Original Contribution

GLUTATHIONE DEPLETION INCREASES NITRIC OXIDE-INDUCEDOXIDATIVE STRESS IN PRIMARY RAT HEPATOCYTE CULTURES:

INVOLVEMENT OF LOW-MOLECULAR-WEIGHT IRON

SOMPADTHANA SINBANDHIT -TRICOT, JOSIANE CILLARD , MARTINE CHEVANNE, ISABELLE MOREL,PIERRE CILLARD , and ODILE SERGENT

Laboratoire de Biologie Cellulaire et Vegetale, Pharmaceutiques et Biologiques, Rennes Cedex, France

(Received 5 August 2002;Revised 13 January 2003;Accepted 14 February 2003)

Abstract—Various drugs and chemicals can cause a glutathione (GSH) depletion in the liver. Moreover, nitric oxide(NO) can be generated in response to physiological and pathological situations such as inflammation. The aim of thisstudy was to estimate oxidative stress when primary rat hepatocytes were exposed to GSH depletion after NOproduction. For this purpose, cells were preincubated with lipopolysaccharide (LPS) and�-interferon (IFN) for 18 h inorder to induce NO production by NO synthase and thenL-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis,was added for 5 h. In hepatocyte cultures preincubated with LPS and IFN before BSO addition, an increase in lipidperoxidation was noted. In those cells, an elevation of iron-bound NO and a decrease in free NO led us to suggest theinvolvement of low-molecular-weight iron (LMW iron) in the enhancement of oxidative stress. Indeed, addition ofdeferiprone, a chelator of LMW iron, reduced iron-bound NO levels and the extent of oxidative stress. Moreover, animportant elevation of LMW iron levels was also observed. As both, N-acetylcysteine, a GSH precursor, andNG-monomethyl-L-arginine, a NO synthase inhibitor, totally inhibited the elevation of LMW iron and oxidative stress,a cooperative role could be attributed to NO production and GSH depletion. © 2003 Elsevier Inc.

Keywords—Nitric oxide, Glutathione depletion, Low-molecular-weight iron, Lipid peroxidation, Rat hepatocytes,Electron paramagnetic resonance, Buthionine sulfoximine, Free radicals

INTRODUCTION

During hepatic inflammation or endotoxemia, liver pa-renchymal and nonparenchymal cells can respond tocytokine or bacterial lipopolysaccharide stimulation byexpressing an inducible form of nitric oxide synthase,which generates large amounts of nitric oxide (NO) fromL-arginine during a long period [1–3]. In the liver, mainactivity has been ascribed to hepatocytes [4]. Still today,it is difficult to predict the role that NO plays in variousoxidative stress because NO often shows paradoxicalaction [5]. Thus, NO has been reported to act as anantioxidant by scavenging alkoxyl [6] and peroxyl radi-cals [7], or by forming inactive complexes with low-

molecular-weight (LMW) iron [8,9]. LMW iron consistsof iron species that are not contained in high-molecular-weight molecules such as ferritin or mitochondrial fer-roproteins. These iron species can trigger oxidative stressby catalyzing the formation of a highly reactive freeradical, the hydroxyl radical, via Fenton or Haber-Weissreactions [10], which need superoxide anion and/or hy-drogen peroxide. Iron has also been reported to directlyinduce lipid peroxidation by leading to the formation offerryl and perferryl species by reaction with H2O2 andO2

•� [11]. In addition, for a high ratio of NO to O2•�,

NO can eliminate O2•� by removing it through the rapid

formation of peroxynitrite subsequently followed by itsdegradation in nonoxidant products [12,13]. However,with equal fluxes of O2

•� and NO [13], NO reacts withsuperoxide anion, forming peroxynitrite, a potent oxidant[12] capable of oxidizing lipids [14], deoxyribose [15],�-tocopherol [16], aminoacids, and proteins [17]. Thus,NO has been recognized to be involved in oxidative

Address correspondence to: Sompadthana Sinbandhit-Tricot, Labo-ratoire de Biologie Cellulaire et Ve´getale, Faculte´ de Pharmacie, 2, avdu Pr Leon Bernard, 35043 Rennes Cedex, France; Tel:�33 (0)223234932; Fax: �33 (0) 223234886; E-Mail: Sompadthana.Sinbandhit-Tricot@tiscali.fr.

Free Radical Biology & Medicine, Vol. 34, No. 10, pp. 1283–1294, 2003Copyright © 2003 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00108-4

1283

stress induced in the liver during hepatic reperfusion [18]or in hepatocytes treated by cytokines and lipopolysac-charide [9].

Owing to its important role in detoxification, the livercan also be exposed to many hepatotoxic chemicals. NOhas been shown to participate in liver injury induced byacetaminophen [19], ethanol [20], carbon tetrachloride[21], or cocaine [22]. What is interesting to note is that inmost cases, such drugs are able to induce glutathione(GSH) depletion, either directly by drug metabolism orconsequently to the oxidative stress induced by suchcompounds [23–28]. GSH, the most abundant thiol inmany cells [29,30], can protect them from oxidativestress by reacting with hydrogen peroxide, superoxideanion [31], singlet oxygen [32], and hydroxyl radical[33] or by participating as a substrate to GSH peroxidaseactivity, which catalyzes the elimination of hydrogenperoxide, lipid peroxides [29,30,34], and peroxynitrite[35]. Moreover, GSH can help to the redox cycling ofantioxidants such as ascorbate and �-tocopherol [36]. Inaddition, GSH has been described to upregulate the ex-pression and activity of nitric oxide synthase [37–39].

Because GSH can protect from oxidative stress, thisstudy was designed to estimate oxidative stress when rathepatocyte cultures are exposed simultaneously to NO athigh levels and GSH depletion. For this purpose, cultureswere supplemented with lipopolysaccharide (LPS) and�-interferon (IFN) in order to induce NO production byNO synthase and also with L-buthionine sulfoximine(BSO), an inhibitor of �-glutamylcysteine synthetase, anenzyme of GSH synthesis. Previously, we demonstratedthat oxidative stress induced by LPS and IFN in rathepatocytes could be attributed to NO formation becausethe supplementation with the NO synthase inhibitor,NG-monomethyl-L-arginine (NMMA), totally suppressedthe induction of lipid peroxidation by LPS and IFN [9].Our present findings showed that, in rat hepatocyte cul-tures, glutathione depletion, even incomplete, led to theenhancement of NO-induced oxidative stress via an in-crease in LMW iron.

MATERIALS AND METHODS

Chemicals

LPS from Escherichia coli serotype 055:B5, N-ace-tylcysteine (NAC), diethyldithiocarbamate (DETC), so-dium nitrite, and ethylenediaminetetraacetic acid(EDTA) were purchased from Sigma (Saint QuentinFallavier, France). BSO was obtained from ICN Bio-medicals (Orsay, France). IFN was provided fromBachem (Voisins-le-Bretonneux, France). NMMA waspurchased from Calbiochem (Fontenay sous Bois,France). 1,2-dimethyl-3-hydroxy-4-pyridone (deferi-

prone) was provided by Acros Organics (Noisy le Grand,France).

Cell isolation and culture

Adult rat hepatocytes were isolated and cultured aspreviously described [40]. Briefly, adult rat hepatocyteswere isolated from 2-month-old Sprague-Dawley rats bycanulating the portal vein and perfusing the liver with aliberase solution (17 �g/ml; Roche Diagnostics, Meylan,France). The cells were collected in Leibovitz medium.Cell suspension was filtered on gauze and allowed tosediment for 20 min to eliminate cell debris, blood, andsinusoidal cells. The cells were washed three times bycentrifugation at 50 � g, tested for viability (�85%) andcounted. Typically, 20 � 106 hepatocytes were plated in175 cm2 Falcon flasks in a medium (ref.H99HMNAO2052; Biomedia, Boussens, France) consti-tuted of 75% Eagle minimum essential medium and 25%medium 199 with Hanks’ salts, and containing strepto-mycin (50 �g), penicillin (5 �g), bovine insulin (5 �g),bovine serum albumin (1 mg), and NaHCO3 (2.2 mg) permilliliter.

Cultures were supplemented with IFN (500 U/ml) andLPS (20 �g/ml) for 23 h. BSO, an inhibitor of GSHsynthesis, was added at a final concentration of 200 �Mfor 5 h after a preincubation with LPS and IFN for 18 h.Hepatocytes treated only with BSO for 5 h or only withLPS and IFN for 23 h were used as controls. In someexperiments, cells were preincubated with LPS and IFNfor 17 h, then supplemented for 1 h with 1 mM NAC, aGSH precursor, or 60 �M deferiprone, a LMW ironchelator, after which 200 �M BSO was added for afurther 5 h incubation time. In other experiments, cul-tures were simultaneously supplemented with LPS andIFN and 500 �M NMMA, an inhibitor of NO synthase.In a set of experiments, NO was exogenously providedby 100 �M S-nitroso-N-acetyl penicillamine (SNAP), aNO donor, which half-life, in rat hepatocytes, is 5.4 h[41]. SNAP was simultaneously added with BSO for 5 h.

Evaluation of lipid peroxidation

Oxidative stress was analyzed by lipid peroxidationmeasurements using two markers as described previ-ously [42]: extracellular free malondialdehyde (MDA)estimated on the ultrafiltrate of culture medium by size-exclusion chromatography and conjugated dienes evalu-ated by the second-derivative ultraviolet spectroscopy ofthe cell lipid extract.

Measurement of NO production

Three indexes were used. First, two markers wereestimated simultaneously, according to a method previ-ously described [9]: dinitrosyl iron complex (DNIC) in

1284 S. SINBANDHIT-TRICOT et al.

cells and nitrite by the Griess color reaction in culturemedium. The signal of DNIC obtained by the binding ofNO to iron-containing molecules was directly detected inintact cells using electron paramagnetic resonance(EPR). The third marker, the mononitrosyl iron complex(MNIC) EPR signal, was obtained in the cells after spintrapping of free NO with iron-DETC by a method pre-viously described [9]. Tsuchiya et al. have shown invitro, that, iron-dithiocarbamate complexes reacted withnitrite to generate NO, leading to misinterpretation ofMNIC as free NO [43]. Therefore, to check whether thepoorly water soluble dithiocarbamate (DETC), used inour experiments, reacted with nitrites, nitrite amountswere measured, both in media and in hepatocytes ofcultures pretreated with LPS and IFN for 21 h and thenwith DETC for 2 h. Nitrites were measured by high-performance liquid chromatography (HPLC) using amodification of a previously described method [44].DETC addition had no effect on nitrite levels measuredin media and in cells of cultures supplemented with LPSand IFN (Table 1).

Determination of LMW iron by electron paramagneticresonance

Measurement of intracellular LMW iron was basedupon the capacity of deferiprone to chelate only LMWiron and to give a paramagnetic chelate, which had theadvantage to be directly detectable by EPR in wholehepatocytes [45].

Measurement of reduced glutathione levels

Hepatocyte glutathione was measured by HPLC usinga modification of previously described assay [46].Briefly, cells were washed and scraped in cold phos-phate-buffered saline. After centrifugation, cell pelletwas lysed in ice-cold 0.6 N perchloric acid containing 2mM EDTA. Precipitated proteins were removed by cen-trifugation (8000 � g for 2 min). Supernatants werediluted (1:1, v/v) in mobile phase buffer and then pH wasadjusted to 2.0–2.3 with 6 N NaOH. Ten microliterswere injected onto the column for chromatography. The

HPLC system (Constametric Model III pump; LDC Mil-ton Roy, Orsay, France) was equipped with a SpherisorbC18 column 250 mm � 4.6 mm (Cluzeau, Sainte Foy LaGrande, France). Eluant was composed of 100 mM chlo-roacetic acid containing 5% methanol (v/v), at a flow rateof 1 ml/min. Detection of GSH was performed by anelectrochemical detector (ESA Model 5010 Coulochem;Environmental Science Association, Sopares, Gentilly,France). The potential settings were �0.80 V and �0.40V for the two electrodes, respectively.

Measurement of protein levels in hepatocytes

The results obtained for the whole indexes, exceptconjugated dienes were corrected for the cellular proteinconcentration according to the Bradford reaction by us-ing the Bio-Rad (Ivry, France) reagent [47]. Proteincontent was determined on cell homogenates after lysisof the cell pellet obtained by centrifugation at 50 � g.

Table 1. Effect of Diethyldithiocarbamate (DETC) on NitriteAmounts in Rat Hepatocyte Cultures

Nitrites (nmol/mg protein)

Media Cells

No DETC DETC No DETC DETC

CONT 13.0 � 1.0 11.7 � 1.0 1.1 � 0.1 1.0 � 0.1LPS � IFN 77.0 � 4.0 78.0 � 3.0 6.6 � 0.1 6.7 � 0.3

Hepatocyte cultures were incubated without any supplementation[CONT] or with LPS and IFN for 23 h [LPS�IFN].

Fig. 1. Effect of L-buthionine sulfoximine (BSO) on lipid peroxidationinduced by lipopolysaccharide (LPS) and �-interferon (IFN) in rathepatocyte cultures. Lipid peroxidation was estimated by the measure-ment of conjugated dienes (A) and malondialdehyde (MDA) (B).Hepatocyte cultures were incubated without any supplementation[CONT], with BSO for 5 h [BSO], with LPS and IFN for 23 h[LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 5 h[LPS�IFN�BSO]. *p � .05 and **p � .01 compared with controlcultures.

1285Effect of BSO on NO-induced lipid peroxidation

Statistical analysis

Values were expressed as mean � SD from threeindependent experiments. Analysis of variance and New-man-Keuls tests were used to identify statistical signifi-cance for multiple comparisons. Differences were con-sidered significant when p � .05.

RESULTS

Enhancement of NO-induced lipid peroxidation byGSH depletion

LPS and IFN treatment of rat hepatocytes for 23 h ledto the increase of conjugated diene and MDA levels (Fig.1). We previously showed that this increase of lipidperoxidation could be ascribed to NO formation becausethe supplementation with the NO synthase inhibitor,NMMA, totally suppressed the induction of lipid peroxi-dation by LPS and IFN (Fig. 2) [9]. An enhancement ofNO-induced lipid peroxidation could be observed whenBSO was added for 5 h after the treatment of cells withLPS and IFN for 18 h (Fig. 1). It should be noted that, incontrol cultures, BSO did not lead to an increase inconjugated diene and MDA levels (Fig. 1). Moreover,BSO addition increased the loss of cell viability in LPS/IFN-treated hepatocytes (65% of viable cells) when com-pared to cultures incubated only with LPS and IFN (84%of viable cells). In BSO-incubated cultures, cell viabilitywas 89%. In order to know the actual involvement of NOand GSH depletion in the enhancement of NO-inducedlipid peroxidation by GSH depletion, hepatocyte cultureswere also supplemented with NAC, a GSH precursor, or

NMMA, a NO synthase inhibitor. In those cells, lipidperoxidation was inhibited when compared with culturestreated with LPS, IFN and BSO (Fig. 2). It should benoted that NAC was ineffective on lipid peroxidationinduced by LPS and IFN. Moreover, when cultures weresimultaneously supplemented with a NO donor (SNAP)and BSO, lipid peroxidation was also enhanced com-pared to SNAP-treated cells (Fig. 3). In an attempt toexplain how GSH depletion can increase NO-inducedlipid peroxidation in rat hepatocytes, a set of cultures,supplemented with LPS and IFN, was exposed to de-feriprone, a LMW iron chelator (Fig. 4). In hepatocyte

Fig. 2. Effect of N-acetylcysteine (NAC) (glutathione precursor) and NG-monomethyl-L-arginine (NMMA) (nitric oxide synthaseinhibitor) on lipid peroxidation in rat hepatocytes supplemented with lipopolysaccharide (LPS) and �-interferon (IFN) prior to theaddition of L-buthionine sulfoximine (BSO). Hepatocyte cultures were incubated without any supplementation [CONT], with BSO for5 h [BSO], with LPS and IFN for 23 h [LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 5 h [LPS�IFN�BSO].*p � .05 and **p � .01 compared with control cultures.

Fig. 3. Effect of L-buthionine sulfoximine (BSO) on lipid peroxidationinduced by S-nitroso-N-acetyl penicillamine (SNAP) in rat hepatocytecultures. Lipid peroxidation was estimated by the measurement ofmalondialdehyde (MDA). Hepatocyte cultures were incubated withoutany supplementation [CONT], with BSO for 5 h [BSO], with SNAP for5 h [SNAP] or simultaneously with SNAP and BSO for 5 h[SNAP�BSO]. *p � .05 and **p � .01 compared with controlcultures.

1286 S. SINBANDHIT-TRICOT et al.

cultures supplemented with BSO after a pretreatmentwith LPS and IFN, deferiprone addition led to a decreaseof lipid peroxidation (Fig. 4). In those cultures, conju-gated diene and MDA levels were not significantly dif-ferent from those obtained in cultures incubated onlywith LPS and IFN. It should be noted that deferipronewas ineffective on lipid peroxidation induced by LPSand IFN.

Changes in NO pools in response to BSOsupplementation

As expected, LPS and IFN were able to induce NOproduction in hepatocytes, as demonstrated by the ele-vation of DNIC, MNIC, and nitrite levels (Fig. 5). Theaddition of BSO to cultures preincubated with LPS andIFN led to an increase in DNIC levels (Fig. 5A), while adecrease in MNIC levels could be observed (Fig. 5B). InLPS/IFN-treated rat hepatocytes, this elevation of DNICwas not achieved as soon as 2 h after adding BSO,because a further increase of 50% was observed between2 h and 5 h after incubation with BSO (2 h: 7.05 � 0.12

� 107 AU/mg protein; 5 h: 10.6 � 0.8 � 107 AU/mgprotein). No significant variation was found for nitrites(Fig. 5C). It should be remembered that MNIC corre-sponded to free NO and DNIC to NO bound to iron-containing molecules. It is the reason why deferiprone, aLMW iron chelator, was tested on those cultures (Fig. 6).In cultures supplemented with LPS and IFN before BSOaddition, the increase in DNIC was inhibited by de-feriprone treatment (Fig. 6). In order to explain why BSOwas added only after an 18 h preincubation time withLPS and IFN, a time-dependent evaluation of NO poolswas performed in absence of BSO (Fig. 7). It should be

Fig. 4. Effect of deferiprone (low-molecular-weight iron chelator) onlipid peroxidation in rat hepatocytes supplemented with lipopolysac-charide (LPS) and �-interferon (IFN) prior to the addition of L-buthi-onine sulfoximine (BSO). Lipid peroxidation was estimated by themeasurement of conjugated dienes (A) and malondialdehyde (MDA)(B). Hepatocyte cultures were incubated without any supplementation[CONT], with BSO for 5 h [BSO], with LPS and IFN for 23 h[LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 5 h[LPS�IFN�BSO]. *p � .05 and **p � .01 compared with controlcultures.

Fig. 5. Effect of L-buthionine sulfoximine (BSO) on nitric oxide (NO)production in rat hepatocyte cultures supplemented with lipopolysac-charide (LPS) and �-interferon (IFN). NO levels were estimated by themeasurement of dinitrosyl iron complex (DNIC) (NO bound to iron)(A), mononitrosyl iron complex (MNIC) (free NO) (B) and nitrites (C).Hepatocyte cultures were incubated without any supplementation[CONT], with BSO for 5 h [BSO], with LPS and IFN for 23 h[LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 5 h[LPS�IFN�BSO]. *p � .05 and **p � .01 compared with controlcultures.

1287Effect of BSO on NO-induced lipid peroxidation

noted that NO formation began only after 12 h of incu-bation with LPS and IFN (Fig. 7).

Increase in LMW iron pool during BSO treatment

The addition of BSO to cells preincubated with LPSand IFN for 18 h led to an increase in LMW iron whencompared to cells treated only with LPS and IFN (Fig.8). This rise began very early, as soon as 2 h of incuba-tion with BSO. In hepatocytes supplemented for 2 h withSNAP, a NO donor. BSO addition also triggered anincrease in LMW iron levels (Fig. 9). It should be notedthat treatment of cells with BSO, LPS/IFN, or SNAPalone did not lead to an elevation of LMW iron whencompared to control cultures (Figs. 8 and 9). In order to

understand the real involvement of NO and GSH deple-tion in this increase in LMW iron, LPS/IFN-treated he-patocyte cultures were also supplemented with NAC, aGSH precursor or with NMMA, a NO synthase inhibitor(Fig. 10). NAC or NMMA addition led to the decrease ofLMW iron pool, when compared to cultures treated withLPS, IFN, and BSO (Fig. 10).

Time courses of GSH depletion after BSO addition

Following BSO treatment, the decrease of GSH levelsin rat hepatocytes was time-dependent (Fig. 11). After5 h of BSO supplementation, GSH levels were dimin-ished of 50% when compared to control cultures. Itshould be noted that supplementation only with LPS andIFN had no effect on GSH levels (data not shown).

DISCUSSION

In this paper, we showed that, in primary rat hepato-cytes, when NO formation was stimulated by a supple-mentation with LPS and IFN, NO-induced oxidativestress was enhanced by glutathione depletion. First, it isimportant to note that a 5 h incubation time with BSOwas sufficient to deplete GSH levels by 50%. While NOhas been described to induce �-glutamylcysteine syn-thetase in hepatocytes [48,49], in cultures incubated withLPS and IFN, but without BSO, no changes in GSHcontent could be observed when compared with controlcultures. These results are in agreement with those ofKuo et al. [48]. Second, it is interesting to emphasize thatour experiments were performed in order to deplete GSHonly after NO formation. By this way, the possibleinhibition by GSH depletion of NO synthase expressionand activity [37–39] was avoided. Indeed, we showedthat, in rat hepatocytes, at least 12 h of incubation withLPS and IFN were necessary to obtain NO formation, in

Fig. 6. Effect of deferiprone (low-molecular-weight iron chelator) ondinitrosyl iron complex (DNIC) (NO bound to iron) levels in rathepatocytes supplemented with lipopolysaccharide (LPS) and �-inter-feron (IFN) prior to the addition of L-buthionine sulfoximine (BSO).Hepatocyte cultures were incubated without any supplementation[CONT], with BSO for 5 h [BSO], with LPS and IFN for 23 h[LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 5 h[LPS�IFN�BSO]. *p � .05 and **p � .01 compared with controlcultures.

Fig. 7. Time course of nitric oxide (NO) pools evaluated by dinitrosyl iron complex (DNIC) (NO bound to iron), mononitrosyl ironcomplex (MNIC) (free NO) and nitrite levels in rat hepatocyte cultures supplemented with lipopolysaccharide and �-interferon for 27 h.

1288 S. SINBANDHIT-TRICOT et al.

accordance with previous results of Nussler et al. [1].Thus, cells were incubated for 18 h with LPS and IFNbefore BSO addition.

Only one paper related a similar enhancement ofNO-induced lipid peroxidation by GSH depletion, but inrat plasma [50]. To our knowledge, our experiment wasthe first to describe such an increase in oxidative stress inprimary cultures of rat hepatocytes. Most of the otherstudies did not observe an elevation of oxidative stressindexes but rather of other markers of toxicity. For

instance, GSH depletion was demonstrated to augmentNO-induced cell mortality in Chinese hamster HA1 fi-broblasts [51], pulmonary V79 fibroblasts [52], in CHO-AA8 ovary cells [53], in rat gastric mucosa cells [54], inmacrophage cell line [55,56], in P815 murine mastocy-toma cell line [57], in rat neurons and oligodendrocytes[58]. Moreover, GSH depletion enhanced the NO-in-duced inhibition of mitochondrial electron transportchain in rat hepatoma cells [59], in rat aortic smoothmuscle cells and in human umbilical vein endothelialcells [60]. An increase in frequency of DNA strandbreakage had also been reported in rat aortic smoothmuscle cells and human umbilical vein endothelial cells[60].

A crucial question was to elucidate mechanismswhereby NO-induced oxidative stress was enhanced byGSH depletion. An elevation of DNIC signal was ob-served in hepatocyte cultures incubated with LPS andIFN before the addition of BSO, compared with thecultures incubated only with LPS and IFN. DNIC werereported to correspond to the binding of NO with non-heme iron protein [61,62] or LMW iron [63,64]. Thus,because deferiprone, a LMW iron chelator, totally inhib-ited this elevation of DNIC, it could be related to anincrease in LMW iron. In return to this rise of DNIC, freeNO levels were reduced. This inhibition by deferiproneled us to examine LMW iron levels using an EPRmethod, based upon the capacity of deferiprone to che-late only LMW iron and to give a paramagnetic chelate

Fig. 8. Time course of low-molecular-weight (LMW)iron pool in rat hepatocyte cultures supplemented with L-buthionine sulfoximine(BSO) for various incubation times after a preincubation with lipopolysaccharide (LPS) and �-interferon (IFN) for 18 h. Hepatocytecultures were incubated without any supplementation [CONT], with BSO for 1, 2, 3, 4, or 5 h [BSO], with LPS and IFN for 19, 20,21, 22 or 23 h [LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 1, 2, 3, 4 or 5 h [LPS�IFN�BSO]. *p � .05 and**p � .01 compared with control cultures.

Fig. 9. Effect of L-buthionine sulfoximine (BSO) on low-molecular-weight iron pool in rat hepatocytes supplemented with S-nitroso-N-acetyl penicillamine (SNAP). Hepatocyte cultures were incubated with-out any supplementation [CONT], with BSO for 2 h [BSO], with SNAPfor 2 h [SNAP] or simultaneously with SNAP and BSO for 2 h[SNAP�BSO]. *p � .05 and **p � .01 compared with controlcultures.

1289Effect of BSO on NO-induced lipid peroxidation

[45]. An important increase of 80% in LMW iron levelswas obtained after 2 h of incubation with BSO in culturespretreated with LPS and IFN for 18 h. Because a con-tinuous increase in DNIC content was observed between2 h and 5 h after adding BSO in the LPS/IFN-treatedhepatocytes, it could be suggested that during this time,LMW iron pool reacted with free NO, explaining thatafter 5 h of incubation with BSO, free NO and LMW ironlevels were reduced (Fig. 12). Moreover, deferipronewas also able to inhibit lipid peroxidation in culturesincubated with LPS and IFN before a further 5 h incu-bation time with BSO. This result suggested that iron

was responsible for the enhancement by BSO of NO-induced oxidative stress. Although the maximum rise inLMW iron was observed at 2 h, lipid peroxidation furtherincreased from 2–5 h likely by a mechanism of propa-gation of lipid peroxidation in membranes [10]. Like-wise, ethanol intoxication of hepatocytes led to a LMWiron release at 1 h and to a maximum oxidative damageat 5 h [65]. Previously, we reported that NO has an

Fig. 10. Effect of N-acetylcysteine (NAC) (glutathione precursor) and NG-monomethyl-L-arginine (NMMA) (nitric oxide synthaseinhibitor) on low-molecular-weight iron pool in rat hepatocytes supplemented with lipopolysaccharide (LPS) and �-interferon (IFN)prior to a 2 h L-buthionine sulfoximine (BSO) incubation time. Hepatocyte cultures were incubated without any supplementation[CONT], with BSO for 2 h [BSO], with LPS and IFN for 20 h [LPS�IFN] or with LPS and IFN for 18 h and then with BSO for 2 h[LPS�IFN�BSO]. *p � .05 and **p � .01 compared with control cultures.

Fig. 11. Time course of glutathione (GSH) levels in rat hepatocytecultures incubated with L-buthionine sulfoximine (BSO) for 5 h.

Fig. 12. Postulated mechanism for the involvement of low-molecular-weight (LMW) iron in the enhancement of nitric oxide (NO)-inducedoxidative stress by glutathione (GSH) depletion in rat hepatocytes. Bya cooperative way, GSH depletion and NO production led to anincrease in LMW iron, which consequently changed NO pools byenhancing NO bound to iron [dinitrosyl iron complex (DNIC)] andreducing free NO [mononitrosyl iron complex (MNIC)]. NAC �N-acetylcysteine; BSO � L-buthionine sulfoximine; LPS � lipopoly-saccharide; IFN � �-interferon; NMMA � NG-monomethyl-L-argi-nine.

1290 S. SINBANDHIT-TRICOT et al.

inhibitory effect on exogenous iron-induced lipid per-oxidation [9]. This apparent discrepancy comparedwith our present work could be explained by the levelsof LMW iron and thereby of lipid peroxidation ob-served in cells cultured without NO formation. Inabsence of NO, both the LMW iron content and theextent of lipid peroxidation were very high in exoge-nous iron-treated cultures [9], whereas, in BSO-addedcultures, these values were almost the same as that ofcontrol cultures. When NO was produced by LPS andIFN supplementation of cultures, NO partially pro-tected from exogenous iron-induced oxidative stressby complexing iron to form DNICs [9], which corre-sponded to a way to inhibit the reactivity of LMW iron[8]. However, the intracellular LMW iron content wasat such a high level that only a fraction of this iron wasinactivated by NO explaining the incomplete inhibi-tion of iron-induced lipid peroxidation [9]. Therefore,in exogenous iron-treated cells, NO-induced lipid per-oxidation was enhanced by the fraction of LMW ironnot complexed by NO. In the present work with BSO,it was quite the same mechanism as above. The addi-tion of BSO to LPS/IFN-treated cultures led to anincrease of 80% in LMW iron content, which is par-ticularly high. Likely, the ability of NO to react withLMW iron was overcome, so that the iron, not com-plexed by NO, could enhance NO-induced lipid per-oxidation. Finally, a balance between the amounts ofNO and of LMW iron should be considered in hepa-tocytes. Thus, in previous studies about the effect ofNO on ethanol-induced oxidative stress in hepato-cytes, the release of LMW iron by ethanol metabolism,in absence of NO, was found to be moderate (only�30%) [65], so that, when NO was produced in thosecells, the levels of LMW iron return to that of controlcultures [45] and ethanol-induced lipid peroxidationwas totally inhibited by NO [9].

In hepatocyte suspensions, Tirmenstein et al. reporteda LMW iron release following GSH depletion alone [66],but, in our case, no increase in LMW iron pool wasobserved in cultures incubated only with BSO. In rathepatocyte cultures, the mechanism of LMW iron in-crease seems to be specific of conditions, for which aGSH depletion is associated to an endogenous NO syn-thesis. Therefore, we tempted to determine the actualinvolvement of GSH depletion and NO in this elevationof LMW iron. Both, the addition of NAC, a GSH pre-cursor, or NMMA, a NO synthase inhibitor, was able tototally inhibit the rise of LMW iron and consequently todecrease lipid peroxidation (Fig. 12). In addition, as Kuoet al. [48] reported that NMMA supplementation de-creased GSH content in hepatocytes, GSH depletioninduced by BSO should be even more pronounced inNMMA-supplemented cultures. Therefore, the protec-

tion that we observed towards lipid peroxidation andLMW iron elevation highlighted the role of NO. More-over, when a NO donor, SNAP, was simultaneouslyadded with BSO to rat hepatocytes, an increase in LMWiron and lipid peroxidation was also observed comparedto cultures treated only with BSO, emphasizing again theinvolvement of NO. With this NO donor, the lower risein LMW iron (�50%) compared to results obtained withendogenous NO (�80%), was accompanied with a lowerincrease in lipid peroxidation, showing the involvementof iron in oxidative stress in these conditions. The com-plete inhibition of the increase in LMW iron by NAC orby NMMA led us to suggest a cooperative role for NOand GSH depletion. Thus, NO and GSH depletion couldpromote both a heme degradation and a decrease inferritin content. First, NO [67,68] and GSH depletion[69,70] are well-known to induce heme oxygenase ex-pression in rat liver and cultured liver cells. Heme oxy-genase catalyzes heme degradation, which leads to theformation of biliverdine and free iron [71,72]. Moreover,NO is recognized to increase heme levels by releasingheme from proteins [67,68]. However, in hepatocytes,Kim et al. reported that the amount of protein-boundheme-iron was nearly 0.25 nmol iron/mg protein [68],which is much less than the increase in LMW iron (�0.5nmol/mg protein) observed in our experiments. There-fore, another source of LMW iron could be suggestedlike ferritin-bound iron. Indeed, IRP-1 could be activatedby NO [73], decreasing the amount of an iron-storageprotein, ferritin [74] and the proportion of ferritin-boundiron [74], which could lead to an enhancement of LMWiron levels. In addition, the activation of IRP-1 was morepronounced in cells with low GSH content [75]. Re-cently, it has been shown that NO mobilized iron fromferritin in a GSH-dependent manner [76]. It should benoted that, in our experiments, GSH depletion was in-complete. Lastly, Mulero et al. reported that NO can alsofacilitate iron release, when iron was delivered by aphagocytic route independently of iron acquired fromtransferrin [77].

In summary, it can be concluded that (1) a GSHdepletion even incomplete can lead to an increase inNO-induced oxidative stress and (2) the mechanism ofsuch an enhancement involved an important elevation ofLMW iron, which can overcome the ability of NO toprotect from iron toxicity (Fig. 12). Although evidenceof a role for heme oxygenase or IRP is still lacking, thetotal protection by NAC or by NMMA encourage thehypothesis of its involvement.

Acknowledgement — This work was supported by Langlois Foundation(Rennes, France).

1291Effect of BSO on NO-induced lipid peroxidation

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1293Effect of BSO on NO-induced lipid peroxidation

ABBREVIATIONS

BSO—buthionine sulfoximineDETC—diethyldithiocarbamateDNIC—dinitrosyl iron complexEPR—electron paramagnetic resonanceGSH—glutathioneIFN—�-interferon

LMW iron—low-molecular-weight ironLPS—lipopolysaccharideMDA—malondialdehydeMNIC—mononitrosyl iron complexNAC—N-acetylcysteineNMMA—NG-monomethyl-L-arginineNO—nitric oxideSNAP—s-nitroso-N-acetyl penicillamine

1294 S. SINBANDHIT-TRICOT et al.