C Basic & Clinical Pharmacology & Toxicology 2004, 94, 213–225.Printed in Denmark . All rights reserved

Copyright C

ISSN 1742-7835

N-Acetylcysteine does not Protect HepG2 Cells againstAcetaminophen-Induced Apoptosis

Irena Manov1, Mark Hirsh2 and Theodore C. Iancu1

1Pediatric Research and Electron Microscopy Unit, 2Laboratory for Shock and Trauma Research, Bruce RappaportFaculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

(Received September 29, 2003; Accepted December 16, 2003)

Abstract: Acetaminophen in large doses is well-known as hepatotoxic, and early therapy with N-acetylcysteine is frequentlylife-saving. However, in later stages of acetaminophen poisoning, treatment with N-acetylcysteine is not always effective.Although some of the pathways of acetaminophen toxicity and the effect of N-acetylcysteine have been elucidated, indepth information on this process is still lacking. Hepatoma-derived HepG2 cultured cells were exposed to acetaminophen(5 and 10 mM), with or without N-acetylcysteine (5 mM), for 24 and 48 hr. For the assessment of oxidative damage,apoptosis and necrosis, we followed redox status, glutathione content, nuclear fragmentation, phosphatidylserine exter-nalization and ultrastructural changes. Variations in Ca2π level and number of mitochondrial dense granules were alsostudied. Acetaminophen treatment of HepG2 cells caused oxidative damage and apoptosis. Significant decrease of cellularredox potential and glutathione content were time- and concentration-dependent. The protective effect of N-acetylcysteinewas expressed by an increase of intracellular glutathione and of the level of metabolic reduction of the redox indicatorAlamar Blue. The apoptogenic effect of acetaminophen was assessed by flow cytometry of annexin V binding, nuclearhypodiploidity, intracellular Ca2π, as well as by ultrastructural examination. Beyond 24 hr of acetaminophen exposure,necrosis was also noticed. We conclude that acetaminophen-induced oxidative damage in HepG2 cultured cells can beprevented by exposure to N-acetylcysteine. However, apoptosis, either early or late, here demonstrated, is not avoided byexposure to N-acetylcysteine. N-Acetylcysteine did not prevent acetaminophen-induced plasma membrane asymmetry,nuclear damage, alterations of Ca2π homeostasis and ultrastructural changes.

Acetaminophen is the drug of choice as an analgesic and anti-pyretic (Prescott 2000). At therapeutic doses adverse effectsare very rare. However, after overdose, the drug has a highpotential toxicity, the most common among these being hepa-totoxicity (Zimmerman & Maddrey 1995; Prescott 2000).The mechanisms of acetaminophen-induced liver cell injuryhave been intensively investigated. The key event of acet-aminophen toxicity is its transformation into the reactivemetabolite N-acetyl-p-benzoquinone imine (NAPQI) bymicrosomal enzymes of the P450 family – CYP2E1, CYP1A2and CYP3A (Dai & Cederbaum 1995; Nelson 1995; Tonge etal.1998; Manyike et al. 2000). NAPQI is detoxified by conju-gation with glutathione (GSH). Once GSH depleted, NAPQIstimulates a wide range of oxidative reactions causing alter-ations in intracellular homeostasis that may result in cell ne-crosis (Adamson & Harman 1993; Pumford et al. 1997). Thebeneficial treatment by N-acetylcysteine, which restores in-tracellular GSH in the early stages of acetaminophen toxicity,demonstrates the importance of this mechanism (Jones1998). It has been shown that in addition to liver cell necrosis,acetaminophen can also induce apoptosis (Ray et al. 1996 &1999; Ray & Jena 2000; Ferret et al. 2001). Apoptotic celldeath has been revealed also in non-hepatic and non-metab-

Author for correspondence: Theodore C. Iancu, B. RappaportFaculty of Medicine, P.O. Box 9649, Haifa 31096, Israel (faxπ972 4 851 4285, e-mail: tiancu/tx.technion.ac.il).

olized cells (Holownia et al. 1997; Boulares et al. 2002). Itthus follows that the mechanism leading to apoptosis doesnot necessarily include oxidative reactions stimulated byNAPQI.

N-Acetylcysteine is a well-established antioxidant used inthe treatment of acute acetaminophen poisoning(Smilkstein et al. 1988; Jones 1998). However, the protectiveeffect is dependent on the time elapsed between acetamino-phen overdose and treatment, i.e. ‘‘time to N-acetylcys-teine’’ (Schmidt et al. 2002). A long time to N-acetylcysteinecan reduce its therapeutic effect (Smilkstein et al. 1988;Schmidt et al. 2002). Whether N-acetylcysteine can preventacetaminophen-induced apoptosis remains unclear. We re-cently found that N-acetylcysteine protected hepatoma-de-rived cells (Hep3B) from acetaminophen-induced oxidativeinjury, but did not prevent the development of nuclear hy-podiploidity, one of the hallmarks of apoptosis (Manov etal. 2002). We propose that an increase of intracellular GSHby N-acetylcysteine is not enough for prevention ofapoptosis. In the present work we addressed this questionusing HepG2 cells. We investigated whether N-acetylcys-teine is capable to prevent acetaminophen-inducedapoptosis or whether its protective effect is due only to itsantioxidant properties. Specifically, we focused on the ef-fects of acetaminophen and acetaminophen π N-acetylcys-teine on the cellular redox potential, plasma membrane, nu-clei, intracellular Ca2π homeostasis and mitochondria. In

IRENA MANOV ET AL.214

addition, we investigated the temporal relationship of oxi-dative damage, apoptosis and necrosis in the course of acet-aminophen overdose in vitro.

Materials and Methods

Chemicals and reagents. All chemicals, Lowry reagent, propidiumiodide and Fluo-3 acetoxymethyl ester were purchased from Sigma-Aldrich Israel Ltd (Rehovot, Israel). Alamar Blue reagent was fromSerotec Ltd (Kidlington, Oxford, UK). AnnexinV- FITC ApoptosisDetection Kit was obtained from Oncogene Research Products(Boston, MA, USA). Cell culture reagents were from ‘BiologicalIndustries’ (Kibbutz Beth HaEmek, Israel).

Cell cultures. The human hepatoblastoma cell line HepG2 wasmaintained in RPMI-1640 medium supplemented with 10% foetalcalf serum (FCS) and incubated in a humidified incubator at 37 æ in5.5% CO2. For the experiments, cells were cultured in the FCS-freemedium and treated by acetaminophen (5 or 10 mM) for 24 and 48hr. In experiments with Fluo-3 loading, time intervals of 6 and 12hr were also used. For investigation of the co-effects of acetamino-phen and N-acetylcysteine, the cells were pretreated with N-acetyl-cysteine (5 mM) for 1 hr prior to the addition of acetaminophen.After incubation, both floating and attached cells were collected foranalysis. Redox status was estimated using the Alamar Blue reduc-tion test (Nakayama et al. 1997) as previously described (Manovet al. 2002). Pretreatment by N-acetylcysteine was used to increaseintracellular GSH thus providing protection against oxidative reac-tions induced by acetaminophen within the first minutes of toxicity(Ferret et al. 2001).

Determination of intracellular glutathione (GSH). Cells were cul-tured, harvested and lysed as described (Manov et al. 2002). TotalGSH concentration was measured by the method of Tietze (1969)with the modification of Vandeputte et al. (1994). Cellular proteinwas measured by the Lowry method (1951).

Propidium iodide staining of cellular DNA. The level of DNA degra-dation was assessed by flow cytometry of propidium iodide-stainednuclei (Nicoletti et al. 1991) as previously described (Manov et al.2002). Briefly, HepG2 cells were treated by acetaminophen oracetaminophenπN-acetylcysteine, stained with hypotonic propidi-um iodide solution (propidium iodide 50 mg/ml in 0.1% sodiumcitrate plus 0.1% Triton X-100). The propidium iodide fluorescenceof individual nuclei was measured by flow cytometry (FACSCalibur,Becton Dickinson, NJ, USA).

Flow-cytometric analysis of annexin V-FITC binding. To visualizephosphatidylserine (PS) externalization on the outer leaflet of thecellular membrane during apoptosis, treated and non-treated cellswere stained with annexin V-FITC and propidium iodide accordingto the manufacturer’s instructions (Oncogene Research Products,protocol for adherent cells). AnnexinVπ/propidium iodideª cellswere defined as early apoptotic, while annexinVπ/propidiumiodideπ cells were defined as late apoptotic or necrotic.

Estimation of Ca2π by flow cytometry. The spectral properties ofFluo-3 as a Ca2π sensitive fluophore have been described by Mintaet al. (1989). HepG2 cells were cultured in 6-well plates, treatedunder different experimental conditions, detached by trypsinizationand re-suspended at a concentration 1¿106 cell/ml in Hepes buffer(137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 1mM CaCl2, 0.5 mM MgCl2, bovine serum albumin 0.5 g/l, Hepes10 mM, pH 7.4). Fluo-3 AM was added to the cells in final concen-tration of 4 mM from the stock solution, containing 2 mM Fluo-3AM and 37.5 g/l Pluronic F-127 in dimethylsulphoxide (Vanden-berghe & Ceuppens 1990). Cells were incubated for 30 min. at 37 æ,washed three times, re-suspended in Hepes buffer and incubated

for 10 min. at 37 æ before analysis. Variations of intracellular Ca2π

concentration were determined by recording the fluorescence inten-sity of intracellular Fluo-3 and percentage of fluorescent cells byflow cytometry. For each analysis 10,000 events were recorded. Noattempts were made to quantify absolute concentrations of freecytosolic calcium. Fluorescence of treated cells was compared withthat of control cells in each experiment.

Transmission electron microscopy and morphometric analysis. Con-trol and treated HepG2 cells were fixed in 2% glutaraldehyde in0.1 M sodium cacodylate buffer, pH 7.2, post-fixed in 2% OsO4,dehydrated in ethanol series and embedded in epoxy. Ultrathin sec-tions were stained with 1% uranyl acetate and 1% lead citrate, view-ed and photographed with a JEOL JEM 100 SX (Tokyo, Japan)electron microscope operated at 80 kV. Five or more 300-mesh cop-per grids from 3 blocks were examined from each sample. A mor-phometric technique was used for estimation of apoptosis and ne-crosis during acetaminophen toxicity. Counts of cells were madedirectly from the viewing screen of the electron microscope at anoriginal magnification ¿5,000. At least 600 cells from two or moreblocks were examined in each sample. Counts of mitochondrialdense granules in HepG2 cells treated and non-treated by acet-aminophen and N-acetylcysteine were performed at an originalmagnification¿10,000 enlarged to ¿100,000 through the binocular.Five hundred or more mitochondria were examined from eachsample.

Statistical analysis. All results were expressed as means∫S.D. offour to six experiments performed in triplicate. Statistical differ-ences were analyzed using Mann-Whitney U test (2-tailed); P valuesof less than 0.05 were considered significant.

Results

Acetaminophen-induced oxidative damage in HepG2 cells andits inhibition by N-acetylcysteine.

The changes in the redox status following exposure ofHepG2 cells to acetaminophen alone and acetaminophenwith N-acetylcysteine are shown in fig. 1. After 24 hr ofexposure to 5- and 10 mM acetaminophen, the cellularredox status decreased (P0.05 versus control) (fig. 1A).N-Acetylcysteine 5 mM maintained the level of oxidativemetabolism in acetaminophen-treated cells. Longer ex-posure to acetaminophen (5, 10 mM for 48 hr) inducedfurther decrease the cellular redox status (fig. 1B). N-Ace-tylcysteine treatment of HepG2 cells exposed to acetamino-phen for 48 hr increased the cellular redox levels (P0.05compared to cultures incubated with acetaminophen alone).

The intracellular GSH content in HepG2 cells treatedand non-treated by acetaminophen with or without N-ace-tylcysteine is shown in fig. 2. After 24 hr, GSH decreasedslightly at 5 mM acetaminophen (P0.05) and twice at 10mM acetaminophen (P0.05). N-Acetylcysteine almostcompletely restored the GSH concentrations of HepG2 cellstreated by 5 and 10 mM of acetaminophen (fig. 2A). Fig.2B shows that the 48 hr acetaminophen-exposure inducedfurther reduction of intracellular GSH. N-Acetylcysteineshowed a significant protective effect expressed by an 1.5-and 2-fold increase of the levels of intracellular GSH, com-pared to cells treated by acetaminophen alone (5- and 10mM respectively).

215ACETAMINOPHEN TOXICITY AND APOPTOSIS

Fig. 1. Effect of acetaminophen (AAP) and AAPπN-acetylcysteine (NAC) on the cellular redox status of HepG2 cells (Alamar Bluereduction test). Cells were cultured in 96-well plates at a density of 2¿104 cells per well. After treatment by AAP or AAPπNAC for 24 hr(A) and 48 hr (B), 20 ml of Alamar Blue were added per well and incubated at 37 æ for 3 hr. Optical density was measured spectrophotometr-ically (570 and 620 nm) by an automated ELISA plate reader. The redox status of the cells was calculated as percent of the differencebetween reduction of Alamar Blue in treated versus non-treated cells. Results presented as % of controls. Mean∫S.D. *P0.05 compared tocontrols; .P0.05 comparison between groups treated by 5 and 10 mM of AAP for the same time period; $P0.05 comparison betweengroups treated by AAP only and AAPπNAC. NΩ10.

Acetaminophen-induced apoptosis and necrosis in HepG2cells and the effect of N-acetylcysteine.

The effect of acetaminophen on the number of hypodiploidnuclei was both time- and dose-dependent (fig. 3A). During

Fig. 2. GSH concentrations in HepG2 cells treated and non-treated by acetaminophen (AAP), with or without N-acetylcysteine (NAC),after 24 hr (A) and 48 hr (B). Results are expressed in mM of GSH per mg of protein. Mean∫S.D. *P0.05 compared with controls; .P0.05comparison between groups treated by 5 and 10 mM of AAP for the same time period; $P0.05 comparison between groups treated byAAP only and AAPπNAC. NΩ6.

the first 24 hr of acetaminophen-treatment, the percentage ofhypodiploid nuclei increased 1.8-fold at 5 mM acetamino-phen and 2.6-fold at 10 mM acetaminophen in comparisonwith control (P0.05). Forty-eight hr of acetaminophen-ex-posure resulted in a further increase in hypodiploid nuclei: 2-

IRENA MANOV ET AL.216

fold at 5 mM and 3-fold at 10 mM (P0.05). The incidenceof hypodiploid nuclei in 24 hr cultures treated withacetaminophenπN-acetylcysteine was not distinct fromthose exposed to 10 mM acetaminophen alone (data notshown). Likewise, the 48 hr exposure to acetaminophenπN-acetylcysteine did not prevent the increase in nuclear hypodi-ploidity (fig. 3B). When used alone, N-acetylcysteine did notenhance the percentage of hypodiploid nuclei.

We assessed PS externalization following exposure of the

Fig. 3. (A) Acetaminophen (AAP) induces increase of hypodiploidnuclei in the cultures of HepG2 cells (time and dose-dependency).After 24 and 48 hr of AAP exposure, the cells were stained by propi-dium iodide and fluorescence of individual nuclei was measured byflow cytometry. For each analysis 10,000 events were recorded. Datais presented as percentage of apoptotic (hypodiploid) nuclei.Mean∫S.D. *P0.05 compared with the controls; .P0.05 com-parison between the groups treated with different AAP concen-trations for the same time period; $P0.05 comparison betweengroups treated for 24 and 48 hr with the same AAP concentration.NΩ6. (B) Effect of N-acetylcysteine (NAC) on the frequency ofapoptotic nuclei of HepG2 cells exposed or not to AAP for 48 hr.The percentages of hypodiploid nuclei (sub G0-G1 DNA content)were determined as described in (A). Values are from one represen-tative experiment. This experiment was repeated three times withsimilar results.

cells to acetaminophen or acetaminophenπN-acetylcysteineby binding of annexin V and uptake of propidium iodide (fig.4). After 24 hr, the percentage of viable cells (annexinVª/pro-pidium iodideª) decreased from 75.1∫4.5% (non-treatedcells) to 63.5∫1.9% at 5 mM acetaminophen (PΩ0.05) and to53.9∫3.1% at 10 mM acetaminophen (P0.05) (fig. 4A). Atthe same time point the number of early apoptotic cells(annexinVπ/propidium iodideª) increased from 6.47∫2.2%to 8.5∫1.1% and 14.8∫4.1% at acetaminophen 5- and 10mM respectively (P0.05). Further 24 h of acetaminophenexposure resulted in (i) an increase of early apoptotic cellsfrom 8.4∫0.9% (control cells) to 20.3∫2.7% and 29.5∫3.8%at 5 and 10 mM respectively (P0.05); (ii) marked increase inthe level of late apoptoticπnecrotic cells (annexin Vπ/propi-dium iodideπ) from 19.9∫3.7% to 35.5∫1.4% (5 mM,P0.05) and to 50.0∫3.6% (10 mM, P0.05); (iii) significantdecrease in the percentage of viable cells, from 61.9∫4.6 to35.5∫4.0 and 15.5∫1.4 at 5- and 10 mM acetaminophen re-spectively (P0.05) (fig. 4B). N-Acetylcysteine, when addedto acetaminophen (10 mM), did not prevent the acetamino-phen-induced increase in apoptotic and necrotic cells. N-Acetylcysteine alone did not produce significant changes inphosphatidylserine externalization.

The levels of intracellular Ca2π were measured by flowcytometry of Fluo-3 loaded cells. The mean fluorescenceintensity and percentage of Fluo-3 positive cells were com-bined to an index (Dyugovskaya et al. 2002) and used as asurrogate marker reflecting the level of intracellular ionizedCa. As displayed in fig. 5, the level of intracellular Ca2π inHepG2 cells exposed to 10 mM acetaminophen for 12 hrincreased to 137.0∫7.5% of the control level (P0.05).Thereafter, the concentration of Ca2π decreased and after48 hr was only 52.2∫7.4% of the control value (P0.05).At 5 mM acetaminophen (data not shown) the level of in-tracellular Ca2π increased slightly to 106.0∫6.9% at 12 hr.After 24 hr of 5 mM acetaminophen treatment, a maximalshift of Fluo-3 fluorescence index was observed(142.2∫10.4%, P0.05). Subsequently, the Ca2π level de-creased and after 48 hr was 87.7∫5.0% of control (P0.05).No significant differences were found when the cells wereexposed to acetaminophen 5- and 10 mMπN-acetylcys-teine. Treatment with N-acetylcysteine alone did not pro-duce significant changes in Ca2π homeostasis.

Transmission electron microscopy. Control cells were oc-casionally found to form rosettes. Slender microvilli wereseen on the surface and within pseudo-lumens (fig. 6A andB). Mitochondria showed a normal appearance, includingregular cristae and mitochondrial dense granules. Othercytoplasmic organelles (peroxisomes, lysosomes) and glyco-gen particles were scarce. The nuclei, although irregular inshape as is the case with most malignant cells, displayednormally distributed heterochromatin. Some cells con-tained a few non-membrane-bound lipid droplets, similarto triglyceride droplets seen in hepatocytes.

During the time course of acetaminophen toxicity a num-ber of cell types were observed: 1) normal cells, similar to

217ACETAMINOPHEN TOXICITY AND APOPTOSIS

Fig. 4. Plasma membrane changes in HepG2 cells during acetaminophen (AAP) exposure and effect of N-acetylcysteine (NAC) (annexin V-FITC binding assay). Changes in plasma membrane as an indication of the PS externalization were determined by flow cytometry after 24hr (A) and 48 hr (B) of treatment. Cells were incubated with annexin V-FITC and propidium iodide as described in Materials and Methods.The number of viable (annexinVª/propidium iodideª), early apoptotic (annexinVπ/propidium iodideª) and late apoptotic or necrotic (an-nexin Vπ/propidium iodideπ) cells, was determined and expressed as percentage of the total cell number (100%) Values are presented asmean∫S.D. NΩ5.

controls; 2) cells with discrete abnormalities including reduc-tion or absence of surface microvilli and of pseudo-lumens,as well as increased frequency of lipid droplets; 3) cells withmorphological changes characteristic of apoptosis, i.e.chromatin condensation and margination (fig. 7A), pro-nounced nuclear lobulation and nuclear fragmentation withformation of apoptotic bodies typical of advanced stages ofapoptosis (fig. 7B); shrinking of the cytoplasm and absenceof microvilli were conspicuous in these cells; 4) cells exhibit-ing features typical of necrosis, such as extensive degenera-tion of the cytoplasmic structure, vacuolization and disrup-

Fig. 5. Alterations of intracellular Ca2π in HepG2 cells incubatedwith acetaminophen (AAP) and effect of N-acetylcysteine (NAC).After 6, 12, 24 and 48 hr of treatment cells were loaded by Fluo-3.Mean fluorescence intensity and percentage of Fluo-3 positive cellswere measured by flow cytometry. Data presented as Fluo-3 fluor-escence index (mean fluorescence intensity¿% of Fluo-3 positivecells/100) and expressed as % of control. $P0.05 in comparisonwith controls. NΩ6.

tion of the plasma membrane (fig. 7B); and 5) so-called ‘‘sec-ondary necrotic’’ cells with morphological elements of bothapoptosis and necrosis (fig. 7C).

We used a morphometrical technique for evaluation ofthe degree of apoptosis and necrosis in HepG2 cells duringthe time course of treatment with acetaminophen andacetaminophenπN-acetylcysteine. After exposure for 24 hrto10 mM acetaminophen, 70.07∫3.65% of cells were intactshowing ultrastructural features of normal cells, similar tocontrol. The percentage of typical apoptotic cells increasedto 17.96∫2.09%, in comparison with non-treated cells(4.67∫0.28%, P0.05) (fig. 8A). When cells were exposedto acetaminophen plus N-acetylcysteine for 24 hr, no pro-tective effect was noticed, the changes and the level ofapoptosis being similar to those observed after exposure toacetaminophen only (20.4∫0.92%) (fig. 8B). With continu-ing acetaminophen exposure (48 hr), more cells exhibitedtypical morphology of necrosis (22.42∫0.08%). Exposureto acetaminophenπN-acetylcysteine for 48 hr resulted in asignificant increase in the degree of necrosis (36.69∫1.14%)and secondary necrosis (12.7∫2.76%) in comparison withcells treated by acetaminophen only (P0.05). N-Acetylcys-teine alone did not induce apoptotic or necrotic changes inHepG2 cells. The above changes and results of counting aresummarized in Table 1 and illustrated in fig. 8A and B.

We noticed variability in the frequency of mitochondrialdense granules in HepG2 cells exposed or not to acetamino-phen and acetaminophenπN-acetylcysteine (fig. 9). In con-trol cells most mitochondria contained matrical granules(table 2 and fig. 9A). The quantitative analysis revealed thatthe proportion of mitochondrial dense granules-positivemitochondria in non-treated cells was 60.4∫2.7%. Incuba-tion with acetaminophen 10 mM for 24 hr resulted in anincrease of mitochondrial dense granules-containing mito-

IRENA MANOV ET AL.218

chondria to 69.0∫4.2% (P0.05, compared to control).After 48 hr of acetaminophen treatment, a reduction inmitochondrial dense granules was found (table 2 and fig.9B): the percentage of mitochondrial dense granules-posi-tive mitochondria decreased to 29.5∫8.7% with acetamino-phen alone and 28.1∫2.2% with acetaminophenπN-acetyl-cysteine (control 62.5∫3.0%, P0.05). Exposure to N-ace-tylcysteine alone did not change the frequency ofmitochondrial dense granules-containing mitochondria innon-treated cells (table 2 and fig. 9C).

Discussion

We here report our investigation of the apoptotic changesinduced by acetaminophen in HepG2 cells and the effect ofN-acetylcysteine. As recently demonstrated, the HepG2 cellline constitutively express CYP2E1, CYP1A2 and CYP3A4,all involved in acetaminophen metabolism (Sumida et al.2000). The acetaminophen concentrations (5 and 10 mM)used in this study have been previously tested in cell culturesystems and revealed acetaminophen-induced cytotoxicity(Shear et al. 1995; Nicod et al. 1997; Bruno et al. 1998;

Fig. 6. Transmission electron microscopy. Electron micrograph of control HepG2 cells showing normal appearance. (A) Rosette formation.Note the presence of numerous microvilli both on the outer surface of the cells and towards the central pseudo-lumen [PL]. Some translucentlipid droplets are also evident. (B) Higher magnification showing details of the fine structure including the presence of normal cytoplasmaticorganelles (mitochondria, rough endoplasmic reticulum). Microvilli are present both on the cell surface and in a pseudo-lumen formedadjacent to another cell. Original magnification (A) ¿2,500; (B) ¿5,000.

Boulares et al. 2002; Manov et al. 2002). In patients, acet-aminophen plasma concentrations associated with hepato-toxicity are 0.5–2.0 mM (according to the nomogram de-veloped by Rumack et al. 1981). The differences betweenthe toxic doses of acetaminophen in vitro and in vivo maybe due not only to the decreased activity of the metabolizingsystems in liver-derived cell lines (Testai 2001): acetamino-phen has been shown to induce toxicity also in an unmetab-olized form, in non-hepatic cells such as HL-60 (Wiger etal. 1997); PC-12 (Holownia et al. 1997) and Jurkat T cells(Boulares et al. 2002). The relative resistance of tumor-de-rived liver cells and immortalized hepatocytes to acetamino-phen (McCloskey et al. 1997) and other compounds, maybe associated with differences in the drug transport path-way and intensity of its efflux (Payen et al. 2000). Xenobiot-ic efflux is mediated by several membrane proteins such asP-glycoprotein and multidrug resistance protein subfamily(LeBlanc 1994; Payen et al. 2000). It is well established thattumor cells frequently overexpress P-glycoprotein (Krish-na & Mayer 2000). Recent studies suggest that the levels ofexpression of multidrug resistance protein, low in normalhepatocytes, are markedly increased in tumour-derived

219ACETAMINOPHEN TOXICITY AND APOPTOSIS

Fig. 7. Various stages of cell injury following exposure to acet-aminophen (AAP). (A) A typical apoptotic cell displays conden-sation, margination and clumping of heterochromatin (arrow).Remnants of necrotic cells are also visible (curved arrow). (B) An-other apoptotic cell displays absence of microvilli, nuclear fragmen-tation and numerous lipid droplets. Adjacent to this cell, an apop-totic body can be seen (arrow) as well as parts of a necrotic cell(curved arrow). (C) The nucleus of this cell shows marginatedchromatin (arrow) and granular nucleoplasm. The remnants of thecytoplasm are vacuolated and normal organelles have been lost(secondary necrosis). Original magnification (A) ¿5,000; (B)¿4,000; (C) ¿5,000.

IRENA MANOV ET AL.220

(HepG2) and immortalized hepatocytes (Roelofsen et al.1997; Schippers et al. 1997). In our recent experiments, pre-treatment of HepG2 and Hep3B cells with the P-glyco-protein inhibitor verapamil, resulted in a significant increaseof acetaminophen-induced toxicity (unpublished data).Therefore, using a higher concentration of acetaminophento induce in vitro toxic effects comparable to the situationin vivo, may be justified.

We found that acetaminophen treatment of HepG2 cellsresulted in a significant decrease of cellular redox potentialand GSH content in a time- and concentration-dependentmanner. It is commonly considered that cellular redox,which is largely determined by the ratio between oxidizedand reduced GSH, plays a significant role in cell survivaland death (Hammond et al. 2001). Low GSH levels are fre-quently associated with mitochondrial dysfunction and mayactivate apoptosis (Coppola & Ghibelli 2000). However, ithas been also reported that intracellular GSH depletion isnot sufficient to generate apoptosis (Lepri et al. 2000).

Significant increase in the frequency of hypodiploid nu-clei was noticed after acetaminophen exposure. AlthoughDNA cleavage is a hallmark of apoptosis, it can be alsofound during necrotic cell death (Dong et al. 1997). Caspa-se-3 activation is considered a pivotal point in the escalationof events leading to apoptosis (Cohen 1997). Lawson et al.(1999) observed that after intraperitoneal injection of 500

Fig. 8. (A): This electron micrograph illustrates the abundance of apoptotic cells (arrows) following exposure to 10 mM acetaminophen(AAP) for 24 hr; (B) The addition of N-acetylcysteine (NAC) 5 mM did not decrease the incidence of apoptotic cells (arrow) induced byAAP. Original magnification (A) ¿2,000; (B) ¿2,500.

mg/kg acetaminophen, DNA fragmentation of mouse livercells does not require activation of caspase-3 and thereforedirect activation of endonucleases is leading to necrosis.However, the caspase-3 activity has been determined onlyup to 6 hr after acetaminophen injection (Lawson et al.1999). Using a similar experimental model, Ferret et al.(2001) found a two-fold and four-fold increase in caspase-3activity in liver extracts from mice 24 hr after acetamino-phen 500 mg/kg and 1000 mg/kg treatment respectively.Likewise, maximal activity of caspases-3 was detected 24 hrafter 10 mM acetaminophen treatment of SK-Hep1 cells(Boulares et al. 2002).

Additional signs of apoptosis following acetaminophenoverdose have been detected both in vivo and in vitro: dis-ruption of mitochondrial integrity and cytochrome c release(Ferret et al. 2001; Boulares et al. 2002), modulation of theexpression of Bcl-XL and Bcl-XS proteins (Ray et al. 1999;Ray & Jena 2000; Boulares et al. 2002) and disturbances ofintracellular Ca2π homeostasis (Ray et al. 1993). Morpho-logical changes associated with apoptosis such as cellshrinkage, chromatin condensation and margination andapoptotic bodies were also observed in the liver after highdoses of acetaminophen (Ray et al. 1999; Ray & Jena 2000;Gujral et al. 2002) Quantitative analysis performed by Rayet al. (1996 & 1999) on mouse liver indicated that 40% ofcells died by apoptosis 24 hr after acetaminophen adminis-

221ACETAMINOPHEN TOXICITY AND APOPTOSIS

Fig. 9. Mitochondrial dense granules (MDG). (A) Control cells dis-play frequent electron-dense (osmiophilic) matrical granules in mostmitochondria, as illustrated in part of two HepG2 cells (curved ar-rows). (B) After 48 hr exposure of the cells to 10 mM acetamino-phen (AAP), most mitochondria are devoid of MDG as seen in partof this HepG2 cell. The large and numerous lipid droplets [L] areconsistent with the changes seen in this stage of damage. (C) Cellsexposed to N-acetylcysteine (NAC) (48 hr) without the presence ofAAP, show normal ultrastructural appearance. Note prominent,large MDG (curved arrows), normal rough endoplasmic reticulumand a well-developed Golgi complex (large empty arrow). Originalmagnification (A, B, C) ¿15,000.

IRENA MANOV ET AL.222

Table 1.

Effect of acetaminophen (AAP), N-acetylcysteine (NAC) and AAPπNAC on the level of apoptosis and necrosis in HepG2 cells (quantitativeelectron microscopy).

24 hr 48 hr

Secondary SecondaryTreatment Normal Apoptosis Necrosis necrosis Normal Apoptosis Necrosis necrosis

Control cells 94.07∫0.35 4.67∫0.28 1.27∫0.08 – 85.82∫0.49 5.20∫0.63 8.46∫1.08 0.54∫0.04AAP 10 mM 70.07∫3.65* 17.96∫2.09* 5.96∫1.21 6.02∫0.4 58.56∫1.96* 11.56∫1.85* 22.42∫0.08* 7.47∫0.03*NAC 5 mM 95.59∫0.21 2.34∫0.28 1.73∫0.58 0.36∫0.51 92.34∫1.0 5.45∫0.91 1.94∫0.48 0.27∫0.38AAPπNAC 74.11∫2.81* 20.4∫0.92* 4.5∫1.43 1.0∫0.46 42.52∫2.95* 5.11∫1.35 36.69∫1.14. 12.7∫2.76.

Note. Data is presented as percentage of cells (means∫S.D.). No less than 600 cells from two or more blocks were examined in each sample.*P0.05 (compared to control for the same time period)..P0.05 (comparison between the groups treated by AAP and AAPπNAC for the same time period).

tration. In contrast, Gujral et al. (2002) showed that ne-crosis, but not apoptosis, is the predominant mechanismof cell death after acetaminophen overdose in vivo. Sinceapoptotic cells in vivo are rapidly removed from the tissueby phagocytes, this data may be an underestimate. Accord-ing to our in vitro findings, both apoptosis and necrosis areobserved in HepG2 cells after acetaminophen treatment. Todifferentiate apoptotic from necrotic cells, we used ultra-structural morphometry and cell staining with annexin Vand propidium iodide. We found that after 24 hr of acet-aminophen-exposure, HepG2 cells died predominantly byapoptosis (table 1 and fig. 8A), while necrosis was promi-nent after 48 hr. In this late stages of toxicity the apoptoticprocess may switch to ‘‘secondary necrosis’’ and apoptoticcells may become indistinguishable from necrotic ones (Lev-in et al. 1999). Additionally, apoptosis in vitro, in the ab-sence of phagocytes, may normally degenerate to ‘‘second-ary necrosis’’ (Gomez-Lechon et al. 2002). Secondary ne-crosis or incomplete apoptosis has been suggested to occurin acetaminophen-treated HL-60 cells (Wiger et al. 1977).

We also investigated the role of intracellular calcium sig-nals in cell death induced by acetaminophen. The rise ofCa2π in the cytosol and its subsequent mitochondrial up-take may contribute to the opening of mitochondrial per-meability transition pores, which depolarize mitochondriaand cause release of pro-apoptotic factors (Kroemer &Reed 2000; Yu et al. 2001). On the other hand, decrease ofCa2π concentration has been observed in apoptotic myeloid

Table 2.

Quantitative analysis of mitochondrial dense granules in HepG2cells exposed to 10 mM acetaminophen (AAP) with or without 5mM N-acetylcysteine (NAC) (transmission electron microscopy).

24 hr 48 hr

Control cells 60.48∫2.71 62.51∫3.04AAP 10 mM 69.05∫4.18 29.49∫8.67*NAC 5 mM 62.87∫5.79 56.72∫4.48AAPπNAC 48.24∫3.59 28.06∫2.23*

Note. Data is presented as % of mitochondrial dense granules(mean∫S.D.). No less than 500 mitochondria from two or moreblocks were examined in each sample.*P0.05 compared with controls.

cells (Gallo et al. 1987) and neurones (Yu et al. 2001). Inour experiments, the level of intracellular Ca2π, evaluatedas an index of Fluo-3 fluorescence, increased significantlyafter 12 hr of 10 mM acetaminophen exposure. Further on,the concentration of Ca2π decreased and after 48 hr was 2-fold lower then the control level, suggesting that during thetime-course of the apoptotic cascade an increase in intra-cellular Ca2π may be followed by its depletion. This findingis in agreement with the concept presented by Yu et al.(2001) illustrating the relationship between Ca2π levels andapoptosis.

Based on our previous experience (Manov et al. 2002),we extended our ultrastructural investigation to the HepG2cells used in this experimental design and focused onchanges in mitochondrial ultrastructure. No abnormalitieswere noticed in shape, size, conformation, cristae or matrixof mitochondria in control cells. Their mitochondrial densegranules, conspicuous in controls, showed variations afterexposure to acetaminophen and acetaminophen plus N-ace-tylcysteine. It is generally accepted that mitochondrialdense granules are intramitochondrial calcium-containingdeposits and may vary in number and size during the shiftor loss of cytoplasmic Ca2π (Saetersdal et al. 1977; Wrob-lewski et al. 1991; Ghadially 1997). Disappearance of mito-chondrial dense granules has been included among featuresof reversible changes in acute hepatoxicity (Desmet & Vos1984). However, their absence can be observed in a numberof other conditions (Ghadially 1997) including excessiveproliferation of abnormal mitochondria (Mandel et al.2001). The time-point comparison between the level of in-tracellular Ca2π and the frequency of mitochondrial densegranules in acetaminophen-exposed HepG2 cells, suggestsa role of mitochondria as a buffering system for regulationof the local Ca2π content (Duchen 2000). While the levelof intracellular Ca2π, following initial rise, decreased after24 hr of exposure to acetaminophen 10 mM, the number ofmitochondrial dense granules-positive mitochondria in-creased. After a longer incubation with acetaminophen (48hr), a significant reduction of mitochondrial dense granuleswas found. Fujimura et al. (1995) obtained similar resultswith isolated rat hepatocytes exposed to acetaminophendemonstrating loss of mitochondrial dense granules by elec-

223ACETAMINOPHEN TOXICITY AND APOPTOSIS

tron microscopy. The role of mitochondrial dense granules,their relationship with calcium metabolism and the effectsof xenobiotics, awaits further clarification.

The study of the influence of 5 mM N-acetylcysteine onacetaminophen-exposed HepG2 cells revealed its protectiveeffect against oxidative damage. A concentration of N-ace-tylcysteine (5 mM) was used earlier for in vitro investigationof its effect against oxidative injury and apoptosis (Shen etal. 1999 & 2000; Manov et al. 2002). We reported that N-acetylcysteine did not protect Hep3B cells from the acet-aminophen-induced nuclear hypodiploidity (Manov et al.2002). The present report provides further evidence that N-acetylcysteine did not prevent acetaminophen-induced fluc-tuations of intracellular Ca2π, externalization of PS andemergence of cells with typical features of apoptosis. Thefact that N-acetylcysteine protects HepG2 cells from acet-aminophen-induced oxidative damage but not fromapoptosis may be explained by the following observations:1. GSH depletion is not sufficient to produce apoptosis andconversely, its supplementation does not exert protection(Lepri et al. 2000). Furthermore, the apoptotic cascade maybe disabled when GSH is depleted and, in contrast,apoptosis may be promoted when GSH is intact (Hentze etal. 2003). Recently we reported that N-acetylcysteine in-creased nuclear hypodiploidity when added to acetamino-phen-exposed Hep3B cells (Manov et al. 2002). It is likelythat GSH levels restored by N-acetylcysteine may promoteacetaminophen-induced apoptosis in our in vitro test-sys-tems; 2. We and others have shown that the antioxidanteffect of N-acetylcysteine is not related to alterations of cal-cium homeostasis (Villagrasa et al. 1997; Xie et al. 1999).It is well established that the change of intracellular Ca2π

is one of the main events leading to apoptosis induced bydifferent stimuli (Kroemer & Reed 2000; Yu et al. 2001) andby acetaminophen in particular (Ray et al. 1993; Ray &Jena 2000).

The beneficial in vivo effect of N-acetylcysteine in earlyacetaminophen poisoning was significantly reduced whenN-acetylcysteine treatment started after 16 hr from the acet-aminophen overdose (Jones 1998; Smilkstein et al. 1988;Schmidt at al. 2002). In a study on mice, Ferret et al. (2001)found that N-acetylcysteine protected against acetamino-phen-induced acute liver failure when administered preven-tively, but not curatively. From these findings we can specu-late that N-acetylcysteine exerts protection against acet-aminophen toxicity only within the early reversible phase ofexposure when acetaminophen conversion to NAPQI andoxidative damage take place. When intracellular damagereached apoptosis and/or necrosis, the effect of N-acetylcys-teine decreases.

In summary, we found that after acetaminophen exposure,HepG2 cells undergo several sequences associated withapoptosis, including: 1) phosphatidylserine externalization;2) increase of hypodiploid nuclei; 3) rise of intracellular Ca2π

in initial stages of acetaminophen treatment followed by itssubsequent depletion; 4) increased frequency of cells withtypical apoptotic morphology and 5) disappearance of mito-

chondrial dense granules in the advanced stages of acet-aminophen toxicity. The study also revealed the protective ef-fect of N-acetylcysteine against oxidative damage. However,in our experimental condition, apoptosis, either early or late,is not avoided by exposure to N-acetylcysteine.

AcknowledgementsThis work was supported in part by the Milman Fund

for Pediatric Research and the Dan David Foundation. Dr.Manov was supported by the Kamea Fund. The authorswish to thank J. Regev and R. Hirshberg for their contri-bution with the photographic work.

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