Role of galectin-3 in acetaminophen-induced hepatotoxicity and inflammatory mediator production.
ABSTRACT Galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in the regulation of macrophage activation and inflammatory mediator production. In the present studies, we analyzed the role of Gal-3 in liver inflammation and injury induced by acetaminophen (APAP). Treatment of wild-type (WT) mice with APAP (300 mg/kg, ip) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was associated with increased hepatic expression of Gal-3 messenger RNA and protein. Immunohistochemical analysis showed that Gal-3 was predominantly expressed by mononuclear cells infiltrating into necrotic areas. APAP-induced hepatotoxicity was reduced in Gal-3-deficient mice. This was most pronounced at 48-72 h post-APAP and correlated with decreases in APAP-induced expression of 24p3, a marker of inflammation and oxidative stress. These effects were not due to alterations in APAP metabolism or hepatic glutathione levels. The proinflammatory proteins, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, and MIP-3α, as well as the Gal-3 receptor (CD98), were upregulated in livers of WT mice after APAP intoxication. Loss of Gal-3 resulted in a significant reduction in expression of iNOS, MMP-9, MIP-3α, and CD98, with no effects on IL-1β. Whereas APAP-induced increases in MIP-2 were augmented at 6 h in Gal-3(-/-) mice when compared with WT mice, at 48 and 72 h, they were suppressed. Tumor necrosis factor receptor-1 (TNFR1) was also upregulated after APAP, a response dependent on Gal-3. Moreover, exaggerated APAP hepatotoxicity in mice lacking TNFR1 was associated with increased Gal-3 expression. These data demonstrate that Gal-3 is important in promoting inflammation and injury in the liver following APAP intoxication.
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ABSTRACT: Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology characterized by progressive fibrosis. Activated fibroblasts are mainly responsible for fibrosis in SSc. Galectin-3, a β-galactoside-binding lectin, plays many important regulatory roles in both physiological and pathological processes including proliferation, apoptosis, inflammation, and fibrosis. The purpose of this study was to assess the serum galectin-3 levels in patients with SSc. Thirty-seven SSc patients, 23 systemic lupus erythematosus (SLE) patients (serving as patient control group), and 28 healthy volunteers were enrolled in this study. Disease activity and severity scores were detected with Valentini disease activity index and Medsger disease severity scale in the SSc group and SLE disease activity index and Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index in the SLE group. The serum levels of galectin-3, vascular endothelial growth factor, transforming growth factor-β, and interleukin-6 were determined. Compared to the control group, the galectin-3 levels were higher in the SSc and SLE groups. The galectin-3 levels were not correlated with the disease activity and severity indexes in both patient groups. But, the serum galectin-3 levels were higher in the active SSc and SLE subgroups than in the inactive SSc (4.6 ± 5.8 vs. 1.3 ± 1.1 ng/ml, p = 0.015) and SLE (17.4 ± 11.3 vs. 6.5 ± 8.9 ng/ml, p = 0.019) subgroups. These results suggest that galectin-3, which is associated with fibrosis and inflammation by previous studies, may be a prominent biomarker of disease activity in SSc.Clinical Rheumatology 08/2013; · 1.77 Impact Factor
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ABSTRACT: Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400mg/kg) and then challenged 48hr later with 600mg APAP/kg. Livers were obtained 4 or 24hr later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430_2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection.Toxicology and Applied Pharmacology 10/2013; · 3.98 Impact Factor
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ABSTRACT: Hepatocellular carcinoma (HCC) represents a global health problem. Infections with hepatitis B or C virus, non-alcoholic steatohepatitis disease, alcohol abuse, or dietary exposure to aflatoxin are the major risk factors to the development of this tumor. Regardless of the carcinogenic insult, HCC usually develops in a context of cirrhosis due to chronic inflammation and advanced fibrosis. Galectins are a family of evolutionarily-conserved proteins defined by at least one carbohydrate recognition domain with affinity for β-galactosides and conserved sequence motifs. Here, we summarize the current literature implicating galectins in the pathogenesis of HCC. Expression of "proto-type" galectin-1, "chimera-type" galectin-3 and "tandem repeat-type" galectin-4 is up-regulated in HCC cells compared to their normal counterparts. On the other hand, the "tandem-repeat-type" lectins galectin-8 and galectin-9 are down-regulated in tumor hepatocytes. The abnormal expression of these galectins correlates with tumor growth, HCC cell migration and invasion, tumor aggressiveness, metastasis, postoperative recurrence and poor prognosis. Moreover, these galectins have important roles in other pathological conditions of the liver, where chronic inflammation and/or fibrosis take place. Galectin-based therapies have been proposed to attenuate liver pathologies. Further functional studies are required to delineate the precise molecular mechanisms through which galectins contribute to HCC.World Journal of Gastroenterology 12/2013; 19(47):8831-8849. · 2.43 Impact Factor
TOXICOLOGICAL SCIENCES 127(2), 609–619 (2012)
Advance Access publication March 29, 2012
Role of Galectin-3 in Acetaminophen-Induced Hepatotoxicity and
Inflammatory Mediator Production
Ana-Cristina Dragomir,* Richard Sun,* Vladimir Mishin,* LeRoy B. Hall,† Jeffrey D. Laskin,‡ and Debra L. Laskin*,1
*Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854; †Drug Safety Sciences,
Janssen Research & Development, Raritan, New Jersey 08869; and ‡Department of Environmental and Occupational Medicine, University of Medicine and
Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
1To whom correspondence should be addressed at Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University,
160 Frelinghuysen Road, Piscataway, NJ 08854. Fax: (732) 445-2534. E-mail: email@example.com.
Received November 8, 2011; accepted March 17, 2012
Galectin-3 (Gal-3) is a b-galactoside-binding lectin implicated
in the regulation of macrophage activation and inflammatory
mediator production. In the present studies, we analyzed the
role of Gal-3 in liver inflammation and injury induced by
acetaminophen (APAP). Treatment of wild-type (WT) mice with
APAP (300 mg/kg, ip) resulted in centrilobular hepatic necrosis
and increases in serum transaminases. This was associated
with increased hepatic expression of Gal-3 messenger RNA and
protein. Immunohistochemical analysis showed that Gal-3 was
predominantly expressed by mononuclear cells infiltrating into
necrotic areas. APAP-induced hepatotoxicity was reduced in Gal-
3-deficient mice. This was most pronounced at 48–72 h post-APAP
and correlated with decreases in APAP-induced expression of
24p3, a marker of inflammation and oxidative stress. These effects
were not due to alterations in APAP metabolism or hepatic
glutathione levels. The proinflammatory proteins, inducible nitric
oxide synthase (iNOS), interleukin (IL)-1b, macrophage inflam-
matory protein (MIP)-2, matrix metalloproteinase (MMP)-9, and
MIP-3a, as well as the Gal-3 receptor (CD98), were upregulated
in livers of WT mice after APAP intoxication. Loss of Gal-3
resulted in a significant reduction in expression of iNOS, MMP-9,
MIP-3a, and CD98, with no effects on IL-1b. Whereas APAP-
induced increases in MIP-2 were augmented at 6 h in Gal-32/2
mice when compared with WT mice, at 48 and 72 h, they were
suppressed. Tumor necrosis factor receptor-1 (TNFR1) was also
upregulated after APAP, a response dependent on Gal-3.
Moreover, exaggerated APAP hepatotoxicity in mice lacking
TNFR1 was associated with increased Gal-3 expression. These
data demonstrate that Gal-3 is important in promoting in-
flammation and injury in the liver following APAP intoxication.
Key Words: acetaminophen; macrophages; inflammation;
galectin-3; liver; TNFR1.
Acetaminophen (APAP)-induced hepatotoxicity is the major
cause of acute liver failure in the United States (Lee et al.,
2008). Tissue injury is initiated by covalent binding of the
reactive intermediate, N-acetyl-p-benzoquinoneimine (NAPQI),
to critical cellular proteins in the liver (Dahlin et al., 1984;
Jollow et al., 1973). Evidence suggests that proinflammatory/
cytotoxic mediators released by activated macrophages play
a role in promoting APAP-induced hepatotoxicity (reviewed in
Laskin, 2009). The factors that induce macrophage activation
and inflammatory mediator production in the liver after APAP
intoxication have not been clearly established. In previous
studies, we demonstrated that mediators released from injured
hepatocytes, including high-mobility group box-1 (HMGB1),
are important in the activation process (Dragomir et al., 2011;
Laskin et al., 1986). A question arises, however, about the role
of macrophages themselves as a source of activating factors.
Macrophages are potent secretory cells, releasing a myriad of
mediators known to be important in nonspecific host defense and
adaptive immunity, as well as inflammation and wound repair.
These diverse activities are mediated by distinct subpopulations
that develop in response to mediators macrophages encounter in
their microenvironment (reviewed in Laskin et al., 2011). Two
major phenotypically distinct subpopulations have been identi-
fied: classically activated proinflammatory macrophages and
alternatively activated anti-inflammatory/would repair macro-
phages. The ability of macrophages to release activating factors
that act in an autocrine and paracrine manner to induce classical
and alternative activation represents an important mechanism
regulating inflammatory responses to tissue injury.
Galectins comprise a family of lectins with affinity for
b-galactoside-containing carbohydrates. Gal-3 is the only
member of this family with a chimeric structure consisting of
a conserved carbohydrate recognition domain and a nonlectin
domain (Henderson and Sethi, 2009). Whereas Gal-3 is
expressed at low levels in monocytes, it is upregulated during
their maturation into macrophages (Liu et al., 1995). Proin-
flammatory cytokines, including tumor necrosis factor-alpha
(TNF-a), further upregulate Gal-3 in macrophages and also
stimulate its release into the extracellular environment (Nishi
et al., 2007). Gal-3 binds to macrophages via CD98, stimulating
? The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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the production of additional proinflammatory cytokines and
chemokines (Norling et al., 2009). Intra-articular administration
of Gal-3 has been reported to induce inflammation in mice
(Janelle-Montcalm et al., 2007). Moreover, mice with a targeted
mutation of Gal-3 exhibit an impaired ability to mount an acute
inflammatory response to thioglycollate (Hsu et al., 2000) or
ovalbumin (Zuberi et al., 2004). These data suggest that Gal-3 is
important in promoting inflammation. Because macrophage-
derived inflammatory mediators contribute to liver injury fol-
lowing APAP intoxication (Laskin, 2009; Laskin et al., 1995),
we speculated that Gal-3 may be involved in the pathogenesis of
hepatotoxicity and this was investigated.
MATERIALS AND METHODS
Animals. Male specific pathogen-free C57Bl/6J wild-type (WT) mice,
Gal-3?/?mice, and TNFR1?/?mice (8–12-weeks old) were obtained from
The Jackson Laboratory (Bar Harbor, ME). Mice were housed in micro-
isolation cages and allowed free access to food and water. All animals
received humane care in compliance with the institution’s guidelines, as
outlined in the Guide for the Care and Use of Laboratory Animals, published
by the National Institutes of Health. Mice were fasted overnight prior to ip
administration of APAP (300 mg/kg) or pyrogen-free PBS control. After
3–72 h, mice were euthanized with nembutal (200 mg/kg) and blood collected
from the abdominal vena cava for determination of aspartate and alanine
transaminases using diagnostic assay kits (ThermoFisher Scientific, Waltham,
MA). Liver samples (100 mg aliquots) were collected and stored at ?20?C in
RNAlater (Sigma-Aldrich, St Louis, MO) until RNA isolation. The remaining
tissue was snap frozen in liquid nitrogen.
Preparation of liver microsomes and measurement of cytochrome P450
2e1 (Cyp2e1) activity. Frozen liver samples (1–2 g) were homogenized at 4?C
in four volumes of buffer (50mM Tris-hydrochloride, 1.15% potassium chloride,
and 0.5mM phenylmethylsulfonylfluoride, pH 7.4) and then centrifuged at
12,000 3 g for 20 min. Supernatants were collected and centrifuged at
105,000 3 g for 90 min. Microsomes were then washed in buffer containing
1.15% potassium chloride and 10mM EDTA (pH 7.4), resuspended in 10mM
potassium phosphate buffer containing 0.25M sucrose, and stored at ?80?C until
analysis. Cyp2e1 activity was measured by the generation of p-nitrocatechol from
p-nitrophenol (Chang et al., 1998). Microsomes were incubated with 100lM
p-nitrophenol and 500lM b-Nicotinamide adenine dinucleotide 2#-phosphate
reduced tetrasodium salt at 37?C for 20 min. The reaction was stopped by the
addition of trichloroacetic acid. The mixture was then centrifuged (13,000 3 g, 5
min, 4?C), supernatants collected, and mixed with 2M NaOH. Changes in
absorbance were measured spectrophotometrically at 535 nm. Concentrations of
p-nitrocatechol in the samples were determined based on a standard curve
generated with authentic p-nitrocatechol.
Measurement of hepatic glutathione. Frozen livers (50 mg) were
homogenized in ice-cold 5% metaphosphoric acid (1:10) and centrifuged at
3000 3 g for 10 min. Supernatants were collected and reduced glutathione
determined using a colorimetric assay kit (OxisResearch, Portland, OR).
Glutathione concentrations in the samples were calculated based on a standard
curve and expressed as lmol/g wet liver.
Histology and immunohistochemistry. Livers were collected, and 5 mm
samples of the left lateral lobes immediately fixed overnight at 4?C in 3%
paraformaldehyde/2% sucrose. Tissue was washed three times in PBS/2%
sucrose and then transferred to 50% ethanol. After embedding in paraffin, 5 lm
sections were prepared and stained with hematoxylin and eosin (Goode
Histolabs, New Brunswick, NJ). Histopathological evaluation was performed
by a board certified veterinary pathologist (L.B. Hall). Findings were graded on
a scale of 0–4, where 0 ¼ none, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, and
4 ¼ severe changes. For immunohistochemistry, sections were rehydrated and
stained with antibody to Gal-3 (1:25,000; R&D Systems, Minneapolis, MN) or
IgG control (ProSci, Poway, CA). Binding was visualized using a Vectastain
Elite ABC kit (Vector Laboratories, Burlingame, CA). Three to five random
sections of each liver were examined.
Immunofluorescence. Livers were collected and 5 mm samples of the left
lateral lobes were immediately snap frozen in liquid nitrogen–cooled
isopentane and embedded in OCT medium (Sakura Finetek, Torrance, CA).
Six micrometer sections were prepared and fixed in 90% acetone/10%
methanol. Sections were stained with antimyeloperoxidase antibody (1:100)
(Dako, Carpinteria, CA), followed by isotype-specific Alexa Fluor488-
conjugated secondary antibody (Molecular Probes, Carlsbad, CA). Images
were acquired using a Leica SP 5 confocal microscope.
Western blotting. Liver samples (50 mg) were lysed in four volumes of
buffer Real-Time PCR System containing 20mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid pH 7.4, 150mM NaCl, 10% glycerol, 1% Triton
X-100, 1.5mM MgCl2, 1mM diethylene triamine pentaaacetic acid, 1mM
phenylmethylsulfonylfluoride, 10mM sodium pyrophosphate, 50mM sodium
fluoride, 2mM sodium orthovanadate, and protease inhibitor cocktail (Sigma-
Aldrich). Protein concentrations were measured using the Bradford assay (Bio-
Rad, Hercules, CA). Proteins were separated on 10.5–14% Tris-glycine
polyacrylamide gels (Bio-Rad) and then transferred to nitrocellulose mem-
branes. Nonspecific binding was blocked by incubation of the blots for 1 h at
room temperature with buffer containing 5% nonfat milk, 10mM Tris-base,
200mM sodium chloride, and 0.1% polysorbate 20 (pH 7.6). Membranes were
then incubated overnight at 4?C with anti-inducible nitric oxide synthase (iNOS;
1:4000; BD Biosciences, San Jose, CA), anti-cyclooxygenase-2 (COX-2; 1:2000;
Abcam, Cambridge, MA), or anti-glyceraldehyde-3-phosphate dehydrogenase
(1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies,
conjugated secondary antibodies (1:10,000) for 1 h at room temperature.
Binding was visualized using an ECL Plus chemiluminescence kit (GE
Healthcare, Piscataway, NJ).
Real-time PCR. Total RNA was isolated from liver tissue using an
RNeasy kit (Qiagen, Valencia, CA). RNA purity and concentration were
measured using a NanoDrop spectrophotometer (ThermoFisher Scientific,
Wilmington, DE). RNA was converted into complementary DNA (cDNA) using
a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster
City, CA) according to the manufacturer’s directions. Standard curves were
generated using serial dilutions from pooled randomly selected cDNA samples.
Real-time PCR was performed using SYBR Green PCR Master Mix (Applied
Biosystems) on a ABI Prism 7900HT Sequence Detection System (Applied
Biosystems). All PCR primer pairs were generated using Primer Express 2.0
(Applied Biosystems) and synthesized by Integrated DNA Technologies (Coral-
ville, IA). For each sample, gene expression changes were normalized relative to
18S rRNA. Data are expressed as fold change relative to control. Primer sequences
were: Gal-3, CACAATCATGGGCACAGTGAA and TTCCCTCTCCTGA
AATCTAGAACAA; lipocalin 2 (24p3), AGGAACGTTTCACCCGCTTT and
TGTTGTCGTCCTTGAGGCC; macrophage inflammatory protein-2 (MIP-2),
AGGCTTCCCGATGAAGAG and CAGGATAAGAGCGAGAGCCTACA; in-
CAGGTCAAAGG; MIP-3a, TGGCCGATGAAGCTTGTGA and AGCGCA
CACAGATTTTCTTTTCT; matrix metalloproteinase-9 (MMP-9), CAAGT
CD98, GAAGCTCTGAGTTCTTGGTTGCA and CTTTCCCACATCCCG-
GAAT; TNF receptor-1 (TNFR1), CAGACTTGCATGGTGAGCTCTT and
AGCCCAGTTACCCAACAGACA; 18S rRNA, CGGCTACCACATCCAAG-
GAA and GCTGGAATTACCGCGGCT.
Statistical analysis. Experiments were repeated two to three times. Data
were analyzed using Student’s t-test or one-way ANOVA followed by Dunn’s
post hoc analysis. A p value of ? 0.05 was considered statistically significant.
DRAGOMIR ET AL.
Effects of APAP on Expression of Gal-3 in the Liver
In initial studies, we analyzed the effects of APAP
intoxication on Gal-3 expression in the liver. Treatment of
WT mice with APAP resulted in a time-dependent increase in
hepatic Gal-3 messenger RNA (mRNA) expression, which was
evident at 24 h, becoming more pronounced at 48 and 72 h
(Fig. 1, upper panel). This was correlated with an increase in
hepatic Gal-3 protein expression, which was predominantly
localized in inflammatory macrophages infiltrating into ne-
crotic areas (Fig. 1, middle panel). No Gal-3 expression was
observed in neutrophils (Fig. 1, middle panel inset). In contrast,
a transient decline in serum Gal-3 levels was noted 6–24 h after
APAP administration; subsequently, serum levels began to
increase and by 72 h were at or above control levels (Fig. 1,
Role of Gal-3 in APAP-Induced Hepatotoxicity
To investigate the role of Gal-3 in the pathogenesis of
APAP-induced hepatotoxicity, we used mice with a targeted
deletion of the lgals3 gene. In WT mice, APAP administration
resulted in centrilobular hepatic necrosis, which was evident
within 3 h (Fig. 2 and Gardner et al., 2010). At 6 h post-APAP,
mild degeneration, necrosis, and hemorrhage were noted in
centrilobular areas, with no evidence of neutrophils (Table 1).
By 24 h, moderate coagulative centrilobular necrosis was
present, as well as minimal to mild hemorrhage within the
necrotic areas; minimal infiltration of neutrophils was also
noted in centrilobular areas. Moderate coagulative necrosis
persisted in centrilobular regions of the liver for 48 h and was
accompanied by mild neutrophil infiltration. At 72 h post-
APAP, mild necrosis and moderate neutrophil infiltration were
evident (Figs. 2 and 3, Table 1). Mitotic figures were also
observed in surviving hepatocytes surrounding necrotic areas
at 48–72 h, indicating liver regeneration (Fig. 2, inset).
Histopathologic changes in the liver were associated with
a time-related increase in serum transaminases, which peaked
6–24 h post-APAP treatment (Table 2). APAP-induced hepa-
totoxicity was significantly blunted in Gal-3?/?mice when
compared with WT mice, as evidenced by more rapid
decreases in serum transaminases and attenuated histologic
alterations. Thus, by 72 h post-APAP, only minimal necrosis
was observed in livers of Gal-3?/?mice relative to mild/
moderate necrosis in WT mice (Fig. 2, Tables 1 and 2).
Although the extent of neutrophil infiltration into the liver was
similar in Gal-3?/?and WT mice 24–48 h post-APAP, as
determined histologically and by myeloperoxidase immunos-
taining (Table 1 and Fig. 3), by 72 h, there were significantly
fewer neutrophils in livers of Gal-3?/?mice when compared
with WT mice.
24p3 is an acute phase protein and a marker of oxidative
stress and inflammation released by neutrophils, macro-
phages, and epithelial cells (Borkham-Kamphorst et al., 2011;
Roudkenar et al., 2007; Sunil et al., 2007). In WT mice, APAP
intoxication was characterized by a dramatic increase in
expression of 24p3 mRNA, which was most prominent after
24 h (Fig. 4). Subsequently, levels began to decline toward
collected 6–72 h after treatment of WT mice with APAP (300 mg/kg, ip) or
control (CTL). Upper panel: Gal-3 expression was analyzed by real-time PCR.
Data were normalized to 18S rRNA. Each bar represents the mean ± SE (n ¼
Sections were stained with anti-Gal-3 antibody or IgG control, as described in
the Materials and Methods section. One representative section from three
independent experiments is shown. Original magnification, 3100. Inset,
neutrophils. Lower panel: Serum was collected 6–72 h after treatment of WT
mice with APAP or control (CTL). Gal-3 expression was analyzed by Western
blotting. One representative blot from five independent experiments is shown.
Effects of APAP intoxication on Gal-3 expression. Livers were
aSignificantly different (p < 0.05) from CTL. Middle panel:
Gal-3 REGULATES APAP-INDUCED INFLAMMATION
control. Loss of Gal-3 resulted in a significant attenuation of
Effects of Loss of Gal-3 on APAP-induced Expression of
In further studies, we analyzed the effects of loss of Gal-3
on APAP-induced expression of pro- and anti-inflammatory
proteins implicated in hepatotoxicity (Laskin, 2009). APAP
administration to WT mice was associated with a time-related
increase in mRNA expression for the proinflammatory media-
tors MIP-2, MIP-3a, and IL-1b. Whereas MIP-2 expression
increased within 6 h and persisted for 48 h after APAP, MIP-3a
upregulation was delayed until 24 h and remained elevated for
72 h. In contrast, IL-1b expression was transiently increased at
24 h post-APAP. MMP-9 mRNA expression also increased in
the liver 24 h after APAP, remaining elevated for at least 72 h
(Fig. 4). Protein expression of iNOS, the enzyme mediating
macrophage production of nitric oxide (Laskin et al., 2010),
also increased 48–72 h after APAP administration to WT mice
(Fig. 5). Loss of Gal-3 blunted the effects of APAP on MIP-3a,
MMP-9, and iNOS but had no effect on expression of IL-1b.
MIP-2 expression was also reduced in Gal-3?/?mice, relative
to WT mice, at 48–72 h post-APAP. In contrast, expression of
this chemokine was significantly increased at 6 h in Gal-3?/?
mice. Expression of the anti-inflammatory cytokine IL-10
was also upregulated in WT mice following APAP intoxication
(Fig. 4). This was observed within 6 h and remained elevated
for 48 h, although at reduced levels. Loss of Gal-3 resulted in
decreased IL-10 expression at 48 h post-APAP. COX-2 is a key
enzyme regulating the biosynthesis of both pro- and anti-
inflammatory eicosanoids (Cook, 2005). Constitutive COX-2
protein was detected in the livers of both WT and Gal-3?/?
mice; however, expression of this protein was significantly
reduced in Gal-3?/?mice (Fig. 5). Whereas in WT mice,
APAP had no major effect on COX-2 protein expression,
a significant decrease was noted in Gal-3?/?mice 48 h after
CD98 has been proposed as a macrophage receptor for Gal-3
(Dong and Hughes, 1997). In WT mice, APAP administration
resulted in a time-dependent increase in CD98 mRNA
expression, which was maximal after 24 h (Fig. 4). Loss of
Gal-3 blunted the effects of APAP on CD98 expression.
Reciprocal Regulation of Gal-3 and TNFR1 Expression in the
Liver Following APAP Intoxication
TNF-a signaling via TNFR1 has been shown to be important
in the production of mediators involved in tissue repair and
antioxidant defense during APAP-induced hepatotoxicity
(Chiu et al., 2003a,b). In agreement with earlier studies (Ishida
et al., 2004), we found that APAP administration to WT mice
resulted in a significant increase in hepatic TNFR1 mRNA
expression, which was maximal after 24–48 h (Fig. 4). This
was reduced in APAP-treated Gal-3?/?mice. To investigate
the role of TNF-a signaling via TNFR1 in APAP-induced
the liver. Livers were collected 6–72 h after treatment of WT and Gal-3?/?
mice with APAP or control (CTL). Sections were stained with hematoxylin
and eosin. One representative section from three independent experiments is
shown. Original magnification, 320. Insets, 360.
Effects of loss of Gal-3 on APAP-induced structural alterations in
Histopathological Evaluation of Hepatic Necrosis and
Neutrophilic Infiltrates in WT and Gal-32/2Mice After APAP
02.0 ± 0.0
3.0 ± 0.0
3.0 ± 0.0
2.3 ± 0.3
2.0 ± 0.0
2.7 ± 0.3
2.0 ± 0.6
0.3 ± 0.3
1.0 ± 0.0
2.0 ± 0.0
3.3 ± 0.3
1.3 ± 0.3
2.0 ± 0.6
0.7 ± 0.3
Notes. WT and Gal-3?/?mice were treated with 300 mg/kg APAP or PBS
control (CTL). Liver sections were prepared 6–72 h later for histopathological
analysis. Findings were graded on a scale of 0–4, where 0 ¼ none, 1 ¼
minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ severe changes. Data are expressed
as mean ± SE (n ¼ 3 mice).
DRAGOMIR ET AL.
Gal-3 expression, we used mice with a targeted deletion of the
gene encoding TNFR1, which we have previously reported
to be more susceptible than WT mice to APAP-induced
hepatotoxicity (Chiu et al., 2003b; Gardner et al., 2003).
Whereas loss of TNFR1 had minimal effects on APAP-induced
CD98 expression, Gal-3 mRNA levels were significantly
greater in TNFR1?/?mice relative to WT mice 72 h post-
APAP (Fig. 6). Gal-3 protein expression was also increased
in livers of TNFR1?/?mice when compared with WT mice
(Fig. 7). This was noted within 6 h and became more prominent
after 48–72 h.
Effects of Loss of Gal-3 on Hepatic Glutathione and Cyp2e1
To determine if reduced hepatotoxicity in Gal-3?/?mice was
due to altered APAP metabolism, we measured the activity of
hepatic Cyp2e1, the major enzyme mediating the generation of
the reactive APAP metabolite NAPQI (Lee et al., 1996). No
significant differences were noted in the activity of Cyp2e1
between WT and Gal-3?/?mice (0.91 ± 0.05 versus 0.97 ± 0.05
nmol/min/mg protein, respectively). We also measured hepatic
glutathione levels. APAP administration to mice resulted in
a rapid and transient decline in hepatic-reduced glutathione
levels in WT, which was evident within 3 h; subsequently,
glutathione levels increased and by 72 h were above control
levels (Fig. 8). Loss of Gal-3 had no significant effect on APAP-
induced alterations in hepatic glutathione levels.
The present studies demonstrate that Gal-3 plays a role in
promoting late proinflammatory responses and perpetuating
injury in the liver following APAP intoxication. This is based
on our findings that Gal-3 is markedly upregulated in macro-
phages infiltrating into the liver 48–72 h after APAP admin-
istration and that loss of Gal-3 results in reduced hepatotoxicity
at these times and decreased expression of the proinflammatory
proteins, 24p3, MMP-9, MIP-3a, iNOS, and CD98. Neutrophil
influx into the liver is also suppressed. These findings are novel
and may have therapeutic implications for developing new
approaches to treating APAP overdose.
Effects of APAP on Serum Transaminases in WT and Gal-32/2Mice
ALT (U/l) AST (U/l)
32.1 ± 2.6
7797.0 ± 508.1a
10,905.8 ± 825.5a
1194.0 ± 126.1a
247.6 ± 41.4
45.4 ± 7.2
6363.1 ± 1266.9
7673.6 ± 1022.9a,b
454.2 ± 53.5b
116.6 ± 15.1b
60.6 ± 5.6
7473.0 ± 481.8a
6391.1 ± 911.4a
522.9 ± 51.8a
261.4 ± 29.3
82.5 ± 10.6
3908.2 ± 723.6a
265.7 ± 26.5b
93.8 ± 15.4b
Notes. ALT, alanine aminotransferase; AST, aspartate aminotransferase. WT and Gal-3?/?mice were treated with 300 mg/kg APAP or PBS control (CTL). Sera
were collected 6–72 h later and analyzed for ALT and AST. Data are expressed as mean ± SE (n ¼ 5–12 mice).
aSignificantly different (p < 0.05) from CTL.
bSignificantly different from WT.
into the liver. Livers were collected 24–72 h after treatment of WT and Gal-3?/?
mice with APAP or control (CTL). Sections were stained with anti-
myeloperoxidase antibody. One representative section from two independent
experiments is shown.
Effects of loss of Gal-3 on APAP-induced neutrophil emigration
Gal-3 REGULATES APAP-INDUCED INFLAMMATION
APAP administration resulted in a dramatic increase in Gal-3
expression in the liver, which was most prominent after 48–72 h.
Moreover, the major cell population expressing Gal-3 consisted
of mononuclear cells accumulating in necrotic areas, which have
previously been shown to display features of classically acti-
vated macrophages (Laskin and Pilaro, 1986). These findings are
in agreement with previous studies showing that Gal-3 is
upregulated in leukocytes infiltrating the liver during carbon
tetrachloride-induced fibrosis and Toxoplasma gondii infection
(Bernardes et al., 2006; Henderson et al., 2006). Our observation
that serum Gal-3 levels declined rapidly following APAP
administration is consistent with increased Gal-3 accumulation
in the liver and suggests a local, intrahepatic role for Gal-3 in this
model of injury. Blood monocytes have been shown to synthesize
Gal-3 (Weber et al., 2009); reduced serum levels after APAP may
also be due to increased emigration of these cells into the liver.
Gal-3 has been reported to be increased in various models
of tissue injury where it functions to stimulate macrophage
production of proinflammatory mediators (Norling et al.,
2009). Consistent with this activity are findings that loss of
Gal-3 is protective in antigen-induced arthritis (Forsman
et al., 2011), renal ischemia-reperfusion injury (Fernandes
with APAP or control (CTL). Samples were analyzed by real-time PCR. Each bar represents the mean ± SE (n ¼ 3–8 mice).aSignificantly different (p < 0.05) from
CTL.bSignificantly different (p < 0.05) from WT mice.
Effects of loss of Gal-3 on APAP-induced expression of inflammatory markers. Livers were collected 6–72 h after treatment of WT and Gal-3-/-mice
DRAGOMIR ET AL.
Bertocchi et al., 2008), neonatal hypoxic-ischemic brain injury
(Doverhag et al., 2010), streptozotocin-induced diabetes
(Mensah-Brown et al., 2009), diet-induced steatohepatitis
(Iacobini et al., 2011), and concanavalin A–induced liver
injury (Volarevic et al., forthcoming). Moreover, protection in
each of these models correlates with decreased levels of
proinflammatory mediators. Similarly, we found that APAP-
induced hepatotoxicity, as assessed histologically, by serum
transaminases and by expression of 24p3, was significantly
reduced in Gal-3?/?mice at 48–72 h, a time coordinate with
the accumulation of Gal-3-positive macrophages in livers of
WT mice. These findings together with the observation that
APAP-induced expression of iNOS, MIP-3a, and MMP-9,
proinflammatory proteins implicated in tissue injury (Gardner
et al., 2002; Ito et al., 2005; Schutyser et al., 2003), was
reduced at these times in Gal-3?/?mice, suggest that Gal-3
plays a role in promoting late inflammatory responses and
the persistence of hepatic injury. This is supported by our
finding that hepatotoxicity resolved more rapidly in Gal-3?/?
mice relative to WT mice. However, the observation that
APAP-induced hepatotoxicity was not completely prevented
by loss of Gal-3 indicates that factors released early after
injury, including damage-associated molecular patterns such as
HMGB1 and heat shock protein 70, contribute to the
pathogenic response to APAP (Dragomir et al., 2011;
Martin-Murphy et al., 2010).
with APAP or control (CTL). iNOS (upper panel) and COX-2 (lower panel) expression were analyzed by Western blotting. Densitometric analysis was performed
using ImageJ. Each bar represents the mean ± SE (n ¼ 3–5 mice).aSignificantly different (p < 0.05) from CTL.bSignificantly different (p < 0.05) from WT mice.
Effects of loss of Gal-3 on APAP-induced expression of iNOS and COX-2. Livers were collected from WT and Gal-3?/?mice 6–72 h after treatment
Gal-3 REGULATES APAP-INDUCED INFLAMMATION
Reactive nitrogen species generated via iNOS have been
shown to contribute to APAP-induced oxidative stress and
tissue injury (reviewed in Laskin, 2009). APAP intoxication
resulted in increased iNOS expression in the liver at 24–72 h.
Findings that decreased hepatotoxicity in Gal-3?/?mice
correlated with reduced iNOS expression suggest that Gal-3-
positive macrophages may be a source of reactive nitrogen
species. This is supported by reports that Gal-3 upregulates
iNOS expression in brain macrophages (Jeon et al., 2010).
MMP-9 is an extracellular matrix–degrading enzyme that
plays a role in microvascular injury induced by APAP (Ito
et al., 2005). As previously reported (Gardner et al., 2003),
MMP-9 expression increased in the liver following APAP
administration, a response most notable at 24 h. This was
significantly attenuated in Gal-3?/?mice and may contribute to
reduced toxicity in these animals. These data are in agreement
with reports that protection from hypoxic-ischemic brain injury
in the absence of Gal-3 was associated with lower MMP-9
levels (Doverhag et al., 2010).
MIP-3a (chemokine [C-C motif] ligand 20) is a chemokine
produced by macrophages and epithelial cells in response to
proinflammatory stimuli such as TNF-a, interferon-c, and
lipopolysaccharide, as well as Gal-3 (Papaspyridonos et al.,
2008; Schutyser et al., 2003). Expression of MIP-3a has also
been reported to increase in vivo during liver and brain
inflammation (Sugita et al., 2002; Utans-Schneitz et al., 1998).
Similarly, we found that expression of MIP-3a increased in the
liver following APAP intoxication and that this correlated with
increased numbers of Gal-3-positive macrophages. The fact
that loss of Gal-3 blunted the effects of APAP on MIP-3a
expression provides additional support for a role of Gal-3 in
promoting inflammation in the liver (Iacobini et al., 2011;
Volarevic et al., forthcoming).
In agreement with previous studies, we found that APAP
administration to WT mice resulted in increased expression of
MIP-2 and IL-1b in the liver (Dambach et al., 2006; Imaeda
et al., 2009; Liu et al., 2004). MIP-2 is a potent neutrophil
chemokine (Clarke et al., 2009). Whereas in WT mice, in-
creases in MIP-2 expression correlated with increased numbers
mRNA expression. Livers were collected 6–72 h after treatment of WT and Gal-
3-/-mice with APAP or control (CTL). Samples were analyzed by real-time PCR.
Each bar represents the mean ± SE (n ¼ 3–5 mice).aSignificantly different (p <
0.05) from CTL.bSignificantly different (p < 0.05) from WT mice.
Effects of loss of TNFR1 on APAP-induced Gal-3 and CD98
expression. Livers were collected 6–72 h after treatment of WT and TNFR1?/?
mice with APAP or control (CTL). Sections were stained with anti-Gal-3 antibody
as described in the Materials and Methods section. One representative section from
three independent experiments is shown. Original magnification, 320.
Effects of loss of TNFR1 on APAP-induced Gal-3 protein
DRAGOMIR ET AL.
of neutrophils in the liver, this was not observed in Gal-3?/?
mice; in these mice, a marked increase in MIP-2 expression
was noted 6 h after APAP intoxication, with no neutrophil
emigration into the liver at this time. These data suggest that
MIP-2 does not contribute significantly to early neutrophilic
responses to APAP. It has been suggested that MIP-2 may play
a protective role in the liver following APAP intoxication by
promoting hepatocyte regeneration (Hogaboam et al., 1999).
Our findings that MIP-2 expression is upregulated in Gal-3?/?
mice relative to WT mice and that this was associated with
reduced hepatotoxicity are in accord with this idea. The
observation that MIP-2 levels decline more rapidly in Gal-3?/?
mice is most likely due to reduced need for tissue repair pro-
cesses. In contrast to MIP-2, APAP-induced IL-1b expression
was unaffected by the loss of Gal-3. These results are
consistent with recent studies suggesting that IL-1b does not
play a role in the pathogenic response to APAP (Williams
et al., 2010). Gal-3 has been reported to upregulate IL-1b
expression in microglia (Jeon et al., 2010); moreover, tissue
IL-1b levels are reduced in Gal-3?/?mice after renal ischemia-
reperfusion injury (Fernandes Bertocchi et al., 2008). It may be
that IL-1b plays distinct roles in different models of tissue
The anti-inflammatory cytokine IL-10 has previously been
shown to be upregulated in the liver following APAP intoxi-
cation and to play a hepatoprotective role in the pathogenic
response (Bourdi et al., 2007; Dambach et al., 2006; Gardner
et al., 2002). Increases in IL-10 mRNA levels were observed in
both WT and Gal-3?/?mice 24 h after APAP administration.
Surprisingly, by 48 h, IL-10 levels were reduced in Gal-3?/?
mice relative to WT mice. This may be a consequence of
decreased inflammation and hepatotoxicity in these mice,
resulting in a reduced requirement for IL-10.
COX-2 catalyzes the biosynthesis of both pro- and anti-
inflammatory eicosanoids (Stables and Gilroy, 2011). We found
that COX-2 was constitutively expressed in livers of WT and
Gal-3?/?mice. This is likely due to continuous exposure to
endotoxin in the portal circulation (Ahmad et al., 2002; Naito
et al., 2004). Previous studies have shown that mice lacking
COX-2 are hypersensitive to APAP, suggesting that COX-2 is
important in limiting hepatotoxicity (Reilly et al., 2001). This is
thought to be due to increased generation of anti-inflammatory
prostaglandins. Interestingly, hepatic COX-2 expression was
reduced in Gal-3?/?mice relative to WT mice, suggesting
a positive regulatory role for Gal-3 in hepatic prostanoid
production. This is supported by our findings that constitutive
COX-2 levels declined in the livers of Gal-3?/?mice treated
with APAP. In contrast, Gal-3 appears to be negatively regulated
by COX-2 as evident from reports that Gal-3 is downregulated in
mice overexpressing COX-2 (Shen et al., 2007). COX-2 mRNA
has been reported to be upregulated in the liver following APAP
intoxication (Reilly et al., 2001). Conversely, we found no major
effects of APAP on COX-2 protein expression in WT mice.
These differences may be due to strain-specific responses and/or
analysis of protein expression in our studies versus mRNA
expression in earlier reports.
CD98 is a transmembrane glycosylated protein thought to
function as a receptor for Gal-3 on macrophages (Dong and
Hughes, 1997). Like Gal-3, CD98 is upregulated during tissue
injury and inflammation and contributes to disease pathogen-
esis (Nguyen et al., 2011). Following APAP intoxication,
CD98 increased in livers of WT mice; moreover, loss of Gal-3
blunted this response. This may contribute to the decreased
sensitivity of Gal-3?/?mice to APAP.
TNF-a signaling via TNFR1 plays a key role in limiting the
production of proinflammatory mediators and promoting
antioxidant generation and tissue repair in the liver following
APAP-induced toxicity (Chiu et al., 2003a,b; Gardner et al.,
2003). Consistent with this activity is our finding that APAP
administration to WT mice resulted in increased expression of
TNFR1, which paralleled the development of hepatotoxicity.
Surprisingly, reduced hepatotoxicity in Gal-3?/?mice was cor-
related with decreased levels of TNFR1, potentially reflecting
the reduced need for activation of protective signaling path-
ways in these mice. We previously reported that loss of TNFR1
results in an exaggerated hepatotoxic response to APAP (Chiu
et al., 2003a,b; Gardner et al., 2003). The present studies show
that this is associated with increased Gal-3 expression in the
liver. These data suggest that Gal-3 contributes to the increased
susceptibility of TNFR1?/?mice to APAP. This is likely due
to increased production of cytotoxic and proinflammatory me-
diators by Gal-3 positive–activated macrophages. In contrast to
loss of Gal-3, loss of TNFR1 had no effect on APAP-induced
expression of CD98. These results indicate that Gal-3 avail-
ability, and not receptor expression levels, is critical for the
development of toxicity. It may also be that a different receptor
mediates Gal-3-dependent responses in these mice.
Cyp2e1-dependent metabolism of APAP to NAPQI is a
critical step in the onset of hepatotoxicity (Lee et al., 1996;
collected 3–72 h after treatment of WT and Gal-3-/-mice with APAP or control
(CTL) and total glutathione levels assayed. Each bar represents the mean ± SE
(n ¼ 3–6 mice).aSignificantly different (p < 0.05) from CTL.
Effects of APAP on hepatic glutathione levels. Livers were
Gal-3 REGULATES APAP-INDUCED INFLAMMATION
Potter et al., 1973). Glutathione plays an important role in
limiting toxicity by forming a nonreactive conjugate with
NAPQI (Mitchell et al., 1973). Our data demonstrate that loss
of Gal-3 did not alter hepatic Cyp2e1 activity or result in
changes in hepatic glutathione levels in response to APAP.
Taken together, these findings indicate that reduced suscepti-
bility of Gal-3?/?mice to APAP-induced hepatotoxicity is not
due to effects on NAPQI formation or inactivation.
In summary, the present studies identify Gal-3 as a novel
regulator of late inflammatory responses in the liver following
APAP intoxication and an important contributor to hepatotox-
icity. Further studies are needed to identify the mechanisms
involved in Gal-3-dependent inflammatory responses during
the pathogenesis of APAP-induced liver injury.
Ahmad, N., Chen, L. C., Gordon, M. A., Laskin, J. D., and Laskin, D. L.
(2002). Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic
macrophages during acute endotoxemia. J. Leukoc. Biol. 71, 1005–1011.
Bernardes, E. S., Silva, N. M., Ruas, L. P., Mineo, J. R., Loyola, A. M.,
Hsu, D. K., Liu, F. T., Chammas, R., and Roque-Barreira, M. C. (2006).
Toxoplasma gondii infection reveals a novel regulatory role for galectin-3 in
the interface of innate and adaptive immunity. Am. J. Pathol. 168, 1910–1920.
Borkham-Kamphorst, E., Drews, F., and Weiskirchen, R. (2011). Induction of
lipocalin-2 expression in acute and chronic experimental liver injury
moderated by pro-inflammatory cytokines interleukin-1b through nuclear
factor-kappaB activation. Liver Int. 31, 656–665.
Bourdi, M., Eiras, D. P., Holt, M. P., Webster, M. R., Reilly, T. P.,
Welch, K. D., and Pohl, L. R. (2007). Role of IL-6 in an IL-10 and IL-4
double knockout mouse model uniquely susceptible to acetaminophen-
induced liver injury. Chem. Res. Toxicol. 20, 208–216.
Chang, T. K. H., Crespi, C. L., and Waxman, D. J. (1998). Spectrophotometric
analysis of human CYP2E1-catalyzed p-nitrophenol hydroxylation. In
Methods in Molecular Biology. Cytochrome P450 Protocols (I. R. Phillips
and E. A. Shephard, Eds.), Vol. 107, pp. 147–152. Humana Press, Inc.,
Chiu, H., Gardner, C. R., Dambach, D. M., Brittingham, J. A., Durham, S. K.,
Laskin, J. D., and Laskin, D. L. (2003a). Role of p55 tumor necrosis factor
receptor 1 in acetaminophen-induced antioxidant defense. Am. J. Physiol.
Gastrointest. Liver Physiol. 285, G959–G966.
Chiu, H., Gardner, C. R., Dambach, D. M., Durham, S. K., Brittingham, J. A.,
Laskin, J. D., and Laskin, D. L. (2003b). Role of tumor necrosis factor
receptor 1 (p55) in hepatocyte proliferation during acetaminophen-induced
toxicity in mice. Toxicol. Appl. Pharmacol. 193, 218–227.
Clarke, C. N., Kuboki, S., Tevar, A., Lentsch, A. B., and Edwards, M. (2009).
CXC chemokines play a critical role in liver injury, recovery, and
regeneration. Am. J. Surg. 198, 415–419.
Cook, J. A. (2005). Eicosanoids. Crit. Care Med. 33, S488–S491.
Dahlin, D. C., Miwa, G. T., Lu, A. Y., and Nelson, S. D. (1984). N-acetyl-p-
benzoquinone imine: A cytochrome P-450-mediated oxidation product of
acetaminophen. Proc. Natl. Acad. Sci. U.S.A. 81, 1327–1331.
Dambach, D. M., Durham, S. K., Laskin, J. D., and Laskin, D. L. (2006).
Distinct roles of NF-jB p50 in the regulation of acetaminophen-induced
inflammatory mediator production and hepatotoxicity. Toxicol. Appl.
Pharmacol. 211, 157–165.
Dong, S., and Hughes, R. C. (1997). Macrophage surface glycoproteins binding
to galectin-3 (Mac-2-antigen). Glycoconj. J. 14, 267–274.
Doverhag, C., Hedtjarn, M., Poirier, F., Mallard, C., Hagberg, H., Karlsson, A.,
and Savman, K. (2010). Galectin-3 contributes to neonatal hypoxic-ischemic
brain injury. Neurobiol. Dis. 38, 36–46.
Dragomir, A. C., Laskin, J. D., and Laskin, D. L. (2011). Macrophage
activation by factors released from acetaminophen-injured hepatocytes:
Potential role of HMGB1. Toxicol. Appl. Pharmacol. 253, 170–177.
Fernandes Bertocchi, A. P., Campanhole, G., Wang, P. H., Goncalves, G. M.,
Damiao, M. J., Cenedeze, M. A., Beraldo, F. C., de Paula Antunes
Teixeira, V., Dos Reis, M. A., Mazzali, M., et al. (2008). A role for
galectin-3 in renal tissue damage triggered by ischemia and reperfusion injury.
Transpl. Int. 21, 999–1007.
Forsman, H., Islander, U., Andreasson, E., Andersson, A., Onnheim, K.,
Karlstrom, A., Savman, K., Magnusson, M., Brown, K. L., and Karlsson, A.
(2011). Galectin 3 aggravates joint inflammation and destruction in antigen-
induced arthritis. Arthritis Rheum. 63, 445–454.
Gardner, C. R., Gray, J. P., Joseph, L. B., Cervelli, J., Bremer, N., Kim, Y.,
Mishin, V., Laskin, J. D., and Laskin, D. L. (2010). Potential role of
caveolin-1 in acetaminophen-induced hepatotoxicity. Toxicol. Appl.
Pharmacol. 245, 36–46.
Gardner, C. R., Laskin, J. D., Dambach, D. M., Chiu, H., Durham, S. K.,
Zhou, P., Bruno, M., Gerecke, D. R., Gordon, M. K., and Laskin, D. L.
(2003). Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor
necrosis factor receptor-1. Potential role of inflammatory mediators. Toxicol.
Appl. Pharmacol. 192, 119–130.
Gardner, C. R., Laskin, J. D., Dambach, D. M., Sacco, M., Durham, S. K.,
Bruno, M. K., Cohen, S. D., Gordon, M. K., Gerecke, D. R., Zhou, P., et al.
(2002). Reduced hepatotoxicity of acetaminophen in mice lacking inducible
nitric oxide synthase: Potential role of tumor necrosis factor-a and
interleukin-10. Toxicol. Appl. Pharmacol. 184, 27–36.
Henderson, N. C., Mackinnon, A. C., Farnworth, S. L., Poirier, F., Russo, F. P.,
Iredale, J. P., Haslett, C., Simpson, K. J., and Sethi, T. (2006). Galectin-3
regulates myofibroblast activation and hepatic fibrosis. Proc. Natl. Acad. Sci.
U.S.A. 103, 5060–5065.
Henderson, N. C., and Sethi, T. (2009). The regulation of inflammation by
galectin-3. Immunol. Rev. 230, 160–171.
Hogaboam, C. M., Simpson, K. J., Chensue, S. W., Steinhauser, M. L.,
Lukacs, N. W., Gauldie, J., Strieter, R. M., and Kunkel, S. L. (1999).
Macrophage inflammatory protein-2 gene therapy attenuates adenovirus- and
acetaminophen-mediated hepatic injury. Gene Ther. 6, 573–584.
Hsu, D. K., Yang, R. Y., Pan, Z., Yu, L., Salomon, D. R., Fung-Leung, W. P.,
and Liu, F. T. (2000). Targeted disruption of the galectin-3 gene results
in attenuated peritoneal inflammatory responses. Am. J. Pathol. 156,
Iacobini, C., Menini, S., Ricci, C., Fantauzzi, C. B., Scipioni, A., Salvi, L.,
Cordone, S., Delucchi, F., Serino, M., Federici, M., et al. (2011). Galectin-3
ablation protects mice from diet-induced NASH: A major scavenging role for
galectin-3 in liver. J. Hepatol. 54, 975–983.
Imaeda, A. B., Watanabe, A., Sohail, M. A., Mahmood, S., Mohamadnejad, M.,
Sutterwala, F. S., Flavell, R. A., and Mehal, W. Z. (2009). Acetaminophen-
induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3
inflammasome. J. Clin. Investig. 119, 305–314.
Ishida, Y., Kondo, T., Tsuneyama, K., Lu, P., Takayasu, T., and
Mukaida, N. (2004). The pathogenic roles of tumor necrosis factor
receptor p55 in acetaminophen-induced liver injury in mice. J. Leukoc.
Biol. 75, 59–67.
DRAGOMIR ET AL.
Ito, Y., Abril, E. R., Bethea, N. W., and McCuskey, R. S. (2005). Inhibition of
matrix metalloproteinases minimizes hepatic microvascular injury in
response to acetaminophen in mice. Toxicol. Sci. 83, 190–196.
Janelle-Montcalm, A., Boileau, C., Poirier, F., Pelletier, J. P., Guevremont, M.,
Duval, N., Martel-Pelletier, J., and Reboul, P. (2007). Extracellular
localization of galectin-3 has a deleterious role in joint tissues. Arthritis
Res. Ther. 9, R20.
Jeon, S. B., Yoon, H. J., Chang, C. Y., Koh, H. S., Jeon, S. H., and Park, E. J.
(2010). Galectin-3 exerts cytokine-like regulatory actions through the JAK-
STAT pathway. J. Immunol. 185, 7037–7046.
Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R., and
Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. II. Role of
covalent binding in vivo. J. Pharmacol. Exp. Ther. 187, 195–202.
Laskin, D. L. (2009). Macrophages and inflammatory mediators in chemical
toxicity: A battle of forces. Chem. Res. Toxicol. 22, 1376–1385.
Laskin, D. L., Gardner, C. R., Price, V. F., and Jollow, D. J. (1995).
Modulation of macrophage functioning abrogates the acute hepatotoxicity of
acetaminophen. Hepatology 21, 1045–1050.
Laskin, J. D., Heck, D. E., and Laskin, D. L. (2010). Nitric oxide pathways in
toxic responses. In General and Applied Toxicology, Chapter 17, pp. 425–
438, 3rd ed. (B. Ballantyne, T. Marrs, T. Syversen, Eds.) Wiley-Blackwell,
Laskin, D. L., and Pilaro, A. M. (1986). Potential role of activated macrophages
in acetaminophen hepatotoxicity. I. Isolation and characterization of
activated macrophages from rat liver. Toxicol. Appl. Pharmacol. 86,
Laskin, D. L., Pilaro, A. M., and Ji, S. (1986). Potential role of activated
macrophages in acetaminophen hepatotoxicity. II. Mechanism of macro-
phage accumulation and activation. Toxicol. Appl. Pharmacol. 86, 216–226.
Laskin, D. L., Sunil, V. R., Gardner, C. R., and Laskin, J. D. (2011).
Macrophages and tissue injury: Agents of defense or destruction? Annu. Rev.
Pharmacol. Toxicol. 51, 267–288.
Lee, S. S., Buters, J. T., Pineau, T., Fernandez-Salguero, P., and Gonzalez, F. J.
(1996). Role of CYP2E1 in the hepatotoxicity of acetaminophen. J. Biol.
Chem. 271, 12063–12067.
Lee, W. M., Squires, R. H., Jr, Nyberg, S. L., Doo, E., and Hoofnagle, J. H.
(2008). Acute liver failure: Summary of a workshop. Hepatology 47,
Liu, F. T., Hsu, D. K., Zuberi, R. I., Kuwabara, I., Chi, E. Y.,
Henderson, W. R., Jr (1995). Expression and function of galectin-3, a
b-galactoside-binding lectin, in human monocytes and macrophages. Am. J.
Pathol. 147, 1016–1028.
Liu, Z. X., Govindarajan, S., and Kaplowitz, N. (2004). Innate immune system
plays a critical role in determining the progression and severity of
acetaminophen hepatotoxicity. Gastroenterology 127, 1760–1774.
Martin-Murphy, B. V., Holt, M. P., and Ju, C. (2010). The role of damage
associated molecular pattern molecules in acetaminophen-induced liver
injury in mice. Toxicol. Lett. 192, 387–394.
Mensah-Brown, E. P., Al Rabesi, Z., Shahin, A., Al Shamsi, M.,
Arsenijevic, N., Hsu, D. K., Liu, F. T., and Lukic, M. L. (2009). Targeted
disruption of the galectin-3 gene results in decreased susceptibility to
multiple low dose streptozotocin-induced diabetes in mice. Clin. Immunol.
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B.
(1973). Acetaminophen-induced hepatic necrosis. IV. Protective role of
glutathione. J. Pharmacol. Exp. Ther. 187, 211–217.
Naito, M., Hasegawa, G., Ebe, Y., Yamamoto, T., et al. (2004). Differentiation
and function of Kupffer cells. Med. Electron Microsc. 37, 16–28.
Nguyen, H. T., Dalmasso, G., Torkvist, L., Halfvarson, J., Yan, Y., Laroui, H.,
Shmerling, D., Tallone, T., D’Amato, M., Sitaraman, S. V., et al. (2011).
CD98 expression modulates intestinal homeostasis, inflammation, and
colitis-associated cancer in mice. J. Clin. Invest. 121, 1733–1747.
Nishi, Y., Sano, H., Kawashima, T., Okada, T., Kuroda, T., Kikkawa, K.,
Kawashima, S., Tanabe, M., Goto, T., Matsuzawa, Y., et al. (2007). Role of
galectin-3 in human pulmonary fibrosis. Allergol. Int. 56, 57–65.
Norling, L. V., Perretti, M., and Cooper, D. (2009). Endogenous galectins and
the control of the host inflammatory response. J. Endocrinol. 201, 169–184.
Papaspyridonos, M., McNeill, E., de Bono, J. P., Smith, A., Burnand, K. G.,
Channon, K. M., and Greaves, D. R. (2008). Galectin-3 is an amplifier of
inflammation in atherosclerotic plaque progression through macrophage
activation and monocyte chemoattraction. Arterioscler. Throm. Vasc. Biol.
Potter, W. Z., Davis, D. C., Mitchell, J. R., Jollow, D. J., Gillette, J. R., and
Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. 3. Cytochrome
P-450-mediated covalent binding in vitro. J. Pharmacol. Exp. Ther. 187,
Reilly, T. P., Brady, J. N., Marchick, M. R., Bourdi, M., George, J. W.,
Radonovich, M. F., Pise-Masison, C. A., and Pohl, L. R. (2001). A protective
role for cyclooxygenase-2 in drug-induced liver injury in mice. Chem. Res.
Toxicol. 14, 1620–1628.
Roudkenar, M. H., Kuwahara, Y., Baba, T., Roushandeh, A. M., Ebishima, S.,
Abe, S., Ohkubo, Y., and Fukumoto, M. (2007). Oxidative stress induced
lipocalin 2 gene expression: Addressing its expression under the harmful
conditions. J. Radiat. Res. (Tokyo) 48, 39–44.
Schutyser, E., Struyf, S., and Van Damme, J. (2003). The CC chemokine
CCL20 and its receptor CCR6. Cytokine Growth Factor Rev. 14, 409–426.
Shen, J., Pavone, A., Mikulec, C., Hensley, S. C., Traner, A., Chang, T. K.,
Person, M. D., and Fischer, S. M. (2007). Protein expression profiles in the
epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel
electrophoresis and mass spectrometry. J. Proteome Res. 6, 273–286.
Stables, M. J., and Gilroy, D. W. (2011). Old and new generation lipid
mediators in acute inflammation and resolution. Prog. Lipid Res. 50, 35–51.
Sugita, S., Kohno, T., Yamamoto, K., Imaizumi, Y., Nakajima, H.,
Ishimaru, T., and Matsuyama, T. (2002). Induction of macrophage-
inflammatory protein-3a gene expression by TNF-dependent NF-jB
activation. J. Immunol. 168, 5621–5628.
Sunil, V. R., Patel, K. J., Nilsen-Hamilton, M., Heck, D. E., Laskin, J. D., and
Laskin, D. L. (2007). Acute endotoxemia is associated with upregulation of
lipocalin 24p3/Lcn2 in lung and liver. Exp. Mol. Pathol. 83, 177–187.
Utans-Schneitz, U., Lorez, H., Klinkert, W. E., da Silva, J., and Lesslauer, W.
(1998). A novel rat CC chemokine, identified by targeted differential display,
is upregulated in brain inflammation. J. Neuroimmunol. 92, 179–190.
Volarevic, V., Milovanovic, M., Ljujic, B., Pejnovic, N., Arsenijevic, N.,
Nilsson, U., Leffler, H., and Lukic, M. L. (Forthcoming). Galectin-3 deficiency
prevents concanavalin A-induced hepatitis in mice. Hepatology.
Weber, M., Sporrer, D., Weigert, J., Wanninger, J., Neumeier, M., Wurm, S.,
Stogbauer, F., Kopp, A., Bala, M., Schaffler, A., et al. (2009). Adiponectin
downregulates galectin-3 whose cellular form is elevated whereas its soluble
form is reduced in type 2 diabetic monocytes. FEBS Lett. 583, 3718–3724.
Williams, C. D., Farhood, A., and Jaeschke, H. (2010). Role of caspase-1 and
interleukin-1b in acetaminophen-induced hepatic inflammation and liver
injury. Toxicol. Appl. Pharmacol. 247, 169–178.
Zuberi, R. I., Hsu, D. K., Kalayci, O., Chen, H. Y., Sheldon, H. K., Yu, L.,
Apgar, J. R., Kawakami, T., Lilly, C. M., and Liu, F. T. (2004). Critical role
for galectin-3 in airway inflammation and bronchial hyperresponsiveness in
a murine model of asthma. Am. J. Pathol. 165, 2045–2053.
Gal-3 REGULATES APAP-INDUCED INFLAMMATION