Blood Glutathione as an Index of Radiation-Induced Oxidative Stress in Mice and Humans

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Original Contribution

BLOOD GLUTATHIONE AS AN INDEX OF RADIATION-INDUCEDOXIDATIVE STRESS IN MICE AND HUMANS

Jose Navarro, Elena Obrador, Jose A. Pellicer, Miguel Asensi, Jose Vina, and Jose M. EstrelaDepartamento de Fisiologıa, Universidad de Valencia, Facultad de Medicina, Av. Blasco Ibanez 17, 46010 Valencia, Spain

(Received 17 June 1996; Revised 4 October 1996; Accepted 18 October 1996)

Abstract—The effect of x-rays on GSH and GSSG levels in blood was studied in mice and humans. An HPLCmethod that we recently developed was applied to accurately determine GSSG levels in blood. The glutathione redoxstatus (GSH/GSSG) decreases after irradiation. This effect is mainly due to an increase in GSSG levels. Mice receivedsingle fraction radiotherapy, at total doses of 1.0 to 7.0 Gy. Changes in GSSG in mouse blood can be detected 10min after irradiation and last for 6 h within a range of 2.0–7.0 Gy. The highest levels of GSSG (20.1 { 2.9 ), amM4.7-fold increase as compared with controls) in mouse blood are found 2 h after radiation exposure (5 Gy). Breastand lung cancer patients received fractionated radiotherapy at total doses of 50.0 or 60.0 Gy, respectively. GSH/GSSG also decreases in humans in a dose–response fashion. Two reasons may explain the radiation-induced increasein blood GSSG: (a) the reaction of GSH with radiation-induced free radicals resulting in the formation of thyl radicalsthat react to produce GSSG; and (b) an increase of GSSG release from different organs (e.g., the liver) into theblood. Our results indicate that the glutathione redox ratio in blood can be used as an index of radiation-inducedoxidative stress. q 1997 Elsevier Science Inc.

Keywords—Glutathione, Blood, Radiation, Breast cancer, Lung cancer, Free radicals, Oxidative stress

INTRODUCTION

Glutathione (gamma-glutamyl-cysteinyl-glycine;GSH), the most prevalent nonprotein thiol in mam-malian cells, protects against radiation-induced celldamage.1,2 Several mechanisms of radioprotection byGSH have been identified. These include radical scav-enging, restoration of damaged molecules by hydrogendonation, reduction of peroxides, and maintenance ofprotein thiols in the reduced state.3 Ionizing radiationinteracts with water giving rise to HOi, H/, and an elec-tron. HOi radicals are highly reactive and can interactwith thiols giving RSi or GSi free radicals, which candimerize to give RSSR, GSSG or RSSG.4 Thus, themeasurement of glutathione disulfide (GSSG), in tis-sues and biological fluids, is used as an index of theoxidative stress that occurs under different physiolog-ical and pathological conditions.5–7 Indeed, we have re-ported that physical exercise-induced oxidative stress,

Address correspondence to: Dr. Jose M. Estrela, Departamento deFisiologıa, Facultad de Medicina, Avenida Blasco Ibanez 17, 46010-Valencia, Spain.

in humans and rats, can be detected by measuring theblood GSH/GSSG ratio.8

Erythrocytes are frequently used in studies of oxidativestress.9 However, a major problem is the measurement ofGSSG because spontaneous or catalyzed GSH oxidationis very rapid and, consequently, high GSSG levels can beerroneously obtained due to oxidation of GSH duringsample preparation.10 In order to solve this problem, wehave developed an HPLC method for measurement ofGSSG in blood which, by using N-ethylmaleimide as thethiol quenching agent, allows an accurate calculation ofthe glutathione redox ratio (GSH/GSSG).10,11 The aim ofthis article was to study whether the radiation-inducedoxidative stress can be correlated with changes of gluta-thione status in blood. We propose the blood GSH/GSSGratio as a rapid indicator for radiation-induced biologicaldamage.

MATERIALS AND METHODS

Animals and irradiation procedure

Adult mice OF1 from IFFA CREDO (Barcelona,Spain), fed ad libitum on a stock laboratory diet (Letica,

1204 J. NAVARRO et al.


Barcelona, Spain), were kept on a 12-h light/12-h darkcycle with the temperature maintained at 227C. All ex-periments were carried out between 10.00 and 12.00 h.Whole mice were irradiated with x-rays using a 6 KeVSL75 linear accelerator from Philips. For this purpose,animals were placed in a perspex box divided by 0.5cm perspex plates into small chambers of 7 1 7 1 10cm (one mouse per chamber). Single fraction radio-therapy, at total doses ranging from 1.0 to 7.0 Gy (be-low the LD50 for mice12), was administered at a rate of5.0 Gy/min.

Humans and radiation therapy

We studied two groups of patients attending an out-patient oncology clinic. There were 12 women withstage I or II (International Union against Cancer:UICC) breast carcinoma which were scheduled to un-dergo surgery and received preoperative irradiation;and, in addition, seven men and women with a stage II(UICC) nonsmall cell lung cancer who received pri-mary radiotherapy. Irradiation treatment was per-formed using a 25 MeV CGR linear accelerator fromSagittaire. The dose to the breast was of 50 Gy givenat 2 Gy per day for 5 days each week, whereas the totaldose to gross disease in lung cancers was of 60 Gygiven at 2 Gy fractions per day in a continuous course.

Blood collection and processing

Blood was collected from the heart (mice) or fromthe vena mediana cubiti (humans) into 1 ml syringescontaining sodium heparin (0.05 ml of a 5% solutionin 6.9% NaCl). Plasma was obtained by low-speed cen-trifugation of whole blood (800 1 g for 5 min at 47C).Erythrocytes were obtained by centrifugation of wholeblood (15001 g for 5 min at 47C), followed by removalof plasma and the buffy coat, and then the pelletederythrocytes were washed in ice-cold Krebs-Henseleitbicarbonate medium (pH 7.4) to yield the original he-matocrit. For GSH measurement, whole blood (0.3 ml)was treated, at 47C, with 0.3 ml trichloroacetic acid(30%) containing 2 mM EDTA. For GSSG determi-nation, whole blood, erythrocytes, or plasma (0.5 ml)were treated, at 47C, with 0.5 ml ice-cold perchloricacid (12%) containing 40 mM NEM (N-ethylmaleim-ide; Sigma Chem. Co., St. Louis, MO), to prevent GSHoxidation, and 2 mM BPDS (bathophenanthroline di-sulfonic acid, Sigma Chem. Co., St. Louis, MO), asdescribed by Asensi et al.10,11 To measure total gluta-thione, plasma (0.3 ml) was treated, at 47C with 0.3 mltrichloroacetic acid (30%). Samples were centrifugedat 15000 1 g for 5 min, at 47C, and the acidic super-

natants were used for GSH, GSSG, and total glutathi-one measurements.

Enzymatic measurement of GSH

GSH was analyzed by the glutathione-S-transferaseassay described by Akerboom and Sies,13

Determination of GSSG by HPLC

GSSG was measured by HPLC as previously de-scribed.10,11 The acidic supernatants (see above) werederivatized by adding 50 of 1 mM -glutamyl-glu-ml gtamate (Sigma Chem. Co.) prepared in 0.3% perchloricacid to 500 of the acidic supernatant. Then, samplesmlwere taken to pH 8.0 with KOH (2 M) / morpholi-nopropane sulfonic acid (0.3 M), centrifuged, and thenan aliquot of 25 of the supernatant was mixed withml50 of 1% 1-fluoro-2,4-dinitrobenzene (Sigma Chem.mlCo.). After this, derivatization was completed in 45 minand dessicated samples remained stable at 0207C forseveral weeks until injection. This procedure reducesGSH oxidation in blood to about 1%.

Measurement of total glutathione

Total glutathione, expressed as the sum of the re-duced and oxidized forms (GSH / 2 GSSG), was de-termined in plasma by a kinetic assay in which a cat-alytic amount of GSH or GSSG and glutathionereductase cause the continuous reduction of 5,5*di-thiobis(2-nitrobenzoic acid) (Sigma Chem. Co.) byNADPH.13

Measurement of glutathione-related enzyme activities

Enzyme activities were determined in fresh eryth-rocytes. After centrifugation, the erythrocytes were re-suspended in distilled water and lysed for 2 h at 47C.The lysate was diluted to a concentration of approxi-mately 50 mg of hemoglobin per ml and, then used forassays. Hemoglobin values were estimated as describedby Van Kampen and Zijlstra.14

Glutathione reductase activity was determined as de-scribed by Akerboom and Sies,13 glucose-6-phosphatedehydrogenase as described by Bergmeyer et al.,15 andglutathione S-transferase as described by Habig et al.16

Glutathione peroxidase (selenium-dependent) activ-ity was measured as follows. The diluted lysate wasmixed with an equal volume of 2 1 1002 M KCN in0.1 M phosphate buffer pH 7.0. The method of Floheand Gunzler,17 using tert-butyl hydroperoxide insteadof H2O2, was utilized.

1205Radiation-induced changes in blood glutathione


Table 1. Effect of Radiation on GSH and GSSGLevels in Mouse Blood

Time AfterIrradiation (h) GSH (mM) GSSG ( )mM GSH/GSSG

0 1.24 { 0.14 4.3 { 1.2 290 { 831 1.15 { 0.06 14.3 { 1.2* 81 { 47*2 1.08 { 0.12 20.1 { 2.9* 54 { 33*4 1.14 { 0.04 16.9 { 3.1* 67 { 28*6 1.23 { 0.09 11.2 { 1.9* 110 { 52*

12 1.25 { 0.13 8.7 { 1.2* 144 { 6524 1.22 { 0.15 6.4 { 1.3 189 { 77

The radiation dose administered was of 5 Gy. Results are expressedas means { SD for 10 different animals.

* Significantly different from controls (time 0 after irradiation) (põ .05).

Table 2. Dose–Response Relationship of Radiation-InducedChanges in Blood GSH and GSSG Levels in Mice

Dose (Gy) GSH (mM) GSSG ( )mM GSH/GSSG

0 1.27 { 0.11 4.89 { 2.01 260 { 891 1.22 { 0.15 5.29 { 1.62 231 { 572 1.10 { 0.07 8.55 { 3.82 129 { 28*3 1.10 { 0.13 13.21 { 4.18* 83 { 17*4 1.05 { 0.10 17.56 { 3.46* 60 { 18*5 1.07 { 0.09* 19.71 { 3.05* 54 { 20*

GSH and GSSG levels in blood were measured 2 h after irradi-ation. Results are expressed as means { SD for 10 different animals.

* Significantly different from controls (0 Gy) (p õ .05).

Expression of results and statistical significance

Results are expressed as means { SD for the indi-cated number of different experiments. The statisticalsignificance was assessed by Student’s t-test.

RESULTS

Effect of radiation on GSH and GSSG levels inmouse blood

As shown in Table 1, when mice receive a singledose of irradiation (equivalent to 5.0 Gy), the glutathi-one redox ratio (GSH/GSSG) in whole blood de-creases. This effect is significant up to 6 h after irra-diation (Table 1). The decrease in GSH/GSSG ismainly due to an increase in the concentration ofGSSG, because GSH levels do not change significantly(Table 1). High levels of GSSG, as compared to con-trols, can be detected in whole blood very quickly (10min after irradiation GSSG concentration in blood was11.07 { 2.12 ; n Å 10; p õ .05), but the highestmMlevels of GSSG are found about 2 h after irradiation(Table 1).

Dose–response relationship of changes in GSH andGSSG by radiation in mouse blood

Dose–response studies were performed 2 h after ir-radiation and, as shown in Table 2, the glutathione re-dox ratio in whole blood decreases progressively in adose-dependent fashion. For all doses, the highestchange in the glutathione status was found around 2 hafter irradiation (results not shown). The decrease inthe GSH/GSSG ratio is significant using a dose of 2.0Gy or higher (Table 2). The maximum GSH/GSSGdecrease that could be achieved with a single dose ofirradiation was obtained by administering 5.0 Gy(Table 2), which led to values of about 20% of thecontrols. Values found using doses of 6.0–7.0 Gy(close to the LD50 for mice12) (results not shown)were not significantly lower than those obtained with5.0 Gy (Table 2).

Effect of radiation on GSH and GSSG levels indifferent animal tissues

Oxygen radicals that damage tissue can be formedfrom reactions initiated by radiation.18 Indeed, oxida-tive stress has been shown to result in GSSG formationand depletion of GSH in various organs.2 Moreover,cells keep their GSSG content very low19 (Table 3) anda rise in the intracellular levels of this disulfide is ac-companied by its release to the extracellular space.6,20

Therefore, the radiation-induced increase of GSSG lev-

els in blood could be due, at least in part, to exportfrom other organs.

GSH depletion may occur very quickly after the ra-diation pulse21 (see above). However, we studied tissuelevels of GSH and GSSG in mice 2 h after irradiationbecause, at that moment, the decrease in whole bloodGSH/GSSG is more profound. As shown in Table 3,radiation (5.0 Gy) tends to decrease GSH and increaseGSSG levels in all tissues studied; nevertheless, ourresults were only statistically significant in a few cases.We found that GSH levels are decreased in the liver,whereas GSSG levels are increased in liver, heart, andpancreas (Table 3). This finding is of special relevancein the case of the liver, because this organ is a majorreservoir of GSH in the body, plays a central role inthe interorgan homeostasis of GSH22,23 and can exportlarge quantities of GSH and GSSG into the blood.18

Interestingly, in two cases (liver and heart) a reboundeffect can be detected resulting in net increase in organGSH levels 24 h after the initial stress (Table 3). Asimilar effect has been previously reported in liver cellsof refed rats after a 48 h-starvation period,24 and in lungcells where GSH can be significantly elevated abovecontrol levels after 24 h of exposure to hyperoxia.18



Table 3. Effect of Radiation on GSH and GSSG Levels in Different Tissues in Mice

Time After Irradiation (h)

0 2 24

Tissue GSH (nmol/g) GSSG (nmol/g) GSH (nmol/g) GSSG (nmol/g) GSH (nmol/g) GSSG (nmol/g)

Brain 1554 { 226 19 { 6 1328 { 159 32 { 17 1817 { 190 24 { 15Lung 1672 { 441 30 { 17 1314 { 224 59 { 16 1730 { 123 40 { 12Heart 719 { 207 15 { 7 700 { 176 38 { 11* 1023 { 156* 14 { 5Liver 6790 { 859 21 { 10 4394 { 633* 87 { 23* 8493 { 575* 39 { 18Pancreas 592 { 133 12 { 5 508 { 117 37 { 13* 616 { 159 15 { 9Spleen 3017 { 359 31 { 19 2531 { 318 46 { 23 2599 { 272 25 { 13Kidney 2468 { 404 28 { 9 2373 { 265 45 { 18 2502 { 234 35 { 12Skeletal muscle 525 { 117 16 { 8 346 { 88 33 { 19 660 { 101 20 { 8Bone marrow 347 { 52 17 { 6 278 { 94 29 { 9 302 { 88 21 { 13

The radiation dose administered was of 5.0 Gy. Results are expressed as means { SD for six different animals.* Significantly different from controls (time 0 after irradiation) (p õ .05).

Table 4. Effect of Radiation on Glutathione-Related Enzyme Activitiesin Mouse Erythrocytes

Time After Irradiation (h)

Activity (U/g Hemoglobin) Controls 2 24

Glutathione reductase 6.0 { 1.6 2.4 { 0.7* 3.8 { 0.6*Glucose-6-phosphate dehydrogenase 9.7 { 1.7 4.5 { 0.9* 6.4 { 1.2*Glutathione peroxidase 162 { 34 77 { 25* 108 { 22*Glutathione S-transferase 5.8 { 1.2 3.7 { 0.6* 3.8 { 0.9*

The radiation dose administered was of 5 Gy. Results are expressed as means { SD forseven different animals.

* Significantly different from controls (p õ .05).

Glutathione-related enzyme activities in blood fromirradiated mice

In order to further investigate the possible reasonsfor the change in glutathione status in whole blood, wemeasured in erythrocytes, from controls and irradiatedmice, the activity of the main enzymes involved in theglutathione redox cycle. These were glutathione reduc-tase, glucose-6-phosphate dehydrogenase, glutathioneperoxidase (selenium-dependent), and glutathione S-transferase (Table 4). We found that all these activitiesper gram hemoglobin are decreased to a similar extent,as compared to controls, 2 h after irradiation exposure(Table 4). Therefore, individual changes in GSH-re-generating or GSH-consuming activities cannot be ar-gued to explain the increase in GSSG levels in wholeblood.

In addition, it is noteworthy that the radiation-in-duced decrease in glutathione-related enzyme activitiesis not unique. In fact, other activities, such as hexoki-nase or pyruvate kinase are also significantly decreased2 h after irradiation exposure (5 Gy) to 59 or 56% ofcontrols, respectively (results not shown). Besides,other activities (i.e., glutamic oxaloacetic transami-

nase) were not affected by radiation (results notshown).

Effect of radiation on GSH and GSSG levels inhuman blood

The results obtained in mice prompted us to inves-tigate whether ionizing radiation can also alter bloodglutathione status in humans. To answer this question,we studied two groups of cancer patients who receivedstandard radiation therapy. We selected these types ofpatients because they were not receiving chemother-apy, and thus, radiation-induced effects could be mea-sured without possible chemical-derived interferences.As described under Materials and Methods, radiationwas administered in 2-Gy fractions to reach a final doseof 50 or 60 Gy. We measured GSH and GSSG levelsin whole blood at different moments during the periodof irradiation. As shown in Table 5, the GSH/GSSGratio in both groups of cancer-bearing patients de-creased progressively as the total radiation dose accu-mulates. As occurs in mice (see above), this effect ismainly due to an increase in GSSG levels in blood (Ta-



Table 5. Dose–Response Relationship of Radiation-Induced Changes in blood GSHand GSSG Levels in Humans

Breast Cancer Lung Cancer

Dose (Gy) GSH ( )mM GSSG ( )mM GSH/GSSG GSH ( )mM GSSG ( )mM GSH/GSSG

0 995 { 193 25 { 6 42 { 14 1037 { 98 26 { 5 42 { 812 874 { 174 25 { 5 36 { 10 944 { 101 30 { 3 32 { 6†

24 917 { 110 38 { 12* 27 { 11* 920 { 76 34 { 8 29 { 8†

48 874 { 202 112 { 33* 8 { 3* 887 { 69† 78 { 21* 12 { 3*

GSH and GSSG levels in blood were measured 2–3 h after irradiation. Control values in healthy people were930 { 127 (GSH; n Å 12); 26 { 7 (GSSG; n Å 12); and 34.4 { 10 (GSH/GSSG; n Å 12). Results aremM mMexpressed as means { SD for the number of patients indicated under Materials and Methods.

* Significantly different from controls (0 Gy) (*p õ .01; †p õ .05).

ble 5). Indeed, remarkable changes occurred in bloodGSSG levels in after irradiation. In breast-cancer pa-tients they rose from 25 to 112 (more than a 400%mMincrease), whereas in lung-cancer patients they rosefrom 26 to 78 (a 300% increase).mM

DISCUSSION

Ionizing radiation is toxic to organisms because itinduces deleterious structural changes in essential mac-romolecules.25 These changes can be the result of directinteraction with radiation. However, it is generally con-sidered that, because of the abundance of water in liv-ing organisms, a more important mechanism is the in-teraction with free radicals formed by photolysis ofwater.25 Thiols and disulfides are among the substances,which if administered before exposure to radiation, areable to confer protection.4 GSH plays a key role in pro-tecting cells against electrophiles and free radicals.5

This is due to the nucleophilicity of the SH group andto the high reaction rate of thiols with free radicals.26

Radiation resistance of many cells is associated withhigh intracellular levels of GSH.2,27,28 Cells containinglow levels of GSH were found to be much more sen-sitive to the effect of irradiation than controls.2 Indeed,GSH can act directly as a free radical scavenger byneutralizing HOi, or indirectly by repairing initial dam-age to macromolecules inflicted by HOi.5 There is am-ple evidence that thiols protect molecules from radia-tion injury, mainly by hydrogen donation, which canrestore damaged molecules to their original state.29 In-deed, GSH is essential in the maintenance of enzymeSH groups in the proper redox state.30 Moreover, GSHis a substrate or a cofactor for a number of protectiveenzymes, such as GSH peroxidase, the GSH S-trans-ferases, or the glayoxalase.5

Radiation-induced oxidative stress may causechanges in the glutathione redox state of different tis-sues and, in addition, increase the rate of GSSG releasefrom cells.4,18 In fact, increases in GSSG levels inside

cells and/or in GSSG efflux from cells are signals ofoxidative stress.5,6 Moreover, cellular GSH levels indifferent tissues decrease under conditions of shock,stress, or peripheral inflammation.20 GSH and GSSGefflux from rat liver is stimulated by various stress-related hormones, including vasopressin, phenyleph-rine, and adrenaline.31 Indeed, interorgan flows of glu-tathione have been proposed.32 Therefore, GSH andGSSG levels in blood may reflect changes in glutathi-one status in other less accessible tissues.8

Different studies have pointed out the interest ofmeasuring blood glutathione for both pathological andphysiological purposes. Herebergs et al.33 show thatcancer patients are more likely to respond to treatmentif their erythrocyte GSH and, by inference, tumor GSHconcentrations are low. There are alterations of GSHreductase activity in peripheral-blood erythrocytes insuch diseases as hypothyroidism34 and riboflavin defi-ciency.35 Exhaustive physical exercise causes changesin the glutathione status of blood, liver and muscle.8,36,37

Moreover, glutathione status may be used as a bio-logical marker of aging.38 Recently, elegant studies byLang et al.8 showed considerable intra- and interindi-vidual variability in normal GSSG levels in humanblood. Naturally, this fact could complicate interpre-tation of changes in blood GSSG levels under clinicalconditions. However, we and others10,39 have shownthat determination of GSH and GSSG levels in normalblood is highly dependent upon sample processing. Asshown in Tables 1, 2, and 5, by using our methodology,reproducible and stable results can be obtained withlow SD. Our present results show a dose-dependentincrease of GSSG concentration in blood after irradi-ation exposure (Tables 1, 2, and 5).

It is well known that red cell glutathione is trans-ported outward through the membrane into the plasmaas either GSSG or thioester conjugates. This is the onlyproven mean by which the erythrocyte disposes of glu-tathione. Insideout vesicles (inverted fragments of redcell membranes), allowed kinetic studies of the gluta-



thione transport system. These experiments showedthat transport occurs and that it requires ATP. In thissystem no transport of GSH occurs at all (for a review,see Beutler and Dale40). In plasma of healthy humans,we found that GSSG levels were 1.3 { 0.5 (n ÅmM7) and GSH (total glutathione–GSSG) levels 7.7 { 1.9

(n Å 7). Besides, in plasma of irradiated patientsmM(breast cancer patients with a total dose of 48 Gy)GSSG levels rose to 6.1 { 1.7 (n Å 7; p õ .05),mMalthough no significant differences were found in GSHlevels as compared to controls (results not shown; con-trol values were similar to those displayed in Table 5).This suggests that the radiation-induced increase inwhole blood GSSG is due to GSH oxidation within theerythrocytes and/or an increase of GSSG release fromdifferent tissues into the blood. Indeed, in fresh eryth-rocytes obtained from mice and resuspended, after cen-trifugation, in a volume of Krebs-Henseleit bicarbonatemedium (pH 7.4) that yields the original haematocrit,GSSG rises from 4.1 { 0.8 (n Å 5) to 19.7 { 1.6mM

(n Å 5; p õ .01) 1 h after irradiation (5 Gy). NomMhemolysis occurred in these experiments and about30% of the total GSSG found in irradiated suspensionsof RBCs was found in the extracellular compartment.This proves that radiation induces GSH oxidation andthat GSSG is transported outward the erythrocytes. Be-sides, irradiation also induces detectable changes in theglutathione status of some tissues (Table 3) and, in-deed, the increase of GSSG levels in whole blood couldbe the consequence of GSH oxidation in this mediumor in different tissues. Therefore, regulation of gluta-thione metabolism in vivo must be considered in termsof the entire organism.18 For instance, the liver is botha synthesizer and user of GSH and also exports largequantities of GSH and, proportionally, of GSSG intothe blood.4 Moreover, GSH and GSSG present inplasma can react with gamma-glutamyl transpeptidase,an enzyme located at the surface of the plasmamembrane of different cells (e.g., kidney), which hy-drolyzes the tripeptide and its disulfide to glutamate andcysteinylglycine (and its disulfide).2 Cleavage of cys-teinylglycine (and its disulfide) is catalyzed by a di-peptidase and the products formed (free amino acids,

-glutamyl amino acids) are transported into cells.2 Sogfar, GSSG released from tissues to the blood can bedegraded by this activity.18 This would explain why inplasma GSH remains apparently unchanged and GSSGlevels are kept very low. Moreover, as recently hy-pothesized, GSSG may occur normally in whole bloodat higher concentrations than commonly believed, andthat low GSSG levels are due to GSSG-reductase ac-tion.7 Consequently, the radiation-induced increase ofGSSG levels in blood is the net result of several factorsacting in vivo at the same time.

At present, chromosomal alterations are studied todetect radiation-induced cell damage.41 In this article,we show that the glutathione redox ratio in blood maybe used as a rapid index of oxidative damage inducedby radiation. This methodology can complement cur-rent cytogenetic studies.

Acknowledgements — This research was supported by grants fromthe Comision Interministerial de Ciencia y Tecnologıa (SAF310-92)and the Consejo de Seguridad Nuclear (Spain). J.N. and E.O. heldfellowships from the Conselleria de Educacion y Ciencia de la Ge-neralitat Valenciana (Spain). We thank Mrs. J. Belloch for her skillfultechnical assistance.

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Blood Glutathione as an Index of Radiation-Induced Oxidative Stress in Mice and Humans