Kupffer cell oxidant production is central to the mechanism of peroxisome proliferators
Michelle L. Rose1,
Chantal A. Rivera2,
Blair U. Bradford2,
Lee M. Graves2,
Russell C. Cattley4,
Robert Schoonhoven3,
James A. Swenberg1,3 and
Ronald G. Thurman1,2,5
1 Laboratory of Hepatobiology and Toxicology, CB#7365, MEJB, Curriculum in Toxicology,
2 Department of Pharmacology and
3 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599-7365 and
4 Chemical Industry Institute of Toxicology, Research Triangle Park, NC, USA
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Abstract
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Increased cell proliferation most likely plays a key role in peroxisome proliferator-induced liver cancer. Recently, Kupffer cells were shown to be responsible for Wy-14,643-induced cell proliferation. However, the mechanism by which peroxisome proliferators activate Kupffer cells is unknown. Since gut-derived endotoxin is a known activator of Kupffer cells, the hypothesis that it is involved was evaluated. Increased cell proliferation and peroxisome induction were unaffected by gut sterilization. Moreover, endotoxin was not detectable in portal blood following treatment with Wy-14,643. Therefore, it is concluded that gut-derived endotoxin is not responsible for Kupffer cell activation. To test the hypothesis that Kupffer cells are activated by Wy-14,643 directly, Kupffer cell superoxide production was measured following treatment in vitro. Wy-14,643 increased superoxide production in a dose-dependent manner (0.1 and 50 µM) with half-maximal stimulation at 2.5 µM. Diethylhexylphthalate (DEHP) and ethylhexanol did not increase superoxide production even at doses 50 times higher than Wy-14,643; however, monoethylhexylphthalate (MEHP) activated superoxide production as effectively as Wy-14,643 with half-maximal stimulation at 5 µM. Treatment with Wy-14,643 for 21 days caused a 2-fold increase in Kupffer cell superoxide production while DEHP did not. Pretreatment of Kupffer cells with staurosporine (0.0110 pM) completely blocked generation of superoxide demonstrating that protein kinase C is required. Moreover, Wy-14,643 increased Kupffer cell protein kinase C activity 3-fold. Pretreatment of Kupffer cells with the amino acid glycine (0.013 mM), which blunts calcium signaling, inhibited Wy-14,643-stimulated superoxide production and increased protein kinase C activity completely. These data are consistent with the hypothesis that potent peroxisome proliferators (Wy-14,643 and MEHP) directly activate Kupffer cell production of oxidants via mechanisms involving protein kinase C. Further, peroxisome proliferator treatments that sustain elevated rates of cell proliferation (e.g. Wy-14,643) activate Kupffer cell superoxide production following long-term dietary treatment supporting the hypothesis that Kupffer cell-derived oxidants are involved in peroxisome proliferator-induced neoplasia.
Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; DEHP, diethylhexylphthalate; FBS, fetal bovine serum; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; MEHP, monoethylhexylphthalate; PKC, protein kinase C; PPAR
, peroxisome proliferator-activated receptor
.
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Introduction
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Peroxisome proliferators are a group of non-genotoxic carcinogens which include hypolipidemic drugs, phthalate esters used in the manufacture of plastics, solvents and several herbicides (1). While these chemicals are structurally dissimilar, they are all lipophilic, cause an increase in both the number and size of peroxisomes and induce peroxisomal enzymes (1). Moreover, they cause liver cancer in rodents by mechanisms which remain unclear (1). Considerable evidence suggests that increased cell proliferation caused by these compounds is central to their carcinogenicity since the most potent carcinogen in this class, Wy-14,643, elevates rates of proliferation for as long as it is administered, while less carcinogenic peroxisome proliferators do not (2). Kupffer cells, the resident hepatic macrophages and a rich source of cytokines in liver (3), were shown recently to be causally responsible for Wy-14,643-induced increases in cell proliferation. This conclusion is based on the finding that inactivating Kupffer cells with methyl palmitate or dietary glycine completely prevented stimulation of hepatocyte replication (4,5). Moreover, Wy-14,643-stimulated increases in the hepatocyte mitogen TNF
were also blocked by inactivating Kupffer cells (4,5), and antibodies to TNF
prevented increases in cell proliferation caused by Wy-14,643 (6). Furthermore, the transcription factor NF
B, which is central in TNF
production, was shown to be activated almost exclusively in the Kupffer cell fraction in liver following treatment with Wy-14,643 (7). Taken together, these data are consistent with the hypothesis that TNF
originating in Kupffer cells is the key mitogen responsible for Wy-14,643-induced hepatocyte proliferation.
Phagocytosis by Kupffer cells is activated by peroxisome proliferators in vivo (8); however, the mechanisms by which peroxisome proliferators activate Kupffer cells and increase TNF
production are not known. Endotoxin, which originates from the cell wall of Gram-negative bacteria, is a known activator of macrophages, including Kupffer cells, and many biological effects of endotoxin are mediated by activated macrophages (9). Inhibition of Kupffer cells with dietary glycine largely prevented TNF
production and blocked mortality in rats treated with lethal doses of lipopolysaccharide (LPS) (10). Similarly, dietary glycine prevented TNF
production and blocked increased rates of cell proliferation in rats treated with Wy-14,643 (5). Therefore, it is possible that increases in gut-derived endotoxin are responsible for Kupffer cell activation and TNF
production following treatment with Wy-14,643. On the other hand, Wy-14,643 may activate Kupffer cells directly since it is very lipophilic and may accumulate in cell membranes (1). Also, aggregates of Wy-14,643 or lipoprotein-bound Wy-14,643 may be taken up by Kupffer cells via phagocytosis (8). Therefore, these studies were designed to determine if Kupffer cells are activated directly by peroxisome proliferators or indirectly via gut-derived bacterial products such as endotoxin.
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Materials and methods
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Animals and treatment
For short-term studies, male SpragueDawley rats (225250 g; Charles River Laboratories, Raleigh, NC) were maintained on a 12 h daynight cycle and given free access to chow and water. Rats were pretreated with polymixin B (150 mg/kg) and neomycin (450 mg/kg) or water vehicle (1.0 ml) i.g. daily for 5 days to sterilize the gut as described elsewhere (11). This protocol has been used successfully in this laboratory to demonstrate that gut-derived endotoxin is responsible for the development of liver injury in a rodent model of alcoholic liver disease (11). Wy-14,643 (100 mg/kg i.g.) or olive oil vehicle (0.4 ml) was given daily for 4 days, 6 h after treatment with antibiotics or water vehicle. Rats were given 5-bromo-2'-deoxyuridine (BrdU) daily in the drinking water (80 mg/100 ml) to quantitate cell proliferation. Animals were killed under pentobarbital anesthesia, and liver sections were fixed in 10% neutral buffered formalin for immunohistochemical analysis, or snap frozen in liquid nitrogen and stored at 80°C for analysis of the peroxisomal marker enzyme acyl CoA oxidase.
For long-term studies, male F344 rats (190220 g; Charles River Laboratories, Raleigh, NC) were acclimated for 10 days and then fed ground NIH-07 diet containing 0.1% Wy-14,643 or 1.2% diethylhexylphthalate (DEHP).
Cell proliferation
Immunohistochemical detection of BrdU was performed essentially as described elsewhere (4,5). Briefly, tissue sections were deparaffinized, rehydrated and stained immunohistochemically for BrdU using anti-BrdU antibodies (Clone Bu20a; Dako, Carpinteria, CA) and the Envision Peroxidase Staining Kit (Dako). Ten random high-power fields (x400) were counted per slide and cell proliferation was expressed as the percentage of cells stained positively for BrdU. A section of duodenum, a rapidly proliferating tissue, was taken from each animal as a positive control for BrdU incorporation (12).
Assays for acyl CoA oxidase activity and plasma endotoxin
Acyl CoA oxidase is an accepted measure of induction of peroxisomes (1). The generation of hydrogen peroxide from palmitate metabolism by acyl CoA oxidase was measured by coupling it to the peroxidation of methanol via catalase (13). Formaldehyde formed from methanol metabolism was measured colorimetrically using Nash reagent (14) and acyl CoA oxidase activity was expressed relative to protein concentration (15). For endotoxin measurements, blood was collected from the portal vein in heparinized syringes and centrifuged to collect plasma in glass tubes. All materials used for the collection and storage of plasma were pyrogen-free. Plasma was stored at 20°C until measurement of endotoxin with a Limulus Amebocyte Lysate test kit (Kinetic-QCL; BioWhittaker, Walkersville, MD).
Kupffer cell and hepatocyte culture and superoxide and catalase assays
Kupffer cells and hepatocytes were isolated using brief collagenase perfusion and Percoll density gradients as described in detail elsewhere (16). Kupffer cells were seeded at a density of 106 cells/ml RPMI 1640 containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 IU/ml and 100 µg/ml, respectively) in 24 well plates, while hepatocytes were seeded at a density of 2x105/ml. One hour later media was aspirated and replaced with fresh RPMI 1640 containing 30% FBS and penicillin/streptomycin, and cells were cultured for 24 h. To measure superoxide, media was aspirated and Kupffer cells were washed twice with Hank's balanced salt solution (HBSS). Kupffer cells were incubated in HBSS, and superoxide was determined as reduction of cytochrome c inhibitable by superoxide dismutase as described elsewhere (17). Superoxide production was stimulated by addition of 1 µM phorbol myristate acetate (PMA) as a positive control in each preparation of Kupffer cells (10 ± 1.0 nmol/106 cells/30 min). Superoxide production by Kupffer cells was linear for up to 30 min of treatment in vitro and at a plating density of up to 1 million cells/ml for all treatments (data not shown).
For measurement of catalase activity, Kupffer cells were plated at a density of 1020x106/10 ml media in 100 mm dishes. Cells were harvested in 1.0 ml of 0.07 M phosphate buffer, pH 7.0 and catalase activity was determined using the method of Luck as described by Bergmeyer (18). Hydrogen peroxide was added and the linear decline in absorbace per unit time was measured spectrophotometrically at 240 nm.
Kupffer cell protein kinase C (PKC) activity
Kupffer cells were isolated and cultured as described above at a density of 20x106 cells/10 ml media in 100 mm dishes. Cells were treated with Wy-14,643 (10 µM), ethanol vehicle (0.1%) or PMA (1 µM) as a positive control in each preparation (12 000 c.p.m./mg protein/15 min). Following treatment, cells were washed twice with ice-cold lysis buffer (20 mM HEPES, pH 7.5, 10 mM EGTA, 2 mM EDTA, 2 mM DTT) and then scraped into 1.0 ml of lysis buffer containing protease inhibitors (40 µg/ml bestatin, 0.5 mM pefabloc, 0.7 µg/ml pepstatin A, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 5.5 µg/ml PMSF). Cell suspensions were sonicated and the lysate was centrifuged at 100 000 g for 20 min at 4°C. The pellet (membrane fraction) was resuspended in 0.5 ml of lysis buffer containing 1% NP-40 and the protease inhibitor mix described above. After 30 min on ice, PKC activity was purified by applying the membrane fraction to DEAE columns. PKC was eluted using column buffer containing 0.15 mM NaCl. To determine PKC activity, 10 µl of phospholipid (0.3 mg/ml phosphatidylserine, 0.03 µg/ml diolein) or water, 5 µl of the PKC-specific MARCKS peptide (60 µM; Biomol, Plymouth Meeting, PA), and 10 µl of reaction buffer (175 mM HEPES, pH 7.5, 35 mM MgCl2, 3.5 mg /ml bovine serum albumin, 540 µM ATP, 15 mM DTT, 14 mM calcium, 3 µCi [
-32P]ATP) was added to plastic tubes on ice. To initiate the reaction, 5 µl of sample was added to each tube and incubated at 30°C for 15 min. Each sample was run in duplicate in the presence and absence of calcium/phosphotidylserine/diolein. The difference between these two activities was defined as PKC activity. The assay was terminated by transferring 25 µl of the reaction mixture to Whatman phosphocellulose paper and placing it in a stirring solution of 150 mM phosphoric acid. Papers were washed three times for 15 min each, dried and incorporation of radioactivity was determined using liquid scintillation counting. Results were expressed per mg protein (15).
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Results
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Gut-derived endotoxin is not responsible for Wy-14,643-induced cell proliferation
To test the hypothesis that activation of Kupffer cells by endotoxin is involved in the mechanism by which Wy-14,643 increases cell proliferation, animals were treated with non-absorbable antibiotics to sterilize the intestine (11). This procedure diminishes endotoxin from Gram-negative bacteria (11). The increase in hepatocyte proliferation (Figure 1A
) and induction of peroxisomes (Figure 1B
) caused by 4 days of treatment with Wy-14,643 were unaffected by sterilizing the gut with polymixin B, which binds endotoxin, and neomycin, which eliminates Gram-negative bacteria, demonstrating that gut-derived endotoxin is not responsible for Wy-14,643-induced cell proliferation and induction of peroxisomes.

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Fig. 1. Effect of sterilization of the gut on Wy-14,643-induced cell proliferation (A) and induction of peroxisomes (B). Cell proliferation and acyl CoA oxidase activity were determined as described in Materials and methods in livers from rats treated with 100 mg/kg Wy-14,643 (WY) or olive oil vehicle (control) for 4 days following pretreatment with antibiotics or water vehicle for 5 days. Data shown are means ± SEM. Asterisks (*) denote statistical differences from control (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 5 each group).
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Endotoxin levels in portal blood were measured to determine if Wy-14,643 treatment caused an increase in endotoxin which could activate Kupffer cells. Since Kupffer cells are known to be activated as early as 5 h following treatment with Wy-14,643 in vivo (8), plasma endotoxin levels in portal blood were measured for up to 24 h (Figure 2
). Basal levels of plasma endotoxin (LPS) range between 0 and 30 pg/ml. Rats receiving 4 weeks of chronic ethanol treatment, where endotoxin has been shown to be elevated (19), were used here as positive controls. Indeed, plasma endotoxin in male rats treated with ethanol was ~50 pg/ml and in female rats nearly 100 pg/ml. Treatment with Wy-14,643 did not increase endotoxin in portal blood above basal levels at any time studied (Figure 2
). Taken together, these data are consistent with the hypothesis that endotoxin is not responsible for activation of Kupffer cells by Wy-14,643.

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Fig. 2. Effect of Wy-14,643 on plasma endotoxin (LPS) levels. Rats were treated with Wy-14,643 (100 mg/kg, i.g.) or olive oil vehicle (0.4 ml), and portal blood was collected at the times indicated. Plasma endotoxin was measured using the Limulus Amebocyte Lysate assay as described in Materials and methods. Data shown are means ± SEM for n = 4 in each group. Average plasma endotoxin levels for male and female rats receiving a chronic ethanol diet for 4 weeks are shown as positive controls.
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Peroxisome proliferators activate Kupffer cells directly
To determine if Wy-14,643 activates Kupffer cells directly, cells were isolated, cultured and superoxide production was assessed. Activation of NADPH oxidase results in superoxide production, a characteristic of stimulated Kupffer cells (3). Wy-14,643 increased superoxide production by Kupffer cells in a dose-dependent manner with half-maximal stimulation at ~2.5 µM (Figure 3
). Wy-14,643 (10 µM) increased superoxide production maximally nearly 7-fold over control values, indicating that it activates Kupffer cells directly. Since both Wy-14,643 and the weaker carcinogen diethylhexylphthalate (DEHP) both elevate hepatocyte replication within the first few days of treatment, the effect of DEHP on superoxide production was also measured. Neither DEHP nor its metabolite ethylhexanol stimulated superoxide production, even at doses 50 times greater than Wy-14,643 (Figure 4A and B
). However, monoethylhexylphthalate (MEHP), a key lipophilic metabolite of DEHP, activated superoxide production nearly as well as Wy-14,643 (half-maximal effect = 5 µM MEHP; Figure 4C
).

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Fig. 3. Effect of Wy-14,643 on Kupffer cell superoxide production. Kupffer cells were harvested and cultured for 24 h as described in Materials and methods. Wy-14,643 or an equal volume of ethanol vehicle (0.1%, 0 µM treatment) was added at the concentrations indicated for 30 min, and superoxide production was measured as described in Materials and methods. Data shown are means ± SEM. Asterisks (*) denote statistical differences from control (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 56 in each group).
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Fig. 4. Effect of diethylhexylphthalate (DEHP), ethylhexanol and monoethylhexylphthalate (MEHP) on Kupffer cell superoxide production. Cells were harvested and cultured as described in Figure 3 . DEHP (A), ethylhexanol (B) and MEHP (C) were added at the concentrations indicated for 30 min, and superoxide production was measured as described in Materials and methods. Data shown are means ± SEM. Asterisk (*) denotes statistical difference from control (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 56 each group).
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Since dietary glycine has been shown to inhibit Wy-14,643-induced cell proliferation and TNF
production (5), Kupffer cells were incubated with glycine prior to stimulation with Wy-14,643 to test the hypothesis that glycine inhibits superoxide production. Indeed, glycine prevented Wy-14,643-stimulated superoxide production by Kupffer cells (Figure 5
). A significant inhibition occurred at 0.1 mM glycine, and superoxide production was completely prevented with 1.0 mM glycine. Inhibition by glycine was half-maximal at ~0.05 mM (Figure 5
).

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Fig. 5. Effect of glycine on Wy-14,643-induced superoxide production by Kupffer cells. Kupffer cells were harvested and cultured as in Figure 3 . Cells were pretreated with the concentrations of glycine indicated or an equal volume of buffer vehicle for 15 min prior to stimulation with 10 µM Wy-14,643. Superoxide production was measured as described in Materials and methods. Data shown are means ± SEM. Asterisks (*) denote statistical differences from control (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 56 in each group).
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Involvement of PKC
PKC is involved, at least in part, in LPS-stimulated superoxide production by Kupffer cells via phosphorylation of specific subunits of NADPH oxidase which causes the assembly of the active NADPH oxidase complex and the production of superoxide (17,20). Here, cells were pretreated with the PKC inhibitor staurosporine to test the hypothesis that PKC is involved in Wy-14,643-stimulated superoxide production. Staurosporine inhibited Wy-14,643-stimulated superoxide production in a dose-dependent manner with an IC50 of ~0.05 pM (Figure 6
).

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Fig. 6. Effect of the PKC inhibitor staurosporine on Wy-14,643-induced superoxide production by Kupffer cells. Kupffer cells were harvested and cultured as in Figure 3 . Prior to stimulation with 10 µM Wy-14,643, cells were treated with the concentrations of staurosporine indicated or an equal volume of DMSO vehicle (<0.00001%) for 15 min. Superoxide production was measured as described in Materials and methods. Data are means ± SEM. Asterisks (*) denote statistical differences from control (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 56 each group).
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The involvement of PKC in Wy-14,643-stimulated signaling in Kupffer cells was evaluated further by measuring PKC activity in Kupffer cells. Kupffer cells were treated with 10 µM Wy-14,643, a dose which caused maximal stimulation of superoxide production, for 20 min and PKC activity was determined using incorporation of [
-32P]ATP into MARCKS peptide, a specific PKC substrate (21). Indeed, Wy-14,643 increased calcium-dependent PKC activity in Kupffer cells nearly 3-fold (Figure 7
), an effect which was ~25% of the activation caused by PMA. Moreover, Wy-14,643-stimulated PKC activation was blocked by pretreatment of Kupffer cells with 1.0 mM glycine, a concentration of glycine which also prevented superoxide production (Figure 7
).

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Fig. 7. Effect of Wy-14,643 on Kupffer cell PKC activity. Kupffer cells were harvested and cultured as described in Materials and methods. Cells were pretreated with 1.0 mM glycine (GLY) or water vehicle (1%) for 15 min prior to stimulation with 10 µM Wy-14,643 (WY) or an equal volume of ethanol vehicle (0.1%, CON) for 20 min. Membranes were harvested and PKC was purified as described in Materials and methods. PKC activity was assessed as calcium- and lipid-dependent incorporation of [ -32P]ATP into the PKC specific MARCKS peptide. Results were expressed per mg protein in the assay, and data are means ± SEM. Asterisk (*) denotes statistical difference from control (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 5 in each group).
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Dietary treatment with Wy-14,643 increases Kupffer cell superoxide production
Although both Wy-14,643 (0.1% in the diet) and DEHP (1.2% in the diet) elevate hepatocyte replication during the first few days of treatment, only Wy-14,643 sustains proliferation following 21 days of treatment (2). Consistent with a role for oxidants in signaling the initial burst in cell proliferation caused by peroxisome proliferators, both Wy-14,643 and MEHP, the active metabolite of DEHP, increased superoxide production following acute treatment (Figures 3 and 4C
). To determine if oxidants (e.g. superoxide) may be involved in signaling sustained cell proliferation in vivo, Kupffer cells were isolated from rats treated with 0.1% Wy-14,643 or 1.2% DEHP in the diet for 21 days and rates of superoxide production were measured. Indeed, Kupffer cells isolated from rats fed Wy-14,643 generated superoxide at rates 2-fold greater than cells from controls (Figure 8
). However, superoxide production was not stimulated by feeding DEHP for 3 weeks.

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Fig. 8. Effect of 21 days of dietary Wy-14,643 and DEHP on Kupffer cell superoxide production. Male F344 rats (225290 g) were fed 0.1% Wy-14,643 or 1.2% DEHP in the diet (NIH 07 chow) for 21 days. Kupffer cells were harvested and cultured as in Figure 3 and rates of superoxide production were measured as described in Materials and methods. Data shown are means ± SEM. Asterisk (*) denotes statistical difference from control and DEHP groups (P < 0.05, ANOVA with Bonferroni post-hoc tests, n = 5 in each group).
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Discussion
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Peroxisome proliferators activate Kupffer cells directly
Increased rates of hepatocyte proliferation are hypothesized to play a key role in the mechanism by which peroxisome proliferators cause liver cancer in rodents (2). Recently, increases in cell proliferation caused by Wy-14,643 were shown to require Kupffer cells, since inactivating them with either methyl palmitate or dietary glycine completely prevented elevated rates of cell turnover (4,5). Although Kupffer cell phagocytosis is activated by peroxisome proliferators in vivo (8), how they signal increases in cell proliferation remains unclear.
Gut-derived endotoxin is a known activator of Kupffer cells (9); however, sterilization of the gut with antibiotics did not prevent the increase in cell proliferation or the increase in acyl CoA oxidase activity characteristic of peroxisome proliferators (Figure 1
). Moreover, plasma endotoxin levels in portal blood were not elevated following treatment with Wy-14,643 in vivo at times when Kupffer cells are known to be activated (Figure 2
). Therefore, it is concluded that gut-derived endotoxin is not involved in Kupffer cell activation by peroxisome proliferators.
However, Wy-14,643 and MEHP stimulated production of superoxide by Kupffer cells (Figures 3 and 4C
). Therefore, it is concluded that peroxisome proliferators activate Kupffer cells directly. Oxidants, including superoxide, are important signaling molecules which activate the transcription factor NF
B (22), an essential factor in the production of TNF
(23). Moreover, NF
B is rapidly activated by Wy-14,643 almost exclusively in Kupffer cells (7). TNF
, which is derived largely from Kupffer cells in liver, has been demonstrated to be responsible for Wy-14,643-induced cell proliferation since antibodies to TNF
blocked increases in hepatocyte replication (24). Additionally, inactivation of Kupffer cells with either methyl palmitate or dietary glycine prevented the increase in TNF
mRNA caused by Wy-14,643 (4,5). Therefore, Kupffer cell TNF
production and increased hepatocyte proliferation may be signaled via activation of NF
B by oxidants (e.g. superoxide). In support of this hypothesis, Marsman et al. demonstrated that both Wy-14,643 (0.1% in the diet) and DEHP (1.2% in the diet) cause an initial burst in hepatocyte proliferation (2), and Wy-14,643 and MEHP, the active metabolite of DEHP, activated Kupffer cell superoxide production following acute exposure in these studies (Figure 3
and Figure 4C
). Marsman et al. further showed that 21 days following dietary treatment, only Wy-14,643 sustained elevated rates of hepatocyte proliferation (2), and here superoxide generation by Kupffer cells was elevated only by Wy-14,643 after 21 days (Figure 8
). Furthermore, Wy-14,643 did not increase superoxide production in isolated hepatocytes even at doses 15 times greater than those which activated Kupffer cells (data not shown). Taken together, these data support the hypothesis that Kupffer cell-derived oxidants signal increases in hepatocyte proliferation following treatment with Wy-14,643.
Oxidant stress caused by leakage of hydrogen peroxide from peroxisomes was hypothesized initially as the mechanism by which these compounds cause liver tumors (25). It seems unlikely that oxidants of peroxisomal origin explain the mechanism of action of peroxisome proliferators since treatment with these compounds in vivo does not lead to increased hydrogen peroxide production (26). Moreover, consideration of enzyme kinetics makes it very unlikely that hydrogen peroxide could diffuse out of the intact peroxisome since degradation via catalase is over five orders of magnitude faster than the rate at which it is produced (27). Furthermore, formation of oxidized DNA bases following treatment with peroxisome proliferators remains controversial raising questions about the role of peroxisomal hydrogen peroxide in the mechanism by which these compounds cause liver cancer in rodents (28,29). Interestingly, catalase is induced ~2-fold in liver following treatment with peroxisome proliferators (1); however, measurement of catalase activity in liver cell fractions demonstrated that it is expressed only in hepatocytes and not Kupffer cells (3265.8 ± 777.3 U/g protein in hepatocytes versus 11.6 ± 4.2 U/g protein in Kupffer cells). Therefore, Kupffer cell-derived oxidants, including superoxide, may play a key role in initiating TNF
production which leads to hepatocyte proliferation since mechanisms for detoxifying reactive oxygen species are deficient in these macrophages.
PKC is required for peroxisome proliferator-stimulated superoxide production by Kupffer cells
Stimulation of superoxide production by Kupffer cells has been shown to involve activation of PKC (17,20). Here, inhibition of PKC with staurosporine prevented Wy-14,643-induced superoxide production (Figure 6
). Moreover, PKC activity, which is calcium dependent, increased following treatment of isolated Kupffer cells with Wy-14,643 (Figure 7
). Therefore, it is concluded that PKC is required for Wy-14,643-stimulated Kupffer cell superoxide production.
Pretreatment of Kupffer cells with glycine completely prevented both Wy-14,643-stimulated PKC activity and superoxide production (Figures 5 and 7
). In addition, dietary glycine has been shown to prevent Wy-14,643-induced hepatocyte replication by inhibiting Kupffer cell production of TNF
(5). Glycine inhibits TNF
production by Kupffer cells via activation of a glycine-gated chloride channel which hyperpolarizes the cell membrane and blunts intracellular calcium signaling (30). Since PKC activity is dependent on calcium and superoxide production requires PKC (Figures 6 and 7
), it is possible that glycine prevents Wy-14,643-stimulated superoxide production by Kupffer cells by hyperpolarizing the Kupffer cell membrane, inhibiting calcium signaling, and preventing activation of PKC (Figure 7
). These data are consistent with the hypothesis that peroxisome proliferators directly stimulate production of oxidants by Kupffer cells which activate the transcription factor NF
B. This leads to TNF
production and elevated rates of cell proliferation via mechanisms involving PKC.
The mechanism by which Wy-14,643 activates PKC is not known; however, several possibilities exist. Wy-14,643 may activate PKC directly. This seems unlikely, however, since administration of peroxisome proliferators directly to subcellular fractions did not alter PKC activity either in these studies (data not shown) or in work by Bronfman et al. (31). A second possibility is that Wy-14,643 increases unsaturated free fatty acids which are known to activate PKC directly (32). In support of this idea, Wy-14,643 competitively inhibits acyl CoA synthetase, the enzyme responsible for activation of long-chain fatty acids into fatty acyl CoA compounds, an essential step in the oxidation, elongation and esterification of fatty acids (33). Since this enzyme plays a central role in lipid metabolism, inhibition of acyl CoA synthetase by peroxisome proliferators could lead to an elevation of free fatty acids which would activate PKC (33). In fact, a correlation between the ability of various peroxisome proliferators to inhibit acyl CoA synthetase and activate PKC exists. (33). A third possibility is that Wy-14,643 displaces fatty acids bound to the peroxisome proliferator-activated receptor
(PPAR
) or to fatty acid binding protein, increasing free fatty acids and activating PKC (32). In support of this idea, peroxisome proliferators have been shown to bind to and displace fatty acids from fatty acid binding protein (34). Moreover, fatty acids are known to bind and activate the PPAR
(35).
Taken together, these data are consistent with the hypothesis that PKC is involved in Wy-14,643-stimulated superoxide production by Kupffer cells. Oxidants, including superoxide, could lead to activation of NF
B and increased TNF
production, which stimulates cell proliferation characteristic of this class of non-genotoxic hepatocarcinogens.
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Notes
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5 To whom correspondence should be addressed Email: thurman{at}med.unc.edu 
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Acknowledgments
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The authors wish to thank Ms Victoria Wong for performing diet analyses and Mr Paul Ross for oversight of animal care during the long-term study. This work was supported, in part, by a grant from NIEHS (ES-04325).
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References
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Received July 29, 1998;
revised September 4, 1998;
accepted September 25, 1998.