Cellular redox changes and response to drugs and toxic agents

FUNDAMLNTAI. AND APPLIED TOXICOLOGY 3:200-208 (1083)

Cellular Redox Changes and Response to Drugs and Toxic Agents HELMUT SIES', REGINA BRIGELIUS, HERIBERT WEFERS, ARMIN MULLER and ENRIQUE CADENAS Institut fiir Physioiogische Chemie !, Universit;it Diisseldorf, Diisseldorf, Germany

ABSTRACT Cellular Redox Changes and Response to Drugs and T o x i c Agents. Sies, I!., Brigelius, R., Wefers, i t . , M i i l l e r , A. and Cadenas, E. (1983). F u n d a m . A p p l . lb.vicol. 3:200-208. Cellular metabolism and, in particular, o x i d a t i o n - r e d u c t i o n systems are linked to responses Io drugs and toxic agents in several ways. M a j o r connections are given by the N A D P H / NADP" system and the G S I I / ( ; S S G system, intracellular reductive pathways generally use N A D P I i as the electron donor. F r o m a toxicological point of view, N A D P H can be considered both as a " d e t u x i c a n t " and as a "toxicant". in the former case, N A D P I I s u p p o r t s the glutathione redox cycle by maintaining a negative redox potential of G S H to permit its detoxication functions to occur. N A D P H is also the main d o n o r for reducing equivalents in drug o x i d a t i o n s by the cytochrome P.450-dependent monooxygenase system which, with some notable exceptions, serves i m p o r t a n t purposes in detoxication. The sources of N A D P H reducing equivalents depend on the nutriti0nl state: major sources in the fed state are represented by the cytosolic pentose phosphate shunt dehydrogenases, whereas mitochondrial sources linked to isocitrale d e h y d r o g e n a s e provide the bulk of N A D P H reducing equivalents in the fasted state. As a "'toxicant", N A D P l i s u p p o r t s redox cycling reactions involving various drugs and other c o m p o u n d s of quinoid structure, aromatic nitro c o m p o u n d s and iron chelates with f o r m a t i o n o f s u p e r oxide anion radicals and subsequent f o r m a t i o n of other oxygen derived radical species. This presentation focuses on recent work carried out with isolated hepatocytes and perfused rat liver with respect to "oxidative stress". The noninvasive techniques of measurement of low-level chemiluminescence and of volatile h y d r o c a r b o n s (ethane, pentane) as well as glutathione release and calcium release have been employed. °Postal address: Prof. Dr. H. Sies. Insiitut fi.lr l'hysioiogische Chemic l, Moorensira~se 5, O-4000-Diisseldorfo W. Germay.

INTRODUCTION In a number of cases cellular responses to drugs and toxic agents are mediated by redox changes and it is now wellestablished that the cellular balance between oxidants and reductants is crucial. This pertains to the expression of toxicity as well as to the expression of certain therapeutic effects. In general, drugs or toxic compounds are reduced by reductases of various kinds in one-electron transfer reaction at the expense of nicotinamide nucleotides, notably NADPH, before they, in turn. reduce oxygen to the superoxide anion radical. This constilutes the so-called redox cycling in which, overall, 'there is an oxygen uptake to generate active oxygen species at the expense of cellular reducing equivalents (see Kappus and Sies, 1981, for review). This concept implies that reducing equivalents in tile form of NADPH may not only be used in detoxication as in the case in the cytochrome P-450-1inked monooxygenase reactions, but also, in a sense, in toxication by sustaining redox cycling. Apart from membrane damage and other effects w h i c h occur subsequent to the formation of the superoxide anion radical, there is also a loss of metabolic energy drawn upon for NADPH generation. In this presentation, we will first examine the redox potentials of nicotinamide nucleotides in the liver and then discuss another reductant, depending on NADPH. This is the glutathione system with its diverse roles in toxicological respects. Paraquat is taken as an example of redox cycling and its toxicological effects are discussed in terms of the relationships to NADPH and glutathione status. Further, we will present recent experiments on chemiluminescence as a noninvasive indicator for activated oxygen species, notably singlet oxygen. In addition, some data on hydrocarbon production by liver as an indicator for lipid peroxidation will be presented. Finally, we will give information on the relationship between NADPH consumption and GSSG release as well as calcium release.

TABLE 1 Redox Potentials of NADH and NADPH Systems in Rat Liver" Mitochondrial Matrix (pH 7.4)

Cytosol (pH 7.0)

Indicator Systems

mV

Indicator Systems

mV

NADHINAD"

- 241

Lactatelpyruvate

- 318

fl-Hydroxybu tyratelacetoaceta te

NADPH/NADP"

-- 393

lsocitratel2-oxoglutarate X COx Malatelpyruvate X CO2 o-P-Gluconatelribulose-5-P X CO:~

- 415

Isocitrate/2-oxoglutarateX COx Glutamatel2-oxoglutarate X NH.~

"Redox potentials as calculated from the indicator melabolite systems are presented for the pH values in cytosol and mitochondrial matrix space. Mitochondrial NADH and NADPH systems are connected by energy-linked tra nshydrogenase. Cytosolic and mitochondrial NADH systems are linked by the malatelaspartate shuttle. Cytosolic and mitochondrial NADPH systems are linked by the permeant tricarboxylates and dicarboxylates. See Sies (1982a) for further discussion. Copyright 1983, Sociely of Toxicology

200

Fundam. Appl. Toxicol. (3)

July/August, 1983

THE ROLE OF CELLULAR REDOX BALANCE 1N TOXICITY

/ /

Reduclants ~C:~-.:: T.~ (NADPH) //4-~:::~5=.'S~z

I~ i~

Uyloso,

I /

I X ,

/

2 '

I

I~

//'DH2~/~"NADP'~',,/~(Fe 'J""~l ~

/C

~'/~ k

1

\

V',"7,i

l\

o, oo~,

/::--.i-'-:~-/

EndopasmlC/ / /tehculum /

\

.................. j o ~ _ ~ . . . . - 7 / Maochondt,a

i

~J-~-ATJ

\

k

I)~OH~/

",,

I/

\"~k

/

/

J

SOD /

/1

-":/

1/~

l

I

. - ......

..........

I/ ~-'_ ~ 7 7. I .'+~P. N%P/ /

...

/-

;~vty ",.--7

.---

;

.

t

2,-,,o os,,-P. "-. ~

~ /

¢,oD

L.-'"

/

NADPH+H'

i

I

~ . f~, . ,

2GSH

/-%.,

NADP"

{

"

-~

~"'

"~

o't~ j

\ ~ k . 7 / ,~ ,.x-

o o,

C,7~

Pemxisomes

\

Cm,-H2O2

t

H20+ROH FIG. 1. Schematic diagram of oxygen reduction by one electron transfer (redox cycling) in different subcellular compartments. Also shown are NADPH supply reactions which maintain, on the one hand, redox cycling as indicated here for iron or drugs and, on the other hand, a protective system working through glutathione peroxidase and glu ta thione reductase. For reasons of space, redox cycling is represented in detail only in the cytosol but is known to occur at membranes of endoplasmic reticulum and mitochondria possibly at higher rates. Also shown is the peroxisomal H,~O.~metabolism. Modified from Kappus and Sies (1981).

MATER/ALS A N D M E T H O D S Hemog/obin-free perfusion o f rat liver This was done as previously described (Sies, 1978). The perfusion system was open, i.e. non-recirculating. The perfusion medium was bicarbonate-buffered Krebs-Henseleit solu-

Taken together, these new methods may provide a useful base for the study of redox problems in biochemical aspects of toxicology and pharmacology as has also been pointed out in a recent study on lipid peroxidation with isolated hepatocytes by Smith et al. (1982).

DithioerythriIol -10 _

1

o

qc

A12u~

S

u~

c o LP r-,

8-

.7.5

'5 E x o

5,0

L) r"

bt

E ,-

¢-

.c A-

-2,5 '6 =,3

E

c o

E

~r 0

0 I'

o

|

I

30

60

9'0

..............

1~o

incubation Time ( m i n ) FIG. 2. Time course of low level chemiluminescence and of thiobarbituric acid-reactive material (O) in isolated hepatocytes. A permeable thiol, dithioerythritol was added (!), and chemiluminescence intensity rapidly decreased. From Cadenas e! aL (1981). Fundamental and Applied Toxicology

(3) 7..8/83

201

"FABLE 2 Glutathione Status in Hemoglobin-free Perfused Rat Liver Under the Influence of Paraquat" Total Glutathione

Mixed Disulfides

GSH

,umnl per gram liver wet weight

GSSG nmol per gram liver wet weight

Cimtrl,I, 20 rain perfusitm

7.54±0.20

5.O0±0.24

1.80±0.11

18.3"+'0.5

1.10 rain per[usilm

o.20:t:O.l 7

4.o.1±0.1o

1.71±0.23

17.0±3.7

5.00±0.13

3.54.T.0.17

2.54±0.08

38.1:kl.7

I'araqu,~t (l raM), 140 m m perfusicm

"[.=ver~, were perfumed tor 1-10 nun. When par,lquat was added, the perfusate ctmtained paraqu,H fr~m 20 I~ l-t0 rain. l.)at,i are given as the mean±S.E.M. (n=8 different perfu~l~,ll~. From BriRt'~iusel al. (I o~2L

lion maintained at 37°C. Perfusate flow was 4 mL per min per gram liver wet weight, which was kept constant throughout the individual experiment. Assays GI utathione levels were determined according to the method of Akerboom and Sies (1981) and references therein. Chemiluminescence measurements were made according to Cadenas et a l (1981) and the exposure of the surface of the perfused liver for chemiluminescence measurement was done as described by Boveris et al. (1980). Measurement of ethane and pentane production by perfused liver was done as described by M011er er al. ( 1981 }. Calcium measurement in the effluent perfusate of perfused liver was done according to Sies et aL (1981 ).

state levels of singlet oxygen and related compounds (Cadenas et aL, 1981). As shown in Fig. 2, the addition of a permeable thiol to such isolated hepatocytes leads to a rapid decrease in c h e m i l u m i n e s c e n c e intensity and also the accumulation of malonaldehyde in the incubation medium is leveling off. Thus the harmful effects of hyperoxygenation can be counterbalanced to a certain ¢xtunt by intracellular protective systems. Conversely hyperoxygenationof glutathione-depleted hepatocytes (after treatment with phorone) led to a threefold increase in both chemiluminescence and malondialdehyde accumulation.

RESULTS AND DISCUSSION Redox systems of major interest: nicotinamide nucleotides and glutathione As shown in Table 1 there are considerable differences in the intracellular redox potentials of NADPH and NADH. The redox potential for NADPH in mitochondria and cytosol of liver is in the vicinity of - 4 0 0 mY, whereas the redox potential of NADH is substantially more positive. Therefore, intracellular reductive pathways generally use NADPH as the electron donor. A detailed discussion on the compartmentation of nicotinamide nucleotides and on the differences for various organs is available (Sies, 1982a). As already indicated in the introduction, NADPH serves both as a toxicant and as a detoxicant. This is given in some detail in Fig. 1. Here the top reaction shows NADPH taking part in redox cycling to produce superoxide anion radicals, whereas at the bottom NADPH is part of the so-called glutathione redox cycle which reduces hydroperoxides, either H20.~or ROOH, at the expense of GSH reducing equivalents. Due to the specificity of GSSG reductase, the utilization of NADPH is selective and there is practically no NADH utilization in the GSH peroxidase reaction. The various states in which reduced glutathione can take part in detoxication, have been discussed in detail recently (Reed and Beatty, 1979; Meister, 1981; Sies, 1982b). Role o f thiols a n d N A D P H in o x i d a t i v e stress In isolated hepatocytes, the effect of oxygenation with the usual CO,,/02 mixture (95/5) leads to accumulation of products of lipid peroxidation such as malondialdehyde. In a similar time course there is an increase in chemiluminescence intensity which is attributable to an increase in the steady 202

~ 300~, ~, ._.->. E~ .~x o f= ~ 200+eL E3 < Z

I

[

(~ -r (3.. 100E3 < Z

0-

I

30

I

. . . .

f'

"

60 90 120 Perfusion Time (rain)

FIG. 3. T h e cellular c o n t e n t s of N A D P H and" N A D P in p e r f u s e d liver in the absence (O) and p r e s e n c e (Q) of paraquat. Paraquat was added at 1 m M c o n c e n t r a t i o n a f t e r Z0 m i n u t e s of perfusion. The N A D P H / N A D P ratio w e r e 5.1 + 0.1 (n = 5) ad the c o n t r o l s were 2.3 4" 0.3 (n = 6) w h e n p a r a q u a t was p r e s e n t , m e a s u r e d at 140 m i n u t e s of perfusion time. Data from Brigelius el al. (1982.).

Fundam..AppL ToxicoL (3)

July/August, 1983

THE ROLE OF CELLULAR REDOX BALANCE IN TOXICITY

t-Butylhydroperoxide (raM) I 0.2

I 0.S 1 1.01 I

1 1 ~O

[ii

I A

101

o

l

10

1

.

q~ u~ o

Phorone o G o ~o

.c E E r-

2 Liver in posit ion

-"----..d _- ~ , , ' / ~ ' ~

N. Control Phenobarbital

DC BG 1

10

20 3~3 Perfusion Time (mini

40

FIG. 4. Chemiluminescence as detected from the surface of perfused liver, elicited by infusion of t-bu ty[ hydroperoxide. The figure shows lh ree different perfusions of livers from control, phenoba rbital pretreated, and phorone pretreated animals. Stable steady states are obtained at lower rates of infusion of hydroperoxide (concentrations as indicated in upper panel). Data from Wefers, unpublished results.

As an example of a compound capable of intracellular redox cycling, we will briefly discuss the cellular responses to the addition of paraquat (methyl viologen). Perfusion of liver with paraquat will lead to significant redox changes. Table 2 demonstrates a substantial decrease in the level of cellular GSH and a concomitant increase in the cellular content of mixed disulfides and GSSG levels, Fig. 3 shows the decrease in NADPH and the concomitant increase in NADP. It is noteworthy that intracellular protein-mixed disulfide sites can be related to the GSSG level. The occupancy of such sites is saturable and the data from different perfusion experiments extrapolate to a maximum value of near 4 #tool of potential cellular mixed disulfide sites (Brigelius et al., 1982). The toxicological interest in mixed disulfide formation is emerging. It has become increasingly clear that a number of proteins can be modified in their activity by mixed disulfide formation (see lists of such enzymes in Mannervik and Axelsson, 1980; Sies, 1983). in particular, enzymes of carbohydrate metabolism are influenced in such a way that upon an increase in mixed disulfide formation there is an increase in gluconeogenesis and an increase in glycogenolysis, both resulting in enhanced provision of glucose-6-phosphate as a source for NADPH reducing equivalents. In addition, the enzymes of the pentose phosphate pathway are activated by disulfide transitions. Fundamental and Applied Toxicology

(3) 7..8/83

Several other important effects of disulfides are known, e.g. inhibition of protein synthesis (Kosower et aL, 1972; see also Kosower and Kosower, 1978, for review), The sulfhydryldisulfide ratios of glutathione were reported to be under the control of cyclic AMP (Isaacs and Binkley, 1977). The role of cellular protective systems in dealing with oxidative stress can be demonstrated clearly in model experiments using t-butyl hydroperoxide as oxidant. As shown in Fig. 4 perfused liver emitted increasing intensities of chemiluminescence upon increasing rates of infusion of t-butyl hydroperoxide, Also shown in Fig. 4 is the increase in photoemission observed when glutathione content was decreased to values of less than 5% of the controls by pretreatment with phorone. Conversely, the amount of chemiluminescence is substantially decreased when animals were pretreated with phenobarbital. At the initial concentration of 0.2 mM t-butyl hydroperoxide, there is no detectable increase in chemiluminescence in livers obtained from phenobarbital-pretreated animals. Glutathione peroxidase as One of the major protective systems consists of the selenium-dependent enzyme and the selenium-independent enzyme activity, As shown in Fig, 5, the chemiluminescence intensity is substantially increased in the state of selenium deficiency and this can be counteracted by pretreatment with phenobarbital. Phenobarbital treatment is known to lead to an increase in the non-selenium-dependent 203

T - B ~ t f!hyd'to(3~,,e o,,,,Ge | ~IM )

t-Elut/lm/drop~,o,

,de i m *,~ J

0,:, t o3o F ....

!

!

.......... T .......... !

o6o ;

I

!

!

t

t

t

!

!

j

.

/

/

Se"

ported by similar experiments in w h i c h an inhibitor of mitochondrial energy dependent transhydrogenase activity, rhein (anthraquinone-2-carboxylate), is employed (data not shown).

Volatile hydrocarbon production during flux through alcohol dehydrogenase Discussions of the involvement of ;ipid peroxidation in several toxicological conditions such as ethanol-induced liver damage or carbon-tetrachloride-induced liver damage are relatively voluminous. Therefore, in the present context we will only present a new technique, hydrocarbon production, w h i c h might be helpful in further elucidation of such problems; we ........ Menodione 20iuM

[

g

L~

r~ r

U

Se"

P h e ~ o b G r b~tol

~f'~-

t j

= °

._ff

__1- ...... 10m,'~

[:IC;, 5. Low level d~emiluminescence as obtained from the surface of perfused rat livers in different states of selenium deficiency. l'resentalion similar to that of Fig..I. Selenium deficiency (Se) was obtained by feeding Torula-based diet for four wk. GSH peroxidase activity in Se -rat livers was less than 5% of controls. Data from Wefers, unpublished.

E c3

tb ..........

2b

3'o

..........

do

Perfusion Time (Pain) "T (o%J et)

Antimycin A 20/uM

x

activity. Also s h o w n in Fig. 5 is a very dramatic increase in the chemiluminescence intensity when glutathione depletion by phorone pretreatment is combined with selenium deficiency. As the chemiluminescence is mainly attributable to the formation of excited species upon interaction of oxygen centered radicals, this experimental system demonstrates that the glutathione-dependent protective system can be tested with the intact organ in terms of its capacity to counteract oxidative stress. A compound known to undergo redox cycling is menadione (vitamin Ka). As shown in Fig. 6A, there is an increase in chemiluminescence intensity with menadione, and this is sensitive to the inhibitor of the respiratory chain, antimycin A. Antimycin A alone does not have any effect and the reversed order of additions does not lead to menadione-induced chemiluminescence (Fig. 6B). At present, the dependence of menadione supported chemiluminescence on mitochondrial energy production is not completely understood. However, it seems that the requirement for NADPH in the maintainance of menadione-dependent redox cycling is involved. This is sup204

m tO C3

Menodione 20MM

g (lJ u c u ut c

E

0

1'o

. .2'o ....

-' 30

Perfusion Time (min) FIG. 6. Menadione-dependent chemiluminescence as detected f r o m the surface of perfused ra t liver. The addition of antimycin A suppresses chemiluminescence when given either after (A) or before (B) menadione,

Fundam. AppL ToxicoL (3)

July/A ugust, 1983

"I'I4E ROLE OF CELLULAR REDOX BALANCF IN TOXICITY which, in turn, elicit enhanced alkane release. Preliminary experiments show that the simple reduction of cytosolic NAD is not in itself sufficient to cause these effects, but the increase in ethane or pentane release can be obtained by infusion of acetaldehyde in presence or absence of pyrazole derivatives to inhibit alcohol dehydrogenase.

Ethanol (3 2 n'.M) n- Prol~pyrazo~ IIO~M) Cy~nJdanol(2~}

~

.o,

-l.00 R ~,

2-

-

O_

-0 I ~0 6~0 Perlus=0n'13rne(mini FIG. 7. Increase o~" ethane release from perfused rat liver during addition of ethanol (time as indicated), and decrease by N-Propylpyrazole and (+)-cyanidanol. From MUller and 5ies (1982). I

20

will not discuss related changes in glutathione such as increased biliary GSSG release (Sies etaL, 1979) or changes in intracellular glutathione (Videla et aL, 1980; Vi6a etaL, 1980). Hydrocarbon production by intact animals was introduced by Riely et aL (1 974) and it has been applied by several groups working with intact animals (KLister et aL, 1977; Litov etaL, 1978; Burk and Lane, 1979; see Wendel and Dumelin, 1981, for review). The adaptation of hydrocarbon measurement to the isolated perfused liver may form a useful simplification, because extra-hepatic contributions, e.g. from the gut flora, are excluded. We will briefly discuss, as an example, the effects of ethanol. As shown in Fig. 7, the rate of ethane release from the isolated perfused rat liver increases w i t h i n a few minutes upon the addition of ethanol (MLiller et aL, 1981 ; MiJIler and Sies, ; 982). This increased rate of ethane production is sensitive to an inhibitor of alcohol dehydrogenase and it is also sensitive to a radical quencher, cyanidanol (Fig. 7). Similarly, pentane formation is increased in a pyrazole-sensitive fashion (MLiller and Sies, 1982). Therefore flux through the alcohol dehydrogenase reaction is responsible for the formation of products

Cytosol r

H20

Isocitrate

t4on~oxygenase" r.JADP" System , , , . ~ DPH - H"

@

'

02

However, it may be mentioned briefly that the NADPH supply is sufficient to maintain drug oxidations in the fed and fasted state. This was demonstrated by direct measurement of '4CO.2 release from (HC dimethylamino)-aminopyrine (Weigl and Sies, 1977) and it was also shown by measurements of 02 uptake (Thurman and Scholz, 1969; Sies and Brauser, 1970). Similarly organ absorbance spectrophotometry of perfused rat liver showed no limitation of NADPH supply (Sies and Brauser, 1970). In a recent study, Oyanagi et aL, 1981) came to a different conclusion, based on a more reduced state of cytochrome P-450 during metabolism of hexobarbital in perfused

Mitochondrion

Fetty Acids] t4alete t Oxalocetote ADP.Pi AcetyI-CoA ,, ~/ f\Cltrote

R0H

M e t a b o l i c consequences o f drug oxidations The metabolic consequences arising from drug oxidations by the cytochrome P-450-dependent monooxygenase system of the endoplasmic reticulum are transmitted through redox changes. They arise from the utilization of NADPH and from the utilization of O.~. During drug oxidations there is a steady state decrease of the N A D P H / N A D P ' ratio. The [free NADPH]/[free NADP'] ratio is decreased to approximately onehalf of the controls, as has been found with hexobarbital or aminopyrine as substrate (Sies and Brauser, 1970). As s h o w n in Fig. 8, a major linkage to reactions of intermediary metabolism is given in the NADP'-dependent isocitrate dehydrogenase reaction, so that biosynthetic pathways can be affected by drug oxidations. For example, in perfused livers from phenobarbital-pretreated rats, lipogenesis from glucose was partially restricted during aminopyrine metabolism (Thurman and Scholz, 1973) and also gluconeogenesis from lactate was inhibited in a similar fashion (Scholz et aL, 1973). In the present context, not all interrelationships between intermediary metabolism and drug oxidations can be given, and more detailed presentations are available (Sies et aL, 1979b; Thurman and Kauffman, 1980).

", M a l a t e

Citrote

1[

Isocitrme

CO2

Glutomote

-

.

2-Oxoglutorote Molote

2-Oxoglutarate -

, ~r~r~,}

Molote

FIG. 8. Relationship of NADPH utilization by the monooxygenase system to cellular tricarbox-,latel dicarboxylate metabolism. NADP-dependent isocitrate dehydrogenase equilibrates cytosolic and mitochondrial NADPH redox systems. Relationship to fatty acid synthesis via :-itrate. cleavage enzyme and ureogenesis is also presented. Modified from Weigl and Sies (1977). Fundamental and Applied Toxicology

(3) 7.8/83

205

NexobQrbitQt (0 L5mM) _

NH CI (2,5mM) O CO £'3 !

C~ L~ C'3

-0.025t -ButylHydroperoxide (0./.6raM)

< <3

-0.050-

10 15 IncubationTime (rain) FIG. o. Oxidation of nicotinanlide nuclL'otides in isolated hepatocytes from phenobarbital pret reared rat. The dihydro band is measured by dual wavelength absorbance photometry, liver from fasted rats w h e n sorbitol was present. However, the extra oxygen uptake observed in the presence of sorbitol may have led to hypoxic conditions, as indicated by higher degrees of reduction of cytochrome oxidase and cytochrome c. The sub-lobular heterogeneity in liver cab now be studied by using flexible light guides (Ji et aL, 1980, 1981). Such methods w i l l be useful in the evaluation of redox effects in responses to drugs and toxic agents at the periportal and pericentral areas of the liver lobe. Therefore, new insight into the problem of pericentral necrosis can be anticipated. As the NADPH-supplying reactions are provided with substrates in the fed as well as in the fasted state, it is only the relative proportion of contributory reactions w h i c h will change during the transition from fed to fasted. In the fed state the liver will mainly rely on the pentose phosphate shunt dehydrogenases because of the supply of glycogen and, therefore, glucose-6-phosphate. In the fasted state, the mitochondrial production of NADPH, as transmitted through isocitrate dehydre,genase, will become of major importance. Fig. 9 demonstrates the degree of oxidation of NADPH attainable during the various NADPH-requiring reactions, Isolated hepatocytes from phenobarbital-pretreated rats oxidize reduced nicotinamide

206

nucleotide upon the addition of the drug substrate, hexobarbital. Further oxidation was obtained by stimulation of ureogenesis w i t h ammonium chloride, requiring NADPH through glutamate dehydrogenase (Fig. 9). An even further oxidation was obtained by addition of a substrate for gtutathione peroxidase, t-butyl hydroperoxide. These states of oxidation were correlated w i t h decreases of the NADPH/NADP" ratio in similar experiments (Sies et aL, 1976). Ca/cium re~ease during drug oxidations During substrate flux through the cytochrome P-450-dependent monooxygenase system, intact perfused liver responds with calcium efflux (Sies et al., 1981 ). This is also shown in Fig. 10A where extra oxygen uptake is elicited by ethylmorphine in a metyrapone sensitive manner. Calcium release, s h o w n in the bottom part of Fig. t0A, shows a similar time course. That calcium release during drug oxidations is not due to energy depletion by possible hypoxic transitions due to the oxygen uptake is shown in Fig. 10B. Here, aminopyrine serves to elicit calcium efflux, and a subsequent hypoxia as effected by mixing nitrogen at an appropriate percentage into the gas mixture does not cause changes in effluent calcium concentration. While at present the exact cellular source for the calcium w h i c h is leaving the hepatocytes is unknown, the effect has been attributed to an oxidation of NADPH during drug oxidations (Sies et aL, 1981 ). Glutathione disulfide release during drug oxidations There are several functions of glutathione in drug metabolism (see Orrenius and Jones, 1978; Reed and Beatty, 1979; Orrenius and Sies, 1982, for reviews). For the present purposes, we may briefly mention that during demethylation of aminopyrine an increase of glutathione release was observed in perfused liver (Oshino and Chance, 1977), and this was attributed to an increase in glutathione disulfide release by separate measurements of GSSG and GSH (Sies et aL, 1978). The increase in GSSG release during drug oxidations was attributed to an increased flux through ~Iutai.hione peroxidase as a rasult of enhanced formation of hydroperoxides. While hydrogen peroxide was made responsib!e for this effect (Jones et al., 1978, 1981), other hydropero.xides may also be produced. This possibility exists because the aminopyrine dependent GSSG release was observed also in livers from selenium deficient animals where H.~O=-dependent GSSG release was not observed (Sies et aL, 1978). Furthermore, the increase in catalase-H~O~ in isolated hepatocytes (Jones et aL, 1978) failed to show in our hands (Sies and Graf, 1982) whereas the increase in catalase-H~O2 w i t h benzylamine was readily detectable, Therefore, the exact source of GSSG during drug oxidations remains to be elucidated. Glutathione disulfide efflux occurs into the biliary space, and recently a relationship between the rate of biliary GSSG efflux and the intracellular GSSG content was demonstrated (Akerboom et aL, 1982a). Transport across the canalicular membrane may occur via a transport system because the estimated concentration ratio between bile and cytosolic water is around 50. Studies on the properties of the transport system (Ekl6w et al., 1981; Akerboom et aL, 1982b) indicated that glutathione conjugates might be transported in a closely linked way, possibly even on the same carrier system. It may be mentioned that in the intact animal under different conditions of "oxidative stress" there can be increased biliary GSSG concentrations, possibly of use as indicator for intracel-

Fundam. AppL Toxicol. (3)

duly/Augus& 1983

THE ROLE OF CELLULAR REDOX BALANCE 1N TOXICITY

lular hydroperoxide metabolism (see Sies and Summer, 1975). For example, in rats treated chronically with ethanol, biliary GSSG was significantly increased whereas GSH remained unaltered as compared to controls (Sies et al., 1979a).

Ethylmorphine ....

~

E

apone

A CKNO WLEDGEMENT

Supported by Deutsche Forschungsgemeinschaft, Schwerpunktsprogramm "Mechanismen toxischer Wirkungen von Fremdstoffen" and by Ministerium fur Wissenschaft und Forschung, Nordrhein-Westfalen. E.C. is recipient of. an A.-v.-Humbolt fellowship.

0.1

-4,--"

r" 130

0,2

LU ¢-

0,3O

% L)

7 '0

' 80

Perfusion

Aminopyrine

E

90 Time (min)

Hypoxia

0

"6 0,1 LM

_c 02"

o"

:3.. ('w

.H

~D (D t_

=_~

t0 min FIG. 10. Increase in oxygen uptake and in calcium release by addition of drug substrate (Type I), ethylmorphine (A) or anainopyrine (B). In A, dependence of calcium efflux on electron flow through the P-450 system is shown by the inhibitory effect of metyrapone. In 13, the interval of hypoxi,: (obtained by increasing Nz in the ~as mixture at the expense of O~) demonstrates that cakium efflux during drug oxidations is not due to oxygen limitation. For further discussion, see Sies e/al. (1981). From Sies and Graf (1982). Fundamental and Applied Toxicology

(J) 7.8/83

REFERENCES Akerboom, T.P.M. and Sies, H. (1981). Assay of Glutathione, Glutathione Disulfide and Glutathione Mixed Disulfides in Biological Sa mples. In Detoxication and Drug Metabolism: Conjugation and Related S.vstems, W.B. Jakoby, ed., pp. 373-382, Meth. in Enzymology, 77. Academic Press, New York, NY. Akerboom, T.P.M., Bilzer, M. and Sies, H. (1952a). The Relationship of Biliary Glutathione Disulfide (GSSG) Efftux and lntracellular GSSG Content in Perfused Rat Liver. J. Biol. Chem. 257:4248-4252. Akerboom, T.P.M., Bilzer, M. and Sies, H. (1982b). Competition Between Transport of Glutathione Disulfide (GSSG),and Glutathione S-Conjugates from Perfused Rat Liver into Bile. F E B S l.x,tt. 140:73-76. Boveris, A., Cadenas, E., Reiter, R., Filipkowski, M., Nakase, Y. and Chance, B. (1980). Organ Chemiluminescence: Noninvasire Assay for Oxidative Radical Reactions. Proc. Natl. Aca~L Sci. USA 77:347-351. Brigelius, R., Lenzen, R. and Sies, H. (1982). increase in Hepatic Mixed Disulfide and Glutathione Disulfide Levels Elicited by Paraquat. 13iochem. Pharmacol. 31:1637-1041.

Burk, R.F., Nishiki, K., Lawrence, R.A. and Chance, B. (1978). Peroxide Removal by Selenium-dependent and Seleniumindependent Glutathione Peroxidases in Hemoglobin-free Perfused Rat Liver. J. BioL C71em. 253:43-40. Burk, R.F. and Lane, J.M. (1979). Ethane Production and Liver Necrosis in Rats After Administration of Drugs and Other Chemicals. ToMcol. Appl. PharmacoL ,50:467-478. Cadenas, E., Wefers, H. and Sies, H. (1981 ). Low Level Chemiluminescence of lsola ted Hepatocytes./:~tr. J. Biochem. 119:531-536. EkliJw, L., Thor, H, and Orrenius, S. Formation and Efflux of Gluthione Disulfide Studied in Isolated Rat Hepatocytes. FEBS Lett. I27:125-128. lsaacs, J.T. and Binkley, F.: Cyclic AMP-dependent Control of the Rat Hepatic Glutathione Disulfide-sutfhydryl Ratio. lYiochhn. Biophys. Acta 498:29-38. lyanagi, T., Suzaki, T. and Kobayashi, S. (1981). Oxidationreduction States of Pyridine Nucleotide and Cytochrome P-450 During Mixed-function Oxidation in Perfused Rat Liver. J. BioL Chem. 256:12933-12939. Ji, S., Lemasters, j.J. and Thurman, R.G. (1980). A Non-invasive Method to Study Metabolic Events Within Sublobular Regions of Hemoglobin-free Perfused Liver. FEBS Lett. 113:37-4 I. It, S., Lemasters, J.J. and Thurman, R.G. (1981). A Fluorometric Method to Measure Sublobular Rates of Mixed-function Oxidation in the Hemoglobin-free Perfused Rat Liver. Molecular l~harmacoL 19:513-516. Jones, D.P., Eklbw, L., Thor, H. and Orrenius, S. (1981). Metabolism of Hydrogen Peroxide in Isolated Hepatocytes; Relative Contributions of Catalase and Glutathione Peroxidase in Decomposition of Endogenously Generated H2Oz. Arch. Biochem. Biophjw. 2 ] 0:505-516. 207

Jones, D.P., Thor, H., Andersson, B. and Orrenius, S. (1078). Detoxification Reactions in Isolated Hepatocytes. Role of f.;lutathione Peroxida.,,e, Calalase, and I,orm,ddehyde Dehydrogenase in Reactions Relating to N-Demethylation by the Cytochrome I'-450 System. J. BioL CTwnt. 253:6031-o037. Kappus, |-I. and Sies H. ( 108 I). Toxic Drug Effects Associated with Oxygen Metabolism: Rednx Cycling and Lipid l'eroxidatitHa. E.~Twrient ia 37:1233-1241. Kfister, U., Albrecht, D. and Kappus, H.(10771. Evidence for Carbon "letrachloride-and Ethanol-induced Lipid l'ert~,idation In I'iv, Demonstrated by Ethdne I'rt)ductlon of Mice and Rats. "l'oxtcol. AppL Pharmacol. -1I:03o-o.18. Kosower, N.S., Vanderhoff, G.A. and Kosower, E.M. (1072). The Effects of Glutatione Disulfide on Inhibition of Protein Synthesis. Biochim. Biophys. Acta 272:023-037. Kosower, N.9. and Kosower, E.M. (1078). The Glutathione Status of Cells. hlternational Rev. Qvtol. 54:109-100. Litov, R.E., Irving, D.H., Downey, I.E. and Tappel, A.L. (1978L Lipid l'eroxidation: A Mechanism'lnvtdved in Acute Elhant~l Toxicity as Demonstrated by/n I'ivo Ethane I'roductit~n in the Rat. I.ipid.~ 13:305-307. Mannervik, B. and Axelsson, K. (1080). Role of Cytoplasmic Thioltransfera:,e in Celhdar Regulation by Thiol-disulfide Interchange. Biochem. J. 100:125-130. Meister, A. ( I o81 ). Metabt~lism and Functions of glutalhit~ne. 77BS c,(o):231-23.1. Miiller, A., Graf, !'., Wendel, A. and Sies, H. (1081). Ethane Prt~ductitwl by Ist~lated I'erfused Rat Liver. A System to Study Metabolic Effects Related to Lipid I'eroxidation. FEBS l.ett. 12o:2.1 1-2.14. Miiller, A. and Sies, H. (1082). Role of Alcohol Dehydrogenase Activity and of Acetaldehyde in Ethanol-induced Ethane and Pentane Production by Isolated Perfused Rat Liver. Biochem. J. 20o:153- 15o. Orrenius, S. and Jones, D.P. (1078). Functions of Glutathione in Drug Metabolism. In I:um'tions o.f Glutathione in Liver and Khlney, H. Sies and A. Wendel, eds., pp. 11~4-175.Springer-Verlag Berlin, Heidelberg, NY. Orrenius, S. and Sies, H. (1982). Compartmenta tion of Detoxification Reactions. In Metabolic Compartmentation, H. Sies, ed., pp. 485-520. Academic Press, London, N e w York, NY. Oshino, N. and Chance, B. (1977). Properties of Glutathione • Release Observed During Reduction of Organic Hydroperoxide, Oemethylation of Aminopyrine and Oxidation of Sorne Substances in l'erfused Rat Liver and Their Implication for the Physiological Function of Catalase. Biochem. J. 162:509-525. Reed, D.J. and Beatty, P.IV. {1979). Biosynthesis and Regulation of Glutathione: Toxicological Implications. Rev. Biochem. 7bxicol. 2:213-241. Riely, C.A., Cohen, G. and Lieberman, M. (1974). Ethane Evolution: A New Index of Lipid Peroxidation. Science 183:208-210. Scholz, R., Hansen, IV. and Thurman, R.G. (1973). Interaction of Mixed-function Oxidation with Biosynthetic Processes. 1. Inhibition of Gluconeogenesis by Aminopyrine in Perfused Rat Liver. Eur. J. Biochem. 38:64-72. Sies, H. (1978). The Use of Perfusion of Liver and Other Organs for the Study of Microsomal Electron Transport and Cytochrome P-450 Systems. In Methods #~ En~l'mology. S. Fleischer and L. Packer, eds., Vol. 52, pp. b,8-59. Sies, H. (1982a). Nicotinamide Nucleotide Compartmentation. In Metabolic Compartmentation, H. $ies, ed., pp. 205-231. Academic Press, London, New York.

208

Sies, H. (1983). Reduced and Oxidized Glutathione Efflux from Liver. In Glututhione - Storage. Tran.wort and Turnover in Mammals. Y. Sakamoto, T. Higashi, and N. Tateishi, eds., pp. 51-70. lapan Sci. Soc. Press, Tokyo. files, H. and Brauser, B. (1970). Interaction of Mixed Function Oxidase with its Substrates and Associated Redox Transitions of Cytochrome P-450 and Pyridine Nucleotides in Perfused Rat Liver. European J. Biochem. 15:531-540. files, H. and Summer, K.H. (1075). Hydroperoxide Metabolizing Systems in Rat Liver. I'ttr. J. Biochem. 57:503-512. Sies, H., Summer, K.H. and Grosskopf, M. (1970). Hydroperoxidelinked I'erturbation of Glutathinne Redox State and its Use in the Study of NADPH-dependent Pathways in Liver Cells. In ~Ae o f Isolated l.iver Cells and Kidne.r Tubules in Metabolic Studies, .I.M. Tager, H.D. $[.~lingand J.R. Wi[liamson, eds., pp.

317-322. North-Holland, Amsterdam. Sies, H., Bartoli, G.M., Burk, R.F. and Waydhas, C. (1978). Glutathione Efflux from l'erfused Rat Liver After Phenobarbital Treatment, During Drug Oxidations, and in Selenium Deficiency. Eur. J. Biochem. 89:113-118. files, H., Koch, O.R., Martino, E. and Boveris, A. (1979a). Increased Biliary Glutathione Disulfide (GSSG) Release in Chronically Ethanol-treated Rats. t"EBS Lett. 103:287-290. Sies, H., Iveigl, K. and Ivaydhas, C. (1979b). Metabolic Consequences of Drug Oxidations in Perfused Liver and in Isolated Hepatocytes from l~henobarbital-prelreated Rats. In 7he huh,'lion o f Drug Metaholisol. R.W. Estrabrook and E. Lindenlaub, eds., pp. 383-400. Symposia Medica Hoechst 14, I,.K. Schattauer \"erlag, Stuttgart and New York, NY. Sies, H., Graf, P. and Estrela, J.M. (19/51). Hepatic Calcium Efflux During Cytochrome I'-450-dependent Drug Oxidation ,at the Endoplasmic Reticulum in Intact Liver. Proc. Natl. Acad. &'i. USA 78:3358-3302. Sies, H. and Graf, P. (1982). Redox Relations Associated with Drug Metabolism in l'erfused Liver. In Microsomes, Drug Oxidations aml Drug Toxieith,s. R. Sato and R. Kato, eds. Wiley Interscience, New York, NY. Smith, M.T., Thor, H., Hartzell, P. and Orrenius. S. (1982). The Measurement of Lipid Peroxidation in Isolated Hepatocytes. Biochem. Pharmaeol. 31:19-26. Thurman, R.G. and Scholz, R. (196-9). Mixed-function Oxidation in Perfused Rat Liver. The Effect of Aminopyrine on Oxygen Uptake. l'Sttr. J. Biochem. 10:459-467. Thurman, R.G. and Scholz, R. (1973). The Role of Hydrogen Peroxide and Catalase in Hepatic Microsomal Ethanol Oxidation. Drttg Metab. Disp. 1:441-448. Thurman, R.G. and Kauffman, F.C. (1980). Factors Regulating Drug Metabolism in Intact Hepatocytes. Pharmacol. Rev. 31:229-251. Videla, L.A., Gernandez, V., Ugarte, G. and Valenzuela, A. (1980). Effect of Acute Ethanol Intoxication on the Content of Reduced Glutathione of the Liverin Relation to its Lipoperoxidative Capacity in the Rat. FEBS Lett. 111:6-10. Vina', J., Estrela, J.M., G uerri, C. and Romero, F..i. (1980). Effect of Ethanol on Glutathione Concentration in Isolated Hepatocytes. Biochem. J. 188:549-552. Weigl, K. and Sies, H. (1977). Drug Oxidations Dependent on Cytochrome P-450 in Isolated Hepatocytes. The Role of the Tricarboxylates and the Aminotransferases in NADPH Supply. Eur. J. Biochem. 77:401-408. Ivendel, A. and Dumelin, E.E. (1981). Hydrocarbon Exhalation. Meth. Enzymol. 77:10-15.

Fundam. AppL ToxicoL (3)

duly/A ugust, 19&~