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The reaction of cytochrome c with the electrophilic compounds (glutathion-S-yl)-1,4-benzoquinone (GSBQ) and iodoacetamide was studied using mass spectrometry. GSBQ is a nephrotoxic quinol-thioether metabolite of benzoquinone, while iodoacetamide is an alkylating agent targeting cysteine thiols. Both chemicals formed covalent adducts with cytochrome c. GSBQ formed adducts with cytochrome c at pH 6 on several histidine and lysine residues. At a pH >7, the initial product rearranged to a disubstituted cyclic quinone species preferentially found at two sites on the protein, Lys25-Lys27 and Lys86-Lys87, via quinol amine linkages. These two sites were previously determined to be the targets of benzoquinone adduct formation [Person et al. (2003) Chem. Res. Toxicol. 16, 598−608]. Cyclic reaction products are preferentially formed at two sites on the protein because of the presence of multiple basic residues in a conformationally flexible region whereas noncyclic products bind to a broad spectrum of available lysine and histidine nucleophiles. Iodoacetamide was a less selective alkylating agent able to form adducts on the majority of the nucleophilic sites of the protein. MS/MS spectra were used to identify signature ions for GSBQ-adducted peptides from the characteristic fragmentation patterns. Neutral losses of the 129 Da γ-glutamate residue and of the 273 Da glutathione moiety were found in both cysteine thiol- and lysine amine-linked GSBQ adduct MS/MS. Characteristic fragment ions were used in conjunction with the scoring algorithm for spectral analysis to search for adducted species present at low levels in the sample, and the analysis is applicable generally to detection of glutathione conjugates by MS/MS. Parallel analysis using matrix-assisted laser desorption/ionization-MS to compare spectra of control and treated samples allowed identification of peptide adducts formed by direct addition of GSBQ and by the subsequent loss of the glutathione moiety in a pH-dependent cyclization reaction.

Protein identification by MS is well-established. Mixtures of proteins from cell extracts are separated by either one- or two-dimensional gel electrophoresis, and specific bands or spots are subjected to in-gel digestion and subsequent analysis by MS. The two most common types of ionization used in MS are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). When ESI is used, the sample is typically analyzed by inline HPLC-ESI-MS/MS with fragmentation of individual digest peptides, followed by database comparison between theoretical and experimental fragmentation patterns. MALDI-MS analysis is based on peptide mass mapping, with mass measurements of the digest peptides searched against a database of theoretical digests. We give here the results of a comparison between ESI-ion trap and MALDI-TOF (time-of-flight) analysis of 11-deoxy,16,16-dimethyl prostaglandin E2 (DDM-PGE2) inducible proteins. Individual peptides identified by the two techniques differed, in general, but the resulting protein identification was the same. Slightly higher coverage of each protein was obtained by MALDI-TOF, but the MS/MS data were more definitive by requiring fewer peptides to assign a positive identification. Both methods effectively identified two proteins in the same gel band. The samples here are derived from a renal epithelial cell line (LLC-PK1) established from the New Hampshire minipig, a species poorly represented in the current database, and strategies and limitations for analyzing such species are discussed.

Identification of multiple and novel posttranslational modifications remains a major challenge in proteomics. The present approach uses comparative analysis by matrix-assisted laser/desorption ionization (MALDI) MS of proteolytic digests from control and treated proteins to target differences due to modifications, without initial assumption as to type or residue localization. Differences between modified and unmodified digest MS spectra highlight peptides of interest for subsequent tandem mass spectrometry (MS/MS) analysis. Targeted HPLC-electrospray ionization (ESI)-MS/MS is then used to fragment peptides, and manual de novo sequencing is used to determine the amino acid sequence and type of modification. This strategy for identifying posttranslational modifications in an unbiased manner is particularly useful for finding modifications produced by exogenous chemicals. Successful characterization of chemically induced posttranslational modifications and novel chemical adducts is given as an example of the use of this strategy. Histone H4 from butyrate-treated LLC-PK1 cells is separated on a gel into bands representing different overall charge state. Bands are analyzed by comparative MALDI-MS and LC-MS/MS to identify the sites of methylation and acetylation. Previous attempts to identify chemically adducted proteins in vivo have been unsuccessful in part due to a lack of understanding of the final adduct form. Cytochrome c is adducted in vitro with benzoquinone, an electrophilic metabolite of benzene capable of interacting with nucleophilic sites within proteins. De novo sequencing identifies a novel cyclized diquinone adduct species as the major reaction product, targeting Lys and His residues at two specific locations on the protein surface. This unpredicted reaction product is characterized using our unbiased methods for detection and demonstrates the important influence of protein structure on chemical adduction.

Prostaglandins (PGs) act locally to maintain cellular homeostasis and stimulate stress response signaling pathways. These cellular effects are diverse and are tissue-dependent. PGE2, and the synthetic analogue, 11-deoxy,16,16-dimethyl PGE2 (DDM-PGE2), protect renal proximal tubular epithelial (LLC-PK1) cells against cellular injury induced by the potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, 2,3,5-tris-(glutathion-S-yl)hydroquinone. Although this cytoprotective response (in LLC-PK1 cells) is mediated through a thromboxane or thromboxane-like receptor coupled to AP-1 signaling pathways, the mechanism of cytoprotection is unknown. In this study, we utilized HPLC-electrospray ionization tandem mass spectrometric (ESI MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometric (MALDI TOF) analysis of proteins isolated from DDM-PGE2-stimulated LLC-PK1 cells to identify candidate cytoprotective proteins. DDM-PGE2 selectively stimulated the synthesis of several proteins in LLC-PK1 cells. Peptide sequencing by ESI-MS/MS of in-gel tryptic protein digests revealed the identity of eight proteins:  endothelial actin binding protein, myosin, elongation factor 2 (EF-2), elongation factor 1α-1 (EF-1α), heat shock protein 90β (HSP90β), glucose-regulated protein 78 (GRP 78), membrane-organizing extension spike protein, and actin. Both ESI-MS/MS and MALDI-MS analysis resulted in the same protein identification. Western analysis confirmed the temporal induction of the majority of these proteins, including EF-2, EF-1α, HSP90β, GRP78, and actin. The collective expression of these proteins suggests that DDM-PGE2-mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an endoplasmic reticulum (ER) stress response. The present studies provide insights into potential downstream targets of PG signaling.

Extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) were all rapidly activated in a ROS-dependent manner during 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-mediated oxidative stress and oncotic cell death in renal proximal tubule epithelial cells (LLC−PK1). TGHQ-induced phosphorylation of ERK1/2 and JNK MAPKs required epidermal growth factor receptor (EGFR) activation, whereas p38 MAPK activation was EGFR independent. In contrast to their established roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK (but not JNK) appear to contribute to cell death, since inhibition of ERK1/2 or p38 MAPKs with PD098059 or SB202190 respectively, attenuated TGHQ-mediated cell death. TGHQ increased AP-1 and NFκB DNA-binding activity, but whereas pharmacological inhibition of ERK1/2 or p38 MAPKs attenuated AP-1 DNA binding activity, it potentiated TGHQ-mediated NFκB activation. Consistent with a role for NFκB activation in the cytoprotective response to ROS in renal epithelial cells, an anti-NFκB peptide SN50 suppressed the protective effects of ERK inhibition (PD098059 treatment). The data provide evidence that the activation of MAPKs by ROS in renal epithelial cells plays an important role in oncotic cell death, and NF-kB is involved in the cytoprotective effects of PD098059.

2,3,5-Tris(glutathion-S-yl)hydroquinone [TGHQ] is a potent nephrotoxicant and nephrocarcinogen, and induces a spectrum of mutations in human and bacterial cells consistent with those attributed to reactive oxygen species (ROS). Studies were conducted to determine whether the oxidative stress induced by TGHQ in renal proximal tubule epithelial cells (LLC-PK1) modulates transcriptional activities widely implicated in transformation responses, namely 12-O-tetradecanoyl phorbol 13-acetate (TPA) responsive element (TRE)- and nuclear factor kappa B (NF-κB)-binding activity. TGHQ increased TRE- and NF-κB-binding activity in a concentration- and time-dependent manner. Catalase fully inhibited peak TGHQ-mediated TRE- and NF-κB-binding activity. In contrast, although deferoxamine fully inhibited TGHQ-mediated TRE-binding activity, it had only a marginal effect on NF-κB-binding activity. Collectively, these data indicate that TGHQ modulates TRE- and NF-κB-binding activity in an ROS-dependent fashion. Cycloheximide and actinomycin D fully inhibited TGHQ-mediated TRE-binding activity, but in the absence of TGHQ increased NF-κB-binding activity. Although protein kinase C (PKC) is widely implicated in stress response signaling, pretreatment of cells with PKC inhibitors (H-89, calphostin C) did not modulate TGHQ-mediated DNA-binding activities. In contrast, pretreatment of cells with (PD098059), a mitogen activated protein kinase kinase (MEK) inhibitor, markedly reduced TGHQ-mediated TRE-binding activity, but enhanced TGHQ-mediated NF-κB-binding activity. We conclude that TGHQ-mediated TRE- and NF-κB-binding activities are ROS-dependent. Although there is a common requirement for hydrogen peroxide (H2O2) in the regulation of these DNA-binding activities, there appears to be divergent regulation after H2O2 generation in renal epithelial cells.

Reactive metabolites play an important role in 3,4-(±)-methylenedioxyamphetamine (MDA) and 3,4-(±)-methylenedioxymethamphetamine (MDMA; ecstasy)-mediated serotonergic neurotoxicity, although the specific identity of such metabolites remains unclear. 5-(Glutathion-S-yl)-α-methyldopamine (5-GSyl-α-MeDA) is a serotonergic neurotoxicant found in the bile of MDA-treated rats. The brain uptake of 5-GSyl-α-MeDA is decreased by glutathione (GSH), but sharply increases in animals pretreated with acivicin, an inhibitor of γ-glutamyl transpeptidase (γ-GT) suggesting competition between intact 5-GSyl-α-MeDA and GSH for the putative GSH transporter. γ-GT is enriched in blood−brain barrier endothelial cells and is the only enzyme known to cleave the γ-glutamyl bond of GSH. We now show that pretreatment of rats with acivicin (18 mg/kg, ip) inhibits brain microvessel endothelial γ-GT activity by 60%, and potentiates MDA- and MDMA-mediated depletions in serotonin (5-HT) and 5-hydroxylindole acidic acid (5-HIAA) concentrations in brain regions enriched in 5-HT nerve terminal axons (striatum, cortex, hippocampus, and hypothalamus). In addition, glial fibrillary acidic protein (GFAP) expression increases in the striatum of acivicin and MDA (10 mg/kg) treated rats, but remains unchanged in animals treated with just MDA (10 mg/kg). Inhibition of endothelial cell γ-GT at the blood−brain barrier likely enhances the uptake into brain of thioether metabolites of MDA and MDMA, such as 5-(glutathion-S-yl)-α-MeDA and 2,5-bis-(glutathion-S-yl)-α-MeDA, by increasing the pool of thioether conjugates available for uptake via the intact GSH transporter. The data indicate that thioether metabolites of MDA and MDMA contribute to the serotonergic neurotoxicity observed following peripheral administration of these drugs.

Hydroquinone (HQ) is a potential human carcinogen to which many people are exposed. HQ generally tests negative in standard mutagenicity assays, making it a “nongenotoxic” carcinogen whose mechanism of action remains unknown. HQ is metabolized to 2,3,5-tris(glutathion-S-yl)HQ (TGHQ), a potent toxic and redox active compound. To determine if TGHQ is a carcinogen in the kidney, TGHQ was administered to Eker rats (2 months of age) for 4 or 10 months. Eker rats carry a germline mutation in the tuberous sclerosis 2 (Tsc-2) tumor suppressor gene, which makes them highly susceptible to the development of renal tumors. As early as 4 months after the initiation of treatment (2.5 μmol/kg, ip), TGHQ-treated rats developed numerous toxic tubular dysplasias of a form rarely present in vehicle-treated rats. These preneoplastic lesions are believed to represent early transformation within tubules undergoing regeneration after injury by TGHQ, and adenomas subsequently arose within these lesions. After treatment for 10 months (2.5 μmol/kg for 4 months followed by 3.5 μmol/kg for 6 months), there were 6-, 7-, and 10-fold more basophilic dysplasias, adenomas, and renal cell carcinomas, respectively, in TGHQ-treated animals than in controls. Most of these lesions were in the region of TGHQ-induced acute renal injury, the outer stripe of the outer medulla. Loss of heterozygosity (LOH) at the Tsc-2 locus was demonstrated in both the toxic tubular dysplasias and tumors from rats treated with TGHQ for 10 months, consistent with TGHQ-induced loss of tumor suppressor function of the Tsc-2 gene. Thus, although HQ is generally considered a nongenotoxic carcinogen, our data suggest that HQ nephrocarcinogenesis is probably mediated by the formation of the quantitatively minor yet potent nephrotoxic metabolite TGHQ, which induces sustained regenerative hyperplasia, loss of tumor suppressor gene function, and the subsequent formation of renal adenomas and carcinomas. In addition, our data demonstrate that assumptions regarding mechanisms of action of nongenotoxic carcinogens should be considered carefully in the absence of data on the profiles of metabolites generated by these compounds in specific target organs for tumor induction.

In this study, we show that 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a putative metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Prior to the onset of apoptosis, TGHQ depletes intracellular glutathione (GSH) in a reactive oxygen species (ROS)-independent manner. Neutral, Mg²⁺-dependent sphingomyelinases, which are normally inhibited by GSH, are subsequently activated, as evidenced by increases in intracellular ceramide and depletion of sphingomyelin. As ceramide levels rise, effector caspase (DEVDase) activity steadily increases. Interestingly, while catalase has no effect on TGHQ-mediated depletion of GSH, this hydrogen peroxide (H2O2) scavenger does inhibit DEVDase activity and apoptosis, provided the enzyme is added to HL-60 cells before an increase in ceramide can be observed. Since ceramide analogues inhibit the mitochondrial respiratory chain, these data imply that ceramide-mediated generation of H2O2 is necessary for the activation of effector caspases-3 and/or -7, and apoptosis. In summary, these studies indicate that TGHQ, and perhaps many quinol-based toxicants and chemotherapeutics, may induce apoptosis in hematopoietic cells by depleting GSH and inducing the proapoptotic ceramide-signaling pathway.

Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explores the varied cytotoxic effects of quinones using specific examples, including quinones produced from benzene, polycyclic aromatic hydrocarbons, estrogens, and catecholamines. The evidence strongly suggests that the numerous mechanisms of quinone toxicity (i.e., alkylation vs oxidative stress) can be correlated with the known pathology of the parent compound(s).

Cellular stress can initiate prostaglandin (PG) biosynthesis which, through changes in gene expression, can modulate cellular functions, including cell growth. PGA2, a metabolite of PGE2, induces the expression of stress response genes, including gadd153 and hsp70, in HeLa cells and human diploid fibroblasts. PGs, gadd153, and hsp70 expression are also influenced by the cellular redox status. Polyphenolic glutathione conjugates retain the ability to redox cycle, with the concomitant generation of reactive oxygen species. One such conjugate, 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), is a potent nephrotoxic and nephrocarcinogenic metabolite of the nephrocarcinogen, hydroquinone. We therefore investigated the effects of TGHQ on PGE2 synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK1). TGHQ (200 μM, 2 h) increases PGE2 synthesis (2−3-fold) in LLC-PK1 cells with only minor (5%) reductions in cell viability. This response is toxicant-specific, since another proximal tubular toxicant, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), stimulates PGE2 synthesis only after massive (68%) reductions in cell viability. Consistent with the ability of TGHQ to generate an oxidative stress, both deferoxamine mesylate and catalase protect LLC-PK1 cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE2 synthesis, implying a major role for hydrogen peroxide in this response. TGHQ induces the early (60 min) expression of gadd153 and hsp70. However, while inhibition of cyclooxygenase with aspirin prevents TGHQ-induced PGE2 synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE2 analogue, 11-deoxy-16,16-dimethyl-PGE2 (DDM-PGE2), which protects LLC-PK1 cells against TGHQ-mediated cytotoxicity, modestly elevates the levels of gadd153 and hsp70 expression. In addition, catalase and, to a lesser extent, deferoxamine mesylate block TGHQ-induced gene expression. Therefore, although TGHQ-induced generation of reactive oxygen species is required for PGE2 synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE2 synthesis.

Direct injection of either 3,4-(±)-methylenedioxymethamphetamine (MDMA) or 3,4-(±)-methylenedioxyamphetamine (MDA) into the brain fails to reproduce the serotonergic neurotoxicity seen following peripheral administration. The serotonergic neurotoxicity of MDA and MDMA therefore appears to be dependent upon the generation of a neurotoxic metabolite, or metabolites, the identity of which remains unclear. α-Methyldopamine (α-MeDA) is a major metabolite of both MDA and MDMA. We have shown that intracerebroventricular (icv) injection of 2,5-bis(glutathion-S-yl)-α-methyldopamine [2,5-bis(glutathion-S-yl)-α-MeDA] causes decreases in serotonin concentrations in the striatum, cortex, and hippocampus, and neurobehavioral effects similar to those seen following MDA and MDMA administration. In contrast, although 5-(glutathion-S-yl)-α-methyldopamine [5-(glutathion-S-yl)-α-MeDA] and 5-(N-acetylcystein-S-yl)-α-methyldopamine [5-(N-acetylcystein-S-yl)-α-MeDA] produce neurobehavioral changes similar to those seen with MDA and MDMA, and acute changes in brain 5-HT and dopamine concentrations, neither conjugate caused long-term decreases in 5-HT concentrations. We now report that direct intrastriatal or intracortical administration of 5-(glutathion-S-yl)-α-MeDA (4 × 200 or 4 × 400 nmol), 5-(N-acetylcystein-S-yl)-α-MeDA (4 × 7 or 4 × 20 nmol), and 2,5-bis(glutathion-S-yl)-α-MeDA (4 × 150 or 4 × 300 nmol) causes significant decreases in striatal and cortical 5-HT concentrations (7 days following the last injection). Interestingly, intrastriatal injection of 5-(glutathion-S-yl)-α-MeDA or 2,5-bis(glutathion-S-yl)-α-MeDA, but not 5-(N-acetylcystein-S-yl)-α-methyldopamine, also caused decreases in 5-HT concentrations in the ipsilateral cortex. The same pattern of changes was seen when the conjugates were injected into the cortex. The effects of the thioether conjugates of α-MeDA were confined to 5-HT nerve terminal fields, since no significant changes in monoamine neurotransmitter levels were detected in brain regions enriched with 5-HT cell bodies (midbrain/diencephalon/telencephalon and pons/medulla). In addition, the effects of the conjugates were selective with respect to the serotonergic system, as no significant changes were seen in dopamine or norepinephrine concentrations. The results indicate that thioether conjugates of α-MeDA are selective serotonergic neurotoxicants. Nonetheless, a role for these conjugates in the toxicity observed following systemic administration of MDA and MDMA remains to be demonstrated, and requires further experimentation.

The metabolism and pharmacokinetics of intravenously administered theophylline and aminophylline (theophylline ethylenediamine) have been studied in 3 volunteers, using 14C-labelled theophylline. Both compounds were metabolized extensively and 1, 3-dimethyl-uric acid, 1-methyluric acid, 3-methylxanthine and two unknown minor metabolites were excreted in the urine, in addition to theophylline. The elimination of theophylline, 1, 3-dimethyluric acid, 1-methyluric acid and the unknown metabolites followed first-order kinetics, but that of 3-methylxanthine followed Michaelis-Menten kinetics. When given as aminophylline, theophylline was metabolized more rapidly and extensively than when given alone. The recovery of 14C in the urine was significantly higher after aminophylline than after theophylline. Abstention from intake of dietary methylxanthines for 7 days resulted in more rapid and extensive metabolism of aminophylline compared with results from the same subjects on their usual diets. The results indicate that, from a metabolic and pharmacokinetic viewpoint, aminophylline and theophylline are not equivalent.

α-Methyldopamine (α-MeDA) is a metabolite of the serotonergic neurotoxicants 3,4-(±)-(methylenedioxy)amphetamine (MDA) and 3,4-(±)-(methylenedioxy)methamphetamine (MDMA). α-MeDA readily oxidizes, and in the presence of glutathione (GSH) it forms 5-(glutathion-S-yl)-α-methyldopamine [5-(glutathion-S-yl)-α-MeDA]. Since GSH conjugates of many polyphenols are biologically (re)active, we investigated the role of 5-(glutathion-S-yl)-α-MeDA in the acute and long-term neurochemical changes observed after administration of MDA. Intracerebroventricular (icv) administration of 5-(glutathion-S-yl)-α-MeDA (720 nmol) to male Sprague-Dawley rats produced behavioral changes similar to those reported after subcutaneous adminstration of MDA. Thus, animals became hyperactive and aggressive and displayed forepaw treading and Straub tails, behaviors usually seen after administration of serotonin (5-HT) releasers, and consistent with a role for 5-(glutathion-S-yl)-α-MeDA in some of the behavioral alterations seen after administration of MDA and MDMA. In addition to the behavioral changes, 5-(glutathion-S-yl)-α-MeDA also caused short-term alterations in the dopaminergic, serotonergic, and noradrenergic systems. An increase in dopamine synthesis appears to be a prerequisite for the long-term depletion of brain 5-HT following MDMA administration. However, although 5-(glutathion-S-yl)-α-MeDA reproduced some of the effects of MDA on the dopaminergic system and was capable of causing acute increases in 5-HT turnover, a single icv injection of 5-(glutathion-S-yl)-α-MeDA did not result in long-term serotonergic toxicity. Thus, although acute stimulation of dopamine turnover may be necessary for long-term serotonergic toxicity, such changes are not sufficient to produce these effects. The effects of a multiple dosing schedule of 5-(glutathion-S-yl)-α-MeDA will therefore require investigation before we can define a role for this metabolite in MDA and MDMA mediated neurotoxicity. MDA also produces a pressor response that is related to its ability to release neuronal norepinephrine stores, and 5-(glutathion-S-yl)-α-MeDA caused comparable depletions of brain norepinephrine concentrations, indicating that both compounds produce similar effects on the noradrenergic system.

tert-Butyl-4-hydroxyanisole (BHA) and its demethylated analog, tert-butyl-hydroquinone (TBHQ), are antioxidants used in food. Both BHA and TBHQ have been shown to promote kidney and bladder carcinogenesis in the rat. We have previously demonstrated that glutathione (GSH) conjugates of a variety of hydroquinones are nephrotoxic and proposed that GSH conjugation serves to target these compounds to the kidney. In the present study, we examined the metabolism of TBHQ, focusing on the formation of potentially nephrotoxic sulfur-containing metabolites. 2-tert-Butyl-5-glutathion-S-ylhydroquinone, 2-tert-butyl-6-glutathion-S-ylhydroquinone, and 2-tert-butyl-3,6-bisglutathion-S-ylhydroquinone were identified as biliary metabolites of TBHQ (1.0 mmol/kg, ip) in male F344 rats, accounting for 2.2% of the dose. Liquid chromatography/mass spectroscopic analysis of urine also revealed the presence of additional sulfur-containing metabolites, tentatively identified as 2,5-dihydroxy-3-tert-butylthiophenol, 2,5-dihydroxy-4-tert-butylthiophenol, and their S-methyl derivatives. No mercapturic acids of TBHQ were found in the urine. The major biliary and urinary metabolites were TBHQ−glucuronide and TBHQ−sulfate, with a trace of TBHQ excreted unchanged. The results indicate that TBHQ undergoes oxidation and GSH conjugation in vivo in the male F344 rat. These conjugates are excreted into bile and undergo further metabolism prior to excretion in urine. Formation of the S-containing metabolites of TBHQ may occur in amounts sufficient to play a role in the toxicity of TBHQ to kidney and bladder.

We have developed a simple experimental technique which allows the determination of the relative rates of intracellular inactivation of chemically reactive metabolites and their diffusion out of isolated rat hepatocytes. By using bromobenzene as a model compound we have demonstrated that bromobenzene-3, 4-oxide generated within hepatocytes is sufficiently stable to leave the endoplasmic reticulum in which it is formed, traverse the cytoplasm and cross the cell membrane to the external environment. The addition of varying amounts of protein, which serves as an external sink to trap the epoxide as a covalently bound adduct, permits the calculation of the relative rates at which the epoxide is inactivated within the cells and diffuses out of the cells. As much as 35% of bromobenzene-3,4-oxide is capable of leaving hepatocytes and being trapped as a covalently bound adduct to glutathione (GSH)-transferase B. The extensive diffusion of bromobenzene-3,4-oxide may play an important role in the intercellular toxicity of this compound within the liver and perhaps may contribute to extrahepatic toxicity. The addition of GSH-transferase B to isolated hepatocyte suspensions caused a decrease in the formation of the 3,4-dihydrodiol, p-bromophenol and o- and p-bromophenol glucuronides, an increase in the formation of bromobenzene GSH conjugates, but did not affect intracellular covalent binding. Kinetic analyses of the data revealed that, in the absence of GSH-transferase B, nearly all of the bromobenzene GSH conjugates are formed within hepatocytes as the epoxide is formed, whereas rearrangement of bromobenzene-3,4-oxide to p-bromophenol and hydration to bromobenzene-3,4-dihydrodiol occurs almost exclusively outside the hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Bromohydroquinone was identified as a metabolite of both bromobenzene and o-bromophenol in the rat in vivo and in vitro. Identification was based on high-pressure liquid chromatography and gas chromatography-mass spectrometry. Formation of 2-bromohydroquinone by rat liver microsomes from both bromobenzene and o-bromophenol was increased by treatment of rats with either phenobarbital or 3-methylcholanthrene. Covalent binding of o-bromophenol to rat liver microsomes was inhibited by glutathione and ascorbate but not by superoxide dismutase or catalase. Liver microsomes converted o-bromophenol to 2-bromohydroquinone and covalently bound material, whereas kidney and lung microsomes metabolized o-bromophenol less rapidly. Administration of 2-bromohydroquinone to rats caused a dose- and time-dependent decrease in hepatic and renal glutathione levels, an increase in blood urea nitrogen levels and histopathological changes in kidney without causing any alterations to the liver. The histological changes in the kidney were indistinguishable from those observed after either bromobenzene or o-bromophenol administration. However, the dose of 2-bromohydroquinone required to elicit a similar nephrotoxicity was less than 10% of that of bromobenzene. Thus, 2-bromohydroquinone may play a role in the nephrotoxicity observed after bromobenzene administration. Although the nature of the nephrotoxic metabolite of 2-bromohydroquinone is not known, our present results suggest that 2-bromohydroquinone or a conjugate thereof may be formed in the liver and transported to the kidney where it elicits toxicity.

Although the conjugation of quinones with glutathione is associated with the process of detoxication, the reaction frequently facilitates quinone-induced toxicity. Thiol conjugates of quinones retain the ability to redox cycle and generate reactive oxygen species (ROS), contributing to the biological (re)activity of a variety of polyphenolic compounds. 2-Bromo-bis(glutathion-S-yl) hydroquinone (2-Br-bis(GSyl)HQ) and 2-bromo-6-(glutathion-S-yl) hydroquinone [2-Br-6-(GSyl)HQ] are potent nephrotoxicants in rats, inducing rapid karyolysis in vivo and DNA single-strand breaks in cultured renal proximal tubular epithelial cells (LLC-PK1). We investigated the cellular and molecular responses initiated after exposure of LLC-PK1 cells to 2-Br-bis(GSyl)HQ and 2-Br-6-(GSyl)HQ. Both quinone thioethers cause the concentration-dependent formation of DNA single-strand breaks, rapidly (2-10 min) inhibit DNA synthesis, and increase the expression of gadd153, a gene responsive to growth arrest and DNA damage. The addition of catalase to LLC-PK1 cells exposed to 2-Br-6-(GSyl)HQ or 2-Br-bis(GSyl)HQ effectively prevents gadd153 induction, which is consistent with findings that the gadd153 gene is subject to redox modulation and that ROS play an important role in quinone thioether-mediated cytotoxicity. Deferoxamine pretreatment also diminishes gadd153 induction, suggesting that in renal proximal tubular epithelial cells, decreased expression of gadd153 is not dependent on the removal of hydrogen peroxide per se but rather on preventing the generation of hydroxyl radical. Chelation of intracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester also reduces gadd153 induction by 2-Br-6-(GSyl)HQ and 2-Br-bis(GSyl)HQ, suggesting a role for calcium in the signaling process. Thus, 2-Br-6-(GSyl)HQ and 2-Br-bis(GSyl)HQ activate a genomic stress response via a signaling pathway that may include ROS, Ca2+, and DNA damage.

The irreversible inhibition of the rat glutathione S-transferase (GST) isoenzyme 1-1 by a series of halogenated 1,4-benzoquinones and their GSH conjugates was studied quantitatively by analysing the time course of enzyme inactivation. With increasing numbers of chlorine substituents, the rate of inhibition greatly increased. Incorporation of a GSH moiety in all cases increased the rate of inactivation compared with the non-substituted compound, and this was due to the increased affinity of the inhibitor for the active site. The ratio between the rates of inhibition for a given quinone with and without GSH substituent was largest for the three dichlorobenzoquinones, with the 2,6-isomer showing a 41-fold increase in rate of inhibition upon conjugation with GSH. The time courses of inhibition could be fitted either to a bi-exponential function (for the GSH conjugates and the higher chlorinated quinones) or to a mono-exponential function (all other quinones). It is concluded that the second component describes the affinity part of the reaction. GST 1-1 possesses two cysteine residues, with modification of one of these, probably located in the vicinity of the active site, having a major impact on the enzyme activity. Compounds with affinity towards the active site preferentially react with this residue. Non-specific quinones react equally with both cysteine residues. This was confirmed by the observation that complete inactivation of GST 1-1 by 2,5-dichlorobenzoquinone was achieved only after modification of two residues, whereas the corresponding GSH conjugate already completely inhibited the enzyme after modification of one residue.

Hydroquinone, an intermediate used in the chemical industry and a metabolite of benzene, is a nephrocarcinogen in the 2-year National Toxicology Program bioassay in male Fischer 344 rats. Current evidence suggests that certain chemicals may induce carcinogenesis by a mechanism involving cytotoxicity, followed by sustained regenerative hyperplasia and ultimately tumor formation. Glutathione (GSH) conjugates of a variety of hydroquinones are potent nephrotoxicants, and we now report on the effect of hydroquinone and 2,3,5-(tris-glutathion-S-yl)hydroquinone, on site-selective cytotoxicity and cell proliferation in rat kidney. Male Fischer 344 rats (160-200 g) were treated with hydroquinone (1.8 mmol/kg or 4.5 mmol/kg, p.o.) or 2,3,5-(tris-glutathion-S-yl)hydroquinone (7.5 micromol/kg; 1.2-1.5 micromol/rat, i.v.), and blood urea nitrogen (BUN), urinary gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase (ALP), glutathione-S-transferase (GST) and glucose were measured as indices of nephrotoxicity. Hydroquinone (1.8 mmol/kg, p.o.) is nephrotoxic in some rats, but not others, but cell proliferation (BrDU incorporation) in proximal tubular cells of the S3M region correlates with the degree of toxicity in individual rats. At 4.5 mmol/kg, hydroquinone causes significant increases in the urinary excretion of gamma-GT, ALP and GST. Pretreatment of rats with acivicin prevents hydroquinone-mediated nephrotoxicity, indicating that toxicity is dependent on the formation of metabolites that require processing by gamma-GT. Consistent with this view, 2,3,5-(tris-glutathion-S-yl)hydroquinone, a metabolite of hydroquinone, causes increases in BUN, urinary gamma-GT and ALP, all of which are maximal 12 h after administration of 2,3,5-(tris-glutathion-S-yl)hydroquinone. In contrast, the maximal excretion of GST and glucose occurs after 24 h. By 72 h, BUN and glucose concentrations return to control levels, while gamma-GT, ALP and GST remain slightly elevated. Examination of kidney slices by light microscopy revealed the presence of tubular necrosis in the S3M segment of the proximal tubule, extending into the medullary rays. Cell proliferation rates in this region were 2.4, 6.9, 15.3 and 14.3% after 12, 24, 48 and 72 h, respectively, compared to 0.8-2.4% in vehicle controls. Together with the metabolic data, the results indicate a role for hydroquinone-thioether metabolites in hydroquinone toxicity and carcinogenicity.

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ) is nephrotoxic in male Fischer 344 rats (20 μmol/kg) and albino guinea pigs (200 μmol/kg), but not BALB/c or B6C3F1 mice or Golden Syrian hamsters (200 μmol/kg). Since quinones are known to alkylate proteins, and because such macromolecular damage may play a role in cytotoxicity, we investigated the covalent binding of TGHQ to kidney (target tissue) and liver (nontarget tissue) of rodents “sensitive” or “resistant” to the nephrotoxic effects of TGHQ. Immunohistochemical staining of tissue obtained 2 h after administration of TGHQ, with rabbit anti-2-bromo-N-(acetyl-l-cystein-S-yl)HQ antibodies, correlated with the subsequent region of necrosis observed 19 h after dosing in Fischer 344 rats and guinea pigs. Immunohistochemical staining was localized to the S3 segment of the renal proximal tubules, at the corticomedullary junction along the medullary rays, and in the outer stripe of the outer medulla. Immunostaining was also observed in the same region in hamsters, but subsequent necrosis did not develop. In contrast, no immunostaining was observed in mice. Moreover, immunostaining was not detected in the livers of any species. Western blot analysis revealed numerous immunoreactive renal proteins in TGHQ-treated animals. The most distinctive immunostaining renal proteins were observed in Fischer 344 rats at ~34 kDa (mitochondria), ~35 kDa (nuclei) which comigrated with histone H1, and ~73 kDa (urine) which comigrated with γ-glutamyl transpeptidase. These adducted proteins were not detected in other species. Qualitative differences in alkylated proteins may therefore contribute to species susceptibility to TGHQ.

Glutathione (GSH) conjugates of hydroquinone (HQ) and 2-bromohydroquinone (2-BrHQ) produce severe renal proximal tubular necrosis in rats. Since the reactivity of quinones lies, in part, in their ability to alkylate proteins, our goal was to develop an immunochemical method with which to investigate the role of protein adduct formation in quinone−thioether-mediated toxicity. An immunogen was synthesized by coupling 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone (2-BrHQ-NAC) to keyhole-limpet hemocyanin (KLH). Anti-2-BrHQ-NAC−KLH antibodies were raised in rabbits and purified by affinity chromatography. Antibody binding to the 2-BrHQ-NAC epitope was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with a bovine serum albumin conjugate of 2-BrHQ-NAC. Affinity-purified anti-2-BrHQ-NAC−KLH antibodies recognized adducted proteins in the kidneys of rats treated with HQ, 2-BrHQ, 2-bromo-bis(glutathion-S-yl)hydroquinone, 2-(glutathion-S-yl)hydroquinone, 2,5-bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone. Immunoreactive proteins were found in all renal subcellular fractions of 2-BrHQ-treated rats, and the distribution of adducts was similiar to that obtained by quantifying 2-Br[¹⁴C]HQ covalent adducts. Western blot analysis revealed that three proteins, at 42, 46, and 79 kDa, were adducted by all the compounds examined. The identification of these adducted proteins will be required to assess their significance in quinol−thioether-mediated nephrotoxicity.

Metabolism of benzene is required to produce the classical hematological disorders associated with its exposure. After coadministration of hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip) to male Sprague−Dawley rats and DBA/2 mice, 2-(glutathion-S-yl)hydroquinone was identified in the bone marrow of both species. 2,5-Bis(glutathion-S-yl)hydroquinone, 2,6-bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone were also observed in the bone marrow of rats but were detected only sporadically in mice. Both species produced 2-(cystein-S-ylglycinyl)hydroquinone, 2-(cystein-S-yl)hydroquinone, and 2-(N-acetylcystein-S-yl)hydroquinone, indicating the presence of a functional mercapturic acid pathway in bone marrow. The ability of bone marrow to acetylate 2-(cystein-S-yl)hydroquinone and deacetylate 2-(N-acetylcystein-S-yl)hydroquinone was confirmed in vitro. Total quinol thioether concentrations were higher in, and eliminated more slowly from, the bone marrow of mice. Intravenous injection of 100 μmol/kg 2-(glutathion-S-yl)hydroquinone to rats gave rise to substantially lower bone marrow Cmax and AUC values compared to values found following coadministration of hydroquinone/phenol, suggesting that the major fraction of the GSH conjugates present in bone marrow are formed in situ. Finally, the erythrotoxicity of several of these conjugates was determined in rats using the erythrocyte ⁵⁹Fe incorporation assay. Administration of 2,3,5-tris(glutathion-S-yl)hydroquinone (17 μmol/kg, iv), 2,6-bis(glutathion-S-yl)hydroquinone (50 μmol/kg, iv), and benzene (11 mmol/kg, sc) significantly decreased ⁵⁹Fe incorporation into reticulocytes to 45 ± 6%, 28 ± 3%, and 20 ± 9% of control values, respectively. Although the doses of 2,3,5-tris(glutathion-S-yl)hydroquinone and 2,6-bis(glutathion-S-yl)hydroquinone represented only 0.2% and 0.4% of the dose of benzene, both conjugates reduced ⁵⁹Fe incorporation to the same degree as benzene. 2-(Glutathion-S-yl)hydroquinone had no effect at the dose tested (200 μmol/kg, iv). In summary, these data suggest that hydroquinone−glutathione conjugates are erythrotoxic and may contribute to benzene-mediated hematotoxicity.

Catechol estrogens, the cytochromes P450 mediated metabolites of 17β-estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17β-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17β-estradiol, in hamster hepatic microsomes. 17β-Estradiol was readily converted to 2- and 4-hydroxy-17β-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 μM−1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17β-estradiol were identified:  2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β-estradiol, and 2-hydroxy-4-glutathion-S-yl-17β-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17β-estradiol was identified:  4-hydroxy-1-glutathion-S-yl-17β-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation linked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17β-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17β-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by ~50% when compared to the value obtained when GSH is used to trap the o-quinones in the absence of ascorbate. A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen-induced carcinogenesis.

The effects of GSH (gamma-glutamylcysteinylglycine) and GSSG on intracellular calcium levels ([Ca2+]i) were investigated using fura-2-loaded dissociated brain cells from newborn rat pups. Both produced concentration-dependent increases in [Ca2+]i (EC50 values of 914.3 +/- 190.5 and 583.0 +/- 97.2 microM for GSH and GSSG, respectively), similar to that observed with N-methyl-D-aspartate (NMDA) and other agonists at the NMDA receptor. Maximum response (expressed as percentage change in [Ca2+]i relative to basal) was significantly greater for GSSG (37.5 +/- 1.6%) than for GSH (25.3 +/- 1.6%). The response to both agents was prevented or reversed by competitive (100 microM) (-)-2-amino-5- phosphonovalerate and noncompetitive (400 nM) MK-801 or 1.0 mM Mg2+ antagonists of NMDA receptor-mediated calcium entry, even at concentrations of GSH and GSSG normally producing maximal response. The idea that these effects are mediated, at least in part, by interaction with the NMDA receptor was supported by the effects of GSH and GSSG on the binding of the NMDA receptor ligand [3H]CGP-39653 to membranes isolated from hippocampal and cortical homogenates. Both GSH and GSSG displaced bound [3H]CGP-39653, with IC50 values of 0.93 +/- 0.18 and 11.02 +/- 1.22 microM, respectively, and produced an increase in the apparent Kd of binding (control, 8.92 +/- 0.83 nM, and GSH, 13.31 +/- 1.19 nM; control, 11.59 +/- 0.35 nM, and GSSG, 18.73 +/- 0.66 nM). However, both also produced modest reductions in Bmax (control, 1265 +/- 69 fmol/mg of protein, and GSH, 901 +/- 73 fmol/mg of protein; control, 1068 +/- 30 fmol/mg of protein, and GSSG, 730 +/- 18 fmol/mg of protein) and Hill slopes (GSH, 0.66 +/- 0.02; GSSG, 0.62 +/- 0.04). This suggests complex kinetics for the interaction of GSH and GSSG with the NMDA receptor. Taken together, the results suggest the potential for modulation of the NMDA receptor complex by GSH and GSSG.

Several lines of evidence suggest that the renal-specific toxicity of quinol-linked GSH conjugates is probably a result of their metabolism by gamma-glutamyl transpeptidase and selective accumulation by proximal tubular cells. Transport of the resultant quinol-cysteine and/or cystein-S-ylglycine conjugate followed by oxidation to the quinone may be important steps in the mechanism of toxicity of these compounds. Factors modulating the intracellular and/or intralumenal concentration of the cystein-S-yl and cystein-S-ylglycine conjugate will, therefore, be important determinants of toxicity. We have now studied the gamma-glutamyl transpeptidase-mediated metabolism of 2-bromo-3-(glutathion-S-yl)hydroquinone. The product of this reaction, 2-bromo-3-(cystein-S-ylglycyl)hydroquinone, undergoes an intramolecular cyclization to yield a 1,4-benzothiazine derivative that retains the glycine residue. A similar cyclization reaction occurs with 2-bromo-3-(cystein-S-yl)hydroquinone, which is unstable in aqueous solutions and undergoes a pH-dependent rearrangement that requires initial oxidation to the quinone. UV spectroscopy revealed that, at neutral pH, further reaction results in the formation of a chromophore, consistent with 1,4-benzothiazine formation. This product arises via cyclization of the cysteine residue via an intramolecular 1,4 Michael addition. Further reaction results in the precipitation of a pigment that exhibits properties of a pH indicator. The pigment undergoes a marked pH-dependent bathochromic shift (approximately 100 nm); it is red in alkali (lambda max, 480 nm) and violet in acid (lambda max, 578 nm). These properties are similar to those of the trichochrome polymers that are formed during melanin biosynthesis from S-(3,4-dihydroxyphenylalanine)-L-cysteine. Because the intramolecular cyclization reactions remove the reactive quinone moiety from the molecules, they may be regarded as detoxication reactions. 1,4-Benzothiazine formation represents a novel pathway that diverges from the usual route of mercapturic acid synthesis and may represent previously unrecognized and important products of quinone metabolism in vivo.

The formation of potentially reactive thiols has been postulated to play a role in the nephrotoxicity caused by a number of glutathione and/or cysteine conjugates. However, the inherent reactivity of such compounds has precluded both their identification in biological systems and a determination of their actual toxicity. To this end we have synthesized 6-bromo-2,5-dihydroxy-thiophenol as a putative metabolite of nephrotoxic 2-bromohydroquinone-glutathione conjugates. The compound was prepared by the addition of sodium thiosulfate to 2-bromo-1,4-benzoquinone followed by reduction of the S-arylthiosulfate to the thiophenol. 2,5-Dihydroxy-thiophenol was similarly prepared. Structural identification was confirmed by mass spectroscopy and nuclear magnetic resonance spectroscopy. Administration of 6-bromo-2,5-dihydroxy-thiophenol to rats (0.35 mmol/kg; intraperitoneally) caused an increase in blood urea nitrogen and histological alterations similar to those observed after 2-bromo-(diglutathion-S-yl)hydroquinone administration. 2,5-Dihydroxy-thiophenol was also nephrotoxic but at a dose of 0.6 mmol/kg. In contrast, no effects on liver pathology were observed after administration of either 6-bromo-2,5-dihydroxy-thiophenol or 2,5-dihydroxy-thiophenol and serum glutamate pyruvate transaminase levels were normal. Neither 2-, 3-, nor 4-bromothiophenol had any effect on blood urea nitrogen at doses between 0.2 and 0.8 mmol/kg (intraperitoneally) and no apparent alterations were seen in kidney slices prepared from bromothiophenol-treated rats. These findings suggest that the quinone function of 6-bromo-2,5-dihydroxy-thiophenol is necessary for the expression of toxicity. In this respect, the lower activity of NAD(P)H quinone oxidoreductase (EC 1.6.99.2) in renal cortex may be of toxicological significance.

2-Bromo-(diglutathion-S-yl)hydroquinone [2-Br-(diGSyl)HQ] causes severe necrosis of the proximal renal tubules in the rat, elevations in blood urea nitrogen (BUN) and increased urinary excretion of protein, glucose, and lactate dehydrogenase. In contrast, 2-Br-3-(GSyl)HQ, 2-Br-5-(GSyl)HQ, and 2-Br-6-(GSyl)HQ caused differentially less toxicity than the diglutathionyl conjugate. None of these conjugates had any apparent effect on liver pathology and serum glutamate-pyruvate transaminase remained within the normal range. Pretreatment of rats with probenecid, an organic anion transport inhibitor, offered only slight protection against 2-Br-(diGSyl)HQ-mediated elevations in BUN, proteinuria, or glucosuria. In contrast, quinine, an organic cation transport inhibitor, potentiated the nephrotoxicity of 2-Br-(di-GSyl)HQ. Thus, in contrast to other nephrotoxic sulfur conjugates, probenecid-sensitive organic ion transport systems do not contribute to the kidney-specific toxicity of 2-Br-(diGSyl)HQ. However, inhibition of renal gamma-glutamyl transpeptidase by AT-125 completely protected rats from the nephrotoxic effects of 2-Br-(diGSyl)HQ. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, caused a 20-25% decrease in 2-Br-(diGSyl)HQ-mediated elevations in BUN and urinary excretion parameters. The isomeric 35S conjugates covalently bound to rat kidney 10,000 x g homogenate in the order 2-Br-6-(GSyl)HQ greater than 2-Br-5-(GSyl)HQ greater than 2-Br-3-(GSyl)HQ greater than 2-Br-(diGSyl)HQ. AT-125 (0.4 mM) decreased covalent binding by 25%, 17%, 33%, and 28%, respectively. Aminooxyacetic acid (0.1 mM) inhibited covalent binding by 26%, 10%, 17%, and 17% respectively. Ascorbic acid (1.0 mM) inhibited covalent binding by 63%, 87%, 62%, and 28%, respectively, and this inhibition correlated, inversely, with the redox potential of the conjugates. Thus, the covalent binding is mediated preferentially by oxidation of the quinol moiety, although the formation of reactive thiols cannot be excluded. In addition, the initial conjugation of 2-BrHQ with GSH does not result in the formation of a less redox-active species. However, the subsequent addition of a second molecule of GSH results in the formation of a more redox-stable compound, which, paradoxically, enhances toxicity. The metabolism of 2-Br-(diGSyl)HQ by renal proximal tubular gamma-glutamyl transpeptidase and trans-membrane transport of the cysteine conjugate(s) followed by oxidation of the quinol moiety is probably responsible for the target organ toxicity of this compound.

The chemical reaction of 1,4-benzoquinone with glutathione results in the formation of adducts that exhibit increasing degrees of glutathione substitution. Purification of these adducts and analysis by 1H and 13C nuclear magnetic resonance spectroscopy revealed the products of the reaction to be 2-(glutathion-S-yl)hydroquinone; 2,3-(diglutathion-S-yl)hydroquinone; 2,5-(diglutathion-S-yl)hydroquinone; 2,6(diglutathion-S-yl)hydroquinone; 2,3,5-(triglutathion-S-yl)hydroquinone; and 2,3,5,6-(tetraglutatathion-S-yl)hydroquinone. The initial conjugation of 1,4-benzoquinone with glutathione did not significantly affect the oxidation potential of the compound. However, subsequent oxidation and glutathione addition resulted in the formation of conjugates that, dependent upon the position of addition, become increasingly more difficult to oxidize. Increased glutathione substitutions, which resulted in an increase in oxidation potentials, paradoxically resulted in enhanced nephrotoxicity. The triglutathion-S-yl conjugate was the most potent nephrotoxicant; the diglutathion-S-yl conjugates exhibited similar degrees of nephrotoxicity; the mono- and tetraglutathion-S-yl conjugates were not toxic. Thus, with the exception of the fully substituted isomer, the severity of renal necrosis correlated with the extent of glutathione substitution. The lack of toxicity of the fully substituted isomer is probably a consequence of its inability to alkylate tissue components. Thus, the conjugation of glutathione with quinones does not necessarily result in detoxification, even when the resulting conjugates are more stable to oxidation. The inhibition of gamma-glutamyl transpeptidase by AT-125 protected against 2,3,5-(triglutathion-S-yl)hydroquinone-mediated nephrotoxicity. It is suggested that other extra-renal sites expressing relatively high levels of gamma-glutamyl transpeptidase might therefore also be susceptible to hydroquinone-linked glutathione conjugate toxicity. This pathway might also contribute to the carcinogenicity and mutagenicity of certain quinones.

Cells respond to a variety of stresses by activating the transcription of a battery of "acute phase" or "stress response" genes. The nature of this response is tailored to the nature of the stress. The extent to which physiologically and pathophysiologically induced growth arrest share common genomic responses is unclear. We therefore compared the effects of a physiologically induced (serum and nutrient depletion) and a chemically induced (2-Br-bis-(GSyl)HQ and 2-Br-6-(GSyl)HQ) stress in renal tubular epithelial cells (LLC-PK1). The response to physiological stress, induced by serum depletion, involves growth arrest characterized by an inhibition of DNA synthesis that occurs in the absence of a decrease in histone mRNA or an increase in gadd153 mRNA, one of the growth arrest and DNA damage inducible genes. In contrast, the chemical-induced stress involves growth arrest accompanied by a decrease in histone mRNA, particularly core histone H2B and H2A mRNA, and the induction of gadd153. Chemical-induced changes in histone mRNA inversely correlate to changes in the expression of a stress gene, hsp70, whose expression is dependent upon the maintenance of appropriate nucleosomal structure.

Chronic exposure of male Syrian hamsters to a variety of estrogens has been linked with a high incidence of renal carcinoma. The basis of this species and tissue specificity remains to be resolved. We have recently shown that (i) 17beta-estradiol is nephrotoxic in the hamster in a manner dependent upon the activity of gamma-glutamyl transpeptidase and (ii) 17beta-estradiol is metabolized to a variety of catechol estrogen glutathione conjugates (Butterworth et al., Carcinogenesis, 18, 561-567, 1997). We report that the catechol estrogen glutathione conjugates exhibit redox properties similar to those of the catechol estrogens, and maintain the ability to generate superoxide radicals. Administration of 2-hydroxy-4-glutathion-S-yl-17beta-estradiol or 2-hydroxy-1-glutathion-S-yl-17beta-estradiol (0.27-5.0 micromol/kg) to Syrian hamsters, produces mild nephrotoxicity. Repeated daily administration of 2-hydroxy-4-glutathion-S-yl-17beta-estradiol causes a sustained elevation in urinary markers of renal damage and in the concentration of renal protein carbonyls and lipid hydroperoxides. Catechol estrogen oxidation and conjugation of glutathione in the liver, followed by the selective uptake of the redox active conjugates in tissues rich in gamma-glutamyl transpeptidase may contribute to 17beta-estradiol-induced renal tumors in the hamster.