PMC

A schematic representation of the cytoprotective actions of ET. Shown is a representation of a eukaryotic cell. ET is accumulated into different cellular compartments via its specific transporter ETT. Reactive oxygen species, viz superoxide , hydroxyl radical OH generated in the mitochondria by respiration are directly scavenged and blocked (indicated by red lines) by ET, and prevented from damaging cellular components. ET present in the cytoplasm also scavenges these free radicals. ET absorbs UV radiation and prevents DNA breaks and mutations. ET may stimulate certain enzymes involved in antioxidant response and repair mechanisms and also have a role in cell proliferation. In addition, ET protects against a variety of other ROS and reactive nitrogen species providing cytoprotection at multiple levels

Ergothioneine protects HeLa cells against pyrogallol, a superoxide generator. (a) HeLa cells were treated with 150 µM pyrogallol for 16 h in the presence of increasing concentrations of ET (0.1–1 mM). ET-protected cells in a dose-dependent manner. (b) Comparison of ET with other water-soluble antioxidants such as glutathione and ascorbate (0.1 mM each). ET was marginally better than the antioxidants glutathione and ascorbate in protecting against pyrogallol-induced cytotoxicity. (c) Morphology of control and ETT cells treated with pyrogallol. RNAi-depleted cells (shown in the bottom panel) undergo severe morphological deformation such as cell shrinkage and membrane blebbing whereas control cells (top panel) retain their normal cellular architecture. (d) MTT Cell viability assays reveal that control cells (gray bars) are more resistant to pyrogallol-induced damage. Data show mean ± S.E. and are derived from three independent experiments, **P<0.01

Ergothioneine protects against apoptosis induced by hydrogen peroxide. (a) Cultured HeLa cells were treated with 1 mM hydrogen peroxide for 16 h after a 24-h pretreatment with 1 mM ET and assayed for cell viability using the MTT Assay. Data are expressed as percentage of viability compared with untreated (designated as U) cells. The ET-treated cells are better protected against H₂O₂ toxicity. Data shown are means ± S.E. of five independent experiments, ***P<0.001 using Student’s t-test. (b) Depletion of the ETT RNA results in increased apoptosis in response to H₂O₂. Cells were transfected with ETT siRNA and 72 h post transfection, treated with or without 1 mM ET for 24 h and then challenged with 0.5 mM H₂O₂ for 16 h. Results are expressed as percentage of viability compared with control, untreated cells. The ETT-depleted cells (black bars) were more susceptible to oxidative stress and showed a marked decrease in viability, P<0.001 using Student’s t-test

(a) Structure of l-Ergothioneine: ET is a betaine of the amino acid histidine with a sulfhydryl group attached to the carbon of the imidazole ring. (b) ETT knockdown in HeLa cells. Total RNA was isolated from control and RNAi-transfected cells and 500 ng of RNA was used for the RT-PCR. The top panel shows the RT-PCR product corresponding to the ETT transcript. The bottom panel shows the levels of the GAPDH transcript used as an endogenous loading control. (c) A quantitative representation of the gel shown in B, which shows approximately 75% knockdown of the ETT transcript. (d) [³H]Ergothioneine uptake assay was performed essentially as described earlier. At 72 h post transfection, cells were incubated with 3 µM ET for an hour at 37°C and uptake monitored. The RNAi-depleted cells show about 75% decrease in ET uptake compared with the control cells. The data shown are the mean ± S.E. of three independent experiments ***P<0.001 using the Student’s t-test

Depletion of the ET transporter leads to increased damage to cellular proteins and lipids. (a) Control- and ETT-depleted HeLa cells were treated with 0.5 mM H₂O₂ or 16 h after preincubation with 1 mM ET and assayed for protein oxidation using 2,4-dinitrophenylhydrazine (DNPH), which reacts with protein carbonyl groups generated by oxidation. The DNPH-derivatized samples were electrophoresed and subjected to western blotting using anti-DNPH antibodies. The signals were quantified using densitometry and expressed as a fold increase in protein carbonylation compared with the untreated control. The data shown are from a representative experiment repeated at least three times. The ETT-depleted cells (black bars) show higher levels of oxidation compared with control cells (gray bars). (b) ETT cells show a higher level of lipid peroxidation. Cells were pretreated with 1 mM ET 72 h post transfection and lipid peroxidation induced using a mixture of 30 µM FeSO₄ and 100 µM H₂O₂ for 2 h at 37°C. The cells were then assayed using the Thiobarbituric Acid Reactive Substances (TBARS) Assay to quantify the levels of Malondialdehyde (MDA), which is a product of lipid peroxidation. The ETT-depleted cells undergo higher oxidation than control cells (Black versus gray bars). Data shown is a representative of three independent experiments

Ergothioneine prevents DNA damage. (a) ET protects mitochondrial DNA (mtDNA) from damage induced by reactive oxygen species. Control and ETT cells were treated with 0.5 mM H₂O₂ for 4 h with or without pretreatment with 1 mM ET for 24 h, and total DNA (genomic and mitochondrial) were isolated. Ten nanograms of the DNA were used as a template for quantitative real-time PCR (qPCR) using probes specific for the D-loop, a hotspot for mtDNA damage and normalized to β-actin, the probe for nuclear DNA. The ETT RNAi-transfected cells (black bars) yielded a decreased signal in the PCR, indicating increased DNA damage even in the presence of ET. The control cells were more resistant to mtDNA damage (gray bars). The data shown are the mean of duplicates of three independent experiments, which yielded similar results. **indicates p<0.01 by Students t test. (b) ET prevents nicking induced by UV and H₂O₂. Supercoiled plasmid DNA (1 µg) was treated with 2.5 mM H₂O₂ in the presence of increasing concentrations of ET (0.25–5 mM), irradiated at 254 nm for 5 min and electrophoresed on a 1% agarose gel in 1 × Tris Acetate EDTA (TAE). The lower band represents supercoiled plasmid DNA. The more slowly migrating band is the open circular DNA arising from nicking of DNA. ET protects against this oxidative stress in a dose-dependent manner