Quantification of glutathione with high throughput live-cell imaging.

Reduction oxidation (redox) reactions are central in life and altered redox state is associated with a spectrum of human diseases. Glutathione (GSH) is the most abundant antioxidant in eukaryotic cells and plays critical roles in maintaining redox homeostasis. Thus, measuring intracellular GSH level is an important method to assess the redox state of organism. The currently available GSH probes are based on irreversible chemical reactions with glutathione and can't monitor the real-time glutathione dynamics. Our group developed the first reversible reaction based fluorescent probe for glutathione, which can measure glutathione levels at high resolution using a confocal microscope and in the bulk scale with a flow cytometry. Most importantly it can quantitatively monitor the real-time GSH dynamics in living cells. Using the 2 nd generation of GSH probe, RealThiol (RT), this study measured the GSH level in living Hela cells after treatment with varying concentrations of DL-Buthionine sulfoximine (BSO) which inhibits GSH synthesis, using a high throughput imaging system, Cytation™ 5 cell imaging reader. The results revealed that GSH probe RT at the concentration of 2.0 µM accurately monitored the BSO treatment effect on GSH level in the Hela cells. The present results demonstrated that the GSH probe RT is sensitive and precise in GSH measurement in living cells at a high throughput imaging platform and has the potential to be applied to any cell lines.

Fluorescent Probes and Mass Spectrometry-Based Methods to Quantify Thiols in Biological Systems.

Significance: Fluorescent probes and mass spectrometry are the two most popular and complementary methods to quantify thiols in biological systems. In this review, we focus on the widely used and commercially available methods to detect and quantify thiols in living cells and the general approaches applied in mass spectrometry-based thiol quantification. We hope that this review can serve as a general guide for redox biologists who are interested in thiol species. Sulfur, one of the most important elements in living systems, contributes to every aspect of physiology and pathology. Thiols, including cysteine, homocysteine, glutathione, hydrogen sulfide, and hydropersulfides, are the main players in the redox biology system. Therefore, quantifying these thiol species in biological systems is one of the important steps to understand their roles in biology. Recent Advances: Fluorescent probes and mass spectrometry-based methods have been developed to detect and/or quantify thiols in biological systems. Mass spectrometry-based methods have been the gold standard for metabolite quantification in cells. Fluorescent probes can directly detect or quantify thiol species in living cells with spatial and temporal resolutions. Additionally, organelle-specific fluorescent probes have been widely developed. These two methods are complementary to each other. Critical Issues: Reliable quantification of thiol species using fluorescent probes remains challenging. Future Directions: When developing fluorescent probes, we suggest using both the fluorescent probes and mass spectrometry-based thiol quantification methods to cross-check the results. In addition, we call on chemical biologists to move beyond qualitative probes and focus on probes that can provide quantitative results in live cells. These quantitative measurements based on fluorescent probes should be validated with mass spectrometry-based methods. More importantly, chemical biologists should make their probes accessible to the biology end users. Regarding mass spectrometry-based methods, quantification of the derivatized thiol specifies should fit into the general metabolomics workflow. Antioxid. Redox Signal. 36, 354-365.

Glutathione Quantification in Live Cells with Real-Time Imaging and Flow Cytometry.

Glutathione (GSH) is a highly dynamic, high abundance molecule regulating redox homeostasis in most mammalian cells. Traditional methods could not achieve quantification of glutathione in live cells with high spatial and temporal resolution. Here, we provide protocols on how to use reversible reaction-based ratiometric fluorescent probes, RealThiol (RT) and its derivatives, to quantify GSH globally or in specific organelles. The protocols are applicable to cultured or harvested cells through confocal imaging and flow cytometry. For complete details on the use and execution of this protocol, please refer to Chen et al. (2017) and Jiang et al. (2015, 2017, 2018a).

Author Correction: Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sophoricoside is a selective LXRß antagonist with potent therapeutic effects on hepatic steatosis of mice.

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by the accumulation of triglycerides and associated with obesity, hyperlipidemia and insulin resistance. Currently, there is no therapy for NAFLD. Emerging evidences suggest that the inhibition of liver X receptor (LXR) activity may be a potential therapy for hepatic steatosis. Here, we identified that sophoricoside is a selective antagonist of LXRß. Sophoricoside protected against obesity and glucose tolerance, and inhibited lipid accumulation in the liver of high-fat diet-induced obesity (DIO) mice and methionine and choline-deficient diet-induced nonalcoholic steatohepatitis mice. Furthermore, sophoricoside inhibited malondialdehyde, and increased superoxide dismutase and glutathione in the liver of the mice. In HepG2 cells, pretreatment with sophoricoside rescued GSH concentration decrease induced by H2 O2 treatment. Our data suggest that sophoricoside is a novel LXRß selective antagonist and may improve glucose and lipid dysfunction, and attenuate lipid accumulation in the liver of DIO mice via anti-oxidant properties, which may be developed as a therapy for NAFLD.

Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.

Cardiac-specific ablation of glutaredoxin 3 leads to cardiac hypertrophy and heart failure.

Growing evidence suggests that redox-sensitive proteins including glutaredoxins (Grxs) can protect cardiac muscle cells from oxidative stress-induced damage. Mammalian Grx3 has been shown to be critical in regulating cellular redox states. However, how Grx3 affects cardiac function by modulating reactive oxygen species (ROS) signaling remains unknown. In this study, we found that the expression of Grx3 in the heart is decreased during aging. To assess the physiological role of Grx3 in the heart, we generated mice in which Grx3 was conditionally deleted in cardiomyocytes (Grx3 conditional knockout (CKO) mice). Grx3 CKO mice were viable and grew indistinguishably from their littermates at young age. No difference in cardiac function was found comparing Grx3 CKO mice and littermate controls at this age. However, by the age of 12 months, Grx3 CKO mice exhibited left ventricular hypertrophy with a significant decrease in ejection fraction and fractional shortening along with a significant increase of ROS production in cardiomyocytes compared to controls. Deletion of Grx3 also impaired Ca2+ handling, caused enhanced sarcoplasmic reticulum (SR) calcium (Ca2+ ) leak, and decreased SR Ca2+ uptake. Furthermore, enhanced ROS production and alteration of Ca2+ handling in cardiomyocytes occurred, prior to cardiac dysfunction in young mice. Therefore, our findings demonstrate that Grx3 is an important factor in regulating cardiac hypertrophy and heart failure by modulating both cellular redox homeostasis and Ca2+ handling in the heart.

Quantitative Real-Time Imaging of Glutathione with Subcellular Resolution.

AIMS: Quantitative imaging of glutathione (GSH) with high spatial and temporal resolution is essential for studying the roles of GSH in redox biology. To study the long-standing question of compartmentalization of GSH, especially its distribution between the nucleus and cytosol, an organelle-targeted quantitative probe is needed. RESULTS: We developed a reversible reaction-based ratiometric fluorescent probe-HaloRT-that can quantitatively measure GSH dynamics with subcellular resolution in real time. Using HaloRT, we quantitatively measured the GSH concentrations in the nucleus and cytosol of HeLa cells and primary hepatocytes under different treatment conditions and found no appreciable concentration gradients between these two organelles. Innovation and Conclusion: We developed the first reversible ratiometric GSH probe that can be universally targeted to any organelle of interest. Taking advantage of this new tool, we provided definitive evidence showing that GSH concentrations are not significantly different between the nucleus and cytosol, challenging the view of nuclear compartmentalization of GSH.

Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications.

SIGNIFICANCE: The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. CRITICAL ISSUES: In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. FUTURE DIRECTIONS: The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 29, 518-540.

Sulfur mustard resistant keratinocytes obtained elevated glutathione levels and other changes in the antioxidative defense mechanism.

BACKGROUND: Sulfur mustard (SM) is a potent blistering chemical warfare agent, which was first used in 1917. Despite the Chemical Weapons Convention, a use was recently reported in Syria in 2015. This emphasizes the importance to develop countermeasures against chemical warfare agents. Despite intensive research, there is still no antidote or prophylaxis available against SM. METHODS: The newly developed SM-resistant keratinocyte cell line HaCaT/SM was used to identify new target structures for drug development, particularly the adaptations in protective measures against oxidative stress. For this purpose, glutathione (GSH) and NAD(P)H levels, the effect of glutathione S-transferase (GST) inhibition as well as activation and expression of Nrf2, GST, glutamate cysteine ligase (GCL) and glutathione-disulfide reductase (GSR) as well as multi-drug resistance (MDR) proteins 1, 3 and 5 were investigated. RESULTS: The HaCaT/SM cells showed not only a better survival after treatment with SM or cytostatic drugs, but also hydrogen peroxide (H2O2). They exhibit more GSH even after SM treatment. Nrf2 levels were significantly lower. Inhibition of GST led to significantly decreased, activation to slightly higher IC50 values after SM treatment and a lower expression of GST was observed. The cells also expressed less GCLC and GSR. Expression of MDR1, MDR3 and MDR5 was higher under control conditions, but less stimulated by SM treatment. An increased NADP+/NADPH ratio as well as higher NAD+ levels were shown. CONCLUSION: In summary, an improved response of the resistant cell line to oxidative stress was observed. The underlying mechanisms are elevated GSH levels as well as lower expression of Nrf2 and its targets GCLC and GST as well as GSR and MDR1, MDR3 and MDR5. GST is an especially interesting target because its inhibition already induced a significant SM sensitivity. SM resistance also caused redox equivalent level differences. Taken together, these findings provide further insight into the mechanism of SM resistance and may open a window for novel therapeutic targets in SM therapy.

Corrigendum: Quantitative real-time imaging of glutathione.

This corrects the article DOI: 10.1038/ncomms16087.

Reversible Reaction-Based Fluorescent Probe for Real-Time Imaging of Glutathione Dynamics in Mitochondria.

We report a mitochondria-specific glutathione (GSH) probe-designated as Mito-RealThiol (MitoRT)-that can monitor in vivo real-time mitochondrial glutathione dynamics, and apply this probe to follow mitochondrial GSH dynamic changes in living cells for the first time. MitoRT can be utilized in confocal microscopy, super-resolution fluorescence imaging, and flow cytometry systems. Using MitoRT, we demonstrate that cells have a high priority to maintain the GSH level in mitochondria compared to the cytosol not only under normal growing conditions but also upon oxidative stress.

Quantitative real-time imaging of glutathione.

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe-designated as RealThiol (RT)-that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can be used for both confocal microscopy and flow cytometry based high-throughput quantification of glutathione levels in single cells. We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.

Loss of glutaredoxin 3 impedes mammary lobuloalveolar development during pregnancy and lactation.

Mammalian glutaredoxin 3 (Grx3) has been shown to be important for regulating cellular redox homeostasis in the cell. Our previous studies indicate that Grx3 is significantly overexpressed in various human cancers including breast cancer and demonstrate that Grx3 controls cancer cell growth and invasion by regulating reactive oxygen species (ROS) and NF-κB signaling pathways. However, it remains to be determined whether Grx3 is required for normal mammary gland development and how it contributes to epithelial cell proliferation and differentiation in vivo. In the present study, we examined Grx3 expression in different cell types within the developing mouse mammary gland (MG) and found enhanced expression of Grx3 at pregnancy and lactation stages. To assess the physiological role of Grx3 in MG, we generated the mutant mice in which Grx3 was deleted specifically in mammary epithelial cells (MECs). Although the reduction of Grx3 expression had only minimal effects on mammary ductal development in virgin mice, it did reduce alveolar density during pregnancy and lactation. The impairment of lobuloalveolar development was associated with high levels of ROS accumulation and reduced expression of milk protein genes. In addition, proliferative gene expression was significantly suppressed with proliferation defects occurring in knockout MECs during alveolar development compared with wild-type controls. Therefore, our findings suggest that Grx3 is a key regulator of ROS in vivo and is involved in pregnancy-dependent mammary gland development and secretory activation through modulating cellular ROS.

Genetically anchored fluorescent probes for subcellular specific imaging of hydrogen sulfide.

Imaging hydrogen sulfide (H2S) at the subcellular resolution will greatly improve the understanding of functions of this signaling molecule. Taking advantage of the protein labeling technologies, we report a general strategy for the development of organelle specific H2S probes, which enables sub-cellular H2S imaging essentially in any organelles of interest.

Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells.

Density functional theory (DFT) was applied to study the thermodynamics and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31G(d) with the SMD solvation model can reliably predict the Gibbs free energy changes (ΔG) of thiol-Michael addition reactions with an error of less than 1 kcal·mol(-1) compared with the experimental benchmarks. Taking advantage of this computational model, the first reversible reaction-based fluorescent probe was developed that can monitor the changes in glutathione levels in single living cells.

Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe.

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe-ThiolQuant Green (TQ Green)-for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies.