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.

Methionine Deprivation Reveals the Pivotal Roles of Cell Cycle Progression in Ferroptosis That Is Induced by Cysteine Starvation.

Ferroptosis, a type of iron-dependent necrotic cell death, is triggered by the accumulation of excessive lipid peroxides in cells. Glutathione (GSH), a tripeptide redox molecule that contains a cysteine (Cys) unit in the center, plays a pivotal role in protection against ferroptosis. When the transsulfuration pathway is activated, the sulfur atom of methionine (Met) is utilized to generate Cys, which can then suppress Cys-starvation-induced ferroptosis. In the current study, we cultured HeLa cells in Met- and/or cystine (an oxidized Cys dimer)- deprived medium and investigated the roles of Met in ferroptosis execution. The results indicate that, in the absence of cystine or Met, ferroptosis or cell cycle arrest, respectively, occurred. Contrary to our expectations, however, the simultaneous deprivation of both Met and cystine failed to induce ferroptosis, although the intracellular levels of Cys and GSH were maintained at low levels. Supplementation with S-adenosylmethionine (SAM), a methyl group donor that is produced during the metabolism of Met, caused the cell cycle progression to resume and lipid peroxidation and the subsequent induction of ferroptosis was also restored under conditions of Met/cystine double deprivation. DNA methylation appeared to be involved in the resumption in the SAM-mediated cell cycle because its downstream metabolite S-adenosylhomocysteine failed to cause either cell cycle progression or ferroptosis to be induced. Taken together, our results suggest that elevated lipid peroxidation products that are produced during cell cycle progression are involved in the execution of ferroptosis under conditions of Cys starvation.

Carnosine dipeptidase II (CNDP2) protects cells under cysteine insufficiency by hydrolyzing glutathione-related peptides.

The knockout (KO) of the cystine transporter xCT causes ferroptosis, a type of iron-dependent necrotic cell death, in mouse embryonic fibroblasts, but this does not occur in macrophages. In this study, we explored the gene that supports cell survival under a xCT deficiency using a proteomics approach. Analysis of macrophage-derived peptides that were tagged with iTRAQ by liquid chromatography-mass spectrometry revealed a robust elevation in the levels of carnosine dipeptidase II (CNDP2) in xCT KO macrophages. The elevation in the CNDP2 protein levels was confirmed by immunoblot analyses and this elevation was accompanied by an increase in hydrolytic activity towards cysteinylglycine, the intermediate degradation product of glutathione after the removal of the γ-glutamyl group, in xCT KO macrophages. Supplementation of the cystine-free media of Hepa1-6 cells with glutathione or cysteinylglycine extended their survival, whereas the inclusion of bestatin, an inhibitor of CNDP2, counteracted the effects of these compounds. We established CNDP2 KO mice by means of the CRISPR/Cas9 system and found a decrease in dipeptidase activity in the liver, kidney, and brain. An acetaminophen overdose (350 mg/kg) showed not only aggravated hepatic damage but also renal injury in the CNDP2 KO mice, which was not evident in the wild-type mice that were receiving the same dose. The aggravated renal damage in the CNDP2 KO mice was consistent with the presence of abundant levels of CNDP2 in the kidney, the organ prone to developing ferroptosis. These collective data imply that cytosolic CNDP2, in conjugation with the removal of the γ-glutamyl group, recruits Cys from extracellular GSH and supports redox homeostasis of cells, particularly in epithelial cells of proximal tubules that are continuously exposed to oxidative insult from metabolic wastes that are produced in the body.

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages.

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.

Iron loading exerts synergistic action via a different mechanistic pathway from that of acetaminophen-induced hepatic injury in mice.

Acetaminophen (APAP) overdose is a major cause of drug-induced acute liver failure. In such cases, free iron is released from lysosomes and is transported to mitochondria where it plays a pivotal role in APAP-induced liver injury. We previously reported that ascorbic acid (Asc) markedly mitigates APAP-induced hepatic damage in aldehyde reductase (Akr1a)-knockout (KO) mice that produce about 10% Asc as wild-type (WT) mice. However, the issue of the protective mechanism of Asc in association with the status of iron remains ambiguous. To gain additional insights into this issue, we examined effects of APAP (500 mg/kg) on female KO mice under conditions of iron loading. While the KO mice without AsA supplementation were more sensitive to APAP toxicity than the WT mice, FeSO4 loading (25 mg/kg) to WT mice aggravated the hepatic injury, which was a similar extent to that of the KO mice. Supplementation of Asc (1.5 mg/ml in the drinking water) ameliorated KO mice irrespective of iron status but did not change the iron-mediated increase in the lethality in the WT mice. Hepatic cysteine and glutathione levels declined to similar extents in all mouse groups at 3 h irrespective of the iron status and largely recovered at 18 h after the APAP treatment when liver damage was evident. Asc prominently mitigated APAP toxicity in KO mice irrespective of the iron status but had no effect on the synergistic action of iron and APAP in the WT mice, suggesting that the mechanism for the deteriorating action of loaded iron is different from that of APAP toxicity.

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.