Oxidative stress and mitochondrial glutathione in human lymphocytes exposed to clinically relevant...

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Original Contributions

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Professor of Anesthesia, “La Sapienza” Uni-ersity, Rome

Staff Anesthesiologist, “La Sapienza” Uni-ersity, Rome

Immunologist, Department of Infectiousiseases, “La Sapienza” University, Rome

Cell Biologist, Department of Anesthesia,La Sapienza” University, Rome

Immunologist, Department of Internaledicine, “San Camillo” Hospital, Rome

ddress correspondence and reprint re-uests to Dr. Delogu at the Department ofnesthesia and Intensive Care, Policlinicomberto 1°, 00161 Rome, Italy. E-mail:[email protected]

his study took place at University Hospital,oliclinico Umberto 1°.upported in part by a grant from “la Sapi-nza” Unversity, Rome, Italy.

eceived for publication February 24, 2003;evised manuscript accepted for publicationuly 14, 2003.
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ournal of Clinical Anesthesia 16:189–194, 20042004 Elsevier Inc. All rights reserved.

60 Park Avenue, South, New York, NY 10010

xidative Stress anditochondrial Glutathione

n Human Lymphocytesxposed to Clinicallyelevant Anestheticrug Concentrations

iovanna Delogu, MD,* Adriana Antonucci, MD,†onia Moretti, MD,‡ Maurizio Marandola, MD,†uglielmo Tellan, MD,† Michele Signore, PhS,§iuseppe Famularo, MD�

epartment of Anesthesia and Intensive Care, University Hospital, Policlinicomberto 1°, Rome, Italy

tudy Objective: To evaluate the potential of compounds commonly used in anesthesiaractice to affect the intracellular oxidant-antioxidant homeostasis of peripheral bloodymphocytes at clinically relevant concentrations; and to study the changes in reactivexygen species production and measure the mitochondrial glutathione content.esign: Prospective, in vitro study.etting: Experimental medical research laboratory at a University Hospital.easurements: Lymphocytes were isolated from the peripheral blood of 15 healthy donors

nd incubated for 12 hours at 37°C with the following drug concentrations: thiopentalodium 20 mmoL/mL, droperidol 130 �mol/mL, propofol 60 mmoL/mL, and succinyl-holine 17 mmoL/mL. Reactive oxygen species (ROS) generation was determined byydroethidine and 2�,7�-dichlorofluorescein diacetate methods. Mitochondrial glutathioneevel was assessed using monobromobimane staining.

easurements and Main Results: Thiopental-treated lymphocytes exhibited an overgen-ration of ROS, but no change was detected in mitochondrial glutathione quantity.ropofol and droperidol could not induce any perturbative effect on the oxidative state ofcells, whereas succinylcholine was found to markedly affect lymphocyte oxidative state

oth by impairing glutathione content and promoting exaggerated production of ROS.onclusion: Drugs commonly used in anesthesia practice may significantly alter the

xidative state of peripheral T cells. This mechanism could contribute to the immuneuppression that occurs transiently in the early postoperative period. © 2004 by Elseviernc.

eywords: Glutathione; lymphocyte; oxidative stress; succinylcholine;
hiopental sodium.
0952-8180/04/$–see front matterdoi:10.1016/j.jclinane.2003.07.007

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ntroduction

n unregulated production of reactive oxygen speciesROS) associated with a shift in the intracellular oxidant-ntioxidant balance towards a pro-oxidant state, may haveeleterious consequences for cellular homeostasis.1,2 Mi-

ochondria play a central role in maintaining this bal-nce,1,2 yet cells are protected from free radical-inducedamage by a network of radical scavenging antioxidantroteins, enzymes, and chemical compounds, includingeduced glutathione (GSH).3,4 An imbalance betweenro-oxidants, including ROS and the scavenging systems,riggers serious damage in cellular membranes and DNAeading to “oxidative stress,” the well characterized condi-ion implicated in the pathogenesis of a wide range ofuman disorders such as critical illness and surgical trau-a.5,6 In fact, a loss of oxidative balance control associatedith a greatly increased oxidative state was found in

ubjects undergoing operative procedures.1,7,8 In a recenteport, we demonstrated that lymphocytes from surgicalatients exhibited alterations in mitochondrial transmem-rane potential paralleled by ROS overproduction and

ncreased, programmed cell death. Such dysfunction ofhe oxidative balance could be a crucial factor involved inhe apoptotic loss of T cells following surgery.9

In spite of contributions of several studies exploring theelationship between anesthetic drugs and oxidative stress,he role of anesthesia itself in this context is unclear.urthermore, previous investigations frequently haveielded contradicting results. For example, experimentserformed in vitro and animal models have shown thatxposure of cells to an inhalational anesthetic such asesflurane alters the intracellular oxidative state toward aro-oxidative milieu. In contrast, when cells are exposedo halothane, significant increases in the biologic activityf endogenous antioxidants can be measured.10 Even afterxposure to propofol, cells exhibit enhanced endogenousntioxidant activity.11,12

The aim of the present study was to evaluate, under initro conditions, the capacity of drug compounds used innesthesia practice to directly affect intracellular oxidant-ntioxidant homeostasis. We determined the productionf ROS and measured mitochondrial GSH levels in lym-hocytes isolated from healthy donors who were exposedo clinically relevant concentrations of anesthetic drugs.or the purpose of our experiments, we selected four

ntravenous (IV) drugs: thiopental sodium, droperidol,ropofol, and succinylcholine, which we considered to beepresentative of different classes of drugs (e.g., barbitu-ates, butyrophenones, depolarizing muscle relaxants, andlkylphenol compounds) that are commonly used by ourtaff in anesthesia practice.

aterials and Methods

ymphocyte Isolation and Drug Treatment

ter receiving approval from the Ethics Committee ofoliclinico Umberto I, University “La Sapienza”, Rome,
nd informed consent from the study participants, we t
90 J. Clin. Anesth., vol. 16, May 2004

btained lymphocytes from the peripheral blood of 15ealthy volunteers who were hospital and laboratory per-onnel. Blood samples were collected in cell preparationubes with sodium heparin (Immunocytometry Systems,ecton Dickinson and Co., San Jose, CA). Peripherallood mononuclear cells (PBMC) were isolated from thelood samples after centrifugation on a Lymphoprepensity gradient (Nycomed, Oslo, Norway), as describedy Boyum.13 The PBMC were then washed twice withhosphate-buffered saline (PBS) and resuspended inPMI 1640 medium (Gibco, Paisley, Scotland) that was

upplemented with 10% heat-inactivated fetal calf serumFCS; Life Technologies Inc., Paisley, Scotland), 10U/mL penicillin with streptomycin, 10 mmoL HEPESSigma Chemical Co., St Louis, MO), and 1 mmoL/L of-glutamine (complete medium; Sigma Chemical Co.).

nitial cell enumeration and cell viability of fresh PMBCere performed by conventional light microscopy using

rypan blue exclusion in a Neubauer chamber. Viability ofells was always greater than 95%. After isolation, separateliquots of cells (5 � 105mL) maintained in their cultureedium, were exposed to four different drugs: thiopental

TPS; Pharmacia S.p.a., Milano, Italy), droperidol (Phar-acia S.p.a.), propofol (AstraZeneca UK Ltd., Maccles-

ield, UK), and succinylcholine (Pharmacia & Upjohn.p.a., Milano, Italy). The drugs were purchased from theuppliers, with 100% purity, with no preservative prepara-ions. Before exposure, each drug was diluted in Krebs-inger phosphate and placed in a measuring cuvette to a

otal volume of 1 mL, giving the final concentrations of 20moL/mL for thiopental, 130 �mo:/mL for droperidol,

0 mmoL/mL for propofol, and 17 mmoL/mL for succi-ylcholine. Lymphocytes were incubated for 12 hours at7°C in 5% of carbon dioxide (CO2) humidified atmo-phere with the above drug concentrations, which corre-ponded to clinically relevant plasma concentrations.14,15

fterward, incubation cells were taken for detection ofOS production and mitochondrial GSH amount assess-ent in each lymphocyte subset of interest. Cells that were

btained from the same sample and incubated in thebsence of the drugs served as study controls.

etermination of ROS Generation and GSHitochondrial Level

s a result of mitochondrial membrane permeability, cellsroduce large amounts of ROS, which, in turn, oxidizesitochondrial inner membrane cardiolipin. Therefore, it

s possible to determine ROS production using hydroethi-ine probe (HE; Molecular Probes, Eugene, OR), a sub-tance that is oxidized by superoxide anion to becomethidium bromide (EthBr) and to emit red fluorescence.urthermore, 2�,7�-dichlorofluorescein diacetate (DCFH-A; Molecular Probes) measures H2O2 formation. For the

imultaneous assessment of surface markers and mito-hondrial ROS generation, such as superoxide and hy-roxyperoxide, cells were washed once with PBS, stainedith PE-labeled, anti-hCD4 or anti-hCD8 antibodies (Bec-

on Dickinson), and then exposed for 15 minutes at 37°C

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o 2 nmoL/L hydroethidine and for 1 hour at 37°C to 5mol/L 2�,7�-dichlorofluorescein diacetate, respectively.16

n control experiments, cells were labeled after preincu-ation with the uncoupling agent carbonyl cyanide m-hlorophenyl-hydrazone (m ClCCP; 50 mmoL/L, 37°C, 30in; Sigma-Aldrich Corp, St. Louis, MO), or the ROS-

enerating agent menadione (1 mmoL/L, 37°C, 1 hour;igma-Aldrich Corp). For DCFH-DA, a positive controlcells kept 2 min in 15 mmoL/L H2O2 and washed threeimes) was inserted. Monobromobimane (MBB; Molecularrobes) stains GSH. In the presence of glutathione-S-ransferase, MBB combines nonenzymatically with GSH,esulting in GSH-specific fluorescence.17 Briefly, T cellsere pelleted and resuspended in 1 mL medium contain-

ng 40 �M MBB for 10 minutes in the dark at roomemperature. The cell suspensions were mixed gently andlaced on ice before analysis, performed on a FACScanytofluorometer (Becton Dickinson). The samples wereeasured within 15 minutes on the flow cytometer. In all

xperiments, FITC and PE isotype controls were includeds appropriate. FSC and SSC parameters were gated on theajor population of normal-sized lymphoid cells. After

uitable compensation, fluorescence was recorded at dif-erent wave lengths: FITC and DCFH-DA at 525 nmFL-1), PE at 575 nm (FL-2), and HE at 600 nm (FL-3).

tatistical Analysis

ata analysis was performed with Graphpad Prism statisti-al software (Graphpad Software, Inc., San Diego, CA).esults were validated using the paired t-test. A p-value less

han 0.05 was considered significant. Experiments wereepeated at least five times, and data are expressed aseans � SD.

esults

xperiments with Thiopental

e observed a significant increase in HE- and DCFH-DA-ositive cells among lymphocytes exposed to thiopentalhen compared with unexposed cells (35.71 � 1.74 vs.1.18 � 1.01 and 29.24 � 1.91 vs. 7.21 � 0.18 for HE- andCFH-DA-positive CD4 cells , respectively; 19.97 � 2.95 vs..37 � 0.84 and 32.78 � 3.05 vs. 10.89 � 1.01 for HE- andCFH-DA-positive CD8 cells, respectively; p � 0.05 forverall above comparisons). This finding indicated an

ncreased generation of mitochondrial ROS among T cellsncubated with thiopental. When we measured the GSH

itochondrial level, we found no difference in the rate ofSHlow positive cells between the thiopental-treated lym-hocytes and the control group lymphocytes, nor in CD4

r the CD8 subsets. The results of these experiments arehown in Figure 1.

xperiments with Droperidol

esults of these experiments showed that exposure toroperidol did not affect ROS generation or GSH amount

n either the CD4 or CD8 subset cells. In fact, no significant 8

ifference was observed in the frequency of HE- or DCFH-A-positive cells or in GSHlow cells of exposed lympho-

ytes compared with untreated cells.

xperiments with Succinylcholine

xperiments with succinylcholine showed that the oxi-ant-antioxidant state was greatly impaired after lympho-ytes were exposed to this drug. The rate of GSHlow cellsas increased among the CD4 and CD8 cells exposed to

uccinylcholine, compared with the control group lym-hocytes (34.91 � 2.31 vs. 9.25 � 1.05 and 36.91 � 3.04 vs.

igure 1. Effect of thiopental sodium on the production ofeactive oxygen species and mitochondrial glutathioneGSH) content. Data (means � SD) are expressed as percent-ges of hydroethidine (HE), 2�,7�-dichlorofluorescein diac-tate (DCFH-DA), and GSHlow positive A. CD4 and B. CD8

ells, in the absence of drug and at drug concentrations of 20moL/mL. *Significant difference vs. control.

.15 � 0.96 for the CD4 and CD8 cells respectively; p �

191J. Clin. Anesth., vol. 16, May 2004

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.05 for all comparisons). Furthermore, the frequency ofE- and DCFH-DA-positive cells was increased among

ymphocytes exposed to the drug with respect to thentreated lymphocytes (31.18 � 2.06 and 28.94 � 1.19 vs..24 � 0.16 and 10.04 � 1.22 for HE- and DCFH-DA-ositive CD4 cells, respectively; 39.01 � 2.15 and 31.01 �.01 vs. 6.18 � 0.12 and 4.91 � 0.39 for HE- andCFH-DA-positive CD8 cells, respectively; p � 0.05 for all

omparisons). Data are shown in Figure 2.

xperiments with Propofol

ropofol proved to be unable to promote ROS overpro-uction or to affect GSH mitochondrial content. In fact,

igure 2. Effect of succinylcholine on production of reactivexygen species and mitochondrial glutathione (GSH) con-ent. Data (means � SD) are expressed as percentages ofydroethidine (HE), 2�,7�-dichlorofluorescein diacetateDCFH-DA), and GSHlow positive A. CD4 and B. CD8 cells inhe absence of drug and at drug concentrations of 17

moL/mL. *Significant difference vs. control.

e found no change in the frequency of labeled cells i


mong treated lymphocytes compared with the controlroup lymphocytes.

iscussion

he results of this study show that drugs that are com-only used by the IV route in anesthesia practice may

ave an almost different potential—at least under in vitroonditions when peripheral blood lymphocytes are used asarget cells—to affect the oxidant state and energy mito-hondrial metabolism. Thiopental was shown to inducexaggerated generation of ROS but this capacity was notaralleled by a change in endogenous GSH content.either propofol nor droperidol exhibited any perturba-

ive action on the oxidant-antioxidant state of T cells,hereas succinylcholine was found to have the strongestotential to disrupt the mitochondrial oxidant and energyetabolism of target cells.The pro-oxidative activity of thiopental that we showed

n our experiments should be confirmed and furthernvestigated, because previous reports have yielded con-licting results. In in vitro oxidative stress models, thiopen-al provided a moderate scavenging effect on red bloodells, and this property has been attributed to its ability tonhibit lipidic peroxidation through an unknown mecha-ism.18 On the contrary, other researchers have supported

he claim that thiopental not only was unable to contrastntracellular oxidative damage in platelets from surgicalatients,19 but was even capable of activating lipidic per-xidation cascade in red cells from experimental ani-als.20,21

This latter finding seems to be in line with our findingf an increased generation of oxygen metabolites aftereripheral blood lymphocytes were exposed to thiopental.he ultimate mechanism responsible for this effect re-ains to be elucidated. In this regard, it has been assumed

hat thiopental, like phenobarbital and secobarbital, canlter the cellular oxidant-antioxidant balance by interfer-ng with the antioxidant enzymatic defenses, namely bynactivating the superoxide desmutase (SOD) system ornducing P-450 cytochrome.22,23 In our study, because weid not find any change in mitochondrial GSH content of

hiopental-treated lymphocytes, we are unable to confirmpossible perturbative effect of thiopental on scavenging

pparatus. It is clear that our results are apparently at oddsith the results of other researchers, but this discrepancyemains difficult to explain. Further studies are needed tostablish a finely tuned correlation between exposure tohiopental and the expected changes in the intracellularxidant-antioxidant balance.

As regards the oxidant-mediating activity of propofol,everal reports have underlined the free-radical scaveng-ng properties of this drug. Our data are partly in agree-

ent with those observations. Even though we did nothow any direct antioxidant properties of propofol, weevertheless found that ROS production and GSH stateere not affected when lymphocytes were exposed to thisrug. Previous investigations have suggested that the anti-xidant activity of propofol should be related to its chem-

cal similarity with other well-known scavengers such as

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utylated hydroxytoluene or �-tocopherol,24 which exertheir antioxidant activity through binding membranehospholipids and entrapping oxygen toxic metabolites.n the same way, propofol could trap free-radical electronscting as a relatively stable intermediate substrate.25–27

ropofol could also directly increase the antioxidant ac-ivity of GSH.28

The literature is lacking in reports about the mediatingffect of droperidol on the intracellular oxidant-antioxi-ant balance. In a study performed in vitro on the antioxi-ative properties of a few drugs used in the perioperativeeriod, droperidol was classified as a compound providingome protection against ROS-induced cellular damage.29

s far as our experiment is concerned, we found thatncubation with this drug did not promote any relevantlteration in oxidative stress or GSH consumption inxposed lymphocytes. In the same study,29 succinylcholineas included in a group of substances that were unable toxert antioxidative functions. However, we observed inur study that exposure to succinylcholine induced aysregulation of the oxidative state on T cells by enhanc-

ng generation of oxygen toxic metabolites and decreasinghe mitochondrial GSH pool.

The molecular mechanism by which succinylcholineriggers such an action needs to be clarified. An intriguingssumption concerns a possible link between the succinyl-holine pro-oxidative action and the Ca2�-dependentathway. Changes in the calcium ion concentration rep-esent a crucial event in the neuromuscular block inducedy muscle relaxant drugs such as succinylcholine.30 On thether hand, perturbations in Ca2� homeostasis are associ-ted with an altered mitochondrial transmembrane poten-ial which, in turn, may lead to mitochondrial depolariza-ion, loss of oxidative phosphorylation, and generation ofOS.31,32 The reduced amount of GSH that we observed

n lymphocytes exposed to the drug may represent theesult of increased consumption of the endogenous scav-nger owing to oxygen metabolite overproduction.

The implications of our study for the practice ofnesthesia should be evaluated further. One importantbservation of the study is that exaggerated oxidant stresseems to be responsible for the increased lymphocyteommitment to apoptosis that is ultimately responsible forhe lymphocytopenia and the impaired immune responseeen in the early postoperative period.9,33,34 Even thoughssay of lymphocyte apoptosis was not an endpoint of ourtudy, we did observe a greatly increased frequency ofpoptotic cells among lymphocytes cultured in the pres-nce of succinylcholine, whereas exposure to droperidolr propofol had no impact on the commitment to apo-tosis (data not shown; manuscript in preparation). Thisinding indirectly confirms that a shift in the intracellular

ilieu towards a pro-oxidative state is probably an impor-ant mechanism through which the drugs used duringnesthesia procedures could contribute to immune sup-ression seen in the early postoperative period.

In conclusion, this study indicates that drugs commonlysed in anesthesia practice could significantly affect thexidant-antioxidant milieu of immune cells such as pe-
ipheral blood lymphocytes. The consequent oxidative
tress gives rise to cellular damage, including acceleratedpoptosis, which is a main contributing factor for postop-rative lymphocytopenia and immunological deficit. Thus,erioperative control of oxidative stress in using the IVnesthetic compounds with minimal impact on the oxi-ant-antioxidant balance of lymphocytes could be of clin-

cal interest and importance, especially when the anesthet-cs are used continuously for long-duration surgery. At theame time, treatment with drugs possessing antioxidantctivity might improve the post-surgery/anesthesia recov-ry period.

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