GSH Conjugates Research Articles

PfGSTF2 endows resistance to quizalofop-p-ethyl in Polypogon fugax by GSH conjugation.

Populations of Polypogon fugax have developed resistance to many acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST-based metabolic resistance to the widely used ACCase-inhibiting herbicide quizalofop-p-ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop-p-ethyl and haloxyfop-R-methyl. This reflects similar cross-resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop-p-ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop-p-ethyl. LC-MS analysis of in vitro herbicide metabolism by Escherichia coli-expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH-quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD-Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD-Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.

Identification of the metabolites of nimbolide in rat by liquid chromatography combined with quadrupole/orbitrap mass spectrometry.

Nimbolide is a major furanoid compound isolated from Azadirachta indica. The aim of this study was to characterize the metabolites of nimbolide in rats and to propose the metabolic pathways. The metabolites were generated by incubating nimbolide (10μM) with rat liver microsomes, nicotinamide adenine dinucleotide phosphate (NADPH), and nucleophiles (glutathione [GSH] or N-acetyl-lysine [NAL]) at 37°C for 60 min. For the in vivo study, nimbolide was intravenously administered to rats at a single dose of 10mg/kg, and the bile and urine were collected. The metabolites were identified by ultra-high-performance liquid chromatography-quadrupole/orbitrap mass spectrometry (UPLC-Q/Orbitrap-MS) using electrospray ionization in positive ion mode. Totally, nine metabolites were detected, and their identities were characterized by accurate MS and MS/MS data. In GSH-supplemented liver microsomes, GSH conjugation was the primary elimination pathway. The furan ring was bioactivated into cis-butene-1,4-dial that can be trapped by GSH. In NAL-supplemented liver microsomes, two NAL conjugates (M4 and M5) derived from cis-butene-1,4-dial were observed. In rat bile and urine, N-acetyl-cysteine, cysteine-glycine, and GSH conjugate were also found. The current study provides an overview of the metabolism and the bioactivation profiles of nimbolide in rats, which aids in understanding its safety and activity.

The Interaction of Myeloperoxidase with the Industrial Contaminant 6-PPD: A Potential Pathway for Reactive Metabolites.

6-PPD (N-[1,3-dimethylbutyl]-N'-phenyl-p-phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/H2O2. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.

Interactions between prosulfocarb and trifluralin metabolism in annual ryegrass (Lolium rigidum).

Cross-resistance between pre-emergence herbicides is developing in Australian populations of annual ryegrass (Lolium rigidum Gaud.). A previous study has reported that selection with prosulfocarb (a pro-herbicide requiring bioactivation to its phytotoxic sulfoxide) can decrease metabolic resistance to trifluralin. Metabolism of prosulfocarb and trifluralin was investigated in L. rigidum populations with different levels of resistance to prosulfocarb, trifluralin and also pyroxasulfone, which is detoxified by glutathione (GSH) conjugation. Coleoptiles and radicles of herbicide-treated seedlings responded differently to the same herbicide. Radicles had a lower capacity for bioactivation of prosulfocarb, and this was correlated with a lower ability to metabolise trifluralin within and among populations. Coleoptile resistance to prosulfocarb sulfoxide was negatively correlated with abundance of a major polar metabolite. There was no evidence of GSH conjugation with the sulfoxide, making any potential links between prosulfocarb and pyroxasulfone resistance less obvious. Activation and metabolism of prosulfocarb in L. rigidum is complex and differentially regulated in different tissues. Selection with prosulfocarb may ameliorate trifluralin metabolism in the radicles, but the relationship between prosulfocarb and pyroxasulfone resistance is not GSH-mediated. When applying pre-emergence herbicides, care should be taken with the composition of mixtures and rotations to avoid selection of cross-resistance between pyroxasulfone and prosulfocarb. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites.

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumour induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette (ABC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the GSH conjugate of the highly toxic monomethylarsonous acid (MMAIII), MMA(GS)2, and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in HEK293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV, despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ~3-fold more resistant to arsenate than Abcc4(-/-) MEFs; however, further characterization indicated this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to differences in human and mouse arsenic toxicokinetics. Significance Statement Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. The human ATP-binding cassette transporter hABCC4 is a high affinity transporter of toxicologically important arsenic metabolites. Here we used multiple cell models to demonstrate that mouse Abcc4 does not protect cells against, or transport, any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

Correlation of Vanillin-Induced Cytotoxicity with CYP3A-Mediated Metabolic Activation.

Vanillin (VAN) is a common flavoring agent that can cause liver damage when ingested in large amounts. Nevertheless, the precise processes responsible for its toxicity remain obscure. The present research aimed to examine the metabolic activation of VAN and establish a potential correlation between its reactive metabolites and its cytotoxicity. In rat liver microsomes incubated with VAN, reduced glutathione/N-acetylcysteine (GSH/NAC), and nicotinamide adenine dinucleotide phosphate (NADPH), two conjugates formed from GSH and one conjugate derived from NAC were identified. We also discovered one GSH conjugate in both the bile obtained from rats and the rat primary hepatocytes that were subjected to VAN exposure. Additionally, the NAC conjugate exerted in the urine of VAN-treated rats was observed. These results indicate that a quinone intermediate was produced from VAN both in vitro and in vivo. Next, we identified CYP3A as the main enzyme that initiated the bioactive pathway of VAN. After the activity of CYP3A was selectively inhibited by ketoconazole (KTZ), the generation of the GSH conjugate declined in hepatocytes exposed to VAN. Furthermore, the vulnerability to VAN-induced toxicity was alleviated by KTZ in hepatocytes. Thus, we propose that the cytotoxicity of VAN may derive from metabolic activation triggered by CYP3A.

Screening and identification of reactive metabolic compounds of Cortex Periplocae based on glutathione capture-mass spectrometry.

As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed onHepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.

Glutathione conjugation of sesquimustard: in vitro investigation of potential biomarkers

Sesquimustard (Q) is a powerful blistering agent that contains additional sulfur atoms. Sulfur mustard causes covalent bonding by alkylating nucleophilic groups of biologically important macromolecules such as lipids, proteins, DNA, or RNA. Most cells maintain relatively high amounts of a unique tripeptide called glutathione (GSH) (γ-glutamyl-cysteinyl glycine), which possesses a free thiol group, to prevent unwanted reactions caused by reactive chemical entities. Moreover, these thiol groups on cysteines (Cys) are the main target for alkylation. Although Q is the most potent vesicant among sulfur mustards, research studies identifying biomarkers of Q are very limited. Therefore, here in this study, we aimed to identify the GSH and Cys conjugates of Q using mass spectrometric methods and to observe the formation of these conjugates in HaCat cell culture following exposure to different doses. We identified four different conjugates of Q, which are bis-glutathionyl ethylthioethylthioethyl conjugate (GSH-ETETE-GSH), hydroxyethylthioethylthioethyl glutathione conjugate (HETETE-GSH), bis-cysteinyl ethylthioethylthioethyl conjugate (Cys-ETETE-Cys), and hydroxyethylthioethylthioethyl cysteine conjugate (HETETE-Cys). The identity of the conjugates was elucidated using liquid chromatography–high-resolution mass spectrometry (LC-HRMS). We also investigated changes in conjugate formation with exposure concentration and time elapsed after exposure in the cell culture. After exposure, GSH conjugates decreased until 1st hour, while Cys conjugates increased until 6th hour. We also observed that conjugate formation depended on the concentration of Q. This is the first study to elucidate the conjugates of Q dependent on GSH conjugation. As biomarkers are essential tools for evaluating exposure to Q, this study contributes to the limited number of studies identifying biomarkers for Q.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Abstract 4650: RalBP1, a novel approach for ovarian cancer therapy

Abstract Ovarian cancer (OC) is the most devastating gynecologic cancer. The ever-climbing incidence rate of OC makes it the most lethal female reproductive disorder. Triggering apoptosis is one of the potential strategies to overcome chemoresistance. Recent evidence suggests that the failure of drug-induced apoptosis may be an underlying cause of drug-resistance. Molecular mechanisms of failed apoptosis in chemoresistant OC cells include p53 mutations, abnormal expression of the Bcl2 family proteins, and upregulation of other inhibitors of apoptosis that block caspases and stabilize the mitochondrial permeability pore. RalBP1 (Ral-binding protein) is a required stress-defense protein for OC, and its specific depletion or inhibition will cause apoptosis in cancer cells expressing RalBP1. RalBP1 is a multifunctional 76 kDa protein encoded as a splice variant in the human RALBP1 gene (18p11.22). RalBP1 is an ATP dependent efflux transporter for glutathione (GSH) conjugates (GS-E) of products of oxidative stress, like 4HNE and GSH-conjugates of chemotherapy drugs. Our recent studies indicate that upon depletion/inhibition of RalBP1, malignant cells are significantly more sensitive to apoptosis than non-malignant cells, suggesting that the stress-protective, anti-apoptotic activity of RalBP1 is necessary for malignant cells. This differential sensitivity to RalBP1 translates into tumor regression, a lack of toxicity, and the prolonged survival of mice bearing subcutaneous OC cells in which RalBP1 is inhibited by anti-RalBP1 IgG or depleted using RalBP1 antisense oligonucleotides. These studies demonstrate that inhibition or depletion of RalBP1 exerts anti-neoplastic effects in OC and indicate that this GS-E and xenobiotic transporter occupies an important position in the hierarchy of stress defense mechanisms necessary for cancer cell survival. Overall, the present research focuses on hypothesis-driven studies to define novel relationships between RalBP1 and signaling pathways, and to determine the suitability of RalBP1 as an anticancer target for OC. Citation Format: Sharad S. Singhal, Sravani Ramisetty, Atish Mohanty, Prakash Kulkarni, David Horne, Sanjay Awasthi, Ravi Salgia. RalBP1, a novel approach for ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4650.

In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200μM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10mg/kg or 25mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400μM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.

Evidence for cytochrome P450 3A4-mediated metabolic activation of SCO-267.

SCO-267 is a potent G-protein-coupled receptor 40 agonist that is undergoing clinical development for the treatment of type 2 diabetes mellitus. The current work was undertaken to investigate the bioactivation potential of SCO-267 in vitro and in vivo. Three SCO-267-derived glutathione (GSH) conjugates (M1-M3) were found both in rat and human liver microsomal incubations supplemented with GSH and nicotinamide adenine dinucleotide phosphate. Two GSH conjugates (M1-M2) together with two N-acetyl-cysteine conjugates (M4-M5) were detected in the bile of rats receiving SCO-267 at 10mg/kg. The identified conjugates suggested the generation of quinone-imine and ortho-quinone intermediates. CYP3A4 was demonstrated to primarily catalyze the bioactivation of SCO-267. In addition, SCO-267 concentration-, time-, and NADPH-dependently inactivated CYP3A in human liver microsomes using testosterone as a probe substrate, along with KI and kinact values of 4.91μM and 0.036min-1 , respectively. Ketoconazole (a competitive inhibitor of CYP3A) displayed no significant protective effect on SCO-267-induced CYP3A inactivation. However, inclusion of GSH showed significant protection. These findings revealed that SCO-267 undergoes a facile CYP3A4-catalyzed bioactivation with the generation of quinone-imine and ortho-quinone intermediates, which were assumed to be involved in SCO-267 induced CYP3A inactivation. These findings provide further insight into the bioactivation pathways involved in the generation of reactive, potentially toxic metabolites of SCO-267. Further studies are needed to evaluate the influence of SCO-267 metabolism on the safety of this drug in vivo.

Comparative metabolism of aschantin in human and animal hepatocytes.

Aschantin, a tetrahydrofurofuran lignan with a 1,3-benzodioxole group derived from Flos Magnoliae, exhibits antioxidant, anti-inflammatory, cytotoxic, and antimicrobial activities. This study compared the metabolic profiles of aschantin in human, dog, mouse, and rat hepatocytes using liquid chromatography-high-resolution mass spectrometry. The hepatic extraction ratio of aschantin among the four species was 0.46-0.77, suggesting that it undergoes a moderate-to-extensive degree of hepatic metabolism. Hepatocyte incubation of aschantin produced 4 phase 1 metabolites, including aschantin catechol (M1), O-desmethylaschantin (M2 and M3), and hydroxyaschantin (M4), and 14 phase 2 metabolites, including O-methyl-M1 (M5 and M6) via catechol O-methyltransferase (COMT), six glucuronides of M1, M2, M3, M5, and M6, and six sulfates of M1, M2, M3, M5, and M6. Enzyme kinetic studies using aschantin revealed that the production of M1, a major metabolite, via O-demethylenation is catalyzed by cytochrome 2C8 (CYP2C8), CYP2C9, CYP2C19, CYP3A4, and CYP3A5 enzymes; the formation of M2 (O-desmethylaschantin) is catalyzed by CYP2C9 and CYP2C19; and the formation of M4 is catalyzed by CYP3A4 enzyme. Two glutathione (GSH) conjugates of M1 were identified after incubation of aschantin with human and animal liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate and GSH, but they were not detected in the hepatocytes of all species. In conclusion, aschantin is extensively metabolized, producing 18 metabolites in human and animal hepatocytes catalyzed by CYP, COMT, UDP-glucuronosyltransferase, and sulfotransferase. These results can help in clarifying the involvement of metabolizing enzymes in the pharmacokinetics and drug interactions of aschantin and in elucidating GSH conjugation associated with the reactive intermediate formed from M1 (aschantin catechol).

SMURF2 predisposes cancer cell toward ferroptosis in GPX4-independent manners by promoting GSTP1 degradation

Ferroptosis is a non-apoptotic form of regulated cell death. Glutathione (GSH) peroxidase 4 (GPX4) and GSH-independent ferroptosis suppressor protein 1 (FSP1) have been identified as major defenses. Here, we uncover a protective mechanism mediated by GSH S-transferase P1 (GSTP1) by monitoring proteinomic dynamics during ferroptosis. Dramatic downregulation of GSTP1 is caused by SMURF2-mediated GSTP1 ubiquitination and degradation at early stages of ferroptosis. Intriguingly, GSTP1 acts in GPX4- and FSP1-independent manners by catalyzing GSH conjugation of 4-hydroxynonenal and detoxifying lipid hydroperoxides via selenium-independent GSH peroxidase activity. Genetic modulation of the SMURF2/GSTP1 axis or the pharmacological inhibition of GSTP1's catalytic activity sensitized tumor responses to Food and Drug Administration (FDA)-approved ferroptosis-inducing drugs both invitro and invivo. GSTP1 expression also confers resistance to immune checkpoint inhibitors by blunting ferroptosis. Collectively, these findings demonstrate a GPX4/FSP1-independent cellular defense mechanism against ferroptosis and suggest that targeting SMURF2/GSTP1 to sensitize cancer cells to ferroptosis has potential as an anticancer therapy.

AOP Report: Glutathione Conjugation Leading to Reproductive Dysfunction via Oxidative Stress.

We propose an adverse outcome pathway (AOP) for reproductive dysfunction via oxidative stress (OS). The AOP was developed based on Organisation for Economic Co-operation and Development (OECD) Guidance Document 184 and on the specific considerations of the OECD users' handbook supplement to the guidance document for developing and assessing AOPs (no. 233). According to the qualitative and quantitative experimental data evaluation, glutathione (GSH) conjugation is the first upstream key event (KE) of this AOP to reproductive dysfunction triggering OS. This event causes depletion of GSH basal levels (KE2 ). Consequently, this drop of free GSH induces an increase of reactive oxygen species (KE3 ) generated by the natural cellular metabolic processes (cellular respiration) of the organism. Increased levels of these reactive species, in turn, induce an increase of lipid peroxidation (KE4 ). This KE consequently leads to a rise in the amount of toxic substances, such as malondialdehyde and hydroxynonenal, which are associated with decreased quality and competence of gamete cell division, consequently impairing fertility (KE5 and adverse outcome). The overall assessment of the general biological plausibility, the empirical support, and the essentiality of KE relationships was considered as high for this AOP. We conclude that GSH conjugation is able to lead to reproductive disorder in fishes and mammals, via OS, but that the amount of stressor needed to trigger the AOP differs between stressors. Environ Toxicol Chem 2023;42:2519-2528. © 2023 SETAC.

In Vitro Enzyme Kinetics and NMR-Based Product Elucidation for Glutathione S-Conjugation of the Anticancer Unsymmetrical Bisacridine C-2028 in Liver Microsomes and Cytosol: Major Role of Glutathione S-Transferase M1-1 Isoenzyme.

This work is the next step in studying the interplay between C-2028 (anticancer-active unsymmetrical bisacridine developed in our group) and the glutathione S-transferase/glutathione (GST/GSH) system. Here, we analyzed the concentration- and pH-dependent GSH conjugation of C-2028 in rat liver microsomes and cytosol. We also applied three recombinant human GST isoenzymes, which altered expression was found in various tumors. The formation of GSH S-conjugate of C-2028 in liver subfractions followed Michaelis-Menten kinetics. We found that C-2028 was conjugated with GSH preferentially by GSTM1-1, revealing a sigmoidal kinetic model. Using a colorimetric assay (MTT test), we initially assessed the cellular GST/GSH-dependent biotransformation of C-2028 in relation to cytotoxicity against Du-145 human prostate cancer cells in the presence or absence of the modulator of GSH biosynthesis. Pretreatment of cells with buthionine sulfoximine resulted in a cytotoxicity decrease, suggesting a possible GSH-mediated bioactivation process. Altogether, our results confirmed the importance of GSH conjugation in C-2028 metabolism, which humans must consider when planning a treatment strategy. Finally, nuclear magnetic resonance spectroscopy elucidated the structure of the GSH-derived product of C-2028. Hence, synthesizing the compound standard necessary for further advanced biological and bioanalytical investigations will be achievable.

Redox Regulation of Xenobiotics by Reactive Sulfur and Supersulfide Species.

Significance: Routine exposure to xenobiotics is unavoidable during our lifetimes. Certain xenobiotics are hazardous to human health, and are metabolized in the body to render them less toxic. During this process, several detoxification enzymes cooperatively metabolize xenobiotics. Glutathione (GSH) conjugation plays an important role in the metabolism of electrophilic xenobiotics. Recent Advances: Recent advances in reactive sulfur and supersulfide (RSS) analyses showed that persulfides and polysulfides bound to low-molecular-weight thiols, such as GSH, and to protein thiols are abundant in both eukaryotes and prokaryotes. The highly nucleophilic nature of hydropersulfides and hydropolysulfides contributes to cell protection against oxidative stress and electrophilic stress. Critical Issues: In contrast to GSH conjugation to electrophiles that is aided by glutathione S-transferase (GST), persulfides and polysulfides can directly form conjugates with electrophiles without the catalytic actions of GST. The polysulfur bonds in the conjugates are further reduced by perthioanions and polythioanions derived from RSS to form sulfhydrated metabolites that are no longer electrophilic but rather nucleophilic, and differ from metabolites that are formed via GSH conjugation. Future Directions: In view of the abundance of RSS in cells and tissues, metabolism of xenobiotics that is mediated by RSS warrants additional investigations, such as studies of the impact of microbiota-derived RSS on xenobiotic metabolism. Metabolites formed from reactions between electrophiles and RSS may be potential biomarkers for monitoring exposure to electrophiles and for studying their metabolism by RSS. Antioxid. Redox Signal. 40, 679-690.

Glutathione: Lights and Shadows in Cancer Patients.

In cases of cellular injury, there is an observed increase in the production of reactive oxygen species (ROS). When this production becomes excessive, it can result in various conditions, including cancerogenesis. Glutathione (GSH), the most abundant thiol-containing antioxidant, is fundamental to re-establishing redox homeostasis. In order to evaluate the role of GSH and its antioxi-dant effects in patients affected by cancer, we performed a thorough search on Medline and EMBASE databases for relevant clinical and/or preclinical studies, with particular regard to diet, toxicities, and pharmacological processes. The conjugation of GSH with xenobiotics, including anti-cancer drugs, can result in either of two effects: xenobiotics may lose their harmful effects, or GSH conjugation may enhance their toxicity by inducing bioactivation. While being an interesting weapon against chemotherapy-induced toxicities, GSH may also have a potential protective role for cancer cells. New studies are necessary to better explain the relationship between GSH and cancer. Although self-prescribed glutathione (GSH) implementation is prevalent among cancer patients with the intention of reducing the toxic effects of anticancer treatments and potentially preventing damage to normal tissues, this belief lacks substantial scientific evidence for its efficacy in reducing toxicity, except in the case of cisplatin-related neurotoxicity. Therefore, the use of GSH should only be considered under medical supervision, taking into account the appropriate timing and setting.

Reactivity of Acrylamides Causes Cytotoxicity and Activates Oxidative Stress Response.

Acrylamides are widely used industrial chemicals that cause adverse effects in humans or animals, such as carcinogenicity or neurotoxicity. The excess toxicity of these reactive electrophilic chemicals is especially interesting, as it is mostly triggered by covalent reactions with biological nucleophiles, such as DNA bases, proteins, or peptides. The cytotoxicity and activation of oxidative stress response of 10 (meth)acrylamides measured in three reporter gene cell lines occurred at similar concentrations. Most acrylamides exhibited high excess toxicity, while methacrylamides acted as baseline toxicants. The (meth)acrylamides showed no reactivity toward the hard biological nucleophile 2-deoxyguanosine (2DG) within 24 h, and only acrylamides reacted with the soft nucleophile glutathione (GSH). Second-order degradation rate constants (kGSH) were measured for all acrylamides with N,N'-methylenebis(acrylamide) (NMBA) showing the highest kGSH (134.800 M-1 h-1) and N,N-diethylacrylamide (NDA) the lowest kGSH (2.574 M-1 h-1). Liquid chromatography coupled to high-resolution mass spectrometry was used to confirm the GSH conjugates of the acrylamides with a double conjugate formed for NMBA. The differences in reactivity between acrylamides and methacrylamides could be explained by the charge density of the carbon atoms because the electron-donating inductive effect of the methyl group of the methacrylamides lowered their electrophilicity and thus their reactivity. The differences in reactivity within the group of acrylamides could be explained by the energy of the lowest unoccupied molecular orbital and steric hindrance. Cytotoxicity and activation of oxidative stress response were linearly correlated with the second-order reaction rate constants of the acrylamides with GSH. The reaction of the acrylamides with GSH is hence not only a detoxification mechanism but also leads to disturbances of the redox balance, making the cells more vulnerable to reactive oxygen species. The reactivity of acrylamides explained the oxidative stress response and cytotoxicity in the cells, and the lack of reactivity of the methacrylamides led to baseline toxicity.