Copper chelation by d-penicillamine alleviates melanocyte death induced by rhododendrol without inhibiting tyrosinase.

Oxidative metabolism of rhododendrol (RD), a skin-whitening ingredient, by tyrosinase has caused leukoderma in a certain population of Japanese consumers. Toxic RD metabolites and reactive oxygen species are proposed causes for the melanocyte death. However, the mechanism by which reactive oxygen species are produced during RD metabolism remains elusive. Some phenolic compounds are known to act as suicide substrates for tyrosinase, resulting in release of a copper atom and hydrogen peroxide during its inactivation. We hypothesized that RD may be a suicide substrate for tyrosinase and that the released copper atom may be responsible for the melanocyte death through hydroxyl radical production. In line with this hypothesis, human melanocytes incubated with RD showed an irreversible decrease in tyrosinase activity and underwent cell death. A copper chelator, d-penicillamine, markedly suppressed the RD-dependent cell death without significantly affecting the tyrosinase activity. Peroxide levels in RD-treated cells were not affected by d-penicillamine. Given the unique enzymatic properties of tyrosinase, we conclude that RD acted as a suicide substrate and resulted in release of a copper atom and hydrogen peroxide, which would collectively impair melanocyte viability. These observations further imply that copper chelation may alleviate chemical leukoderma caused by other compounds.

Consequences of a peroxiredoxin 4 (Prdx4) deficiency on learning and memory in mice.

Peroxiredoxin 4 (Prdx4) is responsible for the oxidative folding of new proteins that are synthesized in the endoplasmic reticulum (ER). It has recently been suggested that increased ER stress is associated with neurodegenerative diseases, including Alzheimer's disease. Prdx4 is widely distributed throughout the brain, and is also expressed in hippocampal neurons and oligodendrocytes, suggesting that it is associated with learning and memory. We previously established Prdx4-knockout (KO) mice but did not examine the behavioral phenotypes. In the present study, we report on the learning and memory abilities of Prdx4-KO mice based on Morris water maze and the Y-maze tests. The findings indicate that Prdx4-KO mice showed a lower spatial memory ability in both tests. In contrast, the results of the open field test indicated that locomotor activity is significantly increased in Prdx4-KO mice. We then performed mRNA analyses of the brains of Prdx4-KO mice and found an increased expression of genes related to the ER-associated degradation (ERAD) mechanism, which is an important protein quality control system for the maintenance of ER homeostasis. Finally, proteomic analyses of the brains of Prdx4-KO mice showed an aberrant expression in the proteins, which have been suggested to be related to calcium homeostasis and synaptogenesis in neurons. Our collective results suggest that the Prdx4 ablation perturbs oxidative protein folding in the ER, thus leading to aberrant ER homeostasis in neuronal cells, ultimately leading to impaired spatial memory formation.

d-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione.

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

Superoxide produced by mitochondrial complex III plays a pivotal role in the execution of ferroptosis induced by cysteine starvation.

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by cysteine starvation or by the inhibition of glutathione peroxidase 4 (GPX4) activity with the accompanying elevation in lipid peroxidation product levels. Despite the central role of mitochondria in oxidative metabolism and hence, as main sources of superoxide, the issue of whether mitochondrial superoxide participates in the execution of ferroptosis remains unclear. To gain additional insights into this issue, we employed suppressors of the site IQ electron leak (S1QEL) and suppressors of the site IIIQo electron leak (S3QEL), small molecules that suppress mitochondrial superoxide production from complex I and III, respectively. The findings indicate that S3QEL, but not S1QEL, significantly protected mouse hepatoma Hepa 1-6 cells from lipid peroxidation and the subsequent ferroptosis induced by cysteine (Cys) starvation (cystine deprivation from culture media or xCT inhibition by erastin). The intracellular levels of Cys and GSH remained low irrespective of life or death. Moreover, S3QEL also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die under conventional cultivating conditions due to the absence of intracellular Cys and GSH. Although it has been reported that erastin induces the hyperpolarization of the mitochondrial membrane potential, no correlation was observed between hyperpolarization and cell death in xCT-knockout cells. Collectively, these results indicate that superoxide production from complex III plays a pivotal role in the ferroptosis that is induced by Cys starvation, suggesting that protecting mitochondria is a promising therapeutic strategy for the treatment of multiple diseases featuring ferroptosis.

Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction.

Ferroptosis is a type of iron-dependent necrotic cell death, which is typically triggered by the depletion of intracellular glutathione (GSH), which is associated with increased lipid peroxidation. Nitric oxide (NO) is a highly reactive gaseous radical mediator with anti-oxidation properties that terminates lipid peroxidation reactions. In the current study, we report the anti-ferroptotic action of NOC18, an NO donor that spontaneously releases NO, in cells under various ferroptotic conditions in vitro. Our results indicate that, when mouse hepatoma Hepa 1-6 cells are incubated with NOC18, cell death induced by various ferroptotic stimuli such as cysteine (Cys) starvation, the inhibition of glutathione peroxidase 4 (GPX4) and treatment with tertiary-butyl hydroperoxide (TBHP) is significantly reduced. Treatment with NOC18 failed to improve the decrease in the levels of Cys or GSH and the accumulation of ferrous iron upon ferroptotic stimuli. The fluorescent intensity of C11-BODIPY581/591, a probe that is used to detect lipid peroxidation products, was increased somewhat by treatment with NOC18 under conditions of Cys starvation, and the accumulation of lipid peroxidation end-products, as evidenced by the levels of 4-hydroxynonenal, were effectively suppressed. The pre-incubation of TBHP with NOC7, a short-lived NO donor completely eliminated its ability to trigger ferroptosis. These collective results indicate that NO exerts a cytoprotective action against various ferroptotic stimuli by aborting the lipid peroxidation chain reaction.

Prion protein interacts with the metabotropic glutamate receptor 1 and regulates the organization of Ca2+ signaling.

Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca2+ signaling. In this study, we evaluated the binding of PrP to metabotropic glutamate receptor 1 (mGluR1) and found that wild-type PrP (PrP-wt) and mGluR1 co-immunoprecipitated in dual-transfected Neuro-2a (N2a) cells. Fluorescence resonance energy transfer analysis revealed an energy transfer between mGluR1-Cerulean and PrP-Venus. In order to determine whether PrP can modulate mGluR1 signaling, we performed Ca2+ imaging analyses following repetitive exposure to an mGluR1 agonist. Agonist stimulation induced synchronized Ca2+ oscillations in cells coexpressing PrP-wt and mGluR1. In contrast, N2a cells expressing PrP-ΔN failed to show ligand-dependent regulation of mGluR1-Ca2+ signaling, indicating that PrP can bind to mGluR1 and modulate its function to prevent irregular Ca2+ signaling and that its N-terminal region functions as a molecular switch during Ca2+ signaling.

Ascorbic acid and CoQ10 ameliorate the reproductive ability of superoxide dismutase 1-deficient female mice†.

Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q10, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44-75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

Type I interferon protects neurons from prions in in vivo models.

Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.

Edaravone, a free radical scavenger, protects against ferroptotic cell death in vitro.

Ferroptosis is characterized by an iron-dependent cell death with increased lipid peroxidation and is typically induced by either a decrease in glutathione (GSH) levels due to an insufficient supply of cysteine (Cys) or the inhibition of phospholipid hydroperoxide glutathione peroxidase (Gpx4). While lipid peroxides are the direct trigger for ferroptosis, the issue of how radical species involve in the cytocidal process remains unclear. To gain insights into this issue, we employed edaravone, a free radical scavenger that is clinically approved for the treatment of acute ischemic strokes and amyotrophic lateral sclerosis (ALS), against ferroptotic cell death caused by various situations, notably under cystine deprivation. We initially investigated the effects of edaravone on ferroptosis in mouse hepatoma Hepa 1-6 cells cultivated in cystine-free medium and found that edaravone largely suppressed ferroptosis. Ferroptosis that was induced in the cells by the use of inhibitors for xCT or Gpx4 was also suppressed by edaravone. Moreover, edaravone also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die in normal cultivating conditions due to the depletion of intracellular Cys and GSH. Although the edaravone treatment had no effects on the intracellular levels of Cys and GSH, both of which remained low in Hepa 1-6 cells under conditions of cystine deprivation, the causative factors for ferroptosis, including ferrous iron and lipid peroxide levels, were significantly suppressed. Collectively, these results indicate that radical species produced at the initial stage of the cytocidal process under Cys-deprived conditions trigger ferroptosis and scavenging these radicals by edaravone represents a promising treatment.

Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide.

Singlet oxygen causes a cytotoxic process in tumor cells in photodynamic therapy (PDT) and skin photoaging. The mechanism responsible for this cytotoxicity is, however, not fully understood. 1-Methylnaphthalene-4-propionate endoperoxide (MNPE) is a cell-permeable endoperoxide that generates pure singlet oxygen. We previously reported that cell death induced by MNPE did not show the typical profile of apoptosis, and the cause of this cell death remains elusive. We report herein on an investigation of the mechanism for MNPE-induced cell death from the view point of ferroptosis. The findings indicate that the MNPE treatment decreased the viabilities of mouse hepatoma Hepa 1-6 cells in vitro, and that this decrease was accompanied by increases in the concentrations of both intracellular ferrous iron and the level of lipid peroxidation, but that the caspase-mediated apoptotic pathway was not activated. The intracellular levels of cysteine and glutathione were not affected by the MNPE treatment. Importantly, an assay of lactate dehydrogenase activity revealed that the cell death caused by MNPE was suppressed by ferrostatin-1, a ferroptosis-specific inhibitor. Collectively, these results strongly indicate that ferroptosis is the main cell death pathway induced by singlet oxygen.

Ascorbic acid insufficiency impairs spatial memory formation in juvenile AKR1A-knockout mice.

AKR1A, an aldo-keto reductase, is involved in the synthesis of ascorbic acid as well as the reduction of a variety of aldehyde compounds. AKR1A-/- mice produce considerably less ascorbic acid (about 10%) compared to AKR1A+/+ mice and require ascorbic acid supplementation in order to breed. To elucidate the roles played by AKR1A in spatial memory, AKR1A-/- male mice were weaned at 4 weeks of age and groups that received ascorbic acid supplementation and no supplementation were subjected to a Morris water maze test. Juvenile AKR1A-/- mice that received no supplementation showed impaired spatial memory formation, even though about 70% of the ascorbic acid remained in the brains of the AKR1A-/- mice at day 7 after weaning. To the contrary, the young adult AKR1A-/- mice at 13-15 weeks of age maintained only 15% of ascorbic acid but showed no significant difference in the spatial memory compared with the AKR1A+/+ mice or ascorbic acid-supplemented AKR1A-/- mice. It is conceivable that juvenile mice require more ascorbic acid for the appropriate level of formation of spatial memory and that maturation of the neural system renders the memory forming process less sensitive to an ascorbic acid insufficiency.

Potential involvement of ubiquitin-proteasome system dysfunction associated with oxidative stress in the pathogenesis of sickle cell disease.

The ubiquitin-proteasome system (UPS) is an important intracellular proteolytic pathway responsible for the degradation of proteins and oxidative damage; hence it plays a central role in maintaining homeostasis of red blood cells (RBCs). The present study investigated the levels of polyubiquitination, the function of proteasomes and effect of hydroxycarbamide (HC) therapy in RBCs from sickle cell disease (SCD) patients. Polyubiquitinated proteins were found to be elevated in untreated SCD (UT-SCD) patients compared to those in HC-treated SCD patients (HC-SCD) and controls. Activities of ß1 and ß2 subunits were a little higher in UT-SCD patients, and much higher proteolytic activities were observed in all three subunits (ß1, ß2 and ß5) of RBCs in HC-SCD patients compared to those of UT-SCD patients and controls, although the protein levels of these subunits remained approximately the same. It is notable that, despite HC therapy, some patients showed persistent complications and accumulation of polyubiquitinated proteins. The enhanced proteasomal activity among HC-treated patients might remove the polyubiquitinated protein and could be one of the important mechanisms of therapeutic action. These findings could be useful to understand the pathophysiology of SCD and its clinical heterogeneity and identify a suitable therapeutic target for the better management of these patients.

Unveiling systemic organ disorders associated with impaired lipid catabolism in fasted SOD1-deficient mice.

Oxidative stress triggers the formation of lipid droplets in the liver by stimulating lipogenesis and simultaneously suppresses lipoprotein secretion under hypernutritional conditions. Herein we report on the observation of systemic organ failure that is associated with lipid droplet accumulation in fasting, SOD1-knockout (KO) mice. Upon a three-day fasting period, the KO mice were observed to be vulnerable, could not be rescued by refeeding and had largely died, while wild-type mice were totally recovered. Visceral fat was rapidly consumed during fasting, which resulted in energy shortage and increased fatality in the KO mice. Lipid droplets had accumulated and continued to remain in KO mouse organs that routinely catalyze fatty acids via ß-oxidation, even though the levels of free fatty acids and ß-hydroxybutyrate, a ketone body, in blood plasma were less in KO mice compared to WT mice during the fasting period. The fasting-triggered organ failure in the KO mice was effectively mitigated by feeding a high calorie-diet for 2 weeks prior to fasting, even though the mice had an excessive accumulation of lipid droplets in the liver. These collective data suggest that the lipid-catabolizing system is the sensitive target of oxidative stress triggered by fasting conditions in the KO mice.

Cystine/glutamate transporter, system xc-, is involved in nitric oxide production in mouse peritoneal macrophages.

The amino acid transport system xc- is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc-, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24-48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.

The viability of primary hepatocytes is maintained under a low cysteine-glutathione redox state with a marked elevation in ophthalmic acid production.

Extracellular cystine, the oxidized form of cysteine (Cys), is taken up by cells via the cystine transporter xCT. xCT is not expressed in the liver but is induced in primary hepatocytes under conventional cultured conditions. However, compared to wild-type hepatocytes those from the xCT-knockout mouse showed no evidence of an abnormality and the levels of both Cys and glutathione (GSH) remained unchanged. The levels of ophthalmic acid (OPT), which is produced as an alternative compound by the GSH-synthesizing pathway, became increased during the culturing of hepatocytes. It therefore appears that, in primary hepatocytes, Cys is provided by systems other than xCT, most likely via the transsulfuration pathway, but the levels that are produced are not sufficient. We also employed mouse hepatoma-derived Hepa1-6 cells, which constitutively express xCT. When Hepa 1-6 cells were cultivated in Cys-free media, the levels of intracellular Cys and GSH were decreased, compared to cells cultured in conventional media, leading to cell death accompanied by an increase in the levels of reactive oxygen species and lipid peroxidation products with characteristics similar to ferroptosis. While OPT levels were increased by only to a limited extent in Hepa 1-6 cells, primary hepatocytes cultured in Cys- and Met-free media showed a marked elevation in OPT, reaching levels nearly equivalent to the GSH levels when the cells were cultured in conventional media. Thus, OPT may become a marker for Cys insufficiency and might be used to predict pathological conditions of cells with elevated oxidative stress.

Heat stress promotes the down-regulation of IRE1α in cells: An atypical modulation of the UPR pathway.

Heat stress induces intracellular protein denaturation and endoplasmic reticulum (ER) stress, which elicits unfolded protein response (UPR) in cells. UPR involves three ER-localized sensor proteins: the inositol-requiring protein 1α (IRE1α), the dsRNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor-6 (ATF6). However, the precise mechanism by which cells deal with heat stress remains to be elucidated. We report herein that heat stress effectively activates all branches of the UPR. While splicing of the XBP-1 mRNA is usually triggered by activated IRE1α, the level of this protein was found to be decreased immediately after the occurrence of heat stress and the down-regulation gradually recovered thereafter. The protein levels of other UPR sensors or ER luminal proteins were unaffected. The down-regulation of IRE1α was independent of cellular viability. It thus appears that the heat-stress induced down-regulation of the IRE1α protein may lead to the termination of the IRE1α signaling pathway in an attempt to avoid excessive responses. We investigated the machinery causing IRE1α down-regulation and found that heat stress effectively promoted the activation of autophagy. Importantly, the inhibition of either proteasomes or autophagic flux failed to rescue the loss of IRE1α following heat stress. These collective results suggest that heat stress simultaneously activates both the UPR and autophagy, followed by the activation of a negative feedback system in UPR by modulating the responses related to the IRE1α-XBP-1 axis.

Oxidative stress caused by a SOD1 deficiency ameliorates thioacetamide-triggered cell death via CYP2E1 inhibition but stimulates liver steatosis.

We investigated the responses of mice that are defective in the superoxide-scavenging enzyme SOD1 to thioacetamide (TAA)-induced hepatotoxicity. When a lethal dose of TAA (500 mg/kg) was intraperitoneally injected, the wild-type (WT) mice all died within 36 h, but all of the SOD1-knockout (KO) mice survived. Treatment with an SOD1 inhibitor rendered the WT mice resistant to TAA toxicity. To elucidate the mechanism responsible for this, we examined the acute effects of a sublethal dose of TAA (200 mg/kg) on the livers of WT and KO mice. The extent of TAA-induced liver damage was less in the KO mice, but, instead, lipogenesis was further advanced in the SOD1-KO livers. The levels of proteins modified with acetyllysine, a marker for TAA-mediated injury, were lower in the KO mice than the WT mice upon the TAA treatment. The KO mice, which were under oxidative stress per se, exhibited a lower CYP2E1 activity, and this appeared to result in a decrease in the production of reactive oxygen species (ROS) during TAA metabolism. Both cleaved ATF6, a transcriptional regulator that is activated by endoplasmic reticulum (ER) stress, and CHOP, a death signal mediator, were highly elevated in the WT mice as the result of the TAA treatment and consistent with the liver damage. We conclude that elevated TAA metabolites and reactive oxygen species that are produced by CYP-mediated drug metabolism trigger lipogenesis as well as liver damage via ER stress and determine the fate of the mice.

Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function.

The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine ß-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

Ablation of aldehyde reductase aggravates carbon tetrachloride-induced acute hepatic injury involving oxidative stress and endoplasmic reticulum stress.

Aldehyde reductase (Akr1a) has been reported to be involved in the biosynthesis of ascorbic acid (AsA) in the mouse liver. Because Akr1a is expressed at high levels in the liver, we aimed to investigate the role of Akr1a in liver homeostasis by employing a carbon tetrachloride (CCl4)-induced hepatotoxicity model. Akr1a-deficient (Akr1a(-/-)) and wild-type (WT) mice were injected intraperitoneally with CCl4 and the extent of hepatic injury in the acute phase was assessed. Liver damage was heavier in the Akr1a(-/-) mice than in the WT mice. Furthermore, severe hepatic steatosis was observed in the livers of Akr1a(-/-) mice compared to WT mice and was restored to the levels in WT mice by AsA supplementation. Since the presence or absence of AsA had no effect on the decrease in CYP2E1 activity after the CCl4 treatment, it appears that AsA plays a role in the process after the bioactivation of CCl4. Biomarkers for oxidative stress and ER stress were markedly increased in the livers of Akr1a(-/-) mice and were effectively suppressed by AsA supplementation. Based on these collective results, we conclude that Akr1a exerts a protective effect against CCl4-induced hepatic steatosis by replenishing AsA via its antioxidative properties.

Ascorbic acid prevents acetaminophen-induced hepatotoxicity in mice by ameliorating glutathione recovery and autophagy.

Aldehyde reductase (AKR1A) plays a role in the biosynthesis of ascorbic acid (AsA), and AKR1A-deficient mice produce about 10-15% of the AsA that is produced by wild-type mice. We found that acetaminophen (AAP) hepatotoxicity was aggravated in AKR1A-deficient mice. The pre-administration of AsA in the drinking water markedly ameliorated the AAP hepatotoxicity in the AKR1A-deficient mice. Treatment of the mice with AAP decreased both glutathione and AsA levels in the liver in the early phase after AAP administration, and an AsA deficiency delayed the recovery of the glutathione content in the healing phase. While in cysteine supply systems; a neutral amino acid transporter ASCT1, a cystine transporter xCT, enzymes for the transsulfuration pathway, and autophagy markers, were all elevated in the liver as the result of the AAP treatment, the AsA deficiency suppressed their induction. Thus, AsA appeared to exert a protective effect against AAP hepatotoxicity by ameliorating the supply of cysteine that is available for glutathione synthesis as a whole. Because some drugs produce reactive oxygen species, resulting in the consumption of glutathione during the metabolic process, the intake of sufficient amounts of AsA would be beneficial for protecting against the hepatic damage caused by such drugs.