ROS Quantification Research Articles

Atorvastatin Prevents the Neuron Loss in the Hippocampal Dentate Gyrus Region through its Anti-Oxidant and Anti-Apoptotic Activities.

Atorvastatin is a member of statins, which has shown positive vascular effects, anti-oxidant, anti-platelet, and anti-apoptotic properties. In this study, we hypothesized that atorvastatin could prevent the neurons lost in the hippocampal dentate gyrus region after transient global Ischemia/Reperfusion (I/R) through its anti- oxidant and anti-apoptotic activities. Twenty-four male Wistar rats, 12-13 weeks old and weighing 250-300 g, were divided randomly into four groups: control, I/R, vehicle (I/R treated with NaCl) and experiment (I/R treated with atorvastatin, 10 mg/kg); rats were sacrificed 96 hours after I/R. Quantitative expression of genes (caspase 8, p53, bax, bcl2, cytochrome c) was studied. The MDA level, SOD, CAT, and GPx activities were measured with biochemical tests. To detect apoptotic cells, TUNEL and Nissl staining were performed. Mitochondria were prepared from the hippocampus rats and used for the quantification of mitochondrial ROS, ATP level, GSH content, membrane potential, cytochrome c release, and determination of mitochondrial swelling. Atorvastatin attenuated the overexpression of bax, cytochrome C, p53, and caspase8 mRNAs and induced expression of bcl-2 mRNA (P<0.001). Atorvastatin treatment increased anti-oxidant enzyme levels (P<0.01). Treatment with atorvastatin reduced the number of TUNEL-positive cells. It could decrease the cytochrome c release (P<0.01), inhibit the decrease of MMP (P<0.001) and increase the ATP level (P<0.001) in hippocampal mitochondria compared with the I/R group. Atorvastatin treatment in I/R rats decreases oxidative stress, production of ROS, apoptosis rate in neuronal cells, and improves the mitochondrial function. Hence, atorvastatin has a proper neuronal protective effect against the I/R injury in the brain.

Abstract 1457: Akt phosphorylation of mitochondrial Lonp1 protease enables oxidative metabolism and advanced tumor traits

Abstract Introduction: The control of protein homeostasis, or proteostasis is essential for cellular adaptation to disparate environmental cues and relies on protein (re)folding or proteolytic removal of aggregated or misfolded proteins. Defective proteostasis can irreversibly impair organelle functions and activate cell death through an unfolded protein response. Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. In this study, we investigated the mechanisms of stress-regulated LonP1 (ATP-dependent protease) in tumor mitochondria and their implications for advanced cancer traits. Experimental procedures: We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, mitochondrial ROS quantification in live cells, mitochondrial electron transport chain (ETC) activity, mitochondria time-lapse video microscopy, protein folding, proteomics analysis, mitochondrial bioenergetics, mitochondrial trafficking and tumor cell motility, and tumor growth and metastasis. A xenograft prostate cancer model was used to study antitumor activity in vivo. Results: We showed that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo. Conclusion: These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target. Citation Format: Jagadish C. ghosh, Jae Ho Seo, Ekta Agarwal, Yuan Wang, Andrew V. Kossenkov, Hsin-Yao Tang, David W. Speicher, Dario C. Altieri. Akt phosphorylation of mitochondrial Lonp1 protease enables oxidative metabolism and advanced tumor traits [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1457.

Proapoptotic Effects of triazol-1,4-Naphthoquinones Involve Intracellular ROS Production and MAPK/ERK Pathway in Human Leukemia Cells.

The natural products constitute an important source of antitumor and cytotoxic agents. Naphthoquinones are effectively quinones present in different plants, with demonstrated anticancer activities. A recent study conducted by our group demonstrated the antileukemic potential of two novel triazol-1,4- naphthoquinones derivatives, PTN (2-(4-Phenyl-1H-1,2,3-triazol-1-yl)-1,4-naphthoquinone) and MPTN (2-[4- (4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-1,4-naphthoquinone). Although, the mechanisms underlying the proapoptotic effects of PTN and MPTN have not been fully elucidated so far. The aim of this study was to evaluate the proapoptotic mechanism of PTN and MPTN in human acute leukemia cells. We used fluorescence microscopy to observe acridine orange and annexin V staining cells. Flow cytometry assay has also been used for ROS quantification, BAX and cytochrome c proteins expression and apoptosis analysis. MTT assay and western blotting technique have been performed as well for MAPK pathway analysis. By using the acridine orange and annexin V staining with fluorescence microscopy, we have characterized the proapoptotic effects of PTN and MPTN in HL-60 cells involving the intrinsic mitochondrial pathway, since these compounds promoted an increase in the intracellular BAX and cytochrome c protein levels (p<0.05). We further demonstrated that apoptosis induction in HL-60 cells was mediated by increasing intracellular ROS levels via ERK but not p38 MAPKs pathway. Taken together, these results have demonstrated that PTN and MPTN are promising tools for the development of new anti-leukemic drugs.

One-pot phytosynthesis of nano-silver from Mentha longifolia L.: their characterization and evaluation of photodynamic potential

The present study deals with the ecofriendly one-pot synthesis and stabilization of silver nanoparticles (AgNPs) by using aqueous extract of Mentha longifolia branches. Spectrophotometric analysis of different ratios of reactants revealed that a 1 to 9 ratio of plant extract and silver salt solution respectively is the most suitable proportion for synthesis. Synthesis of AgNPs was confirmed initially by the observation of change in the color of the reaction mixture which was carried out at 60 °C by using 3 mM of silver salt and the pH of the reaction medium was maintained at 5.22. A characteristic surface plasmon resonance (SPR) band was observed at 495 nm of light wavelength. SEM images revealed that the nanoparticles are in ∼20–80 nm and are anisotropic and nearly spherical while EDX analysis showed the presence of elemental Ag with ∼90% signal intensity. Size distribution analysis of AgNPs was performed by dynamic light scattering technique and AgNPs were found in the range of ∼8–30 nm. ROS quantification revealed that the AgNPs have a quantum yield of 0.09 Φ which provides them the ability to proteolytically treat cancer and other microbial pathogenic cells. AgNPs did not report any photothermal activity to be used as photodynamic agents. These findings explain the redox potential of M. longifolia to bio-fabricate AgNPs and their abilities to generate ROS may help to curb dreading diseases.

Understanding the potential of bio-fabricated non-oxidative silver nanoparticles to eradicate Leishmania and plant bacterial pathogens

The present study was aimed to use an aqueous extract of Mentha arvensis L. to bio-fabricate silver nanoparticles (AgNPs) and evaluation of the potential of non-oxidative nanoparticles in photodynamic treatment of pathogens. Spectrophotometric analysis of colloidal AgNPs represented a characteristic SPR band at 485 nm of the light wavelength. Nanoparticles were structurally anisotropic and nearly spherical and observed between ~ 20 and 100 nm by using SEM and AFM images. The dynamic light scattering technique determined particle size between ~ 14 and 32 nm. Biological applications manifested that the AgNPs are highly biocompatible and caused rupturing of RBCs at an exceptionally high dose estimated as ~ 214 µg/ml, while 10 µg/ml of AgNPs killed nearly ~ 50% of Leishmania tropica. AgNPs were also found effective to curb the growth of various plant bacterial pathogens and the dose-dependent response was observed in between ~ 2 and 12 µg/ml and MIC was recorded at 12 µg/ml. ROS quantification revealed that the AgNPs are not potent to produce free radicles and did not report any quantum yield. The elemental composition analysis of AgNPs by EDX confirmed that these nanoparticles are non-oxidative. ROS quantification and photothermal activity evaluation showed that these nanoparticles cannot be used in photodynamic therapy. The experimental outcomes of this study clarify the use of biocompatible AgNPs into pharmaceutical formulations to treat Leishmaniasis and plant bacterial pathogens.

First insights into the role of PQQ cofactor in the modulation of bacterial redox state and in the early interaction with peanut (Arachis hypogaea L.)

The objective of this work was to study the role of pyrroloquinolinequinone (PQQ) of Serratia sp. S119 strain in the maintenance of the bacterial redox state and in the early interaction with peanut. Lipid peroxidation and antioxidant enzymatic activities were analyzed in Serratia sp. S119 and in its PQQ minus mutant Serratia sp. RSL, exposed to hydrogen peroxide (H2O2). In the plant-bacteria interaction histochemical detection and quantification of H2O2 content was determined in peanut roots inoculated with the strains. The response to the oxidant agent showed significant differences between both studied bacteria. RSL showed growth decrease, lipid peroxidation increase and catalase enzyme activity reduction when the strain was exposed to H2O2. Also, a decrease in peroxidase activity was observed, regardless the presence or absence of this reactive species in this strain. The histochemical staining and quantification of ROS in peanut roots indicated an increase of H2O2 at 60 and 240 min postinoculation (pI) when plants were inoculated with RSL and S119 strains, respectively. In conclusion, PQQ would participate in the maintenance of cellular redox balance during oxidative stress in Serratia sp. S119 and, in the early recognition steps between this strain and peanut plant.

Cytotoxic effects of Ceiba pentandra L. mediated silver nanoparticles on HCT-116 colon cancer cell lines through ROS generation and cell membrane damage

In this study, we assessed the biological effect of Ceiba pentandra bark silver nanoparticles (CP-AgNPs) on HCT-116 cancer cells with an emphasis on cell cytotoxicity, quantification of ROS and determination of mitochondria membrane potential. The synthesized Ceiba pentandra bark silver nanoparticles were characterized by UV-vis spectrometer, High-resolution transmission electron microscope (HR-TEM), X-ray diffraction (XRD) and Selected area electron diffraction (SAED). The synthesized silver nanoparticle cytotoxic and apoptotic effects were studied on colorectal cancer cells (HCT-116). The nanoparticles exhibited an inhibitory concentration (IC50) at (60μg/mL). CP-AgNPs significantly inhibited cell viability and changed the morphology of HCT-116 colon cancer cells. Moreover, CP-AgNPs increased the level of reactive oxygen species, induced cell apoptosis in HCT-116 cell lines through the interference of the mitochondrial membrane potential. These results indicated that Ceiba pentandra bark silver nanoparticles (CP-AgNPs) might act as prospective anticancer agents in colon cancer cells.

Bacterial viability after antimicrobial photodynamic therapy with curcumin on multiresistant Staphylococcus aureus.

Staphylococcus aureus are multiresistant pathogens that causes superficial and systemic infections. Antimicrobial photodynamic therapy (APDT) is an alternative in the treatment of diseases caused by these bacteria. Aim: In this study the APDT response on growth, viability, formation of reactive oxygen species and adhesion of methicillin-sensitive strains of Staphylococcus aureus, strains of methicillin-resistant S. aureusand American-type culture collection (ATCC) of S. aureus were evaluated in vitro, after incubation with curcumin for 20min, and irradiated with LED. Materials & methods: Bacterial growth was assessed by the number of colony-forming units, viability and adhesion were evaluated by confocal microscopy and ROS quantification was performed by fluorimetry. Results: Was observed increase in the production of ROS in APDT groups, besides a decrease in the 4 log growth and loss of the bacterial adhesion. Conclusion: APDT with Curcumin may be an interesting therapeutic alternative, due to its in vitro response, in the control multiresistant clinical S. aureus strains.

Mitochondrial superoxide anions induced by exogenous oxidative stress determine tumor cell fate: an individual cell-based study.

Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli. Modified microfluidics and imaging techniques were used to determine O2 •- levels and construct an O2 •- reaction network. To elucidate the consequences of increased O2 •- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca2+ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. Results from a modified microchip demonstrated that 1 mmol/L H2O2 induced a rapid increase in cellular O2 •- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca2+ and mitochondrial Ca2+ by confocal laser scanning microscopy and confirmed that Ca2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca2+ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.

Structure Guided Design and Development of High Affinity Selective Kinase Inhibitor: A Newer Therapeutic Approach to Attenuate Hepatocelular Carcinoma

Introduction: Microtubule affinity regulating kinase 4 (MARK4) is Ser/Thr kinase, recently exploited as important drug target for neurodegenerative, cancer and other related metabolic disorders. This protein is associated with the centrosome throughout mitosis and may be involved in cell cycle control, specifically in the G1/S checkpoint. Overexpression of MARK4 has been reported in the hepatocellular carcinomas. Method: Structure of human MARK4 was deeply investigated to identify potent inhibitors in existing library. Initial screening of all these compounds was done using molecular docking in the active-site cavity of kinase which were further experimentally validated by fluorescence binding studies and ITC measurements. Compounds showing significant binding affinity were further checked for the extent of enzyme inhibition. Cell proliferation, ROS quantification and apoptotic potential of selected compounds in cancerous (HeLa, HepG2, HuH7 and MCF-7) and normal (HEK-293-T) cell lines were performed to select best inhibitors. Inspired from the structural activity of these natural products, we have synthesized many series of potent inhibitors with improved selectivity towards the MARK4/VAMKIV. Mechanism of regulation of cell proliferation by these selected inhibitors were also studied in detail. Result: Studied acridone derivatives significantly bind to the active site cavity of MARK4 that might be responsible for enzyme inhibition. The selected lead molecules also show the specificity towards MARK4. Inhibition of MARK4 hinders the proliferation of cancerous cells, enhance ROS generation and induce apoptosis. A series of Isatin-triazole hydrazones were also designed and synthesised as MARK4 inhibitor which showed better binding affinity and enzyme inhibition potential in sub micromolar range. This compound also inhibited cell proliferation, cell migration and induces apoptosis in cancerous cells and showed MARK4 specific profile. Conclusion: The structure-guided design and development of high affinity selective inhibitors of human kinase may be further implicated in for therapeutic management of cancer progression and metastasis especially for hepatocellular carcinomas.

Birch Sap (&amp;lt;i&amp;gt;Betula alba&amp;lt;/i&amp;gt;) and Chaga Mushroom (&amp;lt;i&amp;gt;Inonotus obliquus&amp;lt;/i&amp;gt;) Extracts Show Anti-Oxidant, Anti-Inflammatory and DNA Protection/Repair Activity &amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt;

OBJECTIVE: The skin interacts strictly with the surrounding environment. Despite an efficient system of protection, its integrity is continuously assaulted by a massive group of external stresses. UV irradiations represent one of the most harmful factors for the cutaneous tissue. Both UV-A and UV-B can induce deep modifications of the different layers of the skin, including a weakening of its barrier function properties, DNA damages and degradation of the extracellular matrix. The aim of this project was to assess the UV protection activity of two natural compounds, the birch sap from Betula alba and organic extract from Inonotus obliquus (chaga mushroom) used separately or in a complex. METHODS: The anti-oxidant (ROS and MDA quantification, catalase and SOD activity measurement), anti-inflammatory (IL-1β, IL-6, IL-8, IL-10, TNF-α and INF-γ dosages) and the DNA protection/repair activities (DNA lesion site analysis) of birch sap and chaga mushroom extracts tested separately or in a complex containing organic birch sap 5% and Inonotus obliquus extracts 2% were evaluated in vitro after exposure of cultured keratinocytes and fibroblasts or reconstructed epidermis to UV-A/UV-B irradiations. RESULTS: We observed that birch sap from Betula alba and extracts from Inonotus obliquus prevent the formation of ROS and decrease the oxidative stress induced under UV irradiations, suggesting a strong anti-oxidant activity. In addition, the tested products showed an immunomodulatory effect by reducing the quantity of pro-inflammatory cytokines upon UV irradiations. UV-induced DNA damages of keratinocytes were also reduced by birch sap and chaga mushroom extracts. CONCLUSION: Here, for the first time, we have shown the photo-protection activity of extracts obtained from Betula alba and Inonotus obliquus mushroom on skin cells exposed to UV-A and UV-B irradiations. Due to their anti-oxidant, anti-inflammatory and DNA protection/repair activities, the tested products represent promising candidates in the development of cosmetic products with anti-photo-aging activity.

Synthesis, molecular docking and inhibition studies of novel 3-N-aryl substituted-2-heteroarylchromones targeting microtubule affinity regulating kinase 4 inhibitors

A series of 3-N-aryl substituted-2-heteroarylchromones was efficiently synthesized via Pd-mediated oxidative coupling under the influence of hetero-atoms neighboring group participation. Synthesized molecules were evaluated against human microtubule affinity regulating kinase 4 (MARK4) enzyme inhibitors, a recently identified anti-cancer drug target. Among 21 synthesized molecules, compounds 2, 3, and 14 exhibited excellent in vitro inhibitory effect against MARK4 with IC50 value (50% of ATPase activity) at 2.12 ± 0.22 μM, 1.98 ± 0.34 μM and 5.56 ± 0.42 μM respectively. The fluorescence binding and dot blot assay of these compounds were found in μM range, indicating a better binding affinity. In vitro study of these compounds against cancerous cells (MCF-7 and HepG2) inhibited the cell viability, induced apoptosis and lowered the tau-phosphorylation. Cell viability studies of compounds 2, 3, and 14 showed inhibition of cancerous cells growth with IC50 values of 3.22 ± 0.42, 4.32 ± 0.23 μM and 16.22 ± 1.33 μM for human breast cancer cells (MCF-7) and 6.45 ± 1.12, 5.22 ± 0.72 μM and 19.12 ± 1.43 μM for human liver carcinoma cells (HepG2) respectively. ROS quantification of these compounds showed oxidative stress to cancerous cells and molecular docking study showed hydrogen bonding, charge or polar and van der Waals interactions with the active site residues of MARK4. Owning to high binding fit nicely in the active site, offering the possibilities to be used as novel therapeutic molecules in the drug discovery against MARK4-related diseases.

Baccharis trimera protects against ethanol induced hepatotoxicity in vitro and in vivo

Ethnopharmacological relevanceBaccharis trimera has been traditionally used in Brazil to treat liver diseases. Aim of the studyTo evaluate the protective effect of Baccharis trimera in an ethanol induced hepatotoxicity model. Materials and methodsThe antioxidant capacity was evaluated in vitro by the ability to scavenged the DPPH radical, by the quantification of ROS, NO and the transcription factor Nrf2. Hepatotoxicity was induced in animals by administration of absolute ethanol for 2 days (acute) or with ethanol diluted for 28 days (chronic). The biochemical parameters of hepatic function (ALT and AST), renal function (urea and creatinine) and lipid profile (total cholesterol, triglycerides and HDL) were evaluated. In addition to antioxidant defense (SOD, catalase, glutathione), oxidative damage markers (TBARS and carbonylated protein), MMP-2 activity and liver histology. ResultsBaccharis trimera promoted a decrease in ROS and NO, and at low concentrations promoted increased transcription of Nrf2. In the acute experiment it promoted increase of HDL, in the activity of SOD and GPx, besides diminishing TBARS and microesteatosis. Already in the chronic experiment B. trimera improved the hepatic and renal profile, decreased triglycerides and MMP-2 activity, in addition to diminishing microesteatosis. ConclusionWe believe that B. trimera action is possibly more associated with direct neutralizing effects or inhibition of reactive species production pathways rather than the modulation of the antioxidant enzymes activity. Thus it is possible to infer that the biological effects triggered by adaptive responses are complex and multifactorial depending on the dose, the time and the compounds used.

Diosgenin a phytosterol substitute for cholesterol, prolongs the lifespan and mitigates glucose toxicity via DAF-16/FOXO and GST-4 in Caenorhabditis elegans

Caenorhabditis elegans is a sterol auxotroph requires minute amount of exogenous sterol for their growth and development. To culture the C. elegans, cholesterol was given as sterol molecule to maintain the optimum survival of worms. Diosgenin (DG), a plant derived steroidal saponin, structurally similar to cholesterol has been used as a precursor for the synthesis of steroidal hormones. In this study, worms were cultured with cholesterol (Cho+) and cholesterol-free (Cho−) medium with DG (5, 10 and 50μg/mL) at 20°C. It was observed that worms cultured in (Cho−) exhibits late egg production, reduced lipid level and short lifespan, while addition of DG overcomes all defective facts. Combinations of both cholesterol and DG further extend the lifespan (20.8%), hinder lipid level and resistance to oxidative, thermal and high glucose stress. The intracellular ROS quantification was done by flouroscenic probe H2DCF-DA and confirmed that DG had significantly reduced ROS level (35.85%). Increased lifespan of worms were observed in the medium treated with DG which activates the nuclear translocation of DAF-16/FOXO transcription factor, followed by downstream antioxidant gene sod-3 as evidenced by GFP tagged strain. The expression of Phase II detoxification enzyme GST-4 significantly (p<0.001) increased in transgenic worms exposed to DG with 50mM glucose, and storage of lipid in intestinal cells was reduced in N2 wild type worms. Genetic requirement of DG induced longevity was studied with different mutant strains of mev-1, daf-16, skn-1, and eat-2. These studies have proved that DG is a sterol source to worms and modulate the DAF-16, SOD-3 and GST-4 expression levels to extend the lifespan of worms. The present study has also highlighted the use of phytosterols as an alternative to cholesterol.

Elucidation of Dietary Polyphenolics as Potential Inhibitor of Microtubule Affinity Regulating Kinase 4: In silico and In vitro Studies

Microtubule affinity regulating kinase 4 (MARK4) is a Ser/Thr kinase belonging to AMPK-like family, has recently become an important drug target against cancer and neurodegenerative disorders. In this study, we have evaluated different natural dietary polyphenolics including rutin, quercetin, ferulic acid, hesperidin, gallic acid and vanillin as MARK4 inhibitors. All compounds are primarily binds to the active site cavity of MARK4. In silico observations were further complemented by the fluorescence-binding studies and isothermal titration calorimetry (ITC) measurements. We found that rutin and vanillin bind to MARK4 with a reasonably high affinity. ATPase and tau-phosphorylation assay further suggesting that rutin and vanillin inhibit the enzyme activity of MARK4 to a great extent. Cell proliferation, ROS quantification and Annexin-V staining studies are clearly providing sufficient evidences for the apoptotic potential of rutin and vanillin. In conclusion, rutin and vanillin may be considered as potential inhibitors for MARK4 and further exploited to design novel therapeutic molecules against MARK4 associated diseases.

Sensitivity of the green algae Chlamydomonas reinhardtii to gamma radiation: Photosynthetic performance and ROS formation

The aquatic environment is continuously exposed to ionizing radiation from both natural and anthropogenic sources, making the characterization of ecological and health risks associated with radiation of large importance. Microalgae represent the main source of biomass production in the aquatic ecosystem, thus becoming a highly relevant biological model to assess the impacts of gamma radiation. However, little information is available on the effects of gamma radiation on microalgal species, making environmental radioprotection of this group of species challenging. In this context, the present study aimed to improve the understanding of the effects and toxic mechanisms of gamma radiation in the unicellular green algae Chlamydomonas reinhardtii focusing on the activity of the photosynthetic apparatus and ROS formation. Algal cells were exposed to gamma radiation (0.49–1677mGy/h) for 6h and chlorophyll fluorescence parameters obtained by PAM fluorometry, while two fluorescent probes carboxy-H2DFFDA and DHR 123 were used for the quantification of ROS. The alterations seen in functional parameters of C. reinhardtii PSII after 6h of exposure to gamma radiation showed modifications of PSII energy transfer associated with electron transport and energy dissipation pathways, especially at the higher dose rates used. Results also showed that gamma radiation induced ROS in a dose-dependent manner under both light and dark conditions. The observed decrease in photosynthetic efficiency seems to be connected to the formation of ROS and can potentially lead to oxidative stress and cellular damage in chloroplasts. To our knowledge, this is the first report on changes in several chlorophyll fluorescence parameters associated with photosynthetic performance and ROS formation in microalgae after exposure to gamma radiation.

The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes.

Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer.

Thymol and Thymus Vulgaris L. activity against UVA- and UVB-induced damage in NCTC 2544 cell line

Many authors focused on the research of natural compounds in order to protect skin from indirect (UVA) and direct (UVB) ultraviolet radiation side effects. The aim of this study to evaluate the protective effect of a dry extract from T. vulgaris L. and of its major synthetic compound thymol (about 60%), against oxidative and genotoxic UVA- and UVB damage. Experiments were reproduced in a low differentiated keratinocytes cell line (NCTC 2544) Cells were pretreated for 1h, in serum-free medium, with thymol (1μg/mL) or T. vulgaris L. (1.82μg/mL) then exposed to different UVA (8–24J/cm2) or UVB doses (0.016–0.72J/cm2). Immediately after the UV exposure the intracellular redox status was evaluated by ROS quantification and by LPO. Genotoxic aspects were evaluated 24h after the end of irradiations using the alkaline comet assay, the micronucleus formation assay and the immunostaining of phosphorylated H2AX histone protein (detected 1h after the end of UV exposure). Thymol and T. vulgaris L. extract inhibited ROS generation in UVA and UVB-irradiated cells. On the contrary, MDA formation was reduced only in UVA treated cells. Both agents decreased the DNA damage evaluated by the alkaline comet assay, but not in the micronucleus and H2AX tests probably because of the severity of damage (double strands) detected.

Number of Hydroxyl Groups on the B-Ring of Flavonoids Affects Their Antioxidant Activity and Interaction with Phorbol Ester Binding Site of PKCδ C1B Domain: In Vitro and in Silico Studies.

Although flavonoids have been reported for their benefits and nutraceutical potential use, the importance of their structure on their beneficial effects, especially on signal transduction mechanisms, has not been well clarified. In this study, three flavonoids, pinocembrin, naringenin, and eriodictyol, were chosen to determine the effect of hydroxyl groups on the B-ring of flavonoid structure on their antioxidant activity. In vitro assays, including DPPH scavenging activity, ROS quantification by flow cytometer, and proteins immunoblotting, and in silico analysis by molecular docking between the flavonoids and C1B domain of PKCδ phorbol ester binding site were both used to complete this study. Eriodictyol (10 μM), containing two hydroxyl groups on the B-ring, exhibited significantly higher (p < 0.05) antioxidant activity than pinocembrin and naringenin. The IC50 values of eriodictyol, naringenin, and pinocembrin were 17.4 ± 0.40, 30.2 ± 0.61, and 44.9 ± 0.57 μM, respectively. In addition, eriodictyol at 10 μM remarkably inhibited the phosphorylation of PKCδ at 63.4% compared with PMA-activated RAW264.7, whereas pinocembrin and naringenin performed inhibition activity at 76.8 and 72.6%, respectively. According to the molecular docking analysis, pinocembrin, naringenin, and eriodictyol showed -CDOCKER_energy values of 15.22, 16.95, and 21.49, respectively, reflecting that eriodictyol could bind with the binding site better than the other two flavonoids. Interestingly, eriodictyol had a remarkably different pose to bind with the kinase as a result of the two hydroxyl groups on its B-ring, which consequently contributed to greater antioxidant activity over pinocembrin and naringenin.

744 Genome-Wide Association of Composition and Diversity of the Intestinal Bacterial Phyla in Healthy First Degree Relatives (FDR) of Crohn's Disease (CD) Subjects

Figure 1: Sepp1-/enteroids display an increase in stem cell characteristics and increased ROS production, proliferation, and decreased survival in response to hydrogen peroxide administration. (A) Relative Sepp1 mRNA expression. (B) Enteroid growth properties. (C) ROS quantification after 2 hours of treatment (left). Representative images (100x magnification, right). (D) Proliferation quantification as determined by EdU+ cells per crypt area. (100x magnification, right). (E) Survival curves for WT (red), Sepp1 (blue), and Sepp1 (black) enteroids after daily treatment with 400 μM hydrogen peroxide. *P<0.05, **P<0.01, ***P<0.001.