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Changes in leukotrienes and prostaglandins in the liver tissue of rats in the experimental massive hepatic cell necrosis model
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Prostaglandins Leukotrienes and Essential Fatty Acids (1990) 40,149-155 @ Longman Group UK Ltd 1990
Changes in Leukotrienes and Prostaglandins in the Liver Tissue of Rats’ in the Experimental Massive Hepatic Cell Necrosis Model N. Kawada, Y. Mizoguchi, Y. Sakagami, K. Kobayashi,
S. Yamamoto
and S. Morisawa
Third Department of Internal Medicine and First Department of Biochemistry, Osaka City University Medical School and Osaka So&o-Medical Center Hospital, Osaka, Japan (Reprint requests to YM, Third Department of internal Medicine, Osaka City University Medical School, l-5-7 Asahimachi, Abeno-ku, Osaka 545, Japan) ABSTRACT.
When heat-killed Propionibucterium acnes is intravenously injected into rats followed by an intravenous injection of a small amount of Gram-negative lipopolysaccharide (LPS) 7 days later, massive hepatic cell necrosis is induced and most of the rats die within 24 hours of LPS injection. Using this experimental model, we studied the changes in the levels of leukotrienes (LTs) and prostaglandms (PGs) in the liver tissue and bile of rats with experimentally-induced massive hepatic cell necrosis. Both the levels of LTs and PGs in the liver tissue and LTs in the bile increased before the microscopic-appearance of hepatic cell necrosis. These results suggest that arachidonic acid metabolites may play an important role in the induction of liver cell injury.
plays a significant role in the induction of liver cell injury. In this study, we examined the changes in the levels of arachidonic acid metabolites in the liver tissue and bile of rats using this experimentally-induced massive hepatic cell necrosis model.
INTRODUCTION heat-killed Propionibacterium acnes (P. a Gram-positive anaerobe, is intravenously injected into mice and rats followed by an intravenous injection of a small amount of lipopolysaccharide (LPS), a Gram-negative endotoxin, 7 days later, most of the animals die of massive hepatic cell necrosis within 24 hours of LPS injection. In this experimental model, the adherent cells are accumulated in the liver and activated by the two-stage stimulation of P. acnes and LPS, producing the cytotoxic factor (1). We have also reported that this experimental induction of massive hepatic cell necrosis can be significantly suppressed by lipocortin, lipoxygenase inhibitors, leukotriene (LT) inhibitors and other agents which inhibit the production and release of LTs (2). Furthermore, we have shown that when the isolated hepatocytes of rats are incubated with LTC4, the cell viability decreases. On the other hand, prostaglandins (PGs), which are produced via the cyclooxygenase pathway from arachidonic acid, have cytoprotective effects, and experimental model, we using our have demonstrated that PGE, remarkably inhibits the release of transaminase and reduces the histological changes and necrosis of the liver cells (3). These results suggest that the arachidonic acid cascade
When
acnes),
Date received Date accepted
MATERIALS AND METHODS Experimental induction of massive hepatic cell necrosis Four-week-old male Wistar rats (Clea Japan Inc., Japan) were intravenously injected with 1 mg of heat-killed P. acnes through a tail vein followed by an intravenous injection of 10 Fg of Salmonella enteritidis-derived LPS (Difco Laboratories, Detroit, Michigan) 7 days later. Serum transaminase levels were measured, and histological examination was done using an electron microscope after hematoxylin-eosin staining. P. acnes used in this study was donated by the Department of Bacteriology, Osaka City University Medical School. Measurement of LTs in the liver tissue LTC, and LTB4 levels were measured in the liver tissue obtained before and at 30, 60 and 120 min after LPS injection as follows. The rats were anesthetized by an intraperitoneal injection of 40 m$kg of pentobarbital. After laparotomy, the liver was extracted, immersed in cold ethanol (-70°C) in an
30 November 1989 4 January 1990 149
150 Prostaglandins Leukotrienes and Essential Fatty Acids
acetone dry ice bath and frozen. The ethanol-added frozen liver tissue was homogenized in ice-cold ethanol containing 10 PM of AA861 (5 lipoxygenase inhibitor, donated by Takeda Pharmaceuticals, Osaka, Japan) and kept at -20°C overnight. This was deproteinized by centrifugation (3,000 rpm, 4°C 15 min), and the supernatant obtained was evaporated to dryness with nitrogen gas and suspended in 10 ml of water adjusted to pH 5.1 with 1 N HCl according to a slight modification of Powell’s method (4). This solution was then applied to the SEP-PAK C1s cartridge column (Water Associates, Milford, Massachusetts) pretreated with ethanol and water. After the cartridge was washed with 10 ml each of water and petroleum ether, LTs were eluted with 10 ml of methanol. This solution was evaporated to dryness with nitrogen gas, suspended in 40% methanol and used as the sample for reversed-phase high-performance liquid chromatography (RP-HPLC) performed according to the modified method of Borgeat et al. (5). The above sample was applied to the analytical column (Develosil ODS 5K, Nomura Chemicals Co. Ltd., Aichi, Japan) using the mobile phase acid = (acetonitrile : methanol : water : acetic 15 : 5 : 15 : 0.01 adjusted to pH 5.6 with triethylamine) at a flow rate of 0.8 ml/min. The absorbance of the eluted LTs was measured at 280 nm using the UV detector (SPD-6A, Shimazu Industries Co. Ltd., Kyoto, Japan). The fractions corresponding to the standard preparations of LTC4 and LTB4 (donated by Ono Pharmaceuticals, Osaka, Japan) were obtained, which were then evaporated to dryness and suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 0.1% gelatin. LTCd and LTB4 levels were measured using the radioimmunoassay (RIA) kit (Amersham, UK) (6, 7). As a control, physiological saline was administered to the rats instead of LPS.
Measurement of LTs in the bile LTC4 and LTB4 levels were measured in the bile obtained at various time intervals before and after LPS injection. The rats injected with P. acne.s 7 days before were anesthetized by an intraperitoneal injection of 40 mg/kg of pentobarbital. After a polyethylene tube (1 mm in laparotomy, diameter) was inserted into the common bile duct, and the bile was collected into 5 ml of ice-cold ethanol for 30 min. LPS was then injected through a tail vein, and the bile was collected into 5 ml of ice-cold ethanol for various time periods. This was deproteinized by centrifugation (3,000 rpm, 4”C, 15 min), and the supernatant was obtained. LTC4 and LTB4 levels were measured as described above by RP-HPLC and RIA. As a control, physiological saline was administered to the rats instead of LPS.
Administration of AA861 The effects of AA861 on LTC4 and LTB4 levels in the liver tissue and bile were studied. The rats were intraperitoneally injected with 20 mg/kg of AA861 suspended in 5% Arabic gum solution 2 hours before LPS injection. For comparison, rats were injected only with 5% Arabic gum solution. LTC4 and LTB4 levels in the liver tissue as well as the bile were measured according to the method described above. Measurement of PGs in the liver tissue PGF2 and 6-keto-PGF1, levels were measured in the liver tissue obtained before and at 30, 60 and 120 min after LPS injection. The rats were anesthetized, and the liver was extracted. The liver tissue prepared as described above was homogenized in ice-cold ethanol containing 0.1 mM of indomethacin and kept at -20°C overnight. This was deproteinized by centrifugation (3,000 rpm, 4”C, obtained was 15 min), and the supernatant evaporated to dryness with nitrogen gas and suspended in 10 ml of 15% methanol adjusted to pH 3.0 with 1 N HCI. This solution waS then applied to the SEP-PAK C1s cartridge column pretreated with ethanol and water. After the cartridge was washed with 10 ml each of 5% ethanol, water and petroleum ether, PGs were eluted with 10 ml of methyl formate. This solution was evaporated to dryness with nitrogen gas, suspended in 40% methanol and used as the sample for RP-HPLC. was applied to the analytical The sample phase mobile the using column 35 : acid = (acetonitrile : water : acetic 65 : 0.1) at a flow rate of 0.8 ml/min. The absorbance of the eluted PGs was measured at 195 nm using the UV detector. The fractions corresponding to the standard preparations (donated by Ono Pharmaceuticals, Osaka, Japan) were obtained, which were then evaporated to dryness and suspended in 0.1 M phosphate buffered saline containing 0.1% gelatin. PGE2 and 6-keto-PGF1, levels were measured by RIA (7). As a control, physiological saline was administered to the rats instead of LPS. Statistical analysis All results are indicated as mean * S.D. Significant differences were calculated by analysis of variance test (~~0.05) and further by Student’s test.
RESULTS Experimental induction of massive hepatic cell necrosis When the rats were injected with P.acnesfollowed
Leukotrienes
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by LPS 7 days later, massive hepatic cell necrosis was induced in most of them. The movements of the rats began to slow down at one hour after LPS injection, and at 2 to 3 hours, they barely responded, when touched. At 4 hours, the rats began to have convulsions and died, and the survival rate 24 hours after LPS injection was 20%. Changes in serum GOT and GPT levels As shown in Figure 1, serum transaminase levels were normal at 30 min after LPS injection. However, both serum GOT and GPT levels started to increase at 2 hours, and in the rats still surviving after 24 hours, these levels increased to 9500 f 1120 IU and 2520 * 773 IU, respectively.
Histological changes of the liver from a rat with massive hepatic cell necrosis (H&E stain x 100).
Fig. 2
Histological changes of the liver At 24 hours after LPS injection, changes in the liver tissue were noted and necrosis was seen to a large extent in all of the rats (Fig. 2). However, at 30 min after LPS injection, although permeation of macrophages, lymphocytes and plasma cells was observed in the lobules, no changes in the liver cells nor necrosis could be seen in any of the rats. LTC4 and LTB4 levels in the liver tissue As shown in Figure 3, LTC4 level in the liver tissue before LPS injection was 3.37 + 3.25 rig/g liver tissue, while at 30, 60 and 120 min after LPS injection, it was 14.34 + 11.70, 5.39 + 3.06 and 18.34 + 5.93 rig/g liver tissue, respectively, which the
0 y
0
I
I
30
60
I
120(min)
Fig. 3 Time course of leukotriene C, level in the liver tissue of Propionibacterium acnes-treated rats after lipopolysaccharide injection (n = 5). *-**p
showed that the LTC4 level increased after LPS injection, and was significantly higher than that of the control rats at 120 min (~~0.01). LTB4 level also tended to increase after LPS injection and tended to be higher than that of the control rats (Fig. 4). Both LTC4 and LTB4 levels in the liver tissue did not change at all in the control rats administered with physiological saline instead of LPS (Fig. 3,4). The recovery rate was 51 + 3% for LTC, and 84 & 2% for LTB+ LTCd and LTB4 levels in the bile
00.5
2
4
24 (hr)
Fig. 1 Changes in serum GOT and GPT levels in Propionibacrerium acnes-treated rats after lipopolysaccharide injection (II = IO).
LTC, level in the bile before LPS injection was 0.13 rt 0.16 nmol/30 mm/kg weight, but it increased to 0.54 f 0.20 nmol/30 min/kg weight at 120 to 150 min after LPS injection, which was significantly higher than that of the control rats (~~0.05) (Fig. 5). LTB, level was 0.19 rf: 0.17 nmol/30 mm/kg weight before LPS injection, and although it in-
152 Prostaglandins Leukotrienes and Essential Fatty Acids
creased to 0.23 f 0.18 nmol/30 mm/kg weight at 0 to 30 min after LPS injection, it tended to decrease thereafter. However, LTBj level still tended to be higher than that of the control rats (Fig. 6). Neither LTC4 nor LTB4 levels in the bile changed at all in the control rats. Effects of AA861 on LTC4 and LTB, levels in the liver tissue and bile
30
0
120 hid
60
Fig. 4 Time course of leukotriene B, level in the liver tissue of Propionibacterium acnes-treated rats after lipopolvsaccharide injection (n = 5). a-0: LPS-injected rats o-o: control rats
-i
3 1.0,
When AA861 was administered, LTC4 and LTB4 levels in the liver tissue remarkably decreased after LPS injection, and both levels were significantly lower compared to those of the rats not administered with AA861 at 2 hours (p
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3b
i0
9'0 120 150
lh0 (mid
Fig, 5
Time course of leukotriene C, level in the bile of acnes-treated rats after lipopolysaccharide injection In = 5). *-**p
PGE2 level in the liver tissue before LPS injection was 446 f 53 pg/g liver tissue. However, at 30 min after LPS injection, it increased to 1836 f. 865 pg/g liver tissue and was significantly higher compared to that of the control rats (p
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.; z X z 0.5.
c ._A 0 5 O-
!i4zbaI
Fig. 6
0
30
60
90
120
150
180(min)
Time course of leukotriene B, level in the bile of Propionibacterium acnes-treated rats after lipopolysaccharide injection (n = 5). O-O: LPS-injected rats 0-O: control rats
1
IC”eS
.PS
AA8611-i
P.ac”es
LPS
AABSlHi
Fig. 7 Effects of AA861 on leukotriene C, and leukotriene B, levels in the rat liver tissue at 2 hours after lipopolysaccharide injection (n = 5).
Leukotrienes
I
0
30
60
90
and Prostaglandins
in Massive Hepatic Cell Necrosis
153
120 150 180(min) 0
30
60
120 (min)
Time course of 6-keto-prostaglandin F,, level in the hver tissue of Propionibacterium acnes-treated rats after lipopolysaccharide injection (n = 5). *-**p
DISCUSSION
-30
0
30
60
90
120 150 18O(min)
Fig. 8 Effect of AA861 on leukotriene C, and leukotriene B, levels in the rat bile (n = 5). *-**p
.
I
0
**
T
30
60
120 hid
Fig. 9 Time course of prostaglandin E, level in the liver tissue of Propionibacren’um acnes-treated rats after lipopolysaccharide injection (n = 5). *-**p
of the control rats (~~0.01) (Fig. 10). Neither PGE2 nor 6-keto-PGFi, levels in the liver tissue changed at all in the control rats. The recovery rate of PGs was 64 f 5% for PGE2 and 58 k 3% for 6-ketoPGF,..
The role of arachidonic acid and its metabolites in the induction of massive hepatic cell necrosis has been studied using our experimental model (1). In vitro, lipocortin suppressed the induction of liver cell injury caused by the cytotoxic factor produced from liver adherent cells. AA861, [2-( 12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-l,Cbenzoquinonel, which is a 5-lipoxygenase inhibitor (2), Azelastine hydrochloride, 4-[(+chlorophenyl) methyl]-2(hexahydro-l-methyl-1 H-az-epine-4-yl)which monohydrochloride, inhibits the production and release of LTs (2), and Gomisin A, [(+)-(6s,7s,R-biar)-5,6,7,8-tetrahydro-l,2,3,12-tetr amethoxy-6,7-dimethyl-lO,ll-methylenedioxy-6-dib enzo[a,c]cyclooctenol], which induces the production of lipocortin (8), also remarkably suppressed the induction of massive hepatic cell necrosis. LTC4 was shown to induce liver cell injury in vitro. LTB4, LTC4, LTD4 and LTE4 are chemical mediators metabolized from arachidonic acid via the 5-lipoxygenase pathway (9). LTB4 is a potent mediator of inflammation, which enhances the adhesion of leukocytes to vascular endothelial cells and causes the chemotaxis of leukocytes and infiltration of inflammatory cells (lo), and it has been reported to be produced from neutrophils, eosinophils, monocytes, macrophages and mast cells. LTB4 also affects the immune responses, and it induces suppressor cells (ll), enhances natural killer cell activity (12), increases the production of interleukin-1 (13) and y-interferon (14) and partially substitutes helper T cell function (15). LTC4, LTD4 and LTE4 are known to contract the vascular smooth muscle, increase the permeability of postcapillary venules as well as capillaries, cause the
154 Prostaglandins Leukotrienes and Essential Fatty Acids
extravasation of plasma and induce tissue edema, which is a characteristic of the acute inflammatory reaction (9). LTC4, is metabolized into LTD4 and LTDJ, is in turn changed into LTE4, but LTC4 is known to have the strongest physiological activity (9). On the other hand, we have shown that PGs, which are produced from arachidonic acid via the cyclooxygenase pathway, remarkably improve the survival rate and histological changes of the liver in our experimental model (3). Various studies have also indicated that PGs exhibit their cytoprotective actions by stabilizing the cell membrane and lysosome membrane. For example, Ishii et al. (16) reported that in chenodeoxycholic acid-induced liver cell injury, PGEt becomes cytoprotective by stabilizing the plasma membrane. That is, when the cell is stimulated, phospholipase A2 is activated and arachidonic acid is released, inducing the producing the production of PGs. As a result, the amount of arachidonic acid in the cell membrane decreases and the fluidity of the plasma membrane is reduced. When PGs are exogenously administered, they prevent’ the decrease in the amount of arachidonic acid in the cell membrane and restore the fluidity of the plasma membrane. In this way, cell metabolism and cell function are maintained. Stachura et al. (17) reported that the percutaneous administration of PGEz remarkably reduces carbon tetrachloride-induce liver cell necrosis in rats by protecting the endoplasmic reticulum from damage due to carbon trichloride, the metabolite of carbon tetrachloride, or by stabilizing the cell membrane. Sikujara et al. (18) demonstrated in vivo that in ischemia-induced hepatic cell injury using rats, PGI2 exhibits its cytoprotective actions by increasing the amount of cyclic 3’-5’ adenosine monophosphate in the liver. Araki et al. (19) reported that in hypoxiainduced liver injury using cat isolated hepatoxytes, PG12 stabilizes the lysosome membrane and protects the liver cells by inhibiting the release of LDH and Cathepsin D from the liver cells due to hypoxia. Since these studies suggested that the arachidonic acid cascade may be involved in the induction of liver cell injury, we examined the levels of LTs and PGs in the liver tissue and LTs in the bile. As a result, in the liver tissue, the levels of PGs increased at an early stage followed by the increase in the levels of LTs. We should note that these changes in the levels of PGs and LTs were observed as early as 30 min after LPS injection, when serum transaminase levels were still normal and histological changes in the liver were not observed. In addition, we found that the levels of LTs and PGs increased again at 2 hours, after the increase in serum transaminase levels to 100 to 600 IU and the microscopic appearance of necrosis in the liver tissue. Although a more detailed study is necessary, these results sug-
gest that liver cell injury may first be triggered by the changes in the arachidonic acid cascade. This may explain the biphasic time course of LTs and PGs in the present study, in contrast to a concomitant increase in the two levels. As for the levels of LTs in the bile, LTC4 and LTB4 levels increased at an early stage. Keppler et al. (20) have reported that because the levels of LTs increase in the bile of normal rats injected with LPS, the changes in the levels of LTs in the bile are due to LPS and not related to liver cell injury. However, we found that when AA861 was administered to rats in our experimental model, liver cell injury was suppresded (2), and that the levels of LTs decreased in the liver tissue as well as the bile. We therefore conclude that although LTs may not directly cause the induction of liver cell injury, this does not mean that LTs are not involved in liver injury at all. That is, another factor probably plays a role along with LTs in the induction of liver cell injury. Further studies must be made to elucidate this other factor. In any case, there is no doubt that the changes in the arachidonic acid cascade are induced in the liver tissue before the biochemical or histological appearance of liver cell injury. Therefore, these results confirmed that the arachidonic acid cascade plays a significant role in the induction of liver cell injury. Among the arachidonic acid metabolites, LTs have cytotoxic effects while PGs have cytoprotective effects, so that the destruction in the balance of LTs and PGs may lead to the induction of liver cell injury. References 1. Tsutsui H. Mizoguchi Y, Yamamoto S, Morisawa S. Studies on experimentally-induced acute hepatic failure: possible involvement of activated liver adherent cells. p 307-314 in Cells of the Hepatic Sinusoid (Kirn A. Knook D L, Wisse E edsj. The Kuoffer Cell Foundation, The Netherlands, 1986. 2. Miioguchi Y, Sakagami Y, Seki S, Kobayashi K, Yamamoto S, Morisawa S. Protective effect of a leukotriene inhibitor in an experimental massive hepatic cell necrosis model. Gastroenterologia Japonica 23: 263-267, 1988. 3. Mizoguchi Y, Tsutsui H, Miyajima K, Sakagami Y, Seki S, Kobayashi K, Yamamoto S, Morisawa S. The protective effect of prostaglandin E, in an experimental massive hepatic cell necrosis model. Hepatology 7: 11841188, 1987. 4. Powell W. Raoid extraction of oxveenated metabolites of’arachidonic acid from biological samples using octadecylsilyl silica. Prostaglandins 20: 947-957,198O. 5. Borgeat P, Fruteau de Laclos B, Rabinovitch H, Picard S, Braquet P, Hebert J, Laviolette M. Generation and structures of the lipoxygenase products. Eosinophil-rich human polymorphonuclear leukocyte preparations characteristicallv release leukotriene C, on ionophore A23I87 challenge. Journal of Allergy and Clinical Immunology 74: 310-315, 1984. 6. Salmon J A, Simmons P M, Palmer R M J. A radioimmunoassay for leukotriene B,. Prostaglandins 24: 225-235, 1982.
Leukotrienes 7. Salmon J A. Analysis of cycle-oxygenase and lipoxygenase products in inflammation exudate. Prostaglandins 27: 364-366, 1984. 8. Ohkura Y, Mizoguchi Y, Sakagami Y, Kobayashi K, Yamamoto S, Morisawa S, Takeda S, Aburada M. Inhibitory effect of TJN-101 [(+)-(6s,7s,R-biar)-5,6,7,8-tetrahydro-1,2,3,12tetramethoxy-6,7-dimethyl-lO,ll-methylenedioxy-6dibenzo[a,c]cyclooctenol] on immunologically induced liver injuries. Japanese Journal of Pharmacology 44: 179-185. 1987. 9. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220: 568-575. 1983. 10. Ford-Hutchinson A W, Bray M A, Doig M V, Shipley M E, Smith M J H. Leukotriene B, a potent chemotactic and aggregating substance released from polymorphonuclear leukocytes. Nature 286: 264-269, 1980. 11. Rola-Pleszczynski M. Borgeat P, Sirois P. Leukotriene B, induces human suppressor lymphocytes. Biochemical Biophysical Research Communications 108: 1531-1537, 1982. 12. Rola-Pleszczynski M, Gagnon L. Sirois P. Leukotriene B, augments human natural cytotoxic cell activity. Biochemical Biophysical Research Communications 113: 531-537, 1983. 13. Rola-Pleszczynski M, Lemaire I. Leukotrienes augment interleukin 1 production by human monocytes. Journal of Immunology 135: 39583961, 1985.
and Prostaglandins
in Massive Hepatic Cell Necrosis
14. Murray H W, Spitalny G L, Nathan C F. Activation of mouse peritoneal macrophages in vitro and in vivo by interferon y. Journal of Immunology 134: 1619-1622, 1985. 15. Jordan M L, Hoffman R A, Simmons R L. Leukotriene B, augments IL-2-dependent proliferation of T lymphocyte clones. Transolantation Proceedings 18: 224-227. 1986. 16. Ishii Y. The cytotoxicity aufchenodeoxydhohc acid on primary cultured rat hepatocytes and cytoprotective action of prostaglandin E, Japanese Journal of Gastroenterology (in Japanese) 82: 108-117. 1985. 17. Stachura J, Tarnawski A. Ivey K J. Mach T. Bogdal J, Szczudrawa J, Klimczyk B. Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in the rat. Gastroenterology Xl: 211-217, 1981. 18. Sikujara 0, Monden M, Toyoshima K, Okamura J, Kosaki G. Cytoprotective effect of prostaglandin I, on ischemia-induced hepatic cell injury. _ Transulantation 36: 238-243. 1983. 19. Araki’ H, Lefer A M. Cytoprotective actions of prostacyclin during hypoxia in the isolated perfused cat liver. American Journal of Physiology 238: 176-181,198O. 20. Hagmann W. Denzhnger C, Rapp S, Weckbecker G. Keppler D. Identification of the major endogenous leukotriene metabohte in the bile of rats as N-acetyl leukotriene E,. Prostaglandins 31: 239-251. 1986.
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Prostaglandins Leukotrienes and Essential Fatty Acids (1990) 40,149-155 @ Longman Group UK Ltd 1990
Changes in Leukotrienes and Prostaglandins in the Liver Tissue of Rats’ in the Experimental Massive Hepatic Cell Necrosis Model N. Kawada, Y. Mizoguchi, Y. Sakagami, K. Kobayashi,
S. Yamamoto
and S. Morisawa
Third Department of Internal Medicine and First Department of Biochemistry, Osaka City University Medical School and Osaka So&o-Medical Center Hospital, Osaka, Japan (Reprint requests to YM, Third Department of internal Medicine, Osaka City University Medical School, l-5-7 Asahimachi, Abeno-ku, Osaka 545, Japan) ABSTRACT.
When heat-killed Propionibucterium acnes is intravenously injected into rats followed by an intravenous injection of a small amount of Gram-negative lipopolysaccharide (LPS) 7 days later, massive hepatic cell necrosis is induced and most of the rats die within 24 hours of LPS injection. Using this experimental model, we studied the changes in the levels of leukotrienes (LTs) and prostaglandms (PGs) in the liver tissue and bile of rats with experimentally-induced massive hepatic cell necrosis. Both the levels of LTs and PGs in the liver tissue and LTs in the bile increased before the microscopic-appearance of hepatic cell necrosis. These results suggest that arachidonic acid metabolites may play an important role in the induction of liver cell injury.
plays a significant role in the induction of liver cell injury. In this study, we examined the changes in the levels of arachidonic acid metabolites in the liver tissue and bile of rats using this experimentally-induced massive hepatic cell necrosis model.
INTRODUCTION heat-killed Propionibacterium acnes (P. a Gram-positive anaerobe, is intravenously injected into mice and rats followed by an intravenous injection of a small amount of lipopolysaccharide (LPS), a Gram-negative endotoxin, 7 days later, most of the animals die of massive hepatic cell necrosis within 24 hours of LPS injection. In this experimental model, the adherent cells are accumulated in the liver and activated by the two-stage stimulation of P. acnes and LPS, producing the cytotoxic factor (1). We have also reported that this experimental induction of massive hepatic cell necrosis can be significantly suppressed by lipocortin, lipoxygenase inhibitors, leukotriene (LT) inhibitors and other agents which inhibit the production and release of LTs (2). Furthermore, we have shown that when the isolated hepatocytes of rats are incubated with LTC4, the cell viability decreases. On the other hand, prostaglandins (PGs), which are produced via the cyclooxygenase pathway from arachidonic acid, have cytoprotective effects, and experimental model, we using our have demonstrated that PGE, remarkably inhibits the release of transaminase and reduces the histological changes and necrosis of the liver cells (3). These results suggest that the arachidonic acid cascade
When
acnes),
Date received Date accepted
MATERIALS AND METHODS Experimental induction of massive hepatic cell necrosis Four-week-old male Wistar rats (Clea Japan Inc., Japan) were intravenously injected with 1 mg of heat-killed P. acnes through a tail vein followed by an intravenous injection of 10 Fg of Salmonella enteritidis-derived LPS (Difco Laboratories, Detroit, Michigan) 7 days later. Serum transaminase levels were measured, and histological examination was done using an electron microscope after hematoxylin-eosin staining. P. acnes used in this study was donated by the Department of Bacteriology, Osaka City University Medical School. Measurement of LTs in the liver tissue LTC, and LTB4 levels were measured in the liver tissue obtained before and at 30, 60 and 120 min after LPS injection as follows. The rats were anesthetized by an intraperitoneal injection of 40 m$kg of pentobarbital. After laparotomy, the liver was extracted, immersed in cold ethanol (-70°C) in an
30 November 1989 4 January 1990 149
150 Prostaglandins Leukotrienes and Essential Fatty Acids
acetone dry ice bath and frozen. The ethanol-added frozen liver tissue was homogenized in ice-cold ethanol containing 10 PM of AA861 (5 lipoxygenase inhibitor, donated by Takeda Pharmaceuticals, Osaka, Japan) and kept at -20°C overnight. This was deproteinized by centrifugation (3,000 rpm, 4°C 15 min), and the supernatant obtained was evaporated to dryness with nitrogen gas and suspended in 10 ml of water adjusted to pH 5.1 with 1 N HCl according to a slight modification of Powell’s method (4). This solution was then applied to the SEP-PAK C1s cartridge column (Water Associates, Milford, Massachusetts) pretreated with ethanol and water. After the cartridge was washed with 10 ml each of water and petroleum ether, LTs were eluted with 10 ml of methanol. This solution was evaporated to dryness with nitrogen gas, suspended in 40% methanol and used as the sample for reversed-phase high-performance liquid chromatography (RP-HPLC) performed according to the modified method of Borgeat et al. (5). The above sample was applied to the analytical column (Develosil ODS 5K, Nomura Chemicals Co. Ltd., Aichi, Japan) using the mobile phase acid = (acetonitrile : methanol : water : acetic 15 : 5 : 15 : 0.01 adjusted to pH 5.6 with triethylamine) at a flow rate of 0.8 ml/min. The absorbance of the eluted LTs was measured at 280 nm using the UV detector (SPD-6A, Shimazu Industries Co. Ltd., Kyoto, Japan). The fractions corresponding to the standard preparations of LTC4 and LTB4 (donated by Ono Pharmaceuticals, Osaka, Japan) were obtained, which were then evaporated to dryness and suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 0.1% gelatin. LTCd and LTB4 levels were measured using the radioimmunoassay (RIA) kit (Amersham, UK) (6, 7). As a control, physiological saline was administered to the rats instead of LPS.
Measurement of LTs in the bile LTC4 and LTB4 levels were measured in the bile obtained at various time intervals before and after LPS injection. The rats injected with P. acne.s 7 days before were anesthetized by an intraperitoneal injection of 40 mg/kg of pentobarbital. After a polyethylene tube (1 mm in laparotomy, diameter) was inserted into the common bile duct, and the bile was collected into 5 ml of ice-cold ethanol for 30 min. LPS was then injected through a tail vein, and the bile was collected into 5 ml of ice-cold ethanol for various time periods. This was deproteinized by centrifugation (3,000 rpm, 4”C, 15 min), and the supernatant was obtained. LTC4 and LTB4 levels were measured as described above by RP-HPLC and RIA. As a control, physiological saline was administered to the rats instead of LPS.
Administration of AA861 The effects of AA861 on LTC4 and LTB4 levels in the liver tissue and bile were studied. The rats were intraperitoneally injected with 20 mg/kg of AA861 suspended in 5% Arabic gum solution 2 hours before LPS injection. For comparison, rats were injected only with 5% Arabic gum solution. LTC4 and LTB4 levels in the liver tissue as well as the bile were measured according to the method described above. Measurement of PGs in the liver tissue PGF2 and 6-keto-PGF1, levels were measured in the liver tissue obtained before and at 30, 60 and 120 min after LPS injection. The rats were anesthetized, and the liver was extracted. The liver tissue prepared as described above was homogenized in ice-cold ethanol containing 0.1 mM of indomethacin and kept at -20°C overnight. This was deproteinized by centrifugation (3,000 rpm, 4”C, obtained was 15 min), and the supernatant evaporated to dryness with nitrogen gas and suspended in 10 ml of 15% methanol adjusted to pH 3.0 with 1 N HCI. This solution waS then applied to the SEP-PAK C1s cartridge column pretreated with ethanol and water. After the cartridge was washed with 10 ml each of 5% ethanol, water and petroleum ether, PGs were eluted with 10 ml of methyl formate. This solution was evaporated to dryness with nitrogen gas, suspended in 40% methanol and used as the sample for RP-HPLC. was applied to the analytical The sample phase mobile the using column 35 : acid = (acetonitrile : water : acetic 65 : 0.1) at a flow rate of 0.8 ml/min. The absorbance of the eluted PGs was measured at 195 nm using the UV detector. The fractions corresponding to the standard preparations (donated by Ono Pharmaceuticals, Osaka, Japan) were obtained, which were then evaporated to dryness and suspended in 0.1 M phosphate buffered saline containing 0.1% gelatin. PGE2 and 6-keto-PGF1, levels were measured by RIA (7). As a control, physiological saline was administered to the rats instead of LPS. Statistical analysis All results are indicated as mean * S.D. Significant differences were calculated by analysis of variance test (~~0.05) and further by Student’s test.
RESULTS Experimental induction of massive hepatic cell necrosis When the rats were injected with P.acnesfollowed
Leukotrienes
and Prostaglandins
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151
by LPS 7 days later, massive hepatic cell necrosis was induced in most of them. The movements of the rats began to slow down at one hour after LPS injection, and at 2 to 3 hours, they barely responded, when touched. At 4 hours, the rats began to have convulsions and died, and the survival rate 24 hours after LPS injection was 20%. Changes in serum GOT and GPT levels As shown in Figure 1, serum transaminase levels were normal at 30 min after LPS injection. However, both serum GOT and GPT levels started to increase at 2 hours, and in the rats still surviving after 24 hours, these levels increased to 9500 f 1120 IU and 2520 * 773 IU, respectively.
Histological changes of the liver from a rat with massive hepatic cell necrosis (H&E stain x 100).
Fig. 2
Histological changes of the liver At 24 hours after LPS injection, changes in the liver tissue were noted and necrosis was seen to a large extent in all of the rats (Fig. 2). However, at 30 min after LPS injection, although permeation of macrophages, lymphocytes and plasma cells was observed in the lobules, no changes in the liver cells nor necrosis could be seen in any of the rats. LTC4 and LTB4 levels in the liver tissue As shown in Figure 3, LTC4 level in the liver tissue before LPS injection was 3.37 + 3.25 rig/g liver tissue, while at 30, 60 and 120 min after LPS injection, it was 14.34 + 11.70, 5.39 + 3.06 and 18.34 + 5.93 rig/g liver tissue, respectively, which the
0 y
0
I
I
30
60
I
120(min)
Fig. 3 Time course of leukotriene C, level in the liver tissue of Propionibacterium acnes-treated rats after lipopolysaccharide injection (n = 5). *-**p
showed that the LTC4 level increased after LPS injection, and was significantly higher than that of the control rats at 120 min (~~0.01). LTB4 level also tended to increase after LPS injection and tended to be higher than that of the control rats (Fig. 4). Both LTC4 and LTB4 levels in the liver tissue did not change at all in the control rats administered with physiological saline instead of LPS (Fig. 3,4). The recovery rate was 51 + 3% for LTC, and 84 & 2% for LTB+ LTCd and LTB4 levels in the bile
00.5
2
4
24 (hr)
Fig. 1 Changes in serum GOT and GPT levels in Propionibacrerium acnes-treated rats after lipopolysaccharide injection (II = IO).
LTC, level in the bile before LPS injection was 0.13 rt 0.16 nmol/30 mm/kg weight, but it increased to 0.54 f 0.20 nmol/30 min/kg weight at 120 to 150 min after LPS injection, which was significantly higher than that of the control rats (~~0.05) (Fig. 5). LTB, level was 0.19 rf: 0.17 nmol/30 mm/kg weight before LPS injection, and although it in-
152 Prostaglandins Leukotrienes and Essential Fatty Acids
creased to 0.23 f 0.18 nmol/30 mm/kg weight at 0 to 30 min after LPS injection, it tended to decrease thereafter. However, LTBj level still tended to be higher than that of the control rats (Fig. 6). Neither LTC4 nor LTB4 levels in the bile changed at all in the control rats. Effects of AA861 on LTC4 and LTB, levels in the liver tissue and bile
30
0
120 hid
60
Fig. 4 Time course of leukotriene B, level in the liver tissue of Propionibacterium acnes-treated rats after lipopolvsaccharide injection (n = 5). a-0: LPS-injected rats o-o: control rats
-i
3 1.0,
When AA861 was administered, LTC4 and LTB4 levels in the liver tissue remarkably decreased after LPS injection, and both levels were significantly lower compared to those of the rats not administered with AA861 at 2 hours (p
-‘30 lj
3b
i0
9'0 120 150
lh0 (mid
Fig, 5
Time course of leukotriene C, level in the bile of acnes-treated rats after lipopolysaccharide injection In = 5). *-**p
PGE2 level in the liver tissue before LPS injection was 446 f 53 pg/g liver tissue. However, at 30 min after LPS injection, it increased to 1836 f. 865 pg/g liver tissue and was significantly higher compared to that of the control rats (p
,--
pq
7
z
P
l.O-
I .O /
.; z X z 0.5.
c ._A 0 5 O-
!i4zbaI
Fig. 6
0
30
60
90
120
150
180(min)
Time course of leukotriene B, level in the bile of Propionibacterium acnes-treated rats after lipopolysaccharide injection (n = 5). O-O: LPS-injected rats 0-O: control rats
1
IC”eS
.PS
AA8611-i
P.ac”es
LPS
AABSlHi
Fig. 7 Effects of AA861 on leukotriene C, and leukotriene B, levels in the rat liver tissue at 2 hours after lipopolysaccharide injection (n = 5).
Leukotrienes
I
0
30
60
90
and Prostaglandins
in Massive Hepatic Cell Necrosis
153
120 150 180(min) 0
30
60
120 (min)
Time course of 6-keto-prostaglandin F,, level in the hver tissue of Propionibacterium acnes-treated rats after lipopolysaccharide injection (n = 5). *-**p
DISCUSSION
-30
0
30
60
90
120 150 18O(min)
Fig. 8 Effect of AA861 on leukotriene C, and leukotriene B, levels in the rat bile (n = 5). *-**p
.
I
0
**
T
30
60
120 hid
Fig. 9 Time course of prostaglandin E, level in the liver tissue of Propionibacren’um acnes-treated rats after lipopolysaccharide injection (n = 5). *-**p
of the control rats (~~0.01) (Fig. 10). Neither PGE2 nor 6-keto-PGFi, levels in the liver tissue changed at all in the control rats. The recovery rate of PGs was 64 f 5% for PGE2 and 58 k 3% for 6-ketoPGF,..
The role of arachidonic acid and its metabolites in the induction of massive hepatic cell necrosis has been studied using our experimental model (1). In vitro, lipocortin suppressed the induction of liver cell injury caused by the cytotoxic factor produced from liver adherent cells. AA861, [2-( 12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-l,Cbenzoquinonel, which is a 5-lipoxygenase inhibitor (2), Azelastine hydrochloride, 4-[(+chlorophenyl) methyl]-2(hexahydro-l-methyl-1 H-az-epine-4-yl)which monohydrochloride, inhibits the production and release of LTs (2), and Gomisin A, [(+)-(6s,7s,R-biar)-5,6,7,8-tetrahydro-l,2,3,12-tetr amethoxy-6,7-dimethyl-lO,ll-methylenedioxy-6-dib enzo[a,c]cyclooctenol], which induces the production of lipocortin (8), also remarkably suppressed the induction of massive hepatic cell necrosis. LTC4 was shown to induce liver cell injury in vitro. LTB4, LTC4, LTD4 and LTE4 are chemical mediators metabolized from arachidonic acid via the 5-lipoxygenase pathway (9). LTB4 is a potent mediator of inflammation, which enhances the adhesion of leukocytes to vascular endothelial cells and causes the chemotaxis of leukocytes and infiltration of inflammatory cells (lo), and it has been reported to be produced from neutrophils, eosinophils, monocytes, macrophages and mast cells. LTB4 also affects the immune responses, and it induces suppressor cells (ll), enhances natural killer cell activity (12), increases the production of interleukin-1 (13) and y-interferon (14) and partially substitutes helper T cell function (15). LTC4, LTD4 and LTE4 are known to contract the vascular smooth muscle, increase the permeability of postcapillary venules as well as capillaries, cause the
154 Prostaglandins Leukotrienes and Essential Fatty Acids
extravasation of plasma and induce tissue edema, which is a characteristic of the acute inflammatory reaction (9). LTC4, is metabolized into LTD4 and LTDJ, is in turn changed into LTE4, but LTC4 is known to have the strongest physiological activity (9). On the other hand, we have shown that PGs, which are produced from arachidonic acid via the cyclooxygenase pathway, remarkably improve the survival rate and histological changes of the liver in our experimental model (3). Various studies have also indicated that PGs exhibit their cytoprotective actions by stabilizing the cell membrane and lysosome membrane. For example, Ishii et al. (16) reported that in chenodeoxycholic acid-induced liver cell injury, PGEt becomes cytoprotective by stabilizing the plasma membrane. That is, when the cell is stimulated, phospholipase A2 is activated and arachidonic acid is released, inducing the producing the production of PGs. As a result, the amount of arachidonic acid in the cell membrane decreases and the fluidity of the plasma membrane is reduced. When PGs are exogenously administered, they prevent’ the decrease in the amount of arachidonic acid in the cell membrane and restore the fluidity of the plasma membrane. In this way, cell metabolism and cell function are maintained. Stachura et al. (17) reported that the percutaneous administration of PGEz remarkably reduces carbon tetrachloride-induce liver cell necrosis in rats by protecting the endoplasmic reticulum from damage due to carbon trichloride, the metabolite of carbon tetrachloride, or by stabilizing the cell membrane. Sikujara et al. (18) demonstrated in vivo that in ischemia-induced hepatic cell injury using rats, PGI2 exhibits its cytoprotective actions by increasing the amount of cyclic 3’-5’ adenosine monophosphate in the liver. Araki et al. (19) reported that in hypoxiainduced liver injury using cat isolated hepatoxytes, PG12 stabilizes the lysosome membrane and protects the liver cells by inhibiting the release of LDH and Cathepsin D from the liver cells due to hypoxia. Since these studies suggested that the arachidonic acid cascade may be involved in the induction of liver cell injury, we examined the levels of LTs and PGs in the liver tissue and LTs in the bile. As a result, in the liver tissue, the levels of PGs increased at an early stage followed by the increase in the levels of LTs. We should note that these changes in the levels of PGs and LTs were observed as early as 30 min after LPS injection, when serum transaminase levels were still normal and histological changes in the liver were not observed. In addition, we found that the levels of LTs and PGs increased again at 2 hours, after the increase in serum transaminase levels to 100 to 600 IU and the microscopic appearance of necrosis in the liver tissue. Although a more detailed study is necessary, these results sug-
gest that liver cell injury may first be triggered by the changes in the arachidonic acid cascade. This may explain the biphasic time course of LTs and PGs in the present study, in contrast to a concomitant increase in the two levels. As for the levels of LTs in the bile, LTC4 and LTB4 levels increased at an early stage. Keppler et al. (20) have reported that because the levels of LTs increase in the bile of normal rats injected with LPS, the changes in the levels of LTs in the bile are due to LPS and not related to liver cell injury. However, we found that when AA861 was administered to rats in our experimental model, liver cell injury was suppresded (2), and that the levels of LTs decreased in the liver tissue as well as the bile. We therefore conclude that although LTs may not directly cause the induction of liver cell injury, this does not mean that LTs are not involved in liver injury at all. That is, another factor probably plays a role along with LTs in the induction of liver cell injury. Further studies must be made to elucidate this other factor. In any case, there is no doubt that the changes in the arachidonic acid cascade are induced in the liver tissue before the biochemical or histological appearance of liver cell injury. Therefore, these results confirmed that the arachidonic acid cascade plays a significant role in the induction of liver cell injury. Among the arachidonic acid metabolites, LTs have cytotoxic effects while PGs have cytoprotective effects, so that the destruction in the balance of LTs and PGs may lead to the induction of liver cell injury. References 1. Tsutsui H. Mizoguchi Y, Yamamoto S, Morisawa S. Studies on experimentally-induced acute hepatic failure: possible involvement of activated liver adherent cells. p 307-314 in Cells of the Hepatic Sinusoid (Kirn A. Knook D L, Wisse E edsj. The Kuoffer Cell Foundation, The Netherlands, 1986. 2. Miioguchi Y, Sakagami Y, Seki S, Kobayashi K, Yamamoto S, Morisawa S. Protective effect of a leukotriene inhibitor in an experimental massive hepatic cell necrosis model. Gastroenterologia Japonica 23: 263-267, 1988. 3. Mizoguchi Y, Tsutsui H, Miyajima K, Sakagami Y, Seki S, Kobayashi K, Yamamoto S, Morisawa S. The protective effect of prostaglandin E, in an experimental massive hepatic cell necrosis model. Hepatology 7: 11841188, 1987. 4. Powell W. Raoid extraction of oxveenated metabolites of’arachidonic acid from biological samples using octadecylsilyl silica. Prostaglandins 20: 947-957,198O. 5. Borgeat P, Fruteau de Laclos B, Rabinovitch H, Picard S, Braquet P, Hebert J, Laviolette M. Generation and structures of the lipoxygenase products. Eosinophil-rich human polymorphonuclear leukocyte preparations characteristicallv release leukotriene C, on ionophore A23I87 challenge. Journal of Allergy and Clinical Immunology 74: 310-315, 1984. 6. Salmon J A, Simmons P M, Palmer R M J. A radioimmunoassay for leukotriene B,. Prostaglandins 24: 225-235, 1982.
Leukotrienes 7. Salmon J A. Analysis of cycle-oxygenase and lipoxygenase products in inflammation exudate. Prostaglandins 27: 364-366, 1984. 8. Ohkura Y, Mizoguchi Y, Sakagami Y, Kobayashi K, Yamamoto S, Morisawa S, Takeda S, Aburada M. Inhibitory effect of TJN-101 [(+)-(6s,7s,R-biar)-5,6,7,8-tetrahydro-1,2,3,12tetramethoxy-6,7-dimethyl-lO,ll-methylenedioxy-6dibenzo[a,c]cyclooctenol] on immunologically induced liver injuries. Japanese Journal of Pharmacology 44: 179-185. 1987. 9. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220: 568-575. 1983. 10. Ford-Hutchinson A W, Bray M A, Doig M V, Shipley M E, Smith M J H. Leukotriene B, a potent chemotactic and aggregating substance released from polymorphonuclear leukocytes. Nature 286: 264-269, 1980. 11. Rola-Pleszczynski M. Borgeat P, Sirois P. Leukotriene B, induces human suppressor lymphocytes. Biochemical Biophysical Research Communications 108: 1531-1537, 1982. 12. Rola-Pleszczynski M, Gagnon L. Sirois P. Leukotriene B, augments human natural cytotoxic cell activity. Biochemical Biophysical Research Communications 113: 531-537, 1983. 13. Rola-Pleszczynski M, Lemaire I. Leukotrienes augment interleukin 1 production by human monocytes. Journal of Immunology 135: 39583961, 1985.
and Prostaglandins
in Massive Hepatic Cell Necrosis
14. Murray H W, Spitalny G L, Nathan C F. Activation of mouse peritoneal macrophages in vitro and in vivo by interferon y. Journal of Immunology 134: 1619-1622, 1985. 15. Jordan M L, Hoffman R A, Simmons R L. Leukotriene B, augments IL-2-dependent proliferation of T lymphocyte clones. Transolantation Proceedings 18: 224-227. 1986. 16. Ishii Y. The cytotoxicity aufchenodeoxydhohc acid on primary cultured rat hepatocytes and cytoprotective action of prostaglandin E, Japanese Journal of Gastroenterology (in Japanese) 82: 108-117. 1985. 17. Stachura J, Tarnawski A. Ivey K J. Mach T. Bogdal J, Szczudrawa J, Klimczyk B. Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in the rat. Gastroenterology Xl: 211-217, 1981. 18. Sikujara 0, Monden M, Toyoshima K, Okamura J, Kosaki G. Cytoprotective effect of prostaglandin I, on ischemia-induced hepatic cell injury. _ Transulantation 36: 238-243. 1983. 19. Araki’ H, Lefer A M. Cytoprotective actions of prostacyclin during hypoxia in the isolated perfused cat liver. American Journal of Physiology 238: 176-181,198O. 20. Hagmann W. Denzhnger C, Rapp S, Weckbecker G. Keppler D. Identification of the major endogenous leukotriene metabohte in the bile of rats as N-acetyl leukotriene E,. Prostaglandins 31: 239-251. 1986.
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