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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]

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ) is a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. TGHQ generates reactive oxygen species (ROS), causing DNA-strand breaks, hyperactivation of PARP-1, increases in intracellular calcium ([Ca2+]i), and cell death. PARP-1 catalyzes the attachment of ADP-ribose polymers (PAR) to target proteins. In human kidney proximal tubule cells, ROS-mediated PARP-1 hyperactivation and elevations in [Ca2+]i are reciprocally coupled. The molecular mechanism of this interaction is unclear. The aim of the present study was to identify ROS-induced PAR-associated proteins to further understand their potential role in cell death. PAR-associated proteins were enriched by immunoprecipitation, identified by LC-MS/MS, and relative abundance was obtained by spectral counting. A total of 356 proteins were PAR-modified following TGHQ treatment. A total of 13 proteins exhibited gene ontology annotations related to calcium. Among these proteins, the general transcription factor II-I (TFII-I) is directly involved in the modulation of [Ca2+]i. TFII-I binding to phospholipase C (PLC) leads to calcium influx via the TRPC3 channel. However, inhibition of TRPC3 or PLC had no effect on TGHQ-mediated cell death, suggesting that their loss of function may be necessary but insufficient to cause cell death. Nevertheless, TGHQ promoted a time-dependent translocation of TFII-I from the nucleus to the cytosol concomitant with a decrease in tyrosine phosphorylation in α/β-TFII-I. Therefore it is likely that ROS have an important impact on the function of TFII-I, such as regulation of transcription, and DNA translesion synthesis. Our data also shed light on PAR-mediated signaling during oxidative stress, and contributes to the development of strategies to prevent PAR-dependent cell death., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

3, 4-Methylenedioxymethamphetamine (MDMA) is a hallucinogenic amphetamine derivative. The acute effects of MDMA are hyperthermia, hyperactivity, and behavioral changes, followed by long-term serotonergic neurotoxicity in rats and primates. However, the underlying mechanisms of MDMA neurotoxicity remain elusive. We reported that pretreatment of rats with Ro 4-1284, a reversible inhibitor of the vesicular monoamine transporter 2 (VMAT2), reduced MDMA-induced hyperactivity in rats, abolished the hyperthermic response, and the long-term neurotoxicity. Current studies focused on the effects of co- and/or postinhibition of VMAT2 on the acute and chronic effects of MDMA and on the dose-response relationship between MDMA-induced elevations in body temperature and subsequent reductions in indolamine concentrations. Sprague Dawley rats were treated with MDMA (20, 25, or 27.5 mg/kg sc), and either co- and/or posttreatment with the VMAT2 inhibitor (10 mg/kg ip). Rats simultaneously treated with Ro 4-1284 and MDMA exhibited a more rapid increase in body temperature compared to just MDMA. However, the duration of the elevated body temperature was significantly shortened (approximately 3 h vs approximately 8 h, respectively). A similar body temperature response was observed in rats posttreated (7 h after MDMA) with Ro 4-1284. Despite decreases in the area under the curve (Δtemp X time) of body temperature caused by Ro 4-1284, there were no significant differences in the degree of indolamine depletion between any of the MDMA-treated groups. The results suggest that the neuroprotective effects of VMAT2 inhibition is likely due to the indirect monoamine depleting effects of the Ro 4-1284 pretreatment, rather than by the direct inhibition of VMAT2 function., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

2,3,5-tris(Glutathion-S-yl)hydroquinone, a potent nephrotoxic and nephrocarcinogenic metabolite of benzene and hydroquinone, generates reactive oxygen species (ROS) causing DNA strand breaks and the subsequent activation of DNA repair enzymes, including poly(ADP-ribose) polymerase (PARP)-1. Under robust oxidative DNA damage, PARP-1 is hyperactivated, resulting in the depletion of NAD+ and ATP with accompanying elevations in intracellular calcium concentrations (iCa2+), and ultimately necrotic cell death. The role of Ca2+ during PARP-dependent necrotic cell death remains unclear. We therefore sought to determine the relationship between Ca2+ and PARP-1 during ROS-induced necrotic cell death in human renal proximal tubule epithelial cells (HK-2). Our experiments suggest that store-operated Ca2+ channel (SOC) entry contributes to the coupling of PARP-1 activation to increases in iCa2+ during ROS-induced cell death. Poly(ADP-ribose)glycohydrolase (PARG), which catalyzes the degradation of PARs to yield free ADP-ribose (ADPR), is known to activate Ca2+ channels such as TRPM2. However, siRNA knockdown of PARG did not restore cell viability, indicating that free ADPR is not responsible for SOC activation in HK-2 cells. The data indicate that PARP-1 and iCa2+ are coupled through activation of SOC mediated Ca2+ entry in an apparently ADPR-independent fashion; alternative PAR-mediated signaling likely contributes to PARP-dependent necrotic cell death, perhaps via PAR-mediated signaling proteins that regulate iCa2+ homeostasis., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.)

Background: Proteomic approaches identifying biomarkers have been applied to asthma to only a very limited extent., Methods: With an antibody array (RayBiotech, Norcross, GA, USA), the relative intensity and rank differences of 444 proteins were compared in 24 plasma samples obtained at age 3, 11 from children with and 12 without asthma diagnoses at ages 5 and 9. Protein candidates identified by antibody array were quantitated by ELISA in an enlarged sample. Proteins found to differentiate children with and without asthma were also examined for association with known Year 1 asthma risk factors, eczema, and wheeze., Results: In the antibody array, four proteins had rank differences between asthma and non-asthma groups (FDR <0.1). By ELISA, mean log (±s.e.m.) erythropoietin (EPO) level (IU/l) was lower (0.750 ± 0.048 vs. 0.898 ± 0.035; p = 0.006) and mean (±s.e.m.) soluble GP130 (sGP130) level (ng/ml) was higher in the asthma vs. the non-asthma group (302 ± 13 vs. 270 ± 8; p = 0.041). The other 2 array proteins (galactin-3 and eotaxin-3) did not differ by ELISA by asthma. EPO related to the asthma risk factor, first year eczema, whereas sGP130 related to first year wheeze., Conclusions: Through two independent assessments, age 3 plasma levels of EPO and sGP130 were found related to childhood asthma., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Synucleinopathies, including Parkinson's disease (PD), are neurodegenerative diseases characterized by accumulation of α-synuclein (SYN), a small neuronal protein with prion like properties that plays a central role in PD pathogenesis. SYN can misfold and generate toxic oligomers/aggregates, which can be cytotoxic. Environmental arsenic (As)-containing pesticide use correlates with increased incidence of PD. Moreover, because As exposure can lead to inhibition of autophagic flux we hypothesize that As can facilitate the accumulation of toxic SYN oligomers/aggregates and subsequent increases in markers of autophagy. We therefore examined the role of As in the oligomerization of SYN, and the consequences thereof. Chronic exposure of SH-SY5Y cells overexpressing SYN to As caused a dose-dependent oligomerization of SYN, with concomitant increases in protein ubiquitination and expression of other stress markers (protein glutathione binding, γ-GCS, light chain 3 (LC3)-I/II, P62, and NAD(P)H dehydrogenase quinone 1), indicative of an increased proteotoxic stress. Immunocytochemical analyses revealed an accumulation of SYN, and it's colocalization with LC3, a major autophagic protein. Mice exposed to As (100 ppb) for 1 month, exhibited elevated SYN accumulation in the cortex and striatum, and elevations in protein ubiquitination and LC3-I and II levels. However, tyrosine hydroxylase (TH), an indicator of dopaminergic cell density, was upregulated in the As exposed animals. Because SYN can inhibit TH function, and As can decrease monoamine levels, As exposure possibly leads to compensatory mechanisms leading to an increase in TH expression. Our findings suggest that susceptible individuals may be at higher risk of developing synucleinopathies and/or neurodegeneration due to environmental As exposure., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Methylglyoxal (MG) is a highly reactive dicarbonyl compound involved in the formation of advanced glycation endproducts (AGE). Levels of MG are elevated in patients with type-2 diabetes mellitus (T2DM), and AGE have been implicated in the progression of diabetic complications. The antihyperglycemic drug metformin (MF) has been suggested to be a scavenger of MG. The present work examined and characterized unequivocally the resulting scavenged product from the metformin-MG reaction. The primary product was characterized by (1)H, (13)C, 2D-HSQC, and HMBC NMR and tandem mass spectrometry. X-ray diffraction analysis determined the structure of the metformin and MG-derived imidazolinone compound as (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)guanidine (IMZ). A LC-MS/MS multiple reaction monitoring method was developed to detect and quantify the presence of IMZ in metformin-treated T2DM patients. Urine from >90 MF-treated T2DM patients was analyzed, with increased levels of MF directly correlating with elevations in IMZ. Urinary MF was detected in the range of 0.17 μM to 23.0 mM, and simultaneous measurement of IMZ concentrations were in the range of 18.8 nM to 4.3 μM. Since plasma concentrations of MG range from 40 nM to 4.5 μM, the level of IMZ production may be of therapeutic significance. Thus, in addition to lowering hepatic gluconeogenesis, metformin also scavenges the highly reactive MG in vivo, thereby reducing potentially detrimental MG protein adducts, with subsequent reductions in diabetic complications.

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan-HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

3,4-(±)-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT). Conversely, the long-term effects of MDMA manifest as prolonged depletions in 5-HT, and reductions in 5-HT reuptake transporter (SERT), indicative of serotonergic neurotoxicity. MDMA-induced 5-HT efflux relies upon disruption of vesicular monoamine storage, which increases cytosolic 5-HT concentrations available for release via a carrier-mediated mechanism. The vesicular monoamine transporter 2 (VMAT2) is responsible for packaging monoamine neurotransmitters into cytosolic vesicles. Thus, VMAT2 is a molecular target for a number of psychostimulant drugs, including methamphetamine and MDMA. We investigated the effects of depressed VMAT2 activity on the adverse responses to MDMA, via reversible inhibition of the VMAT2 protein with Ro4-1284. A single dose of MDMA (20 mg/kg, subcutaneous) induced significant hyperthermia in rats. Ro4-1284 (10 mg/kg, intraperitoneal) pretreatment prevented the thermogenic effects of MDMA, instead causing a transient decrease in body temperature. MDMA-treated rats exhibited marked increases in horizontal velocity and rearing behavior. In the presence of Ro4-1284, MDMA-mediated horizontal hyperlocomotion was delayed and attenuated, whereas rearing activity was abolished. Finally, Ro4-1284 prevented deficits in 5-HT content in rat cortex and striatum, and reduced depletions in striatal SERT staining, 7 days after MDMA administration. In summary, acute inhibition of VMAT2 by Ro4-1284 protected against MDMA-mediated hyperthermia, hyperactivity, and serotonergic neurotoxicity. The data suggest the involvement of VMAT2 in the thermoregulatory, behavioral, and neurotoxic effects of MDMA., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia/reperfusion injury, and many other pathological conditions. DNA strand breaks caused by ROS lead to the activation of poly(ADP-ribose)polymerase-1 (PARP-1), the excessive activation of which can result in cell death. We have utilized a model in which 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes ROS-dependent cell death in human renal proximal tubule epithelial cells (HK-2), to further elucidate the role of PARP-1 in ROS-dependent cell death. TGHQ-induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca(2+) concentrations, and subsequent nonapoptotic cell death in both a PARP- and Ca(2+)-dependent manner. Thus, inhibition of PARP-1 with PJ34 completely blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers and NAD consumption, and delayed HK-2 cell death. In contrast, chelation of intracellular Ca(2+) with BAPTA completely abrogated TGHQ-induced cell death. Ca(2+) chelation also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca(2+) homeostasis. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Thus, pathways coupling PARP-1 hyperactivation to cell death are likely to be context-dependent, and therapeutic strategies designed to target PARP-1 need to recognize such variability. Our studies provide new insights into PARP-1-mediated nonapoptotic cell death, during which PARP-1 hyperactivation and elevations in intracellular Ca(2+) are reciprocally coupled to amplify ROS-induced nonapoptotic cell death., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

3,4-(±)-Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT), inducing thermogenesis and hyperactivity (5-HT syndrome). The long-term effects of MDMA manifest as a prolonged depletion in 5-HT, and structural damage to 5-HT nerve terminals. MDMA toxicity is in part mediated by an ability to inhibit the presynaptic 5-HT reuptake transporter (SERT). Using a SERT-knockout (SERT-KO) rat model, we determined the impact of SERT deficiency on thermoregulation, locomotor activity, and neurotoxicity in SERT-KO or Wistar-based wild-type (WT) rats exposed to MDMA. WT and SERT-KO animals exhibited the highest thermogenic responses to MDMA (four times 10 mg/kg, sc at 12 h intervals) during the diurnal (first and third) doses according to peak body temperature and area under the curve (∑°C × h) analysis. Although no differences in peak body temperature were observed between MDMA-treated WT and SERT-KO animals, ∑°C × h following the first MDMA dose was reduced in SERT-KO rats. Exposure to a single dose of MDMA stimulated horizontal velocity in both WT and SERT-KO rats, however, this effect was delayed and attenuated in the KO animals. Finally, SERT-KO rats were insensitive to MDMA-induced long-term (7 days) depletions in 5-HT and its metabolite, 5-hydroxyindole acetic acid, in both cortex and striatum. In conclusion, SERT deficiency modulated MDMA-mediated thermogenesis, hyperactivity and neurotoxicity in KO rats. The data confirm that the SERT is essential for the manifestation of the acute and long-term toxicities of MDMA.

Metabolism of 3,4-(±)-methylenedioxymethamphetamine (MDMA) is necessary to elicit its neurotoxic effects. Perturbations in phase I and phase II hepatic enzymes can alter the neurotoxic profile of systemically administered MDMA. In particular, catechol-O-methyltransferase (COMT) plays a critical role in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites. Thus, cytochrome P450 mediated demethylenation of MDMA, or its N-demethylated metabolite, 3,4-(±)-methylenedioxyamphetamine, give rise to the catechols, N-methyl-α-methyldopamine and α-methyldopamine, respectively. Methylation of these catechols by COMT limits their oxidation and conjugation to glutathione, a process that ultimately gives rise to neurotoxic metabolites. We therefore determined the effects of modulating COMT, a critical enzyme involved in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites, on MDMA-induced toxicity. Pharmacological inhibition of COMT in the rat potentiated MDMA-induced serotonin deficits and exacerbated the acute MDMA-induced hyperthermic response. Using a genetic mouse model of COMT deficiency, in which mice lack a functional COMT gene, such mice displayed greater reductions in dopamine concentrations relative to their wild-type (WT) counterparts. Neither WT nor COMT deficient mice were susceptible to MDMA-induced decreases in serotonin concentrations. Interestingly, mice devoid of COMT were far more susceptible to the acute hyperthermic effects of MDMA, exhibiting greater increases in body temperature that ultimately resulted in death. Our findings support the view that COMT plays a pivotal role in determining the toxic response to MDMA.

3,4-(±)-Methylenedioxymethamphetamine (MDMA) and 3,4-(±)-methylenedioxyamphetamine (MDA), a primary metabolite of MDMA, are phenylethylamine derivatives that cause serotonergic neurotoxicity. Although several phenylethylamine derivatives activate microglia, little is known about the effects of MDMA on glial cells, and evidence of MDMA-induced microglial activation remains ambiguous. We initially determined microglial occupancy status of the parietal cortex in rats at various time points following a single neurotoxic dose of MDMA (20mg/kg, SC). A biphasic microglial response to MDMA was observed, with peak microglial occupancy occurring 12- and 72-h post-MDMA administration. Because direct injection of MDMA into the brain does not produce neurotoxicity, the glial response to MDMA metabolites was subsequently examined in vivo and in vitro. Rats were treated with MDA (20mg/kg, SC) followed by ex vivo biopsy culture to determine the activation of quiescent microglia. A reactive microglial response was observed 72 h after MDA administration that subsided by 7 days. In contrast, intracerebroventricular (ICV) administration of MDA failed to produce a microglial response. However, thioether metabolites of MDA derived from α-methyldopamine (α-MeDA) elicited a robust microglial response following icv injection. We subsequently determined the direct effects of various MDMA metabolites on primary cultures of E18 hippocampal mixed glial and neuronal cells. 5-(Glutathion-S-yl)-α-MeDA, 2,5-bis-(glutathion-S-yl)-α-MeDA, and 5-(N-acetylcystein-S-yl)-α-MeDA all stimulated the proliferation of glial fibrillary acidic protein-positive astrocytes at a dose of 10 µM. The findings indicate that glial cells are activated in response to MDMA/MDA and support a role for thioether metabolites of α-MeDA in the neurotoxicity.

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.

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway.

The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase signaling cascades have been implicated in a number of human cancers. The tumor suppressor gene tuberous sclerosis-2 (Tsc-2) functions as a negative regulator of mTOR. Critical proteins in both pathways are activated following treatment of Eker rats (Tsc-2(EK/+)) with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ), which also results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. Western blot analysis of kidney tumors formed following treatment of Tsc-2(EK/+) rats with TGHQ for 8 months revealed increases in B-Raf, Raf-1, pERK, cyclin D1, 4EBP1, and p-4EBP1-Ser65, -Thr70, and -Thr37/46 expression. Similar changes are observed following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (quinol-thioether rat renal epithelial [QTRRE] cells) that are also null for tuberin. These cells exhibit high ERK, B-Raf, and Raf-1 kinase activity and increased expression of all p-4EBP1s and cyclin D1. Treatment of the QTRRE cells with the Raf kinase inhibitor, sorafenib, or the MEK1/2 kinase inhibitor, PD 98059, produced a significant decrease in the protein expression of all p-4EBP1s and cyclin D1. Following siRNA knockdown of Raf-1, Western blot analysis revealed a significant decrease in Raf-1, cyclin D1, and all p-4EBP1 forms noted above. In contrast, siRNA knockdown of B-Raf resulted in a nominal change in these proteins. The data indicate that Raf-1/MEK/ERK participates in crosstalk with 4EBP1, which represents a novel pathway interaction leading to increased protein synthesis, cell growth, and kidney tumor formation.

The loss of tuberin, the tuberous sclerosis-2 (Tsc-2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/B-Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27-cyclin D1 in tuberous sclerosis gene-regulated-renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27-cyclin D1 cytosolic stabilization during renal tumor formation.

Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c.

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.)

2,3,5-Tris(glutathion-S-yl)-hydroquinone (TGHQ), a metabolite of hydroquinone, is toxic to renal proximal tubule epithelial cells. TGHQ retains the ability to redox cycle and create an oxidative stress. To assist in elucidating the contribution of reactive oxygen species (ROS) to TGHQ-induced toxicity, we determined whether the antioxidant, N-acetyl-L-cysteine (NAC), could protect human kidney proximal tubule epithelial cells (HK-2 cell line) against TGHQ-induced toxicity. NAC provided remarkable protection against TGHQ-induced toxicity to HK-2 cells. NAC almost completely inhibited TGHQ-induced cell death, mitochondrial membrane potential collapse, as well as ROS production. NAC also attenuated TGHQ-induced DNA damage and the subsequent activation of poly (ADP-ribose) polymerase and ATP depletion. Moreover, NAC significantly attenuated c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase phosphorylation induced by TGHQ. In contrast, NAC itself markedly increased extracellular regulated kinase1/2 (ERK1/2) activation, and the upstream mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor, PD-98059, only partially inhibited this activation, suggesting that NAC can directly activate ERK1/2 activity. However, although NAC is frequently utilized as a glutathione (GSH) precursor, the cytoprotection afforded by NAC in HK-2 cells was not a consequence of increased GSH levels. We speculate that NAC exerts its protective effect in part by directly scavenging ROS and in part via ERK1/2 activation.

The environmental toxicant hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis via a combination of redox cycling and the covalent adduction of proteins within the S₃ segment of the renal proximal tubules in the outer stripe of the outer medulla (OSOM). Following administration of 2-(glutathion-S-yl)HQ (MGHQ) (400 μmol/kg, i.v., 2 h) to Long Evans (wild-type Eker) rats, Western analysis utilizing an antibody specific for quinol-thioether metabolites of HQ revealed the presence of large amounts of chemical-protein adducts in both the OSOM and urine. By aligning the Western blot film with a parallel gel stained for protein, we can isolate the adducted proteins for LC-MS/MS analysis. Subsequent database searching can identify the specific site(s) of chemical adduction within these proteins. Finally, a combination of software programs can validate the identity of the adducted peptides. The site-specific identification of covalently adducted and oxidized proteins is a prerequisite for understanding the biological significance of chemical-induced posttranslational modifications (PTMs) and their toxicological significance.

Biological reactive intermediates can be created via metabolism of xenobiotics during the process of chemical elimination. They can also be formed as by-products of cellular metabolism, which produces reactive oxygen and nitrogen species. These reactive intermediates tend to be electrophilic in nature, which enables them to interact with tissue macromolecules, disrupting cellular signaling processes and often producing acute and chronic toxicities. Quinones are a well-known class of electrophilic species. Many natural products contain quinones as active constituents, and the quinone moiety exists in a number of chemotherapeutic agents. Quinones are also frequently formed as electrophilic metabolites from a variety of xeno- and endobiotics. Hydroquinone (HQ) is present in the environment from various sources, and it is also a known metabolite of benzene. HQ is converted in the body to 1,4-benzoquinone, which subsequently gives rise to hematotoxic and nephrotoxic quinone-thioether metabolites. The toxicity of these metabolites is dependent upon their ability to arylate proteins and to produce oxidative stress. Protein tertiary structure and protein amino acid sequence combine to determine which proteins are targets of these electrophilic quinone-thioether metabolites. We have used cytochrome c and model peptides to view adduction profiles of quinone-thioether metabolites, and have determined by MALDI-TOF analysis that these electrophiles target specific residues within these model systems.

Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283-1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized. MuDPIT was first described by Link et al. as a new technique useful for protein identification from a complex mixture of proteins (Link et al., Nat Biotechnol 17:676-682, 1999). MuDPIT utilizes two different HPLC columns to further enhance peptide separation, increasing the number of peptide hits and protein coverage. The technology is extremely useful for proteomes, such as the urine proteome, samples from immunoprecipitations, and 1D gel bands resolved from a tissue homogenate or lysate. In particular, MuDPIT has enhanced the field of adduct hunting for adducted peptides, since it is more capable of identifying lesser abundant peptides, such as those that are adducted, than the more standard LC-MS/MS. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response they elicit.

Biologically reactive intermediates are formed following metabolism of xenobiotics, and during normal oxidative metabolism. These reactive species are electrophilic in nature and are capable of forming stable adducts with target proteins. These covalent protein modifications can initiate processes that lead to acute tissue injury or chronic disease. Recent advancements in mass spectrometry techniques and data analysis has permitted a more detailed investigation of site-specific protein modifications by reactive electrophiles. Knowledge from such analyses will assist in providing a better understanding of how specific classes of electrophiles produce toxicity and disease progression via site-selective protein-specific covalent modification. Hydroquinone (HQ) is a known environmental toxicant, and its quinone-thioether metabolites, formed via the intermediate generation of 1,4-benzoquinone (1,4-BQ), elicit their toxic response via the covalent modification of target proteins and the generation of reactive oxygen species. We have utilized a model protein, cytochrome c, to guide us in identifying 1,4-BQ- and 1,4-BQ-thioether derived site-specific protein modifications. LC-MS/MS analyses reveals that these modifications occur selectively on lysine and glutamic acid residues of the target protein, and that these modifications occur within identifiable "electrophile binding motifs" within the protein. These motifs are found within lysine-rich regions of the protein and appear to be target sites of 1,4-BQ-thioether adduction. These residues also appear to dictate the nature of post-adduction chemistry and the final structure of the adduct. This model system will provide critical insight for in vivo adduct hunting following exposure to 1,4-BQ-thioethers, but the general approaches can also be extended to the identification of protein adducts derived from other classes of reactive electrophiles.

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.)

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a widely misused synthetic amphetamine derivative and a serotonergic neurotoxicant in animal models and possibly humans. The underlying mechanism of neurotoxicity involves the formation of reactive oxygen species although their source remains unclear. It has been postulated that MDMA-induced neurotoxicity is mediated via the formation of bioreactive metabolites. In particular, the primary catechol metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 3,4-dihydroxyamphetamine (HHA), subsequently cause the formation of glutathione and N-acetylcysteine conjugates, which retain the ability to redox cycle and are serotonergic neurotoxicants in rats. Although the presence of such metabolites has been recently demonstrated in rat brain microdialysate, their formation in humans has not been reported. The present study describes the detection of 5-(N-acetylcystein-S-yl)-3,4-dihydroxymethamphetamine (N-Ac-5-Cys-HHMA) and 5-(N-acetylcystein-S-yl)-3,4-dihydroxyamphetamine (N-Ac-5-Cys-HHA) in human urine of 15 recreational users of MDMA (1.5 mg/kg) in a controlled setting. The results reveal that in the first 4 h after MDMA ingestion approximately 0.002% of the administered dose was recovered as thioether adducts. Genetic polymorphisms in CYP2D6 and catechol-O-methyltransferase expression, the combination of which are major determinants of steady-state levels of HHMA and 4-hydroxy-3-methoxyamphetamine, probably explain the interindividual variability seen in the recovery of N-Ac-5-Cys-HHMA and N-Ac-5-Cys-HHA. In summary, the formation of neurotoxic thioether adducts of MDMA has been demonstrated for the first time in humans. The findings lend weight to the hypothesis that the bioactivation of MDMA to neurotoxic metabolites is a relevant pathway to neurotoxicity in humans.

Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics, and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. It is likely that topological, chemical, and physical features combine to determine which proteins become targets for chemical adduction. Chemical-induced post-translational modification of certain critical proteins causes a change in structure/function that contributes to the toxicological response to chemical exposure. In this study, we have identified a number of proteins that are modified by quinone-thioethers after administration of 2-(glutathion-S-yl)HQ. Parallel one-dimensional gel electrophoresis was performed, and the Coomassie-stained gel was aligned with the corresponding Western blot, which was probed for adductions. Immunopositive bands were then subjected to trypsin digestion and analyzed via liquid chromatography/tandem mass spectrometry. The proteins that were subsequently identified contained a higher than average (9.7 versus 5.5%) lysine content and numerous stretches of lysine run-ons, which is a presumed electrophile binding motif. Approximately 50% of these proteins have also been identified as targets for electrophilic adduction by a diverse group of chemicals by other investigators, implying overlapping electrophile adductomes. By identifying a motif targeted by electrophiles it becomes possible to make predictions of proteins that may be targeted for adduction and possible sites on these proteins that are adducted. An understanding of proteins targeted for adduction is essential to unraveling the toxicity produced by these electrophiles.

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.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a synthetic recreational drug of abuse that produces long-term toxicity associated with the degeneration of serotonergic nerve terminals. In various animal models, direct administration of MDMA into the brain fails to reproduce the serotonergic neurotoxicity, implying a requirement for the systemic metabolism and bioactivation of MDMA. Catechol-thioether metabolites of MDMA, formed via oxidation of 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine (HHMA and HHA) and subsequent conjugation with glutathione (GSH), are selective serotonergic neurotoxicants when administered directly into brain. Moreover, following systemic administration of MDMA, the thioether adducts are present in rat brain dialysate. MDMA contains a stereogenic center and is consumed as a racemate. Interestingly, different pharmacological properties have been attributed to the two enantiomers, (S)-MDMA being the most active in the central nervous system and responsible for the entactogenic effects, and most likely also for the neurodegeneration. The present study focused on the synthesis and stereochemical analysis of the neurotoxic MDMA thioether metabolites, 5-(glutathion-S-yl)-HHMA, 5-(N-acetylcystein-S-yl)-HHMA, 2,5-bis-(glutathion-S-yl)-HHMA, and 2,5-bis-(N-acetylcystein-S-yl)-HHMA. Both enzymatic and electrochemical syntheses were explored, and methodologies for analytical and semipreparative diastereoisomeric separation of MDMA thioether conjugates by HPLC-CEAS and HPLC-UV, respectively, were developed. Synthesis, diastereoisomeric separation, and unequivocal identification of the thioether conjugates of MDMA provide the chemical tools necessary for appropriate toxicological and metabolic studies on MDMA metabolites contributing to its neurotoxicity.

Members of the multidrug resistance protein (MRP/ABCC) subfamily of ATP-binding cassette proteins transport a wide array of anionic compounds, including sulfate, glucuronide, and glutathione (GSH) conjugates. The present study tested the ATP-dependent vesicular transport of leukotriene C(4) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) mediated by the MRP1 and MRP2 transporters in the presence of six potential modulators from three different classes of GSH-conjugated catechol metabolites: the ecstasy metabolite 5-(glutathion-S-yl)-N-methyl-alpha-methyldopamine (5-GS-N-Me-alpha-MeDA), the caffeic acid metabolite 2-(glutathion-S-yl)-caffeic acid (2-GS-CA), and four GSH conjugates of 2-hydroxy (OH) and 4-OH estrogens (GS estrogens). MRP1-mediated E(2)17betaG transport was inhibited in a competitive manner with a relative order of potency of GS estrogens (IC(50) <1 microM) > 2-GS-CA (IC(50) 3 microM) > 5-GS-N-Me-alpha-MeDA (IC(50) 31 microM). MRP2-mediated transport was inhibited with a similar order of potency, except the 2-hydroxy-4-(glutathion-S-yl)-estradiol and 4-hydroxy-2-(glutathion-S-yl)-estradiol conjugates were approximately 50- and 300-fold less potent, respectively. Transport activity was unaffected by N-acetylcysteine conjugates of N-Me-alpha-MeDA and CA. The position of GSH conjugation appears important as all four GS estrogen conjugates tested were potent inhibitors of MRP1 transport, but only the 2-hydroxy-1-(glutathion-S-yl)-estradiol and 2-hydroxy-1-(glutathion-S-yl)-estrone conjugates were potent inhibitors of MRP2-mediated transport. In conclusion, we have identified three new classes of MRP1 and MRP2 modulators and demonstrated that one of these, the estrogen conjugates, shows unanticipated differences in their interactions with the two transporters.

The serotonergic neurotoxicity of 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) appears dependent upon systemic metabolism because direct injection of MDMA into the brain fails to reproduce the neurotoxicity. MDMA is demethylenated to the catechol metabolite N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA). Thioether (glutathione and N-acetylcysteine) metabolites of N-Me-alpha-MeDA are neurotoxic and are present in rat brain following s.c. injection of MDMA. Because multidose administration of MDMA is typical of drug intake during rave parties, the present study was designed to determine the effects of multiple doses of MDMA on the concentration of neurotoxic thioether metabolites in rat brain. Administration of MDMA (20 mg/kg s.c.) at 12-h intervals for a total of four injections led to a significant accumulation of the N-Me-alpha-MeDA thioether metabolites in striatal dialysate. The area under the curve (AUC)(0-300 min) for 5-(glutathion-S-yl)-N-Me-alpha-MeDA increased 33% between the first and fourth injections and essentially doubled for 2,5-bis-(glutathion-S-yl)-N-Me-alpha-MeDA. Likewise, accumulation of the mercapturic acid metabolites was reflected by increases in the AUC(0-300 min) for both 5-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA (35%) and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA (85%), probably because processes for their elimination become saturated. Indeed, the elimination half-life of 5-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA increased by 53 and 28%, respectively, between the first and third doses. Finally, although the C(max) values for the monothioether conjugates were essentially unchanged after each injection, the values increased by 38 and approximately 50% for 2,5-bis-(glutathion-S-yl)-N-Me-alpha-MeDA and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA, respectively, between the first and fourth injections. The data indicate that neurotoxic metabolites of MDMA may accumulate in brain after multiple dosing.

Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein-protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile-protein adducts produce structural alterations that influence biological function.

Much is known about the distal DNA damage repair response. In particular, many of the enzymes and auxiliary proteins that participate in DNA repair have been characterized. In addition, knowledge of signaling pathways activated in response to DNA damage is increasing. In contrast, comparatively less is known of DNA damage-sensing molecules or of the specific alterations to chromatin structure recognized by such DNA damage sensors. Thus, precisely how chromatin structure is altered in response to DNA damage and how such alterations regulate DNA repair processes remain important unanswered questions. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after double-strand break formation, extends over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. We have shown that reactive oxygen species (ROS)-induced DNA single-strand breaks induce the incorporation of 32P specifically into histone H3. ADP-Ribosylation of histones may stimulate local chromatin relaxation to facilitate the repair process, and, indeed, histone ribosylation preceded DNA damage-induced histone H3 phosphorylation. However, H3 phosphorylation occurred concomitant with overall chromatin condensation, as revealed by decreased sensitivity of chromatin to digestion by micrococcal nuclease and by DAPI staining of nuclei. Inhibitors of the ERK and p38MAPK pathways and inhibition of poly(ADP-ribose) polymerase all reduced ROS-induced H3 phosphorylation, chromatin condensation, and cell death. Precisely how changes in the post-translational modification of histone H3 regulate the survival response remains unclear. Attempts to determine the precise site of histone H3 phosphorylation, putative histone H3 kinases, and histone H3 interacting proteins are underway.

In the present study the effects of a neurotoxic regimen of (+)-MDMA (20 mg/kgx4, s.c.) in 4- and 10-week-old C57Bl/6J mice during treatment and 7 days post-treatment were examined. Rectal temperatures monitored between (+)-MDMA injections (30 min post-injection/2 h intervals) revealed hyperthermic responses in both age groups, with the magnitude of the response significantly greater in older mice. Seven days post-treatment, immunoblot analyses of the vesicular monoamine transporter 2 (VMAT2), and tyrosine hydroxylase (TH) revealed significant reductions (-37 and -58%, respectively) in the older animals, but not in the younger group, compared to age-matched controls. Dopamine transporter (DAT) expression was significantly reduced in both 4- and 10-week-old animals (26 and 69.7%, respectively). (+)-MDMA-treated animals also exhibited significantly lower levels of striatal dopamine, and 3,4-dihydroxyphenylacetic acid than controls, again the effect being more pronounced in the older animals. Although both age groups showed evidence of (+)-MDMA-induced toxicity, our data revealed that older animals exhibited a greater hyperthermic response to (+)-MDMA and were also are more susceptible to subsequent dopaminergic damage than the younger animals.

2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. However, the mechanisms by which TGHQ induces apoptosis are unclear, and they were the focus of the present investigation. TGHQ stimulated the rapid formation (30 min) of reactive oxygen species (ROS) in HL-60 cells, and co-treatment with catalase or the antioxidant N-acetylcysteine (NAC) completely blocked TGHQ-induced apoptosis, implicating a causative role for ROS in HL-60 cell death. Western blot analysis revealed the complete disappearance of pro-caspase 9 between 1 and 2 hours after exposure of HL-60 cells to TGHQ, concomitant with the appearance of cleaved caspase 9 and increases in caspase 9 activity. The appearance of two cleaved forms of caspase 3 occurred subsequent to increases in caspase 9 activity. Levels of the anti-apoptotic Bcl-2 protein remained constant during TGHQ-induced apoptosis of HL-60 cells, but Bcl-2 S70 phosphorylation decreased. In contrast, changes in the subcellular localization of the pro-apoptotic molecule Bax were observed, with a rapid (15-60 min) increase in the ratio of cytosolic to mitochondrial Bax. Cytochrome c release from mitochondria to the cytosol occurred after Bax translocation and the dephosphorylation of pS70 Bcl-2. However the mitochondrial inner transmembrane potential (deltapsi(m)) was maintained, even after cytochrome c was released from the mitochondria. Cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition pore (PTP), did not completely rescue HL-60 cells from apoptosis. Taken together, we conclude that TGHQ facilitates ROS production, alters the post-translational modification of Bcl-2 and subcellular localization of Bax, culminating in the release of cytochrome c and caspase activation.

The selective serotonergic neurotoxicity of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) depends on their systemic metabolism. We have recently shown that inhibition of brain endothelial cell gamma-glutamyl transpeptidase (gamma-GT) potentiates the neurotoxicity of both MDMA and MDA, indicating that metabolites that are substrates for this enzyme contribute to the neurotoxicity. Consistent with this view, glutathione (GSH) and N-acetylcysteine conjugates of alpha-methyl dopamine (alpha-MeDA) are selective neurotoxicants. However, neurotoxic metabolites of MDMA or MDA have yet to be identified in brain. Using in vivo microdialysis coupled to liquid chromatography-tandem mass spectroscopy and a high-performance liquid chromatography-coulometric electrode array system, we now show that GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA are present in the striatum of rats administered MDMA by subcutaneous injection. Moreover, inhibition of gamma-GT with acivicin increases the concentration of GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA in brain dialysate, and there is a direct correlation between the concentrations of metabolites in dialysate and the extent of neurotoxicity, measured by decreases in serotonin (5-HT) and 5-hydroxyindole acetic (5-HIAA) levels. Importantly, the effects of acivicin are independent of MDMA-induced hyperthermia, since acivicin-mediated potentiation of MDMA neurotoxicity occurs in the context of acivicin-mediated decreases in body temperature. Finally, we have synthesized 5-(N-acetylcystein-S-yl)-N-methyl-alpha-MeDA and established that it is a relatively potent serotonergic neurotoxicant. Together, the data support the contention that MDMA-mediated serotonergic neurotoxicity is mediated by the systemic formation of GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA (and alpha-MeDA). The mechanisms by which such metabolites access the brain and produce selective serotonergic neurotoxicity remain to be determined.