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1 doi: 1.193/toxsci/kft184

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4 1 I 5 II (DPH) II II II-2-3 GSH GSSG 2 II II-2-5 HMGB1 IL-1β IL II-2-6 RNA 22 II-2-7 PCR mrna 22 II II II-3-1 DPH 24 ALT AST 25 II-3-2 DPH5 ALT 25 II-3-3 DPH BSO GSH GSSG 27 II II-3-5 CYP DPH 28 II-3-6 CYP GSH 29 II II-3-8 HMGB1 HMGB1 TLR4 DPH 33 II-3-9 Th mrna 34

5 II-3-1 IL-17 IL-17 DPH 36 II-3-11 MPO 37 II-3-12 PGE 1 DPH III DPH CYP III III III-2-3 DPH GSH Cys NAC 53 III-2-4 DPH 54 III III-2-6 SDS- 55 III III-3-1 DPH 56 III-3-2 DPH 57 III-3-3 DPH 6 III-3-4 ABT ALT GSH 61 III-3-5 ABT DPH 62 III-3-6 DPH KTZ 63 III-3-7 KTZ CYP 64

6 III-3-8 Cyp2c Cyp3a 66 III-3-9 PB DPH IV

7 ABT 1-aminobenzotriazole 1- APAP acetaminophen BSO L-buthionine (S,R)-sulfoximine L- (S,R)- DEPC diethylpyrocarbonate DNP 2,4-dinitrophenylhydrazine 2,4- DTNB 5,5'-dithiobis(2-nitrobenzoic acid) 5,5-2- DPH diphenylhydantoin ( ) DW distilled water EDTA ethylene diamine tetraacetic acid EGTA ethylene glycol tetraacetic acid KTZ ketoconazole MDZ midazolam NADP + (H) nicotinamide adenine dinucleotide oxidized (reduced) form dntp deoxyribonucleotide triphosphates 1 -OH MDZ 1 -hydroxymidazolam 1-4-OH MDZ 4-hydroxymidazolam 4-4-OH TOL 4-hydroxytolbutamide 4- PB phenobarbital PBS phosphate buffered saline SDS sodium dodecylsulfate TOL tolbutamide Tris tris(hydroxymethyl)aminomethane 1

8 CAR constitutive androstane receptor CYP cytochrome P45 P45 Cys cysteine DPH-glu DPH N-glucuronide N- HPLC high performance liquid chromatography 4 -HPPH 4 -hydroxy DPH 4-4 -HPPH- 4 -HPPH O-glucuronide 4 - O- -O-glu GS- glutathione conjugated GST glutathione S-transferase LC-MS/MS liquid chromatography with tandem mass spectrometry NAC- N-acetylcysteine conjugated N- PXR pregnane X receptor X UGT UDP-glucuronosyltransferase UDP- ALT alanine aminotransferase AST aspartate aminotransferase T.Bil total bilirubin GR glutathione reductase GSH reduced glutathione GSSG oxidized glutathione Nrf2 NF-E2-related factor 2 NF-E2 2 ROS reactive oxygen species SOD superoxide dismutase 2

9 DAMPs damage-associated molecular pattern molecules Foxp3 forkhead box p3 3 GATA3 GATA-binding protein 3 GATA 3 HLA human leukocyte antigen HMGB1 high-mobility group box 1 1 IFN interferon IgG immunoglobulin G G IL interleukin KC Kupffer cell LPS lipopolysaccharide MCP1 monocyte chemoattractant protein1 1 MIP2 macrophage inflammatory 2 protein 2 MPO myeloperoxidase RAGE receptor of advanced glycation endproducts ROR retinoic acid-related orphan receptor T-bet T-box expressed in T cells T-bet Th helper T cell T Treg regulatory T cells T NALP3 NACHT-, LRR- and PYD NLR 3 PYD-containing protein 3 TLR Toll-like receptor Toll TGF transforming growth factor TNF tumor necrosis factor 3

10 ANOVA analysis of variance cdna complementary DNA DNA DILI drug-induced liver injury DNA deoxyribo nucleic acid ELISA enzyme-linked immunosorbent assay FP forward primer GAPDH glyceraldehyde 3-phosphate -3- dehydrogenase γ-gtp γ-glutamyltransferase γ- γ-gcs γ-glutamylcysteine synthetase γ- HE hematoxylin and eosi HRP horseradish peroxidase LD lethal dose mrna messenger RNA RNA ND not detected NS not significant NT non-treated PCR polymerase chain reaction PVDF polyvinylidene difluoride 2 RNA ribonucleic acid RP reverse primer RT reverse transcription SEM standard error of the mean 4

11 I (Qureshi et al, 211) (APAP) (idiosyncratic) (, 1999) 5

12 HLA II HLA (Aithal and Daly, 21) HLA HLA (Pirmohamed et al., 1996; Park et al., 2; Uetrecht, 2; Deng et al., 29) (DILI) (Wilke et al., 27) (1998 ) (2 ) (24 ) DILI (Fig. 1) DNA (GSH) in vitro (Gan et al., 25) in vivo GSH (LPS) (Shaw et al., 27; Tukov et al., 27; Lu et al., 212) LPS 6

13 GSH (Shimizu et al., 29; 211; Nishiya et al., 28) DILI DILI Fig. 1 DILI DILI ( ) DILI! P45 GSH!!!!!"# $!!!!!!! Fig. 1. Proposed mechanisms of drug-induced toxicity induced by immunemediated or metabolic idiosyncrasy. 7

14 DILI DILI DILI Balb/c (IL)-17 (Kobayashi et al., 29) Balb/c IL-4 (Kobayashi et al., 212) DILI C57BL/6 (Kobayashi et al., 29) cytochrome P45 (CYP) 2E1 Balb/c C57BL/6 (You et al., 26) DILI DILI DILI 8

15 (diphenylhydantoin, DPH) DPH (Dhar et al., 1974; Haruda, 1979; Taylor et al., 1984) DPH DPH 1-8 1, 1 (Mullick and Ishak, 198) DPH (Mullick and Ishak, 198) DPH (, 214) DPH DPH CYP2C9 CYP3A4 in vitro (Munns et al., 1997; Cuttle et al., 2) DPH CYP2C9 in vitro DPH CYP2C9 (Leeder et al., 1992) CYP DPH Fig. 2 9

16 O HO O DPH HN NH O UGT P45 HO HN Dihydrodiol O O NH HN DPH-Glu O N Glu O O Epoxide hydrolase HN NH Glu O HN 4 -HPPH O-Glu O O NH Arene oxide O P45 UGT O HO HN NH HO! HO! HN! O! NH! DPH catechol O! P45 spontaneous oxidation 4 -HPPH O O! O! HN! O! NH! DPH o-quinone O! Fig. 2. Major metabolic pathways of DPH in humans. Dihydrodiol 5-(3,4 -dihydroxy-1,5 -cyclohexadien-1-yl)-5- phenylhydantoin; UGT: UDP-glucuronosyltransferase; Glu: glucuronide; 4 -HPPH: 5-(4-hydroxyphenyl)-5-phenylhydantoin; DPH catechol, 5-(3,4 -dihydroxyphenyl)-5-phenylhydantoin; DPH o-quinone, 5-(3,4-dioxocyclohexa-1,5-dien-1-yl)-5-phenylimidazolidine-2,4-dione; DPH-Glu, DPH N-glucuronide; 4 -HPPH O-Glu, 4 HPPH O-glucuronide. 4 DPH (5-(4 -hydroxyphenyl)-5- phenylhydantoin, 4 -HPPH) CYP2C9 CYP2C11 (Komatsu et al., 2; Yamazaki et al., 21) 4 -HPPH 3,4 (5-{7-oxabicyclo[4.1.]hepta-2,4-dien-3-yl}-5-phenylimidazolidine-2,4-dione, 1

17 ) 5-(3,4-dihydroxycyclohexa-1,5-dien-1-yl)-5-phenylimidazolidine-2,4-dione ( ) 4 -HPPH 3 5-(3,4 -dihydroxyphenyl)-5-phenylhydantoin ( ) ( ) 5-(3,4-dioxocyclohexa-1,5-dien-1-yl)-5-phenylimidazolidine-2,4-dione (o- ) (Munns et al., 1997) 4 -HPPH CYP GSH (Munns et al., 1997; Roy and Snodgrass, 1988; 199) DPH 4 -HPPH N- O- N- glucuronide (DPH-Glu) 4 -HPPH O-glucuronide (4 -HPPH O-Glu) GSH GSH GSH (GST) 4 2 (Grover and Smis, 1964; Eoodhouse et al., 1983; Watanabe et al., 23) in vitro in vivo DPH 11

18 DPH DPH in vitro DILI DPH DPH DILI DILI GST GST-M1 GST-T1 (Ueda et al., 27; Watanabe et al., 23) GSH GSH DPH GSH DILI GSH DPH mrna DPH DPH CYP DPH DPH CYP CYP 12

19 II DPH 1 DPH (Dhar et al., 1974; Haruda et al., 1979) DPH CYP (Munns et al., 1997; Roy and Snodgrass, 1988) GSH Cys DPH GSH DPH GSH GSH DPH GSH DPH DPH 1-8 (Mullick and Ishak, 198) DPH DPH DPH in vivo DPH Kupffer T NK (Winnock et al., 1995; Tiegs et al., 1992) Kupffer (LPS) LPS 13

20 Kupffer (Bilzer et al., 26) Kupffer Kupffer high mobility group box protein1 (HMGB ) Kupffer (Bianchi et al., 27; Scaffidi et al., 22) Kupffer HMGB1 Kupffer Kupffer Toll (TLR) Kupffer (Schwabe et al., 26) TLR Kupffer (damage-associated molecular pattern molecules, DAMPs) DAMPs HMGB1 HMGB1 DILI APAP (Antonine et al., 29) NLR family, pyrin domain containing 3 (NALP3) NALP3 RNA ROS (Church et al., 28; Bryant and Fitzgerad, 29; Martinon et al., 29) 1 (Latz, 21; Schroder and Tschopp, 21) IL-1β IL-18 NALP3 DILI APAP (Williams et al., 211) IL-1β (Yano et al., 14

21 212) NALP3 DILI DILI DILI TLR NALP3 (Ramadori and Armbrust, 21) MCP-1 MIP-1 (Hsieh et al., 28) (Ramalah and Jaescke, 27) Kupffer APAP Kupffer (Fisher et al., 213) DILI DPH (Dhar et al., 1974; Haruda, 1979) DILI Kupffer T (Adams et al., 21) T CD4+CD8- T (Th) T Fig. 3 Th1, Th2, T (T-reg) Th17 (Kidd, 23; Zhu and Paul, 28) Th1 15

22 T-box expressed in T cels (T-bet) (IFN)-γ Th2 GATA 3 (GATA3) IL-4 IL-5 Th17 IL-17 IL-23 (ROR) γt T-reg IL-1 (TGF)-β Foxp3 DILI IFN-γ APAP (Ishida et al., 22) IL-4 (Higuchi et al., 211; Kobayashi et al., 212) IL-17 (Kobayashi et al., 29; Higuchi et al., 212) DILI Th DILI DAMPs Kupffer Th IL-17 Th17 IL-4 IL-5 Th2 GATA3 STAT6 ROR!t STAT3 IL-4 TGF-"# IL-6 Thp IL-12 T-bet STAT1 Th1 IFN-! TGF-" Foxp3 Treg IL-1 TGF-" Fig. 3. Differentiation mechanism of CD4+ Th cells subsets. 16

23 DPH in vitro GSH DPH GSH γ- (γ-gcs) L-buthionine (S,R)-sulfoximine (BSO) GSH DPH mrna mrna 2 II-2-1 DPH S- BSO ( ) 1-aminobenzotriazole (ABT) ( ) RNAiso SYBR Premix Ex Taq ( ) ReverTra Ace ( ) TLR4 E 1 (PGE 1 ) ( ) HMGB1 (Ig)Y ( ) IL-17 IgG2a R&D system (Minneapolis, MN, USA) Envision + for mouse (MPO) ( ) IL-1β IL-17 ELISA ebioscience (San Diego, CA, USA) Ready-SET-GO! Mouse 1L-1β IL-17 HMGB1 17

24 HMGB1 ELISA Kit ( ).9% 121 C 2 (DEPC) DEPC.1% 37 C C 2 1 (PBS) 8 g 2 g (12 ) 29 g 2 g 1 L 121 C PBS II C57BL/6J Balb/c SLC 12 (23 ± 2 C) (55 ± 2 C) C57BL/6J (1 ml/kg) DPH 5, 1 2 mg/kg DPH LD mg/kg (, 214) DPH 1 (1 ml/kg) 18

25 BSO (7 mg/kg) BSO (Shimizu et al., 29) DPH 24 ALT (AST) Dri-Chem 4 ( ) 5 Balb/c C57BL/6 (1 ml/kg) DPH 5 mg/kg 2 3 (1 ml/kg) DPH 1 mg/kg 3 DPH 1 (1 ml/kg) BSO (7 mg/kg) DPH BSO DPH (DPH BSO ) DPH S- (Zimmerman, 1999) BSO DPH DPH 4 DPH 6 DPH 5 (DPH ) DPH, 6, DPH 72 ALT AST (T.Bil) Dri-Chem 4-8 DPH GSH CYP DPH ABT 1 mg/kg DPH 1 ABT Shimizu (29) ABT (ABT 19

26 BSO) DPH (ABT DPH DPH/ABT DPH/BSO ) ALT DPH 24 ALT GSH -8 HMGB1 DPH HMGB1 (PBS) 2 µl/mouse HMGB1 IgY 2 µl/mouse TLR4 DPH 5 µg/mouse (2 µl/mouse) (Higuchi et al., 212) mrna IL-17 DPH 3 IL-17 1 mg/mouse PBS (2 µl/mouse) IgG2a PGE 1 PGE 1 (5 µl/mouse) DPH 3 5 mg/mouse HMGB1 IL-17 PGE 1 DPH 24 ALT II-2-3 GSH GSSG Tietze (1969) GSH GSSG 1 mg 5% 1 ml 8, g 4 C mm β- (NADPH) 14 µl 2

27 4.8 mm 5,5-2- (DTNB) 25 µl 5 2 µl GSSG 1 µl 2-2 µl 1 (4 Units/mL) 25 µl 5 45 nm Biotrak II plate reader (GE Healthcare) GSH GSSG II-2-4 Protein Carbonyl ELISA kit (Enzo LifeScience) 1 mg 3 (.1 M (Tris)-HCl,.1 M 1 mm (EDTA), ph 7.4) Protein Assay kit (Bio-Rad) (4 mg/ml) 5 µl 2,4- (DNP) 2 µl 45 2 enzyme immunoassay buffer 2 µl (ELISA) 96 4 C 12 (PBS) wash buffer 5 blocking solution 1 25 µl 3 PBS wash buffer 5 -DNP 1 2 µl 37 C 1 PBS wash buffer 5 (HRP) 1 2 µl 1 PBS wash buffer 5 chromatin reagent 1 2 µl 2 Stopping reagent 1 1 µl 45 nm Biotrak II plate reader 21

28 II-2-5 HMGB1 IL-1β IL-17 HMGB1 HMGB1 ELISA Kit IL-1β IL-17 Ready-SET-GO! Mouse IL-1β IL-17 not detected (ND) II-2-6 RNA RNAiso RNA 26 nm Nano Drop (Nano Drop Technologies) RNA RNA 4 µg 15 ng/ml 1 µl (DEPC) 23 µl ( ) µl 2.5 M dntps 8 µl ReverTra Ace (1 unit/µl) 1 µl 3 C 6 9 C 1 cdna II-2-7 PCR mrna Mx3P RT-PCR Table 1 cdna 1 µl 1 um (FP) (RP).8 µl SYBR Premix EX Taq buffer 1 µl ROX.25 µl 2 µl PCR FasL, Foxp3, Gapdh, IL-1β, IL-6, IL-23p19, NALP3, RAGE, S1A8, S1A9, TLR2, TLR4 TLR9 95 C 3 94 C 4 64 C T-bet 95 C 6 95 C C

29 MIP-2 MCP-1 GATA3 95 C 6 94 C 4 (GATA3 15 ) 62 C C ROR-γt 95 C 6 95 C C C PCR PCR 23

30 Table 1 Sequence of primers used for real-time RT-PCR analyses. Gene FasL Foxp3 Gapdh GATA3 IL-1β IL-6 IL-23 p19 MIP-2 MCP-1 NALP3 RAGE RORγt S1A8 S1A9 T-bet TLR2 TLR4 TLR9 FP RP FP RP FP RP FP RP FP RP FP RP FR RP FP RP FP RP FP RP FP RP FP RP FP RP FP RP FP RP FP RP FP RP FP RP Sequence AGA AGG AAC TGG CAG AAC TC GCG GTT CCA TAT GTG TCT TC CTA GCA GTC CAC TTC ACC AAG GCT GCT GAG ATG TGA CTG TC AAA TGG GGT GAG GCC GGT ATT GCT GAC AAT CTT GAG TGA GGA GGA CTT CCC CAA GAG CA CAT GCT GGA AGG GTG GTG A GTT GAC GGA CCC CAA AAG AT CAC ACA CCA GCA GGT TAT CA CCA TAG CTA CCT GGA GTA CA GGA AAT TGG GGT AGG AAG GA CCA GTG TGA AGA TGG TTG TG CTA GTA GGG AGG TGT GAA GT AAG TTT GCC TTG ACC CTG AAG ATC AGG TAC GAT CCA GGC TTC TGT CAT GCT TCT GGG CTT G CCT CTC TCT TGA GCT TGG TG GTT GAC GGA CCC CAA AAG AT CAC ACA CCA GCA GGT TAT CA GTG CTG GTT CTT GCT CTA TG ATC GAC AAT TCC AGT GGC TG ACC TCC ACT GCC AGC TGT GTG CTG TC TCA TTT CTG CAC TTC TGC ATG TAG ACT GTC CC GAG TGT CCT CAG TTT GTG CAG TAG ACA TAT CCA GGG ACCCAG GAT GGC CAA CAA AGC ACC TT CCT CAA AGC TCA GCT GAT TG TGC CCG AAC TAC AGT CAG GAA C AGT GAC CTC GCC TGG TGA AAT G GAA AAG ATG TCG TTC AAG GAG TTG CTG AAG AGG ACT GTT ATG TTC TTC TCC TGC CTG ACA CC CCA TGC CAT GCC TTG TCT TC ATT CTC TGC CGC CCA GTT TGT C ACG GTT GGA GAT CAA GGA GAG G FP: Forward primer, RP: Reverse primer. II-2-8 DPH 24 4 µm 1% ( ) 24

31 (HE) MPO DPH 24 4 µm.1% (w/v) 37 C 1 3% H 2 O 2 15 MPO 37 C 3 Envision + for mouse (DAKO) 4 H 2 O 2 (DAB) 3 MPO II Student s t-test (ANOVA) Tukey P <.5 3 II-3-1 DPH 24 ALT AST DPH/BSO 24 ALT Fig. 4A DPH 1 2 mg/kg DPH ALT II-3-2 DPH 5 ALT DPH/BSO ALT DPH 4 ( U/l) DPH

32 (P <.1) ( U/l) (Fig. 4B) ALT DPH DPH/BSO DPH % 5% 7% DPH DPH/BSO ALT DPH/BSO ( U/l) ALT DPH 24 ALT, AST T.Bil Fig. 4B DPH/BSO BSO DPH 24 Fig. 4C DPH DPH/BSO Balb/c DPH ALT (Fig. 4D) DPH DPH S- Fig. 4E S- /BSO ALT, AST T-Bill (Fig. 4E) DPH 26

33 (A) Single administration (B) mg/kg, p.o. mg/kg, i.p. mg/kg, p.o. mg/kg, i.p. ** (Day) 7 ALT 3 Mephenytoin ALT (U/l) (U/l) DPH +BSO DPH + BSO BSO DPH AST (E) ALT *** *** 1.5 ** (D) CTL BSO DPH+BSO 2 * ** (C) ** 8 ** T.Bil T-Bil 2 1 ** CTL BSO DPH DPH + BSO (mg/dl) (U/l) ALT (U/l) 4 ALT (U/l) C57BL/6 Balb/c Time from the last administration (h) DPH + BSO Fig. 4. Time-dependent changes in plasma ALT and AST levels in DPH-induced liver injury. (A) In a single administration experiment, mice were given an oral dose of DPH at 2 or 1 mg/kg and an intraperitoneal injection of 1 or 5 mg/kg. BSO (7 mg/kg) was intraperitoneally injected 1 h prior to the DPH treatment, and blood was collected 24 h after DPH administration. Values represent the mean ± SEM of four animals. (B) Female C57BL/6 mice were intraperitoneally given DPH at 5 mg/kg for 2 days, and afterwards on days 3-5, DPH was orally given at 1 mg/kg. BSO (7 mg/kg) was intraperitoneally injected 1 h prior to each DPH administration. Each vehicle was used as a control. At and 6 h after DPH administration on days 1-4 and at, 3, 6, 24, 48, and 72 h after the final DPH treatment, blood was collected to measure plasma ALT levels. The days 6, 7, and 8 correspond to 24, 48, and 72 h after the final DPH treatment. In the second panels the plasma ALT, AST and T-Bil levels were measured 24 h after the final DPH treatment. Values represent the mean ± SEM of four to six animals. (C) Liver tissue sections from 24 h after the final DPH treatment were stained with H&E. (D) Female Balb/c mice were treated with DPH and BSO described in (B). (E) As the negative control, mice were given mephenytoin and BSO on the same dosing regimen as in (B). At 24 h before (-24) and, 6, and 24 h after the final mephenytoin treatment, blood was collected to measure plasma ALT levels (E). Values represent the mean ± SEM of four animals. Values represent the mean ± SEM of four to five specimens. The differences relative to the BSO-treated mice (ALT, AST and T-Bil) were considered significant at *p <.5, **p <.1, and *** p <.1. II-3-3 DPH および BSO 投与が肝臓中 GSH および GSSG 含量に及ぼす影響 DPH 最終投与後から肝臓中 GSH および GSSG 含量を経時的に測定した (Fig. 5A) GSH 含量は BSO 単独投与群では投与から 時間後にかけて溶媒対照群と比較して有意な低 27

34 DPH DPH GSH DPH 3-6 DPH/BSO DPH -24 GSSG DPH/BSO BSO BSO 2.5 GSH (GSH + GSSG) GSH DPH BSO DPH BSO GSH GSSG II-3-4 DPH DPH -24 (Fig. 5B) DPH/BSO DPH 6 II-3-5 CYP DPH DPH 24 ALT AST (Fig. 5C) ABT ALT AST DPH II-3-2 ALT DPH/BSO ABT ABT 28

35 II-3-6 CYP GSH DPH 24 GSH (Fig. 5D) II-3-3 DPH DPH/BSO GSH DPH/BSO ABT ABT GSH 29

36 (U/l) (A) ( µmol/g tissue) (B) (nmol/mg protein) (C) *** * ** ALT DPH CTL! ABT! DPH + DPH + DPH + DPH + +BSO +BSO BSO!!! +! +ABT+ ABT! +!! + + GSH * *** ** ** * ** ** * (U/l) * *** (h) Protein carbonyls * (h) CTL BSO DPH DPH+BSO ( µ mol/g tissue) CTL DPH DPH+BSO * AST * *** GSSG *** CTL ABT 1 DPH 2 DPH 3 DPH 4 DPH 5!! BSO +BSO!!! +! +ABT+! +!! + + * (D) Fig. 5. Time-dependent changes in hepatic GSH, GSSG, and oxidative stress marker in DPH-induced liver injury. Mice were intraperitoneally given DPH at 5 mg/kg for 2 days, and afterwards on days 3-5, DPH was orally dosed at 1 mg/kg. BSO was intraperitoneally injected 1 h prior to each DPH administration. Each vehicle was used as a control. At, 1.5, 3, 6, 12, and 24 h after the final DPH treatment, the liver was collected to measure the hepatic GSH, GSSG, and GSH+GSSG levels, the GSH/GSSG ratio (A), and its level of hepatic protein carbonyls (B). ABT (1 mg/kg), a non-specific inhibitor of P45, was intraperitoneally given 1 h prior to the final DPH treatment. Each vehicle was used as a control. At 24 h after the final DPH treatment, the liver and plasma were collected to measure plasma ALT and AST levels (C), hepatic GSH and GSH+GSSG levels, and the GSH/GSSG ratio (D). The data are shown as the mean ± SEM of the results from four mice. The differences relative to the control mice were considered significant at *p <.5, **p <.1, and *** p <.1, and the differences between ABT-treated and vehicle-treated mice were considered significant at p <.5, p <.1. * (µmol/g tissue) *** (h) ( µ mol/g tissue) N.S GSH ** *** N.S. *** CTL! ABT! DPH + DPH + DPH + DPH + +BSO +BSO!!! +! +ABT+ GSH+GSSG ** * * *** ** ** (h)! +!! + + * * ** (µmol/g tissue) * ** ** *** GSH+GSSG ** *** ***!! CTL ABT DPH DPH DPH DPH +BSO +BSO!!! +! +ABT+! +!! + + II-3-7 TLRs RAGE S1A8/9 mrna 3

37 Fig. 6A DPH/BSO TLR9 DPH 3 12 TLR4 S1A8/9 DPH 1.5 S1A8 S1A9 DPH 12 RAGE DPH mrna DPH/BSO RAGE DPH/BSO BSO mrna TLRs NALP3 NALP3 IL-1β mrna (Fig. 6B) DPH/BSO DPH 1.5 NALP3 IL-1β mrna DPH DPH/BSO BSO mrna IL-1β 1 IL-1β IL-1β mrna IL-1β DPH DPH/BSO IL-1β (Fig. 6C) DPH/BSO DPH 3 DPH 4 (DPH h) DPH 3 IL-1β DPH/BSO 31

38 (A) Relative expression Relative expression (B) Relative expression! * TLR2 CTL BSO DPH DPH+BSO * ** (h) (h) 3! 2! 1!! S1A8 * NALP3! **! -24!! 1.5! 3! 6! 12! 24! (h)! * TLR4 * (h) ! 5! 4! 3! 2! 1!! (C) Fig. 6. Time-dependent changes in the hepatic mrna expression levels of DAMP related genes, NALP3 and IL-1. (A and B) Mice were intraperitoneally given DPH at 5 mg/kg for 2 days, and afterwards on days 3-5, DPH was orally dosed at 1 mg/kg. BSO was intraperitoneally injected 1 h prior to each DPH administration. Each vehicle was used as a control. At 24 h before (-24) and, 1.5, 3, 6, 12, and 24 h after the final DPH treatment, liver and plasma samples were collected to measure the expression of both the hepatic mrnas of DAMP-related genes NALP3 and IL-1. The expression levels of hepatic mrnas were normalized to that of Gapdh. The data are shown as the mean ± SEM. The results of the time-dependent study are taken from three to five mice, and the other data are taken from four to five mice. The differences relative to the control mice were considered significant at *p <.5, **p <.1, and *** p <.1. * ** S1A9 ** * * ** (h) IL-1!! ***! -24!! 1.5! 3! 6! 12! 24! (h)! (h) (pg/ml) TLR9 ** ** RAGE * IL-1! ** (h) DPH DPH+BSO (h) 32

39 II-3-8 HMGB1 HMGB1 TLR4 DPH DPH DPH/BSO HMGB1 (Fig. 7A) DPH/BSO DPH 3-24 DPH DPH/BSO HMGB1 DPH 24 ALT AST (Fig. 7B) IgY ALT DPH/BSO TLR4 DPH 24 ALT AST (Fig. 7C) 33

40 (A) (ng/ml) (C) (U/l) DPH DPH+BSO HMGB1 protein (h) DPH +BSO +Vehicle ALT DPH +BSO +Eritoran (U/l) (U/l) (B) DPH +BSO DPH +BSO +Vehicle ALT DPH DPH +BSO +BSO +IgY +Anti- HMGB1 AST DPH +BSO +Eritoran (U/l) DPH +BSO AST DPH DPH +BSO +BSO +IgY +Anti- HMGB1 Fig. 7. Time-dependent changes of plasma HMGB1 protein levels, and results of the neutralization studies in DPH-induced liver injury. (A) Mice were intraperitoneally given DPH at 5 mg/kg for 2 days, and afterwards on days 3-5, DPH was orally dosed at 1 mg/kg. BSO was intraperitoneally injected 1 h prior to each DPH administration. Each vehicle was used as a control. At 24 h before (-24) and, 1.5, 3, 6, 12, and 24 h after the final DPH treatment, plasma samples were collected to measure the plasma HMGB1 protein. (B) The effect of an anti-hmgb1-antibody. Mice were intravenously administered the anti-mouse HMGB1-antibody (2 g anti-hmgb1-antibody in.2 ml sterile PBS) simultaneously with the final DPH treatment. Blood was collected 24 h after the final DPH treatment. (C) The effect of eritoran, a TLR4 antagonist, on DPH-induced liver injury. Mice were intravenously given eritoran (5 g/mouse in.2 ml sterile saline) simultaneously with the final DPH treatment. Blood was collected 24 h after the final DPH treatment. The data are shown as the mean ± SEM. The results of the time-dependent study are taken from three to five mice, and the other data are taken from four to five mice. The differences relative to the control mice were compared to the -24 h mice were considered significant at p <.1 in ELISA; and compared to isotype IgY or vehicle-treated mice were considered significant at p <.5 and p <.1 in neutralization or antagonist studies, respectively. II-3-9 Th mrna Th mrna Fig. 8A T-bet DPH/BSO DPH DPH/BSO 34

41 DPH 24 GATA3 DPH/BSO DPH 6 24 DPH DPH/BSO DPH 3 RORγt DPH/BSO DPH 1.5 DPH mrna FoxP3 DPH/BSO DPH DPH DPH 3 IL-23p19 DPH/BSO DPH 12 (Fig. 8B) DPH DPH IL-6 DPH/BSO DPH DPH (Fig. 8B) FasL DPH/BSO DPH -24 DPH DPH/BSO DPH MIP-2 DPH/BSO DPH 6-24 (Fig. 8C) DPH DPH 3, MCP-1 DPH/BSO DPH 24 DPH mrna BSO 35

42 (A) 2! T-bet! CTL BSO DPH DPH+BSO 2! GATA3! 5! ROR!!t! 2! Foxp3! Relative expression! 1.5! 1!.5!! (B) Relative expression! 5! 4! 3! 2! 1! **! *! *! *! **! **! -24!! 1.5! 3! 6! 12! 24! (h)! IL-23 p19! *! 1.5! 3! 1! 2! 1!.5!! 7! 6! 5! 4! 3! 2! 1! *! **! *! -24!! 1.5! 3! 6! 12! 24! (h)! IL-6! *! *! *! *! 4! 1!! 2.5! 2! 1.5! 1!.5! **! -24!! 1.5! 3! 6! 12! 24! (h)! FasL! *! ***! *! *! **! *! **! **! 1.5!.5!! *! **! *! *! ***! *! -24!! 1.5! 3! 6! 12! 24! (h)!! (C) Relative expression! 16! 14! 12! 1! 8! 6! 4! 2!! -24!! 1.5! 3! 6! 12! 24! (h)! MIP-2! **! ***! *!! 2! 15! 1! 5!! -24!! 1.5! 3! 6! 12! 24! (h)! MCP-1! -24!! 1.5! 3! 6! 12! 24! -24!! 1.5! 3! 6! 12! 24! (h)! (h)! Fig. 8. Time-dependent changes of hepatic mrna expression levels of proinflammatory cytokines and chemokines in DPH-induced liver injury. (A - C) The experimental conditions for DPH treatments and blood and liver collection were the same as those in Fig. 6. The data are shown as the mean ± SEM. The results of the time-dependent study are taken from three to five mice, and the results from the neutralization study are taken from four to five mice. The differences relative to the control mice were considered significant at *p <.5, **p <.1, and *** p <.1.! -24!! 1.5! 3! 6! 12! 24! (h)! II-3-1 IL-17 IL-17 DPH DPH DPH/BSO IL-17 Fig. 9A DPH/BSO DPH 6-24 DPH 36

43 IL-17 DPH/BSO DPH 24 ALT AST IgG (Fig. 9B) IgG II-3-11 MPO DPH 24 MPO (Fig. 9C) DPH DPH/BSO MPO BSO (Fig. 9C) DPH DPH/BSO 37

44 (pg/ml) (A) (h) (C) 25 IL-17 MPO-staining DPH DPH+BSO (U/l) (B) DPH +BSO ALT DPH +BSO +IgG DPH +BSO +Anti- IL-17 (U/l) DPH +BSO AST DPH +BSO +IgG DPH +BSO +Anti- IL-17 MPO-positive cells ** *** *** CTL DPH DPH +BSO Fig. 9. A neutralization study of IL-17 and assessment of neutrohil infiltration in DPH-induced liver injury. (A) The experimental conditions for DPH treatments and blood collection were the same as those in Fig. 6. (B) For the neutralization study, mice were intravenously injected with an anti-mouse IL-17 antibody (1 g anti-mouse IL-17 antibody in.2 ml of sterile PBS) 3 h after the final DPH treatment. As a control, the IgG isotype was used. At 24 h after the final DPH treatment, plasma was collected to measure its ALT and AST levels. (C) Neutrophil infiltration was assessed by immunostaining for MPO. Data indicate the number of MPO-positive cells in mice treated with DPH and BSO, DPH alone, or vehicle. The data are shown as the mean ± SEM. The results of the time-dependent study are taken from three to five mice, and the results from the neutralization study are taken from four to five mice. The differences relative to the control mice were considered significant p <.5 in ELISA; and those compared to isotype IgG-treated mice were considered significant at p <.5 in neutralization study, and the differences relative to the control mice (MPO-stained) were considered significant at **p <.1, and *** p <.1. II-3-12 PGE 1 DPH PGE 1 (Talpain et al., 1995) (Kobayashi et al., 29; Higuchi et al., 212) DPH 38

45 Fig. 9C で示されたことから PGE1 処置により肝障害が緩和されるかどうか 血漿中 AST および ALT 値ならびに好中球遊走ならびに浸潤に関与するケモカインである MIP-2 および MCP-1 mrna 発現量を評価した その結果 PGE1 投与により DPH 最終投与 24 時間後の血漿中 AST および ALT 値が溶媒対照群と比較して有意な低値を示すことを明らかにした (Fig. 1A) また DPH 最終投与 24 時間後の MIP-2 および MCP-1 mrna 発現量は溶媒対照群 (CTL) と 同等の低値を示した (Fig. 1B) PGE1 が DPH/BSO 投与マウスの肝臓における好中球浸潤に 与える影響を検討するため PGE1 併用処置マウスの DPH 最終投与 24 時間後の肝臓を用いて MPO 陽性細胞を計測した その結果 DPH/BSO 投与マウスの MPO 陽性細胞数は溶媒処置 群と比較しては有意な低値を示した (Fig. 1C) ALT MPO-staining 12 (U/l) (U/l) 12 (C) AST 8 * 4 8 ** 4 DPH+BSO+PGE1 (B) DPH +BSO +Vehicle MIP-2 1 Rerative expression DPH +BSO +PGE1 DPH +BSO +Vehicle 15 1 * 5 DPH +BSO +PGE1 MPO-staining DPH +BSO +Vehicle DPH +BSO +PGE1 MCP MPO-positive cells (A) DPH+BSO+Vehicle * CTL DPH DPH +BSO +BSO +Vehicle +PGE1 1 CTL DPH DPH +BSO +BSO +Vehicle +PGE1 Fig. 1. Effects of PGE1 on DPH-induced liver injury. The experimental conditions for DPH administration were the same as those in Fig. 4. Mice were intraperitoneally injected with PGE1 (5 µg/mouse, dissolved in.5 ml sterile saline) 3 h after the final DPH treatment. Each vehicle was used as a control. At 24 h after the final DPH treatment, the plasma and liver were collected to measure ALT and AST levels (A), hepatic mrna levels of MIP-2 and MCP-1 (B) and for immunohistochemistry (C). The expression level of hepatic mrna was normalized to that of Gapdh. Mononuclear cell infiltration was assessed by immunostaining for MPO. The number of MPO-positive cells in DPH and BSO-treated or DPH, BSO, and PGE1-treated mice is shown in (C). The arrows indicate MPO-positive cells. The data are shown as the mean ± SEM of the results from four to five mice. The differences compared to the DPH-, BSOand vehicle-treated mice were considered significant at *p <.5 and **p <.1. 39

46 4 DPH LD mg/kg DPH 2 mg/kg ALT (214) DPH 1 mg/kg 2 GSH BSO DPH C57BL/6 BSO GSH γ-gcs in vivo GSH (Griffith and Meister, 1979; 1982) BSO CYP (UGT) (Drew and Miners, 1984; Watanabe et al., 23) BSO DPH 2 mg/kg ALT (Fig. 4A) GSH DPH DPH 1-8 DPH (Mullick and Ishak, 198) DILI DILI BSO DPH 5 DPH 5 (Figs. 4B and 4C) DPH 5 DPH/BSO ALT (Figs. 4B and 4C) GSH DPH 4

47 DPH DPH 5 DPH 5 1 mg/kg ALT (15-2 U/l) ALT DPH 24 75% DPH 2-3 DPH CYP (Hagemeyer et al., 21) CYP mg/kg DPH DPH (Lolin et al., 1994; Burstein et al., 1999) DPH 1-2 ALT 7 U/l 25% GSH APAP N- p- (NAPQI) S- (Zhou et al., 25) GSH GST (Grover and Smis, 1964; Eoodhouse et al., 1983; Watanabe et al., 23) APAP BSO DILI 41

48 (Nishiya et al., 28; Shimizu et al., 29;211; Kobayashi et al., 212) (Lauterburg and Veltez, 1988) (Hernanz et al., 2) (Staal et al., 1992) GSH GSH γ-gcs (Nakamura et al., 22) GSH DPH DILI BSO DPH BSO ALT DPH BSO (Figs. 4A and 4B) DPH GSH DILI (Masubuchi et al., 29; Welch et al., 26; You et al., 26) Balb/c BSO DPH 5 ALT (Fig. 4D) Balb/c (Kobayashi et al., 29; Yano et al., 212; Higuchi et al., 212; Matsuo et al., 214) C57BL/6 APAP Balb/c C57BL/6 (Kobayashi et al., 29) APAP (Masubushi et al., 29) DPH C57BL/6 DPH 42

49 DPH GSH DPH GSH (GSH + GSSG) (Fig. 5A) GSH GSH/GSSG GSH GSH BSO DPH GSH (DPH 3-6 ) GSH (Fig. 5A) BSO GSH BSO GSH Fig. 4A (Fig. 4B) DPH CYP (Munns et al., 1997; Cuttleet al., 2; Roy and Snodgrass, 1988;199) DPH CYP ABT DPH/BSO DPH ALT (Fig. 5C) GSH DPH CYP CYP ABT DPH GSH ABT DPH GSH (Fig. 5D) CYP GSH (BSO ) DPH (Figs. 5C and 5D) CYP DPH GSH 43

50 (Takakusa et al., 28) / DILI APAP NF-E2 (Nrf2) (Goldring et al., 24) ROS H 2 O 2 (SOD) DILI APAP SOD2 (Fujimoto et al., 29; Ong et al., 26) DPH/BSO (Fig. 5B) GSH ROS SOD GSH SOD2 (Fujimoto et al., 29; Ong et al., 26) SOD2 DILI SOD ROS Kupffer LPS RNA CpG DNA TLR (Bianchi, 27) NF-κB (Schwabe et al., 26; Kita et al., 21; Oo and Adams, 21) RAGE (AGE) TLRs 44

51 (Zeng et al., 29) TLRs mrna S1A8/9 mrna (Fig. 6A) TLR RAGE TLRs mrna TLRs Kupffer mrna mrna TLRs mrna TLRs HMGB1 (Schwabe et al., 26) HMGB1 APAP (Antoine et al., 29; Higuchi et al., 212) DPH/BSO HMGB1 (Fig. 7A) HMGB1 ALT AST (Fig. 7B) HMGB1 DPH HMGB1 S1A8/9 DAMPs TLRs TLR4 ALT AST (Fig. 7C) HMGB1 S1A8/9 DAMPs TLR4 IL-6 TNF-α IL-1β Yano (212) IL-1β IL-1β NALP3 APAP (Williams et al., 211) TLRs NALP3 45

52 NALP3 RNA ROS (Bryant and fitzgerad, 29; Martinon et al., 29) DILI ROS NALP3 Fig. 4B ROS DPH NALP3 (Figs. 6B and C) DAMPs S1A8/9 mrna HMGB1 ALT (Figs. 6A-C and 7A) TLR4 eritoran (LPS) A (Mullarkey et al., 23) DPH APAP TLR4 (Kupffer ) IL-6 IL-1β IL-6 IL-23 Th (Kita et al., 21; Oo and Adams, 21) Fig. 5 Th Th17 RORγt mrna (Fig. 8A) Th1, 2 Treg T-bet, GATA3 FoxP3 mrna (Fig. 8A) DPH Th17 Th1 FasL TNF-α Th2 46

53 IL-4 IL-5 Treg IL-1 (Zhu and Paul, 28) Th17 IL-17 IL-17 MIP-2 MCP-1 IL-23 IL-6 (Zhu and Paul, 28) MPO (Jaescke, 26) MPO (Fig. 9C) DPH Th17 DPH IL-17 IL-17 (Fig. 9B) Th17 IL-17 IL-17 IL-1β IL-17 (Latz, 21) IL-17 IL-17 DPH (Fig. 9B) TLR4 HMGB1 (Figs 7B and C) IL-17 HMGB1 (Figs. 7B, C and 9B) Th17 47

54 PGE 1 (Kobayashi et al., 29; Higuchi et al., 212) PGE 1 PGE 1 AST ALT (Fig. 1A) MPO (Fig. 1C) MIP-2 MCP-1 (Fig. 1B) PGE 1 PGE 1 DPH DPH TLR4 Th17 48

55 III DPH CYP 1 II GSH BSO DPH 5 DPH DILI (Zhou et al., 25) APAP (Adams et al., 21, Fig. 11) DPH DPH CYP2C CYP3A (Chaudhry et al., 21; Fleishaker et al., 1995) CYP DILI CYP 49

56 Neutrophil TNF-!" IL-1#" MIP-2 etc. Kupffer cell Y TLRs HMGB1 S1A8/9 etc. H 2 O 2 HOCl Cell Necrosis Hepatocytes Reactive metabolite APAP Fig. 11. Mechanisms of APAP-induced liver injury. This figure was created by modification of a report by Adams et al (21). DPH in vitro (Komatsu et al., 2; Yamazaki et al., 21) DPH (Fig. 2) 4 -HPPH CYP2C9 CYP2C11 4 -HPPH CYP2C9 CYP3A4 o- (Munns et al., 1997) 4 -HPPH (Roy and Snodgrass, 1988; 199) H 2 O DPH CYP (Munns et al., 1997; Leeder et al., 1992; Cuttle et al., 2) CYP2C9 CYP3A4 DPH CYP2C9 (Leeders et al., 5

57 1992) DPH CYP Cys NAC GSH (Munns et al., 1997; Roy and Snodgrass, 1988;199) II DPH BSO (Fig. 4B) DPH GSH GSH GSH in vitro (Gan et al., 25) DPH GSH (DEM) DPH DEM (Roy and Snodgrasss, 1988) CYP DPH GSH (Munns et al., 1997) GSH DPH GSH in vitro GSH DPH DPH DPH DPH CYP DPH DPH 1-8 CYP APAP APAP CYP2E1 CYP3A CYP APAP 51

58 (Kostrubsky et al., 1997; Sinclair et al., 1998) APAP NAPQI CYP DILI DPH CYP CYP DPH 2 III-2-1 ABT, BSO DPH I-2-2 LKT Laboratories (St. Paul, MN, USA) 6 (G6P) 6 (G6PDH) β- (NADP + ) ( ) Cyp3a (sc-3621) (Santa Cruz, CA) CYP2C13 ( Cyp2c ) ( ) (PVDF) -P ( ) ( ) HPLC III-2-2 II-2-3 GSH II-2-4 DPH 5 DPH 3 PE-5 DPH 6 Cys NAC II 52

59 DPH/BSO DPH 24 Cyp DPH Day DPH Day1, 2 4 II C KTZ DPH 1 KTZ 5 mg/kg (1 ml/kg) KTZ (Soo et al., 21) DPH 24 II-2-4 ALT GSH DPH KTZ 1 Cyp PB CYP PB 8 mg/kg (1 ml/kg) 4 BSO 7 mg/kg (1 ml/kg ) BSO 1 DPH 1 mg/kg (1 ml/kg) DPH 6 24 ALT PB CYP PB 4 III-2-3 DPH GSH Cys NAC III µl 13, g 3 DPH 2 µl 4 µl 13, g 3 - (LC-MS/MS) HP12 (HPLC, Agilent Technologies, Santa Clara, CA, USA) Q-TOF Ultima (Micromass, Manchester, UK) Sun Shell 2.6 µm, 15 mm 2.1 mm I.D. ( ) 1 53

60 mm (95-65% % % 1 95% 7.9 ).2 ml/min 12 C 2.8 kv 3.2 kv 5 ev 35 V 25 C 8 l/h 1 l/h Q-TOFMS Metabolynx (Waters, Milford, MA, USA)) MS/MS 1-4 ev MS/MS III-2-4 DPH DPH 5 µl 2 µl 2 ul (1 nm ) 13, g 3 - (LC-MS/MS) Acquity UPLC (Waters) MS/MS Waters XevoTQ (Waters) Acquity UPLC BEH C18 (1.7 mm, 3 mm 1.1 mm, I.D., Waters) DPH, 4 -HPPH, Cys-DPH NAC-DPH 1 mm / (98%.5 8% %.4 2%.5 98%.5 ).5 ml/min DPH DPH-Glu 4 -HPPH O-Glu (MRM) m/z Table 2 Xevo TQ MS.5 kv, 15 C 6 C 1 l/h 12 l/h 54

61 .15 ml/min DPH 4 -HPPH ( ) DPH 4 -HPPH MS/MS DPH 4 -HPPH MS/MS Table 2 MS/MS conditions for the Xevo TQ MS instrument. Compound Mass MRM transition Ion mode (ES) CV (V) CE (ev) DPH 4 -HPPH DPH-Glu 4 -HPPH O-Glu Dihydrodiol Cys-DPH NAC-DPH IS (verapamil) > > > > > > > > 165. positive positive negative negative negative negative negative positive Abbreviations: CE, collision energy; CV, cone voltage; Cys-, cysteine-conjugated; DPH, phenytoin; ES, electrospray source; Glu, glucuronide; IS, internal standard; MRM, multiple reaction monitoring; NAC-, N-acetylcysteine-conjugated III-2-5 Emoto (2) γ- (Bradford, 1976) (MDZ) (TOL) Emoto (2) III-2-6 SDS- Laemmli (197) SDS 55

62 Cyp2c Cyp3a 1 µg 4 µg 7.5% SDS- Immobilon P PVDF Odyssey blocking buffer (Li-COR Biosciences, Lincoln, NE, USA) ( Cyp2c Cyp3a ) (IRDye 68 IgG, IRDye 68 IgG Li-COR Biosciences) Odyssey Infrared Imaging system (Li-COR Biosciences) III-2-7 II III-3-1 DPH DPH (DPH ) DPH 3-6 LC-MS/MS 129 u GSH 9.87 (Fig. 12A) MS Fig. 13A DPH [M-H] - m/z 556 m/z 128 GSH C-S m/z 272 GSH m/z 383 DPH DPH GSH DPH 56

63 III-3-2 DPH DPH (DPH/BSO ) DPH 24 GSH 129 u (Baillie and Davis, 1993) (data not shown) GSH γ- GSH (Meister and Anderson, 1983) GSH N- NAC DPH m/z 251 Cys (121 u) NAC (163 u) m/z selected reaction monitoring (SRM) (Figs. 12B and 12C) Cys m/z 37 MS/MS C-S m/z 283 Cys S m/z 19 (Fig. 13B) NAC m/z 413 MS/MS NAC C-S m/z 283 NAC m/z 24 NAC S m/z 19 (Fig. 13C) 3 GSH NAC DPH DPH Cys NAC (Fig. 14) 57

64 (A) GS-DPH (9.87 min) % of maximum count Elution time (min) (B) Cys-DPH (9.7 min) Elution time (min) (C) NAC-DPH (1.76 min) Elution time (min) Fig. 12. LC-MS/MS analysis of GS-DPH adducts in bile (A), and Cys-DPH (B) and NAC-DPH adducts (C) in plasma of the DPH-induced liver injury mice. MS spectrum of GS-DPH, Cys-DPH and NAC-DPH at 9.87 min, 9.7 min and 1.76 min of the elution time, respectively. 58

65 (A) 143 H 2N COOH % of maximum count O HN S O O HN NH NH O COOH 383, 254 (-C 5 H 7 NO 3 ) 556 -H m/z (B) 24 m/z (u) % of maximum count H 2 N S O HN OH 19 (-C 3 H 6 NO 2 ) 37 m/z 37 O NH O -H (C) 24 m/z (u) % of maximum count H 3 C 283 O HN S O HN OH 24, 19 (-C 5 H 8 NO 3 ) 413 m/z 413 O -H O NH 19 m/z (u) Fig. 13. LC-MS/MS analysis of GS-DPH in bile, and Cys-DPH and NAC-DPH adducts in plasma of the DPH-induced liver injury mice. Mice were intraperitoneally injected DPH at a dose of 5 mg/kg for 2 days and then orally administered at a dose of 3 days. The plasma was collected at 24 h after the final DPH administration. The bile was collected at 3 to 6 h after the final DPH administration. The bile pooled of 4 mice was used for the LC-MS/MS analysis. MS spectrum of the parent M - ion m/z 556, m/z 413 and m/z 37 were observed at 9.8, 1.7 and 9.7 min of the elution time, respectively. Product ion spectrum of the each of parent M - ion was detected in the bile (A) and plasma (B and C). 59

66 DPH-Glu HO O HO HN NH Dihydrodiol O HN N Glu O O HO 4 -HPPH HN NH Ugt O Ugt O P45 HN NH Glu O DPH O HN 4 -HPPH O-Glu P45 GSH -H 2 O GS O meh HN NH O O O O HN NH GS-DPH O O NH Arene oxide O H 2 N S O H 3 C HN O S O O OH Cys-DPH HN O NH O OH HN NH NAC-DPH O Fig. 14. Proposed metabolic pathways of DPH in mice with DPH-induced liver injury. III-3-3 DPH Day 3 Day 5 DPH Fig. 15 DPH Day 3 Day 5 DPH 3-6 Day 5 DPH Day 3 4 -HPPH Day 3 Day 5 DPH 24 Day 5 Day 3 Day 3 Day 5 4 -HPPH O-Glu Day 3 6

67 Day 5 DPH-Glu Day 3 Day 5 NAC-DPH Cys-DPH Day 3 Day 5 Day 3 Day 5 DPH Concentraion (µg/ml) Area/IS area DPH ** Day 5 Day 3 ** (ng/ml) (h) Time Time after after the DPH administration '-HPPH-O-Glu 2 Day 3: N.D (h) Area/IS area Time after the DPH administration x x1 4 1x1 4 4'-HPPH (h) ** ** * DPH-Glu *** (h) * *** Area/IS area Area/IS area 25 2 Dihydrodiol *** *** 15 *** 1 *** (h) 2 25 NAC-DPH Day 3: N.D (h) Area/IS area Cys-DPH Day 3: N.D (h) Fig. 15. Changes in the plasma concentration of DPH and its metabolites in DPH-induced liver injury mice. Mice were intraperitoneally injected DPH at a dose of 5 mg/kg for 2 days and then orally administered at a dose of 3 days. BSO was intraperitoneally injected at a dose of 7 mg/kg at 1 h prior to the each of DPH administration. At, 1.5 3, 6, 12, and 24 h after the 2 days intraperitoneal DPH administration (indicated as day 3) and after the final DPH administration (indicated as day 5), the blood was collected for assessment of DPH and its metabolites in plasma. The data are shown as the means ± SEM of the results from 6 to 12 mice. Differences compared with mice on the day 3 in each time points were considered significant at **P <.1 and ***P <.1. III-3-4 ABT ALT GSH ABT DPH/BSO DPH ALT (Fig. 16A) ABT ALT ABT DPH 24 61

68 ABT DPH DPH 24 GSH Fig. 16B ABT DPH/BSO CYP III-3-5 ABT DPH ABT DPH DPH (Fig. 16C) DPH ABT DPH 3-24 ABT 4 -HPPH ABT DPH 3-24 ABT 6-24 DPH-Glu ABT 4 -HPPH O-Glu DPH 24 ABT ABT Cys-DPH NAC-DPH DPH 24 ABT CYP DPH DPH 62

69 (U/l) (A) ABT -ABT ALT (h) Time Time after the last DPH administration (C) (ng/ml) Area/IS area *** ****** DPH *** +ABT -ABT 4'-HPPH-O-Glu * *** (B) (ng/ml) (h) Time Time after the last DPH administration (µmol/g tissue) (h) Time after the DPH administration * (Area/IS area area) Hepatic GSH contents ** CTL ABT DPH DPH +BSO +BSO +ABT 4'-HPPH *** ****** *** *** (h) 5x1 4 4x1 4 3x1 4 2x1 4 1x1 4 DPH-Glu Area/IS area (h) Area/IS area Dihydrodiol * ** *** (h) 2 25 *** NAC-DPH ** (h) Area/IS area Cys-DPH 1 *** *** *** * ** ** (h) Fig. 16. Effects of ABT treatment on DPH-induced liver injury in mice. Mice were intraperitoneally given DPH at a dose of 5 mg/kg for 2 days followed by oral administration of 1 mg/kg DPH on days 3 through 5. BSO (7 mg/kg) was intraperitoneally injected 1 h prior to each DPH administration. ABT (1 mg/kg), a non specific inhibitor of P45, was intraperitoneally given 1 h prior to the final DPH administration. At, 3, 6, 9, 12, and 24 h after the final DPH administration, the blood was collected for measure the plasma ALT levels (A) and DPH and its metabolites concentrations (C). The livers were collected at 24 h after the final DPH administration to measure the hepatic GSH contents (B). The data are shown as the means ± SE of the results from 4 to 12 mice. The differences compared with non-abt-treated mice in each time point were considered significant at *P <.5, **P <.1 and ***P <.1 (A and C), and the differences compared with the non-abt-treated mice were considered significant at **P <.1 (B). III-3-6 DPH KTZ DPH Cyp Cyp2c Cyp3a KTZ DPH DPH 24 ALT KTZ 63

70 (Fig. 17A) DPH 24 GSH Fig. 17B BSO BSO 24 GSH 8 µg/g tissue GSH DPH/BSO BSO GSH KTZ KTZ Cyp GSH III-3-7 KTZ CYP KTZ DPH Cyp2c Cyp3a KTZ Cyp2c TOL Cyp3a MDZ (Fig. 17C) TOL DPH/BSO BSO DPH Cyp2c KTZ BSO DPH/BSO KTZ TOL KTZ DPH/BSO TOL BSO DPH Cyp2c KTZ MDZ 1 -OH DPH/BSO BSO DPH Cyp3a KTZ BSO MDZ 1 -OH DPH/BSO MDZ 4-OH DPH/BSO 64

71 BSO BSO KTZ MDZ 4-OH DPH/BSO KTZ DPH Cyp3a DPH/BSO Cyp3a (A) 25 ALT (B) Hepatic GSH contents 1 (U/l) (C) (pmol/min/mg protein) * KTZ TOL hydroxylation *** BSO BSO +DPH ** BSO BSO +DPH KTZ (µmol/g tissue) (pmol/min/mg protein) BSO DPH +BSO ** DPH +BSO +KTZ MDZ 1'-hydroxylation 4 *** BSO BSO +DPH *** *** BSO BSO +DPH KTZ (pmol/min/mg protein) MDZ 4-hydroxylation ** *** N.D. BSO BSO BSO BSO +DPH +DPH KTZ Fig. 17. Effects of ketoconazole on DPH-induced liver injury in mice. Mice were intraperitoneally injected DPH at a dose of 5 mg/kg for 2 days and then orally administered at a dose of 2 days. BSO was intraperitoneally injected at a dose of 7 mg/kg at 1 h prior to the each of DPH administration. Cyp inhibitor, KTZ (5 mg/kg in corn oil) was intraperitoneally injected 1 h prior to the final DPH administration. At 24 h after the final DPH administration, plasma and livers were collected for assessment of plasma ALT levels and hepatic GSH contents, respectively (A and B). To assessment for the effects of Cyp inhibitor on hepatic Cyp enzyme activities, hepatic microsomes were prepared from Mice immediately before the finale DPH administration. Microsomal Cyp2c and 3a activities were assessed by the method described as Fig. 16. The data are shown as the means ± SEM of the results from 4 to 5 mice. Differences compared with the non-inhibitor treated mice or BSO alone treated mice were considered significant at **P <.1 and ***P <.1. P <.1, N.D., Not detected. 65

72 III-3-8 Cyp2c Cyp3a DPH Cyp2c Cyp3a DPH/BSO Day, 1, 2 4 CYP (Fig. 18A) DPH Cyp2c Cyp3a Day (DPH ) DPH Cyp2c Cyp3a TOL Day 1, 2 4 Day (DPH ) Fig. 18B Cyp2c Cyp3a MDZ 1 DPH Day 1, 2 4 Day Day 2 4 Cyp3a MDZ 4 Day 1, 2 4 (2 pmol/min/mg protein) DPH Cyp2c DPH CYP Cyp2c 66

73 (A) Relative protein level (B) (pmol/min/mg protein) Cyp2c protein ** *** *** Administration period (day) TOL hydroxylation ** ** *** Times Administration of DPH+BSO period administration (day) (pmol/min/mg protein) Relative protein level Cyp3a protein *** *** *** Administration period (day) MDZ 1'-hydroxylation 4 *** *** Times Administration Number of DPH+BSO of period administration (day) (pmol/min/mg protein) MDZ 4-hydroxylation N.D. N.D. N.D Administration period (day) Fig. 18. Effects of DPH administration on Cyp2c and 3a protein expressions and its enzyme activities. Mice were intraperitoneally injected DPH at a dose of 5 mg/kg for 2 days and then orally administered at a dose of 2 days. BSO was intraperitoneally injected at a dose of 7 mg/kg at 1 h prior to the each of DPH administration. Hepatic microsomes were prepared 24 h after the, 1, 2, and 4 times after DPH administration. Microsomal Cyp2c and 3a contents were assessed by Western blotting analysis. The quantitative analysis of protein expression was performed using densitogram (A). Microsomal Cyp2c activity and 3a activity were evaluated by tolbutamide hydroxylation, and midazolam 1 - and 4-hydroxylation using high-performance liquid chromatography, respectively (B). The data are shown as the means ± SEM of the results from 4 to 5 mice. Differences compared with the times of DPH administration mice were considered significant at **P <.1 and ***P <.1. N.D.: Not detected. III-3-9 PB DPH DPH BSO (Fig. 4) DPH Cyp Cyp DPH (Fig. 19A) PB DPH 24 PB 67

74 ALT ( 3 U/l) DPH ( 8 U/l) PB TOL Fig. 19B PB PB Cyp2c DPH Cyp DPH (A) ALT (U/l) Single administration study PB+DPH+BSO DPH+BSO ** (h) Time after the DPH administration (B) (pmol/min/mg protein) PB TOL hydroxylase activity - ** + Fig. 19. Potentiate effects of Cyp2 inducer phenobarbital on development of DPH-induced hepatotoxicity by single DPH administration. Mice were intraperitoneally injected PB (8 mg/kg) for 4 days and orally administered DPH (1 mg/kg) on the day 5. BSO was intraperitoneally injected 1 h prior to the DPH administration. At, 6, and 24 h after the DPH administration, the plasma were collected for assessment of plasma ALT levels. To assessment of hepatic Cyp2c activities, the livers were collected at 24 h after the 4 times PB treatment (8 mg/kg for 4 days). Cyp2c activities were determined according to the method same as Fig. 18. The data are shown as the means ± SEM of the results from 4 to 5 mice. Differences compared with the non-pb treated mice were considered significant at **P <.1. 4 in vivo DPH GSH (TCPO) (Roy and Snodgrass, 1988) 68

75 CYP DPH 4 -HPPH 4 -HPPH o- (Munns et al., 1997) GSH Cys LC-MS/MS DPH GS-DPH Cys-DPH NAC-DPH (Figs. 13A-C) DPH DPH o- GSH 3 4 GSH 3 4 GSH DPH GSH GSH GSH (Ross et al., 1996; Nishiya et al., 28) o- 2 5 GSH MS GSH GS-DPH Cys-DPH NAC-DPH II-3-4 DPH GSH (Fig. 4A) BSO DPH (Fig. 3B) GSH BSO 69

76 DPH DPH 8 µg/ml 4 -HPPH.36 µg/ml (Fig. 15) 52 µg/ml 1.64 µg/ml (Vasko et al., 198; Kerb et al., 21) DPH 4 -HPPH DPH 4 -HPPH DPH CYP DPH 1 DPH DPH 5 DPH BSO (Fig. 4) DPH II-3-6 DPH DPH 1-8 (Mullick and Ishak, 198) DPH DPH CYP2C CYP3A (Chaudhry et al., 21; Fleishaker et al., 1995; Yamazaki et al., 21; Hagemeyer et al., 21) CYP DPH DPH DPH CYP DILI DPH Day 5 Day 3 (Fig. 15) DPH CYP 7

77 DPH UGT1A DPH DPH-Glu DPH Ugt1a (Sytherland et al., 1993) Cys-DPH NAC-DPH Day 3 Day 5 DPH DPH DPH CYP ABT DPH (Fig. 16A) ABT DPH DPH-Glu ABT (Fig. 16C) DPH ABT ABT 2 DPH Cyp ABT 4 -HPPH O-Glu ABT 4 -HPPH 4 -HPPH O-Glu (Fig. 16C) DPH CYP CYP BSO DPH KTZ CYP3A4 CYP2C (Emoto et al., 23) Cyp2c DPH KTZ (Fig. 17A) GSH (Fig. 17B) KTZ DPH 71

78 KTZ CYP Cyp2c TOL KTZ (Fig. 17C) Cyp2c DPH DPH/BSO Cyp3a MDZ 1 -OH 4-OH KTZ (Fig. 17C) BSO KTZ MDZ 1 -OH 4-OH (Fig. 17C) KTZ Cyp3a DPH/BSO KTZ MDZ 1 -OH 4-OH DPH MDZ 1 -OH 4-OH (Fig. 17C) DPH Cyp3a mechanism based inhibition (MBI) (Fig. 18B) KTZ DPH MBI Cyp3a KTZ Cyp3a Cyp2c DPH CYP2C9 CYP2C19 DPH (Pachkoria et al., 27) CYP GSH 2 DPH CYP GST γ-gcs DPH DPH DPH Cyp2c Cyp3a DPH Cyp2c 72

79 Cyp3a DPH (Fig. 18A) Cyp2c Day Day 4 3 Cyp3a 4 DPH Cyp2c Cyp3a Cyp DPH DPH X (PXR) (CAR) CYP3A CYP2C (Jackson et al., 24; Chaudhry et al., 21; Luo et al., 22) Cyp2c Cyp3a (Fig. 18A) MDZ 1 -OH Cyp3a Cyp2c Cyp2c (Waterschoot et al., 28) MDZ 4 -OH Cyp3a DPH TOL MDZ 1 -OH DPH Cyp2c Cyp3a (Fig. 18B) Cyp3a MDZ 4-OH DPH (Fig. 18B) DPH Cyp2c Cyp3a Cyp2c Cyp3a DPH (Figs. 17A and B) DPH CYP CYP3A (Munns et al., 1997) DPH CYP3A MBI 2 DPH CYP3A1/2 (Yamazaki et al., 21) Cyp3a MBI Cyp3a DPH CYP3A o- (Munns et al., 1997) o- 73

80 4 -HPPH CYP3A Yamazaki et al., 21 Cyp3a MBI o- o- o- CYP o- MBI MBI time-dependent inhibiton (TDI) CYP MBI CYP (Riley et al., 27) Cyp3a MBI DPH DPH BSO (Fig. 4) Cyp DPH Cyp2b, Cyp3a Cyp2c PB DPH (Fig. 19A) CYP DPH DPH Cyp PB Cyp2c (Fig. 19B) Cyp2c APAP CYP (Yue and peng, 29) CYP CYP 74

81 IV in vivo DILI in vivo DILI DILI DPH II DPH DPH DPH BSO GSH DPH TLR4 eritoran TLR4 III DPH Cyp Cyp2c GSH in vivo 75

82 DILI CYP Cyp PB Cyp DPH Cyp DPH DILI BSO GSH CYP DILI DILI 76

83 ( ) 77

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