Download Inhibitio¡ of GLutathione S-Õransferases Studies

Inhibitio¡ of GLutathione S-Íransferases
Studies with quinones and et,hacrynic acid
Re--ingi van Glutathion S-lransferases
Studies net chinonen en etacrynezuur
(met een samenvatting in het Nederlands)
Proefschrift ter verkrijging van de graad van doctor
aan de Universiteit Utrecht
op gezag van de Rector Magnificus, Prof.Dr. J.A. van Ginkel
ingevolge het besluit van het College van Decanen
in het openbaar te verdedigen
op vrijdag 14 januari 1994 des middags te 2.30 uur
door
Joannus Hubertus Toussaint Ma¡ia Ploenen
geboren op
te Beek (L)
1
BIELIOTHEEK
I
FË8. 1gg4
ZEIST
Promotoren: Prof.Dr. v.J. Feron
Prof.Dr. P.J. van Bladeren
Co-promotor: Dr. B. van
Ornmen
proefschrift
Het in dit
beschreven onderzoek werd
uitgevoerd bij de afdeling Biologische loxicologie van het
Instituut voor Toxicologie en Voeding TNO.
Dit proefschrift werd mogelijk gemaakt met financiêle
ondersteuning door de Nederlandse Organisatie voor
wetenschappelijk Onderzoek (NWO/ gebied
Medische
wetenschappen, subsidie 900-527-124) .
Het drukken van het proefschrift werd voor een deel
mogelijk door financiëIe steun van het Instituut voor
Toxicologi-e en Voeding TNO en de Nederlandse OrganisatJ-e
voor wetenschappelijk Onderzoek.
CIP-GEGEVENS KONINKLIJKE BTBLTOTHEEK, DEN HAÀG
Ploemen, Joannus Hubertus Toussaint Maria
Intribition
of glutathione
S-transferases: studies with
quinones and ethacrynic acid ,/ Joannus Hubertus Toussaint
Maria Ploemen. - Utrecht : Universiteit UÈrecht,
Faculteit Diergeneeskunde
Proefschrift Universitei-t Utrecht. - Met lit.
samenvatting in het Nederlands.
rsBN 90-393-0449-7
opg. -
Met
Trefw.: gLutathion S-transferases ; remming / toxicologie.
contents
page
Chapter 1- General Introduction
Part I
4
Halogenated quinones and catechol-derived quinones
Chapter 2- Irreversible inhibition of human
glutathione S-transferases by tetrachloro1r4-benzoquinone and its glutathione
conjugate
Chapter 3- In vitro and in vivo reversible and
irreversible Ínhibition of rat gluÈathione
S-transferase isoenzlanes by caffeic acid
and its 2-S-glutathionyl conjugate
Chapter 4- Inhibition of human glutathione Stransferases by doparnine, a-methyldopa and
their S-S-glutathionyl conjugates
Chapter 5- S-12,4-Dinitrophenyl)glutathione excretion
into the medium of rat hepatoma cells.
Effects of ethacrynic acid and tetrachloro1r4-benzoquinone and derivatives.
Chapter 6- Summary part I
Part II Ethacrynic acid and derivatives
Chapter 7- Inhibition of rat and human glutathione Stransferase isoenzymes by ethacrynic acid
and its glutathione conjugate
Chapter 8- Isoenz]¡me selective irreversible inhibition
of rat and human glutathione S-transferases
by ethacrynic acid and two brominated
derivatives.
Chapter 9- Reversible conjugation of ethacrynic acid
with glutathione and and human glutathione
S-transferase P1-1
chapter 10- Sumnary part II
Part IIr
Chapter 11- Generaf discussion and
Samenvatting
su¡nmary
23
34
51
70
86
89
100
116
131
L34
L45
List of publications
Curriculum vitae
150
Nawoord
t_5 3
Abbreviations
155
L52
voar JacqueTine
-4-
Chapter
1
ceneral Introduction
1.1 Glutathione s-transferases:
expression, and catalytic aspects
properties,
function,
In the course of evolution, organisms have developed
enzfrme-systems that protect against toxic effecÈs of
xenobiotics. In g'eneral, these enzymes increase the
hydrophilic properti-es of compounds, thus transforming them
into products capable of being transported and excreted Ín
the urine or bile. The chemical reactions xenobiotics
undergo, can be conveniently divided into two major
phase I-reactions in which oxidation,
classes, vtz.
reduction, and hydrolysis reactions often serve to provide
a chemical handle on the xenobiotic molecule and phase IIreaction in which conjugation reactions further append
hydrophilic groups to the xenobiotics (Àrmstrong 1987).
The glutathione S-transferases (GST) (EC 2.5.1.18)
catalyae the conjugation reaction of the tripeptide
glutathione (y-GIu-Cys-GIy) to numerous electrophilic
substrates, the first step in the mercapturic acid pathway
(Jakoby 1978). This conjugation reaction has been widely
recognized to be crucial for the detoxification of several
agents of both endogenous and exogenous origin.
Several
classes of
substrates are known, such as epoxides,
guinones, arB-unsaturated aldehydes and ketones, alkyl- and
aryl halides (Mannervik 1985). The glutathione conjugate
formed is usually less toxic than the parent compound,
several cases bioactivation occurs (Van
although in
Bladeren 1988). In addition to the enzyme function, it is
proposed that the cST serve as bincling protein by both
covalent and non-covalent interaction (Vos and Van Bladeren
1990). Both the abundance of the enzyme, comprising about
4e" of the soluble protein in human liver (Van Ommen et
a7.
1990) and the high concentrations of glutathione 1up to 510 mM) indicate the importance of glutathione and cST in
the maintenance of health (Àrmstrong 7987). For a
historical perspective and as well for more recent reviev¡s
-5-
the reader is referred to Jakoby (f978), Mannervik (1995),
Mannervik and Danielson (1988), Coles and Ketterer (1990),
and Armstrong (1991).
GST have been isolated and characterized from various
organs in a variety of species, including mammals and
vertebrates (Mannervik 1985 ) . Most of the enzl¡mes are
presenÈ in the cytosol, although a microsomal form has been
characterized
(Morgenstern and Depierre 19gg). In
parÈicular,
rat
and human cytosoJ-ic GST have been
extensively studied, and this introduction will further
concentrate on these cytosolic GST. The cytosolic enzl¡mes
found in ma¡nmalian species exist as homodimeric and
heterodimeric proteins, with molecular masses of Èhe
subunits varying from about 24.5 Lo 2j kDa. Sofar, 15 rat
subunits and l-0 human subunits have been idenÈified (Table
L). The existence of muttiple isoenzymes of GST probably
signifies their biological importance. As a group of
enzymes the GST catalyse a broader spectrum of substrates
tharr could be achieved wit.h a single enzyme form. Although
individual enzymes tolerate a wide range of substrates,
each enzyme displays an unique preference for certain
electrophilic
aroups and exhibits a high degree of
stereoselectivity
for some compounds. The GST appear to be
organized in four distinct gene families designated alpha,
Rü¡ pi (t4annervik 1985) and theta (Meyer et aL. 1991-).
Sequence homology among subunits within the same gene class
is quite high, normally in the range of 60-80%, while
inter-gene class homology displays a relatively low degree
of sequence identity (about 3O%). Heterodimers have only
been isolated from subunits of the same gene class/
suggesting that there are specific structural requirements
for familial subunit-subunit interactions (Ji et ai-. I9g2).
The expression of GST is cel-I- and tissue-specific, in
which the liver has the highest activities
and number of
molecular forms (Tsuchida and Sato L992). Other organs
share some, but not a1I forms are expressed. For instance
the human liver contains only trace amounts of pi-class
GST, which on the other hand is found in relatively
high
amounts in several other organs, in particular in placenta,
kidneys and intestine. The notable phenomenon of increased.
levels of cST expression in certain drug resistant ceII
lines and tumors wiII be discussed separately in section
1.3. Developmental changes in cST expression have been
noted, e.g. the fetal liver expresses the normally almost
absent pi-class GST. The regulation of
-6-
Table 1. Classification
of cytosolic
homodimeric
glutathione S-transferases from rat and human* .
CIass
Rat
Human
Alpha
1-1
2-2
A1-1
A2-2
À3-3
8-8
L0-10
Mu
aa
,
M1a-1a
M1b-t b
4-4
6-6
9-9
11-
skin '9.9
M2-2
M3-3
t-
1
Yb2-Yb2
Pi
7-7
P1-1
Theta
5-5
T1-1
T2-L2
13- 13
Yrs-Yrs
adapted from Tsuchida and Sato 1992, Mannervik l_992,
Harris et al_. 1991.
and
the GST expression is dependent on both hormonal and
environmentar factors (Mannervik 1gg5). Besides a rarge
variability in tissue distribution,
GST isoenzymes may
demonstrate a differentiar
localization within certain
tissues or even within cells (vos and van Braderen 1990).
Genetic variation of cST expression has been observed, in
particular the genetic pollzmorphism of human GST Mla_l_a,
which is due to deletion of the cST Mla_l_a gêne. About 50%
of all indi-viduar-s rack the expression of csr M1a-1a (van
Onmen et al". 1990).
Until recently, little
direct information on the
structure of the active site was available. Indirect
-7-
evidence was used to postulate that the active site is
composed of both a gl"utathione binding site (G-site) and
adjacent to this site a second substrate binding site (H(Mannervik 1985). The G-site has a very high
síÈe)
specificity for glutathione. On the basis of studies with a
series of glutathione analogues, it has been postulated
that the y-glutamyl part of glutathione is the main bi_nding
determinant of glutathione to the G-site, while the glycyl
part of gluÈathione is the least, essenÈial for the binding
(Adang et al. 1990). Hoh¡ever, this could not be confirmed
j-n X-ray diffraction studies (Ji et aJ-. L992).
The analysis of the kinetic mechanism of cST has led to
a generall-y accepted model, in which GST has tr^¡o distinct
subunits each bearing a catalytically
independent site
(Mannervik 1985). The isolated monomers are completely
inactive (Dirr and Reinemer L99L; Aceto et a7. 19921 |
although there is no obvious inÈeraction between the sites.
The enzl¡me kinetics of GST display a slight deviation from
Michaelis-Menten rate behaviour when the concentration of
glutathione
is varied (Mannervik 1985). Most studies
support the steady-state random mechanism in which
substrates
bind
sequentially
(Mannervik 1985).
Nevertheless, in several cases the kinetics
may be
conveniently described as a unireactant system by the use
of one substrate at saturating conditions (Mannervik and
Danielson 1988 ) .
In early studies, chemical modification was used to
elucidate the possible involvement of various amino acid
residues in the cataJ-ytic activity. From these studies, it
has been suggested that cysteine residues (Àskelöf et a7.
1975, Carne et aL. 1978) and histidine
(Awasthi et a7.
1987 ) v¡ere present either
in the active site or in the
vicinity of the active site and that these amino acid
residues may play an essential role in the catalysis. The
use of site-directed mutagenesis unambiguously ruled out
the involvement of both cysteine (Chen et al-.7.992;
Widersten et af. 1991¡ and histidine (Zhang et al-. I99L;
Kong e¿ a7. 1991; Wang et af. 1,992) in the catalysis. The
chemical modification of arginine residues also inactivated
GST (Schäffer et af. J-988; Asaoka 1989), while replacement
of certain individual arginine residues by site-directed
mutagenesis dj-minished the enzymatic activity, indicating
that these arginines might play an essential role in the
catalysis (Kong et a7. L992).
More recently, an essential role of a tyrosine residu in
the catalytic mechanism was recognized (Stenberg et aL.
-8-
199L; Liu et aJ. 1992,. Kong et al . tgg2,. KoIm et aJ. . Igg2l
À tyrosine residue near the N-terminus of Gsr is conserved.
in the sequences of all of the cytosolic enzymes found. It
h/as shown that the sulfhydryt proton of enzyme bound
glutathione has a pKa of 6.6 which is about 2_3 pf units
less than in aqueöus solution lGraminski.t
19g9).
Sincep*" of the glutathione conplexes of a cST
"f. mutant
lng
rny.Hhr_ch tyrosine was replaced by a phenylalanine residue
(E ) Ís 7.8, it was suggested that the hydrogen bond
between this
tyrosine
residue and the enzyme_bound
glutathione herps to rov¡er the pK. of grutathione in the
binary enzyme substrate conrplex 1fliu et al. L992).
Recently, significant progress in the knowledge of GST
has been made, since suitable crystals io. X_ray
diffracÈion studies have become avairable (Reinemer et at.
L99L; Ji et a7- 1-gg2). The three-dimensionar- structure of
the GST from pig class pi and rat class have very similar
fording toporogy. Both the subunits of the proteins have a
smaller a,/B domain (domain I, residues 1_82 and !_74, for
GST 3-3 and GST p1-1 respectively) which can be consid.ered
to be the glutathione binding site, while the larger
helicar domain rr (residues 9o-2r"7 and g1-207 tor csr arrg-¡
and GST p1-1, respectively) is predominantly involved
in
the xenobiotic substrate binding (Ji
al.. L992) .
"t contacts in
Moreover, the overarr feature of the subunit
both dimers are quite similar. The active site of the mu_
class GST is located in a deep ( 19_A,) cavity which is
composed of three mobile structural elements (Ji et aJ..
1"992). The three-di:nensional structure revears the hyitrogen
bond of the hydroxyl group of tyrosine 6 with the sulphur
of glutathione, which j-s the only d.irect hydrogen bond,
confirming its essential role (Ji et aJ. 1992).
1.2 Glutathione s-transferases and drug resistance.
The effectiveness of many crinically usefur- anticancer
drugs is severely limited by drug resistance.
Drug
resistance can be either intrinsic, in which the resisÈance
is present even during the start of the therapy, or
acquired, in which resistance arises during the course of
the therapy (waxman 1990). Murtidrug resi-stance in human
cancer occurs when malignant tumors display cross_
resistance to a broad spectrum of structuralry unrerated
drugs (Murren and Hait 1-gg2). The classical and most
intensely studied form of multidrug resistance
phenotype) is mediated by overexpression of(MDR_I
an
-9-
approximately 170 kDa membrane glycoProtein' an energydependent drug efflux PumPr which has been called Pglycoprotein (Tirikainen and Krusius 1991,). In many cases¡
however, the etiology of drug resistance is clearly
multicausal, and many biochemical mechanisms are involved
(for reviews see: Hayes and rüolf 1990; Borst 1991; Black
and Wolf l-99L). Increased l-evels of GSÎ, especially the piclass, have been identified in some P-glycoprotein positive
MDR ceII lines, however transfection experiments have not
demonstrated a role for GST in these ceII lines (Tol,tnsend
et al-. L992; Murren and Hait L992).
on the other hand, several findings suggest the
involvement of GSTf besides other mechanisms, in a
phenotype of drug resistance, viz resistance to
different
alkylating agents such as chlorarnbucil, melphalan, and
(Morro\^t and Cowan f990). These findings
nitrosoureas
include cST overexpression of especially the pi-class in
tumors (Wang and Te\^t' 1985; Sato 1989 ) , the direct
conjugation of alkylating agienÈs by GST (Dulik et a7. 1986;
Ciacco et aL. 1991), and the expression of GST in yeast and
marnmalian cell lines by genetic engineering which confers
to these organism resistance to alkylating agents (Le\^/is e¿
al.. l-988; Black et a7. 1990). Espeeially the alpha-class
GST has been correlated to this alkylating resistance
phenotype (Ciaccio et af. 1991-) / although also the mu- and
pi-classes of GST have been implicated (Black and Wolf
1991).
A role of GST in the resistance to alkylating agents has
these GST
also been shown by the use of GsT inhibitors.
increased the cytotoxic action of alkylating
inhibitors
agenÈs (Te\^¡ et al. 1988; HalI et a7. 1989). A promising
strategy to overcome this alkylator-resistance phenotype
may be based on inhibition of GST.
1.3 Inhibition
of glutathione s-transferases.
Àny substance that reduces the velocity of an enz]¡mecaÈalyzed reaction can be considered to be an enzlr¡neinhibitor. The inhibition of enzymes nay be divided in two
types: reversible and irreversible inhibition-
-10-
1.3-1 Reversible inhibition
of glutathione s-t,ransferases.
Reversible inhibition
requires that the enzl¡me and
inhibitor form a reversible noncovarent comprex. Às stated
in section 1.1 Èhe enzl¡me kinetics of GST and thus Èhe
reversibre inhibition of GST may be adequately described by
a sirnple unireactant
system (with one substrate
saturating). In this simple case, there are three main
trpes of reversibre inhibition:
competitive (inhibitor
combines with free enz¡rme in a manner that prevents
substrate binding) / non-competitive ( inhibit,or binds
reversibly, randomly on a different
site
than the
substrate), and uncompetitive inhibition (inhibitor binds
not Èo the free enzyme, but to the enzyme_substrate
complex). For more complicated systems the reader is
referred to Segel (L9741 in which e.g. the partial
(non)_
competitive and mixed type inhibition are discussed.
A1l the above mentioned tlpes of reversibre inhibition
of cST have been observed (Clark et al. Lg67; Tipping et
al- L979; Díericks 1983). The reversible inhibition can be
expressed by an inhibition constant (K. ) for arr of these
types of inhibition mechanism (Segel nlù¡.
Hoh¡ever, most
sÈudies express the reversible inhibition as a I_^_value,.
which is the concentration of inhibitor
resurting5uin 50%
inhibition
in a defined experiment (unless othe^¿ise
stated: 1 mM glutathione and 1 mM CDNB). This varue does
not need a mechanistic interpretation,
and thus needs
relatively few measurements. The relation between ,50 and
K. for 3 types of inhibition is given in Figure 1_. :
Competitive Inhibit ion
rso:(1+[s]/r*)*r.
Àroncompet
Unconpetit ive Inh ibit ion
rSO= ( l-+Km/[s] )*Ki
j t ive Inh ibit ion
r5o = Ki
Figure. 1 The Relation between IUO and K. (Seget 1974).
-11 -
Àn extensive Iist of inhibitors of cST (with I-^-val-ues
between <0.1 to 1000 pM) has been published lu"ntr.iVif
and
Danielson 1988), and this list is still growing (Vos and
Van Bladeren 1990; Van Bladeren and Van Ommen 1991).
Several tlpes of inhibitors are known, like e.g. the
glutathione derivatives, the glutathione analogs, the
chlorophenoxy alkyl acid herbicides, diuretic drugs, bile
plant
phenols,
dyes,
metal
compounds,
acids,
hypolipidemic
drugs,
halogenated
isothiocyanates ,
steroid
hormone derivatives,
antianaesthetics,
inflammatory drugs, chalcones and others. The inhibitory
potency ranking of these compounds is difficult,
and
depends e.g. on the species, class and even the GST
isoenzyme used. In general, especially the mu GsT are very
The glutathione
susceptible to reversible inhibition.
derivatives and analogues are amoung the most potent
inhibitors of many GST.
competitive
Recently, an intereJting and efficient
inhibitor (toi.rards glutathione) of the rat mu-class GST/ yL-glut.amyI-D-a-aminoadipi-c acid, has been synthesized
(Adang et a7. l-991). This dipeptide meets many reguirements
of GST. The
for a good in vivo applicable inhibitor
hydrolyzable peptide bond between cysteine and glycine is
ydeleted, the thiol group is replaced and the critical
against y-glutamyl
glutamine
is
made resistant
transpeptidase degradation.
The flavonoids, a class of plant-derived phenolic
effect on rat
substances, have a considerable inhibitory
cST (Merlos et al. 1991). Àmong the most potent flavonoids
as reversibl-e inhibitors \"/ere quercetin, kaempferol, morin
(with I<^ ranging from 0.03-0.07 pM). since
and Iuteolin
large
the flavonoids are preséät in the diet in relatively
amounts, further studies about the in vivo effect of
fLavonoids are desirable.
Several studies were addressed to the well-known binding
and hemin to GST
of nonsubstrate ligands like bilirubin
(Mannervik 1985). Fatty acids and fatty acid esters/
classes of compounds which are relatively non-toxic, caused
of rat and mouse GST (Mítra et a7.
a marked inhibition
(I-^
1991-). Especially arachidonic acid \^¡as very effective
of I7 UM). The fatty acid binding site involved in tËË
inhibition of GST P1-1 was identifj-ed, as the residues 141188 (Nishihira et al. . L992). This inhibition of GST by the
fatty acids needs further exploration in vivo.
-12-
I.3.2
lrreversible
transferases.
inhibition
of
glutathione
s-
frreversible inhibit.ion is characterized by a covalent
modification of an enzyme which leads to an irreversible
loss of activity.
Inactivation may be the result of
modification of an essential residue involved in catarysis,
by sterically impedi_ng the substrate binding, by distorting
the protein, or by impairing its mobility (Ferhnst 1984).
Several irreversible
inhibitors of GST are known.
Askelöf et a7. (1975) was the first to show that cysteine
modification of GST may result in irreversibre inhibition.
Table 2 summarizes several studies in which the target
amino acid was identified directly. In particutar the piclass GST is very susceptible to SH-reagenis.
Àn interesting class of irreversible inhibitors are the
active site-directed or at-finity labels, whi_ch are designed
to resemble a substrate of GST to ',target" the inhibitor
specificarl-y to the active site and to form covalent bonds
v/ith cST.
E.g. the affinity labeling of rat cST isoenzl¡mes by S_
( 4-bromo-2,3-dioxobutyl )glutat,hj_one
reveals
some
interesting features (Katusz and Colman 1991,. Katusz et aL.
L992). In the mu-class this compound reacted r"¡ith
tyrosines, while in the rat alpha-class a cysteine \^ras the
targeÈ amino acid.
our rab has addressed several studies to the glutathione
conjugates of halogenated quinones, since these conjugates
are very potent active-si-te directed inhibitors (van omrûen
et al-. 1988; Van OÍmen et al. 1989; Vos et at. 1999).
Quinones react as a ruLe efficiently
with sulfhydryl
groups, either by arylation or oxidation of thiols to a
disulfide. Tetrachloro-lr4-benzoquinone (TCBQ), has been
shown to react with and inactivate rat GST very efficiently
(Van Ommen et af. l-989). The readily formed glutathione
conjugate of TCBQ (GSTCBQ), reacted even more efficiently
with rat cST (Van Ommen et aL. L988). The involvement of
the aetive site was suggested by using S-hexylglutathione,
a compound with known affinity to the GST active site. The
t,argeting effect of GSTCBQ seems to be based on the
affinity of the glutathione moiety for the active site.
-13-
Table 2. Irreversible
r-soenzyme
inhibitionl
target site
S- ( 4-bromo-2, 3 -dioxobutyf
Tyr
GST 4-4
csr
3-3
GST 1-1
Mafeimide derivatives
GST P1-lGST
7
7-7
)
P1-1
cST 7-7
human GST
reference list
glutathione
Katusz l-991
Katusz 1992a
Katusz 7992b
(cysz)
Tyr (Cys2)
Cys
Cys
Desideri 1991; Lo Bel-Io
Cys
1-990,' Tamai
Tamai 1990
-chToro-2 | 4-dinitîobenzene
GST
of rat of
Caccuri
199O
1992
Cys
Cys
Àdams 1992
Cys
Cys
Shen 199L, i-993
Shen L991
Cys
Terada l-993
Adang 1991
H
"o,
GST
P1-1
GSI 7-7
Disuffides
GST
PL-1
cST 3-3
cys
) at least 504 inhibition with concentration inhibitor
less than 0.5 mM, at 25oC within t hour. 2) regarded as not
essential.
1
1.4 Aim of the study
of GST has been studied for a variety of
Inhibition
reasons. From a mechanistic point of view, the kinetic
mechanism of the reaction, the architecture of the active
site, and the discrimination between isoenzymes of GST have
atl been studied using more or less selective inhibitors.
of GST may
From section l-.2 it is clear that inhibition
also be a subject of relevance in relation to the drug
resisÈant alkylator-phenotype. Especially for this latter
reasor¡, several groups have examined potential GST
inhibitors.
-L4-
years ago, we concluded that TCBQ
in particular
night be used as a starting point toand
deveråp
in vivo
applicabre i*eversible inhibitors. The effecti.veness
of
GSTCBg was based on the combination
of
a
reactive
moiety
(TCBQ) with a group with affinity
(grutathione) - The investigations describedfor the enzl¡me
in this thesis
were designed to further elucidate this concept:
several classes of compounds were studied fookii.rg Firstly,
capacity to serectivery irreversibly inhibit csr for the
in vitro.
Next, on the longer term, we set out to assess
whether
these ttæes of inhibitors of csr rnight be applicable
in
vivo.
The first
primarily designed to investigate,
"l:U, was
whether these TcBg.and
rerated qu-inone-based inhibitors are
capable to inhibit human cST i_n vitro. The in
inhibitory potential of several naturally viÈro cST_
occurring
catechors
Some
GSTCBQ
and catechoramines was subsequently
These compounds might provide a ,,pro_drug,, investigated.
concept, since
their oxidatj-on
dênêrÂrôa
rl
tiili"
usares ro.*"å
nrTf ï.nT:;:ïï:::
""3;"::;;:i".
incr-uded in these
studies, since the glutathione
moiety
might provide a targetting effect.
Ethacrynic acid, an a,B_unsaturated ketone, is
another
since ir is a known porenr inhibiror
ïa":::atlg_inhibiror,
vr
(,ÞL
rcrL
rsoenzymes (Àhokas et
19g5). Moreover,
ethacrynic acid readily reacts with aJ.
sulfhydryl
groups.
Potentialry, covarent interaction with csr milnt
inactivate
the enzrrme' studies were designed to assess the
mechanism
and extent of inhibition oi ttr" individual 5-soenzymes
of
both rat and human GST in vitro.
Finarly, in the past many studies on the inhibition
of
con j
in cell lines have been harnpered by the lack
of a
suitable toor to assess the total in¡¡iuition.
lrle deveroped
a system to assess the total amount of cST inhibition
cel! Iines. TCBQ, and ethacrynic acid and derivatives in
were
studied using this system.
this thesis consists of tv¡o parts, to enable
a rapid
overviev¡ of the studies with the structural-rf
rather
unrelated quinones,
cST
and ethacrynic acid.
-15-
References
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-23-
Chapter
2
Irreversible iuhibition of human glutathione S-transferase
isoenzyoes by tetrachloro-J.r4-benzoquinone and
its
glutathione conjugate
J.H.T.M. Ploemen, B. van Ommen and p.J. van Bladeren.
BiochemicaL PharmacoTogy 4L: l-665-L669, L99\.
Abstract
The quinones tetrachloro-lr4-benzoquinone (TCBQ) and its
glutathione conjugate (cS-TCBg) are potent irreversible
inhibitors of most human glutathione S-transferase (cST)
isoenzymes. Human Pl--l-, MLb-l-b, and Ml_a-l_a are almost
completely inhibited aÈ a molar ratio TCBQ/GST = 2/1. The
isoenzyme A1-L was inhibited up to 7SZ, and higher
concentrations (TCBQ/CST = 6/t¡ were needed to reach this
maximum effect. For these isoenzlmes 75-95e" of the maximal
amount of inhibition was already reached on incubation of
equimolar ratios
of TCBQ and sçþunit GST, while
approximately 1- nmol (0.82-0.95) 1f4-[U---C]TcBe per nmol
subunit GST could be covalently bound. These results
suggest that these GST isoenzlzmes posses only one cysteine
in or near the active siÈe of GST, which is completely
responsible for the inhibiÈion. fn agreement, human
isoenzyme A2-2 which possesses no cysteine, was not
inhibited and no TCBQ was bound to it. The rate of
inhibition
was studied at 0o: TCBQ, trichloro-lr4benzoquinone and GSTCBQ all inhibit
cST very fast.
Especially for A1-1, the inhibition by the glutathione
conjugate is significantly faster Èhan inhibition by TCBe:
the glutathione moiety seems to target the quinone to the
enzyme. For the other isoenzlnnes only minor differences are
observed between TCBQ and its gtutathione conjugate under
the conditions used.
-24-
Introduction
The glutathione S-transferases (cSTs¡ arè a group of
isoenzymes that catalyze the reaction of elecÈrophilic
compounds with glutathione [1]. ñlong the broad substrate
spectrum are ørB-unsaÈurated carbonyl derivates, epoxides,
quinones and a range of other alkytating agents t1l. In
addition, cSTs also detoxify electrophilic netaboJ_ites by
serving as targets for alkylation or arylation t2-51.
Ho\Àrever, the effect of Èhis reaction on the acÈivity of
GSTs has not been studied in deÈail.
The cSTs of rat, mouse and man are divided in three
distinct cl-asses: the a-, U-, and n-class. They vary widely
in tissue distribution. The highest concentrations of GSTs
are found in the liver, with the exception of the n-class
which is most abundant in the kidney and placenta. fhe fclass cSTs are expressed in only 50? of human individuals
t6t.
Inhibitors of GSfs are of considerable interest:
firstly,
it has been proposed that inhibition of cSTs could
overcome thè resistance to some antineoplastic drugs that
certain tumor cefls display Í.7 , 81. Secondly, csts are
involved in the biosynthesis of
leukotrienes and
prostaglandins [9]. Modulation of the biosynthesis of these
compounds by inhibition
of cSTs could be of potential
therapeutic benefit in the treatment of related disorders
tel.
A large number of inhibitors of GSTs are known [6]. Most
of these however act in a competitive manner, i.e. their
effects
are reversible.
Recently, tetrachloro-I,4benzoquinone (TCBQ) has been shorlrn to inhibit
rat
glutathione S-transferase very strongly in an irreversible
fashion []-01. This compound has been shown to react with
cysteine residues in the vicinity of the active site []_11.
Using rat isoenzymes, several characteristics of the
reaction were studied. Most importantly, the glutat.hione
conjugate of TCBQ (cSTCBg) has been shown to inhibit even
more strongly: the glutathione moieÈy seems to target the
conjugate to the active site
of the enzl¡me. The
corresponding B-nercaptoethanol conjugate, showed a much
slorì/er rate of inhibition [10]. Lastly, in contrast with
other reag:ents [11], the reaction of Èhe quinone as well as
the quinone conjugate with the first cysteine of cST, has
the major effect on Èhe inhibition [ 12 ] .
-25-
The present study has been designed to determine the
inhibition of the human isoenzymes by TCBQ, the rate of
inhibition by TCBQ, trichloro-1r4-benzoguinone, and GSTCBe,
as well to determine the number of cysteine residues that
react hrith TCBQ.
Materials and
Methods
ChemicaLs and radiochemicals. pentachloro-[u-14C]phenol
was fron CEÀ (Gif sur yvette, France, 36.9 ycí/ymol). TCBQ
h/as from Merck (Darmstadt, F.R.c. ),
glutathione
its
conjugate \4ras synthesized as described etsewhere [10].
Trichloro-1-r4-benzoquinone (1r4-TriClBQ) h¡as a generous
gift of B. Spenkelink (Agr+lultura1 University V,rageningen,
The Netherland?). 1,4-[U---C]TCBQ h¡as prepared from
pentachloro-[U--'C]phenol by microso¡nes from dexamethasoneinduced male rats/ as described previously t131. The
conditions used hrere: incubation for t hr at 37o with 1
mg/mL microsomal protein/ 1 mM NADPH, with After 30 min and
extra 1 mM addition, 2 pmol pentachloro-¡u-raclphenol (36.9
UCi/Umol) in 1.5 mL of acetone, 0.1 M potassium phosphate
buffer (pH 7.4]l, 3 mM Mgcl^ and 2 mM ascorbic acid. The
total volume was 100 mL. The réaction hras stopped with 1 mI,
of î4N HCI and extra ascorbic aclS was added to i.0 mM. 1,4_
lU- CITCHQ and pentachloro-[U-t'C]phenol were extracted
lthree times with ]_40 mI of acetone: ethylacetate (l_:2)/
and HrO was removed with Na?SO4. The solvent was removed by
evaporation. 1,4-TCHQ was- púrified by preparat.ive HPLC
(zorbax ODS, DuponE, 2I.2 mm x 25 cn) using a gradient of
50-l-00% methanol against 0.05% formic acid in 45 min,
followed for 45 min at 1O0% methanol, with a flow of 3
ml,/min, and UV detectiogo at 296 nm. The conversion \^/as
approximateJ-y 37%. It4-[U---C]TCBQ was freshly prepared for
each experiment by quantitative oxidation for 5 min at 25o
\.rith an excess of 2r3-dichloro-Sr6-dicyano-1r4-benzoquinone
in methanol and purified by HpLC as described above, and
collected on i-ce (k' : 1.9 | 2.4, and 3.5, respectively, for
LIA-TCHQ: fCBg and pentachlorophenol).
Purification. GST isoenzymes were purified from liver
and
placenta
using
S-hexylglutathione
affinity
chromatography. Separation of the GST isoenzl.nes was
achieved with chromatofocusing with a mono p_column
(Pharnacia, The Netherlands), as previously described [14].
The purity was confirmed by HPLC analysis t15l and
isoerectric focusing [14]. specific activities with CDNB as
-26-
second substrate (see below) \¡/erez 37.4t 23.8, 89.8, 85.4
and 69.9, respectively, for human À1-1, A2-2, M1a-1a, MLb1b and PL-L. ÀIl enzlnne concentrations are expressed as the
concentration of the subunit.
Enzge assays. To determine the amount of inhibition at
different molar ratios of TCBQ/GST, incubations of 25 pmol
enzlrme (M_. 25,9OO, 25,9OOt 26,7OO, 26t6OO and 24r800,
respectively, for human À1-1, A2-2 | M1a-l-a, MLb-l"b and P1-1
t16l) with 6.25 to 250 pmol TCBQ !'rere performed for l-5 urin
at 25o, in 0.L M potassium phosphate buffer (pH 6.5),
supplemented with 1 mM EDTA, after which GST activity
towards 1-chloro-2r4-dinitrobenzene (CDNB) was measured at
25o (pH 6.5), according to Habig et aL. [17].
In order to det,ect a time-dependent inhibition of GST by
TCBQf L,4-TriClBQ and GSTCBQ, 25 pmol of enzyme r{¡as
incubated with 75 pnol of quinone in a cuvette in 110 UL at
intervals 850 1rt, containing
Oo. At various tine
glutathione (l- pmol) and þotassium phosphate buffer (final
concentration: 0.1" M, with L nM EDTA, pH 6.5) at 25o $/as
added, whereafter 40 FL CDNB (1- ynol) $ras added and the
inhibition
of enzymatic CDNB conjugation qras measured at
340 n¡n at 25o.
All enzlme assays were performed in duplicate, while
controls r¡¡ere treated in the sane way.
Measurement of covaient binding of TCBQ to hu\An GSr.
The specific activity of freshly prepared l-r4-[U--'c]fcBg
was decreased to 101000 dpm/nmol. One nanomole of enzyme
v¡as incubated in a microconcentration tUþe (centricon TM
10, Aroicon, U.s.À.) with l-0 nmol 1r4-[U-r+c]TcBQ for 30 min
at 25o in 1" mL of 0.L M potassium phosphate (pH 6.5) with
mM EDTA, followed by incubation for 5 min with 2 rnM
ascorbic acid. Solvent containing unreacted (hydro)quinone
was removed by centrifugation for 30 min at 5000 g (at
L00), followed by three washing steps: addition of 0.5 mI
of methanol-/H)O (1:L) and centrífugation for 40 min at 5000
g. The dry filter
was dissolved overnight in 1 mL of
soluene 350 (Packard, U.S.A.) at 37o and the sample was
liquid
screened for radioactivity in 15 mT, of scintillation
(Hionic fluor, Packard).
Statjstjcal methods- Nonlinear regression analysis $ras
performed hrith the statistical package Genstat,s. Parallel
curve analysis was pçrformed using the equation of the
Í.
curve:Y=a.+b.e
II
1-
-27-
Results
The inhibition of human GSTs by
-Bxtent of inhibition.
is shown in Fig. 1. Alnost complete inhibition (up to
983) for the isoenzyrues of the U- and n-classes was
observed at a molar ratio TCB9/GST = 2/1. Inhibition of the
representatives of the a-class r¡/as not complete: the
isoenzlnne A1-L was inhibited up to 75%, and a higher
concentration was needed to reach the maximum effect
(TCBQ/GST = 6/I). Human À2-2 \.¡as not inhibited
at all.
InÈerestingly, for all these isoenzlmes (with the exception
of A2-2 ), the major part of the inhibition
(about 75 to
85%) has been reached at TCBQ/GST = l/1.
TCBQ
>
1e0
I
,,to
ts
g
(Ê
o
100
2
=
E"o
U
*Bo
t
60
50
40
x-x
F1-1
A-A
P
o-o fr?-?
+-+
1-1
M1a-'1
X_X M1b-
a
1b
30
e0
10
o
R.tro TCBo/nonoo!r
Fig. L Renaining Activity at different molar ratios of
TCBQ/enzyme, after incubation of 25 nM subunit GST Al--1,
A2-2t MJ-a-1a, MLb-l-b, and Pl-L, with 6.25-250 nM TCBQ for
l-5 min at 25o, after which GST actívity towards CDNB h¡as
measured at 25" . the results are the average of tr^¡o
incubations.
-28-
Timecourse of inhibition. the ti.mecourse of inhibition
with TCBQ and GSfcBg for all isoenzlznes is shown in Fig. 2.
In order to slow down the reaction, the incubaÈions were
performed at Oo. Both TCB9 and GSTCBQ inhibited the GST
isoenzymes À1-1, P1-1, M1a-1a, and P1-1 very quickly. Some
interesting differences were observed: the rate of
M1a-1a
n1 -1
I rurtnlr¡
I rEltd¡g
rcttvtt¡
ælrvr lg
n2-¿
M1b- 1b
100.
¡ ülñ¡i!
rcttvttg
50
¡ rutñlDe
*ltvrtg
o
I melntn¡
rc{tvtly
Fig. 2. Timedependenr inhibition of huma¡ GST by l,zl--TCBQ (O), l,+TriClBQ (+), and its
glutathionc conjugate (A), 25 pmol cnzyme was incubatcd wilh 75 pmol quinone in a orettc in 110.4L
(l ¡mol) and potassium Phos?hate buffcr
(final concentration:0.1M, wilh 1mM EDTA, pHó.5) at 25" was added, whcrcaftet ¿l0PL CDNB
ãt 0'. At various time intenats 850 pL, containing glutathionc
(1 prnol) was added and the inhibition of cnzymatic CDNB conjugation was mcasucd at 340 nm. The
results are the average of two incubations.
-29-
inhibition by csrcBQ was significantly higher than Èhe rate
of inhibition by TCBQ, only for the human A1-1. Under the
condi-tions used only minor differences between TcBe and the
glutathione conjugate were observed for MLa-1a and M1b_1b,
while for human p1-r the parent quinone withouL the
glutathione moiety inhibited faster. A2-2 hras again not
inhibited (FiS. 2). The inhibitory effect of 1,4_TTiCIBQ
was also investigated for the isoenzymes Al-L and p1_1.
1r4-TriClBQ still inhibited cST Àt_-1 and p1-l_ very quickly,
but somewhat slower than TCBQ. The targeting effect of the
gluÈathione moiety for the isoenzyme Àl--l hras some\^rhat more
obvious, while l-r4-TriclBe stirr inhibits csr p1-1 some$¡hat
faster than the glutathione conjugate.
covaJ-ent binding. GST isoenzymes h¡ere incubated wiÈh
radiorabelled TCBQ to study the extent ít covarentry binds.
The maximal binding is presented in Tabte J_. All cst
isoenzlanes bind about 1 nmol (0.92-0.95) per nmol GST, wiÈh
the exception of A2-2, where no binding was observed.
Tabre i- - covarent binding of
transferases
Isoenzyme
A1-1
A2-2
Pl_-1
MLa-La
Mlb-1b
TCBQ
to
human
grutathione s-
Covalent binding
(nmol TCBQ,/nnoI cST subunit)
0.83 + 0.07
0.08 + 0.00
0.89 + o.27
0.9s ! 0. l_2
0.82 ! 0.L2
nan?[ole lsubunit ) enzyme r.ras incubated with 10 nmol
1f4-[U-*'C]TCBQ for 3O min at 25o, for experimental details
see Materials and Methods. Va1ues are the average t SD of
duplicate incubations.
One
-30-
Discussion
In the present study it has been shown that human GST,
ÀL-l-, Ml-a-l-a, MLb-lb and P1-1 are inhibited strongly as a
result of covalent nodificaÈion. Quinones are known to
react very rapidly with sulfhydryl groups t10l. Às
expected, the human A2-2 which possesses no cysteine
residues [1-8], is thus not inhibited by TCBQ, and only 0.08
nmol TCBQ could be bound per nmol subunit A2-2. The low
amount of binding observed presumably reflects the
relatively slow reaction of quinones with amino groups
[]-9I. The isoenzymes A1-1", M1a-la, Mlb-lb and P1-L have
been shown to be inhibited to about 75-85% of the maximum
TcBQ/csT - 1/I,
while
amount at a molar ratio,
modified
by
1r4-Lucysteine
could
be
one
çgproximately
-'CITCBQ. fhis suggests that human GST A1--l-, MLa-14, MLb-lb
and Pl-l- possess one cysteine residue in or near the
vicinity of the active site, which is completely
responsible for the inhibition. These phenomena have
already been reported for some isoenz]¡mes of rat and human
GST: the modification of only one cysteine of rat isoenzlme
4-4 and 7-7 by respectively TCBQ or lv-ethylmaleimide, and
of only one cysteine of human n by N-(4-anilino-L-naphthyl)
maleimide or CDNB, resulted in compleÈe inhibition [2, L2,
2O, 2J-l. Hohrever, the quinones differ in their inhibitory
characteristics from these compounds: Iower concentrations
are needed and in contrast to N-ethylmaleimide, they also
inhibit the human a- and ¡.r-classes [2]. The total amount of
cysteine residues per GsT subunit ranges from 1 (41) to 4
(M1a, M1b, P1) 122-24). Thus, the binding of only one nmol
TCBQ per nmol cST furÈher indicates that complete
modifícation of all cysteine residues does not occur for
human MLa-la and Pl--1. In accordance with the result, it
has been shown that N-(4-anilino-1-naphthyl) maleimide
reacÈs only with one cysteine residue per human P1-1
subunit l,2O), the other three cysteines are probably
Iocated inside the hydrophobic core, and,/or form disulphide
bonds. Using rat isoenzl'me 4-4, aII three available
cysteine residues could be modified by TCBQ [1-2], thus the
cysteine residues of rat isoenzl'me 4-4 (¡r-class) are better
accessible than those of human Mla-la.
The glutathione conjugate of TCBQ, which retains its
oxidized structure [10], inhibited a mixture of rat GST
isoenzlmes at a much higher rate than Èhe corresponding Pmercaptoethanol conjugate. The glutathione moiety
seems
to
- 31 -
targeÈ the quinone to the enzyme. In the present study, the
rate of inhibition by TCBQ and its glutathione conjugate
v¡ere mutually compared. In general only minor differences
!ì¡ere observed. Thus, intracellullarly
formed GSfCBQ does
not slow the rate of inhibition.
In the case of the
isoenzyme A1-1, this glutathione conjugate even accelerated
the inhibition.
In conclusion, in yjyo the cysteine residues of human
GST can become a Èarget for quinones, as weII as for other
alkylating and arylating agents such as ethaòrynic acid,
acrolein,
maleimide derivates and netabolites
of
providing a second
bromobenzene
thereby
1.2-51,
detoxification pathway by cST. However, with the exception
of A2-2, all these subunited possess one cysteine residue
that is important for the conjugating activity.
If that
residue is modified, the enzl¡me will be inhibited.
References
l-. Mannervik B. The isoenzymes of glutathione transferase.
Adv Enzwol. Rel. Areas MoJ BioL STz 357-417 | L985.
2. Tamai Kr Satoh K, Tsuchida S, Hatayama I, Maki T and
Sato K. Specific
inactivation
of glutathione
Stransferases in class pi by SH-modifiers. Biochen
Biophys Res Cornm 167: 33L-338, 1990.
3. Aniya Y, Mclenithan JC and Ànders Mw. Isoenzyme
selective
arylat{gn
of cytosolic
Alutathione Stransferase by [-'C]bromobenzene metabolites. Biochen
Pharmacol- 372 25L-257t L988.
4. Yamada T and Kaplowitz N. Binding of ethacrynic acid to
hepatic glutathione S-transferases in vivo in the rat.
Biochem Pharnacol 292 L2A5-t208, 1980.
5. Berhane K and Mannervik. Inactivation of the genotoxic
aldehyde acrolein by human glutathione transferases of
classes alpha, mu, and pi. Mo7 PharmacoJ- 372 257-2541
1990.
6. Mannervik g and Danielson UH. clutathione transferasesstructure and catalytic activity. CRC Crit Rev Biochem
23: 283-336, 1988.
7. Buller ÀL, Clapper ML and Tew KD. GLutathione Stransferases in nitrogen mustard-resistant and sensitive
cell line. Mo7 PhatmacoJ- 3L: 575-578, 1988.
8. Sato K. Glutathione transferases as markers of
preneoplasia and neoplasia. Adv Cancer Res 522 2Q5-255,
1989.
-32-
Leung KH. Selective inhibition of leukotriene C4
synthesis in human neutrophils by ethacrynic acid.
Biochem Biophys Res Conn 7372 L95-200, 1986.
10. Van Onrnen B, Den Besten C, Rutten ALM, Ploemen JHTM,
Vos RI{E, Muller F and Van Bladeren PJ. Acti-ve sitedirected irreversible
inhibition of glutathione
Stransferases by the glutathione conjugate of tetrachloro1r4-benzoguinone. J BioT Chem 2632 t2939-t2942, L988.
11. Carne Ct lipping E and Ketterer B. The binding and
catalytic actvities
of forms of ligandin
after
nodification of its thiol groups . Biochem J L77 z 433-439,
9.
L979.
L2. Van Orûmen B, Ploemen JHTM, Ruven HJ, vos RME, Bogaards
JJP, Van Berkel WJH and Van Bladeren PJ. Studies on the
acÈive site of rat glutathione S-transferase isoenzymes
4-4. Eur J Biochem 18Lt 423-429, L989.
13. Van Omnen 8., Voncken JW/ Mu1ler F/ and Van Bladeren
PJ. the oxidation of tetrachloro-1r4-hydroquinone by
microsomes and purified cytochrome P-450b. Chem BioT
Interactions 552 247-25O, L988.
1,4. Vos RME, Snoek MC, Van Berke1 WJH/ MüIler F and Van
Bladeren PJ. Differential
induction of rat
hepatic
glutathione S-transferase isoenzymes by hexaehlorobenzene
and benzyl isothiocyanate: comparison with induction of
phenobarbital and 3-methylcholanthrene. Biochem PharmacoT
37 z tO77 -t082, 1-988.
15. Bogaards JJP, Van Orìmen B and Van Bladeren PJ. An
inproved method for Èhe separation and quantification
of
gluÈathione S-transferase subunits in rat Èissue using
high-performance liguid chromatography. J Chromatogr 4742
435-440, L989.
16. Hayes JD, Pickett CB and Mantle TJ. GLutatione Stra¡sferases
and Drug Resistance. p. 11. Taylor &
Francis, London.1990.
J-7. Habig' WH, Pabst MJ and Jakoby !,¡8. Glutathione Stransferases, The first
step in mercapturic acid
formation. J BioT Chen 249: 7130-7L39 t 1-9'74.
18. Rhoads DM, Zarlengo RP and Tu CPD. The basic
glutathione
S-transferases from human livers are
products of separate genes. Biochem Biophys Res Comm
L45¿ 4'l 4-481/ 1987.
19. Liberato DJ, Byers VS, Dennick RG and Castagnoli N.
Regiospecific attack of nitrogen and sulfur nucleophiles
on quinones derived from oak/ivy qatechols and analogues
as model for urushiol-protein conjugate formation. J Med
Chem 24t 28-33, 1981.
-33-
20. Lo Bello M, Petruzzelli R, De Stefano E, Tenedini C,
Barra D and Federici c. Identification
of a highly
reactive sulfhydryl group in human placental glutathione
transferase by site-direcÈed fluorescent reagent. FEBS
Lett 263: 389-391-, L990.
2l-. Adams PA and Sikakana CNT. Factors affecting the
inactivation of human placental P1-L. Biochem PharmacoT
39: 1883-L889, 1990.
22. Tu CPD and Qian B. Human liver glutathione Stransferases: complete primary seguence of a HA subunit
cDNA. Biochem Biophys Res Comm L4I: 229-231, 1987.
23. Seidegard J, Vorachek R, Pero Rw and Pearson wR.
Hereditary differences
in expression of the human
glutathione transferase active on transstilbene oxide
are due to a gene deletion. Proc NatJ- Aead Sci USÄ 85:
'7293-729'7, l_988.
24. CoweII IG, Dixon KH, Pemble SE, Ketterer B and Taylor
JB. The structure of the human glutathione S-transferase
n gene. Biochem J 2552 79-83, 1988.
-34-
Chapter
3
In vitro and in yiyo reversible and irreversible inhibition
of rat glut,at,bione s-transferase isoenzlmes by caffeic acid
and its 2-S-glutatbionyl conjugate
J.H.T.M. Ploemen, B. van Ommen, À.M. de Haanf
Schefferlie and P.J. van Bladeren.
J.G.
Food and Chenical- ToxicoTogy 3I.. 475-482t 1993.
Abstract
The reversible
and
irreversible
inhibition
of
glutathione S-transferases (cST) by caffeic acid [3-(3,4dihydroxyphenyl)-2-propenoic acidl was studied in vitro
using purified rat isoenz]¡mes, and in vjr¡o in male Wistar
(VnJ) rats.
The concentrations of caffeic acid that
inhibited reversibly 50% of the activity of dj_fferenÈ GST
isoenzymes towards 1-chloro-2r4-dinitrobenzene (CDNB) (I_^
values) were 58 (GST 4-4),360 (cST 3-3) and 470 pM (cST ?y
7),
and higher than 640 pM for cST isoenzymes of the a
class (GST 1-l- and 2-2). rhe major glutathione conjugate of
caffeic acid, 2-S-glutathionylcaffeic acid (2-GSCA), was a
much more potent reversible
inhibitor of cSTf with I_^
values of 7.L (cST 3-3)/ 13 (cST 1-1), 26 (cST ¿-¿1, 38
(cST 7-7) and more than L25 pM (GST 2-2). On the other
hand, caffeic acid was a much more efficient irreversible
inhj-bitor of GST than 2- GSCA. In this respect, cST 7-7 h¡as
by far the most sensitive enzlane. The remaining activity
towards CDNB (expressed as percentage of control) after
incubating 1.25 1IM-GST with 100 pM caffeic acid for 6 hr at
37"C was 34 (GST 2-21 , 24 (cST t-1) | 23 (cST 4-4) / 10 (cST
3-3) and 5% (GST 7-7). Almost no irreversible inhibition of
cST L-1 and 3-3 occurred during incubation with 2-cSCÀ.
Incubation of caffeic acid with liver microsomes from
dexamethasone-induced rats catalyzed the oxidation
of
caffeic acid about 18 tines more effectively as compared
with the spontaneous oxidation, as determined by the
formation of GSH conjugates from caffeic acid. In vivo, tine
ac
effect of single oraL doses of caffeic acid ( 50-500 ¡fflg/kg
body weight ) on the cytosolic GST activity tovtards CDNB r^¡as
kidney and
studied 18 hr after dosing in the liver,
linear
intestinal
mucosa. A marginal but significant
relaÈionship was found bethreen the amount of caffeic acid
dosed and the irreversible inhibition of cST activity in
in the
the liver, with a maximum of about 14% inhibition
highest dose group. This Ínhibition coincided. with a small
decrease in the ¡r-class cST subunits, which r,ias only
significant for GST subunit 4.
Introduction
Caffeic acid, a natural phenolic antioxidant, is widely
kingdom (Herrmann, 1956).
in the plant
distributed
Chtorogenic acid, the ester of caffeic acid and quinic
acid, is thought to be the most widespread caffeic acidcontaining compound (Herrmann, 1956). As an example, coffee
beans contain up to 6% of this compound (Herrmann, l-956).
Caftaric acid, another major ester of caffeic acid, is
found in grapes as the rnajor phenol (up to 100 mg/titre in
fresh juice from white grapes; Cheynier et a7., 1986). Much
attention is given to the auto- and enzymic oxidation of
because these undesirable
acid derivatives
caffeic
and aesthetic
reactions result in the loss of nutritional
values of food products from plants (Cillier and Singleton,
1991). The rate of oxidation is dependent on many factors
such as pH and oxygen concentration (Cheynier and Van
and Singleton, l-989). The enzyme
Hulst, 1988; Cillier
polyphenoloxidase can considerably increase the rate of
oxidation (Cheynier et a7., 1986). As a consequence of both
auto- and enzyme-catalyzed reactions, a reactive o-quinone
metabotite is formed (Cillíer and Singleton, 1990). This oquinone can either be reduced or reacÈ with nucleophiles by
hray of a Michaelis-type addition. In the case of
glutathione (GSH), both reduction with the concomitant
formation of oxidized GSH and formation of a GSH adduct of
caffeic acid occur (Cheynier and Van Hulst, 1988; Cillier
and Singleton, 1990). The relative importance of these two
reactions is dependent on the concentrations of GSH and
caffeic acid (cillier and Singleton, 1990).
Quinones, especially halogenated quinones, are al-so
inhibiÈion
strong irreversible
of
known for their
glutathione S-transferases (GsT) (van Ommen et al-., 1988).
GST exist as a family of dimeric isoenzymes, of which the
-36-
most important function is the conjugation of various
electrophilic
xenobiotics with the co-substrate csH
(Armstrong, l99L; Mannervik, 19g5; Mannervik and Danielson,
1988)- Most csr isoenzlanes possess a nucreophilic amino
acid which is rapidly modified by quinones, a reaction
which inactivates the enzl¡me. This amino acid is presumably
a cysteine residue (proenen et ar., 1991), which is rocated
in or near the active site of GST (Van Ommen et aJ., 19g9).
Interestingly,
the GSH conjugaÈes of several quinones
react nore rapidly with cST (thereby inactivating the
enzyme), presumably as a result of the higher affinity of
the GSH part of the molecule for the active site and,/or the
reaction with a different amino acid lVan Ommen et aJ.,
1989). In that respect, attention has recently been draw to
tyrosine since this residue is the target site for a
closely related
compound, S_4_bromo_2r3_dioxobutyJ.)
glutathione (Katusz and Colman, 1,99L; Katusz et a7.t lg92).
In addition to the discussed irreversible
inhibitory
potential, these csH conjugates also inhibit csr reversibry
because of the affinity of the csH moiety for the active
site.
Inhibition of GST may have serious consequences, because
the enzyme prays a key rore in many detoxification
reactions as werr as a number of endogenous processes. on
the other hand, inhibitors of GST can arso be a usefur
device in the investigation of the rore of GST in specific
reactions sueh as the undesirable inactivation of some
drugs (Brack and Ρ¡orf , 1991). caffeic acid and rerated
prant phenors are known as reversibre inhibitors of rat Gsr
mixtures, with concentrations that inhibit 50% of Gsr
activity
(lSO values) of t4O UM (Das et aJ., :.9B4).
Ho$/ever' the potential
irreversible
inhibition
of
individual GST isoenzymes by caffeic acid-derived quinones
has not been studied- The present study was designJted to
study both the reversibre and irreversibr_e inhibition of
GST isoenzymes in vitro and in vivo by caffeic acid anit
its
major glutathione conjugate.
Materials and
Methods
ChemicaLs. Caffeic
acid [3_(3,4_dihydroxyphenyl¡_2_
propenoic acidl, S-hexyJ_glutathione, NADPH, GSH¿ tragacanth
and tyrosinase (3'130 u/mg sorid) rdere from sigma chenicar
co. (st Louis, Mo, usÀ). Hplc-grade trifruoroacetic
acid
-37-
r{as obtained from Baker (Deventer, The Netherlands), HPLCgrade methanol v¡as from Rathburn Chemicals Ltd (walkerburn,
ScoÈland, UK). HPLC-grade acetonitrile was from Vùestburg
(Leusden, The Netherlands). Epoxy-activated Sepharose 68
was purchased from Pharmacia (Uppsala, S\^¡eden).
Synt/:esis of 2-S-gLutathionyJcafteic acid.
2-Sglutathionylcaffeic
acid (2-GSCÀ) was prepared using
in
analogy to the synthesis of
tyrosinase
2acid (Cillier
cysteinylcaffeic
and Singleton, 1990). 0.1
mmol caffeic acid was ineubated with 10 mmol cSH in the
presence of 2000 U tyrosinase/rnl in 0.L M-sodium citrate
(pH 5.3) for 30 min at 15oC (total vol. L00 nI).
The
reaction was followed by HPLC, using a Zorbax ODS column
(4.6 x 250 rüu). Elution was performed at a flow rate of 1
ml/rnin, with 0.1% Èrifluoroacetic acid lsolvent A) and 0.J-%
trifluoroacetic
acid in acetonitrile (solvent B), and with
a linear gradient of 2-2OZ B in 13 min followed by a
gradient to i-00% B in 7 min (k' = 6.9 and 7.5 for the major
product and caffeic acid, respectively¡. the incubate was
lyophilized, purified repeatedly by preparative HPLC, using
a Zorbax ODS column (2L.2 x 250 nm), and eluted
at a flow rate of 4 ml/min with
isocratically
L2e"
in a 0.05% aqueous acetic acid solution. À
acetonitrile
product of more than 95% purity, as judged by I tHj nuclear
magnetic resonance (400 mhz ) , \¡ras obtained and \./as
identified as 2-GSCA with the following information: for
the caffeic acid part, 6 8.3 (d, J = L6.4 Hz), 7.3 (d, J =
8.4 Hz), 7.0 (d, J = 8.8 Hz) and 6.4 (d, J = L6.0 Hz), as
published for 2-cysteinylcaffeic acid (Cillier, 1990); and
for the GSH part 6 2.1, (mt 2Ht GIu B), 2.4 ll|-t 2Ht J = 7.6,
clu y), 3.1 (dd/ 1H, J = 14.6, 9.8, Cys F H_), 3.3 (dd, 1H,
J = 14.6, 4.2t Cys B H*)r 3.70 (l-H GIu a), 3.73 (2t1 Gly a),
4.2 (dd, 1H, J = 9.é, 4.0, Cys a). The UV spectrum of 22522320 = 0.85)
cSCÀ (À_^_- 252 and 320 nm; maximum ratio
h¡as iA8Êëicaf to 2-cysÈeinylcaffeic acid (citlier
and
Singleton, 1990). À 5% impurity, eluting as a shoulder of
2-GSCÀ, r^¡as tentatively identified as S-GSCA because of the
spectral resemblance to s-cysteinylcaffeic acid tÀ ___ 257
and 322 run,' maximum ratio 257 2322 - L; 1cifTlër ana
Singleton, 1990) l.
and assay. cST isoenzy'mes hrere purified
GST purification
from the liver and kidney (cST 7-7) using affinity
chromatography (S-hexylglutathione-Sepharose 68), and
separation of Lhe various isoenzlrmes was achi-eved by
chromatofocusing or ionexchange chromatography as described
-38-
previously (Ploemen et a7., 1993; Vos et al.. l_9gg). purity
was
confirmed by
sodium dodecyl sulphate gel
electrophoresis, iso-erectric focusing and HpLc analysis as
described previously (Bogaards et aI ., L9g9¡ Vos et a-1..,
1988). ÀlJ_ enzyme concentrations were expressed as the
concentration of the subunit tM-z 25r5OO, 27,SOOt 26t3OOl
26 t3OO, and 24,800, respectivelyr'for
GST !, 2 | 3 t4 and
(Hayes et â1., 1990) l. protein content was determined by
the method of Lowry et aL. (l_951), using bovine serum
albumin as standard.
lnhibition
studies.
Determinations of reversible
inhibition
rvere carried out in triplicaÈe by mixing in
cuvettes used for
the enzyme assay ( in
final
concentrations) 25 nM-GST with either caffeic acid (5, l_0,
20, 40, 80, l-60, 32O or 640 UM) or 2-cSCÀ (2, 5, 25, SO or
725 1rM), after which the enzymatic activity towards 1_
chloro-2r4-dinitrobenzene (CDNB) rvas neasured directry.
Time-dependent irreversibre
inhibition r^ras measured in
duplicate at a minimum of six time points by incubating
I.25 UM-GST (1-1, 2-2, 3-3, 4-4 and 7-7) e¡ith 0.1 mM_
caffeic acid or 2-cscA in 25 mM-potassium phosphate buffer
(pH 7.4, vol. 0.3 m1) at 37oC, after which the activity
towards CDNB was determined; in the case of 2_GSCÀ only GST
1-1 and 3-3 $/ere studied. The same experiments were arso
performed in the presence of 0.1 mM-GSH. The remaining
activity
was expressed as percentage of brank incubation
(enz]¡me without inhibitor).
Although the dirution wiÈhout
loss of inhibi-tion already shows the irreversibitity
of t,he
reaction, the irreversibre nature of the chemicar reaction
of enzl¡me and inhibitor hras arso estabrished by storage of
time-dependent inhibited enzyme (as described above) \^/ith
csH (1 trìM GSH, to remove the reactive metaborite derived
from caffeic acid; storage up to 6 hr), which did not
affect the amount of inhibition.
Reaction of caf feic acid and. GSz. Caffej_c acid (0..1 mM)
-nras incubated at 37oc in 25 mM-potassium phosphat-e buffer
(pH 7.4) with a mixture of cST isoenzymes (about 25_4A US
GST 1-1, 2-2, 3-3, 4-4 and 7-7; voJ-. 1 ml). The reaction
was followed by HPLC by injecting 0.1 mI on a Vydac Tp5
column (3 x 200 nrn; 0.6 nt/min; using solvent À and B; for
gradient see below¡.
einding of caffeic acid-nodified. GS? to bioaffinity
matrix. cST 1-2 (75 UM) , 3-4 (76 UM) and 4-4 (1-9 ÌlM) were
treated with 800 U tyrosinase (5 min at 25oC¡ in the
absence (cont.rols) or presence of 1 m¡f-caffeic acid (vol.
-7
-39-
o.4 ml). The incubate was loaded on a s-hexyrgrutaÈhione
bioaffinity
matrix, whereafter the csr was eruted as
described (vos et aJ.., 1988). euantification of csr in the
eluate was performed by injecting 0.1 mI on a Vydac Tp5
wide-pore reversed-phase column (4.6 x 250 mm), which was
eluted at a flow rate of 1 mr/min with sorvents A and B
(see above) with a rinear gradient of 3g-472 B in 18 min
followed by a gradient to 60% B in 5 min and finally
by a
gradient to 62% g in 7 min.
MicrosomaT incubations.
0.1 mM-caffeic acid vras
incubated at 37oc with 0.25 n¡,r-csH in the presence of river
microsomes, derived from dexamethasone-induced rats at a
concentration of 1 mg protein,/ml and prepared as described
previously (Van Ommen et al., 19g5), and l_ mM_NADPH (extra
addition of I
after 15 min). The finar vor. was
'M-NADPH
500 ¡rl (buffer: 0.1
M-potassium phosphate, pH 7.4t with 3
mM-MgCt2). After 30 min, 25 pt HCI (6 M) r/ras added to stop
the reaõtion. The incubation was centrifuged for 4 mÍn at
l-0'000 g, after which 0.1 mr of the supernatant vras
injected on HPLC [Zorbax ODS column (4.6 x 2SO rm), flow
rate 0.9 ml/min; 2% aceÈic acid sorution and methanor, with
a linear gradient of 35-762 methanor in 10 minl. under
these conditions the conjugates (2-cscÄ and s-cÀce) were
separated from caffeic acid 1k' = L.2 and 1.7, for the
conjugates and caffeic acid, respectively). The conversion
of caffeic acid to grutathione conjugates \4ras measured by
peak-area integrat.ion at 252 nm. rncubations were performed
in triplicate.
AnimaLs and doses. fhe effect of a single oral dose of
caffeic acid on the activity of GST isoenzl¡mes \^¡as studied
with Wistar rats (WU). 40 male Ífistar rats (g_9 wk old)
were housed individually 3 days before the start of the
experiment in wire-bott.omed stainless-steer cages in a room
at 2o-24"ct Ao-ioz rerative humidity, with a ventiration
rate of 1O air changes/hr and a light/dark cycle of 12 hr.
They were fed ad r.i.b. with a TNo open-formula basar diet,
and water r¡ras arso provided ad r.ib. All'cation i-nto five
groups was performed at random. The mean body weight (t
SEM) for the groups was 332 t L5, 329 + 11, 356 t l-3, 334 t
1l- and 347 + 10 for groups À, B, C, D and E, respectively
(see below) - These groups v/ere oralry dosed v¡ith
respectively 0, 50, J-00, 250 and 5OO mg caffeic acid/ng
body weight. Caffeic acid rras administered as a 0.5%
tragacanth suspension (10 mf/kg body weight). No clinical
symptoms during the experiment hrere observed in any of the
-40-
treatment groups. After 18 hr the animals r¡tere killed by
decapitation under ether anaesthesia. From all rats the
Iiver, kidney, and the mucosa of the small intestine were
collected. The small intestine r¿as washed with homogenizing
buffer (0.1 M-Tris-HCIt O.L4 M-KCI, pH 1.4), after which
the mucosa was collected.
Preparation of cytoso-ls. Tíssues \r¡ere homogenized with a
Potter-Elvehjem tissue homogenizer in ice-coId honogenizing
buffer (see above). AfI following procedures were performed
at 0-6"C. cytosol was Prepared by centrifugation for 75 min
at 1051000 g. The fat layer was removed, and the cytosol
was stored at -80oC in l-ml portions. Isolation, separation
and quantification of GST $¡ere performed as described
previously (Bogaards et af., 1989 and 1990).
of
GTutathione S-tra¿sferase assay. The activity
with
determined
\^¡as
isoenzymes
GST
cytosolic or individual
CDNB as second substrate, using the spectrophoÈometric
method of Habig et a7. (1974\.
Statistical anaTysis. Levels of significance were tested
by one-way analysis of variance (ANOVÀ) - The relationship
between dose and effect was determined by linear-regression
analysis. I"^ values were determined using the probit
function: p 3'bo + blrn (c) (with c = concentration)
(Finney, L97I\.
Results
Rerzersjbfe inhibition. Reversible inhibition, expressed
as Itrñ (uM), is shown in Table 1- GST 4-4 is the isoenzlnne
whicñ" was most strongly inhibited by caffeic acid. The
reversible inhibition decreased in the order of GST 3-3,
cST 7-i I GSI 1-1 and GS'l 2-2, with the I-^ for the a-class
used. An
enzymes outside the concentration i9"9"
values:
predict
individual
to
fittecl
was
curve
exponential
for 140 pM-caffeic acid (I-^ for a mixture of GST; Das et
al., 1984), the calculatËä renaining activity ranged from
25t 65, 70, 90 and 95%, for GST 4-4' GST 3-3 | Gs'8 7-7 ' GST
1-1 and GST 2-2, respectively. As expected, because of the
GSH moiety 2-GScÀ \^tas a much more potent reversible
inhibitor of rat GST. For all but the a-class GST 2-2t 1i-h.e
I-^ vras below 50 pM. GSf 2-2 showed 38% inhibition at
rlU
uu.
-4rTable 1. Concnetrations of caffeic
acid and
2-s_
glutathionylcaffeic
acid
(2-GSCA) resulting
in 503
inhibition of the catalytic activity of different classes
of raÈ glutathione S-transferase isoenzymes to!,rards CDNB.
Caffeic acid
2-GSCA
GST 2-2
>
>
>
GST 4-4
360 (295-455)* 7.r (6.2-8.0)
s8 (s1-66)
26 (23-31_)
Enzyme
q-class
GST 1-].
p-class
csT 3-3
¡r-c1ass
GST 7-7
640
640
470 (380-640)
L3 ( 11-ls
)
1,25
36 (32-42)
* 95% confidence interval given in parentheses.
25 nM-Gsr was incubated with different concentrations of
inhibitor,
after
which the enzymatic activity
was
determined towards CDNB (as described in Materiars and
Methods ) . Àssays \4¡ere carried out in triplicate
with at
least five concentrations of caffeic acid or 2-GscÀ. The
concentrati-on of inhibitor resulting in 50% inhibition of
activity to$rards CDNB (r",.,) was carculated with the probit
function: .p = b^ + b- ln'Ycl.
UI
-Reaction of caffeic acid with Gs¡Ì. Tyrosinase efficiency
cataryzed the oxidation of caffeic to an o-quinone
intermediate, which react with thiol-s
(Cillier
and
singleton, 1990). The add.ition of csH occurs preferentiarry
at the 2-position of the benzene ring. Addition at Èhe 5_
posi-tion arso occurs, however, at a l0wer rate in anarogy
to the addition of cysteine (Ciller and Singleton/ 1990).
Microsomar incubation of o.r- mM-caffeic acid for 30 min
at 37oc with 0.25 mM-csH increased the rate of oxidation of
caffeic acid about 18 times compared $/ith the spontaneous
oxidation, as measured by conjugate formation (E ãonversion
by integration of peak area aL 252 nm (i SD): 1.3 I 0.2 and
23-6 + L-o for brank incubations and incubations with
microsomes, respectively) .
-42-
rrreversibTe inhibitio.¡?. The irreversible inhibition
of
the individual cST isoenzymes by incubaÈing with caffeic
acid and 2-GSCA is shown in Fig. 1. GST 1-7 was by far the
most sensitive isoenzlrme, followed by GST 3-3. Slower and,
in general, similar rates of inactivation could be observed
for the other cST isoenzymes (Fig- 1¡. This inactivation
could be completely prevented by incubation of cST with
caffeic acid in the presence of 0.1 mM-cSH. No significant
irreversible inhibition of cST 1-1 and 3-3 with 2-GSCÀ was
observed (Fig. 1).
=
(t
ó
CD
s
Ê,
E
E
o
Ρme (hr)
Fig. t. Time-dependent ineversible inhibition of glutathione ,S-transferase (GSÐ. 1.25¡u'GST was
inã¡bated with 0.1 mM-catreic acid (solid lines) or 2-gtutathionylcaffeic acid (dashed lines) at 37"C' after
which the activiry towards l-chloro-2,4dinitrobenzeni was detérmined: GST l-l 1l); GST2-2 (A); GST
3-3 (O); GST ¿4 (O); GST 7-7 (l). The remaining activity was expressed ¿rs Percentage of blank
iqcuùation. The maximal loss of activity in the blank was less than 10%, with the cxception of a 16 and
24o/o loss for GST 3-3 and 7-7, respectively. Each time point is the average of two incubations (for
For experimenøl details see Materials
individuat points, the coefficient of variation
åï iff:,i:îi.r%).
-43-
Reaction of caffeic
acid and. GST. The covalent
interaction of caffeic acid with GST \À¡as arso studied using
HPLC separation of the individual subunits after incubation
of a mixture of GST isoenzymes with caffeic acid. Gsr 7--j
hras the most reactive isoenzyme (Fig. 2). The decrease of
GST (À = 274 nm) \4ras accompanied by an increase of enzlzme_
bound caffeic acid (as detected at
= 32a nm). Àfter
prolonged incubation peak-broadening^ was observed,
suggesting a change in the structural properties of the
subunits. rn a contror incubation \dithout caffeic acid, ony
a small loss of the peak area (at 2i4 nm) of the GST
subunit 7 was observed (10-L5% loss after 1g0 min).
Fig. 2. Typiel HPLC chromarograms of GST incubared at 37"C wirh 0.1 mu-effeic acid. The GST
subunits wclc elutcd in the fo¡lowing older: subunit 3. subunit 4. subuni t 7, subunit 2 atrd subunit l. caffeic
acid wæ iDcubated for 30 min (A) or 120 min (B) wilh approx. 25-40 ¡g GST t-t, 2-2, 3-3, U aîd 't-'l
in a 6nal volme of I ml. after which 0.1ml was injecred on Vydac TP5 (3 mm x 20 m) [214 nm (solid
lincs) and 320 nm (dæhcd lines)1. For cxperimental de¡ails se Mate¡ials and Merhods.
-44-
of the S-hexylglutathione bioaffinity
The affinity
maxtrix tov¡ards caffeic acid-modified GST was studied using
GST 1-2, 3-4 and 4-4. In this study, oxidation of caffeic
acid was achieved with tyrosinase according to CiIIier and
Singleton (L99O). For this purPose' GST $¡as incubated with
caffeic acid in the presence of tyrosinase, which resulÈed
in about 60-80g inhibition of the catalytic activity
to\^¡ards CDNB (n - 3t data not shown¡, whereafter the
incubate was purified on the bioaffinity column. The eluate
was analysed using a Vydac column. The normal GST subunit
peaks almost completely disappeared from the chromatograms,
while virtually no new peaks rtere present (data not shown¡ '
and subunit eomposition of the 7iver,
cST activity
kidney and smaJ-I intestinaJ- mucosa. The effect of a single
measured with
dose of caffeic acid on totaf GST activity
With CÐNB as
in
Fig.
3.
presented
is
CDNB as substrate
linear
second substrate I a smatl but significant
relationship between the dose of caffeic acid and the level
of GST was observed in the rat liver'
of inhibition
Administration of caffeic acid
c
o
a
o
q
1
400
1
aoo
Ø1oo Ãe/ri
t
I OOO
E
800
E
t
;¡
]oo .¿rs
Ñ25o .c/re
Øso ^./te
-l o .e/r¡
600
400
e
INTESTINE KIONEY
LIVER
Fiq. 3. Cwosolic GST activity in the livcr. kidncy and small
i"l?.iinul'.u"o* of rats trated with a singlc oml dosc of
Ctoups of eight male wistar (wU) Éts reccived
*ä"i"
""i¿.Zso ot'Soo mg caffeic acid/kg body wcight Aficr
ó. SO. fæ,
iöfri, Csf activity wai detemined in the c¡osol, uing
ò-oÑ¡ the wónd substrate. Signifiøt difrerenæs in
Ããor Uv^ oneway analysis of variane wcre ob*rued only
The rclationship betwæn GST
æ¡ivity (nmoì/mi¡/mg) and dos of caffeic acid (mg/kg)
*e¡e áetãimined by lincar-regrdsion analysis
i"'t
"'riì"t Darameters: ægræsion cæfficient of effeic
i*ai-.i"d
I"iJ -0.¡si (P=0.012), with a constant tem of 1269
innollmintmgjl. For cxperimental dctails w Matcrials and
i.-,¡" Ií*t 1r io.ozs).
Methods.
-45-
resurted in a decrease in csr activity of approximatery L4?
in the highest dose group (as estimated by reqression
anarysis). This coincided v¡ith a rinear decrease in ¡.r-crass
subunit concentration, which was however only significant
for cST subunit 4 (Iable 2). fn the highest dose groupr â
decrease in csr subunit 4 of approximatery 17%
ãbserved
(as estimated by regression analysis). No'^ras
significant
inhibition of the catarytic activity towards õor¡s râ¡as
observed in the kidney or small intestine mucosa.
Tabre 2- Effect of a singre dose of caffeíc acid on rat
Iiver
glutathione S_transferase (GST) subunit
content
Subunit
concentration
Dose of caffeic acid
(mg/k body r4'eight)
csT 1_1
0 (control)
50
100
250
so0
(Uglmg cytosolic protein)
70.8!2.4
9.7!2.8
to.4!2.O
9 .7lL.7
10
.8+1.
3
3-3
csr 4-4*#
GST 2-2
GST
9.O+2.6
12.o+2.6
l-1.3+t-.9
1L.612.0
t2.L+1.9
11.7+1.8
9.911.5
9 .8+ 1.0
9.5+1.1
9 . 7+i..8
8.5+1. 7
8.8+1.6
1t_.0r1.6
L1.2+0.8
Ll.7+r.7
* Significant by one-vray analysis of variance (p < 0.075).
# The relationship between GST subunit A $g/r¡rg) and dose
of
caffeic acid (mS/kS) \â/as deÈermined by linear_
regression anarysis Iestimated parameters: regression
coefficient
of caffied acid -0.00392 (p = 0.013); with a
constant term of 1l_.51 (Uølrng¡¡.
GST subunits were separated and quantified according to the
procedures described in Materials and Methods.
Discussion
So tar, most studies on the effects
of various
inhibitors
of Gsr have concentrated on inhibition of the
-46-
reversible type. Especially in vivo, changes in Gsr
activity by irreversible binding of compounds have been the
subject of only few investigations. The aim of our study
of GST
v¡as to invesÈigate both tyPes of inhibition
isoenzymes by caffeic acid and its major GSH conjugate (2cScA), both in vitro and i¡ vivo.
In vitro, 2-GSCÀ r¿as clearly a more effective reversible
inhibiÈor than the parent compound, in analogy with other
GSH conjugates (Mannervik and Danielson, 1988; Ploemen et
GSI 2-2 was insensitive to both
a7., 1990). Strinkingly,
compounds. with caffeic acid, Das et al. (1984) found an
I.., value of 140 pM using a mixture of rat GsT. Froln our
dáËa, it is clear that with this concentration only GST 4-4
is inhibited strongly, indicating the necessity of the use
of individual GST isoenzymes.
on the other hand, caffeic acid is a much more potent
irreversible inhibitor than 2-GSCA. For its tine-dependent
caffeic acid presumably has to be oxidized to
inhibition,
the GST
which inactivates
an o-quinone structure/
isoenzymes, in analogy with tetrachloro-1r4-benzoquinone
which inhibits GST by covalent modification of the cysteine
residues of the enzfrme (Ploemen et a7., I99l; van Onmen et
a7., L989). In accordance with this hypothesisr âû excess
of cSH protects against inactivation of GST by caffeic
acid, since GSH efficiently traps the unstable o-quinone
metabolite and eiÈher reduces it back to the parent
compound or generaÈes 2-GSCÀ (Cillier and Singleton, 1990).
tempting to conclude that a slower rate of
is
It
spontaneous oxidation of 2-GSCA to an o-quinone structure
may explain the observed lack of irreversible inhibition of
related
cST 1-1 and 3-3 by 2-GSCÀ, since the structurally
acid is more resistant to
conjugate 2-gluthionylcaftaric
oxidation than its parent compound (Cheynier et a7.r 1988).
Moreover, the addition of cysteine at the S-position of the
quinone derived from 2-GSCÀ presumably occurs at a slov¡er
rate,
in analogy with the strucÈurally related 2-Scysteinylcaffeic acid (Cillier and Singleton, 1990). On the
other hand, the binding of 2-GSCÀ to the target amino acid
of the enzyme may be sterically impeded.
GST 7-7 is by far the most sensitive isoenzyme to
inactivation by incubation with caffeic acid, as shown by
the enzymatic activity tor¡¡ards CDNB. This coincides with
the formation of adducts between GST 7-7 and caffeic acid
derivatives, as shown by HPIC. Recently/ it has become
clear that this n-class isoenzyme possesses a highly
-47-
reactive cysteine residue/ the modification of which Ieads
to inactivation (Ricci et a7., L99I; Tamai et a7., l-990).
In addition, cST 7-7 is known for its unique susceptibility
to oxidation, which also inactivates the enzyme (Shen et
al.,
1991). The rate of inactivation of GST 7-7 strongly
correlates with the published rate of autoxidation of
caffeic
acid lCiIIier
and Singleton,
1989). This
susceptibility to oxidation of cST 7-7 and the strong
correlation between its inactivation and caffeic acid
oxidation suggest that, besides the observed covalent
modification of cST 7-7 by caffeic acid derivatives,
oxidation of the reactive cysteine of GST 7-7 by caffeic
acid night conÈribute to inactivation.
Caffeic acid is efficiently absorbed in rats after oral
administration (Camarasa et a7., L988). In vivo, the
reversible inhibition of cST by caffeic acid is dependent
on the concentration of the inhibitor in the cell. This
reversible inhibition cannot be determined using the
catalytic
activity tor¡rards CDNB because of the dilution
factors of about l-0r000 times (as a result of tissue
homogenization and dilution in t.he enzymic assay). On the
other hand, irreversible
inhibition can be measured
directly using the activity towards CDNB or by determining
subunit content because the caffeic acid-rnodified enzl¡me
subunits are only very poorly purified on the Shexylglutathione
bioaffinity
column. Only
marginal
irreversible inhibition could be seen in rat l-iver cytosols
(up to approx. 14%) concomitantly with a slight decrease in
the U-class subunits (only significant for GST 4-4). cST
subunit 4 is unigue for its sensitivity to both reversible
(in vitro ) and irreversible inhibiÈion (in vjyo and vitro).
It is likely that because of the relatively high content of
oxidizing enzl¡mes (e.9. mixed-function oxidases) in the
liver,
relatively
quinone
high concentrations of
intermediates can be formed. This was supported by the fact
that microsomal incubations catalysed the oxidation of
caffeic acid. However, GSH is an effective scavenger of the
quinone. Because of the lack of strong irreversible
inhibition in vivo and the low I"n value (especially for 2cSCÀ), it is reasonable to conclúðe that further in vivo
studies have Èo concentrate on the reversibl-e inhibition of
GST by caffeic acid and its derivatives (e.9. by measuring
their actual concentrations in the cell).
-48-
Refereûces
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-51 -
Chapt,er 4
Inhibition of hr¡man glut,aÈhione S-transferases by dopanine,
a-methyldopa and their s-S-glutathíonyl conjugates
J.H.T.M. Ploemen, B. Van Ommen/ A.M. de Haan, J.C.
P.J. Van Bladeren.
Venekamp,
Chemico-nioTogicaT Interactions,
in press.
nbstract
The reversible and irreversible
inhibition
of human
glutathione S-transferases (GST) by dopamine, a-methyldopa
and their 5-S-glutathionylconjugates (termed s-GSDÀ and 5was studied using purified
GSMÐOPA, respectively)
using CDNB as
isoenzlrmes. The reversibJ.e inhibition,
substrate and expressed as I-^/ ranged from 0.18-0.24 (cST
Mla-1a) , O.lg-O.24 (GST MLÉYlb) to O.5-0.54 mM (GST A1-1)
About 2Oz
for s-GSDA and s-GSMDOPA, respectively.
inhibition was observed for cST À2-2 and P1-1-, using 0.5 mM
reversible
of both S-GSDA and 5-GSMDoPÀ. No significant
inhibition was observed with dopamine and a-methyldopa.
Tyrosinase was used to generaÈe ortåo-quinones fro¡n
dopamine and a-methyldopa which may bind covalently to cST
and thereupon irreversibly inhibit GST. ln this respect,
GST P1-1 was by far the most sensitive enzfrme. The
inhibition (expressed as 3 of control) after incubating 0.5
pM GST in the presence of 100 units/ml tyrosinase with 5 ¡M
of the catecholamines for 10 min at 25"C, was 99% and 67e"
for
dopamine and a-methyldopa, respectively. Moderate
irreversible inhibition of GST A1-1 by both dopamine and ømethyldopa (33% and 25%, respectively), and of GST Mlb-1b
by dopamine (45e") !,ras also observed. GST Pl-1 is also the
only isoenzyme susceptible to irreversible inhibition by 5while no significant inhibition was
GSDA (33% inhibition),
observed v¡ith s-GSMDOPA.
A minor part of the inhibition by dopamine (23e"\, and
the complete inhibition by s-cSDÀ was restored by reduction
-52-
hrith dithiotreitol.
This suggests that cST pL_1 is
inhibited by disulfide formation in the case of s-csDÀ,
whire this oxidative pathway also substantially contributes
to the inactivation by dopamine. This lt¡as supported by the
HPlC-profile of the csr pr.-1 subunit which h¡as strongly
affected by dopamine, whire for 5-GSDÀ after reducÈion with
dithiotreitor the original erution profile of the subunit
returned.
Iutroduction
The usual
metabolisn
of
catecholamines in
catechoraminergic neurons of the central and peripheral
nervous system proceeds through tlvo major enzymatic
pathways, involving monoamine oxidase and catechol-omethyltransferase t1l. In addition, especially in non_
nervous tissue, oxidative pathways play a significant ro1e,
since the catechol moiety is readily oxidized
It is
wel-l known that e.g. the oxidation products[1].
of 3,4_
dihydroxyphenylalanine (dopa) are involved in meranogrenesis
[2]. The oxidation of catechoramines is a comprex process
which proceeds by one-electron oxidation, in which the
catecholamines are first converted to ortåo-semiquinones,
which after
disproportionation
give rise 1"
the
corresponding ortiro-quinones and the parent compounds
t1l.
rn addition. the quinones of the catechoramines are also
formed by two-electron oxidation catalyzed e.g.
by
tyrosinase
t2l. The unstable quinone may rapidly be
reduced, or form covalent adducts v¡ith sulfhydryl groups of
either
small molecures rike the abundant tripeptide
glutathione, or proteins [3]. rn (eu)meranine biosynthesis,
this
nucleophilic addition reaction competes with an
intramolecular cycrization of the ortiro-quinones with an
amino group in the side chain, which is followed by a
complex cascade of intramolecular rearrangements in which
redox reactions are invorved [2]. The covalent interaction
of quinones with accessible and essential
protein
sulfhydrils may result in (un)desirable enzyme inhibition
[4]. The family of the glutathione S_transferases (GST) is
very susceptible to this type of inhibition [5,6].
The predominantly cytosolic GST catalyze the conjugation
of glutathione with electrophiric compounds of both
endogenous and xenobiotic origin
such as e.g. epoxides,
a,B-unsaturated aldehydes and ketones, alkyl and aryl
halides [7rB]. The catalytic rate enhancement is in general
-53-
relatively
low compared with other enzlzmes, although this
might be compensated by their abundance t9l. Besides the
function as catalyst/ cST also acts as binding protein
rn higher organisms the cytosoric GST have been divided [7].
in
4 gene families, Èermed alpha, mu, pi, and theta [9/10].
A large variety of reversibre inhibitors of the csr is
known t9l. rn general, these inhibitors can be divided in
glutathione derivatives, second substrate analogs, and
nonsubstrate binding ligands [g]. As stated above, GST is
al-so sensitive to an irreversibte inactivation by quinones
as result of covarent modification of cysteine residues
t6l. This type of inactivation was enhanced for the
grutathione conjugates of a range of structurarry rerated
quinones [6r11]. Evidence is accumulating that
the
glutathione moiety binds non-covarentry to the active síte,
thereby "targeting" the quinone moiety to the enzlme to
form covarent bonds presumably with a cysteine residue of
the protein. Moreover, the rate of inactivation of the mu_
class GST by the glutathione conjugates of quinones may
al-so be enhanced by reaction with a different target amino
acid, since the structurally related compound S_(4_Bromo_
2,3-dioxobutyr)glutathione, has been shown to react rapidly
and specifically with a tyrosine of rat GSf 4_4 Í1,21.
Recently' there has been a growing interest for in vivo
appricable inhibitors of GST, since evidence indicates that
the GST are involved in cellular drug resistance t 13_15 l.
The present study has been designed to assess
the
individual reversibre and irreversible inhibitory capacity
for human GST by the neurotransmitter dopamine and the
structurally
related
a-methyldopa and their
5_S_
glutathionyl conjugates (Fig. 1). a-Methyrdopa (arso known
as Aldomet) was chosen because its relative large side
chain, and since this drug is commonly used as an
antihypertensive agenÈ.
Materials and Methods.
Chemicals. a-methyldopa
( 3- ( 3.4-dihydroxyphenyl ) -2methyl-L-alanine),
dopamine hydrochloride (3_hydroxy_
tyramine hydrochloride; 99"ø pure) were purchased from
Janssen Chj_mica (GeeI, Belgium). clutathione
and
dithiotreitol
r"rere from Boehringer (Mannheim¡ Germany). S_
hexylglutathione
and tyrosinase lfrorn mushroom; 3/130
units/mg solid¡ were from Sigma Cheni_cal Co. (St. Louis,
USA). HPLC-grade TFA was obtained from J.T. Baker Inc.
-54-
(À)
(B)
NH2
Ho.,-^"/^1,ÀH2
lllr
/\' ,/)
HOY
COOH
SG
(c)
(D)
Fig. 1. The molecular structure of dopamine (A), d.methyldopa (B), 5-S-glut.aÈhionyldopamine (C) and 5-Sglutathionyl-a-methyldopa (D). cS = glutathione (y-cLU-CySGLY
)
(Phillipsburg, NJ, USA), HPLC-grade methanol vras from
Rathburn Chemicals Limited (Walkerburn, Scotland). npoxyactivated Sepharose 68 v¡as purchased from Pharmacia
(Uppsala/ Sweden).
Syntåesis of s-S-glutathionyT conjugates of dopamine and
a-nethyTdopa (5-GSDA and 5-GSMDOPA). The glutathione
conjugates were prepared using tyrosinase.
0.2 mmol
dopamine or a-methyldopa \^ras incubated with 5 mmol
glutathione in the presence of 100 units of tyrosinase/m1
in 0.1 M ammonium acetate buffer pH 5.8 for 4 hours at 25oC
(total volume of 100 ml). The reaction was followed on RpHPLC, using Zorbax ODS (4.6 * 250 m¡n). Elution was
performed at a flow of 1 ml/min with 0.1% TFÀ (solvent A)
and 0.1% TFA in methanol (solvent B), 1 min isocratically
at 1% B, followed with a linear gradient of 1*75% B in Ll
min, finally
followed by a gradient to 100% B in 2.5 min,.
(k'= 2.3, 2.8, 3.2, 3.4) for dopamine, s-cSDÀ, q.methyldopa, and S-GSMDOPÀ, respectively). the incubates
were lyophilized, dissolved in 10 m1 of 40 mM sodium
-55-
acetate buffer pH 4.7, filtered through a 0.45 pM Millexrfilter (Millipore, Molsheim, France), and 2 mI fractions
\^/ere purified by preparatíve Rp-HpLC, using zorbax ODS
(21 .2 x 250 mm), eluted isocratically
at a flow of 4 ml,/min
with 6Z methanol in 10 ÍìM anmonium acetate buffer pH 4.7
(k'= 7.7,
and 0.8.
for
5-cSDÀ, and s-cSMDOpÀ/
respectively).
Fractions containing the glutathione
conjugates were lyophilized, dissolved in l0 mI of the
sodium acetate buffer and repeatedly purified using the
preparative RP-HPLC method (see above). Glutathione
conjugates rof >99% purj-ty, as judged by HplC-analysis (see
above) and -H-NMR (4OO MHz, D^O, 6=4.7 ppm) wçre obtained.
The H-NMR spectrum of S-éSoe was assiqnéA ." follows:
signals for the catecholamine part ð 6.90 (d, 1H, J=I.j
Hz), 6.80 (d, 1H, J=L.1 Hz), 3.20 (t | 2H, J= 7.2 Hz) | 2.93
(Et 2Ht J=7.2 Hz), and for the glutathione part 6 2.15 (m,
2H, GIu þ\, 2.49 (L, 2Ht J=7.5 Hz, Gl-u y) | 3.2O (m/ tH, Cys
B (H.)), 3.32 (ddf LH, J= 14.5 and 5.0 Hz, cys B (H-)),
3.83* 1s, 2Ht cly d), 4.00 (È/ !H, J= 6,16 Hz, GIu a¡, l.+Z
(dd, 1H, J= 8.3 and 5.0 Hz, Cys a).Îhe *H-NMR spectrum of
s-GSMDOPA gave the
following resonances: for the
catecholamine part ó 6.86 (d, lH/ J=2.O Hz) | 6.75 (d, 1H,
J=2.0 Hz)t 3.22 (d, 1H, J= L4.S Hz, CH? (Hh)), 2.89 (d, l_H,
J= L4.5 Hz, CH., (H^) ), 1.58 (s, 3H¿ -CH"i, and for the
glutathione pait 6*2.13 (mt 2Ht Glu Bl | 2:47 (t, 2H, J= 7.7
Hz, Glu y), 3.18 (dd, IH, ¡= 14.3 and 1.9 Hz, Cys Ê (Ha) ),
3.36 (dd, 1H, J= 74.3 and 7.9 Hz, Cys,B (HÌ^)), 3.85 (s,"2H,
Gly a), 3.89 (t, l-H, J= 6.5 Hz, c1u d), 4.4O (dd, tH, J=
7.9 and 4.8 Hz, Cys c). The coupling of l_.7-2.O Hz betv/een
the protons in the aromatic region corresponds to meta
positions of these protons. Àssignments have been confirmed
by a 2D-COSY experiment. The Uv-spectrum of both
glutathione conjugates were identical, with À
256 and
max
291 nm; €"o.= 1850 M-1.cm-1.
Stabilit! of S-S-glutathionyL conjugates of dopamine and
a-methyldopa. The stability of 5-GSDA and s-cSMDOpÀ, under
conditions IikeIy giving autoxidation, was studied by
incubation of 100 yM of S-GSDA and S-GSMDOPÀ at 37"C in 50
mM potassium phosphate buffer pH 7.4 (with 10% methanol;
final volume 4 ml), by inject.ing 12 samples of 100 UI of a
time-series (0 to 12 h) on Rp-HpLC (see above). The
stability was compared v¡ith the parent compounds incubated
in the same way. The peak areas were integrated with Nelson
Analytical Model 2600 Chromatography Software at zgL nm and
279 ûrr for the glutathione conjugates and the parent
HÀ
-56-
compounds, respectively, and expressed as % of t=0. Tl (h)
values v¡ere determined using logaritbm probit anáIysis
[16].
The formation of glutathione adducts of 5-cSDÀ and 5csMDoPA v¡as studied by incubati-on of 0.5 nM of the
catechols with or without 3.5 mM glutathione (as trapping
agent¡ in the presence of 100 units,/ml tyrosinase in 0.1 M
potassium phosphate buffer pH 7.4 for l-0 min at 25oC (final
volume 0.25 m1). 50 ¡rI samples were injected on RP-HPLC
(Zorbax ODS, 25O r, 4.6 mm, for elution see above). Blanc
incubations without tyrosinase were perforrned in the sarne
way. The conversion was measured by peak area integration
at 29L nm (see above) and expressed as 3 of the blanc
These experiments were performed in
incubations.
No significant conversion h¡as seen in the
triplicate.
absence of tyrosinase.
The stability of 5-GSDA and S-GSMDOPA was also studied
in microsomal incubatioñs. 0.5 ml,t conjugate vras incubated
at 37 "C with 3.5 mM glutathione in the presence of rat
liver mícrosomes at a concentration of 1- mg protein/ml,
prepared as described previously [17], and l- mM NÀDPH. The
final volume \,¡as l- ml (buffer: 0.L M potassium phosphate pH
7.4 with 3 mM MgCl?). Àfter 30 min, 200 pI of 2Oe" acetic
acid was added fo stop the reaction. The incubates \'tere
filtered using a 0.22 ¡M filter
lMiIlexr-GS, Millipore),
after which 30 ¡rI of the filtrate was injected on HPLC
(zorbax ODS column, see above). The conversion of the
conjugates was measured by peak area integration at 291 nm.
Incubations \¡/ere performed in triplicate.
and assay. GsT isoenzymes \^rere purified
GST purification
from liver and placenta (GST P1-1) using affinity
chromatography (S-hexylglutathione-Sepharose 6B), and
separat j-on of the various isoenzlanes \4tas achieved by
chromatofocusing as described previously [18]. Purity \^¡as
confirmed by SDS gel electrophoresis, isoelectric focussing
and HPlc-analysis as described. AII enzlme concentrations
are expressed as the concentration of the subunit (Mrt
25,9OO, 25,9OO, 26t1OO, 26,600, and 24800, respectively fõr
GST subunit 41, A2, M1a, M1b, PL t 191 ) . Protein h¡as
d.etermined by the method of Lowry [20], using bovine serum
albumin as standard.
The reversible inhibition was
rnhibition
studies.
determined by mixing in the cuvettes of the enzymic assay
1in final concentrations) of 10 nM GsT (in triplicate) with
50, 1OO, 200, and 500 UM of 5-GSDA and S-GSMDOPÀ' and 2.5
-57-
mM of the parent compounds, after which the enzymatic
activity tor''ards .DNB was measured imnediately (see bel-ow).
rhe tirne-dependent irreversibre inhibiÈion hTas measured
by incubating 0.5 uM GST with 5 yM of the catechoramines
and their gluÈathione conjugates in the presence of 100
units/ml tyrosinase for 10 min at 25"c in o.i M potassium
phosphate buffer pH 7.4 (tripticate,. final volumè 50 pl).
No significant ross of catarytic activity was observed
with
tyrosinase in the absence of catecholamines (blanc
incubations). The reaction was terminated
an excess of
glutathione (50 UL of 2 mM glutathione),with
after which the
samples r^rere stored on ice. Blanc
\,rere
performed in the same wdy, with theincubations
onission of the
catecholamines. After storage for 2 hours (to assess
the
irreversibility
by trapping the potentially released
catecholamines v¡ith an excess of glutathiãne),
the
catarytic activity was determi-ned towards .DNB (see be10w)
by transferring J_O ¡rI samples into a 250
cuvettes.
Remaining activity !ì/as expressed as % of blancUI
incubation.
The inhibition of cST pj_1 by dopamine and S_(;SDA.
To
study the effect of the strong reducing agent dithiotreitol
on Èhe extent of time-dependent inhibition of csr p1-1
by
doparnine and s-csDA, 0.5 pM GST p1-1 was inactivated
with
the catecholamines in the presence of tyrosinase
as
described above (in duplicate, final votumå 10O pI). The
reaction was terminated with 100 irr of 2 mM glutathione
(blanc incubations) or 100 ¡rl of 0.1 M dithiotråito:_
in O.1 M potassium phosphate pH g.4), whereafter (both
the
samples were incubated for 10 min at 37"c. The catatytic
activity was determined tovrards CDNB (see below¡.
The
enzymatic activity
h/as also measured after prolonged.
incubation with either glutathione or dithiotreitot
( tg
h
at 6oC).
To study the adduct formation of dopamine with cST p1_l_,
2 pM of cST P1-1 was incubated irrith 5 lrM dopamine
for 10
min at 25oC in 110 ¡rl of 0.1 M potassium phosp-hate pH
.7.4
in the presence or absence oi tyrosinase (iOO units/ml¡,
foll0wed by incubation for r-0 min at 370c with 1r.
¡rt of 0.5
M dithiotreitor.
QuantificaÈion of native and modified csr
P1-1 lras performed by injecting 0.1 mr on a vydac protein
Peptide
column (4.6 ¡, 250 run), eluted at a flow of &1
9fS
mr/ml_n, r¡rrth 0.1% TFA in deionized rn/ater (solvent
À) and
0.1% TFÀ in acetonitrile
(sotvent B), with a Linear
gradient of 35-55% B in 20 min, followed for 5 min
at 55A
-58-
B. Peak area was integratecl at 2]-4 nm. Incubations were
performed in duplicate.
GTutathione S-transferase assay. The activity of
individual GsT isoenzymes etas determined with 1 mM
glutathione and 1 mM CDNB as second substrate at 25"C in
0.1 M potassium phosphate buffer pH 6.5, using the
spectrophotometric method of Habig [21].
StatisticaT anaTysis. Levels of significance were tested
by one-\r¡ay ANOVA (P < 0.05 ) ' while comparison bet\47een
groups were made using Dunnett control grouþ comparison
test (P < 0.01-).
Results
of the 5-GSDÀ and S-GSMDOPA and their
The stability
parent compounds were studied at 3'7"C. In general, the
glutathione conjugates \4tere less stable than the parent
compounds, while the a-mdthyldopa rnoiety htas less stable
than dopamine. The tL (h) values (vtith bet\¡/een brackets the
95% confidence intérval ) htere 10 .5 (9 -2 - .. 12 .5 ) , 5 . 6
and 1.8 h (1.6---2.1), for
5.3 (4.8...5.9),
(5.1...6.3),
5-GSMDOPÀ,
and
s-GSDA,
a-methyldopa,
dopamine,
respectively. Since no trapping (thiol) agent was added to
the incubations, the breakdoq¡n of the compounds is most
Iikely due to cyclization of the quinones formed upon
autoxidation. These result may indicate that either the
autoxidation or the rate of inÈramolecular
rate
of
cyclization of the glutathione conjugates s¡as faster than
for the parent catecholamines.
The 5-s-glutathionylconjugates htere incubated v¡ith
tyrosinase and glutathione, to study whether tyrosinase is
able to oxidize the S-S-glutathionylconjugates and whether
subsequent nucleophilic addition of thiol may occur. À
small but significant (P < 0.01) part of s-GSDA was turned
over in the presence of glutathione (Iable L). No
conversion of 5-GSDÀ occurred in the absence of
glutathione, suggesting that under the conditions used
addition occurred, without significant
nucleophilic
cyclization. Interestingly, tyrosinase catalyzed conversion
of s-GSMDOPA was not observed. FinaIIy, the tyrosinase
incubations (with glutathione) resulted in more than 90%
conversion of d-methyldopa and dopamine within 10 min (data
not shown).
Using microsomes (which contain mixed function oxidases¡
to oxidize the S-S-glutathionylconjugates, the same trend
-59-
as for the tyrosinase (+ glutathione) incubations was
observed: only relatively poor but significant (p < 0.01)
conversion of s-GSDA.
lable 1.
Substrate consumption of s-GSDA and 5-GSMDOPA
(expressed as t recovery of blanc incubations) after
incubation vrith tyrosinase and microsomes.
tyrosinase
without
glutathione
tyrosinase
5-GSDA
94 ! L3.4
80 1 3.9
73 ! 2.2
5-GSMDOPÀ
98 1 0.6
98 t 0.8
94 ! 4.9
compound
microsomal
incubation
0.5 mM of 5-cSDÀ and S-GSMDOPÀ \4ras incubated in the
presence of 3.5 mM glutathione for l-0 ¡nin at 25oC with 100
units/ml tyrosinase or for 30 min at 37oC with 1 mg/ml rat
liver microsomes. Incubations without glutathione were also
performed with tyrosinase. The recovery (expressed as 3 of
blanc incubations withouÈ tyrosinase or microsomes,
respecÈively) was measured by peak area integration at 291çm. Values are the average of 3 incubations (! SD).
Statistical
difference (P < 0.01-) beth¡een control group
and experimental group with Dunnett comparison test,
after
significant ANOVÀ (P < 0.05).
Rer¡ersjb-l.e inhibition.
Dopamine andø-methyldopawere
insubstantial reversible inhibiÈors of the human cST. The
catalyÈic activity
to!,lards CDNB in the presence of 2.5 mM
of both compounds r¡¡as inhibited less than 15% for GST A1-1,
A2-2, Mla-la, M1b-l-b, and PL-l- (result not shown) . Às
expected, the reversible inhibition
was enhanced by the
glutathione moiety (Table 2). In general, only small
differences between S-GSDA and S-GSMDOPÀ existed. The muclass isoenzymes Mla-la and Ml-b-lb \,/ere Èhe most strongly
inhibited GST isoenzymes by both conjugates. Within the muclass virtually no difference beth¡een M1a-la and Mlb-lb was
noticeable. On the other hand, the alpha-class GST À1-L was
-60-
significantly
more inhibited than À2-2. GST A2-2 and pl_-1
hrere the least inhibited isoenzymes.
*
Table 2. I<rì (mM) -values toh¡ards CDNB of rat glutaÈhione
S-transferasé"incubated with 5-csDÂ or 5-cSMDOpÀ.
enzym
5-GSDÀ
5-GSMDOPA
alpha-class
GST Àl_-L
GST À2-2
0.50
>0.50
mu-cIass
GST MLa-1a
esT Mt_b-1b
pi-class
GSl Pl-l_
( 18% )
o.24
o.24
>0.50
0.54
>O.5O (22e")
0.18
0.1_9
( 15%
)
>o.5o
(22e")
l-0 nM enzyme was incubated wi.th different concentrations of
inhibitor,
after
which the enzlznatic activity was
determined towards CDNB. At least 4 concentrations in
triplicate
$rere used. Between brackets the ? inhibition
Eith 0.5 mM inhibitor.
The concentration of inhibitor resurting in 5o% inhibition
of acÈivity to$¡ards CDNB (I5O).
Irreversibl-e inhibition.
The experimental conditions
that were shown to convert the parent catecholamines and 5GSDA, were used to generate quinones of the catecholamines
in situ, to study Èhe irreversible
inhibiÈion of the
individual
GST isoenzlzrnes. The concentration
of
catecholamine \^¡as reduced to 5 UM, to prevent reversible
inhibition
(the concentration of catecholamine will
subsequently be reduced in the CDNB-assay to a final
concentration of 0.1 UM). cST p1-1 was by far the most
sensitive isoenzyme for the quinones derived of dopanine
and a-methyldopa, and !ìlas the only GST which t/as
significantly
inhibited by incubation with 5-cSDÀ (p<O.Ot)
(Fig. 2). Furthermore, a noticeable sensitivity of cST M1b1b for dopamine was present, while cST A1-l_ was sensitive
to inhibition by both a-methyldopa and dopamine. GST È2_2
and Mla-la v/ere not inhibited by any of the compounds used.
- 61 -
120
't
()
(ú
o,
.s
E
'õ
f
100
dopamine
80
60
WW s-esoa
40
WZZ
E
o
s
s-esMDopA
20
0
A1-1 A2-2
M1a-1a M1b-1b p1-1
Fíq- 2. rrreversibre inhibition of human GST by dopamine,
d-netbyldopa (MDOPÀ), s-cSDÄ., and s_cSMDOpÀ in Lh" pr"".rr..
of tyrosinase. 0.5 ¡rM GST hras incubated with 5
of
inhibitor in the presence of 100 uniÈs/mr tyrosinase UM
for 10
min at 2SoC. The reaction v¡as terminated with 1 rM
grutathione- Branc incubations were performed in the same
way, with the onission of the catecholamines. The catarytic
activity
r{¡as determined towards CDNB. varues are the
average of three incubations and expressed as % remainíng
activity
of blanc incubation (t SEM). For experimental
see
Materials and Methods.
Çetails
statistical
difference (p < o.or-) bet\,reen control group
and experimental group with DunnetÈ comparison test, after
significant ANOVA (p < O.O5).
With the strong reducing agent dithiotreitol,
the
inhibition
of cST pl_-l_ by 5-cSDÀ could be completely
aborished (Tabre 3) - A considerable extent of the
inhibition
of csr p1-1 by dopaurine courd arso be restored
by dithiotreitol
(about 23% recovery of Èhe inhibition).
Vtith prolonged (overnight)
incubation,
no further
restoration of activity was achieved (results not shown),
-62-
Table 3. The effect of the reducing agent dithiotreitol
(DTT) on the e" remaining activity of cST P1-1 inactivated
by dopamine and S-GSDA
+
enzlme
-
dopamine
0.4 1 L.0
23.4 ! 3.0
5-GSDA
76.4 ! 4.6
L02.8 ! 2.2
DTT
DTT
0.5 UM GST P1-1 was inhibited with 5 UM of the
catechol¡mines in the presence of 100 units,/m1 tyrosinase
for 1O min at 25"C, followed by incubation for 10 min at
37"C with either 1 mM glutathione (- DTT) or 50 mM DTT. The
catalytic activity
was determined tor¡/ards CDNB, and
expressed as % remaining activity
of blanc incubation
incubaÈed in the same hray without the catecholarnines.
values are the average of two incubations (+SD).
indicating that a consistent part of the inhibition is not
reversible by reduction.
The covalent interaction of dopamine and s-GSDA with GST
Pl-1 r4/as also studied using an HPlC-separation of the
individual subunits of cST PL-l, incubated with the
catecholamines in the presence of Èyrosinase and
subsequently treated hrith dithiotreitol
to reduce
(Fig. 3). Àfter incubation with doparoine, peak
disulfides
broadening and'a new peak was detected, suggesting a major
change in the the structural properties of the subunits. On
the other hand, incubation with S-GSDÀ did not reveal this
phenomenon.
-63-
Fig. 3. TypicaL fipLc-chromatograms of cST pj-l su.bunj¿
incubated with dopamine and í-GSDA after incubation with
tyrosinase and subsequentTy treated with d.ithiotreitoJ- to
reduce disuLfides- 2 uM of csr p1-1 was incubated with
tyrosinase (100 units,/rnl) and 5 pM of the catecholamines,
forrowed by incubation with 0.05 M dithiotreitor.
The GST
P1-1 subunit was separated using Rp-HpLC. Uv-detection at
at 2I4 nm. Incubations v¡ere performed in duplicate. For
experimental details see Materials and Methods. lA) cST pl_
1 + 5-GSDÀ +tyrosinase; lB) GST p1-l_ + tyrosinase (C) cS,I
+ dopamine; lD, cST p1-1 + dopamine f tyrosinase.
Discussion
The potential for covalent binding to essential
sulfhydryl groups of enzymes with subsequent inactivation
of these enzlzmes by quinones derived from catecholamines
has been noted before 1.2,41. previously, it has been
reported that guinones derived from catecholamines inhibit
housefly GST efficiently l27l. T.n the present study it has
been shown that the human pi-crass GST p1-1 is a serective
and sensitive target site for quinones derived from
dopamine and a-rnethyldopa, which thereupon inhibit
the
enzl¡me. The GST p1-1 is also, albeit to a lesser extent,
susceptible to inactivation
by the 5-S-glutathionyl
conjugate of dopamine. Ihe cST A2-2, which does not possess
any cysteine residues [23], was not inactivated by the
catecholamines used, again indicating that a cysteine
residue is presumably the target site.
ft is weII established that the cST of pi-class possess
a highly reactive cysteine residue, modification of which
-64-
results
in enzyme inactivation l24,25l. The cysÈeine
residup-involved in this inactivation has been located at
the 47L¡r position 124,251, close to the G-site of cST [26].
The three-dimensional structure revealed however that this
cysteine is not a structural part of the active site [26].
The non-essential role of cysteines in the catalytic
activity has also been shown with site-directed mutagenesis
of this cysteine presumably
127 ,281. The nodification
inactivates the enzyme either by conformation changes, or
by blocking the substrate binding 126,291. The cysteine
residues of the GST pi-class also may undergo a
(reversible) oxidative inactivation, by the formation of an
intersuþgnit disutfide bethreen the cysteines at the 47En
and L01-"-position [30r31]. Since a notable part of the
activity
of GST P1-1 inactivated with dopamine-quinone was
restored after reduction with dithiotreitol,
it can be
concluded that the quinones derived from dopamine interact
with GST P1-1 by both the reversible oxidation reaction
(mJ-nor pathway) and the nucleophilic addition (major
paÈhvray). This was confirmed by the HplC-profile of the
subunit of cST P1-1", which r{as strongly affected by
dopamine even after reduction with dithiotreitol.
On the oÈher hand, the quinones derived from 5-cSDÀ
inactivate cST P1-1 completely via disulfide formation,
since the inactivation hras fully abolished by reduction
with dithiotreitol.
This was also confirmed by the HPLCchromatogram of the GST subunit of p1-1, which was
unaffected after reduction. It is unclear why the extent of
inactivation is relatively
low and \"rhy only disulfide
formation occurred. Most likely, the relatively low rate of
quinone formation from S-GSDA, as well as the low chemical
reactivity of the nucleophilic addition of a second thiol
to S-GSDA both contribute to this phenomenon. This
postulate r^/as supported by the present study in which only
poor conversion of S-GSDA with both microsomal incubations
and tyrosinase was observed, as well as by earlier
studies
in 'which was shown that the nucleophilic addition of thiol
to quinones (derived with relative large concentrations of
tyrosinase from structurally related cysteinylconjugates of
catecholamines) occurred at a low rate [2r3t32]. Thus in
the case of s-cSDÀ, most likely the oxidation of cysteine
47 .of cST P1-1 occurred more rapidly than nucleophilic
addition to this cysteine. The oxidation of cysteine 47 is
accompanied by disulfide
formation, presumably with
cysteine 10i-.
-65-
The significance of the reversibre inhibition of GST by
glutathione derivatives has been noted before [9]. E.g. the
potent reversible inhibitors S-hexylglutaÈhione, S_(p_
bromobenzyl)grutathione and the grutathione conjugate of
ethacrynic acid have l.n-values .ranging from <0.1 to 20 UM
for the human alpha-i"mu-, and pi-class [9r33]. Virtually
no reversible inhibition of the human Gsr r,fas observed. with
the catecho,Lamines themselves, nevertheress the grutathione
conjugates of the catechoramines reversibry inni¡itea arl
of the studied human csr. However, compared with the above
mentioned compounds, these conjugates are relatively
moderate inhibitors of csr. rnterestingry, in the case of
the glutathione conjugates of ethacrynic acid,
S_(p_
bromobenzyr) and now the catecholamines, the human mu-crass
is much more sensj_Èive to reversible product (analog)
inhibition
than the arpha- and pi-class, indicating that
especiarly this csr crass might be very susceptibre to
product-bindinq.
The in vivo occurrence of catechoramine oxidation
products has been demonstrated by the recovery of 5_S_
cysteinyldopamine and s-s-cysteinyrdopa in the human brain
[34], and in the tyrosinase catalyzed synt,hesis of red
brown melanin pigrnent in normal and marignant meranocytes
Under normal physiological conditions it seems
t 35 I .
unlikely that significant inactivation of Gsr pi--1 occurs,
since the protective
agenÈ grutathione is abundant
throughout the body in l--10 mM concentration [gr9], and
e.g. in the human brain at 1 mM t361. t{henever the
protective mechanism is disrupted I37l or whenever the
catecholamines are aùninistered in rerative large doses,
the possibitity exists hohrever that the quinones derived
from catechol¡rnines might significantly react hTith GsT p1_
1. These enzf¡mes are present in human brain and meranoma
cells [9138]. Dopamine and analogs v/ere shown to inhibit
melanoma growth by inhibition of DNA pol]¡merase q, probably
by interaction with a sulfhydryl group t391. In this
respect, it will be interesting to assess the role of GST
inhibition
by these compounds in Èhe observed grovr-th
inhibition of melanoma cells, since GST inhibition may be
involved in the inhibition of the cell proliferation [40].
-66-
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18. Ploemen JHTM, Bogaards JJP, Veldink GA, Van Ornmen Bf
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Protein measurement with the Folin phenol reagent. J BioI
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2L. Habig WH, Pabst MJ and Jakoby wB. clutathione Stransferases, the first
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23. Rhoads DM. Zarlengo RP and Tu CPD. The basic
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24. Tamai K, Satoh K, Tsuchida S, Hatayama T, Maki T and
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inactivation
of glutathione Stransferases in class pi by SH-modifiers. Bjociren Biophys
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25. Lo Bello M. PetÍuzzelli R, De Stefano E, Tenedini Cl
Barra D and Federici G. Identification
of a highly
reactive sulphydryl group in human placental glutathione
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transferase by site-directed fluorescent agent. FEBS Lett
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26. Reinemer P, Dirr Hw, Ladenstein R and Huber R. Threedimensional structure of class n glutathione
SÈransferase from human placenta in complex with Shexylglutathione at 2.8 .å, resolution. J Mot Biol 2272
21_4-226 , 1992.
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Yasui À, Oikawa A and Sato K. RoIe of cysteine residues
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28. Kong KHf Inoue H and Takahashi K. Non-essentiality of
cysteines and histidine residues for the activity of
human glutathione S-transferase. Biochem Biophys .Res Comm
lSLz 748-755, 1991-.
29. Caccuri AM, polizio F, piemonte F, TagÌiatestâ p,
Federici G and Desideri À. Investj.gation of the active
site of human placenta glutathione transferase r by means
of a spin-Iabelled glutathione analogue. Biochim Biophys
Acta LL22z 265-268, 1992.
30. Ricci G, Del Boccio G, pennelli A, Lo Bello M,
Petruzzelli R/ Caccuri ÀM, Barra Ð and Federici G. Redox
forms of human pÌacenta glutathione transferase. J Bjol.
Chen 266¡ 2I4Q9-2I415, L99t.
31. Shen H, Tsuchida S, Tamai K and Sato K. Identification
of cysteine residues involved in disuLfide formation in
the inactivation of glutathione transferase p-form by
hydrogen peroxide. Arch Biochem Biophys 3OO: I37-LlL,
L993.
32. Patel N, Kumagai y, Unger SE, Fukuto JM and Cho AK.
Transformation of dopamine and a-methytdopamine by NG10g_
15 cells: formation of thiol adducts. Chem Res Toxicol. 4:
42t-429, L99L.
33. Ploemen JHTM, Van Ommen B and Van Bladeren pJ.
Inhibition of rat and human glutathione S-transferase
isoenzymes by ethacrynic acid and its
glutathione
conjugate. Biochem pharmaeoT 4O: L631-1635¿ 1990.
34. Fornstedt B, Bergh I, Rosengren E and Carlsson A. An
improved HPlC-electrochemical detection method for the
measuring brain levels of 5-S-cysteinyldopamine, 5-Scysteinyl-3,4-dihydroxyphenylalanine, and 5-S-cysteinyl_
3,4-dihydroxyphenylacetic acid. J Neurocåem 34¿ 579-5g6,
1990.
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35. Inoue S, fto S, Wakamatsu K, Jirnbo K and I'ujita K.
Mechanism of growth inhibition of melanoma cells by 4-Scysteinylaminylphenol and its
analogues.
Biochem
PharmacoJ. 39 ¡ 1077-1083, 1990.
36. Slivka A, Spina MB and Cohen c. Reduced and oxidized
glutathione in human and monkey brain. Neuroscjence Lett
742 LL2-I7-8, 1987.
37. Maker HS, Vieiss C and Brannan TS. The effects of
dopamine, norepinephrine,
5-hydroxytryptamj_ne, 6hydroxydopamine, ascorbate, glutathione and peroxide on
the in vitro activities of creatine and adenylate kinases
in the brain of the rat. IeuropharmacoJ. 252 25-32, 19g6.
38. Ramachandran C, Yuan ZK and Krishan À. Doxorubicin
resistance in human melanoma cells: MDR-1 and glutathione
S-transferase r¡ gene expression. Biochem pharmacol 45:
743-75L, l_993.
39. Wick MM. Levodopa and dopamine analogs as DNA
pollanerase inhibitors and antitumor agents in hurnan
melanoma. Cancer Res 4O: 1,4L4-L4LB, 1980.
y and Kaneko T.
40. Sato Yf Fujii
S, Fujii
Àntiproliferative effects of glutaÈhione S-transferase
inhibit.ors on the K562 cell line. Biochem pharmacoL 39¿
L263-L266, 1990.
-70-
Chaptêr
5
s-(2'4-dinitropbenyl)glutathione excretion into the medium
by rat hepatoma cells. Effects of ethacrynic acid and
tetrachloro-1, 4-benzoquinone and derivatives
J.H.T.M. Ploemen, À.8. Keyzer,
and P.J. van Bladeren.
Ommen,
J.G. Schefferlie, B.
van
Submitted
Àbstract
Àn assay is presented to study glutathione
conjugation
1-chroro-2,4-dinitrobenzene (CDNB) in rat H-35 Reuber
hepatoma celIs, by the quantification of the excretion of
S-(2,4-dinitrophenyl¡glutathione
(DNPSG) on HPLC. Ce1ts
exposed to 1-0 uM CDNB in pBs, displayed rinear excretion of
DNPSG up to 10-20 min. The amount of conjugate Left
intracelrularry was minimal (at the end of the assay 5.0 to
8.1 prnol,/nmol protein), compared to the amount excreted
(about L9 nmol).
The effects of two types of inhibitors of purified
glutathione S-transferases (GST) isoenzymes $rere studied
with this assay, using a preincubation of I hr. A
significant
(p<0-025) rinear rerationship between the
concentrati-on (0-50 uM) of both ethacrynic acid (EA) and
its dibromo dihydro derivative (diBrEA) and the DNpsG
excretion (after 10 min exposure to CDNB) was observed.,
with a maximum of 30% and 50% reduction of DNpsG excretion
for
EA and diBrEÀ, respectively. The intracellular
concentration of DNpsG at the end of the experiment was not
affected, thus excruding an effect of EA on the glutathione
conjugate transport. rntracerrurar glutathione revels were
also similar to contror varues. From the fact that Gsr
activity in celrs lysed at the end of the experiment vras
arso similar to controls, it can be concr-uded that EÀ
inhibits intracelrular GST in a reversibte manner. with the
other type of inhibitors,
tetrachloro_1r4_benzoquinone
(TCBQ) and its glutathione conjugate, of which the glycine
with
- 7L -
carboxyl group was esterified to improve Èhe absorption, no
such concenÈration dependent inhibition of the DNPSG
excretion was found, indicating that these compounds are
not usable as GST inhibitors in cells systems.
Introduction
Extensive lists
of reversible
and irreversible
inhibitors
of glutathione S-transferases (GST) activity
have been published (Mannervik and Danielson, l-988,' Van
Bladeren and Van Onmen, 1991). In general, inhibitors of
enzlntres can be used to study the mechanism of the catalysis
or the architecture of the active site. In the case of the
GST, use has also been made of inhibitors
to distinguish
various isoenzymes (Mannervik 1985). Hob/ever, the role of
the cytosolic cST in the alkylating
drug resistant
phenotype, observed in the treatment of tumors (wa:<man,
1990,. Black and wolf, 1991¡ potentially makes inhibition of
useful.
GST clinically
The reversible inhibition of cST is usualJ-y reporÈed in
Iqô-values. Fortunately, this Ian-value has predoninantly
béén determined with one co-Éübstrate ( l-chloro-2,4dinitrobenzene, CDNB) under established experimental
condition (Habig et a7.t t9'74), which enables comparison of
I"^-values obtained from structurally unrelated inhibitors.
oñ"the other hand, the comparison of the irreversible
inhibition
data of even structurally related compounds is
similarity
strongly hampered since in general little
betrtreen these studies exists (Van Onmen et aL., 1-988;
Berhane and Mannervik, 1-990, Tamai et aJ-., I99Q¡ Desideri
et a7., l-991-).
The extrapolation of inhibition data, obtained with pure
enzlrme preparations, to the in vitro cell or in vivo
situation is relatively complicated. the pharmacokinetics
and meÈabolisn of the inhibitor determine its intracellular
may change
concentration
concentration and this
continuously. Moreover, the GST concentration jn situ is
nuch higher than the enzyme concentration used in the usual
enzl¡rie assays (Mannervik¿ 1985 ) .
Although cST inhibition in biological systems can easily
be determined in cytosolic preparations, using this method,
is always very much
the extent of reversible inhibition
underestimated, since the non-covalently linked inhibitor
is extremely diluted in both the cytosol preparation and in
the enzl¡mic assay.
-72-
The direct measurement of
the glutathione conjugation
a moder subsÈrate in intact ceI1 curtures has the
advantage that conjugaÈion and inhibiÈion are studied,
without dilution of enzymes or inhibitors. Both tlT)es of
inhibition lirreversibre and reversible) are determined,
and moreover the potentiar contribution of grutathione
depletion by the inhibitor
is included. The glutathione
conjugate excretion by isolated celrs has been determined
directly with e.g. bromosulphthalein and a-bromoisovarerate
(for review see: Mulder and Te Koppele, 19gg). A system to
determine the inhibition of the grutathione coniugalion in
cell lines may be based on any of these substrates.
The present study describes a rapid method to determine
the glutathione conjugation with CDNB, by the measurement
of excretion of the glutathione conjugate òt col¡g (s_(2t4_
dinitrophenyl)glutathione,
DNpSc) in Èhe meaium, in the
(Reuber H-35) rat hepatoma cerl line. GDNB was chosen
as
model substrate since it is the commonly used co-substrate
of GST in enzyme assays. The effects of potent inhibitors
of GST isoenzlznes (halogenated quinones and ethacrynic
acid¡ on the DNpSc excretion was studied.
with
Materials and
Methods
Materials and syntheses. Grutathione and dithiotreitor
h¡ere obtained from Boehringer, Mannheim, Germany.
Tetrachloro-J_r4-benzoquinone (TCBQ) was from Merckf
Darmstadt, Geraany. 1-Chloro_2r4_dinitrobenzene (CDNB),
Ethacryni-c
acid,
3-[4r5-dimethylthiazol_2_yl]_2,5_
diphenyttetrazorium bromide (MTT), s-hexyrgrutathione, Nacetyl-L-cysteine, and 2rS-dimethoxycinnamic acid \¡/ere
obtained from sigma Chemical Co., St. Louis, Mo,
Dibromo, dihydro ethacrynic acid v/as synthesized USA.
as
previously described (ploemen et aJ., L993). Hplc_grade
trifruoroacetic acid was obtained from Baker (Deventer, The
Netherlands), Hplc-grade methanor \^ras from Rathburn
Chemicals Limited
(Walkerburn, Scotland). Hplc_grade
acetonitrile
was from !ùestburg (Leusden, The Netherlands).
Epoxy-activated sepharose 6B v/as purchased from pharmacia
(uppsala, srâ/eden). DNpsG was synthesized according
tindwall and Boyer ( l_987 ) . The monoethyl ester to
of
glutathione
[(y-L-9tuÈamyt-L-cysteinyflethyf glycÍnate)]
was synthesized according to Anderson and Meister (19g9).
The glutathione
adduct of the monoethyl esÈer of
glutathione and TCBQ [cS(ethyl)TCBQ] was prepared
in
- 73 -
analogy to the synthesis of the 2-S-glutathionyl-3,4'5trichloro-l,4-benzoquinone (GSTCBQ) (Van Orunen et a7.,
O.26 mmol of the monoethyl ester of
1988). Briefly,
glutathione in 20 ml methanol \^tas added dropwise to 24 mmol
of TCBQ in 5OO ml of methanol during one minute, and
stirred for one minute. After evaporation and repeated
extractions with ethyl acetate to remove unreated TCBQ, the
resulting conjugate was purified by preparative RP-HPLC
(zorbax ODS, 250*2I.2 run)' eluted isocratically (flow 4
ml/min) with 60% methanol and 40% of a 0.1% (v/v) agueous
formic acid sotution (k'= 3.0). rhe identity and purity of
the product $/as established by the Uv-spectrum' r'¡hich was
identical to GSTCBQ (l.Iv-maxima aE 249 and 288 nm cf . Van
OÍunen et a7. 1990), FAB mass sPectrometry (n/z = 544.9)t
and analytical RP-HPLC (purity >95%) using the system
described in Van Ommen et a7' (l-9BB).
Ðîug exposure and cytotoxicity assays- Rat Reuber H-35
hepatoma cells were cultúred in Ham's FL2 medium (Flow
Laboratories, Irvine, Scotland), supplemented with 1-0%
fetal calf serum | 50 ÍÍJ/L penicillin, 50 mg/L streptomycin,
50 mg/L gentamicin, ât 37"c in a humid atmospherÊ
containing 5e" CO^. For each experiment, approximately 8*10"
cells v¡ere plaÉed onto a petri dish and cultured until a
semiconfluent monolayer \^tas obtained (in about 2 days).
Cells \irere exposed to agents in triplicate for one hour at
37oC in the culture medium (final volume 10 ml). AII test
prepared in DMSO (final
compounds were freshly
concentration in the rnedium always 0-5%) Ethacrynic acid 150 UM) and TCBQ (25 ¡-rM) were incubated
for one hour in the culture medium or PBS to study the
Sarnples (0.1 ml) were injected on HPLC to
stability.
quantify the amount of parent compounds, usinq the HPLCseparation as described (Van Orunen et af. | 1988; Ploemen et
a7., I99O). Ethacrynic acid was far more stable in culture
medium than TCBQ, since more than 90% and less than 5e' of
the unchanged parent compound was detected aftêr one hour
of incubation, of respectively ethacrynic acid and TCBQ
(data not shown).
In order to exclude cytotoxic effects during the assay
for glutathione conjugation, only concentrations of the
test compounds giving at least 90% viable cells were used
and
in the experiments. Drug effects on cell proliferation
MTT
microculture
the
by
determined
were
cytotoxic effects
assay, and expressed as ? viable cells lMickisch et a7.,
L99O). Àt least l0 concentrations in octuplicate were used
- 74 -
to determine the 90% survival value (Mickisch et
âf.,
1990) ' The 90% cerr survivar- concentrations
determined were
25, 12.5, >80, 80, and 1L.1 pM; for TCBQ/ GS(ethyI)TCBe,
ethacrynic acid, dibromo dihydro ethacrynic acid,
and CDNB/
respectively) .
The intracellular glutathione conjugation was estimated
by the DNPSG excretion in the medium. CeIIs lyere
cul-tured
and exposed to the test compounds (see above). Next/
the
cel-ls were washed rvith pBS (to remove the test compounds
from the mediurn), whereafter l_0 yM of CDNB in pBS (10
mI)
was added. One ml aliquots \^¡ere taken from the
medium
at
7Ot 20, and 30 min, N-acetyl_L_cysteine (in 25
of
water)
¡rI
was added inmediatery to these sampres (final concentration
1 mM) to remove unreacted CDNB.100
US of Z,S_
dimethoxycinnamic acid (in r0o pr of ethanor) was
aaded as
internal standard. The samples hrere stored on i_ce.
DNPSG
h¡as separated from the N_acetyl_L_cysteine
conjugate
of
CDNB by injecting
0.1 mr on a zorbax oDs reversed phase
column (4.6 t 250 mm), eluted at a flow rate of 1
m1/min,
with 0.1% v/v trifluoro acetÍc aeid (solvent
À)
and
v,/v trifluoro acetic acid in methanol (solvent B) with0.1%
a
Iinear gradient of 30_90% B in 10 min, followed
by
a
gradient to 1OO% in 7 min (k,= 2.g, and
3.g,
for
respectively DNPSG, and the N_acetyl_L_cysteine
of CDNB). euantification of DNPSG r¡/as performed conjugate
by peak
area integration at 340 nm, using concentration/absorbance
curves of the DNPSG standard. The nev¡ peak visible in
the
HPlc-chromatogram (peak II, Fig.l), is likely
conjugate
of N-acetyr-L-cysteine and CDNB, since it has the
a simirar uv_
spectrum as DNpsG (À____= 340 nm) and its retention
identically to the pr8åü.t formeá upon incubations ofti_ne is
only
qlutathione and N-acetyr-L-cysteine. DNpsG in
the
sampres
(stored at 0-6.C) v\¡as stable for at l_east 2 weeks.
The
intracellular
DNpSc and the glutathione
concentration \^rere determined in control
and
following exposure to the test compounds andcells
subsequent
CDNB exposure, as described (Lindwall
and
Briefly, after trypsinization the cells ,.." Boyer, L9e7).
(5
min 800 rpn) in ice-cold pBs, the perret v¡as
"e.rtrifuged
carefurry
rinsed with ice-coId pBS and resuspended in pBS. Aliquots
were used to determine the protein concentration (Lov7ry
et
a7., 1951). The cells r¡¡ere disrupted with 5å phoshoric
acid, and denatured protein vras removed by filtration.
The
HPLC analysis was used to quantify DNpSc,
while glutathione
was determj_ned as described (Reed et al. 19gO).
/
-75*
O
peak
I
ilms
(mln)
l5
Figure L. TypicaT HPLc-chtomatogram of medium of rat H-35
hepatoma celts exposed to CDNB- The large peak present (at
1l-.8 nin¡ ís 2,S-dimethoxycinnamic acid which was added as
internal standard. Peak I= DNPSG,' Peak II= N-acetyl-tconjugate of CDNB. Y-axis: fuII scale O.02 A at 340 nm'
GSr^subunit composition of rat H-35 hepatoma' !9 dishes
175 cmz) vrere cultured with rat H-35 hepatoma cells until a
semiconfluent monolayer v¡as obtained. Àfter trypsinization
the cells \¡/ere centrifuged (5 min 800 rPm) in PBS
and disrupted by
(containing 1 mM dithiotreitol),
centrifuged (30 min
were
sonification. The cell hourogenates
r¡tere used to
supernatants
the
and
L5000 g t at 6"C )
S-hexylglutathione
by
composition
subunit
GST
the
determine
Sepharose 68 affinity chromatography followed by wide pore
RP-HPLC separation as described (Bogaards et al' L989)'
GST assay in supernat¿nt. After exposure (3 hr) to Èhe
test compounds (see above), the cells were washed (wíth
about 1O-L5 mI PBS) and harvested by scraping \^tith a rubber
policeman in l" mI PBS, \^rhereafter the cells were disrupted
by sonification and the suPernatant $tas prepared (see
above). The GST activity \¡¡as measured in the supernatant
with CDNB according to Habig et aJ-. (L974)'
between
relationship
\hethods- The
Statistical
concentration and effect h¡as determined by linearregression analysis. Levels of significance v¡êre tested by
one-$ray ANOVÀ (P < o.o5), while comparison between groups
hrere made using Dunnett control group comparison test (P <
0.01)
.
-76-
Results
GST subunit conposition of rat H-35 hepatoma. The cST
subunit composition was determined with bioaffinity
chromatography, followed by Rp-HpLC separation of the GST
subunits. Rat H-35 hepatoma expressed almost exclusively
the mu-class GST subunits 3 and 4, in a ratio
of
approximately l:4. À small unknown peak (about 6å of the
total GST subunits, assuming a similar molar extinction
coefficient) $ras observed, eluting between the peaks of cST
subunit 1 and 2, which might be subunit 6 (Coles and
Ketterer, 1990).
llIeasurenent of glutathione conjugation vith CDNB. The
moder substrate CDNB $ras used to determine the glutathione
conjugation in rat H-35 hepatoma cells, by following the
excretion of its glutathione conjugate in the mediurn by
HPLC analysis. The excretion of DNpsG was in general linear
over a time span of lO-2O nin (see control, Fig.2-4).
However, since a deviation of the linearity
after 15 min
$/as observed in some cases, only the values at 10 min were
used to determine the relationship betr^reen concentration
125
4
o
o
o
o
o
o
1oo
---å-
conlrol
õ75
25
c
o
37.5 yM
E
o,
Es0
o
50 pM
o
f,
.L
¡rM
2s
o
0
10
15
tim6 (min)
Figure 2. The ÐMSG excretion of rat H-35 hepatoma cells
expo.sed to ethacrynic acid. Cells were exposed to drugs for
one hour, washed with pBS and subsequent exposed to 10 gM
CDNB. The glutathione conjugation was determined by the
excretion of DNPSG into the medium. Values are the average
(N=3) t sEM.
-77-
125
2
o
o
o
o
o
o
100
o
75
---*-
control
25 pM
E
rl
c
37.5
o
o
x
o
50 uM
o
(,
¡rM
25
(n
fL
o
10
15
20
25
t¡me (min)
Figure 3. The DNPSG excretion of rat H-35 hepatoma cells
exposed to dibrono dihydro ethacrynic acid. Cells were
exposed to drugs f or one hour, \^tashed with PBS and
subsequent exposed to 10 UM CDNB. The glutathione
conjugation was determined by the excretion of DNPSG into
the medium. Values are the average (N=3) t SEM.
---å-
G
ã60
o
o
o
control
5¡tMG
3so
IOpMG
E¿o
e
5 pM TCBQ
c
€30
o
o
ázo
10 ¡:M TCBQ
U'
25
o
À
ztv
o
r.¡M
TCBA
o
10
15
1¡m6 (min)
Figure 4. The ÐNPSG exctetion of rat E-35 hepatoma ce77s
exposed to TCBQ and GS(ethyL)fcBQ. Cel1s $/ere exposed to
drugs for one hour, $/ashed v'¡ith PBS and subsequent exposed
to L0 UM CDNB. The glutathione conjuqation \"/as determined
by the excretion of DNPSG into the medium. Values are the
average (N=3) t SEM. À.bbreviation: G= GS(ethyI)TCBQ
-78-
and effect (see below). At this time-point, the excreti_on
of DNPSG in the control incubations varied between the
various assays from 19 to 54 pmol/lO*cells 1Fig.4 and
F.í9.2, respecÈivety). Because of this
variation
the
expression as % of control incubations is indicated. fhe
maximal consumption of CDNB (calculated from the excretion
after 30 mip (Fig.2) and the number of cells per dj_sh
(about l-.7*L0' cells) was about 19 nmol, which is 19% of
the total amount of CDNB present per dish.
Inhibition studies. The effect of several GST inhibitors
on the DNPSG excretion in H-35 hepatoma cells was sÈudied.
The relationship between concentration of inhibitor
(UM)
and excretion of DNPSG $¡as determined by linear regression
analysis at 10 min lsee above). For boÈh ethacrynic acid
and its dibrono dihydro derivative a significant linear
relationship was found Iestinated parameters: regression
coefficient of ethacrynic acid
constant
term
of
-O.264 Ã(P=O.O22l with
43.9
for dibromo
lpmol/1-O'cells) , and regression coefficient
dihydro ethacryni-coacid -0.578 (p=0.013) with constant term
of 57.7 lpmol/lo'cells)1. Àt the highest concentration, a
decrease of approximately 30% and 50% was observed (as
esÈimated by regression analysis¡, for ethacrynic acid and
dibromo
dihydro
ethacrynic
acid,
respectively.
GS(ethyl)TCBQ was used instead of the cST inhibitor cSrCBe/
since the monoethylester of glutathione has improved
absorption characteristics (Ànderson and Meister, 1989).
For TCBQ and cS(ethyl)TCBQ no significant relationship
between concentration of inhibitor and DNPSG excretion was
observed (Fig.4).
For comparison, the GST activity v¡as deÈerrnined in the
supernatant of simular numbers of rat hepatoma cells (Table
l-). An increased exposure time (3 versus t hr) and an
increased concentration of eÈhacrynic acid (80 versus 40
LrM) was used as compared hrith the DNpSG method. Even under
these conditions, no significant decrease in GST activity
was observed for either ethacrynic acid or TCBQ and their
derivatives. Thus irreversible inhibition can be excluded.
-79-
Table 1. Effect of exposure Èo test compounds on the GST
activity tohrards CDNB in the supernatant of rat H-35
hepatona cells.
GST
activity
nmol/min.ng
Compound
(
blank
25 pM TCBQ
L2.5 ¡rM cS(ethyl)rcBQ
105
blank
80 UM ethacrynic acid
80 UM dibromo dihydro
ethacrynic acid
1,L2
)
t 4
130r9
134 t 16
t
r
to2 t
L10
4
12
26
q
8*10- çat H-35 hepatoma cells were plated onto a petri dish
( 56 cm- ) and cuLtured until
a semiconfluent monolayer \4¡as
obtained 1in about 2 days), followed by incubation with the
test compounds for 3 h at 37"C in culture
medium,
vrhereafter supernatant h¡as prepared. Values are the average
of 3 measurements (1 SEM).
To study the observed inhibition of the DNPSG excretion
in more detail we studied the effecÈ of the addiÈion of the
tr.¡o parent inhibiÈors (Ethacrynic acid or TCBQ) on either
the glutathione
concentration or the intracellular
concentration of DNPSG. Those two parameters were
determined at the end of the assay (Table 2\.
No
(significant) effects l¡¡ere observed. Ho\^rever, the variation
in the DNPSG levels was high, presumably as a result, of the
Ior¿ intracellular
concentration of DNPSG (close to the
detection liutit of the UV-detection).
-80-
Table 2. Intracellular DNPSG and glutathione concentration
in rat H-35-hepatoma cells exposed to test compounds and
subsequent exposure to CDNB.
DNPSG
compound
blank
25 UM TCBQ
5O ¡.rM ethacrynic
acid
(pnol/mg protein)
6.9 ! L.52
8. 1 t 5.00
5 .0 ! 1.59
glutathione
(runot,/mg Prot,ein)
.9 t 0.57
4.3 t 0.40
4.8 + 0.68
3
Rat H-35 hepatoma hras incubated with test compounds in
culture medium. After t hr at 37oC the cells were washed
with PBS and exposed to 10 UM CDNB (in PBS) for 30 min at
37"c. CeII extracts were made and DNPSG and glutathione
were determined. Values are the average t SEM with N=3.
Díscussion
Despite the large number of
inhibitors
of pure
GST
isoenzymes known, only few attempts have been made to study
in biological systems. the aim of our
GST inhibition
present study was to develop a simple assay to determine
the glutathione conjugation in rat hepatoma cells, and to
study the ef fects of t!'to promising classes of GST
inhibitors (ethacrynic acid and TCBQ) with this assay.
In the assay presented in this paper (HPLC separation of
DNPSG from serum-free medium, after preincubation with GST
inhibitors),
the concentration of the substrates may vary.
The preincubation with the GST inhibitor \"ras performed in
cornplete culture medium. In this way the glutathione
content can be augmented by the presence of the sulfurcontaining precursors cysteine and methionine (according to
the manufacturer respectively 0.2 mM and 30 ¡.tM) to maximize
the glutathione biosynthesis (Reed, 1990). The co-substrate
CDNB is a hydrophobic cotnpound that very rapidly enters the
cetl probably via sirnple diffusion (Oude Elferink et a7.,
(major)
1989). Using hepatocytes, the absence of
glutathione depletion using a higher CDNB concentration (40
UM) had been reported (Lindwall and Boyer, 1987). The
-81 -
linearity
of the DNpSc excretion suggests that no
glutathione depletion occurred. On the other hand, using
the analysis at time-point l_0 min at which the excretion of
DNPSG is linear, effects of product inhibition of the cST
(by the glutathione conjugate formed) may be mininized.
In many different organs and cell types, DNpSc is
efficiently
excreted by an ATp-dependent glutathione Sconjugate export pump (Ishikawa, 1992). Several glutathione
conjugates have been shown to competitively inhibit this
pump, whiLe the efflux r¡/as a saturable process (Lindwall
and Boyer, J-987; Kobayashi et a7., L99O; Àkerboom et aJ.,
1991; Oude Elferink et aJ., 1989). The reported K.,s in
general range from 50 UM to to 1 mM (Lindwall andrBoyer,
1987; Àkerboom et a7., L99L). From the fact that the vast
majority of the CDNB conjugates was excreted from the
hepatoma ce11s, it might be concluded that these also
possess the conjugate pump. A small increase in the
intracellular concentration of DNPSG was observed after
treatment with ethacrynic acid, however this difference
does not reach statistical
significance. Even with SO UM
ethacrynic acid., the intracellular accumulation of DNPSG
(viz. 8.1 pmol,/mg protein¡ is sti1l much smaller than the
accumulated excretion 1viz. 11300 pmol, calculgted after 30
min exposure with number of cells is 1.7*10Þ). However,
since considerable underestimation of the extent of DNPSG
formation might occur when strong inhibition of the export
pump occurs, this phenomenon may not to be overlooked.
Ethacrynic acid has been shown to inhibit pure cST
isoenzymes very efficiently in a reversible manner (Ahokas
et a7., 1985,. Ploemen et al-., L99O). In the present study,
a concentration dependent inhibition of the glutathione
conjugation by ethacrynic acid was observed. This coincides
with no (major) depletion of glutathione (with up to 50 ¡rM
ethacryni-c acid¡, as reported for other cell lines lTew et
a1., 1988; Rhodes and T\,¡entyman, L992,. Singh et aL., I9g2).
GST of the pi-c1ass was shovrn to be sensitive
to
irreversible inhibition by ethacrynic acid lploemen et al. I
1993). In rat H-35 hepatoma cells no such irreversible
inhibition was observed, as expected since this cell line
almost exclusively expresses the GST subunit 3 and 4 of the
mu-class. Using pure enzymes, I.^-values of about 1 UM Í/ere
observed for the mu-class CSC" lploemen et af., 1990,.
Hansson et a7. | l-99I), while in the rat hepatoma cell line
about 30% inhibition is observed by incubation with 50 UM
ethacrynic acid. This comparison again indicates the
-82-
diffieulty
in extrapolating data obtained from enzl¡me
assays to ceII systems.
Dibromo dihydro eÈhacrynic acid has an enhanced
irreversj-ble inhibitory potential of pure cST enzfrmes,
while the reversible inhibitory capacity was comparable to
ethacrynic acid iÈself (Ploenen et a7., L993). Again. no
ireversible
inhibiÈion was observed. Àn increased cellul-ar
absorption and/or the occurrence of glutathione depletion
might cause the decreased glutathione conjugation as
compared with et,hacrynic acid itself.
The halogenated quinones irreversibly
inhibited almost
every GST isoenzlnne studied ( Van Ommen et aJ. . , 1988,.
Ploemen et a7., 1991-). The GSÎ, of especially the alphaclass, $rere inactivated even faster by the glutathione
conjugate of TCBQ (Van Ommen et af., I99L; Ploemen et a7.l
l-991-). It is assumed that the glutathione moiety targets
the compound to the the active site of cST. Many cell types
wiII however not readily take up glutathione, thus the
absorption properties need to be improved. To this end a
new derivative was synthesized, GS(ethyl)TCBQ/ in which the
glycine carboxyl group of the glutathione moiety hras
This glutathione monoethyl ester is readily
esterified.
transported into cells (Anderson and Meister, 1989), while
the glycyl carboxylate group modification does not affect
the affinity for GST (Adang et âf., f990). Nevertheless/
neither TCBQ nor GS(ethy1)TCBQ displayed inhibition of the
conjugation in rat hepatoma cells. Using complete medium,
TCBS readily reacted with medium compounds, presumably e.g.
r,¡ith the above mentioned cysteine, moreover at 37oC TCB9
will undergo also significant hydrolysis (Kutyrev, 1991).
For j¡ vitro application, halogenated quinones as such arê
not suitable. A prodrug conceptf in which non-toxic and
chemically unreactive precursors are synthesized, seems the
indicated route of investigation.
In conclusion, an assay is presented which was used to
estimate the intracellular glutathione conjugating activity
in rat H-35 hepatoma cells. Using the assay it was shown
that ethacrynic acid and the dibromo dihydro derivative are
good intracellular
inhibitors, and the halogenated quinones
are not.
-83-
References
Adang ÀEP, Brussee J I van der Gen À and Mulder G.I. The
glutathione-binding
site
glutathione
in
S_
transferases. BiocJ:en J 269, 4'7-54t L990.
Ahokas JTr Nicholls FÀ, Ravenscroft p.J. and Ernmerson B.T.
Inhibition
of purified
rat
1iver glutathione Stransferase isoenzymes by diuretic
drugs. Biochem
Phamacol 34t 2L57-2i_61, 1995.
Akerboom TPM, Narayanaswami V, Kunst M and Sies H. ATpdependent S-(2,4-dinitrophenyl)glutathione
transport in
canalicular plasma menbrane vesicles from rat liver.
J
BioJ- Chen 266¿ 1,3147-1,3L52, 1991.
Anderson ME and Meister A. Glutathione monoesters. Anal
Bjoc.hem183 z t6-2O, 1989.
Armstrong RN. GlUÈathione S-transferases:
reaction
mechanism, structure, and function. Cåem Res Toxicol_ 4¿
L31-139 | l_991.
Berhane K and Mannervik B. Inactivation of the genotoxic
aldehyde acrolein by human glutathione transferases of
classes alpha, ou, and pi-. Mol pharmacoJ 37; 251--254,
1990.
Black SM and wolf CR. The role of glutathione-dependent
enzlrnes in drug resistance. pharmacol Therap 5J.: 139-154,
1991.
Bogaards JJP, Van Onmen B and Van Bladeren pJ. An improved
meÈhod for
the separation and quantification of
glutathione S-transferase subuniÈs in rat tissue using
high-performance liquid chromatography. J Chromatogr 474t
435-440, L989.
Coles B and Ketterer The role of glutathione and
glutathione transferases in chemical carcinogenesis. Crjt
Rev Biochem I4o7 Bio725z 47-70, 1ggo.
Desideri À, Caccuri AM, polizio F, Bastoni R and Federici
G. Electron paramagnetic resonance identification of a
high1y reactive thiol group in the proximit.y of the
catalytic site of human placenta glutathione transferase.
J Biol- Chen 2662 2063-2066t L99L.
Habig WH, Pabst MJ and Jakoby WB. Glutathione Stransferases, The first
step in mercapturic acid
formation. J BioT Chen 249: 713O-7139t 1,974.
Hansson J I Berhane K, Castro W, Jungnelius lJ t
and
Mannervik B. and Ringborg U. Sensitization of human
melanoma cells to the cytotoxic effect of melphalan by
the glutathione transferase inhibitor ethacrynic acid.
Cancer Res 51: 94-98, L99L.
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Kobayashi K, Sogame Y, Hara H and Hayashi K- Mechanism of
glutathione S-conjugates transPort in canalicular and
basolateral rat liver plasma membranes - J Biol Chem 265¡
7737-7741 , l-985.
Kutyrev AÀ. Nucleophitic reactions of quinones- Tetrahedron
472 8043-8065, 1991.
Ishikawa 1. The ÀTP-dependent glutathione S-conjugate
export pump. Trends Biochem Sciences L7z 463-468t 1992Lindwall G and Boyer TD. Excretion of glutathione
conjugates by primary cultured rat hePatocytes- J BioT
Chen 262: 51-51-5158' J-98'1 .
Lowry OH, Rosebrough NJ, Farr ÀT, and Randall RJ. Protein
measurement with the Folin phenol reagent- J BioI Chen
L93z 265-275 | L95!.
Mannervik B. The isoenzlnaes of glutathione transferase - Adv
EnzW Rel Areas Mol BioJ- 57l. 357-417t ]-985t',lannervik B and Danielson UH. Glutathione transferases Structure and catalytic activiEy. Cîit Rev Biochem MoJ
Biol 232 283-337 / 1988.
Mickisch G, Fajta S, Keilhauer G/ Schlick E. Tschada R and
Alken P. Chemosensitivity testing of primary human cell
carcinoma by tetrazolium based microculture assay (MTT).
llrol Res 18: 131--136 | I99O.
Mulder GJ and Te Koppele JM. Glutathione conjugation in
in perfused organs and in isolated cells:
vivo,
pharmacokinetic aspects. In.' GLutathione conjugation.
Edited by H. Sies & B. Ketterer- pp. 357-389.
Àcademic Press Limited, London, L988oude Elferink PJ, ottenhoff R' Liefting W, de Haan J and
Jansen PLM. Hepatobiliary transport <¡f glutaÈhione and
with hereditary
in rats
conjugate
glutathione
hyperbilirubinemia. J Clin Invest 84¿ 4'16-4A3, 1989.
Ploemen JHTM, Van Ommen B and Van Bladeren PJ- Inhibition
of rat and human glutathione S-transferase isoenzyrnes by
eÈhacrynic acid and its glutathione conjugate- Biochem
Pharmacol 4O: 1631-1635' 1990.
Ploemen JHTM, Van Onmen B and Van Bladeren PJ. Irreversible
inhibition of human glutathione S-transferase isoenzymes
by tetrachloro-1,,A-benzoquinone. Biochem PharmacoL 4L?
1665-1669, L99r.
Ploemen JHTM, Bogaards, JJP, Veldink GÀ, Van om¡nen B,
Jansen DHM and van Braderen PJ' rsoenzfrme selective
of rat and human glutathione Sinhibition
irreversible
transferases by ethacrynic acid and t$¡o brominated
derivatives. Bioehem Pharmacoi 45: 633-639' 1'993-
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DJ, Babson JR, Beatty p!ìf, Brodie AE, Ellis úM and
Potter DVù. High-performance liguid chromatography
analysís of nanomole levels of glutathione, glutathione
disulfide,
and related thiors and disurfides . AnaI
Biochem 1O6: 55-62, L99O.
Reed ÐJ. Grutathione: Toxicorogicar irnplications. .å¡n Rey
PharmdcoJ- Toxicol 2O: 603-631", 1990.
Rhodes T and Twentyman pR. À study of ethacrynic acid as a
potentiar nodifier of nrerphalan and cisplatin sensitivity
in human lung cancer parental and drug_rèsistant ceII
Iines. Br J Cancer 65¿ 684-690, l-gg2.
Singh SV, Xu BH, Maurya AK and Mian AM. Modulation of
mitomycin C resistance by glutathione transferase
inhibitor
ethacrynic acid. Biochim Biophys Acta Lr37
257-263,]-992.
Tamai Kf Satoh H, Tsuchida S, Hatayama I, Maki T and Sato
H. specific inactivation of grutathione s-transferases in
class pi by SH-rnodif iers . Biochem Biophys Res Conun 167..
331-338,1990.
Tew KD, Bomber ÀM and Hoffman SJ. Ethacrynic acid and
piriprost as enhancers of cytotoxicity in drug resistant
and sensitive celr rines - cancer Res 4gz 3622-3625, r-9gg.
Van Bl_aderen pJ and Van Onmen B. The inhibition
of
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S-transferases:
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toxic
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pharmacoJ.
Therapeut 51: 35-46 | 1-991.
Van Ommen B, Den Besten C, Rutten ALM, ploemen JHTM| Vos
RME' Müller F and van Bladeren pJ. Àctive site-directed
irreversibre inhibition of glutathione s-transferases by
the
glutathione
conjugate
of
tetrachloro_I,4_
benzoquinone. J BioL Chem 263: L2939_I2g42, 19gg.
Van Ommen B, ploemen JHTM, Bogaards JJp/ Monks TJ, Lau SS
and van Bl-aderen pJ. rrreversibre
inhibition of rat
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Reed
z
-86-
Chapter
6
Sumary part I
The first part of this thesis describes studies on the
inhibitory effecÈ of quinones and some catechors on human
and rat GST. Àn in vitro system was developed to studly the
inhibition of the grutathione conjugation in intact cerrs.
rn chapter 2 | .']'e irreversible inhibition of human Gsr
of the alpha-¿ ru-, and pi-class by TCBQ and GSTCBQ is
described. À considerabre irreversible inhibition
(>7oz
with tenfold excess of inhibitor over enzyme) of GST \4ras
observed. Using these conditions, incorporation of about
one nmol TCBQ per nmol subunit GST was observed. GSI
|
which is the only GST which does not possess cysteine, ^2_2
was
not inhibited and did not bind TCBQ. rt is tempting to
conclude that those human GST which were irreversibly
inhibited, contain a cysteine j-n or near the active site,
which is completely responsible for the inactivation by
TCBQ.
GSTCBQ inactivated human GST isoenzymes very fast.
However, onry in the case of csr A1-1 the inactivation
proceeded significantly faster than for TCBQ: in this case
the glutathione moiety seemed to target Èhe quinone to the
enzf.¡ne.
chapter 3 describes the reversible and irreversibre
inhibition of rat cST by the catechol caffeic acid.
Moderate reversible inhibition
courd be reached with
caffeic acid (I.n-values ranging from 58 to >640 UM). fhe
major glutathíðne conjugate of caffeic
2_S_
glutathionyrcaffeic acid 12-cscÀ) was a much more
".id, potent
reversible inhibitor of GST (with f_^_values rangins from 7
to >125 UM). on rhe other hand, sigf,Yficant timã_aåpendent.
irreversible
inhibition
of rat csr, especiarly of the picrass Gsr 7-7, was observed in incubations wiÈh caffeic
acid, whire no significant irreversibre inactivation with
2-GSCA was
found.
-87-
chapter 3 also describes the in vivo effect of single
body weight) on
oral doses of caffeic acid 150-500
GST acÈivity in rats L8
the irreversible inhibition of the'r.¡.S/kg
kidney and intestinal mucosa.
hr after dosíng in liver,
only in liver,
a marginal but significant irreversible
inhibition was observed (about 14% in Èhe highest dose
group), which coincides with a decrease of subunit GST 4.
FurÈher studies should concentrate on the reversible
inhibition by caffeic acid and its glutathione conjugate.
Chapter 4 deals with the reversible and irreversible
inhibition
of human cST by the catecholamines dopamine, cÀs
5-S-glutathionylconjugates.
methyldopa and their
compared with caffeic acid, only moderate reversible
inhibition by the glutathione conjugates vtas observed (r5Ovalues ranging from O.l-8-O.54 mM) , whj-le the paréñt
inhibit
GST
catecholamines did noÈ significantly
reversibly.
Using tyroslnase to generate quinones in situ
the pi-class GST P1-1 was by far the most susceptible
enz]me towards irreversible inhibition (67-993 inhibit.ion
under the conditions used) by both the Parent
catecholamÍnes, while only GST PL-1 is irreversibly
inhibited (33å inhibition) by the glutathione conjugate of
dopanine.
The nature of the time-dependent inactivation of GST
PL-1 by doparnine and its gtutathione conjugate was studied
i.n more detail. It was shown that a minor part of the
inactivation r^ras due to disulfide formation in the enzyme
in the case of dopamine, while the inactivation by its
glutathione
formation.
conjugate vtas fully
the result of disulfide
of a non-toxic
Chapter 5 describes the effect
concentration of TCBQ and its glutathione conjugate, of
which Èhe gtycine carboxyl group was esterified to improve
the absorption¡ oR the DNPSG excretion in rat hepatoma
cells. In these cells which contain mainly the mu-class GST
3 and 4t no significant reduction of this excretion \^zas
observed, concomiÈant htith the absence of an effect on the
to$/ards CDNB in the supernatant, and the
cST activity
concentration of gluÈathione in ce1ls. Thus lcBQ and its
derived conjugate do not seem to inhibit GST in cellular
systems.
PART II
-89-
Chapter
rnhibition
of
rat
7
and hu-man grutathione s-transferase
isoenzlmes by ethacrynic acid and its glut.athione conjugate
J.H.T.M. Ploemenf B. van Ommen and p.J. van Bladeren.
Biochemical_ pharmacology 40: 1631-j-635, 1990.
Abstract
Ethacrynic acid, a potent inhibitor of grutathione Stransferases (GST), has been shown to enhance the
cytotoxiciÈy of chlorambucir in drug resistant celr lines,
but a definite mechanism has not been estabrished. Both
coval-ent binding to GST and reversible inhibition of csr
have been reported. rn the present study no irreversibre
inhibition
hras observed: for
a1I rat GST tested,
inactivation hTas comprete within 15 sec at oo, and d.iarysis
of GST after incubation with ethacrynic ac!-d gave comprete
recovery of enzyme activity
for aII isoenzymes tested.
Moreoverr the inhibition f¡/as competitive to\,/ards l-chroro2,4-dinitrobenzene and non-competitive toh/ards glutathione
for rat isoenzyme 1-1- strong inhibition of both human and
rat GST of the a-, p- and r¡_classes rrras obtained with
ethacrynic acid, whire conjugation of ethacrynic acid with
grutathione díd not aborish its inhibiting properties. For
the a- | p- and ¡r-class I.r.ì values (UM) vrere 4.6_6. O, 0.3_
1.9 and 3.3-4.9t respectivéïy for ethacrynic acid, ánd 0.g_
2.8, <0.1-1.2 and 11.0, respectively for its glutathione
conjugate. Of a1l isoenzymes tested the hunan isoenzyme U
is most sensitive to the action of both ethacrynic acid and
its glutathione conjugate.
Introduction
Recently it has been postulated that glutathione S_
transferase (GST) isoenzymes play a role in the intrinsic
and acquired resistance to cytostatic drugs IL_2). Many
electrophilic
carcinogens and mutaqfens v¡ere found to be
-90-
substrates of the GST [3], and also some cytostatics [4].
GST can also bind a wide range of xenobiotics covalently'
thus showing a second mode of action by which reactive
j-ntermediates may be detoxified [5'6]. Moreover' GST plays
a role in the biosynthesis of eicosanoids, which are
to ceII
in
involved
several processes related
hyperplasia and
proliferation,
cell
differentiaÈion,
neoplasia l't l. It has been shown that GST activity i s
resistant
to
enhanced in human breasÈ cancer cells
Adriamycint [t], while levels of glutathione have been
found to be elevated in arsenic-resistant Chinese hamster
ovary cells t9l and cisptatin-resistant rat ovarian cell
lines [10]. Inhibition of GST would thus be potentially
beneficial in the treatment of tumors.
Ethacrynic acid, a diureÈic drug, enhances the
of chlorambucil in Vùalker 256 rat breast
cytotoxicity
carcinoma cells with aequired resistance to nitrogen
mustards as well as in two human colon carcinoma ceII lines
[].11. Interestingly. ethacrynic acid has been shown to be a
very potent inhibitor of rat isoenzlrynes of GST 16 t!2 'I3l I
while it can also decrease glutathione levels due to a
conjugation reaction, both spontaneously and GsT-catalyzed
li-41. The nature of the inhibition of GST by ethacrynic
acid is not v/ell documented: uncertainty exists about the
of the inhibition. It has been reported that
reversibility
in vivo some covalent binding to rat isoenzyme 3-4 occurs
\¡tas not
[ 15 ] , but its relevance to the enzymatic activity
investigated. Irreversible inhibition would correlate GST
activity Èo de nouo synthesis of GST. On the other hand,
reversible inhibition would relate the inhibition to the
pharmacokinetics of ethacrynic acid.
The present paper describes the naÈure and the
by ethacrynic
concentraÈion dependency of the inhibition
of GSÎ. The
isoenzymes
rat
and
human
both
for
acid,
inhibition characteristics of the glutathione conjugate of
ethacrynic acid were separately investigated, since
conjugation with glutathione will almost certainly occur in
the target cells.
Materials and Methos
ChemicaLs. Ethacrynic acid (Sigma Co.' St Louis, MO,
USA) vras dissolved in absolute ethanol; its glutathione
conjugate in 0.1 M sodium phosphate buffer pH 6.5 with 1 mM
EDTA (Merck, FRG), both stored (not longer than 3 h) at 0o.
-91 -
Syntåesis of the gTutathione conjugate of ethacrynic
acid. One hundred mg of ethacrynic acid in 5 mL of
ethanol/water (1:1) was added to 1OO mg of glutathione in 5
mf, of ethanol/water (l-:L) with 10 droplets of a saturated
solution of NaHCO3, and stirred at room temperature for 49
hr. The solvents -!üere evaporated and the residue was
dissolved in 2 mL of saturated KHCO?, and a solution ot;W
H?PO¿ was added, until
the solut.ioñ became cloudy. The
résulting precipitate h¡as filtrated and dried. The identity
v¡as confirmed by fast atom bombardment mass spectrometry
(n/z = 609.9), the conjugate \^ras more than 99% pure as
judged by HPtC-analysis (Rp18 Hypersil ODS column, elution
with a solution of L% acetic acid in water and a linear
gradient from 10 to 100% acetonitrile in 20 min, k, = 4.4).
Purification
of
GST. Glutathione S-transferase
isoenzymes were purified from liver
and kidney of rats
[phenobarbital-induced male Sprague-Dawley rats, 15 weeks
of ê9ê, treated for 7 days with 0.1å (w/v) sodium
phenobarbital in drinking waterl and human liver and
placenta using S-hexylglutathione affinity
chromatography.
Separation of the GST isoenzymes of human liver (Ài--2, Mla1a), human placenta (pl-1) and rat kidney (7-7\ was
achieved with chromatofocusi-ng on a pharmacia FPLC system
equipped with a mono p-column, as previously described
[16]. GST isoenzymes of rat liver ürere separated by ionexchange chromatography using CM-sepharose fast
flow
(Pharmacia) t141. PurÍty was confirmed by HpLC analysis
Specific activities
[ 17 ] , and isoelectric focusing t 16 I .
with CDNB as second substrate lsee below) hrere 39.0, 10.0,
35.1, 32.7 | 17.L and 14.0, respectively, for rat 1-1, 2-2,
3-3. 3-4, 4-4t 7-7 and l-9.3t 73.5 and 69.3, respectively,
for human A1,-2, Mla-la and p1-1.
Incubations.
In order to detect a time-dependent
covalent inhibition of GST by ethacrynic acid, 0.25 pM cST
\t¡as j-ncubated with 5 pM ethacrynic acid at Oo: At various
time intervals a 0.1-mL sample \¡¡as transferred into a
cuvette containing CDNB and glutathione (both 1 mM) and the
inhibition of enzymatic CDNB conjugation $¡as measured at
340 nm (see below).
Reversibility
of the inhibition
was analysed by
dialyzing experiments: 5 yM GST \¡ras incubat.ed with 500 ¡.rM
ethacrynic acid for L5 min at room Èemperature. Activity
hras determined, and t.he incubate (approx. O.Z mT,) r,ras
dialyzed overnight at 40 against 4OO mT, of 0.1 M sodium
/ACI/"
-92-
phosphate buffer pH 6.5 with 1 mM EDTÀ, after which the
specific activity was determined.
To determine the nature of the reversible inhibition 25
nM rat 1-1 \n¡as incubated with 0.3-L.2 nM CDNB and 50 nM rat
1-1 with 0.O25-l- ml,l glutathione with t.O, 2.5, 5.0 or L0 ¡.rM
ethacrynic acid in duplicate (for enzyme assay see below).
Fifty nM enzyme was incubated (triplicate) with 0.1, 0.5/
1.0, 5.0, l-O and 200 ¡rM ethacrynic acid or its glutathione
conjugate to study the concentration dependency of the
L/fracLional
inhibition. To determine maximal inhibition,
vras plotted versus 1/[inhibitor],
for both
inhibition
ethacrynic acid and its glutathione conjugate inhibition
was complete. I5O values were obtained from plots.
Enzpe assays. The activity of GST with CDNB \¡/as
determined according to Habig et af. [14], in 0.1- M sodiu¡n
phosphate buffer pH 6.5 with I mM EDTA; the conjugation was
initiated
by adding glutathione in order to minimize
effects from the glutathione conjugate of ethacrynic acid
on the initial rate of conjugation. To determine apparent,
1.0 UM enzyme v¡as
K,_ and v__-, with ethacrynic acid,
iflcubatednlã Aupficate with t2.5-2oo uM ethacrynic acid AND
1 mM glutathione in 0.1 M sodiurn phosphate buffer pH 6.5
with
L mM EDTA. The conjugate vi/as determined
at 27O nm (extinction coefficient
specÈroplgtom?trically
h¡ere made for chemical
5.0 nM -cm * ¡ , conections
Apparent Ktrn and vn8{periments
were obtained from
reactivity.
the control
Liner,reaver-Burk plots.
atf
samples \nrere treated in the same h¡ay. Enz]¡me concenÈrations
are expressed as subunit concentrations.
Results
Reversj.bLe or irreversibJ-e inhibition. Two assays were
performed in order to discriminate bet$reen reversible and
irreversible inhibition of cST by ethacrynic acid. Firstly,
the time course of inhibition !'ras determined at 25"
\¡/as complete within L5 sec (rat isoenzymes 4-4
inhibition
25 twlt and l--l- 50 nM both with a 200-fold molar excess).
Since the initial rate of the reaction might be very fast,
experiments h¡ere subsequently performed at 0o. However, for
all rat GST isoenzymes Èested, inactivation was still
time interval
complete r.¡ithin 15 sec, i.e. the first
measured (FiS. 1-). Secondly, incubations of GST with
ethacrynic acid were performed, after which unreacted
r^taS
removed
by
acid
dialysis.
ethacrynic
z
-93-
u
=
G
ct
=
=
g
r¡¡
È,
t
ttm.
{¡ac,
Fig. 1. Time course of inhibition of rat GST isoenzymes lt (tr), 2-2 (O), 3-3 (A), 34 (+) or 4-4 (x). Éve ¡rM
etlacrynic acid and 0.25 ¡rM enzyme were mixeã at f, ånd
a z5-pmol enzyme sample (50 pmol for l-l) was transferred
rnto. a cuvetre containing 1 mM CDNB, and lmM glu_
tathione was added and conjugation *"s me"sur"õ at
340nm. The results are the aveiage of two incubations.
120
Þ
10o
->
Ëso
so
Ew
E
:40
&
råt
1.1
7.7
Fig. 2- Effect of dialysis on the remaining acrivity of rat
G-S-T isoenzymes. Five ¡rM enzyme was -incubatåd with
5æ4Y-ethacrynic acid at roorn temperature, and a 5ù
pmol (for 1-1,2-2,3-3 and 3-4) or a l(i)-pmol (for 44 and
7-7) erlzyme sample was transferred inio a cuvette with
1 mM CDNB_and 1m,lvf glutathione was added. Approxi_
mately 0-7 mL was dialysed overnight ar 40 in a00 mL
0_.1 M sodium phosphate buffer pH 6.5 with 1 mM EDTA.
Controls were treated similarly. Results are expressed as
the- per cent of control (rSE) of 2-4 incubations, loss of
activity during this dialysing procedure was always less than
XVo.
-94-
Although during the incubation the Ísoenzlznes \¡¡ere
inhibited uP to 9OZ ' Èhe acÈivity of GST $'as fully
(Fig. 2): the remaining activity
recovered after dialysis
rançfes between 85 and 118?, with no significant differences
fron the controls (P = 0.05, Student's t-test).
Nâ¿ure of the reversibTe inhibition. To obtain
information about the nature of the reversible inhibition,
enzyme kinetics experiments were conducted with rat
isoenzlaoe 1-1 and ethacrynlc acid. with CDNB as the
increased, while V ,
variable substrate, the apParent K*
mthat
ethaårynic
did not change (Fig. 3), indicating
"3îä
competes with CDNB. ltith glutathione as the variable
v
substrate, the apparent K- remained unchanged while
decreased (Fig. 3¡, indTcating non-cornpetitive innibitÏSã
torl¡ards glutathione.
0.r0
0.09
0,04
0.08
Ê
E
o.o7
0,03
0-06
Ê
E
0.05
0.04
0.d¡
0.02
23
,4cot¡gl
46
(ru't)
14csH¡ (mM'l)
Fie. 3. Lineweaver-Burk plot showing competitive inhibition of ¡â1 GST isoenzyme 1-1 towards CDNB
bv-5 l.) or l0¡lM (A) ethacrynic acid (lefo or non-compeútive inhibition towards glutathione (righl)
Uí i.ò Í.1, 2.5 (O) ¡M ethasynic acid. Conrrol (E). The values are the average of three incubations.
Experiments were perfomed as describ€d in Materials and Methods.
Table
l.
ljo (pM). values fo¡ tbe inhibition of tbe major isoeüym€s glutathione S+ransferases, of both rat and human,
with ethacrynic acid and its glutathione conjugate
pclass
a-class
Rat
Rat
Ethacrynic acid
Glutathione @n.iugate
.
6.0
t.2
Human
B182
1-1
4.6
2.8
ó.0
0.8
Rat
Rat
3-3UU
Ral
1.9
1.2
0.8
0.3
1.0
<0.1
i.9
1.0
Human
Ral
tt
'7-1
4.8
11.0
Human
Í
3.3
11.0
The conceotration of e¡hasynic acid or its glutathione conjugate resultingin 50Vo inhibition of the enzymic activity
der€mined by incubating 50 nM of GST with a concentration range of the inhibitoß. For indiv¡dual values, the
coefñcient of variation was less thm 157¿.
(¡50) was
-95-
Isoenzyme selectivity of the inhibition
by ethacrynic
acid and its gTutathione conjugate. For both ethacrynic
acid and its glutathj-one conjugate the I.^ values rÀ,ere
determined tor¡ards all najor human and raË"GST isoenzlmes
(Table 1).
Strong inhibition
of both human and rat cST of the a-,
y- and n-classes was obtained with ethacrynic acid, as well
as with its gtutathione conjugate. Human and rat isoenzymes
of the F-¡ n-, and a-classes in general showed the same
trends of
inhibition.
There are some interesting
differences between the various classes: the glutathione
conjugate of ethacrynic acid inhibiÈs the a- and ¡.r-class
more strongly than eÈhacrynic acid itself
(vrith the
exception of rat 4-4), while it inhibits the n-class to a
lesser extent than ethacrynic acid itself.
Of all
isoenzlmes tested the human isoenzlme U is the most
strongly inhibited isoenzyme, in particular
by the
glutathJ-one conjugate of ethacrynic acid.
The apparent KD values of the rat GST isoenzlmes with
ethacrynic ac'Ïd e¡ere determined. Some correlation exists,
beth¡een K, and I5O (Table 2), with the exception of rat
Table 2. Àpparent K * (UM) and V
1pnol/min/mg¡ of the
major cst lsoànzí*"" ort€ñ" rat to!'¡ard.s ethacrynic
acid
Isoenzl¡mes
l_-1-
K
m
V
max
90
0. 13
2-2
3-3
200
1. 33
50
50
0.39
o.44
4-4
* For details of the assay, see Materials and Methods.
7-7
t20
2.50
-96-
Discussion
Recently there has been considerable interest in the use
non-toxic inhibitors of cST activity to
of relatively
enhance the effect of cytostatics in the treatment of
tumours. The present study indicates that ethacrynic acid
and its glutathione conjugate are promising in vivo
inhibitors of both rat and human isoenzymes of GST,
especially of human y. There has been some discussion about
In vivo,
of the inhibition
the reversibility
[15].
ethacrynic acid was found to bind covalently to isoenzyme
3-4 with a maximum of one molecule per three molecules of
heterodimeric rat isoenzl¡me 3-4, 90 min after injection in
rats [15]. Àfter dialysis of rat GST isoenzymes incubating
with ethacrynic acid the enzymic action is completely
restored, and furthermore, there is no time-dependent
both findings indicate a reversible mechanism
inhibition:
This is confirmed by the kinetic data
of inhibition.
obtained with rat GST isoenzyne 1-1 and ethacrynic acid:
with respect to CDNB and noncompeÈitive inhibition
with respect to glutathione was
competitive inhibition
observed, as may be expected for a compound which is an
substrate of GST. Our results indicate that
electrophilic
in
covalent binding, if it occurs, does not result
Nevertheless,
of the enzlzmic acÈivity.
inhibition
ethacrynic acid and its glutathione conjugate strongly
inhibit rat and human GST. There are some differences
between the various classes of isoenzltmes, while in general
there are only small differences between human and rat GST
within the same class. Tt is interesting that the n-class,
which has the highest activity to\"/ards ethacrynic acid as a
substrate/ also is inhibited to the smallest extent by the
glutathione
conjugate of ethacrynic acid. The high
enzymatic activity of the r¡-class towards ethacrynic acÍd,
could perhaps be explained by the relative affinity of the
glutathione conjugate for the enz]¡me, causing the product
to leave the active site rel-atively quickly. In general,
the p-class is Èhe most strongly inhibited class of
isoenzymes, both by ethacrynic acid and its glutathione
conjugate/ the human ¡r isoenzyme being even more strongly
inhibited than its rat analogs. In agreement with this
resu.Lt, it has been shown that ethacrynic acid inhibits the
leukotrien. co production in human neutrophils [L8]. The
human y isoerizyme is involved in the conjugation of
-97-
glutathione to leukotriene A¿l resulting in the infor¡nation
of leukotriene C, I191.
Non-toxic coñcentrations of ethacrynic acid have been
shown to potentiate the cytotoxic activity of chlorambucil
in Walker 256 rat breast carcinoma cells with acquired
resistance to nitrogen mustards, and in human colon
carcinoma cell lines []-l-1. Two hypotheses v¡ere formulated
to explain Èhe enhanced cytotoxicity.
Firstly,
inhibition
of cST may result in a longer lifetime of chlorambucil, and
secondly synergetic effects of ethacrynic acid and
chlorambucil on Èhe biosynthesis of prostaglandins rnay
enhanced the cytotoxicity
Í7,L1-,2O1. Indomethacin, an
inhibitor
of cST and an anÈi-inflammatory drug has been
shown to potentiate the cytotoxicity of chlorambucil in CHO
cells
resistant
nitrogen mustards, while
to
acetylsalicyclic acid, an anti-inflammatory agent, caused
no potentiation of chlorambucil cytotoxicity, suggesting
that the potentiation is not due to the effects on
prostaglandin synthesis I211. A modest inhibition of GST
activity in t.he nitrogen mustards resistant ceII lines has
been observed with ethacrynic acid t l-11. The actual
inhibition of cST by ethacrynic acid and its conjugate may
be larger than Èhe observed inhibition,
because the
glutathione concentration .Iî?
intracellular
decreased
[]-21. Within 4 hr about 60-70% of a dose of [--C]ethacrynic
acid (5 or 5O mS/kg¡ \^¡as excreted into the bile of rats,
while approximately 40% was in the form of the glutathione
conjugate [22], indicating a high turn-over.
Thus, in r¡ir¡o ethacrynic acid temporarily inhibits GST
directly; this effect is enhanced by cSH depletion; and
is prolonged by the
lastly
the inhibitory
effect
glutathione conjugate.
References
1. Buller AL, Clapper ML and Tev,/ KD, Glutathione Stransferases in nitrogen mustard-resistant and
sensitive ceII line. Mol Pharmacol 3L; 575-578t l-987.
2. Sato K, Glutathione transferases as markers of preneoplasia and neoplasia. Adv Cancer Res 52t 205-2551
1989.
3. Ketterer B, Coles B and Meyer DJ, The role
of
glutathione
in
detoxification.
Environ
HeaLth
Perspect 49 259-69, 1,983 .
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4.
Dulik DM. Fenselau C and Hilton J, Characterization of
melphalanglutathione adducts whose informatj-on is
catalyzed by glutathione transferases. Biochem PharmacoT
35: 3405-3409, 7986.
5. Mannervik B, The isoenzynes of glutathione transferase.
Adv EnzymoL 57¿ 357-4a7, 1985.
6. Mannervik B, Danielson and Danielson UH, Glutathione
transferases: structure and catalytic activity. CRC Crit
Rev Biochem 232 283-337, 7988.
7. Chambers DÀ and Cohen RI,, Eicosanoids and turnor
promotion. In:
Biochemistry of Arachidonic Acid
Metabolism(Ed. Lands ffiM), pp. 343-373. Martinus Nijhoff
Publishing, Boston, 1985.
8. Batist G, Tulpule A, Sinha BK, Kathi Ac, Myers CE and
Cowan KH, Overexpression of a novel anionj-c glutathione
transferase in nultidrug-resistant
human breast cancer
cells. J BioJ- Chem 26L: L5544-15549, 1986.
9. Lee TC, wei ML, Chang WJ, Ho IC, Lo JF, Jan KY and Huang
Hr Elevation of glutathione levels and glutathione Stransferase activity in arsenic-resistant chinese hamster
ovary cells. .r¿ Vitro C?TJ- DeveTop BioJ 252 442-448,
1_989.
l-0. Chen G,
intracellular
activities
experimental
Frei E and ZeIIer WJ, Determination of
glutathione and glutathione related enzl¡me
in
cisplaÈin-sensiÈive
and resisÈant
ovarian carcinoma cell lines. Cancer Lett
46¿ 2O7-211,1989.
11. Tew KD, Bomber AM and Hoffman SJ, Ethacrynic acid and
piriprost
as enhancers of cytotoxicity in drug resistant
and sensitive ceII lines. Cancer Res 48: 3622-3625, 1988.
12. Ahokas, Jl, Nicholls FA, Ravenscroft PJ and Emmerson
ltÎ, Inhibition of purified rat liver glutathione Stransferase isoenzymes by diuretic drugs. Biochem
Phamacol 342 2157-2161, 1985.
13. Vessey DA and Boyer TD, Characterization of the
activation of rat liver glutathione S-transferases by
nonsubstrate ligands. Tox Appl PharmacoL g3z 275-28Q,
1988.
1-4. Habig WH, Pabst WJ and Jakoby WB, Glutathione Stransferases. The first
step in mercapturic acial
formation. J BioT Chem 249: 7130-7139t L974.
15. Yamada T and KaplowiÈz N, Binding of ethacrynic acid to
hepaÈic alutathione S-transferases in vivo in the rat.
Biochem Pharmacol 292 t2O5-1208, l-980.
-99-
16. Vos RME, Snoek MC, Van Berkel VüJH, Muller F and Van
Bladeren PJ, Differential
induction of rat
hepatic
glutathione S-transferase isoenzymes by hexachlorobenzene
and benzyl isothiocyanate: comparison vrj-th induction by
phenobarbital and 3-methylcholanthrene. Biochem Pharmacol
37 z LO17-LO82, L988.
L7. Bogaards JJP, Van Onmen B and Van Blad.eren pJ, An
improved method for the separation and quantification
of
glutathione S-transferase subunits in rat tissue highperformance liquid chronatography. J Chromatog 4742 43544O, L989.
18. Leung KH, Selective inhibition of leukotriene C4
synthesis in human neutrophilis by ethacrynic acid.
Biochem Biophys Res Co¡nmun L37: 195-200, L986.
19. Tsuchida S, Izumumi T, Ischikawa T, Hatayama I and
Satoh K, Purification of a ne\^r acidic glutathione Stransferase, GST-YnlYnl-, with a high leukotriene Co
synÈhetase activity from rat brain. E'ur J Biochem 170í
759-L64, L987 .
20. Tisdale MJ, Inhibition of prostaglandin synthetase by
anti-tumour agents. Chen BioL lnteract 18: 91-L00 | ]-977.
21. HaIl A, Robson CN, Hickson ID, Harris ÀLf Proctor SJ
and Cattan AR, Possible role of inhibition of glutathione
S-transferase in the partial reversal of chlorambucil
resistance by indomethacin in a chinese hamster ovary
cell line. Cancer -Res 492 6265-6268, 1"989.
22. Klaassen CD and Fit,zgerald ÎJ, Metabolism and biliary
excretion of ethacrynic acid. J Pharmacol Exp Ther L91:
548-556, L974.
-100-
Chapter I
rsoenzl¡me
serective irreversible inhibitiou of rat
and
glutathione S-transferase isoenzlmes by ethacrynic
acid and two brominated derivatives
human
J.H.T.M. Ploemen, J.J.p. Bogaards, c.À. Veldink,
Ommen, D.H.M. Jansen and p.J. van Bladeren.
B.
van
Biochemical pharmacoTogy 45: 633-639 | Lgg3
Abstract
fn the present study it has been shown that ethacrynic
acid can inhibit ctutatflone S-transferase (cST) of the pi_
crass irreversibry.
¡'=clethacrynic acid, 0.g nmor/n¡nol
human P1-L and O.B nmol/nmoÌ rat GSI 7_7 could be
incorporated, resurting
in 6s-g3z inhibition
of the
activity
tov¡ards l-chloro-2,4-dinitrobenzene
(CDNB).
Ilgenzymes of the alpha- and mu_class also bound
I c]ethacrynic acid, however without J-oss of catarytj_c
activityrncorporation ranged from o.3 to 0.6 ancr 0.2
nmol/nmol enzyrne for the mu- and alpha_class GST
isoelafmes, respectively. For allisoenz]¡mes, incorporation
of [--c]ethacrynic acid courd be prevented by preinãubation
\dith tetrachloro-1r4-benzoquinone, suggesting, that a
cysteine residue is Èhe target site. protection of csr p1-1
against inhibition
by ethacrynic acid by the substrate
analog S-hexylglutathione, indicates an active
site_
direcÈed modification. The monobromo and dibromo dihydro
derivatives of ethaerynic acid hrere synthesized in an
effort to produce more reactive compounds. The monobromo
derivative did not exhibit enhanced irreversible inhibitory
capacity. However, the dibromo dihydro derivative inhibited
both human and raÈ Gsr isoenzymes of the pi-class very
efficiently, resurting in 90-96z inhibition of the activity
towards CDNB. Interestingly,
this conpound is also a
powerfur irreversible
inhibitor of the mu-crass csr
isoenzymes, resulting in 52-zot inhibition. The two bromine
-
101_
-
affecÈ the strong reversible
atoms only narginally
inhibitory capacity of ethacrynic acid, with rC"n (UM) of
0.4-0.6 and 4.6-10 for the mu- and pi-class GST'isoenzf¡mes,
respectively.
Introductio¡l
The gtutathione S-transferases (GST) are a multigene
of isoenzlmes which caÈalyze the reaction between
numerous etectrophilic compounds and glutathione (GSH) [].31. In addition, GST can also act as a peroxidase and as a
binding protein for a variety of organic compounds t1l.
Maûmalian cells contain both cytosolic and membrane-bound
isoenzymes. The cytosolic GsT have been divided into four
gene families, the alpha-r olt-r pi- and theta-class [4].
They exist as dimers, with heterodimers occurring within
family
the same class [1].
Considerable evidence îndicates that the GST are' in
addition to many other factors, involved in cellular drug
resistance t5-10]. Drug resistance to cytostatics used in
cancer treatment can emerge at the start of the theraPy
or as a response to therapy (acquired). GST are
lintrinsic)
implicated in parÈicular in the resistance to alkylating
agents such as chlorambucil, melphalan and nitrosoureas,
and to redox cycling drugs such as Adriarnycin. Further
exploration of the role of GSr in drug resistance, and
potentially,
in modulation of the chemotherapy could be
¡nodifications of the GsH/GsT system [7]on
selective
based
The diuretic drug ethacrynic acid has been shown to be an
excellent competitive inhibitor of the GST system: the GST
substrate ethacrynic acid depletes GSH and inhibits GST
strongly [ 11r 1.2 ] . Moreover, the GSH conjugate formed
inhibits GST as efficientty as ethacrynic acid itselt lLzl IC-^ values ranged from 0.1 to 11 UM for ethacrynic acid
it" csH conjugate, and increased in the order mu-,
"rrå'
alpha- and pi-class [].2 1. Tew et af. t13 l \¡/ere the first to
sensitization of cultured tumor cells to
show that
alkylating drugs can be achieved by ethacrynic acid, and
several similar studies have been reported since [14r15].
Ethacrynic acid can react in a Michael-type reaction
with cysteine, and already in l-978, ethacrynic acid was
to rat GST 3-4
shown to bind to GST [16], in particular
inactive enzlme
result
in
an
not
does
this
Hohrever,
t1?1.
[]-21. In general, cysteine modification does not always
inactivate the GST, the effects are known to vary for each
-tozisoenzyme [18-21]. Since ethacrynic acid and its cSH
conjugate are known to act directly as strong competitive
inhibitors of cST, the present study vras designed to
investigate
the irreversibLe inhibitory capacity for
individual GST isoenzymes. In addition, ts¡o derivatives of
ethacrynic acid were synthesized which would atso disptay
alkylating ability, namely the monobromo derivative, which
could undergo a Michael-type addition-erimination reaction
and the dibromo dihydro derivative which possesses an
electrophilic alpha-halogeno ketone rnoiety.
Materials and
Methods
Chemicals and synthesis. ¡14c¡ntn."rynic acid was from
Àmershrm (Àmersham, U.K. ) (15 mCi/rnrool¡. Di_bromo dihydro
ethacrynic acid rras prepared by treatment of ethacrynic
acid with bromine as follows: 500 mg of ethacryni_c acid was
dissolved in 20 mL of methylene chloride in a 100 mL flask,
thoroughly flushed with N, and protected from light.
Under
a N2-atmosphere 68 pL of Ér, in 20 mL of methylene chloride
was-added dropwise until thé brown color of Br. stopped
disappearing. The solvents \Àrere removed by erraporátion. The
disappearance of ethacrynic acid \^ras checked with TtC
(stationary phase: silica gel; mobile phase: methylene
chloride/nethanol,/acetic acid (SO/SO/L, by vol.);
and
detecÈion with a 1% KMrlO^/22 Na^CO. solution). The
monobromo ethacrynic acid was fireparedza"'follor" t 657 mg
of dibromo dihydro ethacrynic acid was added to 50 mI, of
dry dinethyl formamide (DMF). Àir hras removed under vacuum,
and the flask was brought into a N.-aÈmosphere. K^CO- (460
mg) hras added and Èhe solution was Étirred overnigÉt in the
dark. K?CO? and DMF v¡ere removed by acid extraction: 50 n¡
of methylené chloride and 2OO mL of deionized H^O $¡ere
added, while shaking the pH of the waterphase was ádjusted
with 1 N Hcr to 5-6- The waterphase v¡as further extracted
with 25 nI, methylene chloride. The combined nethylene
chloride phases were lr¡ashed twice with 4OO mI- of a HCI
solution (pH 4-5). The methylene chloride was removed by
evaporation until a visêous oil hras obtained. TLC (see
above) was used to follow the fornation of a double bondMonobromo ethacrynic acid was purified by preparative HpLc,
using a Zorbax ODS (250 mm x 2L.2 ¡nm i.d.) column, eluted
with a gradient of 50-95% in l_00 min with a formic acid
solution pH 3 (eluens A) and methanol (eluens B), at a flow
rate of 4 rnt/min, and detection at 270 nm, with injections
-
l_o3
-
åb,r
\.,.8 ./)r,.\"í\
'
lr I( )l
'"^"¿\"y'Y\o
d.ilI
o
_ilJ
uoo
l*
Y
\^^.4n**("
î*"ri"loAð"'
coq
I
I qco3/Dm¡
Y
**.,í\".
o.'!
Fig. L - The structure of ethacrynic acid and its dibromo
dihydro and monobromo derivative. The letters at the
protons correspond v¡ith the NMR data.
of 5O-100 mg product in ethanol. The eluent t¡as collected
on ice, whereafter methanol íras removed by evaporation and
the residue rtras rinsed with methylene chloride and dried
under N2. The compounds \¡¡ere pure (> 99%) as judged with rH
N¡,tR (seõ belovr) and HPLC, using Hypersil oDS (100 x 3 nm
eluted with a gradient of 2O-95e" in 20 min (for
i.d.)
eluens see above), at a flow rate of 0.4 mr,,/min (J<'
6.2,7.5
and 8.2 for, respectively, ethacrynic acid,
monobromo ethacrynic acid and dibromo dihydro ethacrynic
acid). The th/o bromo derivatives $/ere identified and
characterized by lH NMR (300 MHz) and mass spectrometry.
NMR (cDcI2) dibromo dihydro ethacrynic acid 1rig. 1): 6
3.83/4.32 Ìdd, J = ro.4 Hz, proton a), õ 2.Lo/2.37 (tu,
proton c), 6l-.10 (8, J = 7.L Hz, proton d), ó 7.67 1d, ,t =
8.6 Hz, proton e), ó 6.80 (drJ = A.7 Hz, proton f ) , and ó
4.82 (s, proton S). Both enantiomers of dibromo dihydro
ethacrynic acid are present, as could be derived from the
-104-
proton signals for proÈon a and c, showing doubre doubrets
and nultiplets. NMR (CDCI") monobromo ethacrynic acid (Fig.
1): 6 7.05 (s, proton a] and õ 2.65 (q,J = j.S Hz, proron
c), 6 1.L3 (t,J = 7.5 Hz, proton d), 6 j.5g (ô.,J = g.5 Hz,
proton e), 6 6.82 (drJ = 8.5 Hz, proton f) and ó 4.g1 (s,
proton S). As expected from the sterical
hindrance,
presumably only -E'-monobromo ethacrynic acid is present,
since the measured 6 for proton a is closest to the
calculated 6 (calculated ó = 6.94 and 6.95 Hz, for
respecÈiveLy, Z- and .E-monobromo ethacrynic acid L22J).
Vùith pass spectrometry (Finnigan MÀT g2OO, electron impact)
the M (n/z 38O) is visible in the spectrum. Moreover,
ions are presefr: m/z 32L tM ll.::^..T+j.t n/z,fragment
CH2COOHI,
247 tM - BrHC=CC"H.ì and a fragrment
corresponding Eo n/z lg9 [c"H"o.cL"1 i rne dibromo dihydro
eÈhacrynic acid spectrum $ral'iåeátiÉar to the spectrum from
ethacrynic acid with one Br'-peak extra (presumably by Br*_
abstraction with formation of a double bond, folrowed by
Br- elimination).
cST purification and assay. GST isoenzl¡mes !,rere purified
from liver, kidney (rat cSI 7-7) and placenta (human GST
P1-1) using affinity
chromatography (S-hexylglutathione_
Sepharose 68), as described previously [23j. the separation
of the different
isoenzymes vras achieved
by
chromatofocusing on porybuffered exchangers lpharmacia,
Uppsala, Sweden), with pBE 119 for GST J--!, 2_2, 3_3, 4_4,
A1-1, À2-2 and M1a-1a, and pBE 94 for cST 7-7 and pl__]-. pBE
L18 was equilibrated with 0.025 M triethylamine_HCl (pH
11),
and elution was performed with (J.:45 dilut,ed)
pharmalyte (pH 8-10.5)-Hcl (pH 8) (phannacia), white pBE 94
was quilibrated with 0.025 M ethanolamine-CH,COOH (pH 9.4),
and eluted with polybuffer 96-HCI (pH 7t (pharmacia).
Purity
hras confirmed by SDS gel electrophoresis,
isoerectric focussing and HpLc analysis as described
previously [23 t24). protein was determined according to
Lowry et aJ.. l25l with bovine serum albumin as standard.
Gsr activity was assayed using l--chl-oro-2r4-dinitrobenzene
(CDNB) as the substrate [26], specific
activity
(pmol/mi_n
ûrg ) v/ere 54, 22 ,
44, t6 , L7 , 3L, 22 , 150 and jO ,
respectively, for GST 1-L, 2-2, 3-3, 4-4, 7-jt AL_Lt A2_2,
M1a-1a and P1-1. ÀIt enzyme concentrations were expressed
as the concentration of the subunit (M_: 25,SOO, 2TtSOOl
26t3OO, 26t3OO, 24,BOO, 25,gOO, 25,gOA; 26t7OO anð.24,gOO,
respectively, for GSf subunits 1¡ 2, 3t 4, -l , AI , A2, Mla
and P1 l27l). AI1 steps were performed at O-Bo, with oxygen
-105-
protection by 0.1 mM dithiotreitol
in all steps, with the
exception of the chromaÈofocusing and the final dlalysis,
which were performed, respectively, with degassed buffer
and under nitrogen.
1a.
Labeling of GST by t"Clethacrynic acif;. GST (10 UM) \r'as
incubated for l- hr at 37o with 100 ¡,tM l-'C]ethacrynic acid
(total volume 25 UL) in 0.1 M potassium phosphate buffer pH
7 .4,
to determine the binding capacity of ethacrynic acid
under drastic conditions. The enzlrme \¡ras preciPitated with
0.4 mL of 2Oz (w/v) trichloroacetic acid and 0.4 ng bovine
serum al-bumin were added to increase the protein
concentration and to facilitate the work up procedure. the
enzfrme was centrifuged 5 min at 101000 g. We carefully
washed the pel1et three times with ice-cold acetone [with
acid solutionl, which
Lz of a 65? (w/v) trichloroacetic
removes ethacrynic almost completely from the blank
samples. The enz]¡me v¡as dissolved with 0.l- M potassiun
phosphate buffer pH 7.4 (three extractions with 0.3 nL) and
with 10 mL of
the sample h/as screened for radioactivity
liquid. The recovery of protein was about
scintillation
80?. These incubations were performed in duplicate.
Quinones can label cST very efficiently/ Presumably by
reaction with cysteine residues of GSÍ. The same
experiments hrere performed, after preincubation for 5 min
at 25o with 5O UM tetrachloro-1-r4-benzoquinone, to compare
ethacrynic acid labeling capacity after preincubation with
qçinones.
The time course of the incorporation of
L+
[-'C]ethacrynic acid in human GST P1-1- and GST 7-7 \4tas
investigated in more $çtail at 20' by incubating L.25 pM
enzyme with 6.25 pM [-'C]ethacrynic acid in 0.1 M potassium
phosphate pH 7.4 (total volume 0.16 mL). For washing
procedure see above.
inhibition
and
rnhibition
studies.
To correlate
labeling, the same experimental conditions as described for
the tabeling experiments were used for determining the
towards CDNB according to Habig [26].
enzymatic activity
fn an. independent experiment 1.25 UM GST P1-1 was also
incubated for 20 min at 20o with 6.25 pM ethacrynic acid in
the presence of 100 pM S-hexylglutathione 1in triPlicate)'
towards CDNB r^las
which the catalytic activity
after
determined. In an independent experiment the inhibition of
GST 4-4, J-7, l,ttLa-La and Pl--l htere compared by incubating
0.5 UM enzyme with 10 UM ethacrynic acid, monobromo
ethacrynic acid and dibromo dihydro ethacrynic acid for 2
hr at 20" in 0.1 M potassium phosphate PH 7.4. The
-L06-
inhibitor h¡as added, immediately before measuring the
activity, to aII blank incubations in all tiroe course
studies. In an earlier study only L5% irreversible
inhibition of cST 7-7 could be detected using the dialysis
method [12]. These experiments were perforrned at a lower pH
(6.5) and lower temperature (]-7'). !ùhen these conditions
were repeated, using the assay described above, the
inhibition found was 38 + 4.O%. The difference with the
dialysis experiment could presumably be attributed to the
loss of activity in Èhe blank during the dialysis, which is
especially high for cST 7-7. At the present. time it is
known that protecting cysteine residues of GST 7-7 by
reducing agents avoj-ds Èhis drawback of dialysis [28].
(UM) values for ethacrynic acid and its bromo
IC.^
_
. JV.
derivãtives
were determined by mixing 7.5 nM GST with a
concenÈration range of Èhe inhibitors. Àt least six
concentrations (in duplo) were used to determine the ICÉ^
values. ICÉ^ values r./ere obtained from plots. t'taximåÏ
inhibition'"1> 95*) was checked using plots as deserilced
previously [12].
Results
1A
-'C-Labeled
ethacrynic acid was used to determine
covalent labeling of GST under rigorous condÍtions (Table
J-). SÍgnificant labeling of the pi-class cST \4ras observed.
Table 1.
þv{cnt
bìndìng
towards CDNB and effect
Enzyme
GST I.1
GST 2-2
GST 3-.3
GST,|.4
GST 7-7
of fl'C]ethacrynic acid to rat GST isocnzymes, remaining activity
of preincubation *'ith tctrachloro-1,4-bcnzoquinone
covalent binding of |¡C]cthacrynic acid
Covalcnt
binding
enryme)
(nmol label/nmol
0.19
!
0.m+
r
r
0.04
0.V2
0.08
0.61
0.31 0.04
0.74 È o.ffi
7a
Remaining
activity
99*11
113È6
Itg + t2
88+14
7*l
(TCBQ) oir
Covalent binding
after preincubation
wirh TCBQ
(nmol label/nmol enzyme)
0.08 a 0.01
0.11 + 0.01
0.04 a 0.û2
0.04 * 0.û2
0.m É 0.00
Enzyme (10pM) was incubated $'fth lmpM [t4C]ethac'rynic acid for 60min at 37 (N = 2),
after which the labeling of GST was determincd. The labeling was also determined under the
same conditions after preinctbation with S0plvl TCBQ for 5 min at 25'. The enzymatic activity
towa¡ds CDNB was determined after incubation of l0 pM cnzyme with 100 pM éthacrynic acid
fo¡ 60min at 3?. (N = 2), and expressed ali pcr c€nt of control incubation (loss of enzymatic
activity in controls <107o, witl lhe exc€ptiotr of GST 7-7: a20Vo lo*s).
Rcsults are means É SD.
-107-
mu-class isoenzlnnes hrere also significantly
labeled, especially rat GST 3-3 (up to 0.6 nmol/nmot
enzlme). Considerably lower labeling of the rat alpha-class
isoenzlmes was observed. However, significant inhibition of
the enzt¡mic activity was only observed in the pi-class
(rable 1).
The GST of
lc¡ctton rcltvr
nnol t¡brU nnol mnon¡r
t.0
0.8
0.6
o.4
o.e
o.o
tln¡
(m¡n)
tt¡.
(arñ)
1.O
o.8
0.6
0.4
0.2
o.o
Fig. 2. The time course of the labeling of the GST
isoenzymes human P1-1 and rat GST 7-7 was investigated
at 2V by incubating l.2íttM enzyme with 6.2:pM
ethacrynic acid, after which the enzymatic activity towards
CDNB and the amount of radioactivity incorporated wcre
determined.
Remaining fraction catalytically active;
(f)
(O) nmol |aClethacrynic
acid/nmol monomer (for
experimental details see Materials and Methods).
-
t_o8
-
The time course of labeling versus inhibition h¡as
determined for the pi-class GST, of both human and rat,
under mild conditions (Fig. 2). Under these conditions,
both human and rat GST isoenzymes of pi-class
ürere
inhibited in about 100-300 min to their maximum value,
concomitant with maximal- Iabeling of about 0.8 nmol/nmol
enzyme. There is a clear comelation betrn/een tabeling and
inhibition. Preincubation of rat GST with tetrachloro-l-,4benzçguinone very efficiently inhibited the incorporation
of [-'C]ethacrynic acid in allrocST isoenzynes tested (Table
1). The incorporation of [-'C]eÈhacrynic acid in cST 7-7
was completely inhibited, while also the incorporation in
Èhe mu-class e/as inhibited remarkably. The competitive
inhibitor S-hexylglutathione (IC-^ = 20 ¡rM [2]), conpletely
abolished the inhibition of dBr pf-f by ethacrynic acid
(Table 2).
Table 2. Effects of S-hexylglutathione on the inhibition
GST P1-1 by ethacrynic acid
Incubation
cST + ethacrynic acid
cST + S-hexylglutathione
ethacrynic acid
of
å Remaining activity
+
70 + 8.0
to4 ! 6.'7
cST (1.25 UM) was incubated with 6.25 UM ethacrynic acid
for 20 min at 20o in the presence or absence of 1O0 UM Safter which the catalytic
hexylglutathione (in triplicate),
activity
lexpressed as per cent of control incubations)
toqrards CDNB vras measured. Results are means t SD.
The monobromo and dibromo dihydro derivatives of
eÈhacrynic acid were synthesized in an effort to develop
related compounds with increased alkylating
structurally
ability. Mu- and pi-class cST isoenzymes of both human and
rat \^rere incubated with these compounds ( Table 3 ) ; a
preliminary experiment has shown that the alpha-class is
either not affected by the dibromo dihydro derivative (GST
-109-
Table 3. Irrevcnible inactivation (exprcsscd ¡ìs per ccnt rcmaining activity) of rat and human GST,
and reversible inhibition [expressed as rca (pM)'valucs] with ctñacryniiacid, a¡d its monobromo
and dibromo dihydro dcrivatives
Ethacrynic acid
Monobromo
7o Remaining
Enryme
GST
GST
GST
GST
7-7
P1-1
¿l-4
Mla-la
ICs
4.8't
4.0
0.7
0.2
7o Rcmaining
lcs
acriviry
29
19
111
7n
Dibromo dihydro
>50
>50
>50
18
activi¡y
59
79
1r0
104
7o Rcmaining
ICso
4.6
10
0.6
0.4
activity
4
l0
48
30
Tbe irreversible inactivation was determined by incubating 0.5¡M en:yme u'ith 10f¡M inhibiror
for 120 min at 2tr (N _=-2), after which the enzymatic activity (as per ccnt oi controt incubations) was
determined towards CDNB.
Reversible inhibition was determined by mixing 7.5 nM enzyme with inhibitor, after which the
enzymatic activiry was immediately determined towards CDNB.
' The conccntration of cthacrynic acid or ils bromo derivativcs resulting in 507¿ inhibition of the
enzymic aaivity towa¡ds CDNB (Ica), For indúdúal values, the cocfficicnr of variarion was less
lha¡207o.
t rcs of GST 7-7 f¡om Ploemen
et al.
ll2l.
Experiments were performcd as described in Matcrials and Methods.
A2-2't ot very inefficiently
(cST j_-1, 2-2 and À1-1 only -30_
4OZ inhibition
after incubation under very rigorous
conditions, results not shown). Às kras found for ethacrynic
acid' the monobromo derivative did not irreversibly inhibit
cST of the mu-class, \À¡hile for Èhe pi-elass the monobromo
derivative was much less efficient
than ethacrynic acid
itself.
The dibromo dihydro derivative, on the other hand,
inhibits both human and rat GST isoenzlmes pi-class more
efficiently
(note that
the conditions are slightty
different from the ones used in Tab1e 1). Interestingly,
this compound is also an irreversible inhibitor of the muclass GST isoenzl¡mes.
Lastly, the competitive inhibiÈion \./as determined (Tabte
3): the t!'ro bromo atoms only marginally affect the strong
conpetitive (reversible¡ inhibitory capacity of ethacrynic
acid, only human cST p1-1 is inhibited to a 1esser extent.
oiséussiou
The catalytic activity of the pi-class isoenzymes rat
cST 7-7 and human p1-1 v/as strongly inhibited in an
irreversible manner by ethacrynic acid.
In a previous
studyr we have shown that ethacrynic acid is also a strong
competitíve inhibitor of rat and human cST of alpha-r Rltand pi-class, with 50% inhibition
at 0.3-6 UM. The
inactivation of the pi-class isoenzlmes correlates strongly
-LLo-
r^rith the incorporation of ¡14C1"th."rynic acid, with
maximal inhibition with incorporation of about 0.g
nmol,/nmot enzfrme- This suggests that one singre amino acid
is responsj-ble for the inhibition of GST of the pi-class.
Preincubation with tetrachloro-L,A-benzoquinone prevented
the incorporation of label courpletely for cST 7_j,
suggesting the involvement of a cysteine residue 1291. The
study with S-hexyJ_glutathione (a substrate analog, with
considerable affinity for the active site of GST [2])
indicates that the modification takes place in the active
site. Ethacrynic acid presumably reacts with the highly
reactive thiol group in the proximiÈy of the catarytic site
of GST 7-7 and human cST pL-l_, which has been identified as
the cysteine at the 47Eh position by several groups
[30r3]-1. This reactive cysteine seems to be conserved
within pi-class isoenzlmes of various speciesr e.g. pig and
bovine GST pi-class isoenzymes also conÈain a
preferentiarry modified highly reactive cysteine residue,
chemicar modification of which reads to enzyme inactivation
132,33 I . These cysteine residues of the pi-class are also
unique in their sensÍtivity to i-nactivation by oxidation
t28l and SHlss exchange reaction reagents [34], both
resulting in (intramolecular or mixed) disulphides. It is
widery recognized that oxygen protection is cruciar whire
working with the pi-class isoenzymes to assure that the
enzfrme has the ,Xative form Í2gr3Ll. Significant
incorporaÈion of [-'C]ethacrynic acid was also observed
with representatives of the mu-class GST. rn particurar the
mu-class GST 3-3 and to a lesser extent GST 4-4 were
labeled with ethacrynic acid. However, no concomitant loss
of catalytic activity was observed. This property is not
restricted to ethacrynic acid: e.g.
bromobenzene
netabolites are also, incorporated in rat GST subunit 1 and
4 without inhibiÈion [35] and studies with iodoacetamide
and GST 3-3, showed that only the cysteine without loss of
activity [36]. Preincubation of GST 3-3 h¡ith tetrachloro_
1tâ-benzoquinone prevented the
incorporation of
I C]ethacrynic acid very strongly, again suggesting the
involvement of one cysteine residue, presumably cysteine
86. Interestingly, nodification of the enzyme by
tetrachloro-1r4-benzoquinone and its gtutathione conjugate
does lead to inactivation. The question whether this is due
to the structure of the reagent, or to the labeling of the
other cysÈeine residues (at the 114th and 173rd position¡
remaíns to be answered.
-
t_]_l-
-
The alpha-class GST isoenzymes are only marginally
susceptible to
labeling
with ethacrynic acid, and
tçlrachloro-1r4-benzoquinone prevents iricorporation
of
[-'C]ethacrynic acid for about 50%, thus indicating that
this might be due to non-specific reactions in which
residues other than cysteines are involved.
One of the goals of this work was to improve the
irreversible
inhibitory capacity of ethacrynic acid by
newly synthesized derivatives with enhanced chemical
reactivity.
To Èhis end two derivatives \,rere synthesized,
the monobromo and dibrono dihydro ethacrynic acid. The
monobrorno derivative, with the intact arB-unsaturated
carbonyl bond noiety, irreversibly inhibited pi-class GST
to a lesser extent than ethacrynic acid itself. Ho\4¡ever/
the dibromo dihydro derivative which is expected to react
at the alpha rather than the beta carbon, inactivates piclass GST more efficiently than the parent compound.. This
compound, which undergoes a substitution-type reaction,
also irreversibly inhibits GST of mu-class, and to a much
lesser extent cST of alpha-class. The cST A2-2 is the only
enz]¡me not inhibited and is also the only GST isoenzyme
which does not contain cysteine residues, suggesting again
that the inactivation is a result of cysteine modification.
In contrast with the monobromo derivat.ive, the dibromo
díhydro compound also retains its
strong reversible
inhibitory capacity. Thus, this compound combines the
properties of a strong irreversible
and reversible
inhibitor.
Recently, Kuzmich et al-. [37] showed that ethacrynic
acid in cellular systems can induce GST of pi-class at the
Èranscriptional level. Ethacrynj-c acid is also a substrate
for pi-class GST. Thus, presumably, a distinction has to be
made between short and longer term effects in the processes
induced by ethacrynic acid in a cell. Àt present, the
following train of events seems likety:
(i)
direct
competitive inhibition by ethacrynic acid itself, followed
by'(ii) GSH depletion with concomitant formation of the GSH
conjugate of ethacrynic acid which can result (iii) in a
cornpetitive inhibition
by the conjugate while
( iv)
irreversible
inhibition can occur at low intracellular cSH
concentration, and finally (v) the cST are Índuced. It wil1
be a challenge to unravel the complex molecular mechanism
controlling inhibition and activation in cellular systems
and in vivo, in this respect in particular the dibromo
dihydro derivative might be a useful device.
-LI2-
References
1. Mannervik B. The isoenzlznes of glutathione transferase.
Adv EnzymoT Relat Areas Mo7 BioJ- 57: 357-4L7, L985.
2. Mannervik B and Dani-elson UH. clutathione transferases Structure and catalytic activity. CRC Crit Revs Biochem
232 283-337 t 1988.
3. Armstrong RN. Glutathione S-transferases: Reaction
Mechanism, Structure, and Function. Cåem Res ToxicoT 4:
l_3L-140 | L99L
4. Mannervik B, Awasthi YC, Board Pcr Hayes JD, DI IIio Cl
Ketterer B, Listowsky I, Morgenstern R, Muramatsu M,
Pearson vrlR, Pickett CB, Sato K/ Widerstern M and Vlotf CR.
Nomenclature for human glutathione transferases. Biochem
J 282: 305-308, 1992.
5. Morror^¡ S and Cowan KH. Glutathione S-transferases and
drug resistance. Cancer ce77s 2: 75-22t L99O.
6. Hayes JD and Wolf CR. Review article - Molecular
mechanisms of drug resistance. Biochem J 272¿ 281-2951
1990.
7. waxman DJ. Glutathione S-transferases: role
in
alkylating agent resistance and possible target for
modulation chemotherapy - À review. Cancer Res 50 z 64496454t 1990.
8. Borst P. Genetic mechanisms of drug resistance - A
review. Revjer¿s in AncoTogy 4: 87-1O5 | L99L.
9. Black SM and Wolf CR. The role of glutathione-dependent
enzlrmes in drug resistance. Pharmac Ther 51-: 1-39-154,
1991_.
10. Tiirikainen MI and Krusius 1. Multidrug resistance.
AnnaTs of l4edicine 23¿ 5O9-52Ot L99L.
11. Àhokas JI, Nicholls FA, Ravenscroft PJ and Emmerson BT.
Inhibition
of purified
rat
liver
glutathione
Stransferase
isoenzymes by diuretic drugs. Biochem
Phazmacol 342 2L57-21-61-, l-985.
12. Ploemen JHTM, Van Ommen B and Van Bladeren pJ.
Inhibition of rat and human glutathione S-transferase
isoenzl'mes by ethacrynic acid and its glutathione
conjugate. Biochem Pharmacol- 4O: 1631-1635, l-990.
13. Tew KD, Bomber AM and Hoffman SJ, Ethacrynic acid and
piriprost as enhancers of cytoÈoxj-city in drug resistance
and sensitive cell lines. Cancer Res 482 3622-3625, 1-988.
14. Nagourney R.è,, Messenger JC, Kern DH and Weisenthal tM.
Enhancement of anthracycline and alkylator cytotoxicity
-L13-
by ethacrynic acid in primary cultures of human tissues.
Cancer Chemother pharmacol 26: 319-322t L9gO.
15. Hansson J, Berhane K, Castro VM, ilungnelius lJ,
Mannervik B and Ringborg U. Sensitization of human
melanoma cells to cyÈotoxic effect of melphalan by
glutathione transferase inhibitor ethacrynic acid. cancer
Res 51: 94-98, 1_991
16. Wallin JD, Clifton G and Kaplowitz N. The effect of
phenobarbital, probenecid and diethyl maleate on the
pharmacokinetics and biriary excretion of ethacrynic acid
in rat. J Phaxmdcol Exp Therapeutics 205: 4i.l.-47g, Lg7g.
L7. Yamada T and Kaplowitz N. Binding of ethacrynic acid to
hepatic glutathione S-transferases in vivo in the rat.
Biochem PharmacoT 29t I2O5-L2O8, 1990.
18. Van Onmen B, Den Besten C, Rutten ALM, ploemen JHTM,
Vos RME, MüIler F and Van Bladeren pJ. Active site_
directed irreversible
inhibition
of glutathione
S_
transferases by the glutathione conjugate of tetrachloro_
1,4-benzoquinone. J BioJ Chem 263: L2939-L2942, L9gg.
i-9. Ploemen JHTM, Van Ommen B and van Bladeren pJ.
Irreversible
inhibition
of human gluÈathione S_
transferase isoenzlanes by tetrachloro-1_ r4benzoquinone. Eioct¡em pharmacoJ 41: 1665-1669, Lgg1.
20. Tamai K, Satoh K, Tsuchida S¿ Hayayama I, Maki T and
Sato K. Specific
inactivation
of glutathione
S_
transferases in class pi by SH-modifj_ers.
Biochem Biophys Res Co¡nmun L67: 331-339, L99O.
21 . Chang LH, I¡Iang Ly and Tam MF. The single cysteine
residue on an alpha family chick liver glutathione S_
transferase cL 3-3 is not functionally important. Biocåe¡n
Biophys Res Connux 180: 323-329, ]-99L.
22 Pretsch E, Clerc T I Seibl J , and Sj:non vt.
StrukturaufkTärung Organische verbindungen. Springer_
Verlag, Berlin, i-976.
23. Vos RME, Snoek MC, Van Berkel WJH, Müller F and Van
Bladeren pJ. Differential
induction of rat hepatic
glutathione s-transferase isoenzymes by hexachrorobenzene
and benzyl isothiocyanate: comparison with induction by
phenobarbital and 3-rnethylchol_anthrene . Biochem
PharmacoL 3Tz LO77-L082, 1989.
24. Bogaards JJP, Van Ommen B and Van Bladeren pJ. An
improved method for the separation and quantification of
glutathione S-transferase subunits in rat tissue using
high-performance J_iquid chromatography. J Chromatography
474¿ 435-440, 1989.
-rt4-
25. Habig WH, Pabst MJ and Jakoby WB. cluÈathione Stransferases, The first
step in mercapturic acid
formation. J BioT Chen 249: 7L3o-7L39, L974.
26. Hayes JD, Pickett CB and Mantle TJ. GLutathione Stransferases and Drug Resistance. Taylor & Francis,
London, 1990.
27. Shen Ht Tamai K, Satoh K, Hatayama I, Tsuchida S and
Sato K. Modulation of class pi glutathione transferase
activity
by sulfhydryl group modification. .qrcir Biochem
Biophys 2862 l-.78-t82t L99L.
28. Van Ommen B, Ploemen JHTM, Ruven HJ, Vos RME, Bogaards
JJP. Van Berkel WJH and Van Bladeren, Studies on the
active site of rat glutathione S-transferase isoenzyne 44. Chemical modification by tetrachloro-L, -benzoquinone
and its glutathione conjugate. Eur J Biochem tSLz 423429, L989.
29. Tamai K, Shen H, Tsuchida S, Hatayama I, Satoh K, Yasui
À, Oikawa A and SaÈo K. Role of cysteine residues in the
glutathione transferase P (7-71:
activity
of
rat
elucidaÈion by oligonucleotide site-directed mutagenesis.
Biochen Biophys Res Co¡nmun L79z 790-797, I99L.
30. Ricci G, Del Boccio c, Pennelli À, Lo Bello M¿
Petruzzelli R, Caccuri AM, Barra D and Federici G. Redox
forms of human placenta glutathione transferase. J BioJ
Chen 2662 2t4O9-2t4L5t 1991.
31. Schäffer J, Gallay O and Ladenstein R, Glutathione
placenta.
transferase
from
bovine
Preparation,
biochemical characterization,
crystallization,
and
preliminary crystallographic analysis of a neutral
class pi enzyme. J BioL Chen 263: 1"7405-17411-, 1988.
32. Dírr H!'I, Mann K, Huber R, LadensÈein R and Reinemer P.
Class n glutathione S-transferase from pig
lung.
primary
Purification,
biochemical characterization,
Eur J Biochem 196: 693structure and crystallization.
698,1991.
33. Nishihara T, Maeda H, Okamoto KI, Oshida T, Mizoguchi T
and Terada T. Inactivation of human placenta glutathione
S-transferase by SH/SS exchange reaction with biological
Biochem Biophys Res Conmun 174: 580-585,
disulfides.
1991.
34. Aniya y, Mclenithan JC and Anders MW. Isoenzyme
aryll!ion
selective
of cytosolic
Salutathione
transferase by [ -'C]bromobenzene metabolites.
Biochem PharmacoT 372 25L-257t L988.
-115-
35. Hsieh JC, Huang SC, Chen W!, Lai TC and Tam ¡tF.
Cysteine-86 is not need.ed for the enzl¡mic activÍÈy of
glutathione S-transferase 3-3. Biachem J 27Bz 293-297,
1991_.
36.
Kuzmich S, Vanderveer LÀ, I{alsh ES, IJacret.a Fp and Tew
KD. Increased levels of glutathione S-transferases n
transcript as a mechanism of resistance to ethacrynic
acid. Biochem ,t 28lt 2L9-224, 1-992.
-
l_t_6
Chapter
-
9
Reversible conjugat,ion of ethacrynic acid with
and human glutatbione S-transferase P1-L
gl.utathione
J.H.T.M. Ploemen, A. Van Schanke, B. van Onmen, and P.J.
van Bladeren.
Sub¡nitted
Abstract
The reversibility
of the conjugation reaction of the
diuretic drug ethacrynic acid (EÀ), an arB-unsaturated
ketone, with glutathione and glut.athione S-transferase p1-1
(GST Pl--l-) has been studied. When the glutathione conjugate
of EÀ was incubated with a fivefold molar excess of Nacetyl-L-cysteine or GST Pl--1, a tine-dependent transfer of
EA to N-acetyl-L-cysteine or GST P1-1 was observed. With
increasing pH, the pseudo first
order rate constants of
tr4nsfer of EA to N-acetyl-L-cysteine increased from_0.010
h-' (pti 6.4), to o.o4o n-t (pn 7.4), and 0.076 h-I (pH
8.4).
From the fact that preincubation of cST p1-1 with 1c$]oro-2,4-dinitrobenzene reduced the incorporation of
[-'C]EÀ from 0.94 ! O.2L to 0.16 I O.O2 mol EA per mol
subunit, and from automated Edrnan degradation of the major
rlflioactive peptide isolated after pepsin digestion of the
[-'C]EA-Iabeted enzfrme, it was concluded that the reaction
of EÀ takes place with cysteine 47 of cST p1-1.
When, cST P1-l- v¡as inactivated with a fivefold molar
excess of EÀ, adding an excess of gluÈathione resulted in
full restoration of the catal-ytic activity in about 120 h.
These findings may have several i-mplications: although,
under norrnal physiological condiÈions the inhibition of cST
P1-1 by covalent binding of EA would be reversed by
glutathione, Ieaving reversible inhibition by the the
glutathione conjugate of EA and EÀ itself
as the main
mechanism of inhibition.
lthen glutathion levels are low the
-1L7-
covalent inhibition might be predominant resulting in
completely different ti-me course of inhibition.
a
I¡troduction
Conjugation with
the
tripeptide
glutathione is
considered to be an important detoxification reaction for
electrophilic xenobiotics, inctuding numerous cytgstatic
agents. In general this reaction is catalysed by cST'(1_3).
Most GST occur in cyt,osol and belong to one of four
multigene families, termed alpha, mu, pi, and theta (L,41.
Evidence has accumulated thaÈ increased cST activity may be
one of the causes of drug resistance, especially with
respect to alkylating agents (5).
The diuretic drug ethacrynic acid, an drB-unsaÈurated
ketone, is a potent rer¡ersi.bLe inhibitor of GST isoenzl¡mes
(6,7), and has been used to study the role of cST in drug
resistance in vitro, usini cell lines (g), and in a phase f
clinical study with the cytostatic agent thiotepa (9).
Moreover, a concentration-dependent inhibition by
ethacrynic acid of the enzfzme-catalyzed conjugat,ion of
glutathione with the clinically important alkylating agent
chlorambucil, has been reported ( lO) . The reversibJ"e
inhibition would further be enhanced by the formation of
the glutathione conjugate of ethacrynic acid, which is an
even stronger inhibitor for all cST but the pi-class (7).
For both human and rat GST of the pi-class, covalent
modification of csr concomitant with an irreversibl.e loss
of activity, could be achieved using slightly more drastic
incubation conditions (L1).
Conjugation with glutathione does not always leads to
the detoxification of electrophilic xenobiotics (j_2). In
addition to gtutathione conjugates that are reacÈive by
themselves, other types of glutathione conjugates may
undergo further metabolism to a reactive species (a2). A
speciaÌ case involves glutathione conjugates that exert
their toxic effects through release of reactive species:
the glutathione conjugates serve as transport and targeting
agents. This situation occurs when the glutathione
conjugation reacÈion is reversible, as found e.g. for some
methyl isocyanates ( L3 ) and isothiocyanaÈes ( 14) . The
Michael addition of glutathione with arB-unsaturated
aldehydes and ketones is another well-known reversible
reaction (15). À reversible Michael reaction has e.g. been
-118-
shov/n to be involved in the covalent bindj-ng of the
veterinary drug furazolidone (16).
Since ethacrynic acid also contains an arB-unsaturated
ketone moiety, the present study !'/as designed to
of
covalent interaction
investigate the reversibfe
ethacrynic acid with glutathione as well as with GST P1-L.
The interaction of ethacrynic acid with this enzyme was
included, since the inactivation of the GST of t,he pi-class
in several cases has been shown to be the result of the
modification of a highly reactive cysteine residue (17,18),
and since pi-class, next to alpha, is one of the primary
GST classes that are involved in drug resistance (5).
Materials and
Methods
ChemicaTs and enzymes. Ethacrynic acid 12r3-dichloro-4(2-nethylene-1-oxobutyl)phenoxy)acetic acid,
S-hexylglutathione, N-acetyl-L-cysteine, and TrisIhydroxl¡methyl]aminomeÈhane \^/ere from Signa ChemicaL Co., St. Louis, MO.
Epoxy-activated Sçgharose 68 was purchased from Pharmacia,
Uppsala, Sweden. [-'C]Ethacrynic acid was purchased from
(15 mCi,/runol ) .
Buckinghamshire, U.K.
Amersham,
lrifluoroacetic acid was from Baker Inc./ Philipsburg, NJ.
Pepsin (from porcine gastric mucosa) was obtained from
Boehringer, Mannheim, Germany.
conjugate of ethacrynic acid \itas
The radioactive
prepared by adding 6 ¡.rmoles of glutathione in 180 pl of 0.1
M potassiuqophosphate buffer pH I with 50? ethanol, to 1.3
pmoles of [*'c]ethacrynic acid. After overnight incubation,
the glutathi-one conjugate of ethacrynic acid was purified
by preparative RP-HPLC using zo¡bax ODS (21.2 * 250 m),
eluted at a flow rate of 4 ml,/min with 0.0L? formic acid
(solvent I) and meÈhanol (solvent II), with a linear
gradient of 40-100% II in 60 nin (k'= 2.0 and 3.1 for the
conjugate and ethacrynic acid respectively). About 70%
conversion of ethacrynic acid to the glutathione conjugate
was obtained. Methanol was removed under N^, after which a
stock-solution (of 136 UM) of the glutathiáne conjugate was
storéd at -30oC. À product of 95+ % purity was obtained as
judged with RP-HPLC analysis, with an identical retention
time to the non-radioactive conjugate (7).
The N-acetyl-L-cysteine conjugate of ethacrynic acid was
prepared, in analog¡¡ to the synthesis of the glutathione
conjugate (7) - The -H-NMR (400 MHz, D.rO) spectrum of the Nacetyl-L-cysteine conjugate are cónsistent v,¡ith the
-119-
expected structure (11-); with the following information for
the ethacrynic acid part: 6 7.e1/7.79 (ddt 1H, J= 8.8 Hz) ,
6 7.23 (d, LH, J= 8.8 Hz) | 4.99 (st 2H) , ó 3.8 (m, 1H), ð
L.95/L.81- (dm, 2H), 6 1.1 (m, 3H); and for the N-acetyJ_-Lcysteine part 6 2.L9 (s, 3H, -CH"). The proton signals of
cys a overlap with DrO (ó= 4.7), \"rdile the signals of cys B
and the protons next'to the sulphur-atom (of the ethacrynic
acid moiety) v/ere found in the region of 6 2.9-3.Lt as a
complicated multiplet pattern.
GST P1-1 was purified as described (11). Protein \^ras
determined by the method of Lov/ry, using bovine serum
albumin as standard (19).
Incubations. The glutathione conjugate of ethacrynic
acid (0.5 mM) was incubated at 2O"c with N-acetyl-Lcysteine (2.5 mM), in 0.4 ml of 0.1 M potassium phosphate
buffer with 0.1 mM EDTÀ at three pH-levels (6.4, 7.4, and
8.4). For each pH, 22 independent samples were prepared. Àt
each Èime point, 20 ¡r1 waê injected on RP-HPLC, using a
Zorbax ODS (250 * 4.6^mm) column, eluted at a flor^/ rate of
1 ml,/min with 0.1% TFAZ in deionized \,r'ater (solvent A) and
in methanol (solvent B), with a linear gradient of 30-95% B
in 18 min, followed by 2 min at 95% B (k'= 4.5 | 5.!, and
6.0 for the glutathione conjugate, N-acetyl-L-cysteine
conjugate, and ethacrynic acid respectively). Peak areas at
270 nm were integrated \,üith Ne1son Analytical Model 2500
Chromatography Software. EDTA was added to the incubations
Èo prevent the trapping agent N-acetyl-L-cysteine and the
free glutathione from oxidation to Èheir disulfides. Since
no free ethacrynic acid could be detected, it is concluded
that EDTA protected suffiçiently against oxidation.
Covalent binding of l*'C]ethacrynic acid was studied in
a volume of 75 yI 0.1 M potassium phosphate buffer pH 7.4
with 0.1 mM EDTÀ (buffer A), after preincubation of 25 yM
cST P1-1 for 75 min at rogm temperature with (n=3) or
without (n=2) 1 mM CDNB-, qlereafter the enzf¡me hras
incubated for l-10 min with [*'C]ethacrynic acid (final
concentration/ 100 UM). Enzyme-bound ethacrynic acid was
separated from ethacrynic acid by RP-HPLC (vydac TP5
column, 200*3 mm). Elution r¡/as performed with a flow of 0.6
ml/min, with solvent À (see above) and 0.1% lFA in
(solvent C), with a linear gradient of 30-60%
acetonitrile
C in 30 min (k'= 4.0, and 6.3 for ethacrynj-c acid and
enzyme with bound ethacrynic acid respectively). UVdetection (at 2L4 nm) was used to identify the enzyme peak,
-120-
hThile simulÈaneously the radioactivity was measured using
an on Ii1¡ radiochemical detector.
The I C]ethacrynic acid labeled GST p1-1 (0.25 mg) was
digested with pepsin lenzyme to protein ratio l/2O (w/w)]
j-n 0.05 M lris,/H?po¿ (pH L.8) for j_8 hr at 37oC. The pepsin
peptide mixture-waé purified on a Rp column (Vydac protein
& pepÈide CLS. 250 * 4.6 mm), eluted with solvent A and C
(see above), 5 min isocratically at 1OO% À, followed by a
linear gradient from 0-60? C in 70 min ( ftow rate 1
mt/min¡. fhe main radioactive peak was repeatedly purified
on the same column. The peptide was degraded using
automated Edman degradation on an Appried Biosystems Model
475 peptide sequencer on-ì_ine connected to Model 120À pTHanalyzer.
The catalytic activity of cST p1-1, inactivated with
ethacrynic acid, \^ras moniÈored after the addition of
glutathione. L ¡rM cST pl_-L vras preincubated in buffer A
(see above) \"/ith or wiÈhout 1_0 ¡.rM of ethacrynic acid (final
volume, 200 pI), after which glutathione hras added (final
concentration: 0, 10, 1OO, and 1OOO UM). These incubations
were performed in triplicate,
ât room temperaÈure. At
various time points , 20 pmol enzyme samples \^rere
transferred to cuvettes, after which the catalytic activity
towards CDNB hTas measured (20). A time series was stopped,
when the remaining catalytic activity in the corresponding
blank incubation was less than 7O%.
To study the interaction ,gf cST pl"-l with
the
glutathione
conjugate of ¡'=Clethacrynic acid, seven
independent samples of L0 ¡.rM GST p1-1 vTere incubated at
room temperature with 2 pM of the radioactive glutathione
conjugate in buffer À (see above), (finat volume: 50 UI).
To separate the glutathione conjugate and free ethacrynic
acid from the enzyme with bound ethacrynic acid, 30 ¡.rI \^¡as
injected on the Vydac Tp5 column (see above).
Results
The occurrence of the retro Michael cleavage of the
glutathione conjugate of ethacrynic acid r¡¡as studied by
incubation of the glutathione conjugate of ethacrynic acid
with an excess of N-acetyl-L-cysteine (FiS. l_). The
transfer of the ethacrynic acid moiety to N-acetyl_L_
cysteine was folLowed with time by quantification of the
glutathione
and
N-acetyl-L-cysteine
conjugates of
ethacrynic acid on Rp-HpLc. The rate of Èransfer increased
-L2L-
fraction of conjugates
1.00
0.80
0.60
0.40
0.20
0.00
50
100
150
200
time (h)
FIc. I Tîansfer of ethacrynic acid from its gfutathione
conjugate to N-acetyT-L-cysteine (NAC). The glutathione
conjugate of ethacrynic acid (0.5 mM) was incubated at 20oC
with a fivefold excess of NAC. The transfer of the
ethacrynic acid moiety to NAC \^ras followed with time by
quantification of the conjugates on RP-HPLC by integration
of the peak areas aE 27O nm. The reaction was studied at
three pH-Ievels; pH 6.4 (L),7.4 (ar, and 8.4 (¡). Open
symbols reflect the newly formed NAC conjugates, while the
closed symbols reflect the gluÈathione conjugate of
eÈhacrynic acid.
increasiqg pH, with pseçdo first order rates of 0.010
wifh
h *, 0.040 h -, and 0.076 h *, for pH 6.4, pH1.4, and8.4,
respectively. Àfter l-80 h of incubation aÈ pH 8.4, an
equilibrium was reached betr^zeen the glutathione and the Nconjugate,
suggesting
acetyl-L-cysteine
that
the
disÈribution of ethacrynic acid over glutathione and N-
-r22-
acetyr-L-cysteine is mainly determined. by their rerative
concentrations.
Retro l'lichael creavagre can arso occur with GsT p1-1
bound ethacrynic acid, if the assumption is righÈ that
ethacrynic acid reacts with a cysteine residue ot cÀr p1-1
(1t)fn In order to check this assumption, the incorporation
of [--c]ethacrynic acid in csr p1-l after preinãubation
\'rith CDNB was studied, which is known to inactivate GST p1L by modification of cysteine 47
O.g4 + O.2t nmol
label per nmor Gsr p1-1 courd11,7).
be incorporated in branc
incubations, idenÈical to an earlier stuay (11).
Às
expected, CDNB protects against incorporation of ethacrynic
acid: 0.L6 t 0.02 nmot labet per nmol cST p1_1 could be
incorporated after preincubation with CDNB, supporting the
hypothesis that ethacrynic acid reacts with cysiãine az of
cST PL-l-. fn order to identify the amino acid involved. in
the reaction, the csr p1-L with bound ethacrynic acid h¡as
digested with pepsin and the resulting peptides were
separated on HpLc. À main radioactive peak was identified,
which contained >got of the radioactivity, eluting at 45
min (Fíg 2). The amino acid seç[uence indicated that it
spans residues 44-46 in the primary amino acid sequence of
GST Pl-l- (Lys-Àla-Ser) (zL-?e), while an unknown residue
r^/as observed in the 4""_cycle (presuraably the cys_
ethacrynic aci-d adduct). Thus it was again eoncluded that
Cys 47 is the main target site.
Then, cST pL-1 (l UM) was incubated with ethacrynic acid
( 10 fM), resulting in 903 ross of activity
towards CDNB,
and glutathione ¡¡as added. The catarytic activity tov¡ards
CDNB h¡as measured over a L2O hr period (Fig. 3). FuII
restoration of the catarytic activity occurs with 0.i. nM
and 1 mI"1 glutathione (insert Fig. 3). The 10 ¡.rM incubation
initiauy shows partial restoration of catarytic activity,
but after prolonged incubation ross of catarytic activity
is observed, probably due to oxidation. rtti" is arso
observed in the corresponding control incubation (a 3Oe
loss of activity in about 30 h; result not shown). Without
a trapping agent for free ethacrynic acid, no restoration
of activity was observed (Fig. 3).
-L23-
CPM
9000
8000
7000
6000
5000
4000
3000
2000
1
000
0
0
10
20
30
40
50
60
70
time (min)
FIG. 2 EPLC analysis of the pepsin digest of ethacrynic
acid-TabeLed GSTPI-J. From one minute fractions,
samples
(40 UI) !'rere screened for radioactivity. fnsert: peptides
monitored fron 0-75 min at 21-4 nm, peak at 44.8 min
indicated (fu1l scale 0.3 aufs).
In order to investigate whether retro Michael cleavage of
the glutathione conjugate of ethacrynic acid concomitant
with incorporation of ethacrynic acid in GsT P1-1 occurs/ a
S-fold molar excess of the.,Ênzyme was incubated with the
gJ-utathione conjugate of [--C]ethacrynic acid. À time
dependent increase of enzyme bound label was observed (Fig.
4), in accordance with the reversible nature of the
reactions.
-r2460
t00
80
60
ao
.=
.l
æ
40
0
C)
(ú
ot
.c
c
'(ú
E
o
s
20
aa_a.a
A
0
0
10
20
30
time (h)
FIG. 3 Restoratjon of the catalytic activity of ethacrynic
acid-inactivated
human cST P7-7, by incubation with
gTutathione. GST P1-l- (1 UM) was incubated with ethacrynic
acid ( 10 UM), resulting in 90% loss of catalytic activity
to!,/ards CDNB. Glutathione (GSH) was added [0 (^)/ 0.01 (O),
O.l- (o) , and 1mM (a)1. The catalytic activiÈy to\n¡ards CDNB
was measured over a l-20 h period, and expressed as per cent
of control incubations with non ethacrynic acid modified
enzyme (note: incubations were stopped when the remaining
activity
in the corresponding control was less than 70%).
Insert: effect of prolonged incubation.
Individual points are the averaqe of three measurements,
with coefficients of variation less than 153.
-L25-
Discussion
arB-unsaturated aldehydes and ketones have long been
both
form conjugates with glutathione'
known to
spontaneously and enzl¡me-catalyzed (24). The extent to
which the enzyme plays a role differs widely amoung members
of this class of compounds (25). The chemical reaction
the conjugation of ethacrynic acid and
involved in
sÈructurally related compounds, a Michael addition, is
reversible. In the present study, it v¡as shown that this
retro Michael cleavage of ethacrynic acid and glutathione
indeed occurs. Thus, ethacrynic acid may be transferred
from one low molecular weight compound to another, or to
and äccessible cysteines in proteins t e.9.
reactive
47
of GST P1-1 as observed. This phenomenom is
cysteine
probably a conmon featurce of arB-unsaturated aldehydes and
ketones: transport via a thiol conjugate and subsequent
regeneration of the reactive agent thus may be involved in
the biological activity of such adducts ( 15 ) .
The nature of the inhibition of GST by ethacrynic acid
has been studied in detail , since it r¡/as reported that
ethacrynic acid in vivo bound covalently to rat GST 3-4
(26).
Previously we showed that ethacrynic acid and its
glutathione conjugate were potent reversible inhibitors of
cST isoenzlzmes, with r-^-values in the range of 1--10 ¡M
(7). This reversible inhiËYtion was suggested to be the
mechanism in vivo, since the
predominant inhibitory
incorporation of ethacrynic acid in GST 3-4 did not appear
to inactivate the enzlnae (7 tLLl. In the case of GST PL-1'
it was shown recently that the mechanisms of reversible
inhibition by ethacrynic acid and its glutathione conjugate
non-comPetitive,
and
(competitive
were distinct
(27). For the alpha- and mu-class the
respectively)
conjugate of ethacrynic acid \^tas an even more potent
the pi-class some
while for
inhibitot,
reversible
results have been reported (7 t27 \ .
"onilirtir,g
GST of the pi-class are inhibited by covalent binding of
arp-unsaturated aldehydes and ketones: acrolein, a toxic
atdehyde that occurs as environmental pollutant (28), and
also ethacrynic acid 111), specifically inactivated GST of
It is now clear that the inhibition is only
the pi-class.
transitory, since the chemical reaction is reversible: full
restoration of the catalytic activity can be achieved by
prolonged incubation with an excess of gluÈathione. In an
-L26-
100
.ll -
EÀ
.= ìt)
€o
*o
60
RI
ËP
40
Þs
20
time (h)
Frc. 4 The +ûteraction of GST P1-1 with Ëåe glutq¿hione
conjugate of [--C]ethacrynic acid. 10 UM cST pL
incubated with 2 pM of the glutathione conjugat.,-ttnill
labeled in the ethacrynic acid noiety. To separa¡. tne
glutathione conjugate and free ethacrynic acid fro* ¡¡"
enzlnue-bound fraction,
a RP-HPLC method was ssad as
described under "Materials and Methods". The transfer of
the ethllrynic acid moiety is expressed as per cerrt of
total [--C]-IabeI.
-t27^
earlier study, only marginal inactivation of GST 7-7 by
ethacrynic acid r^ras observed using overnight dialysis
of the
experiments (7). Presumably the reversibility
of this
failure
the
to
reaction
contributed
chenical
of
dialysis experiment to detect time-dependent inhibition
GST P1-1 by ethacrynic acid.
The inptications of the present findings may be severalthis mode of action, reversible covalenÈ binding
Firstly,
to GST P1-1 and glutathione, may have some significance for
the use of ethacrynic acid as in vivo inhibiÈor for GST Pl-f- in drug resistance. Under normal psysiological conditions
(glutathione concentration 1-10 nM, (1))r glutathione may
be expected to reverse any covalent binding of ethacrynic
acid to GST P1-1, and the inhibition of GST would only
occur reversibly, through the glutathione conjugate of
ethacrynic acid and ethacrynlc acid itself. However, in
those cells with either high levels of GST P1-1 and/or low
of GST P1-1
1eve1s of glutathione, covalent inhibition
would
might be predominant. The time course of inhibition
be completely different in these two cases.
Recently/ it was shown that chronic exposure of human
colon carcinoma cells to ethacrynic acid led to a 2-3-fold
by an induction of the
increase of GST Pl--l- activity,
enz)rme at the transcriptional level (29). This phenomenon
has been proposed by Talalay and co-workers to be a general
one (30): comPounds that contain a Michael acceptor or froru
which a Michael accePtor can be formed during metabolisn
are usually inducing agenÈs for GST. The contrary effects
observed for Michael acceptors, i.e. inhibition of GST by
covalent modification, and induction of GST merit more
aÈtention.
some arB-unsaturated aldehydes are
Furthermore,
established inhibitors of growE.h. In the case of 4hydroxynonenal and related compounds it was shown that
inhibition of DNA synthesis was involved, presumably as a
result of a reaction with a functional sulfhydryl group of
DNÀ potymerase (15). More recently' another type of growEh
inhibition has beeq^ reported, which involves the a,Bunsaturated ketone at'-PGJ,, a cyclopentenone prostaglandin
which readily forms glu€athione conjugates (31). These
conjugations should in principle also be able to undergo
retro Michael cleavage to reform the parent compounds.
Interestingly, it has been shown that ethacrynic acid,
of GST, also has
with other inhibitors
along
-L28-
antiproliferative
be reversible.
effects on cell lines (32), which
seem
to
References
L. Mannervik B. The isoenzymes of glutathione transferase.
Adv Enzymol. Rel.at Areas Mol Biof 572 357-4L7,1985.
2. Mannervik B and Danielson UH. Glutathione transferases Structure and catalytic acÈivity. cRC Crit Rev Biochem
232 283-337, L988.
S-transferases:
Glutathione
RN.
3. Àrmstrong
reaction mechanism, structure, and function. Chem Res
Toxicol 4. l-31--140, 1991'.
4. Meyer DJ, Coles B, Pemble SE, Gilmore KS' Fraser GM and
Ketterer B. Theta, a new class of glutathione transferase
purified from rat and man. Biochem J.274t 4O9-4I4, 199L.
in
5. waxman DJ. Glutathione S-transferases: role
alkylating agent resistance and possible target for
modulation - A reviev¡. cancer Res 5O: 6449-6454t L99O.
6. Ahokas JT, Nicholls FÀ, Ravenscroft PJ and Emmerson BT.
glutathione
Srat
liver
of purified
Inhibition
isoenzymes by diuretic drugs. Biochem
transferase
PhatmacoT 34: 2t57-2i61, 1985.
7. Ploemen JHTM, van Ommen B and Van Bladeren PJ.
Inhibition of rat and human glutathione S-transferase
isoenzymes by ethacrynic acid and its glutathione
conjugate. Biochem Pharmacof 4O: L63l--1-635, 1990.
8. Tel\r KD, Bomber AM, and Hoffman sJ. Ethacrynic acid and
piriprost as enhancers of cytotoxicity in drug resistance
and sensitive cell lines. CanceÍ Res 482 3622-3625, l-988.
9. o'Ðwyer PJ, Lacreta F, Nash S, Tinsley Pw' Schilder R'
Clapper ML, Te\^7 KD, Panting L, Litwin S' Comis RL and
Ozols RF. Phase I study of thiotepa in combination with
the glutathione transferase inhibitor ethacrynic acid.
Cancer Res 51: 6059-6065, 1991.
10. Ciaccio PJ, Tew KD and Lacreta FP. Enzymatic
conjugation of chlorarnbucil with glutathione by human
glutathione S-transferases and inhibition by ethacrynic
acid. Biochem PharmacoT 422 L5O4-L5O7 | t991'.
11. Ploemen JHTM, Bogaards JJP' Veldink GA' van ommen Bf
Jansen DHM and van Bladeren PJ' rsoenzyme selective
inhibition of rat and human glutathione Sirreversible
transferases by ethaerynic acid and tvto brominaÈed
derivatives. Biochem PharmacoT 45: 633-639' 1993.
-t29-
l-2. Monks lJ, Ànders MW, Dekant W, Stevens JL, Lau SS and
Van Bladeren pJ. Glutathione conjugate med.iated
toxicities. Tox AppT pharmacol lO6: 1_19, lrgg}.
L3. Bailrie TÀ and sratter ,fc. crutathione: a vehicre for
the transport of chemicalry reactive metabolites in vivo.
Acc Chem Res 24¿ 264-27Ot LggL.
L4. Bruggeman IM, Tenmink JHM and Van Bladeren pJ.
Glutathione- and cysteine-mediated cytotoxicity of altyl
and benzyl isothJ-ocyanate. Toxicol_ Appl Pharmacol 83:
349-359, L986.
15. vgitz G. Biological interactions of arB_unsaturated
aldehydes. J Free Rad Biol Med 7: 333_349, 1989.
16. Vroomen LHM, Berghmans MCJ, Groten Jp, Koeman JH and
Van Bladeren pJ. Reversible interaction of a reactive
intermediate derived from furazoridone wíth glutathione
and protein. ?oxjco-l AppJ pharnacot 93: 53_60f 19gg.
17. Caccuri AM, petruzzelli R, polizio F, Federici G, and
Desideri A. rnhibition of grutathione transferase z from
human placenta by l_-chloro-2r4-dinitrobenzene occurs
because of covalent reaction with cysteine 47. Areh
Biochem Biophys 297¿ LL9-L22t Lggz.
18. Tamai K, Satoh K, Tsuchida S, Hatayama I, Maki T and
SaÈo K. Specific
inactivation
of glutathione
S_
transferases in class pi by SH_modifiers. BjocJ:em Biophys
Res Cor¿mun 167: 331-339, 1990.
19. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ.
Protein measurement r¡ith the Forin phenor reagent. J Biol
Chem 193: 265-275, 1-95L.
20. Habig WH, pabst MJ and Jakoby tVB. Glutathione S_
transferases- Ihe first
step in mercaturic acid
formation. J BioL Chen 249:. 7I3O-7i..3}, 1974.
2L. Kano 1t salcai M and Muramutsa M. structure and
expression of a human n glutathione
S_transferase
messenger RNÀ. Cancer Res L7 z 5626_5636, l-gg7 .
22- ÄLin P, Mannervik B and Jiirnvarr H. structurar evidence
for three different tl1)es of glutathione transferase in
human tissues. FEBS Lett 7822 3lg_32g, 1gg5.
23. Dao DD, partridge CA, Kurosky À and Àvüasthi yC. Human
glutathione s-transferases. characterization
of the
anionic forms from lung and placenta. Biocåem J 221; 33_
43, L984.
24. Boyland E and Chasseaud LF. Enzl¡mes catalysing
conjugations of glutathione with crB_unsaturated carbonyl
compounds. Biochem J LO9: 651_66j., j.96g.
-130-
25. ÅIin P, Danielson H and Mannervik B. 4-hydroxyalk-2enals are substtates for glutathione transferase. ¡'EaS
Lett L97: 267-270, l-985.
26. Yamada T and Kaplowitz N. Binding of ethacrynic acid to
hepatic Alutathione S-transferases in vivo in the rat.
Biochem Pharmacol 292 L2O5-1-208, 1980.
27. Awasthi S, SrivasÈava SK, Àhmad F, Ahmad H and Ànsari
cAs. Interactions of glutathione s-transferase-z wj-Èh
ethacrynic acid and its glutathione conjugate. Bíochin
Biophys Acta LL64z 173-t78, L993.
the
28. Berhane K and Mannervik B. Inactivation of
genotoxic aldehyde acrolein by human glutathione
transferases of classes alpha, mu, and pj-. Mol PharmacoL
37¿ 25L-257,1990.
29. Kuzmich S, vanderveer LÀ, Walsh ES, Lacreta FP and Tew
KD. Increased levels of glutathione S-transferase ît
transcript as a mechanism of resistance to ethacrynic
acid. Eiochem J 28tt 219-224t 1992.
30. Tatalay P, De Long MJ and Prochaska HJ. Identification
of a cornmon chemical signal regulating the induction of
enzl'mes that protect against chemical carcinogenesis.
Proc NatJ. Acad sci t/sÄ 852 826L-8265, 1988.
effect
31-. Koizumi T, Negeshi M and Ichikawa Atrlnhibitory
A---prostaglandin
glutathione
on
Jrintracellular
of
índuced proteín syntheses in porcine aortic endotheliål
cel1s. Biochem PharmacoL 44: L597-L6O2, L99232. Sato Y, Fujii S, Fujii Y and Kaneko T.
effects of glutathione S-transferase
Àntiproliferative
inhibitors on the K562 cell Iine. Biochem PharmacoJ- 39:
L263-1266, l-990.
-131-
Chapter
Summary
10
part fI
fn part
fI
of this thesis, the reversible and
irreversible inhibition
of Gsr by ethacrynic acid
derivatives is described. Moreover/ an attempt was madeand
to
esÈimate the relevance of the reverse Michael creavage of
ethacrynic acid bound to cST. Finally, the effects on the
DNPSG excretion were estimated in intact cells.
chapter 7 dears with the reversibre inhibition
of rat
and human cST by ethacrynic acid. Ethacrynic acid is a very
potent reversible inhibitor with lor.,_values in the range
of
<0.1-6 UM. fn particular the human"mu_class GST is very
susceptible to reversible inhibition.
The reversible
inhibitory capacity is retained after conjugation with
glutaÈhione. In the case of the alpha_ and mu_class the
reversibre inhibition is even stronger with the conjugate.
chapter 7 arso demonstrates that the susceptibirit; of GSr
for the irreversible
inhibition by ethacrynic acia is
relatively
lort7. No significant irreversible inhibition was
observed, using conditions similar to the TCBQ experiments
described in chapter 2.
Chapter 8 describes that incubation of cST of the pi_
class with ethacrynic acid using slightly more drastic
conditions (one hour at 370c) resurts in . Li..-a.pendent
inhibition.
About 0.g nrnor ethacrynic acid per n or h.r*.r,
P1-1 and 0.9 nmol per nmol rat cST 7_j could be
incorporated, resulting in 65-93* inhibition. Isoenzl¡mes of
the alpha- and mu-c1ass also bound ethacrynic acid
from 0.2-0.6 runot/runo1 enzyme), however without lranging
loss of
catalytic activity.
chapter g also dears wit.h the enhanced irreversible
inhibitory capacity of a newly synthesized dibromo dihydro
derivative of ethacrynic acid. This compound inhibits the
pi-class very efficiently,
resurting in 90-96u inhibition.
Interestingly,
this
compound is
also a powerful
-L32-
irreversible inhibitor of the mu-class
resulting in 52 to 70% inhibition-
GST
isoenzlmes,
of the conjugatj'on
Chapter 9 descrÍbes the reversibility
reaction of ethacrynic acid with glutathione and GST P1-1'
Prolonged incubation of the glutathione conjugate of
ethacrynic acid (up to 120-15O hr) with a fivefold molar
excess of N-acetyl-L-cysteine or GST Pl-f indeed showed a
time-dependent transfer of êthacrynic acid to N-acetyl-Lcysteine or GST P1-L. Moreover, ethacrynic acid bound to
GST P1-1, transferred to an excess of glutathione (within
l-20 hr), with concomitant fuII restoration of the catalytic
Íhus, depending on e.g- the relative
activity.
concentration of the reactants, transfer reactions may
occur in either direction. From the fact that Preincubltion
of GST P1-l- with CDNB reduced the incorporation of t clethacrynic acid, and from automated Edman degradation of
pepsine
the major radioactiye þeptide isolated after
it
enzlme,
labeled
acid
digestion of the ¡'=c1-ethacrynic
takes
acid
ethacrynic
of
reaction
the
that
was concluded
place with cysteioe 47 of GST P1-1.
The effects of ethacrynic acid and its dibro¡no dihydro
derivative on the DNPSG excretion in rat hepatoma cells was
linear relationship
presented in chapter 5. À significant
bet!,reen the concentration (0-50 uM) of ethacrynic acid and
its dibromo dihydro derivative and the DNPSG excretion was
observed, with a naximum of 30% and 50ts reduction of DNPSG
excretion for ethacrynic acid and its dibromo dihydro
derivative, respectively- At the end of the experiment the
intracellular DNDSG and glutathiOne levels f^tere similar to
control values. From the fact that GST activity in cells
lysed at the end of exposure period vtas also si-rnilar to
control_ values, it can be concluded that ethacrynic acid
inhibits intracellular GST in a reversible manner'
PART III
-134-
Chapter lL
General discussion and Sumary
The studies described in this thesis focused on
inhibitors of hunan and rat GST isoenzlmes. several
strategies can be envisaged for the discovery and design of
such agents- The GST have two sites for substrates binding,
one for glutathione, one for the second substrate. severa.l_
groups have concentrated on glutathione analogues, which
serectively bind GST isoenzlzmes in vitro (Graminski et ar.
1,989¡ Adang et a7. J.991; Castro et aJ. 1993). tte have
concentrated on compounds binding to the second substrate
binding site, which inactivate or irreversibly nodify the
enz]¡mes. Two groups of compounds were studied, fcBe/GsTCBe
and a series of other quinones and related catechols, and
ethacrynic acid and derivatives.
11.1 puiuones
our studies wiÈh rcBe originated from our investigations
the microsomar metaborism of hexachrorobenzene.
Hexachlorobenzene is mainly converted to pentachlorophenol,
while a small amount of tetrachloro-1r4-hydroquinone \,{as
also detected (Ommen et aI. 1996,. Ommen et al.. l9gga). From
the latter a reactive compound was identified which reacted
very efficientJ-y with glutaÈhione. This compound, viz. TCBQ
and in particular GsrcBe, inactivated arI studied rat csr
isoenzymes to a considerable extent (Onrien et aJ. 1gggb).
several experiments suggested that the quinone reacted with
a cysÈeine residue of GST (Van Om¡nen et al,. l9g9). In an
earlier sÈudy, in vitro inhibition
of GST mixtures by
3 t4,5,6-tetrachloro-Lr2-benzoquinone
(ort¡o-TcBe)
had
already been observed lDierickx, 1993). Most strikingly,
however, the gtutaÈhione conjugate inactivated the Gsr with
an increased rate as compared with the parent quinones.
this night indicate that these grutathione conjugates have
of
-135-
for the active site, thereby increasing the
affinity
selectiviÈy of the compound (the so-called
"targetting"
Indeed, data were obtained which suggested that
effect).
GSTCBQ reacted acÈive site-directed with rat GST (Van Ommen
et af . l-988b).
related
tr4The effect of several structurally
benzoquinones and 1r4-naphthoquinones !'tas subsequently
sLudied (vos et a7. L989). For the benzoquinones and the
naphthoquinones the extent of inhibition increased with an
increasing number of halogen substituents. Neither the tlpe
of halogen nor the position of the chlorine-atoms was of
major importance. The GST 3-3 was found to be the most
sensitive towards a whole series of inhibitors, while Èhe
activity of GsT 2-2 was least affected (vos et a7. l-989).
a series of halogenated glutathione conjugates of
Finally,
1r4-benzoquinones was studied using GST 1-l-. Again with
increasing numbers of chlorine substituents, the rate of
inhibition greatly increased (Van OrûÍien et af. l-991a). The
targetting effect, described as the ratio between the rates
of inhibition for a given guinone with and without the
f or
the
\^tas largest
glutathione
substituent,
GST
1-1,
l¡tas
that
which
suggested
It
dichtorobenzoquinones.
possesses tv¡o cysteine residues, htas inactivated by the
modification of one of these cysteine residues, which is
probably located near the active site. Compounds with
the glutathione
for the active site, like
affinity
conjugates, modify the one located near the active site.
complete
This was supported by the observation that
inactivation of GsT 1-l- by 2'S-dichlorobenzoquinone was
achieved only afÈer modification of tr./o residues, \4thereas
the corresponding glutathione conjugate already inhibited
after the modification of one (van ommen et al. L99La). The
target amino acid of the conjugate might be cysteine 11L,
s-(4-bro¡no-2,3since another irreversible GST inhibitor,
GST 1-1 by the
inactivated
dioxobutyl)glutathione
modification of this cysteine (Katusz et a7. 7992a).
In this thesis it was shown that TCBQ can be used to
inactivate human GST in vitro. Further indirect evídence
\.¡as provided that lCBg reacted with a cysteine residue,
since the cST A2-2 which does not contain cysteinesr was
the only isoenzyrne which did not incorporate TCBQ and thus
remained catalytically active. GSTCBQ only unambiguously
displayed the targetting effect wiÈh GST ÀL-1. In contrast.
inactivated by GSTCB9.
GST P1-1 was less effectively
results lrere obtained using the structurally
Similar
-
l_36
-
related
compounds, trichloro-benzoquinone
and
the
glutathione conjugate of 2,5-dichloro-1-, 4-beozoquinone lVan
Ommen et a7. L991b). The absence of a targetting effect
in
the pi-class might be explained by the fact that the GST of
the pi-class contains an accessible and reactive cysteine
residue (Reinemer et a-l . L99L; Caccuri et al. . L992 ), which
reacts more rapidly with the che¡nically more reactive TcBg
(the pure ¡mino acid cysteine reacts more rapidly with TCBQ
than with GSTCBQ). In this Èhesis it was also demonstrated
that despite Èhe effective cST inhibition in vitro, no
significant inhibition of the excreÈion of the glutathione
conjugate (DNPSG) by rat hepatoma cells was observed.
Horr¡ever, iÈ should be recognized that cSTCBg and TCBQ are,
despite their high reactivity \^rith cST, still very distinct
from the ideal so-called suicide inhibitor or trojan-horse
(Fersht L984), which should be chemically unreactive in the
absence of the target GST. Nevertheless¡ because of their
high potency of inactivation of GST isoenzymes. future
attempts to synthesize irreversible inhibitors may be based
on quinone structures. Recently, the three dimensional
structure of some GST isoenzymes became available (Reinemer
et a7. 1-991; Ji et aJ-. L992), which might facilitate a more
rational inhibitor design. In cooperation with prof.Dr. RN
Àrmstrong (University
of Maryland, USA) studies are
currently in progress to identify the target site of GSTCBQ
on GST 3-3. From the three dimensional structure, target
amino acid were identified which might be involved in the
binding of cSTCBg to cST 3-3. These amino acids (Tyr6,
Cys86, Cys11-4, Tyr1l5, CI73) have been replaced by serine
or phenylalanine with site-direcÈed mutagenesis. These cST
mutants may be helpful in identifying the target site. The
studies of Katusz and co-workers (Katusz et al_. 1991-,
1992a, L992b) with the strucÈurally related compound S-(4_
bromo-2,3-dioxobutyl)gluLathione clearly demonstrate Èhat
the target amino acid might vary (a cysteine, tyrosine
and,/or tyrosine and cysteine, for GST l--1, GST 4-4, and GST
3-3, respectively). Thus, in an effort to prepare isoenzl,me
selective inhibitors,
aII the isoenzymes may have to be
studied individually.
In this thesis, the in vitro irreversibty inhibitory
potential for GST by catechol derived quinones and the
reversibLe inhibitory poÈential of the parent sÈructures
vtere determined. These compounds might provide a "pro-drug"
concept, since their oxidation generates quinones. The
-L37-
studied catechols, ví2. caffeic acid and dopamine and qnethyldopa are relatively non-toxic and thus might be
applicable in some cells.
Caffeic acid is widely
distributed in the plant kingdorn, while the catecholamines
are present in high concentration in the nervous systern.
Catecholamines are alo known for
their
role in
melanogenesis. Especially those cells
with
high
concentrations of the cST pi-class and with optimal
conditions for quinone formation mighÈ be sensitive to
irreversible inactivation, since the GST of the pi-class is
by far the most sensitive to the inactivation by the
quinones derived from both groups of compounds. Hohrever,
unlike the catecholamines, caffeic acid was also an
effective reversible inhibitor of the major GST isoenzlnnes
studied. Both the glutathione conjugate of either eaffeic
acid and the catecholamines display reversibly inhibitory
capacity of all GST isoenzlzmes studied. This productinhibition
of the enzyme, seems Èo be a conmon property of
almost any glutathione conjugate studied sofar
(Van
Bladeren and Van Om¡nen, L99L).
The pharmacokinetics of these compounds wiII
strongly
influence the in vivo inhibition of GST. In thi-s conLext it
was shown that a single oral dose of caffeic acid, gave no
major irreversible inhibition of GST in intestinat mucosa,
kidney, or liver.
The use of radioactive
labelled
compoundsr ês well the estimation of the glutathione
conjugation with model substrates (to determine the total
inhibition
by glutathione depletion, reversible and
irreversible inhibition) is indicated.
LL.2 Etbacrynic acid
Ethacrynic acid is a potent diuretic drug, introduced as
therapeutic agent in the middle sixties (I¡Iillianson L977).
This compound has recently been studied in a phase I
clinical
trial,
for its application in the treatment of
drug resistance of the alkylat,or phenotlpe (O'D\isyer et aJ..
1991). Relatively high doses of ethacrynic acid could be
administered without toxic effects occurringr.
Ethacrynic acid may react spontaneously or GST cat.alyzed
with gluÈathione. For most isoenzymes the catalytic
activity
of GSf tolrards ethacrynic acid is not high. This
may be compensated by the high concentration of several
isoenzlnnes (Mannervik L985, Van Ommen et a-l. 1990). From
acid,
ethacrynic
in principle
tr,¡o diastereomeric
-138-
glutathione conjugates may be formed, due to the formation
of a new optically active center during Èhe conjugation
reaction.
However, NMR studies indicated that both
d.iastereomers are formed in about equal amounts
(unpublished results),
indicating that the enzymatic
reaction
does not proceed with
an
appreciable
stereoselectivity.
In vivo, in the rat dur+2g the first
60 minutes afÈer
intravenous injection,
[*-C]ethacrynic acid is rapidly
excreted, with 80-90% of the label appearing in bile and
the remainder in urine (Klaassen and Fitzgerald 1_974,
$raIlin et aL. 1978). In bile, the major metabolite is the
glutaÈhione conjugate, comprising about 6O% of the labelled
material (Klaassen and Fitzgerald L974, Wallin et aL.
1978). Induction of hepatic GST by phenobarbital increased
biliary excretion of the glutathione conjugate (ltallin et
a7. 1978). This supports the view that the enzymatic
catalysis is predominant.
Ethacrynic acid has been shown Èo inhibit rat GST
reversibly
(,Ahokas et aL. l_985 ) . f n this thesis the
concentrations of ethacrynic acid resulting in 5O?
inhibition of the enzlzmatic activity
(I_^-values) to$¡ards
CDNB were determined. Strong inhibitioìuof both human and
rat GST isoenzyrnes of the alpha-, ßu-, and pi-class $ras
obtained with ethacrynic acid. The mu-class was most
sensitive to the reversible inhibition by ethacrynic acid,
as has also been sho$¡n by Hansson et aL. (i-991).
The dibromo dihydro derivative of ethacrynic acid shov¡ed
a similar potential for reversible inhibition as ethacrynic
acial itself. This suggests that the a,B-unsaturated ketone
moiety is not very critical
for the inhibitory action.
Presumably, the inrportant structural element in the
molecule is the 2,3-dichloro-phenoxyacetic acid moiety. Tvio
compounds with this moiety, tienilic
acid and indacrynic
acid, have been shown to be strong inhibitors as well
(Àhokas et al-. 1-985). On the other hand, the closely
related 2,4,S-Erichlorophenoxyacetic acid (2t4,5-T) and
some structural analogs did not efficiently
inhibit cST
(Vessey and Boyer 1988). Thus, newly developed derivatives
of ethacrynic acid should contain this critical element.
The glutathione conjugate of ethacrynic acid reversibly
inhibited GST isoenzymes of alpha- and mu-class even more
efficiently
than the parent compound, especially the human
isoenzymes. In human liver cytosol,
the glutathione
conjugate was also more potent (approxinately one order of
-139-
magnitude) than ethacrynic acid ltakamatsu and Inaba Igg2).
The inhibitory
effects of the cysteine conjugate and the
mercapturate produced by further metabolism of the initial
gluÈathione
conjugate
of
ethacrynic acid,
\.rere
approximately t!,/o orders of magnitude less than the parent
ethacrynic acid in human liver cytosol (Takamatsu and Inaba
1992). This suggest that the glutathione moiety is critical
for the inhibitory
action of the glutathione conjugate.
Since ethacrynic acid is very efficiently
conjugat,ed with
glutathione, this compound will be responsible for the
major part of the (reversibte) inhibition in vivo.
Àlready in 1,978, it hras shov¡n that a small but
consistent çgrtion (approximately 1%) of an administered
dose of [-'C]ethacrynic acid covalently binds to both rat
liver and kidney cytosol (Wallin et al. t97g). Upon furÈher
purification,
it
$/as shohrn that
ethacrynic acid
specificalJ-y binds to rat mu-class GST isoenzl'me g-4
(Yamada and Kaplowitz 1980).
f,Ie performed studies to assess the relevance of this
covalent binding to the enzl¡matic activity, which revealed
no significant inactivation of cST 3-4 by covalent binding
of ethacrynic acid, or for any of the other isoenzymes
studied. On the other hand, using stightly more drastj_c
conditions, the catalytic activity of the cST of the piclass isoenzymes lr¡ere strongJ-y inactivated by covalent
binding. It vras shown that the reaction of ethacrynic acid
takes place with cysteine 47 of cST p1-1. Thus ethacrynic
acid reacts with a cysteine residue, which has been
recognized as a highly reactive thiol in Lhe proximity of
the catalytic site of cST 7-7 and human cST pl-l (Tamai et
aL. L99O, Ricci et a7. 1991). The structural integrity
of
this cysteine seems to be crucial for maintaining an
active-enzyme conformation, despite its none-involvement in
either substrate binding or catalysis (Reinemer et aL.
79921.
The dibromo dihydro derivative of ethacrynic acid was
synthesized in an effort t.o enhance the covalent binding
characteristics .
Dibromo dihydro
ethacrynic acid
inactivated pi-class cST more efficiently than the parent
compound, while reversible inhi_bitory capacity of the
courpound was similar to ethacrynic acid (see above). Thus,
this
compound combines the properties of a strong
Iipophilic irreversible and reversible inhibitor, making it
a potential tool- to inhibit GST in biological systems. This
was confirmed in rat hepatoma cells,
in which the DNPSG
-L40-
excretion vras more efficiently inhibited by dibromo dihydro
derivaÈive Èhan ethacrynic acid itself.
Normally, in irreversible inhibition the inhibitor is
covalently línked to the enzlzrne so tightly
that its
dissocj-ation from the enzl¡me is very slow. Horvever, the
Michael addition of glutathione with the a,B-unsaturated
aldehydes and ketones is a well-known reversible reaction
(Witz 1989). Via this so-called retro Michael cleavage, the
inactivation of the pi-class GST may be reversed by release
of ethacrynic acid, when the concentration of free
ethacrynic acid decreases (e.9. as a result of transport or
conjugation with glutathione). We sho\^/ed that
full
restoration of the catalytic activity of cST p1-1 r"rhich was
inactivated by ethacrynic acid (>9Ot), could actualLy
occur by prolonged incubaÈion in the presence of
glutathione. The velocity of the retro Michael cJ-eavage at
room temperature vras relatively low. However the rate of
this reaction will be increased by the higher temperature
in vivo. This regeneraÈion of the unmodified native cST p11 enzyme by release of a covalently bound inhibitor may
have
several implications.
ÀIthough under normal
physiological cond.itions the inhibition
of cST pL-1 by
covalent binding of ethacrynic acid would be reversed by
glutathione,
leaving reversible
inhibition
by the
glutathione conjugate and ethacrynic acid itself as the
main mechanism of inhibition, when glutathione levels are
low, the covalent inhibition might be predominant resulting
in a completely different time course of inhibition.
In conclusion, ethacrynic acid is a particularly
interesting inhibitor of GST, since it inhibits all cST
reversibTy and cST of pi-class irreversibly. A major part
of the inhibition due to covalent binding of ethacrynic
acid to cST Pl--l- may be reversible in vivo. As also
concluded for the quinones, studies using model substrates
to asselt glutaÈhione conjugation in vivo as well as the
use of [*'c]ethacrynic acid are clearly indicated.
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-L45-
Sanenvatting
Glutathion S-transferases
(GST) vervullen
een
belangrijke ro1 in de biotransformatie van een groot aantar
reaktieve verbindingen. De GST van rat en mens komen voor
als een groep van isoenzymen/ die ingedeeld kan worden in 4
klassen: alpha, mu, pi en theta.
De belangrijkste functie van de GST isoenzymen is de
katalyse van de conjugatie van electrofiele,
hydrofobe
verbindingen met het t,ripeptide glutathion. Deze
reaktie
kan in het argemeen beschouwd worden als een detoxificatiestap.
De inhibitie
van GST wordt om een aantal
bestudeerd. Zo worden remmers van cST gebruikt redenen
om het
katalytisch mechanisme en de opbouw van heÈ akti-eve centrum
te bestuderen. Isoenzfrm selektieve remmers worden eveneens
gebruikt om de indeting van csr in verschirrende krassen
onderbouwen. Daarnaast kan remming van GST mogelijk te
ook
relevantie hebben voor bepaalde vormen van geneesmiddel_
resistentie.
Met name de resistenti.
t.g"r, alkylerende
cytostatica (zoals chlorambucil, melphatan e.d.¡ wårat in
verband gebracht met GST. Zo wordÈ een verhoogale GST
expressie vraargenomen i-n tumorcerlen resistent tegen deze
alkyrerende cytostatica- Deze cytostatica blijken
feschikte
substraten van cST te zijn, \¡¡aardoor GST deze cyiostatica
kan inaktiveren.
De studies beschreven in dit proefschrift
z'jn gericht
op de inhibitie
van Gsr van rat en mens. verschirlende
benaderingen kunnen gekozen worden voor de ontwikketing
van
remmers - zo bestuderen verschirlende groepen
onderzoekers
de glutaÈhion analoga. rn dit proefschrift is de aandacht
gericht
op de zogenaamde "t$reede substraten,,, de
xenobiotica, en dan met name stoffen die cST remmen middels
een covarente interaktie.
Tf^7ee groepen van verbindingen
werden bestudeerd: de gehalogeneerde chinonen
en
gerelateerde catechoren, en etacrynezuur (EA) en derivaten.
In hoofdstuk l- wordt een overzicht gegeven van de
eigenschappen, funkties, expressie en katarytische
aspekten
-L46-
van GST isoenzymen, alsmede de rol van GSI in geneesmiddel
resistentie. Verder worden enkele theoretische aspekten van
reversibele en irreversibele rernning, toegespitst op GSTf
beschreven.
De verbinding tetrachloor-1-, -benzochinon (TCBQ) is in
onze werkgroep gebruikt als modelstof om de irreversibele
remming van cST Èe bestuderen. TCBQ bindt aan alle
bestudeerde rat GST isoenzf¡menf waardoor GST geï.naktiveerd
v¡ordt. Het glutathionconjugaat van TCBQ (GSTCBQ) bfijkt GST
van het
nog sneller te inaktiveren dan TCBQ' De affiniteit
glutathiongedeelte voor heÈ aktieve centrum verhoogt de
van de remmer aanzienlijk.
selektiviteit
In hoofdstuk 2 wordt beschreven dat TCBQ en GSTCBQ ook
humane GST isoenzymen kunnen remmen. Vrijwel volledige
irreversibele renming van GsT van alpha-. ßu-r en piklassen werd bereikt met tienmaal overmaat chinon. Deze
inhibitie ging gepaard met de inbouw van één molecuul TCBQ
per cST subunit. Het isoeñzlm GST À2-2 bevat a1s enige geen
cysteines, en bleek ook het enige bestudeerde isoenzym te
zj,jo dat niet geremd werd en geen TCBQ inbouwde. Chinonen
reagleren over het algemeen snel met cysteines. Het fijkt
dus aannemelijk dat in de buurt van het aktieve centrum een
cysteineresidu aanwezig is.
Bij de humane GST werd alleen voor het alpha-klasse
snellere inaktivatie door
isoenzym À1-1 een aanzienlijk
GSTCBQ hraargenomen.
Hoofdstuk 3 handelt over de irreversibele remming van rat
GST isoenzymen door chinonen gevormd uit het catechol
caffeic acid, alsmede de reversibele remming door caffeic
acid zeLf. Deze verbinding kan beschouwd worden als een
"pro-drug'", daar de oxidatie van caffeic acid het chinon
genereert. Caffeic acid is relatief niet-gifÈig en komt
planten in
hoge
relatief
alle
voor in vrijwel
Caffeic acid bleek een redelijk goede
concentratíes.
reversiÞ,ele remmer van GSf te zijn. Het glutathionconjugaat
van caffeic acid 12-GSCÀ) \¡tas een nog sterkere reversibele
remmer. Aan de andere kant, resulteerde alleen de oxidatie
van caffeic acid in sterke irreversibe-Ze remmer van GST,
met name van de pi-klasse.
fn Hoofdstuk 3 wordt ook het effekt van een eenmalige
orale dosis van caffeic acid op de irreversibeJ-e remning
in lever, nier en darmmucosa
van de GST aktiviteit
beschreven. ÀIIéén in de lever werd een marginale
irreversibele remming waargenomen. Geadviseerd werd om in
schenken aan
toekomstige studies vooral aandacht te
-r47-
reversibele
reruning
door caffeic
acid en zijn
glutathionconjugaat .
Hoofdstuk 4 behandelt de remming van humane GST door de
catecholamines dopamine en methyldopa. Ook oxidatie van
catechoramines leidt tot de vorming van reaktieve chinonen.
catecholamines spelen een ror a1s neurotransmitters i-n het
zenuwstelsel, maar zíjn ook bekend van\¡¡ege hun rol in de
pigmentvorming in de huid. Methyldopa wordt ook toegepast
als anti-hypertensium. De catecholamines zelf bleken GST
niet reversibel te renmen, terwijl
hun gtutathion
conjugaten we1 over reversibele remmingseigenschappen
bleken te beschikken. De chinonen gevormd uit
de
catecholamines remde het cST van de pi-klasse (pl-j_) sterk
irreversibel.. Ook het glutathionconjugaat van dopamine ( 5GSDA) bleek dit isoenzym (in mindere mate) irreversibel te
remmen. Àanwijzingen werden verkregen dat de remming van
csr P1-1 door dopamine deers het gevorg r/sas van disulfide
vorming in het enzl¡m, terwijl
de remming door 5_cSDÀ
volledig het gevolg hras van de vorming van disulfides.
Hoofdstuk 5 behandelt de effekten van niet_giftige
concentraties TCBQ en GSTCBQ (waarvan de glycine
carboxyrgroep veresterd was om de absorptie te verbeteren)
op de uitscheiding in het medium van heÈ intracellulair
gevormd.e
glutathionconjugaat
van
1-chloro-2 r 4dinitrobenzeen (DNPSG) bij rat hepatoma cellen. In deze
cellen werd geen significante
reductie van deze
uitscheiding gevonden en geen effekt op de GST aktiviteit
in het supernatant. Dus TCBQ en zíjn
afgeleide
glutathionconjugaat zijn niet geschikt om GST te remmen in
celsystemen.
Hoofdstuk 6 vat kort arle studies met de chinonen samen.
De studies beschreven in hoofdstuk 7, g en 9 handelen
over de rernming van GST door etacrynezuur (EA). Sinds de
zestiger jaren wordt EÄ als diureticum toegepast. In 19g5f
werd aangetoond dat EÀ over goede remmingskarakteristieken
van rat GST beschikt. Bovendien kan EA in relatief
hoge
dosis aan nensen worden toegediend.
In de literatuur bestond enige discussie over de aard
van de remming: zower reversibele remming ars covalente
binding aan rat Gsr 3-4 was beschreven. rn hoofdstuk 7 v¡erd
de aard van de remming onderzocht. Er werd geen
significante rer'ring van GST door EÀ opgespoord onder
condities waarbij TCBQ de csr isoenzymen irreversi-ber
remde. Vastgesteld werd dat EÀ vooral een sterke
reversibele renmer van rat en humane GSf is. Het
-148-
glutathionconjugaat van EA (GSEA) bleek de alpha- en muklasse GST zelfs nog sterker reversibel te remmen. GSEÀ
blijkt een van de meest potente reversibele remmers van GST
te zijn.
Door gebruik te maken van meer drastische ornstandigheden
(hogere temperatuur, langere incubatietijd¡
werd in
hoofdstuk 8 vastgesteld dat selektieve
irreversibeJ-e
remming van GST van de pi-klasse door EÀ toch kan optreden.
Ca. 0.8 nmol EA per nmol humaan of rat GST van de pi-klasse
kon worden ingebouwd, hetgeen gepaard ging met 65-95%
remming.
Hoofdstuk I handelt ook over de remmingseigenschappen
van een gesynthetiseerd dibromo dihydro EÀ derivaat. Dit
derivaat bleek de cST pi-klasse zeer efficiënt irreversibel
te remmen, terwijl bovendien ook de mu-klasse geremd werd.
De chemische reactie betrokken in de conjugatie van EA
en aanverv¡ante arp-onverzadigde ketonen, een Michael
additie,
is
in prinCipe reversibel. Daarom werd de
reversibiliteit
van de conjugatie reactie van EA met
glutathion en cST P1-1 bestudeerd (hoofdstuk 9). Door lange
tijd te incuberen (tot 150 uur), werd aangetoond dat EA
gebonden aan zowel glutathion als cST P1-1, volledig kan
overstappenr op een overmaat van respectievefijk
N-acetylL-cysteine of glutathion. Afhankelijk van de concentraÈie
van de stoffen, kunnen transfer
reacties
in beide
richtingen optreden.
Middels Edman degradatie van het peptide \^¡aaraan EA
gebonden was, werd het cysteine residu h¡aarmee EÀ reageert,
geïdentificeerd (Cysteine 47).
De effekten van EA (en zijn dibromo dihydro derivaat) op
de uit.scheiding van DNPSG in rat hepatoma cellen, werd
beschreven in hoofdstuk 5. EA en dibromo d.ihydro derivaat
bleken deze uitscheiding effectief
te remmen, met een
maximum van 303 en 50% reductie voor respectievelijk EÀ en
zj-jn derivaat. Aanwijzingen werden verkregen dat deze
remming mj-ddels reversibele GST remming tot stand kornt.
Hoofdstuk 10 vat kort aIle experimenten met EÀ samen.
In hoofdstuk 11 worden de resultaten met de chinonen en EÀ
bediscussieerd en vergeleken met andere studies.
Geconcludeerd hrordt dat de gehalogeneerde chinonen vaû^¡ege
hun goede remmingseigenschappen van humane GST gebruikt
kunnen worden als basisstof voor de ontwikkelingen van een
in vivo werkzame suicide-inhibitor. Bij de chinonen zullen
toekomstige studies o.a. gericht zijn op het vaststellen
van het aminozuur van GST waarmee GSTCBQ reageertr om
-r49-
zodoende een op chinonen gebaseerde rationere drug-design
te vereenvoudigen. De toepasbaarheid van irreversibere
rernming van csr van de pi-krasse in biorogische systemen
door de catecholen, kan onderzocht worden in cellen rijk
aan dit GST en,/of met optimare condities voor chinonvorming
(zoals b.v. in melanoma ce1len). Op grond van de bepaalde
remmingsconstantes kan verwacht worden dat caffeic acid en
zijn glutathionconjugaat ook geschikt zíjn
om cST
reversibel te remmen in vivo. voor EA kan geconcludeerd
worden dat het een belangwekkende remmer van GSI is, omdat
het reversibel aIIe GST remt en irreversibel GST van de pi_
klasse. De reversibele remming kan in vivo nog vergroot
worden door de vorming van het grutathionconjugaat van EÀ.
Een belangrijk gedeelte van de inhibitie
als gevolg van
covalente binding van EA aan GST pl--1, kan in vivo
reversibel zijn. Het gebruik van radioaktief geraberde
stoffen
van de catecholen, chinonen en/of EA wordt
aanbevolen om de remming in vivo te onderzoeken.
-150-
List of publicat,ions
Ploemen JHTM, van ommen B and van Bladeren pJ. rnhibition
of the rat and human glutathione s-transferase isoenzymes
by eÈhacrynic acid and its grutathione conjugate. Biochem
Pharmacol- 40: 1631--1635 , 1990 .
Proemen JHTM, van oin¡nen B and Van Bladeren pJ. rrreversible
inhibition
of human grutathione s-transferase isoenzymes
by tetrachloro-l-,A-benzoquinone and its
glutathione
conjugate. Biochem pharmacol 41: 1665-1669t Lggt.
Ploemen JHTM, Bogaards JJp, Veldink GA, Van Orunen B, Jansen
DHM and Van Bladeren pJ. Isoenz]¡mes selective
irreversible inhibition of rat and human glutathione stransferases bt ethacrynic acid and two brominated
derivatives. Biocåem pharmacoJ_ 45: 633-639t Lgg3.
Ploemen JHTM, Van Ommen B, de Haan À, Schefferlie Jc and
van Braderen pJ. rn vitro and i-n vivo irreversibre and
reversibl-e inhibition of rat grutathione s-transferase
isoenzymes by caffeic acid and its 2*S_glutathionyt
conjugate. Fd Chen Toxic 3j.: 475-482, l-gg3.
Proemen JHTM, van omnen B/ Bogaards JJp and van Braderen
PJ. Ethacrynic acid and its glutathione conjugaÈe as
inhibitors of glutathione s-transferases. xenobiotica
232 9L3-923t 7993.
Ploemen JHTM/ Van Ommen B, de Haan ÀM, Venekamp JC and Van
Bladeren pJ. Inhibition
of human glutathione S_
transferases by dopamine, a-methyldopa and their 5_S_
glutathionyl conjugates. Cåem-BioJ- Interactions In press.
Às co-author
Van Ommen B, Den Besten C/ Rutten ALM, ploemen JHTM/ Vos
RME, Müì_Ier F, and Van Bladeren pJ. Àctive site directed
irreversible
inhibition of grutathione s-transferases by
glutathione
the
conjugate
of
tetrachloro_I,4_
benzoquinone. J BioI chen 263: I2939_t2942, l_9gg.
Van Ommen B, Ploemen JHTM, Ruven HJ, Vos RME, Bogaards JJp,
Van Berkel- WJH, and Van Bladeren pJ. Studies on the
active site of glutathione S-transferase isoenzyme 4_4.
chemical- modifications by tetrachroro-1,4-benzoquinone
and its glutathione conjugate. Eur J Biochem 1g1: 423_
429 , t_989.
Van Ommen B, Ploemen JHTM, Bogaards JJp/ Monks TJ and Van
Bladeren PJ- rrreversible inhibition of rat grutathione
-
151"
-
S-transferase 1-1 by quinones and thej-r glutathione
relationship and
structure-activity
conjugates:
mechanism. Biochem J 276: 66l--666, I99L.
Van Ornmen Bt Bogaards JJP, Ploemen JHTM' Van der Greef J
and Van Bladeren PJ. Quinones and their glutathione
conjugates as irreversibte inhibitors of glutathione Stransferases. In: Vlitmer CM (Ed. ) et al. Biological
reactive intermediates IV. Ne$¡ York, Plenum Press t ]-99]-.
Advances in experimental medicine and biology, vol. 283
van Bladeren PJ, Ploenen JHTM and van Ommen B. Glutathione
s-transferase polymorphism in relation to toxicity.
(Ed. ).
Developments and ethical
MI
Weitzner
considerations in toxicology. Cambridge: The royal
society of chemistry, L993, p.69-85.
Nagengast FM, van den Ban G, Ploemen JHTM' Leenen R¡ Zock
PL, Katan MB, Hectors MPC| de Haan AFJ, van Tongen JHM.
The effect of a natural high-fibre diet on faecal and
biliary bile acids, faecal pH and whole-gut transmit time
in man. A controlled study. Eur J Cfin Invest 47 z 63L639, 1993-
-r52-
Curriculum vitae
Jan-Peter Ploenen hrerd op 1_ november 1963 t,e Beek (L)
geboren. De middelbare school periode werd doorlopen op de
Scholengemeenschap "groenewald" te Stein, hraarop in 1982
het diploma Àtheneum B werd behaald. In dat zelfde jaar
\"/erd begionnen met de sÈudie Humane Voeding aan de
Landbouwuniversiteit l{ageningen. Àls specialisatie-richting
binnen de studie werd de oriêntatie loxicologie gekozen.
Tijdens de doctoraalfase werden de hoofdvakken Humane
Voeding (Prof.Dr. M.B. Katan), Toxicologie (Prof.Dr. J.H.
Koeman), en Biochemie (Prof.Dr. F. MüIIer) afgelegd. Een
stage Toxicologj-e (Dr. J.Vü.c.M. V'iilmer¡ werd afgelegd op de
afdeling Biologische loxicologie van het TNO Instituut voor
Toxicologie (TNO-ITV) te Zeist. Het doctoraaÌexamen werd in
juni 1988 afgelegd. Àansluitend werd de mj-l-itaire dienst
vervuld.
Op 1 september 1989 trad hij in dienst als onderzoeker
in
opleiding op een project gefinancieerd door de
Nederlandse Organisatie voor wetenschappelijk Onderzoek
(NvüO, gebied Medische fùetenschappen) / bij de afdeling
Biologische ToxicoLogie van TNO-ITV, alwaar het in dit
proefschrift
beschreven onderzoek werd uitgevoerd onder
Ieiding van Prof.Dr. P.J. van Bladeren. Vanaf september
1993 is hij aldaar werkzaam als onderzoeker op een
Stichting Technisch V'Ietenschappen gefinancieerd project
onder leiding van Prof.Dr. P.J. van Bladeren en Prof.Dr.
N.P.E. Vermeulen.
-
r-53
-
Nanroord
En eindelijk kun je dan het laatste gedeelte schrijven.
Vrijwel de laatste produktieve handeling leidt tot de
waarschijnlijk meest gelezen pagina's van dit proefschrift!
Op de achtergrond hoor ik de muziek van MTv, en in het
bijzonder één regel valt me op: "...r¡¡as it worth it?..."
(Petshop Boys). Ja, het was het waard!
Aan dit proefschrift heeft een aantal mensen een
cruciale bijdrage geleverd.
Peter van Bladeren, jouw bijdrage h¡as van zeer grote
waarde. De tekst van aIIe artikelen werd kritisch maar
vooral ook opbouwend van conmentaar voorzien. Onze
werkgroep stuur je op voortreffelijke wijze. NieÈ alleen
jouw vaak geroemde v/etenschappelijke kennis garandeert een
hoge produkt.iviteit,
doch ook je prettige persoonlijke
omgang.
Ben van Ommen, ook jouw bijdrage $ras van essentieel
belang. Zeker in het begin zorgde je voor een bliksemstart.
En het bleef me iedere keer weer verbazen dat je creatieve
maar vooral ook razendsnelle hersenkronkels, zo vaak de
juiste ingevingen waren om een probleem op te lossen.
Vic Feron, onze frequente meetings hadden een niet te
onderschatten waarde voor mij: je hield de voortgang
perfect in de gaten, en later scherpte je de uiteindelijke
vraag- en doelstelling aan. Mijn opleiding tot Toxicoloog
handen.
was in voortreffefijke
VeIe mensen hebben labwerkzaarnheden voor dit project
verricht. De studenten: Dilian Jansen¿ Annne¡narie de Haan,
Johan Schefferlie, Arne van Schanke en Annelj-es Keijzer,
wil ik allen bedanken voor hun essentiële participatie
aan
dit onderzoek. Jan Bogaards wil ik bedanken voor de
synthese van enkele verbindingen. En natuurlijk
wil ik
iedereen van mijn sectie (Moleculaire ToxicoÌogie) bedanken
voor de goede (vierk)sfeer.
Prof.Dr. R.N. Armstrong (University of Maryland, USA) it
\4ras a real privilege to work at your laboratory.
À very
significant part of the rapid recent progress in the
knowledge of the mechanisms underlying the catalysis of GST
to your lab. I hope that our
can be attributed
collaboration will continue in t,he future, and eventually
will Iead to the identification of the target amino acids
for GSTCBQ in rat GST 3-3.
-754-
BilI
Johnson, v/orking together r¡/as not only very
productive, but also big fun. Dear Bill,
moreover the
barbaric Dutch changed his view on politics much more than
he admitted to the best race-biking republican!
Sonja Jespersen (afdeling Structuuropheldering¡ much
progress was made by the great job you did in the analysis
of the peptides.
Jack Vogels en Joke venekamp bedankt voor jullie
voortreffelijke hulp bij de analyse van de NMR-spectra.
ook het secretariaat was altijd
zeex behulpzaam, dank
aan Karin Hoefs en i.h.b. aan Jolanda de Bruin voor het
tlpen van 4 hoofdstukken (naar ik begrijp heb je erg
genoten van de speciale karakters! ).
De sectie cenetische Toxicologie v/as altijd
behulpzaam,
en in het bijzonder Nico de Vogel en Winfried teeman
verzagen mij van de nodige goede adviezen over celkweektechnieken.
ÀIle AIO's van de afdeling zorgden voor de optimale mix
tussen wetenschap/ teamgeest en ontspanning. De getoonde
interesse en betrokkenheid voor dit project was van grote
waarde. En wellicht kan de frequentie van de stapavonden
over ca. 1 jaar (post-doc's)
dan eindelijk
worden
opgevoerd
!
Karel Kagie, dank voor jouw hulp bij het totstandkomen
bij dit boekje. zoals altijd, heb je alles snel én perfect
geregelil.
Iedereen die ik vergeet te vermelden, excuses maar
vooral ook bedankt!
Jacqueline, dit proefschrift zou nooit voltooid zijn
zonder jouw steun. Ook in periodes waarin ik volledig
gegrepen werd door de wetenschap, kon ik steeds op jouw
begrip rekenen. Daarnaast zorgde je ervoor dat mijn leven
niet alleen uit onderzoek bestond, waarvoor ik je erg
dankbaar ben. En voor de toekomst, qéên overschot aan
vakantiedagen meer: India, here we come!
- 155 -
Alrbreviations
CDNB:
DNPSG:
2-GSCÀ¡
5-GSDÀ:
cS ( ethyl ) TCBQ:
l-chLoro-2 ,4-dinitrobenzene
( 2 , -dinitrophenyl ) glutathione
2-S-glutathionylcaffeic acid
s-S-glutathionyldopanine
2-S- t ( y-L-glutanlrl-L-cysteinyl ( ethyl
glycinate¡ )-3 | 4 | s-trichloro-1", 4-
S-
benzoqulnone
GSH:
5-GSMDOPA:
GST:
GSTCBQ:
glutathione
5-S-glutathionyl-a-methyldopa
glutaÈhione s-transferases
2-s-glutathionyl-3 , 5 , 6-trichlorobenzoquinone
TCBQ:
:
TFA:
TCHQ
tetrachloro- 1 r 4-benzoquinone
tetrachloro- 1 , 4-hydroqu inone
trifluoroacetic acid
1r
4-