GSH-depleting Agent Research Articles

Facile synthesis of an acid-responsive cinnamaldehyde-pendant polycarbonate for enhancing the anticancer efficacy of etoposide via glutathione depletion.

Glutathione (GSH) is an important antioxidant that maintains cellular redox homeostasis and significantly contributes to resistance against various chemotherapeutic agents. To address the challenge of GSH-mediated drug resistance in etoposide (ETS), we developed a facile synthetic method to prepare a biocompatible acid-responsive polycarbonate (PEG-PCA) containing cinnamaldehyde (CA), a potent GSH-depleting agent, as a side chain using nontoxic raw materials. This polymer self-assembled in aqueous solutions to form nanoparticles (ETS@PCA) that encapsulated ETS, enhancing its water solubility and enabling tumor-targeted delivery. In vitro studies demonstrated that ETS@PCA could respond to the acidic tumor microenvironment, releasing CA to rapidly deplete GSH levels. Consequently, ETS@PCA exhibited superior cytotoxicity compared to free ETS. Furthermore, in vivo experiments corroborated the enhanced tumor inhibitory effects of ETS@PCA.

STEM-09. EXPLORING THE ROLE OF TP53 IN REDOX REGULATION AND RESPONSE OF GLIOBLASTOMA STEM CELLS TO ROS-INDUCING AGENTS

Abstract Glioblastoma, the most malignant primary tumor of the central nervous system in adults presents a major therapeutic challenge due to the presence of chemo-radioresistant glioblastoma stem cells (GSCs). These GSCs adapt the thioredoxin (Trx) and glutathione (GSH) antioxidant systems to counteract treatment-induced reactive oxygen species (ROS) and promote their survival. The tumor suppressor gene wild-type (wt)TP53 regulates the expression of pro-oxidant or antioxidant genes in response to chemoradiotherapy based on p53 DNA-binding activity in a reducing microenvironment. Given the importance of p53 as a redox-sensitive protein, we hypothesized that p53 might protect GSCs against ROS-inducing agents. Auranofin (Au), a FDA-approved gold(I)-containing compound inhibits thioredoxin reductase 1 (TrxR1), a key antioxidant protein. We investigated the effects of Au on different GSCs including wtp53 GSC line and its counterpart p53-knockdown. We assessed viability, neurosphere formation, ROS levels and expression of p53 and TrXR1 to evaluate the cytotoxicity of Au, alone and in combination with L-buthionine sulfoximine (L-BSO), a GSH-depleting agent. We conducted TCGA analysis to explore the correlation between TrxR1 and p53. Our findings revealed a positive significant correlation between p53 and TrxR1 expression, consistent with lower levels of TrxR1 in p53-knockdown GSCs compared to their wt-p53 counterparts. P53-knockdown GSCs displayed a higher basal level of ROS, which further increased upon treatment with Au. P53-knockdown significantly increased the sensitivity of cells to Au, with pronounced effects for Au/L-BSO combination. Our findings reveal that expression levels of wtp53 affect the role of p53 in TrxR1-redox regulation and sensitivity to ROS-inducing agents in GSCs.

Glutathione/Glucose-Depleting Nanoparticles with NO Generation for Ferroptosis/Starvation/NO-Induced Cancer Therapy

Depleting intracellular glutathione (GSH) has emerged as a potent strategy to combat cancer. However, the existing GSH-depleting agents are too toxic or ineffective and standalone GSH depletion fails to yield a satisfactory curative effect. Herein, we present an intelligent nanoparticle that possesses GSH depletion, glucose consumption accompanied with H2O2 production, and NO generation properties for multimodal cancer therapy. The nanoparticle is constructed by synthesis of tetrasulfide bond-doped mesoporous silica nanoparticles followed by conjugating glucose oxidase (GOx) on the surface and loading l-arginine (l-Arg) into the mesopores. In this nanoparticle, the doped tetrasulfide bonds can quickly deplete GSH, which increases the cellular reactive oxygen species concentration to induce ferroptosis and meanwhile triggers particle biodegradation to expose the loaded l-Arg. Moreover, the elevated H2O2 level activates l-Arg to release NO for NO therapy. GOx consumes glucose to initiate starvation therapy and simultaneously produces a large amount of H2O2. Importantly, the produced H2O2 can not only potentiate ferroptosis but also promote NO release to enhance NO therapy. Besides, NO could in turn improve the efficacy of starvation therapy by damaging the mitochondria to block energy supply. In vitro and in vivo studies demonstrate that the nanoparticles show a great synergistic effect of ferroptosis/starvation/NO therapy, which can significantly kill cancer cells and remarkably inhibit tumor growth without obvious side effects. Therefore, we think that the designed nanoparticles may provide a promising paradigm for synergistic cancer therapy and hold a prospect in clinical trials.

Mitochondria-targeted photosensitizer based nanoplatform loading glutathione inhibitor for enhanced breast cancer photodynamic therapy

Photodynamic therapy (PDT) is an approved photoactivated technique that has been widely used for anti-tumor therapy through the generation of excessive ROS. However, the poor water solubility and targeting ability of photosensitizer and ROS depleting by high concentrations of glutathione (GSH) in the tumor microenvironment will limit the efficiency of PDT and synergistic therapy. Herein, we report a mitochondria targeted heterodimeric photosensitizer (TPP-TK-PPa) that can form co-delivery nanoplatform together with DSPE-PEG2000 and GSH-depleting agent DEM through the self-assembly. The TPP-TK-PPa/DEM NPs reveals excellent mitochondrial targeting ability and can efficiently deliver the therapeutic agents to tumor cells and release them into the mitochondria in an ROS-responsive manner. Additionally, The TPP-TK-PPa/DEM NPs can induce in situ production of ROS during PDT and reduce ROS consumption by decreasing intracellular GSH levels, which is attributed to the progressive release of DEM after cellular internalization. In addition, generation of ROS during PDT leads to distinct changes in mitochondrial membrane potential and morphology, which ultimately leads to apoptosis. More importantly, the in vitro antitumor activity results demonstrate that TPP-TK-PPa/DEM NPs showed remarkable phototoxicity and the IC50 values of TPP-TK-PPa/DEM NPs in MDA-MB-231 breast cancer cells was as low as 250 nM after PDT. This work provides a strategy for the development of photosensitizers with specific organelle targeting ability, which is of great importance for precise and effective PDT.

Electrochemical Characterization of Auranofin in Aqueous Media

Detection of antibiotic compounds in various media is necessary in solving the public health crisis of antibiotic resistance[1]. Auranofin (AF) is a repurposed antibiotic first used as an antirheumatic in the 1950s. It is useful against various bacteria, fungi, virus infections and cancers either by itself or synergistically with other antibiotics[2–5]. AF is composed of aurothioglucose and phosphine moieties, and therefore, has lipophilic and hydrophilic properties, making its characterization in aqueous media particularly difficult. In the presented study, electrochemistry was employed to characterize and detect AF in aqueous solutions using modified glassy carbon electrodes (GCEs), coated with Carbon Black (CB). CB coating was optimized, oxidation and reduction potentials were identified, and pH dependence, as well as Limits of Detection (LOD) and Quantification (LOQ) were determined. Interferences with ions and other antibiotics was studies and are important factors for the detection of AF in the environment. This study reports on the sensitivity, stability, and selectivity of the CB-GCE sensors, proving useful for future field detection and/or bioelectrochemical investigations.CITATIONS[1] M.R. Islam, S. Kuss, F. Schweizer, Characterization of Antibiotic Compounds By Electrochemistry, ECS Meeting s. MA2020-01 (2020). https://doi.org/10.1149/ma2020-01442531mtgabs.[2] R. Díez-Martínez, E. García-Fernández, M. Manzano, Á. Martínez, M. Domenech, M. Vallet-Regí, P. García, Auranofin-loaded nanoparticles as a new therapeutic tool to fight streptococcal infections, Scientific Reports. 6 (2016). https://doi.org/10.1038/srep19525.[3] K.J. Habermann, L. Grünewald, S. van Wijk, S. Fulda, Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death, Cell Death & Disease. 8 (2017). https://doi.org/10.1038/cddis.2017.412.[4] E. v. Capparelli, R. Bricker-Ford, M.J. Rogers, J.H. McKerrow, S.L. Reed, Phase I clinical trial results of auranofin, a novel antiparasitic agent, Antimicrobial Agents and Chemotherapy. 61 (2017). https://doi.org/10.1128/AAC.01947-16.[5] T. Onodera, I. Momose, M. Kawada, Potential anticancer activity of auranofin, Chemical and Pharmaceutical Bulletin. 67 (2019). https://doi.org/10.1248/cpb.c18-00767.

The Inhibitory Effect of Sulforaphane on Bladder Cancer Cell Depends on GSH Depletion-Induced by Nrf2 Translocation

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18–11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.

Glutathione-Depleting Nanomedicines for Synergistic Cancer Therapy.

Cancer cells frequently exhibit resistance to various molecular and nanoscale drugs, which inevitably affects the drugs' therapeutic outcomes. Overexpression of glutathione (GSH) has been observed in many cancer cells, and solid evidence has corroborated the resulting tumor resistance to a variety of anticancer therapies, suggesting that this biochemical characteristic of cancer cells can be developed as a potential target for cancer treatments. The single treatment of GSH-depleting agents can potentiate the responses of the cancer cells to different cell death stimuli; therefore, as an adjunctive strategy, GSH depletion is usually combined with mainstream cancer therapies for enhancing the therapeutic outcomes. Propelled by the rapid development of nanotechnology, GSH-depleting agents can be readily constructed into anticancer nanomedicines, which have shown a steep rise over the past decade. Here, we review the common GSH-depleting nanomedicines which have been widely applied in synergistic cancer treatments in recent years. Some current challenges and future perspectives for GSH depletion-based cancer therapies are also presented. With the understanding of the structure-property relationship and action mechanisms of these biomaterials, we hope that the GSH-depleting nanotechnology will be further developed to realize more effective disease treatments and even achieve successful clinical translations.

Engineering nanomedicine for glutathione depletion-augmented cancer therapy.

Glutathione (GSH), the main redox buffer, has long been recognized as a pivotal modulator of tumor initiation, progression and metastasis. It is also implicated in the resistance of platinum-based chemotherapy and radiation therapy. Therefore, depleting intracellular GSH was considered a potent solution to combating cancer. However, reducing GSH within cancer cells alone always failed to yield desirable therapeutic effects. In this regard, the convergence of GSH-scavenging agents with therapeutic drugs has thus been pursued in clinical practice. Unfortunately, the therapeutic outcomes are still unsatisfactory due to untargeted drug delivery. Advanced nanomedicine of synergistic GSH depletion and cancer treatment has attracted tremendous interest because they promise to deliver superior therapeutic benefits while alleviating life-threatening side effects. In the past five years, the authors and others have demonstrated that numerous nanomedicines, by simultaneously delivering GSH-depleting agents and therapeutic components, boost not only traditional chemotherapy and radiotherapy but also multifarious emerging treatment modalities, including photodynamic therapy, sonodynamic therapy, chemodynamic therapy, ferroptosis, and immunotherapy, to name a few, and achieved decent treatment outcomes in a large number of rodent tumor models. In this review, we summarize the most recent progress in engineering nanomedicine for GSH depletion-enhanced cancer therapies. Biosynthesis of GSH and various types of GSH-consuming strategies will be briefly introduced. The challenges and perspectives of leveraging nanomedicine for GSH consumption-augmented cancer therapies will be discussed at the end.

Abstract A110: Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells

Abstract Androgen receptor (AR) over-expression, mutation and the emergence of AR splice variants (AR-Vs) promote disease-driving AR signaling in castration-resistant prostate cancer (CRPC). In addition to the sustained AR activation, apoptosis evasion adds extra hurdle for treating hormone refractory prostate cancer (PCa). To overcome these challenges, we firstly designed isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and then rationally combined ITC-ARi with glutathione (GSH) biosynthesis inhibitor buthionine sulphoximine (BSO) to efficiently downregulate AR/AR-V and induce ferroptosis in enzalutamide (Enz)-resistant PCa cells. Naturally occurring ITCs (e.g., sulforaphane and phenylethyl isothiocyanate) display pleotropic anti-PCa activities. A representative ITC-ARi 2-63 is designed by incorporating ITC into an AR ligand scaffold. The sulfhydryl reactivity of ITC is transiently masked as N-acetyl cysteine (NAC) conjugate that gradually releases parental free ITC in aqueous solution. 2-63 antagonizes AR transactivation, downregulates AR/AR-V7, and upregulates cellular stress markers Hsp70 and heme oxygenase-1 (HO-1), indicating the disruption of heat shock proteins (Hsps) and the activation of Nrf2 pathway. Because the anticancer effects of ITC/ITC-NAC conjugate are attenuated by GSH-participated conjugation or thiol exchange, we rationally combined 2-63 with BSO for enhanced anti-PCa activities. 2-63 and BSO synergistically reduce viability of multiple PCa cell lines (VCaP, C4-2 and 22Rv-1), and the noncancerous prostatic RWPE-1 cell is much less affected. Because 2-63 plus BSO does not upregulate typical apoptosis markers, we investigated alternative cell death mechanism in combinatorial treatment. Drug combination-caused cell viability loss is effectively rescued by iron chelator deferoxamine (DFO), antioxidants α-tocopherol (α-Toc) and ferrostatin-1 (Fer-1), or the inhibitor of HO-1 Zinc (II) protoporphyrin IX (ZnPP). Both Fer-1 and α-Toc are potent radical scavengers blocking lipid hydroperoxide generation, and ZnPP suppresses HO-1-catalyzed upregulation of intracellular Fe2+. The reverse of growth suppression by DFO, antioxidants and ZnPP supports the induction of ferroptosis, a regulated non-apoptotic cell death caused by reactive oxygen species (ROS)- and iron-dependent lipid peroxidation. 2-63 and BSO also more efficiently downregulate AR/AR-V than 2-63 alone in Enz-resistant PCa cells. Interestingly, ferroptosis-preventing DFO and Fer-1 do not rescue AR/AR-V7 and are unable to suppress the upregulation of Hsp70 and HO-1. Our results suggest that the synergism of 2-63 and BSO occurs through increasing drug accessibility to cellular targets, expanding availability of iron and potentially affecting glutathione peroxidase 4 (GPX4) activity, the most important cellular defense to eliminate lipid hydroperoxide. Combining ITC-ARi and GSH-depleting agents could be a new concept leading to effective CRPC treatment. Citation Format: Zhihui Qin, Liping Xu, Siyu Ou. Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A110. doi:10.1158/1535-7163.TARG-19-A110

Vasodilator oxyfedrine inhibits aldehyde metabolism and thereby sensitizes cancer cells to xCT-targeted therapy.

The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis, an iron‐dependent form of cell death triggered by the aberrant accumulation of lipid peroxides. Inhibitors of the cystine‐glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, are thus potential anticancer agents. However, the efficacy of xCT‐targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. Identification of drugs for combination with xCT inhibitors that are able to overcome resistance to xCT‐targeted therapy might thus provide the basis for effective cancer treatment. We have now identified the vasodilator oxyfedrine (OXY) as a sensitizer of cancer cells to GSH‐depleting agents including the xCT inhibitor sulfasalazine (SSZ). Oxyfedrine contains a structural motif required for covalent inhibition of aldehyde dehydrogenase (ALDH) enzymes, and combined treatment with OXY and SSZ was found to induce accumulation of the cytotoxic aldehyde 4‐hydroxynonenal and cell death in SSZ‐resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase‐2 expression as a potential biomarker for the efficacy of such combination therapy. Furthermore, OXY‐mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation.

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.

Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death

Rhabdomyosarcoma (RMS) cells have recently been reported to be sensitive to oxidative stress. Therefore, we investigated whether concomitant inhibition of the two main antioxidant defense pathways, that is, the thioredoxin (TRX) and the glutathione (GSH) systems, presents a new strategy to trigger cell death in RMS. In this study, we discover that GSH-depleting agents, i.e. γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine (BSO) or the cystine/glutamate antiporter inhibitor erastin (ERA), synergize with thioredoxin reductase (TrxR) inhibitor auranofin (AUR) to induce cell death in RMS cells. Interestingly, AUR causes accumulation of ubiquitinated proteins when combined with BSO or ERA, in line with recent reports showing that AUR inhibits the proteasome besides TrxR. Consistently, AUR/BSO or AUR/ERA cotreatment increases ubiquitination and expression of the short-lived proteins NOXA and MCL-1, accompanied by increased binding of NOXA to MCL-1. Notably, NOXA knockdown significantly rescues RMS cells from AUR/BSO- or AUR/ERA-induced cell death. In addition, AUR acts together with BSO or ERA to stimulate BAX/BAK and caspase activation. Of note, BSO or ERA abolish the AUR-stimulated increase in GSH levels, leading to reduced GSH levels upon cotreatment. Although AUR/BSO or AUR/ERA cotreatment enhances reactive oxygen species (ROS) production, only thiol-containing antioxidants (i.e., N-acetylcysteine (NAC), GSH), but not the non-thiol-containing ROS scavenger α-Tocopherol consistently suppress AUR/BSO- and AUR/ERA-stimulated cell death in both cell lines. Importantly, re-supply of GSH or its precursor NAC completely prevents AUR/ERA- and AUR/BSO-induced accumulation of ubiquitinated proteins, NOXA upregulation and cell death, indicating that GSH depletion rather than ROS production is critical for AUR/BSO- or AUR/ERA-mediated cell death. Thus, by demonstrating that GSH-depleting agents enhance the antitumor activity of AUR, we highlight new treatment options for RMS by targeting the redox homeostasis.

The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells

Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.

Investigation of the active components in Tripterygium wilfordii leading to its acute hepatotoxicty and nephrotoxicity

Ethnopharmacological relevanceThe traditional herbal medicine Tripterygium wilfordii Hook. f. (TW) has been widely used for the treatment of rheumatoid arthritis and autoimmune disease in the clinic. However, adverse reactions of TW including hepatotoxicity and nephrotoxicity have been frequently reported. Terpenes and alkaloids are among the most important active components in TW. Triptolide (TP), a major terpene in TW, has been found to induce toxicity, and metabolic pathways could lead to detoxification of TP. In this study, whether other major terpenes or alkaloids in TW contribute to its toxicity was investigated. The role of metabolic eliminations in their potential detoxification process was also evaluated. Materials and methodsThe toxicity of TW and its five major active components (one terpene and four alkaloids) in mice was evaluated in terms of mortality and blood biochemical levels (ALT, AST, BUN and CREA). TP was used as a positive control. Metabolic pathways leading to potential detoxification of TW or its two representative components (triptonide and wilforgine) were evaluated in glutathione (GSH)-depleted (treated with L-buthionine-S,R-sulfoxinine, BSO) and aminobenzotriazole (ABT; a nonspecific inhibitor for P450s)-treated mice. ResultsIn normal mice, the major metabolic pathways for the terpene compounds TP and triptonide (TN) were hydroxylation and cysteine conjugation, and the alkaloid wilforgine (WG) mainly underwent oxidative metabolism and hydrolysis. In ABT/BSO-treated mice, the hydroxylated metabolites of TP, TN and WG were found at a lower level than normal mice, and the level of cysteine conjugates of TN increased probably due to the stress response. Compared with normal mice, mortality and levels of ALT (but not BUN) were significantly higher (P<0.01) in TW (or TP)-treated mice (1.2mgkg−1), indicating the acute toxicity (may not nephrotoxicity) of TW and its active component TP. Pretreatment with ABT and/or BSO increased the acute toxicity (including hepatotoxicity and nephrotoxicity) caused by TW or TP. No significant toxicity was found for TN or four alkaloids in normal mice or ABT/BSO-treated mice. ConclusionsTP was probably the main contributor to the toxicity of TW, and the terpene TN and alkaloids in TW may be of no toxicological concern at dosage levels up to 20-fold of the therapeutic dose. Metabolic eliminations to less reactive metabolites implied a high potential for detoxification of TW, and caution should be taken for TW clinical use during co-administration with other CYP inhibitors or GSH-depleting agents.

Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.

Effect of Glutathione Depletion on Nrf2/ARE Activation by Deltamethrin in PC12 Cells

Transcription factor NF-E2-related factor 2 (Nrf2) is important for cell protection against chemical-induced oxidative stress. Previously, we have reported that in PC12 cells, Nrf2 can be triggered by deltamethrin (DM), a commonly used pyrethroid insecticide. Molecular mechanisms behind Nrf2 activation by DM are still unclear. Here we studied the effects of cell glutathione (GSH) depletion on Nrf2 activation by DM. We found that DM enhanced Nrf2 expression at the mRNA and protein levels and increased nuclear Nrf2 levels. Activation of Nrf2 was associated with activation of its downstream targets, such as heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC). In contrast, DL-buthionine-[S,R]- sulfoximine (BSO), a known GSH-depleting agent, did not increase Nrf2 protein expression or cause its nuclear accumulation. However, pre-treatment with BSO triggered mRNA expression of HO-1 and GCLC. Furthermore, BSO pre-treatment suppressed DM-induced Nrf2 upregulation and activation and lowered mRNA expression of HO-1 and GCLC upon DM treatment. These data demonstrate that GSH depletion is not necessary for the activation of Nrf2/ARE by DM in PC12 cells, and that GCLC and HO-1 expression can increase through other signalling pathways.

Role of glutathione in lung retention of 99mTc-hexamethylpropyleneamine oxime in two unique rat models of hyperoxic lung injury

Rat exposure to 60% oxygen (O(2)) for 7 days (hyper-60) or to >95% O(2) for 2 days followed by 24 h in room air (hyper-95R) confers susceptibility or tolerance, respectively, of the otherwise lethal effects of subsequent exposure to 100% O(2). The objective of this study was to determine if lung retention of the radiopharmaceutical agent technetium-labeled-hexamethylpropyleneamine oxime (HMPAO) is differentially altered in hyper-60 and hyper-95R rats. Tissue retention of HMPAO is dependent on intracellular content of the antioxidant GSH and mitochondrial function. HMPAO was injected intravenously in anesthetized rats, and planar images were acquired. We investigated the role of GSH in the lung retention of HMPAO by pretreating rats with the GSH-depleting agent diethyl maleate (DEM) prior to imaging. We also measured GSH content and activities of mitochondrial complexes I and IV in lung homogenate. The lung retention of HMPAO increased by ≈ 50% and ≈ 250% in hyper-60 and hyper-95R rats, respectively, compared with retention in rats exposed to room air (normoxic). DEM decreased retention in normoxic (≈ 26%) and hyper-95R (≈ 56%) rats compared with retention in the absence of DEM. GSH content increased by 19% and 40% in hyper-60 and hyper-95R lung homogenate compared with normoxic lung homogenate. Complex I activity decreased by ≈ 50% in hyper-60 and hyper-95R lung homogenate compared with activity in normoxic lung homogenate. However, complex IV activity was increased by 32% in hyper-95R lung homogenate only. Furthermore, we identified correlations between the GSH content in lung homogenate and the DEM-sensitive fraction of HMPAO retention and between the complex IV/complex I activity ratio and the DEM-insensitive fraction of HMPAO retention. These results suggest that an increase in the GSH-dependent component of the lung retention of HMPAO may be a marker of tolerance to sustained exposure to hyperoxia.

Multidrug Resistance Associated Protein 1 together with Glutathione Plays a Protective Role against 4-Hydroxy-2-nonenal-Induced Oxidative Stress in Bovine Aortic Endothelial Cells

4-Hydroxy-2-nonenal (HNE), an aldehyde produced by lipid peroxidation, induces cytotoxicity and oxidative stress. Glutathione (GSH) protects against the cytotoxicity of HNE. However, the protective mechanism of GSH has not been fully examined. We examined the protective role played by the relationship between GSH and multidrug resistance associated protein 1 (MRP1) against the HNE-induced oxidative stress in bovine aortic endothelial cells (BAECs). HNE induced the loss of viability of BAECs. Exogenous GSH, which is membrane-impermeable, prevented the loss of viability induced by HNE by inhibiting HNE uptake in BAECs, probably due to the formation of the HNE-SG complex in the extracellular space. We demonstrated that HNE induced the expression of MRP1 protein, which can transport the HNE-SG complex. The induction of MRP1 protein expression by HNE disappeared in BAECs pretreated with L-buthionine sulfoximine, a GSH-depleting agent. This result suggests that HNE, together with intracellular GSH, contributes to the regulation of MRP1 protein expression. Moreover, we found that MK571, an MRP1 inhibitor, promoted the HNE-induced oxidative stress and cell death. Taken together, these findings suggest that MRP1, together with GSH, plays a protective role against the HNE-induced oxidative stress in BAECs.

Preclinical Genotoxicology of Nor-β-lapachone in Human Cultured Lymphocytes and Chinese Hamster Lung Fibroblasts

Nor-β-lapachone has shown several biological properties. Regarding cytotoxic activity against cancer cell lines, it has been recognized as an important prototype. However, quinonoid drugs present a major challenge because of their toxicity. In this study, we evaluated the cytotoxicity and genetic toxicity of nor-β-lapachone in human lymphocytes and HL-60 leukemia cells and murine V79 fibroblasts, to shed some light on its selectivity toward cancer cells. As measured by MTT test, exposure of V79 cells to nor-β-lapachone resulted in a weak cytotoxicity (IC(50) = 13.41 μM), and at a concentration up to 21.9 μM, no cytotoxic effect was observed in lymphocytes, while in HL-60 cells, nor-β-lapachone elicited significantly greater cytotoxicity (IC(50) = 1.89 μM). Cultures coexposed to GSH-OEt showed an increased viability, which may indicate a neutralization of ROS generated by quinonoid treatment. In fact, only the highest concentrations of nor-β-lapachone (10 or 20 μM) caused an increase in oxidative stress in nontumor levels cells as measured by TBARS and nitrite/nitrate detection. This was accompanied by an alteration in intracellular thiol content. However, NAC pre-exposure restored the redox equilibrium of the cells and the concentration of thiol levels to control values. Nor-β-lapachone at 2.5 and 5 μM failed to induce DNA damage in nontumor cells, but at the highest concentrations tested, it induced single and double DNA strand breaks and increased the frequency of chromosomal aberrations. Interestingly, these damages were prevented by NAC pretreatment or exacerbated by prior exposure to the GSH-depleting agent 1-bromoheptane. In electrochemical experiments, nor-β-lapachone at the same concentrations as those used in genotoxic tests did not damage DNA directly, but at the highest concentration tested (200 μM), it caused a very weak DNA interaction. Corroborating electrochemical data, oxidative modifications of DNA bases were observed, as checked by DNA repair enzymes EndoIII and FPG, which reinforced the indirect actions caused by nor-β-lapachone through ROS generation and not via DNA intercalation. The DNA repair capacities were higher for nontumor cells than for leukemia cells, which may be related to the selective cytoxicity of nor-β-lapachone toward cancer cells. Our data suggest that ROS play an important role in nor-β-lapachone toxicity and that its DNA-damaging effect occurs only at concentrations several times higher than that needed for its antiproliferative effect on cancer cells.

D-Amphetamine-induced cytotoxicity and oxidative stress in isolated rat hepatocytes

Amphetamines (AMP) are potent psychostimulants and commonly used drugs of abuse. Its chronic administration creates tolerance and addiction and also associated with neurotoxicity and hepatocellular damage through oxidative stress. The present study was designed to evaluate the cytotoxic effects as well as the oxidative stress induced by d-amphetamines in isolated rat hepatocytes. Hepatocytes were isolated by collagenase perfusion technique and were exposed to different concentrations of AMP (0.2, 0.4, 0.8 and 1.6 mM) in a time-course experiment for up to 2 h. AMP exposure induced a significant decrease in cell viability and a significant increase in the leakage of hepatic enzymes {lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and asparate aminotransferase (AST)} in a concentration and time-related manner. In the same experiment, GSH content and thiobarbituric acid reactive substances (TBARS) generation were determined as indices of oxidative stress and lipid peroxidation respectively. AMP exposure results in a significant decrease in cellular GSH content as well as a significant enhancement of TBARS accumulation in a concentration and time-related manners. The obtained results suggested that 2-h exposure of hepatocytes to AMP (0.8 mM) was accompanied by submaximal responses. Therefore, a subsequent dose–response experiment was designed to evaluate the role of GSH modulation and oxidative stress in AMP toxicity in hepatocytes at 2 h. LDH release and TBARS generation were used as indicators in this experiment. Pretreatment with the GSH-depleting agents, chlorodinitrobenzene (CDNB), buthionine sulfoximine (BSO), or bis(chloroethyl)-nitrosurea (BCNU) enhanced the cytotoxicity of AMP. Conversely, pretreatment with GSH or sulfhydryl compounds such as methionine (MT), cysteine (CYS) or dithiothreitol (DTT) attenuated AMP toxicity. Similarly, co-incubation with enzymatic antioxidants, superoxide dismutase (SOD) or catalase (CAT) or iron chelator, desferroxiamine (DFO) or the hydroxyl radical scavengers, dimethylsulfoxide (DMSO) exhibited significant protection against AMP cytotoxicity. The present results indicate that AMP has a potential cytotoxic effect in isolated rat hepatocytes. AMP cytotoxicity is concentration-dependent. GSH depletion and oxidative stress play an important role in enhancing hepatotoxic potential of AMP in isolated rat hepatocyte. Thiol group-donors, antioxidants, free radical scavengers and iron chelators can play a critical role against AMP-induced cellular damage.