The Keap1‐Nrf2 Pathway in Toxicology
Curtis Klaassen University of Kansas Medical Center Cd tolerance from Cd pretreatment
Time after Cd pretreatment n Mortality (%) No pretreatment 10 100 2 hr 10 100 4 hr 10 100 6 hr 10 60 8 hr 10 50 24 hr 10 0 2 day 10 0 4 day 10 0 8 day 10 0 16 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 hepatotoxicity
No pretreatment 1000000 Cd pretttreatment
100000 ml) //
units 10000
(S
DH * * * S 1000
100 Control 2.0 3.0 4.0 5.0 mg Cd/kg
Rats were pretreated with CdCl2 ((gg,2.0 mg Cd/kg, sc) and 24 hrs later were 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. Goering and Klaassen TAAP 74:308, 1984 Cd pretreatment alters subcellular Cd distribution
80 No pretreatment 70 * Cd pretreatment liver 60 the
nn
i 50
40 30 stribution ii d 20 *
Cd * *
% 10 0 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 dis tr ibu tion was de term ine d 2 hrs ltlater. D Dtata are M ean ± SE o f 6 rat s.
Goering and Klaassen TAAP 70:195, 1983 Cd pretreatment alters cytosolic Cd distribution
No pretreatment 353.5 Cd pretreatment 3 2.5 er vv li
2
Cd/g 1.5
ug 1 0.5 0 0.6 1.0 1.4 1.8 2.2 2.6 Ve/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 dis tr ibu tion was de term ine d 2 hrs ltlater.
Goering and Klaassen TAAP 70:195, 1983 MT-null mice are highly susceptible to chronic Cd-induced nephrotoxicity
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.
Liu……Klaassen ToxSci 46: 197, 1998 The relationship between human exposure to cadmium and renal injjyury
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 against liver injury.
OA Oleanolic acid UA Ursolic acid UV Uvaol α‐H α‐Hederin HD Hederagenin GL Glycyrrhizin Jie Liu 87‐98 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 3000 CCl4 2500
2000 ∗
1500 ∗ ALT (U/L) ALT 1000 ∗ ∗ ∗ 500 ∗
0 25000
20000
15000 ∗ ∗
10000 SDH (U/ ml) ∗ ∗ 5000 ∗
0
2.0 de)
aa ∗ ∗ 1.5 ∗
∗ 1.0
NECROSIS (gr NECROSIS 0.5
0.0 CON Jgs Ngs Ful OA Swe Om DDB SDH (kIU/L) ALT (U/L) 20000 25000 10000 10000 15000 4000 6000 8000 5000 2000 Mortality (%) Necrosis (grade) 30 40 10 20 0 1 2 3 0 0 0 B r o S m a o l Saline Saline i Saline A b n c e e Bromobenzene Bromobenzene Bromobenzene
e n ∗ t a z m e n i n e Acetaminophen Acetaminophen o Acetaminophen ∗ ∗ p ∗ C h a e T r n h b ∗ ∗ i o ∗ Carbon tetCarbon tetCarbon
o tetCarbon n a
c t e e t Thioacetamide Thioacetamide t Thioacetamide ∗ a ∗ F m ∗ u i r d o e s ∗ ∗ e ∗ Furosemide Furosemide m Furosemide P i d h e a ∗ ∗ l ∗ l o Phalloidin Phalloidin C i Phalloidin d o i l n c ∗ ∗
h ∗ Colchicine Colchicine i Colchicine c i C n a e
d OA Vehicle ∗ ∗ ∗ D m Cadmium Cadmium Cadmium - i G u m a l ∗ ∗
N ∗ D-GalN/LPS D-GalN/LPS D-GalN/LPS / L P S ∗ ∗ ∗ Oleanolic acid How?
We spent a considerable amount of time tying to determine how Oleanolic Acid protects against this large panel of hepatotoxicants, unfortunately, with little success. Precarcinogens activation Anticarcinogenic enzyme inducers are of two types: (a) bifunctional inducers (e.g ., TCDD, polycyclic aromatics) that elevate both Phase II enzymes, NAD(P)H: quinone oxidddoreductase, and certain Phase I enzymes; (b) monofunctional inducers ((g,e.g., diphenols, thiocarbamates, 1,2‐dithiol‐3‐ thiones, beta‐ naphthoflavone) that elevate priilimarily Phase II enzymes. Cancer prevention Prochaska and Talalay Cancer Res 1988 Phenolic antioxidants Pheno licantiox idan ts
Nrf2 Maf GST‐Ya GST‐Ya ARE ARE GST‐Pi Nqo1 Antioxidants Itoh et al., BBRC 1997.
Nrf2 Maf Ho‐1 (Alam et al., JBC 1999) ARE Gclc (Moinova et al., BBRC 1999) Oxidative stress and activators
CUL3 Keap1 Keap1
Nrf2
Nrf2
Anti‐oxidant and inflammation Nrf2 Cell survival and proliferation Maf Drug metabolism Lipid metabolism ARE Keap1 cysteine Nrf2 activator Structure residues Reference
(Dinkova‐ dexamethasone 21‐ 257, 273, 288, 297, Kostova et al., mesylate and 613 (human) 2002)
196, 226, 241, 257, iodoacetyl‐N‐ (Hong et al., 288, and 319 biotinylhexylenediamine 2005b) (mouse) 77, 226, 249, 257, (Hong et al., sulforaphane 489, 513, 518 2005) (human) 151, 319, and 613 (Luo et al., xanthohumol (human) 2007) Michael Sporn, “the father of chemoprevention” asked for our Oleanolic Acid so he could make more potent analogues. Oleanolic acid
CDDO CDDO‐Me CDDO‐Im
2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oic acid (CDDO) CDDO compounds are potent Nrf2 activators
140000 3500 WT-Vehicle 120000 WT-CDDO-Im ∗ 3000 * Nrf2-null Vehicle Nrf2-null-CDDO-Im 2500 100000 * 2000 80000 ALT (IU/L) l liver RNA l liver 1500 aa
∗ mm 1000 g tot 60000 ∗ μ Seru 500 40000 *
RLUs/2 0 Nrf2 WT WT null null 20000 CDDO-Im - + - + 0 Acetaminophen + +++ Nqo1 Gclc Ho-1
A single dose of CDDO‐Im (1mg/kg, i.p.) CDDO‐Im protection against increases Nrf2‐dependent genes in wild‐ acetaminophen hepatotoxicity is type but not Nrf2‐null mice Nrf2‐dependent
Reisman and Klaassen. Toxi Appl Pharm. 2009. Oleanolic acid is also a Nrf2 activator
18 Nf2Nrf2 N1Nqo1 GlGclc H1Ho-1 8 16 7 * 14 * 6 * -actin ontrol)
β 12 ression ontrol 5 ein oo tt CC CC pp 4 10 3 Nrf2 Pro 8 *
Relative to to Relative 2 Normalized t 1 6
ND ND r mRNA Ex rmalized to * 0 ee Wild-type Nrf2-null oo 4 (N Oleanolic Acid - + - + Liv 2
0 WT null WT null WT null WT null 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‐Nrf2 Pathway Alter? Generation of “Gene dose‐response” Model
Nrf2‐null WT Keap1‐KD Keap1‐HKO Keap1 β‐actin 1.2 Keap1 1.4 Nrf2‐null Wild‐type Keap1‐KD Keap1‐HKO 1.0 1.2 Keap1 0.8 1.0 ression pression xx 0.6 pp 080.8
0.4 0.6 ∗ 0.4
mRNA e mRNA 0.2 ∗ ∗
Protein ex 0.2 0.0 ∗ l ul pe 0.0 -n ty -KD HKO f2 d- p1 1- ll e D O il a p nu yp -K K Nr W Ke a 2- -t 1 -H Ke f ild ap 1 Nr W e ap K Ke
Col 2 4 Nrf2 ∗ on ii 3 ∗
2
1
Protein express Protein N.A. 0 ll e D O nu yp -K K 2- -t 1 -H rf ild ap 1 N W e ap K Ke
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” Model
Nrf2‐null WT Keap1‐KD Keap1‐HKO 20 Nqo1 Nqo1 ∗ 15 Gclc 10
expression Β‐actin AA ∗ 5 20 ∗ mRN Nqo1 ∗ 12 15 0 Cytosol GSH ∗ ll e D O 10 ∗ nu yp -K K 2- -t 1 -H rf ild ap 1 10 ∗ N W e ap 8 K Ke ∗ er tissue er ein expression tt
5 vv 6 Pro N.A. 4 0 ll e u p KD KO -n ty - mol/g li f2 d- p1 -H 2 r il a 1 μ 4 N W e ap K Ke Gclc ∗ 0 ll e u p KD O 3 -n ty 1- HK sion 2 - - rf ild ap 1 ss 252.5 N W e ap K Ke ∗ Gclc ∗ 2 2.0
1.5 1
mRNA expre 1.0
0 expression otein
rr 050.5 ll e
D O P nu yp -K K 2- -t 1 -H rf ild ap 1 N W e ap 0.0 K e ll e D O K nu yp -K K 2- -t 1 -H rf ild ap 1 N W e ap K Ke Transcriptional Profiling by Microarray Analysis
Nrf2
Nrf2‐null Wild‐type Keap1‐KD Keap1‐HKO
n=3 n=3 n=3 n=3
Liver
Total RNA
Microarray Data Analysis from Microarray
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 packages Constitutively Induced or Suppressed Genes
WT>Nrf2‐null Keap1‐KD>WT WT 17 25 45 48 97 124 115 80 81 61 113 90 294 329 Keap1‐HKO>Keap1‐KD 8021 Keap1‐HKO Suppressed genes 6 Induced genes 6 ∗ ∗ ∗ ∗ 4 4 ∗ ∗ old-induction 2 old-induction 2 FF FF 0 0 ll e D O ll e D O nu yp -K K nu yp -K K 2- -t 1 -H 2- -t 1 -H rf ild ap 1 rf ild ap 1 N W e ap N W e ap K Ke K Ke Annotation clustering using DAVID analysis Term Enrichment Score Gene Symbols Top annotation cluster for the set of genes induced through Nrf2 activation Glutathione transferase and Gstm1, Gss, Gsta2, Gstm2, Gsta3, Gstm3, 8.2 glutathione metabolism Gclc, Gstm4, Gsta4, Gpx4, Gstm6 Cyp2g1, Xdh, Ptgr1, Htatip2, Ugdh, Coq7, Oxidation reduction and NADPH 6.0 Fth1, Akr1c13, Akr1a4, Cryl1, Fmo1, Gpx4, bind enzymes Aox1, Cyp2a5, Txnrd1, Nqo1, Srxn1 Carboxylesterase 3.9 Ces2, Ces1, BC015286, Ces5 Xdh, Ftl1, Gclc, Fech, Ftl2, Hexa, Hexb, Chemical and iron homeostasis 3.2 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 232.3 A1Adi2CAAvpr1a, Adipor2, Cp, Apom, Cbs extracellular stimulus What Drug Processing Genes are Altered by Nrf2 activa tion • Uptake transporters • Phase‐I enzymes • Phase‐II enzymes • Efflux transporters Nrf2 Nf2Nrf2‐null Wild‐type K1Keap1‐KD KK1eap1‐HKO Nrf2- null WT Keap1KD Keap1HKO Cyp2c70 Ugt2a3 Dpyd EG13909 Gmps Fmo5 Itpa Ugt2b1 Adh5 Cyp2c29 Cyp2a12 2 Tpmt Ugt2b34 Cyp2e1 Mgst1 Cyp2c37 Es22 Ces3 Cyp3a25 Maob Cyp2d10 Gstt1 Cyp3a13 Cyp3a11 Gstk1 Impdh2 Gstm5 Gstm7 Gusb 2210023G05Rik Fmo1 Cyp2d22 Gsta4 Gstm2 Cda Cyp2a5 Gsta2 Gstm3 Gstm1 Gstm4 Gsto1 Mgst3 Cyp1a2 Cyp2b10 Adh1 Cyp2d9 Gstt2 Gstz1 Aox1 Xdh Ces5 Gstm1 2 Gstm6 Gstp1 Dpys Gsta3 Ugt1a6a Ugt1a9 Cyp2d26 Umps Cyp2c44 Cyp2c50 Uck1 Ugt2b5 Ugt2b36 Cyp2c54 Hprt1 Cyp2c55 Upb1 Tpmt Adh4 Nat2 Es1 Drug metabolizing genes Major P450 Drug Metabolizing Genes in Human Human Gene Mouse Inducers Nuclear ortholog receptor CYP1A2 Cyp1a2 Omeprazole AhR CYP2A6 Cyp2a4 Barbiturates CAR CYP2B6 Cyp2b10 N.A. CYP2C8 Cyp2c65 N.A. CYP2C9 N.A. Rifampin PXR CYP2C19 Cyp2c50 Rifampin PXR CYP2D6 Cyp2d22 N.A. CYP2E1 Cyp2e1 Ethanol CYP3A4 Cyp3a41a Rifampin PXR Phase‐I Drug Processing Enzymes (Cytochrome P450) 20 18 Nrf2-null Wild-type 16 Keap1-KD 14 Keap1-HKO 12 ∗ ∗ 10 8 3 ∗ d-induction ∗∗ Fol 2 1 ∗ ∗ ∗ ∗ ∗ 0 Cyp2a5 Cyp2c50 Cyp2c54 Cyp2g1 Cyp2u1 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 Non‐P450 Phase‐I Enzymes: Aldo‐keto Reductase 3.0 Nrf2-null ∗ Wild-type 2.5 Keap1-KD Keap1-HKO n 2.0 ∗ oo ∗ 1.5 ∗ ld-inducti oo F 101.0 ∗ ∗ 0.5 0.0 Akr1a4 Akr1b3 Akr1c13 Akr1c19 Akr7a5 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 CbCarboxy ltlesterase Carbonyl Reductase Carboxylesterase 1600 Nrf2-null ∗ 5 Wild-type ∗ Nrf2-null 1400 Keap1-KD Wild-type Keap1-HKO 4 ∗ Keap1-KD Keap1-HKO 1200 ction ction uu 3 uu 1000 ∗ 2 ∗ 10 ∗ Fold-ind Fold-ind ∗ 5 1 ∗ ∗ ∗ 0 0 Cbr1 Cbr3 Ces1 Ces2 Ces5 Isoform Substrates Isoform Substrates Cbr1 Doxorubincin, S‐ Ces1 ACE inhibitors nitroglutathione (imidapril) Cbr3 Doxorubincin Ces2 Propionyl propranolol Non‐P450 Phase‐I Enzymes: Other ∗ 50 3 Nrf2-null Nrf2-null ∗ Wild-type Wild-type Keap1-KD Keap1-KD Keap1-HKO 40 Keap1-HKO ction 2 uction uu ∗ ∗ 5 ∗ 4 Fold-ind ∗ Fold-ind 1 ∗ 3 ∗ ∗ 2 ∗ ∗ 1 ∗ ∗ ∗ ∗ 0 0 Aldh1a1 Aox1 Ephx1 Fmo1 Nqo1 Xdh Gene Enzyme Substrate Aldh1a1 Aldehyde dehydrogenase Acetaldehyde Aox1 Aldehyde oxidase Famciclovir Eh1Ephx1 EidEpoxide hdhydro lase PlPolycyc lic aromatic hydrocarbons Fmo1 Flavin monoxygenase 1 Cimetidine Nqo1 NAD()(P)H quinone oxidddoreductase Quinones Xdh Xanthine dehydrogenase 6‐mercaptopurine Phase‐II enzymes: Glutathione Conjugation Glutathione S-transferase 34 Nrf2-null ∗ Wild-type Keap1-KD 32 Keap1-HKO ion tt 30 ∗ ∗ 10 ∗ Fold-induc 8 ∗ ∗ 6 ∗ ∗ ∗ ∗ 4 ∗ ∗ ∗ 2 ∗ ∗ ∗ ∗ ∗ ∗ ∗ 0 Gsta2 Gsta3 Gsta4 Gstm1 Gstm2 Gstm3 Gstm4 Gstm6 Gstp1 Gstt3 Mgst3 Not altered: Gstk1, Gstm5, Gstm7, Gsto1, Gstp1, Gstt1, Gstt2 Phase‐II enzymes: Glucuronidation Cytosol ER Ugp2 Glucose UDP‐glucuronic acid Slc35d1 Glucuronidation Ugdh Ugts 4.0 ∗ 3.5 Nrf2-null Wild-type 303.0 Keap1-KD Keap1-HKO 2.5 ∗ ∗ 2.0 nduction ∗ ∗ 1.5 ∗ ∗ ∗ ∗ ∗ ∗ ∗ Fold-i ∗ 1.0 ∗ ∗ ∗ ∗ ∗ 0.5 ∗ 0.0 Ugp2 Ugdh Slc35d1 Ugt1a6a Ugt1a9 Ugt2b35 Ugt2b36 Ugt2b5 Ugt3a1 Uptake and Efflux Transporters Uptake Transporters Efflux Transporters 150 2 Nrf2-null ∗ Wild-type Nrf2-null Keap1-KD 100 Wild-t ype Keap1-HKO Keap1-KD Keap1-HKO 50 ∗ 5 ∗ induction induction -- ∗ 1 ∗ ∗ 4 Fold ∗ Fold- ∗ 3 ∗ ∗ ∗ 2 ∗ ∗ ∗ ∗ ∗ ∗ 1 ∗∗ ∗ 0 0 Mrp2 Mrp3 Mrp4 Mrp9 Bcrp Abcg5 Abcg8 Oatp1a1 Atp8b1 Ntcp Oat2 Revised from Klaassen and Lu, Toxicol Sci. 2008;101:186. BIOTRANSFORMATION ÏHydrophilicity Conjugates UPTAKE Nucleophiles EFFLUX Phase I Phase II Cyps UtUgts, SltSults Phase I Nqo1 Mrps ification xx Eh-1 Diffusion Deto Electrophiles Oxidative Stress and Formation of Gsts Adducts GSH Conjugation Cysteine Glycine Glutamate γ‐glutamylcysteine GSH Gcl Gs Nrf2 AhR and Nrf2 interactions Yeager et al., Toxi Sci 2009. Expression of Which Antioxidant Genes are altered by Nrf2 Antioxidant Genes were Induced with Graded Nrf2 Activation 1) GSH synthesis and regeneration 2) Reduction of hydrogen peroxide 4 30 ∗ Nrf2-null ∗ Nrf2-null WT WT Keap1-KD 3 Keap1-KD Keap1-HKO Keap1-HKO 25 ∗ ∗ ∗ 2 3 ∗ ∗ ∗ ld-induction old-induction ∗ oo FF 2 F ∗ 1 ∗ 1 ∗ 0 0 Gclc Gss Gsr Gclc Gss Gsr Gpx2Gpx2 Gpx4 Gpx4 Prdx6Prdx6 3) Reduction of oxidized protein 4) Reduction of bilirubin and ion sequester 14 ∗ ∗ Nrf2-null 8 Nrf2-null WT WT Keap1-KD Keap1-KD Keap1-HKO 6 Keap1-HKO ∗ 4 12 ∗ ∗ ∗ 2 4 ∗ ∗ ∗ ∗ Fold-induction ∗ Fold-induction ∗ ∗ ∗ ∗ ∗ ∗ ∗ 2 ∗ ∗ ∗ 1 ∗ ∗ ∗ 0 0 Glrx Glrx5 Srxn1 Txnrd1 Glrx1 Glrx5 Srxn1 Txnrd1 BlvrbBlvrb Fech Fech Fth1 Ftl1 Ftl1 Ftl2 Ftl2 . H2O2 OH Fe2+ NADPH Txn (red) Prdx (red) Ferritin Td1Txnrd1 NADP+ Txn (oxi) Prdx (oxi) GSSG NADP+ Gpx Gr Prdx (red) GSH NADPH Sulfiredoxin Gcl, Gs Prdx (oxi) glutamate, cysteine, glucine Biliverdin NADP+ Bvrb Bilirubin NADPH H2O Does Nrf2 activation alter the toxicity of chemicals? Diquat: ‘Model Chemical’ to Study Oxidative Stress • Diquat does not bind covalently with biological molecules, and minimally metabolized in rodent. • Diquat treatment can cause lipid peroxidation, Diquat dibromide idincreased ALT lllevel, BUN lllevel and necrosis in rats and mice. • The induction of toxicity by diquat was even more enhanced in mice deficient of Thioredoxin‐2 + . + O NADP Diquat 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 reductase (Rogers et al Toxicol Appl Pharmacol 217(3):289,2006). 2+ ‐ NADPH Diquat O2 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 mRNA Survival Wild-type 120 Nrf2-null Keap1-kd 100 80 vival (%) 60 rr Su 40 20 0 0123456 Time (()h) Lipid Peroxidation 70 Wild-type 60 Nrf2-null Keap1-kd 50 ∗ ∗ 40 RS nmol/L 30 ∗ ∗ TBA 20 10 0 0123456 Time (()h) Liver Injury Control Diquat SALTSerum ALT Nrf2-null ∗ ∗ ∗ 600 ∗ 400 LT (U/L) LT ∗ AA Wild-type 200 Wild-type Nrf2-null Keap1-kd 0 0 1 2 3 4 5 6 Time (h) Keap1-kd GSH and GSSG Concentrations in Liver ∗ GSH Catalase ∗ 10 ∗ ∗ 8 6 ∗ ∗ ol/g tissue mm μ ∗ . 4 ‐ Peroxireductase Wild-type O2 H20 2 Nrf2-null Keap1-kd 0 101.0 Wild-t ype GSSG Nrf2-null Keap1-kd Gpx 0.8 0.6 ∗ tissue ∗ gg 0.4 ∗ mol/ μ GSH GSSG 0.2 0.0 01234567 Time (h) Lung Toxicity Control Diquat Relative Lunggg Weight 1.2 ∗ ∗ Nrf2-null 1.0 ht % gg 0.8 0.6 0.4 g weight/body wei Wild-type Lun WT 0.2 Nrf2-null Keap1-kd 0.0 0123456 Time (h) Keap1-kd Conclusions on diquat • Diquat treatment induced lung and liver injury. • This 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 of total GSH and GSH synthesis. Acute Alcoholic Liver Disease: when oxidative stress meets di sr upt ed lippdid meeabostabolism ROS Alcoholic liver disease Disrupted lipid metabolism Aldh1a1 Nrf2 Antioxidant Decrease lipid synthesis Hypothesis: Nrf2 activation prevents alcohol‐induced hepatic oxidative stress and lipid accumulation. Does Nrf2 alter the ethanol disappearance in mice ? 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 tail at 1, 2, 3, 4, 5, 6 h after treatment Serum ethanol concentration Ethanol disappearance 3.0 Nrf2-null 2.5 WT Keap1-KD l) Keap1- HKO 2.0 nol (mg/m aa 1.5 1.0 Serum Eth 0.5 000.0 01234567 Time (h) Does Nrf2 prevent ethanol‐induced liver injury? Nrf2 Nrf2‐null Wild‐type Keap1‐KD Keap1‐HKO “Binge drinking” model (5g/kg ethanol) Or isocaloric glucose solution Sacrifice after 6 hours Serum Liver ALT, TBARS, Histology, GSH, and lipid profile and lipid profile Markers of liver injury 140 ∗ ALT 120 100 Control Ethanol ∗ 80 L // U † 60 ∗ † 40 20 0 ∗ LDH 1000 ∗ Control 800 Ethanol 600 † U/L 400 † 200 0 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. Markers of oxidative stress in vivo 80 ∗ ∗ TBARS Control 60 Ethanol † 40 ARS (mg/L) ARS BB T 20 0 Cytosol GSH 10 Control Ethanol † 8 6 4 ol/g liver tissue liver ol/g mm μ 2 0 Mitochondrial GSH 10 Control Ethanol 8 † ∗† 6 ∗ 4 ∗ † mol/g liver tissue liver mol/g μ 2 0 Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Indicator of oxidative stress in vitro 2',7'‐dichlorodihydrofluorescein cellular esterase ROS H2DCF DCF (green fluorescent) diacetate, acetyl ester (H2DCFDA) Control Ethanol 4 Nrf2-null Wild-type Keap1-KD Nrf2‐null 3 Keap1-HKO ontrol) tive to tive cc aa 2 ROS (Rel 1 wild-type wild-type Wild‐type Nrf2 0 0h 3h 6h 24h Time 4 Nrf2-null Keap1‐KD Wild-type ol) to rr Keap1-KD 3 Keap1-HKO 2 Keap1‐HKO ROS (Relative 1 wild-type cont wild-type Nrf2 0 Nf2Nrf2 0mM 30mM 100mM 300mM Concentration 12 3.5 Nqo1 ‐ Gclc ntrol control d 3.0 oo 10 Control to Control wil 2.5 8 Ethanol Ethanol † to ∗ control 2.0 ∗ † 6 control relative 1.5 type ‐ relative 4 type A ild RNA el over wild-type vel over wild-type c wild-type over vel 1.0 NN vv ee ww mm † ∗† 2 mR 0.5 † ∗ 0 l mRNA 0.0 mRNA le mRNA Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Nrf2 Control Ethanol Control Ethanol Nqo1 Gclc Β‐actin Β‐actin Nrf2 Nrf2 Nrf2 Nrf2 12 1.8 Nqo1 1.6 Gclc 10 ∗† l l 1.4 pe control pe control Control † Control to to oo oo ∗ yy yy 8 Ethanol 1.2 Ethanol ∗† contr contr 1.0 ∗ 6 relative relative 0.8 † type type ‐ 4 ‐ 0.6 0.4 wild wild Protein 2 Protein in level over wild-t over in level n level over wild-t over n level † 0.2 ee 0 0.0 Prot Nrf2-null Wild-type Keap1-KD Keap1-HKO Protei Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Nrf2 Lipid profiles in serum and liver 60 100 Serum TG Control ∗ Liver TG ∗ † Ethanol Control 50 80 Ethanol ∗ ∗ /L) /g liver) /g gg 40 60 30 40 20 Serum TG (m TG Serum 20 Hepatic TG (mg 10 0 0 Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Nrf2 0.08 Serum FFA Liver FFA 0.5 Control Ethanol † 0.06 ∗ Control 0.4 ∗ ∗ Ethanol ids (mEq/gliver) Acids (mEq/L) Acids ∗ 030.3 cc 0.04 ∗ 0.2 0.02 Col 1 0.1 erum Free Fatty erum Free Fatty epatic Free Fatty A Free Fatty epatic SS H 0.0 0.00 Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Nrf2 Srebp1 mRNA, protein, and target gene expression 4 *† Srebp1 mRNA 3 Control * Ethanol 2 type controltype A relative to -- † NN † wild mR 1 * 2.5 ∗ † Scd1 mRNA 2.0 0 Control Nrf2-null Wild-type Keap1-KD Keap1-HKO Ethanol ld-type control ld-type 151.5 Nrf2 1.0 † † Control Ethanol 0.5 NA relatvie to wi to relatvie NA Srebp1 RR 0.0 m H3 Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Nrf2 1.8 1.6 † Srebp1 Protein 1.4 Control 1.2 Ethanol 1.0 0.8 †† 0.6 ild-type controlild-type rotein relative to relative rotein ww P 0.4 0.2 0.0 Nrf2-null Wild-type Keap1-KD Keap1-HKO Nrf2 Summary GSH Gclc Nqo1 Oxidative stress Nrf2 Lipid accumulation Srebp1 • Keap1‐Nrf2 pathway plays an important role in protecting against ethanol‐induced hepatic alterations in vitro and in vivo. • The protective effect of Nrf2 may result from elevation of cellular GSH concentrations and suppression of the Srebp1 signaling pathway. • We showed earlier that MT protects against Cd hepatotoxicity • Might Nrf2 also protect against Cd toxicity Summary Nrf2 protects against: • Diquat • Ethlhanol • Cd Conclusion: • Nrf2 appears to an important pathway to protect against many chemicals. • Presently: Take antioxidants • Future: Have body make antioxidants Nrf2 Maf ARE Acknowledgements • KUMC • Dr Jie Liu • Dr Lauren Aleksunes • Dr Jonathan Maher • Dr Ronnie Yeager • Dr Scott Reisman • Kai Connie Wu • Nrf2‐null and Keap1‐KD mice • Dr Jefferson Chan • Dr Massayuki Yamamoto • Grant Support • ES‐013714, ES‐09716, DK‐ 081461, RR‐021940