Research Article The Protection of Hepatocyte Cells from the Effects of Oxidative Stress by Treatment with Vitamin E in Conjunction with DTT

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1 Hindwi Pulishing Corportion Journl of Biomediine nd Biotehnology Volume 21, Artile ID , 7 pges doi:1.1155/21/ Reserh Artile The Protetion of Heptoyte Cells from the Effets of Oxidtive Stress y Tretment with Vitmin E in Conjuntion with DTT Jen-Hsing Tsi, 1 Hw-Wen Chen, 2 Yi-Wn Chen, 3 Jer-Yuh Liu, 4, 5 nd Chong-Kuei Lii 2 1 Deprtment of Physil Therpy, Shool of Medil nd Helth Sienes, Fooyin University, Kohsiung 8312, Tiwn 2 Shool of Nutrition nd Institute of Nutrition, College of Helth Cre, Chin Medil University, Tihung 442, Tiwn 3 Shool of Nutrition, College of Helth Cre nd Mngement, Chung Shn Medil University, Tihung 423, Tiwn 4 Grdute Institute of Cner Biology, College of Mediine, Chin Medil University, Tihung 442, Tiwn 5 Center for Moleulr Mediine, College of Mediine, Chin Medil University, Tihung 442, Tiwn Correspondene should e ddressed to Jer-Yuh Liu, jyl@mil.mu.edu.tw nd Chong-Kuei Lii, klii@mil.mu.edu.tw Reeived 16 Novemer 29; Aepted 25 Mrh 21 Ademi Editor: George E. Plopper Copyright 21 Jen-Hsing Tsi et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. We investigted the effet of vitmin E on memrne protein thiols under oxidtive stress, whih we indued y treting heptoytes with tert-utyl hydroperoxide (TBH) for 6 mins. Those ells whih we pretreted with vitmin E formed fewer les (22.3% ompred to 6.% in nonvitmin E-treted ells) nd mintined ytosoli lium onentrtion nd the numer of memrne protein thiols insted of showing the usul symptoms in ells undergoing oxidtive stress. Dithiothreitol (DTT) lso ommonly redues le formtion in heptoytes ffeted y TBH. However, our experiments lerly demonstrte tht DTT does not prevent the hnges in ytosoli lium nd memrne protein thiols in the leing ells. Consequently, we deided to pretret ells with oth DTT nd vitmin E nd found tht the influene of TBH ws entirely prevented. These findings my provide us with new spet for investigting the mehnism of le formtion under oxidtive stress. 1. Introdution Formtion of les on the surfe of heptoytes is not only n erly sign of toxi injury under ishemi onditions or oxidtive stress ut lso hs signifint ssoition with poptosis or nerosis [1]. This morphologil normlity hs een ttriuted to hnge in intrellulr lium homeostsis [2, 3]. An inrese in the onentrtion of intrellulr lium my indue series of lium-dependent retions tlyzed y the lium-dependent proteses, phospholipses, or endonuleses [4]. These enzymes my disrupt the integrity of the ytoskeleton nd led to le formtion nd growth. The ourse of plsm memrne leing on heptoytes hs een divided into three stges: formtion, shedding nd fusion, nd finlly rupture [5]. Injuries to heptoytes in the first two stges re reversile wheres le rupture is irreversile nd results in ell lysis [6]. In ddition to its nutritionl importne, vitmin E (αtoopherol) is lso nturl ntioxidnt whih n prevent lipid peroxidtion in ellulr nd suellulr memrne phospholipids under oxidtive stress [7]. Lipid peroxidtion my use dmge of the plsm memrne nd n inrese in the numer of ytosoli-free lium ions. This n result in the hnge in the verpmil nd nifedipine-sensitive C 2+ hnnels [8] or n inresed possiility of rhidoni idindued toxiity of CYPE1-expressing ells [9]. This inrese in ytosoli C 2+ onentrtion n e prevented y vitmin E; moreover, we hve lso demonstrted tht vitmin E my prevent le formtion nd the loss of protein thiols in tertutyl hydroperoxide-(tbh-) treted heptoytes [1, 11]. Sine vitmin E only protets protein thiols whih hve een depleted due to intertion with endogenously generted lipid peroxidtion produts [12], it is possile tht the ttenution of plsm memrne protein thiols my e relted to the mintenne of intrellulr lium homeostsis.

2 2 Journl of Biomediine nd Biotehnology In order to determine the role of vitmin E in this mehnism, we employed onfol mirosopy, high-pressure liquid hromtogrphy (HPLC), nd spetrophotometry to investigte the hnges in the onentrtion of intrellulr lium ions nd the numer of plsm memrne protein thiols of rt heptoytes under oxidtive stress indued y TBH. 2. Mterils nd Methods 2.1. Isoltion nd Culture of Heptoytes. All niml experiments were onduted with pprovl from Chung Shn Medil University Animl Cre nd Use Committee. Mle Sprgue-Dwely rts (8 weeks) were purhsed from the Ntionl Animl Breeding nd Reserh Center, Tipei, Tiwn. Heptoytes were isolted from the liver of these nimls y ollgense perfusion [1], nd >9% were found to e vile y the trypn lue exlusion test. The ells were then plted to ollgen-preoted 3-mm plsti tissue ulture dishes (Flon Lwre, USA) with totl of ells in L-15 ulture medium (ph 7.6) ontining 18 mm N-2-hydroxyethylpiperzine-N -2- ethnesulfoni id (HEPES), 2.5% fetl ovine serum, 5 mg/l eh of insulin nd trnsferrin, 5 μg/l sodium selenite, 1g/L gltose, 1μmol/L dexmethsone,, IU/L peniillin, nd mg/l streptomyin. After ulturing in 37 C humidified inutor in mient ir for 4 hours, untthed nd ded ells were removed from the ulture. The ells were then ultured in the L-15 ulture medium with.2% ovine serum lumin without fetl ovine serum t 37 C for 4 hours; ells were inuted t 37 C without tretment or treted with μm vitmin E for 2 hours. Cultures with vitmin E tretment were then treted with 5 mm dithiothreitol (DTT) for 15 min or without this tretment. Those without vitmin E tretment were treted with 5 mm DTT nd/or 15 mm ethylene glyol tetreti id (EGTA) for 15 min or without ny tretment. These ultures were treted with indited onentrtions of TBH, nd hnges in the ells were deteted Confol Mirosopy. Alterntions in intrellulr lium were determined y onfol mirosopy with lium-sensitive fluoresent dye (fluo 3-AM) nd video mirosopi imging using the method of Burnier et l. [13] with modifitions. Fluo 3-AM (5 μm) ws dded to ulture medium, nd the heptoytes were inuted t 37 Cfor 3 min in the drk. The pluroni id (2 μl/ml) ws dded to fluo 3-AM for dispersing the dye. After removing the ulture medium, the ells were wshed with L-15 ulture medium without ovine serum lumin nd then ultured with 1 ml of this medium in 3-mm ulture dish. After leling with fluo 3-AM, the ulture dish ws pled into thermostti stge mintined t 37 C. Heptoytes with vrious tretments or without tretment were snned under onfol mirosope (LSM 41 invert, Zeiss, Germny). Confol mirosopy ws performed ording to the proedures s previously desried [13] Cell Morphology Exmintion. Tissue ulture dishes were pled on heted mirosope stge (37 C). Following the ddition of TBH, ell memrne le formtion ws monitored under phse-ontrst inverted mirosope (Nikon, Tokyo, Jpn) equipped with CCD mer monitor. The perentge of heptoytes-ering les ws determined on pitures tht were tken t 15, 3, 45, nd 6 min, respetively. At lest 15 ells were ounted in eh nlysis. The perentge of ells-ering les ws used to express the extent of memrne leing in eh group High-Pressure Liquid Chromtogrphy. These ells were llowed to stnd for 3 min to dissolve glutthione (GSH) into perhlori id. To the id solvent ontining GSH (4 μl), 4μL iodoeti id (12mg/mL) nd potssium ironte (KHCO 3 ) were dded nd pled in the drk for 15 min efore dding 44 μl 3% 2, 4-dinitrofluoro enzene in ethnol. The mixture ws then vigorously shken nd stored t 4 C for 8 hours. After entrifuged t 6, gfor 5 min, the superntnt ws filtered through.45-μm filter. Conentrtions of GSH were determined y HPLC using the method s previously desried [14] Spetrophotometry. To determine lipid peroxidtion, heptoytes were wshed twie with old phosphte-uffered sline (PBS, ph 7.4) fter removl of the ulture medium. The ells were extrted with 2 μl of 5 mm potssium phosphte uffer (ph 7.4). Lipid peroxidtion ws determined s thiorituri id retive sustnes (TBARS) [15]. The fluoresene of the smples ws deteted t n exittion wvelength of 515 nm nd n emission wvelength of 555 nm in F45 fluoresene spetrophotometer (Hithi, Jpn) nd 1, 1, 3, 3-tetrmethoxypropne ws used s TBARS stndrd. For the determintion of memrne protein thiols, the heptoytes were wshed twie with PBS, nd 6 μl of 2 mm potssium phosphte uffer (ph 7.4) ws dded, fter removing the ulture medium. The ells were then srped nd entrifuged t 8 g for 1 min. The superntnt ws entrifuged gin t 15 g to otin the ytosoli frtions (superntnt) nd the memrne frtions (pellet). The memrne frtions were then mixed thoroughly with the sme uffer (8 μl) ontining 5% SDS. The totl memrne protein thiols were mesured fter the inution with 5,5 -dithio-is-nitroenzoi id s previously desried [16], nd the totl protein onentrtions were determined y the method s previously desried [17]. To express the ell viility, the ltte dehydrogense (LDH) lekge ws nlyzed ording to the method s previously desried [18] Sttistil Anlysis. Dt were expressed s men ± stndrd devition. Signifint differenes mong the groups were nlyzed y one-wy nlysis of vrine. Dunn s multiple tests were used to determine the differene mong groups, nd Student s t-test ws used in se of the two group omprison. P<.5 ws onsidered to hve sttistil signifine.

3 Journl of Biomediine nd Biotehnology 3 3. Results 3.1. Initition of Heptoyte Bleing y TBH nd Chnges in the Intrellulr Clium. Under the onfol mirosope, the lotions of les oserved under the trnsmission mode orresponded to their intensities (Figures 1(), 1(), 1(e), 1(g), nd 1(i)). The fluoresene intensity from the heptoytes treted with 2. mm TBH inresed with time (Figures 1(), 1(d), 1(f), 1(h), nd1(j)), suh s ell,,, d, nd e of Figure 1() whose onentrtion of intrellulr lium rpidly inresed from 12 min nd rehed the mximum t 18 min (Figure 2). A signifint inrese in fluoresene intensity nd le in heptoyte were oserved t 18 min fter TBH tretment, s rrow indited (Figures 1(e) nd 1(f)). These hnges eme more severe t 3min, s rrow indited (Figures 1(g) nd 1(h)). The fluoresene intensity disppered t 6 min euse of le rupture (Figures 1(i) nd 1(j)) Effets of Vitmin E on the Intrellulr Clium in TBH-Treted Heptoytes. In heptoytes treted with 1. or 2. mm TBH for 6 min under phse-ontrst inverted mirosope, 18% ± 4.2% (n = 3) or 6.6% ± 1.1% (n = 4), respetively, formed les on the ell memrne. These phenomenons were similr to the oservtion of le formtion from onfol mirosope. Signifintly lower perentge of 22.3% ± 4.2% (n = 4) in 2. mm TBH-treted heptoytes ws otined y the pretretment with vitmin E(P<.5). Moreover, pretretment with EGTA in 2. mm TBH-treted heptoytes lso yielded signifintly lower of 27.4 ± 5.8 (n = 4). However, no signifint differenes were found etween the pretretment with vitmin E nd EGTA (P >.5). Although the fluoresene response in 1. mm TBHtreted heptoytes ws not oserved (dt not shown), the positive response ws deteted t 12 min fter tretment with 2. mm TBH (ontrol) nd inresed to 2 folds t 18 min nd grdully deresed from 4 min. In 2. mm TBH-treted heptoytes pretreted with vitmin E, the response ws in stedy level nd signifintly lower thn ontrol in the middle period. Wheres, pretreted with EGTA in 2. mm TBH-treted heptoytes, the onentrtion of intrellulr lium ws grdully deresed from 15 min, nd to zero t 3 min (Figure 3()) Effets of Vitmin E nd DTT on the Intrellulr Clium in TBH-Treted Heptoytes. In ddition to vitmin E, DTT is lso n importnt memer of the ntioxidtive gent. Pretretment with DTT signifintly deresed the perentge of leing from 62.2%±1.2% in the heptoytes onlytretedwith2mmtbhfor6minto25.% ± 2.2% (P <.5). However, fter dding vitmin E with DTT to the TBH-treted ells, the leing perentge ws signifintly redued to zero. The onentrtion of intrellulr lium response from the 2. mm TBH-treted ells with pretretment of DTT inresed with time in the leing ells ut no signifint differene ws found in the prior period (Figure 3()) Effets of Vitmin E nd DTT on Totl Glutthione (GSH), LDH Lekge, nd Lipid Peroxidtion in TBH- Treted Heptoytes. Intrellulr totl GSH onentrtion signifintly deresed fter treting the heptoytes with 1. or 2. mm TBH for 6 min, lthough the GSH onentrtion in 2. mm TBH-treted ells ws signifintly lower thn tht of the 1. mm TBH-treted ones. Pretretment with vitmin E or DTT mintined GSH in 2. mm TBH-treted heptoytes; the levels of GSH were signifintly lower thn those of the untreted group. However, there ws no signifint differene in the GSH level etween the vitmin E plus DTT-treted group nd the untreted group (Tle 1). The levels of LDH lekge in heptoytes treted with 1. or 2. mm TBH, EGTA nd 2. mm TBH, or DTT nd 2. mm TBH were signifintly higher thn the untreted group. However, there ws no signifint differene in the lekge etween the untreted group nd 2. mm TBHtreted ells with pretretment of vitmin E or vitmin E plus DTT (Tle 1). Lipid peroxidtion ws mesured y TBARS prodution in heptoytes. TBARS prodution ws signifintly higher in the ells treted with 1. or 2. mm TBH, DTT or EGTA with 2. mm TBH thn the untreted group. However, there ws no signifint differene in the prodution etween 2. mm TBH-treted ells with the untreted group nd pretretment of vitmin E or vitmin E plus DTT (Tle 1) Effets of Vitmin E nd DTT on the Loss of Memrne Protein Thiols Indued y TBH. In oth memrne nd ytosol, the levels of memrne protein thiols in heptoytes treted with 1. or 2. mm TBH or EGTA nd 2. mm TBH for 6 min were signifintly lower thn the untreted group. In the presene of vitmin E, there ws no signifint differene in the level of protein thiols of the memrne frtion, wheres this level remined signifintly lower thn the untreted group in the ytosoli frtion. In the pretretment with DTT in 2. mm TBH-treted heptoytes, lthough the levels of protein thiols of the memrne frtion were signifintly lower thn these of the ontrol, there ws no signifint differene in the ytosoli frtion. However, no signifint differene ws found in the level of protein thiols of oth the memrne nd ytosoli frtions in the vitmin E plus DTT pretreted ells (Tle 2). In the ells without the supplement of vitmin E, tretment of 2. mm TBH used rpid loss of the memrne protein thiols nd 37% of the thiols were lost within 15 min. Theperentgeoflossthenemelessseverefter15min, nd totl loss of 41% ws oserved t 6 min fter TBH tretment. In the presene of vitmin E, the perentge of loss ws lso more severe in the first 15 min. However, the totl loss of thiols ws only 15% t 6 min. 4. Disussion The formtion of les in TBH-treted heptoytes hs een ttriuted to the elevtion of intrellulr lium onentrtion [19, 2]. Using onfol mirosopy, we visully demonstrted the importnt role of intrellulr

4 4 Journl of Biomediine nd Biotehnology d e () 5 () () (d) (e) (f) (g) (h) (i) (j) Figure 1: Chnges in the fluoresene intensity of intrellulr lium in TBH-treted heptoytes. Using onfol mirosopy, the hnges of ell morphology were lso photogrphed efore (), 12 (), 18 (e), 3 (g), nd 6 min (i) fter 2. mm TBH tretment. At the sme time, the hnges of fluoresene intensity of intrellulr lium were photogrphed efore (), 12 (d), 18 (f), 3 (h) nd 6 min (j) fter 2. mm TBH tretment. Pseudodensity sle indites fluoresene intensity in ritrry units. Arrows indite the ells with le. Br, 2 μm.

5 Journl of Biomediine nd Biotehnology 5 Tle 1: Effet of vitmin E nd DTT on totl GSH ontent, LDH lekge, nd TBARS prodution in rt heptoytes with TBH tretment. Tretment Totl GSH (nmol/mg protein) LDH lekge (%) TBARS (nmol/mg protein) Untreted 47.7 ± ±.6.66 ±.9 TBH (1. mm) 19.5 ± ± ±.41 TBH (2. mm) 4.1 ± 1.2 d 76.2 ± ±.31 d VitminE(μM)+TBH(2.mM) 9.1 ± ± ±.11 EGTA (15 mm) + TBH (2. mm) 2.1 ±.1 d 62.8 ± ±.51 e DTT (5 mm) + TBH (2. mm) 29.7 ± 3.5 e 26.6 ± 1.7 e 1.75 ±.2 VitminE(μM)+DTT(5mM)+TBH(2.mM) 36.9 ± 4.2 e 2.8 ± ±.6 Vlues re expressed s mens ± SD (n = 3-4). Mens in the sme olumn not shring the sme supersripts differ signifintly (P <.5). Tle 2: Effet of vitmin E nd DTT on the loss of memrne protein thiols in TBH-treted heptoytes 6 min fter tretment. Tretment Protein thiol level (%) Memrne Cytosol Untreted TBH (1. mm) 78.7 ± ± 6.9 TBH (2. mm) 59. ± ± 7.9 VitminE(μM)+TBH(2.mM) 85.4 ± ± 2.9 EGTA (15 mm) + TBH (2. mm) 76.1 ± ± 2.1 DTT (5 mm) + TBH (2. mm) 75.7 ± ± 5.5 VitminE(μM)+DTT(5mM)+TBH(2.mM) ± ± 1. Vlues re expressed s men ± SD (n = 3-4). Mens in the sme olumn not shring the sme supersripts differ signifintly (P <.5). Fluoresene intensity (.u.) Time (min) Figure 2: Kinetis of hnges in the onentrtion of intrellulr lium in ell,,,d,nd e of Figure 1 efore nd fter treted with 2. mm TBH. lium in the formtion of les on the ell memrne of heptoytes treted with TBH. A signifint inrese in fluoresene intensity nd multiple le formtion in single heptoyte were oserved nd the intensity of fluoresene ws proportionl to the size of les. By pretreting heptoytes with EGTA to remove the extrellulr lium, we found tht no fluoresene intensity ws oserved nd tht the perentge of leing signifintly deresed from 61% in the ontrol group to 27%. Moreover, tretment with EGTA fter le formtion lso redues the perentge of leing. These dt onfirmed tht le formtion is d e ssoited with the inrese in onentrtion of intrellulr lium. However, sine lipid peroxidtion uses dmge of the plsm memrne whih results in n inrese in the numer of ytosoli free lium ions [8], the prevention of leing my e due to the omintion of EGTA with the intrellulr iron whih is required for lipid peroxidtion [21]. In order to rule out this possiility, we nlyzed the effet of EGTA on the lipid peroxidtion used y TBH nd found tht EGTA did not derese lipid peroxidtion under oxidtive stress. Although EGTA does not ffet lipid peroxidtion under oxidtive stress, tretment with this ompound protets TBH-treted ells from deth y preventing the inrese in onentrtion of intrellulr lium [22, 23]. These findings onfirm tht the intrellulr lium inrese used y TBH is exlusively due to lium influx from the extrellulr site [22, 24] nd indite the importne of intrellulr lium in the formtion of plsm memrne leing. There is positive orreltion etween lipid peroxidtion in the memrne nd the loss of memrne protein thiols [25]. Our previous study demonstrted tht protetion of ell morphology y vitmin E is ssoited with protein thiols [1, 11]. Vitmin E prevents the deth of ultured heptoytes treted with TBH [26, 27]. It hs lso een reported tht lium umultion used y lipid peroxidtion is ompletely prevented y vitmin E [8]. In this study, we demonstrted tht vitmin E not only loks the elevtion of intrellulr lium onentrtion ut lso prevents the loss of protein thiols in the memrne of TBH-treted heptoytes. These findings indite tht the integrity of ell memrne onserved y vitmin E my e importnt in the mintenne of intrellulr lium homeostsis.

6 6 Journl of Biomediine nd Biotehnology Fluoresene intensity (% of time ) Fluoresene intensity (% of time ) Time (min) Control (n = 56) +vite(n = 4) + EGTA (n = 52) () Time (min) Control (n = 46) +DTT(n = 38) () Figure 3: The effet of vitmin E nd DTT on the onentrtion of intrellulr lium in TBH-treted heptoytes. () Chnges in onentrtion of intrellulr lium were determined in the ells treted with 2. mm TBH (ontrol), with 2. mm TBH y the pretretment with μm vitmin E for 2 h, or with 15 mm EGTA for 15 min. () Chnges in onentrtion of intrellulr lium were determined in the ells treted with 2. mm TBH (ontrol) or with 2. mm TBH y the pretretment with 5. mm DTT for 15 min. The results were sed on three seprte experiments, nd the vlues re expressed s men ± SD. Tretment mens in the sme time not shring the sme supersripts differ signifintly (P <.5). Although there is n ssoition etween memrne leing nd intrellulr lium onentrtion, leing my lso e indued y other mehnisms, sine les were found in 22% of the heptoytes pretreted with vitmin E, fter TBH tretment. It hs een reported tht the ltertion of ytosoli free lium my not e required for le formtion [28, 29]. Moreover, Hg 2+ -treted heptoytes lso form les on the ell memrne, nd the level of leing is independent of the onentrtions of intrellulr lium [3]. In this study, we found tht lthough DTT redues the loss of ytosoli protein thiols nd dereses le formtion in TBH-treted heptoytes, it n not prevent n inrese in the onentrtion of intrellulr lium in ells whih do form les. However, pretretment with oth vitmin E nd DTT entirely loks le formtion, mintins intrellulr lium homeostsis, nd prevents totl protein thiol loss, lipid peroxidtion, nd the onsumption of GSH. This indites tht plsm memrne leing is reltively omplex nd my e due to mny ftors. Although it hs een reported tht DTT is effetive in preserving the homeostsis of intrellulr lium nd the integrity of the ell memrne [31, 32], the ontroversil results my e due to different ell onditions, different time ourses nd vrying tretment doses. Bsed on the oservtions in this study, vitmin E speifilly prevents the loss of protein thiols in the plsm memrne, while DTT speifilly prevents the loss of protein thiols in the intrellulr site. Thus, these dt indite tht vitmin E my preserve the integrity of the ell memrne y the protetion of memrne protein thiols nd hene mintin intrellulr lium homeostsis of heptoytes under oxidtive stress. These findings suggest tht the different effets of vitmin E nd DTT my provide us with new spet for investigting the mehnism of le formtion under oxidtive stress nd for developing new preventtive strtegy. Aknowledgment This work ws supported y Grnt from the Ntionl Siene Counil (NSC B-4-2 nd NSC B-4-1), Tiwn. Referenes [1] H. A. Ar, F. Ssni, M. H. Rfiee, A. Ftemi, nd A. Jvheri, Histologil nd iohemil ltertions in erlystge lor ishemi-reperfusion in rt liver, World Journl of Gstroenterology, vol. 15, no. 16, pp , 29. [2] S. A. Jewell, G. Bellomo, nd H. Thor, Ble formtion in heptoytes during drug metolism is used y disturnes in thiol nd lium ion homeostsis, Siene, vol. 217, no. 4566, pp , [3] D. J. MConkey nd S. Orrenius, The role of lium in the regultion of poptosis, Journl of Leukoyte Biology, vol. 59, no. 6, pp , [4] M. J. Berridge, M. D. Bootmn, nd P. Lipp, Clium life nd deth signl, Nture, vol. 395, no. 673, pp , [5] B. Hermn, A. L. Nieminen, G. J. Gores, nd J. J. Lemsters, Irreversile injury in noxi heptoytes preipitted y n rupt inrese in plsm memrne permeility, The FASEB Journl, vol. 2, no. 2, pp , [6] G. J. Gores, B. Hermn, nd J. J. Lemsters, Plsm memrne le formtion nd rupture: ommon feture of heptoellulr injury, Heptology, vol. 11, no. 4, pp , 199. [7] E. Niki, Ation of sori id s svenger of tive nd stle oxygen rdils, Amerin Journl of Clinil Nutrition, vol. 54, no. 6, supplement, pp. 1119S 1124S, [8] E. Alno, G. Bellomo, M. Prol, R. Crini, nd M. U. Dinzni, Stimultion of lipid peroxidtion inreses the intrellulr lium ontent of isolted heptoytes, Biohimi et Biophysi, vol. 191, no. 3, pp , 1991.

7 Journl of Biomediine nd Biotehnology 7 [9] A. A. Cro nd A. I. Cederum, Role of intrellulr lium nd phospholipse A2 in rhidoni id-indued toxiity in liver ells overexpressing CYP2E1, Arhives of Biohemistry nd Biophysis, vol. 457, no. 2, pp , 27. [1] S.-T. Wng, J.-H. Kuo, R.-G. Chou, nd C.-K. Lii, Vitmin E protetion of ell morphology nd protein thiols in rt heptoytes treted with tert-utyl hydroperoxide, Toxiology Letters, vol. 89, no. 2, pp , [11] H.-W. Chen, T. Ching, C.-Y. Wng, nd C.-K. Lii, Inhiition of tert-utyl hydroperoxide-indued ell memrne le formtion y α-toopherol nd glutthione, Food nd Chemil Toxiology, vol. 38, no. 12, pp , 2. [12] P. Dogterom, G. J. Mulder, nd J. F. Ngelkerke, Lipid peroxidtion-dependent nd -independent protein thiol modifitions in isolted rt heptoytes: differentil effets of vitmin E nd disulfirm, Chemio-Biologil Intertions, vol. 71, no. 2-3, pp , [13] M. Burnier, G. Centeno, E. Burki, nd H. R. Brunner, Confol mirosopy to nlyze ytosoli nd nuler lium in ultured vsulr ells, Amerin Journl of Physiology, vol. 266, no. 4 prt 1, pp. C1118 C1127, [14] D. J. Reed, J. R. Bson, nd P. W. Betty, High-performne liquid hromtogrphy nlysis of nnomole levels of glutthione, glutthione disulfide, nd relted thiol nd disulfides, Anlytil Biohemistry, vol. 16, no. 1, pp , 198. [15] C. G. Frg, B. E. Leiovitz, nd A. L. Tppel, Lipid peroxidtion mesured s thiorituri id-retive sustnes in tissue slies. Chrteriztion nd omprison with homogentes nd mirosomes, Free Rdil Biology nd Mediine, vol. 4, no. 3, pp , [16] A. F. Boyne nd G. L. Ellmn, A methodology for nlysis of tissue sulfhydryl omponents, Anlytil Biohemistry, vol. 46, no. 2, pp , [17] O.H.Lowry,N.J.Roserough,A.L.Frr,ndR.J.Rndll, Protein mesurement with the Folin phenol regent, The Journl of Biologil Chemistry, vol. 193, no. 1, pp , [18] P. Moldeus, J. Hogerg, nd S. Orrenius, Isoltion nd use of liver ells, Methods in Enzymology, vol. 52, pp. 6 71, [19] H. Miyoshi, K. Umeshit, M. Skon, et l., Clpin tivtion in plsm memrne le formtion during tert-utyl hydroperoxide-indued rt heptoyte injury, Gstroenterology, vol. 11, no. 6, pp , [2] L. M. Pérez, P. Milkiewiz, J. Ahmed-Choudhury, et l., Oxidtive stress indues tin-ytoskeletl nd tightjuntionl ltertions in heptoytes y C 2+ -dependent, PKC-medited mehnism: protetive effet of PKA, Free Rdil Biology nd Mediine, vol. 4, no. 11, pp , 26. [21] G. Minotti nd S. D. Aust, The role of iron in oxygen rdil medited lipid peroxidtion, Chemio-Biologil Intertions, vol. 71, no. 1, pp. 1 19, [22] I. Skid, A. P. Thoms, nd J. L. Frer, Inreses in ytosoli lium ion onentrtion n e dissoited from the killing of ultured heptoytes y tert-utyl hydroperoxide, Journl of Biologil Chemistry, vol. 266, no. 2, pp , [23] J. Heo, G. H. Kim, K. S. Lee, et l., Effet of C 2+ hnnel lokers, externl C 2+ nd phospholipse A2 inhiitors on t- utyl hydroperoxide-indued lipid peroxidtion nd toxiity in rt liver slies, The Koren Journl of Internl Mediine, vol. 12, no. 2, pp , [24] J.-A. Kim, Y. S. Kng, Y. O. Kim, S. H. Lee, nd Y. S. Lee, Role of C 2+ influx in the tert-utyl hydroperoxideindued poptosis of HepG2 humn heptolstom ells, Experimentl nd Moleulr Mediine, vol. 3, no. 3, pp , [25] A. Pompell, A. Romni, A. Benedetti, nd M. Comporti, Loss of memrne protein thiols nd lipid peroxidtion in llyl lohol heptotoxiity, Biohemil Phrmology, vol. 41, no. 8, pp , [26] P. A. Glsott Jr., E. Gilfor, nd J. L. Frer, Effets of vitmin E on the killing of ultured heptoytes y tert-utyl hydroperoxide, Moleulr Phrmology, vol. 41, no. 6, pp , [27] J.-H. Kuo, H.-W. Chen, R.-G. Chou, nd C.-K. Lii, Vitmin E protetion of ell morphology under oxidtive stress is relted to ytoskeletl proteins in rt heptoytes, Arhives of Toxiology, vol. 71, no. 4, pp , [28] J. J. Lemsters, J. DiGuiseppi, A.-L. Nieminen, nd B. Hermn, Bleing, free C 2+ nd mitohondril memrne potentil preeding ell deth in heptoytes, Nture, vol. 325, no. 699, pp , [29] A.-L. Nieminen, G. J. Gores, B. E. Wry, Y. Tnk, B. Hermn, ndj.j.lemsters, Cliumdependeneofleformtion nd ell deth in heptoytes, Cell Clium, vol. 9, no. 5-6, pp , [3] A.-L. Nieminen, G. J. Gores, T. L. Dwson, B. Hermn, nd J. J. Lemsters, Toxi injury from meruri hloride in rt heptoytes, Journl of Biologil Chemistry, vol. 265, no. 4, pp , 199. [31] M. Kretzshmr nd W. Klinger, The hepti glutthione system-influenes of xenoiotis, Experimentl Pthology, vol. 38, no. 3, pp , 199. [32] P. Nioter, D. MConkey, S.-A. Svensson, G. Bellomo, nd S. Orrenius, Correltion etween ytosoli C 2+ onentrtion nd ytotoxiity in heptoytes exposed to oxidtive stress, Toxiology, vol. 52, no. 1-2, pp , 1988.

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