Ultrastructural Changes during Acute ... - SAGE Journals

TOXICOLOGIC PATHOLOGY ISSN:O192-6233 Copyright 0 1987 by the Society of Toxicologic Pathologists

Volume IS, Number 4, 1987 Printed in U.S.A.

Ultrastructural Changes during Acute Acetaminophen-Induced Hepatotoxicity in the Mouse: A Time and Dose Study* MICHAEL E. PLACKE,GARYL. GINSBERG, D. STUARTWYAND, AND STEVEN D. COHEN Department of Pathobiology, College of Agricirltirre aiid Natural Resources, and Section of Pfiaritiacologyarid Toxicology, School of Pharmacy, University of Connecticut,Storrs, Conriecticiit 06268

ABSTRACT This study was undertaken to evaluate the early ultrastructural changes during the development of acetaminophen hepatotoxicity. Doses at or near the threshold for hepatotoxicity were selected to permit comparison of early reversible effects to those which ultimately progressed to necrosis in the absence of early agonal effects or drug-induced mortality. Both 300- and 600-mg/kg doses resulted in similar declines in hepatic glutathione levels to 14 and 22% ofcontrol values, respectively, by 2 hours, with more rapid recovery after the low dose. Plasma sorbitol dehydrogenase activity was elevated after 600 mg/kg but not after 300 mg/kg. During the first 2 hours after acetaminophenthere was cytomegalywith rapid progression to necrosis after 600 mg/kg but minimal progression after 300 mg/kg. Ultrastructurally, vesiculation, vacuolation and mitochondria1and plasma membrane degeneration culminated in scattered single cell death by 4 hours and widespread centrilobular necrosis by 8 hours after 600 mg/kg. The time course of lesion development was slower after 300 mg/kg with damage restricted to the first two to three rows of centrilobularcells and limited numbers of isolated necrotic cells by 8 hours. By 18 to 24 hours livers of mice given 300 m a g appeared normal. Results are consistent with the endoplasmic reticulum being the site of acetaminophen activation and initial attack. However, early ultrastructural changes in mitochondria and plasma membrane observed after the high dose were not prominent after the low dose. This suggests that early acetaminophen damage to these organelles may play a critical role in acetaminophen hepatotoxicity.

hepatotoxicity to the covalent binding (e.g., 17, 22, 25), studies in hamsters suggested that acetaminophen hepatotoxicity was reflective of lipid peroxidative changes (4). The underlying mechanisms responsible for hepatic necrosis have not been clearly defined. The primary lesion of acute acetaminophen toxicity in man is confluent, centrilobular necrosis (24). Mice and hamsters also develop similar lesions at comparable doses (7). For most of the studies cited above, the early, and perhaps more critical, morphological changes were not thoroughly investigated. Furthermore, dosages employed may have been so high as to confound morphological discrimination of specific acetaminophen effects with secondary effects of congestion-induced hypoxia (8,9,2932). Therefore, the present study was undertaken to provide a detailed examination ofthe ultrastructural changes associated with doses of acetaminophen which were near orjust above the threshold for overt necrosis. Emphasis was placed upon early time points

INTRODUCTION Acetaminophen (N-acetyl p-aminophenol) is considered a relatively safe analgesic when taken in therapeutic doses. However, in excess, acetaminophen produces potentially lethal hepatotoxicity (5, 6). Investigations into the mechanisms of acetaminophen’s hepatotoxicity have demonstrated that the drug is activated by microsomal mixed-function oxidases (MFO)to an electrophilic intermediate which is subsequently inactivated through conjugation with glutathione. When glutathione reserves become severely depleted, the remaining electrophile is free to react by covalently binding with nucleophilic sites on proteins or other macromolecules (for reviews see 2,15). While the majority of reports have linked

* Send correspondence to: Dr. Steven D. Cohen, Section of Pharmacology and Toxicology, School of Pharmacy, U-92, University of Connecticut, Storrs, Connecticut 06268. Dr. Placke’s current address is: Battelle Columbus Laboratories, 505 King Avenue, Columbus, Ohio 43201. 43 1

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TABLE I.-Hepatic glutathione (GSH) and plasma sorbitol dehydrogenase (SDH) activity after acetaminophen (APAPP Hours after APAP

GSHb 300 m g k g

600 mgkg

100

100

SDHc

600 m d k g

24 29 1.5 12 2 49 4 1O d 66d 354 6 6I d 17d 7 10 88 12 67 2,l 63d Fasted mice were killed at selected times after 300 or 600 mg APAP/kg, PO. Values in table are means from groups of 4 or 0 0.5

84 3 Id 14d

71 23d 22d

more mice.

In %- of control. Control GSH content = 4,518 f 238 nmoYg X A SE (n = 15). Plasma SDH (in U/ml) was not elevated after 300 mg/kg at any of the times studied. d p 5 0.05 versus control (0 hours aRer APAP).

and dose selection represented an attempt to distinguish reversible from irreversible effects. The intent was to identify specific organelles as early targets for acetaminophen-induced changes which might be mechanistically involved in the hepatotoxicity.

METHODS Adult, male, Cr1:CD- 1(1CR)BR mice (Charles River, Wilmington, MA) 10 to 12 weeks old, 30 to 35 g, were used for both pilot and definitive studies. They were housed in groups of 10 in stainless steel cages with grid flooring above pans containing sawdust. Mice were allowed free access to food (Purina Laboratory Chow) and water. They were kept in airconditioned (25°C) animal quarters on a 12-hour light/dark cycle and given at least 1 week to acclimate prior to use. A pilot study was conducted to establish dose levels for the detailed time course studies. Groups of 2 to 4 mice were killed 2 or 18 hours after 100, 300,400,600 or 800 mg/kg of acetaminophen given

TOXICOLOGIC PATHOLOGY

by gavage. Liver damage was assessed by histopathology and plasma SDH activity. In the definitive study, mice were fasted 18 hours before dosing. Acetaminophen was dissolved in 50% propylene glycol in water in concentrations adjusted to provide 300 and 600 mg of drugkg in a single oral injection volume of 10 mVkg. Groups of 5 to 10 mice were killed at selected times between one half and 24 hours after treatment. Fasted, vehicletreated controls were included at each time point. One group each of unfasted or fasted, non-treated mice were additional controls. All mice were killed by decapitation between 10 and 1 1 am to minimize diurnal effects. Livers were immediately removed from all mice and sections from the median and caudate lobes were taken for light and electron microscopic examination. Liver for histopathologic evaluation was fixed in 10%buffered formalin, sectioned at 5 to 6 microns and stained with hematoxylin and eosin (H&E). Neutral lipids were demonstrated with oil-red-0 staining and glycogen by the periodic acid Schiff (PAS) method. Tissue for transmission electron microscopic studies was fixed in a cold solution of 2% glutaraldehyde-2% paraformaldehyde prepared in 0.1 M phosphate buffer. After 6 hours the tissue was postfixed in 2% osmium tetroxide for 1 additional hour and embedded in a polymer resin (Epi-rez 507 1). Ultrathin sections were prepared, placed on copper mesh grids, stained with 2% uranyl acetate and Reynold's lead citrate, and examined using a Philips 300 electron microscope (1 4). The remaining liver was homogenized in 5% trichloroacetic acid, centrifuged, and DTNB (5,5-dithiobis-2-nitrobenzoic acid) was added to the supernatant for measurement of glutathione as non-protein sulfhydryls as described (1). Plasma sorbitol dehydrogenase (SDH) activity was determined as described (10). For the biochemical data, one-way analysis of variance and the studentized range test (26) determined significant (p < 0.05) differences between treated and control groups.

FIG.1.-(Top, left) Acetamiqophen (600 mg/kg) 1 hour. Centrilobular region of liver. Loss of density in hepatocytes immediately adjacent to the central vein (CV).x 2,240. FIG.2.-(Top, right) Acetaminophen (600 mg/kg) 1 hour. Centrilobular hepatocyte with detached ribosomes diffusely scattered throughout the cytoplasm. There are frequent myelin figures, apparently originating from degenerating mitochondria (arrowheads). Absence of intracristal calcium granules. x 35,600. FIG. 3.-(Bottom, left) Acetaminophen (600 mg/kg) 2 hours. Border between 2 centrilobular cells with multiple membranes accumulated along the plasmalemma @lack arrow), accumulation of dense flocculant material (F) and mitochondria with'loss of double limiting membrane (M). Normal bile canaliculus at bottom. x 28,500. FIG.4.-(Bottom, right) Acetaminophen (600 mg/kg) 2 hours. Perisinusoidal border of a central hepatocyte. Dense mitochondria (white arrow) and large degenerative mitochondria (M) and compressed space of Disse (black arrow). x 11,500.

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PLACKE ET AL RESULTS

Pilot St tidy A dose of 800 mg/kg produced early congestion and high mortality without significant hepatic necrosis; whereas, 600 mg/kg caused maximal glutathione depletion and diffuse hepatic necrosis with minimal mortality. In contrast, 300 mg/kg produced little necrosis but the same level of glutathione depletion by 2 hours as 600 mg/kg; whereas, 100 mg/ kg caused no glutathione depletion and no visible lesions. Histopathologic findings in the pilot study were consistent with previously published results from this laboratory (13). From these results 300 and 600 mg/kg were selected for the following time course study.

Detailed Time Course Study Biochenristry. Results of the selected biochemical measurements are presented in Table I. Glutathione was significantly decreased between 1.5 and 6 hours after administration of 600 mg acetaminophen/kg and returned to 67% of control levels by 12 hours. In contrast, after 300 mg/kg, glutathione was decreased 86% by 2 hours yet had returned to 66% of control levels by 4 hours and 88% by 12 hours. Plasma SDH activity was not elevated after 300 mg/kg; however, it increased almost 15-fold by 4 hours with 600 mgkg and continued to rise to 90-fold by 12 hours. Histopathologv. In mice given 600 mg/kg, alterations began with hydropic degeneration, followed by hepatocellular atrophy, pyknosis, and coagulative necrosis. Lesions started in the centrilobular zone and in time extended into portal areas. In contrast, mice given 300 mg/kg had much less severe hepatic damage. Two to 4 hours after acetaminophen, hydropic degeneration and cytomegaly were seen in centriIobular hepatocytes. By 8 hours many hepatocytes had cytoplasmic vacuoles, increased eosinophilia, and there were occasional necrotic centrilobular hepatocytes. At 18 and 24 hours after 300 mg/kg livers appeared nearly normal. No lesions


were observed in livers of vehicle- or untreated controls. There were no drug related deaths after either dose through 24 hours. PAS staining demonstrated that, in comparison to fasted controls, both dose groups exhibited centrilobular glycogen depletion by 2 hours and this extended to the midzonal region by 4 hours. Glycogen depletion preceded the development of necrosis. After both doses the oil-red-0 stain showed drug related fatty change characterized by marked increase in the size of intracellular lipid droplets. The fatty change was seen in centrilobular hepatocytes by 8 hours after the low dose and by 4 hours after the high dose, and persisted to the time of necrosis. Ultrastriictiiral Pathology (600 itig/kg). Livers from control mice were similar to previous ultrastructural descriptions (4, 16, 20, 26). Livers examined at 30 minutes after acetaminophen resembled controls. The first hepatocyte changeswere seen at 60 minutes and involved only the first row of hepatocytes adjacent to central veins. These cells had decreased density, mild cytomegaly, and dispersion of organelles (Fig. 1). Mitochondria were frequently compressed.and were occasionally fused with small myelin figures (Fig. 2). The small intracristal calcium granules normally associated with mitochondria were not observed. Most of the endoplasmic reticulum (ER) was mildly dilated, elongated or round, and had dissociation of ribosomes (Fig. 2). Myelin figures and membrane fragments were numerous within degenerating hepatocytes. Livers from mice killed 1.5 to 2 hours after drug exposure had comparable changes of slightly greater seventy with cytotoxic effects extending several cell layers from the central vein. Cell densities increased with distance from central veins. The cytoplasm contained free ribosomes, dilated ER and Golgi, and membranous fragments. Mitochondria appeared to have lost portions of their double limiting membranes (Fig. 3) and were swollen and distorted (Fig. 4). Plasma membranes were thickened due to multiple layers of membrane and accumulation of dense

+ FIG.5.--(Top, left) Acetaminophen (600 mg/kg) 6 hours. Degenerative hepatocytes of the midzonal region with lipid accumulation. x 2,800. FIG.6.-(Top, right) Acetaminophen (600 mg/kg) 6 hours. Centrilobular hepatocyte with several bizarre-shaped mitochondria(M). Two different membrane bound vacuoles; 011) bound by a single membrane and relatively clear, and (V2) bound by a double membrane with a matrix similar in density to nearby mitochondria, possibly formed by fusion of several mitochondria. x 12,075. FIG.7.-(Bottom, left) Acetaminophen (300 mg/kg) 4 hours. Hydropic degeneration of cells around a central vein (CV). There is dispersion of organelles. x 2,800. FIG.8.-(Bottom, right) Acetaminophen(300 mg/kg) 8 hours. Advanced degenerationis limited to hepatocytes adjacent to the central vein (CV).Undulating plasma membrane (arrowhead). There is loss of cytoplasmic matrix and karyolysis. x 2,800.

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flocculant material (Fig. 3). There was cytomegaly and the space of Disse was collapsed. Additional changes after 2 hours included decreased density of nucleoplasm, occasional clumping and margination of chromatin, and loss of the perinuclear space. Centrilobular hepatocytes from mice killed 4 to 6 hours after acetaminophen were usually necrotic and degeneration had extended into midzonal and periportal zones. The more peripherally affected hepatocytes resembled cells adjacent to central veins at 1 hour after dosing. By 6 hours in the central cells, nearly all microvilli had disappeared and were seen as small rounded fragments in the space of Disse. Kupffer cells and endothelial lining cells appeared normal and sinusoids did not appear congested. Accumulation of intracellular lipid was prominent in midzonal hepatocytes (Fig. 5). Vesicles of possible ER origin appeared to fuse, forming multimembrane bound vacuoles (Fig. 6). There were enlarged distorted mitochondria which appeared to have formed by mitochondria1 fusion. At 8 to 12 hours, cells in necrotic regions were fragmented with efllux of cytoplasm. Livers examined 18 and 24 hours after acetaminophen had increased radial progression of the lesion. Sinusoidal endothelium lost its structural integrity with escape and pooling of red blood cells. By this time changes in periportal hepatocytes resembled those in centrilobular cells at 2 to 4 hours. Ultrastnictural Pathologv (300 nig/kg;). The earliest changes were noted 2 hours after drug administration and consisted of minimal hydropic degeneration and mild segmental vesiculation of the endoplasmic reticulum with ribosomal detachment. Hepatocytes from the 4- and 8-hour groups had cytomegaly and decreased density (Fig. 7). Lesions were slower in developing than in the 600-mglkg group and were restricted to the first 2 or 3 rows of central cells; There were limited numbers ofnecrotic cells in the 8-hour group and the loss of microvilli, membrane fragments, alterations of the plasma membrane, etc. were similar to the findings in the high dose group (Fig. 8). By 12 hours necrosis had not progressed and there were fewer degenerating hepatocytes than in the 4- and 8-hour groups. Midzonal and periportal hepatocytes appeared unaffected. The hepatocytes from the 18- and 24-hour groups appeared essentially normal. DISCUSSION The present study describes the .time course of morphological changes in mouse liver after administration of acetaminophen. The purpose of the study was to compare morphologic changes caused by both hepatotoxic and nonhepatotoxic doses of acetaminophen with the intent of distinguishing those changes which were reversible from those which were irre-


versible and associated with full lesion development. The lower dose produced occasional single cell necrosis, however this was not great enough to cause detectable leakage of SDH into the plasma. In contrast, plasma SDH activity after 600 mg/kg indicated that this dose was overtly hepatotoxic; yet there were no deaths through 24 hours. Hepatic glutathione concentrations were decreased to the same extent by both treatments but recovered rapidly after 300 mg/kg. This is consistent with the lower dose being near the threshold required for hepatotoxicity while the higher dose clearly exceeded that threshold (2 1,22). That sinusoidal congestion and blood pooling were not observed until 18 to 24 hours after 600 mg/kg suggests that agonal or hemodynamic factors reported by others (32) did not play an important role in the earlier acetaminophen effects observed in this study. Hydropic changes preceded most other changes caused by either dose of acetaminophen. Such changes may reflect early disturbances in cellular osmoregulatory function. However, since most hepatocytes appeared to recover after the lower dose, it would seem unlikely that the hydropic changes play a key role in the hepatotoxicity. Carbon tetrachloride also causes necrosis subsequent to hydropic degeneration (1 8) yet many of the biochemical and pathologic. changes induced by carbon tetrachloride and acetaminophen are different. Thus, early hydropic disturbances may reflect a reversible, nonspecific stage in the toxic process. Careful comparison of the ultrastructural effects caused by the two doses indicates the major difference to be that many more cells were affected and the progression of effects was more rapid after the high dose. However, some early changes were predominantly associated with the 600-mg/kg treatment. The most prominent of the discriminating effects were those on the mitochondria and plasma membrane. The swelling and distortion of mitochondria with loss of calcium granules and the "layered" appearance of the plasma membranes were widely evident between 1 and 2 hours after 600 mg/ kg (e.g., Figs. 3, 4) but were detected rarely or not at all after 300 mdkg and were not noted in controls. Therefore, these may represent prenecrotic changes induced by the drug. In contrast, other changes such as dilatation and vesiculation of the endoplasmic reticulum and decreased cytoplasmic density were common to both dosage treatment groups and may represent reversible effects of acetaminophen. Many studies have implicated covalent binding of acetaminophen-derived electrophile in hepatotoxicity (see reviews 2, 15). However, binding may merely reflect the formation of an electrophile and may not necessarily mediate the toxicity (1 1). De-

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tection ofearly ultrastructural modifications of plasma membrane, mitochondria a n d endoplasmic reticulum in the present study is in close agreement with earlier demonstration of significant covalent binding in plasma membrane, mitochondria and microsomes within the first 2 hours after an hepatotoxic dose of acetaminophen (12). The highest specific activity was found in plasma membrane. Acetaminophen also inhibited specific enzymes in mitochondria and plasma membrane a t times when other enzymes in these as well as other hepatocyte organelles were unaffected (1 2). This is indicative of an apparent selectivity at both the cellular and organelle level. Others have also demonstrated biochemical changes in plasma membrane early after acetaminophen (23, 28) and we recently reported inhibition of respiration in mitochondria isolated from acetaminophen-treated mice (1 9). Thus, early disturbances in these specific organelles (i.e., mitochondria and plasma membrane) most likely play a critical role in acetaminophen toxicity. ACKNOWLEDGMENTS Supported in part by NIH Grant GM31460 and Stauffer Chemical Company Predoctoral Fellowship in Toxicology to G. L. Ginsberg. Presented in part at the annual meeting of the Society of Toxicology, Las Vegas, Nevada, March 1983.

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17.

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