inducers of cytochrome p450 2e1 enhance methotrexate-induced hepatocytotoxicity

Inducers of Cytochrome P450 2E1 EnhanceMethotrexate-Induced Hepatocytotoxicity

MANUELA G. NEUMAN,1 ROSS G. CAMERON,2 JULIA A. HABER,1 GADY G. KATZ,1

IZABELLA M. MALKIEWICZ,1 and NEIL H. SHEAR1

1Division of Clinical Pharmacology, Sunnybrook and Women’s College Health Sciences Centre,2Department of Pathology, Toronto Hospital, Departments of Pharmacology, Pathology and

Medicine, University of Toronto, Toronto, Ontario, Canada

Objectives: To study the effect of cytochrome P450 2E1-inducerson methotrexate (MTX)-induced cytotoxicity in human hepatocytes,and investigate the role of silymarin in preventing this toxicity.Design and methods: Cells were exposed to MTX in the presenceof either ethanol (EtOH) or acetaminophen (APAP), or either com-bined with silymarin (S). Apoptosis and necrosis were measured byanalyzing 6000 cells/sample using transmission electron micros-copy, while cytokine release and apoptosis were quantitated byELISA. Cytokine expression was measured by RT-PCR. Gluthatione(GSH) content was determined in cytosolic (c) and mitochondrial (m)fractions.Results: MTX1EtOH and MTX1APAP increased MTX cytotoxicity2.9-fold and 1.9-fold, respectively. S abolished this toxicity. MTX 1EtOH increased the release of IL 6, IL 8 and TNF a by 1.0, 1.2, and1.1 times, respectively. Cytokine expression was upregulated ver-sus control for IL 6 (22%), IL 8 (38%), and TNF a (29%). Addition of0.5 mmol/L S downregulated TNF a expression and reducedcytokine release. TNF a increased cytotoxicity by 22%, whileanti-TNFa antibody eradicated it. MTX1EtOH depleted 45% mGSH(p , 0.001) while S replenished it to 87% (p , 0.001), when bothwere compared to control levels.Conclusions: Cytochrome P450 2E1-inducers contribute to in-crease oxidative stress in MTX-exposed cells by increasing TNF aand depleting both cGSH and mGSH. This enhances MTX-cytotox-icity and promotes apoptosis. Copyright © 1999 The CanadianSociety of Clinical Chemists

KEY WORDS: methotrexate; cytochrome P450 2E1;apoptosis; necrosis; acetaminophen; ethanol; Hep G2; nor-mal human primary hepatocytes.

Introduction

Methotrexate, or aminopterin, is an antagonist offolic acid used to treat psoriasis, psoriatic and

rheumatoid arthritis, leukemia, and various otherconditions. Its use as a chemotherapeutic agentbegan in the late 1940s (1), and in 1951 it wasreported as a useful treatment for psoriasis (2).

Methotrexate (MTX), a weak bicarboxylic organicacid, is transported into cells by reduced folatecarriers (3), higher levels of which are found inpsoriatic plaques than in normal skin (4). Bybinding to dihydrofolate reductase with greateraffinity than folic acid, MTX limits the conversionof folic acid to tetrahydrofolate, a molecule neces-sary for the synthesis of DNA during S-phase(5,6). MTXs inhibition of the synthesis of purineand pyrimidine thymidylate results in a de-oxynucleotide pool imbalance, which could explainthe improper DNA synthesis and subsequent ap-optosis of cells (6). However, due to its lack ofspecificity toward malignant cells, MTX can alsocause liver hepatotoxicity, including steatosis,cholestasis, fibrosis, and cirrhosis (7,8).

The conversion of MTX to its major extracellularmetabolite, 7-hydroxymethotrexate, takes place inthe liver, where it is oxidized by a soluble enzymaticsystem (9). Inside cells, MTX is stored in polygluta-mated form (10). Long-term drug administrationcan cause accumulation of MTX polyglutamates anddecrease folate levels (11,12). Galivan et al. (10)found an in vitro reciprocal relationship betweenfolyl and MTX polyglutamate accumulation in H35hepatoma cells. The presence of higher levels ofpolyglutamates causes a longer intracellular pres-ence of the drug, and has been suggested as amechanism for MTXs hepatotoxicity (11,12). Newerinvestigations into the mechanism of MTX-inducedliver damage focus on apoptosis and pro-apoptoticgenes and factors.

Apoptosis is programmed cell death beginningwith chromatin condensation and DNA fragmen-tation. Apoptotic bodies with intact membranesare phagocytosed by macrophages, allowing un-needed cells to be eliminated without provokingan inflammatory response. Apoptosis can be pro-moted in a number of ways; one suggested mech-anism is the interaction between Fas (CD95/APO-1) and Fas-ligand (13–15). P53, a gene which

Correspondence: M. G. Neuman, Ph.D., Division Clini-cal Pharmacology, E-240, Sunnybrook HSC, 2075 Bay-view Avenue, Toronto, Ontario, M4N 3M5, Canada. E-mail: [email protected].

Manuscript received July 20, 1999; accepted July 20,1999.

Clinical Biochemistry, Vol. 32, No. 7, 519–536, 1999Copyright © 1999 The Canadian Society of Clinical Chemists

Printed in the USA. All rights reserved0009-9120/99/$–see front matter

PII S0009-9120(99)00052-1

CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999 519

can sense DNA damage and cytogenic stress, reg-ulates apoptosis and other factors involved inapoptosis, including Fas, Bax, Bcl-2, and TGF-a(16). When wild type p53 is present, MTX is able toupregulate Fas receptors in vitro, up to 70% ofHepG2 cells (14,15).

Caspase activity has also been linked to apopto-sis. Los et al. (17) report that the activation ofcaspases (ICE/Ced-3 proteases) is needed forMTX-induced apoptosis. The addition of a caspaseinhibitor in vitro will inhibit MTX-induced apopto-sis (17).

In vivo reports of hepatotoxicity from MTX arewell-documented (7,18,19). Histologic abnormalitiescan include binucleated cells, anisonucleosis and cellloss thought to be related to folic acid depletion (20).Morphologic changes can include dilatations ofsmooth endoplasmic reticulum (ER), proliferation ofmicrovesicular ER, mitochondrial changes and thepresence of scattered apoptotic bodies (21). A meta-analysis of rheumatoid and psoriatic arthritis pa-tients taking MTX found that the incidence of pro-gression of liver abnormality by at least onehistologic grade was 27.9% (18). The chance ofworsening by at least one grade was higher forpsoriatic patients (7.7%) than for rheumatoid arthri-tis patients (2.7%), the putative reason being thedifference in dosing (18,22,23). Kevat et al. (22) alsosuggest that increased hepatotoxicity is associatedwith smaller, more frequent dosing as opposed to alarger weekly dose, and that length of therapycorrelates with hepatotoxicity.

Along with large cumulative dose and highdosing frequency, there are several other riskfactors for MTX-induced hepatotoxicity: increasedage, obesity, diabetes mellitus, concomitant med-ications and especially alcohol use (18,20,24).Neuman et al. (25) and Landau et al. (26) showedthat in vitro, EtOH increased MTX-induced toxic-ity when added to HepG2 cells. Several studiesagree that alcohol consumption aggravates liverdamage due to MTX (18,22), while Malatjalian etal. (27) found that occasional alcohol consumption(# 3 drinks/week) did not significantly correlatewith MTX-induced liver damage. Neuman et al.(21) have suggested a mechanism for the in-creased hepatotoxicity resulting from a combina-tion of MTX and EtOH. They propose that oxida-tive stress produced by EtOH lowers theconcentration of MTX needed to produce damage,and that EtOH also lowers the level of GSH, animportant detoxification molecule.

In 1977, Nyfors et al. (28) reported that theincidence of fibrosis and cirrhosis in psoriasis pa-tients taking MTX showed a marked and rapidincrease after a cumulative dose of 2–4 g, andsuggested that liver biopsy was necessary for treat-ment beyond this range. Guidelines now dictate thatbiopsy should be performed at baseline and afterevery 1.5 g cumulative dose of MTX (27). Liverbiopsy remains the most reliable method of diagnos-ing liver abnormality, but there is still debate con-

cerning the use of enzyme levels as an indicator ofdamage. Some reports show that the levels of ala-nine aminotransferase and aspartate aminotrans-ferase do not always correlate with the grade of liverhistology (23,29), while other reports found thatserial enzyme abnormalities agreed with degree ofdamage (30,31). In addition, Jaskiewicz et al. (32)propose that increased deposition of laminin, fi-bronectin and collagen III, IV correlates with cumu-lative dose of MTX. In our investigation, we proposethat the presence of proinflammatory cytokines canserve as a possible marker for monitoring MTX-induced liver damage.

MTX-induced hepatotoxicity appears to be a con-sequence of the interaction of many factors: dosingschedule and length of treatment, patient risk fac-tors, type of disease, and presence of genetic andmolecular apoptotic factors. Our investigation at-tempts to measure the cytotoxic effect of MTX onhepatocytes, using the in vitro model of HepG2 cellsand normal human primary hepatocytes (NHPH)developed by Neuman et al. (33) and Neuman andTiribelli (34). We used human cell lines in order toavoid inter-species differences in gene products, andchose in vitro methods to avoid the individual differ-ences (gender, race, environment) which complicatein vivo studies. We examined the modulatory effectof cytochrome P450 2E1 inducers EtOH and APAPon MTX-induced cell damage by measuring thepresence of cytokines and level of cytotoxicity andapoptosis, as well as GSH content. Using light andelectron microscopy, we looked at changes in cellmorphology.

In addition, we studied the role of Disulfiram(DS), a 2E1 inhibitor, and Silymarin (S), a naturalantioxidant, in preventing MTX cytotoxicity. S is astandardized bioflavenoid extracted from the seedsof the thistle Silybium marianum (Gartner) or Car-duus marianus (L). S has 4 isomeric components:silybinin, iso-silybinin, silydianin and silychristin.Eichler and Hahn (35) first reported the hepatopro-tective effect of milk-thistle extract when used pro-phylactically in experimental carbon tetrachlorideand trinitrotoluene poisoning. Since then, manystudies have reported S-induced hepatoprotectiveeffects in vivo and in vitro (36–39). In 1995, Shear etal. (40) showed that S reduced APAP-induced toxic-ity in HepG2 cells and in epidermoid A431 cells, invitro, which suggests that the mechanism of Silyma-rin’s cellular protection is the enhancement of intra-cellular GSH, a phenomenon which occurs in themitochondrion.

In its reduced form, GSH is necessary for thedetoxification of xenobiotics. The enzyme g-glu-tamylcysteine synthetase catalyzes the first step inthe production of GSH, and is inhibited by L-buthi-onine-(S,R)-sulfoximine (BSO). Its release from cellsis a sensitive marker for monitoring mitochondrialintegrity, which could be correlated with histologicalabnormalities (41,42).

NEUMAN ET AL.

520 CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999

Materials and methods

DRUGS AND CHEMICALS

EtOH was purchased from Alcohol Ltd. (Toronto,ON, Canada). Plain a-MEM, Hanks balanced so-dium solution and calcium chloride were obtainedfrom Gibco (Burlington, ON, Canada). Trypsin waspurchased from Difco (Detroit, MI, USA) and wasprepared as a 1% solution. APAP was obtained fromSigma Chemical Company (St. Louis, MO, USA), aswas BSO, tetrazolium salt MTT (3-{4,5-dimethylthiazol-2-yl}2,5 diohenyltetrazolium bromide) forthe cytoviability assay, and haematoxylin-eosinstain for light microscopy. S was purchased fromAldrich Chemical Co. (Milwaukee, WI, USA) anddimethylsulfoxide (DMSO) was obtained fromFisher Chemical (Fisher Scientific, Toronto, ON,Canada). PBS (phosphate buffered saline withoutCa21 or Mg21) was used to wash cells and to removemedium. All plastic ware for cell cultures was ob-tained from Falcon (Becton Dickinson, Oxnard, CA,USA). All of the remaining reagents were of analyt-ical grade, obtained from Sigma Chemical Co.

For quantitative determination of DNA frag-ments, we used the Nucleosome Enzyme-LinkedImmuno Sorbent Assay (ELISA) kit from OncogeneResearch Products CN Biosciences (Cambridge, MA,USA) (43). The Nucleosome kit of low molecularDNA associated with histone is considered to be asensitive tool for DNA detection at the length of180–200 bp. The ELISA applies a Digoxigenin de-tector antibody and a Streptavidin conjugate withhorseradish peroxidase (SA-HRP) Sheep anti-digoxigenin antibody to quantitate the apoptoticnucleosomes.

HEP G2 CELLS LINE

Hep G2 cells were obtained from Wistar Institute(Philadelphia, PA, USA). Cells were seeded in flasks(1 3 106 cells/mL) (33,40). The cell counts weremonitored using a Coulter counter (Coulter Elec-tronics Inc., Hialeah, FL, USA). Cells in long-termcultures were grown in a-MEM supplemented with10% v/v heat inactivated fetal bovine serum (FBS).At the beginning of the experiment, when cellsreached 70% confluence, the growth medium wasremoved from the culture flasks. The cultures werewashed twice with phosphate buffered saline (PBS)and fresh serum free medium was used as base forall the treatments. The viability of cells not treatedwith EtOH or APAP was not altered by culturingthem for up to 6 days in serum free medium (33).

SILYMARIN PREPARATION

Because S is an anhydrous substance that doesnot dissolve in water, it was first dissolved in DMSOand then added to the a MEM. S solution was usedon the day it was dissolved. A control experiment forthe effect of DMSO on the cytoviability of HepG2

cells was carried out by Shear et al. (40), whichshowed that at the concentration we used to dissolveS, DMSO did not affect cell viability.

PRIMARY HUMAN HEPATOCYTES CULTURE

The hepatocyte cultures were developed from nor-mal liver tissue obtained after lobectomy from organdonors, where only a part of the liver was used fortransplantation, by cultivating in a highly enrichedmedium. Our method for preparing hepatocyte sus-pensions was based originally on that of Ballet et al.(44). The liver graft was perfused with ice-coldUniversity of Wisconsin solution (45) and kept on ice(2–10 h) until cells were isolated. The material waswashed with HEPES media. The hepatocytes wereisolated using the two step perfusion technique. Theaverage yield was 1.5 billion cells/preparation andthe average viability, assessed by trypan blue exclu-sion test, was 94%. Hepatocytes were washed threetimes in a culture medium (1/1 Ham F12 and Lei-bovitz L-15 supplemented 5 mmol/L glucose, 50U/mL penicillin, 50 mg/L streptomycin, 1028 mol/Linsulin). Cells were then plated (1 3 106/mL) incollagen coated Falcon flasks (3 mg /cm2 ) andcultured under conventional conditions in mediumsupplemented with FBS. One hour later, the me-dium was changed to remove the floating, unat-tached hepatocytes. The medium was replaced after12 h. Every 24 h thereafter, the media was changedwith a fresh one in the presence of FBS. Non-hepatocellular cells were not present. Cells weretreated with a MEM with or without the addtion ofEtOH or APAP, as described in the experimentaldesign. The dilutions were done in media in theabsence of FBS. We have been able to maintain thecells exhibiting typical morphological characteris-tics of hepatocytes in culture continuously for 10days. For ELISA, cells were plated directly in 96well plates not coated with collagen.

EXPERIMENTAL DESIGN

Control cells were plated in 96 well plates and in75 Falcon flasks incubated with a MEM. Cells werealso incubated with medium and either 40 mmol/LEtOH or 10 mmol/L APAP for 24 h. These levels ofEtOH and APAP were reported as non-toxic to cellsby Shear et al. (40) Treated cells were incubated for24 h with medium and either 10 mmol/L MTX, or 10mmol/L MTX 1 40 mmol/L EtOH, or 10 mmol/LMTX 1 5 mmol/L APAP.

The above experiment was repeated, with 0.5mmol/L S added to each control and correspondingtreatment. HepG2 and NHPH cells were pretreatedwith 0.5 mmol/L BSO, in order to induce glutathionedepletion in cells (40). The experiment was also runwith DS added to each control and correspondingtreatment.

All components were filtered-sterilized, and theentire procedure was conducted under aseptic con-ditions. The cells were routinely maintained in a

METHOTREXATE-INDUCED HEPATOCYTOTOXICITY


humidified incubator in 95% air/5% CO2, at 37°C.(Additional details of the individual experiments areprovided in the figure legends.)

MEASUREMENT OF APOPTOSIS

After a period of 18–20 h storage at 220°C, thecells underwent lysis and were exposed to the detec-tor antibody for 1 h at room temperature. The cellswere washed and incubated for another 30 min withSA-HRP conjugate. The chromogene reaction wasstopped and the absorbance was read using a spec-trophotometer plate reader with dual lengths 450/595 nm. The intensity of the yellow color is propor-tional to the number of nucleosomes in the sample.For each treatment, 6 wells/plate in 5 differentplates were quantitated. The results are reported as% of apoptosis vs. control, non-treated cells taken as0% apoptosis. For the standard curve, we ran repli-cates of six in each plate, using two different plates.The sensitivity of the assay was measured by assay-ing the non-treated cells at time zero. The meansignal and the standard deviation were calculated.The assay can distinguish 0.3 % from zero.

The Maxline Microplate Reader, from MolecularDevice Corp. (Menlo Park, CA, USA) was connectedto a computer using SOFT MAX software 2.3 forWindows (Molecular Devices Corp.), which allowedus to template the plate according to the experimen-tal needs, and perform the statistical analysis di-rectly on the template format.

GLUTATHIONE MEASUREMENT

For GSH determination, the cells were collected(46) on HEPES Kalium phosphate buffer (pH 7.4, 1mM EDTA and 0.1% bovine serum albumin). Ini-tially, the pellet was homogenized on ice, using aBranson model B-12 sonicator (2 3 10 s). All thesolutions were made the day assay was run. Afterdiscarding the 3,000 rpm fraction, the rotor wasaccelerated to 16,000 rpm to obtain the mitochon-drial pellet using a JS-20 rotor, with a refrigeratingultracentrifuge (Optima L7 Beckman InstrumentsInc., Fullerton, CA, USA). The supernatant wascentrifuged again at 45,000 rpm (TJ-70 rotor) for 60min at 4°C; this supernatant was considered thecytosolic fraction. The GSH was determined usingthe recycling assay of Tietze (47). After isolating theorganelles by centrifugation at high speeds, we mea-sured the specific biochemical markers that charac-terized the major function for each entity (48,49). Tocheck the fractions for cross contamination, we de-termined specific activities of succinic dehydroge-nase (SDH; EC. 1.3.99.1) for mitochondrion (50),lactic dehydrogenase (LDH; EC. 1.1.1.27), for cy-tosol (51), and glucose-6-phosphatase (G6P ase;EC.3.1.3.9), for microsomes (52,53). Enzyme activi-ties were measured at 37°C, 30°C, and 37°C, respec-tively. SDH activity was measured by reduction of2,6-dichlorophenol/indophenol (50). The final mito-chondrial preparation was typically enriched in

SDH vs. homogenate, 3.2 times. G6Pase activity wasmeasured by coupling to a glucose oxidase system(52,53). G6Pase activity in microsomes was found tobe 0.3 6 0.015 mM P/min/mg protein, while in themitochondrial fraction the activity was 0.001 60.0005 mM P/min/mg protein (a cross contaminationof 0.33%). cGSH and mGSH were determined aftercorrection for recovery of each specific marker en-zyme. Protein content was measured using a Bioradkit (54) using bovine serum albumin as standard.

CYTOTOXICITY ASSAY

For the MTT assay, HepG2 and NHPH cells wereseeded directly into 96-well plates at a density of 106

per well. MTT (100 ml of a 1 mg/mL solution) wasadded to each well of the 96-well plate and incubatedfor 1 h at 37°C, protected from light. At the end ofthe incubation the untransformed MTT was re-moved from the well by aspiration; 100 ml of DMSOwas then added to each well, and cells incubated for1 h while protected from light. The plate was thenshaken vigorously (Microshaker II, Dynatech, Dyna-Med, Toronto, ON, Canada) at speed setting 10movements/min in order to ensure that the blueformazan was fully solubilized. The optical densityof each well was measured at two wavelength mode(560 nm and 690 nm) using the automatic multiwellmicroplate spectrophotometer. Cytoviability was ex-pressed as the percentage of succinate dehydroge-nase activity in the treated cells as compared tocontrols. Each measurement was done in sextuplets(33).

CYTOKINE MEASUREMENT

Cytoscreen™, Immunoassay Kits for human IL1a, IL 6, and TNF a, and ELISA (Biosource Inter-national, Camarillo, CA, USA) were used for thequantitative determination of cytokines in serum.The principle of the method the use of a solid phaseassay (ELISA), and the assay is designed to recog-nize both natural human and recombinant humanIL 1a, IL 6, and TNF a. The wells of a 96-microtiterplate were coated with antibody specific for eachcytokine, and readings were done at 450 nm. Thecorrelation coefficient was linear (r 5 0.989) in aconcentration range between 2 and 500 pg/mL (Fig-ure 1).

STATISTICAL ANALYSIS

All data are expressed as means 6 standarddeviation (SD). Differences between groups wereanalyzed using an ANOVA test for repeated mea-surements with a Bonferroni test to correct formultiple comparison. All statistical analyses wereperformed with the statistical software package Mi-crocal Origin 301 (Microcal Inc., Northampton, MA,USA).

NEUMAN ET AL.


LIGHT AND ELECTRON MICROSCOPY

The cells were prepared for light (LM) and trans-mission electron microscopy (TEM) studies using astandard procedure as outlined subsequently (55).For each group of control (a MEM only, 40 mmol/LEtOH, 5 mmol/L APAP) and treated cells (10mmol/L MTX, 10 mmol/L MTX 1 40 mmol/L EtOH,10 mmol/L MTX 1 5 mmol/L APAP), six flasks ofeither NHPH or HepG2 were used. After the periodof incubation, the media was removed and cells werewashed twice with PBS. Five mL of 1% trypsin wasadded for 2 min to each flask. Cells were washedagain with PBS and then resuspended in plainmedia. Cell suspensions were centrifuged at 50 g for10 min. Pellets were immediately fixed in 2.5% v/vglutaraldehyde for a minimum of 24 h. Blocks ofcells were separated, post-fixed in 1% v/v osmiumtetroxide, dehydrated with a graded series of ace-tone concentrations and embedded in Araldine. Sec-tions (1 micron thick) were viewed by LM. For LMstudies an Olympus microscope equipped with Leco2005, Image Processing and Analysis System (LecoInstruments) with Microsoftt Visual Basic™ soft-ware (Toronto, ON) were used. Cells were consid-ered apoptotic if the classic features of pyknoticnuclei, cytoplasmic condensation and nuclear chro-matin fragmentation could be observed.

Representative blocks were selected, subjected toultra-thin sectioning and stained with uranyl ace-tate and lead citrate for TEM. Electron micrographswere taken with a transmission electron microscopeJEOL 1200 E x II (JOEL Institute Inc., MA, USA).Ultrastructural findings were examined in five dif-ferent grids/flask in each experiment. On each grid,200–400 cells were examined. An average of 9000(300 cells/grid 3 grids/flask 3 6 flasks/treatment)

cells were analyzed for each treatment. We usedstandard criteria for the morphological identifica-tion of cellular structures (56). When cells wereassessed by electron microscopy, cell shrinkage,electron dark cytoplasm, and apoptotic bodies wereconsidered criteria for classic apoptosis (55,57).

MORPHOMETRIC ANALYSIS

Only intact hepatocytes with nuclei were assessedboth for LM and TEM. The system used for LMmorphometry was a modulator high-performanceimage processing and analysis system, extendedwith high resolution camera which gave a true colorimage processing. For each block, 5 slides werestudied and the 60 hepatocytes/slide were mea-sured. The morphological dimensions (particle siz-ing) were implemented by a combination of hard-ware and software to ensure an optimizedperformance of Microsoft Visual Basic.

Results

We measured the percentage toxicity of MTX, aswell as toxicity caused by treating NHPH andHepG2 cells with either MTX 1 EtOH or MTX 1APAP. In addition, we studied the effects of S inmediating this toxicity. The results are given inFigure 2.

Treatment with 10 mmol/L MTX resulted in tox-icity to NHPH, but was considered non-toxic toHepG2 cells. Values for % toxicity under 12% wereconsidered non-toxic. Treatment with 40 mmol/LEtOH alone was not toxic to either NHPH or HepG2cells, but there was a significant difference in the

Figure 1—Standard curve for IL 6. Cytoscreen™, Immu-noassay Kits, IL 6 and Enzyme-Linked-Immuno-SorbentAssay (Biosource International, Camarillo, CA) were usedfor the quantitative determination of IL 6 in cell culturemedia. The assay is designed to recognize both naturalhuman and recombinant human IL 6 and TNF a. Thereadings were done at 450 nm. The correlation coefficientwas linear (r 5 0.989) in a concentration range between 2and 500 pg/mL.

Figure 2—Effect of EtOH and APAP on MTX-inducedcytotoxicity. NHPH and HepG2 cells were compared forMTX-induced cytotoxicity. For each experiment, 5 plateswere seeded and in each one 6 wells were treated/expo-sure. Each value is mean 6 SD of 30 wells. Formula fortoxicity: viability control 100%—viability treated—ap ,0.001 higher vs. control; bp , 0.001 lower thanMTX1EtOH; cp , 0.001 lower than the cells receiving thesame treatment but without S; dp , 0.05 lower than thetoxicity manifested by NHPH.



viability between the 2 cell types (Figure 2). Thesame was true for treatment with 5 mmol/L APAP,with p , 0.05 higher viability for HepG2 cellscompared with NHPH.

Treatment with MTX 1 EtOH resulted in signif-icantly higher toxicity to both cell types (p , 0.01)compared with MTX alone; toxicity for HepG2 cellswas lower in this case than for NHPH (p , 0.05).Similarly, MTX 1 APAP was significantly moretoxic to cells than MTX alone (p , 0.001), althoughit was less toxic than MTX 1 EtOH (p , 0.001).Again, a difference between cell types was noted,with toxicity of NHPH higher than HepG2 cells (p ,0.05). When silymarin was added to MTX 1 APAPor MTX 1 EtOH, Figure 2 shows that both exhibitedsignificantly less toxicity than the same treatmentwithout S (p , 0.001). Note that treatment withEtOH 1 S or APAP 1 S along with MTX reducedtoxicity levels to below those of MTX alone (p ,0.001).

Of the percentage of NHPH and HepG2 cells thatdied in the previous experiment, we were interestedto know how many of these had undergone apopto-sis. Therefore, we measured percentage of apoptosis,as seen in Figure 3.

Percentage apoptosis remains low (#3%) whencells are exposed to either EtOH or APAP. MTXexposure causes a much higher % apoptosis in bothNHPH and HepG2 cells. However, it is the deadlycombination of MTX 1 EtOH which produces thelargest % apoptosis: 22% and 18% for NHPH and

HepG2 cells respectively. The % apoptosis was sig-nificantly higher for cells exposed to MTX 1 EtOHthan MTX alone (p , 0.001), with less apoptosis inHepG2 cells than NHPH (p , 0.05). MTX 1 APAPwas not as lethal to cells; it produced % apoptosis inNHPH comparable to MTX alone (p , 0.05). HepG2cells were significantly more prone to apoptosiswhen treated with MTX 1 APAP than with MTXalone (p , 0.05).

One cause of hepatotoxicity and apoptosis may bethe reduction of intracellular GSH. We measuredcGSH and mGSH in cells exposed to EtOH, APAP,BSO, Silymarin and their combinations.

The levels of EtOH and APAP used (40 mmol/Land 5 mmol/L, respectively) did not lower GSHlevels when compared with control values (Table 1).Addition of BSO, a known glutathione depleter,lowered both cGSH and mGSH when compared withthe control (p , 0.001). When both BSO and Sily-marin were added to cells, GSH levels were higherthan when BSO alone was added (p , 0.001).MTX 1 EtOH and MTX 1 APAP decreased cGSHwhen compared with control (p , 0.05), while mGSHwas also decreased (p , 0.001). Incubation withMTX 1 EtOH 1 S increased cGSH and mGSH levels(p , 0.001) as compared to treatment with MTX 1EtOH. Exposure to MTX 1 APAP 1 S increasedGSH levels significantly when compared to MTX 1APAP (p , 0.001).

In order to determine the existence of an addi-tional mechanism by which the combinations of

Figure 3—MTX-induced apoptosis. In NHPH and HepG2cells treated for 24 h with MTX, EtOH, APAP or theircombinations, apoptosis was measured by quantitativedetermination of DNA fragments of the 200 bp, by ELISAkit. For each treatment, 6 wells/plate in 5 different plateswere quantitated. The results are reported as % of meanapoptotic cells vs. controls. Results are expressed asmean 6 SD. Statistical analysis was performed using oneway analysis of variance (ANOVA) with Bonferroni cor-rection. a p , 0.001 higher than MTX; b p , 0.05 higherthan MTX alone; c p , 0.05 lower than MTX1EtOH; d p ,0.05 lower % apoptosis than NHPH.

TABLE 1MTX-Induced GSH Depletion; Role of S on GSH

Depletion

ExposureCytosolic

GlutathioneMitochondrialGlutathione

(cGSH) (mGSH)(nanoMol/mg

protein)(nanoMol/mg

protein)

Control 14.06 6 0.60 1.58 6 0.07EtOH (40 mmol/L) 14.00 6 0.08 1.38 6 0.09APAP (5 mmol/L) 13.85 6 0.40 1.45 6 0.05BSO (0.5 mmol/L) 11.00 6 0.62* 0.70 6 0.03*BSO 1 S (0.5 mmol/L) 13.02 6 0.42** 1.45 6 0.03**MTX (10 mmol/L) 13.20 6 0.30 1.20 6 0.04MTX 1 EtOH 12.44 6 0.30a 0.87 6 0.03b

MTX 1 APAP 12.12 6 0.50a 0.78 6 0.03b

MTX 1 EtOH 1 S 13.73 6 0.10c 1.38 6 0.02c

MTX 1 APAP 1 S 13.19 6 0.60d 1.05 6 0.01d

For glutathione determination, the cells were collectedand immediately homogenized on ice as described inMaterials and Methods. The homogenate was fractionatedby ultracentrifugation and the cGSH and mGSH weremeasured in the respective fractions. All the results arepresented in mean 6 standard deviation.

*(p , 0.001); lower as compared with control.**(p , 0.001); higher as compared with BSO.a(p , 0.05); as compared with control.b(p , 0.001); lower than control.c(p , 0.001); higher than MTX 1 EtOH.d(p , 0.001); higher than MTX 1 APAP.

NEUMAN ET AL.


MTX 1 EtOH or MTX 1 APAP exert their toxicity,we measured the release of cytokines into mediawhen HepG2 cells were exposed to either combina-tion. The results are shown in Table 2.

Exposure to EtOH, APAP, or MTX did not signif-icantly alter cytokine release when compared withcontrol values (Table 2). The most notable increasein cytokine release resulted from HepG2 cell expo-sure to MTX 1 EtOH, where IL 8 and TNF a levelsin media increased significantly as compared toMTX alone (p , 0.001). IL 6 levels increased also(p , 0.05). In the case of MTX 1 APAP, only IL 8levels increased to a significant extent comparedwith addition of MTX alone (p , 0.05); however, thisincrease was lower than that caused by exposure toMTX 1 EtOH (p , 0.001). The addition of either S or

DS to MTX 1 EtOH decreased release levels for allthree cytokines compared with release levels fromexposure to MTX 1 EtOH; this decrease was signif-icant for IL 6 levels (p , 0.05) and for IL 8 and TNFa levels (p , 0.001). Despite the addition of S or DSalong with MTX 1 EtOH, TNF a release was signif-icantly higher than from exposure to MTX alone:p ,0.05 for S and p , 0.001 for DS. The combination ofMTX 1 EtOH 1 DS caused lower release levels thanMTX 1 EtOH 1 S (p , 0.05).

In order to discern whether the release of cyto-kines into media is correlated with their expressionat the cellular level, we examined the mRNA levelsof IL 6, IL 8, and TNF a in NHPH and HepG2 cells(Table 3).

Control expression of cytokines was given 100%

TABLE 3Effects of MTX on Cytokine Expression

Exposure IL 6 IL 8 TNF a

Control 100 100 100EtOH (40 mmol/L) 110 6 2.0 106 6 5.0 112 6 14APAP (5 mmol/L) 108 6 8.0 100 6 4.0 104 6 2.0MTX (10 mmol/L) 118 6 8.0 118.0 6 3.5 115 6 10.0MTX 1 EtOH 122 6 3.0# 138 6 2.0 128.5 6 2.0#*MTX 1 APAP 115 6 10.0 113.5 6 2.5 115 6 3.0MTX 1 EtOH 1 S 107 6 10.0## 132.5 6 3.0** 119.0 6 3.0#,**MTX 1 EtOH 1 DS 116 6 6.0## 128.5 6 1.5** 112.5 6 2.0**

Cells were seeded in flasks (1 3 106 cells/mL) and were exposed for 24 hours only toplain medium (control), 40 mmol/L EtOH, 5 mmol/L APAP, 10 mmol/L MTX or theircombination in the presence or in absence of 0.5 mmol/L S or 40 mmol/L DS. Totalcellular RNA was isolated from cells. Cytokine gene expression was evaluated byRT-PCR. The amount of DNA was normalized for the amount of G3PDH. Analysis ofthe expression was performed in triplicate in at least 3 different cell populations andresults of the cytokine expression was measured by densitometry as shown in materialsand methods. The results are presented in percentage, giving controls 100% value.

For statistical analysis, ANOVA with Bonferroni correction was used: #p , 0.05higher than control; ##p , 0.05 lower than MTX1EtOH; *p , 0.001 higher thanMTX; **p , 0.001 lower than MTX1EtOH.

TABLE 2Cytokine Release into Media by Hep G2 Cells

Exposure IL 6 IL 8 TNF a

Control 17.7 6 1.50 17.00 6 1.2 8.0 6 1.0EtOH (40 mmol/L) 16.70 6 0.80 18.00 6 1.5 7.5 6 1.0APAP (5 mmol/L) 15.85 6 0.90 15.00 6 1.5 6.5 6 1.0MTX (10 mmol/L) 18.20 6 1.30 18.50 6 3.5 9.5 6 1.0MTX 1 EtOH 22.40 6 0.30# 38.2 6 7.5* 22.5 6 2.0*MTX 1 APAP 20.10 6 0.50 23.5 6 2.5**# 9.5 6 3.0MTX 1 EtOH 1 S 18.70 6 0.10## 27.5 6 3.0** 19.0 6 3.0#,**MTX 1 EtOH 1 DS 16.10 6 0.60## 18.5 6 1.5** 12.5 6 2.0*,**

1 3 106 cells/mL were incubated with 10 mmol/L MTX, 40 mmol/L EtOH, 5 mmol/LAPAP or a combination of MTX 1 EtOH given concomitantly in the presence or absenceof S or DS, or MTX 1 APAP for 24 hours. Cytokines released into media (pg/mL) weremeasured in 5 different flasks in triplicates. The results represent Mean 6 SD.CytoscreenTM, Immunoassay Kits, Human IL 6, IL 8 and TNF a (ELISA) were used forthe quantitative determination of cytokines in cell culture media.

For statistical analysis of multiple treatments ANOVA with Bonferroni correctionwas used: #p , 0.05 higher than MTX; ##p , 0.05 lower than MTX 1 EtOH; *p ,0.001 higher than MTX; **p , 0.001 lower than MTX1EtOH.



value. Expression of IL 6, IL 8, and TNF a in cellsexposed to either EtOH or APAP were close tocontrol levels, as seen in Table 3. Cytokine expres-sion in cells exposed to MTX alone was higher thancontrol. When cells were incubated with MTX 1EtOH, expression of all three cytokines was higherthan control (p , 0.05), however, only the expressionof IL 8 and TNF a was higher than in cells exposedto only MTX (p , 0.001). None of the values forcytokine expression in cells exposed to MTX 1 APAPwere significantly higher than expression in cellsexposed to MTX alone. When either S or DS wasadded to the combination of MTX 1 EtOH, expres-sion was lower for IL 6 (p , 0.05) and IL 8 and TNFa (p , 0.001) than when cells were incubated withMTX 1 EtOH. Only TNF a expression was greaterthan control in cells treated with MTX 1 EtOH 1 S(p , 0.05).

To ascertain that the cytotoxicity was due to thepresence of pro-inflammatory cytokines in media,we exposed the cells to human recombinant IL 6, IL8, and TNF a and measured the percentage toxicity,which is shown in Figure 4.

When exposed to MTX 1 EtOH, HepG2 cellsreleased 22.40 6 0.30 pg/mL and 38.2 6 7.5 pg/mL ofIL 6 and IL 8 respectively (Table 2). We addedhuman recombinant IL 6 and IL 8 at the same

levels, and Figure 4 shows that we observed lowlevels of cytotoxicity in both NHPH and HepG2 cells.The combination of these two cytokines did notsignificantly increase % toxicity. When exposed tohuman recombinant TNF a at a concentration of22.5 6 2.0 pg/mL, a significant increase in cytotox-icity occurred (p , 0.001). Although incubation withTNF a 1 IL 6 resulted in toxicity higher than control(p , 0.001), it did not affect % cytotoxicity comparedwith exposure to TNF a alone. Exposure to TNF a 1IL 8 resulted in higher cytotoxicity levels thanexposure to TNF a only, especially in NHPH cells ascompared with HepG2 cells (p , 0.05).

These results led us to suspect that TNF a plays arole in MTX-induced toxicity. To test this hypothe-sis, we treated cells with human recombinant TNFa. The amount used to treat cells (22.5 6 2.0 pg/mL)was the same as the levels of TNF a released by cellswhen they were treated with MTX 1 EtOH (Table2). We also examined the putative protective effectsof S on cytotoxicity induced by the combination ofMTX 1 APAP and MTX 1 EtOH.

The addition of human recombinant TNF a to cellsresulted in a significantly higher % toxicity to bothNHPH and HepG2 cells compared with controls, asseen in Figure 5 (p , 0.001). We also treated NHPHand HepG2 cells with anti-TNF a to see if this was

Figure 4—Cytokine-induced toxicity. To define the role ofeach cytokine, and their combinations, in producing liverdamage, human recombinant cytokines were added to thecells and the subsequent cytotoxicity measured. Cellswere exposed to IL 6 (22.40 6 0.30 pg/mL) or IL 8 (38.2 67.5 pg/mL) or TNF a (22.5 6 2.0 pg/mL) at concentrationssimilar to that observed after exposure to MTX1EtOH(Table 2). Some cells were exposed to a combination of 2cytokines. The cytoviability was measured in 5 differentplates in triplicates (15 wells/treatment) using MTT as-say. For statistical analysis, the results were comparedusing one way analysis of variance ANOVA with Boferronicorrection; p , 0.05 was consider as significant. a p ,0.001 higher than control; b p , 0.001 higher than TNF a;c p , 0.05 higher than TNF a and p , 0.05 lower than thelevel of toxicity measured in NHPH cells treated with thesame dose of TNF a 1 IL 8.

Figure 5—Role of TNF and S in hepatocytoprotectionagainst MTX1EtOH-induced toxicity. NHPH and Hep G2cells were first treated with anti-TNF a antibody (22pg/mL) for 24 h, and then incubated with recombinantTNF a (22 pg/mL). Cells exposed to TNF a present ahigher cytotoxicity ap , 0.001 higher than the control.Pretreatment with anti-TNF a antibody significantly (p ,0.001) reduced the cytotoxic effect of TNF a. Cells exposedto MTX1EtOH presented a higher toxicity than the tox-icity induced by TNF a: bp , 0.05. The significance of c isof note: p , 0.001 reduction (vs. MTX1EtOH) in the toxiceffect induced by 10 mmol/L MTX 1 40 mmol/L EtOHwhen cells were pretreated with the antibody against theTNF a. Cytotoxicity of the cells exposed to MTX1EtOH1Sfurther decreased d p , 0.05 vs. MTX1EtOH1Anti-TNFa. Exposure of HepG2 cells to same treatment resulted ina lower toxicity: ep , 0.05 vs. when compared with NHPH.

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toxic to cells, and at the concentration we used(22.5 6 2.0 pg/mL) it was found to be non-toxic(Figure 5). By adding TNF a 1 anti-TNF a, we wereable to block the action of TNF a and achieve %toxicity values which were close to control levels.The previously measured value of MTX 1 EtOH %toxicity was higher (p , 0.05) than toxicity producedby the addition of TNF a alone. Addition of anti-TNFa to MTX 1 EtOH resulted in toxicity lower thaneither MTX 1 EtOH or TNF a alone (p , 0.001).Toxicity for NHPH was higher (p , 0.05) in this casethan for HepG2 cells. MTX 1 EtOH 1 S wasconsidered non-toxic to cells, with viability signifi-cantly higher than with either MTX 1 EtOH, orTNF a (p , 0.001) or MTX 1 EtOH 1 anti-TNF a (p, 0.05). There was also a difference in viabilitybetween NHPH and HepG2 cells, with viabilityhigher for HepG2 cells (p , 0.05).

Figure 6 shows the mRNA expression of TNF a.Presented in lane I is the expression of TNF a in thenon-treated control cells (considered 100%). Lane IIrepresents the elevated TNF a expression in cellstreated with MTX alone (115%), when comparedwith control. Lane III shows the additional elevationin expression of TNF a when cells were incubatedwith MTX1 EtOH (128%), and lane IV reveals thatTNF a expression is reduced by the addition of Swhen cells are treated with MTX 1 EtOH (119%).

TEM MORPHOMETRY STUDIES

Cells that have not been exposed to drugs shownormal organelles: abundant mitochondria andrough and smooth ER and occasional small lipidvesicles. Bile canaliculus-like structures can be ob-served at the confluence of the cells (Figure 7). Withthe addition of 40 mmol/L EtOH or 5 mmol/L APAP,cells do not change much (Figures 8 and 9). Whencells are treated with 10 mmol/L MTX, an enlarge-ment of some cells can be seen, with swollen mito-chondria and some disappearance of mitochondrialcristae. Endoplasmic reticulum undergoes vesicula-tion or dilatation, with either slight or more exten-sive enlargement, respectively (Figure 10). Lipidaccumulation is seen. Lipid vacuoles are roundedinclusions with a smooth homogeneous surface oflow electron density, and these exhibit a sharpdelimitation. In cells treated with MTX1 APAP,some of the cells are enlarged (Figure 11), present-ing variations in size, shape and number of mito-chondria, or mitochondria with rarefied matrix.There is a heterogeneity of size and electron densitybetween the individual fatty droplets (macro andmicrovesicular steatosis), a phenomenon that mayalso be observed by LM. Endoplasmic reticulumpresents vesiculation, dilatation and ballooning.

Exposure to 40 mmol/L EtOH and 10 mmol/LMTX increases the number of lipid droplets and themitochondrial damage. Some cells preserve a nor-mal nucleus but exhibit “giant” mitochondria withfewer cristae. Others are apoptotic cells with anirregular nucleus and dark condensed cytoplasm

(Figure 12). Exposure to S prevents the morpholog-ical damage produced by the MTX1EtOH combina-tion (Figure 13). Cells incubated with S presentnormal mitochondria and normal smooth ER. Con-comitant exposure to MTX 1APAP 1S results innormal-looking cells with normal mitochondria, ex-hibiting only slight enlargement of ER (Figure 14).

LM MORPHOMETRY STUDIES

The image processing of slides using the fieldmeasurements of intact cells showed no significantdifferences between cells treated with either APAP,

Figure 6—MTX-induced TNF a expression in Hep G2cells. Cells were exposed to plain media only for 24 h:control line I was considered to have mRNA expression ofTNF a of 100%; Line II: MTX; Line III: MTX1 EtOH; LineIV: MTX1EtOH in presence of S. RNA was extracted andthen amplified by PCR. The PCR product was electropho-resed on 2% agarose gel. The expression of each specificcytokine was quantitated by densitometry and given as %,with control cell expression considered 100%. Data ispresented as mean 6 standard deviation. For data anal-ysis, ANOVA with Bonferroni corection was used. Theresults are given in Table 3.



EtOH, MTX, or plain media only, but revealedsignificant differences between the area of cellsexposed to MTX 1 APAP (4912 6 175 mm2) orMTX1EtOH (5725 6 215 mm2) versus the cellsexposed only to plain media (4425 6 525 mm2) (p ,0.05). Cells treated with MTX1APAP1S andMTX1EtOH1S showed a cell surface of 3995 6 215mm2 .

In the intact cells taken into consideration for themorphometric studies, nuclear area is not changedsignificantly, i.e., 860 6 122 mm2 in control cells vs.780 6 135 mm2 in cells exposed to MTX 1 EtOH.Ratio of the cell surface/nuclear surface in controlcells was 5.145, while in treated cells the ratio was7.339 (p , 0.05). Concomitant exposure to S andMTX 1 EtOH or MTX1APAP gave cell surface/nuclear surface ratios of 5.122 and 4.981, respec-tively (p , 0.05).

Discussion

We have previously reported that the HepG2 cellline and NHPH are reliable in vitro models for thestudy of drug and alcohol-induced hepatotoxicity(33–35). In the present work we studied the factorsthat may contribute to MTX-induced cytotoxicity

and the possible prevention of such toxicity. Thedamage produced by MTX is most severe when cellsare exposed to 40 mmol/L EtOH 1 10 mmol/L MTXfor 24 h. The toxicity is partly due to the direct effectof MTX; EtOH, an inducer of cytochrome P450 2E1,may also be responsible as it can enhance theproduction of reactive oxygen species (ROS). This isindicated by the fact that concomitant exposure toDS and MTX1ETOH reduces the cytotoxicity ofMTX1EtOH.

In terms of ultrastructural changes, cells exposedto MTX1EtOH shared a specific trait: mitochondriawere enlarged and appeared as swollen or elongatedstructures with disrupted cristae, lacking normalorganization (33–35). Interestingly, S can protectcells from MTX1EtOH-induced damage, althoughthe mechanism of this effect is still not fully under-stood. Cells treated with MTX1EtOH1 S presentedintact mitochondria, similar in shape and size tomitochondria in control cells. Identical beneficialchanges in mitochondria have been observed in ratstreated with S during EtOH-intoxication (58).

A number of studies have shown that cachectin—TNF a, IL 1 a, and IL 6 are elevated in the plasmaof patients with alcoholic hepatitis (59–61). How-ever, it is unclear if this elevation is associated with

Figure 7—Transmission electron micrograph (TEM) of Hep G2 cells (control, non-treated). Cells were plated 75 Falconflasks at a density of 106 cells/mL and were grown in media supplemented with FCS. At 70% confluence, cells wereincubated in plain media for 24 h. After the media was removed, the cells were prepared for TEM as described in materialand methods. The TEM shows normal looking cells with normal centrally-located nucleus (N), a bile-canaliculus (BC) andnumerous normal mitochondria (M). Magnification 3 5,000.

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hepatic damage or is instead one of the causes oftoxic effects. In our previous work (62), we reportedthat high levels of EtOH (80 mmol/L) enhanced TNFa and IL 6 production, and provoked hepatocytotox-icity. In the present study, in vitro exposure of cellsto MTX1APAP resulted in an elevation in cytokinerelease, particularly for IL 8, as shown in Table 2.When cells were exposed to MTX1 EtOH, there wasan elevation in IL 6, IL 8, and TNF a levels. Theincrement of cytokine release was accompanied byan over-expression of the 3 cytokines, at the mRNAlevel (Table 3 and Figure 6). Exposure to MTX1EtOH resulted in significant over-expression of IL 6,IL 8, and TNF a. This fact suggests that EtOH, aP450 2E1-inducer, causes cells treated with MTX tosynthesize and release cytokines. This effect couldbe either directly or indirectly caused by ROS, whichmay induce cytokine overexpression (62).

To define the role of each cytokine in the produc-tion of cellular damage, we added human recombi-nant cytokines in the amount released by cellstreated with the drugs (Table 2). We also measuredcytotoxicity produced by exposing cells to each cyto-kine, as well as their combinations (Figure 5). Theaddition of IL 6 or IL 8, alone or in combination, atconcentrations similar to that observed after expo-sure to MTX1EtOH, did not result in an increased

toxicity. On the other hand, incubation of the cellswith TNF a resulted in a significant reduction inviability. When TNF a was given together with IL 6,no addtional increase in cytotoxicity was observed.

These results implicate TNF a as a potentialcandidate for the EtOH-induced toxicity measuredin our in vitro model. Additional support for thisconclusion is the observation that when cells areincubated with MTX1EtOH1anti-TNF a antibody,the cytotoxic effect of TNFa may be significantlyreduced (Figure 5). The protective effect of anti-TNFa antibody, demonstrated by its ability to block TNFa bioactivity, suggests that active TNFa is toxic toliver cells in vitro. The fact that anti-TNF a antibodydid not abolish the toxicity produced by MTX1EtOHmay show that other factors play a role in thistoxicity. The mechanism(s) of hepatotoxicity of ca-chectin is not clear. Though low levels of TNF a areactually required for cell proliferation (63), highlevels have been shown to induce hepatotoxicityduring in vitro experiments (64–66). However, thecytotoxic effect observed on addition of TNF a waslower than that observed when cells were exposed toEtOH. This indicates that TNF a is only partlyresponsible for the cytotoxic effect of MTX1EtOH,and that metabolic alterations should be also con-sidered (67–69).

Figure 8—Transmission electron micrograph of NHPH exposed to 40 mmol/L EtOH for 24 h. Cells were plated in 75 Falconflasks at a density of 106 cells/mL and were grown in media supplemented with FCS. At 70% confluence, cells wereincubated in media containing 40 mmol/L EtOH. There is a large-sized cell at the center of the micrograph.. The nucleus(N) is normal looking and presents a normal nucleolus. The cell shows a considerable amount of normal looking, functionalmitochondria (M). A bile canaliculus (BC) can be observed between the cells.



The data on GSH content support our hypothesisthat metabolic factors may be partly responsible forcytotoxicity caused by MTX1EtOH. The mitochon-drial pool of GSH is a cell defense important in themaintenance of vital cell functions (62,70–72). Inthis study we have shown that a combination ofeither MTX1APAP or MTX1EtOH selectively de-creases the mitochondrial pool of GSH in cells (Table1). This observation concurs with previous results ofexperiments using intact animals in vivo, and rathepatocyte cultures in vitro (62,67,72). In thesestudies, it has been suggested that the depletion in

mGSH content could be a result of impaired trans-port from cytosol (73,74).

Previously, we described structural and func-tional changes of mitochondria exposed to EtOH inan in vitro model (62,75). We exposed cells to BSO, aknown depleter of gluathione, at the same time thatwe exposed the cells to each drug and each drug incombination with EtOH; our aim in this was toclarify the effect of non-toxic doses of EtOH incombination with MTX on the reduction of mGSH.We were also interested in the relationship of lowmGSH and cell viability. The important role of

Figure 9—Transmission electron micrograph of Hep G2 cells treated with 5 mmol/L APAP. When cells reached 70%confluence, they were washed with PBS and media containing 5 mmol/L APAP was added (time 0). After a period of 24 h,cells were collected as described in material and methods. Two cells with normal looking nuclei (N) can be seen in themicrograph; one has an enlarged endoplasmic reticulum (ER). One lipid droplet (LD) can be seen. Mitochondria (M) arenormal.

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mGSH in cytoviability is clear: because mGSH de-pletion occurs at 12 h (62), prior to loss of cytoviabil-ity, mGSH depletion decreases cell viability andmay be considered a cause of cytotoxicity. No differ-ences in cytoviability were observed when cells wereincubated with 40 mmol/L EtOH or 5 mmol/L APAP(Figure 2). High cytoviability also corresponded withnormal cGSH and mGSH. However, a significantreduction in cell viability was observed after 24 hexposure to MTX1EtOH or MTX 1APAP, therefore,corresponding with a significant reduction inmGSH. The importance of mGSH in the develop-ment and progression of MTX1EtOH-induced dam-age is supported by the notable cytotoxicity whichoccurs when mGSH is reduced by 24%, and by adecrease in cytosolic GSH.

Our results point to a reduced sensitivity to EtOHwhen the GSH depletion has been averted by S. This

antioxidant also enhances mGSH levels. We previ-ously demonstrated the role of S in APAP-inducedtoxicity in vitro, in HepG2 cells and in an epidermoi-dal cell line (40). This experiment showed that S hadcytoprotective effects, especially in enhancing GSHlevel. The potential mechanism of S-mediated stim-ulation of intracellular GSH may include regulationof cell volume (morphometic measurements). Asshown by TEM in Figure 13, cells treated withMTX1EtOH1S look normal, with prevention of cellshrinkage, nuclear condensation and subsequentcell death (apoptosis) produced by MTX1EtOH (Fig-ure 12). We suggest that S’s protective effects maybe due to its role in augmenting mGSH content incells (Table 1), which stabilizes the mitochondrialmembrane and therefore reduces the mitochondrialpermeability transition. As a result, apoptotic sig-naling is blocked.

Figure 10—Transmission electron micrograph of HepG2 cells treated with 10 mmol/L MTX for 24 h. Cells present largenuclei (N) and normal organelles, including mitochondria (M). There is enlargement of endoplasmic reticulum (ER).Magnification 3 5,000.



Figure 11—Transmission electron micrograph of NHPH treated with 10 mmol/L MTX 1 5 mmol/L APAP for 24 h. At thecenter of the micrograph is a cell presenting a normal nucleus (N), enlargement of endoplasmic reticulum (er) and normalmitochondria (M). In the left hand corner, part of a cell presenting a ballooning of endoplasmic reticulum (ER) is seen, andin the right corner of the same micrograph is a cell with lipid droplets (LD).

Figure 12—Transmission electron micrograph of a representative apoptotic cell. NHPH treated with 10 mmol/L MTX 140 mmol/L EtOH for 24 h present different degrees of morphological changes. A cell which preserves the form of ahepatocyte is visible in the micrograph. Mitochondria are also present. The nucleus (N) has a higher density than normal,and clumps of chromatin (CR) are condensed. The cell exhibits enlargement of endoplasmic reticulum (ER).

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Figure 14—Transmission electron micrograph of HepG2 cells treated for 24 h with 10 mmol/L MTX 1 5 mmol/L APAP 10.5 mmol/L S Cells present normal nuclei (N) and abundant, normal mitochondria (M). One of the cells exhibitsenlargement of endoplasmic reticulum (er). In the other cells, the endoplasmic reticulum appears normal.

Figure 13—Transmission electron micrograph of NHPH exposed for 24 hrs to 10 mmol/L MTX 1 40 mmol/L EtOH 1 0.5mmol/L S. Cells with normal-looking mitochondria (M), normal nuclei (N), and small vesiculation of endoplasmic reticulum(er) are present. Between 3 cells in the left hand corner is a bile caniliculus (BC).



However, by reducing ROS and acting as a freeradical scavenger, S may react with the toxic metab-olites generated by MTX1APAP to form metaboliclow activity products. These products are capable ofreducing lipid peroxidation, which may translateinto morphological changes such as reduction of theballooning of ER observed in MTX1APAP treatedcells (Figure 11). Cells treated with MTX1EtOH1APAP present normal organelles (Figure 14).

There are multiple ways in which TNF a mayproduce cytotoxicity, and it appears that differentpathways cause injury to different cell types (62,76).We describe a highly reproducible model of TNF acytotoxicity, which was reversible by adding humanantibodies to TNF a. The system uses human-de-rived liver parenchymal cells, in isolation. The TNFa in our model is partly responsible for theMTX1EtOH-induced apoptosis and cytotoxicityseen in Figures 3 and 5. This translates morpholog-ically into the presence of apoptotic cells with chro-matin condensation, which preserve their cellularmembrane and the integrity of organelles (Figure12). To our knowledge, this is the first report inwhich non-toxic doses of EtOH have triggered MTX-induced apoptosis.

In murine hepatocyte cultures, TNF a cytotoxicityis associated with DNA fragmentation and apoptosis(62,77). This observation was confirmed in ourmodel (Figures 3 and 4). TNF a significantly in-creased cytotoxicity. This phenomenon was pre-vented when cells were exposed to anti-TNF a anti-body. All together, these data indicate that apoptosisinduced by MTX1EtOH in liver cells may be partlyrelated to the effects of TNFa, a cytokine which alsoplays an important role in EtOH-induced toxicity invitro.

Treatment of cells with either MTX1EtOH orMTX1 APAP and 0.5 mmol/L S resulted in a re-duced cytotoxicity (Figure 5). Interestingly enough,NHPH are more sensitive to both MTX1EtOH andMTX1APAP induced toxicity, a phenomenon thatmay be due to a higher content of cytochrome P4502E1 found in NHPH. S significantly decreased therelease of IL 6, IL 8, and TNF a, release levels ofwhich were previously increased by MTX1EtOHexposure. DS, a P450 2E1 inhibitor, also contributesto a reduction in the release of cytokines. Therefore,the MTX1EtOH increase in proinflammatory cyto-kines may be related to the induction of cytochromeP450 2E1 and the production of ROS, which togetherwith the decrease of mGSH, will contribute to celldamage. Caution should be taken in extrapolatingdata obtained in an in vitro model to in vivo condi-tions; however, our results may indicate that adownregulation of TNF a and an increase in mGSHmay play a beneficial role in the prevention andtreatment of liver damage induced by either multi-ple medication, e.g., chronic use of MTX and APAP,or chronic alcohol consumption and treatment withMTX.

Acknowledgements

We would like to thank the Canadian Foundation ofDermatology for their financial support.

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NEUMAN ET AL.


inducers of cytochrome p450 2e1 enhance methotrexate-induced hepatocytotoxicity

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