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Carcinogenesis |
Laboratory of Hepatobiology and Toxicology, Department of Pharmacology [I. R., S. Y., R. G. T.], Curriculum in Toxicology [I. R., J. A. S., R. G. T.], and Department of Environmental Sciences and Engineering [R. S., J. A. S.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892 [B. H. S., S. M. H.]; and Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709 [R. C. C.].
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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B (NF-B) in
Kupffer cells in vivo and activated superoxide
production by isolated Kupffer cells. Here, we tested the hypothesis
that NADPH oxidase (NADPH OX) is the source of oxidants increased by
Wy-14,643. Indeed, both activation of NF-B and increases in cell
proliferation due to a single dose of Wy-14,643 (100 mg/kg) were
prevented completely when rats were pretreated with diphenyleneiodonium
(1 mg/kg), an inhibitor of NADPH OX. p47phox is a critical
subunit of NADPH OX; therefore, p47phox knockout mice were
used to specifically address the hypothesis of NADPH OX involvement. In
livers of wild-type mice, Wy-14,643 activated NF-B, followed by an
increase in mRNA for tumor necrosis factor 
. Importantly, these
changes did not occur in p47phox knockouts. Moreover, when
Kupffer cells were treated with Wy-14,643 in vitro,
superoxide production was increased in cells from wild-type but not
p47phox-null mice. Finally, when mice were fed a
Wy-14,643-containing (0.1%) diet for 7 days, the increase in liver
weight and cell proliferation caused by Wy-14,643 in wild-type mice was
blocked in p47phox-null mice. Combined, these results are
consistent with the hypothesis that Wy-14,643 activates NADPH OX, which
leads to NF-B-mediated production of mitogens that causes
hepatocellular proliferation characteristic of this class of
nongenotoxic carcinogens. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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B3
activation in Kupffer cells plays a critical role in increased
hepatocyte proliferation caused by Wy-14,643 (2)
and
ciprofibrate (3)
suggest that these mechanisms may not be
mutually exclusive (i.e., that oxidants could play a role in
signaling production of mitogens in specialized cells in the liver). Cell proliferation has been linked experimentally and conceptually to carcinogenesis induced by both genotoxic and nongenotoxic carcinogens as a crucial event in converting DNA damage to heritable mutations and causing clonal expansion of mutated cell populations (4) . Increased proliferation of hepatocytes observed during continued treatment with peroxisome proliferators is thought to contribute to the carcinogenic process. Indeed, it was found that sustained increases in cell proliferation correlated with tumor number (5) . The idea that spontaneously initiated cells are promoted by this class of compounds was supported by the observation that more tumors develop in older than in younger rats fed nafenopin or Wy-14,643, although tumors were found in both of these groups (6 , 7) . Furthermore, these chemicals induce preferential growth of altered hepatocytes in preneoplastic foci (8) .
It was recently suggested that low levels of oxidants may play a role
in signaling increases in cell proliferation caused by peroxisome
proliferators via a Kupffer cell-mediated mechanism involving TNF
and NF-B (9)
. Indeed, hepatocyte proliferation caused
by Wy-14,643 was prevented by inactivation of Kupffer cell production
of TNF with methyl palmitate (10)
, antibodies to TNF
(11)
, or dietary glycine (12)
. Furthermore,
activation of NF-B by Wy-14,643 occurs rapidly in Kupffer cells and
precedes changes in hepatocytes (2)
. Moreover, Wy-14,643
caused superoxide anion production in isolated Kupffer cells
(13)
. Oxidants are known to play a major role in the
activation of NF-B. Indeed, pretreatment of rats with allopurinol, a
free radical scavenger (14)
, abolished Wy-14,643-induced
activation of NF-B (2)
. Furthermore, when catalase was
overexpressed in mouse liver, the activation of NF-B and increase in
cell proliferation caused by treatment with the peroxisome proliferator
ciprofibrate was decreased significantly (3)
.
Collectively, these observations support the concept that oxidants play
a significant role in the peroxisome proliferator-induced proliferative
response. Because it is unclear how peroxisome proliferators increase
production of reactive oxygen species, these studies were designed to
determine whether NADPH OX, a major superoxide-producing enzyme in
macrophages, is involved in the oxidant-dependent activation of NF-B
by the peroxisome proliferator Wy-14,643 in Kupffer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays.
Nuclear protein extracts were prepared on ice as described by Dignam
et al. (16)
with minor modifications
(2)
. Protein concentration was determined using the
Bradford protein concentration assay kit (Bio-Rad, Hercules, CA; Ref.
17
). A gel mobility shift assay was used in this study to
assess the amount of active protein involved in protein-DNA
interactions. Binding conditions for NF-B were characterized, and
electrophoretic mobility shift assays were performed as described in
detail elsewhere (18)
. Briefly, equal amounts (40 µg) of nuclear extract were preincubated 10 min on ice with 1 µg of
poly (dI-dC) and 20 µg of BSA (both from Pharmacia Biotech,
Piscataway, NJ) and 2 µl of a 32P-labeled DNA
probe (10,000 cpm/µl; Cerenkov) containing 1 ng of double-stranded
oligonucleotide in a total volume of 20 µl. Mixtures were incubated
20 min on ice and resolved on 5% polyacrylamide (29:1 cross-linking)
and 0.4x Tris-borate EDTA gels. After electrophoresis, gels were dried
and exposed to Kodak film. In supershift experiments, 1 µl of rabbit
antisera against p50 and p65 protein (a kind gift from Dr. N. R. Rice,
National Cancer Institute, Bethesda, MD) was added to the reaction
mixture after incubation with a labeled probe that was further
incubated at 25°C for 30 min. Labeled and unlabeled oligonucleotides
contained the consensus sequence for NF-B (19)
. Data
were quantitated by scanning autoradiograms with GelScan XL (Pharmacia
LKB, Uppsala, Sweden).
Isolation of RNA and RNase Protection Assay.
Samples of liver tissue were homogenized in 2 ml of RNAzol B solution,
and total cellular RNA was extracted according to standard procedures
(20)
. RNA was dissolved in Tris-EDTA buffer [10
mM Tris-HCl and 1 mM EDTA (pH 8.0)]. Rodent
cytokine multinucleotide RNA probe template sets (rCK-1 for rat tissue
and mCK-3b for mouse tissue; PharMingen, San Diego, CA) were used for
synthesis of radiolabeled ([32P]UTP) antisense
RNA probes, and RNase protection assays were performed on 30 µg of
individual liver RNA samples using a RiboQuant multiprobe RNase
Protection assay kit (PharMingen). Protected fragments were separated
on 6% polyacrylamide QuickPoint (NOVEX, San Diego, CA) gels, dried,
and exposed to X-ray film. Intensities of protected bands were
quantified using an image analyzer.
Superoxide Production by Kupffer Cells.
Rat and mouse livers were perfused as described elsewhere
(21)
, and the nonparenchymal cell fractions were separated
by centrifugation through Percoll gradients based on the method of
Smedsrod and Pertoft (22)
. Nonparenchymal cells were
seeded at a density of 106 cells/well in 24-well
plates and cultured in RPMI 1640 containing low glucose and 10% fatty
acid-free BSA. After 1 h, nonadherent cells were removed by
replacing the culture media and adherent cells were verified to be
Kupffer cells by phagocytosis of FITC-labeled latex beads (1 µm
diameter; Polysciences, Warrington, PA) by fluorescent microscopy
(23)
. Cells were cultured for 24 h prior to all
experiments. Subsequently, media was removed and Kupffer cells were
washed twice with HBSS. Wy-14,643 (10 µM) was added
to cells and incubated in HBSS at 37°C. After 30 min, cytochrome
c (50 µM) was added to each well and
the reaction was allowed to proceed for an additional 30 min at 37°C.
Superoxide generation was assessed as reduction of cytochrome
c inhibitable by superoxide dismutase, as described
elsewhere (13)
.
Cell Proliferation.
Rats were given BrdUrd (100 mg/kg i.p.; Sigma Chemical Co.) 1 h
before sacrifice. In experiments with mice, cell proliferation was
assessed with osmotic pumps (Alzet model 2001, 1 µl/hr; Alza Corp.,
Palo Alto, CA), which were implanted s.c. and contained 200 µl of 16
mg/ml BrdUrd. After sacrifice, livers were rinsed with HBSS and fixed
with 4% paraformaldehyde for subsequent paraffin embedding. A section
of duodenum, a tissue that proliferates rapidly, was collected as a
positive control for BrdUrd incorporation. Tissue sections (5 µm)
were deparaffinized, rehydrated, and hydrolyzed in 4 N HCl for 20 min
at 37°C. Immunohistochemical staining was performed using a DAKO
Envision System Peroxidase Staining Kit (DAKO, Carpinteria, CA) and a
primary monoclonal antibody to BrdUrd (clone Bu20a; DAKO), as described
elsewhere (12)
. Cel1 proliferation was quantitated by
determining the percentage of BrdUrd-positive hepatocytes in 10 random
high-power fields/slide (1000 hepatocytes/slide).
Acyl-CoA Activity.
Acyl-CoA oxidase is localized in peroxisomes, and its activity,
measured as formaldehyde formed from hydrogen peroxide generated by
peroxisomal ß-oxidation, is a measure of induction of peroxisomes
(24)
. Liver samples (
100 µg) were homogenized in 10
volumes of 0.25 M sucrose buffer. A reaction mixture (1.4 ml; for
details see Ref. 12
) was warmed to 37°C and mixed with
200 µl of homogenate. The reaction was terminated after 10 min with
40% trichloracetic acid. Trichloracetic acid was added before
homogenate to the blanks. The solution was centrifuged to pellet
protein, and 0.5 ml of supernatant was added to 0.2 ml of Nash Reagent
to measure formaldehyde (25)
. After 60 min of incubation
at 37°C, absorbance was read at 405 nm. Protein concentration was
determined by the method of Bradford (17)
.
Statistics.
Results are reported as means ± SEM with
n = 4 to 5 in each group. Treatment groups
were compared using one-way ANOVA, followed by Student-Neuman-Keuls
post-hoc test, or two-way ANOVA using Student-Neuman-Keuls post-hoc
test, where appropriate. A P < 0.05 was
selected before the study to determine statistical differences between
groups.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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B,
TNF, and Cell Proliferation in Rat Liver Are Blocked by Inhibiting
Generation of Superoxide.B in Kupffer cells
from rats treated with the peroxisome proliferator Wy-14,643 was free
radical dependent because it was blocked by the radical scavenger
allopurinol (2)
. Because NADPH OX is a major source of
reactive oxygen species in activated macrophages, such as Kupffer
cells, DPI, an inhibitor of the NADPH OX (26)
, was used to
test the hypothesis that reactive oxygen species are involved in
peroxisome proliferator-induced stimulation of growth of parenchymal
cells. Rats were pretreated with DPI (1 mg/kg/day s.c.) for 4 days
before administration of Wy-14,643 [100 mg/kg intradermally (i.g.)].
The dose of DPI and route of administration were selected based on:
(a) the IC50 value of 6 mg/kg for rat
(27)
; and (b) the proven pharmacological
effectiveness of DPI in a long-term study in rats (28)
.
In a whole liver, Wy-14,643 causes a 3.5-fold increase in NF-B
activity as early as 2 h after treatment (2
; Fig. 1
). This activation was blocked completely by pretreatment with DPI (Fig. 1)
. Similarly, WY-14,643 increased TNF mRNA about 2-fold, which is
regulated by NF-B, an effect also prevented by DPI (data not shown).
It is known that NF-B is activated by peroxisome proliferators first
in Kupffer cells (2)
. Moreover, Kupffer cells are the main
source of TNF in the liver (29)
, and TNF is a direct
hepatocyte mitogen (30)
. To test whether Kupffer cell
NADPH OX is inhibited by DPI, Kupffer cells were isolated from
untreated rats and cultured in the presence or absence of DPI
(15 µM). LPS (100 ng/ml), a known activator of NADPH OX
in macrophages, was added. Indeed, LPS increased superoxide anion
production by Kupffer cells by 5.5-fold (control, 0.4 ± 0.2 nmol/106 cells/15 min; LPS,
2.2 ± 0.2 nmol/106 cells/15 min),
and DPI blocked this increase completely (0.2 ± 0.1
nmol/106 cells/15 min). Therefore, it is
concluded that DPI blocks superoxide-generating capacity of Kupffer
cells by inhibiting NADPH OX (see below).
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B, and TNF have been suggested as the
signaling pathway activated by peroxisome proliferators in rodent
liver, which leads to increased hepatocellular proliferation (9
, 31)
. Indeed, when cell proliferation was assessed 24 h
after gavage with Wy-14,643, a 5-fold increase in DNA synthesis caused
by WY-14,643 was prevented by DPI (Fig. 2A
, WY and WY+DPI). Treatments with
olive oil, a vehicle for Wy-14,643, and 5% glucose, a vehicle for DPI,
or DPI alone had no effect on either cell proliferation (CON
and DPI, respectively) or activity of NF-B (data not
shown). Importantly, a 3-fold induction of peroxisomes, which was
assessed by measuring activity of the marker enzyme of peroxisomal
proliferation, acyl-CoA-oxidase, was not affected by DPI (Fig. 2B)
. These results support the hypothesis (5
, 31)
that up-regulation of peroxisomal enzymes occurs
independently of activation of proliferation of hepatocytes.
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NADPH OX Is the Molecular Source of Oxidants Produced by Kupffer
Cells Due to Peroxisome Proliferators.
Peroxisomal ß-oxidation of fatty acids is unique because it
produces H2O2 instead of
NADH; therefore, it was initially thought that these compounds caused
oxidative stress leading to oxidized DNA bases (32)
.
Despite the fact that oxidized bases have been detected
(33)
, this idea has been challenged (34)
.
First, several studies failed to detect DNA adducts or modified DNA
bases after chronic exposure to plasticizers or other peroxisome
proliferators (35)
. Furthermore, given the extremely high
rate at which peroxisomal catalase converts
H2O2 into
H2O,
H2O2 should not escape
peroxisomes (36)
. In fact, it was shown that treatment
with ciprofibrate and perfluorooctanoate increased
H2O2 in vitro but not
in the perfused liver where fatty acid supply is rate limiting for
H2O2 generation via
peroxisomal ß-oxidation (37)
. Moreover, spontaneous
liver tumors occur in mice lacking peroxisomal fatty acyl-CoA oxidase
(38)
. On the other hand, recent evidence that reactive
oxygen species are produced by Kupffer cells and are involved in
triggering cell proliferation suggests a new role for oxidants in the
molecular mechanism of peroxisome proliferators.
To specifically address the question of whether NADPH OX is the
molecular source of oxidants produced due to Wy-14,643, knockout mice
that lack the p47phox component of this enzyme
were used (15)
. First, p47phox-null
(-/-) and wild-type (+/+) mice were gavaged with Wy-14,643 (100
mg/kg) for different times up to 24 h, and NF-B activity was
assessed in whole liver nuclear extracts (Fig. 3)
. The activation of NF-B in liver of wild-type mice was time
dependent with a peak at 5–8 h (2-fold) after Wy-14,643
administration, followed by a steady decline toward basal levels.
However, no activation of NF-B was observed in
p47phox (-/-) mice. Similarly, a significant
increase in activity of NF-B was observed in liver of wild-type mice
given Wy-14,643 for 5 h; however, no changes were detected in
knockout mice (data not shown). Furthermore, mRNA for TNF increased
in a time-dependent manner and was nearly 3-fold greater after
treatment with Wy-14,643 in p47phox (+/+) mice in
24 h (Fig. 4)
. Importantly, Wy-14,643 failed to up-regulate TNF mRNA levels in
p47phox (-/-) mice.
|
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.
|
. Importantly, this increase in relative mean liver weight
due to Wy-14,643 administration did not occur in
p47phox (-/-) mice (Fig. 6A)
.
Moreover, the increase in liver weight in wild-type mice fed a
Wy-14,643 diet was accompanied by a significant 6-fold increase in
BrdUrd labeling index compared with wild-type controls (Fig. 6B)
. However, hepatocellular BrdUrd labeling index was not
increased by Wy-14,643 in p47phox (-/-) mice.
In contrast, acyl-CoA oxidase was increased equally in livers of
wild-type and knockout mice treated with Wy-14,643 for either 24 h
(data not shown) or 7 days (Fig. 6C)
.
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, thus triggering hepatocyte
proliferation (see Fig. 7
). Moreover, evidence that Kupffer cells are involved in the mechanism
of action of phthalates has also been presented (9)
. In
this study, it was demonstrated that Kupffer cell NADPH OX is activated
leading to production of superoxide
(O2·-), which activates nuclear factor
NF-B, a transcription factor that regulates TNF production;
however, it is not clear how Wy-14,643 activates NADPH OX.
Phosphorylation of p47phox and other proteins
that comprise this multiunit oxidant-producing enzyme leads to the
assembly of the complex on the membrane and production of superoxide
anion (40)
. It is hypothesized that peroxisome
proliferators activate NADPH OX via PKC, an enzyme involved in
increased cell proliferation and tumor promotion (41)
.
Indeed, PKC is known to activate NADPH OX in macrophages via
phosphorylation of p47phox, a regulatory subunit
of this enzyme (42)
. Recently, convincing evidence for
activation of PKC in Kupffer cells by peroxisome proliferators has been
presented (13)
.
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FOOTNOTES |
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1 Supported in part by Grant ES-04325 from the
National Institute of Environmental Health Sciences. 
2 To whom requests for reprints should be
addressed, at Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, CB #7365, University of North Carolina, Chapel Hill, NC
27599-7365. Phone: (919) 966-4745; Fax: (919) 966-1893; E-mail: thurman{at}med.unc.edu
3 The abbreviations used are: NF-B, nuclear
factor B; Wy-14,643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio
acetic acid; DPI, diphenyleneiodonium sulfate; PKC, protein kinase C;
TNF, tumor necrosis factor ; NADPH OX, NADPH oxidase; DEHP,
di(2-ethylhexyl) phthalate; BrdUrd, 5-bromo-2'-deoxyuridine; LPS,
lipopolysaccaride.
Received 12/28/99. Accepted 7/ 6/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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B in Kupffer cells before hepatocytes. Carcinogenesis (Lond.), 19: 1217-1222, 1998.B activation and DNA synthesis induced by the peroxisome proliferator ciprofibrate. Carcinogenesis (Lond.), 19: 631-637, 1998. prevent increases in cell replication in liver due to the potent peroxisome proliferator, WY-14,643. Carcinogenesis (Lond.), 18: 669-674, 1997.B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition. J. Biol. Chem., 266: 252-260, 1991.D subunit of active NF-B is required for inhibition of NF-B by I B. Genes Dev., 3: 1689-1698, 1989. is a direct hepatocyte mitogen in the rat. Biochem. Mol. Biol. Int., 29: 1-4, 1993.[Medline]
. Drug Metab. Rev., 29: 235-260, 1997.[Medline]
is restricted to hepatic parenchymal cells, not Kupffer cells: implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis. Carcinogenesis (Lond.), 21: 823-826, 2000.This article has been cited by other articles:
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  C. G. Woods, A. M. Burns, B. U. Bradford, P. K. Ross, O. Kosyk, J. A. Swenberg, M. L. Cunningham, and I. Rusyn WY-14,643 Induced Cell Proliferation and Oxidative Stress in Mouse Liver are Independent of NADPH Oxidase Toxicol. Sci., August 1, 2007; 98(2): 366 - 374. [Abstract] [Full Text] [PDF]
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R. A. Roberts, P. E. Ganey, C. Ju, L. M. Kamendulis, I. Rusyn, and J. E. Klaunig Role of the Kupffer Cell in Mediating Hepatic Toxicity and Carcinogenesis Toxicol. Sci., March 1, 2007; 96(1): 2 - 15. [Abstract] [Full Text] [PDF]
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 K. Bedard and K.-H. Krause The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology Physiol Rev, January 1, 2007; 87(1): 245 - 313. [Abstract] [Full Text] [PDF]
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 E. Teissier, A. Nohara, G. Chinetti, R. Paumelle, B. Cariou, J.-C. Fruchart, R. P. Brandes, A. Shah, and B. Staels Peroxisome Proliferator-Activated Receptor {alpha} Induces NADPH Oxidase Activity in Macrophages, Leading to the Generation of LDL with PPAR-{alpha} Activation Properties Circ. Res., December 10, 2004; 95(12): 1174 - 1182. [Abstract] [Full Text] [PDF]
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 I. Rusyn, S. Asakura, B. Pachkowski, B. U. Bradford, M. F. Denissenko, J. M. Peters, S. M. Holland, J. K. Reddy, M. L. Cunningham, and J. A. Swenberg Expression of Base Excision DNA Repair Genes Is a Sensitive Biomarker for in Vivo Detection of Chemical-induced Chronic Oxidative Stress: Identification of the Molecular Source of Radicals Responsible for DNA Damage by Peroxisome Proliferators Cancer Res., February 1, 2004; 64(3): 1050 - 1057. [Abstract] [Full Text] [PDF]
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 R. C. Cattley Peroxisome Proliferators and Receptor-Mediated Hepatic Carcinogenesis Toxicol Pathol, February 1, 2004; 32(2_suppl): 6 - 11. [Abstract] [PDF]
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D. Saha, K. R. Sekhar, C. Cao, J. D. Morrow, H. Choy, and M. L. Freeman The Antiangiogenic Agent SU5416 Down-Regulates Phorbol Ester-Mediated Induction of Cyclooxygenase 2 Expression by Inhibiting Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity Cancer Res., October 15, 2003; 63(20): 6920 - 6927. [Abstract] [Full Text] [PDF]
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 S. P. Anderson, C. S. Dunn, R. C. Cattley, and J.C. Corton Hepatocellular proliferation in response to a peroxisome proliferator does not require TNF{alpha} signaling Carcinogenesis, November 1, 2001; 22(11): 1843 - 1851. [Abstract] [Full Text] [PDF]
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 M. A. Koay, J. W. Christman, B. H. Segal, A. Venkatakrishnan, T. R. Blackwell, S. M. Holland, and T. S. Blackwell Impaired Pulmonary NF-{kappa}B Activation in Response to Lipopolysaccharide in NADPH Oxidase-Deficient Mice Infect. Immun., October 1, 2001; 69(10): 5991 - 5996. [Abstract] [Full Text] [PDF]
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 I. Rusyn, M. B. Kadiiska, A. Dikalova, H. Kono, M. Yin, K. Tsuchiya, R. P. Mason, J. M. Peters, F. J. Gonzalez, B. H. Segal, et al. Phthalates Rapidly Increase Production of Reactive Oxygen Species in Vivo: Role of Kupffer Cells Mol. Pharmacol., April 1, 2001; 59(4): 744 - 750. [Abstract] [Full Text]
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) or p47phox-null (
) mice and seeded at a
density of 106 cells/ml in 24-well plates. To measure
superoxide, Kupffer cells were incubated in HBSS (CON)
with either 10 µg/ml LPS, 10 µM Wy-14,643
(WY), or vehicle control (VEH; DMSO,
0.1% final concentration) and cultured for 30 min at 37°C.
Superoxide was determined as reduction of cytochrome c
inhibitable by superoxide dismutase, as described in "Materials and
Methods." Data are presented as means ± SE.
a, statistical differences (P < 0.05) from control group by two-way ANOVA, followed by
Student-Newman-Keul’s post-hoc test (n = 4 preparations in each group).
