Dithiolethiones D3T and ACDT Protect Against Iron Overload-Induced Cytotoxicity and Serve as Ferroptosis Inhibitors in U-87 MG Cells.

Iron overload-induced oxidative stress is implicated in various neurodegenerative disorders. Given the numerous adverse effects associated with current iron chelators, natural antioxidants are being explored as alternative therapeutic options. Dithiolethiones found in cruciferous vegetables have emerged as promising candidates against a wide range of toxicants owing to their lipophilic and cytoprotective properties. Here, we test the dithiolethiones 3H-1,2-dithiole-3-thione (D3T) and 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) against ferric ammonium citrate (FAC)-induced toxicity in U-87 MG astrocytoma cells. Exposure to 15 mM FAC for 24 h resulted in 54% cell death. A 24-h pretreatment with 50 µM D3T and ACDT prevented this cytotoxicity. Both dithiolethiones exhibited antioxidant effects by activating the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor and upregulating levels of intracellular glutathione (GSH). This resulted in the successful inhibition of FAC-induced reactive oxygen species, lipid peroxidation, and cell death. Additionally, D3T and ACDT upregulated expression of the Nrf2-mediated iron storage protein ferritin which consequently reduced the total labile iron pool. A 24-h pretreatment with D3T and ACDT also prevented cell death induced by the ferroptosis inducer erastin by upregulating the transmembrane cystine/glutamate antiporter (xCT) expression. The resulting increase in intracellular GSH and alleviation of lipid peroxidation was comparable to that caused by ferrostatin-1, a specific ferroptosis inhibitor. Collectively, our findings demonstrate that dithiolethiones may show promise as potential therapeutic options for the treatment of iron overload disorders.

Reversal of genetic brain iron accumulation by N,N'-bis(2-mercaptoethyl)isophthalamide, a lipophilic metal chelator, in mice.

N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) is a novel lipophilic metal chelator and antioxidant used in mercury poisoning. Recent studies have suggested that NBMI may also bind to other metals such as lead and iron. Since NBMI can enter the brain, we evaluated if NBMI removes excess iron from the iron-loaded brain and ameliorates iron-induced oxidative stress. First, NBMI exhibited preferential binding to ferrous (Fe2+) iron with a negligible binding affinity to ferric (Fe3+) iron, indicating a selective chelation of labile iron. Second, NBMI protected SH-SY5Y human neuroblastoma cells from the cytotoxic effects of high iron. NBMI also decreased cellular labile iron and lessened the production of iron-induced reactive oxygen species in these cells. Deferiprone (DFP), a commonly used oral iron chelator, failed to prevent iron-induced cytotoxicity or labile iron accumulation. Next, we validated the efficacy of NBMI in Hfe H67D mutant mice, a mouse model of brain iron accumulation (BIA). Oral gavage of NBMI for 6 weeks decreased iron accumulation in the brain as well as liver, whereas DFP showed iron chelation only in the liver, but not in the brain. Notably, depletion of brain copper and anemia were observed in BIA mice treated with DFP, but not with NBMI, suggesting a superior safety profile of NBMI over DFP for long-term use. Collectively, our study demonstrates that NBMI provides a neuroprotective effect against BIA and has therapeutic potential for neurodegenerative diseases associated with BIA.

N,N'bis-(2-mercaptoethyl) isophthalamide (NBMI) exerts neuroprotection against lead-induced toxicity in U-87 MG cells.

N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) is a novel lipophilic heavy metal chelator and thiol redox antioxidant. This study was designed to investigate the neuroprotective activity of NBMI in U-87 MG cells exposed to lead acetate (PbAc). Cells were pretreated with NBMI for 24 h prior to a 48 h exposure to PbAc. Cell death (55%, p < 0.0001) and reduction of intracellular GSH levels (0.70-fold, p < 0.005) induced by 250 µM Pb were successfully attenuated by NBMI pretreatment at concentrations as low as 10 µM. A similar pretreatment with the FDA-approved Pb chelator dimercaptosuccinic acid (DMSA) proved ineffective, indicating a superior PKPD profile for NBMI. Pretreatment with NBMI successfully counteracted Pb-induced neuroinflammation by reducing IL-1ß (0.59-fold, p < 0.05) and GFAP expression levels. NBMI alone was also found to significantly increase ferroportin expression (1.97-fold, p < 0.05) thereby enhancing cellular ability to efflux heavy metals. While no response was observed on the apoptotic pathway, this study demonstrated for the first time that necrotic cell death induced by Pb in U-87 MG cells is successfully attenuated by NBMI. Collectively these data demonstrate NBMI to be a promising neuroprotective compound in the realm of Pb poisoning.

Neuroprotective effects of disubstituted dithiolethione ACDT against manganese-induced toxicity in SH-SY5Y cells.

Dithiolethiones are lipophilic, organosulfur compounds that activate the Nrf2 transcription factor causing an upregulation of various phase II antioxidant enzymes. A disubstituted dithiolethione 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) retains the functional pharmacophore while also containing modifiable functional groups. Neuroprotection against autoimmune encephalomyelitis in vivo and 6-hydroxy dopamine (a model for Parkinson's disease) in vitro have been previously reported with ACDT. Manganese (Mn) is a metal essential for metabolic processes at low concentrations. Overexposure and accumulation of Mn leads to a neurological condition called manganism which shares pathophysiological sequelae with parkinsonism. Here we hypothesized ACDT to be protective against manganese-induced cytotoxicity. SH-SY5Y human neuroblastoma cells exposed to 300 µM MnCl2 displayed approximately 50% cell death, and a 24-h pretreatment with 75 µM ACDT significantly reversed this cytotoxicity. ACDT pretreatment was also found to increase total GSH levels (2.18-fold) and the protein levels of NADPH:quinone oxidoreductase-1 (NQO1) enzyme (6.33-fold), indicating an overall increase in the cells' antioxidant defense stores. A corresponding 2.32-fold reduction in the level of Mn-induced reactive oxygen species was also observed in cells pretreated with ACDT. While no changes were observed in the protein levels of apoptotic markers Bax and Bcl-2, pretreatment with 75 µM ACDT led to a 2.09-fold downregulation of ZIP14 import transporter, indicating a potential reduction in the cellular uptake of Mn as an additional neuroprotective mechanism. These effects did not extend to other transporters like the divalent metal transporter 1 (DMT1) or ferroportin. Collectively, ACDT showed substantial neuroprotection against Mn-induced cytotoxicity, opening a path for dithiolethiones as a potential novel therapeutic option against heavy metal neurotoxicity.