The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H₂O₂. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.     

Redox‐Active Phenolic Compounds Mediate the Cytotoxic and Antioxidant Effects of Carpodesmia tamariscifolia (=Cystoseira tamariscifolia)

Carpodesmia tamariscifolia is a brown alga rich in (poly)phenols with important cytotoxic and antioxidant effects. However, the relationship between its chemical composition and its effects is unknown. The aim of this study is to identify the potential compounds and mechanisms responsible for its main effects. The alga was extracted consecutively with hexane, dichloromethane and methanol and further fractionated using Sephadex LH‐20 and silica gel columns when appropriate. The fractions were subjected to thin‐layer chromatography and liquid chromatography‐mass spectrometry analysis and evaluated for their total phenolic content (Folin‐Ciocalteu assay), radical scavenging activity (DPPH assay), cytotoxic activity (MTT assay on the SH‐SY5Y cell line), and ability to generate H₂O₂ (Amplex Red assay). Chromatographic and phenolic analyses of the fractions indicate that abundant redox‐active phenols are present in all the fractions and that a high amount of prenylated hydroquinone derivatives is present in the apolar ones. In the hexane and dichloromethane fractions, the cytotoxic and antioxidant activities are closely related to their phenolic content, whereas in the methanol fractions, the cytotoxicity is negatively related to the phenolic content and the antioxidant activity is positively related to it. In the same tests, hydroquinone behaves as both strong cytotoxic and antioxidant agent. H₂O₂ assay shows that C. tamariscifolia fractions and hydroquinone can autoxidize and generate H₂O₂. Our results suggest that redox‐active phenols produce the pharmacological effects described for C. tamariscifolia and that the hydroquinone moiety of prenylated hydroquinone derivatives is responsible for both cytotoxic (through a pro‐oxidant mechanism secondary to its autoxidation) and antioxidant effects of the apolar fractions.     

Tyrosinase‐catalyzed oxidation of rhododendrol produces 2‐methylchromane‐6,7‐dione,the putative ultimate toxic metabolite: implications for melanocyte toxicity

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a skin‐whitening agent until it was reported to induce leukoderma in July 2013. To explore the mechanism underlying its melanocyte toxicity, we characterized the tyrosinase‐catalyzed oxidation of RD using spectrophotometry and HPLC. Oxidation of RD with mushroom tyrosinase rapidly produced RD‐quinone, which was quickly converted to 2‐methylchromane‐6,7‐dione (RD‐cyclic quinone) and RD‐hydroxy‐p‐quinone through cyclization and addition of water molecule, respectively. RD‐quinone and RD‐cyclic quinone were identified as RD‐catechol and RD‐cyclic catechol after NaBH₄ reduction. Autoxidation of RD‐cyclic catechol produced superoxide radical. RD‐quinone and RD‐cyclic quinone quantitatively bound to thiols such as cysteine and GSH. These results suggest that the melanocyte toxicity of RD is caused by its tyrosinase‐catalyzed oxidation through production of RD‐cyclic quinone which depletes cytosolic GSH and then binds to essential cellular proteins through their sulfhydryl groups. The production of ROS through autoxidation of RD‐cyclic catechol may augment the toxicity.     

CREB‐mediated Bcl‐2 expression contributes to RCAN1 protection from hydrogen peroxide‐induced neuronal death

Regulator of calcineurin 1 (RCAN1) is located on the Down syndrome critical region (DSCR) locus in human chromosome 21. In this study, we investigated the functional role of RCAN1 in the reactive oxygen species (ROS)‐mediated neuronal death signaling. We found that RCAN1 was able to protect the cells from H₂O₂‐induced cytotoxicity. The expression of RCAN1 caused an inhibition of the H₂O₂‐induced activation of mitogen‐activated protein kinases (MAPKs) and AP‐1. In contrast, RCAN1 significantly enhanced the activity of cAMP response element‐binding protein (CREB). Furthermore, RCAN1 induced the expression of the CREB target gene, Bcl‐2. Consistently, knockdown of endogenous RCAN1 using shRNA down regulated the phosphorylation of CREB and the expression of Bcl‐2, which protects the cells from H₂O₂‐induced cytotoxicity. Our data provide a new mechanism for the cytoprotective function of RCAN1 in response to oxidant‐induced apoptosis. J. Cell. Biochem. 114: 1115–1123, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition of bromodomain‐containing protein 4 ameliorates oxidative stress–mediated apoptosis and cartilage matrix degeneration through activation of NF‐E2–related factor 2‐heme oxygenase‐1 signaling in rat chondrocytes

During the progression of osteoarthritis, dysregulation of extracellular matrix (ECM) anabolism, abnormal generation of reactive oxygen species, and proteolytic enzymes have been shown to accelerate the degradation process of cartilage. The purpose of the current study was to investigate the functional role of bromodomain‐containing protein 4 (BRD4) in hydrogen peroxide (H₂O₂)–stimulated chondrocyte injury and delineate the underlying molecular mechanisms. We observed that the expression BRD4 was markedly elevated in rat chondrocytes after H₂O₂ stimulation. Additionally, inhibition of BRD4 using small interfering RNA or JQ1 (a selective potent chemical inhibitor) led to repression of H₂O₂‐induced oxidative stress, as revealed by a decrease in the reactive oxygen species production accompanied by a decreased malondialdehyde content, along with increased activities of antioxidant markers superoxide dismutase, catalase, and glutathione peroxidase on exposure of chondrocytes to H₂O₂. Meanwhile, depletion of BRD4 led to repress the oxidative stress–induced apoptosis of chondrocytes triggered by H₂O₂ accompanied by an increase in the expression of anti‐apoptotic Bcl‐2 and a decrease in the expression of pro‐apoptotic Bax and caspase 3 as well as attenuated caspase 3 activity. Moreover, knockdown of BRD4 or treatment with JQ1 markedly attenuated ECM deposition, reflected in a marked upregulation of proteoglycans collagen type II and aggrecan as well as downregulation of ECM–degrading enzymes matrix metalloproteinase 13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5). More importantly, inhibition of BRD4‐activated NF‐E2–related factor 2 (Nrf2)–heme oxygenase‐1 signaling. Mechanistically, the protective effect of BRD4 inhibition on H₂O₂‐stimulated apoptosis and cartilage matrix degeneration was markedly abrogated by Nrf2 depletion. Altogether, we concluded that the protective effect of BRD4 inhibition against oxidative stress–mediated apoptosis and cartilage matrix degeneration occurred through Nrf2–heme oxygenase‐1 signaling, implying that BRD4 inhibition may be a more effective therapeutic strategy against osteoarthritis.     

Degree of polymerization of 5,6‐dihydroxyindole‐derived eumelanin from chemical degradation study

Eumelanin is a brown‐black pigment comprising 5,6‐dihydroxyindole (DHI) and its 2‐carboxy derivative (DHICA), but the detailed structure of eumelanin is unclear. Chemical degradation is a powerful tool for analyzing melanin. H₂O₂ oxidation degradation of eumelanin affords pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pyrrole‐2,3‐dicarboxylic acid (PDCA). The ratio of PDCA to PTCA provides information about the eumelanin structure. In this article, we propose simple equations on the basis of previous experimental results on dimer yields for evaluating the yields of PTCA and PDCA from any DHI oligomers. Assuming the chemical disorder model of DHI‐melanin, we solve an equation where a theoretical expression for the ratio of PDCA to PTCA is set to the corresponding experimental value to obtain a plausible Poisson distribution of DHI oligomers. The results demonstrate that the main contributors to DHI‐melanin are tetramers and pentamers as shown by the mass spectrometry.     

SILAC analysis of oxidative stress‐mediated proteins in human pneumocytes: New role for treacle

To better understand lung oxidant stress responses, we examined A549 lung cells exposed to H₂O₂ using “stable isotope labeling by amino acids.” We identified 466 cytosolic and 387 nuclear proteins; H₂O₂ exposure produced ≥twofold differences in 31, all were downregulations. None were previously reported as oxidant stress response proteins, although they share common functions. One of the responders, treacle, was linked to p53, an important oxidative stress response. The Treacher Collins–Franceschetti syndrome can result from treacle mutation and insufficiency was suggested to cause increased p53 leading to the syndrome. However, results here indicate p53 and treacle responses to H₂O₂ are independent: treacle remains suppressed after p53 recovery; the threshold for treacle reduction is well above that for p53 induction; and treacle suppression by short interfering RNA does not modify the p53 response. Evidence of treacle antioxidant activity include reduction being driven by proteasome degradation independently of mRNA, typical for oxidant‐absorbing proteins, and increased sensitivity to H₂O₂ consequent to short interfering RNA suppression. Data here show a link between oxidative stress and treacle reduction, demonstrate that treacle does not control p53, provide evidence of a treacle oxidant defense role, support the hypothesis that oxidant stress plays a role in the Treacher Collins–Franceschetti syndrome, and raise the possibility that treacle plays an anti‐oxidant role in lungs.     

Triosephosphate isomerase tyrosine nitration induced by heme–NaNO2–H2O2 or peroxynitrite: Effects of different natural phenolic compounds

Peroxynitrite and heme peroxidases (or heme)–H₂O₂–NaNO₂ system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways‐induced protein nitration and oxidation are controversial. Both nitrating systems can dose‐dependently induce triosephosphate isomerase (TIM) nitration, however, heme–H₂O₂–NaNO₂ was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3‐morpholinosydnonimine hydrochloride (SIN‐1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme–H₂O₂–NaNO₂ and SIN‐1 and protein oxidation induced by SIN‐1, but promoted heme–H₂O₂–NaNO₂‐induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN‐1‐induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme–H₂O₂–NaNO₂‐induced nitration. Our results indicated that peroxynitrite and heme–H₂O₂–NaNO₂‐induced protein nitration was different, and the later one could be a better model for anti‐nitration compounds screening.     

Sulfur and nitrogen co‐doped graphene quantum dot‐assisted chemiluminescence for sensitive detection of tryptophan and mercury (II)

A simple one‐step thermal treatment to prepare strong fluorescent sulfur and nitrogen co‐doped graphene quantum dots (SN‐GQD) using citric acid and l ‐cysteine as precursors was developed. The ultra‐weak chemiluminescence (CL) from the reaction of hydrogen peroxide (H₂O₂) and periodate (IO₄^?) was significantly enhanced by SN‐GQD in acidic medium. The enhanced CL was induced by excited‐state SN‐GQD (SN‐GQD*), which was produced from the transfer energy of (O₂)₂* and ¹O₂ to SN‐GQD and recombination of oxidant‐injected holes and electrons in SN‐GQD. In the presence of tryptophan (Trp), the CL intensity of the SN‐GQD–H₂O₂–KIO₄ system was greatly diminished. This finding was used to design a novel method for determination of Trp in the linear range 0.6–20.0 μM, with a limit of detection (LOD) of 58.0 nM. Furthermore, Hg²⁺ was detectable in the range 0.1–9.0 μM with a LOD of 64.0 nM, based on its marked enhancement of the SN‐GQD–H₂O₂–KIO₄ CL system. The proposed method was successfully applied to detect Trp in milk and human plasma samples and Hg²⁺ in drinking water samples, with recoveries in the range 95.7–107.0%.     

Tyrosinase‐catalyzed metabolism of rhododendrol (RD) in B16 melanoma cells: production of RD‐pheomelanin and covalent binding with thiol proteins

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD‐quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4‐amino‐3‐hydroxy‐n‐butylbenzene as a specific indicator, we detected moderate levels of RD‐pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD‐quinone to non‐protein thiols and proteins through cysteinyl residues. The covalent binding of RD‐quinone to proteins was 20‐ to 30‐fold greater than dopaquinone. These results suggest that the tyrosinase‐induced metabolism of RD causes melanocyte toxicity.     

Rapid in vivo screening system for anti‐oxidant activity using bacterial redox sensor strains

Aim: To develop a faster and easier in vivo method to screen compounds for anti‐oxidant activity using a microbial system. Methods and Results: Bacterial redox sensor‐based assay systems were applied. The activities of SoxR and OxyR, the bacterial redox sensors, were monitored to probe the intracellular redox status through two reporter strains, Escherichia coli soxS_p‐lacZ and oxyS_p‐lacZ fusions, which specifically respond to paraquat, a superoxide generator, and H₂O₂, respectively, with practically no cross reactivity. For the test screening, 27 natural compounds including phenolics and flavonoids that are putatively considered anti‐oxidant nutritional supplements were collected and assayed for their capability to alleviate oxidative stress in these bacterial systems. Among them, rutin, kaempferol and quercetin had significant anti‐H₂O₂ activity, and betaine, glycyrrhizic acid and baicalin had weak anti‐superoxide activity. While rutin, kaempferol and quercetin significantly reduced the H₂O₂ stress at low concentrations, betaine, glycyrrhizic acid and baicalin required higher concentration for their anti‐superoxide effects. In vitro, only quercetin protected DNA in a metal‐catalysed oxidation system, suggesting that the other compounds might indirectly exert their anti‐oxidant activities through other biological functions. Finally, quercetin, rutin and kaempferol significantly restored the viability of a superoxide dismutase mutant that has limited viability because of defective defence against oxidative stress. Conclusion: These bacterial systems could provide a more efficient method for measuring the activity of compounds affecting cellular oxidative stress and viability. Significance and Impact of the Study: The demand for anti‐oxidant and anti‐ageing activities is increasing in one of the fastest growing segments of the functional food market, but the screening for these activities is currently very laborious, expensive and time consuming. This study suggests a basis for a high throughput screening method for these activities.     

Heat stress hardening of oriental armyworms is induced by a transient elevation of reactive oxygen species during sublethal stress

Pre‐exposure to mild heat stress enhances the thermotolerance of insects. Stress hardening is a beneficial physiological plasticity, but the mechanism underlying it remains elusive. Here we report that reactive oxygen species (ROS) concentrations were quickly and transiently elevated in the armyworms, Mythimna separata, by exposing them to 40°C, but not other tested temperatures. Larvae exposed to 40°C had subsequently elevated antioxidant activity and the highest survival of all tested heating conditions. The elevation of ROS after lethal heating at 44°C for 1 h was approximately twofold compared to heating at 40°C. Injection of an optimal amount of hydrogen peroxide (H₂O₂) similarly caused sequential elevation of ROS and antioxidant activity in the test larval hemolymph, which led to significantly enhanced survival after lethal heat stress. The H₂O₂‐induced thermotolerance was abolished by coinjection of potent antioxidants such as ascorbic acid or N‐acetylcysteine. Both preheating at 40°C and H₂O₂ injection enhanced expression of genes encoding superoxide dismutase 1, catalase, and heat shock protein 70 in the fat body of test larvae, indicating the adequate heat stress induced a transient elevation of ROS, followed by upregulation of antioxidant activity. We infer that thermal stress hardening is induced by a small timely ROS elevation that triggers a reduction–oxidation signaling mechanism.     

Acid hydrolysis reveals a low but constant level of pheomelanin in human black to brown hair

We previously reported a constant ratio of the benzothiazole pheomelanin marker thiazole‐2,4,5‐tricarboxylic acid (TTCA) to the eumelanin marker pyrrole‐2,3,5‐tricarboxylic acid (PTCA) in eumelanic, black human hair. A constant level (20%–25%) of benzothiazole‐type pheomelanin was recently demonstrated in human skin with varying concentrations of melanin. Therefore, in this study, we aimed to investigate the origin of pheomelanin markers in black to brown human hair by developing a method to remove protein components from hair by heating with 6 M HCl at 110°C for 16 hr. For comparison, synthetic melanins were prepared by oxidizing mixtures of varying ratios of dopa and cysteine with tyrosinase. Hair melanins and synthetic melanins were subjected to acid hydrolysis followed by alkaline H₂O₂ oxidation. The results show that the hydrolysis leads to decarboxylation of the 5,6‐di‐hydroxyindole‐2‐carboxylic acid moiety in eumelanin and the benzothiazole moiety in pheomelanin and that eumelanic human hair contains 11%–17% benzothiazole‐type pheomelanin.     

Nrf2‐Mediated Resistance to Oxidant‐Induced Redox Disruption in Embryos

Events that control developmental changes occur during specific windows of gestation and if disrupted, can lead to dysmorphogenesis or embryolethality. One largely understudied aspect of developmental control is redox regulation, where the untimely disruption of intracellular redox potentials (E_h) may alter development, suggesting that tight control of developmental‐stage–specific redox states is necessary to support normal development. In this study, mouse gestational day 8.5 embryos in whole embryo culture were treated with 10 μM dithiole‐3‐thione (D3T), an inducer of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). After 14 hr, D3T‐treated and ‐untreated conceptuses were challenged with 200 μM hydrogen peroxide (H₂O₂) to induce oxidant‐induced change to intracellular E_hs. Redox potentials of glutathione (GSH), thioredoxin‐1 (Trx1), and mitochondrial thioredoxin‐2 (Trx2) were then measured over a 2‐hr rebounding period following H₂O₂ treatment. D3T treatment increased embryonic expression of known Nrf2‐regulated genes, including those responsible for redox regulation of major intracellular redox couples. Exposure to H₂O₂ without prior D3T treatment produced significant oxidation of GSH, Trx1, and Trx2, based on E_h values, where GSH and Trx2 E_h recovered, reaching to pre‐H₂O₂ E_h ranges, but Trx1 E_h remained oxidized. Following H₂O₂ addition in culture to embryos that received D3T pretreatments, GSH, Trx1, and Trx2 were insulated from significant oxidation. These data show that Nrf2 activation may serve as a means to protect the embryo from chemically induced oxidative stress through the preservation of intracellular redox states during development, allowing normal morphogenesis to ensue.     

Atmospheric hydrogen peroxide and Eoarchean iron formations

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean–Early Paleoproterozoic (2.7–2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H₂O₂), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H₂O₂ that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO₂, O₂, and CH₄ mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H₂O₂ rainout was calculated to be <10⁶ molecules cm^?2 s^?1. Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H₂O₂ was insufficient by several orders of magnitude to account for IF deposition (requiring ~10¹¹ H₂O₂ molecules cm^?2 s^?1). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)‐oxidizing micro‐organisms the most likely mechanism responsible for Earth's oldest IF.     

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication

UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of ¹O₂ by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of ¹O₂ by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.     

Nobiletin protects human retinal pigment epithelial cells from hydrogen peroxide–induced oxidative damage

Nobiletin (3′,4′,5,6,7,8‐hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, has been reported to have antioxidant effect. However, the effect of nobiletin on human retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H₂O₂) is still unclear. Therefore, we investigated the protective effect of nobiletin against H₂O₂‐induced cell death in RPE cells. Our results demonstrated that nobiletin significantly increased cell viability from oxidative stress. Nobiletin inhibited H₂O₂‐induced ROS production and caspase‐3/7 activity in ARPE‐19 cells. Furthermore, nobiletin significantly increased Akt phosphorylation in ARPE‐19 cells exposed to H₂O₂. Meanwhile, LY294002, an inhibitor of PI3K/Akt, abolished the protective effect of nobiletin against H₂O₂‐induced decreased cell viability and increased caspase‐3/7 activity in ARPE‐19 cells. In summary, these data show that nobiletin protects RPE cells against oxidative stress through activation of the Akt‐signaling pathway. Thus, nobiletin should be an oxidant that attenuates the development of age‐related macular degeneration.     

Cinnamtannin B-1, a natural antioxidant that reduces the effects of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on CCK-8-evoked responses in mouse pancreatic acinar cells

This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen peroxide (H₂O₂) in mouse pancreatic acinar cells. We have studied Ca²⁺ mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence of various concentrations of H₂O₂. We found that H₂O₂ (0.1–100 μM) increased CM-H₂DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H₂O₂-induced oxidation of CM-H₂DCFDA. CCK-8 induced oxidation of CM-H₂DCFDA in a similar way to low micromolar concentrations of H₂O₂, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H₂O₂ induced a slow and progressive increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Cinnamtannin B-1 reduced the effect of H₂O₂ on [Ca²⁺]_c, but only at the lower concentrations of the oxidant. H₂O₂ inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H₂O₂ on enzyme secretion. Finally, H₂O₂ reduced cell viability, and the antioxidant protected acinar cells against H₂O₂. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca²⁺ overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.     

Graphene‐amplified electrogenerated chemiluminescence of CdTe quantum dots for H2O2 sensing

Electrogenerated chemiluminescence (ECL) of thiol‐capped CdTe quantum dots (QDs) in aqueous solution was greatly enhanced by PDDA‐protected graphene (P‐GR) film that were used for the sensitive detection of H₂O₂. When the potential was cycled between 0 and ?2.3 V, two ECL peaks were observed at ?1.1 (ECL‐1) and ?1.4 V (ECL‐2) in pH 11.0, 0.1 M phosphate buffer solution (PBS), respectively. The electron‐transfer reaction between individual electrochemically‐reduced CdTe nanocrystal species and oxidant coreactants (H₂O₂ or reduced dissolved oxygen) led to the production of ECL‐1. While mass nanocrystals packed densely in the film were reduced electrochemically, assembly of reduced nanocrystal species reacted with coreactants to produce an ECL‐2 signal. ECL‐1 showed higher sensitivity for the detection of H₂O₂ concentrations than that of ECL‐2. Further, P‐GR film not only enhanced ECL intensity of CdTe QDs but also decreased its onset potential. Thus, a novel CdTe QDs ECL sensor was developed for sensing H₂O₂. Light intensity was linearly proportional to the concentration of H₂O₂ between 1.0 × 10^?5 and 2.0 x 10^‐7 mol L^?1 with a detection limit of 9.8 x 10^?8 mol L^?1. The P‐GR thin‐film modified glassy carbon electrode (GCE) displayed acceptable reproducibility and long‐term stability. Copyright © 2012 John Wiley & Sons, Ltd.