V:,. ution of copper in mice . Eur.389-392.

I . Obrusnik : Copper and zinc-ve tissues of rats with experi de neuropathy . Brit. J. indusir.

elements - a selective survey ..76-500 .ion disulphide and the nervousntresearch .XVIIIInternationalial Health, Brighton, . England

lism of disulfiram and diethyl-ts with demonstration of ani'' 'bition of the glucuronicie .:,iol . Biochem . Pharmacol.

el and copper mobilization by.rbamate . J. New Drugs 1964,

selos : Changes in the hepaticment with foreign compounds .ippl. 1, 247-249 .

i pharmacol. e t toxicol 1979,45, 45-51 .

From the Department of Forensic Medicine, Karolinska Institutet, S-104 01 Stockholm 60, Sweden

glutathione Depletion .in Isolated Hepatocytes: Its Relation to LipidPeroxidation and Cell Damage

ByIrene Anundi, Johan Hdgberg and A. Howard Stead

(Received January 22, 1979 ; Accepted February 22, 1979)

Abstract. The effects of glutathione depletion in isolated hepatocytes have been studied . A list of compoundswhich depleted glutathione and induced lipid peroxidation and cell lysis is given . The effects of halogenatedacetamides were studied in more detail and results of studies on the interaction of iodoacetamide with cellularconstituents are presented . A single metabolite of iodoacetamide, tentatively identified as the glutathioneconjugate, was excreted from the cells while less than one percent of the "parent compound" was retained,tightly bound to macromolecules . This bound component could not be associated with the cellular damage .Methionine, cysteine and a-tocopherol, as well as paracetamol and ethylmorphine, were found to prevent lipidpcroxidation and lysis . It is concluded that GSH deficiency per se can lead to lipid peroxidation and that thisreaction caused the observed hepatocellular lysis .

Key-words : Isolated hepatocytes - diethylmaleate - halogenated acetamides - .glutathione depletion - lipidpcroxidation - rat.

rnobiotics or their metabolicallyy activated inter-Iitcdiates may form water soluble and excretablei t njugates with hepatic glutathione (GSH) and canb Isodeplete the GSH reservoir (Gillette et al. 1974) .(ISH is also a co-factor for the selenium dependentiSH-peroxidase which reduces lipid peroxidases

,1nd hydrogen peroxide (Burk et a!. 1978) and hasIhucn implicated in the defence against lipid peroxi-Jfltion (Christophersen 1968 ; O'Brien & Little968; McCay et al. 1976). Thus, it may be proposed

,lhttt drug metabolism can lead to lipid peroxidation .by depleting hepatic GSH . We have used isolatedeputocytes to study GSH turnover in the liver andlound that diethylmaleate, which readily depletes091 in vivo (Boyland & Chasseaud 1970) inducedipid peroxidation in this model system . A prelimi-

nary report on the relationship between GSHlavcls, lipid peroxidation, and cell lysis has been

published (Anundi et al. 1978) . In this work wehave further elucidated the role of lipid peroxida-tion as the possible mechanism of toxicity .Compounds are known which interact with

lipids and proteins in such a way that both lipidperoxidation and protein alkylation have beenconsidered as a cause of toxicity. Well knownexamples are carbon tetrachloride (DeFerreyra etal. 1974; Recknagel et al. 1977; Lindstrom et al.1978), halothane (Rao 1977; Wood et al. 1976), 6-hydroxydopamine, dialuric acid and alloxan (Sachs& Jonsson 1975) . During recent years it has becomeevident that GSH protects against protein alkylalion (Gillette et al. 1974) and that electrophiliccompounds which deplete GSH may alkylate pro-teins . The main point in this communication is thatcellular damage following GSH depletion can beexplained by lipid peroxidation which destroys the

46

IRENE ANUNDI ET AL .

cell before the alkylation of proteins, as a compo-nent of cellular damage, is expressed .

Materials and Methods

Male Spraugue-Dawley rats (190 to 220 g) fed ad libitum,were used throughout the study . All chemicals werecommercially available and were used without furtherpurification. [1- 14 Cj-labeled iodoacetamide, specific ac-tivity 57 mCi/mmol, was purchased from The Radio-chemical Centre, Amersham, England.

Hepatocytes were isolated by collagenase perfusionand incubated essentially as described previously (Hog-berg & Kristoferson 1977) . The cell suspension wasdiluted to a final concentration of 1 X 106 cells/ml in KrebHenseleit buffer supplemented with Hepes (N-2-hydroxy-ethylpiperazine N'-2-ethane sulphonic acid) (Sigma) (25mM), penicillin (50 .000 IE), heparin (5 .000 IE) and horseserum (5%) and incubated in rotating flasks . Detailsabout further additions are given in figure legends . Cellmembrane permeability, measured as the latency ofNADH oxidation, was recorded at different time inter-vals (Hogberg & Kristoferson 1977) .Reduced glutathione was determined in sedimented cellsusing the colorimetric thiol method described by Saville(1958) and lipid peroxidation was measured as theaccumulation of malondialdehyde in the total incubatewith the thiobarbituric reaction (Hogberg et a!. 1975a) .Uptake of '4C-iodoacetamide into cells was determined inI ml aliquots of sedimented cells and the radioactivitymeasured in a Beckman LS-150 Liquid ScintillationCounter (counting efficiency _>90%) . Protein bindingwas estimated in 1 X 10 6 cells after precipitation with6.5% trichloroacetic acid and washing with 80% me-thanol (Reid et a!. 1973). Standards were prepared forquench correction . Separation of "C-iodoacetamide andits reaction product with GSH was performed as follows :10l aliquots of supernatant from the total incubate wereapplied to thin-layer chromatography plates (0 .2 mmsilica gel N-HR, Polygram pre-coated sheets, Macherey

Table 1.Effect of glutathione depleting agents on malondialdehyde (MDA) accumulation and cell lysis .

Nagel and Co., Duren, G.F .R .) as well as a similar aliqun :of a solution containing standards of both iodoacetamid,and its GSH conjugate . The plates were developed a:room temperature with a solvent system of methanolacetic acid (1 : 4) and standards were located (Ri s 0 .78 an,.0 .40 respectively) with ninhydrin spray reagent . Silica pr:from the corresponding areas of chromatographed sups,natant fractions was scraped directly into vials and tinscintillator added . The radioactivity was measured alirdispersal of the silica gel by sonication. Appropria++samples scraped from the TLC plates served as blankand were treated in a similar manner .

Results

In order to obtain relatively short incubation time'compounds that rapidly depleted GSH were usedin this study . A list of compounds, all of which ma'deplete GSH in isolated liver cells within tw. F

hours, is given in table 1 . Ethylmorphine has beet:reported previously to deplete GSH without affcc #ting cell membrane permeability (Jones et al. 1978)The other compounds . produce effects similar I those described previously for diethylmaleate (Anundi et al. 1978), that is, malondialdehyde accu.mulation in the cells coupled with cell lysis . Will.the exception of ethylmorphine, all the compound :listed in table I can be expected to conjugaldirectly to GSH, and at least chioroacetamidciodoacetamide and diethylmaleate did not inductlipid peroxidation in isolated microsomes fortifies ;;with NADPH . The effects of chloro- and iodoace -tamide on isolated hepatocytes were further iuvestigated .

The changes in GSH level, rate of malondialdchyde accumulation and membrane permeabilit :

* The compounds were added to the incubation medium in concentrations found to deplete GSII . "Yes" denoteeffects similar to those illustrated in fig . I and 4 . "No" denotes abscence of effects as compared to, conu, ,(also illustrated in fig . 1 and 4) .

Geier046.99

FF8 t 11Gnn+, 6

itvll

II :

- t, n,

Concentration(MM)*

GSHdepletion

MDAaccumulation

Celllysis

2-(Ethylmercurimercapto .benzoic acid (thimerosol) 0.11 yes yes yesChloroacetamide 0 .2 yes yes yesp-Phenylendiamine 1 .0 yes yes yesDiethylmaleate 0.6 yes yes yesIodoacetamide 0.075 yes yes yesEthylmorphine 1 .0 yes no no

)well as a similar aliquotan6rds of both iodoacetamidtThe plates were developed at.i solvent system . of methanol;ards were located (Rf's0.78 andhydrin spray reagent. Silica geleas of chromatographed superdped directly into vials and thedioactivity was measured after:1 by sonication . AppropriateTLC plates served as blanks

liar anner .

tesults

ively short incubation times,ly depleted GSH were used:ompounds, all ofwhich mayated liver' cells within two1 . Ethylmorphine has beendeplete GSH without affec.meability (Jones et al. 1978),s produce effects similar to)usly for diethylmaleate (A .Lt is, malondialdehyde accu;oupled with cell lysis . Withnorphine, all the compoundsi be expected to conjugated at least chloroacetamide,e . ` jnaleate did not induct,solaced microsomes fortifiedfects of chloro- and iodoacctepatocytes were further in-

I to deplete GSII. "Yes" dentseffects . a s compared to contr

N0 50

,,uto0

xN

0Ec

EFFECT OF GLUTATHIONE DEPLETION IN HEPATOCYTES .

47

M

oP50-

0 30

a

10

OkOh 0.2 mM chloroacetamide are shown in fig . 1 .%tnlondialdehyde levels increased during the thirdI r of incubation and preceded the increase intjiaxma membrane permeability by about 30 min . ;titnlondialdehyde did not accumulate in cells lysed!~y aonication or by detergents . This order of eventsrNa seen for all the compounds which induced

2-A

I

I

2

I1 2 3 4

1 2

5

1

4time (hours)

M. 1 . Effect of chloroacetamide on isolated hepatocytes . Cells were incubated in a medium containing 0 .2 mM chloro-rtamide (0 0) or 0.2 mM chloroacetamide plus 0.2 mM methionine(O- 0). A : intracellular levels of reduced

(l ll . B : cell membrane permeability and C : malondialdehyde levels .

100-

a

50-

E

x dCL

malondialdehyde accumulation (table 1) . Whenmethionine, an amino acid known . to stimulateGSH synthesis in liver cells (HSgberg & Kristofer-son 1978) was included in the medium, the GSHlevel increased during the third and fourth hour ofincubation (fig . 1A) and malondialdehyde accumu-lation and cell lysis were inhibited (fig . lB and C) .

e

B

0 1,

u,Mrn

>ta

,.

1

2

3

4

1

2

3

4time (hours)

I'ig, 2 . Effect of paracetamol and ethylmorphine in iodoacetamide induced cell damage . Cells were incubated with, loontinuous lines) or without (dotted line) iodoacetamide (0 .075 mM). Paracetamol (0.5 mM; (X=X) and ethyl-

a0chine (1 mM; *-*)were added to iodoacetamide-containing incubation mixtures . A : malondialdehyde contenthi,cell membrane permeability .

2

3

4time (hours)

Fig . 3 . lodoacetamide metabolism in isolated hepato-cytes . Cells were incubated in a medium containing "C-iodoacetamide (0.05 mM) at 0 during the first hour andthen at 37 . At indicated time points, aliquots of theincubate were taken and cells separated from medium bycentrifugation (80 g) . The cell pellet was washed once(which caused a loss of radioactivity corresponding to 2-3nmol metabolites/106 cells) and GSH concentration(0

0) and radioactivity (- 0) determined. Themetabolite (0-0) was isolated from the supernatantby thin-layer chromatography .

Cell damage was similarly prevented by the addi-tion of cysteine, which also stimulates GSH synthe-sis (Hogberg & Kristoferson 1978) .

The diverging . effect of ethylmorphine, that isGSH depletion without lipid peroxidation andlysis, was further studied . It was found that ethyl-morphine could prevent iodoacetamide-inducedlipid peroxidation and that the degree of inhibitionof malondialdehyde accumulation varied with theconcentration and the time of addition of ethyl-morphine. Ethylmorphine (2 mM) also preventedall damage during a time period of four hours . Fig .2 shows that 1 mM added at zero time delayedmalondialdehyde accumulation and lysis about 1 .5 .hours. Paracetamol (0 .5 mM), which like ethyl-morphine is a substrate for the microsomal mono-oxygenase system, delayed these effects about onehour.The metabolism of '4C-iodoacetamide was exa-

mined to elucidate possible receptors in the cell .The fate of a subtoxic dose of this compound (0 .05 .mM) is shown in fig . 3 ; the same metabolic pattern

was also seen during the first two hours with a toxicdose (0.075 mM) of the compound. The uptakephase was not readily detectable at 37, but incuba .tion at 0 during the first hour revealed a rapidincrease in intracellular levels of the radiolabeledcompound. No free iodoacetamide was detected inthe medium at the end of the first hour . Thetemperature was then raised to 37 0 and an excretionphase followed, indicated by a redistribution ofradioactivity from the cells to the medium (fig . 3).One single metabolite, the GSH conjugate, andtrace amounts of free iodoacetamide were isolatedfollowing thin-layer chromatography of the mc dium (fig. 3). Part of the iodoacetamide wasretained in, the cells (fig. 3), tightly bound tomacromolecules . The relationship of this bindingto the toxic response observed in the cells wasfurther studied .

The extent of binding to macromolecules wafound to be reduced by incubating at 0 instead ofat 37 (fig . 4C) although incubation at 0 during thefirst hour did not significantly change the pattern oftoxic effects (fig. 4A and B) . Furthermore, bychanging the medium after the first hour to one freeof iodoacetamide, additional binding to macro .molecules could be prevented (fig . 4). When, in.cluded in the iodoacetamide free medium, a .tocopherol proved to be an effective antidote foriodoacetamide induced toxicity (fig . 4). Methioninealso inhibited lipid peroxidation when added after,(.,one hour, indicating that cell damage could be'fprevented by treatments which apparently, did notinterfere with the covalent binding to macromole .cules .

Control experiments were performed where lipidperoxidation was not preceded by GSH depletionChloramine-T is commonly used to halogenateproteins (Montelaro & Rueckert 1977), and assu ming similar effects as obtained with other haloge nating compounds it could be expected to inducelipid peroxidation (Welton & Aust 1972; Kumar elal. 1977) . Chloramine was found to induce malon .dialdehyde accumulation without prior GSH de.pletion and without the lag seen in fig . 1 and 4 (fig.5). The rate with which GSH decreased in theseexperiments was much slower than that observedfor the halogenated . acetamides and comparablewith the rate observed when lipid peroxidation wasinduced by iron (Hogberg et al. 1975b). A signifi

4 . lodoacetamide-in(cetamide (0 .075 mM

(dotted lines) . The ttpherol (0.1 mM; sohndialdehyde content,

EFFE'

a

( i 3 4t

5 . Chloramine-T-indtI hopatocytes .were incubated with (Iamine-T in the mediusnccntration of 50 pAdintely before incubilevels of reduced GS : :

increase in cellularI after three hours, 1

i4ntion was not furth

tl; 'st two hours with a toxicf the" compound. The uptakey detectable at 37', but i ncuba.se first hour revealed a rapidular levels of the radiolabelediodoacetamide was detected inend of the first hour. The

n raised to 37 and an excretionlicated by a redistribution ofhe cells to the medium (fig. 3).lite, the GSH conjugate, andto iodoacetamide were isolatedr chromatography of the me-t of the iodoacetamide was[Is (fig . 3), tightly bound toie relationship of this bindingtse observed in the cells was

riding to macromolecules wasI by. incubating at 0 instead ofugh incubation at 0 during thenificantly change the pattern oflA and B). Furthermore, bym - after the first hour to one free

. additional binding to macro.. 4prevented (fig . 4) . When in-oacetamide free medium, a-ti'l ,) an effective antidote forcececoxtctty (fig. 4) . Methionineperoxidation when added afterig that cell damage could betents which apparently did not)valent binding to macromole-

:nts were performed where lipidof preceded by GSH depletion .ottimonly used to halogenateo & Rueckert 1977), and assu-as obtained with other halogeit could be expected to induceWelton & Aust 1972 ; Kumar eine was found to induce malon ration without prior GSH de-t the lag seen in fig. 1 and 4 (fig.vhich .GSH decreased in theseuch slower than that observedd.-acetamides and comparableed when lipid peroxidation walogberg et al. 1975b). A signifi-

EFFECT OF GLUTATHIONE DEPLETION IN HEPATOCYTES

0

Q5- B

10-t)-0-0

113 I

2 3 4time (hours)

1, Chloramine-T-induced lipid peroxidation in iso-il,hcpatocytes.lb were incubated with(*-*) or without (0-0)wamine-T in the medium . Chloramine was diluted to

,nincentration of 50 sM from a 5% stock solutiontodiately before incubation was started. A: intracel-

im!~ levels of reduced GSH. B: malondialdehyde levels .

i increase in cellular permeability became evi-tit,nfter three hours, but its relationship to lipidlotion was not further elucidated .

'~g . 4, iodoacetamide-induced changes in isolated hepatocytes . Cells were incubated in a medium containing 14C-I r :icetamide (0.075 mM). The temperature was either 37 throughout (continuous line) or 0 for one hour and then(dotted lines). The temperature was raised by changing to a preheated and iodoacetamide free medium . a-

;istiphcrol (0 .1 mM; solved in dimethylsulfoxide) was added to one of the incubates after one hour (0-0). A :6,ndialdehyde content, B : cell membrane permeability and C : iodoacetamide binding to macromolecules .

Discussion

49

In this study a series of compounds has been used totest whether GSH depletion caused cellular lysis bylipid peroxidation. We have found that, in fact,several GSH depleting compounds promoted lipidperoxidation and subsequent cellular lysis . Offundamental importance for the proposed toxico-logical mechanism is the belief that GSH protectsagainst lipid peroxidation in the metabolicallyactive cell . Earlier studies with isolated hepatocytesindicate that this is the case (Hogberg et al. 1975a)and further support is presented in this paper . Aclose correlation between low GSH levels in non-leaking cells and the accumulation of malondial-dehyde is documented, as well as the antidotaleffects of amino acid precursors for GSH synthesis .Of further importance was the apparent time lagbetween addition of the test compounds (anddepletion of GSH) and the accumulation of malon-dialdehyde. This lag distinguishes lipid peroxida-tion induced by the halongenated acetamides ordiethyl maleate from that induced in isolated cellsby iron salts (Hogberg et al 1975a), cumenehydroperoxide (Hogberg el al. 1975b), carbontetrachloride (Lindstrom et a1. 1978) or chlora-mine. It can be assumed that this . time periodreflects a phase of accumulation of peroxides or

I

Geier04702

oxygen radicals in the GSH deficient hepatocytes,and that such activated oxygen species eventuallywill trigger a random lipid oxidation (Fong et al.

1973). The. lag can thus be taken as a futher.indication that GSH prevents lipid peroxidation.

However, it also indicates that lipid peroxidationinduced by compounds such as halogenated ace-tamides or diethyl maleate is a consequence of

GSH deficiency per se.A causal relationship between lipid peroxidation

and cellular lysis was indicated by the tight coup-ling between these two events . This is clearly-documented in fig. 2, where ethylmorphine andparacetamol could be shown not only to inhibitlipid peroxidation but also to delay cellular lysis .Similar effects were obtained with a-tocopherol . Byaddition of this naturally occurring antioxidant itwas possible to preserve the cells in a seeminglyintact state for up to four hours, despite the factthat GSH was depleted.The metabolism of iodoacetamide was largely

brought to an end within four hours as judged bythe fate of the 14C-label. Of importance was the

observation that the binding of this electrophile tomacromolecules could be decreased and stoppedduring the first hour without significant changes in

the effect on cellular permeability . This pheno-.menon permitted us to show that a-tocopherol,when added after the first hour, could prevent lipidperoxidation and lysis, apparently without affec-ting the acetamide binding to presumed cellularreceptors such as proteins. A similar conclusioncan be drawn from the experiments where methio-nine was added after the first hour . The fate of thehalogen ions was not investigated, primarily be-cause diethylmaleate and other non-halogens in-duced similar effects as the halogenated aceta-mides . It can thus be summarized that our resultsstrongly support the thesis that GSH deficientisolated hepatocytes can undergo lipid peroxidation rapidly enough to destroy the cells before

alkylation of proteins (or other macromolecules)has been expressed as a cell destructive reaction .

The detailed nature of the apparent increase inselectivity for GSH conjugation obtained by lowtemperature incubation is not clear to us . Theobserved effect may relate to the increase inselectivity of iodoacetamide for certain SH-groupsat low temperature described by Oesterhelt et al. .

(1977) . However, the activity of GSH S-transferalsin intact hepatocytes may also contribute to thtchanged pattern (cf. ref. Jakoby et al.1976) . Ipreliminary experiments, also performed at lovtemperature, a transient effect on CoA levels wasobserved, but the loss was small (less than . ont';

percent) as compared to the loss of GSH . Thitechnique of utilizing low temperature incubationmay thus be a useful tool in future efforts 1characterize in more detail toxic effects of thin,reagents in biological systems .A novel mechanism for toxicity can be discern

in this work. GSH depletion, lipid peroxidatit*'and cellular lysis stand . out as key events in IIItoxicity of several disparate compounds. Howeverthe antidotal effects of the drugs ethylmorphiroand paracetamol indicate important limits for thrtin vivo significance of such a mechanism .

AcknowledgementsThe authors . wish to thank Annika Kristofersor

for technical assistance and Professor Paul Hochstein for valuable discussion on the manuscriptThe work was supported by grants from Karnlinska Institutet (No 390-5) and Arbetarskydt

fonden (No . 77-295) . One of the authors (A .H.Sr

was the recipient of a Council of Europe Fellow

ship.

References

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Boyland, E . & L . F. Chasseaud : The effect of socarbonyl compounds on rat liver glutathione levo ;Biochem. Pharmacol 1970,19,1526-1528 .

Burk, R. F., K . Nishiki, R . A. Lawrence & B . CharPeroxide removal by selenium-dependent and sehnium-independent glutathione`peroxidases in hemglobin-free perfused rat liver . J. BioL Chem . 1971253,43-46 .

Christophfersen, B . 0 .: Formation of monohydroipolyenic fatty acids from lipid peroxides by a glutsthione peroxidase. Biochim . Biophys. Acta 1968, 135-46 .

DeFerreyra, E . C ., J . A . Castro, M . I . Diaz Gomc .N. D'Acosta, C. R. DeCastro & O. M. DeFenePrevention and treatment of carbon tetrachlorhepatotoxicity by cysteine : studies about its mechi,nism . Toxicol AppL PharmacoL 1974, 27, 558-51

08, K .-L ., P . B . M,.fir 11 . Misra : Evidencee' mbranes is initiatedoecd during flavine1073 . 248, 7792-7797

`ttlyntte, J . R ., J . R. IvrVilonl mechanisms ofetd. 1974, 14, 271-281

;1l4yborg, J ., S . Orreniu "rwtlon in isolated hep!8,595-602 .

Jnslwrg, J ., S . Orrer'_ludics on lipid pero:'Jkytcs. Eur . J. Bioch,

1,16hcrg, J . & A . Krisltlutnthione levels artepitIocytes . Eur. J. I

14barM, ,1 . & A . KristFevlutcd hepatocytes .

- ;J1, 271-274 ./11uhy . W . B ., W. H . V'

J . Pabst : Gluts'~pccts . In: Glutath

fix,: J. M. Arias atw York, 1976, pp .D . P ., H . Thor

)eioxification reacti(rsf blutathione peroxi(ehydrogenase in rer110n by the cytochrot197K,6031-6039 .

)kl-hor, K . S ., R . Walls0on .and hemolysis iihvorkf hormones . Ar61. 14-521 .ldhtrom, T. D ., M .t>t phcnobarbital and

Jhloride toxicity in is(ttrthol. 1978, 28, 48-''oy . P . B ., D . D. C

Brook : Effect of glu

v.. . ie activity of GSH S-transfers .)afbcytes may also contribute to 1114.tem .(cf. ref. Jakoby et aL1976). I .experiments, also performed at fora transient effect on CoA levels wn t

at the loss was small (less than ontcompared to the loss of GSH. Th utilizing low temperature incubatione a useful tool in future efforts It in more detail toxic effects of third

,iological systems .techanism for toxicity can be discerns .

lgementsits wish to thank Annika Kristofersu.1 assistance and Professor Paul Ho chJuable discussion on the manuscript',was supported by grants from Karitutet (No 390-5) and Arbetarskyd(I :-

. .77-295) . One of the authors (A .H .S

ipient of a Council of Europe Fellow

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:ompounds,on rat liver glutathione level .'harmacol. 1970,19, 1526-1528.K. Nishiki, R. A . Lawrence & B . Chan,.

.removal by, selenium-dependent and stipendent glutathione=peroxidases in hemperfused rat liver . J. Biol. Chem. 19,

i .

'sen, B . 0 . : 'Formation of monohydroxatty acids from lipid, peroxides by a glut-oxidase . Biochim. Biophys. Acia .1968, let

E. C ., J . A. Castro, M . I. Diaz Gomesta, C . R. DeCastro & 0. M. DeFenrr and treatment of carbon tetrachlorlticity by cysteine : studies about its mec.lcol. Appl. Pharmacol. 1974, 27, 558-57

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plt, K .-L ., .P. B . McCay, J . L. Poyer, B . B. KeeleH. Misra: Evidence that peroxidation of lysosomal

Ihembranes is initiated by hydroxyl free radicals pro-,flNeed during flavine enryme activity J Biol Ch em. .1,973, 248, 7792-7797 .t)atte, J . R ., J . R . Mitchell & B . B . Brodie : Bioche-lillcal mechanisms of drug toxicity. Ann. Rev. Pharma-tol 1974, 14, 271-288.

1)4sberg, J ., S . Orrenius & R . E . Larsson: Lipid peroxi-dl,tion in isolated hepatocytes . Eur. J. Biochem. 1975a,W 595-602 .gbcrg, J ., S . Orrenius & P . J . O'Brien : FurtherMpdics on lipid peroxide formation in isolated hepa-

` lkytes . Eur. J. Biochem. 1975b, 59, 449-455,~1c . GSH depletion, lipid peroxidatix>t -. ~dtcrg, J . & A . Kristoferson : A correlation between

events in thy1

f1ulnthione levels and cellular damage in isolatedysis stand out as key

Ikpntocytes . Eur. J. Biochem. 1977, 74, 77-82 .;veral disparate compounds . Howevs

ilhgberg, J . & A . Kristoferson: Glutathione tumover intl effects of the drugs ethylmorphin;

401ated hepatocytes . Acta pharmacol. et toxicol. 1978,amol indicate important limits for t

271-274.

ficance of such a mechanism

by, W. B ., W. H. Habig, J. H . Keen, J . W. Ketley &. .

k1 J Pabst: Glutathione Stransferass Ctlti, .-e:aaycWapects. In : Glutathione: Metabolism and function .Iktlw. : J. M. Arias and W. B. Jakoby. Raven Press,,`!ow York, 1976, pp. 189-211 .

, D. P., H . Thor, B. Andersson & S . Orrenius:1}aloxification reactions in isolated hepatocytes; Role'I 8lutathione peroxidase, catalase, and formaldehydetkhydrogenase in reactions relating to N-demethyla-l fxn by the cytochrome P-450 system . J. Biol. Chem .

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,1)4xn and hemolysis induced by lactoperoxidase andih yorid hormones. Arch. Biochem. Biophys . 1977, 180,114-521 .

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51

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Oesterhelt, D ., H. Bauer, G.-B . Kresze, L. Steber &F. Lynen: Reaction of yeast fatty acid synthetasewith iodoacetamide . Eur. J. Biochem. 1977,79,173-180 .

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