metabolism ARELipid metabolism

Nrf2 activator StructureKeap1 cysteine

residues Reference

dexamethasone 21‐mesylate

257, 273, 288, 297, and 613 (human)

(Dinkova‐Kostova et al.,

2002)2002)

iodoacetyl‐N‐biotinylhexylenediamine

196, 226, 241, 257, 288, and 319 (Hong et al.,

2005b)biotinylhexylenediamine (mouse) 2005b)

sulforaphane77, 226, 249, 257, 489, 513, 518

(human)

(Hong et al., 2005)(human)

xanthohumol 151, 319, and 613 (human)

(Luo et al., 2007)

Michael Sporn “the father ofMichael Sporn, the father of chemoprevention” asked for our Oleanolic Acid

so he could make more potent analoguesso he could make more potent analogues.

Oleanolic acid

CDDO CDDO Me CDDO ImCDDO CDDO‐Me CDDO‐Im

2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oic acid (CDDO)

f

1400003500

CDDO compounds are potent Nrf2 activators

al li

ver R

NA

80000

100000

120000WT-Vehicle WT-CDDO-Im Nrf2-null Vehicle Nrf2-null-CDDO-Im

∗

m A

LT (I

U/L

)

1500

2000

2500

3000 *

*

RLU

s/2 μ

g to

ta

20000

40000

60000 ∗

∗

Seru

m

0

500

1000

1500

*

Nrf2 WTWT nullnull

Nqo1 Gclc Ho-10

20000CDDO-Im - + - +

Acetaminophen + + + +

A single dose of CDDO‐Im (1mg/kg, i.p.) increases Nrf2‐dependent genes in wild‐

type but not Nrf2‐null mice

CDDO‐Im protection against acetaminophen hepatotoxicity is

Nrf2‐dependent

Reisman and Klaassen. Toxi Appl Pharm. 2009.

Oleanolic acid is also a Nrf2 activatorOleanolic acid is also a Nrf2 activator

18 H 1N 1 G lN f2

tein

C

ontr

olo β-

actin

5

6

7

8

*

pres

sion

Con

trol

)

12

14

16Ho-1Nqo1 GclcNrf2

**

Nrf

2 Pr

otR

elat

ive

to C

Nor

mal

ized

to

0

1

2

3

4

ND ND er m

RN

A E

xpor

mal

ized

to C

4

6

8

10

*

*

0

Oleanolic Acid - + - +Wild-type Nrf2-null

Live

(No

0

2

4

Oleanolic Acid + - +nullWT

+ - +nullWT

- + - +nullWT

- + - +nullWT

Oleanolic Acid - + - + - + - + - + - + - + - +

A single dose of oleanolic acid (30mg/kg, i.p.) increases Nrf2 translocation into nucleus and induce Nrf2 target genes in wild‐type but not Nrf2‐null mice

What Pathways Might the Keap1 Nrf2What Pathways Might the Keap1‐Nrf2 Pathway Alter?

Nrf2‐null WT Keap1‐KD Keap1‐HKO

Generation of “Gene dose‐response” Model

Keap1

xpre

ssio

n

0.8

1.0

1.2

Keap1

pres

sion

0 8

1.0

1.2

1.4

β‐actinKeap1

Nrf2‐null Wild‐type Keap1‐KD Keap1‐HKO

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

mR

NA

ex

0.0

0.2

0.4

0.6

∗

∗

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

Prot

ein

exp

0.0

0.2

0.4

0.6

0.8

∗∗

Col 2 Col 2 Col 2 Col 2 Nrf2io

n 4 ∗

ll e D O

Prot

ein

expr

essi

0

1

2

3

N.A.

∗

Nrf2

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO


Messenger RNA and Protein Levels of Nrf2 Target Genes, as well as GSH Content were Increased in “Gene dose‐response” Modelp

Nrf2‐null WT Keap1‐KD Keap1‐HKO

Β‐actin

Nqo1

Gclc

Nqo1

A e

xpre

ssio

n

10

15

20

∗

Nqo1

tein

exp

ress

ion

5

10

15

20∗

∗

Cytosol GSH

ver t

issu

e

6

8

10

12

∗

∗∗

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

mR

NA

0

5 ∗

∗

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

Prot

0

5

N.A.

2 5 Nrf2-null

Wild-type

Keap1-KD

eap1-HKO

μmol

/g li

v

0

2

4

6

Gclc

ssio

n

3

4∗

Gclc

rote

in e

xpre

ssio

n

0 5

1.0

1.5

2.0

2.5∗

N W KeKeap

ll e D O

mR

NA

exp

res

0

1

2 ∗

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

Pr

0.0

0.5

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

Transcriptional Profiling by Microarray Analysisp g y y y

f


Nrf2

n=3 n=3 n=3 n=3

Liver

Total RNA

Microarray

Data Analysis from Microarray

imported into “R” program using affy package

Raw data from microarray analysis (CEL file)

imported into R program using affy package

Processed by Robust Multichip Averaging (gcRMA) package

The mean probe signal intensities higher than log2100 in at least one genotype group were selected for further analysis

Analysis of variance by Linear Models for Microarray Data (Limma) Package

Gene symbols and other annotations were obtained from the mouse 4302 and anaffy

kpackages

Constitutively Induced or Suppressed Genes

WT>Nrf2‐null Keap1‐KD>WT WT<Nrf2‐null Keap1‐KD<WT

97 2517

61115

124 4845

9080

294

6181

329

90113

8021Keap1‐HKO>Keap1‐KD 8021Keap1‐HKO<Keap1‐KD

Induced genes6 Suppressed genes6

Fold

-indu

ctio

n

2

4∗

∗∗

Fold

-indu

ctio

n

2

4

∗∗

∗

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

F

0

Nrf2-null

Wild-type

Keap1-KD

Keap1-HKO

F

0

Annotation clustering using DAVID analysisg g y

Term Enrichment Score Gene Symbols

Top annotation cluster for the set of genes induced through Nrf2 activation

Glutathione transferase and glutathione metabolism 8.2 Gstm1, Gss, Gsta2, Gstm2, Gsta3, Gstm3,

Gclc, Gstm4, Gsta4, Gpx4, Gstm6

Oxidation reduction and NADPH bind enzymes 6.0

Cyp2g1, Xdh, Ptgr1, Htatip2, Ugdh, Coq7, Fth1, Akr1c13, Akr1a4, Cryl1, Fmo1, Gpx4, Aox1, Cyp2a5, Txnrd1, Nqo1, Srxn1

Carboxylesterase 3.9 Ces2, Ces1, BC015286, Ces5

Chemical and iron homeostasis 3.2 Xdh, Ftl1, Gclc, Fech, Ftl2, Hexa, Hexb, Bnip3, Afg3l2, Fth1, Abcg8, Anxa7

Cofactor biosynthetic process 2.4 Gss, Fech, Gclc, Coq7, Gch1

Top annotation cluster for the set of genes suppressed through Nrf2 activation

Response to nutrient and 2 3 A 1 Adi 2 C A Cbpextracellular stimulus 2.3 Avpr1a, Adipor2, Cp, Apom, Cbs

What Drug Processing Genes are Altered by Nrf2 ti tiactivation

• Uptake transporters• Phase‐I enzymes• Phase‐II enzymes• Efflux transporters

N f2 ll Wild K 1 KD K 1 HKO

Nrf2

Nrf2 null


Nrf2-nullWTKeap1KDKeap1HKO

2210

023G

05R

ik

Adh1

Adh4

Adh5

Aox

1

Cda

Ces

3

Ces

5

Cyp

1a2

Cyp

2a12

Cyp

2a5

Cyp

2b10

Cyp

2c29

Cyp

2c37

Cyp

2c44

Cyp

2c50

Cyp

2c54

Cyp

2c55

Cyp

2c70

Cyp

2d10

Cyp

2d22

Cyp

2d26

Cyp

2d9

Cyp

2e1

Cyp

3a11

Cyp

3a13

Cyp

3a25

Dpy

d

Dpy

s

EG13

909

Es1

Es22

Fmo1

Fmo5

Gm

ps

Gst

a2

Gst

a3

Gst

a4

Gst

k1

Gst

m1

Gst

m1

2

Gst

m2

Gst

m3

Gst

m4

Gst

m5

Gst

m6

Gst

m7

Gst

o1

Gst

p1

Gst

t1

Gst

t2G

stz1

Gus

b

Hpr

t1

Impd

h2

Itpa

Mao

b

Mgs

t1

Mgs

t3

Nat

2

Tpm

t

Tpm

t 2

Uck

1

Ugt

1a6a

Ugt

1a9

Ugt

2a3

Ugt

2b1

Ugt

2b34

Ugt

2b36

Ugt

2b5

Um

ps

Upb

1

Xdh

Drug metabolizing genes

Major P450 Drug Metabolizing Genes in Humanj g g

Human Gene Mouseortholog

Inducers Nuclearreceptorortholog receptor

CYP1A2 Cyp1a2 Omeprazole AhRCYP2A6 Cyp2a4 Barbiturates CARCYP2A6 Cyp2a4 Barbiturates CARCYP2B6 Cyp2b10 N.A.CYP2C8 Cyp2c65 N.A.CYP2C8 Cyp2c65 N.A.CYP2C9 N.A. Rifampin PXRCYP2C19 Cyp2c50 Rifampin PXRCYP2D6 Cyp2d22 N.A.CYP2E1 Cyp2e1 EthanolCYP3A4 Cyp3a41a Rifampin PXR

Phase‐I Drug Processing Enzymes (Cytochrome P450)

14161820

Nrf2-nullWild-typeKeap1-KDKeap1-HKO

d-in

duct

ion

38

1012

∗

∗ ∗Fo

l

1

2

∗ ∗ ∗∗

∗ ∗

∗

Cyp2a5 Cyp2c50 Cyp2c54 Cyp2g1 Cyp2u10

Isoform Human ortholog Substrates

Cyp2a5 CYP2A6 Nicotine, halothane, aflatoxin

Cyp2c50 N.A. Arachidonic Acid

Cyp2c54 N.A. Arachidonic Acid

Cyp2g1 CYP2G1P Sex hormones, acetaminophen

Cyp2u1 CYP2U1 Fatty acids

3.0

Non‐P450 Phase‐I Enzymes: Aldo‐keto Reductase

on 2.0

2.5


∗

∗

old-

indu

ctio

1 0

1.5

2.0

∗∗

Fo

0.5

1.0 ∗ ∗

Akr1a4 Akr1b3 Akr1c13 Akr1c19 Akr7a50.0

Isoform Human ortholog Substrates

Akr1a4 AKR1A1 Trans‐muconaldehyde (cytotoxic metabolite of benzene)

Akr1b3 AKR1B1 Acrolein, 4‐hydroxy‐trans‐2‐nonenal (HNE)

Akr1c13 N.A. N.A.

Akr1c19 N.A. N.A.

Akr7a5 AKR7A2 Aflatoxin B1

Non‐P450 Phase‐I Enzymes: Carbonyl Reductase and C b l tCarboxylesterase

Carbonyl Reductase Carboxylesterase

uctio

n 1200

1400

1600Nrf2-nullWild-typeKeap1-KDKeap1-HKO

∗

uctio

n

3

4

5


∗

∗

Fold

-indu

5

10

1000 ∗

∗

Fold

-indu

1

2

3

∗∗

Cbr1 Cbr30

∗

Ces1 Ces2 Ces50

∗∗

Isoform Substrates Isoform Substrates

Cbr1 Doxorubincin, S‐nitroglutathione

Cbr3 Doxorubincin

Ces1 ACE inhibitors (imidapril)

Ces2 Propionyl propranolol

Non‐P450 Phase‐I Enzymes: Other

uctio

n 40


∗

uctio

n

2


∗

∗

Fold

-ind

1

2

3

4

5

∗

∗∗

∗ ∗

∗

∗∗

Fold

-indu

1∗

∗

Ephx1 Fmo1 Nqo1 Xdh0

1∗ ∗

Aldh1a1 Aox10

∗

Gene Enzyme Substratey

Aldh1a1 Aldehyde dehydrogenase Acetaldehyde

Aox1 Aldehyde oxidase Famciclovir

E h 1 E id h d l P l li tiEphx1 Epoxide hydrolase Polycyclic aromatic hydrocarbons

Fmo1 Flavin monoxygenase 1 Cimetidine

( ) d dNqo1 NAD(P)H quinone oxidoreductase Quinones

Xdh Xanthine dehydrogenase 6‐mercaptopurine

Phase‐II enzymes: Glutathione Conjugation

Glutathione S-transferase

tion 32

34 Nrf2-nullWild-typeKeap1-KDKeap1-HKO

∗

Fold

-indu

ct

810

30∗ ∗

∗

0246

∗

∗

∗ ∗∗

∗

∗

∗

∗

∗

∗

∗

∗

∗∗

∗

Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst30

Not altered: Gstk1, Gstm5, Gstm7, Gsto1, Gstp1, Gstt1, Gstt2

Phase‐II enzymes: Glucuronidation

Glucose UDP‐glucuronic acidUgp2 Slc35d1

Cytosol ER

GlucuronidationGlucose UDP glucuronic acidUgdh

Slc35d1 GlucuronidationUgts

3 0

3.5

4.0

Nrf2-nullWild-typeKeap1-KD

∗

nduc

tion

2.0

2.5

3.0 pKeap1-HKO

∗∗

Fold

-i

1.0

1.5

∗

∗

∗

∗∗ ∗ ∗

∗

∗

∗ ∗

∗

∗∗

∗

Ugp2 Ugdh Slc35d1 Ugt1a6a Ugt1a9 Ugt2b35 Ugt2b36 Ugt2b5 Ugt3a10.0

0.5∗

∗ ∗

Uptake and Efflux Transporters

Uptake Transporters Efflux Transporters

150

Nrf2-nullWild t

∗2 Nrf2-nullWild-type

Uptake Transporters

-indu

ctio

n

5

50

100 Wild-typeKeap1-KDKeap1-HKO

∗

∗∗in

duct

ion

1

Keap1-KDKeap1-HKO

Fold

-

1

2

3

4

∗

∗

∗

∗

∗

∗

∗∗

∗

∗

∗ ∗

∗

Fold

- 1

∗

∗

∗

∗

Mrp2 Mrp3 Mrp4 Mrp9 Bcrp Abcg5 Abcg80

∗

Oatp1a1 Atp8b1 Ntcp Oat20

Revised from Klaassen and Lu, Toxicol Sci. 2008;101:186.

UPTAKEPhase I

BIOTRANSFORMATION

Nucleophiles EFFLUXHydrophilicity

Phase IIU t S lt

Conjugates

Cyps

xific

atio

n

Phase INqo1Eh-1

Ugts, Sults

Mrps

Diffusion Det

oxElectrophilesOxidative Stress and GOxidative Stress and

Formation of Adducts GSH Conjugation

Gsts

Glutamate

Cysteine

γ‐glutamylcysteine GSH

Glycine

Gcl Gs Nrf2Gcl Gs

AhR and Nrf2 interactions

Yeager et al., Toxi Sci 2009.

Expression of Which Antioxidant Genes are altered by Nrf2y

Antioxidant Genes were Induced with Graded Nrf2 Activation

4

Nrf2-null WT

∗30

Nrf2-null WT

∗

1) GSH synthesis and regeneration 2) Reduction of hydrogen peroxide

Fold

-indu

ctio

n

2

3Keap1-KD Keap1-HKO

∗∗

∗ ∗∗

∗

old-

indu

ctio

n

3

25

Keap1-KD Keap1-HKO

∗

Gclc Gss Gsr

F

0

1

Gpx2 Gpx4 Prdx6

Fo

0

1

2 ∗

∗

∗

Gclc Gss Gsr Gpx2 Gpx4 Prdx6

6

8 Nrf2-null WT Keap1-KD

∗14

Nrf2-null WT Keap1-KD

∗

3) Reduction of oxidized protein 4) Reduction of bilirubin and ion sequesterGpx2 Gpx4 Prdx6

Fold

-indu

ctio

n

2

4

6 Keap1-HKO

∗

∗

∗∗

∗

∗

∗∗

∗

∗∗Fo

ld-in

duct

ion

4

12

Keap1-HKO

∗

Blvrb Fech Fth1 Ftl1 Ftl20

1∗ ∗ ∗∗

Glrx Glrx5 Srxn1 Txnrd10

2 ∗∗∗

∗

∗

∗∗

Glrx1 Glrx5 Srxn1 Txnrd1 Blvrb Fech Fth1 Ftl1 Ftl2

H O OH.H2O2

Prdx (red)Txn (red)NADPH

T d1

OHFe2+

Ferritin

Prdx (oxi)Txn (oxi)NADP+

Txnrd1

GSSG NADP+

GrGpx

Prdx (red)

Sulfiredoxin

GSH NADPH

Gr

Gcl Gs

Gpx

Prdx (oxi)

Sulfiredoxin

glutamate, cysteine, glucine

Gcl, Gs

Biliverdin NADP+Biliverdin

Bvrb

Bilirubin

NADP+

NADPHH2O

Bilirubin NADPH

Does Nrf2 activation alter the toxicity of chemicals?

Diquat: ‘Model Chemical’ to Study Oxidative StressDiquat: Model Chemical to Study Oxidative Stress

• Diquat does not bind covalently with biological

Diquat dibromide

Diquat does not bind covalently with biological molecules, and minimally metabolized in rodent.

• Diquat treatment can cause lipid peroxidation, i d ALT l l BUN l l d i iDiquat dibromide increased ALT level, BUN level and necrosis in rats and mice.

• The induction of toxicity by diquat was even more

Diquat . + O2NADP+

y y qenhanced in mice deficient of Thioredoxin‐2 (Pérez et al Free Radic Biol Med 44(5):882, 2008), Glutathione peroxidase‐1 (Ho et al Free Radic Biol Med 43(9):1299,2007), and Glutathione

Diquat 2+ O2‐NADPH

( ) )reductase (Rogers et al Toxicol Appl Pharmacol 217(3):289,2006).

Experimental Design

125 mg/kg Diquat (i.p.)

Nrf2‐null Wild‐type Keap1‐KD

Serum Lung Liver 1‐6 hours

ALT H&E Lung GSH H&E Liver GSH Gclc mRNAALT H&E Lung GSH H&E Liver GSH Gclc mRNA

SurvivalSurvival

100

120Wild-type Nrf2-null Keap1-kd

rviv

al (%

)

60

80

Sur

20

40

Time (h)0 1 2 3 4 5 6

0

( )

Lipid PeroxidationLipid Peroxidation

50

60

70Wild-typeNrf2-nullKeap1-kd

RS

nmol

/L

30

40

50∗∗

∗∗

TBA

10

20

30

Time (h)

0 1 2 3 4 5 60

( )

Liver Injury

Control DiquatS ALT

Nrf2-null

qSerum ALT

∗

Wild-typeALT

(U/L

)

400

600∗

∗∗

∗

∗ Wild type

0 1 2 3 4 5 6

A

0

200Wild-typeNrf2-nullKeap1-kd

Keap1-kdTime (h)

0 1 2 3 4 5 6

GSH and GSSG Concentrations in Liver

GSH∗Catalase

6

8

10

mol

/g ti

ssue

∗

∗∗∗

∗

Catalase

0

2

4

Wild-typeNrf2-nullKeap1-kd

μm

GSSG1 0 Wild t

∗

O2

.‐ PeroxireductaseH20

GSSG

0.6

0.8

1.0 Wild-typeNrf2-nullKeap1-kd

∗∗g

tissu

e

Gpx

0 1 2 3 4 5 6 70.0

0.2

0.4 ∗

μmol

/ g

GSH GSSG

Time (h)

Lung Toxicity

Control DiquatRelative Lung Weight

ght % 1.0

1.2 ∗

∗ Nrf2-null

g g

g w

eigh

t/bod

y w

eig

0.4

0.6

0.8

Wild type

Time (h)

0 1 2 3 4 5 6

Lun

0.0

0.2WT Nrf2-null Keap1-kd

Wild-type

Time (h)

Keap1-kd

Conclusions on diquatConclusions on diquat

• Diquat treatment induced lung and liver injuryDiquat treatment induced lung and liver injury.

• This injury was enhanced in Nrf2‐null miceThis injury was enhanced in Nrf2 null mice, and attenuated in Keap1‐kd mice.

• The difference of the injury appears to be caused by the difference in the basal levels ofcaused by the difference in the basal levels of total GSH and GSH synthesis.

Acute Alcoholic Liver Disease: when oxidative stress meets disrupted lipid metabolismee s d s up ed p d e abo s

ROSAlcoholic

Disrupted lipid metabolism

Alcoholic liver disease

AntioxidantNrf2

Aldh1a1

Antioxidant

Decrease lipid synthesis

Hypothesis: Nrf2 activation prevents alcohol‐induced hepaticHypothesis: Nrf2 activation prevents alcohol induced hepatic oxidative stress and lipid accumulation.

Does Nrf2 alter the ethanol disappearance in mice ?


Nrf2


“Binge drinking” model (5g/kg ethanol)Or isocaloric glucose solution

Collect 20 µL of the blood from the tailat 1, 2, 3, 4, 5, 6 h after treatment

Serum ethanol concentration

Ethanol disappearance

3.0

l)

2.5Nrf2-nullWTKeap1-KDKeap1-HKO

anol

(mg/

m

1.5

2.0

Keap1 HKO

Seru

m E

tha

1.0

0 0

0.5

Time (h)

0 1 2 3 4 5 6 70.0

Does Nrf2 prevent ethanol‐induced liver injury?


Nrf2

“Binge drinking” model (5g/kg ethanol)Binge drinking model (5g/kg ethanol)Or isocaloric glucose solution

Sacrifice after 6 hours

LiverSerum

Histology, GSH, and lipid profile

ALT, TBARS, and lipid profile

140

Markers of liver injury

ALT

/L

80

100

120

∗

∗

ControlEthanol

U/

20

40

60 ∗ †

†

0LDH

800

1000 ∗

∗

ControlEthanol

U/L

200

400

600 †

†

0

200

Nrf2-null Wild-type Keap1-KD Keap1-HKO

Nrf2

*: different between control and cadmium treatment in the same genotype.†: different between wild‐type and other genotype groups after ethanol treatment.

TBARS80 ∗

Markers of oxidative stress in vivoTBARS

BAR

S (m

g/L)

40

60

∗

†

ControlEthanol

Cytosol GSH10

TB

0

20

†ControlEthanol

mol

/g li

ver t

issu

e

4

6

8

†Ethanolμm

0

2

Mitochondrial GSH10 Control

Ethanol

μmol

/g li

ver t

issu

e

4

6

8∗

∗∗

††

†

0

2


Nrf2

Indicator of oxidative stress in vitro

2',7'‐dichlorodihydrofluorescein diacetate, acetyl ester (H2DCFDA)

cellular esterase H2DCF

ROS DCF (green fluorescent)

ativ

e to

cont

rol)

3

4 Nrf2-null Wild-type Keap1-KD Keap1-HKO

Control Ethanol

Nrf2‐null

RO

S (R

ela

wild

-type

c

0

1

2

Wild‐type Nrf2

to rol)

4 Nrf2-null Wild-type Keap1-KD

Time

0h 3h 6h 24h

Keap1‐KD

RO

S (R

elat

ive

wild

-type

con

t r

1

2

3eap

Keap1-HKO

Keap1‐HKO

Nrf2 N f2

Concentration

0mM 30mM 100mM 300mM0

Nrf2 Nrf2

Nqo1cont

rol

10

12

Gclc

ontr

ol

3.0

3.5

d‐

vel o

ver w

ild-ty

pe

4

6

8

10ControlEthanol

∗

evel

ove

r wild

-type

co

1.0

1.5

2.0

2.5ControlEthanol

∗

†

††

mRN

A relativ

e to

wild

‐type control

NA relativ

e to wil

type

con

trol

Nrf2-null Wild-type Keap1-KD Keap1-HKOmR

NA

lev

0

2


mR

NA

le

0.0

0.5 ∗∗†

††

Nrf2 Nrf2

m w

mRN

Nqo1

Β‐actin

Control Ethanol Control Ethanol

Gclc

Β‐actin

Nqo1

ype

cont

rol

10

12

Control ∗Gclc

ype

cont

rol

1.4

1.6

1.8

Control ∗

Nrf2 Nrf2 Nrf2 Nrf2

††

to

ol to

ol

n le

vel o

ver w

ild-ty

2

4

6

8 Ethanol

ein

leve

l ove

r wild

-ty

0.2

0.4

0.6

0.8

1.0

1.2 Ethanol ∗∗

†

†

†

Protein relativ

e wild

‐type contro

Protein relativ

e wild

‐type contro

Nrf2-null Wild-type Keap1-KD Keap1-HKOProt

ei

0 Nrf2-null Wild-type Keap1-KD Keap1-HKOProt

e

0.0

0.2†

Nrf2 Nrf2

Lipid profiles in serum and liver

/g li

ver)

40

50

60 ∗

∗ ∗

g/L) 80

100∗

Serum TG†

ControlEthanol

Liver TGControlEthanol

Hep

atic

TG

(mg

10

20

30

40

Seru

m T

G (m

g

20

40

60

0Nrf2-null Wild-type Keap1-KD Keap1-HKO0


Nrf2 Nrf2

cids

(mEq

/g li

ver)

0.06

0.08

∗

∗

Acid

s (m

Eq/L

)

0 3

0.4

0.5

∗ ∗

†

Serum FFA Liver FFA

ControlEthanol

ControlEthanol

Col 1

epat

ic F

ree

Fatty

Ac

0.02

0.04 ∗

Seru

m F

ree

Fatty

0.1

0.2

0.3

H 0.00Nrf2-null Wild-type Keap1-KD Keap1-HKO

S

0.0Nrf2-null Wild-type Keap1-KD Keap1-HKO

Nrf2 Nrf2

4

*†

Srebp1 mRNA, protein, and target gene expressionSrebp1 mRNA

NA

rela

tive

to

-type

con

trol

2

3*

* ControlEthanol

†

†

Scd1 mRNA

ld-ty

pe c

ontr

ol

1 5

2.0

2.5∗

mR

Nw

ild-

0

1


*

ControlEthanol

††

†

RN

A re

latv

ie to

wi

0.5

1.0

1.5

Srebp1

Control Ethanol

††

Nrf2

mR 0.0


Srebp1 Protein1.6

1.8†

Srebp1H3

Nrf2 Nrf2

rote

in re

lativ

e to

wild

-type

con

trol

0.6

0.8

1.0

1.2

1.4 ControlEthanol

† †


P w

0.0

0.2

0.4

Nrf2

Summary

GSHGclcGSHGclc

Nrf2

GclcNqo1GclcNqo1 Oxidative stress

Nrf2

Lipid accumulation

Srebp1Srebp1

• Keap1‐Nrf2 pathway plays an important role in protecting against ethanol‐induced hepaticalterations in vitro and in vivo.

• The protective effect of Nrf2 may result from elevation of cellular GSH concentrations andsuppression of the Srebp1 signaling pathway.

• We showed earlier that MT protects against Cd hepatotoxicity

• Might Nrf2 also protect against Cd• Might Nrf2 also protect against Cd toxicity

SummarySummary

Nrf2 protects against:Nrf2 protects against:• Diquat

h l• Ethanol• CdConclusion:• Nrf2 appears to an important pathway toNrf2 appears to an important pathway to

protect against many chemicals.

• Presently: Take antioxidants

• Future: Have body make antioxidants

MafNrf2

ARE

Acknowledgements

KUMC• KUMC• Dr Jie Liu• Dr Lauren Aleksunes• Dr Jonathan Maher• Dr Jonathan Maher• Dr Ronnie Yeager• Dr Scott Reisman• Kai Connie WuKai Connie Wu

• Nrf2‐null and Keap1‐KD mice• Dr Jefferson Chan• Dr Massayuki Yamamoto

• Grant Support• ES‐013714, ES‐09716, DK‐

081461, RR‐021940

The Keap1 Nrf2 Pathway Toxicology · The Keap1‐Nrf2 Pathway in Toxicology Curtis Klaassen...

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