566

Agents and Actions vol. 9, 5/6 (1979) Birkh~iuser Verlag, Basel

Protection of Mice Against Endotoxin-Induced Liver Damage by Anti-Inflammatory Drugs by J. FERLUGA 1, A. KAPLUN2 and A.C. ALLISON 1 Division of Cell Pathology, Clinical Research Centre, Harrow, Middlesex, UK

Abstract Mice were injected with Corynebacterium parvum, which induces multiple granulumas in liver and renders animals hyper-reactive to the lethal effect of bacterial lipopolysaccharlde (LPS). Such animals when challenged with LPS developed also extensive liver parenchymal cell damage as estimated by elevated blood aspartate transamlnase levels and a hypoglyeaemia. Treatment with indomethacin, hydro, cortisone, dexamethasone, promethazlne, metiazinic acld and (+)catechln ameliorated the liver damage. Hydrocortisone, dexamethasone, promethazine and metlazinic acid also reduced the mortality rate in mice challenged with LPS. Diarrhoea, accompanying the LPS-lnduced shock, was prevented by the drugs used. Possible agents mediating the hepatotoxic and shock effects of LPS are discussed.

A variety of agents stimu!ating the mononuclear phagocytic system were found to render experimental animals very susceptible to bacterial lipopolysaccharide (LPS, endotoxin). They include bacteria and viruses such as Myeobacterium boris [1], mouse hepatitis virus [2] and Corynebaeterium parvum [3]. In addition, mice rendered hyper-reactive with Myeobaeterium boris [4], killed C. parvum [5] or Sehistosoma mansoni - infection [6] were shown to develop severe liver parenchymal cell lesions when challenged with LPS, accompanied by a pronounced hypoglycaemia and elevated aspartate transaminase blood levels. Because of the similarity with naturally occurring hepatitis we thought that such a model would be suitable for studying the pathogenesis of hepatitis. This is based on a working hypothesis that mononuclear phagocytic cells (macrophages) recruited into the liver Present addresses: ~International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya; 2Israel Institute f o r Biological Research, Ness-Ziona, Israel.

lobules by the pretreatment with inflammatory agents, release soluble factors toxic for hepatocytes when exposed to LPS. In the present study the C. parvum-LPSinduced hepatitis model in the mouse was used to analyse the effect of various drugs on liver damage as well as on mortality rate. Among the pharmacologically active agents released by macrophages are prostaglandins [7] and therefore the effect of indomethacin, a potent inhibitor of prostaglandin synthetase [8], has been tested. Hydrocortisone, dexamethasone, two phenothiazine drugs and a flavonoid, (+)-catechin, were also included. Another property of activated macrophages is the development of procoagulant activity due to tissue thromboplastin production [9]. It is therefore possible that intravascular coagulation may be triggered by LPS, thus contributing to the general shock. Therefore some mice were depleted of their circulatory fibrinogen with a thrombin-like enzyme from Malayan pit viper venom, ancrod, before challenging with LPS. A further possibility is that hypoglycaemia, together with fluid and electrolyte loss, is the major cause of death, so the effects of supplementation with glucose and a balanced salt solution have been investigated. Materials and methods Killed Corynebaeterium parvum preparation was obtained from Wellcome Reagents Ltd., Lipopolysaccharide B, E. eoli 055 :B5 (LPS) from Difco Lab., promethazine hydrochloride (Phenergan| from May & Baker Ltd., Hydrocortisone succinate and dexamethasone sodium phosphate from Organon Lab. Ltd., glucose (Dextrose Injection B.P., 5% solution) from Travenol Lab. Ltd., and Ringer's Solution for Injection from Boots Co. Ltd. Metiazinic acid (Soripal | was a gift from Rh6ne-Poulenc,

Protection Against Endotoxin Liver Damage by Drugs indomethacin from Merck Sharp & Dome Ltd. and (+)catechin (or (+)-cyanidanol-3) from Dr T.B. Pulvertaft, Zyma Ltd. Human lZ~l-fibrinogen (100 /aCi-mg) and 1251serum albumin (2.5 aCi/mg) were from The Radiochemical Centre, Amersham, U.K., and ancrod (Arvin~) from Berk Pharmaceuticals Ltd. Mice of BALB/c strain, 2 to 4 months old, were bred at the Clinical Research Centre. Mice were injected intravenously with 0.7 mg/mouse of C. parvum suspended in 0.2 ml saline. Seven days later the male animals received intravenously 25/ag and female animals 12.5/ag of LPS dissolved in 0.2 ml saline or saline only and were kept at an ambient temperature between 26 to 28~ C. In some mice the mortality was recorded during 5 days. The others were 3 to 4 h after challenge with LPS exsanguinated from axillary blood vessels under ether anaesthesia, blood collected in tubes on ice containing 20 IU/ml blood of heparin and the ceils sedimented at 4~ by centrifugation. Part of plasma was added to tubes with 10 mg/ml of sodium fluoride and examined for the glucose content using a glucose oxidase method [ 10], and in the other part the aspartate transaminase content was determined by the method of KARMEN [11]. In some experiments part of the blood was also collected in capillary tubes for haematocrit estimation. In protection experiments, 2 parts of 5% glucose were mixed with 1 part of Ringer's solution and 1.25 ml of the mixture or of Ringer's solution only, injected intraperitoneally 5 to 10 min before LPS, and two and a half hours later. Promethazine, hydrocortisone and dexamethasone were dissolved in saline, metiazinic acid in 0.15 M NaOH water solution and pH adjusted to 7.4 with 0.15 M HCI solution. Indomethacin was dissolved in phosphatebuffered saline of pH 8 (1.25 mg/ml) by warming to 50~ for half a minute. These drugs were administered intraperitoneally or subcutaneously usually in 0.2 ml volumes 30 to 60 min before LPS injection. (+)-Catechin was suspended in 0.5% methylcellulose water solution by sonication and given in 0.5 ml volume by a stomach tube as indicated. Ancrod, 25 Twyford Units/kg in 0.2 ml saline was injected intravenously 3 to 4 b before LPS. This dose was found to deplete about 90% of circulatory fibrinogen [12]. Four animals per group were usually used and mean values and S.E. are shown.

567 Results M i c e w e r e injected with C o r y n e b a c t e r i u m p a r v u m and, 7 d a y s later, tested for their hyperreactivity to a small d o s e ( 2 5 / 2 g ) o f E. coli lipop o l y s a c c h a r i d e . E i g h t out o f 13 mice died within few h o u r s manifesting seizures w h e r e a s n o n e o f n o r m a l mice, receiving this dose o f L P S , died ( T a b l e 1). T o e s t i m a t e liver p a r e n c h y m a l cell d a m a g e , a s p a r t a t e t r a n s a m i n a s e and glucose levels were d e t e r m i n e d in circulating b l o o d f r o m v a r i o u s g r o u p s o f mice, which were found to be highly elevated and l o w e r e d respectively in mice treated with C. p a r v u m and L P S . T h e r e was little or less c h a n g e in these values in n o r m a l mice, even w h e n injected with a m u c h higher d o s e o f 400/2g LPS.

In further e x p e r i m e n t s the effect o f v a r i o u s agents on m o r t a l i t y and h e p a t o c y t e d a m a g e was assessed in mice injected with C. p a r v u m and L P S (Table 2). P r o m e t h a z i n e alone r e d u c e d the n u m b e r o f fatal cases to a b o u t 75%, but w h e n p r o m e t h a z i n e was c o m b i n e d with g l u c o s e or R i n g e r ' s solution the m a j o r i t y o f m i c e survived. Similarly, m e t i a z i n i c acid, w h e n used with the t w o solutions p r o t e c t e d a b o u t 7 5 % o f animals. G l u c o s e and R i n g e r ' s solutions t h e m s e l v e s w e r e only m o d e r a t e l y effective, as was p r e - t r e a t m e n t with ancrod. P r o m e t h a z i n e a m e l i o r a t e d a s p a r t a t e t r a n s a m i n a s e levels in one e x p e r i m e n t and glucose levels in two, w h e r e a s m e t i a z i n i c acid affected o n l y g l u c o s e levels. I n t r a p e r i t o n e a l administration of glucose raised the b l o o d g l u c o s e levels but did not l o w e r circulating t r a n s a m i n a s e levels. H o w e v e r , in a s e p a r a t e e x p e r i m e n t the a m o u n t o f the latter was r e d u c e d by ad-

Table 1 Effect of E. coli lipopolysaccharide (LPS) on male mice pretreated with Corynebacterium parvum.

Treatment

Mortality (No. dead/total)

C.parvum + Saline C.parvum + LPS 25 ,ug Nil + Saline Nil + LPS 25/.tg Nil + LPS 400/lg

0/10 8/13 0/10 0/10 7/10

Aspartate transaminase a (IU/I plasma) 146 + 6 1139 + 392 76 + 8 107 + 24 428 _+ 68 b

Glucosea (mmol/1 plasma) 6.7 _+0.5 1.5 _+0.1 7,6 +_ 0.5 4.5 _+0.6 4.1 _+0.9 b

Treatment: C. parvum, 0.7 rag/mouse i.v. and 7 days later the LPS dose/mouse or 0.2 ml saline i.v. a Mean values and S.E. from 4 animals/group, blood samples taken 3-4 h and b 10 h after LPS injection.

568

Protection Against Endotoxin Liver Damage

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Protection of mice against endotoxin-induced liver damage by anti-inflammatory drugs.

566 Agents and Actions vol. 9, 5/6 (1979) Birkh~iuser Verlag, Basel Protection of Mice Against Endotoxin-Induced Liver Damage by Anti-Inflammatory D...
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