Borle, AB

Fagiuoli, S

Farghali, H

Francavilla, A

Gasbarrini, A

Nalesnik, MA

Starzl, TE

Van Thiel, DH

PubMed

Name not available

FK 506 modulates D-galactosamine-induced hepatitis in rats

D-Scholarship@Pitt

FK 506 Modulates D-Galactosamine-Induced Hepatitis in Rats H. Farghali, A. Gasbarrini, A.B. Borle, M.A. Nalesnik, A. Francavilla, S. Fagiuoli, T.E. Starzl, and D.H. Van Thiel THE immunosuppressive agent FK 506 has been inves-tigated for its adverse effects on the liver, kidney, and the pancreas. I FK 506, however, has multiple biological effects which may be beneficial for the liver. For instance, pretreatment of rats with FK 506 has been shown to both protect the liver from ischemic injury2 and to ameliorate the hepatic injury associated with ischemia and reperfu-sion in rats. 3 Other immunomodulators having different chemical structures have been shown to exhibit similar hepatoprotective effects with different types of hepatic injury.4-6 The aim of the present study was to assess the effect of FK 506 pretreatment on D-galactosamine-induced hepatitis. METHODS Male, adult Sprague-Dawley rats weighing 220 to 270 g were divided into four groups of six to twelve each. Groups I and 3 were used as controls and were gavaged with water, while animals in groups 2 and 4 were given 0.3 mg/kg of FK 506 suspended in water orally. Twenty-four hours later, acute hepatic failure was induced by a single intraperitoneal injection of D-galactosamine hydrochloride dissolved in 0.9% NaCI at dose of 2 glkg for groups I and 2 and 2.5 g/kg for groups 3 and 4. All rats were observed for \0 days for survival. Blood was collected from the tail vein, and serum aspartate aminotransferase (AST) and alanine aminotrans-ferase (ALT) levels were estimated at 24,48, and, when possible, 72 hours after GA treatment. Seventy-two hours after the GA insult. 3 rats from groups 3 and 4 were anaesthesized, and the right lobe of the liver was removed and freeze-clamped with liquid nitrogen for A TP enzymatic analysis. The residual left hepatic lobe was utilized for histopathologic examination after the speci-mens were fixed in 10% formalin, dehydrated, embedded in paraffin, cut at 5 /-Lm, and stained for histological examination with hematoxylin and eosin. The extent of the liver necrosis was estimated semiquantitatively. The number of frankly necrotic hepatocytes per 10 random high-power microscopic fields (Olym-pus BH-2 microscope, 4oox) was counted. A portion of the frozen liver was ground in liquid nitrogen and 4% (wt/vol) precooled perchloric acid (I x 5 wtlvol) and then homogenized. The homog-enized samples were centrifuged at 4°C at 3000 rpm for 10 minutes. The supernatant was brought to pH 7 using 6 mollL K2C03 and respun at 4°C at 3000 rpm for 5 minutes. The resultant samples were analyzed for ATP by the Sigma kit (Sigma Chemical Co, St. Louis, Mo). All data are reported as mean ± SEM. The From the Department of Surgery (H. F., A.G., MAN., A.F., S.F., T.E.S., D.H.V.T.) and Department of Physiology (A.B.B.), Univer-sity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Address reprint requests to D.H. Van Thiel, PhD, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213. © 1991 by Appleton & Lange 0041-1345/91/$3.00/+0 2000 I- 1000 CJ) < E :3 u.. CJ) --I-.... < 1000 800 600 400 200 A 20 B 20 ---0-- ContrOl ----..- FK 506-treated 40 60 80 Time (hours) --Control FK 50S-treated 40 60 80 Time (hours) Fig 1. (A) Plasma AST (U/L) levels in control rats (GA, 2 g/kg) or FK 506-treated ones (FK 506 + GA) as a function of time after GA; (8) plasma AL T (U/L) levels in control rats (GA, 2/kg) or FK 506-treated ones (FK 506 + GAl as a function of time after GA. Points are means ± SEM. Transplantation Proceedings, Vol 23, No 6 (December), 1991: pp 2809-2811 2809 2810 4000 ---Control A ----+-- FK SaG-treated 3000 -E 3 u.. 2000 ~ I-en < 1000 l~----r-~~-----r----~--~ o 10 20 30 40 50 Time (hours) -Control 4000 B --.- FK 506-treated 3000 -E 3 u.. 2000 en --I-~ < 1000 Time (hours) Fig 2. (A) Plasma AST (U/l) levels in control rats (GA, 2.5 g/kg) or FK 506-treated ones (FK 506 + GA) as a function of time after GA; (8) plasma Al T (U/l) levels in control rats (GA, 2.5 g/kg) or FK 506-treated ones (FK 506 + GA) as a function of time after GA. Points are means ± SEM. Student's t test was used for statistical analysis. P < .05 was considered significant. RESULTS FK 506 pretreatment increased the survival of groups 2 and 4, where survival was 90% versus 100% and 50% versus 67% for groups 1, 2, 3, and 4, respectively. The FARGHALI, GASBARRINI, BORlE ET AL 4 3 -~ o ~ 2 -o Control FK 506 Treatment Fig 3. Hepatic ATP contents (jLmol/g of liver weight) in control rats (GA-treated) or FK 506-treated ones (FK 506 + GA). Values are means of 3 animals ± SEM. effect of FK 506 pretreatment on the GA (2 g/kg) induced increases in AST and AL T in groups 1 and 2 is given in Fig 1 and 2, respectively. FK 506 pretreatment was associated with a significant reduction in the plasma levels of AST and ALT at all time intervals tested (P < .05). AST and ALT levels for groups 3 and 4 were estimated only after 24 and 48 hours of GA administration, because rats were too ill at 72 hours. Nevertheless, FK 506 pretreatment reduced both the AST and AL T levels at different time intervals as illustrated in Fig 3 and 4, respectively (P < .05). Moreover, the ATP levels within the liver of the FK 506-treated rats in group 4 was 3.4 ± 0.5 p,mollL per gram as compared to 2.6 ± 0.2 p,mollL per gram for the controls in group 3 (P < .01). The number of frankly necrotic cells in the livers of the FK 506-treated rats of group 4 was significantly re-duced (P < .01) versus that seen in the control livers of group 3 where they were 27 ± 7.6 versus 61 ± 8.2, respectively. DISCUSSION Recently, an interplay between pharmacologic immuno-modulation and hepatic function has been shown to exist in several types of hepatic injury, which can be induced experimentally. In recent years, several reports demon-strating a hepatotrophic effect of FK 506 when adminis-tered before or immediately after a variety of hepatic injuries have been published.2•3 The study herein reported was directed to determine the effect of FK 506 pretreat-D-GALACTOSAMINE-INDUCED HEPATITIS rII a; u u :;: o .. u Q) c -o 80 60 40 20 o Control Treatment FK 506 Fig 4. Number of hepatic parenchyma demonstrating necrosis in GA-treated rats (control, 2.5 g/kg) and FK 506-treated ones (FK + GA) 72 hours after GA administration. ment on yet another type of hepatic injury produced by o-galactosamine (GA). GA is a highly specific hepatotoxin that causes a dose-dependent depletion of uridine metab-olites.7 A hepatoprotective effect of several immunomod-ulators against chemically induced liver injury has been documented previouslyK~·UK9 The present data indicate a similar effect of FK 506 pretreatment in modulating the GA-induced hepatotoxicity at both dose levels used. He-patoprotection was demonstrated by reduced plasma lev-els of aminotransferases, increased survival, conservation of the energy status of the liver as evidenced by measure-ment of hepatic ATP levels, and a reduced necrotic index as assessed histologically. 2811 A somewhat similar immunosuppressive agent, cyclo-sporine, was also a hepatotrophic agent in rats or mice after partial hepatectomy, 1~1O and acts as chemopro-tectant against the potent hepatotoxin microcystin, LR. \3 It appears that FK 506 has a similar cytoprotective effect, as it is also protective against various kinds of hepatic insults ranging from surgical to chemical injury, and is shared by other immunomodulators, not all of which are immunosuppressive. The molecular mechanisms responsi-ble for their cytoprotective action are not apparent and deserve further investigation. REFERENCES 1. Nalesnik MA, Todo S, Murase N, et al: Transplant Proc 19:89, 1987 2. Mazzaferro V. Scotti-Foglieni CL. Porter KA, et a1: Trans-plant Proc 22:93, 1990 3. Sakr MF, Zetti ON. Hassanein TI, et al: Hepatology 13:947, 1991 4. Yoshikawa T, Furukawa Y, Wakamatsu Y, et al: Experien-tia 38:501,1982 5. Farghali H. Machkova Z, Kamenikova L, et al: Exp Clin PharmacoI6:449. 1984 6. Farghali H. Buchar E, Machkova Z, et al: Exp Clin Phar-macoI8:469, 1986 7. Rogers VK, Wu OY: Cancer Chemother Pharmacol 26:93, 1990 8. DeBrabander M, Van Belle H, Aerts F, et al: Int Immunop-harmacol1:93,1979 9. Farghali H, Buchar E, liricika Z, et al: In Kotyk, et al (eds): Highlight of Modem Biochemistry. Zeist, VSP International. 1989, p 1539 10. Kim YI, Caine RY, Nagasue N: Surg Oynecol Obstet 166:317, 1988 11. Kahn D, Lai HS, Romovacek H, et al: Transplant Proc 20:850, 1988 12. Yoshimura S, Kamada N: Transplant Proc 21:911, 1989 13. Hermansky SJ. Casey Pl, Stohs Sl: Toxieol Lett 54:279. 1990 

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FK 506 modulates D-galactosamine-induced hepatitis in rats

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