Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition that involves the targeted degeneration of motor neurons. The precise pathogenetic mechanisms are still largely unclear. In this study, we utilized the SOD1-G93A mouse model of ALS to investigate the effects of folic acid (FA), an important factor involved in homocysteine metabolism. Our results indicated that a little FA prolong the lifespan of the SOD1-G93A mice and medium and high dose FA significantly shortened the lifespan of the SOD1-G93A mice. Furthermore, we observed that overdose FA significantly elevated inflammation levels in the cerebellum, as evidenced by increased concentrations of tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), Cluster of Differentiation 68 (CD68), Cluster of Differentiation 86 (CD86), and Monocyte Chemoattractant Protein-1 (MCP-1), facilitating microglial activation. The low dose FA were the opposite of the overdose FA. Additionally, high dose FA increased the levels of phosphorylated p65 (p-p65)/p65, thereby promoting the NF-κB signaling pathway. Conversely, high dose FA inhibited the expression of total superoxide dismutase (T-SOD) while increasing the level of malondialdehyde (MDA) in the cerebellum; these effects were not observed in muscle tissue. Moreover, high dose FA elevated the levels of Bax2/Bcl-2. Concurrently, high dose FA induced increased apoptosis in SOD1-G93A mice, as demonstrated by TUNEL staining. Notably, the brain iron content of SOD1-G93A mice remained unchanged following FA treatment. These findings provide evidence that overdose FA supplementation may exert pro-inflammatory effects by promoting the NF-κB pathway, oxidative stress, and apoptosis in the context of ALS.
Peroxynitrite (ONOO-) is a reactive nitrogen species implicated in oxidative injury and impaired calcium handling. We examined whether ONOO- inactivates the cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and whether heat shock protein 70 (Hsp70) preserves SERCA2a function during ONOO- exposure. Human embryonic kidney (HEK-293) cells were co-transfected with SERCA2a and either empty pMT2 vector (S2a-pMT2) or Hsp70 (S2a-Hsp70). Cells were untreated or exposed to ten nominal 250 μM additions of ONOO-, delivered once per minute for 10 min. Hsp70 abundance was greater in S2a-Hsp70 cells, although the magnitude of this difference varied by treatment (interaction, p = 0.0015). SERCA2a abundance was unaffected by transfection condition or ONOO- exposure. Co-immunoprecipitation showed that Hsp70 and SERCA2a were present in the same protein complex under the conditions examined. ONOO- reduced maximal SERCA2a activity by approximately 20% in S2a-pMT2 cells (p = 0.03), whereas activity was preserved in S2a-Hsp70 cells. ONOO- also increased SERCA2a-associated reactive carbonyl content by approximately 80% in S2a-pMT2 cells (p = 0.01), with no increase in S2a-Hsp70 cells. SERCA2a 3-nitrotyrosine content and FITC-binding capacity did not differ among groups. These findings show that Hsp70 overexpression preserves SERCA2a activity during acute ONOO- exposure and that this protection is associated with reduced SERCA2a carbonyl accumulation.
Mitochondria are essential organelles responsible for cellular ATP production and contain their own mitochondrial DNA (mtDNA), which encodes key components of oxidative phosphorylation. Because mitochondria continuously generate reactive oxygen species (ROS), mtDNA is particularly susceptible to oxidative damage. Although DNA repair enzymes are present in mitochondria, the regulation of mtDNA repair and its impact on cellular responses to oxidative stress remain incompletely understood. Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key enzyme in the base excision repair (BER) pathway, and the mitochondrial isoform hOGG1-2a contributes to the maintenance of mtDNA integrity. In this study, HeLaS3 cell lines stably overexpressing hOGG1-2a were established to examine responses to oxidative stress. hOGG1-2a overexpression was associated with reduced survival following H2O2 treatment, γ-ray exposure, heat shock, and ultraviolet C (UVC) irradiation. Apoptotic cell death increased after oxidative stress. Mitochondrial membrane potential assessed by JC-1 staining was significantly reduced in hOGG1-2a-overexpressing cells. Long-range PCR analysis revealed reduced mtDNA amplification efficiency, and oxidative stress was accompanied by a greater reduction of the mitochondrial enzyme Aconitase 2. These cells exhibited elevated basal ATP levels and altered ATP responses under oxidative stress conditions. In addition, mitochondrial superoxide-associated fluorescence detected by MitoSOX™ was significantly increased. Combined long-range PCR and Sanger sequencing indicated reduced mtDNA amplification after H2O2 exposure without a marked increase in point mutations. Collectively, these findings suggest that hOGG1-2a overexpression sensitizes cells to oxidative stress and is associated with mitochondrial redox dysregulation, reduced mitochondrial membrane potential, altered ATP responses, and reduced mtDNA amplifiability during prolonged stress.
Iron overload‑induced osteoporosis (IOO) is a growing concern, yet the underlying mechanisms remain obscure. Ferroptosis, an iron‑dependent lipid peroxidation‑driven cell death, and NADPH oxidase 4 (NOX4), a major source of reactive oxygen species (ROS), have been implicated separately, but their interplay in bone marrow stromal cells (BMSCs) under iron overload is unknown. Here we investigated whether melatonin, an endogenous indole with antioxidant properties, protects BMSCs from iron overload‑induced ferroptosis and osteogenic dysfunction by regulating NOX4 transcription. BMSCs were exposed to ferric ammonium citrate (FAC, 200 µM) with or without melatonin (100 µM) or the ferroptosis inhibitor ferrostatin‑1 (Fer‑1). FAC suppressed cell viability, elevated Fe2+ and ROS, triggered classical ferroptotic mitochondrial damage (shrinkage, cristae loss), and impaired osteogenic differentiation (ALP activity, mineralization, Alp, Runx2, Col1a1, Osterix). Fer‑1 attenuated ROS and mitochondrial injury but did not reduce Fe2+ accumulation, confirming ferroptosis downstream of iron loading. Melatonin did not lower Fe2+ levels either; however, it effectively quenched ROS, restored mitochondrial ultrastructure, and rescued osteogenic function. Mechanistically, melatonin transcriptionally suppressed NOX4, as evidenced by reduced NOX4 mRNA and diminished NOX4 promoter activity in dual‑luciferase reporter assays, without affecting Fe2+ levels. Collectively, melatonin alleviates ferroptosis and restores osteogenesis in iron‑overloaded BMSCs, an effect that is strongly associated with the transcriptional suppression of NOX4, independent of iron chelation. Our findings extend the established NOX4-ferroptosis mechanism to BMSCs and identify, for the first time, transcriptional suppression of NOX4 as a novel mechanism that is strongly indicated to contribute to melatonin's protective effects against iron overload-induced osteogenic dysfunction.
Oxidative stress arising from excessive generation of free radical species in biological systems remains a major concern associated with several pathological conditions, thereby driving continuous interest in the development of effective antioxidants. Among various natural polyphenol-rich sources, Aloe vera is recognized as an important reservoir of bioactive antioxidant compounds. In the present study, five major polyphenols derived from Aloe vera were structurally modified through Sulfur and Selenium substitution at the hydroxyl and carbonyl functionalities to enhance their antioxidant potential. The changes in the thermodynamic parameters such as bond dissociation enthalpy, ionization potential, proton dissociation enthalpy, and electron transfer enthalpy were evaluated using density functional theory to elucidate the antioxidant mechanisms of the designed derivatives through the Hydrogen Atom Transfer, Single Electron Transfer followed by Proton Transfer, and Sequential Proton Loss followed by Electron Transfer pathways. Electronic properties and reactivity were further explored using frontier molecular orbital, molecular electrostatic potential, natural bond orbital, total density of states, transition state, and spin density distribution analyses. In addition, pharmacokinetic profiling was performed to evaluate the drug-likeness and biological suitability of the compounds. Molecular docking studies were conducted against the allosteric site of the antioxidant enzyme superoxide dismutase to investigate possible interactions that could enhance enzymatic activity through allosteric modulation. The computational results consistently demonstrated that Selenium-substituted derivatives exhibited the highest antioxidant activity, followed by Sulfur-substituted compounds, outperforming the parent molecules as well as standard antioxidant references including trans-Resveratrol, Gallic acid, and Fumaric acid. These findings reveal that chalcogen substitution can significantly improve the antioxidant efficiency of Aloe vera polyphenols and highlights the potential of Selenium-containing derivatives as promising candidates for the development of next generation antioxidants.
The phenolic phytocannabinoids frequently exhibit a prooxidant activity; however, its significance in their toxic/therapeutic action remains unclear. On the other hand, the predominant prooxidant character of mammalian cell cytotoxicity of hydroxybenzenes is demonstrated by a negative dependence of the cytotoxicity on the midpoint redox potential of phenoxyl radical/phenol couple (E(Ph-O•/Ph-OH) or E27 at pH 7.0). However, the E27 values of phytocannabinoids are unknown. Here using quantum mechanical calculations and cyclic voltammetry, the E27 values of 10 phytocannabinoids containing resorcinol, 1-hydroxychromene, or 1-hydroxychromane moieties were estimated to be 0.72-0.82 V, i.e. close to or below that of resorcinol, 0.81 V. Using the above E27 values, it was found that the cytotoxicity of phytocannabinoids in MH22a cells follows the same two-parameter regression as that of model hydroxybenzenes, i.e. it increased with decreasing E27 and increasing octanol/water partition coefficient at pH 7.0 (log.D). The prooxidant nature of phytocannabinoid cytotoxicity was evidenced by the protective effects of antioxidants, and the potentiating effect of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). On the other hand, the in vitro activity of phytocannabinoids against Plasmodium falciparum FcB1 strain was much greater than that of model hydroxybenzenes, and did not follow the same correlation. Unlike the lipophilicity-independent activity of hydroxybenzenes, the antiplasmodial activity of phytocannabinoids showed a trend to increase with their log D. These findings enabled to suggest that the mammalian cell cytotoxicity of phytocannabinoids may be largely determined by their induced oxidative stress, but their antiplasmodial activity may be determined by other, as yet poorly characterized, factors.
Caulerpa lentillifera is an edible green seaweed widely consumed in Southeast Asia and has been recognized for its nutritional and antioxidant properties. Previous studies demonstrated that its ethyl acetate extract (CLEA) exhibits strong free radical-scavenging and metabolic regulatory activities; however, its potential protective role against ultraviolet (UV)-induced oxidative stress has not yet been elucidated. Therefore, this study aimed to evaluate the photoprotective effects of CLEA on UV-induced cellular damage in keratinocytes (HaCaT) and fibroblasts (L929), and explore its underlying mechanisms. The results revealed that CLEA significantly improved cell viability following UV irradiation and attenuated intracellular reactive oxygen species (ROS) accumulation in both keratinocytes and fibroblasts. Mechanistically, CLEA enhanced nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated the mRNA expression of downstream antioxidant enzymes, particularly heme oxygenase-1 (HMOX1). In addition, CLEA suppressed UV-induced expression of matrix metalloproteinases, MMP1 and MMP3, while promoting procollagen synthesis, suggesting its role in preserving skin integrity. Collectively, these findings suggest that CLEA confers photoprotective effects against UV-induced oxidative stress and extracellular matrix degradation through activation of the Nrf2/HMOX1 pathway and inhibition of MMPs. This study highlights the potential of C. lentillifera extract as a natural photoprotective agent, warranting further in vivo and clinical investigations to validate its efficacy and safety.
Reactive oxygen species (ROS), produced by NADPH oxidase, promote contraction of the arteries. We hypothesized that (1) procontractile influence of NADPH oxidase derived ROS differs between α1- and thromboxane A2-receptor activation and (2) that such an influence is realized by the activation of different signaling pathways. Mesenteric arteries of rats were studied using wire-myography, Ca2+-fluorimetry and qPCR. Pan-NADPH oxidase inhibitor VAS2870 weakened contractile responses and [Ca2+]i rise to methoxamine (α1-adrenoceptor agonist) and U46619 (thromboxane A2 receptor agonist), this effect was more pronounced when U46619 was used. Suppression of Rho-kinase activity by Y27632 did not eliminate the effect of VAS2870 on both U46619- and methoxamine-induced contraction. The effect of VAS2870 persisted in the presence of LTCC (L-type voltage-gated Ca2+ channels) blocker nimodipine during U46619-induced, but not methoxamine-induced contraction. In contrast, the effect of VAS2870 persisted in the presence of protein kinase C inhibitor GF109203X during methoxamine-induced, but not U46619-induced contraction. Among all NADPH oxidase isoforms mRNAs of Nox2 and Nox4 were the most abundant. NOX2 inhibitor GSK2795039, but not NOX1/4 inhibitor GKT137831, weakened significantly both methoxamine- and U46619-induced contraction. This effect was more pronounced in case of U46619-induced contraction. Thus, procontractile influence of ROS, produced by NADPH oxidase (mainly, by NOX2), is greater during contraction induced by thromboxane A2 than α1-adrenoceptor activation in rat mesenteric arteries. Such influence is realized by activation of LTCC when α1-adrenoceptors are stimulated and by activation of protein kinase C when thromboxane A2 receptors are stimulated.
Acrylamide (ACR) is an environmental toxicant reported to have nephrotoxic effects. Edaravone is a free radical scavenger proven to have antioxidant properties. However, the role of edaravone in regulating endoplasmic reticulum stress (ER stress) in ACR-induced nephrotoxicity remains unclear. Thus, the present study aims to explore the role of ER stress and edaravone in an ACR-induced nephrotoxicity rat model. In this study, 24 male albino Wistar rats were randomly divided into four groups. The control group received normal saline orally for 28 days. The acrylamide group received acrylamide (10 mg/kg) for 28 days. Additionally, two treatment groups received 10 mg/kg ACR for 28 days; afterwards, edaravone at 5 mg/kg and 10 mg/kg was administered for the last 14 days (15-28 days). On the 29th day, all animals were sacrificed, and blood and kidney samples were collected. Renal function test, oxido-nitrosative stress parameters, and histopathological analysis, along with the western blotting analysis, were performed. Results suggest that ACR exposure significantly elevated the levels of BUN, creatinine, and urea. In addition, levels of MDA and nitrite were significantly increased with a significant drop in GSH levels. Further, upregulated protein expressions of GRP78, CHOP, and Caspase-12 were observed in ACR group, indicating activation of ER stress. Histopathological changes in the kidney were also seen along with these alterations. Interestingly, edaravone treatment mitigated all of these detrimental effects. These findings suggest novel mechanistic insights into the therapeutic potential of edaravone against ACR-induced nephrotoxicity by regulating oxidative stress and ER-stress mediated apoptosis.
Oral squamous cell carcinoma (OSCC) requires therapeutic strategies that preserve oral function while providing effective tumor control. Here we demonstrate that non-thermal plasma (NTP) irradiation of a clinically established povidone-iodine solution generates a potent cytotoxic formulation termed plasma-activated povidone-iodine (PAI). PAI selectively reduced viability of OSCC cell lines (SAS, HSC-2, HSC-4) compared with normal fibroblasts. Mechanistically, PAI activity strictly depended on its acidic pH (3.0 or below) and hydrogen peroxide (H2O2) generated during NTP irradiation. Neutralization or freeze-drying abolished both H2O2 content and cytotoxicity, indicating that reactive oxygen species are essential mediators. Unlike plasma-activated media, whose effects are predominantly iron-dependent, PAI-induced cytotoxicity required intracellular monovalent copper (Cu+). The Cu+ chelator tetrathiomolybdate suppressed ROS accumulation, glutathione depletion, lipid peroxidation, and cell death, whereas classical ferroptosis or apoptosis inhibitors failed to rescue viability. PAI increased intracellular Cu+ levels and triggered oxidative damage including γH2AX induction; however, the resulting death program was mechanistically distinct from apoptosis, ferroptosis, and cuproptosis. Our findings reveal a previously unrecognized Cu+-dependent oxidative pathway initiated by PAI. This study establishes an accessible iodine-based platform that harnesses metal-driven redox chemistry for selective OSCC elimination.
BACKGROUND:Bronchopulmonary dysplasia is a common chronic lung disease in preterm infants with a complex pathogenesis, and it is necessary to search for the potential pathogenesis and therapeutic strategies of bronchopulmonary dysplasia at the cellular molecular level. The present study investigated the role of reactive oxygen species (ROS)/sirtuin 1 (SIRT1) axis in hyperoxia-induced BEAS-2B cells injury. METHODS:Cell counting kit-8, cell scratch, ROS assay, immunofluorescence, mitochondrial membrane potential, transmission electron microscopy assay and Western blot were performed to investigate the impairment of BEAS-2B by hyperoxia as well as the roles of SIRT1 and ROS in hyperoxia-induced BEAS-2B injury. RESULTS:(1) Hyperoxia increased ROS in a time-dependent manner and decreased the levels of SIRT1 and mitochondria-associated proteins in BEAS-2B. (2) The SIRT1 agonist reduced ROS and improved mitochondrial membrane potential levels, attenuated mitochondrial fragmentation and mitochondrial morphological damage, and reversed the hyperoxia-induced decrease in mitochondria-associated proteins expression in BEAS-2B. (3) The ROS scavenger N-Acetylcysteine reduced ROS levels, improved mitochondrial membrane potential levels, attenuated mitochondrial morphological damage, and reversed the hyperoxia-induced decrease in SIRT1 and mitochondria-associated proteins levels in BEAS-2B. CONCLUSION:The ROS/SIRT1 axis is involved in hyperoxia-induced mitochondrial injury in BEAS-2B cells, and SIRT1 may be a potential therapeutic target for bronchopulmonary dysplasia.
Catechin is a natural compound abundant in green tea and is known as an antioxidant. We previously synthesized a catechin analog with a planar structure (planar catechin, PCat) and reported that it has about 10-fold stronger radical-scavenging activity than the parental (+)-catechin. We also showed that chemical conjunction of two different types of antioxidants has the potential to create an efficient antioxidant network for scavenging reactive oxygen species. In this study, we synthesized a PCat conjugated with teprenone (Tep), which is also known as a potential antioxidant, and found that the PCat-Tep conjugate exhibited remarkable cytotoxicity in cancer cells. On the other hand, the scavenging activity toward superoxide anion and DNA-protective effect against X-ray irradiation in aqueous solution were not enhanced by Tep conjugation. However, kinetic analysis of radical-scavenging activity in an organic solvent indicated that the PCat-Tep conjugate was approximately twice as effective as PCat alone. Moreover, the results of quantum chemical calculations imply that the association of subtle kinetic effects, such as differences in reorganization energy, solvation, or steric and conformational factors arising from the long hydrophobic side chain, rather than thermodynamic factors, are associated with the reactivity of the PCat-Tep conjugate.
Ferroptosis is a regulated form of cell death implicated in a wide range of pathological conditions. Iron-dependent lipid peroxidation driven by reactive oxygen species is a central feature. Because ferroptosis contributes to oxidative stress-induced tissue injury, including ischemia-reperfusion damage and neurodegenerative disorders, the suppression of this process has attracted considerable interest. Through a phenotypic screen of a unique chemical compound library from the University of Osaka, we identified a series of polycyclic quinone compounds containing a six-membered silacycle that exhibited protective effects against ferroptosis in vitro. These compounds selectively suppressed ferroptosis induced by various distinct ferroptosis inducers, while showing no protective effects against apoptosis. The ferroptosis-suppressive effects of these compounds were associated with reduced lipid peroxidation and decreased accumulation of ferrous iron in the mitochondria. Direct comparison with a carbon-substituted analog revealed that silicon substitution within the polycyclic quinone scaffold markedly enhanced the ferroptosis-suppressive activity. These results indicate that silicon-containing polycyclic quinone compounds represent a selective class of ferroptosis inhibitors and that silicon substitution confers a functional advantage for ferroptosis suppression.
Coenzyme Q10 (CoQ10) is an essential lipid-soluble antioxidant and a key component of the mitochondrial electron transport chain, playing critical roles in cellular redox homeostasis and energy metabolism. Although organ-specific differences in CoQ10 levels have been reported in humans, the mechanisms underlying tissue-specific regulation of CoQ10 remain poorly understood. Moreover, species differences in the predominant CoQ isoform limit the suitability of conventional rodent models for studying human CoQ10 metabolism, aging, and disease. In this study, we aimed to explore factors contributing to organ-specific CoQ10 levels and to establish fundamental reference data for medaka (Oryzias latipes), a vertebrate model that endogenously synthesizes CoQ10. CoQ10 concentrations in multiple organs were quantified by high-performance liquid chromatography. Levels of vitamin E and free cholesterol were also measured. Mitochondrial DNA (mtDNA) content was assessed as an index of mitochondrial abundance, and expression of CoQ10 biosynthetic enzymes, the CoQ-binding protein prosaposin (Psap), and 3-hydroxy-3-methylglutaryl-CoA reductase was analyzed by RT-qPCR. CoQ10 was detected in all organs examined, with the highest levels observed in the heart and liver, followed by the kidney and brain, and lower levels in skeletal muscle and the digestive tract. No significant sex-dependent differences were observed. CoQ10 levels were positively associated with PDSS2 expression and mtDNA content, while Psap expression showed strong positive correlations with multiple CoQ-related genes. These findings provide insight into factors associated with tissue-specific CoQ10 distribution and support medaka as a physiologically relevant model for investigating CoQ10 metabolism, oxidative stress, aging, and disease.
Superoxide (O2-), generated by mitochondrial respiration and redox enzymes, may act as a cellular aging trigger (CAT) when dysregulated. This study investigated whether mitochondrial (rotenone-induced) and cytoplasmic (paraquat-induced) O2- imbalance produces similar or distinct oxi-inflammatory activation in human peripheral blood mononuclear cells (PBMCs). Two in vitro protocols were performed. First, PBMCs were exposed to rotenone (ROT) or paraquat (PQT) for 6 or 24 h, using phytohaemagglutinin (PHA) as a positive control. Second, 48-h cultures from 21 donors, including healthy controls (HC) and individuals with Parkinson's disease (PD), were treated with 30 μM ROT or PQT. Cell viability was evaluated by the MTT assay (metabolic activity) and trypan blue exclusion (membrane integrity). Cytokines, O2-, nitric oxide (NO) and morphometric parameters were quantified. ROT and PQT induced distinct early responses: ROT increased IL-1β and NO, whereas PQT caused delayed elevations of IL-1β, O2- and NO. Because no baseline differences were detected between HC and PD donors, data were analyzed jointly. After 48 h, both oxidants elevated IL-1β, IL-6, TNF-α and NO. Morphometric analysis revealed ROT-associated fragmentation and PQT-induced hyperchromatic aggregates, consistent with pro-inflammatory damage-associated molecular patterns (DAMPs). These findings indicate that mitochondrial and cytoplasmic O2- imbalance initiates oxi-inflammatory activation in PBMCs, supporting superoxide dysregulation as a potential CAT and a hormetic modulator of aging-related signaling.
To investigate the role of peroxiredoxin isoforms (Prdx) in the radioresistance of cancer cells, the expression of Prdx1-6, DNA repair genes, and apoptosis regulators was studied in human cancer cell lines with varying radiosensitivities (A549, Caco-2, and MCF-7) after exposure to ionizing radiation. A correlation was found between high constitutive Prdx1-6 expression levels and increased radioresistance. Predominantly cytosolic isoforms Prdx2 and Prdx6 demonstrated pronounced induction after irradiation, indicating their critical role in protecting against radiation-induced oxidative stress. Most radiosensitive A549 cells exhibited the lowest baseline Prdx expression and the most pronounced transcriptional changes after irradiation, whereas MCF-7 and Caco-2 cells had higher constitutive expression and a weaker response to radiation. Mitochondrial Prdx3 and Prdx5, as well as ER-localized Prdx4, exhibited relatively stable expression. A549 cells demonstrated the highest induction of DNA repair genes, which may indicate more severe DNA damage. In contrast, MCF-7 cells were characterized by high basal expression of repair genes and elevated γH2AX levels before irradiation, which may reflect their "readiness" for repair and explain their higher radioresistance. Furthermore, radioresistant MCF-7 cells had increased expression of anti-apoptotic genes (BCL2, MCL1, BIRC5), suppressing the mitochondrial apoptotic pathway. Meanwhile, A549 cells showed higher induction of pro-apoptotic genes (PUMA, NOXA, BAK1) and activation of caspase-3, which correlates with their increased radiosensitivity. Therefore, peroxiredoxins protect cells from radiation exposure, either by being constitutively expressed or by being highly inducible in response to radiation, and promote cell survival after irradiation. This makes them attractive targets for overcoming cancer cell radioresistance.
Sodium hypochlorite (NaOCl) is widely used as a disinfectant and antiviral agent because of its strong oxidative properties. The principal antimicrobial species in NaOCl solutions are hypochlorite ion (OCl-) and hypochlorous acid (HOCl), whose relative proportions vary with pH. The iodometric titration method (KI method) is commonly used to evaluate disinfecting efficacy by measuring available chlorine; however, it cannot distinguish specific oxidizing species. In this study, we investigated the NaOCl-derived oxidizing species quantified by the KI method using absorption spectroscopy, pH measurements, and electron spin resonance (ESR) analysis to determine whether the KI method appropriately reflects disinfecting activity. NaOCl solutions were prepared by dissolving solid NaOCl in ultrapure water, and portions were acidified with sulfuric acid (H2SO4). Absorption spectra showed peaks corresponding to OCl- in NaOCl solutions and HOCl in acidified NaOCl solutions, indicating that most OCl- was converted to HOCl after acidification. However, the KI method yielded nearly identical oxidizing capacities for both solutions, contradicting reports that HOCl has much higher bactericidal activity than OCl-. Because the KI method enforces strongly acidic conditions through H2SO4 addition, OCl- is likely converted to HOCl during measurement, meaning the original chemical composition is not preserved. ESR analysis during the KI procedure further suggested the generation of highly reactive hydroxyl radicals (•OH) while HOCl concentrations remained largely unchanged. These findings indicate that OCl- and HOCl may not be directly measured by the KI method. Our results suggest that the conventional KI-based evaluation of disinfecting efficacy may need to be reconsidered.
The antioxidant properties of Nickel(II) 5,10,15,20-tetraphenylporphyrin (NiTPP) and Zinc(II) 5,10,15,20-tetrakis(4-methylphenyl)porphyrin (ZnTMPP) were examined using complementary experimental and computational approaches. Radical-scavenging activity was evaluated in the DPPH system by cyclic voltammetry and UV-visible spectroscopy, using α-tocopherol as a benchmark. Electrochemical measurements showed EC50 values of 139.1 ± 1.3 µM for NiTPP and 269.9 ± 4.5 µM for ZnTMPP, while α-tocopherol exhibited an EC50 of 90.99 ± 0.61 µM. In contrast, spectrophotometric analysis yielded EC50 values of 164.1 ± 2.3 µM for NiTPP and 142.1 ± 0.8 µM for ZnTMPP, indicating more efficient radical quenching by ZnTMPP in solution. Both metalloporphyrins displayed stronger binding affinities toward DPPH than α-tocopherol, with NiTPP showing the highest association constant (Kb = 7.49 × 104 L·mol-1; ΔG = -27.95 kJ·mol-1), followed by ZnTMPP (Kb = 3.14 × 104 L·mol-1; ΔG = -25.08 kJ·mol-1). Protein-level interactions relevant to antioxidant regulation were explored through molecular docking and 300 ns molecular dynamics simulations targeting the Keap1 Kelch domain. Both porphyrins occupied the canonical binding site, with NiTPP exhibiting the most favorable docking score (-9.4 kcal·mol-1). Molecular dynamics and MM-GBSA analyses confirmed greater structural stability and stronger binding for the NiTPP-Keap1 complex. These findings highlight a complementary antioxidant behavior, where ZnTMPP favors direct radical scavenging, while NiTPP shows enhanced protein recognition and stabilization.
