Reciprocal regulation of oxidative stress and mitochondrial fission augments parvalbumin downregulation through CDK5-DRP1- and GPx1-NF-κB signaling pathways.

Loss of parvalbumin (PV) expressing neurons (PV neurons) is relevant to the underlying mechanisms of the pathogenesis of neurological and psychiatric diseases associated with the dysregulation of neuronal excitatory networks and brain metabolism. Although PV modulates mitochondrial morphology, volume and dynamics, it is largely unknown whether mitochondrial dynamics affect PV expression and what the molecular events are responsible for PV neuronal degeneration. In the present study, L-buthionine sulfoximine (BSO, an inhibitor of glutathione synthesis) did not degenerate PV neurons under physiological condition. However, BSO-induced oxidative stress decreased PV expression and facilitated cyclin-dependent kinase 5 (CDK5) tyrosine (Y) 15 phosphorylation, dynamin-related protein 1 (DRP1)-mediated mitochondrial fission and glutathione peroxidase-1 (GPx1) downregulation in PV neurons. Co-treatment of roscovitine (a CDK5 inhibitor) or mitochondrial division inhibitor-1 (Mdivi-1, an inhibitor of mitochondrial fission) attenuated BSO-induced PV downregulation. WY14643 (an inducer of mitochondrial fission) reduced PV expression without affecting CDK5 Y15 phosphorylation. Following status epilepticus (SE), CDK5 Y15 phosphorylation and mitochondrial fission were augmented in PV neurons. These were accompanied by reduced GPx1-mediated inhibition of NF-κB p65 serine (S) 536 phosphorylation. N-acetylcysteine (NAC), roscovitine and Mdivi-1 ameliorated SE-induced PV neuronal degeneration by mitigating CDK5 Y15 hyperphosphorylation, aberrant mitochondrial fragmentation and reduced GPx1-mediated NF-κB inhibition. Furthermore, SN50 (a NF-κB inhibitor) alleviated SE-induced PV neuronal degeneration, independent of dysregulation of mitochondrial fission, CDK5 hyperactivation and GPx1 downregulation. These findings provide an evidence that oxidative stress may activate CDK5-DRP1- and GPx1-NF-κB-mediated signaling pathways, which would be possible therapeutic targets for preservation of PV neurons in various diseases.

Proteomics reveals dynamic metabolic changes in human hematopoietic stem progenitor cells from fetal to adulthood.

BACKGROUND: Hematopoietic stem progenitor cells (HSPCs) undergo phenotypical and functional changes during their emergence and development. Although the molecular programs governing the development of human hematopoietic stem cells (HSCs) have been investigated broadly, the relationships between dynamic metabolic alterations and their functions remain poorly characterized. METHODS: In this study, we comprehensively described the proteomics of HSPCs in the human fetal liver (FL), umbilical cord blood (UCB), and adult bone marrow (aBM). The metabolic state of human HSPCs was assessed via a Seahorse assay, RT‒PCR, and flow cytometry-based metabolic-related analysis. To investigate whether perturbing glutathione metabolism affects reactive oxygen species (ROS) production, the metabolic state, and the expansion of human HSPCs, HSPCs were treated with buthionine sulfoximine (BSO), an inhibitor of glutathione synthetase, and N-acetyl-L-cysteine (NAC). RESULTS: We investigated the metabolomic landscape of human HSPCs from the fetal, perinatal, and adult developmental stages by in-depth quantitative proteomics and predicted a metabolic switch from the oxidative state to the glycolytic state during human HSPC development. Seahorse assays, mitochondrial activity, ROS level, glucose uptake, and protein synthesis rate analysis supported our findings. In addition, immune-related pathways and antigen presentation were upregulated in UCB or aBM HSPCs, indicating their functional maturation upon development. Glutathione-related metabolic perturbations resulted in distinct responses in human HSPCs and progenitors. Furthermore, the molecular and immunophenotypic differences between human HSPCs at different developmental stages were revealed at the protein level for the first time. CONCLUSION: The metabolic landscape of human HSPCs at three developmental stages (FL, UCB, and aBM), combined with proteomics and functional validations, substantially extends our understanding of HSC metabolic regulation. These findings provide valuable resources for understanding human HSC function and development during fetal and adult life.

Mechanisms Mediating the Combined Toxicity of Paraquat and Maneb in SH-SY5Y Neuroblastoma Cells.

Epidemiological and experimental studies have demonstrated that combined exposure to the pesticides paraquat (PQ) and maneb (MB) increases the risk of developing Parkinson's disease. However, the mechanisms mediating the toxicity induced by combined exposure to these pesticides are not well understood. The aim of this study was to investigate the mechanism(s) of neurotoxicity induced by exposure to the pesticides PQ and MB isolated or in association (PQ + MB) in SH-SY5Y neuroblastoma cells. PQ + MB exposure for 24 and 48 h decreased cell viability and disrupted cell membrane integrity. In addition, PQ + MB exposure for 12 h decreased the mitochondrial membrane potential. PQ alone increased reactive oxygen species (ROS) and superoxide anion generation and decreased the activity of mitochondrial complexes I and II at 12 h of exposure. MB alone increased ROS generation and depleted intracellular glutathione (GSH) within 6 h of exposure. In contrast, MB exposure for 12 h increased the GSH levels, the glutamate cysteine ligase (GCL, the rate-limiting enzyme in the GSH synthesis pathway) activity, and increased nuclear Nrf2 staining. Pretreatment with buthionine sulfoximine (BSO, a GCL inhibitor) abolished the MB-mediated GSH increase, indicating that MB increases GSH synthesis by upregulating GCL, probably by the activation of the Nrf2/ARE pathway. BSO pretreatment, which did not modify cell viability per se, rendered cells more sensitive to MB-induced toxicity. In contrast, treatment with the antioxidant N-acetylcysteine protected cells from MB-induced toxicity. These findings show that the combined exposure of SH-SY5Y cells to PQ and MB induced a cytotoxic effect higher than that observed when cells were subjected to individual exposures. Such a higher effect seems to be related to additive toxic events resulting from PQ and MB exposures. Thus, our study contributes to a better understanding of the toxicity of PQ and MB in combined exposures.

Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion.

Bcl2-associated athanogene-1 protein (Bag1) acts as a co-chaperone of heat shock protein 70 and heat shock cognate 70 and regulates multiple cellular processes, including cell proliferation, apoptosis, environmental stress response, and drug resistance. Since Bag1 knockout mice exhibited fetal lethality, the in vivo function of Bag1 remains unclear. In this study, we established a mouse line expressing Bag1 gene missing exon 5, which corresponds to an encoding region for the interface of heat shock protein 70/heat shock cognate 70. Despite mice carrying homoalleles of the Bag1 mutant (Bag1Δex5) expressing undetectable levels of Bag1, Bag1Δex5 homozygous mice developed without abnormalities. Bag1Δex5 protein was found to be highly unstable in cells and in vitro. We found that the growth of mouse embryonic fibroblasts derived from Bag1Δex5-homo mice was attenuated by doxorubicin and a glutathione (GSH) synthesis inhibitor, buthionine sulfoximine. In response to buthionine sulfoximine, Bag1Δex5-mouse embryonic fibroblasts exhibited a higher dropping rate of GSH relative to the oxidized glutathione level. In addition, Bag1 might mitigate cellular hydrogen peroxide levels. Taken together, our results demonstrate that the loss of Bag1 did not affect mouse development and that Bag1 is involved in intracellular GSH homeostasis, namely redox homeostasis.

Feasibility study of multimodal imaging for redox status and glucose metabolism in tumor.

Understanding the tumor redox status is important for efficient cancer treatment. Here, we noninvasively detected changes in the redox environment of tumors before and after cancer treatment in the same individuals using a novel compact and portable electron paramagnetic resonance imaging (EPRI) device and compared the results with glycolytic information obtained through autoradiography using 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). Human colon cancer HCT116 xenografts were used in the mice. We used 3-carbamoyl-PROXYL (3CP) as a paramagnetic and redox status probe for the EPRI of tumors. The first EPRI was followed by the intraperitoneal administration of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, or X-ray irradiation of the tumor. A second EPRI was performed on the following day. Autoradiography was performed after the second EPRI. After imaging, the tumor sections were evaluated by histological analysis and the amount of reducing substances in the tumor was measured. BSO treatment and X-ray irradiation significantly decreased the rate of 3CP reduction in tumors. Redox maps of tumors obtained from EPRI can be compared with tissue sections of approximately the same cross section. BSO treatment reduced glutathione levels in tumors, whereas X-ray irradiation did not alter the levels of any of the reducing substances. Comparison of the redox map with the autoradiography of [18F]FDG revealed that regions with high reducing power in the tumor were active in glucose metabolism; however, this correlation disappeared after X-ray irradiation. These results suggest that the novel compact and portable EPRI device is suitable for multimodal imaging, which can be used to study tumor redox status and therapeutic efficacy in cancer, and for combined analysis with other imaging modalities.

A ROS storm generating nanocomposite for enhanced chemodynamic therapy through H2O2 self-supply, GSH depletion and calcium overload.

Catalytic generation of toxic hydroxyl radicals (˙OH) from hydrogen peroxide (H2O2) is an effective strategy for tumor treatment in chemodynamic therapy (CDT). However, the intrinsic features of the microenvironment in solid tumors, characterized by limited H2O2 and overexpressed glutathione (GSH), severely impede the accumulation of intracellular ˙OH, posing significant challenges. To circumvent these critical issues, in this work, a CaO2-based multifunctional nanocomposite with a surface coating of Cu2+ and L-buthionine sulfoximine (BSO) (named CaO2@Cu-BSO) is designed for enhanced CDT. Taking advantage of the weakly acidic environment of the tumor, the nanocomposite gradually disintegrates, and the exposed CaO2 nanoparticles subsequently decompose to produce H2O2, alleviating the insufficient supply of endogenous H2O2 in the tumor microenvironment (TME). Furthermore, Cu2+ detached from the surface of CaO2 is reduced by H2O2 and GSH to Cu+ and ROS. Then, Cu+ catalyzes H2O2 to generate highly cytotoxic ˙OH and Cu2+, forming a cyclic catalysis effect for effective CDT. Meanwhile, GSH is depleted by Cu2+ ions to eliminate possible ˙OH scavenging. In addition, the decomposition of CaO2 by TME releases a large amount of free Ca2+, resulting in the accumulation and overload of Ca2+ and mitochondrial damage in tumor cells, further improving CDT efficacy and accelerating tumor apoptosis. Besides, BSO, a molecular inhibitor, decreases GSH production by blocking γ-glutamyl cysteine synthetase. Together, this strategy allows for enhanced CDT efficiency via a ROS storm generation strategy in tumor therapy. The experimental results confirm and demonstrate the satisfactory tumor inhibition effect both in vitro and in vivo.

Propyl gallate induces cell death in human pulmonary fibroblast through increasing reactive oxygen species levels and depleting glutathione.

Propyl gallate (PG) exhibits an anti-growth effect on various cell types. The present study investigated the impact of PG on the levels of reactive oxygen species (ROS) and glutathione (GSH) in primary human pulmonary fibroblast (HPF) cells. Moreover, the effects of N-acetyl cysteine (NAC, an antioxidant), L-buthionine sulfoximine (BSO, a GSH synthesis inhibitor), and small interfering RNA (siRNAs) against various antioxidant genes on ROS and GSH levels and cell death were examined in PG-treated HPF cells. PG (100-800 µM) increased the levels of total ROS and O2·- at early time points of 30-180 min and 24 h, whereas PG (800-1600 µM) increased GSH-depleted cell number at 24 h and reduced GSH levels at 30-180 min. PG downregulated the activity of superoxide dismutase (SOD) and upregulated the activity of catalase in HPF cells. Treatment with 800 µM PG increased the number of apoptotic cells and cells that lost mitochondrial membrane potential (MMP; ΔΨm). NAC treatment attenuated HPF cell death and MMP (ΔΨm) loss induced by PG, accompanied by a decrease in GSH depletion, whereas BSO exacerbated the cell death and MMP (ΔΨm) loss without altering ROS and GSH depletion levels. Furthermore, siRNA against SOD1, SOD2, or catalase attenuated cell death in PG-treated HPF cells, whereas siRNA against GSH peroxidase enhanced cell death. In conclusion, PG induced cell death in HPF cells by increasing ROS levels and depleting GSH. NAC was found to decrease HPF cell death induced by PG, while BSO enhanced cell death. The findings shed light on how manipulating the antioxidant system influence the cytotoxic effects of PG in HPF cells.

The cadmium tolerance enhancement through regulating glutathione conferred by vacuolar compartmentalization in Aspergillus sydowii.

Aspergillus was found to be a vital hyperaccumulation species for heavy metal removal with admirable tolerance capacity. But the potential tolerance mechanism has not been completely studied. This study quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH), and reactive oxygen species (ROS) in the protoplasts and vacuoles of mycelium. We modulated GSH synthesis using buthionine sulfoximine (BSO) and 2-oxothiazolidine-4-carboxylic acid (OTC) to investigate the subcellular regulatory mechanisms of GSH in the accumulation of Cd. The results confirmed that GSH plays a crucial role in vacuolar compartmentalization under Cd stress. GSH and GSSG as a redox buffer to keep the cellular redox state in balance and GSH as a metal chelating agent to reduce toxicity. When regulating the decreased GSH content with BSO, and increased GSH content with OTC, the system of Cd-GSH-ROS can change accordingly, this also supported that vacuolar compartmentalization is a detoxification strategy that can modulate the transport and storage of substances inside and outside the vacuole reasonably. Interestingly, GSH tended to be distributed in the cytoplasm, the battleground of redox takes place in the cytoplasm but not in the vacuole. These finding potentially has implications for the understanding of tolerance behavior and detoxification mechanisms of cells. In the future bioremediation of Cd in soil, the efficiency of soil remediation can be improved by developing organisms with high GSH production capacity.

Metallothionein Alleviates Glutathione Depletion-Induced Oxidative Cardiomyopathy through CISD1-Dependent Regulation of Ferroptosis in Murine Hearts.

This study was designed to discern the effect of heavy scavenger metallothionein on glutathione (GSH) deprivation-evoked cardiac anomalies and mechanisms involved with an emphasis on ferroptosis. Wild-type and cardiac metallothionein transgenic mice received GSH synthase inhibitor buthionine sulfoximine (BSO; 30 mmol/L in drinking water) for 14 days before assessment of myocardial morphology and function. BSO evoked cardiac remodeling and contractile anomalies, including cardiac hypertrophy, interstitial fibrosis, enlarged left ventricular chambers, deranged ejection fraction, fraction shortening, cardiomyocyte contractile capacity, intracellular Ca2+ handling, sarcoplasmic reticulum Ca2+ reuptake, loss of mitochondrial integrity (mitochondrial swelling, loss of aconitase activity), mitochondrial energy deficit, carbonyl damage, lipid peroxidation, ferroptosis, and apoptosis. Metallothionein itself did not affect myocardial morphology and function, although it mitigated BSO-provoked myocardial anomalies, loss of mitochondrial integrity and energy, and ferroptosis. Immunoblotting revealed down-regulated sarco(endo)plasmic reticulum Ca2+-ATPase 2a, glutathione peroxidase 4, ferroptosis-suppressing CDGSH iron-sulfur domain 1 (CISD1), and mitochondrial regulating glycogen synthase kinase-3ß phosphorylation with elevated p53, myosin heavy chain-ß isozyme, IκB phosphorylation, and solute carrier family 7 member 11 (SLC7A11) as well as unchanged SLC39A1, SLC1A5, and ferroptosis-suppressing protein 1 following BSO challenge, all of which, except glutamine transporter SLC7A11 and p53, were abrogated by metallothionein. Inhibition of CISD1 using pioglitazone nullified GSH-offered benefit against BSO-induced cardiomyocyte ferroptosis and contractile and intracellular Ca2+ derangement. Taken together, these findings support a regulatory modality for CISD1 in the impedance of ferroptosis in metallothionein-offered protection against GSH depletion-evoked cardiac aberration.

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5-2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed in vivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

The nonessential amino acid cysteine is required to prevent ferroptosis in acute myeloid leukemia.

ABSTRACT: Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing.

Inhibition of the glutamate-cysteine ligase catalytic subunit with buthionine sulfoximine enhances the cytotoxic effect of doxorubicin and cyclophosphamide in Burkitt lymphoma cells.

Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.

The Role of Glutathione and Sulfhydryl Groups in Cadmium Uptake by Cultures of the Rainbow Trout RTG-2 Cell Line.

The aim of this study is to investigate the role of cellular sulfhydryl and glutathione (GSH) status in cellular cadmium (Cd) accumulation using cultures of the rainbow trout cell line RTG-2. In a first set of experiments, the time course of Cd accumulation in RTG-2 cells exposed to a non-cytotoxic CdCl2 concentration (25 µM) was determined, as were the associated changes in the cellular sulfhydryl status. The cellular levels of total GSH, oxidized glutathione (GSSG), and cysteine were determined with fluorometric high-performance liquid chromatography (HPLC), and the intracellular Cd concentrations were determined with inductively coupled plasma mass spectrometry (ICP-MS). The Cd uptake during the first 24 h of exposure was linear before it approached a plateau at 48 h. The metal accumulation did not cause an alteration in cellular GSH, GSSG, or cysteine levels. In a second set of experiments, we examined whether the cellular sulfhydryl status modulates Cd accumulation. To this end, the following approaches were used: (a) untreated RTG-2 cells as controls, and (b) RTG-2 cells that were either depleted of GSH through pre-exposure to 1 mM L-buthionine-SR-sulfoximine (BSO), an inhibitor of glutathione synthesis, or the cellular sulfhydryl groups were blocked through treatment with 2.5 µM N-ethylmaleimide (NEM). Compared to the control cells, the cells depleted of intracellular GSH showed a 25% reduction in Cd accumulation. Likewise, the Cd accumulation was reduced by 25% in the RTG-2 cells with blocked sulfhydryl groups. However, the 25% decrease in cellular Cd accumulation in the sulfhydryl-manipulated cells was statistically not significantly different from the Cd accumulation in the control cells. The findings of this study suggest that the intracellular sulfhydryl and GSH status, in contrast to their importance for Cd toxicodynamics, is of limited importance for the toxicokinetics of Cd in fish cells.

Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.

Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.

Iron-based metal-organic framework co-loaded with buthionine sulfoximine and oxaliplatin for enhanced cancer chemo-ferrotherapy via sustainable glutathione elimination.

BACKGROUND: Emerging ferroptosis-driven therapies based on nanotechnology function either by increasing intracellular iron level or suppressing glutathione peroxidase 4 (GPX4) activity. Nevertheless, the therapeutic strategy of simultaneous iron delivery and GPX4 inhibition remains challenging and has significant scope for improvement. Moreover, current nanomedicine studies mainly use disulfide-thiol exchange to deplete glutathione (GSH) for GPX4 inactivation, which is unsatisfactory because of the compensatory effect of continuous GSH synthesis. METHODS: In this study, we design a two-in-one ferroptosis-inducing nanoplatform using iron-based metal-organic framework (MOF) that combines iron supply and GPX4 deactivation by loading the small molecule buthionine sulfoxide amine (BSO) to block de novo GSH biosynthesis, which can achieve sustainable GSH elimination and dual ferroptosis amplification. A coated lipid bilayer (L) can increase the stability of the nanoparticles and a modified tumor-homing peptide comprising arginine-glycine-aspartic acid (RGD/R) can achieve tumor-specific therapies. Moreover, as a decrease in GSH can alleviate resistance of cancer cells to chemotherapy drugs, oxaliplatin (OXA) was also loaded to obtain BSO&OXA@MOF-LR for enhanced cancer chemo-ferrotherapy in vivo. RESULTS: BSO&OXA@MOF-LR shows a robust tumor suppression effect and significantly improved the survival rate in 4T1 tumor xenograft mice, indicating a combined effect of dual amplified ferroptosis and GSH elimination sensitized apoptosis. CONCLUSION: BSO&OXA@MOF-LR is proven to be an efficient ferroptosis/apoptosis hybrid anti-cancer agent. This study is of great significance for the clinical development of novel drugs based on ferroptosis and apoptosis for enhanced cancer chemo-ferrotherapy.

Buthionine sulfoximine and chemoresistance in cancer treatments: a systematic review with meta-analysis of preclinical studies.

Buthionine sulfoximine (BSO) is a synthetic amino acid that blocks the biosynthesis of reduced glutathione (GSH), an endogenous antioxidant cellular component present in tumor cells. GSH levels have been associated with tumor cell resistance to chemotherapeutic drugs and platinum compounds. Consequently, by depleting GSH, BSO enhances the cytotoxicity of chemotherapeutic agents in drug-resistant tumors. Therefore, the aim of this study was to conduct a systematic review with meta-analysis of preclinical studies utilizing BSO in cancer treatments. The systematic search was carried out using the following databases: PubMed, Web of Science, Scopus, and EMBASE up until March 20, 2023, in order to collect preclinical studies that evaluated BSO, alone or in association, as a strategy for antineoplastic therapy. One hundred nine investigations were found to assess the cytotoxic potential of BSO alone or in combination with other compounds. Twenty-one of these met the criteria for performing the meta-analysis. The evidence gathered indicated that BSO alone exhibits cytotoxic activity. However, this compound is generally used in combination with other antineoplastic strategies, mainly chemotherapy ones, to improve cytotoxicity to carcinogenic cells and treatment efficacy. Finally, this review provides important considerations regarding BSO use in cancer treatment conditions, which might optimize future studies as a potential adjuvant antineoplastic therapeutic tool.

Glutathione depression alters cellular mechanisms of skeletal muscle fatigue in early stage of recovery and prolongs force depression in late stage of recovery.

The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body wt/day) treatment for 5 days, which decreased GSH content to ∼10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N = 17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR.

Inhibition of glutathione generation in hepatic steatotic rats augments oxidative stress.

Fatty liver disease has been strongly associated with a low glutathione (GSH) level in hepatocytes with increased oxidative stress, which is critically involved in the initiation and progression of the disease. The study investigated whether the GSH deficiency induced by buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl cysteine synthetase, can be restored by the administration of GSH ester. We showed that mice fed a diet with cholesterol plus sodium cholate developed steatosis followed by hepatic GSH reduction. Moreover, the GSH level in the cytosol and mitochondria of steatosis plus BSO decreased than that of steatosis alone. Subsequent studies with the liver tissues and plasma of BSO plus steatosis revealed the accumulation of cholesterol in the hepatocytes, downregulating the concentration of GSH, antioxidant enzymes, and GSH metabolizing enzymes with a significant rise in reactive oxygen species (ROS), blood glucose level and plasma lipid profile. The administration of GSH ester in BSO-administered mice, prevented the depletion of GSH by upregulating the GSH concentration, antioxidant enzymes, and GSH metabolizing enzymes, followed by a reduction in ROS and plasma lipid concentration. The histopathological analysis showed a marked increase in inflammation followed by hepatocytes ballooning in BSO-induced group and steatosis control group, which was ameliorated by GSH ester administration. In conclusion, our data suggest that the restoration of GSH in the cytosol and mitochondria through the injection with GSH ester plays a principal role in maintaining the GSH level in the liver, thereby delaying the progression of fatty liver disease.

DNA damage and up-regulation of PARP-1 induced by columbin in vitro and in vivo.

Columbin (CLB) is the most abundant (>1.0%) furan-containing diterpenoid lactone in herbal medicine Tinospora sagittate (Oliv.) Gagnep. The furano-terpenoid was found to be hepatotoxic, but the exact mechanisms remain unknown. The present study demonstrated that administration of CLB at 50 mg/kg induced hepatotoxicity, DNA damage and up-regulation of PARP-1 in vivo. Exposure to CLB (10 µM) induced GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1 and cell death in cultured mouse primary hepatocytes in vitro. Co-treatment of mouse primary hepatocytes with ketoconazole (10 µM) or glutathione ethyl ester (200 µM) attenuated the GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1, and cell death induced by CLB, while co-exposure to L-buthionine sulfoximine (BSO, 1000 µM) intensified such adverse effects resulting from CLB exposure. These results suggest that the metabolic activation of CLB by CYP3A resulted in the depletion of GSH and increase of ROS formation. The resultant over-production of ROS subsequently disrupted the DNA integrity and up-regulated the expression of PARP-1 in response to DNA damage, and ROS-induced DNA damage was involved in the hepatotoxicity of CLB.