Non-cytotoxic nanomolar concentration of arctigenin protects neuronal cells from chemotherapy-induced ferroptosis by regulating SLC7A11-cystine-cysteine axis.

Neurotoxicity is a common side effect of certain types of therapeutic drugs, posing a major hurdle for their clinical application. Accumulating evidence suggests that ferroptosis is involved in the neurotoxicity induced by these drugs. Therefore, targeting ferroptosis is considered to be a reasonable approach to prevent such side effect. Arctigenin (ATG) is a major bioactive ingredient of Arctium lappa L., a popular medicinal plant in Asia, and has been reported to have multiple bioactivities including neuroprotection. However, the mechanisms underlying the neuroprotection of ATG has not been well elucidated. The purpose of this study was to investigate whether the neuroprotection of ATG was associated with its ability to protect neuronal cells from ferroptosis. Using neuronal cell ferroptosis model induced by either classic ferroptosis induces or therapeutic drugs, we demonstrated for the first time that ATG in the nanomolar concentration range effectively prevented neuronal cell ferroptosis induced by classic ferroptosis inducer sulfasalazine (SAS) and erastin (Era), or therapeutic drug oxaliplatin (OXA) and 5-fluorouracil (5-FU). Mechanistically, we uncovered that the anti-ferroptotic effect of ATG was attributed to its ability to activate SLC7A11-cystine-cysteine axis. The findings of the present study implicate that ATG holds great potential to be developed as a novel agent for preventing SLC7A11 inhibition-mediated neurotoxicity.

The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud's phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer's disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects.

Cystine deprivation triggers CD36-mediated ferroptosis and dysfunction of tumor infiltrating CD8+ T cells.

Cancer cells develop multiple strategies to evade T cell-mediated killing. On one hand, cancer cells may preferentially rely on certain amino acids for rapid growth and metastasis. On the other hand, sufficient nutrient availability and uptake are necessary for mounting an effective T cell anti-tumor response in the tumor microenvironment (TME). Here we demonstrate that tumor cells outcompete T cells for cystine uptake due to high Slc7a11 expression. This competition induces T-cell exhaustion and ferroptosis, characterized by diminished memory formation and cytokine secretion, increased PD-1 and TIM-3 expression, as well as intracellular oxidative stress and lipid-peroxide accumulation. Importantly, either Slc7a11 deletion in tumor cells or intratumoral cystine supplementation improves T cell anti-tumor immunity. Mechanistically, cystine deprivation in T cells disrupts glutathione synthesis, but promotes CD36 mediated lipid uptake due to dysregulated cystine/glutamate exchange. Moreover, enforced expression of glutamate-cysteine ligase catalytic subunit (Gclc) promotes glutathione synthesis and prevents CD36 upregulation, thus boosting T cell anti-tumor immunity. Our findings reveal cystine as an intracellular metabolic checkpoint that orchestrates T-cell survival and differentiation, and highlight Gclc as a potential therapeutic target for enhancing T cell anti-tumor function.

Protective effects and mechanism of chemical- and plant-based selenocystine against cadmium-induced liver damage.

Although selenium (Se) and cadmium (Cd) often coexist naturally in the soil of China, the health risks to local residents consuming Se-Cd co-enriched foods are unknown. In the present study, we investigated the effects of chemical-based selenocystine (SeCys2) on cadmium chloride-induced human hepatocarcinoma (HepG2) cell injury and plant (Cardamine hupingshanensis)-derived SeCys2 against Cd-induced liver injury in mice. We found that chemical- and plant-based SeCys2 showed protective effects against Cd-induced HepG2 cell injury and liver damage in mice, respectively. Compared with Cd intervention group, co-treatment with chemical- or plant-based SeCys2 both alleviated liver toxicity and ferroptosis by decreasing ferrous iron, acyl-CoA synthetase long-chain (ACSL) family member 4, lysophosphatidylcholine acyltransferase 3, reactive oxygen species and lipid peroxide levels, and increasing ACSL3, peroxisome proliferator-activated receptor α, solute carrier family 7 member 11 (SLC7A11) and glutathione and glutathione peroxidase 4 (GPX4) levels. In conclusion, chemical- and plant-based SeCys2 alleviated Cd-induced hepatotoxicity and ferroptosis by regulating SLC7A11/GPX4 signaling and lipid peroxidation. Our findings indicate that potential Cd toxicity from consuming foods grown in Se- and Cd-rich soils should be re-evaluated. This study offers a new perspective for the development of SeCys2-enriched agricultural products.

Alkaline Water: Help or Hype for Uric Acid and Cystine Urolithiasis?

PURPOSE: The consumption of alkaline water, water with an average pH of 8 to 10, has been steadily increasing globally as proponents claim it to be a healthier alternative to regular water. Urinary alkalinization therapy is frequently prescribed in patients with uric acid and cystine urolithiasis, and as such we analyzed commercially available alkaline waters to assess their potential to increase urinary pH. MATERIALS AND METHODS: Five commercially available alkaline water brands (Essentia, Smart Water Alkaline, Great Value Hydrate Alkaline Water, Body Armor SportWater, and Perfect Hydration) underwent anion chromatography and direct chemical measurements to determine the mineral contents of each product. The alkaline content of each bottle of water was then compared to that of potassium citrate (the gold standard for urinary alkalinization) as well as to other beverages and supplements used to augment urinary citrate and/or the urine pH. RESULTS: The pH levels of the bottled alkaline water ranged from 9.69 to 10.15. Electrolyte content was minimal, and the physiologic alkali content was below 1 mEq/L for all brands of alkaline water. The alkali content of alkaline water is minimal when compared to common stone treatment alternatives such as potassium citrate. In addition, several organic beverages, synthetic beverages, and other supplements contain more alkali content than alkaline water, and can achieve the AUA and European Association of Urology alkali recommendation of 30 to 60 mEq per day with ≤ 3 servings/d. CONCLUSIONS: Commercially available alkaline water has negligible alkali content and thus provides no added benefit over tap water for patients with uric acid and cystine urolithiasis.

Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size.

Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21Escherichia coli in liquid culture with 50 mM of SeO32- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D-E) lacks an obvious functional role, whereas the other mutation is L-H within 5 Šof the enzyme active site. This mutation places a second H residue within 5 Šof an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG, and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained the ability to reduce SeO42-. This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.

Gold-Promoted Biocompatible Selenium Arylation of Small Molecules, Peptides and Proteins.

A low pKa (5.2), high polarizable volume (3.8 Å), and proneness to oxidation under ambient conditions make selenocysteine (Sec, U) a unique, natural reactive handle present in most organisms across all domains of life. Sec modification still has untapped potential for site-selective protein modification and probing. Herein we demonstrate the use of a cyclometalated gold(III) compound, [Au(bnpy)Cl2 ], in the arylation of diselenides of biological significance, with a scope covering small molecule models, peptides, and proteins using a combination of multinuclear NMR (including 77 Se NMR), and LC-MS. Diphenyl diselenide (Ph-Se)2 and selenocystine, (Sec)2 , were used for reaction optimization. This approach allowed us to demonstrate that an excess of diselenide (Au/Se-Se) and an increasing water percentage in the reaction media enhance both the conversion and kinetics of the C-Se coupling reaction, a combination that makes the reaction biocompatible. The C-Se coupling reaction was also shown to happen for the diselenide analogue of the cyclic peptide vasopressin ((Se-Se)-AVP), and the Bos taurus glutathione peroxidase (GPx1) enzyme in ammonium acetate (2 mM, pH=7.0). The reaction mechanism, studied by DFT revealed a redox-based mechanism where the C-Se coupling is enabled by the reductive elimination of the cyclometalated Au(III) species into Au(I).

Induction of ferroptosis by photodynamic therapy and enhancement of antitumor effect with ferroptosis inducers.

BACKGROUND: Photodynamic therapy (PDT) is an effective tumor treatment that involves the administration of a photosensitizer to generate cytotoxic 1O2 [reactive oxygen species (ROS)] from molecular oxygen that is produced from energy absorption following tumor irradiation at specific wavelengths. Ferroptosis is induced by the disruption of the glutathione peroxidase 4 (GPX4) antioxidant system, leading to lipid peroxidation. We hypothesized that talaporfin sodium-photodynamic therapy (TS-PDT)-generated ROS would lead to ferroptosis via accumulation of lipid peroxidation. METHODS: Cell viability assay in TS-PDT-treated cells in combination with a ferroptosis inhibitor (ferrostatin-1: Fer-1) or ferroptosis inducers (imidazole ketone erastin: IKE, Ras-selective lethal 3: RSL3) was performed. Accumulation of lipid peroxidation, GPX4 antioxidant system and cystine/glutamate antiporter (system xc-) activity in TS-PDT-treated cells was investigated. In xenograft mice, the antitumor effect of TS-PDT in combination with ferroptosis inducers (IKE or sorafenib) was examined. RESULTS: TS-PDT-induced cell death was partly suppressed by Fer-1 and accompanied by lipid peroxidation. TS-PDT combined with IKE or RSL3 enhanced the induction of cell death. TS-PDT inhibited cystine uptake activity via system xc-. In vivo, the combination of TS-PDT and ferroptosis inducers (IKE or sorafenib) reduced tumor volume. CONCLUSION: This study found that the mechanism underlying TS-PDT-induced ferroptosis constitutes direct lipid peroxidation by the generated ROS, and the inhibition of system xc-, and that the combination of a ferroptosis inducer with TS-PDT enhances the antitumor effect of TS-PDT. Our findings suggest that ferroptosis-inducing therapies combined with PDT may benefit cancer patients.

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns-/- larvae, and restoration of the zebrafish pronephros function.

N-acetyl-L-cysteine improves the performance of chronic cyclic heat-stressed finisher broilers but has no effect on tissue glutathione levels.

1. It was hypothesised that dietary N-acetyl-L-cysteine (NAC) in feed, as a source of cysteine, could improve the performance of heat-stressed finisher broilers by fostering glutathione (GSH) synthesis. GSH is the most abundant intracellular antioxidant for which the sulphur amino acid cysteine is rate limiting for its synthesis.2. In the first experiment, four levels of NAC: 0, 500, 1000 and 2000 mg/kg were added to a diet with a suboptimal level of sulphur amino acids in the finisher phase. In the second experiment, NAC was compared to other sulphur amino acid sources at equal molar amounts of digestible sulphur amino acids. Birds were allocated to four groups: control, 2000 mg/kg NAC, 1479 mg/kg L-cystine, and 2168 mg/kg Ca-salt of 2-hydroxy-4-(methylthio)butanoic acid. A chronic cyclic heat stress model (temperature was increased to 34°C for 7 h daily) was initiated at 28 d of age.3. In the first experiment, growth performance and feed efficiency in the finisher phase were significantly improved by graded NAC. ADG was 88.9, 92.2, 93.7 and 97.7 g/d, and the feed-to-gain ratio was 2.18, 1.91, 1.85 and 1.81 for the 0, 500, 1000 and 2000 mg/kg NAC treatments, respectively. However, liver and heart GSH levels were not affected by NAC. On d 29, liver gene transcript of cystathionine-beta-synthase like was reduced by NAC, which suggested reduced trans-sulphuration activity. The second experiment showed that L-cystine and Ca-salt of 2-hydroxy-4-(methylthio) butanoic acid were more effective in improving performance than NAC.4. In conclusion, N-acetyl-L-cysteine improved dose-dependently growth and feed efficiency in heat-stressed finishing broilers. However, this was not associated with changes in tissue GSH levels, but more likely worked by sparing methionine and/or NAC's and cysteine's direct antioxidant properties.

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

Sulfur metabolic response in macrophage limits excessive inflammatory response by creating a negative feedback loop.

The excessive inflammatory response of macrophages plays a vital role in the pathogenesis of various diseases. The dynamic metabolic alterations in macrophages, including amino acid metabolism, are known to orchestrate their inflammatory phenotype. To explore a new metabolic pathway that regulates the inflammatory response, we examined metabolome changes in mouse peritoneal macrophages (PMs) in response to lipopolysaccharide (LPS) and found a coordinated increase of cysteine and its related metabolites, suggesting an enhanced demand for cysteine during the inflammatory response. Because Slc7a11, which encodes a cystine transporter xCT, was remarkably upregulated upon the pro-inflammatory challenge and found to serve as a major channel of cysteine supply, we examined the inflammatory behavior of Slc7a11 knockout PMs (xCT-KO PMs) to clarify an impact of the increased cysteine demand on inflammation. The xCT-KO PMs exhibited a prolonged upregulation of pro-inflammatory genes, which was recapitulated by cystine depletion in the culture media of wild-type PMs, suggesting that cysteine facilitates the resolution of inflammation. Detailed analysis of the sulfur metabolome revealed that supersulfides, such as cysteine persulfide, were increased in PMs in response to LPS, which was abolished in xCT-KO PMs. Supplementation of N-acetylcysteine tetrasulfide (NAC-S2), a supersulfide donor, attenuated the pro-inflammatory gene expression in xCT-KO PMs. Thus, activated macrophages increase cystine uptake via xCT and produce supersulfides, creating a negative feedback loop to limit excessive inflammation. Our study highlights the finely tuned regulation of macrophage inflammatory response by sulfur metabolism.

Clinical study on the efficacy and tolerability of an oral supplement based on arginine, l-cystine, zinc and B6 vitamin (Cystiphane®) in patients with telogen effluvium.

BACKGROUND: Telogen effluvium (TE) is a common cause of non-cicatricial hair loss with no treatment-standardized protocol. The aim of our study was to evaluate the efficacy, tolerability, and patient compliance of a treatment with an oral supplement based on arginine, l-cystine, zinc and B6 vitamin (Cystiphane®, Laboratoires Bailleul, Geneva, Switzerland) with hair-growth properties, administered 4 times daily, in patients affected by TE. METHODS: We recruited 20 patients, aged between 18 and 70 years old, affected by TE. Patients were asked to take the oral supplement as a monotherapy, four tablets daily, in one or two administrations during meals. The study lasted 3 months. We evaluated the efficacy and tolerability of the treatment both qualitatively by collecting the clinician's opinion through a clinical evaluation and clinical-anamnestic form filled in by the researcher, and quantitatively through global photography and trichoscopy. We collected the patient's opinion through a self-assessment test, at the beginning of the recruitment and after 3 months of treatment. RESULTS: Eighteen patients were evaluated. After 3 months of taking the supplement, the researcher rated an average improvement of 2.89 at the clinical evaluation. For what concerns hair quantity, at the control trichoscopy, the mean trichoscopic value had risen to +2.055, whereas for hair diameter the mean trichoscopic diameter value had increased to +1.83. After 3 months of treatment, patients gave an average efficacy opinion of 3.61. CONCLUSIONS: The oral supplement has proved effective as an adjuvant in the treatment of TE in our cohort of patients.

SLC43A2 and NFκB signaling pathway regulate methionine/cystine restriction-induced ferroptosis in esophageal squamous cell carcinoma via a feedback loop.

Studies have indicated dietary restriction of methionine/cystine provided a therapeutic benefit in diseases such as cancer. However, the molecular and cellular mechanisms that underlie the interaction between methionine/cystine restriction (MCR) and effects on esophageal squamous cell carcinoma (ESCC) have remained elusive. Here, we discovered the dietary restriction of methionine/cystine has a large effect on cellular methionine metabolism as assayed in a ECA109 derived xenograft model. RNA-seq and enrichment analysis suggested the blocked tumor progression was affected by ferroptosis, together with the NFκB signaling pathway activation in ESCC. Consistently, GSH content and GPX4 expression were downregulated by MCR both in vivo and in vitro. The contents of Fe2+ and MDA were negatively correlated with supplementary methionine in a dose-dependent way. Mechanistically, MCR and silent of SLC43A2, a methionine transporter, diminished phosphorylation of IKKα/ß and p65. Blocked NFκB signaling pathway further decreased the expression of SLC43A2 and GPX4 in both mRNA and protein level, which in turn downregulated the methionine intake and stimulated ferroptosis, respectively. ESCC progression was inhibited by enhanced ferroptosis and apoptosis and impaired cell proliferation. In this study, we proposed a novel feedback regulation mechanism underlie the correlation between dietary restriction of methionine/cystine and ESCC progression. MCR blocked cancer progression via stimulating ferroptosis through the positive feedback loop between SLC43A2 and NFκB signaling pathways. Our results provided the theoretical basis and new targets for ferroptosis-based clinical antitumor treatments for ESCC patients.

Methionine supplementation spares body protein by regulating the expression of mTORC1 downstream factors in rats fed a soy protein diet with sufficient sulfur amino acids: a pilot study.

Dietary supplementation with methionine and threonine spares body protein in rats fed a low protein diet, but the effect is not observed for other essential amino acids. Although the requirement for sulfur amino acids is relatively high in rodents, the precise mechanisms underlying protein retention are not fully understood. The aim of this study was to explore whether the activation of mammalian target of rapamycin complex 1 (mTORC1) downstream factors in skeletal muscle by supplementation with threonine and/or methionine contributes to protein retention under sufficient cystine requirement. Male Sprague-Dawley rats were freely fed a 0% protein diet for 2 weeks. These experimental rats were then fed a restricted diet (14.5 g/day) containing 12% soy protein supplemented with both cystine and, methionine and threonine (MT), methionine (M), threonine (T), or neither (NA) (n = 8) for an additional 12 days. Two additional groups were freely fed a diet containing 0% protein or 20% casein as controls (n = 6). Body weight and gastrocnemius muscle weight were higher, and blood urea nitrogen and urinary nitrogen excretion were lower, in the M and MT groups than in the T and NA groups, respectively. p70 S6 kinase 1 abundance was higher, and eukaryotic translation initiation factor 4E-binding protein 1 abundance and mRNA levels were lower, in the skeletal muscles of the M and MT groups. These results suggest that methionine regulates mTORC1 downstream factors in skeletal muscle, leading to spare body protein in rats fed a low protein diet meeting cystine requirements.

Effects of Dietary Cystine and Tyrosine Supplementation on Melanin Synthesis in the Pacific Oyster (Crassostrea gigas).

Melanogenesis is a multistep process to produce melanin for dark pigmentation in skin coloration. Previous studies in vertebrates demonstrated that cystine and tyrosine amino acids are involved in the melanin synthesis. However, very little is known about the melanogenesis in bivalve. In this study, cystine supplementation for 30 days significantly upregulated the expression of CgB-aat1, CgCbs and CgTyr and pheomelanin content in the Pacific oyster Crassostrea gigas. Transmission electron microscope (TEM) results revealed more melanosomes in the connective tissue and melanin granules were secreted in epithelium of mantle. In contrast, tyrosine supplementation had no clear effect on melanogenesis except the gene expression changes of CgB-aat1 and CgCbs. In addition, prolonged supplementation of cystine or tyrosine for 60 days had a negative impact on melanogenesis. Indeed, after 60 days, expression of most of the melanin synthesis-related genes under study was decreased, and melanin content was significantly reduced, indicating that cystine and tyrosine might inhibit production of eumelanin and pheomelanin, respectively. In addition, in vitro analysis using primary cell culture from mantle tissue indicated that incubation with cystine, tyrosine, or B-AAT1 polypeptide, CBS/TYR recombinant proteins induced the increase of CgB-aat1 and CgCbs expression in a dose-dependent manner, suggesting the presence of a regulatory network in response to cystine and tyrosine amino acids intakes in pheomelanin synthesis-related gene expression. Taken together, these data indicate that cystine-CgB-aat1-CgCbs-CgTyr axis is a potential regulator of the pheomelanin biosynthesis pathway, and thus plays an important role in the mantle pigmentation in C. gigas. This work provides a new clue for selective cultivation of oyster strains with specific shell colors in bivalve breeding.

Cystine/Glutamate Xc- Antiporter Induction Compensates for Transsulfuration Pathway Repression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) to Ensure Cysteine for Hepatic Glutathione Biosynthesis.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been associated with the induction of oxidative stress and the progression of steatosis to steatohepatitis with fibrosis. It also disrupts metabolic pathways including one-carbon metabolism (OCM) and the transsulfuration pathway with possible consequences on glutathione (GSH) levels. In this study, complementary RNAseq and metabolomics data were integrated to examine the hepatic transsulfuration pathway and glutathione biosynthesis in mice following treatment with TCDD every 4 days for 28 days. TCDD dose-dependently repressed hepatic cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA and protein levels. Reduced CBS and CTH levels are also correlated with dose-dependent decreases in hepatic extract hydrogen sulfide (H2S). In contrast, cysteine levels increased consistent with the induction of Slc7a11, which encodes for the cystine/glutamate Xc- antiporter. Cotreatment of primary hepatocytes with sulfasalazine, a cystine/glutamate Xc- antiporter inhibitor, decreased labeled cysteine incorporation into GSH with a corresponding increase in TCDD cytotoxicity. Although reduced and oxidized GSH levels were unchanged following treatment due to the induction of GSH/GSSG efflux transporter by TCDD, the GSH:GSSG ratio decreased and global protein S-glutathionylation levels in liver extracts increased in response to oxidative stress along with the induction of glutamate-cysteine ligase catalytic subunit (Gclc), glutathione synthetase (Gss), glutathione disulfide reductase (Gsr), and glutathione transferase π (Gstp). Furthermore, levels of ophthalmic acid, a biomarker of oxidative stress indicating GSH consumption, were also increased. Collectively, the data suggest that increased cystine transport due to cystine/glutamate Xc- antiporter induction compensated for decreased cysteine production following repression of the transsulfuration pathway to support GSH synthesis in response to TCDD-induced oxidative stress.

The effects of transitioning from immediate release to extended release cysteamine therapy in Norwegian patients with nephropathic cystinosis: a retrospective study.

BACKGROUND: Nephropathic cystinosis is a rare lysosomal storage disorder in which accumulation of cystine and formation of crystals particularly impair kidney function and gradually lead to multi-organ dysfunction. Lifelong therapy with the aminothiol cysteamine can delay the development of kidney failure and the need for transplant. The purpose of our long-term study was to explore the effects of transitioning from immediate release (IR) to extended release (ER) formulation in Norwegian patients in routine clinical care. METHODS: We retrospectively analysed data on efficacy and safety in 10 paediatric and adult patients. Data were obtained from up to 6 years before and 6 years after transitioning from IR- to ER-cysteamine. RESULTS: Mean white blood cell (WBC) cystine levels remained comparable between the different treatment periods (1.19 versus 1.38 nmol hemicystine/mg protein) although most patients under ER-cysteamine underwent dose reductions. For the non-transplanted patients, the mean estimated glomerular filtration rate (eGFR) change/year was more pronounced during ER-treatment (- 3.39 versus - 6.80 ml/min/1.73 m2/year) possibly influenced by individual events, such as tubulointerstitial nephritis and colitis. Growth measured by Z-height score tended to develop positively. Four of seven patients reported improvement of halitosis, one reported unchanged and two reported worsened symptoms. Most adverse drug reactions (ADRs) were of mild severity. One patient developed two serious ADRs and switched back to IR-formulation. CONCLUSIONS: The results from this long-term retrospective study indicate that switching from IR- to ER-cysteamine was feasible and well tolerated under routine clinical practice. ER-cysteamine allowed satisfactory disease control over the long period considered. A higher resolution version of the Graphical abstract is available as Supplementary information.

Contribution of NRF2 to sulfur metabolism and mitochondrial activity.

NF-E2-related factor 2 (NRF2) plays a crucial role in the maintenance of cellular homeostasis by regulating various enzymes and proteins that are involved in the redox reactions utilizing sulfur. While substantial impacts of NRF2 on mitochondrial activity have been described, the precise mechanism by which NRF2 regulates mitochondrial function is still not fully understood. Here, we demonstrated that NRF2 increased intracellular persulfides by upregulating the cystine transporter xCT encoded by Slc7a11, a well-known NRF2 target gene. Persulfides have been shown to play an important role in mitochondrial function. Supplementation with glutathione trisulfide (GSSSG), which is a form of persulfide, elevated the mitochondrial membrane potential (MMP), increased the oxygen consumption rate (OCR) and promoted ATP production. Persulfide-mediated mitochondrial activation was shown to require the mitochondrial sulfur oxidation pathway, especially sulfide quinone oxidoreductase (SQOR). Consistently, NRF2-mediated mitochondrial activation was also dependent on SQOR activity. This study clarified that the facilitation of persulfide production and sulfur metabolism in mitochondria by increasing cysteine availability is one of the mechanisms for NRF2-dependent mitochondrial activation.

N-acetylcysteine improves impulse control and attenuates relapse-like alcohol intake in long-term drinking rats.

Increasing evidence suggests that individuals with alcohol use disorder (AUD) present with a disrupted glutamatergic system that underlies core components of addictive disorders, including drug relapse and low impulse control. N-acetylcysteine (NAC) is a cystine prodrug that has been found to promote glutamate homeostasis and drug abstinence. However, no studies to date have evaluated NAC's effect on impulsivity in substance use disorders. Here we determined whether NAC would decrease alcohol-intake behaviors, in addition to improving impulse control, in long-term alcohol drinking male Wistar-Han rats. Before the start of the experiments, all rats were exposed to long-term intermittent access to 20% ethanol for at least seven weeks. Next, in different groups of rats, the effect of NAC (60 and/or 90 mg/kg) was evaluated on (i) voluntary alcohol drinking using a two-bottle free choice paradigm, (ii) the motivation to self-administer alcohol under a progressive ratio schedule of reinforcement, and (iii) relapse-like drinking using the alcohol deprivation effect model. Finally, (iv) NAC's effect on impulse control was evaluated using the five-choice serial reaction time task. Results showed that NAC administration at 90 mg/kg significantly reduced relapse-like drinking and improved impulse control. In contrast, NAC had no effect on levels of alcohol drinking or motivation to drink alcohol. In conclusion, our findings continue to support the use of NAC as an adjuvant treatment for the maintenance of abstinence in AUD. Moreover, we provide evidence for NAC's efficacy in improving impulse control following drinking, which warrants further investigation in substance use settings.