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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 Àceto À, Caccuri ÀM, Sacchetta P' Bucciarelli T, Dragani B, Dlssociation and Rosato N, Federici G and Di IIio. unfolding of pi-class glutathione transferases " Biochem J 2852 247-245t t992. Àdams PÀ and Sikakana CNT. 1-Chloro-2r4-dinitrobenzenemediated irreversible inactivation of acidic glutathione S-transferases. Biochem Pharmacol 43¿ t757-1760' 1992. Adang ÀEP, Brussee J, van Der Gen A and Mulder GJ. The glutathione-binding site in glutathione S-transferases. Biochem J 269: 47-54, L99O. Adang AEP, Brussee J' Van Der Gen À and Mu1der GJliver glutathione S-transferase of rat Inhibition isoenzymes by peptides stabilized against degradation by y-glutamyl transpeptidase. J BioT Chem 266: 830-836, 199 1. Àdang AEP, Moree WJr Brussee J, Mulder GJ and Van Der Gen. by S-transferase 3-3 of glutathione Inhibition glutathione derivatives that bind covalently to the active site. Biochem J 27A: 63-68, 1991Àhokas JT, Nicholls FA, Ravenscroft PJ and Enmerson BT. glutathione Sliver rat of purified lnhibition isoenzymes by diuretic drugs ' Biochem transferase Phazmacol 34t 21.57-2L6L' L985. Àsaoka K. Inactivation of bovine liver glutathione Sof arginine modification transferases by specific residues with phenylglyoxal. J Enzyme Inhibition 3: 7780, 1989. Armstrong RN. Enzyme-catalYzed detoxif ication reactions : Mechanisms and stereochemistrY. CRC Crit Rev Biochem 222 39-87, 1987. reaction Armstrong RN. Glutathione S-transferases: mechanism, structure, and function- Chem Res Toxicol 4t 131-140 , 7,99L. Askelöf P, Guthenberg Ct Jakobson I and Mannervik B. Purification and characterization of two glutathione Sfrom rat liver. Biochem J aryltransferase activities 147: 513-522 ' L975. Äwasthi YC, Bhatnagar À and Singh SV- Evidence for the of involvement of a histidine at the active site glutathione S-transferase ry' from human liver. Biochem Biophys Res Comm L43: 965-970t 1987Black SM, Beggs JD, Hayes JD, Bartoszek A, Murumatsu M, Sakai M and wolf CR. Expression of human glutathione Sconfers in Saccharomyce cerevisiae transferases -16- resistance to the anticancer drugs adriamycin and chlorambucLl. Biochen J 26g: 309-31_5, tg9T. Brack sM and wolf cR. The rore of grutathione-dependent enzfrmes in drug resistance. pharmac Ther 5L: 13g-154, 1991_. Borst P. Genetic mechanism of drug resistance. Acta Oncologica 3O: 87-1"05 | L99I. Caccuri ÀM, petruzzell_i R, polizio F, Federici G. InhibiÈion of glutathione. transferase n from human placenta by L-chroro-2r4-dinitrobenzene occurs because of covalent reaction with cysteine 47. Arch Biochem Biophys 2972 La9-I22, Lgg2. Carne Tt Tipping E and Ketterer B. The binding and catalytic activities of forms of ligandin after modification of its thiol groups . Biochem J L77 z 433_ 439, L979. Chen WL, Hsieh JC, Hong JL, Tsai Sp and Tan MF. Site_ directed mutagenesis and chemicar modification of cysteine residues of rat grutathione s-transferase 3-3. Biochem J 2862 2O5-21O, 1992. Ciaccio PJ, Tew KD and taCteta Fp. Enzlrmatic conjugation of chlorambucil with gtutathione by human glutathione stransferases and inhibition by ethacrynic acid. Biochem Phazmacol 422 L5O4-J'SO7, J9gL. clark Ac' Darby FJ and smith JN. species differences in the inhil¡ition of glutathione s-aryltransferase by phthaleins and dicarboxylic acids Biochem J LO3 : 49-54, 1967 . Coles B and Ketterer B. The role of glutathione S_ transferases in cheuricat carcinogenis. cRc crit Re', Biochem 25, 47-80, L99O. Desideri À, Caccuri A[,I, polizio F, Bastoni R and Federici G- Erectron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta grutathione transferase. J BioJ Chem 266= 2063-2066, 1"991,. Dierickxs PJ. rnteraction of benzo- and naphtoquinones with soluble glutathione S-transferases from rat liver. PhazmacoJ- Res Co¡nm 15: 581-591, 1993. Dirr H['I and Reinemer p. nguiribrium unfording of crass rT grutathione s-transferase. Biochem aiophys Res co¡un 1go3 294-300 | L991. Dulik DM, Fenselau c and Hilton J. characterization of melphalan-glutathione adducts whose formation is catalysed by glutathione transferases. Biochem pharmacoL 35: 3405-34O9, 1986. -1,7- X, zÌnan.g P, Armstrong RN and Gilliland GL. The threedimensional structure of a gtutathione S-transferase from the mu qene class. Structural analysis of the binary 2-2-Ã' complex of isoenzyme 3-3 and glutathione at resolution- Biochemistry 3L: 10169-10184, L992. Jakoby WB. The glutathi-one S-transferases: a grouP of multifunctional detoxification proteins. .ådr¡ EnzymoL ReJ Areas Mof BioL 46: 383-4L4, 1-978. Fersht À. Enzwe structure & mechanism. Freeman wH & company, New York, 1984. Graminski GF, Kubo Y and Àrmstrong RN. Spectroscopic and kinetic evidence for the thiolate anion of glutathione at S-transferase. of glutathione site active the Biochemistry 28: 3562-3568, 1989. Hayes JD and violf CR. Molecular mechanisms of drug resistance. Biochem J 272t 28L-295, 1990. Hall- À, Robson CN, Hickson ID, Harris AL' Proctor SJ and Cattan AR. Possible role of inhibition of glutathione Stransferase in the partial reversal of chlorambucil by indomethacin in a chinese hamster ovary ' resistance cell line. Ca¡cer Res 492 6265-6288, f989. Harris JM, Meyer DJ, Coles B and Ketterer B. A novel glutathione transferase ( L3-13 ) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes. Biochem J 278: L37-141. Katusz RM and Colman RF. S-(4-Bromo-2r3-dioxobutyl) glutathione: À ner^t affinity label for the 4-4 isoenzlmes of rat liver glutathione S-transferase. aiochemistry 30: Ji LL23O-LL238t 1991,. Katusz RM, Bono B and Colman RF. Identification of tyrosine (LL5) labeled by S-(4-brono-2,3-dioxobutyl)glutathione in the hydrophobic substrate bind5-ng site of glutathione Stransferase, isoenzyme 3-3. Arch Biochem Biophys 2982 667-677, 1992. Bono B and Colman RF. Àffinity labeling of Katus?.,RM, IIf of glutathione S-transferase. isoenzyme 1-1, by Scys--' Biochemistry 3 1 : ( 4-bromo-2 , 3-dioxobutyl ) glutathione. 8984-8990 | L992. KoIm RH, Sroga GE and Mannervik B. Participation of the phenolic hydroxyl group of tyr-8 in the catalytic activity of human glutathione transferase P1-1- Biochem J 2852 537-540| L992. Kong KH, Inoue H and Takahashi K. Non-essentiality of cysteine and histidine residues for the activity of human class pi glutathione S-transferase. Biochem Biophys Res Con¡n 1812 748-'155t 799L. -18- Kong KH, Inoue H and Takahashi K. Site-directed mutagenesis of amino acid residues Ínvolved in the glutathione binding of human glutathione S-transferase p1-1. J Biochem 712: 725-728, L992. Kong KH, Nishida M, Inoue H, Takahashi K. gzrosine-Z is an essential residue for the catalytic activity of human class pi glutathione S-transferase: chemical modification and site-directed mutagenesis studies. Biochem Biophys Res Comm !822 LI22-LL29, 1992. Lewis AD, Hickson ID, Robson CN, Harris ÀL, Hayes JD, Griffiths SA, Manson MMf Hall ÀE, Moss JE and Wolf CR. Àmplifícation and increased expression of alpha class glutathione S-transferase-encoding genes associated v¡ith resistance to nitrogen mustards. proc Natl Acad Sci USA 85:85LL-8515/ L988. Líu S, Z}j,ang Pt Ji x/ Johnson vtw, Gilliland cL and Armstrong RN. Contribution of Èyrosine 6 to the catalytic mechanism of isoenzlane 3-3 of glutathione S-transferase. J BioL Chem 267: 4296-4299, 7992. Lo Bello M, Petruzzelli R, De Stefano B, Tenedini C, Barra D and Federici c. Identification of a highly reactive sulphydryl group in human placental glutathione transferase by a site-directed fluorescent reagent. FEBS Lett 263: 389-39Lt L99O. Mannervik B. The isoenzlzmes of glutathione transferase. .ådy Enzymol ReJ, .åreas Mol Biol 572 357-477 | !985. Mannervik B and Danielson UH. Glutathione transferasesstructure and catalytic activity. CRC Crit Rerz Biochem 23¿ 283-336, 1988. Mannervik B, Aürasthi YC, Board PG, Hayes JD, Di IIio C, Ketterer B, Listowsky 1, Morgenstern R, Muramatsu M, Pearson !ùR, Pickett CBI Sato K, Vùidersten M and ltolf CR. Nomencl-ature for human glutathione transferases. Ejocåem J L992: 305-308 | L992. Merlos M, Sanchez RM, Camarasa J and Àdzet T. Flavonoids as inhibitors of rat Ij-ver cytosolic glutathione Stransferase. Experienta 47: 616-619 | 799L. Meyer DJ, Coles B, Pemb1e SE, Gilmore KS, Fraser GM and Ketterer B. Theta, a ne\¡r class of glutathione transferases purified fron rat and man. Biochem J 274l. 4O9-4L4t L99L. Mitra A, Govind!ìrar S and Kulkarni Àp. Inhibition of hepatic glutathione S-transferases by fatty acids and fatty acid esters. Toxicol ¿e¿t 58: 135-141, 1991. -19- Morgenstern R and DePierre JW. Membrane-bound glutathione transferases. In: GLutathione Conjugatjon. Eds.: Sies H and Ketterer B, Academic Press, London, L57-I75, 1988. Morro\,r CS and Co\,van KH. Glutathione S-transferases and drug resistance. Cancer Ce77s 2¿ L5-22, 1990. Murren JR and Hait WN. !{hy haven't \^re cured multidrug resistant tumors. OncoTogy Res 4z L-6, L992. Nishihira J, Ishibashi T, Sakai M, Nishi S, Kondo H and Makita A. Identification of the fatty acid binding site on glutathione S-transferase P. Biochem Bjop.¡rys Res Conm L89t t97-2O5, t992. Reinemer P, Dirr HW, Ladenstein R, Schäffer J, Gallay O and Huber R. The three-dimensional structure of class r¡ glutathj-one S-transferase in complex with glutathione sulfonate aÈ 2.3  resolution. EIrBo J 1O: IggT-2OO5, i.991. Sato K. Glutathione transferases as markers of preneoplasia and neoplasia. ¿dy Canceî Res 52 ¿ 205-255, L989. Schäffer J, Gallay O and Ladenstein R. Glutathione transferase from bovine placenta. J Biol Chem 263: 1,74o5-r14L1 , l_988. Segel IH. Enzwe kinetics . Behaviour and anaTysis of rapid equiTibrium and steady-state enzyme systems. Vlileylnterscience, Ner^r Yoxk, L974. Shen H, lamai K, Satoh K, Hatayama I, Tsuchida S and Sato glutathione K. Modulation of class pi transferase activity by sulfhydryl qroup modification. ¿,rcå Biochem Biophys 2862 1-78-L82, I99L. 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. Arcå Biochem Biophys 3OO: L37-LALl 1993. Stenberg G, Board PG and Mannervik B. Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class alpha glutathione transferase. -F¡'.8S Lett 293: 153-155 | L997. Tamai K, Satoh K, Tsuchida S, Hatayama I, Maki T and Sato K. Specific inactivation of glutathione S-transferases in class pi by SH-¡nodifiers. Biochem Biophys .Res Conm 167 : 331-338, l-990. Terada T, Maeda H, Okamoto KI, Nishinaka T, Mizoguchi T, Nishirada T. Modulation of glutathione S-transferase activity by a thiol/disulfide exchange reaction and the involvement of thioltransferase. Arch Biochem Biophys 300: 495-500, 1993 . -20- Ter^¡ KD, Bomber AM and Hoffman SJ. Ethacrynic acid and piriprost as enhancers of cytotoxiciÈy in drug resistance and sensitive cell lines. Cancer Res 48: 3622-3625, 1988. Tirikainen MI and Krusius T. Multidrug resistance. .A¡n liled 232 509-520, l-991. Tipping E, Ketterer B, Christodoulides L, Elliott BM, Àldridge WN and Bridges Jw. The interactions of triethyltin with rat glutathione S-transferases A/ B and c. Chem BioJ Interact 242 317-327 ¡ 1-979. Townsend AJ, Tu cPD and Cowan KH. Expression of human U or a class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents. Mol PhazmacoT 4]-z 23O236, L992. Tsuchida À and Sato K. Glutathione transferases and cancer. CRC Crit Rev Biochem 272 337-3A4t L992. Van Bladeren PJ. Formation of toxic metabolites from drugs and other xenobiotics by glutathione conjugation. TIPS 9: 295-298, 1988. Van Bladeren PJ and Van Ommen B. The inhibition of glutathione mechanisms, S-transferases: toxic consequences and therapeutic benefits. Pharmac Ther 51: 35-46, t99L. Van Ommen B, Den Besten C, Rutten ALM, Ploemen JHTM, Vos RME, Muller F and Van Bladeren PJ. Àctive site-directed irreversible inhibition of glutathione S-transferases by glutathione conjugate of tetrachloro-I14the benzoquinone. J BioT Chen 263: 12939-12942, 1988. Van ommen B, Ploemen JHTM/ Ruven HJ, Vos RME, Bogaards JJP, Van Berkel wJH and van Bladeren PJ. SÈudies on the active site of rat glutathione S-transferase isoenzymes 4-4. Eur J Biochem !8L, 423-429, L989. van Orunen B, Bogaards JJP, Peters wHM, Blaauboer B and Van Bladeren PJ. Quantification of human hepatic glutathione S-transferases. Biochem J 269: 609-613, 1990. Vos RME, van ommen B, Hoekstein MSJ, De Goede JHM and Van Bladeren. Irreversible inhibition of rat glutathione Stransferase isoenzlmes by a series of structurally related quinones. Chem Biol rnteract 7L¿ 38L-392, 1989. vos RME and Van Bladeren PJ. Glutathione S-transferases in relation to their role in the biotransformation of xenobiotics. CJ:em BioT Interact 75¿ 241-265, l-990. wang RW, Newton DJ, Pickett CB and Lu ÀYH. Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants. Arch Biochem Biophys 2972 86-9Lt L992. -2t- Wang AL and Tew KD. Increased glut,athione S-transferase activity in a cell line with acquired resistance to nitrogen mustards. Cancer Treatmen Reports 69: 677-6g2, t_985. VlaJ<man DJ. clutathione S-transferases: role in alkylating agent resistance and possible target for modulation chemotherapy- a review. Cancer Res 50z 6449-6454, l-9g}. Vüidersten M, Holmström E and Mannervik B. Cysteines resj-dues are not essential for the catalytic activity of human class mu glutathione transferase Mj.a-1a. FE'AS Lett 293: L56-159, 199L. zhang P, Graminski cI' and Àrmstrong RN. Àre the histidine residues of glutathione S-transferase i¡nportant for catalysis. J BioJ Chen 266: 1_9745-19479, L99I. -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 Armstrong RN. Glutathione S-transferases: reaction mechanism. sÈructure, and function. ChemicaJ- Res Toxicol 4: 131-139, L99L. Black SM and Wolf CR. The role of glutathione-dependent enz]¡mes in drug resistance. pharmacoL Therapeut 51: L39L54t L99r. Bogaards JJP, Van Onmen B, Falke HE, Willems MI and Van Bladeren PJ. Glutathione S-transferase subunit induction patterns of Brussels sprout, allyl isothiocyanate and giotrin in rat liver and small intest.inal mucosa: a new approach for the identification of inducing xenobiotics. Fd Chetn Toxicol, 28: 81-88, I99O. Bogaards JJP, Van Orunen B and Van Bladeren pJ. Àn improved method for the separation and quantification of glutathione S-transferase subunits in rat tissue using high-performance Iiquid chromatography. J Chromatogr 4742 435-440, L989. Camarasa J, Escubedo E and Adzet T. pharmacokinetics of caffeic acid in rats by a high-performance liquid chromatography method. J pharmac Biomed Anal 6: 503-510, 1988. Cheynier \rF, Trousdale EK, Singleton VL, Salgues MJ and $fyIde R. Characterization of 2-S-gtutathionylcaftaric acid and iÈs hydrolysis in ration to grape wines. J Agric Fd Chem 342 217-221, 1986. Cheynier vF and Van Hulst MWJ, oxidation of trans-caftaric acid and 2-S-glutathionylcaftaric acid in model solutions. J Agric Fd Chem 36: 1O-15, 1988. CiIIier JJL and Singleton \IL. Nonenzymic autoxidative phenolic browning reactions in a caffeic acid model_ system. J Agric Fd Chem 37: 890-896t !989. Cillier JJL and Singleton VL. Caffeic acid autoxidation and the effects of thiol. J Agric Fd Chem 38: L7B9-L796, r.990. Cillier JJL and Singleton \IL. Characterization of the products of nonenzymic autoxidative phenolic reactions in a caffeic acid model system. J Agric Fd Chen 392 \2981303, 1991. Das M, Bickers DR and Mukhtar H. plant phenols as in vitro inhibitors of glutathione S-transferase(s). Biochem Biophys .Res Comm J2O.. 427 -433 , l-984. Finney DJ. Probit AnaLysis. 3rd Ed. pp. 50-97. Cambridge University Press, London, 1971. -49- Habig WH, Pabst MJ and Jakoby WB. Glutathione Stransferases, The first step in mercapturic acid formation. J Biol Chen 2492 7I3O-7I39, L974. Hayes JD, Pickett CB and Mantle IJ. Glutatione Stransferases and Ðrug Resistance. p. 11. Taylor & Francis, London, 1990. Hermann K. tiber Kaffeesäure und Chlorogensäure. pharmazie Ltz 433-449, ),956. Katusz RM and Colman RF. S-(4-Bromo-2,3dioxobutyl) glutathione: a ne\^r affinity label for the 4-4 isoenzlmes of rat liver glutathione S-Èransferase. Biochem 30: LL23O-LL238t L99r. Katusz RM, Bono B and Colman RF. Identification of tyrosine (115) labeled by S-(4-brorno-2,3-dioxobutyl)glutaÈhione in the hydrophobic substrate binding site of glutathione Stransferase, isoenzyme 3-3. Arch Biochem Biophys 2982 667-677 | L992. Lowry OH, Rosebrough NJ/ Farr AL and Randall RJ. protein measuremenÈ wiÈh the Folin phenol reagent.. J niol Chem L93¿ 265-275t 1951. Mannervik B. The isoenzymes of glutathione transferase. .{dy Enzym ReL Àreas Mo7 BioT 572 357-417, 1985. Mannervik B and Danielson UH. Glutathione transferasestructure and catalytic activity. Crit Rev Biochem MoL BioJ 23:. 283-337, L988. Ploemen JHTM, Bogaards JJP, Veldink GA, Van OEunen B, Jansen DHM and Van Bladeren PJ. fsoenzyme selective irreversible inhibition of rat and human glutathione S-transferases by ethacrynic acid and tr"¡o brominated derivatives. Biochem Pharmac 45: 633-639, 1993. 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 Phazmac 40: 1631-1635, 1990. Ploemen JHTM, Van Onmen B and Van Bladeren pJ. Irreversible inhibition of human glutathione S-transferase isoenzymes by tetrachloro-1r4-benzoquinone. Biochem pharmacol 41: L665-1669 | 1991_. Ricci G/ Del Boccio G, Pennelli A, Lo Betlo M, Petruzzelli R, Caccuri AM, Barra D and Federici G. Redox forms of human placenta glutathione transferase. J BioT Chem 2662 2L4O9-214L5, ]-99r. Shen H, Tamai K, Satoh K, Hatayama I, Tsuchida S and Sato K. Modulation of class pi glutathione transferase -50- activity by sulfhydryl group modification. Arch Biochem Biophys 286¿ t'78-L82, L99I. Tamai K, Satoh K, Tsuchida S, Hayaya¡na I, Maki T and SaÈo K. Specific inactivation of glutathione S-Èransferases in class pi by SH-modifiers. Ejocl:em Ejophys Res Cornm L67z 331-338 , r99O. Van Ommen B, Den Besten C, Rutten ALM¿ Ploemen JHTM, Vos RME, Müller F and Van Bl-aderen PJ. Active site directed irreversible inhibition of glutaÈhione S-transferases by glutathione the conjugate tetrachloro-L,4of benzoquinone. J BioJ. Chen 263: L2939-12942, 1988. Van Ommen B, Ploemen JHTM, Ruven HJ, Vos RME, Bogaards JJP, Van Berkel vùJH and Van Bladeren PJ. Studies on the active site of rat glutathione S-transferase isoenzlare 4-4. Chemical modification by tetrachloro-1- t 4-berLzoquinone and its glutathione conjugate. Eur J Biochem LBJ-z 423-429, 1989. Orûmen Van B, Van Bladeren PJ, TeÍunink JHM and Müller F. Formation of pentachlorophenol as the major producÈ of microsomal oxidation of hexachlorobenzene. Biochem Biophys Res Comm J-26: 25-3L, 1985. Vos Rì4E, Snoek MC, Van Berkel- wJH, MüIler F and Van Bladeren PJ. Differential induction of rat hepati-c glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate: comparison with induct.ion of phenobarbital and 3- methylcholanthrene. Biochem Pharmac 37 ¡ 1,077 -L082, 1988. -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-csD 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- References 1. Bindoli A, Rigobello MP and Deeble DJ" Biochemical and toxicological properties of the oxidation products of catecholamines. Free Rad Biol_ Ired L3: 391-405 | 1992. 2. Thompson A, Land EL, Chedekel MR, Subbarao KV and Truscott T. A pulse radiolysis investigation of the oxidation precursors of the melanin 3,4dihydroxyphenylalanine (dopa) and the cysÈeinyldopas. Biochin Biophys Acta 843: 49-57, 1985. 3. fto S, Kato T and Fujita K. CovalenÈ binding of catechols to proteins through the sulphydryl group, Biochem PharmacoT 372 a7O7-1710, 1988. 4. Monks TJ and Lau SS. foxicology of quinone-thioethers. Crit Rev ToxicoL 22z 243-270, L992. 5. Àskelöf P, cuthenberg C, Jakobson I and Mannervik B. Purification and characterization of two glutathione Saryltransferase activities from rat liver. Biochem J L47 513-522, L975. 6. Van Onmen B, Den Besten C, Rutten ÀLM, Ploemen JHTM, Vos RME, Mü1Ier F and Van Bladeren PJ. Àctive sitedirected irreversible inhibition of glutathione Stransferases by the glutathione conjugate of tetrachloro1/4-benzoquinone. J BioJ- Chen 263¿ 12939-72942, l-9BB. 7. Mannervik B and Danielson UH. Glutathione transferases Structure and catalytic activity. Crit Rerr Biochem MoI BioL 23¿ 283-337, 1988. 8. Àrmstrong RN. Glutathione S-transferases: reaction mechanism structure, and function. Cl2em Res ToxicoL 4: 131-140, L99r. 9. l"lannervik B. The isoenzymes of glutathione transferase. Adv Enzwo1 ReL Areas Mo7 Biol- 57: 357-417 | L985. 10. Meyer DJ, Coles B/ Pemble SE, GiLmore KS, Fraser GM and Ketterer B. Theta, a nevr cl-ass of glutathione transferases purified from rat and man. Biocl:em J 274l. 409-4L4, L99L. 11. Van OÍunen B/ Ploemen JHTM, Bogaards JJP/ Monks TJ, Lau SS, and Van Bladeren PJ. IrreversibLe inhibition of rat glutathione S-transferase 1-1 by quinones and their glutathione conjugates. Biocåen J 276: 66]--666t I99:-. 72. Katusz RM and Colman RF. S-(4-Brono-2,3-dioxobutyl)glutathione: A new affinity label for the 4-4 isoenzymes of rat liver glutathione S-transferase. Bioc.hem 30: II23O-]-\238, 1991. z -67- 13. Black enzlrmes 1991. and idolf CR. The role of glutathione-dependent in drug resistance. Pharmacol Therap 51: 139-154, SM 14. Morrow S and Cowan KH. Glutathione S-transferases and drug resistance. Cancer ceLTs 2z 15-22t 1990. l-5. Waxman DJ. Glutathione S-transferases: role in alkylating agent resistance and possible target for modulation chernotherapy - A review. Cancer Ães 5O z 64496454t 1990. 16 Finney DJ. In: Probit analysis, Cambridge University Press, London, L971"t pp. 50-9'7. 17. Van Ommen B, Van Bladeren PJ, lemmink JHM and MüIler F. Formation of pentachlorophenol as the major product of microsomal oxidation of hexachlorobenzene. Biochem Biophys Res Conm L26z 25-3I. 1985. 18. Ploemen JHTM, Bogaards JJP, Veldink GA, Van Ornmen Bf Jansen DHM and Van Bladeren PJ. Isoenzyme selective irreversible inhibition of rat and human glutathione Stransferases by ethacrynic acid and th¡o brominated derivatives. Biocåem PharmacoT 45: 633-639, 7993. 19. Hayes JDf Pickett CB and Mantle TJ. In: Glutathione Stransferases and Drug Resistance, Taylor & Francis, London, L990, p.11. 20. Loh¡ry OH, Rosebrough NJ, Farr AL and Randall RJ. Protein measurement with the Folin phenol reagent. J BioI Chen l93z 265-275, l-951. 2L. Habig WH, Pabst MJ and Jakoby wB. clutathione Stransferases, the first step in mercapturic acid formation. J BioL Chem 249¿ 7130-7139, L974" 22. Kulkarni ÀP, Motoyama N/ Dauterman VùC and Hodgson E. Inhibition of housefly glutathione S-transferase by catecholamines and quinones. BuLL Environm Contam Toxicol 20 z 227 -232 | L9'18 . 23. Rhoads DM. Zarlengo RP and Tu CPD. The basic glutathione S-transferases from human liver are products of separate genes. Biochem Biophys -Res Comn I45z 474-484, L987. 24. Tamai K, Satoh K, Tsuchida S, Hatayama T, Maki T and Sato K. Specific inactivation of glutathione Stransferases in class pi by SH-modifiers. Bjociren Biophys Res Con¡n 167 z 33L-338, l-990. 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 -68- transferase by site-directed fluorescent agent. FEBS Lett 263:389-391-, J-99O. 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. 27. Ta¡nai K, Shen H, Tsuchida S, Hatayama I, Satoh K, Yasui À, Oikawa A and Sato K. RoIe of cysteine residues in the activity of rat glutathione transferase p (7-71. Biochem Biophys Res Conm L79¿ 79e-797, J'ggL. 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. -69- 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. -84- 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- -85- 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 glutathione S-transferases: mechanisms, toxic consequences and therapeutic benefits. 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 glutathione s-transferase 1-l- by quinones and their glutathione conjugates. Bjocåen J 276: 661_666, 1991. 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 . -98- 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. References AEP, Brussee J, Van Der Gen A and Mulder GJ. Inhibition of rat liver glutathione S-transferase isoenzymes by peptides stabilized against degradaÈion by y-glutamyl transpeptidase. J BioJ Chem 266: 830-836, Adang 199 1. -L4L- Àhokas JT, Nicholls FA, Ravenscroft PJ and Emmerson BT. InhÍbition of purified glutathione rat liver Stransferase isoenz]rmes by diuretic drugs. Biochem PharmacoJ- 34:. 2157-21-6L, 1985. Àrmstrong RN. Glutathione S-transferases: reaction mechanism, structure, and function. CÌ?em Res Toxicol 4: 131-140, 799L. Castro WÍ, Kelley MK, Engqvist-Goldstein A, and Kauvar LM. Glutathione analogue sorbents selectively bind glutathione S-transferase isoenzymes. Biochem J 2922 37r-377 | L993. Dierickx PJ. In vitro binding of 3,4,516r-tetrachloro-1r2benzoquinone by rat liver glutathione S-transferases. Res Comm Chem Pathol PharmcoT 4L: 5L7-521,, L983. Fersht À. Enzyme structuÍe & mechanism. Freenan WH & company, Ne$¡ York/ 1984. Graminskj- GF, Zhang P/ Sesay MA, Anmon HL and Àrmstrong RN. Formation of the 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion at the active siÈe of glutathione Stransferase: Evidence for enzymì-c stabilization of ócomplex int.ermediates in nucleophilic aromatic substiÈution reactions. Biochemistry 28: 6252-6258. Hansson J I Berhane K, Castro VM, Jungnelius U, Mannervik B/ and Ringborg U. Sensitization of human melanoma cells to cytotoxic effect of nelphalan by glutathione transferase inhibitor ethacrynic acid. Cancer Res 51: 94-98t L99L. Ji X, Zhang P, Àrmstrong RN and GilIiland cL. The threedinensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzlnae 3-3 and glutathione at 2.2-à resolution. Biochemistry 3L: 10169-10L84r 1992. Kl-aassen CD and Fitzgerald TJ. Metabolisn and biliary excretion of ethacrynic acid. J PharmacoT Experimen Therap 191: 548-556, L974. Katusz RM and Colman RF. S-(4-Bromo-2r3-dioxobutyl) glutathione: À ne\^r affinity label for the 4-4 glutathione isoenzl'mes of rat liver S-transferase. Biochemistry 3O: 11230-11238, 199L. KatusllfM, Bono B and Colman RF. Àffinity labeling of cys of glutathione S-transferase, isoenzyme 1-L, by S- ( -brono-2 , 3-dioxobutyl ) glutathione. Bioehemistry 3 L : 8984-8990, 1,992a. Katusz RM, Bono B and Colman RF. Identification of tyrosine (115) Iabeled by S-(4-bromo-2r3-dioxobutyl)glutathione -L42- in the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 3-3. Arch Biochem Biophys 2982 667-677, L992b. Liu S, Zhang P, Ji Nt Johnson WW, Gilliland GL and Armstrong RN. Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione Stransferase. J BioJ Chen 2672 4296-4299, L992. Mannervik B. The isoenzlanes of glutathione transferase. Adv EnzwoT Re-z. Areas Mo7 BioT 57: 357-4]-7, L985. Mannervik B and Danielson UH. Glutathione transferasesstructure and catalytic activity. C.RC Crit Rey Biochem 23: 283-336, 1988. o'Dr^¡f/er PJ, tacreta F, Nash S, Tinsley PW, Schilder R, Clapper ML, Tew KD, Panting L, Litwin S/ Comis RL and Ozols RF. Phase I study of thiotepa in combinaÈion with Èhe glutathione transferase inhibitor ethacrynic acid. Cancer Res 5L: 6059-6065t 199L. Ploemen JHTM, Bogaards JJP, Van Ommen B and Van Bladeren PJ. Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases. Xenobioticaz in press. Ricci G, Del Boccio G, Pennelli À, Lo Bello M, petruzzelli R, Caccuri AM, Barra D and Federici G. Redox forms of human placenta glutathione transferase. J Biol Chem 2662 2t4O9-2L4t5, 1991. Takamatsu Y and Inaba T. Inhibition of human hepatic glutathione S-transferases by ethacrynic acid and its metabolites. ToxicoT Lett 622 24L-245, 1992. Tamai K, Shen H, Tsuchida S, Hatayama I, Satoh K, Yasui A, Oikawa A and Sato K. Role of cysteine residues in the activity of rat glutathione transferase 7-7: elucidation by oligonucleotide site-direcÈed mutagenesis. Biochem Biophys Res Conn L79¿ 790-797, 1997. Reinemer P, Dirr HW, Ladenstein R, Schäffer J, Gallay O and Huber R. The three-dimensional- structure of class n glutathione S-transferase in complex with glutathione sulfonate at 2.3 Å resolution. EMBO J 10: L9T7-2OO5, l_991. Van Bladeren PJ. Formation of toxic metabolites from drugs and other xenobiotics by glutathione conjugation. ?rps 9: 295-298, L988. Van Bladeren PJ and Van Ommen B. The inhibition of glutathione S-transferases: mechanisms, toxic consequences and therapeutic benefits. Pharmac Ther 512 35-46 , 1_99L. -143- Van Ommen Bf Àdang ÀEp, Brader L, posthumus MA, Muller F, and Van Bladeren. The microsomal metabolism of hexachlorobenzene. Biochem pharmacol 35: 3233-323g1 1986. Van Ommen B, Voncken J!,f, Muller F, and Van Bladeren pJ. The oxidation of tetrachloro-1r4-hydroquinone by microsomes and purified eytochrome p-450b. Chen Aiol Interactions 65t 247-25O, 1"988a. Van Otrìmen B, Den Besten C, Rutten ALM, ploemen JIITM, Vos RME, Muller F and Van Bladeren pJ. Active site_ directed irreversible inhibítion of glutathione Stransferases by the glutathione conjugate of tetrachloro-l,4-benzoguinone. J aioL Chem 2632 1,29391"2942, 1988b. Van Ommen B, ploemen JHTM, Ruven HJ, Vos RME, Bogaards JJp, Van Berkel hIJH and Van Bladeren pJ. Studies on the active site of rat glutathj_one S-transferase isoenzymes 4-4. Eur J Biochem J-Stt 423-429t 1989. Van Ommen B, Bogaards JJp, peters !.IHM, Blaauboer B and Van Bladeren PJ. Quantification of human hepatic Alutathione S-transf erases . Bioc hem J 269 z 6O9-6L3 , j-990 . Van Ommen B, Ploemen JHTM, Bogaards JJp, Monks TJ and Van Braderen PJ. rrreversible inhibition of rat glutathione S-transferase 1_-1 by quinones and their glutaÈhione conjugates: structure-activity relationshj_p and mechanism. Biochem J 276¿ 66t-666, L991a. Van Omlnen B/ Bogaards JJp, ploemen JHTM, Van Der Greef, and Van Bladeren pJ. Quinones and their glutathione conjugates as irreversible inhibitors of glutathione S_ transferases. In: Vtitmer CM (Ed. ) et aI. Biological reactive intermediates IV. New york, plenum press, Adv ExperimentaJ- riedicine e BioIoW: 283, 1991b. Vessey DA and Boyer TD. Characterization of the activation of rat liver glutathione S-transferases by nonsubstrate ligands. ToxicoJ- Applied pharmacoJ- 932 Zj5-280, 19gg. Vos RME, Van Ommen B, Hoekstein MSJ, De Goede JHM and Van Bladeren. Irreversible inhibition of rat glutathione S_ transferase isoenzymes by a series of structurally related quinones. Chem BioL lnteract 7L: 3Bl_-392, 1999. üTallin JD, Clif¿on c and Kaplowitz N. The effect of phenobarbital, probenecid and diethyl maleate on the on the pharmacologykinetics and biliary excretion of ethacrynic cid in the rat. J pharmacoJ- Experimen Therapeut 2O5: 47I-479. Williaurson HE. Furosemide and ethacrynic acid. J Cl.jr2 - Pharmaco] L7 z 663-672, Witz G. Biological 1,44 - 1-977 . interactions of arp-unsatrrratêd aldehydes. Free Rad BioT & Medicine 7 z 333-349, l-991-. Ya¡nada T and Kaplowitz N. Binding of eÈhacrynic acid to hepaÈic Alutatftione S-transferases in vivo in the rat. Biochem Pharmacol 292 L205-t208, l-980. -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-