Role of Tissue Repair in Liver Toxicity Outcomes

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Role of Tissue Repair in Liver Toxicity Outcomes
Harihara M. Mehendale, Ph.D., DABT, Fellow ATS
The Ohio Valley Chapter of Society of
Toxicology September 13, 2004
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Acute Toxic Injury
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Stage I Infliction of Injury
Stage II Progression of Injury
Metabolism Reactive metabolite generation
Injury
Time
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Two-Stage Model of Toxicity
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Injury
Repair
Toxicity
Suppressed tissue repair
Stage I
Chemical
Cellular injury
Progression of Injury
Bioactivation, RONS
Stage II
Stimulated tissue repair
Cytokines, Growth factors, Signaling events
Regression of Injury
Death
Survival
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Dose Response Tissue Repair

• Single Administration
• Acetaminophen (APAP)
• CCl4
• CHCl3
• Thioacetamide (TA)
• Trichloroethylene (TCE)
• Allyl alcohol (AA)
• Chemical Mixtures
• TCE CHCl3
• TCE CHCl3 Allyl alcohol
• CHCl3 TA
• TA TCE CHCl3 AA

• Stimulation of TR
• Autoprotection
• TA TA
• CCl4 CCl4
• Heteroprotection
• TA APAP

• Inhibition of TR
• CD CCl4

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Dose Response Thioacetamide
5
600
600
300
300
150
150
50
50
H after TA administration
Mangipudy et al., Environ. Health Perspect. 103
260-267, 1995
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Antimitosis after Low Dose of TA
Mangipudy et al. Environ. Health Perspect. 104
744-749, 1996
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Tissue Repair governs final outcome
Autoprotection
Mangipudy et al. Pharmacol. Toxicol. 77 182-188,
1995
Species Difference
LD50 35 fold different
Cai and Mehendale. Toxicol. Appl. Pharmacol. 104
511-520, 1990
Strain Differences
LD50 No Difference
Kulkarni et al. Arch. Toxicol. 70 714-723, 1996.

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Physiological Manipulations Protection by Diet
Restriction
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Male Sprague-Dawley rats (275-300 g) DR, 35
for 21 days
Thioacetamide (600 mg/kg)
Groups Death ad
libitum (AL) 90 Diet restriction
(DR) 30
Ramaiah et al. (1998) Toxcol. Sci. 45 233-241
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Cell Cycle and Its Stimulators
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TGF-?, HGF, NO, PPAR- ?
Progression
TNF-? IL-6, NO
S
Priming
G1
(NF-?B, STAT-3, MAPKs)
c-fos, c-myc, c-jun
Go
G2
Quiescence
D
MITOSIS
D
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TGF-? Immunohistochemistry
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N
C
C
N
H
AL 24 h
DR 24 h

• C- centrilobular area, N- necrosis, H- TGF-?
  positive hepatocytes
• Staining around the centrilobular region
• Hepatocytes immediately next to necrosis
• stain positive

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Signaling Pathways
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TNF-?
NO
JAK-STAT
MAPK
HGF/c-MET
iNOS
IL-6
TGF- ?
HGF
TNF-?
EGF-R
c-met
IL-6R
TNFR 1
NO
NF-?B
AP-1
STAT-3
MAPK
GENES
Cell division
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Diabetic rats are highly susceptible to a
non-lethal dose of TA
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Diabetes in Rats
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• Non-lethal dose of thioacetamide causes lethality
  in diabetic rats
• Inhibited tissue repair leads to progression of
  liver injury and death

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NF-?B-DNA Binding
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MAPK Pathway
NDB
DB
0 6 12 24 36
0 6 12 24 36
I?B
NDB
DB
0 C 0 6 12 24
0 6 12 24
0 6 12 24 36
0 6 12 24 36
Cyclin D1
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TNF-?
IL-6

TNFR
EGFR
MAPKs
P
MAPKs
NF-?B
I-?B
P
NF-?B
I-?B
Cyclin D1
NF-?B
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3
5
3
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Type 2 Diabetes Sensitizes Liver to Progression
of Injury
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adeath in 24-48 h bdeath in 48-72 h
Sawant et al. J. Pharmacol. Exp. Ther. 308
694-704, 2004.
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Summary-Type 2 DB
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What are the mechanisms of delayed and inhibited
TR in DB rats?
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DB RAT
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INSULIN SIGNALING PATHWAY
Cytosol
Insulin Signaling
PI3Kinase
Oxidative stress
MAPK
p38 MAPK
IkBa
P
IkBa
Nuclear membrane
NF-kB
Nucleus
NF-kB
pRb
NF-kB
E2F
?
E2F

Cyclin D1
Cyclin D1
G1
cdk6
cdk4
P
S
pRb
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Acute Toxic Injury Paradigm
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Stage I Infliction of Injury
Stage II Progression of Injury
Metabolism Reactive metabolite generation
Injury
Time
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Leading Theories

• Generation of free radicals
• Inflammatory cells
• Leakage of degradative enzymes

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Hypothesis Hydrolytic enzymes or "death
proteins" released from necrosed cells cause
progression of injury.
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Effect of calpain inhibitor CBZ
Group No of No of
Survival
animals Deaths CCl4 alone (3 ml/kg)
12 8 25 CCl4CBZ
12 4
75 CCl4 DMSO 12
8 25 C.O. DMSO 4
0 100 C.O. CBZ 4 0
100
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Effect of CBZ on CCl4-induced Liver Injury
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CCl4 2 ml/kg
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H E Staining
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CCl4
12 h
24 h
48 h
CCl4 CBZ
12 h
24 h
48 h
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Is calpain involved ??
Calpain Progression of injury Upon
injury Appearance in the
extracellular space
Plasma Degradation Breakdown of
calpain by calpain? specific
substrate When released Hepatocytes
death
Limaye et al. Toxicol. Appl. Pharmacol. 191
211-226, 2003.
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Hypothesis
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Higher expression of endogenous inhibitors of
death proteins in new cells stops their lytic
action.
Endogenous Inhibitors
Death protein Inhibitor Calpain
Calpastatin
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Calpastatin
0 h
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CalpastatinandPCNA
72 h
48 h
6 h
PCNA
72 h
48 h
6 h
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Validation of the Hypothesis Generalization of
the concept

• Partial Hepatectomy
• 70 PH male S-D rats
• Time course 4 d, 7 d

CCl4 2.8 ml/kg, ip
Kodavanti et al. Toxicol. Pathol. 17 494-505,
1989.

• Newborns
• 20 days old male S-D rats

CD (10 ppm) CCl4(100 µl)
Cai and Mehendale. Pediatr Res. 33 225-232,
1993. Dalu et al. Toxicology 111 29-42, 1996.
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Calpastatin after PH
4 d
PCNA
4 days
7 days
Sham Control (4 d)
7 d
PCNA
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Newborns
CAST
Adult Control
PCNA
NB
Adult
CAST
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Overexpression of Calpastatin Using Adenovirus
Effect of CAST Overexpression on APAP-induced
Lethality
Ad/CSTN injected via tail vein
Ad/CAST
Ad/LacZ
CAST
APAP 600 mg/kg, ip
GAPDH
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Summary
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• Infliction of injury and Progression (Regression)
  of injury are two stages of toxicity.
• Tissue repair plays a major role in the ultimate
  outcome of toxicity.
• Calpain mediates progression of injury.
• Overexpression of CAST in the new cells underlies
  rapid regression of injury during tissue repair.

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Acknowledgments
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• Tom Bucci John Latendresse, PAI
• Jan K. Reddy, Northwestern University
• Dr. Masatoshi Maki
• Nagoya University, Nagoya, Japan
• Funding agencies, NIEHS, ATSDR, NIDDK
• Many Students, Post Docs and colleagues