The Toxicologist - Society of Toxicology

Kupffer cells, can also be classically and alternatively activated in vitro by inflammatory
cytokines upregulated in the liver after APAP administration. In the present
studies we determined if these cells play a role in the hepatic response to APAP. ECs
were collected from rats 12-72 hr after administration of APAP (600 mg/kg, ip)
and analyzed by real time PCR for expression of classical and alternative activation
markers. Treatment of rats with APAP resulted in a rapid and transient increase in
EC expression of inducible nitric oxide synthase, a marker of classical activation.
This was followed at 72 hr by EC expression of arginase I (ArgI), YM2 and FIZZ1,
markers of alternative activation. Co-culture of control ECs with hepatocytes pretreated
with 5 mM APAP for 2 hr resulted in increased expression of ArgI and mannose
receptor demonstrating that APAP-injured hepatocytes release factors important
in alternative activation of ECs. APAP administration to rats also resulted in
hepatocyte expression of matrix metalloproteinase-9 (MMP9) and connective tissue
growth factor (CTGF), markers of tissue repair, which was most prominent
after 72 hr. Moreover, control hepatocytes co-cultured with alternatively activated
ECs were found to express MMP9 and CTGF. Taken together, these data suggest a
role of alternatively activated ECs in liver repair following APAP intoxication.
Supported by NIH grants GM034310, ES004738, CA132624, AR055073,
ES005022.
456 NEGATIVE REGULATION OF CLASSICAL
MACROPHAGE ACTIVATION BY THE STK RECEPTOR
DURING ACETAMINOPHEN-INDUCED
HEPATOTOXICITY.
C. R. Gardner 1 , Y. Liu 1 , P. A. Hankey 2 , J. D. Laskin 3 and D. L. Laskin 1 . 1 Rutgers
University, Piscataway, NJ, 2 Pennsylvania State University, University Park, PA and
3 UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ.
Classically activated macrophages and mediators they release, including reactive nitrogen
species (RNS), play a critical role in the pathogenesis of acetaminophen
(APAP)-induced hepatic necrosis. Restricting the activity of these macrophages and
production of RNS is key to ameliorating tissue injury and initiating wound healing.
Macrophage stimulating protein (MSP) is a liver-derived protein reported to
bind to the STK receptor on macrophages resulting in inhibition of NF-κB and
down regulation of inducible nitric oxide synthase (iNOS). In the present studies
we analyzed the role of MSP signaling via STK in regulating macrophage cytotoxic
activity during APAP-induced hepatotoxicity. APAP administration (600 mg/kg,
i.p.) to rats resulted in a rapid and transient increase in iNOS mRNA expression in
liver macrophages which was evident at 24 hr. At this time, expression of the antiinflammatory
cytokine, IL-10 was suppressed. These are characteristics of classically
activated cytotoxic macrophages. Treatment of animals with APAP also resulted
in increased expression of STK in liver macrophages. To analyze the role of
STK in APAP hepatotoxicity, we used transgenic mice lacking this receptor. We
found that STK-/- mice were significantly more sensitive to APAP than wild type
mice. Thus while administration of APAP to wild type mice resulted in 8% mortality
at 24 hr, in STK-/- mice, mortality was 44%. Moreover, surviving STK-/- mice
displayed severe hemorrhagic centrilobular hepatic necrosis with injury extending
into the midzonal and portal regions of the liver, which was not evident in wild type
mice. Taken together, these findings suggest that MSP signaling via STK is important
in down regulating inflammatory responses of cytotoxic hepatic macrophages
and promoting repair following APAP intoxication. Supported by NIH
GM034310, ES004738, CA132624, AR055073 and ES005022.
457 ROLE OF MACROPHAGE-DERIVED GALECTIN-3 IN
ACETAMINOPHEN-INDUCED HEPATOTOXICITY.
A. Dragomir 1 , R. Sun 1 , J. D. Laskin 2 and D. L. Laskin 1 . 1 Pharmacology and
Toxicology, Rutgers University, Piscataway, NJ and 2 Environmental and Occupational
Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ.
Toxic doses of acetaminophen (APAP), a widely used analgesic, are known to cause
centrilobular hepatic necrosis. Accumulating evidence suggests that macrophages
and inflammatory mediators they release play a key role in the pathogenic process.
In the present studies, mechanisms regulating macrophage release of inflammatory
mediators were investigated. Galectin-3 (Gal-3) is a beta-galactoside binding lectin
secreted by activated macrophages that stimulates production of proinflammatory
mediators and reactive oxygen species. Treatment of wild-type mice with APAP
(300 mg/kg, i.p.) resulted in a time-dependent increase in Gal-3 expression in the
liver, reaching a maximum after 48-72 hr. Immunohistochemistry indicated that
Gal-3 was predominantly expressed by macrophages infiltrating into necrotic areas.
To analyze the role of Gal-3 in APAP-induced hepatotoxicity, we used Gal-3 -/mice.
In wild-type mice, APAP caused a time-dependent increase in serum ALT
and AST levels, beginning within 6 hr. This was associated with histological evidence
of centrilobular necrosis. These effects were significantly attenuated in Gal-3 -
/- mice. APAP-induced increases in expression of the proinflammatory cytokines
98 SOT 2011 ANNUAL MEETING
TNF-α, and MCP-1, and lipocalin 2, a marker of oxidative stress, were also reduced
in Gal-3 -/- mice. In contrast, expression of the antioxidant enzyme, heme
oxygenase-1, as well as the anti-inflammatory protein galectin-1, were increased in
Gal-3 -/- mice relative to wild-type mice following APAP administration. Taken together,
these results suggest that Gal-3 plays an important role in macrophage activation
and mediator release during the pathogenesis of APAP-induced hepatotoxicity.
Supported by NIH grants GM034310, ES004738, CA132624, AR055073 and
ES005022.
458 TARGETING OF THIOREDOXIN REDUCTASE BY THE
ACETAMINOPHEN METABOLITE N-ACETYL-P-
BENZOQUINONE IMINE.
Y. Jan 1 , D. E. Heck 2 , D. L. Laskin 3 , Y. Liu 3 , A. Dragomir 3 and J. D. Laskin 1 .
1 Environmental & Occupational Medicine, UMDNJ-Robert Wood Johnson Medical
School, Piscataway, NJ, 2 Environmental Health, New York Medical College, Valhalla,
NY and 3 Pharmacology & Toxicology, Rutgers University, Piscataway, NJ.
Acetaminophen (APAP), an analgesic and antipyretic, is bioactivated by cytochrome
P450s in hepatocytes to the reactive electrophilic metabolite N-acetyl-pbenzoquinone
imine (NAPQI). NAPQI is known to bind to glutathione and a variety
of hepatic proteins, an important downstream event mediating
acetaminophen hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a critical
cellular antioxidant which contains a selenocysteine in its C-terminal redox center,
an accessible target for electrophilic modification. In the present studies, we determined
if NAPQI targeted TrxR. APAP (0.5-10 mM) was found to cause a
marked inhibition of TrxR activity in primary cultures of rat hepatocytes, and in reaction
mixes containing liver microsomes or recombinant P450s and purified rat
liver TrxR. To elucidate mechanisms underlying the inhibitory actions of APAP on
TrxR, the effects of NAPQI on purified TrxR were characterized. Treatment of TrxR
with NAPQI was found to inhibit enzyme activity (IC50 = 23 nM). A recombinant
mutant enzyme, in which selenocysteine was replaced with cysteine, was 5000-fold
less sensitive to NAPQI, suggesting that selenocysteine was modified. This is supported
by our findings that alkylation of TrxR with biotin-conjugated iodoacetamide,
which selectively reacts with selenol or thiol groups on proteins, was inhibited
by NAPQI. Moreover, LC-MS/MS analysis demonstrated that NAPQI
selectively modified the cysteine and selenocysteine residues in the C-terminal
redox center of TrxR. Collectively, these data demonstrate that reactive intermediates
of APAP can target TrxR and this may be an important mechanism by which
APAP overdose induces oxidative stress and cellular damage. Supported by NIH
grants GM034310, AR055073, CA093798, CA132624, ES004738, ES005022,
and ES017389.
459 TRIFLUOPERAZINE MEDIATED
HEPATOPROTECTION AND REDUCTION OF HIF-1α
IN ACETAMINOPHEN MEDIATED TOXICITY IN MICE.
S. Chaudhuri 1 , S. S. McCullough 1 , A. Brown 1 , J. A. Hinson 2 and L. P. James 1, 2 .
1 Pediatrics/Clinical Pharmacology, Arkansas Childrens Hospital Research Institute,
Little Rock, AR and 2 Pharmacology and Toxicology, University of Arkansas for
Medical Sciences, Little Rock, AR.
Mitochondrial permeability transition (MPT) and oxidative stress are proposed
mechanisms for APAP mediated hepatotoxicity. Trifluoperazine (TFP) has been
previously shown to reduce APAP mediated hepatotoxicity (Yamamoto et al, 1990;
Dimova et al., 1995) in vivo. In vitro studies of APAP toxicity in freshly isolated hepatocytes
showed that TFP reduced MPT (Reid et al., 2005). We previously reported
the induction of HIF-1α in APAP toxicity in mice and have hypothesized
that HIF-1α induction is secondary to MPT associated oxidative stress. Thus we
examined the effect of TFP on hepatotoxicity and HIF-1α induction in APAP mediated
hepatotoxicity. B6C3F1 male mice were pretreated with TFP (10 mg/kg)
prior to APAP (200 mg/kg IP). The mice were sacrificed at 1, 4 and 24 h. Mean (±
SE) values for ALT were 1786 (± 258) IU/L and 16 (± 2) IU/L for APAP/veh and
APAP/TFP mice at 4 h, respectively (p≤0.05). At 24 h, mean ALT values were
4267 (± 207) IU/L and 3055 (± 928) IU/L for APAP/veh and APAP/TFP mice
(p≤0.05). Histopathological examination of liver sections was consistent with the
biochemical data. Hepatic glutathione depletion and hepatic APAP-protein
adducts were comparable in both groups of mice at all time points, indicating that
TFP did not affect the metabolism of APAP. HIF-1α expression determined by immunoblots
showed that HIF-1α expression was reduced by 45% in APAP/TFP
mice at 1, 4, and 24 h compared to the APAP/veh mice (p≤0.05). The data suggest
that TFP reduces toxicity in APAP treated mice and that HIF-1α induction is secondary
to MPT associated oxidative stress.