Biological Reactivity of PolyphenolicâGlutathione ... - ACS Publications

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Chem. Res. Toxicol. 1997, 10, 1296-1313

Invited Review Biological Reactivity of Polyphenolic-Glutathione Conjugates Terrence J. Monks* and Serrine S. Lau Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712-1074 Received May 30, 1997

1. Introduction

1. Introduction 1.1. Quinones as Oxidants and Electrophiles 1.2. Addition of Sulfur Nucleophiles to Quinones 2. Biological Reactivity of Polyphenolic-Glutathione Conjugates 3. Nephrotoxicity of Polyphenolic-Glutathione Conjugates 3.1. Mechanism of Action and Cellular Targets 3.2. Role of Polyphenolic-Glutathione Conjugates in Chemical-Induced Nephrocarcinogenesis 3.2.1. Hydroquinone 3.2.2. 17β-Estradiol 3.2.3. 3-tert-Butyl-4-hydroxyanisole 4. Neurotoxicity Conjugates

of

Polyphenolic-Glutathione

4.1. 3,4-(()-(Methylenedioxy)amphetamine and 3,4-(()-(Methylenedioxy)methamphetamine 4.1.1. Transport of Glutathione and Glutathione Conjugates across Blood-Brain Barriers 4.1.2. Functional Roles of Glutathione in the Brain 4.2. Neurotoxicity of Endogenous Polyphenolic Conjugates 4.2.1. Oxidation of Dopamine Parkinson’s Disease 4.2.2. Oxidation of Serotonin 4.2.3. Tetrahydroisoquinolines 5. Hematotoxicity Conjugates

of

and

Polyphenolic-Glutathione

5.1. Role for Polyphenolic-Glutathione Conjugates in Benzene-Mediated Hematotoxicity 5.2. Aminophenols and Quinone-ThioetherCatalyzed Methemoglobinemia 6. Conclusions and Future Directions

* Address correspondence to this author at the above address. Tel: (512) 471-6699. Fax: (512) 471-5002. E-mail: [email protected].

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1.1. Quinones as Oxidants and Electrophiles. Quinones are both oxidants and electrophiles. The ability of quinones to redox-cycle and create an oxidative stress and their ability to form covalent adducts with a variety of cellular macromolecules provide the basis for their biological activity. Both of these reactions can result in the destruction of molecules essential for cell survival. The complete reduction of a quinone to a quinol (hydroquinone or catechol) requires two electrons and two protons (1). The one-electron-reduced form of a quinone is a semiquinone, which may react with molecular oxygen to yield the superoxide anion radical (O2•-),1 which subsequently undergoes either spontaneous or enzymecatalyzed dismutation to form hydrogen peroxide. O2•also reduces Fe3+ to Fe2+, and hydrogen peroxide then reacts with Fe2+ to generate the hydroxyl radical, which is probably the reactive species responsible for quinonemediated oxidative damage. 1.2. Addition of Sulfur Nucleophiles to Quinones. Since nucleophilic addition to a quinone represents a formal two-electron reduction, the oxidant and electrophilic properties of quinones are closely related (2). Because of the inherent nucleophilicity of the cysteinyl sulfhydryl, protein and nonprotein sulfhydryls represent major targets for quinones. The addition of a thiol to the double bond of a quinone represents nucleophilic addition to an R,β-unsaturated carbonyl. Glutathione (GSH) is the major nonprotein sulfhydryl present in cells (3), and when GSH is added to quinones the reaction is generally considered cytoprotective, because the thiol function in GSH serves as a “sacrificial” nucleophile, sparing critical nucleophilic sites on cellular macromolecules from irreversible modification. The redox potential of a quinone is strongly influenced by substituent effects (4, 5). Thus, addition of an electronegative substituent to a quinone usually results in a stronger oxidant, and its reduced or hydroquinone form is less easily oxidized. Conversely, addition of electron-donating groups to a quinone usually results in a weaker oxidant, and the hydroquinone forms are more easily oxidized. Because of these influences, the hydroquinone generated when a nucleophile adds to 1Abbreviations: BHA, butylated hydroxyanisole; CSF, cerebrospinal fluid; DOPA, 3,4-dihydroxyphenylalanine; γ-GT, γ-glutamyl transpeptidase; GSH, glutathione; GSSG, glutathione disulfide; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine; MDA, 3,4-(()(methylenedioxy)amphetamine; MDMA, 3,4-(()-(methylenedioxy)methamphetamine; O2•-, superoxide anion radical; TBHQ, tert-butylhydroquinone.

© 1997 American Chemical Society

Invited Review

a quinone can often be reoxidized by the unsubstituted parent quinone (6). In this way a single molecule of a quinone can ultimately form products of multiple nucleophilic additions (7, 8) or generate cross-links between nucleophilic sites on macromolecules (9).

2. Biological Reactivity of Polyphenolic-Glutathione Conjugates There is little difference between the reduction potentials of menadione and its GSH conjugate (10). Indeed, the addition of GSH and N-acetylcysteine to menadione may actually lower the redox potential of the quinone (11). The addition of cysteine to 3,4-dihydroxyphenylalanine (DOPA) also lowers the oxidation potential of DOPA by about 45-50 mV (12, 13). Thus cystein-S-ylDOPA is easier to oxidize than DOPA itself. Consistent with the electrochemical properties of quinol-thioethers, both the GSH and N-acetylcysteine conjugates of menadione retain the ability to redox cycle, with the concomitant formation of reactive oxygen species (14, 15). Because of these findings, the conjugation of menadione with GSH cannot be considered a true detoxication reaction (although excretion of the quinone is facilitated by conjugation with GSH). The formation of reactive oxygen species from menadione and its GSH conjugate is enhanced in the presence of dicumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (DT-diaphorase), suggesting that both compounds are substrates for twoelectron reduction by this enzyme (14). Several quinoneGSH conjugates, including 2-methyl-3-(glutathion-S-yl)1,4-naphthoquinone, are effective substrates for DTdiaphorase (11). Seven GSH conjugates are formed during the peroxidase-catalyzed oxidation of p-phenetidine, several of which exist in both oxidized and reduced forms and are readily interconverted by redox processes (16). Although the peroxidase-mediated covalent binding of p-phenetidine to protein is inhibited by the addition of GSH (17), binding to DNA is enhanced by GSH (18), and a bisglutathionyl adduct of N-(4-ethoxyphenyl)-p-benzoquinonimine binds to calf thymus DNA (19). The GSH conjugate of acetaminophen is also readily oxidized to a free radical intermediate (20) in a reaction catalyzed by peroxidases. The cytotoxicity of 2,6-dimethoxy-1,4-benzoquinone to lens epithelial cells in culture is also related to reactive oxygen generation and the formation of a stable free radical (21). The formation of the latter is dependent upon the conjugation of 2,6-dimethoxy-1,4benzoquinone with GSH and the ESR spectra of the radical are consistent with the one-electron oxidation of 2,6-dimethoxy-3,5-bis(glutathion-S-yl)hydroquinone (21). The redox activity of 2,6-dimethoxy-3,5-bis(glutathionS-yl)hydroquinone is also consistent with the hypothesis that oxidative damage plays an important role in the pathogenesis of human cataracts. The cataractogenicity of naphthalene is also attributed to its metabolism to 1,2naphthoquinone (22, 23) and possibly to the corresponding cysteine and GSH conjugates, which are very effective at catalyzing the oxidation of ascorbic acid (22). Ascorbate levels decrease substantially during cataract formation. ESR studies also provide evidence for the generation of GSH-conjugated semiquinone free radicals from 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone, 3-(glutathion-S-yl)-1,4-naphthoquinone, 2,3-bis(glutathion-Syl)-1,4-naphthoquinone, and 2-(glutathion-S-yl)-1,4-benzoquinone (24, 25). Thus, the ability of a quinone to form

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multiple nucleophilic addition products, and the ability of quinol-thioethers to redox-cycle and generate reactive oxygen species, provides a clear basis for their biological reactivity.

3. Nephrotoxicity of Polyphenolic-Glutathione Conjugates GSH conjugates of a variety of polyphenols are potent nephrotoxicants in Sprague-Dawley and Fischer 344 rats (7, 26-30). The nephrotoxicity of this class of metabolites in rats is a consequence of the relatively high activity of γ-glutamyl transpeptidase (γ-GT) within the brush border membrane of renal proximal tubular epithelial cells. Cells in the S3 segment of the proximal tubule are selective targets of the polyphenolic-GSH conjugates. γ-GT catalyzes the first step in the metabolism of GSH and its S-conjugates (31); it is an ectoenzyme with the active site located on the outer surface of the cell membrane (32). Thus, the hydrolysis or transpeptidation reactions catalyzed by the enzyme take place extracellularly, and the product of the reaction, the cysteinylglycine dipeptide, is a substrate for dipeptidases which are similarly concentrated in the brush border membrane of proximal tubular epithelial cells (33). The cysteine conjugates are then transported across the brush border membrane via the amino acid transport system. In this manner, metabolism of polyphenolic-GSH conjugates by γ-GT within the brush border membrane is coupled to the cellular uptake of the corresponding polyphenolic-cysteine conjugate. The first example of polyphenolic-GSH conjugateinduced nephrotoxicity was the identification of 2-bromobis(glutathion-S-yl)hydroquinone as a nephrotoxic metabolite of bromobenzene (26). 2-Bromo-bis(glutathionS-yl)hydroquinone produces renal toxicity at a dose of about 0.1% of that of bromobenzene and is therefore an extremely potent nephrotoxicant. Thus, 2-bromo-bis(glutathion-S-yl)hydroquinone produces enzymuria and glucosuria at doses of 10 µmol/kg (27) and elevations in blood urea nitrogen at 15-20 µmol/kg (26). Although inhibition of γ-GT with acivicin protects animals from 2-bromo-bis(glutathion-S-yl)hydroquinone-mediated nephrotoxicity (27), species differences in susceptibility to 2-bromo-bis(glutathion-S-yl)hydroquinone do not correlate with differences in renal γ-GT activity (34). Only rats, which express the highest level of renal γ-GT, and guinea pigs, which express the lowest amount of renal γ-GT, are susceptible to 2-bromo-bis(glutathion-S-yl)hydroquinone-induced nephrotoxicity. In the guinea pig, it appears that the high ratio of N-acetylcysteine conjugate N-deacetylase to cysteine conjugate N-acetyltransferase activity predisposes this species to the toxicity of polyphenolic-GSH conjugates (35). 3.1. Mechanism of Action and Cellular Targets. The toxicity of 2-bromo-bis(glutathion-S-yl)hydroquinone is related to its ability to generate reactive oxygen species and cause extensive DNA fragmentation (Figure 1). Thus, the 2-bromo-bis(glutathion-S-yl)hydroquinone-dependent formation of single-strand breaks in DNA in renal proximal tubular epithelial cells (LLC-PK1) is inhibited by scavengers of hydrogen peroxide (catalase) and Fe3+ (deferoxamine) (36). Catalase and deferoxamine also prevent cell death, suggesting that DNA damage and cytotoxicity are related (37). Toxicity is therefore likely to involve the iron-catalyzed HaberWeiss reaction, in which O2•- undergoes dismutation to

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Monks and Lau

Figure 1. Cellular mechanisms of polyphenolic-glutathione conjugate-mediated cytotoxicity in renal tubular epithelial cells. The redox and electrophilic properties of quinol-thioethers probably combine to produce a level of stress that the cell cannot survive. The generation of reactive oxygen species causes DNA damage that initiates a growth arrest response which in the absence of additional stress enables the cell to repair the DNA damage and survive the toxic insult. However, in the presence of a compromised stress response, caused perhaps by the inhibition of key repair enzymes, the damage cannot be effectively repaired and the cell dies.

form hydrogen peroxide and reduces Fe3+ to Fe2+ (see above). Interestingly, superoxide dismutase potentiates 2-bromo-bis(glutathion-S-yl)hydroquinone-mediated cytotoxicity but has no effect on 2-bromo-6-(glutathion-Syl)hydroquinone-induced cytotoxicity, and catalase is more effective at protecting cells from 2-bromo-bis(glutathion-S-yl)hydroquinone-induced toxicity than from 2-bromo-6-(glutathion-S-yl)hydroquinone (36). The reasons for such differences are not known but may involve differences in the mechanisms and kinetics of oxidation, similar to those described for ferrihemoglobin formation from 4-(N-dimethylamino)phenol and its bis-glutathionyl conjugate (38) (also see below). Rapid damage to the nucleus of renal proximal tubular epithelial cells also occurs in vivo, prior to overt signs of cell necrosis, and includes karyorhexis, karyolysis, and extensive DNA fragmentation (39). The finding that the nucleus and DNA are sensitive targets of 2-bromo-bis(glutathion-S-yl)hydroquinone is further supported by the observation that the growth arrest- and DNA damageinducible gene, gadd153, is activated by relatively low

concentrations (50 µM) of 2-bromo-bis(glutathion-S-yl)hydroquinone (40). Concomitant with the upregulation of gadd153, histone H1, H2A, H2B, H3, and H4 mRNA decrease significantly in LLC-PK1 cells treated with 2-bromo-bis(glutathion-S-yl)hydroquinone (41). Synthesis of the replication-dependent histones is tightly coupled to DNA synthesis, and the inverse relationship between gadd153 and histone mRNA provides a sensitive molecular marker of growth arrest. 3.2. Role of Polyphenolic-Glutathione Conjugates in Chemical-Induced Nephrocarcinogenesis. 3.2.1. Hydroquinone. Hydroquinone is used as a developer in the photographic industry, as an antioxidant in the rubber industry, and as an intermediate in the manufacturing of food antioxidants; it has also been identified in relatively high concentrations in the smoke of nonfiltered cigarettes (up to 155 µg/cigarette) (42) and is an important metabolite of benzene (see below). Although hydroquinone is generally nonmutagenic in short-term bacterial mutagenicity assays such as the Ames test (43) and no mutagenic activity has been found

Invited Review

in mouse cells in vivo (44), it causes base-pair changes in the TA1535 Salmonella tester strain (45) and is mutagenic in oxidant-sensitive (TA104) Salmonella tester strains (46), consistent with the mutagenicity of 1,4benzoquinone in several Ames bacterial tester strains (47). Hydroquinone also causes renal tubular cell degeneration in the renal cortex of male and female rats (F344/N) treated for 13 weeks with hydroquinone (1.82 mmol/kg) (48), and in long-term studies it causes marked increases in tubular cell adenomas (48, 49). Although hydroquinone is metabolized primarily via conjugation with glucuronic acid and sulfate, 2-(N-acetylcystein-Syl)hydroquinone, 2-(glutathion-S-yl)hydroquinone, 2,5bis(glutathion-S-yl)hydroquinone, 2,6-bis(glutathion-Syl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone are all in vivo metabolites of hydroquinone (50, 51). In the rat, hepatic cytochromes P450 2E1 and 1A1 catalyze the oxidation of hydroquinone to 1,4-benzoquinone.2 Enriched human microsomes prepared from a human B lymphoblastoid cell line transfected with various cytochrome P450 cDNAs indicate that cytochromes P450 2E1, 1A1, and 3A4 catalyze the oxidation of hydroquinone to 1,4-benzoquinone, but cytochrome P450 2C9 predominantly catalyzes the oxidation of the conjugates.2 The GSH conjugates are relatively potent nephrotoxicants; 2,3,5-tris(glutathion-S-yl)hydroquinone (10-20 µmol/kg, iv) causes severe renal proximal tubular necrosis in male Sprague-Dawley rats (28). Consistent with the species-dependent nephrocarcinogenicity of hydroquinone, rats but not mice are susceptible to 2,3,5tris(glutathion-S-yl)hydroquinone-mediated nephrotoxcity (35). The basis of this species difference is, as yet, unclear but does not involve differences in renal γ-GT, cysteine conjugate N-acetyltranferase, or N-acetylcysteine conjugate deacetylase activity (34, 35). Immunohistochemical analysis of mouse kidney 2 h after administration of 2,3,5-tris(glutathion-S-yl)hydroquinone reveals a lack of covalently adducted proteins, in contrast to the distinct pattern of staining observed in rat kidney.3 Since oxidation of the quinol conjugate to the quinone is a prerequisite for covalent adduction, differences in the ability to oxidize and reduce the conjugates may contribute to the difference in susceptibility between the rat and mouse. Some chemicals may induce carcinogenesis by a mechanism involving cytotoxicity followed by sustained regenerative hyperplasia and ultimately tumor formation. Consistent with this scenario, 2,3,5-tris(glutathion-S-yl)hydroquinone (7.5 µmol/kg; 1.16-1.33 µmol/rat, iv) increases cell proliferation in proximal tubular cells of the S3M region, and the degree of proliferation correlates with the degree of toxicity.4 Coincident with cell proliferation and increases in DNA synthesis, expression of the histone genes is also significantly elevated 48 h after administration of 2,3,5-tris(glutathion-S-yl)hydroquinone.4 These metabolites therefore provide an excellent model with which to investigate the role of cytotoxicity in chemical-induced carcinogenicity. However, polyphenolic-GSH conjugates are also mutagenic.5 Treatment of the pSP189 shuttle vector containing the supF gene with 2-Br-bis(glutathion-S-yl)hydroquinone followed by replication of the plasmid in either human AD293 cells or bacterial (MBL50) cells significantly increased the 2Sawalha,

Monks, and Lau, unpublished data. Monks, and Lau, unpublished data. Monks, and Lau, unpublished data. 5Jeong, Wogan, Lau, and Monks, unpublished data. 3Kleiner, 4Peters,


mutation frequency.5 The major mutations are base substitutions, occurring predominantly at G:C sites. Although mutations occur throughout the target gene, “hot-spots” are observed. Deletions (>10 bp) are also detected. In addition, GSH conjugates of hydroquinone catalyze 8-hydroxydeoxyguanosine formation in calf thymus DNA (52). The cytotoxic and genotoxic properties of these conjugates likely play an important role in hydroquinone-mediated carcinogenicity. 3.2.2. 17β-Estradiol. Exposure to estrogens has been associated with neoplastic changes in both humans and laboratory animals (53). In an established model of estrogen-mediated carcinogenesis, male Syrian golden hamsters develop renal carcinoma following prolonged exposure to estrogens (54-56). Although a number of mechanisms have been proposed to explain estrogenmediated carcinogenesis, carcinogenicity has been linked to their metabolism to reactive catechols or quinones (53). Aromatic hydroxylation of 17β-estradiol at the C-2 or C-4 position generates catechol estrogens, and 4-hydroxy-17βestradiol causes renal tumors upon prolonged administration to hamsters (57). However, 2-hydroxy-17βestradiol and 4-hydroxy-17β-estradiol have limited affinity for estrogen receptors (58). Catechol estrogens are readily oxidized to reactive semiquinones, o-quinones (53), and quinone methides (59). Although the liver is the major site of catechol estrogen formation (60), the kidney is the target of estrogen-mediated carcinogenicity in male Syrian hamsters. Since hamster kidney expresses relatively high levels of the cytochrome P450 catalyzing the 4-hydroxylation of 17β-estradiol, the tissue selectivity of 17β-estradiol in the hamster has been attributed to the in situ synthesis of 4-hydroxy-17βestradiol. In contrast, the delivery of liver-derived catechol estrogens to the kidney should be greater in hamsters because of the low hepatic and blood levels of catechol O-methyltransferase (61). Catechol estrogens also undergo oxidation coupled to conjugation with GSH, and whereas the ability of catechol estrogens to redox cycle is quenched by catechol O-methyltransferase-mediated monomethylation, conjugation with GSH does not eliminate their reactivity (62). Under conditions optimized for catechol estrogen O-methylation (63), catechol estrogen oxidation occurs readily, and in the presence of GSH the o-quinones are trapped as GSH conjugates [2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy1-(glutathion-S-yl)-17β-estradiol, 2-hydroxy-4-(glutathionS-yl)-17β-estradiol, and 4-hydroxy-1-(glutathion-S-yl)17β-estradiol] (64). Although the relative importance of catechol-estrogen-thioether formation to the overall metabolism of estrogens in vivo is unclear, in liver, the most active of the peripheral tissues that metabolize estrogens, the major metabolites of 17β-estradiol are present mainly in the water-soluble fraction (65). It also seems unlikely that catechol estrogens will survive in the free form in the circulation, and thus conjugation with GSH provides an effective means of delivering the catechol selectively to the kidney. The activity of catechol O-methyltransferase is low in hamster tissue in comparison to other rodent species (61) and in hamster liver is 10-fold lower than in mouse liver and 100-fold less than in rat liver. Thus the fraction of catechol estrogen available for oxidation and conjugation with GSH is likely to be greater in the hamster than in other rodent species. Indeed, consistent with this view, only a small fraction (4-7%) of 2-hydroxy-17β-estradiol excreted in hamster


bile is methylated, and no 2-methoxy-17β-estradiol is found in urine (66). Alternatively, catechol estrogens also reach the kidney as sulfate conjugates, which may be released by sulfatases in a mechanism analogous to that described for 1,4-naphthoquinone (67). However, because inhibition of γ-GT attenuates the nephrotoxicity of 17βestradiol in the hamster (see below), it is unlikely that sulfoconjugation contributes to the susceptibility of the hamster to estrogen-induced nephrocarcinogenesis. GSH and cysteine conjugates are also formed in incubations of 17β-estradiol with human liver homogenates. However, estrogen mercapturic acids have yet to be identified in human urine (68), and their formation in humans is questionable (69). Catechol-estrogenthioether conjugates may be metabolized in humans to products which either are not readily excreted or become bound to tissue macromolecules. In support of this view, only 15% of a dose of radiolabeled 2-hydroxy-1-(glutathion-S-yl)-17β-estradiol administered to rats is recovered in the urine and only 5% in the feces (70), and only the N-acetylcysteine metabolite is identified in urine. The reactivity of catechol-estrogen-GSH conjugates is further supported by the finding that in the absence of ascorbate, the pattern of thioether metabolites shifts toward formation of 2-hydroxy-bis(glutathion-S-yl)-17βestradiol, indicating that 2-hydroxy-1-(glutathion-S-yl)17β-estradiol and 2-hydroxy-4-(glutathion-S-yl)-17βestradiol readily oxidize, and the corresponding quinoneGSH conjugates are trapped with GSH (63). The biological reactivity of these metabolites is therefore associated with the catechol moiety, which is readily oxidized to the corresponding o-quinone. Both 2-hydroxy-1-(glutathionS-yl)-17β-estradiol and 2-hydroxy-4-(glutathion-S-yl)-17βestradiol maintain the ability to redox cycle and catalyze the formation of O2•- (71). The one-electron reduction of the o-quinones and their subsequent redox cycling with the concomitant generation of reactive oxygen species may contribute to estrogen-mediated nephrocarcinogenicity in hamsters. Both 2-hydroxy-4-(glutathion-S-yl)-17β-estradiol and 2-hydroxy-1-(glutathion-S-yl)-17β-estradiol produce mild nephrotoxicity in hamsters at doses of 0.27 and 1.5 µmol/ kg, respectively (71), the equivalent of 27 and 150 nmol/ 100 g of hamster. At doses of 17β-estradiol that are estimated to occur in the hamster model of nephrocarcinogenicity (5.4 µmol/kg; equivalent to 147 µg of 17βestradiol or 540 nmol/100 g of hamster) 11.7% of the dose (63 nmol) is recovered as GSH conjugates of the catechol estrogens of either 17β-estradiol or estrone (72). Thus, catechol-estrogen-GSH conjugates are formed in amounts sufficient to produce mild nephrotoxicity in dosing regimens of 17β-estradiol used to produce renal tumors. Importantly, 17β-estradiol (50 µmol/kg) is mildly nephrotoxic in Syrian hamsters in a manner sensitive to inhibition of γ-GT, consistent with a role for catechol estrogen thioethers in the renal effects of 17β-estradiol (72). It is noteworthy that the development of estrogenmediated nephrocarcinogenicity in hamsters is associated with dysplasia of, and cytotoxicity to, the proximal tubule (73) or with the primitive interstitial cells of the proximal tubule (74). 3.2.3. 3-tert-Butyl-4-hydroxyanisole. 3-tert-Butyl4-hydroxyanisole (BHA) and its demethylated analogue, tert-butylhydroquinone (TBHQ), are phenolic antioxidants widely used in foods. Both compounds are generally nonmutagenic in short-term genotoxicity tests. However, chronic dietary administration of BHA results in

Monks and Lau

papilloma and carcinoma formation in the forestomach of rats, mice, and hamsters (75). Although BHA inhibits hepatocellular carcinomas (76, 77), it enhances the development of preneoplastic and neoplastic lesions in rat kidney (76) and urinary bladder (76, 78, 79). TBHQ is a metabolite of BHA in man (80), causes cell proliferation in renal pelvic epithelia (81), and enhances hyperplasia in the urinary bladder (82, 83). Metabolites have been implicated in the toxicity of BHA (84), and TBHQ, tert-butyl-1,4-benzoquinone, 3-tert-butyl-4,5-dihydroxyanisole, and 3-tert-butyl-4,5-quinone induce DNA damage in forestomach epithelium of rats after oral administration (84). TBHQ also induces cell proliferation in renal and urinary bladder epithelia in the rat (85). Rat and human cytochromes P450 (86) and peroxidases (87) catalyze both the O-demethylation of BHA to TBHQ and the subsequent oxidation to the corresponding quinone. Interestingly, depletion of reduced GSH inhibits the proliferative response to BHA, indicating that GSH is involved in the carcinogenic process (88, 89). Tajima et al. (90) identified 5-(glutathion-S-yl)TBHQ and 6-(glutathion-S-yl)TBHQ as metabolites of TBHQ in microsomal incubations and 2-tert-butyl-5-(methylthio)hydroquinone and 2-tert-butyl-6-(methylthio)hydroquinone in the urine of Wistar rats after ip administration of 3-BHA or TBHQ. 5-(Glutathion-S-yl)TBHQ, 6-(glutathion-S-yl)TBHQ, and 3,6-bis(glutathion-S-yl)TBHQ were subsequently identified as in vivo metabolites of TBHQ (91). Whereas about 16% of a single dose of HQ (1.8 mmol/kg, po), the unsubstituted analogue of TBHQ, is excreted as 2-(N-acetylcystein-S-yl)hydroquinone [this was the only sulfur-containing metabolite identified in the urine of rats (90)] mercapturic acids of TBHQ were not found in the urine of rats, suggesting that the tertbutyl substituent either prohibits N-acetylation of the cysteine moiety or facilitates its metabolism by β-lyase(s) (Figure 2) (92). Metabolism of aromatic cysteine Sconjugates by β-lyases usually results in the urinary excretion of stable thiols, S-glucuronides, S-oxides, or S-methyl compounds. Thus, pentachlorothiophenol and benzylthiol, metabolites of S-(pentachlorophenyl)-L-cysteine and S-(benzyl)-L-cysteine, respectively, do not exhibit the mutagenic and nephrotoxic properties of the halogenated alkane and alkene metabolites (93), and nephrotoxicity is not seen when N-acetyl-S-(pentachlorophenyl)-L-cysteine is administered to rats (94, 95). When administered to rats, 5-(glutathion-S-yl)TBHQ, 6-(glutathion-S-yl)TBHQ, and 3,6-bis(glutathion-S-yl)TBHQ produce a mild nephrotoxicity (96), and 3,6-bis(glutathion-S-yl)TBHQ is more toxic than either 5-(glutathion-S-yl)TBHQ or 6-(glutathion-S-yl)TBHQ. This finding is consistent with other studies. Thus, 2-bromobis(glutathion-S-yl)hydroquinone is a far more potent nephrotoxicant than 2-bromo(glutathion-S-yl)hydroquinones; 2,3,5-tris(glutathion-S-yl)hydroquinone is far more toxic than 2,3-bis(glutathion-S-yl)hydroquinone, 2,5-bis(glutathion-S-yl)hydroquinone, or 2,6-bis(glutathion-Syl)hydroquinone, which are all more toxic than 2-(glutathion-S-yl)hydroquinone (7); and 2,3-bis(glutathion-Syl)-1,4-naphthoquinone is more toxic than 2-(glutathionS-yl)-1,4-naphthoquinone (97). The toxicity of mono(glutathion-S-yl) conjugates may be limited by the ability of the corresponding cystein-S-ylglycine and cystein-Syl conjugates to undergo an oxidative cyclization reaction that results in 1,4-benzothiazine formation (98, 99) (Figure 3). The products of the cyclization reaction vary, depending upon the number and position of the thiol

Invited Review


BHA and TBHQ promote second-stage carcinogenesis in the bladder, probably by increasing DNA synthesis, hyperplasia, and tumor formation. Interestingly, 3,6-bis(glutathion-S-yl)TBHQ increases bladder wet weight and causes severe hemorrhaging of the bladder (96), suggesting a mechanism of carcinogenesis in which the initial step involves cytolethality in the target tissue. Structurally related polyphenolic-GSH conjugates do not appear to cause any adverse effects on the bladder. Only phenolic antioxidants with a tert-butyl substituent, including BHA and TBHQ, enhance DNA synthesis and the development of neoplastic lesions in epithelial cells of the urinary bladder. In contrast, phenolic species with hydroxyl substituents alone, such as catechol, resorcinol, and hydroquinone, lack promoting activity in the bladder (83). Differences in the partitioning of the polyphenolicGSH conjugates through either the mercapturic acid or the β-lyase-thiomethyl shunt pathways (Figure 2) (101) may determine whether metabolites of these conjugates cause bladder toxicity.

4. Neurotoxicity of Polyphenolic-Glutathione Conjugates Figure 2. Substituent-directed partitioning of polyphenoliccysteine conjugates through the mercapturic acid or thiomethyl shunt pathway. Following metabolism of polyphenolic-glutathione conjugates by γ-GT (I) and dipeptidases (II), the resulting cysteine conjugates may be either N-acetylated to form the mercapturic acid (III) or converted by cysteine conjugate β-lyase to the corresponding thiol (IV) followed by the thiomethyl transferase-mediated S-methylation of the free thiol (V). Either the size or the charge characteristics of the ortho substituent direct the extent of partitioning through pathway III or IV.

substituent (Figure 3), and differences in the redox properties of these products will influence toxicity. Alternatively, cyclization may be considered an intramolecular detoxication reaction, since it removes the reactive quinone function from the molecule, and may contribute to the difference between the nephrotoxicity of 2-methyl3-(N-acetylcystein-S-yl)-1,4-naphthoquinone, which cannot undergo the cyclization reaction, and the lack of toxicity of 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone, which appears to cyclize readily (15, 97). In the case of GSH conjugates of TBHQ, it would appear that the bulky tert-butyl substituent may hinder intramolecular cyclization but predisposes the compound to dimerization and polymerization reactions. The deposition of polymers in the kidney and the excretion of dark-colored pigments in the urine of rats treated with 5-(glutathionS-yl)TBHQ and 6-(glutathion-S-yl)TBHQ are consistent with this interpretation. The formation of pigments may, in the short term, serve as a detoxication reaction, considering the very moderate acute toxicity of both 5-(glutathion-S-yl)TBHQ and 6-(glutathion-S-yl)TBHQ. In chronic studies, the persistence of insoluble pigments may cause tubular obstruction and irritation, which could contribute to the chronic toxicity of TBHQ. The moderate toxicity of TBHQ-GSH conjugates suggests that they possess attributes necessary to induce cell proliferation without severe organ dysfunction. A constant sublethal, but moderately toxic, insult to cells could lead to increased repair and cell turnover and to the promoting effects documented in long-term feeding studies with BHA and TBHQ (100). The renal carcinogenicity of BHA in rats might thus be related to the renal toxicity of its metabolites.

4.1. 3,4-(()-(Methylenedioxy)amphetamine and 3,4-(()-(Methylenedioxy)methamphetamine. 3,4(()-(Methylenedioxy)amphetamine (MDA) and 3,4-(()(methylenedioxy)methamphetamine (MDMA) are ringsubstituted amphetamine derivatives, structurally related to psychomotor stimulant amphetamines and the hallucinogen mescaline (102). MDA and MDMA are serotonergic neurotoxicants in several animal species, including non-human primates (103-105). The actions of MDA and MDMA are biphasic, initially causing an acute release of 5-hydroxytryptamine (5-HT) (106) followed by prolonged depletion of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) (103, 107) and structural damage to 5-HT terminal and preterminal axons, in various regions of the central nervous system (103, 108). The neurotoxic effects of MDA and MDMA are dependent on the route and frequency of drug administration (105). Direct injection of either MDA or MDMA into the brain fails to reproduce the neurotoxicity following peripheral administration, indicating that the parent amphetamines are unlikely to be responsible for the neurotoxic effects (109) and suggesting that systemic metabolism plays an important role in the development of toxicity. MDA and MDMA are metabolized by cytochrome(s) P450 to R-methyldopamine, and N-methyl-R-methyldopamine, respectively (110-114). The demethylenation of MDMA is also catalyzed by the hydroxyl radical (115). However, intracerebral injection of either 3-O-methyl-Rmethyldopamine or R-methyldopamine fails to induce monoaminergic neurotoxicity (116). In contrast, the 6-hydroxydopamine analogues, 2-(methylamino)-1-(2,4,5trihydroxyphenyl)propane (2,4,5-trihydroxymethamphetamine) and 2-amino-1-(2,4,5-trihydroxyphenyl)propane (2,4,5-trihydroxyamphetamine), putative metabolites of MDMA and MDA, respectively, do cause depletion of both dopamine and 5-HT following intracerebroventricular (icv) and intrastriatal administration to rats (117, 118). However, exactly how these metabolites might gain access to the brain following systemic formation remains to be established. Because (i) neither MDA nor MDMA produces neurotoxicity when injected directly into brain, (ii) icv administration of some major metabolites of MDA


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Figure 3. Intramolecular cyclization and 1,4-benzothiazine formation from glutathione conjugates of 1,4-benzoquinones. Removal of γ-glutamic acid by γ-GT (I) and glycine by dipeptidases (II) facilitates the oxidation of the cysteinylglycine and cysteine conjugates to 1,4-benzoquinones. The cysteine amino group subsequently condenses with the quinone carbonyl (III) and rearranges (IV, H-shift) to the 1,4-benzothiazine. Differences in the position of thiol addition and in the nature of the R substituent determine differences in the structures of the rearrangement products. Rearrangement of (2R)-3-(glutathion-S-yl)hydroquinone (A), (2R)-5-(glutathion-S-yl)hydroquinone (B), and (2R)-6-(glutathion-S-yl)hydroquinone (C) is illustrated.

Invited Review


Figure 4. Proposed model for the delivery of polyphenolic-glutathione conjugate metabolites of 3,4-(()-(methylenedioxy)amphetamine to the brain. Systemic metabolism of 3,4-(()-(methylenedioxy)amphetamine to R-methyldopamine followed by oxidation and GSH conjugation results in the delivery of polyphenolic-GSH conjugates to the blood-brain barrier. The conjugates are transported into brain either via an intact GSH transporter (I) or, to a lesser extent, via metabolism by γ-GT coupled to the uptake of the corresponding cysteine conjugate (II). Oxidation of the cysteine conjugate to the corresponding quinone-thioether (III) and inhibition of endothelial γ-GT (IV) may enhance the uptake of the polyphenolic-GSH conjugate into brain, presumably via pathway I. Uptake of the polyphenolic-cysteine conjugates may also be limited by oxidation within the capillary lumen and alkylation of plasma proteins (V). Within the central nervous system, the polyphenolic-GSH conjugates are metabolized by γ-GT and dipeptidase to the corresponding polyphenolic-cysteine (VI) and N-acetylcysteine (VII) conjugates. The redox (VIII) and electrophilic (IX) properties of the polyphenolic thioether conjugates may then contribute to serotonergic neurotoxicity. The N-acetylcysteine conjugates appear to be slowly eliminated from the brain (X).

fails to reproduce the neurotoxicity, (iii) R-methyldopamine is a metabolite of both MDA and MDMA, (iv) R-methyldopamine and N-methyl-R-methyldopamine are readily oxidized by O2•- to the o-quinones which readily react with both protein and nonprotein sulfhydryls, including GSH (114, 119), and (v) quinone-thioethers exhibit a variety of toxicological activities, it seemed possible that thioether metabolites of R-methyldopamine might contribute to the neurotoxicity of MDA and MDMA.

In addition, γ-GT is present in high concentrations in the brain, particularly on endothelial cells that form the blood-brain barrier (120), and there appears to be a transporter capable of transferring GSH conjugates from the circulation into the brain (121). Systemic formation of catecholic-GSH conjugates followed by uptake into and metabolism by the brain (Figure 4) provides a basis for the role of metabolism in MDA- and MDMA-mediated neurotoxicity.


A single icv injection of 5-(glutathion-S-yl)-R-methyldopamine (720 nmol) to male Sprague-Dawley rats causes short-term changes in neurotransmitter levels similar to those seen after subcutaneous administration of MDA (20 mg/kg; 23 µmol/rat) (122). MDA also produces a pressor response due to its ability to release neuronal norepinephrine stores; the potent adrenergic effect of MDA is long-established (123). Both MDA and 5-(glutathion-S-yl)-R-methyldopamine cause depletions in brain norepinephrine concentrations (122). The behavioral changes seen after 5-(glutathion-S-yl)-R-methyldopamine are also compatible with it contributing to the behavioral and/or neurotoxic effects of MDA and MDMA. Consistent with the behavioral changes, 5-(glutathionS-yl)-R-methyldopamine also causes an increase in the turnover of 5-HT, as evidenced by increases in 5-HT concentrations in the midbrain/diencephalon/telencephalon and by increases in 5-HIAA in the midbrain/diencephalon/telencephalon, cortex, hippocampus, and striatum (122). Both MDA and 5-(glutathion-S-yl)-Rmethyldopamine increase the turnover of brain dopamine, and an increase in dopamine synthesis is considered a prerequisite for the long-term depletion of brain 5-HT following MDMA administration (124, 125). However, although 5-(glutathion-S-yl)-R-methyldopamine reproduces some of the effects of MDA on the dopaminergic system and is capable of causing acute increases in 5-HT turnover, it does not cause long-term serotonergic toxicity (122). Multiple dosing regimens are employed in most models of MDMA and MDA neurotoxicity, and such regimens might produce shifts in the metabolism and/or accumulation of a neurotoxic metabolite in brain tissue. 5-(Glutathion-S-yl)-R-methyldopamine is rapidly cleared from the brain and metabolized via the mercapturic acid pathway to 5-(cystein-S-yl)-R-methyldopamine (126). Concentrations of the cysteine conjugate also decline rapidly, with the concomitant formation of 5-(N-acetylcystein-Syl)-R-methyldopamine (126) which, in contrast to 5-(glutathion-S-yl)-R-methyldopamine and 5-(cystein-S-yl)-Rmethyldopamine, is eliminated relatively slowly from the brain. 5-(Glutathion-S-yl)-R-methyldopamine is also readily oxidized to the corresponding quinone-GSH conjugate and undergoes addition of a second molecule of GSH to form 2,5-bis(glutathion-S-yl)-R-methyldopamine. However, although multiple icv injections of 5-(glutathion-S-yl)-R-methyldopamine (4 × 720 nmol) and 5-(N-acetylcystein-S-yl)-R-methyldopamine (4 × 100 nmol) to Sprague-Dawley rats produce overt behavioral changes similar to those seen following administration of MDA (93 µmol/kg, sc) (126), they do not produce long-term decreases in brain 5-HT concentrations. In contrast, 2,5bis(glutathion-S-yl)-R-methyldopamine (4 × 475 nmol) decreases 5-HT levels in the striatum, hippocampus, and cortex 7 days after injection (127). The relative sensitivity of the striatum, hippocampus, and cortex to 2,5-bis(glutathion-S-yl)-R-methyldopamine is the same as that observed for MDA (the absolute effects are greater with MDA). The effects of 2,5-bis(glutathion-S-yl)-R-methyldopamine are also selective for serotonergic nerve terminal fields, in that 5-HT levels are unaffected in regions of the cell bodies (127). Because 2,5-bis(glutathion-S-yl)R-methyldopamine causes long-term depletion in 5-HT without adversely affecting the dopaminergic system, it also mimics the selectivity of MDA/MDMA. The data imply a role for quinone-thioethers in the neurobehavioral and neurotoxicological effects of MDA/MDMA.

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Since polyphenolic thioethers maintain the ability to redox-cycle (60) and because all the thiol conjugates of R-MeDA remain susceptible to oxidation, the presence and persistence of these metabolites in brain tissue may contribute to the neurotoxicity of MDA and MDMA. The unique bifunctional nature of quinone-thioethers facilitates their interaction with several enzymes that have either quinones as their usual substrates and/or GSH as a cofactor (60). The 5-(S-glutathionyl) conjugates of dopamine and R-methyl-DOPA inhibit human GSH Stransferases (128), and the ability of several GSHconjugated halogenated quinones to inhibit GSH S-transferases is being used as a basis for the development of drugs to augment the efficacy of chemotherapeutic agents, primarily alkylating agents, that are substrates for GSH S-transferases within tumor-resistant cells. The toxicity of 5-(cystein-S-yl)-R-methyldopamine and 5-(N-acetylcystein-S-yl)-R-methyldopamine may also be regulated by intramolecular cyclization reactions that occur after oxidation. Cyclization of 5-(cystein-S-yl)-Rmethyldopamine can occur in one of two ways (Figure 5). Following oxidation, the side chain (alanine-derived) amino group can cyclize to give the 5,6-dihydroxyindoline, in a reaction analogous to the Raper-Mason pathway of eumelanin biosynthesis (129-131). The dihydroxyindoline can be chemically oxidized by the o-quinone to the quinonimine followed by tautomerization to the 5,6dihydroxyindole. Alternatively, the cysteinyl amino group can condense with the quinone carbonyl to give a benzothiazolyl-like compound, in a reaction analogous to pheomelanin synthesis (132). Only the latter reaction removes the reactive quinone function, since the dihydroxyindole can undergo further oxidation to the indolic o-quinonimine, which then polymerizes to pigments characteristic of the melanins. The cysteinyl amino group is blocked in 5-(N-acetylcystein-S-yl)-R-methyldopamine and following oxidation can no longer undergo cyclization. Therefore, in addition to any inherent differences in the redox properties of 5-(cystein-S-yl)-Rmethyldopamine and 5-(N-acetylcystein-S-yl)-R-methyldopamine, the latter is likely to maintain redox activity because it lacks the ability to undergo intramolecular detoxication (benzothiazolyl formation). 4.1.1. Transport of Glutathione and Glutathione Conjugates across Blood-Brain Barriers. CatecholGSH conjugates of MDA and MDMA must be capable of crossing the blood-brain and blood-CSF barriers in order to produce toxicity. Brain microvascular endothelial cells maintain tight junctions (133) and possess a high density of mitochondria (134). The mitochondria supply the high-energy requirements for the transport of water-soluble substances through the endothelial barrier via specific transporters. The directionality of ion transport across the blood-brain barrier is achieved by a polarized distribution of ion channels on the endothelial cell surfaces. Therefore, endothelial cells are polarized in a manner similar to other transport interfaces, such as renal epithelia, with the preferential localization of specific transport systems and receptors on either the luminal or the antiluminal side of vessel walls (135). For amino acids, at least three different carrier systems have been identified within the blood-brain barrier (136), and a variety of neurotoxicants are transported into brain across the blood-brain barrier via these amino acid carriers. For example, β-(N-methylamino)-L-alanine, a purported neurotoxic constituent of the cycad plant responsible for the high incidence of amyotrophic lateral

Invited Review


Figure 5. Cyclization of 5-(cystein-S-yl)-R-methyldopamine. Cyclization of 5-(cystein-S-yl)-R-methyldopamine can occur in one of two ways. Following oxidation (I), the cysteinyl amino group can condense with the quinone carbonyl (II) and, following rearrangement (hydride shift, III), gives a benzothiazine-like compound in a reaction analogous to pheomelanin synthesis. Alternatively, the side chain (alanine-derived) amino group can cyclize to give the 5,6-dihydroxyindoline (IV), in a reaction analogous to the Raper-Mason pathway of eumelanin biosynthesis. The dihydroxyindoline can be chemically oxidized by the o-quinone to the o-quinonimine (V) which tautomerizes to the 5,6-dihydroxyindole (VI). Oxidation to the o-quinone (VII) and condensation (VIII) of either the 5,6dihydroxyindoline or the 5,6-dihydroxyindole give rise to the indole(ine)-linked benzothiazines (IX).

sclerosis (and related parkinsonism dementia) in the western Pacific, is transported into brain via the large neutral amino acid carrier (137). The cysteine conjugate of dichloroacetylene, a potent nephrotoxicant (138) and neurotoxicant (139), is also transported across the bloodbrain barrier by the Na+-independent system L-transporter for neutral amino acids, while uptake of the corresponding GSH conjugate is mediated by an as yet unknown carrier system (140). The saturable, carriermediated transport of GSH across the blood-brain barrier has also been reported (121). Thus, transport systems that can facilitate the uptake of GSH and cysteine conjugates of R-MeDA into the brain have been described. GSH and 5-(glutathion-S-yl)-R-methyldopamine apparently use the same transporter since GSH decreases 5-(glutathion-S-yl)-R-methyldopamine uptake into brain (122). Interestingly, pretreatment of rats with acivicin, which inhibits γ-GT, significantly increases the uptake of 5-(glutathion-S-yl)-R-methyldopamine into brain, presumably by preventing its clearance from blood via metabolism and increasing the fraction available for uptake via the GSH transporter (Figure 4) (122).

4.1.2. Functional Roles of Glutathione in the Brain. Interestingly, direct icv injection of R-methyldopamine does not produce the same behavioral profile as 5-(glutathion-S-yl)-R-methyldopamine (122) suggesting that the behavioral changes caused by 5-(glutathionS-yl)-R-methyldopamine are not simply due to the catecholamine moiety. Although the cytoprotective effects of GSH are well-established (GSH and related enzymes participate in the protection of neurons from a variety of stresses), additional roles for GSH in brain function are being reported and provide a pharmacological basis for the relationship between alterations in GSH homeostasis and the development of certain neurodegenerative processes. For example, several studies have demonstrated the existence of GSH binding sites within the mammalian central nervous system (141, 142) suggesting a role for GSH as a neuromodulator or neurotransmitter, and in rat brain slices depolarization induces GSH secretion into the extracellular space in a Ca2+-dependent manner (143). Both GSH and GSSG modulate the NMDA receptor (144), and GSH also modulates µ-opioid, substance P/neurokinin-1, and kainic acid receptor binding sites


(145). GSH and GSSG also elicit a potent antinociceptive activity (146). Consistent with a role for GSH in receptormediated events, it produces an increase in secondmessenger synthesis (inositol 1,4,5-triphosphate) when added to cultured rat brain astrocytes (147). GSH may also be required to maintain the uptake of catecholamines into synaptosomes (148), and it accelerates sodium channel inactivation in excised rat axonal membranes (149). Thus, GSH (and GSSG) appears to play important functional roles in the central nervous system. The unique structural features of polyphenolic-GSH conjugates such as 5-(glutathion-S-yl)-R-methyldopamine permit toxicological and pharmacological activity as a consequence of either the polyphenolic (catecholamine) or GSH moiety. Toxicological sequelae may result from the electrophilic and redox properties of the quinone, whereas neuropharmacological changes may result from either the catecholamine or GSH moiety. 4.2. Neurotoxicity of Endogenous Polyphenolic Conjugates. 4.2.1. Oxidation of Dopamine and Parkinson’s Disease. Parkinson’s disease is characterized by the loss of nigrostriatal dopaminergic neurons and decreases in concentrations of the neurotransmitter dopamine. The substantia nigra of Parkinson’s pateints also exhibits substantial decreases in the concentration of GSH, with a corresponding increase in the levels of 5-(cystein-S-yl)dopamine relative to dopamine. The addition of cysteine to either dopamine or 3,4-dihydroxyphenylalanine results in the formation of products that are more readily oxidized than the parent catechols (12, 13, 150). Dryhurst and colleagues have investigated the chemistry of these reactions in detail, and their work has provided important insights into the oxidation chemistry of dopamine and its potential role in Parkinson’s disease (150-152). Thus, 2-(cystein-S-yl)dopamine, 5-(cysteinS-yl)dopamine, and 2,5-bis(cystein-S-yl)dopamine readily oxidize, and the corresponding o-quinones undergo intramolecular cyclization in reactions analogous to those described for 2-Br-3-(cystein-S-yl)hydroquinone (99). The dihydrobenzothiazines derived from the cysteine adducts of dopamine are also easily oxidized and appear to be highly toxic. The potent redox properties of these endotoxicants are consistent with the involvement of oxidative stress in the pathogenesis of Parkinson’s disease. 4.2.2. Oxidation of Serotonin. A defect in the metabolism 5-HT has been implicated in various neurodegenerative, neuropsychiatric, and behavioral disorders (153, 154). 5-HT is converted to several polyhydroxylated metabolites including 5,6- and 5,7-dihydroxytryptamine, both of which are potent neurotoxicants (155, 156). Serotonergic abnormalities also occur in mental disturbances of the Alzheimer type (157), and the cerebrospinal fluid of Alzheimer’s patients contains a product with properties consistent with an oxidized form of 5-HT (158) and suggested to be tryptamine-4,5-dione (159). Since tryptamine-4,5-dione causes the release of 5-HT from serotonergic neurons (160), a property shared by the known neurotoxicant 5,6-dihydroxytryptamine (161), tryptamine-4,5-dione might also exhibit neurodegenerative properties. However, GSH is present in relatively high concentrations (2 mM) throughout the brain (162), and at physiological pH, tryptamine-4,5-dione reacts rapidly with GSH to form 7-(S-glutathionyl)tryptamine4,5-dione, which has been suggested to be the species responsible for the neurodegenerative effects of tryptamine-4,5-dione (163). A putative GSH conjugate of tryptamine-4,5-dione has also been identified from an

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acid-soluble extract of rat brain homogenate (160). At physiological pH, 5,6-dihydroxytryptamine is also rapidly oxidized to the o-quinone (164) and reacts with GSH to form 4-(glutathion-S-yl)-5,6-dihydroxytryptamine, which further oxidizes to the corresponding quinone-thioether. Further attack by GSH on the oxidized conjugate gives 4,7-bis(glutathion-S-yl)-5,6-dihydroxytryptamine and 4,4′bis(glutathion-S-yl)-2,7′-bis(5,6-dihydroxytryptamine). Susceptibility of polyphenolic-GSH conjugates to further oxidation and the reactivity of the resultant quinonethioethers toward sulfhydryl groups are consistent with the proposition that endogenous quinone-thioethers contribute to the neurotoxicity of polyphenolic metabolites of 5-HT. 4.2.3. Tetrahydroisoquinolines. A variety of tetrahydroisoquinoline alkaloids are elevated in the brains of chronic alcoholics and may undergo oxidation to neurotoxic metabolites which contribute to the various physical and behavioral changes that accompany chronic alcoholism (165). In particular, concentrations of salsolinol (1,2,3,4-tetrahydro-1-methyl-6,7-isoquinolinediol), formed via condensation of dopamine with acetaldehyde (166), a metabolite of ethanol, are elevated in discrete regions of the brain in rats that chronically consume ethanol (167). Salsolinol is readily oxidized to a variety of products following initial oxidation to the o-quinone. In the presence of GSH, the o-quinone undergoes nucleophilic attack to yield the 5-S- and 8-S-conjugates, both of which are further oxidized to the 5,8-bis-glutathionyl conjugate (168). Because chronic alcohol consumption increases the concentrations of acetaldehyde in the brain, it may inhibit aldehyde dehydrogenase, causing an accumulation of 3,4-dihydroxyphenylacetaldehyde, a dopamine metabolite, which subsequently condenses with dopamine to form tetrahydropapaveroline (169), an alkaloid that demonstrates addictive properties in rats (170). Tetrahydropapaveroline is readily oxidized to a diquinone intermediate, which, in the presence of GSH, gives 1,2,3,4-tetrahydro-1-[[6-(S-glutathionyl)-3,4-dihydroxyphenyl]methyl]-6,7-isoquinolinediol (171). The conjugate is also further oxidized followed by addition of a second molecule of GSH to 1,2,3,4-tetrahydro-1-[[6-(Sglutathionyl)-3,4-dihydroxyphenyl]methyl]-8-(S-glutathionyl)-6,7-isoquinolinediol (171). Thus, oxidation of tetrahydropapaveroline within the brain could cause toxicity either indirectly, via depletion of GSH concentrations, or directly, via the concomitant formation of potentially neurotoxic quinone-thioethers.

5. Hematotoxicity of Polyphenolic-Glutathione Conjugates 5.1. Role for Polyphenolic-Glutathione Conjugates in Benzene-Mediated Hematotoxicity. Benzene is an important industrial chemical, a component of gasoline, and an environmental pollutant (172). Exposure to benzene causes a variety of hematological disorders, including leukopenia, anemia, thrombocytopenia, pancytopenia, aplastic anemia, and leukemia (173, 174). Metabolism of benzene is a prerequisite for toxicity (175), and hepatic cytochromes P450 convert benzene to several phenolic metabolites, including phenol, hydroquinone, catechol, resorcinol, 1,2,4-benzenetriol, and the ring-opened metabolites trans,trans-muconaldehyde and trans,trans-muconic acid (176). None of these metabolites are capable, on their own, of reproducing the myelotoxic effects of benzene (177), but a combination of

Invited Review

hydroquinone and phenol decreases total bone marrow cellularity (178), damages bone marrow pronormoblasts and normoblasts (179), and increases the formation of micronuclei in polychromatic erythrocytes (180). It has been proposed that benzene-induced bone marrow suppression may involve peroxidase- and/or phenoxy radicalmediated oxidation of hydroquinone to 1,4-benzosemiquinone and 1,4-benzoquinone which may either arylate tissue macromolecules and/or redox-cycle with the concomitant formation of reactive oxygen species (181, 182). However, although semiquinone formation from a variety of quinones leads to oxygen consumption, no oxygen consumption occurs in reactions in which the 1,4-benzosemiquinone free radical is formed enzymatically because 1,4-benzo(semi)quinone is so electron-affinic that its rate of reduction by O2•- is over 4 orders of magnitude faster than the reverse reaction (reduction of O2 to O2•-) (25), which is usually responsible for oxygen consumption via redox cycling. Thus, although redox cycling of semiquinone radicals resulting in the generation of ROS is proposed to be of major importance in the toxicity of many quinones, this mechanism is clearly excluded in the case of 1,4-benzoquinone (25, 183). 1,4-Benzoquinone also undergoes nucleophilic addition with GSH, leading to the formation of 2-(glutathion-Syl)hydroquinone, 2,5-bis(glutathion-S-yl)hydroquinone, 2,6-bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone (26). Thioether metabolites of hydroquinone have been identified in the bone marrow of both male Sprague-Dawley rats and DBA/2 mice following coadministration of hydroquinone/phenol (2.0 mmol/kg, ip) (184). Concentrations of quinol-thioethers in bone marrow are higher in mice than in rats (184), consistent with the relative sensitivity of these two species to benzene-induced hematotoxicity (185). Bone marrow stromal cells from mice possess 28-fold less DTdiaphorase activity than those from rats (186), and bone marrow cells with high peroxidase activity relative to DTdiaphorase activity also yield more 1,4-benzoquinonederived protein adducts (187) and by extension may represent cellular targets for the thioether metabolites of hydroquinone. Thus, a greater fraction of the thioether metabolites in mouse bone marrow may be adducted to proteins, limiting the “free” fraction available for detection. Indeed, GSH conjugates of hydroquinone are more chemically (re)active than hydroquinone and are capable of arylating tissue macromolecules (184, 188). The γ-GT- and dipeptidase-catalyzed products of 2-(glutathion-S-yl)hydroquinone, 2-(cysteinylglycine)hydroquinone, and 2-(cystein-S-yl)hydroquinone were also identified in the bone marrow of both rats and mice. Concentrations of the cysteine conjugate are relatively high and persist in bone marrow. The availability of bone marrow γ-GT and dipeptidases for processing of GSH conjugates is important, because cysteinylglycine and cysteine conjugates of hydroquinone are more chemically (re)active than their corresponding GSH conjugates (188). Consequently, such metabolites are potent arylators and redox cyclers, and the GSH conjugates of hydroquinone are far more efficient at catalyzing O2•- formation than hydroquinone.6 2,3,5-Tris(glutathion-S-yl)hydroquinone and 2,6-bis(glutathion-S-yl)hydroquinone are toxic to (pro)normoblasts (59Fe incorporation assay) at doses of 17 and 50 µmol/kg, iv, respectively (184), which represent 0.2% and 0.4% of the dose of benzene required to produce 6Butterworth,

Lau, and Monks, unpublished data.


a similar toxicity. Myeloperoxidase- and prostaglandin H synthase-mediated oxidation of hydroquinone to 1,4benzoquinone in the presence of GSH yields 2-(glutathion-S-yl)hydroquinone (187) and should occur readily in bone marrow (189, 190). The hematotoxicity of benzene may therefore be due to the combined effects of both 1,4-benzoquinone and its GSH conjugates (Figure 6). 5.2. Aminophenols and Quinone-ThioetherCatalyzed Methemoglobinemia. Exposure to a variety of aminophenols causes methemoglobinemia (191), and polyphenolic-GSH conjugates appear to play an important role in this process (38). Thus, when GSH concentrations within red blood cells are high, the binding of the quinonimine, formed via oxidation of 4-(Ndimethylamino)phenol, to hemoglobin decreases, with the concomitant formation of 4-(N-dimethylamino)-2,3,6-tris(glutathion-S-yl)phenol. However, when GSH concentrations are low, the major product is 4-(dimethylamino)2,6-bis(glutathion-S-yl)phenol, which is capable of catalyzing the formation of ferrihemoglobin. Interestingly, substantial differences exist in the kinetics of ferrihemoglobin formation from 4-(N-dimethylamino)phenol and 4-(dimethylamino)-2,6-bis(glutathion-S-yl)phenol. Thus, the reaction between 4-(dimethylamino)2,6-bis(glutathion-S-yl)phenol and hemoglobin and the autoxidation of 4-(dimethylamino)-2,6-bis(glutathion-Syl)phenol generate O2•- which increases ferrihemoglobin formation, a portion of which is due to the hydrogen peroxide produced via dismutation of O2•-. In contrast, O2•- and hydrogen peroxide are not formed in the reaction between 4-(N-dimethylamino)phenol and hemoglobin. The autoxidation of 4-(dimethylamino)-2,6-bis(glutathionS-yl)phenol is also more rapid than its oxidation by oxyhemoglobin, whereas 4-(N-dimethylamino)phenol oxidation by oxyhemoglobin is greater than its rate of autoxidation. 4-(N-Dimethylamino)phenol is also nephrotoxic in rats and cytotoxic to isolated rat kidney tubules (192, 193).

6. Conclusions and Future Directions Polyphenols are ubiquitous; naturally occurring polyphenols and quinones are ingested every day in our diet. They are also formed as metabolites from a variety of drugs and environmental contaminants. The high concentrations of GSH within cells ensures that the body will be exposed to polyphenolic-GSH conjugates. GSH conjugates derived from a variety of polyphenols exhibit an array of biological activity. This biological reactivity is a consequence of the maintenance of the electrophilic and redox properties of the parent polyphenol following the addition of GSH. Indeed, the redox activity of polyphenols is frequently enhanced following conjugation with GSH and cysteine. Thus, the conjugation of quinones with GSH does not a priori result in detoxication. Only when GSH conjugation is efficiently coupled to export of the conjugate from the cell (phase III metabolism) via the GS-X export pump or transporter (194, 195) will the initial detoxication process be complete. However, cells which have the capacity to accumulate the GSH conjugates, such as cells exhibiting high concentrations of γ-GT or high activity of an intact GSH importer, will be exposed to the redox and electrophilic properties of these conjugates. In this respect, physiological similarities exist between the organs and tissues that are susceptible to polyphenolic-GSH conjugate-mediated


Monks and Lau

Figure 6. Role for 1,4-benzoquinone and its glutathione conjugates in hydroquinone-mediated hematotoxicity. Following oxidation of hydroquinone (I), 1,4-benzoquinone may either alkylate tissue macromolecules (II) or form a variety of GSH conjugates, including 2,6-bis(glutathion-S-yl)hydroquinone (III). Redox cycling of either the protein-bound quinone (IV) or the GSH conjugate (IV) may generate reactive oxygen species. Alternatively, oxidation of the protein-bound quinone may result in alkylation of a second protein, generating protein-protein cross-links (V). 2,6-Bis(glutathion-S-yl)-1,4-benzoquinone may also alkylate cellular proteins or react with GSH to form 2,3,5-tris(glutathion-S-yl)-hydroquinone.

toxicity. In particular, similarities between transporting epithelia, such as those of renal proximal tubular epithelial cells, and the blood-brain and blood-CSF barriers are readily apparent. Brain microvascular endothelial cells maintain tight junctions (133) and possess a high density of mitochondria (134), characteristics associated with renal proximal tubular epithelial cells. Endothelial cells are also polarized in a manner similar to other transport interfaces, such as renal epithelia, with the preferential localization of specific transport systems and receptors on either the luminal or the antiluminal side of vessel walls (135). Epithelial cells of the choroid plexus, the major site of cerebrospinal fluid formation, also function in the blood-CSF barrier, and there is considerable similarity between the secretion of CSF by the choroid plexus and that of urine by the renal tubules, as reflected in both structural and functional (physiologi-

cal) characteristics (196). In addition to the physiological similarities between the blood-brain and blood-CSF barriers and renal epithelia, considerable biochemical similarities exist. Alkaline phosphatase, 5′-nucleotidase, Na+/K+-ATPase, guanylate cyclase, adenylate cyclase, aminopeptidase, and γ-GT are all components of endothelial cells of the blood-brain barrier and renal tubular epithelia. The knowledge that GSH is not just involved in cytoprotection but that it appears to possess pharmacological activity offers some unique research opportunities. The distinctive structural features of polyphenolic-GSH conjugates provide a structural basis for both toxicological and pharmacological activity. Toxicological sequelae may result from the electrophilic and redox properties of the quinone, whereas pharmacological changes may result from either the polyphenol or GSH moiety. Whether

Invited Review


GSH and cysteine conjugates derived from, for example, the catechol estrogens or dopamine exert some of their biological effects through interactions with specific receptors is an inviting question that deserves attention.

Acknowledgment. The authors would like to thank Drs. Jos Mertens, Michael Butterworth, Melanie Peters, Barbara Hill, Maria Rivera, Jeongmi Jeong, and Timothy Miller for their substantial contributions to the work described in this review. The research in the authors’ laboratories was supported by grants awarded by the National Institutes of Health (ES04662, ES07359, GM39338, CA58036, and OA10832).

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