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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) 17β-estradiol is nephrotoxic in the hamster in a manner dependent upon the activity of γ-glutamyl transpeptidase and (ii) 17β-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-17β-estradiol or 2-hydroxy-1-glutathion-S-yl-17β-estradiol (0.27-5.0 μmol/kg) to Syrian hamsters, produces mild nephrotoxicity. Repeated daily administration of 2-hydroxy-4-glutathion-S-yl-17β-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 γ-glutamyl transpeptidase may contribute to 17β-estradiol-induced renal tumors in the hamster. [ABSTRACT FROM PUBLISHER]

Patient-derived progenitor cell (PC) dysfunction is severely impaired in diabetes, but the molecular triggers that contribute to mechanisms of PC dysfunction are not fully understood. Methylglyoxal (MGO) is one of the highly reactive dicarbonyl species formed during hyperglycemia. We hypothesized that the MGO scavenger glyoxalase 1 (GLO1) reverses bone marrow-derived PC (BMPC) dysfunction through augmenting the activity of an important endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), resulting in improved diabetic wound healing. BMPCs were isolated from adult male db/db type 2 diabetic mice and their healthy corresponding control db/+ mice. MGO at the concentration of 10 µmol/L induced immediate and severe BMPC dysfunction, including impaired network formation, migration, and proliferation and increased apoptosis, which were rescued by adenovirus-mediated GLO1 overexpression. IRE1α expression and activation in BMPCs were significantly attenuated by MGO exposure but rescued by GLO1 overexpression. MGO can diminish IRE1α RNase activity by directly binding to IRE1α in vitro. In a diabetic mouse cutaneous wound model in vivo, cell therapies using diabetic cells with GLO1 overexpression remarkably accelerated wound closure by enhancing angiogenesis compared with diabetic control cell therapy. Augmenting tissue GLO1 expression by adenovirus-mediated gene transfer or with the small-molecule inducer trans-resveratrol and hesperetin formulation also improved wound closure and angiogenesis in diabetic mice. In conclusion, our data suggest that GLO1 rescues BMPC dysfunction and facilitates wound healing in diabetic animals, at least partly through preventing MGO-induced impairment of IRE1α expression and activity. Our results provide important knowledge for the development of novel therapeutic approaches targeting MGO to improve PC-mediated angiogenesis and tissue repair in diabetes., (© 2019 by the American Diabetes Association.)

2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephroncarcinogenic metabolite of benzene and hydroquinone, retains the ability to redox cycle and create oxidative stress. We have previously detected that TGHQ induces ROS-dependent necrotic or apoptotic cell death in renal epithelial HK-2 and human leukemic HL-60 cells respectively. Herein, we sought to determine the nature of the Nrf2 regulation in HK-2 and HL-60 cells undergoing TGHQ-mediated ROS-dependent cell death, due to the key role of Nrf2 in oxidative stress. Intriguingly, Nrf2 was upregulated in HK-2, but not in HL-60 cells, despite the ROS-dependent nature of cell death in both cell types. The possibility that TGHQ targeted the GSK3β-dependent Nrf2 stabilization pathway in HL-60 cells was discounted, whereas TGHQ-induced decreases in Nrf2 phosphorylation at Ser40 site appears to partially underlie the inability of TGHQ to up-regulate Nrf2 expression in HL-60 cells. Moreover, whereas the TGHQ-induced post-translational stabilization of Nrf2 in HK-2 cells resulted in the expected upregulation of HO1 and NQO1 mRNA, TGHQ actually decreased Nrf2 mRNA in HL-60 cells, with a concomitant decrease in NQO1, but not HO1 mRNA. In summary, we define differences between the two cell types that might contribute to the engagement of the Nrf2 signaling pathways. By extension, these data provide evidence that Nrf2 is not necessarily activated in ROS-dependent cell death, and further delve into the knowledge that Nrf2 regulation sensing by cells might be achieved at solely transcriptional level, not related to its degradation., (Copyright © 2019. Published by Elsevier B.V.)

Although all- trans -retinoic acid (ATRA) provides protection against a variety of conditions in vivo, particularly ischemia, the molecular mechanisms underpinning these effects remain unclear. The present studies were designed to assess potential mechanisms by which ATRA affords cytoprotection against renal toxicants in LLC-PK 1 cells. Pretreatment of LLC-PK 1 cells with ATRA (25 μM) for 24 h afforded cytoprotection against oncotic cell death induced by p -aminophenol (PAP), 2-(glutathion- S -yl)hydroquinone (MGHQ), and iodoacetamide but not against apoptotic cell death induced by cisplatin. Inhibition of protein synthesis with cycloheximide blunted ATRA protection, indicating essential cell survival pathways must be engaged before toxicant exposure to provide cytoprotection. Interestingly, ATRA did not prevent the PAP-induced generation of reactive oxygen species (ROS) nor did it alter glutathione levels. Moreover, ATRA had no significant effect on Nrf2 protein expression, and the Nrf2 inducers sulforaphane and MG132 did not influence ATRA cytoprotection, suggesting cytoprotective pathways beyond those that influence ROS levels contribute to ATRA protection. In contrast, ATRA rapidly (15 min) induced levels of the cellular stress kinases p -ERK and p -AKT at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Consistent with a role for p -ERK in ATRA-mediated cytoprotection, inhibition of p -ERK with PD98059 reduced the ability of ATRA to afford protection against PAP toxicity. Collectively, these data suggest that p -ERK and its downstream targets, independent of ROS and antioxidant signaling, are important contributors to the cytoprotective effects of ATRA against oncotic cell death., (Copyright © 2017 the American Physiological Society.)

Bone marrow-derived progenitor cells (BMPCs) are potential candidates for autologous cell therapy in tissue repair and regeneration because of their high angiogenic potential. However, increased progenitor cell apoptosis in diabetes directly limits their success in the clinic. MicroRNAs are endogenous noncoding RNAs that regulate gene expression at the posttranscriptional level, but their roles in BMPC-mediated angiogenesis are incompletely understood. In the present study, we tested the hypothesis that the proangiogenic miR-27b inhibits BMPC apoptosis in Type 2 diabetes. Bone marrow-derived EPCs from adult male Type 2 diabetic db/db mice and their normal littermates db /+ mice were used. MiR-27b expression (real-time PCR) in EPCs was decreased after 24 h of exposure to methylglyoxal (MGO) or oxidized low-density lipoprotein but not high glucose, advanced glycation end products, the reactive oxygen species generator LY83583, or H 2 O 2 The increase in BMPC apoptosis in the diabetic mice was rescued following transfection with a miR-27b mimic, and the increased apoptosis induced by MGO was also rescued by the miR-27b mimic. p53 protein expression and the Bax/Bcl-2 ratio in EPCs (Western blot analyses) were significantly higher in db/db mice, both of which were suppressed by miR-27b. Furthermore, mitochondrial respiration, as measured by oxygen consumption rate, was enhanced by miR-27b in diabetic BMPCs, with concomitant decrease of mitochondrial Bax/Bcl-2 ratio. The 3' UTR binding assays revealed that both Bax, and its activator RUNX1, were direct targets of miR-27b, suggesting that miR-27b inhibits Bax expression in both direct and indirect manners. miR-27b prevents EPC apoptosis in Type 2 diabetic mice, at least in part, by suppressing p53 and the Bax/Bcl-2 ratio. These findings may provide a mechanistic basis for rescuing BMPC dysfunction in diabetes for successful autologous cell therapy.

The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biologically reactive intermediates formed as endogenous products of various metabolic processes are considered important factors in a variety of human diseases, including Parkinson's disease and other neurological disorders, diabetes and complications thereof, and other inflammatory-associated diseases. Chemical-induced toxicities are also frequently mediated via the bioactivation of relatively stable organic molecules to reactive electrophilic metabolites. Indeed, chemical-induced toxicities have long been known to be associated with the ability of electrophilic metabolites to react with a variety of targets within the cell, including their covalent adduction to nucleophilic residues in proteins, and nucleotides within DNA. Although we possess considerable knowledge of the various biochemical mechanisms by which chemicals undergo metabolic bioactivation, we understand far less about the processes that couple bioactivation to toxicity. Identifying specific sites within a protein, which are targets for adduction, can provide the initial information necessary to determine whether such adventitious posttranslational modifications significantly alter either protein structure and/or function. To address this problem, we have developed mass spectrometry (MS)-based approaches to identify specific amino acid targets of electrophile adduction (electrophile-binding motifs), coupled with molecular modeling of such adducts, to determine the potential structural and functional consequences. Where appropriate, functional assays are subsequently conducted to assess protein function.

Methylglyoxal (MG) is a biologically reactive byproduct of glucose metabolism, levels of which increase in diabetes. MG modification of protein generates neutral hydroimidazolone adducts on arginine residues which can alter functional active sites. We investigated the site-specificity of MG adduction to human serum albumin (HSA) using multiple reaction monitoring (MRM) of 13 MG-modified tryptic peptides, each containing an internal arginine. Seven new sites for MG modification (R257>R209>R222>R81>R485>R472>R10) are described. Analysis of MG-treated HSA showed substantial R257 and R410 modification, with MG-modified R257 (at 100μM MG) in drug site I causing significant inhibition of prostaglandin catalysis. The MG hydroimidazolone (MG-H1) adduct was modeled at R257, and molecular dynamics simulations and affinity docking revealed a decrease of 12.8-16.5kcal/mol (S and R isomers, respectively) for warfarin binding in drug site I. Taken together, these results suggest that R257 is a likely site for MG modification in vivo, which may have functional consequences for prostaglandin metabolism and drug bioavailability., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Metabolism is a prerequisite for the development of benzene-mediated myelotoxicity. Benzene is initially metabolized via cytochromes P450 (primarily CYP2E1 in liver) to benzene-oxide, which subsequently gives rise to a number of secondary products. Benzene-oxide equilibrates spontaneously with the corresponding oxepine valence tautomer, which can ring open to yield a reactive alpha,beta-unsaturated aldehyde, trans-trans-muconaldehyde (MCA). Further reduction or oxidation of MCA gives rise to either 6-hydroxy-trans-trans-2,4-hexadienal or 6-hydroxy-trans-trans-2,4-hexadienoic acid. Both MCA and the hexadienal metabolite are myelotoxic in animal models. Alternatively, benzene-oxide can undergo conjugation with glutathione (GSH), resulting in the eventual formation and urinary excretion of S-phenylmercapturic acid. Benzene-oxide is also a substrate for epoxide hydrolase, which catalyzes the formation of benzene dihydrodiol, itself a substrate for dihydrodiol dehydrogenase, producing catechol. Finally, benzene-oxide spontaneously rearranges to phenol, which subsequently undergoes either conjugation (glucuronic acid or sulfate) or oxidation. The latter reaction, catalyzed by cytochromes P450, gives rise to hydroquinone (HQ) and 1,2,4-benzene triol. Co-administration of phenol and HQ reproduces the myelotoxic effects of benzene in animal models. The two diphenolic metabolites of benzene, catechol and HQ undergo further oxidation to the corresponding ortho-(1,2-), or para-(1,4-)benzoquinones (BQ), respectively. Trapping of 1,4-BQ with GSH gives rise to a variety of HQ-GSH conjugates, several of which are hematotoxic when administered to rats. Thus, benzene-oxide gives rise to a cascade of metabolites that exhibit biological reactivity, and that provide a plausible metabolic basis for benzene-mediated myelotoxicity. Benzene-oxide itself is remarkably stable, and certainly capable of translocating from its primary site of formation in the liver to the bone marrow. However, therein lies the challenge, for although there exists a plethora of information on the metabolism of benzene, and the fate of benzene-oxide, there is a paucity of data on the presence, concentration, and persistence of benzene metabolites in bone marrow. The major metabolites in bone marrow of mice exposed to 50 ppm [(3)H]benzene are muconic acid, and glucuronide and/or sulfate conjugates of phenol, HQ, and catechol. Studies with [(14)C/(13)C]benzene revealed the presence in bone marrow of protein adducts of benzene-oxide, 1,4-BQ, and 1,4-BQ, the relative abundance of which was both dose and species dependent. In particular, histones are bone marrow targets of [(14)C]benzene, although the identity of the reactive metabolite(s) giving rise to these adducts remain unknown. Finally, hematotoxic HQ-GSH conjugates are present in the bone marrow of rats receiving the HQ/phenol combination. In summary, although the fate of benzene-oxide is known in remarkable detail, coupling this information to the site, and mechanism of action, remains to be established., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Hydroquinone (HQ) is a metabolite of benzene, and in combination with phenol (PHE), reproduces benzene myelotoxicity. HQ readily oxidizes to 1,4-benzoquinone (1,4-BQ) followed by the reductive addition of glutathione (GSH). Subsequent cycles of oxidation and GSH addition give rise to a variety of mono-, and multi-GSH substituted conjugates. Following administration of PHE/HQ (1.1 mmol/kg/0.9 mmol/kg, ip) to male Sprague-Dawley (SD) rats, 2-(glutathion-S-yl)HQ [GS-HQ], 2,5-bis-(glutathion-S-yl)HQ [2,5-GS-HQ], 2,6-bis-(glutathion-S-yl)HQ [2,6-GS-HQ], and 2,3,5-tris-(glutathion-S-yl)HQ [2,3,5-GS-HQ] were all identified in bone marrow. 2-(Cystein-S-ylglycine)HQ [2-(CysGly)HQ], 2-(cystein-S-yl)HQ [2-(Cys)HQ], and 2-(N-acetylcystein-S-yl)HQ [2-(NACys)HQ] were also found in the bone marrow of PHE/HQ and benzene treated rats and mice, indicating the presence of an active mercapturic acid pathway within bone marrow. Moreover, 2,6-GS-HQ and 2,3,5-GS-HQ were hematotoxic when administered to rats. All of the HQ-GSH conjugates retain the ability to redox cycle and generate reactive oxygen species (ROS), and to arylate target proteins. Recent in vitro and in vivo studies in our laboratory revealed lysine and arginine residues as primary targets of 1,4-BQ, GS-HQ and 2-(NACys)HQ adduction. In contrast 1,4-BQ-adduction of cysteine residues may be a transient interaction, where physiological conditions dictate adduct stability. The generation of ROS and alkylation of proteins may both contribute to benzene-mediated myelotoxicity, and the two processes may be inter-dependent. However, the precise molecular mechanism by which benzene and HQ-GSH conjugates induce hematotoxicity remains to be determined. Within 18h of administration of PHE/HQ to SD rats a significant decrease in blood lymphocyte count was observed. At this early time point, erythrocyte counts and hemoglobin concentrations remained within the normal range. Concomitant with the decrease in lymphocyte count, western blot analysis of bone marrow lysate, using HQ-GSH and 4-hydroxy-2-nonenal (4HNE) specific antibodies, revealed the presence of HQ-GSH- and 4HNE-derived protein adducts. Identification of these adducts is required before the functional significance of such protein modifications can be determined., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Matrix assisted laser desorption ionization (MALDI) mass spectrum images are created from an array of mass spectra collected over a tissue surface. We have increased the mass range of proteins that can be detected in tissue sections from kidneys, heart, lung and brain of different rodent species by a modification of the sandwich technique, which involves co-crystallizing matrix with analyte. A tissue section is placed upon a drop of sinapinic acid matrix dissolved in 90% ethanol and 0.5% Triton X-100. Once the matrix has dried, a seed layer of sinapinic crystals is added as a dispersion in xylene. Additional layers of sinapinic acid are added as solutions in 90% ethanol followed by 50% acetonitrile. Numerous peaks with signal to noise ratio of four or greater are observed between 25 kDa to 50 kDa. This represents approximately 10 times as many peaks as are detected using traditional matrix spotting and spraying.

Numerous studies have suggested that the lifetime dose of unopposed estrogen is a significant risk factor for breast and uterine cancer. Estradiol (E2) plays a putative role as a tumor promoter through interaction with estrogen receptors but can also be metabolized to redox active and/or mutagenic semiquinones and quinones. Similarly, equine estrogens (components of certain hormone replacement therapy preparations) are converted to quinone metabolites. The use of hormone replacement therapy has also been associated with increased breast and endometrial cancer risk. Recently, metabolites of certain equine estrogens have been shown to inhibit human glutathione S-transferases (hGSTs). Since E2 and equine estrogens share similarities in other biological interactions, we have investigated the inhibitory capacity of endogenously formed E2 metabolites toward various hGSTs. The quinone metabolite of 2-hydroxy-17-beta-estradiol (2-OH-E2) was synthesized, and inhibition of hGST-mediated biotransformation of model substrates was assessed. Inhibition of purified recombinant hGSTM1-1 and hGSTA1-1 occurred in a concentration-dependent manner with IC50-values of approximately 250 and 350 nM, respectively. hGSTs M2-2, P1-1 and T1-1 were significantly less sensitive to inhibition. Specific glutathione-conjugates of the estrogen quinone also potently inhibited hGSTM1-1 and hGSTA1-1. Mass spectrometry data indicate that the inhibition was not mediated via covalent adduction. Although we have demonstrated hGST inhibition via E2 metabolites, our findings indicate that the isoform specificity and potency of GST inhibition by endogenous E2 metabolites is different than that of equine estrogen metabolites.

The cytotoxicity of menadione on hepatocytes was studied by using the substrate generation/tip collection mode of scanning electrochemical microscopy by exposing the cells to menadione and detecting the menadione-S-glutathione conjugate (thiodione) that is formed during the cellular detoxication process and is exported from the cell by an ATP-dependent pump. This efflux was electrochemically detected and allowed scanning electrochemical microscopy monitoring and imaging of single cells and groups of highly confluent live cells. Based on a constant flux model, approximately 6 x 10(6) molecules of thiodione per cell per second are exported from monolayer cultures of Hep G2 cells.

11-Deoxy-16,16-dimethyl PGE(2) (DDM-PGE(2)) protects renal proximal tubule epithelial cells (LLC-PK(1)) against the toxicity induced by 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. We have now determined the ability of DDM-PGE(2) to protect against other renal toxicants and report that DDM-PGE(2) only protects against oncotic cell death, induced by H(2)O(2), iodoacetamide, and TGHQ, but not against apoptotic cell death induced by cisplatin, mercuric chloride, or tumor necrosis factor-alpha. DDM-PGE(2)-mediated cytoprotection is associated with the upregulation of at least five proteins, including the major endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (Grp78). To elucidate the role of Grp78 in oncotic cell death, we used LLC-PK(1) cells in which induction of grp78 expression was disrupted by stable expression of an antisense grp78 RNA (pkASgrp78). As anticipated, DDM-PGE(2) failed to induce Grp78 in pkASgrp78 cells, with a concomitant inability to provide cytoprotection. In contrast, DDM-PGE(2) induced Grp78 and afforded cytoprotection against H(2)O(2), iodoacetamide, and TGHQ in empty vector transfected cells (pkNEO). These data suggest that Grp78 plays an essential role in DDM-PGE(2)-mediated cytoprotection. Moreover, TGHQ-induced p38 MAPK activation is disrupted under conditions of a compromised ER stress response in pkASgrp78 cells, which likely contributes to the loss of cytoprotection. Finally, using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we found that DDM-PGE(2) induced several proteins in pkNEO cells, but not in pkASgrp78 cells, including retinol-binding protein, myosin light chain, and heat shock protein 27. The findings suggest that additional proteins may act in concert with Grp78 during DDM-PGE(2)-mediated cytoprotection against oncotic cell death.

2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ), a reactive metabolite of the nephrotoxicant hydroquinone, induces the ROS-dependent activation of MAPKs, followed by histone H3 phosphorylation and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK(1)). Cell death and histone H3 phosphorylation are attenuated by pharmacological inhibition of p38 MAPK or ERK1/2 pathways. Because TGHQ, but not epidermal growth factor (EGF), induces histone H3 phosphorylation and cell death in LLC-PK(1) cells, we hypothesized that there are differences in the mechanisms by which TGHQ and EGF induce activation of the EGF receptor (EGFR). We therefore compared the relative ability of TGHQ, H(2)O(2), and EGF to activate EGFR and MAPKs and found that p38 MAPK activation is EGFR independent, whereas ERK1/2 activation occurs mainly through EGFR activation. TGHQ, H(2)O(2), and EGF induce different EGFR tyrosine phosphorylation profiles that likely influence the subsequent differential kinetics of MAPK activation. We next transfected LLC-PK(1) cells with a dominant negative p38 MAPK-expressing plasmid (pcDNA3-DNp38). TGHQ failed to induce phosphorylation of p38 MAPK and its substrate, MK-2, in pcDNA3-DNp38-transfected cells, indicating loss of function of p38 MAPK. In untransfected, pcDNA3 or pcDNA3-p38 (native)-transfected LLC-PK(1) cells, Hsp27 was intensively phosphorylated after TGHQ treatment, whereas in pcDNA3-DNp38-transfected cells, TGHQ failed to induce Hsp27 phosphorylation. Thus EGFR-independent p38 MAPK and EGFR-dependent ERK1/2 activation by TGHQ lead to the activation of two downstream signaling factors, i.e., histone H3 and Hsp27 phosphorylation, which have in common the potential ability to remodel chromatin.

The tuberous sclerosis-2 (Tsc-2) gene is a suppressor of renal tumorigenesis and an early target of reactive oxygen species-induced renal cancer. Tuberin, the protein product of the Tsc-2 gene, participates in the regulation of cell proliferation, although the mechanism by which it suppresses proliferation is unknown. Quinol-thioether-transformed rat renal epithelial (QT-RRE) cell lines, derived from quinol-thioether-transformed primary renal epithelial cells from Eker rats, lack tuberin expression due to loss of heterozygosity of the Tsc-2 gene. These cell lines were used to examine the mechanism by which tuberin exerts its antiproliferative action. Loss of tuberin function correlates with high ERK activity (39), which could contribute to the formation of renal tumors. In this study, we sought to identify possible downstream effectors regulated by tuberin, using QT-RRE cells transfected with Tsc-2 cDNA to restore tuberin expression. Constitutively high ERK, B-Raf, and Raf-1 activities were observed in QT-RRE cells. However, restoration of tuberin expression in QT-RRE cells by transient transfection with Tsc-2 cDNA substantially decreased both ERK and B-Raf activity, with only modest changes in Raf-1 activity, suggesting tuberin functions as an upstream negative regulator of the ERK pathway. High ERK activity was not mediated through EGF receptor activation, but treatment with genistein demonstrated that protein kinases are involved in ERK cascade activation. The data indicate that loss of tuberin results in the upregulation of the ERK signaling pathway with subsequent increases in new DNA synthesis, and ultimately, tumor formation.

The Tsc-2 tumor suppressor gene encodes the protein tuberin, a multi-functional protein with sequence homology to the GTPase activating protein (GAP) for Rap1. Mutations in the Tsc-2 gene are associated with the development of renal tumors. The Eker rat (Tsc-2(EK/+)) bears a mutation in one allele of the Tsc-2 gene, which predisposes these animals to renal cancer. Treatment of wild-type (Tsc-2(+/+)) and mutant (Tsc-2(EK/+)) Eker rats with 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ; 7.5 micro mol/kg. i.v.), a potent redox active and nephrotoxic metabolite of hydroquinone increases the incidence of renal tumors only in animals carrying the mutant Tsc-2(EK/+) allele. We now show that the constitutive expression of 8-oxoguanine-DNA glycosylase (OGG1) in Tsc-2(EK/+) rats is three-fold lower than in wild-type Tsc-2(+/+) rats. Moreover, treatment of wild-type and mutant Eker rats with TGHQ greatly increases 8-oxo-deoxyguanosine (8-oxo-dG) levels within the outer stripe of the outer medulla. Tsc-2(EK/+) rats, with lower constitutive renal OGG1 expression, experience substantially higher levels of 8-oxo-dG than do wild type Tsc-2(+/+) rats. Interestingly, whereas OGG1 expression was rapidly (4 h) induced in Tsc-2(+/+) rats following exposure to TGHQ, it was significantly reduced in Tsc-2(EK/+) rats. The combination of the higher constitutive expression of OGG1 in Tsc-2(+/+) rats, and its rapid induction in response to TGHQ treatment, coupled to the initial decrease in OGG1 expression in Tsc-2(EK/+) rats, results in Tsc-2(EK/+) OGG1 protein levels just 5% of those seen in Tsc-2(+/+) rats 8 h after treatment. Coincidentally, 8-oxo-dG levels in Tsc-2(+/+) rats 8 h after treatment with TGHQ are just 5% of those that occur in Tsc-2(EK/+) rats. The results indicate that the Tsc-2 gene influences constitutive OGG1 expression and the ability of OGG1 to respond to an oxidative stress, consistent with the proposal that Tsc-2 is an acute-phase response gene. In keeping with this view, acute TGHQ-induced cytotoxicity was greater in Tsc-2(EK/+) rats than in Tsc-2(+/+) rats. The mechanism(s) coupling tuberin expression to the regulation of OGG1 are not known and are under investigation.

Although 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ; 2.5 micromol/kg ip) markedly increased cell proliferation within the outer stripe of the outer medulla (OSOM) of the kidney in both wild-type (Tsc2(+/+)) and mutant Eker rats (Tsc2(EK/+)), only TGHQ-treated Tsc2(EK/+) rats developed renal tumors, indicating that cell proliferation per se was not sufficient for tumor development. Tuberin expression was initially induced within the OSOM after TGHQ treatment but was lost within TGHQ-induced renal tumors. High extracellular signal-regulated kinase (ERK) activity occurred in the OSOM of Tsc2(EK/+) rats at 4 mo and in TGHQ-induced renal tumors. Cyclin D1 was also highly expressed in TGHQ-induced renal tumors. Reexpression of Tsc2 in tuberin-negative cells decreased ERK activity, consistent with the growth-suppressive effects of this tumor suppressor gene. Thus 1) stimulation of cell proliferation after toxicant insult is insufficient for tumor formation; 2) tuberin induction after acute tissue injury suggests that Tsc2 is an acute-phase response gene, limiting the proliferative response after injury; and 3) loss of Tsc2 gene function is associated with cell cycle deregulation.

The present studies were conducted to determine the pharmacological nature of a cytoprotective 11-deoxy-16,16-dimethyl-PGE(2) (DDM-PGE(2)) receptor in LLC-PK(1) cells. DDM-PGE(2)-mediated cytoprotection against 2,3,5-(trisglutathion-S-yl)hydroquinone (TGHQ)-mediated cytotoxicity can be reproduced using thromboxane A(2) (TXA(2)) receptor (TP) agonists (U46619 and IBOP), and the cytoprotective response to DDM-PGE(2) and TP agonists is inhibited by TP antagonists (SQ-29,548 and ISAP). Western blot analysis using an antipeptide antibody against the human platelet TP receptor (55 kDa) identified a particulate associated 54-kDa protein. DDM-PGE(2)-mediated 12-O-tetradecanoyl phorbol-13-acetate (TPA) responsive element (TRE) binding activity is not inhibited by cyclooxygenase inhibitors (aspirin and indomethacin) or a TXA(2) synthase inhibitor (sulfasalazine), suggesting that the biological response to DDM-PGE(2) is not dependent on de novo TXA(2) biosynthesis. Peak DDM-PGE(2)- and U46619-mediated TRE binding activity and nuclear factor-kappaB (NF-kappaB) binding activity are inhibited by SQ-29,548. The full cytoprotective response to DDM-PGE(2) requires an 8-h pulse with agonist. DDM-PGE(2)-mediated TRE and NF-kappaB binding activity remain elevated in the presence of agonist and rapidly decay following agonist washout, suggesting a direct correlation between DDM-PGE(2)-mediated cytoprotection and persistent DNA binding activities. TPA, a protein kinase C activator, induces cytoprotection and a persistent increase of NF-kappaB binding activity. DDM-PGE(2)-mediated cytoprotection and NF-kappaB binding activity but not TRE binding activity are inhibited by sulfasalazine. We conclude that the DDM-PGE(2) receptor is a TP receptor and that the cytoprotective response may be mediated in part by NF-kappaB.

Hydroquinone is a nephrocarcinogen in rats but generally tests negative in standard mutagenicity assays. However, 2,3,5-tris-(glutathion-S-yl)hydroquinone, a potent nephrotoxic metabolite of hydroquinone, and 2-bromo-bis-(glutathion-S-yl)hydroquinone, another cytotoxic quinol-glutathione (GSH) conjugate, cause extensive single strand breaks in DNA in a manner that is dependent on the formation of reactive oxygen species. We, therefore, investigated whether quinol-GSH conjugates have the potential to behave as genotoxicants. The shuttle vector pSP189, containing the supF gene, was treated with 2,3,5-tris-(glutathion-S-yl)hydroquinone and replicated in both human AD293 cells and Escherichia coli MBL50 cells. The mutation frequency increased 4.6- and 2.6-fold in human AD293 and bacterial MBL50 cells, respectively. Base substitutions were the major type of mutations, and they occurred predominantly at G:C sites in both cell types. A high frequency of deletions (30%), including < 10- and > 10-bp deletions, were observed in AD293-replicated plasmids. The most common types of mutations in AD293 cells were G:C to A:T transitions (33.8%) and G:C to T:A (29.4%) and G:C to C:G (19.1%) transversions. In MBL50 cells, the major mutations were G:C to T:A (33.8%) and G:C to C:G (31.3%) transversions and G:C to A:T transitions (27.5%). The mutation spectra were similar to those reported for *OH-induced mutations, suggesting that *OH generated from polyphenolic-GSH conjugates not only plays a role in cytotoxicity but also provides a basis for their mutagenicity and carcinogenicity.

The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-l-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of alpha-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.

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.

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.

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.

3-tert-Butyl-4-hydroxyanisole and tert-butyl-hydroquinone (TBHQ) are antioxidants known to promote renal and bladder carcinogenesis in the rat, although the mechanisms of these effects are unclear. Because glutathione (GSH) conjugates of a variety of hydroquinones are nephrotoxic, and because 2-tert-butyl-5-(glutathion-S-yl)hydroquinone [5-(GSyl)TBHQ], 2-tert-butyl-6-(glutathion-S-yl)hydroquinone [6-(GSyl)TBHQ], and 2-tert-butyl-3,6-bis-(glutathion-S-yl)hydroquinone [3,6-bis-(GSyl)-TBHQ] have been identified recently as metabolites of TBHQ in the male rat, we investigated the effects of these metabolites in the male rat. At the highest dose tested (400 micromol/kg,i.v.) 5-(Gsyl)TBHQ and 6-(GSyl)TBHQ caused 2-fold increases in the urinary excretion of gamma-glutamyl transpeptidase and alkaline phosphatase, and pigments arising from the polymerization of metabolites were deposited in the kidney. 3,6-bis-(GSyl)TBHQ (200 micromol/kg) was the most potent of the GSH conjugates tested and produced significant increases in the urinary excretion of gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase, and glucose (2-, 2-, 22-, and 11-fold increases, respectively). Alterations in the biochemical parameters correlated with the degree of single cell and tubular necrosis in the S(3)-M segment of the proximal tubule, as observed by light microscopy. In addition to nephrotoxicity, 3,6-bis-(GSyl)TBHQ increased the bladder wet weight 2-fold and caused severe hemorrhaging of the bladder. The half-wave oxidation potentials of 5-(Gsyl)TBHQ and 6-(GSyl)TBHQ were similar to that of TBHQ, whereas the half-wave oxidation potential of 3,6-bis-(Gsyl)TBHQ was approximately 100 mV higher than that of TBHQ. The TBHQ-GSH conjugates also catalyzed the formation of 8- hydroxydeoxyguanosine, indicating that GSH conjugation does not impair the redox activity of TBHQ. Because some chemicals may induce carcinogenesis by a mechanism involving cytotoxicity followed by sustained regenerative hyperplasia, our results suggest that the toxicity of GSH conjugates of TBHQ to kidney and bladder may contribute to the promoting effect of 3-tert-butyl-4-hydroxyanisole and TBHQ in these tissues.

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.

Two bromobenzene-glutathione conjugates have been detected as both in vivo and in vitro metabolites of bromobenzene. Separation and purification by high pressure liquid chromatography (HPLC) and analysis by 13C and 1H-NMR spectroscopy indicated that the metabolites are trans-3-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol and trans-4-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol. The two conjugates are formed in unequal amounts; over a dose range of 25-500 mg/kg the ratio of the two conjugates excreted into bile in 6 h was 1.6 +/- 0.1 (mean +/- S.E.). Pretreatment of rats with either phenobarbital or 3-methyl-cholanthrene did not significantly alter the ratio of the two conjugates excreted into bile. When bromobenzene was incubated with rat liver microsomes and glutathione, the same two conjugates were formed in the presence but not in the absence of 100 000 x g supernatant. Furthermore, in the presence of 100 000 x g supernatant from control animals, microsomes from rats treated with phenobarbital formed both conjugates 6 times more rapidly than did microsomes from control rats, whereas microsomes from rats treated with 3-methylcholanthrene formed both conjugates less rapidly than did those from control rats. Thus, the data suggest that both conjugates are formed via bromobenzene 3,4-oxide and that their formation requires in liver cytosol.

The application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to determine the structure of two bile metabolites isolated from rats injected interperitoneally with bromobenzene is described. The structures of the two molecules are obtained unambiguously from the proton-proton spin coupling constants. This paper discusses the fundamentals of the technique and demonstrates the resolution of small long-range coupling constants.