Original Contribution

CHANGES IN GLUTATHIONE STATUS AND THE ANTIOXIDANT SYSTEMIN BLOOD AND IN CANCER CELLS ASSOCIATE WITH TUMOUR

GROWTH IN VIVO

JOSE NAVARRO,* ELENA OBRADOR,* JULIAN CARRETERO,* I GNACIO PETSCHEN,† JOSE AVINO,** PILAR PEREZ,‡

and JOSE M. ESTRELA**Departamento de Fisiologı´a and **Departamento de Pediatria, Ginecologı´a y Obstetricia, Universidad de Valencia, Facultad deMedicina, Valencia, Spain,†Servicio de Radioterapia, Hospital Universitario La Fe, Valencia, Spain, and‡Fundacio´n Instituto

Valenciano de Oncologı´a, Valencia, Spain

(Received17 April 1998;Revised23 July 1998;Accepted27 July 1998)

Abstract—The relationship among cancer growth, the glutathione redox cycle and the antioxidant system was studiedin blood and in tumour cells. During cancer growth, the glutathione redox status (GSH/GSSG) decreases in blood ofEhrlich ascites tumour-bearing mice. This effect is mainly due to an increase in GSSG levels. Two reasons may explainthe increase in blood GSSG: (a) the increase in peroxide production by the tumour that, in addition to changes affectingthe glutathione-related and the antioxidant enzyme activities, can lead to GSH oxidation within the red blood cells; and(b) an increase of GSSG release from different tissues into the blood. GSH and peroxide levels are higher in the tumourcells when they proliferate actively, however GSSG levels remain constant during tumour growth in mice. Thesechanges associate with low levels of lipid peroxidation in plasma, blood and the tumour cells. The GSH/GSSG ratio inblood also decreases in patients bearing breast or colon cancers and, as it occurs in tumour-bearing mice, this changeassociates with higher GSSG levels, especially in advanced stages of cancer progression. Our results indicate thatdetermination of glutathione status and oxidative stress-related parameters in blood may help to orientate cancer therapyin humans. © 1998 Elsevier Science Inc.

Keywords—Glutathione, Blood, Cancer growth, Ehrlich, Breast carcinoma, Colon carcinoma, Free radicals, Oxidativestress

INTRODUCTION

Increased flux of oxy-radicals and loss of cellular redoxhomeostasis can be tumorigenic [1]. A wide variety ofnormal and malignant cell types generate and releasesuperoxide or hydrogen peroxide in vitro either in re-sponse to specific cytokine/growth factor stimulus orconstitutively in the case of tumour cells [2]. Cancer cellscan generate large amounts of hydrogen peroxide andthis, if it occurs in vivo, may contribute to their ability tomutate and damage normal tissues, and, moreover, facil-itate tumour growth and invasion [3]. Indeed, it has beensuggested that persistent oxidative stress in tumour cellscould partly explain some important characteristics ofcancer, such as activated protooncogenes and transcrip-

tion factors, genomic instability, chemotherapy- or ra-diotherapy-resistance, invasion, and metastasis [4]. How-ever, tumour-induced oxidative stress in vivo and itsconsequences remain open questions.

Resistance of many cells against oxidative stress isassociated with high intracellular levels of glutathione(g-glutamyl-cysteinyl-glycine; GSH) [5–7]. GSH actsdirectly as a free radical scavenger by neutralizing HO•,restores damaged molecules by hydrogen donation, re-duces peroxides, and maintains protein thiols in the re-duced state [8]. GSH and thiol redox status regulateexpression of genes involved in the pathogenesis ofdifferent diseases, including cancer, AIDS, diabetes, oratherosclerosis [9]. Oxidative stress may cause changesin the glutathione redox state of different tissues and,thereby, increase the rate of glutathione disulfide (GSSG)release from cells [10]. In fact, increases in GSSG levelsinside cells and/or in GSSG efflux from cells are signals

Address correspondence to: Dr. Jose´ M. Estrela, Departamento deFisiologıa, Facultad de Medicina, Av. Blasco Iban˜ez 17, 46010 Valen-cia, Spain; E-Mail: jose.m.estrela@uv.es.

Free Radical Biology & Medicine, Vol. 26, Nos. 3/4, pp. 410–418, 1999Copyright © 1998 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/99/$–see front matter

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of oxidative stress [8,11]. Moreover, interorgan flows ofglutathione have been proposed [12]. Therefore, GSHand GSSG levels in blood may reflect changes in gluta-thione status in other less accessible tissues [13]. Indeed,different studies have pointed out the interest of measur-ing blood glutathione for pathological and physiologicalpurposes [13–19]. In addition, the erythrocyte has provedto be a valuable cell method for studying oxidative stress[20]. This is due to the fact that the mammalian eryth-rocyte has a limited life span (55–60 d in mice), isincapable of protein synthesis de novo, has the capacityfor only low extents of proteolysis, and shows decreasedenzymic protection and increased sensitivity to oxidativedamage with aging [21].

Previously, it has been reported that elevation of in-tracellular GSH in tumour cells is associated with mito-genic stimulation [22], that GSH controls the onset oftumour-cell proliferation by regulating protein kinase Cactivity and intracellular pH [23], and that severe tu-mour-induced cachexia in the host is accompanied by adecrease in the rate of cancer cell proliferation and adecrease in GSH in the tumour [24]. GSH and its depen-dent enzymes work in concert with other antioxidantsand antioxidant enzymes to protect cells against reactiveoxygen intermediates (ROIs) [8]. Thus, changes in therate of cancer-cell proliferation are accompanied bychanges in their intracellular GSH levels and, conse-quently, these could be reflected by changes in the anti-oxidant machinery. The aim of this study was to assesswhether tumour-induced oxidative stress and growth arereflected by changes in the glutathione redox cycle andthe antioxidant system in blood and in cancer cells.

MATERIALS AND METHODS

Animals and tumour inoculation

We used adult male mice OF1 from IFFA CREDO(Madrid, Spain). The animals were fed ad libitum on astock laboratory diet (Letica, Barcelona, Spain) and kepton a 12 h-light/12-h dark cycle with the room tempera-ture maintained at 22°C. All experiments were carriedout or started between 10.00 and 12.00 h to minimize theeffects of diurnal variation. The Ehrlich ascites tumour(EAT) was kindly provided by the Department of Pa-thology (Universidad de Valencia) and inoculated intra-peritoneally as previously described [24]. All injectedmice developed an ascites tumour. The animals died15.26 1.0 d (n 5 16) after inoculation of the tumour.

Isolation and compartmentation of tumour cells

Cells were collected from tumour-bearing mice, andthen isolated and separated in intracellular and extracel-lular compartments, as previously reported [24].

Cancer patients

We studied two groups of patients attending an out-patient oncology clinic. Patients were grouped using thetumour-nodes-metastasis (TNM) staging system adoptedby the International Union against Cancer. There were 20women with stage T1 (without regional lymph nodemetastasis and with no evidence of distant metastasis),and 14 with stage T3 breast carcinoma (from N0 to N2;and from M0 to M1); and, in addition, 13 men andwomen with stage T1 (N0 and M0), and 7 with stage T3colon carcinoma (from N1 to N2; and from M0 to M1),which were scheduled to undergo treatment but had notreceived previous surgery, chemotherapy or radiother-apy. Therefore, we could compare, in both groups, initial(T1) and advanced stages (T3) of cancer growth.

Blood collection and processing

Blood was collected from the heart (mice) or from thevena mediana cubiti (humans) into 1 ml syringes con-taining sodium heparin (0.05 ml of a 5% solution in 6.9%NaCl). Plasma and erythrocytes were separated as pre-viously reported [20]. The pelleted erythrocytes werewashed in ice-cold Krebs-Henseleit bicarbonate medium(pH 7.4) to yield the original hematocrit. For GSH,GSSG or total glutathione measurements, whole blood,erythrocytes, or plasma were treated as previously de-scribed [20].

Enzymatic measurement of GSH

GSH was analyzed by the glutathione-S-transferaseassay [25].

Determination of GSSG by h.p.l.c.

Samples treated with N-ethylmaleimide and batho-phenanthroline disulfonic acid (Sigma Chemical Co., St.Louis, MO, USA) were derivatized and analyzed byh.p.l.c. as previously described [20,26]. This procedurereduces GSH oxidation in biological samples to about1%.

Measurement of total glutathione

Total glutathione, expressed as the sum of the reducedand oxidized forms (GSH1 2 GSSG), was determinedin plasma by a kinetic assay in which a catalytic amountof GSH or GSSG and glutathione reductase cause thecontinuous reduction of 5,59dithiobis(2-nitrobenzoicacid) (Sigma Chemical Co.) by NADPH [25].

411Glutathione status and the antioxidant system in blood and in cancer cells

Measurement of enzyme activities

Glutathione-reelated and antioxidant enzyme activi-ties were determined in cancer cells and fresh erythro-cytes. Cancer cells, obtained from tumour-bearing mice,were washed twice at 4°C in Krebs-Henseleit bicarbon-ate medium (pH 7.4) without Ca21 or Mg21 added andcontaining 0.5 mM EGTA, and then suspended and ho-mogenized in 0.1 M phosphate buffer (pH 7.2) at 4°C.After centrifugation, the erythrocytes were resuspendedin distilled water and lysed for 2 h at4°C. The lysate wasdiluted to a concentration of approximately 50 mg ofhemoglobin (Hb) per milliliter and, then, used for assays.Hb values were estimated as previously described [27].

Glutathione reductase (GR) activity was determinedas described by Akerboom and Sies [25], glucose-6-phosphate dehydrogenase (G6PDH) as described byLohr and Waller [28], and glutathione S-transferase(GST) as described by Habig et al. [29].

Glutathione peroxidase (selenium-dependent) (GPX)activity was measured as described by Flohe´ and Gunzler[30]. In tumour cells, H2O2 was used as a substrate. Inthe case of red blood cells, tert-butyl hydroperoxide,instead of H2O2, was utilized; and in addition, to avoidinterference of methemoglobin, the diluted lysate wasmixed with an equal volume of 23 1022 M KCN in 0.1M phosphate buffer pH 7.0.

Superoxide dismutase (SOD) activity was measuredas described by Flohe´ and Otting [31], using 2 mMcyanide in the assay medium to distinguish mangano-type enzyme (MnSOD) from the cuprozinc type (CuZn-SOD). Catalase (CAT) activity was analyzed as de-scribed by Aebi [32].

g-Glutamyltranspeptidase (GGT) activity in tissueswas determined as described by Shaw et al. [33].

Flow cytometry

Cellular suspensions were diluted to approximately400,000 cells/ml. Flow cytometric analysis were per-formed with an EPICS PROFILE II (Coulter Electron-ics, Hieleah, FL, USA). Fluorochromes were fromMolecular Probes (Poortgebouw, Leiden, The Nether-lands), exepting dihydroethidium which was fromSigma. Fluorochromes were excited with an argonlaser tuned at 488 nm. Forward-angle light scatter andright-angle light scatter were mesured and fluores-cence was detected through a 488-nm blocking filter, a550-nm long-pass dichroic and a 525-nm band-pass ora 575-nm long-pass. Samples were acquired for10,000 individual cells. Cell viability was determinedby the fluorescent dye propidium iodide (final concen-tration 10mM) at 630 nm fluorescence emission andby light scatter properties. The rest of the studies were

limited to viable cells. Intracellular peroxide levelswere measured with 29,79-dichloro-dihydro-fluores-cein diacetate [34] (10mg/ml, final concentration) at525 nm fluorescence emission.

Lipid peroxidation

High-performance liquid chromatographic separationof malondialdehyde-thiobarbituric acid (MDA-TBA) ad-duct was utilized to evaluate lipid peroxidation inplasma, erythrocytes, and tumour cells. We used a vari-ation of the technique described by Richard et al. [35].For this purpose, plasma and erythrocytes were obtainedas indicated above. To a 100ml aliquot of fresh eryth-rocytes were added 50ml of cold water and 10ml of a 2%butylated hydroxytoluene solution, followed by depro-teinization with 150 ml of cold trichloroacetic acid(20%). After centrifugation (2000 g) for 10 min thesupernatant was removed for analysis. Tumour cellswere isolated as indicated above and homogenized inhypotonic Tris-HCl buffer (0.05 mM, pH 7.2) containing1 mM EDTA. Then, in each case, 0.1 ml of sample (orstandard solution prepared daily from tetramethoxypro-pane) and 0.7 ml of working solution (0.4% TBA and 6%perchloric acid, 2:1, v/v) were mixed and heated to 95°Cfor 1 h to allow hydrolysis of the lipoperoxides. Afterchilling (10 min in an ice-water bath), the flocculentprecipitate was removed by centrifugation at 3500 g for10 min. The supernatant was neutralyzed with KOH,centrifuged immediately at 1600 g for 3 min, and filtered(0.22 mm) prior to injection on an Ultrasphere ODScolumn (5m) (25 3 0.46 cm) from Teknokroma (Bar-celona, Spain). Mobile phase consisted in 50 mM phos-phate buffer (pH 6.0) and methanol (58:42, v/v). Iso-cratic separation was performed with 1.0 ml3 min21

flow, and detection at 532 nm. Calibration curves wererun daily and assayed using tetramethoxypropane, whichundergoes hydrolysis to liberate stoichiometric amountsof MDA [36].

Expression of results and statistical significance

Results are expressed as means6 S.D. for the indi-cated number of different experiments. The statisticalsignificance was assessed by Student’st-test.

RESULTS AND DISCUSSION

Blood glutathione and tumour growth

Previously, we studied growth-related parametersand glutathione content in EAT growing in vivo andshow that, over a period of 2 weeks between inocula-tion and severe cachexia in the host, the tumour grows

412 J. NAVARRO et al.

exponentially until cell number reaches a plateau ap-proximately 8 –10 d after inoculation [24]. GSH levelsincrease in the tumour cells when they proliferateactively, reaching a maximum approximately on day 7and decreasing gradually over the next week as thetotal cell number increases and the growth rate de-clines [24]. As shown in Table 1, the volume of asciticfluid increased 2.6-fold from day 7 to day 14, althoughtumour cell density decreased significantly. GSH con-centration in cancer cells changed when differentstages of tumour growth were compared, however,GSSG contents did not vary significantly (Table 1).Thus, the change in the GSH/GSSG ratio within thetumour, which is found on day 14, is mainly due to adecrease in the GSH content (Table 1). Interestingly,when GSH and GSSG levels where determined inblood of tumour-bearing mice, we found that the GSH/GSSG ratio decreases progressively as the tumourgrows (Table 1). This finding in blood becomes sig-nificant when the tumour shows the highest rate of cellproliferation (around day 7) and last until host’s death(Table 1). However, in blood, the change in the glu-tathione status is mainly due to an increase in theGSSG level (Table 1). These facts suggest possiblecorrelations between changes in the glutathione statusin blood and in cancer cells.

GSH and GSSG in blood are largely located withinthe erythrocytes. However, it is known that red cellglutathione is transported outward through the mem-brane into the plasma as either GSSG or thioester con-jugates. Indeed, kinetic studies of the glutathione trans-port system, using inverted fragments of red cellmembranes, showed that transport occurs, that it requiresATP, and that GSH is not transported (for a review seeref. [37]). Therefore, we also measured GSSG and GSHin the plasma compartment and found that, in plasma ofnon-tumour-bearing mice GSSG levels were 1.56 0.4mM (n 5 7) and GSH (total glutathione2 GSSG) levels8.66 2.2 mM (n 5 7). Besides, GSSG levels in plasma

of tumour-bearing mice rose to 5.56 1.6 (n 5 7; p ,.01) at day 7after inoculation and stayed around thisvalue, without significant changes, from day 7 to day 14(results not shown). However, GSH values in plasma, atdifferent stages of tumour growth, did not change sig-nificantly when compared to non-tumour-bearing con-trols (results not shown). Besides, it has been reportedthat cellular GSH levels in different tissues decreaseunder conditions of shock, stress, or peripheral inflam-mation [8,11]. Indeed, GSH and GSSG efflux from ratliver is stimulated by various stress-related hormones,including vasopressin, phenylephrine, and adrenaline[38]. Furthermore, we found that, as compared to healthycontrols, GSH levels decrease in liver and kidney ofEAT-bearing mice [23]. Thus, the increase in bloodGSSG, accompaning tumour growth (Table 1), can bedue to GSH oxidation within the red cells and, in addi-tion, an increase of GSSG release from different tissuesinto the blood.

The blood decreases in GSH may also be due to adecrease in available substrate for GSH synthesis.However, in a previous report [24] we showed that thisis not the case because the blood supply of the aminoacids involved in GSH synthesis is not limited inEAT-bearing mice. Moreover, cyst(e)ine (cysteine1cystine) concentration, the rate-limiting amino acid forGSH synthesis [10], increases significantly in blood ofmice bearing the EAT for 14 d compared with non-tumour-bearing controls or mice bearing the tumourfor 7 days [24].

Naturally, possible changes in general hematologi-cal features [39] were taken into account in our cal-culations. A summary of haematological data is givenin Table 2. Interestingly, the hematocrit, the Hb con-centration and the number of erythrocytes were sig-nificantly decreased in mice bearing the tumour for14 d. In fact, anemia is found frequently in cancerpatients [39]. These findings, as well as those shownfor white cells (where although lymphopenia and neu-

Table 1. Changes in Blood GSH and GSSG Levels in Ehrlich Ascites Tumour-Bearing Mice

Parameter

Time after Inoculation of the tumour (d). . .

0 4 7 10 14

Tumour volume (ml) 1.26 0.3 5.76 0.6a 10.36 0.8a 15.16 1.4a

1026 3 tumour cell density (no. of cells/ml) 7256 67 6236 77 5666 52a 4316 47a

Blood GSH (mmol/g Hb) 7.16 0.7 6.96 0.5 6.56 0.8 5.76 0.8 5.06 0.6a

Blood GSSG (nmol/g Hb) 256 6 306 5 506 12a 576 10a 426 12a

Blood GSH/GSSG 3146 73 2386 55 1526 27a 1246 48a 1276 53a

Tumour GSH (mmol/g) 1.36 0.3 3.06 0.5a 1.46 0.5 0.86 0.2a

Tumour GSSG (nmol/g) 186 8 356 12 236 10 226 10Tumour GSH/GSSG 746 21 986 55 626 11 386 16a

Results are expressed as means6 S.D. for 9–10 animals.a Significantly different from day 4 group (measurements in the tumour) or from day 0 group (measurements in the blood) (p , .05).

413Glutathione status and the antioxidant system in blood and in cancer cells

trophilia associated with tumour growth (Table 2), thepercentage of monocytes, eosinophils and basophils intumour-bearing animals did not vary as compared tocontrols (results not shown) or platelets (Table 2), arecommon features in tumour-bearing hosts [39].

Glutathione-related enzyme activities

In order to determine the possible reasons for thechange in glutathione status, we measured, in cancercells and in erythrocytes, the activities of GR, G6PDH,GPX and GST, i.e., the main enzymes involved in theglutathione redox cycle. As shown in Table 3, theactivities of GPX and GST, i.e., the enzymes thatutilize GSH, increase in the tumour when low rates ofcellular proliferation (day 14) are compared with highrates (day 7). In parallel, both activities are also in-creased in erythrocytes of tumour-bearing mice as

compared to non-tumour-bearing controls, being thechange significant with low rates of tumour growth(Table 3). On the other hand, GR and G6PDH, i.e.,enzymes that tend to regenerate GSH, did not changesignificantly in the cancer cells, when different stagesof tumour growth where compared. However, bothactivities tend to increase in erythrocytes of tumour-bearing mice as compared to non-tumour-bearing con-trols (Table 3). It is interesting to notice that therelative activity of the GPX in erythrocytes is muchhigher than the GR activity, thus, in addition to thesupply of NADPH for the GR, changes in GPX activ-ity could also play a role in determining the glutathi-one status within the red blood cells. Therefore, en-zymic changes affecting the glutathione-relatedactivities could explain, at least in part, the shifttoward oxidation of the glutathione status within thetumour and the red blood cells.

Table 2. Hematological Findings in Tumour-Bearing Mice at Different Stages of Tumour Growth

Parameter

Time after Inoculation of the Tumor (d). . .

0 7 14

Hematocrit (%) 39.36 1.7 38.06 4.5 28.26 3.9a

Hemoglobin (g/100 ml) 14.46 1.5 14.36 1.2 11.06 1.3a

Mean corpuscular volume (fl/cell) 46.86 0.7 47.06 1.5 47.16 2.3Mean corpuscular hemoglobin (pg/cell) 17.06 0.6 18.06 0.8 18.66 0.5a

Mean corpuscular hemoglobinconcentration (g/100 mL)

36.26 0.7 38.26 0.5a 39.56 1.1a

1026 3 No. of erythrocytes (perml) 8.5 6 0.3 8.36 0.9 6.36 1.1a

1023 3 No. of leucocytes (perml) 9.1 6 1.1 18.66 3.7a 12.16 3.01023 3 No. of platelets (perml) 4276 112 7196 157a 5366 173Lymphocytes (%) 596 7 256 6a 286 10a

Polymorphonuclear neutrophils (%) 356 6 686 7a 676 10a

Results are expressed as means6 S.D. for 9–10 animals.a Significantly different from controls (day 0 group) (p , .05).

Table 3. Glutathione-Related and Antioxidant Enzyme Activities in Erythrocytes and Cancer Cells at Different Stages of Tumour Growth in Mice

Time after inoculation of the tumour (d). . .

0 7 14

RBC RBC Tumour RBC Tumour

Glutathione reductase 6.36 1.3 7.36 1.5 1.56 0.3 12.76 1.9 1.86 0.5Glucose-6-phosphate dehydrogenase 9.16 1.5 12.36 1.4a 0.56 0.1 13.96 2.0a 0.46 0.1Glutathione peroxidase 1556 27 1476 22 0.86 0.2 2086 43a,b 2.96 0.3a

Glutathione S-transferase 6.06 1.0 5.56 1.7 1.96 0.3 11.06 2.5a,b 2.56 0.4a

Superoxide dismutase 2.56 0.3 3.66 0.5a 3.96 0.3 4.46 0.5a 3.06 0.2a

Catalase 1876 46 3416 58a 2.46 0.5 1906 27 1.26 0.4a

RBC, red blood cells, GR, G6PDH, GPX and GST activities are expressed as U/g of cell (tumour) or U/g of Hb (erthrocytes). SOD activity isexpressed as U/mg of cell or Hb. SOD in RBC represent exclusively the CuZn-type activity. In tumour cells, control MnSOD activity was ofapproximately 5% of the total SOD activity measured on day 0 after inoculation of the tumour. This activity remained unchanged in the tumour untilday 14, therefore, changes observed in total SOD activity during cancer growth refer to changes in CuZnSOD activity. CAT activity is expressed asK/g of cell or Hb (see the “Materials and Methods” section). Results are expressed as means6 S.D. for 6–7 animals.

a Significantly different from day 7 group (tumour) or from day 0 group (RBC) (p , .05).b Significantly different from day 7 group (RBC) (p , .05).

414 J. NAVARRO et al.

Oxidative stress in relation to tumour growth

As shown in Fig. 1, tumour cells produce more per-oxides when they proliferate actively (between days 5–9after inoculation of the tumour [23]. This rise in perox-ides indicates that intensification of oxygen free radicalproduction occurs and that GSH oxidation should beexpected. In addition, the increase in glutathione perox-idase activity (Table 3) should be reflected by an increasein the GSSG content of the tumour cells. However,GSSG levels remain constant in the tumour so long as itgrows (Table 1).

During the onset of severe tumour-related weight lossin the host [24], when the rate of tumour growth de-creases, catalase (CAT) activity decreases by 50%whereas superoxide dismutase (SOD) activity decreasesslightly in the tumour cells (Table 3). As shown in Fig.1, this is accompanied by a significant and progressivedecrease in peroxide levels. In consequence, the rate oflipid peroxidation in the tumour was found lower on day14 (Table 4). In agreement with this finding, reducedrates of lipid peroxidation have been observed in differ-ent experimental tumours [40]. This is important becausea number of observations indicate an apparent inverserelationship between levels of lipid peroxidation, andrates of cell proliferation and/or extent of differentiation[2,41]. In fact, the periodic bursts of DNA synthesis(S-phases) can be linked to corresponding depressions inthe susceptibility of membranes to lipid peroxidation[42]. Furthermore, the results displayed in Fig. 1 are also

in agreement with the idea that tumour cells may producelarge amounts of hydrogen peroxide [3]. However, ourresults demonstrate that the antioxidant defenses changein the cancer cells during tumour growth, and this mayhelp to explain why some cells or cell subsets are resis-tant to oxidative cytolysis [43]. Indeed, although mito-chondrial generation of superoxide can be greatly stim-ulated by TNF-a [44], a cytokine produced bymacrophages/monocytes in response to the invasion ofthe host by a tumour, previous results by our groupdemonstrate that high GSH levels confer tumour resis-tance against TNF-a-induced cytotoxicity in vivo [45].

Hydrogen peroxide generated by the tumour can bereleased to the extracellular space [3]. Interestingly, theincrease in peroxide levels produced in the cancer cells(Fig. 1) is accompanied by an increase of GSSG in blood(Table 1). As shown in Table 1, although peroxide pro-duction decreases when the rate of tumour cell prolifer-ation decreases (Fig. 1), GSSG levels in blood remainelevated, as compared to controls, from day 7 until day14 after inoculation of the tumour. However, as shown inTable 3, GPX activity in erythrocytes increases when therate of tumour growth decreases, and this may explainwhy GSSG levels remain high. Furthermore, we alsofound that GSSG levels in plasma of tumour bearingmice were 1.96 0.6 mM (n 5 10) on day 4 of tumourgrowth, and that this value was not significantly differentto that obtained in healthy mice (see above). Thus,changes in whole blood GSSG, in all our experimentalconditions, reflect the situation within the erythrocytes.

Finally, SOD activity increased within the erythro-cytes during tumour growth, whereas CAT activity onlyincreased during the high-rate period of cancer cell pro-liferation (Table 3). These changes associated with lowlevels of lipid peroxidation, both in blood and in plasma,during tumour growth (Table 4). Indeed, the increase inantioxidant enzyme activities could be a protectivemechanism for the cells due to the tumour-induced hy-perproduction of ROIs. It is noteworthy that in erythro-cytes O2

2• arises also from autoxidation of oxyhemoglo-bin [46] and that elevated SOD activity could imbalance

Fig. 1. Peroxide levels in tumour cellsin vivo at different stages oftumour growth in mice. Kinetics of peroxide production in cancer cellsrelated to the rate of tumour growth in vivo were analyzed, as indicatedunder “Materials and Methods,” by flow cytometry. Intracellular per-oxide levels were measured on the basis of 29,79-dichloro-dihydro-fluorescein oxidation resulting in an increment of the mean 29,79-dichloro-dihydro-fluorescein fluorescence intensity (D FL1) expressedin arbitrary units. MeanD FL1 values were derived from single-parameter fluorescence histograms from samples collected at the cor-responding time points. Results are expressed as means6 S.D. for 6different experimental animals. *Significantly different from day 4group (p , .05).

Table 4. Lipid Peroxidation in Plasma, Erythrocytes and CancerCells During Tumour Growth in Mice

Malondialdehyde

Time after inoculation of the tumour (d)

0 7 14

Plasma (nmol/ml) 4876 116 3656 97 2666 89a

Erythrocytes (nmol/ml) 6526 134 3876 125a 3556 103a

Tumour (nmol/g) 2496 35 1506 48a

Results are expressed as means6 S.D. for 9 mice.a Significantly different from day 7 group (measurements in the

tumour) or from day 0 group (measurements in plasma or red bloodcells) (p , .01).

415Glutathione status and the antioxidant system in blood and in cancer cells

antioxidant defense, resulting in enhanced oxidation dueto accelerated generation of H2O2, the product of O2

2•

dismutation. However, as shown in Table 3, CAT activ-ity in the red blood cells, which is higher than controlsprecisely when the tumour is producing more peroxides(Fig. 1), must work in removing the hydrogen peroxidethat released by the tumour reaches the blood stream.Therefore, changes in glutathione status within the eryth-rocytes may reflect similar changes in the tumour cells,and these effects can be explained, at least in part, bychanges involving glutathione-related and antioxidantenzyme activities during tumour growth. Moreover,these enzymic changes may suggest possible cancer-induced alterations in the regulation of gene expressionin the pluripotent stem cells, or may reflect a more rapidturnover of the red blood cells. Indeed, young erythro-cytes may be higher in antioxidant enzymes than oldererythrocytes [21]. Anemia occurs frequently in cancerpatients. EAT-bearing mice also develop anemia (Table3). However, we did not find in blood of EAT-bearingmice a significant change in the percentage of reticulo-cytes as compared to controls (1–3%; results not shown).Thus, apparently, anemia in our tumour-bearing miceresponds to the typical anemia of chronic malignancy,normocytic, normochromic, with normal reticulocytecount, normal red cell maturation, and slightly dimin-ished red cell survival [39]. Decreased enzymic protec-tion and increased sensitivity to oxidative damage inerythrocytes has been reported as a function of cell anddonor aging [21], however, erythrocyte half-life in EAT-bearing mice (measured by59Fe pulse labeling [21]) isonly slightly shorter (526 4 d, n 5 10) as compared tocontrols (596 3, n 5 10). Therefore, an increase in thepercentage of young erythrocytes can not be argued toexplain the changes in glutathione-related and antioxi-dant enzyme activities in erythrocytes of tumour-bearingmice.

Glutathione homeostasis during cancer progression

The absence of changes in GSSG content within thetumour cells can be explained by excretion of GSSG tothe extracellular medium. Indeed, glutathione efflux(GSH1 GSSG) from tumour cells occurs, is coordinatedwith the rate of GSH synthesis [24], and could be re-flected by an increase of GSH and/or GSSG in plasma.However, in plasma of non-tumour-bearing mice wefound that GSH (total glutathione2 GSSG) levels were9.0 6 2.4 mM (n 5 10), andthat this value did notchange significantly as compared to tumour-bearingmice at any stage of tumour growth (results not shown).Moreover, as explained above, GSSG levels in plasmaare also very stable. GSH and GSSG present in plasmacan react with GGT, an enzyme located at the surface of

the plasma membrane of different cells, which hydro-lyzes the tripeptide and its disulfide to glutamate andcysteinylglycine (and its disulfide) [5]. Cleavage of cys-teinylglycine (and its disulfide) is catalyzed by a dipep-tidase and the products formed (free amino acids,g-glu-tamyl amino acids) are transported into cells [5]. Thisactivity was not detectable in Ehrlich cells [24], never-theless, GSSG molecules reaching the blood stream canbe rapidly removed by the GGT present in the host’sorgans, e.g., the kidney that, under physiological condi-tions, shows the highest GGT activity among all tissuesand whose blood flow is approximately 20% of the totalcardiac output [10]. To investigate if the presence of agrowing tumour may affect indirectly the GGT activityin different tissues, we measured it in kidney, brain andliver of tumour and non-tumour-bearing mice and foundno significant differences (1396 26 U/g, in kidney;1036 38 mU/g in brain; and 286 12 mU/g in liver;n 59). Thus, an increased in GGT activity can not be arguedto explain the removal of the glutathione released by thetumour.

A hypothesis postulates that GSSG levels in wholeblood may arise to higher concentrations than commonlybelieved, and that low GSSG levels are due to GSSG-reductase action [47]. However, our present results showthat pathological conditions such as cancer can be ac-companied by changes of glutathione status in blood, andthat this effect is the net result of several factors acting invivo at the same time.

Clinical relevance

It has been suggested that cancer patients are morelikely to respond to chemotherapy if their erythrocyteGSH are low [14]. However, as shown in Table 1,changes in blood glutathione status in EAT-bearing miceare largely due to the increase in GSSG levels. More-over, Hercbergs et al. [14] state that if erythrocyte GSHconcentration is low, by inference, tumour GSH levelsshould be low; however, this preliminary study did notseek a direct correlation of erythrocyte GSH with tumourGSH. In fact, we have shown in EAT that this may notbe the case if one compares different stages of tumourgrowth (Table 1). Naturally, a necessary step forward isto establish the relationship between our experimentalmodel and the human patients. To answer this question,we investigated whether cancer growth can alter bloodglutathione status in humans. For this purpose, we stud-ied two groups of cancer patients who had not receivedprevious treatment. Thus, cancer-induced effects couldbe measured without possible treatment-derived interfer-ences.

High levels (up to 0.5 nmol/h/104 cells) of hydrogenperoxide are constitutively released from a wide variety

416 J. NAVARRO et al.

of human tumours (melanoma, colon carcinoma, pancre-atic carcinoma, neuroblastoma, ovarian and breast carci-noma) [3]. As shown in Table 5, the GSH/GSSG ratio inblood also decreases in humans and, as it occurs intumour-bearing mice (Table 1), this change associateswith higher GSSG levels and, in advanced stages ofcancer progression, with higher GPX activity. In addi-tion, as compared to controls, it is noteworthy that thecatalase activity also tends to increase at advanced stagesof cancer progression, and that this fact associates withlower levels of lipid peroxidation in erythrocytes (Table5). Together, these findings support the idea of the pro-tective role of the glutathione redox system, in conjunc-tion with antioxidant enzymes, against ROIs hyperpro-duced in cancer patients. In conclusion, taking intoaccount that multidrug and radiation resistance of manytumours is associated with increased cellular GSH levels[5,7], we propose the GSH/GSSG ratio in blood as auseful parameter for cancer therapy.

Acknowledgements— We thank Mrs. M. J. Ortega for her skillfultechnical assistance. This research was supported by grants from theComision Interministerial de Ciencia y Tecnologı´a (SAF236-96) andthe Generalitat Valenciana (GV-3277/95), Spain. J.N., E.O. and J.C.held fellowships from the Generalitat Valenciana, Spain.

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Table 5. Glutathione Status, Glutathione-Related and Antioxidant Enzyme Activities, and Lipid Peroxidation in Blood of Cancer Patients

Controls Breast Cancer Colon cancer

For all Initial Advanced Initial Advanced

GSH 7.46 0.5 7.36 0.8 6.76 1.4 7.16 0.6 6.96 1.0GSSG 1186 30 4036 97a 1,1356 462a 1716 71a 6596 244a

GSH/GSSG 656 27 226 12a 6 6 3a,b 446 15 136 6a,b

Glutathione reductase 6.26 1.8 6.56 0.7 6.06 1.1 6.36 1.6 6.66 1.5Glutathione peroxidase 496 14 556 17 786 15a 436 12 666 10a,b

Superoxide dismutase 1.86 0.3 2.06 0.5 2.86 0.5a 1.66 0.4 1.96 0.3Catalase 2066 49 2136 56 2946 32a 1666 46 2536 25b

Malondialdehyde 9306 257 9056 132 5346 103a,b 9726 312 6876 184

GSH, GSSG and glutathione status values refer to those found in total blood. All other data refer to those calculated in isolated erythrocytes. GSHis expressed asmmol/g Hb; GSSG as nmol/g Hb; glutathione reductase and glutathione peroxidase activities as U/g Hb; superoxide dismutase activityas U/mg Hb; catalase activity as K/g Hb; and malondialdehyde as nmol/ml. Data are expressed as means6 S.D. for the number of patients indicatedunder “Material and Methods,” excepting controls (healthy volunteers), which weren 5 16.

a Significantly different from controls (p , .05).b Significantly different from “initial stages” (p , .05).

417Glutathione status and the antioxidant system in blood and in cancer cells

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