Role of hepatic non-parenchymal cells in the response of rat hepatocytes to the peroxisome proliferator nafenopin in vitro

Susan C. Hasmall1, Douglas A. West, Kine Olsen and Ruth A. Roberts

Cancer Biology Group, Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of liver cancer by peroxisome proliferators such as nafenopin is frequently associated with increased liver growth, increased DNA synthesis and suppression of apoptosis. The cytokine, tumour necrosis factor {alpha} (TNF{alpha}), and non-parenchymal liver cells have been implicated in mediating the hepatic response to peroxisome proliferators. Here, we have investigated the dependency of the hepatocyte response to peroxisome proliferators on non-parenchymal cells, a major source of hepatic cytokines. Addition of non-parenchymal cells, or conditioned medium from non-parenchymal cell cultures, increased DNA synthesis (220% and 270% of control, respectively) and suppressed transforming growth factor ß1-induced hepatocyte apoptosis (32% and 54% of control, respectively). Removal of non-parenchymal cells from normal hepatocyte cultures prevented both the nafenopin- and TNF{alpha}-induced increase in DNA synthesis and suppression of hepatocyte apoptosis; this response was restored by returning non-parenchymal cells to the purified hepatocytes. TNF{alpha} was detected in the medium of non-parenchymal cell (3–15 pg/ml) and normal hepatocyte cultures (25–100 pg/ml) by bioassay using L929 cells. However, the contribution of TNF{alpha} released from non-parenchymal cells was small compared with that released spontaneously by hepatocytes. Nafenopin significantly increased the release of TNF{alpha} from non-parenchymal cells to 56 ± 18 pg/ml, but had little effect on TNF{alpha} release by hepatocytes. However, the concentration of exogenous TNF{alpha} required to elicit a response in hepatocytes was 100 pg/ml and above. These data provide evidence that hepatic non-parenchymal cells are permissive for the growth response of hepatocytes in vitro to peroxisome proliferators and this may be mediated, at least in part by TNF{alpha}. However, the levels of TNF{alpha} released spontaneously or in response to peroxisome proliferators are insufficient per se to induce a growth response.

Abbreviations: EGF, epidermal growth factor receptor; NPC, non-parenchymal liver cell; PP, peroxisome proliferator; PPAR{alpha}, peroxisome proliferator activated receptor {alpha}; TGFß1, transforming growth factor ß1; TNF{alpha}, tumour necrosis factor {alpha}.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The group of non-genotoxic chemicals known as peroxisome proliferators (PPs) includes a diverse range of pharmaceutical and industrial chemicals. Fibrate hypolipidaemic drugs are used to reduce plasma cholesterol in humans, and plasticizers such as diethylhexylphthalate and di-isononylphthalate are used widely for example to impart plasticity to PVC. There are clear species differences in response to PPs. In rodents, peroxisome proliferation is associated with perturbation of liver growth, increased DNA synthesis, suppression of apoptosis and eventual tumour formation (reviewed in 1,2). However, there is little evidence that these adverse growth effects occur in humans (reviewed in 3) although humans do respond to the beneficial effects of fibrate drugs (reviewed in 4). The growth effects of PPs in vivo have been modelled in hepatocytes in vitro. PPs do not stimulate DNA synthesis or suppress apoptosis in cultured human hepatocytes in vitro (5,6). In contrast, in rodent hepatocytes, PPs stimulate DNA synthesis and cause suppression of both spontaneous apoptosis and that induced by the physiological negative growth regulator, transforming growth factor ß1 (TGF-ß1) (7). In the light of potential human exposure to these rodent hepatocarcinogens, a better understanding of the mechanism of rodent carcinogenesis is important in accurate evaluation of the potential risk of PPs to humans.

The rodent response to PPs is mediated by the peroxisome proliferator activated receptor {alpha} (PPAR{alpha}) since in PPAR{alpha}-null mice, PPs were unable to elicit liver growth, DNA synthesis, peroxisome proliferation or liver tumours (810). In addition, PPs did not increase DNA synthesis, suppress apoptosis or induce peroxisome proliferation in PPAR{alpha}-null hepatocytes in vitro (11,12). Liver growth is tightly regulated by growth factors such as hepatocyte growth factor and epidermal growth factor together with cytokines such as interleukin 1{alpha} (IL-1{alpha}), interleukin 6 and tumour necrosis factor {alpha} (TNF{alpha}; 13). TNF{alpha} is essential for liver regeneration; Akerman et al. (14) have shown that blocking TNF{alpha} activity with an anti-TNF{alpha} antibody prevents liver regeneration. Additionally, liver regeneration is reduced in transgenic mice deficient in the receptor TNFR1 (15). However, others suggest that the hepatic response to non-genotoxic carcinogens is unaltered in TNFR-null mice (16). In vitro, the rodent hepatocyte growth response to PPs can be prevented by either neutralizing TNF{alpha} or blocking the receptor TNFR1. Additionally, TNF{alpha} has been shown to mimic the PP-induced growth effects (1719). TNF{alpha} has also been implicated in mediating the rodent response to the PP, Wyeth-14,643 (20; reviewed in 21,22). The major source of cytokines in liver are non-parenchymal cells (NPCs) such as the stellate fat storing cells and Kupffer cells (23,24). Although exogenous TNF{alpha} elicits a growth response in vitro and TNF{alpha} signalling is required for PP response, the role of cytokine production by NPCs and the relationship between TNF{alpha} and PPAR{alpha} in mediating the hepatocyte response to PPs is not clear. We have investigated the effect of NPCs on hepatocyte DNA synthesis and apoptosis and on the hepatocyte response to PPs in vitro. These data provide evidence that TNF{alpha} released from hepatic NPCs may be important in regulating growth of hepatocytes in vitro. TNF{alpha} signalling is required to enable PPs to exert their growth effects; however, TNF{alpha} alone is not sufficient to induce a PP response in rat hepatocytes in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Fetal calf serum was purchased from First Link (Brierley Hill, UK) Ltd. Bromodeoxyuridine (BrdU) labelling reagent, diaminobenzidine tetrahydrochloride (DAB), and native human platelet TGFß1 were purchased from Sigma (Poole, UK), and the anti-BrdU antibody from Boehringer Mannheim (Lewes, UK). The peroxidase linked anti-mouse secondary antibody was from Dako (High Wycombe, UK) and Hoechst 33258 was from Molecular Probes Inc. (Junction City, OR). Recombinant rat TNF{alpha} from Insight Biotechnology Ltd (Wembley, UK) and IL-1{alpha} from R&D systems (Abingdon, UK). All other reagents were of the highest purity available and were purchased from Gibco (Paisley, UK) or Sigma.

Cell preparation
Cells were isolated from the livers of male Fischer 344 rats using a two-stage collagenase perfusion technique as described previously (25). The resultant cell suspension was centrifuged (at 50 x g for 2 min) and the hepatocyte pellet was washed and cells cultured as described previously (7). The cultures are referred to as normal hepatocyte cultures. NPCs were isolated from the supernatant by a method adapted from that of Nagelkerke et al. (26). The supernatant was centrifuged (400 x g for 10 min) and the resultant cell pellet was resuspended in Gey's balanced salt solution. NPCs were prepared by differential centrifugation on a metrizamide gradient [30% (w/v); 400 x g for 15 min] and washed in William's medium. Hepatocytes and NPCs were cultured in William's medium supplemented with 10% fetal calf serum (heat-inactivated), 10 µg/ml insulin, 0.1 mM hydrocortisone, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere. To determine the effect of NPCs and NPC medium on hepatocytes, medium was collected after 3 days' exposure to NPCs and hepatocyte–NPC co-cultures were prepared by adding hepatocytes to established NPC cultures after 4 days.

Separation of pure hepatocytes by Percoll density gradient
The proportion of NPCs in the normal cell suspension prepared by collagenase digestion was determined by light microscopy using a haemocytometer for each cell preparation. Hepatocytes were separated from NPCs, by centrifugation through Percoll (specific gravity 1.074 g/ml) at 400 x g for 10 min. The resultant pellet of pure hepatocytes was washed and the cells were cultured as described for normal hepatocyte cultures. The NPCs were collected from the supernatant by centrifugation (1400 x g, 6 min). To reconstitute the normal hepatocyte preparation, pure hepatocytes and NPCs were mixed together in the same proportion as the original cell preparation (18% ± 3% NPCs).

Measurement of DNA synthesis
DNA synthesis was determined as described previously (7). Cells were dosed after 24 h in culture and DNA synthesis determined by incorporation of BrdU during a 16 h labelling window 32–48 h after plating. Cells were fixed at 48 h and incorporated BrdU was localized using an anti-BrdU antibody followed by a peroxidase-linked secondary antibody and DAB substrate. Replicative DNA synthesis was measured by scoring the percentage of labelled nuclei. One thousand cells were counted per flask.

Measurement of apoptosis
Spontaneous and/or TGFß1-induced apoptosis were determined as described previously (7). Apoptosis was determined after 48 h exposure to test chemical by staining with Hoechst 33258 (5 ng/ml). For TGFß-induced apoptosis, TGFß1 (5 ng/ml) was added after 24 h exposure to chemical. Cells were fixed after 72 h in culture and apoptotic cells were identified as those with brightly staining, condensed chromatin. Where one apoptotic hepatocyte had generated multiple apoptotic bodies, this was scored as one apoptotic cell. One thousand cells were counted per flask.

Measurement of TNF{alpha}
TNF{alpha} activity was determined in culture medium taken from normal hepatocyte, NPCs and pure hepatocyte cultures at a range of time points during the 48 h culture period. TNF{alpha} activity was measured in L929 cells (mouse fibroblast cell line) as described previously (27). The standard curve was modified to include a range of known concentrations (0–1000 pg/ml) of rat TNF{alpha}. L929 cells were preincubated for 1 h with actinomycin D (6 µg/ml) and for a further 24 h in the presence of sample medium or TNF{alpha} standard medium. Cell viability was determined by staining with crystal violet and solublization in sodium dodecyl sulfate; absorbance was determined at 620 nm.

Statistics
Differences between treatment group means and the appropriate control were compared using Student's t-tests. A one-sided t-test was used to test for increased DNA synthesis or reduction of apoptosis in treatment groups. A two-sided t-test was used to test for changes in concentration of TNF{alpha}. Data are expressed as mean ± SD and statistical significance at P <= 0.05 or P <= 0.01 is indicated by * or **, respectively.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
NPCs can influence hepatocyte growth
Normal hepatocyte cultures prepared by the widely used collagenase digestion described by Berry and Friend (25) contain some NPCs which can be identified in hepatocyte monolayer cultures. Figure 1A and BGo shows the presence of Kupffer cells, visualized by phagocytosis of colloidal carbon and stellate cells in hepatocyte monolayers after 48 h in culture. Hepatocyte preparations contained 18% ± 3% NPCs whilst in vivo NPC represent ~40% of the liver cells. We have examined the effect of adding NPCs to hepatocyte cultures to levels approaching those seen in vivo. Addition of NPCs stimulated DNA synthesis and suppressed apoptosis in normal hepatocyte cultures (220% and 32% of control respectively; Figure 2A and BGo). Addition of the medium from NPC cultures increased DNA synthesis and suppressed hepatocyte apoptosis in the absence of additional NPCs (270% and 54% of control respectively), suggesting that a soluble factor is involved.



View larger version (80K):
[in this window]
[in a new window]
 
Fig. 1. Normal and purified hepatocyte cultures. NPCs are visible in normal hepatocyte monolayers. (A) A Kupffer cell, after phagocytosis of colloidal carbon and (B) a stellate cell in normal hepatocyte cultures. NPCs are not visible in cultures of purified hepatocytes (C). All photographs were taken after 48 h in culture.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. NPCs may regulate hepatocyte growth response in vitro. Addition of NPCs or medium from NPCs on DNA synthesis (A) and TGFß1-induced apoptosis (B) in normal hepatocyte cultures (Heps). The percentage of hepatocytes in S-phase (A) and percentage of cells undergoing apoptosis (B) are expressed as mean ± S.D.; * or ** indicate statistical significance at P <= 0.05 or P <= 0.01, respectively.

 
NPCs are required for the hepatocyte growth response to nafenopin
As expected from previous experiments (7), nafenopin stimulated DNA synthesis and suppressed apoptosis in normal hepatocyte cultures (Figure 3Go). Removal of NPCs from this normal hepatocyte preparation by purification on Percoll density gradients before culture increased the background rate of DNA synthesis (Figure 3Go) but had no effect on apoptosis (data not shown). These pure hepatocyte cultures appeared to be normal and are shown in Figure 1CGo. Nafenopin did not increase DNA synthesis or suppress hepatocyte apoptosis in these NPC-depleted hepatocyte cultures (Figure 3Go). Reconstitution of the `original' hepatocyte culture by re-addition of NPCs to the hepatocytes restored the hepatocyte response to nafenopin since nafenopin was able to stimulate DNA synthesis and suppress apoptosis (Figure 3Go). Interestingly, re-addition of NPCs also restored the background rate of DNA synthesis, suggesting that NPCs produce both inhibitory and stimulatory factors to regulate hepatocyte growth. The slightly higher viability of pure hepatocytes may also contribute to the higher rate of spontaneous DNA synthesis in pure hepatocyte cultures. To confirm that DNA synthesis can be induced in pure hepatocytes despite the higher background level we examined the effect of the mitogen, epidermal growth factor (EGF). EGF increased the rate of DNA synthesis in purified hepatocytes, demonstrating their integrity and ability to respond to a mitogenic stimulus (Table IGo). EGF also increased the rate of DNA synthesis in purified hepatocytes after reconstitution with NPCs or addition of medium from NPCs. NPCs are an important component of liver, comprising 40% of liver cells in vivo and <=20% of a normal hepatocyte culture. To understand the effect of nafenopin in the liver, it is important to assess DNA synthesis and apoptosis in control and nafenopin-treated NPCs. In cultures of NPCs the spontaneous rate of DNA synthesis was high and was not increased by EGF or nafenopin; likewise, spontaneous apoptosis was low and was not reduced by EGF or nafenopin (Table II).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3. NPCs are required for hepatocyte response to nafenopin. Role of NPCs on (A) induction of DNA synthesis and (B) suppression of TGFß1-induced apoptosis in response to nafenopin (50 µM, {blacksquare}). DNA synthesis and apoptosis were determined in normal hepatocytes, purified hepatocytes and in a mixture of purified hepatocytes with NPCs to reconstitute the normal hepatocyte preparation. Values are relative to vehicle control; actual control values are shown above the control bars ({square}). * and ** indicate statistical significance at P <= 0.05 and P <= 0.01, respectively.

 

View this table:
[in this window]
[in a new window]
 
Table I. Induction of hepatocyte DNA synthesis in response to EGF
 
The cytokines TNF{alpha} and IL-1{alpha} do not induce DNA synthesis in purified hepatocytes
Addition of TNF{alpha} to NPC-depleted cultures at levels shown to elicit a robust response in normal cultures (25 000 pg/ml) did not induce DNA synthesis or suppress apoptosis (Figure 4A and BGo). In addition, TNF{alpha} in combination with nafenopin did not increase DNA synthesis in pure hepatocytes, (2.0% ± 0.6% and 3.6% ± 0.7% cells in S-phase for TNF{alpha}+IL-1{alpha} and control, respectively). The lack of response to TNF{alpha} is very interesting since a requirement for TNF{alpha} has been demonstrated previously, (18,19); together these data suggest that, in addition to TNF{alpha}, other factors are involved. We have examined the effects of IL-1{alpha} alone and in combination with TNF{alpha} and/or nafenopin on DNA synthesis in pure hepatocytes. IL-1{alpha} either alone or in combination with TNF{alpha} did not induce DNA synthesis in pure hepatocytes (2.1% ± 0.9%, 2.6% ± 0.4% and 3.6% ± 0.7% cells in S-phase for IL-1{alpha} alone, IL-1{alpha} +TNF{alpha} and control, respectively). Furthermore, IL-1{alpha} either alone or in combination with TNF{alpha} did not restore the response to nafenopin (2.0% ± 0.5%, 2.0% ± 0.4% and 3.6% ± 0.7% cells in S-phase for IL-1{alpha} +nafenopin, IL-1{alpha}+TNF{alpha}+nafenopin and control, respectively). NPCs produce a complex mixture of many cytokines and growth factors; however, medium collected from NPC cultures did not restore the response of purified hepatocytes to nafenopin (4.8% ± 1.2% and 4.1% ± 0.4% cells in S-phase for nafenopin and control, respectively).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. Response of purified and normal hepatocytes to TNF{alpha}. Hepatocytes were purified by density gradient centrifugation and the effect of TNF{alpha} on (A) induction of DNA synthesis and (B) suppression of TGFß1-induced apoptosis was determined. (C and D) The response of normal hepatocyte cultures to increasing concentrations of TNF{alpha} in (C) induction of DNA synthesis and (D) suppression of TGFß1-induced apoptosis. The percentage of cells in S-phase (A and C), and percentage of cells undergoing apoptosis (B and D) are expressed as mean ± SD. ** indicates statistical significance at P <= 0.01.

 
Hepatocytes exhibit a threshold response to TNF{alpha}
In normal hepatocyte cultures, endogenous TNF{alpha} at concentrations of >=100 pg/ml induced DNA synthesis and suppressed TGFß1-induced apoptosis (Figure 4C and DGo). There was no further increase in response at higher concentrations of TNF{alpha} and concentrations of <100 pg/ml had no effect on DNA synthesis or apoptosis. These data suggest that the induction of DNA synthesis and suppression of apoptosis by TNF{alpha} may be an on/off response occurring at a threshold of ~100 pg/ml.

TNF{alpha} is released by non-parenchymal cells and by hepatocytes
NPCs released a low background level of TNF{alpha} into the culture medium (<=15 pg/ml medium; Figure 5Go). The concentration of TNF{alpha} was increased by exposure of cultured NPCs to nafenopin. The response appeared to be biphasic; there was a rapid increase within the first 4 h of exposure to nafenopin; levels returned towards control after 16 h and started to increase for the second time after 24 h exposure. TNF{alpha} remained increased until >=48 h after addition of nafenopin. TNF{alpha} release was much higher in untreated normal hepatocyte cultures than in NPCs. In hepatocyte cultures the concentration of TNF{alpha} (25–100 pg/ml) varied between time points and hepatocyte preparation. Nafenopin caused a small increase over control, which was statistically significant at some time points (Figure 5Go). The largest increase, 1.5-fold, was detected 8 h after dosing and therefore preceded the nafenopin-induced increase in DNA synthesis (measured 8–24 h after dosing) and apoptosis (48 h after dosing). In contrast to nafenopin, lipopolysaccharide (50 ng/ml) used as a positive control caused a 4-fold increase in TNF{alpha} concentration at 24 h. Low levels of TNF{alpha} (69 ± 9 pg/ml) were detected in purified hepatocytes, but only after prolonged culture.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. TNF{alpha} concentration in hepatocyte and NPC cultures. Concentration of TNF{alpha} in vehicle control ({blacktriangleup}), and nafenopin ({blacksquare}) treated hepatocyte (solid lines) and NPC (dotted lines) cultures. Bars show TNF{alpha} concentration in untreated (solid bar) and lipopolysaccharide-treated (open bar) hepatocyte cultures after 24 h. TNF{alpha} concentration (pg/ml culture medium) is expressed as mean ± SD; * and ** indicate statistical significance at P <= 0.05 or P <= 0.01, respectively.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of DNA synthesis and suppression of apoptosis by PPs, such as nafenopin, has been associated with subsequent rodent tumourigenesis (reviewed in 1,2). The signalling pathway is not understood, although TNF{alpha} is clearly implicated in the hepatocyte growth response to PPs (20). TNF{alpha} signalling is required for PP-induced growth since blocking either TNF{alpha} or TNFR1 with antibodies prevented the nafenopin-induced growth response in vitro (18,19). In addition, exogenous TNF{alpha} is able to elicit a growth response in hepatocytes in vitro similar to that caused by nafenopin (17). Hepatic NPCs, the Kupffer and stellate cells, are the major site of cytokine production in the liver and have been implicated in the hepatocyte response to PPs. We have examined the role of NPCs in mediating the growth response to PPs and have examined the effect of nafenopin on TNF{alpha} production by both NPCs and hepatocytes.

We have shown that hepatocyte cultures, which are frequently used to study growth responses, contain NPCs in proportions varying between preparations but sometimes as high as 20%. Removal of these NPCs increased DNA synthesis suggesting that NPCs may release inhibitory factors to restrict DNA synthesis in untreated hepatocytes. Addition of extra NPCs to normal hepatocyte cultures that already contain NPCs also increased the background rate of DNA synthesis and suppressed apoptosis. These data demonstrate that changing the ratio of NPCs and hepatocytes can up- or down-regulate hepatocyte growth and suggest that NPCs play an important role in maintaining hepatocyte homeostasis. Since medium from NPC cultures also increased DNA synthesis and suppressed apoptosis in the absence of NPCs, it is probable that soluble factors are involved. In normal hepatocyte cultures, nafenopin increased DNA synthesis and suppressed apoptosis [shown here and previously (7)]. Here we have shown that NPCs were required to permit this increased DNA synthesis and suppression of apoptosis in hepatocytes. In the absence of NPCs, the response to nafenopin was lost but could be restored by reconstitution of the original preparation. Thus, NPCs produce factor(s) which can regulate hepatocyte growth and are required for hepatocytes to respond to nafenopin.

NPCs are the major source of hepatic cytokines (23) and the cytokines IL-1 and TNF{alpha} are required for a growth response to PPs, since antibodies to either cytokine prevent the hepatocyte response to PPs (18). Similarly, the non-parenchymal Kupffer cells are reported to mediate the mitogenic effects of the PP, Wyeth 14,643 (21,28). Therefore it seems likely that NPCs in hepatocyte cultures release cytokines which are required for, but not responsible for, PP-induced growth response. We have shown that nafenopin stimulated the release of the cytokine TNF{alpha} from NPCs but not from hepatocytes. However, the concentration of TNF{alpha} released (<60 pg/ml) was very low compared with that in untreated hepatocytes, and nafenopin did not stimulate TNF{alpha} release by hepatocytes. These results are consistent with the lack of nafenopin-induced TNF{alpha} mRNA in vivo or in vitro (29,30). Furthermore the PP Wyeth-14,643 did not increase hepatocyte mRNA or serum concentration of TNF{alpha} in vivo despite increased TNF{alpha} mRNA levels in Kupffer cells (19,31) and Wyeth-14,643 did induce cell proliferation in TNF{alpha}-null mice (32). Together these data suggest that increased TNF{alpha} production by NPCs is unlikely to mediate the hepatocyte response to nafenopin.

High concentrations of exogenous TNF{alpha} (25 000 pg/ml) can induce DNA synthesis and suppress apoptosis (17); however, the effects of lower levels seen in response to nafenopin had not been examined. We have shown here that a threshold concentration (~100 pg/ml) is required for a response suggesting that the low levels of TNF{alpha} released by NPCs in response to nafenopin are unlikely to elicit a growth response. Furthermore exogenous TNF{alpha} did not induce DNA synthesis or suppress apoptosis in purified hepatocytes in the absence of NPCs, although EGF did stimulate DNA synthesis in these cells. Therefore hepatocytes respond to nafenopin in the presence of NPCs, but not to levels of TNF{alpha} expected to induce a response in normal hepatocytes. These data suggest that although NPCs produce some TNF{alpha} in response to nafenopin, TNF{alpha} alone is not sufficient to induce a response or mediate the hepatocyte response to PPs. A key candidate is IL-1{alpha} since antibodies to either cytokine prevents the hepatocyte response to PPs (18). However, IL-1{alpha} did not induce DNA synthesis in pure hepatocytes and did not restore the response to nafenopin. Furthermore, IL-1{alpha} and TNF{alpha} combined did not induce DNA synthesis in pure hepatocytes or restore the response to nafenopin. It seems likely that NPCs secrete many cytokines and growth factors required to maintain homeostasis and to enable hepatocytes to respond to nafenopin. The inability of medium from NPCs to restore the response to nafenopin suggests that NPCs may require activation by a signal from nafenopin-treated hepatocytes. In addition, local concentration effects and/or stability of cytokines may play a role.

The mechanism of increased release or synthesis of TNF{alpha} reported here for NPCs and previously for Kupffer cells (19) in response to PPs remains unclear. PPs activate the transcription factor PPAR{alpha} and therefore PPAR{alpha}/ mice, which lack a functional PPAR{alpha}, do not respond to PPs (811). However, we (data not shown) and others (33) could not detect PPAR{alpha} mRNA in rat Kupffer cells and future studies will focus on the role of PPAR{alpha}. Our TNF{alpha} dose–response in hepatocytes and the inability of exogenous TNF{alpha} to induce a response in purified hepatocytes, together with the lack of response reported in PPAR{alpha}/ mice, suggest that the amount of TNF{alpha} elicited by PPs is insufficient per se to elicit a growth response. It seems more likely that TNF{alpha} together with other hepatic cytokines are permissive for a PPAR{alpha}-dependent response to PPs. Similarly, it has been reported that EGF requires the presence of TNF{alpha} to prime hepatocytes for DNA synthesis (34,35). Interestingly, EGF did induce DNA synthesis in purified hepatocytes, which suggests that purified hepatocytes do contain sufficient TNF{alpha} to permit a response at least to EGF. Therefore, either PPs require a higher concentration of TNF{alpha} or, more likely, several cytokines and/or growth factors are required to enable the hepatocytes to respond to PPs. In summary, nafenopin stimulates release of TNF{alpha} both from NPCs and from hepatocytes, TNF{alpha} is required for but is not responsible for the growth response to nafenopin. NPCs may be the source of a diversity of cytokines and growth factors that provide survival signals, enabling PPs to induce hepatocyte DNA synthesis and suppress apoptosis.


    Notes
 
1 To whom correspondence should be addressed Email: sue.hasmall{at}ctl.zeneca.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Moody,D.E., Reddy,J.K., Lake,B.G., Popp,J.A. and Reese,D.H. (1991) Peroxisome proliferation and non-genotoxic carcinogenesis: commentary on a symposium. Fund. Appl. Toxicol., 6, 233–248.
  2. Ashby,J., Brady,A., Elcombe,C.R., Elliot,B.M., Ishmael,J., Odum,J., Tugwood,J.D., Kettle,S. and Purchase,I.F.H. (1994) Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. Human Exp. Toxicol., 13, S1–S117.
  3. Williams,G.M. and Perrone,C. (1995) Mechanism-based risk assessment of peroxisome proliferating rodent hepatocarcinogens. Ann. N.Y. Acad. Sci., 804, 554–572.
  4. Tucker,M.J. and Orton,T.C. (1995) Comparative Toxicology of Hypolipidaemic Fibrates. Taylor & Francis, London.
  5. Hasmall,S.C., James,N.H., Soames,A.R. and Roberts,R.A. (1998) The peroxisome proliferator nafenopin does not suppress hepatocyte apoptosis in guinea pig liver in vivo or in human hepatocytes in vitro. Arch. Toxicol., 72, 777–783.[Medline]
  6. Hasmall,S.C., James,N.H., Macdonald,N., West,D., Chevalier,S., Cosulich,S.C. and Roberts,R.A. (1999) Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphlathate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro. Arch. Toxicol., 73, 451–456.[Medline]
  7. James,N.H. and Roberts,R.A. (1996) Species differences in response to peroxisome proliferators correlate in vitro with induction of DNA synthesis rather than with suppression of apoptosis. Carcinogenesis, 17, 1623–1632.[Abstract]
  8. Lee,S., Pineau,T., Drago,J., Lee,E.J., Owens,J.O., Kroetz,D.L., Fernandez-Salguero,P.M., Westphal,H. and Gonzalez,F.J. (1995) Targeted disruption of the {alpha} isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotrophic effects of peroxisome proliferators. Mol. Cell. Biol., 15, 3012–3022.[Abstract]
  9. Gonzalez,F.J. (1997) Recent update on the PPAR{alpha}-null mouse. Biochimie, 79, 139–144.[Medline]
  10. Peters,J.M., Cattley,R.C., Gonzalez,F.J. (1997) Role of PPARalpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator WY-14, 643. Carcinogenesis, 18, 2029–2033.[Abstract]
  11. Hasmall,S.C., James,N.H., Macdonald,N., West,D., Gonzalez,F.J., Peters,J.M. and Roberts,R.A. (2000) Suppression of mouse hepatocyte apoptosis by peroxisome proliferators: role of PPAR{alpha} and TNF{alpha}. Mutat. Res., 448, 193–200.[Medline]
  12. Christensen,J., Gonzales,A., Cattley,R. and Goldsworthy,T. (1998) Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens. Cell Growth Differ., 9, 815–825.[Abstract]
  13. Michalopoulos,G.K. and De Frances,M.C. (1997) Liver regeneration. Science, 276, 60–66.[Abstract/Full Text]
  14. Akerman,P., Cote,P., Yang,S., McClain,C., Nelson,S., Bagby,G. and Diehl,A.M. (1992) Antibodies to tumour necrosis factor-{alpha} inhibit liver regeneration after partial hepatectomy. Am. J. Physiol., 263, 579–585.
  15. Yamada,Y., Kirillova,I., Peschon,J.J. and Fausto,N. (1997) Initiation of liver growth by tumour necrosis factor: deficient liver regeneration in mice lacking type 1 tumour necrosis factor receptor. Proc. Natl Acad. Sci. USA, 94, 1441–1446.[Abstract/Full Text]
  16. Ledda-Columbano,G., Curto,M., Piga,R. et al. (1998) In vivo hepatocyte proliferation is inducible through a TNF and IL-6-independent pathway. Oncogene, 17, 1039–1044.[Medline]
  17. Rolfe,M., James,N.H. and Roberts,R.A. (1997) Tumour necrosis factor {alpha} (TNF-{alpha}) suppreses apoptosis and induces S-phase in rodent hepatocytes: a mediator of the hepatocarcinogenicity of peroxisome proliferators? Carcinogenesis, 18, 2277–2280.[Abstract]
  18. West,D., James,N., Holden,P., Brindle,R., Rolfe,M. and Roberts,R. (1999) Role for tumour necrosis factor {alpha} (TNF{alpha}) receptor 1 (TNFR1) and interleukin 1 receptor (IL1R) in the suppression of apoptosis by peroxisome proliferators. Hepatology, 30, 1417–1424.[Abstract/Full Text]
  19. Bojes,H.K., Germolec,D.R., Simeonova,P., Bruccoleri,A., Schoonhoven,R., Luster,M.I. and Thurman,R.G. (1997) Antibodies to tumour necrosis factor {alpha} prevent increases in cell replication in liver due to the potent peroxisome proliferator, WY-14,643. Carcinogenesis, 18, 669–674.[Abstract]
  20. Rusyn,I., Tsukamoto,H. and Thurman,R.G. (1998) WY-14,643 rapidly activates nuclear factor {kappa}B in Kupffer cells before hepatocytes. Carcinogenesis, 19, 1217–1222.[Abstract]
  21. Rose,M., Rusyn,I., Bojes,H., Germolec,D., Luster,M. and Thurman,R. (1999) Role of Kupffer cells in peroxisome proliferator-induced hepatocyte proliferation. Drug Metab. Rev., 31, 87–116.[Medline]
  22. Roberts,R. and Kimber,I. (1999) Cytokines in non-genotoxic hepatocarcinogenesis. Carcinogenesis, 20, 1397–1401.[Full Text]
  23. Old,L.J. (1985) Tumour necrosis factor (TNF). Science, 230, 630–632.[Medline]
  24. Decker,K. (1990) Biologically active products of stimulates liver macrophages (Kupffer cells). Eur. J. Biochem, 192, 245–261.[Medline]
  25. Berry,M.N. and Friend,D.S. (1969) High yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol., 43, 506–520.[Medline]
  26. Nagelkerke,J., Barto,K. and van Berkel,T. (1983) In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells. J. Biol. Chem., 258, 12221–12227.[Abstract]
  27. Cumberbatch,M. and Kimber,I. (1992) Dermal tumour necrosis factor-{alpha} induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans, cell migration. Immunology, 75, 257–263.[Medline]
  28. Rose,M.L., Germolec,D.R., Schoonhoven,R. and Thurman,R.G. (1997) Kupffer cells are causally responsible for the mitogenic effect of peroxisome proliferators. Carcinogenesis, 18, 1453–1456.[Abstract]
  29. Holden,P., Hasmall,S., James,N., West,D., Brindle,R., Gonzalez,F., Peters,J. and Roberts,R. (2000) Tumour necrosis factor {alpha} (TNF{alpha}): role in suppression of apoptosis by the peroxisome proliferator nafenopin. Cell. Mol. Biol., 46, 29–39.
  30. Menegazzi,M., Carcereri-De Prati,A., Shinozuka,H., Pibiri,M., Piga,R., Columbano,A. and Ledda-Columbano,G.M. (1997) Liver cell proliferation induced by nafenopin and cyproterone acetate is not associated with increases in activation of transcription factors NF-{kappa}B and AP-1 or with expression of tumor necrosis factor-{alpha}. Hepatology, 25, 585–592.[Abstract]
  31. Rose,M.L., Rivera,C.A., Bradford,B.U., Graves,L.M., Cattley,R.C., Schoonhoven,R., Swenberg,J.A. and Thurman,R.G. (1999) Kupffer cell oxidant production is central to the mechanism of peroxisome proliferators. Carcinogenesis, 20, 27–33.[Abstract/Full Text]
  32. Givler,B., Alberts,G., Wollenberg,G., Pitzenberger,M., Deluca,J. and Lawrence,J. (2000) Tumour necrosis factor {alpha} (TNF{alpha}) is not required for WY-14,643 induced cell proliferation in mice. Toxicol. Sci., 48, 420.
  33. Peters,J., Rusyn,I., Rose,M., Gonzalez,F. and Thurman,R. (2000) Peroxisome proliferator-activated receptor {alpha} is restricted to hepatic parenchymal cells, not Kupffer cells: implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis. Carcinogenesis, 21, 823–826.[Abstract/Full Text]
  34. Chevalier,S., Macdonald,N. and Roberts,R.A. (1999) Induction of DNA replication by peroxisome proliferators is independent of both tumour necrosis factor {alpha} priming and EGF-receptor tyrosine kinase activity. J. Cell Sci., 112, 4785–4791.[Abstract]
  35. Webber,E., Bruix,J., Pierce,R. and Fausto,N. (1998) Tumour necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology, 28, 1226–1234.[Abstract/Full Text]
Received April 11, 2000; revised July 13, 2000; accepted August 22, 2000.