Peroxisome proliferators do not increase DNA synthesis in purified rat hepatocytes

Wolfram Parzefall1,, Walter Berger, Eveline Kainzbauer, Olga Teufelhofer, Rolf Schulte-Hermann and Ronald G. Thurman

Institut für Krebsforschung, Universität Wien, Borschkegasse 8a, A-1090 Wien, Austria


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There have been numerous reports that chemicals which induce peroxisomes in rodent liver increase DNA synthesis in isolated hepatic parenchymal cells, but not as well in vitro as in vivo. It is also known that tumour necrosis factor {alpha} (TNF{alpha}) is mitogenic in isolated hepatocytes. Since Kupffer cells are a major source of TNF{alpha} in the liver and have recently been shown to be activated by peroxisome proliferators, the possibility exists that the effect of peroxisome proliferators on DNA synthesis in parenchymal cells is via Kupffer cell contamination of isolated hepatocyte preparations. The purpose of this study was to evaluate this hypothesis by studying the effect of model peroxisome proliferators on purified hepatocyte preparations. Hepatocytes were prepared from rat liver by standard calcium-free and collagenase perfusion. Subsequently, cells were centrifuged through Percoll to remove contaminating non-parenchymal cells. Cells were at least 99.9% pure as assessed by cell counting using specific markers for hepatocytes (resorufin O-glucoside) and Kupffer cells (FITC-labelled latex beads). Hepatocytes were cultured in Williams medium + 10% fetal bovine serum for 24 h followed by culture for 48 h in Williams medium plus or minus drug or mitogen additions. Under these conditions epidermal growth factor stimulated DNA synthesis assessed by incorporation of [3H]thymidine ~5-fold over control levels. The peroxisome proliferators WY,14-643 and nafenopin, however, had no effect on DNA synthesis, although they did increase acyl-CoA oxidase as expected. In contrast, TNF{alpha} increased cell proliferation nearly 10-fold in purified hepatocytes, an effect nearly doubled by WY-14,643. Further, when conditioned medium from purified Kupffer cells incubated with WY-14,643 was added to pure hepatocytes, DNA synthesis was increased over 2-fold in a time-dependent manner. Collectively, these data support the hypothesis that peroxisome proliferators do not influence DNA synthesis in isolated hepatocytes per se. Rather, they stimulate cytokine production by Kupffer cells which in turn increases DNA synthesis in parenchymal cells. An increase in mitogenic cytokine production by Kupffer cells is necessary for stimulation of DNA synthesis in purified rat parenchymal cells.

Abbreviations: DEHP, di (2-ethylhexyl)phthalate; TNF{alpha}, tumour necrosis factor {alpha}.


    Introduction
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 Abstract
 Introduction
 References
 
Peroxisome proliferators are a class of structurally diverse compounds which produce profound changes in livers of rats and mice which include hepatomegaly, induction of peroxisomal enzymes of fatty acid metabolism and increases in size and number of peroxisomes (1). Importantly, continuous exposure to these agents leads to the development of hepatocellular carcinoma (2). Nearly two decades ago several groups began to study the effect of these agents on isolated hepatocytes in vitro. When primary cultures of rat hepatocytes were exposed to a wide range of chemicals known to induce peroxisomes in vivo induction of peroxisomal marker enzymes was observed (3,4). Thus, this work established that hepatic parenchymal cells indeed respond to this class of chemicals in vitro. The response of the liver to these compounds in vivo includes both hyperplasia and hypertrophy. Thus, it was not surprising when it was reported that peroxisome proliferators increase DNA synthesis in isolated hepatocytes in vitro, mirroring the response in vivo, at least qualitatively (514). Most of these studies reported a 2- to 3-fold increase in DNA synthesis, but both lower and higher values have been observed. This is surprising since the response is much more robust in vivo (e.g. 10-fold). On the other hand, attempts to ensure that these preparations were free of contaminating non-parenchymal cells had not been taken. This could be important, since it is now known that non-parenchymal cells such as Kupffer cells are a rich source of mitogens such as tumour necrosis factor {alpha} (TNF{alpha}) (15,16). Indeed, the increase in cell replication caused by WY-14,643 in vivo in the rat was completely prevented by pretreatment with anti-TNF{alpha} antibodies (17). More recently it was demonstrated that WY-14,643 directly stimulates TNF{alpha} production by isolated Kupffer cells (18). Thus, the question arises whether the effect of peroxisome proliferators on DNA synthesis in parenchymal cells in vitro is direct or occurs via production of mitogens from contaminating Kupffer cells. Indeed, this study clearly shows that hepatocytes of high and verified purity exhibit increased DNA synthesis in vitro to TNF{alpha}. In contrast, the model peroxisome proliferators WY-14,643 and nafenopin were without effect. Moreover, WY-14,643 nearly doubles the effect of TNF{alpha} on DNA synthesis in purified rat hepatocytes.

The method of Kreamer et al. was used here to purify hepatocytes (19). After standard preparation of liver cells with collagenase, parenchymal cells were purified by three low speed sedimentation steps (15 g) and subsequent centrifugation over Percoll to remove non-parenchymal cells. Verification of this procedure in our hands is depicted in Figure 1Go. Resorufin O-glucoside is specifically cleaved in the cytosol of hepatocytes while phagocytosis of FITC-labelled latex beads is a unique property of Kupffer cells. Large rectangular hepatocytes were photographed under normal light and are depicted in Figure 1AGo. After incubation with resorufin O-glucoside only the red fluorescence of liberated resorufin was detected (Figure 1BGo). Moreover, when cells were incubated with resorufin O- glucoside and latex beads only red fluorescence was detected (Figure 1CGo). Some beads which were not cell-associated were also observed (Figure 1CGo, arrows). Similar results were obtained in 10 subsequent fields from two separate liver preparations. Indeed, all cells were determined to be hepatocytes since they were resorufin-positive and latex bead- negative. Uptake of multiple FITC-labelled latex beads (arrows) by purified Kupffer cells is depicted in Figure 1DGo as a positive control.



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Fig. 1. Validation of purity of hepatocyte preparations. Hepatocytes were isolated from 200–340 g male Wistar rats (Breeding Facility Himberg, University of Vienna) fed ad libitum (lights off 0:00–12:00). Hepatocytes were collected from the initial cell suspension by centrifugation at 15 g for 5 min and washed twice with MEM medium. A suspension of cells was mixed with an equal volume of 90% isotonic Percoll and centrifuged at 50 g for 10 min (19). Viability assessed from Trypan blue exclusion was 94 ± 2%. Hepatocytes were cultured on collagen gel in Williams medium + 10% FBS at a density of 30 000 cells/cm2 in 6-well plates for 4 h. Hepatocyte purity was assessed with the parenchymal cell-specific marker, resorufin O-glucoside (28). (A) Light field micrograph of cells. (B) Resorufin O-glucoside (0.1 mM) was added to hepatocytes cultured on coverslips in HBSS for 30 min at 37°C. After two washes with HBSS, red fluorescence of liberated resorufin was determined with cyn-3 filters and a Leica image acquisition and analysis system. (C) Addition of FITC-labelled latex beads and resorufin O-glucoside. (D) The non-parenchymal cell fraction was separated by centrifugation through a Percoll gradient based on the method of Pertoft and Smedsrod (29) and enriched Kupffer cell fractions were seeded on collagen-coated supports and cultured for 1 h. Non-adherent cells were removed by replacement with fresh culture medium and adherent cells were verified to be Kupffer cells by phagocytosis of FITC-labelled latex beads by fluorescence (30). Typical experiments. Arrows point to FITC-labelled latex beads. Magnification, 260x.

 
It was shown many years ago that addition of peroxisome proliferators (e.g. clofibrate) to isolated hepatocytes induced peroxisomes, reflected in a time-dependent increase in acyl-CoA oxidase activity (3). This phenomenon has been replicated with a variety of peroxisome proliferators (4). Therefore, it was of interest to determine whether a similar phenomenon occurred here in purified hepatocytes. Activity of the marker enzyme acyl-CoA oxidase was measured as described in detail elsewhere (23). Indeed, WY-14,643 increased acyl-CoA oxidase ~2-fold (data not shown), demonstrating that induction of acyl-CoA oxidase is clearly a property of pure hepatic parenchymal cells. It is known that peroxisomes are induced more robustly in vivo than in vitro (3,21). These findings are consistent with the recent observation that PPAR{alpha}, which is required for induction of peroxisomes, was localized in parenchymal but not in Kupffer cells (20).

On the other hand, considerable evidence supports the hypothesis that the increase in DNA synthesis caused by peroxisome proliferators occurs via a mechanism different from induction of peroxisomes (32). For example, Marsman and Popp (21) fed WY-14,643 and the plasticizer di(2-ethylhexyl)phthalate (DEHP) and observed an initial burst in DNA synthesis and induction of peroxisomes of similar magnitude with both compounds. Subsequently, DNA synthesis declined with both chemicals, only remaining elevated over basal values with WY-14,643 for as long as the compound was given. Under these conditions, however, peroxisomes remained elevated with both compounds. A similar dissociation between increase in DNA synthesis and induction of peroxisomes has subsequently been observed in studies with WY-14,643 using glycine (22) and anti-TNF{alpha} antibody (17). Moreover, WY-14,643 increases DNA synthesis 8- to 10-fold in vivo but only 2- to 3-fold in vitro (15,21). Thus, one would not necessarily conclude that because peroxisome proliferators increase peroxisomes in pure hepatocytes that DNA synthesis would follow (see below).

To account for preparation to preparation variation, rates of DNA synthesis were normalized to control values in this study (Figure 2Go). With hepatocytes purified with Percoll the concentration of epidermal growth factor which gave maximal responses increased replicative DNA synthesis ~5-fold (P < 0.05). In contrast, the model peroxisome proliferators WY-14,643 and nafenopin had no effect (Figure 2Go). In sharp contrast, TNF{alpha} increased DNA synthesis nearly 10-fold in purified hepatocytes (P < 0.05 ). This effect was potentiated by WY-14,643. To test the hypothesis that the responses reported in the literature with peroxisome proliferators could be due to contaminating Kupffer cells, hepatocytes were incubated with conditioned medium from Kupffer cells incubated with WY-14,643 for various times. Under these conditions DNA synthesis was increased over 2-fold in a time-dependent manner when purified parenchymal cells were cultured with conditioned medium from Kupffer cells (Figure 3Go). Thus, it is concluded that increases in DNA synthesis due to peroxisome proliferators reported in the literature are most likely due to parenchymal cells contaminated with Kupffer cells. This finding is consistent with the recent observation that WY-14,643 and mono(2-ethylhexyl)phthalate, a major metabolite of DEHP, increased TNF{alpha} in isolated Kupffer cells (18,24). A reasonable hypothesis to explain the effect of this class of compounds is that peroxisome proliferators act on Kupffer cells to produce mitogenic cytokines which stimulate DNA synthesis in hepatic parenchymal cells (see Figure 4Go). Simultaneously, these agents increase peroxisomes in parenchymal cells. In support of this hypothesis, West et al. recently reported that antibodies to TNF{alpha} receptor 1 block the increase in DNA synthesis in hepatocytes due to nafenopin (6). Thus, these results suggest that the increases in DNA synthesis induced by peroxisome proliferators in purified hepatic parenchymal cells may be due to the production of mitogenic cytokines by Kupffer cells (see Figure 4Go). On the other hand, Peters et al. (25) reported that the increase in DNA synthesis caused by WY-14,643 does not occur in PPAR{alpha} knockout mice. This phenomenon cannot involve PPAR{alpha} in Kupffer cells, since it was shown not to be present therein (20). Thus, parenchymal cell PPAR{alpha} and Kupffer cell TNF{alpha} could both be required for maximal DNA synthesis due to peroxisome proliferators. This hypothesis is supported by the observation that TNF{alpha} and WY-14,643 in combination increased DNA synthesis in purified rat hepatocytes (Figure 2Go).



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Fig. 2. Effects of model peroxisome proliferators and mitogens on DNA synthesis by purified hepatocytes. Conditions were as in Figure 1Go. Hepatocytes were cultured on collagen gel in Williams medium + 10% FBS at a density of 30 000 cells/cm2 in 6-well plates for 24 h and subsequently for 48 h in Williams medium without serum. Medium was supplemented throughout with 1.67 µM insulin and 3 µM dexamethasone. At 24 h WY-14,643 (20 µM) and nafenopin (30 µM) were added in DMSO (final concentration 0.2%) while EGF (10 ng/ml) and TNF{alpha} (50 ng/ml) were added in saline. At 70 h 2 µCi of [3H]thymidine was added and the reaction was stopped at 72 h by placing cultures on ice and aspiration of the medium. Cells were harvested and radioactivity was assessed by scintillation counting. Values are expressed per unit DNA determined as described elsewhere (31). Data are means ± SD, n = 4–5 individual hepatocyte preparations. *P < 0.05 by rank test.

 


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Fig. 3. Effect of conditioned medium from Kupffer cells on DNA synthesis in hepatocytes. Conditions were as in Figure 1Go. Kupffer cells and hepatocytes were purified and Kupffer cells were plated on 6-well plates at a density of 30 000 cells/cm2 in Williams medium + 10% FBS in the presence of 20 µM WY-14,643. Conditioned medium was collected at the times denoted and added to pure hepatocytes. Hepatocytes were cultured in conditioned medium for 48 h as described in Figure 1Go. Typical experiment. *P < 0.05 by rank test.

 


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Fig. 4. Working hypothesis. Based on this work, it is hypothesized that peroxisome proliferators do not affect DNA synthesis in pure hepatocytes per se. Rather, they stimulate Kupffer cells to produce mitogens which stimulate proliferation of parenchymal cells. Model peroxisome proliferators, however, do increase acyl-CoA oxidase in pure preparations of parenchymal cells and enhance the effect of TNF{alpha} on DNA synthesis. This latter phenomenon is depicted by a question mark in the scheme.

 


    Notes
 
1 To whom correspondence should be addressed Back


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 Abstract
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 References
 

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Received July 21, 2000; revised July 21, 2000; accepted November 9, 2000.