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.

Cystine reduces mitochondrial dysfunction in C2C12 myotubes under moderate oxidative stress induced by H2O2.

Moderate oxidative stress induces temporal impairment in mitochondrial ATP production. As glutathione (GSH) content is reduced to eliminate oxidative stress by oxidation-reduction reaction, intracellular GSH content is crucial for maintaining mitochondrial function under oxidative stress. GSH precursors such as N-acetyl cysteine (NAC) and cysteine are known to suppress oxidative stress based on the supply of cysteine residues being rate-limiting for GSH synthesis. However, it remains unclear whether cystine (Cys2) can suppress mitochondrial dysfunction under oxidative stress conditions. Therefore, we examined whether Cys2 could attenuate mitochondrial dysfunction under moderate oxidative stress without scavenging reactive oxygen species (ROS) in the medium. C2C12 myotubes were incubated for 120 min in a Cys2-supplemented medium and subsequently exposed to hydrogen peroxide (H2O2). Heme oxygenase-1 (HO-1) gene expression, intracellular cysteine and GSH content, intracellular ATP level, and maximal mitochondrial respiration were assessed. Cys2 treatment significantly increased GSH content in a dose-dependent manner under oxidative stress. Cys2 treatment significantly decreased HO-1 expression induced by H2O2 exposure. In addition, maximal mitochondrial respiration rate was decreased by H2O2 exposure, but improved by Cys2 treatment. In conclusion, Cys2 treatment mitigates oxidative stress-induced mitochondrial dysfunction by maintaining GSH content under moderate oxidative stress without scavenging ROS in the medium.

Effects of oral cystine and glutamine on exercise-induced changes in gastrointestinal permeability and damage markers in young men.

PURPOSE: Although acute prolonged strenuous exercise has been shown to increase markers of gastrointestinal permeability and damage, little is known regarding the efficacy of nutritional supplement interventions on the attenuation of exercise-induced gastrointestinal syndrome. This study addressed the effects of oral amino acid supplementation on markers of gastrointestinal permeability and damage in response to exercise. METHODS: Sixteen active men aged 22.7 ± 2.6 years (mean ± standard deviation) completed placebo or cystine and glutamine supplementation trials in random order. Participants received either a placebo or cystine and glutamine supplements, three times a day for 5 days, separated by a 2-week washout period. On day 6, participants took their designated supplements 30 min before running at a speed corresponding to 75% of maximal oxygen uptake for 1 h, followed by a 4-h rest period. Blood samples were collected pre-exercise, immediately post-exercise, 30 min post-exercise, and 1, 2 and 4 h post-exercise on day 6. The plasma lactulose to mannitol ratio (L:M) and plasma intestinal fatty acid-binding protein (I-FABP) were used as markers of gastrointestinal permeability and damage, respectively. RESULTS: Plasma L:M (linear mixed model, coefficient ± standard error: - 0.011 ± 0.004, P = 0.0090) and changes (i.e., from pre-exercise) in plasma I-FABP (linear mixed model, - 195.3 ± 65.7 coefficient ± standard error (pg/mL), P = 0.0035) were lower in the cystine and glutamine supplementation trial than in the placebo trial. CONCLUSION: Oral cystine and glutamine supplementation attenuated the markers of gastrointestinal permeability and damage after 1 h of strenuous running in young men. TRIAL REGISTRATION NUMBER: UMIN000026008. DATE OF REGISTRATION: 13 December 2018.

Cystine reduces tight junction permeability and intestinal inflammation induced by oxidative stress in Caco-2 cells.

Intestinal oxidative stress produces pro-inflammatory cytokines, which increase tight junction (TJ) permeability, leading to intestinal and systemic inflammation. Cystine (Cys2) is a substrate of glutathione (GSH) and inhibits inflammation, however, it is unclear whether Cys2 locally improves intestinal barrier dysfunction. Thus, we investigated the local effects of Cys2 on oxidative stress-induced TJ permeability and intestinal inflammatory responses. Caco-2 cells were cultured in a Cys2-supplemented medium for 24 h and then treated with H2O2 for 2 h. We assessed TJ permeability by measuring transepithelial electrical resistance and the paracellular flux of fluorescein isothiocyanate-dextran 4 kDa. We measured the concentration of Cys2 and GSH after Cys2 pretreatment. The mRNA expression of pro-inflammatory cytokines was assessed. In addition, the levels of TJ proteins were assessed by measuring the expression of TJ proteins in the whole cells and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions (IS/S ratio). Cys2 treatment reduced H2O2-induced TJ permeability. Cys2 did not change the expression of TJ proteins in the whole cells, however, suppressed the IS/S ratio of claudin-4. Intercellular levels of Cys2 and GSH significantly increased in cells treated with Cys2. Cys2 treatment suppressed the mRNA expression of pro-inflammatory cytokines, and the mRNA levels were significantly correlated with TJ permeability. In conclusion, Cys2 treatment locally reduced oxidative stress-induced intestinal barrier dysfunction possively due to the mitigation of claudin-4 dislocalization. Furthermore, the effect of Cys2 on the improvement of intestinal barrier function is related to the local suppression of oxidative stress-induced pro-inflammatory responses.

D-Cystine di(m)ethyl ester reverses the deleterious effects of morphine on ventilation and arterial blood gas chemistry while promoting antinociception.

We have identified thiolesters that reverse the negative effects of opioids on breathing without compromising antinociception. Here we report the effects of D-cystine diethyl ester (D-cystine diEE) or D-cystine dimethyl ester (D-cystine diME) on morphine-induced changes in ventilation, arterial-blood gas chemistry, A-a gradient (index of gas-exchange in the lungs) and antinociception in freely moving rats. Injection of morphine (10 mg/kg, IV) elicited negative effects on breathing (e.g., depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive). Subsequent injection of D-cystine diEE (500 µmol/kg, IV) elicited an immediate and sustained reversal of these effects of morphine. Injection of morphine (10 mg/kg, IV) also elicited pronounced decreases in arterial blood pH, pO2 and sO2 accompanied by pronounced increases in pCO2 (all indicative of a decrease in ventilatory drive) and A-a gradient (mismatch in ventilation-perfusion in the lungs). These effects of morphine were reversed in an immediate and sustained fashion by D-cystine diME (500 µmol/kg, IV). Finally, the duration of morphine (5 and 10 mg/kg, IV) antinociception was augmented by D-cystine diEE. D-cystine diEE and D-cystine diME may be clinically useful agents that can effectively reverse the negative effects of morphine on breathing and gas-exchange in the lungs while promoting antinociception. Our study suggests that the D-cystine thiolesters are able to differentially modulate the intracellular signaling cascades that mediate morphine-induced ventilatory depression as opposed to those that mediate morphine-induced antinociception and sedation.

Cyst(e)ine in nutrition formulation promotes colon cancer growth and chemoresistance by activating mTORC1 and scavenging ROS.

Weight loss and cachexia are common problems in colorectal cancer patients; thus, parenteral and enteral nutrition support play important roles in cancer care. However, the impact of nonessential amino acid components of nutritional intake on cancer progression has not been fully studied. In this study, we discovered that gastrointestinal cancer patients who received cysteine as part of the parenteral nutrition had shorter overall survival (P < 0.001) than those who did not. Cystine indeed robustly promotes colon cancer cell growth in vitro and in immunodeficient mice, predominately by inhibiting SESN2 transcription via the GCN2-ATF4 axis, resulting in mTORC1 activation. mTORC1 inhibitors Rapamycin and Everolimus block cystine-induced cancer cell proliferation. In addition, cystine confers resistance to oxaliplatin and irinotecan chemotherapy by quenching chemotherapy-induced reactive oxygen species via synthesizing glutathione. We demonstrated that dietary deprivation of cystine suppressed colon cancer xenograft growth without weight loss in mice and boosted the antitumor effect of oxaliplatin. These findings indicate that cyst(e)ine, as part of supplemental nutrition, plays an important role in colorectal cancer and manipulation of cyst(e)ine content in nutritional formulations may optimize colorectal cancer patient survival.

Cystine supplementation rebalances the redox homeostasis of microenvironment in non-small cell lung cancer cells and reverses their resistance to docetaxel.

Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 µg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.

Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets.

The regulation of glycerol permeability in the gastrointestinal tract is crucial to control fat deposition, lipolysis and gluconeogenesis. Knowing that the amino acid glutamine is a physiological regulator of gluconeogenesis, whereas cystine promotes adiposity, herein we investigated the effects of dietary supplementation with glutamine and cystine on the serum biochemical parameters of piglets fed on amino acid-enriched diets, as well as on the transcriptional profile of membrane water and glycerol channels aquaporins (AQPs) in the ileum portion of the small intestine and its impact on intestinal permeability. Twenty male piglets with an initial body weight of 8.8 ± 0.89 kg were allocated to four dietary treatments (n = 5) and received, during a four week-period, a basal diet without supplementation (control) or supplemented with 8 kg/ton of glutamine (Gln), cystine (Cys) or the combination of the two amino acids in equal proportions (Gln + Cys). Most biochemical parameters were found improved in piglets fed Gln and Cys diet. mRNA levels of AQP3 were found predominant over the others. Both amino acids, individually or combined, were responsible for a consistent downregulation of AQP1, AQP7 and AQP10, without impacting on water permeability. Conversely, Cys enriched diet upregulated AQP3 enhancing basolateral membranes glycerol permeability and downregulating glycerol kinase (GK) of intestinal cells. Altogether, our data reveal that amino acids dietary supplementation can modulate intestinal AQPs expression and unveil AQP3 as a promising target for adipogenesis regulation.

Oral administration of Cystine and Theanine ameliorates oxaliplatin-induced chronic peripheral neuropathy in rodents.

Oxaliplatin frequently causes severe peripheral neuropathy as a dose-limiting toxicity. However, this toxicity lacks a strategy for prevention. Cystine/Theanine is a supplement, which includes precursors for the biosynthesis of glutathione. In this study, we investigated the effects of Cystine/Theanine on oxaliplatin-induced peripheral neuropathy using an in vivo model. Repeated injection of oxaliplatin (4 mg/kg intraperitoneally twice a week for 2 weeks) caused mechanical allodynia, cold hyperalgesia and axonal degeneration of the sciatic nerve in rats. Mechanical allodynia and axonal degeneration, but not cold hyperalgesia, were ameliorated by daily co-administration of Cystine [200 mg/kg orally (p.o.)] and Theanine (80 mg/kg p.o.). Moreover, co-administration of Cystine and Theanine to rats significantly increased the glutathione level in the sciatic nerve compared with the oxaliplatin group. Furthermore, Cystine and Theanine did not attenuate the tumour cytotoxicity of oxaliplatin in C-26 tumour cell-bearing mice. These findings suggest that Cystine and Theanine may be beneficial for preventing oxaliplatin-induced peripheral neuropathy.

Dietary Cystine Ameliorates Defects in Spermatogenesis via Testosterone Production Induced by Protein Deficiency and Darkness in Rats.

Nutrition and light-dark cycle influence rat testicular development. With 9% casein diet (low protein diet) under normal 12 h-12 h lighting cycles (9P), juvenile rat testes undergo normal growth. On the other hand, a low protein diet with constant darkness (D9P) results in a growth arrest of rat testes. Supplementation of cystine to the low protein diet under constant darkness (D9PC) had a tendency to increase testes weight, suggesting an improvement in growth suppression. Whether the growth suppression of testes in D9P is associated with suppression of spermatogenesis has not yet been shown. We aimed to determine the effect of a low protein diet and constant darkness with or without dietary cystine in testes using a histological technique. In the histological assessment, D9P testes showed a decreased number of seminiferous tubules with elongated spermatids, indicating a functional testicular defect in this group. However, cystine supplementation resulted in enhanced spermatogenesis versus control animals (D9PC vs. D9P) implying the importance of cystine to testicular development in this condition. Furthermore, serum testosterone concentration was increased in D9PC suggesting contribution of testosterone to ameliorate spermatogenesis. From these results, we conclude that cystine supplementation to a low protein diet under constant darkness promoted an increase in testosterone which in turn benefitted spermatogenesis.

A general covalent binding model between cytotoxic selenocompounds and albumin revealed by mass spectrometry and X-ray absorption spectroscopy.

Selenocompounds (SeCs) are promising therapeutic agents for a wide range of diseases including cancer. The treatment results are heterogeneous and dependent on both the chemical species and the concentration of SeCs. Moreover, the mechanisms of action are poorly revealed, which most probably is due to the detection methods where the quantification is based on the total selenium as an element. To understand the mechanisms underlying the heterogeneous cytotoxicity of SeCs and to determine their pharmacokinetics, we investigated selenium speciation of six SeCs representing different categories using liquid chromatography-mass spectrometry (LC-MS) and X-ray absorption spectroscopy (XAS) and the cytotoxicity using leukemic cells. SeCs cytotoxicity was correlated with albumin binding degree as revealed by LC-MS and XAS. Further analysis corroborated the covalent binding between selenol intermediates of SeCs and albumin thiols. On basis of the Se-S model, pharmacokinetic properties of four SeCs were for the first time profiled. In summary, we have shown that cytotoxic SeCs could spontaneously transform into selenol intermediates that immediately react with albumin thiols through Se-S bond. The heterogeneous albumin binding degree may predict the variability in cytotoxicity. The present knowledge will also guide further kinetic and mechanistic investigations in both experimental and clinical settings.

Twendee X Ameliorates Phosphorylated Tau, α-Synuclein and Neurovascular Dysfunction in Alzheimer's Disease Transgenic Mice With Chronic Cerebral Hypoperfusion.

BACKGROUND: The pathological impact of chronic cerebral hypoperfusion (CCH) on Alzheimer's disease (AD) is still poorly understood. In the present study, we investigated the role of CCH on an AD mouse model in phosphorylated tau and α-synuclein pathology, neurovascular unit, cerebrovascular remodeling, and neurovascular trophic coupling. Moreover, examined protective effect of a new antioxidant Twendee X (TwX). METHODS: APP23 mice were implanted to bilateral common carotid arteries stenosis with ameroid constrictors to gradually decrease the cerebral blood flow. The effects of the administration of TwX were evaluated by immunohistochemical analysis and Immunofluorescent histochemistry. RESULTS: The present study revealed that the expressions of phospho-tau and phospho-α-synuclein were significantly increased in the APP23 + CCH mice group as compared with wild type and APP23 mice groups (*P < .05 and ⁎⁎P < .01 versus WT; #P < .05 and ##P < .01 versus APP23). In addition, CCH significantly exacerbated MMP-9 activation relating to blood-brain barrier destruction (⁎⁎P < .01 versus WT; #P < .05, and ##P < .01 versus APP23), enhanced neurovascular remodeling, and impaired a neurovascular trophic coupling in the vascular endothelial BDNF expression of the APP23 + CCH group. TwX treatment (20 mg/kg/day, from 4.5 to 12 months) significantly reduced tau and α-synuclein pathologies, ameliorated neurovascular dysfunction compared with APP23 + CCH group. CONCLUSIONS: Our findings indicate that administration of a new antioxidative mixture TwX substantially reduced the above neuropathologic abnormalities, suggesting a potential therapeutic benefit of TwX for AD with CCH.

Cysteine/cystine-rich undenatured whey protein supplement in patients' pressure ulcers outcomes: an open label study.

OBJECTIVE: The prevalence and costs associated with treating pressure ulcers (PU) are at high levels. Frequently, PUs heal slowly or not at all, which may be due to the patient's catabolic state which may include protein energy malnutrition. The objective of this open label clinical trial was to improve healing rates by providing patients with a patented, high-quality protein containing all essential amino acids to ensure positive nitrogen balance. An additional benefit of this protein is the delivery of bioavailable cysteine (cystine) to promote glutathione (GSH) synthesis which supports immune function and heightens antioxidant defences. METHODS: Patients with category II, III and IV PUs were fed 20g BID whey protein dietary supplement for 16-120 days, without change in ongoing 'best practice' PU management and their progress recorded. RESULTS: A total of 10 patients were recruited, with an average age of 77 years. Most had shown no improvement in healing for ≥2 months before treatment and usually had other complications including chronic obstructive pulmonary disease (COPD), diabetes and various cardiovascular diseases. There were a total of 23 PUs, with some patients having more than one. Of these, 44% (n=10) showed complete resolution 83% (n=19) had better than 75% resolution over the observation period. Healing rates ranged from 16.9-0.2cm2/month (healed PUs) and 60.0-1.6cm2/month for resolving PUs. CONCLUSION: By providing the necessary amino acids to rebuild tissues and bioactive cysteine (cystine) to promote synthesis of intracellular GSH and positive nitrogen balance, improvement in PUs healing was achieved.

The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoa†.

Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P < 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation-reduction potential of spermatozoa, with sulfasalazine only preventing this effect on fresh spermatozoa that were incubated for 3 h at 37°C, but not in frozen-thawed spermatozoa. While sulfasalazine reduced the motility of frozen-thawed spermatozoa, it increased motility in fresh samples. The present findings provide new and relevant data on the mechanism regulating the redox status of spermatozoa and suggest that a different redox regulatory mechanism exists in cryopreserved spermatozoa, thus providing new clues to improve current cryopreservation technologies and treat male factor infertility.

Clinical and Pathological Benefit of Twendee X in Alzheimer's Disease Transgenic Mice with Chronic Cerebral Hypoperfusion.

BACKGROUND: Multiple pathogeneses are involved in Alzheimer's disease (AD), such as amyloid-ß accumulation, neuroinflammation, and oxidative stress. The pathological impact of chronic cerebral hypoperfusion on Alzheimer's disease is still poorly understood. METHODS: APP23 mice were implanted to bilateral common carotid arteries stenosis with ameroid constrictors for slowly progressive chronic cerebral hypoperfusion (CCH). The effects of the administration of Twendee X (TwX) were evaluated by behavioral analysis, immunohistochemical analysis, and immunofluorescent histochemistry. RESULTS: In the present study, chronic cerebral hypoperfusion, which is commonly found in aged Alzheimer's disease, significantly exacerbated motor dysfunction of APP23 mice from 5 months and cognitive deficit from 8 months of age, as well as neuronal loss, extracellular amyloid-ß plaque and intracellular oligomer formations, and amyloid angiopathy at 12 months. Severe upregulations of oxidative markers and inflammatory markers were found in the cerebral cortex, hippocampus, and thalamus at 12 months. Twendee X treatment (20 mg/kg/d, from 4.5 to 12 months) substantially rescued the cognitive deficit and reduced the above amyloid-ß pathology and neuronal loss, alleviated neuroinflammation and oxidative stress. CONCLUSIONS: The present findings suggested a potential therapeutic benefit of Twendee X for Alzheimer's disease with chronic cerebral hypoperfusion.

Dietary cystine is important to maintain plasma mercaptalbumin levels in rats fed low-protein diets.

The oxidized/reduced state of plasma albumin in rats is influenced by the quantity of dietary protein. However, the effects of the protein quality on the oxidized/reduced state of plasma albumin are not clear. We hypothesized that the quality of dietary protein might modulate the oxidized/reduced state of plasma albumin. The aim of the present study was to examine whether the amino acid composition of dietary protein modulates the oxidized/reduced state of plasma albumin in rats. Male Sprague-Dawley rats were fed low-protein diets containing 5% casein (CA), 5% egg white (EW), or 6% wheat gluten (WG) for 2 weeks. The plasma albumin concentration gradually decreased in rats fed each diet; however, there was no significant difference among the groups. In rats fed the 5% CA diet, the percentage of mercaptalbumin within the total plasma albumin was significantly lower than in those fed the EW or WG diet. Compared with EW or WG, CA contains lower amounts of glycine and cystine. In rats fed a 5% CA diet supplemented with cystine, the percentage of mercaptalbumin was significantly higher than that in rats fed a 5% CA diet supplemented with glycine. The expression of hepatic eukaryotic initiation factor 4E-binding protein 1 was significantly lower in rats fed the cystine-supplemented diet than in those fed the glycine-supplemented diet. These results suggest that dietary protein with a high cystine content maintains plasma mercaptalbumin levels in rats fed low-protein diets.

Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.

Non-frozen preservation protocols for mature mouse oocytes dramatically extend their developmental competence by reducing oxidative stress.

The objective of this study was to test whether aging induces oxidative stress (OS) during oocyte preservation at different temperatures and whether the oocyte competence can be extended by antioxidant supplementation. The increase in activation susceptibility was efficiently prevented when oocytes were preserved at 37°C for 9 h in HCZB medium with 10.27 mM pyruvate and 10 µM α-tocopherol, at 25°C for 30 h with 20.27 mM pyruvate, and at 15°C for 96 h and at 5°C for 48 h with 10.27 mM pyruvate. Satisfactory blastocyst development was achieved after oocyte preservation at 37°C for 9 h, at 25°C for 30 h, at 15°C for 48 h and at 5°C for 24 h using the above protocols but with cysteamine/cystine supplementation. Transfer of blastocysts obtained from the above protocols showed no difference in pregnancy outcome between newly ovulated and preserved oocytes. Because oocytes preserved at 15°C for 48 h were fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 54 h. Assays for ROS and glutathione indicated that in vitro preservation caused marked OS in oocytes. In conclusion, marked OS was observed following in vitro preservation of mature oocytes at different temperatures. Whereas any protocol that reduced OS could inhibit activation susceptibility, only those protocols that decreased OS while increasing glutathione synthesis could sustain oocyte competence.

Developmental potential of prepubertal mouse oocytes is compromised due mainly to their impaired synthesis of glutathione.

Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca(2+) reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca(2+) storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca(2+) store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.

Extracellular cysteine (Cys)/cystine (CySS) redox regulates metabotropic glutamate receptor 5 activity.

Extracellular cysteine (Cys)/cystine (CySS) redox potential (E(h)) has been shown to regulate diverse biological processes, including enzyme catalysis, gene expression, and signaling pathways for cell proliferation and apoptosis, and is sensitive to aging, smoking, and other host factors. However, the effects of extracellular Cys/CySS redox on the nervous system remain unknown. In this study, we explored the role of extracellular Cys/CySS E(h) in metabotropic glutamate receptor 5 (mGlu5) activation to understand the mechanism of its regulation of nerve cell growth and activation. We showed that the oxidized Cys/CySS redox state (0 mV) in C6 glial cells induced a significant increase in mGlu5-mediated phosphorylation of extracellular signal-regulated kinase (ERK), blocked by an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK), U0126, a nonpermeant alkylating agent, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), and a specific mGlu5 antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), respectively. ERK phosphorylation under oxidized extracellular Cys/CySS E(h) was confirmed in mGlu5-overexpressed human embryonic kidney 293 (HEK293) cells. Oxidized extracellular Cys/CySS E(h) also stimulated the generation of intracellular reactive oxygen species (ROS) involved in the phosphorylation of ERK by mGlu5. Moreover, activation of mGlu5 by oxidized extracellular Cys/CySS E(h) was found to affect expression of NF-κB and inducible nitric oxide synthase (iNOS). The results also showed that extracellular Cys/CySS E(h) involved in the activation of mGlu5 controlled cell death and cell activation in neurotoxicity. In addition, plasma Cys/CySS E(h) was found to be associated with the process of Parkinson's disease (PD) in a rotenone-induced rat model of PD together with dietary deficiency and supplementation of sulfur amino acid (SAA). The effects of extracellular Cys/CySS E(h) on SAA dietary deficiency in the rotenone-induced rat model of PD was almost blocked by MPEP pretreatment, further indicating that oxidized extracellular Cys/CySS E(h) plays a role in mGlu5 activity. Taken together, the results indicate that mGlu5 can be activated by extracellular Cys/CySS redox in nerve cells, which possibly contributes to the process of PD. These in vitro and in vivo findings may aid in the development of potential new nutritional strategies that could assist in slowing the degeneration of PD.