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Vol. 59, Issue 4, 744-750, April 2001
Laboratory of Hepatobiology and Toxicology, Department of Pharmacology (I.R., H.K., M.Y., R.G.T.) and Curriculum in Toxicology (I.R., R.P.M., R.G.T.), University of North Carolina, Chapel Hill, North Carolina; Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (M.B.D., A.D., K.T., R.P.M.); Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (J.M.P., F.J.G.); and Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland (B.H.S., S.M.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The role of oxidants in the mechanism of tumor promotion by peroxisome
proliferators remains controversial. The idea that induction of
acyl-coenzyme A oxidase leads to increased production of
H2O2, which damages DNA, seems unlikely; still,
free radicals might be important in signaling in specialized cell types
such as Kupffer cells, which produce mitogens. Because hard evidence for increased oxidant production in vivo after treatment with peroxisome proliferators is lacking, the spin-trapping technique and
electron spin resonance spectroscopy were used. Rats were given
di(2-ethylhexyl) phthalate (DEHP) acutely. The spin trapping agent
-(4-pyridyl-1-oxide)-N-tert-butylnitrone was also
given and bile samples were collected for 4 h. Under these
conditions, the intensity of the six-line radical adduct signal
increased to a maximum value of 2.5-fold 2 h after administration
of DEHP, before peroxisomal oxidases were induced. Furthermore, DEHP
given with [13C2]dimethyl sulfoxide produced
a 12-line electron spin resonance spectrum, providing evidence that
DEHP stimulates ·OH radical formation in vivo.
Furthermore, when rats were pretreated with dietary glycine, which
inactivates Kupffer cells, DEHP did not increase radical signals.
Moreover, similar treatments were performed in knockout mice deficient
in NADPH oxidase (p47phox subunit). Importantly, DEHP
increased oxidant production in wild-type but not in NADPH
oxidase-deficient mice. These data provide evidence for the hypothesis
that the molecular source of free radicals induced by peroxisome
proliferators is NADPH oxidase in Kupffer cells. On the contrary,
radical adduct formation was not affected in peroxisome
proliferator-activated receptor knockout mice. These observations
represent the first direct, in vivo evidence that phthalates increase
free radicals in liver before peroxisomal oxidases are induced.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Peroxisome
proliferators are potentially hazardous chemicals that are widely used
and persist in the environment (IARC Working Group on Peroxisome
Proliferation, 1995
). These agents, administered to rodents, cause
hepatomegaly, proliferation of peroxisomes in hepatocytes and marked
increases in the enzymes of peroxisomal 
-oxidation of fatty acids.
Importantly, long-term treatment of rodents with these compounds
results in the development of liver tumors (Reddy et al., 1980).
Peroxisome proliferators may increase cancer risk in humans, although
this idea has been challenged and remains controversial (Dalen and
Dalton, 1996; Newman and Hulley, 1996). Whether or not phthalates pose
a health risk to humans is controversial (Wilkinson and Lamb, 1999);
therefore, determining the mechanisms underlying phthalate-induced
effects is important.
Two mechanisms have been proposed for peroxisome proliferator-induced
hepatocarcinogenesis: increased cell proliferation/decreased apoptosis
and oxidative stress [reviewed in Gonzalez et al. (1998)]. The latter
hypothesis is supported by the fact that chemicals of this class
up-regulate enzymes that generate
H2O2 in hepatic peroxisomes. Moreover, increases in 8-hydroxydeoxyguanosine,
lipofuscin, and conjugated dienes have been reported in livers of rats
treated for long periods with peroxisome proliferators (Goel et al.,
1986). On the other hand, some of these points have been challenged. For example, H2O2
production increases in vitro with peroxisome proliferators, but not in
intact cells, in which fatty acid supply is the limiting factor
(Handler et al., 1992). Moreover, several attempts to detect increases
in 8-hydroxydeoxyguanosine after treatment with peroxisome
proliferators have not been successful (Cattley and Glover, 1993).
Alternatively, it has been hypothesized that peroxisome proliferators
first act on Kupffer cells, the resident hepatic macrophages, to
increase production of oxidants that act as signaling molecules to
activate the transcription factor NF-
B, leading to release of
mitogenic cytokines. Indeed, it was recently shown that activation of
the transcription factor NF-B by peroxisome proliferators occurs
first in Kupffer cells and is dependent upon reactive oxygen species
derived from NADPH oxidase (Rusyn et al., 1998, 2000). Indeed,
peroxisome proliferators do this by directly activating Kupffer cells
to produce superoxide anion (Rose et al., 1999a).
However, whether or not phthalates increase oxidants in vivo remains unclear. Here, the hypothesis that phthalates increase reactive oxygen species was studied using the spin trapping technique and ESR, a technique that allows direct detection of reactive oxygen species in vivo. The first physical evidence for rapid production of radicals after administration of DEHP in vivo and a role for Kupffer cell NADPH oxidase is presented.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Animals and Treatments.
Female Sprague-Dawley rats (200-250
g; Charles River, Raleigh, NC), PPAR knockout [wild-type, SV129
(Lee et al., 1995)] and p47phox knockout
[C57BL/6x129 (Jackson et al., 1995)] mice and their correspondent
wild-type counterparts (25-30 g) were used in these experiments.
Phthalates [di(2-ethylhexyl)phthalate (DEHP), 2-ethylhexanol, ethylhexanoic acid, and phthalic acid] and dimethyl sulfoxide (DMSO)
were obtained from Aldrich (Milwaukee, WI),
[13C2]DMSO were obtained
from Isotech (Miamisburg, OH), and -(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN) from Sigma (St. Louis,
MO). All other chemicals and reagents were of the highest available purity from standard suppliers.
Synthesis of 2-Ethyl[1-13C]hexanol and
Di(2-ethyl[1-13C]hexyl)-,'-13C2-phthalate.
Grignard reagent was prepared by the reaction of 3-bromoheptane (TCI
America, OR) and magnesium turnings (Aldrich) in anhydrous diethyl
ether (J.T. Baker, Phillipsburg, NJ) solvent.
13CO2 (Cambridge Isotope
Lab, Inc. MA) was introduced into Grignard reagent with vigorous
stirring at 0°C. Subsequently, 30% sulfuric acid was added to
decompose the excess Grignard reagent and the magnesium turnings, and
to produce 2-ethyl[1-13C]hexanoic acid. The
ether phase was extracted with 30% potassium hydroxide, and the
solution was washed with n-hexane and then acidified with
30% sulfuric acid, pH 1.0. The recovered product was extracted with
diethyl ether, dried over anhydrous sodium sulfate, filtered, and
concentrated at 40°C under reduced pressure to obtain
2-ethyl[1-13C]hexanoic acid. Freshly prepared
ethereal diazomethane (DeBoer and Backer, 1954) was added
dropwise to the 2-ethyl[1-13C]hexanoic acid to
produce the methyl ester. Excess diazomethane was blown off with
nitrogen gas, and the ester was refluxed for 30 min with excess lithium
aluminum hydride (Aldrich) to yield 2-ethyl[1-13C]hexanol. Excess lithium aluminum
hydride was decomposed with water-saturated diethyl ether. The ether
phase was transferred, dried over anhydrous sodium sulfate, filtered,
and then concentrated at 40°C under reduced pressure to obtain the
2-ethyl[1-13C]hexanol.
Di(2-ethyl[1-13C]hexyl)-,'-13C2-phthalate
was synthesized as detailed previously with minor modifications
(Takeshita and Takizawa, 1977). A solution of
2-ethyl[1-13C]hexanol and pyridine in benzene
was added dropwise to
phthaloyl-,'-13C2
chloride (Isotech) in benzene with vigorous stirring for 30 min at 5 to
10°C. Subsequently, the mixture was refluxed on a boiling water bath
for 1 h. After cooling to room temperature, the mixture was
filtered to remove the pyridine hydrochloride formed. The solution was
washed with water, dried over anhydrous sodium sulfate, and then
concentrated at 40°C under reduced pressure. The residue obtained was
dissolved in n-hexane. The solution was washed with 1%
sodium hydrogen carbonate and then with water. The solution was dried
and concentrated as detailed above. The identity of final products was
confirmed by thin-layer chromatography and high-performance liquid
chromatography with commercially available phthalates (Aldrich) as
standards (data not shown).
Detection of Free Radicals.
Animals were anesthetized with
pentobarbital (75 mg/kg), and the gallbladder was cannulated using a
10-cm length of polyethylene 10 tubing. The spin trap POBN (1 g/kg,
i.p.) was injected immediately after treatment with phthalates (see
above) and bile samples were collected into Eppendorf tubes containing
50 µl of Desferal (3.3 mg/ml; Sigma) for 4 h at 20-min intervals
in rats, or as a single 2 h sample in mice. Bile samples were
frozen immediately on dry ice and stored at 
70°C until analyzed by
electron spin resonance (ESR) spectroscopy. ESR spectra were recorded
on a Bruker EMX ESR spectrometer with a super high-Q cavity. Instrument
settings: microwave power, 20 mW; modulation amplitude, 1 G; conversion time, 0.6 s; time constant, 1.3 s. Spectra were recorded on
an IBM-compatible computer interfaced to the spectrometer. Hyperfine coupling constants were determined with a spectral simulation program.
ESR analysis of bile from animals treated with xenobiotics has been
demonstrated to be useful in monitoring hepatic free radical-adduct
formation in vivo (Knecht and Mason, 1988). Furthermore, the radical
adducts detected in bile may be derived from both parenchymal and
nonparenchymal cells. In addition, POBN is rapidly absorbed and
distributed throughout the body after intraperitoneal administration
and is relatively stable in vivo (Liu et al., 1999).
Acyl CoA Oxidase Activity.
Acyl CoA oxidase is localized in
peroxisomes, and its activity, measured as formaldehyde formed from
hydrogen peroxide generated by peroxisomal -oxidation, is a measure
of induction of peroxisomes (Inestrosa et al., 1979). Liver samples
(~100 µg) were homogenized in 10 volumes of 0.25 M sucrose buffer.
A reaction mixture [1.4 ml; for details see Rose et al. (1997)] was
warmed to 37°C and mixed with 200 µl of homogenate and the reaction
was terminated after 10 min with 40% trichloracetic acid, which was
added before homogenate to the blanks. The solution was centrifuged to
pellet protein, and 1.0 ml of supernatant was added to 0.2 ml of Nash Reagent to measure formaldehyde after 30 min of incubation at 37°C.
Protein concentration was determined by the method of Bradford (1976).
Statistics. Data presented are representative ESR spectra from three to five separate experiments per group. For statistical comparisons, one-way analysis of variance with Tukey's multiple comparison test was used. A p value less than 0.05 was selected before the study to determine statistical differences between groups.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Effect of DEHP and Ethyl Hexanol on Radical Adduct Formation.
Here, the effects of DEHP and its metabolites on the production of
reactive oxygen species in rodent liver in vivo were studied. Weak
radical adduct ESR signals were detected in bile from rats injected
intraperitoneally with POBN (Fig. 1A);
however, a significant 2- to 3-fold increase in the free radical adduct
signals was detected in the bile of rats 2 h after intragastric
administration of DEHP (Fig. 1B). Spectral simulation revealed the
presence of two free radical adduct spectra with hyperfine coupling
constants: (I) aN = 15.6 ± 0.2 G, a
H = 2.6 ± 0.2 G; and (II) aN = 15.6 ± 0.2 G,
aH = 3.5 ± 0.1 G (Fig. 1C). Spectrum I is most likely to derive from a family
of carbon-centered radical adducts (POBN/·L, about 90% of the
combined radical adduct) based on the hyperfine coupling constants and
comparatively broad ESR linewidth of 0.8 G. In contrast, the narrow
linewidth (0.35 G) and large hydrogen coupling of adduct II (about
10%) match the properties of
POBN/·CO2
possibly
formed by the abstraction of a hydrogen atom from endogenous formate
(Burkitt et al., 1993).
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,'-13C2-phthalate
and 2-ethyl[1-13C]hexanol). The
13C-labeled carboxyl group was suspected to be
the source of the POBN/·CO2, whereas the
labeled alcohol could have formed the lipid-like radical adduct.
Treatments using each of the compounds gave no spectra with double the
number of ESR lines because of the presence of
13C (Fig. 1D for DEHP, data not shown for
2-ethylhexanol), indicating that neither DEHP nor 2-ethylhexanol
molecules were involved directly in the free radical adduct formation.
This result supports the conclusion that DEHP or its metabolites do not
form reactive species per se; rather, they activate production of
reactive species by oxidant-generating enzymes in the liver.
Because peroxisome proliferators can activate isolated Kupffer cells
directly to produce superoxide anion (Rose et al., 1999a), it was
hypothesized that a superoxide anion-derived radical adduct was
involved. Furthermore, it is possible that the toxicity of superoxide
anion in vivo is potentiated by iron-catalyzed generation of hydroxyl
radical (·OH), which could abstract hydrogen from endogenous
formate (Minotti and Aust, 1989). Because of high reactivity and low
stability of ·OH-derived adducts of POBN, direct spin-trapping
of this radical in biological systems is difficult (Halliwell and
Grootveld, 1987). This experimental difficulty is remedied with the
introduction of DMSO into the systems studied. Hydroxyl radical reacts
with DMSO to yield methanesulfonic acid and methyl radical
(·CH3) (Klein et al., 1981). The latter
reacts rapidly with POBN to form relatively stable ESR-detectable
nitroxides; this approach has been used in vivo to detect ·OH
(Kadiiska et al., 1995). To determine whether DEHP-induced radical
adducts were caused by ·OH, DMSO (0.5 ml/kg, i.g.) was given at
the time of DEHP treatment (Fig. 2).
Indeed, direct evidence for formation of a
·CH3 mediated adduct of POBN in the
presence of DMSO (Fig. 2B) was obtained when
[13C2]DMSO was given with
DEHP and a characteristic 12-line ESR signal of
POBN/·13CH3 was
observed (Fig. 2C; hyperfine coupling constants: species I
(POBN/·CO2):
aN = 15.8 G, and
aH = 3.4 G;
species II
(POBN/·13CH3):
aN = 16.0 G,
aH = 2.8 G,
and a13C = 4.9 G; and species III (POBN/·L):
aN = 15.6 G and
aH = 2.7 G).
Collectively, these results provide direct evidence in support of the
hypothesis that DEHP rapidly causes formation of ·OH in vivo.
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Kupffer Cell NADPH Oxidase Is the Source of Oxidants Due to
Phthalates in Vivo.
To test the hypothesis that Kupffer cells are
causally involved in increased radical production because of the
phthalates observed here, glycine, an agent that inactivates Kupffer
cells (Wheeler et al., 1999), was used. DEHP increased production of POBN/radical adducts in rats fed control diet for 4 days (Fig. 3B). However, when animals were fed
dietary glycine (5% w/w), no increase in POBN/radical signal caused by
DEHP was observed (Fig. 3D). It is concluded that rapid activation of
Kupffer cells by phthalates leads to formation of reactive oxygen
species in vivo.
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). It was recently demonstrated that in mice deficient in
the p47phox subunit of NADPH oxidase, both the
activation of NF-B and the increase in cell proliferation caused by
WY-14,643 were prevented nearly completely (Rusyn et al., 2000).
Therefore, here we tested the hypothesis, using knockout mice deficient
in NADPH oxidase, that NADPH oxidase is causally responsible for
increased oxidant production caused by peroxisome proliferators.
Similar to what was observed in the rat, ESR spectra composed of free
radical adducts I and II were detected in bile from
p47phox wild-type and knockout mice injected
intraperitoneally with POBN (Figs. 4, A
and C, respectively). However, intragastric administration of DEHP
resulted in a 2- to 3-fold increase in formation of POBN/radical adducts in wild-type mice (Fig. 4B). Although this spectrum seems to be
primarily composed of
POBN/·CO2, simulation
reveals that POBN/·CO2
contributes only about 26% to signal intensity. Its presence is
accentuated because of the inverse square relationship between line
width and peak height. Importantly, DEHP failed to increase production
of oxidants in NADPH oxidase-deficient mice (Fig. 4D), thus providing
evidence for the hypothesis that NADPH oxidase is the source of
radicals activated by phthalates in vivo.
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).
Indeed, peroxisomes are markedly increased in both size and number in
liver parenchymal cells of rats and mice treated with phthalates
(Ganning et al., 1984). Increases in hepatic protein levels of acyl CoA
oxidase and other peroxisomal enzymes are evident as soon as ~4 h
after administration of peroxisome proliferators (Reddy et al., 1986).
In this study, activity of acyl CoA oxidase was measured in liver
homogenates from wild-type and p47phox knockout
mice treated with DEHP. An increase of ~2.5-fold in activity of this
marker peroxisomal enzyme was detected 5 h after a single dose of
DEHP in both wild-type (control, 1.6 ± 0.3 nmol/min/mg protein;
5 h DEHP, 4.2 ± 0.2) and knockout mice (1.7 ± 0.1 and 4.2 ± 0.2, respectively). However, no increase was evident after 2 h (wild-type, 2.0 ± 0.3 nmol/min/mg protein; knockout:
2.2 ± 0.4), when radical adducts were detected in wild-type but
not NADPH oxidase-deficient mice (see above).
Peroxisome proliferation involves activation of an intracellular
receptor (i.e., PPAR) that acts as a transcription factor to
up-regulate synthesis of many lipid-metabolizing enzymes [reviewed in
Gonzalez et al. (1998)]. Experiments with PPAR knockout mice showed
unequivocally that hepatocellular proliferation and tumors caused by
peroxisome proliferators require this receptor (Peters et al., 1997).
Because no direct evidence has been presented to address the role of
peroxisomes in increased production of oxidants caused by phthalates,
PPAR knockout mice were treated with DEHP and POBN and bile was
collected for ESR analysis. Importantly, ESR spectra of similar
intensity (2- to 3-times control levels) were detected in both
wild-type and PPAR knockout mice treated with DEHP (Figs.
5B and D). ESR spectra were similar to
free radical adduct species I and II detected in bile from
p47phox wild-type and knockout mice (see above).
These data support the hypothesis that PPAR is not involved in the
rapid increase in oxidants caused by phthalates detected here.
Moreover, because Kupffer cells do not express PPAR (Peters et al.,
2000), this result further supports the idea that Kupffer cells but not
hepatocytes are responsible for early increases in oxidants in response
to phthalates.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Direct Evidence for Production of Hydroxyl Radical after Treatment
with Phthalates.
The role of oxidants in the mechanism of action
of peroxisome proliferators remains controversial despite numerous
efforts to link these compounds to oxidized DNA [reviewed in Rose et
al. (1999b)]. No direct evidence of increased oxidant production in vivo after treatment with these chemicals has yet been presented. On
the other hand, a plethora of indirect evidence both in support and in
opposition to the hypothesis that peroxisome proliferators increase
oxidants has been presented. Recently, the in vivo formation of free
radicals after exposure to several toxic chemicals has been
demonstrated using the spin trapping technique and ESR spectroscopy (Kadiiska et al., 1998). Indeed, in vivo spin trapping is a useful tool
because it allows direct detection and characterization of oxidants in
tissues and body fluids (Liu et al., 1999).
]; however, based on 13C
experiments and coupling constants, the radical adduct detected in this
study was assigned as originating from hydroxyl radical (Fig. 2). It is
most likely that in liver, where both superoxide dismutase and
transition metals are present, superoxide anion was rapidly transformed
to H2O2 and than to
hydroxyl radical via a metal-catalyzed Haber-Weiss reaction [reviewed
in Dunford (1987)]. Another possible mechanism for hydroxyl radical
generation in biological systems is a reaction of superoxide with
nitric oxide to form peroxynitrite, which leads to "hydroxyl-like"
radical formation (Halliwell et al., 1999).
It is not clear how phthalates activate NADPH oxidase in Kupffer cells;
however, one possible mechanism is via increases in calcium and
activation of PKC, which leads to phosphorylation and assembly of NADPH
oxidase subunits. Indeed, supporting evidence for this hypothesis has
been reported. Specifically, it was shown that peroxisome proliferators
increase intracellular free calcium in cultured Kupffer cells (Hijioka
et al., 1992). Furthermore, dietary glycine, an agent that disrupts
calcium signaling via hyperpolarization of cell membrane, blocked
hepatocellular proliferation because of peroxisome proliferators (Rose
et al., 1997). Finally, treatment of Kupffer cells in vitro with
peroxisome proliferators increases activity of PKC 3-fold; both glycine
and staurosporine, an inhibitor of PKC, inhibited superoxide production
and increased PKC activity completely (Rose et al., 1999a).
Furthermore, because oxidants here are produced before
H2O2-generating enzymes are induced by phthalates in hepatocytes and are independent of PPAR, an
intracellular receptor that mediates pleiotropic responses to
phthalates in parenchymal cells (Fig. 5), it is concluded that induction of peroxisomes is not responsible for increases in reactive oxygen species detected here.
Role of Oxidants in the Mechanism of Action of Phthalates: Harmful
Species or Signaling Molecules?
Oxidative stress may regulate cell
proliferation [reviewed in Nakamura et al. (1997)]. Specifically,
oxidants may play a role in tumor promotion by modulating the
expression of a family of prooxidant genes that are related to cell
growth and differentiation. It was recently suggested that low levels
of oxidants might play a role in signaling increases in proliferation
of liver parenchymal cells caused by peroxisome proliferators via a
Kupffer cell-mediated mechanism involving TNF and NF-B (Rose et
al., 1999b). Indeed, taking oxidant production in specific liver cell
types (i.e., Kupffer cells) into consideration may provide important
insights into the mechanisms by which these chemicals elicit
proliferative responses in rodent liver. Importantly, evidence that
supports the concept that oxidants play a significant role in the
peroxisome proliferator-induced proliferative response by participating
in signaling in Kupffer cells has been presented. For example, Kupffer cells are known to be activated by peroxisome proliferators in vivo
(Bojes and Thurman, 1996). Moreover, peroxisome proliferators rapidly
activate the redox-sensitive transcription factor NF-B in Kupffer
cells in an oxidant-dependent manner that results in production of
mitogenic cytokines, such as TNF, and increases in cell
proliferation [reviewed in Rose et al. (1999b)]. Furthermore, monoethylhexylphthalate, a key lipophilic metabolite of DEHP, increased
superoxide anion production in isolated Kupffer cells in a
dose-dependent manner, indicating that phthalates can activate Kupffer
cells directly (Rose et al., 1999a).
stimulates DNA synthesis in primary hepatocytes (Beyer and
Theologides, 1993) and it was shown that TNF from Kupffer cells is
involved in the action of phthalates (Rose et al., 1999b). We have
shown recently that in mice lacking active NADPH oxidase, peroxisome
proliferators fail to increase early cell proliferation in the liver
(Rusyn et al., 2000). This phenomenon is caused by a lack of activation
of transcription factor NF-B and production of mitogenic cytokines
by these chemicals in the absence of active NADPH oxidase. However,
PPAR is required for the increased cell proliferation and tumors
caused by peroxisome proliferators (Peters et al., 1997). How can both
TNF and PPAR be required? One idea is that both factors are
required for maximal stimulation of cell proliferation. In fact, it is
known that peroxisome proliferators increase proliferation of liver
parenchymal cells both in vivo and in vitro; however, the in vitro
effect is much less robust and persistent (i.e., 8- to 10-fold
increases in vivo versus only about 2-fold increases in vitro)
regardless of the dose of the compound used (Marsman et al., 1988).
Moreover, in highly purified hepatocytes, it was shown recently that
peroxisome proliferators alone fail to increase proliferation of
parenchymal cells in vitro; however, when peroxisome proliferator is
added with TNF, cell proliferation is potentiated significantly.
This data supports the hypothesis that both peroxisome proliferator and
TNF are required for maximal stimulation of cell proliferation in
liver parenchymal cells (Parzefall et al., 2001).
Collectively, this work provides the first direct evidence that
phthalates induce formation of radicals in vivo. This finding establishes a role for oxidants in the mechanism of hepatocellular proliferation by this important class of chemicals via the following sequence of events: phthalates rapidly activate Kupffer cells, resulting in production of oxidants from NADPH oxidase, activation of
NF-B, and release of mitogenic cytokines such as TNF. This leads
to proliferation of parenchymal cells. Concomitantly, phthalates activate PPAR in hepatocytes, leading to induction of peroxisomes, a
phenomenon necessary for induction of tumors months later.
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Footnotes |
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Received September 21, 2000; Accepted December 21, 2000
This study was supported in part by National Institutes of Health Grants ES04325 and ES07126.
Send reprint requests to: Ivan Rusyn, M.D., Ph.D., 1124 M.E. Jones Bldg., CB #7365, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: iir{at}med.unc.edu
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Abbreviations |
|---|
NF-B, nuclear factor B;
ESR, electron
spin resonance;
PPAR, peroxisome proliferator-activated receptor
;
DMSO, dimethyl sulfoxide;
DEHP, Di(2-ethylhexyl) phthalate;
POBN, -(4-pyridyl-1-oxide)-N-tert-butylnitrone;
i.g., intragastric;
PKC, protein kinase C;
TNF, tumor necrosis factor
.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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is a direct hepatocyte mitogen in the rat.
Biochem Mol Biol Int
29:
1-4[Medline]. in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator WY-14,643.
Carcinogenesis
18:
2029-2033 is restricted to hepatic parenchymal cells, not Kupffer cells: Implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis.
Carcinogenesis
21:
823-826B in Kupffer cells before hepatocytes.
Carcinogenesis
19:
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