Effect of lindane and phenobarbital on cyclooxygenase-2 expression and prostanoid synthesis by Kupffer cells
Bernd Kroll,
Susanne Kunz,
Thomas Klein1 and
Leslie R. Schwarz2
GSF-National Research Center for Environment and Health, Institute of Toxicology, D-85758 Neuherberg/München and
1 University of Marburg, Department of Pediatrics, 35037 Marburg, Germany
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Abstract
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Prostaglandins (PGs) have been implicated in tumor promotion. In this study, we investigated the effect of the hepatic tumor promoters lindane and phenobarbital (PB) on the PG metabolism of Kupffer cells in vitro and in vivo, in particular on the expression of cyclooxygenase (COX), the leading enzyme in prostanoid synthesis. Exposure of primary cultures of Kupffer cells to lindane for 1 h stimulated the production of the PGs PGE2 and PGD2 markedly (up to 50-fold) and that of PGF2
by >3-fold. This effect was accompanied by an increase in the COX-2 protein, as demonstrated by western blotting. Similarly, PB, which shares several effects with lindane in rat liver, also clearly induced COX-2. Lindane and PB affected the PG synthesis in vitro and in vivo in Kupffer cells of rats that had been treated with the two compounds for 56 days. Kupffer cells, which were isolated at days 2, 5 and 56 of the treatment, showed a significant increase in the levels of COX-2 mRNA and protein. Total COX activity was increased ~2-fold and 3- to 5-fold in Kupffer cell homogenates of PB- and lindane-treated animals, respectively, compared with the untreated controls. These results suggest that paracrine mechanisms may contribute to the tumor-promoting activity of lindane and PB, stimulating the production of PGs by Kupffer cells.
Abbreviations: AA, arachidonic acid; COX, cyclooxygenase (prostaglandin H-synthase); DTT, dithiothreitol; DMEM, Dulbeco's modified Eagle's medium; lindane,
-hexachlorocyclohexane; PB, phenobarbital; PGs, prostaglandins; RTPCR, reverse transcriptasepolymerase chain reaction
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Introduction
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Arachidonic acid (AA) metabolism and, in particular, prostaglandins (PGs) have long been postulated to be involved in many aspects of carcinogenesis, including tumor growth and development (1,2). Many tumor tissues, such as experimental tumors in rats and various tumors in humans, synthesize large amounts of PGs, in particular PGE2 and PGF2
(1). Various studies suggest the involvement of products of prostaglandin H-synthase (cyclooxygenase, COX) in tumor promotion. COX is the rate-limiting enzyme in prostaglandin synthesis. Promotion of mammary tumors by polyunsaturated fats (3), colonic tumors by bile acids (4), urinary bladder tumors by saccharin (5) and skin tumors by tetradecanoylphorbol acetate (TPA; 6,7) were significantly decreased by administering inhibitors of COX. A decisive role of PGF2
and PGE2 has been demonstrated in the tumor promotion by TPA in mouse skin. In accordance with these findings, inhibition of TPA tumor promotion by the COX inhibitor indomethacin could be overcome by the co-administration of PGF2
(7). PGs may also be involved in tumor promotion in rat liver. Inhibitors of COX clearly diminished stimulation of growth of preneoplastic liver cell foci and liver tumors by phenobarbital (PB) or a choline-deficient diet (810).
In the liver, non-parenchymal cells, in particular Kupffer cells and endothelial cells, are the main producers of prostanoids, i.e. PGs, prostacyclins and thromboxanes (1113). In contrast, the parenchymal hepatocytes have only a low capacity to synthesize PGs but have a pronounced capacity to degrade prostanoids (14,15). This raises the question of whether hepatic tumor promoters may stimulate PG synthesis of non-parenchymal liver cells, such as Kupffer cells, which may then act on the hepatocytes. An increased production of prostanoids may be the consequence of either an increased availability of the immediate COX substrate AA, due to the activation of phospholipases A2 and C (16,17), or the induction of isoenzyme cyclooxyenase-2 (COX-2). In contrast to the constitutive cyclooxygenase (COX-1), COX-2 has been shown to be highly expressed by various mitogens and inflammatory ligands (18,19). In this study, therefore, we examined whether the classical tumor promoters in the liver, the barbiturate PB and the insecticide lindane, affect the prostanoid metabolism in Kupffer cells. Experiments were performed in vitro and in vivo, and special emphasis was placed upon clarifying whether PB and lindane induce COX-2.
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Materials and methods
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Chemicals
Aprotinin, Trasylol, was purchased from Bayer (Leverkusen, Germany); RPMI 1640 medium and penicillin/streptomycin (10 000 U/10 000 µg per ml) were from Biochrom (Berlin, Germany); fetal calf serum, collagenase A and DNase I were from Boehringer Mannheim (Mannheim, Germany); prostanoids, prostanoid ELISA-kits, COX-2 antibody and COX-2 standard were from Cayman (Ann Arbor, MI); pronase E was from Merck (Darmstadt, Germany); ED2 antibody was from Dianova (Hamburg, Germany); ECL-system was from Amersham (Braunschweig, Germany); agarose was from Gibco BRL (Paisley, UK) and Nycodenz was from Nycomed (Heidelberg, Germany). All other chemicals and enzymes were obtained from Merck and Sigma (Deisenhofen, Germany) at the highest quality commercially available.
Treatment of animals
Male Wistar rats (in vivo studies: 120140 g body wt; in vitro studies: 180200 g body wt) were obtained from Charles River (Sulzfeld, Germany) and housed four per cage in an environment of 2223°C, 5060% relative humidity and a 12 h lightdark cycle. The animals had free access to food (standard diet; Altromin, Lage, Germany) and tap water and were treated according to the German guidelines for the care and use of laboratory animals. The rats were acclimatized to the facility for 5 days before the start of the experiments. Untreated control rats received water and food without extra additives. Tap water for the PB-treated rats was supplemented with 0.75 g/l PB according to Diwan et al. (20) and Maekawa et al. (21). Lindane-treated rats were fed a standard diet containing 350 mg lindane/kg diet according to Schröter et al. (22). After a treatment period of 2, 5 or 56 days, the animals were used for the isolation of Kupffer cells and hepatocytes.
Isolation of hepatocytes and Kupffer cells
For the in vitro studies, Kupffer cells were isolated according to the collagenase/pronase digestion method of Eyhorn et al. (23) by perfusion of the liver via the portal vein with a modified KrebsHenseleith buffer containing first 0.5 mM Ca2+ (5 min) and subsequently 2 mM Ca2+ and 1.25 mg/ml pronase E (10 min) at a flow rate of 44 ml/min. This was followed by an ex situ perfusion for 15 min with RPMI 1640 medium containing 0.13 U/ml collagenase A and 0.01 mg/ml DNase. After dissection of the liver, the tissue was digested for 12 min in RPMI 1640 medium containing 1 mg/ml pronase E and 0.1 mg/ml DNase. During the incubation, the pH was carefully controlled to 7.357.40 by gassing with air containing 5% CO2 and adding small amounts of 0.1 M NaOH. Subsequently, the cell suspension was filtered through gauze (mesh 80 µm) and centrifuged (100 g, 5 min). The supernatant was centrifuged once again (400 g, 5 min) and the pellet subjected to a density centrifugation in 16.7% Nycodenz (1400 g, 20 min). Afterwards, the cells were collected and centrifuged twice in elutriation medium [RPMI 1640 medium containing 1% heat-inactivated fetal calf serum, DNase I (20 mg/l), penicillin (100 000 U/l) and streptomycin (100 000 µg/l)]. All cells were collected in 10 ml elutriation medium and subjected to centrifugal elutriation according to the method of Eyhorn et al. (23) using the elutriation medium. Approximately 400x106 cells were injected at a rotor speed of 3250 r.p.m. Subsequently, the flow rate was increased step-by-step from 18.5 to 50 ml/min. Kupffer cells were eluted at a flow of 36.247.5 ml/min.
In the in vivo studies, hepatocytes and Kupffer cells were isolated from treated animals as follows. Similar to the published method for the isolation of hepatocytes (24), the liver was digested by perfusion with calcium-free and Ca2+/collagenase-containing media. The resulting cell suspension was filtered twice through gauze (mesh 80 µm and mesh 40 µm) and centrifuged at 50 g for 1 min. Hepatocytes in the pellet were purified from debris and non-parenchymal cells using the percoll density centrifugation method described by Kreamer et al. (25). The supernatant of the initial cell suspension was centrifuged once again (400 g, 5 min) to obtain the non-parenchymal liver cells. The pellet was resuspended in 10 ml elutriation medium and subjected to centrifugal elutriation as described above.
Cell culture
Kupffer cells were seeded at a density of 0.3x106 cells/cm2 in RPMI 1640 medium supplemented with 20% heat-inactivated fetal calf serum. Cells were kept in culture for 48 h prior to the experiments. Cell culture was performed at 37°C in a humidified atmosphere of 5% CO2 in air.
Determination of prostaglandins (ELISA)
PG production was determined in the absence of serum. After incubation with lindane, an aliquot of the medium was removed from the culture and centrifuged briefly. The supernatant was frozen in liquid nitrogen prior to storage at 80°C. The amounts of various PGs in the culture medium were determined using specific ELISA (Cayman) following strictly the recommendation of the supplier.
Reverse transcriptasepolymerase chain reaction (RTPCR)
Isolation of mRNA and RTPCR analysis were performed as we have described recently (26).
Analysis of COX-2 protein (western analysis)
Cell homogenates were mixed with lysis buffer (3x106 cells/50 µl), sonified, boiled for 5 min and centrifuged. The supernatant was subjected to discontinuous SDSPAGE using a 4% collecting and a 10% separating gel. After blotting, the nitrocellulose membranes were incubated in phosphate-buffered saline supplemented with 0.05% Tween-20, 5% dried milk and 5% fetal calf serum for 30 min to prevent non-specific binding. For the identification of the COX-2 protein a COX-2 (murine) polyclonal antiserum from Cayman (no. 160 116) was used as primary antibody (1:500) and a peroxidase (POD)-coupled monoclonal anti-rabbit antibody (1:7500) from Sigma as secondary antibody. The POD activity was visualized by the ECL system.
Analysis of COX activity
The activity of COX in Kupffer cell homogenates was determined spectrofluorimetrically according to Reed et al. (27). In brief, 25 µl of the cell homogenate were added to 2.0 ml of 0.1 M TrisHCl buffer (pH 8) and 20 µl of N,N,N',N'-tetramethylphenylenediamine solution (4 mg/ml). The basic extinction of the samples was recorded for 1 min at 611 nm. Subsequently, the COX reaction was started by the addition of 20 µl of an AA solution (20 mM). The change in the absorption was determined for at least 3 min. The enzyme activity was calculated from the difference of the absorption change in the absence and presence of AA. The substrate AA was present at saturating concentration.
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Results
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Effects on PG metabolism in Kupffer cell cultures
Kupffer cells were isolated from rat liver by collagenase/pronase digestion, purified by centrifugal elutriation and cultured as monolayers for 48 h (recovering period). More than 90% of the Kupffer cells stained using the macrophage-specific antibody ED2 and >75% phagocytized latex beads, showing that they were functionally active.
The insecticide lindane significantly increased the production of various PGs by the Kupffer cells as determined in the culture medium using specific ELISAs. One hour after the addition of lindane (10 µM), the concentrations of PGD2 and PGE2 were increased up to 50-fold and that of PGF2
>3-fold compared with the corresponding solvent controls (Figure 1
). The concentration of PGF2
in the medium rose further to ~8-fold, after increasing the time of exposure to the insecticide to 3 h (data not shown).

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Fig. 1. Effect of lindane on the release of prostaglandins (PGs) in Kupffer cell cultures. Kupffer cells were isolated from male Wistar rats and cultured for 48 h before the addition of lindane as described in Materials and methods. Lindane (10 µM) was incubated for 1 h in serum-free medium. PGs were analysed using specific ELISAs. Data represent the means ± SD of three independent experiments.
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One possible explanation for the stimulation of PG synthesis by lindane may be the expression of COX-2. Thus, cell homogenates of lindane-treated and non-treated Kupffer cell cultures were subjected to a western analysis using COX isoenzyme-specific antibodies for the inducible COX-2 enzyme. As shown in Figure 2
, there was a marked increase in the COX-2 signal 1, 8 and 24 h after the addition of lindane. In further experiments, we showed that PB, another hepatic tumor promoter which shares several effects with lindane in rat liver, also clearly induced COX-2, 8 and 24 h after its addition (Figure 2
).

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Fig. 2. Effect of lindane and PB on the expression of COX-2 in Kupffer cells. Cells were cultured for 48 h and subsequently treated with lindane (10 µM) and PB (1 mM) for various times. COX-2 protein was detected by western analysis as described in Materials and methods. The results show a representative blot selected from three similar experiments.
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In contrast to Kupffer cells, only a faint signal of COX-2 could be detected in homogenates of hepatocytes by western analysis, and COX-2 was not inducible in hepatocytes by a 24 h treatment with lindane or PB (data not shown).
Effects on PG metabolism in Kupffer cells in vivo
The results of the in vitro study raised the question whether the hepatic tumor promoters lindane and PB also induce COX-2 in vivo in the rat and, if so, whether the induction of the enzyme is persistent. Thus, rats were treated with the two compounds at doses used for tumor promotion (2022). After 2, 5 and 56 days, the rats were killed and Kupffer cells were isolated from their livers. The homogenates of the cells were used to determine the steady state levels of COX-2 mRNA and protein as well as the enzyme activity of COX. COX-2 mRNA was analysed using RTPCR. There was a significant increase of the specific message of COX-2 in Kupffer cells of the lindane- and PB-treated animals at all time points as shown in Figure 3a and b
. In accordance with this result, the expression of the COX-2 protein was also markedly increased in Kupffer cells of the treated rats, as shown by western blotting (Figure 4
). Finally, the treatment of the animals also caused an increase in the COX activity (Figure 5
). The stimulation of the enzyme activity was ~2-fold and 3- to 5-fold in the Kupffer cell homogenates of the PB- and lindane-treated animals, respectively.


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Fig. 3. Levels of COX-2 mRNA in Kupffer cells of lindane- and PB-treated rats. Kupffer cells were isolated from rats which had been fed a diet containing 350 mg/kg lindane, or water containing 0.75 g/l PB for 2, 5 and 56 days. (a) The amount of COX-2 mRNA was determined by RTPCR analysis as described in Materials and methods. The expression of ß-actin was taken as an internal control. A representative gel is shown from three similar experiments. (b) Quantification by densitometry of the results shown in (a).
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Fig. 4. Expression of COX-2 in Kupffer cells of lindane- or PB-treated rats. Kupffer cells were isolated from rats which had been fed a diet containing 350 mg/kg lindane, or water containing 0.75 g/l PB. COX-2 protein was examined by western analysis as described in Materials and methods. The blots show the results of the three animals in each of the experimental groups after 2, 5 and 56 days.
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Fig. 5. COX activity in Kupffer cells of lindane- and PB-treated rats. Kupffer cells were isolated from rats which had been fed a diet containing 350 mg/kg lindane, or water containing 0.75 g/l PB for 2, 5 and 56 days. COX activity was determined in the homogenates of the isolated cells as described in Materials and methods. Data represent the means ± SD of three animals in each treatment group. COX activity of Kupffer cells from untreated animals was set as 100%.
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Discussion
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Findings of the past several years indicate an important role of prostanoids in tumorigenesis (see Introduction). This may also include tumor promotion in the liver by PB and related compounds (8,10,28). Our results corroborate this notion, showing that the hepatic tumor promoters lindane and PB induce COX-2 in Kupffer cells in vitro and in vivo. In view of a possible role in tumor promotion, it is particularly interesting that the COX activity in the cells persistently increased during the entire treatment period of the rats (almost 2 months).
COX-2 induction in vitro by lindane was immediate. Within 1 h, we observed a marked increase of COX-2 protein and product formation in primary Kupffer cell cultures. Rapid COX-2 upregulation has also been reported to occur in intestinal epithelial cells following stimulation with phorbol esters (29). In contrast to lindane, we did not detect induction of COX-2 protein (Figure 2
) and PG synthesis (data not shown) 1 and 3 h after stimulation with PB; however, COX-2 protein was significantly upregulated after 8 h (Figure 2
). The different kinetics of COX-2 induction by lindane and PB may indicate different molecular events. The molecular mechanisms underlying the modulation of gene expression by the two tumor promotors are far from being understood. Recently, a functionally active xenobiotic responsive element was identified in the murine COX-2 promotor (30); however, this element is known to mediate the effects of polyhalogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and not of compounds like PB and lindane. With respect to the barbiturate, responsive elements that are inducible by PB have been described in cytochrome P450 genes (3134); however, we are not aware of corresponding data on the COX-2 gene.
In vivo, we found a marked and persistent increase of COX-2 protein and mRNA during the 56 days of treatment with lindane and PB. In view of the fact that COX-2 mRNA contains multiple AUUUA motifs in its 3'-untranslated region consistent with rapid mRNA degradation (35), it is tempting to speculate that mRNA stabilization may significantly contribute to the sustained mRNA expression and lead to the increased protein and product synthesis.
Besides affecting the expression of COX-2, chemicals may also stimulate the synthesis of prostanoids by increasing the availability of AA, the precursor of prostanoid synthesis. This may apply particularly to lindane. This insecticide displays structural homology with inositol and was found to increase the free intracellular calcium concentration and the activity of phospholipases A2, C and D in macrophages and kidney tubular cells (3638). Accordingly, Forgue et al. (39) showed that lindane stimulates the metabolism of AA and the synthesis of prostanoids in mouse peritoneal macrophages. Thus, the insecticide may stimulate prostanoid metabolism by both increasing the amount of COX-2 and increasing the availability of AA.
According to our knowledge, hepatic PG levels have only been determined in PB-treated, but not in lindane-treated, rats. Conflicting results were obtained. Hendrich et al. (28) found increased concentrations of PGF2
in the liver homogenates of PB-treated rats, whereas Peebles and Glauert (40) reported that PGE2 levels were decreased and PGF2
levels were either unchanged or decreased after treatment of rats with the barbiturate. The reason for this discrepancy is not clear. Moreover, it has to be emphasized that the significance of PG levels determined in liver homogenates is limited. Thus, Peebles and Glauert (40) state in their paper: `Since hepatocytes make up the majority of cells within the liver, the concentration of an eicosanoid determined using liver homogenate may not reflect the concentration at the site of production, such as a Kupffer cell, or at the site of action, such as a prostaglandin receptor on a hepatocyte.' This statement gains in importance by taking into account that hepatocytes are the predominant site of degradation of prostanoids in the liver, while Kupffer and liver endothelial cells are the important sites of their synthesis in the organ (1115). Thus, in this study, we deliberately examined the effect of lindane and PB on Kupffer cells and not on the whole liver. In this context, it is interesting to note that preliminary results from our group indicate that the two tumor promoters also induce COX-2 and stimulate the synthesis of PGs in primary cultures of liver endothelial cells. However, the two compounds did not affect the COX-2 levels in hepatocytes (data not shown). The latter observation is consistent with the recent data of Ledwith et al. (41) showing no induction of COX-2 by PB in a murine hepatocyte cell line.
In view of the pleiotrophic biological effects of PGs, no final answer can be given yet to how they may promote tumor growth (1). Generally, an increased cell proliferation and/or a decreased apoptosis are thought to represent key mechanisms underlying tumor promotion (4244). Interestingly, several PGs are known to exhibit both mitogenic and cytoprotective activity. There is evidence that PGs are involved in the signalling of compensatory liver growth after partial hepatectomy (4547) and PGs such as PGF2
and PGE2 have been shown to stimulate DNA synthesis and cell proliferation in various cell types, including primary cultures of hepatocytes (4851). A cytoprotective role of certain PGs and the prostacyclin PGI2, which is also a product of the COX pathway, and their derivatives (Iloprost, Misoprostol, Rioprostil, Enprostil and 16,16'-dm PGE2) has been reported in intestine (5255), liver and liver cell cultures (5662). In addition, recent data from our group clearly show that PGs, in particular PGD2, PGE1 and PGI2, exhibit a strong anti-apoptotic activity in primary cultures of rat hepatocytes treated with transforming growth factor ß1 (63), a cytokine which most likely plays an important physiological role in the regulation of apoptosis in the liver (6467). In this context it should also be mentioned that genetically engineered cells that constitutively overexpress COX-2 show a decreased apoptosis (68,69) and that many tumor cells overexpress COX-2 (1,2,6,70).
Our results suggest that paracrine mechanisms may contribute to the tumor-promoting activity of lindane and PB, stimulating the production of prostanoids by Kupffer cells, biologically active products which are known to exhibit mitogenic and anti-apoptotic activity. The role of Kupffer cells in hepatic tumor promotion may not be restricted to an increased release of prostanoids. Recent data (7175) point to the involvement of Kupffer cells in the tumor-promoting action of peroxisome proliferators, which stimulate the release of TNF
and oxidants.
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Notes
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2 To whom correspondence should be addressed Email: schwarz{at}gsf.de 
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Received March 29, 1999;
revised May 11, 1999;
accepted May 20, 1999.