Hepatic necrosis and glutathione depletion in captopril ... - Europe PMC

Br. J. exp. Path. (I985) 66, 67-78

Hepatic necrosis and glutathione depletion in captopril-treated mice T.R. Helliwell, J.H.K. Yeung* and B.K. Park* Departments of Pathology and *Pharmacology and Therapeutics, University of Liverpool, PO Box 147, Liverpool L69 3Bx

Received for publication

12

April I984

Summary. Captopril (CP) is an angiotensin-converting enzyme inhibitor whose metabolism involves endogenous thiols which may be depleted at high doses of CP. Following intraperitoneal administration of CP (50-300 mg/kg), dose-dependent depletion of hepatic glutathione, increased serum transaminase (SGPT) levels and hepatic necrosis were observed. The hepatic necrosis observed was either subcapsular or parenchymal in distribution. Both types of necrosis showed a dose-dependent increase in severity but with a large inter-animal variation. The patterns of necrosis observed with CP are different from the necrosis caused by paracetamol. Oral CP (300 mg/kg) caused parenchymal necrosis in only one animal. It is suggested that subcapsular necrosis may be due to the direct effect of i.p. captopril whereas parenchymal necrosis may be a consequence of hepatic GSH depletion.

Keywords: glutathione depletion, captopril, hepatotoxicity

Captopril (CP) is an orally active angiotensin-converting enzyme inhibitor used in the treatment of systemic and pulmonary hypertension and congestive heart failure (Atkinson & Robertson I979; Gavras et al. I980; Leier et al. I983). A number of adverse reactions to the drug have been reported, including skin rash and fever (Case et al. I978), loss of taste (Vlasses & Ferguson I979), ulcers (Seedat I979), leucopenia (Van Brummelen et al. I980), serum sickness (Hoorntje et al. I979), agranulocytosis (Amann et al. I980), nephrotic syndrome (Atkinson et al. I980), and decreased renal and hepatic function (Coulie et al. I983; Vandenburg et al. I98I; Hurault de Ligny et al. I982). The mechanism of these adverse reactions is still not known. Studies in man and animals have shown that the principal route of metabolism for CP involves the formation of mixed disulphides

with proteins and endogenous thiols (Kripalani et al. I980; Park et al. I982). At high doses, CP depletes endogenous thiols because of the requirement of cysteine and glutathione for the metabolism of CP and CP-protein conjugates (Yeung et al. I983). Glutathione (GSH) is the major free thiol in most cells and has diverse functions such as the detoxification of xenobiotics, removal of peroxides, protection against effects of ionizing radiation, maintenance of the sulphydryl status of proteins and modulation of enzyme activity by disulphide interchange (Arias & Jakoby I976; Sies & Wendell I978). There are many examples of drug-induced toxicity following GSH depletion in tissues (Mitchell et al. I 9 73b; Jollow et al. I9 74) although it is not established whether depletion of GSH per se leads to cell damage. We have found in previous studies that CP produces the same degree of GSH depletion as paracetamol

67

T.R. Helliwell et al. 68 (Yeung et al. I983). Therefore, the purpose histology and the remainder immediately of this study was to determine whether frozen in liquid nitrogen for determination of CP-induced GSH depletion was associated hepatic GSH on the same day. Some animals with hepatotoxicity, and if so whether the from each group also had the spleen removed resulting histological changes were the same for histology. as those observed with paracetamol. Determination of hepatic GSH. The frozen liver was pulverized and an aliquot (0.25-0.5 g) was homogenized with ice-cold i M perchMaterials and methods loric acid (3 ml) containing 2 mM EDTA. The Reagents. The captopril used in these studies homogenate was then centrifuged (2000 g) was a gift from Dr Z. Horovitz of the Squibb at 40 for I 5 min to obtain a clear supernaInstitute, New Brunswick, NJ, USA. Glyoxa- tant. An aliquot (0.2 ml) of the acidic lase I and methylglyoxal were obtained from supernatant was neutralized with o. I ml of 2 Sigma Chemical Company, London, UK. M potassium hydroxide containing 0.3 M SGPT enzymatic kit and other general re- morpholino-propanesulphonic acid. The agents were purchased from British Drug mixture was centrifuged for 30 s at 2000 9. Houses, UK. CBA/Ca mice were obtained The sample was assayed for GSH, immediafrom Bantin and Kingman, UK. tely after neutralization, by the glyoxalase I method (Akerboom & Sies I 98 I). The formaMethods. Male CBA/Ca mice (25-30 g) were tion of S-lactoyl GSH was monitored at 240 kept in standard conditions. All were given nm in a Pye-Unicam dual wavelength u.v. free access to food and water, except for the spectrophotometer. oral dosing groups, which were starved overnight and food resumed 2 h after dosing. Determination of SGPT activity. Serum was obtained from clotted whole blood centriThe dosing scheme is shown in Table i. Twenty four hours after dosin-g, the ani- fuged at 2000 g for I5 min. The SGPT mals were killed by a blow to the head, the activity was determined with a diagnostic kit abdomen opened and in some cases blood from British Drug Houses. obtained via the aorta. The liver was dissected free, a sagittal slice removed for Histology. The tissues were fixed for 24 h in Table i. Dose regimen to determine the effect of oral and i.p. doses of CP on hepatic GSH, SGPT activity and liver histology Dose

No. of Animals

saline control, i.p. hydrochloric acid (pH 2) control, i.p. 50 mg/kg CP, i.p. I00 mg/kg CP, i.p. 200 mg/kg CP, i.p. 300 mg/kg CP, i.p. 300 mg/kg paracetamol, i.p. saline control, oral 300 mg/kg CP, oral 400 mg/kg CP, oral 6oo mg/kg CP, oral 8oo mg/kg CP, oral

6 7 7 7 7 7 7 6 6 6 6 6

Hepatic necrosis and glutathione depletion 69 io% buffered formalin solution, embedded in dependent increase in SGPT activity (Table paraffin wax and 5-um-thick sections cut. All 2); the increase in SGPT activity correlated sections were stained with haematoxylin with the decrease in hepatic GSH (r=0.59;

and eosin, and selected sections by Gordon and Sweet's method for reticulin fibres or with alizarin red for calcium. Sections were cut from at least two levels in each block of tissue.

The extent of the hepatic necrosis was estimated by measuring the area of necrotic tissue present in one histological section. The image of a section was projected onto paper and the areas of necrosis outlined. The area of the outline was then measured by planimetry using a Kontron MOP-30 image analysis system. Calibration was by similar measurement of a stage micrometer. For comparative purposes the section showing the largest total area of necrosis was used. Statistical analysis. All results are reported as the mean±SD. Differences between means were determined using Student's t-test. Results

Biochemistry As shown in Table 2, CP given i.p. produced a dose-dependent depletion of hepatic GSH at 24 h (5-33%). There was a large inter-animal variation in the depletion of GSH at 24 h and consequently only the groups given 200 and 300 mg/kg CP showed a statistically significant decrease. There was also a dose-

P< o.oo5).

When CP was given orally, a dose-dependent decrease in hepatic GSH (9-40%) was observed (Table 3). However, there was no significant increase in SGPT activity. Histology Liver. The tissues examined from both control groups (saline and acid) showed a normal architecture and well-preserved cells. There was no evidence of surface inflammation

or necrosis.

The paracetamol-treated animals showed zones of eosinophilic coagulative necrosis 0.I-0.2 mm wide surrounding hepatic veins (Fig. i). The necrotic zones were present throughout the liver with no predilection for subcapsular or deep areas. The adjacent tissue showed dilated sinusoids and mild fatty change in hepatocytes. When captopril was given i.p., two types of change were observed: subcapsular and parenchymal necrosis. Subcapsular necrosis was defined as eosinophilic coagulative necrosis of a zone of hepatocytes immediately beneath the peritoneal covering of the liver. This type of necrosis was accompanied by an infiltrate of polymorphonuclear leucocytes. Subcapsular necrosis was observed in all animals given captopril, though the severity varied from necrosis of individual cells (Fig.

Table 2. Effect of i.p. doses of CP on hepatic GSH, SGPT activity and liver histology

(mg/kg CP)

(pmol/g)

SGPT activity (units/litre)

Control

8.42±0.62

139.50± 37.I6

Treatment

50

100 200

300

GSH

8.04±1.03 i63.66±65.98 7.46± 1.19 2IO.13±97.82*** 6.41 ± I.I6** 275.5I ± 52.22***

5.65 ±2.85*

Average grade Average area of of subcapsular parenchymal necrosis (x I04 .m2) necrosis 2.3±0.9

i.9±0.69 4.1 ± i.86

363.72±43.59*** 5.4±0.78

0

20.5±40.0

48.o±44.6

104.9+ i81.5

nf=5-7. Results are given as mean±sd. * P