Nephrotoxicity of quinol/quinone-linked S-conjugates

Toxicology Letters, 53 (1990) 5967 59

Elsevier

TOXLET 02386

Nephrotoxicity S-conjugates

of quinol/quinone-linked

Terrence J. Monks and Serrine S. Lau

Division of Pharmacology and Toxicology. College of Pharmacy, The University of Texas at Austin, Austin,

TX (U.S.A.)

Key words: Quinol/quinone thioethers; SConjugates; Nephrotoxicity; 1,4_Benzothiazines

INTRODUCTION

The reactivity of quinones resides in their ability to undergo ‘redox-cycling’ and to thereby create an oxidative stress [l] and/or to react directly with cellular nucleophiles such as protein and non-protein sulfhydryls [2]. Glutathione (GSH) is the major non-protein sulfhydryl present in cells [3] although there are relatively few studies on the addition of sulfur nucleophiles to quinones [4] and especially of the biological consequences of these reactions. The addition of a thiol to the double bond of a quinone is in fact a special case of nucleophilic addition to an a&unsaturated carbonyl. Cysteine or GSH adds to 2-alkylnaphthoquinones (such as menadione and other vitamin K derivatives) to give a simple adduct that resides in the oxidized quinone form. With benzoquinone derivatives (which exhibit a more positive redox po- tential than naphthoquinones) further reaction leads to reduction of the keto group and disproportionation to yield a conjugate that resides in the reduced hydroquinone form. Alternatively, a free amino group may condense with one of the quinone car- bony1 groups and, by further oxidation with excess quinone, give a 1,Cbenzothiazine derivative [5]. The natural occurrence of quinones of both the benzoquinone and naphthoquinone type makes these reactions of particular biological interest.

The conjugation of potentially toxic electrophiles with GSH - the process of thioether formation - is usually associated with detoxication and excretion. Com- pounds that are conjugated with GSH are usually excreted in urine as their corresponding mercapturic acids, S-conjugates of N-acetyl cysteine. In contrast to the gen-

Addressfor correspondence: Terrence J. Monks, Ph.D., Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, U.S.A.

0378-4274/90/s 3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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erally accepted role of GSH conjugation serving as a detoxication mechanism, several recent studies provide evidence that quinone-thioethers exhibit significant biological activities. For example, the GSH conjugate of menadione can redox-cycle, with the concomitant formation of reactive oxygen species [6]. In addition, Ross et al. [7] have isolated 3 GSH conjugates from the peroxidase-catalyzed oxidation ofp-phenetidine, which exist in both oxidized and reduced forms and which are readily interconverted by redox processes. Potter et al. [8] have also demonstrated that the GSH conjugate of acetaminophen is readily oxidized to a free radical intermediate. It has been sug- gested that the redox activity of quinone-GSH conjugates may play a role in the oxidative damage of cataract [9]. For example, formation of a stable 2,6_dimethoxyquin- one-GSH free radical has been demonstrated in bovine lens epithelial cells and may contribute to the cytotoxicity caused by this dietary quinone [9]. The GSH conjugate of menadione, 2-methyl-3-(glutathion-S-yl)-1,6naphthoquinone, has antimitotic properties in cultures of chick fibroblasts whereas the GSH conjugate of l,Cnaph- thoquinone does not [lo] and the antibacterial activity of the quinonoid fuscin is not altered upon addition of thioglycolic acid [l 11. The GSH conjugates of menadione and toluquinone have been shown to be substrates for NADP-linked 15hydroxy- prostaglandin dehydrogenase and are mixed-type inhibitors of prostaglandin Bi oxidation [12]. The GSH conjugate of tetrachloro-1,4-benzoquinone is an effective inhibitor of the GSH S-transferases [13]. Finally, the GSH conjugate(s) of N-(4- ethoxyphenyl)-p-benzoquinone imine has been shown to bind to DNA [ 141. We have recently shown that conjugation of quinones with GSH results in the formation of potent, and selective, nephrotoxicants [15-l 71. In addition, we have established that the reactivity of benzoquinolLGSH conjugates is a consequence of their oxidation to the corresponding quinone [ 16,181.

RESULTS AND DISCUSSION

Both 2-bromo-(diglutathion-S-yl)hydroquinone and 2,3,5-(triglutathion-S-yl)- hydroquinone are relatively potent and specific renal proximal tubular toxicants. Administration of 10-20 pmol/kg of these compounds to male Sprague-Dawley rats causes severe necrosis of the S3 segment of renal proximal tubules, increases in blood urea nitrogen, enzymuria, and glucosuria. The mechanism of action of these compounds is unclear at present. Although metabolism of the conjugates by y-glutamyl transpeptidase (y-GT) appears to be a prerequisite for toxicity, processing by cysteine conjugate /&lyase, which is essential for the expression of both haloalkenyl- and haloalkane S-conjugate-mediated nephrotoxicity, does not appear to play a major role in the nephrotoxicity of benzoquinol-GSH conjugates [16,17]. In addition, the nephrotoxicity of 6-bromo-2,5-dihydroxythiophenol, a putative p-lyase-catalyzed metabolite of 2-bromo-3-(glutathion-S-yl)hydroquinone, was shown to be dependent upon the quinone function [18]. Thus, the evidence suggests that the toxicity of benzoquinol-GSH conjugates is mediated via their metabolism by y-GT and oxidation to the corresponding quinone.

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The activity of y-GT may be required for one, or both, of two possible functions. The enzyme may be functioning indirectly in a transport capacity, in which formation of the cysteinylglycine and cysteine conjugates is a prerequisite for cellular uptake. In support of this view, AT-125, an inhibitor of y-GT, decreased the accumula- tion of 2-bromo-(diglutathion-S-yl)hydroquinone by isolated renal cortical slices [19]. In contrast, y-GT may be functioning in a metabolic capacity, in which enzyme activity is required for the generation of the ultimate toxic metabolite(s). Indeed, although oxidation of benzoquinol-GSH conjugates appears necessary for the generation of the majority of the reactive metabolites formed from these compounds [16], the GSH conjugates of 2-bromohydroquinone are relatively stable to oxidation [16,20,21]. However, the oxidation of 2-bromo-3-(glutathion-S-yl)hydroquinone is exquisitely regulated via its metabolism through the mercapturic acid pathway [21]. Thus, 2-bromo-3-(cystein-Syl)hydroquinone is easier to oxidize, at pH 7.4, than either 2-bromohydroquinone or its corresponding GSH conjugate, and N-acetylation to form the mercapturic acid regenerates a compound that is relatively stable to oxidation [21]. y-GT activity, therefore, may be required for both transport and activation of the benzoquinol conjugates.

Since y-GT plays such a crucial role in the toxicity of benzoquinol-GSH conjugates, we investigated the y-GT-catalyzed hydrolysis of 2-bromo-3-(glutathion-S-yl)- hydroquinone in detail. The reaction resulted in the formation of multiple products, as analyzed by HPLC, and ultimately in the deposition of a black precipitate that exhibited properties of a pH indicator. Purification and structural analysis of the ini- tial major product of this reaction revealed the formation of a 1,4-benzothiazine [22]. This product arises via the intramolecular cyclization of the cysteinyl amino group on the quinone carbonyl atom (Fig. 1). These reactions are analogous to the formation of the trichochrome polymers during melanin synthesis, which involves the oxidative cyclization of 3,Cdihydroxy-5-(cystein-S-yl)phenylalanine and 3,4-dihydroxy- 6-(cystein-S-yl)phenylalanine [23]. The involvement of this pathway in 2-bromo-3- (glutathion-S-yl)hydroquinone metabolism provides an explanation for our previous inability to identify either cystein-S-y1 or N-acetylcystein-S-y1 conjugates as major in vivo metabolites of i4C-2-bromohydroquinone, even though the corresponding GSH conjugates could be identified in both bile and urine [24]. Thus, 1,Cbenzothiazine formation represents a novel pathway which diverges from the classical mercapturic acid biosynthetic pathway. Although the toxicological significance of this pathway is unclear experiments carried out with L-homocysteine conjugates of 2-bromohydroquinone suggest that it probably constitutes a detoxication reaction [20]. The additional methylene group in the homocysteine conjugates should effectively prohibit the oxidative cyclization reaction. Thus, if cyclization were a prerequisite for toxicity, then the homocysteine analogues should be less toxic than the corresponding cysteine conjugates. However, the homocysteine conjugates caused significant damage to renal proximal tubules, indicating that inhibition of 1 ,Cbenzothiazine formation per- mits the development of toxicity.

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H shlft

glyclne

Br

Fig. 1. Oxidative cyclization and 1,4-benzothiazine formation from 2-bromo-3-(glutathion-S-yl)hydroquinone).

Additional evidence for oxidative cyclization and 1 ,Cbenzothiazine formation mo- dulating the nephrotoxicity of quinone-thioethers has been obtained in studies inves- tigating the toxicity of various, 1,4-naphthoquinone-thioethers. 2-Methyl-3-(N-acetylcystein-S-yl)- 1 +naphthoquinone, the mercapturic acid metabolite of menadione, caused a dose-dependent (50-200 /*mol/kg) necrosis of the terminal portion of the S2 segment and the S3 segment within the medullary ray [25J (The localization of this lesion is different from that seen with the benzoquinol conjugates, the basis for which is unclear.) In contrast to the mercapturate, 2-methyl-3-(glutathion-S-yl)- 1,4- naphthoquinone (200 pmol/kg), the GSH conjugate of menadione, caused no apparent pathological alterations to the kidney 1251. The striking difference between the effects of the N-acetylcysteine and GSH conjugates of menadione may be related to their relative ability to undergo the cyclization reaction. The l,Cbenzothiazines, and

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the di(poly)meric products arising from the oxidative coupling of these compounds, exhibit distinct chromophores, and the formation of such chromophores has been observed during in vitro incubations of isolated rat renal epithelial cells with 2-methyl-3-(glutathion-9yl)-1,4_naphthoquinone but not with 2-methyl-3-(N-acetylcystein- S-yl)-1,Cnaphthoquinone [Jones et al., personal communication]. Thus, administration of the mercapturate precludes removal of the reactive quinone moiety prior to its delivery and transport into renal proximal tubular cells. In contrast, the GSH conjugate requires processing by renal brush-border y-GT to effect its uptake, as the corresponding cysteinylglycine and/or cysteine conjugate, into proximal tubular cells. As emphasized above, this reaction can result in quinone detoxication.

We also investigated the toxicity of the GSH conjugates of 2,5-dichloro-1 ,Cbenzo- quinone and 2,3,5-trichloro-l+benzoquinone. The conjugates formed from these compounds have certain similarities with the 2-bromohydroquinone and hydroquinone conjugates, the main difference being that when reaction of the chloroquinones with GSH occurs at a chlorine-substituted carbon atom, the resulting conjugate resides in the quinone form. Interestingly, when 2,5-dichloro-3-(glutathion-S-yl)- 1,4- benzoquinone and 2,5,6-trichloro-3-(glutathion-S-yl)-l,Cbenzoquinone were co-administered with ascorbic acid, pretreatment of Sprague-Dawley rats with AT- 125, to inhibit y-GT activity, actually potentiated the nephrotoxicity [26]. Although the mechanism of potentiation is unclear, it may, again, be related to effects subsequent to the formation of the cysteinylglycine conjugates. Thus, differences in the rate at which the various quinone-thioethers undergo the cyclization reaction, relative to their rates of, for example, N-acetylation or macromolecular arylation, may well determine whether the inhibition of y-GT either potentiates or protects the nephrotoxicity of these compounds.

It is therefore apparent that factors other than y-GT can regulate quinone-thioether nephrotoxicity. Additional support for this view was obtained from studies on the susceptibility of different species to 2-bromo-(diglutathion-S-yl)hydroquinone-mediated nephrotoxicity. Thus, although rats expressed the highest level of renal y-GT activity and were the most susceptible species to 2-bromo-(diglutathion-S-yl)hydroquinone-mediated nephrotoxicity (Table I), renal y-GT activity did not correlate with susceptibility in the other species examined. For example, the only other rodent species that developed renal proximal tubular necrosis following administration of 2- bromo-(diglutathion-Syl)hydroquinone (200 pmol/kg) was the guinea-pig, and this species exhibited the lowest activity of renal y-GT (Table I).

Interestingly, the lesion observed in the guinea-pig, which involved the lower portions of the S$ into the outer stripe of the outer medulla, displayed two rather unique features. First, many of the injured cells displayed apparent metaphase plates with the chromosomal material in varying stages of dissolution. Secondly, the injured cells were frequently found to be filled with basophilic granules. Other injured cells displayed more typical features of injury, including loss of the brush border (consis- tent with the appearance of y-GT in the urine), vesiculation, eosinophilia and pyknot- ic nuclei.

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TABLE I

SPECIES DIFFERENCES IN RENAL y-GLUTAMYL TRANSPEPTIDASE ACTIVITY AND SUSCEPTIBILITY TO 2-BROMO-(DIGLUTATHION-S-YL)HYDROQUINONE-MEDIATED NEPHROTOXICITY

Relative activity BUN (mg%/24 h)

Treated Control

Fischer 344 rats 100% 33.1 k8.1 12.9k2.4 Sprague-Dawley rats 72% 34.9k4.5 15.5;t2.5 Golden Syrian hamsters 34% 20.4k4.3 19.3k2.8 BALB/c mice 27% 21.3k4.3 20.5k3.7 Albino guinea-pigs 8% 66.1&19.1 21.4f5.6

y-GT activity in male Fischer 344 rats was 2.26 kO.16 units/pg protein (1 unit = 1 nmol of p-nitroaniline formed per minute at 25°C). 2-Bromo-(diglutathion-S-yl)hydroquinone was administered by intravenous injection at a dose of 15 (Sprague-Dawley) and 20 pmol/kg (Fischer 344) to rats and 200 pmol/kg to hamsters, mice and guinea-pigs. Figures represent the mean f SD (n =4).

The reasons for the inter-species variation in susceptibility to 2-bromo-(diglutathion- S-yl)hydroquinone-mediated nephrotoxicity are unclear at present. However, significant physiological differences exist between species in proximal tubular cell structure and function [27]. These differences may contribute, in part, to the inability of 2-bromo-(diglutathion-S-yl)hydroquinone to elicit toxicity when incubated with isolated rabbit renal proximal tubule suspensions [28]. In addition, species differences in the specific activity of the mercapturic acid pathway enzymes may also determine susceptibility to quinol/quinone-linked S-conjugate-induced nephrotoxicity. Differ- ences in the substrate specificity of these enzymes might also contribute to the observed differences in both the relative potency of these compounds and to differences in the consequences of y-GT inhibition. For example, in rats, 2-bromo-6-(N-acetylcystein-9yl)hydroquinone is a more potent renal proximal tubular toxicant than either 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone or 2-bromo-5-(N-acetylcystein-S-yl)hydroquinone (Table II) and is a better substrate for the renal cytosolic AL deacetylase than the inactive isomer. In contrast, differences in the affinity of the various multi-GSH substituted isomers of both 2-bromohydroquinone and hydroquinone for renal y-GT do not appear to determine their differential toxicity [29]. In this case, increasing GSH substitution results in both a decrease in the affinity for y-GT and a decrease in the turnover number; yet 2-bromo-(diglutathion-S-yl)- hydroquinone and 2,3,5-(triglutathion-4yl)hydroquinone are more potent nephrotoxicants than any of the lower substituted isomers. Thus, both physiological and biochemical factors probably determine species- and substrate-specific toxicity of quinol/quinone-thioethers.

The mechanism of quinol/quinol-linked S-conjugate toxicity is unclear, but based

6.5

TABLE II

RELATIVE NEPHROTOXICITY OF 2-BROMO-(N-ACETYLCYSTEIN-SYL)HYDROQUIN- ONES

Positional isomer LDH Y-GT BUN

(units/l9 h) (units/l9 h) (mg%)

Controls 0.07+0.01 0.3kO.10 14.64 1.1

2-Bromo-3-NAC 0.13+0.06 0.9+0.15 17.3k1.5

2-Bromo&NAC 0,84+0.23 5.5+ 1.8 17.613.3

2-Bromo&NAC 4.16k 1.40 18.653.9 28.lk5.6

Compounds were administered by intravenous injection in saline/DMSO (2: 1, v/v) at a dose of 1 SO pmol/ kg Control animals received the vehicle only. Animals were euthanized 19 h after the injection and blood and urine analyzed by established methodology. The figures represent the mean + SE (n =4-8).

upon the known properties of quinones, toxicity could be initiated either via the arylation of cellular macromolecules that are critical for cell structure and function, or via the generation of reactive oxygen species during quinone redox cycling. Where- as the former mechanism may be important in the nephrotoxicity of benzoquinol-S- conjugates, the latter mechanism probably contributes to the toxicity of l,Cnaphtho- quinone S-conjugates. Thus, in contrast to 2,3,5,6-(tetraglutathion-~-yl)hydroquinone, 2,3,5-(triglutathion-S-yl)hydroquinone is a potent nephrotoxicant [17]. The lack of toxicity of the fully substituted isomer could be a consequence of its inability to arylate macromolecules. Moreover, increases in blood urea nitrogen concentrations following “C-2-bromohydroquinone administration to rats correlated with covalent binding to renal macromolecules [24]. At present there is little evidence to support a role for redoxcychng in the nephrotoxicity of benzoquinol S-conjugates. In contrast, both the GSH and mercapturic acid conjugates of menadione undergo redox- cycling with the concomitant formation of reactive oxygen species [6,30]. In addition, inhibition of GSH reductase with N,N-bis(2-chloroethyl)-N-nitrosourea potentiated the toxicity of 2-methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone in isolated renal epithelial cells [31]. Whether redox-cycling plays a role in the in vivo toxicity of this conjugate is under investigation. However, the fully substituted quinone moiety in this compound suggests that macromolecular arylation is an unlikely explanation of toxicity.

In conclusion, there is now ampie evidence attesting to the biological reactivity of quinone thioethers. Further work is required to determine the in vivo disposition of these compounds and to integrate metabolism with the site and mechanism of toxicity.

ACKNOWLEDGEMENTS

Portions of this work were supported by USPHS awards ES 04662 and GM 39338

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and a NATO Collaborative Research Award (No. 542/87). S.S.L. is the recipient of a Pharmaceutical Manufacturers Association Foundation Faculty Development Award. We thank Dr. Tom Jones for histological interpretation of the kidney slices obtained from the guinea-pig, Dr. Stony Lo for the kinetic studies with y-GT, and Maria Rivera, Barbara Hill and Rowena Sioco for expert technical assistance.

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2 Jocelyn, PC. (1972) Biochemistry of the SH Group. Academic Press, London. 3 Reed, D.J. and Meredith, M.J. (1985) Cellular defense mechanisms against reactive metabolites. In:

M.W. Anders (Ed.), Bioactivation of Foreign Compounds. Academic Press, New York, pp. 71-108. 4 Finley, K.T. (1974) The addition and substitution chemistry of quinones. In: S. Patai (Ed.), The Chem-

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8 Potter, D.W., Miller, D.W. and Hinson, J.A. (1986) Horseradish-peroxidase catalysed oxidation of acetaminophen to intermediates that form polymers or conjugate with glutathione. Mol. Pharmacol. 29, 155-162.

9 Wolff, S.P. and Spector, A. (1987) Pro-oxidant activation of ocular reductants. 2. Lens epithelial cell cytotoxicity of a dietary quinone is associated with a stable free radical formed with glutathione in vitro. Exp. Eye Res. 45, 791-801.

10 Friedmann, E., Martian, D.H. and Simon-Reuss, I. (1948). Sulphydryl addition compounds of some quinones and related substances in their action on the growth of normal cells. Br. J. Pharmacol. 3, 335-340.

11 Marcus, S. (1948) Antibacterial activity of fuscin. Addendum to: Studies in the Biochemistry of Micro- organisms 79. Fuscin, a metabolic product of Oidiodendron fuscum Robak. I. Preparation, properties and antibacterial activity (Michael, S.E.), Biochem. J. 43,528-533.

12 Chung, H., Harvey, R.G., Armstrong, R.N. and Jarabak, J. (1987) Polycyclic aromatic hydrocarbon quinones and glutathione thioethers as substrates and inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase. J. Biol. Chem. 262,12448-12451.

13 Van Ommen, B., Den Besten, C., Rutten, A.C.M., Ploemen, J.H.T.M., Voo, R.M.E., Muller, M. and Van Bladeren, P.J. (1988) Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,Cbenzoquinone. J. Biol. Chem. 263, 12939-12942.

14 Larsson, R., Boutin, J. and Moldeus, P. (1988) Peroxidase-catalysed metabolic activation of xenobiot- its. In: J.W. Gorrod, H. Oelschlager and J. Caldwell (Eds.), Metabolism of Xenobiotics. Taylor and Francis, New York, pp. 43-50.

15 Monks, T.J., Lau, S.S., Highet, R.J. and J.R. Gillette (1985). Glutathione conjugates of2-bromohydroquinone are nephrotoxic. Drug Metab. Disposit. 13, 553-559.

16 Monks, T.J., Highet, R.J. and Lau, S.S. (1988) 2-Bromo-(diglutathion-9yl)hydroquinone nephrotoxicity. Physiological, biochemical and electrochemical determinants. Mol. Pharmacol. 34,492-500.

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17 Lau, S.S., Hill, B.A., Highet, R.J. and Monks, T.J. (1988) Sequential oxidation and glutathione addition to 1,Cbenzoquinone: correlation of toxicity with increased glutathione substitution. Mol. Phar- macol. 34,829836.

18 Monks, T.J., Highet, R.J., Chu, P.S. and Lau, S.S. (1988) Synthesis and nephrotoxicity of 6-bromo- 2,5-dihydroxy-thiophenol. Mol. Pharmacol. 34, 15-22.

19 Lau, S.S., McMenamin, M.G. and Monks, T.J. (1988) Differential uptake of isomeric 2-bromohydroquinone glutathione conjugates into rat kidney slices. Biochem. Biophys. Res. Commun. 152,223-230.

20 Lau, S.S. and Monks, T.J. (1990) Glutathione conjugation as a mechanism of targeting latent quinones to the kidney. In: D.J. Jollow, R. Snyder and LG. Sipes (Eds.), Biological Reactive Intermediates, IV. Molecular and Cellular Effects and Human Impact. Plenum Press, New York (in press).

21 Monks, T.J. and Lau, S.S. (1990) Glutathione conjugation, y-glutamyl transpeptidase and the mercapturic acid pathway as modulators of f-bromohydroquinone oxidation. Toxicol. Appl. Pharmacol. 103, 557-563.

22 Monks, T.J., Highet, R.J. and Lau, S.S. (1990) Oxidative cyclization, l&benzothiazine formation and dimerization of 2-bromo-3-(glutathion-9yl)hydroquinone. Mol. Pharmacol. 38, 121-127.

23 Prota, G. (1988) Progress in the chemistry of melanins and related metabolites. Med. Res. Rev. 8, 525 556.

24 Lau, S.S. and Monks, T.J. (1990) The in vivo disposition of 2-bromo-[i4C]-hydroquinone and the effect of y-glutamyl transpeptidase inhibition. Toxicol. Appl. Pharmacol. 103, 121-132.

25 Lau, S.S., Jones, T.N., Highet, R.J., Hill, B.A. and Monks, T.J. (1990) Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of menadione and 1,Cnaphthoquinone. Toxicol. Appl. Pharmacol. 104,334350.

26 Monks, T.J., Lau, S.S., Mertens, J.J.W., Temmink, J.H.M. and Van Bladeren, P.J. (1990) The nephrotoxicity of 2,5-dichloro-3-(glutathion-S-yl)-l,4-benzoquinone and 2,5,6-trichloro-3-(glutathion-S-yl)- 1,4-benzoquinone is potentiated by ascorbic acid and AT-125. In: D.J. Jollow, R. Snyder and I.G. Sipes (Eds.), Biological Reactive Intermediates. IV. Molecular and Cellular Effects and Human Im- pact. Plenum Press, New York (in press).

27 Kriz, W. and Kaissling, B. (1985) Structural organization of the mammalian kidney. In: D.W. Seldin and G. Giebisch (Eds.), The Kidney: Physiology and Pathophysiology. Raven Press, New York, pp. 265-306.

28 Schnellmann, R.G., Monks, T.J., Mandel, L.J. and Lau, S.S. (1989) 2-Bromohydroquinone-induced toxicity to rabbit renal proximal tubules: the role of biotransformation, glutathione and covalent bond- ing. Toxicol. Appl. Pharmacol. 99, 1927.

29 Hill, B.A., Lo, H.-H., Monks, T.J. and Lau, S.S. (1990) The role of y-glutamyl transpeptidase in hydro- quinoneglutathione conjugate mediated nephrotoxicity. In: D.J. Jollow, R. Snyder and LG. Sipes (Eds.), Biological Reactive Intermediates, IV. Molecular and Cellular Effects and Human Impact. Ple- num Press, New York (in press).

30 Brown, P.C. and Jones, T.W. (1988) The toxicity of 2-methyl-1,4_naphthoquinone and two thioether conjugates. Toxicologist 8, 133 (Abstract No. 529).

31 Brown, P.C. and Jones, T.W. (1989) The role of oxidative stress in the isolated renal epithelial toxicity of menadione and its N-acetyl-L-cysteine conjugate. Toxicologist 9, 161 (Abstract No. 644).

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Nephrotoxicity of quinol/quinone-linked S-conjugates