CONTEMPORARY Glutathione

TI:RR~.NCF

J. MONKS.* M. SFRRIN~

ISSUES Conjugate

W. ANUERS,t S. LAII.* AND

IN TOXICOLOGY

Mediated

Toxicities’

WOL.FGANG DEKANT,$ PETER J. VAN BL~DEREN

JAMES

L. STEVENS.~

Glutathione Conjugate Mediated ronicities. bfONhS. .J. J.. I~NDFRS. M. W.. DKANT. W.. SIIV’CM. J. L.., LAO. S. S.. AZUI) V,AN BI AIXRFN. I'. J. (1990). 7i)\-/i~o/ .I/‘/‘/. I’hrr~wt~r~/. 106. IIO. Glutathwne ()-glutam!I-r.-c~stcin!,Igl!cine; GSH) IS present in high concentrations In most living cells and participates in a wriety of vital cellular reactions. In particular. GSH protects cells from potentially tow electrophilcs formed via the metabolism ofxenobiotics, and such reactions ha\c long been associated with the process ofdctoxication (Baumann and Preusse. 1x71); JatTe. 1879). Compounds that form GSH conjugates are processed by )-glutamvl transpeptidase (7-C; I) and dipeptidases to cystclne .Fconjugates. which are usually excretedin urine as their corresponding mercapturic acids (S-substituted A’-acetyl-1 qsteinc conjugates). In addition. GSH pcroxidase actlcitl. whether catalyzed by the selenium-dependent GSH pcroxidase or b! the GSH S-transfcrases. serves to detoxify hydrogen peroxide and organic hydroperosides. However, in recent years. cvidencc indicating that GSH conjugation plays an important role in the formation of to,xic metabolitec from a variety ofchcmicals has accumulated. Thus, several classes of compounds are conlerted. via conjugation with GSH. into either cytotoxic. genotoxic. or mutagenic metabolites. The purposes of the symposium on “Glutathione Conjugate Mediated Toxicities” prcsentcd at the IO90 Sucicty of ‘Iouicolog~ Annual Meeting were to discuss recent tindings in this rapid11 moving tield. to present ideas on the mechanisms and modulation ofGSH conjugatcdependent toxicities. to present a consensus on the broader significance of this work. and to idcntit) directions tbr future research. This paper summarizes these presentations. GSH conjugation reactions arc involved in the bioactivation of several classes of uenobiotics. and four types of GSH-dependent bioactivatlon reactions can be identified: (I ) directly toxic GSH conjugates may he Cormed from bicinal dihaloalkanes via formation ofelectrophilic sulfur mustards: (2) cysteine conjugate +lyasc-dependent hioactivation is involved in the srlcctive ncphrotoxicity of haloalkenes: (3) GSH conjugates of’hydroquinones and isothioqanates may serve as transport and targeting metabolitcs: and (4) GSH-dependent reactions may he involved in the release of toxic agents t’rom precursor organic thiocyanates and nitrosoguanidines f/K-methyl-A”-nitro-N-nitrosguanidine). , ,‘a,, ,c:alrm,r Fkr\. I”
of radiolabel being present in mitochondrial DNA (Schrenk and Dekant. 1989). THE

/-I-LYASE ENZYMES: CHEMICAL. BIOCHEMICAL, AND PHYSIOLOGICAL REGULATION

As summarized above. it is clear that for some cysteine conjugates. particularly those of the halogenated alkane and olefin types. activation to reactive species by the enzymes cysteine conjugate /$lyase is a critical step in toxicity. The term cysteine conjugate fi-lyase

1% MEDIATED

TOXICITIFS

3

was coined by Tateishi ef al. ( 1978) after the initial purification of the hepatic enzyme. In general, all &lyases contain pyridoxal phosphate (Stevens and Jones, 1989). Thus, the use of the pyridoxal phosphate-requiring enzyme antagonist, aminooxyacetic acid, readily differentiates between /I-lyase-dependent and -independent pathways of activation (Elfarra LYal.. 1986; Stevens rt (II.. 1986a). However, though the @-lyase is the penultimate step in activation. there are several other mechanisms by which the rate of reactive intermediate production can be regulated. These mechanisms can be divided into chemical (regulation occurring during the enzyme chemistry), biochemical (regulation by interaction among molecules produced by separate enzymes). and physiological (regulation by the disposition of the elements of the pathway among organs). This section summarizes knowledge about /-Ilyases and mechanisms which might regulate its activity and the toxicity of its products.

The bacterial o-lyases receive little attention with respect to the toxicity of cysteine conjugates. However. it is clear that they play a major role in the metabolism of xenobiotics in rive (Bakke, 1986) due to the presence of these bacterial enzymes in the gut flora (Tomisawa el LII., 1984: Bakke. 1986: Larsen and Stevens, 1986). Unlike the mammalian enzymes, the bacterial enzymes can be true &eliminases since they will catalyze cu.&eliminations from physiological substrates such as cystathionine (Larsen and Stevens 1986). The bacterial filyases will also act on a much broader range of substrates than their mammalian counterparts and are dependent on the presence of a good leaving group at the P-carbon (Tateishi, 1983; ‘Tomisawa t7 trl.. 1984; Stevens, 1985: Larsen and Stevens, 1986). Recent reviews provide summaries of the broad role played by the bacterial B-lyases in drug metabolism (Bakke. 1986: Stevens and Jones. 1989: Stevens and Bakke. 1990).

6

Tile Heputir /~-LJuw and Rrgulution Inactivation (Chemical Recqulation)

MONKS

ET AL..

b?,

Although the liver is not a target for cysteine conjugate toxicity, fi-lyases have been isolated from rat and human liver (Tateishi et al.. 1978: Stevens. 1985; Tomisawa et ni., 1986) and cysteine conjugates are metabolized by hepatic b-lyases: therefore, their regulation may be of some interest. Rat liver P-lyase has been purified and is identical to kynureninase. an enzyme that catabolizes the tryptophan degradation product kynurenine (Stevens, 1985). The chemical mechanisms by which the enzyme acts on kynurenine and cysteine conjugates are very different (Fig. 4). The kynureninase reaction yields the cleavage fragment (anthranilic acid: Fig. 4, Eq. ( 1)) and alanine, while the @-lyase-catalyzed cleavage of cysteine conjugates yields the fragment plus pyruvate and ammonia (Fig. 4. Eq. (2)). These reactions leave a much different intermediate bound to the enzyme-pyridoxal phosphate complex. For the kynureninase reaction, the intermediate has carbanion ion character at the p-carbon (Fig. 4, Eq. (3)) while the P-elimination reaction leaves an eneamine intermediate with carbonium ion character (Fig. 4, Eq. (4)). This difference between the pyridoxal phosphate-enzyme intermediates has important implications for the enzyme. During the catalytic cycle. the rat liver enzyme is inactivated. This type of inactivation can occur by one of two mechanisms: (a) the nucleophilic attack of an enzyme-bound nucleophile on the D-carbon of the eneamine (Stevens. 1985; Stevens and Jones. 1989). and (b) an aldol-type condensation reaction between the eneamine intermediate and the enzyme-Schiff base complex (Likos et ul.. 1987: Uenos et a/., 1982). Inactivation is particularly important for those pyridoxal phosphate-dependent enzymes that catalyze p-elimination as a fortuitous side reaction rather than as its primary metabolic chore.

lat ion The kidney is the target organ for many toxic cysteine conjugates. Rat kidney cysteine conjugate &lyase has been purified from cytosol and mitochondria and both forms are identical to the glutamine transaminase K enzymes found in those cellular compartments (Stevens et al.. 1986b. 1988). Another activity has been described in the outer membrane of mitochondria, but its role in toxicity is not clear (Lash et al.. 1986). Renal p-lyases are pyridoxal phosphate-dependent enzymes, but are not subject to the chemical regulation (inactivation) described above. Regulation of these enzymes does occur, but at a biochemical level through the interaction of metabolic products of different enzymes. The purified rat kidney enzymes are also rapidly inactivated by their substrates (Stevens et al., 1986b, 1988). The basis for this inactivation lies in different mechanisms for transamination. the usual reaction catalyzed by the enzyme. and o-elimination. Transamination is a two-step reaction (Fig. 5): ( 1) donation of the amine to the enzyme with the cu-keto acid departing, and (2) the donation of the amine from the enzyme to a second CYketo acid. Thus, the enzyme can accumulate in its pyridoxamine form after a half-transamination reaction and will remain inactive until a second substrate (the cu-keto acid) accepts the amine from the enzyme. This occurs rapidly with the purified enzymes and it is necessary to add exogenous tu-keto acid to allow either the elimination or the transamination reactions to proceed (Stevens et trl.. I986b: Stevens and Jones, 1989). However, the &elimination reaction is a concerted reaction in which the end products leave the enzyme and no second substrate is required. In crude systems, such as kidney homogenate, there is no requirement for added cu-keto acid (Stevens et al., 1986b, 1989). since there are alternative sources of oc-keto acids intrinsic to the mixture. One ofthese sources is another enzyme, t-tu-hydroxyacid oxidase (t-amino

GLIIT4THIONF

C-W

CONJUGATE

MEDIATED

TOXICITIES

,-CH-COO@ NH,@

R-CH

,--CH--COO@

*Hz0 RH

NH,@

+

2)

(eq.

3)

(eq.

4)

@CH,-i--COO@

CH,=F--COO@ :NH

@NH

@?AJ:3H

@Q$J:,3 H

CH,=C-COO@

@CH>-C-COO@ @{H

@AH

II

LH

CH 00

-

CH,

FIG,. A. Ddkrencu

(eq. NH 4@

CH 3-!--COO@+

111 the mr’chanisms

H

of k)nurenine

acid oxidase). which catalyzes the oxidation of amino acids, including cysteine conjugates (Stevens r’/ trl.. 1986b. 1989). to their corresponding keto acids with the concomitant reduction ofmolecular oxygen to hydrogen peroxide (Fig. 7). In this way, the oxidase can provide the necessary tu-keto acids in a futile cycle in which the tu-keto acid of the substrate is formed and undergoes the second half of the transamination reaction with the pyridoxamine-enzyme complex. Indeed, were it not for the fact that the I.-cu-hydroxyacid oxidase copurihed with the p-lyase for several steps. it would have been most difficult to purify the enzyme at all (Stevens ct [I/.. 1986b). After the oxidasc has been separated from the &lyase,

and

cysteine

CH,

conjugate

mctahdism

by the

hepatic

there is a requirement for added cu-keto acids for enzyme activity. A complete discussion of this mechanism of regulation can be found elsewhere (Stevens rt LI/., 1986b, 1989: Stevens and Jones, 1989). It has been shown that factors other than the I -cu-hydroxyacid oxidase may play equally or more important roles in the biochemical regulation of the ,B-lyase enzymes (Stevens e/ ~1.. 1989).

As emphasized above, the mercapturic acid biosynthetic pathway is the source of cysteine

MONKS

I OH H2N

ET

AL.

fi-ELIMINATION

,-N=C,

LA,-4

4ti

-

+

I

TRANSAMINATION

0

r-5

merhlonlne

NH2

y-MT6

.NH2ToH

SF+

07”” Amino Acfd Oxfdase

R

L-F/ 02

‘NH3

H202

FIG. 5. Mechanism of regulation of rat kidney cysteine conjugate /%lyase transamination function of the enzyme. Reproduced. by permission of the ( 1986b). In this figure. the enzyme is shown schematically CE) and only the the PLP cofactor is shown. As in all PLP enzymes. the cofactor exists on the Schiffbase with a protein bound amine. probably lysine.

conjugates which subsequently cause damage to the kidney. The majority of GSH conjugates are formed in the liver and exit the liver into the bile (Inoue et ~1.. 1981, 1982a.b: Bakke, 1986). Obviously, there must be mechanisms for both the processing of these GSH conjugates to their cysteine conjugates, a necessary step in the @lyase pathway (Stevens et ul., 1986a), and for returning these conjugates to the kidney. Elegant work from several laboratories suggests a complex interorgan shuttling pathway which functions to direct mercapturate biosynthesis toward the terminal metabolite, the IV-acetylcysteine conjugate. and the terminal organ, the kidney. where the mercapturate is excreted. This pathway is summarized schematically in Fig. 6. Briefly, the GSH conjugate, which exits the liver in the bile. is degraded to the cysteine conjugate in the bile cannaliculae, the bile duct, and the

through interaction with the publisher, from Stevens rl al. aldehyde functional group of enzyme surface in an internal

intestinal epithelium. The cysteine conjugate is then subject to transport from the intestine. perhaps by amino acid transport systems for which they are substrates (Schaeffer and Stevens, 1987a,b), and is returned to the liver. The liver extracts the cysteine conjugates as it would an amino acid, again perhaps via amino acid transporters. The liver acetylates the amino acid which then exits to the general circulation. The N-acetylcysteine conjugate is a good substrate for the renal organic anion transporter (Inoue ef al.. 198 1: Lock et LI/.. 1986: Zhang and Stevens, 1989) which resides in the basement membrane of the renal epithelial cells of the proximal tubule, the target cell population in viva. The extraction of the mercapturate by the organic anion transporter underlies the inhibition of cysteine conjugate toxicity by probenecid, an inhibitor of the organic anion transporter (Lock et al.. 1986:

GI LJTATHIONE

CONJUGATE

Pratt and Lock, 1988; Zhang and Stevens. 1989). The cysteine conjugate is not a substrate for the organic anion transporter (Zhang and Stevens. 1989). Therefore, the carefully organized interorgan cooperation directs the cysteine conjugate toward its target organ as the mercapturate. Since the mercapturates of the nephrotoxic cysteine conjugates are not substrates for the @-lyase, they need to be deacetylated prior to activation. A deacetylase activity has been purified from liver (Suzuki and Tateishi, 198 1) and is also present in kidney (Nash et ul., 1984). In isolated kidney cells the rate-limiting step in the activation of the IV-acetylcysteine conjugate is the deacetylation step (Zhang and Stevens, 1989). Thus the physiological disposition of mercapturate biosynthesis places a rate-limiting step upstream from the fi-lyase. This type of physiological regulation may explain the tubular specificity of cysteine conjugate toxicity which may (McFarlane ef al.. 1989) or may not (Jones rf u/.. 1988) be due to the localization of the @-lyase itself. In conclusion, a number of mechanisms exist through which the activity of the @-lyase enzymes can be regulated and which may account for (a) the lack of toxicity of cysteine conjugates to the liver, especially should the hepatic enzyme be inactivated (chemical regulation), (b) the loss of /3-lyase activity during the purification of the kidney enzymes (biochemical regulation), and (c) the specificity of the toxicity within the kidney (physiological regulation). It remains to be seen if any ofthese mechanisms regulate the toxicity if? Spiro.

QLJINOL/QUINONE-LINKED GLIJTATHIONE CONJUGATES The reactivity of quinones resides in their ability to undergo “redox cycling” and to thereby create an oxidative stress (Smith r/ al., 1985) and/or to react directly with cellular nucleophiles such as protein and nonprotein sulfhydryls (Jocelyn, 1972). GSH is the major nonprotein sulfhydryl present in cells (Reed.

MEDIATED

TOXICITIES LIVER

FIG. 6. Interorgan cooperation during mercapturic acid biosynthesis. A simplified version of the interorgan cooperation is shown. A more detailed scheme which includes other reactions available to cysteine conjugates can be found in Stevens and Jones ( 1989).

1985) and although there are relatively few studies on the addition of sulfur nucleophiles to quinones (Finley, 1974) and especially of the biological consequences of these reactions, recent evidence clearly indicates that a variety of quinone-thioethers possess biological activity. For example. the GSH and N-acetylcysteine conjugates of menadione can redox cycle. with the concomitant formation of reactive oxygen species (Wefers and Sies. 1983). In addition. Ross et al. (1985) have isolated three GSH conjugates from the peroxidasecatalyzed oxidation of p-phenetidine. which exist in both oxidized and reduced forms and which are readily interconverted by redox processes. Potter et al. (1986) have also demonstrated that the GSH conjugate of acetaminophen is readily oxidized to a free radical intermediate. A stable 2,6-dimethoxyquinone-GSH free radical has been demonstrated in bovine lens epithelial cells which may be responsible for the cataractogenicity caused by this dietary quinone (Wolff and Spector. 1987). The GSH conjugates of menadione and toluquinone have been shown to be substrates for NADP-linked 15hydroxyprostaglandin dehydrogenase and are mixed-type inhibitors of prostaglandin Bl oxidation (Chung r’t (I/., 1987). The GSH conjugate of tetrachloro- 1.4-

10

MONKS

benzoquinonc is an effective inhibitor of the GSH S-transferases (van Ommen LJ/al., 1988) and the GSH conjugate(s) of N-(4-ethoxyphenyl)-p-benzoquinoneimine has been shown to bind to DNA (Larsson et al., 1988). There is. therefore, ample evidence attesting to the biological reactivity of quinonethioethers. Conjugation of quinones with GSH also results in the formation of potent. and selective, nephrotoxicants (Monks ct al., 1985, 1988a: Lau et al.. 1988a). Interestingly, the relative toxicity of the quinol-GSH conjugates increased as the extent of GSH addition increased. The tissue selectivity of Z-bromo(diglutathion-S-yl)hydroquinone and 2,3.5(triglutathion-Syl)hydroquinone appears to be a consequence of their targeting to renal proximal tubule cells by brush border r-GT. In support of this suggestion. inhibition of yGT by pretreatment of animals with AT-135 protected them against both ?-bromo(diglutathion-S-yl)hydroquinone (Monks CI al., 1988a) and Z-3,5-(triglutathion-Syl)hydroquinone (Lau C/ (I/., 1988a) mediated nephrotoxicity. The activity of y-GT may be required to facilitate the transport of the quinol-GSH conjugates into renal proximal tubule cells as their corresponding cysteinylglycine and/or cysteine conjugates. In support of this view, the accumulation of 3-bromo(diglutathion-S-yl)hydroquinone into isolated renal slices was inhibited by AT- 125 (Lau cv a/.. 1988b). The reactivity of benzoquinol-GSH conjugates appears to be a consequence of their oxidation to the corresponding quinonc (Monks c’t trl.. 1988a; Lau ct LI/.. 1988a) rather than via metabolism by fi-lyase (Stevens and Jakoby. 1983). In support of this view. inhibition of $-lyase with aminooxyacetic acid had only minor effects (17%, inhibition) on either ?-bromo-(diglutathion-[‘5S]-yl)hydroquinone covalent binding to renal homogenate or 2bromo - (di - glutathion - S - yl)hydroquinone nephrotoxicity (Monks et N/.. 1988a). In addition, the nephrotoxicity of 6-bromo-3.5 dihydroxythiophenol. a putative fi-lyase cat-

ET

AL.

alyzed metabolite of 2-bromo-3-(glutathionS-yl)hydroquinone, was shown to be a function of the quinone moiety; thiophenols lacking the quinone group were not nephrotoxic (Monks et al.. 1988b). In contrast to aminooxyacetic acid. ascorbic acid substantially (63-87%) inhibited the covalent binding of 3-bromo-(monoglutathion-Syl)hydroquinone metabolites to renal homogenates. However. the covalent binding of 3bromo-(diglutathion-S-yl)hydroquinone to renal homogenates was only 30-45% of that seen with the homologous 3-bromo-(monoglutathion-S-yl)hydroquinone conjugates and ascorbic acid had only a minor effect (38% inhibition) on Z-bromo-(diglutathion-Syl)hydroquinone binding. The ability of ascorbic acid to inhibit the covalent binding of the various isomers correlated with their oxidation potentials (Monks ri ~1.. 1988a). Thus. differences in the electrochemical properties of the isomeric ?-bromo-(glutathion-.S’yl)-hydroquinone conjugates appear to determine their relative reactivity. Somewhat paradoxically, however, the most toxic conjugate. 2-bromo-(diglutathion-S-yl)hydroquinone. was the most stable to oxidation at pH 7.4 (Monks rt ~1.. 1988a; Lau and Monks. 199Oa). The initial conjugation of Z-bromohydroquinone with GSH is therefore a detoxication rcaction since the resulting conjugates are more difficult to oxidize than Z-bromohydroquinone. The apparent paradox was claritied when the oxidation potentials of the mercapturic acid pathway metabolites were determined. Thus, the activity of r-GT appears to facilitate oxidation of the quinol moiety and metabolism of the quinol-GSH conjugates through the mercapturic acid pathway and has significant effects on the reactivity of the intermediates (Lau and Monks. 1990a; Monks and Lau, 1990). Hydrolysis of 3-bromu-3(glutathion-S-yl)hydroquinone by -+T and formation of the corresponding cysteinc conjugate result in the formation of a compound that is more readily oxidized than 7-bromohydroquinone, whereas h’-acctylation of the cysteine conjugate to give the mercapturatc

GLUTATHIONE

CONJUGATE

regenerates a compound that is more stable to oxidation than 2-bromohydroquinone (Lau and Monks. 1990a; Monks and Lau, 1990). Thus the oxidation of 3-bromohydroquinone is exquisitely regulated by its passage through the mercapturic acid pathway. The potential toxicological significance of this relationship has been discussed (Monks and Lau, 1990). In addition to the production of reactive cysteinyl-quinones. a novel pathway of quinol-GSH metabolism that diverges from the classical route of mercapturic acid synthesis has been identified. Thus. the y-CT mediated hydrolysis of 2-bromo-3-(glutathion-Syl)hydroquinone resulted in the subsequent oxidative cyclization of the cysteinylglycine and cysteine conjugates and in 1.4-benzothiazine formation (Monks ut ul., 1990a). The intramolecular cyclization reaction removes the reactive quinone function from the molecule and might therefore be considered an intramolecular detoxication reaction. This view was supported by studies utilizing the homocysteine derivatives of 7-bromohydroquinone (Lau and Monks, 1990a) in which the additional methylene group should prevent, or at least hinder, the cyclization reaction. Since the 2-bromohydroquinone-homocysteine conjugates were nephrotoxic. this provided additional evidence that an intact quinone nucleus was essential for the toxicity of quinonethioethers and that the pathway leading to formation of the cyclized product(s) is a detoxication pathway. The toxicity of quinol-GSH conjugates will therefore be dependent upon the relative rates of cysteinylglycine and cysteine conjugate cyclization and their rate of macromolecular arylation. The I .4-benzothiazines undergo further oxidative coupling to give rise to dimeric and polymeric products analogous to the reactions observed during phaeomelanin synthesis from cystein-,S-yl-3,4dihydroxyphenylalanine (Prota. 1988). These reactions probably explain the inability to identify either cystein-Syl or N-acetylcysteinS-yl conjugates as major it1 vilv metabolites of 2-bromohydroquinone. despite the formation of the corresponding GSH conjugates

MEDIATED

TOXICITIES

II

(Lau and Monks, 1990b). and offer additional reasons for the inability of both aminooxyacetic acid and probenecid to inhibit 2-bromo(diglutathion-S-yl)hydroquinone nephrotoxicity. REVERSIBLE GLUTATHIONE CONJUGATION When the numerous substrates for GSH conjugation are grouped according to the mechanism by which conjugate formation takes place, two categories can be distinguished: (i) Substrateswhere a substitution reaction occurs. i.e.. a leaving group is replaced by the sulfur atom of the GSH molecule. Thus methyl iodide gives rise to S-methyl GSH and iodide ion. and from ethylene oxide S-2-hydroxyethyl GSH is formed: GSH+RX+GRS+HX (ii) Substrates where an addition reaction takes place, i.e., the whole GSH molecule is added onto the substrate. For example ~$5 unsaturated ketones give 1,4-addition via the so-called Michael reaction: GSH + R Q GSRH Reversibility of the con.jugation reaction can realistically be expected only in this latter category. Addition of GSH to a substrate and elimination of GSH from the conjugate can both occur, depending on conditions of pH and GSH concentration. Since the original electrophihc agent is initially detoxified, but can be releasedagain, the GSH conjugate may serve as a transport or storage form of the alkylating agent. As will be shown below. the net result could be the appearance of reactive electrophilic speciesin unexpected parts of the body. Systemic effects of highly reactive compounds might be explained in this way. The structure of the alkylating moieties of the examplesknown so far have been tabulated (Table 1). The first example of reversibility ofthe conjugation reaction was found for benzyl and

12

MONKS

,Vorc. The distinguishing feature of compounds known to react reversibly with GSH is the fact that they UC/C/ the whole GSH molecule to give a conjugate. To date. isothiocyanates. isocqanates.

GLUTATHIONE

CONJUGATE

falls apart to give acrolein and a number of other products. The lifetime of 4-hydroxycyclophosphamide in plasma can be lengthened considerably by the presence of GSH, and thiol derivatives have actually been synthesized as “depots” for the release of the active metabelite (Brock and Hohorst, 1977). Moreover, Hales ( 198 I ) found that the addition of GSH to incubation mixtures had a dual effect on the mutagenicity: relatively low concentrations of GSH increased the number of revertants whereas an excess of GSH lowered the mutagenicity. The last category of potential substrates for reversible GSH conjugation is the aldehydes themselves. For both formaldehyde and acetaldehyde, the addition of GSH has been studied fairly comprehensively (Vina cjt (/I., 1980: Koivusalo c’t (I/., 1989). The resulting thiohemiacetal can fall apart and reform the aldehyde. but can also go on to react with a second molecule of GSH and perhaps also with nitrogen nucleophiles in DNA (Fennel1 c’f [II.. 1988). This again might have important implications. because the distribution of the GSH conjugates in the body and even in the cell would be totally different from that of the aldehydes themselves and certainly merits further study. In conclusion, a reaction which has long been known as one of the simplest to understand in xenobiotic metabolism. the detoxication of electrophiles by reaction with GSH, is fhst becoming more and more complicated. The realization that GSH conjugation is not always an endpoint. but that the reactive species can be tx$wr~wc/ for some classes of compounds. may have important implications, especially for the explanation of effects at sites distant from the site of initial exposure and/ or initial conjugation. The GSH-dependent bioactivation of xenobiotics has recently been reviewed (van Bladeren, 1988; Monks and L.au. 19X9: Vamvakas and Anders. 1990). SIGNIFICANCE AND DIRECTIONS

FUTURE

(I) Elucidation of the GSH-dependent hioactivation of haloalkanes offers a sound

MFDIATED

TOXICI-TIES

13

mechanism for the mutagenicity and carcinogenicity of vicinal dihaloethanes. Moreover, elucidation of the /3-lyase-dependent bioactivation of nephrotoxic haloalkenes helps to explain the observed organotropic toxicity and. perhaps, nephrocarcinogenicity of this class of compounds. Future studies should be directed toward an understanding of the mechanistic basis underlying the mutagenicity of chloroalkene-derived conjugates and the lack of mutagenicity of tluoroalkene-derived conjugates. The observation that fluoroalkene- and chloroalkene-derived conjugates yield thionoacyl halides and thioketenes, respectively, may provide an important clue to the selective mutagenicity ofthe chloroalkene conjugates. The i3-lyase pathway may also offer a strategy to target therapeutic agents to the kidney (Hwang and Elfarra. 1989). (2) Is the renal carcinogenicity of haloalkenes due to DNA interactions or are chronic toxic effects of major importance? Trichloroethene, tetrachloroethene. and hexachloroI ,3-butadiene increase the incidence of renal neoplasms in rats, but only at doses that also induce severe nephrotoxicity: nontoxic doses are nontumorigenic. In addition, trichloroethene and tetrachloroethene induce a low rate of renal tumors exclusively in male rats. Although the biosynthesis ofgenotoxic S-conjugates and their renal activation by &lyase may be involved in this organ-specific tumor induction. the basis for the sex-specific susceptibility requires investigation. Haloalkenr derived S-conjugates are also highly cytotoxic in renal proximal tubular cells. Hence. the relationship between cell death. cell proliferation. and tumor growth rates in the kidney needs to be determined. (3) Nephrocarcinogenic chloroalkencs may occupy an intermediate position between carcinogens whose activity resides exclusively via direct DNA alterations and those active via epigenetic or nongenotoxic pathways. To establish the relative contribution of DNA interactions and chronic toxicity to the renal carcinogenicity of halogenated alkenes the cxtent of binding of metabolites to renal nuclear

14

MONKS

DNA in vivo and the persistence of the adducts formed needs to be established. Moreover, calcium-dependent mechanisms of DNA damage and radical-induced DNA damage and their contribution to haloalkene carcinogenesis need to be investigated. S-Conjugates induce a disturbance in calcium homeostasis and mitochondrial dysfunction both of which might trigger DNA damage (Lash and Anders. 1986: Wallin c’t ~1.. 1987: Trump et rd.. 1989). (4) Are quinol-linked S-conjugates genotoxic? Does the formation of such Sconjugates contribute to the renal carcinogenesis of aromatics which likely form such metabolites? Quinol-linked S-conjugates may be formed in the metabolism of the renal carcinogen 1,4dichlorobenzene (Hawkins et ul.. 1980) and with some other aromatics. Quinones may be mutagenic (Chesis et al.. 1984) and the binding of benzoquinone to DNA constituents has been observed (Kalf et ul.. 1987: Bauer IX u/.. 1989); hydroxyl radical-mediated DNA damage has been demonstrated (Kasai et ~1.. 1986). Could similar damage result from the redox cycling of quinone-linked S-conjugates and might S-conjugate formation similarly contribute to the renal carcinogenicity observed with some aromatic compounds that form polyphenolic metabolites? (5) Several of the S-conjugates formed are nephrotoxic. Both physiological and biochemical factors contribute to the susceptibility of the kidney to these compounds. For some compounds activated via the ,&lyasc pathway. such as hexachloro- 1,3-butadiene. biliary excretion. and extrarenal processing play an important role in nephrotoxicity (Nash rt cl/., 1Y 84). For other compounds. the ability of the kidney to concentrate amino acid Sconjugates and the distribution and high activity of S-conjugate processing enzymes within the kidney also predispose this organ to S-conjugate mediated toxicity (Monks and Lau. 1987: Dekant et u/.. 1989). The targeting of quinone-GSH conjugates to preneoplastic cells that often expressabnormally high levels

ET

AL.

of T-CT may have potential chemotherapeutic implications (Monks and Lau, 1989). (6) Can GSH conjugation and T-GT act as potential modulators of endogenous quinol oxidation? Are quinone-thioethers important metabolites of the catecholamines and catechol estrogens? These questions are being actively pursued and may provide additional insights into the potential physiological function of y-GT. (7) The generality of the reversibility of some GSH conjugations warrants further investigation since, for example. isocyanates and isothiocyanates of various kinds are used in large amounts in the chemical industries.

ACKNOWLEDGMENTS T.J.M. was supported by Gram ES 04662 from the National Institute ofEnvironmental Health Sciences. T.J.M. and P.J.vB are recipients of a North Atlantic Treaty Organiration Collaborative Research Award (No. 541/X7). M.W.A. was supported by National Institute of Environmental Health Sciences Grant ES 03 I27 and by Air Force Oflice of Scientific Research Grant AFOSR-86-0302. W.D. was suuported bv the Deutsche Forschungsremeinschali (SFB I ii) and the Bundesministerium fur Forschung und Technologie. Bonn. FRG. J.L.S. was supported by Awards DK 3X925 and GM 39604 from the National Institute ol Diabetes. Digestive and Kidney Disorders and the National Institute of General Medical Sciences. respectively. S.S.1.. is the recipient of a Pharmaceutical Manufacturers Association Career Development Award and was supported by Grant GM 3933X from the National Institute ofGeneral Medical Sciences.

REFERENCES AHMED. A. E.. AI\;D ANDERS. M. W. (197x1. Metabolism of dihalomethanes to formaldehyde and inorganic halide. II. Studies on the mechanisms of the reaction Bioc/wm. P/rtrrrm~~~/. 27. 202 I-1025. ANDERS. M. W.. LAsti. 1.. H.. Dt&wr. W.. E~FARRA. A. A.. AND Dow. D. R. (1988). Biosynthesis and biotransformation ofglutathione S-conjugates to toxic mctabolites. C'RC' .

( I VXOa). Identilication ol’.S- I .3.2-trichlorovin?l-‘~-acc~ylc!steinc as a urinary metaholitc of tctrachlorocthylcnc: Bioactivation through glutathionc cI /I/C>/ I!~(c’rtr~‘/ 60. 3 l-45. Dl ~,\h

I. W..

‘C1ZRIl:US.

I

RS, M. u’. ( IYX7h).

01‘ the cytotoxic

DrhAYl. L)ERS,

and neph-

I.?-trifluoroethyl).LC’S. I 81, 7443-7437.

L. H.. ANI)

ANDERS.

M. W. ( 198Xa).

W..

DEKANT.

W..

VAMVAKAS.

S.. HFNSCHI

I~R. 0..

END

AN-

M. U’. (I9XXh). Enzymatic conjugation of hexachloro-I.%hutadicnc with glutathionc: Formation of I(glutathlon-S-yl)-1.2.3.3.3.pent(lchlor[)huta -1.3 dicno and I ,4-his(glutathion-S-yt)I .2.3.4-tetrachlorohutaI .?dienc. fI~r,q :\/~i~rh. /I);.\[uI\ 16, 701-706. B~;RTHOLD.

t(..

VAMVAK~S.

S.. HENK‘H-

M. W. ( 1988~). Thioacylating Intermediates as metabolites ofS-( I .7-dichlorovinyl)-I cystiene and S-( I .2.2-trichlorovinyl)-L-qsteine formed by cysteine conjugate /+lyase. C’lrcw~. KCY. I;)v-i~r>/ I. 175-17x. I FR. D.. AUD

ANDERS.

h’..

B~RI~K)~u).

K..

VhMV4hAS.

S..

AUI)

T-hioacylating agents as ultimate Intcrmediatcs in the +lyasc catalyzed metabolism of S-( Pentachlorohutadienyl)-1 qstcinc. C’/rcw~ /I/o/ /,I/cYu~~/ 67. I3Y- I-IX.

FR. D. (19XXd).

W..

VAMVAK~S.

S.. AUDANDERS.

M. W. (19x9).

Bmxti\ation ofnephrotow haloalbenes h> glutathione conjugation: Formation of toxic and mutagenic intermediates h\ qsteien conjugate I%lyase. nvr~g .If~/~ril K(,V 20, 43~x3. Doily

AND

I5

conjugates and bioactivation ofcysteien S-conjugates by cysteicn conjugate [j-lyase. In (~lnlul/ri~~rw ~~ot~j~~~pitiot~~ I1.r .4/c&uli.wl untl Ll~~~lqyic~rrl S~,yrfi/wri/xv (H. Sies and B. Ketterer. Eds. ). pp. -I I .S447. Academic Press. Orlando.

DrhAwT.

AMISS.

(‘t~lw,. H.. ~IARvF’~. J~K,~H,\I\. J. ( 19X7).

DKANT. W.. LASH. Fate ofglutathione

HLiiS(‘HI

B. N. (IOU). Mutagenicity of quinones: Pathway\ 01‘ metabolic activation and detoxitication. I’rrK Ztr// IMd .SlY l’LY I 81, 169h-1700.

.4k1)

TOXICITIES

rotoxic S-conjugate S-O-chloro-I. cysteine. Pvoc,. .I~(lil. 1~0’ ki.

DLh,\N-I.

J. M.. Tt MMINK. J. M. H.. ANL) VAN P. J. ( 19X6). Glutathionoand cysteinc-me-

diated cvtotoxicit\ or all\1 and henrl /;I\/< (11 .. l/‘/d J’h~mrru~l X3. 349-359.

MEDIATED

D. R.. QUI~RB~MA~~.

4. J.. BORCH.

R. F.. 4r.0

M. W. ( 19Xia). Enzymatic reaction of chlorotritluoroethene with glutathione: “F NMR evidence tiw stereochemical control of the reaction. Uir,r,i~(,r)li\//?, 24? 5137-5143.

ANDERS.

DOIIN. D. R.. LI.ININGFR, M\~N. A. J.. AND ANDY

J. R., LASH,

1.. H.. QlwIw-

KS. hl. W. (19X5h). Nephrotoxicity of S-O-chloro-I. I.?-trifluoroethyl)glutathione and S-(l-chloroI. I .‘-tr-itluoro-ethyl)-r.-c\isteine. the glutathione and cysteine conjugates of chlorotritlu~~roethene. .I JYr~rrr~~r~o/. Cvp J%CT 235. X5 I -X57.

DOIlk. D. R.. AUI) cAslv4. J. E. (lYX7). ~~‘hiimniunl ion intermediates In the formation and reactions of S-(2-

haloethyl)Dtiww‘h. SON, ( IY82). I‘/c/irlu,rl EI I ,IRR~.

-cysteines.

Hir?rwy

(‘xcinogencsis 1/l/l/ 7;J\ilcli A. :2.. 4x1)

nll~l~l,l~t,r

(14X6). induced

IS. I 15-l 2-1.

4..

III.

32.

J4hoHSoN.

Mechanism

ot‘S-(

nephrotosicit>.

zxx. 1:~NNI I l.. I R.. D~AI ( IYXX). Crowlinked

Role

J.. ‘IHOMP~ I . ii. I:.

isothiocynatc.

M. W. (19x4).

coniugatcs:

I’/irrmtr~

A.

hlwssa> of ally1 2, I I & 130.

ANDLRT.

ccssingofglutathione EI I,ARR~.

(‘lrcwf

J. R.. PRI;JI 4~. J. D.. HAslm\h. R. B.. GII I.s. II. D.. .\Nl) M. J. Jollow. R. Snyder, and I. G. Sipes. Eds.). Plenum. New York. in press. I AI’. S. S.. 4ND MONKS. 7. J. ( 1990b). The in vii,0 disposition of 2-hromo-[‘JC]-hydroquinone and the e&t of y-glutamyi transpeptidase inhibition. 7i)\rcr~/. l/?/7/. /‘/krrtHuc~rl/ 103, 13 1- 133. Lthos,

J. J.. Utw.

H..

F~I.DH~IIS.

W..

ANU

METZI.I,R.

1~. E. (19X2). A novel reaction of the cocnrymc ofglutamatc decarboxylase with I.-swine-O-sulfate. U(c)chcrlli.\irt~ 21, 4377-4386. 1oc.h. A. E.. ODUM. J., AND ORMON~X P. ( 1986). Transport of ,~-acetyl-S-pentachloro-I .3butadienylcysteine by rat renal cortex. T~vic~r>/r~,e,t~ 59, I?- 15. Lot h. E. 4. (I 9X7). The nephrotoxicity ofhaloalhanc and haloalhene glutathionc conjugates, In .S&c~/ir.(/j~ nncl .\l~dw~r/ur ,\/c~~hnr~rn\ 01 7’1).wi(l~. pp. 5%X3. Macmillan Press. New York. Lo(‘h. E. 4. ( 19X8). Studies on the mechanism of nephrotoxicity and nephrocarcinogenicity ofhalogenatcd alAcncs. (‘KC‘ C’ri/. Ro 7~~.\rt~oi. 19, 23-42. M,%nc‘tt&N\‘D. D. H.. nw REED. D. J. (19X9). ldentihcation c>f the reactive glutathione conjugate S-(2-chloroethyl)glutathione in the bile of I-bromo-7-chloroethancmated rats by high-pressure liquid chromatography and prccolumn dcrivatization with o-phthalaldchyde. C‘Ircwr R(,\. 7’l~\-lwl 2, 449-454. MC I 4RI A\CE. M..

~‘OSlFR.

J. R.. Greso’!.

G. G..

KIN.HAc;~. R.. AND MOLDFILIS. P. ( IYX5). Characterization and mechanism of formation of reactive products formed during pcrouidase-catalysed oxidation of II-phenetidine. ,Z/rj/ l%mw~~~col.

27. 777-286.

SCHAEFI-hi. V. H.. AND SI‘EVEI\;S. J. 1 (1987a). Mcchanism of transport for toxic cysteinc conjugates in rat kidncq cortex membrane vesicles. 12/o/ Phu~~~~cic~o/ 32. 293-298. SC‘Ii4bFFFR. v. H.. AUD S-IEVFNS. J. 1.. (19X7b). ThC transport of ,y-cystcine conjugates in LLC-PK I cells and it3 role in toxicity. :\fo/. P/www(.o/ 31, 506-i I?. SCHNFI LMhhPu. R. G.. CROSS. T. J.. AND Loch. E. A. (1989). Pentachlorobutadienyl-L-cysteine uncouples oxidativc phosphorylation hy dissipating the proton gradient. 7-0\i0~/ l/1/)1 /‘II~I.I)ILI~,I>/ 100, 49X-505. SCHREbh. D.. 4xD D~%wl. W. (IYXY). Covalent binding of hexachlorobutadienc metaholites to renal and hepatic mitochondrial DNA. (‘~l~c~/nr!~~,r~c~.\ic 10. I 139-I 14 I. SHIII. T.-W.. APUI) HII L. D. L. ( I98 I ). Metabolic actwation of I .?-bromoethane hq glutathione transferasc and h> mlcrosomal mixed function oxidase: Further evidence few formation of two rcactivc metabolites. K(,\ C’tw,,11,1, (‘IllWl I’uri1,1/ Plrurrwld 33, 449-46 I. SMITH. M. T.. FV4hS. I-L-c?;steine lated rat renal cortical mitochondria. .h~/Jlf~~\. 258. M-372. WFF~RS. H.. ANI) SIFS. I-1. (lYX3).

A. E.. COI~~RFAVF. S. (19X7). studied Ire,/). Hepatic

Toxicit) wsith isoD10~~/1(‘117. low-Icvt’l

chemrluminescnce during redox cycling of menadione and the menadione-glutathione conjugate: Rrlation to glutathionc and NAP(P)H: quinone reductase (DT-diaphorase) activrtk. tr~,/r. U/,~C~/)~~/~I B/r~plr~x 224. 56%

W.. wt) HEUSCHLER. D. of haloalhene and haloalkane porcine kidney cells. 7’1~\rc~1/.

V’,\M~ Ah.\% S.. AND ANDFRS. M. W. (JYYO). Formation of I-cactive intermediates hy Phase II enzymes: Glutathione-dependent binactivation reactions. , Jr/r* ZCvfj lid Hirri, in press.

(i.

B., BUYS. W.. AND VAN DER GEU, A. ( 1981). The relation between the structure of vicinal dihalogen compounds and their mutagenic activation via conjugation to glutathione. (‘urc,inc?~~,nc,ti.c 2, 499-505. 4%~ OMMFN. B.. DEN BFST~N. C.. R~JUFN. A. c‘. M..

10.

V4Mv4h45. S.. DEIcANT. W.. SCHIFFMANN. D.. 4ND HFUSCHLEK. D. ( 1988d). Induction 01’ unscheduled DhA synthesis and micronucleus formation in Syrian hamster embryo tihroblasts treated Jugatcs of chlorinated hydrocarbons. 4. 3Y3-403.

19

VAN BLADEREN. P. J.. BREIMER. D. D.. ROT-I-FVEEL-SMIIS, G. M. T.. DE KNIFF. P.. MOHN, G. R., VAN MEET~REN.

V~MV~ICAS. S.. KORDOWIC’H. F. J.. DEKANT. W.. NtriDtChER. T., AND H~NSCHLER. D. (19X&c). Mutagenic@ ofheuachloro-1.3-butadiene and its S-conjugates in the Ames test-Role of activation by the mercapturic acid pathway in its nephrocarcinogenicity. C;rrc,in~~,~~,~~~,.civ 9. 907-9

TOXICITIES

57x. z. G. ( 19X9 ). Biological interactron of cu.ij-unsaturated aldehydes. pt?c Rutlic~rrl Hio/. .21d. I. 333-34’4. WoLr-r. S. P.. ,AND SPFCTOR. A. (1987). Pro-oxidant

WI.I

ac-

tivation of ocular reductants. 2. Lens epithelial cell c?totosicit?; of a dretar) quinone is associated with a stable Tree radical formed with glutathione it) ri/lrj. f,Cv/) !:‘I?

Kc'.\.-IS, 7') I-X(1 I. ZI~AUC;. G.. AUI) Qtvrus. J. 1.. (IYXY). activation of S-( I .3-dichlorovin>l)-I.-cvstcrnc acrtyl-S-( proximal 6I

I.?-dichlorovin~l)-L-clsteine tubules. li~\-i(~r)l.

l/I/?/

-Iransport and

and :f-

in rat hrdnc) I’/)~irrlrtr~~~I/ 100. 5 I -