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
Goering and Klaassen TAAP 70:195, 1983
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
Goering and Klaassen TAAP 70:195, 1983
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
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‐null Wild‐type Keap1‐KD Keap1‐HKO
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‐null Wild‐type Keap1‐KD Keap1‐HKO
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
Nrf2-nullWild-typeKeap1-KDKeap1-HKO
∗
∗
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
Nrf2-nullWild-typeKeap1-KDKeap1-HKO
∗
∗
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
50Nrf2-nullWild-typeKeap1-KDKeap1-HKO
∗
uctio
n
2
3Nrf2-nullWild-typeKeap1-KDKeap1-HKO
∗
∗
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‐null Wild‐type Keap1‐KD Keap1‐HKO
Nrf2
Nrf2‐null Wild‐type Keap1‐KD Keap1‐HKO
“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‐null Wild‐type Keap1‐KD Keap1‐HKO
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-null Wild-type Keap1-KD Keap1-HKO
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
Nrf2-null Wild-type Keap1-KD Keap1-HKO
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-null Wild-type Keap1-KD Keap1-HKO
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
Nrf2-null Wild-type Keap1-KD Keap1-HKO
*
ControlEthanol
††
†
RN
A re
latv
ie to
wi
0.5
1.0
1.5
Srebp1
Control Ethanol
††
Nrf2
mR 0.0
Nrf2-null Wild-type Keap1-KD Keap1-HKO
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
† †
Nrf2-null Wild-type Keap1-KD Keap1-HKO
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