Oxidative Stress In Mammalian Cells Research Articles

Biophysical Analysis of EGCG’s Protective Effects on Camptothecin-Induced Oxidative Stress in Bone-like Cancer Cells Using Electric Cell-Substrate Impedance Sensing (ECIS)

Various medical treatments aim to counteract the impact of oxidants on mammalian cells. One such antioxidant is Epigallocatechin-3-gallate (EGCG), an active ingredient in green tea, which has demonstrated protective effects against cellular oxidants like camptothecin (CAMPT). This study examines how EGCG mitigates CAMPT’s effects on UMR cells, focusing on cell proliferation and biophysical parameters. UMR cells were treated with different CAMPT concentrations and incubated for 72 h. Subsequently, cell proliferation and viability were assessed. In a separate experiment, UMR cells were co-treated with CAMPT and varying EGCG concentrations to evaluate EGCG’s ability to mitigate CAMPT’s oxidative effect. Electric Cell–Substrate Impedance Sensing (ECIS) technology was also used to assess the biophysical parameters of CAMPT-treated UMR cells, including cell monolayer resistance, cell spreading, and cell attachment. The results showed a concentration-dependent decrease in cell proliferation for CAMPT-treated UMR cells. However, co-treatment with EGCG reversed CAMPT’s oxidative effects in a concentration-dependent manner. ECIS technology revealed a decrease in biophysical parameters when UMR cells were treated with CAMPT alone. Statistical analysis indicated significant differences with p-values < 0.05. This study suggests that EGCG effectively protects UMR cells from oxidative stress and highlights its potential role in mitigating oxidative stress in mammalian cells. Additionally, the use of ECIS technology validates its application in corroborating the biological effects of CAMPT and EGCG on UMR cells.

Ebselen Optimized the Therapeutic Effects of Silver Nanoparticles for Periodontal Treatment.

Silver nanoparticles (AgNPs) possess excellent antibacterial effects on periodontal pathogens, but their clinical application is limited mainly due to their cytotoxicity through inducing oxidative stress in human cells. Ebselen disrupts the reactive oxygen species (ROS) scavenging in bacteria and relieves oxidative stress in mammalian cells. This study aimed to assess the antibacterial and anti-inflammatory effects of AgNPs and ebselen as well as the protective effect of ebselen, to further provide the theoretical basis for their future application in periodontal treatment. The antibacterial and anti-biofilm effects of the synthesized AgNPs combined with ebselen were assessed on Porphyromonas gingivalis (P. gingivalis), Streptococcus gordonii (S. gordonii), and Fusobacterium nucleatum (F. nucleatum) in planktonic condition and as biofilms. In addition, the intracellular bactericidal efficiency of AgNPs and ebselen was evaluated in P. gingivalis-infected human gingival fibroblasts (HGFs). The cytotoxicity, intracellular ROS levels, and potential antioxidative enzymes were detected in HGFs treated with AgNPs and ebselen. Further, the anti-inflammatory effects were evaluated by in vitro and in vivo experiments. The combination of AgNPs and ebselen showed excellent antibacterial effects against planktonic P. gingivalis and F. nucleatum and synergistic antibiofilm effects on all mono- and multi-species biofilms. In addition, ebselen significantly enhanced the intracellular bactericidal efficiency of AgNPs. Furthermore, ebselen combined with up to 20 μg/mL AgNPs showed no obvious cytotoxicity to HGFs. Evidently, ebselen alleviated the AgNPs-induced ROS by increasing the levels of glutathione and superoxide dismutase 2. Moreover, AgNPs and ebselen together declined the release of P. gingivalis-stimulated inflammatory cytokines both in vitro and in vivo, and reduced alveolar bone resorption effectively. AgNPs combined with ebselen would be an effective adjuvant for periodontal treatment owing to their synergistic antibacterial and anti-inflammatory effects.

Epigallocatechin Gallate (EGCG) Mitigates The Effect of Camptothecin‐Induced Oxidative Stress on Bone Cancer‐like cells (UMR 106‐01 BSP) by Inhibiting Apoptosis

Many medical treatments have been proposed and utilized to mitigate the effects of oxidants on mammalian cells. Some studies have demonstrated the antioxidative effects of EGCG (an active ingredient in green tea) against cellular oxidants such as camptothecin (CAMPT). However, not many studies have shown how EGCG mitigates the effects of CAMPT on UMR cells. We hypothesized that EGCG mitigates the effects of CAMPT on cell proliferation and biophysical parameters in UMR cells by inhibiting CAMPT‐induced apoptosis. Briefly, UMR cells were routinely passaged, counted, plated at 4.8 x 105 cells/mL in six‐well culture plates, then treated with varying concentrations of CAMPT. The cultures were incubated for 72 hours at 37°C + 5% CO₂ infusion. Cells were subsequently counted to establish the proliferation and viability of the induced cells as compared to controls. In another experiment, designated UMR cells were co‐treated with CAMPT and different concentrations of EGCG then processed, as mentioned above, to evaluate whether or not the EGCG treatment was able to mitigate the oxidative effect of CAMPT through proliferation / viability studies. Apoptosis markers were assessed for each experiment by using a Caspase‐3/CPP32 Colorimetric Assay Kit. Lastly, designated cells were treated with CAMPT alone, or CAMPT +EGCG then assessed using electric cell‐substrate impedance sensing (ECIS) technology to explore the effects of the chemicals used on key biophysical parameters of UMR cells (i.e. cell membrane resistance, rate of cell spreading and cell attachment). Briefly, CAMPT‐treated and CAMPT+EGCG‐treated UMR cells were plated at 2.5 x 105 cells/mL in eight‐well ECIS array plates. The plates were placed into the ECIS array holder and incubated at 37°C with 5% CO₂. The biophysical parameters (cell membrane resistance/impedance, cell spreading, cell attachment) were monitored and measured in real‐time for 48 hours, using the ECIS instrument. F test ANOVA was used to compare variances between all trials and all values in the results were expressed as means ±SD. The results of the proliferation study showed a decrease in CAMPT‐treated UMR cell proliferation in a concentration‐dependent manner (Figure 1). Cells co‐treated with CAMPT + EGCG revealed that EGCG was able to reverse the oxidative effects of CAMPT on the UMR cells in a concentration‐dependent manner *Figure 2). The Caspase‐3/CPP32 Colorimetric Assay showed an increase in apoptotic marker activity from the CAMPT‐induced samples in contrast with the uninduced controls. The ECIS technology documented decreases in biophysical parameters in the UMR cells when CAMPT alone was used. Co‐treatment of the cells with CAMPT+EGCG showed a reverse effect on the biophysical parameters. F‐Test ANOVA yielded statistically significant values with p values <0.05. The results of this research suggest that EGCG is effective in protecting UMR cells from oxidative stress by inhibiting apoptosis. Moreover, results from the ECIS instrument validates this technology as a corroborator of the biological effects of CAMPT and EGCG on UMR cells. This study suggests that EGCG has a protective role in mitigating oxidative stress in mammalian cells.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS.

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Genotoxicity, oxidative stress, and inflammatory response induced by crack-cocaine: relevance to carcinogenesis.

Crack-cocaine is a cocaine by-product widely consumed by general population in developing countries. The drug is low cost and is associated with more intense effects when compared to other illicit drugs. Genotoxicity, oxidative stress, and inflammatory response are considered crucial events in carcinogenesis, since they actively participate in the multistep process. The purpose of this paper was to provide a mini review regarding the relationship between carcinogenesis and genotoxicity, oxidative stress, and inflammation induced by crack-cocaine. The present study was conducted on search of the scientific literature from the published studies available in PubMed, MEDLINE, Scopus, and Google Scholar for all kind of articles (all publications to November 2020) using the following key words: crack-cocaine, DNA damage, genotoxicity, cellular death, cytotoxicity, mutation, oxidative stress, inflammation, and mutagenicity. The results showed that published papers available were almost all in vivo test system being conducted in humans or rodents. Crack-cocaine was able to induce genotoxicity and oxidative stress in mammalian cells. However, the role of inflammatory response after exposure to crack-cocaine was not conclusive so far. In summary, this study is consistent with the notion that crack-cocaine is a chemical carcinogen as a result of genotoxicity and oxidative stress induced in mammalian and non-mammalian cells.

Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems

Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.

Bioactive constituents of Emblica officinalis overcome oxidative stress in mammalian cells by inhibiting hyperoxidation of peroxiredoxins.

Emblica officinalis (Amla) is a renowned fruit having nutritional and medicinal traits mostly linked to its antioxidants content. In the current study, the methanolic crude extract of amla fruit is subjected to sequential fractionation to get its partially purified fractions. The ethyl acetate (EA) and butanol (BUT) fractions of amla showed maximum antioxidant potential. The ferric reducing capability and nitric oxide scavenging activity were highest in EA fraction. One of the highlights of the study is the cellular antioxidant assay conducted in HeLa cells. Additionally, HeLa cells pre-treated with EA and BUT fractions were able to combat oxidative stress via total reduction in hyperoxidation of intracellular peroxiredoxin enzyme. Gallic acid, ascorbic acid, ellagic acid, rutin, quercetin, and catechol are the major compounds present in these fractions as identified by LC-ESI-MS followed by their quantification by HPLC. These findings indicate that components of E. officinalis can protect intracellular oxidative stress-mediated degeneration. PRACTICAL APPLICATIONS: The study highlighted that E. officinalis is a promising source of phenolics and flavonoids acting as natural antioxidants, which showed varied potential to scavenge ROS. Also, the plant fractions were able to fight intracellular oxidative stress via total reduction in hyperoxidation of the human peroxiredoxin. In conclusion, we can say that the regular intake of such food supplements that affect important antioxidant enzymes can be of special interest in the management of oxidative stress-mediated human ailments.

PCB11 Metabolite, 3,3'-Dichlorobiphenyl-4-ol, Exposure Alters the Expression of Genes Governing Fatty Acid Metabolism in the Absence of Functional Sirtuin 3: Examining the Contribution of MnSOD.

Although the production of polychlorinated biphenyls (PCBs) is prohibited, the inadvertent production of certain lower-chlorinated PCB congeners still threatens human health. We and others have identified 3,3’-dichlorobiphenyl (PCB11) and its metabolite, 3,3’-dichlorobiphenyl-4-ol (4OH-PCB11), in human blood, and there is a correlation between exposure to this metabolite and mitochondrial oxidative stress in mammalian cells. Here, we evaluated the downstream effects of 4OH-PCB11 on mitochondrial metabolism and function in the presence and absence of functional Sirtuin 3 (SIRT3), a mitochondrial fidelity protein that protects redox homeostasis. A 24 h exposure to 3 μM 4OH-PCB11 significantly decreased the cellular growth and mitochondrial membrane potential of SIRT3-knockout mouse embryonic fibroblasts (MEFs). Only wild-type cells demonstrated an increase in Manganese superoxide dismutase (MnSOD) activity in response to 4OH-PCB11–induced oxidative injury. This suggests the presence of a SIRT3-mediated post-translational modification to MnSOD, which was impaired in SIRT3-knockout MEFs, which counters the PCB insult. We found that 4OH-PCB11 increased mitochondrial respiration and endogenous fatty-acid oxidation-associated oxygen consumption in SIRT3-knockout MEFs; this appeared to occur because the cells exhausted their reserve respiratory capacity. To determine whether these changes in mitochondrial respiration were accompanied by similar changes in the regulation of fatty acid metabolism, we performed quantitative real-time polymerase chain reaction (qRT-PCR) after a 24 h treatment with 4OH-PCB11. In SIRT3-knockout MEFs, 4OH-PCB11 significantly increased the expression of ten genes controlling fatty acid biosynthesis, metabolism, and transport. When we overexpressed MnSOD in these cells, the expression of six of these genes returned to the baseline level, suggesting that the protective role of SIRT3 against 4OH-PCB11 is partially governed by MnSOD activity.

Protective Effects of Fibroblast Growth Factor 21 Against Amyloid-Beta1-42-Induced Toxicity in SH-SY5Y Cells.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive loss of cholinergic neurons. Amyloid beta is a misfolded protein that represents one of the key pathological hallmarks of AD. Numerous studies have shown that Aβ1-42 induces oxidative damage, neuroinflammation, and apoptosis, leading to cognitive decline in AD. Recently, fibroblast growth factor 21 (FGF21) has been suggested to be a potential regulator of oxidative stress in mammalian cells. FGF21 has been shown to improve insulin sensitivity, reduce hyperglycemia, increase adipose tissue glucose uptake and lipolysis, and decrease body fat and weight loss by enhancing energy expenditure. In this study, we investigated the effect of FGF21 Aβ1-42 toxicity in SH-SY5Y neuroblastoma cells. Our data shows that FGF21 significantly decreased Aβ1-42-induced toxic effects and repressed oxidative stress and apoptosis in cells exposed to Aβ1-42 peptide. Our investigation also confirmed that FGF21 pretreatment favorably affects HSP90/TLR4/NF-κB signaling pathway. Therefore, FGF21 represents a viable therapeutic strategy to abrogate Aβ1-42-induced cellular inflammation and apoptotic death in the SH-SY5Y neuroblastoma cells.

Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis.

Cell apoptosis is one of the main pathological alterations during oxidative stress (OS) injury. Previously, we corroborated that nuclear factor-κB (NF-κB) transactivation confers apoptosis resistance against OS in mammalian cells, yet the underlying mechanisms remain enigmatic. Here we report that microRNA-19a (miR-19a) transcriptionally regulated by reactive oxygen species (ROS) production and NF-κB deactivation prevents OS-initiated cell apoptosis through cylindromatosis (CYLD) repression. CYLD contributes to OS-initiated cell apoptosis, for which NF-κB deactivation is essential. MiR-19a directly represses CYLD via targeting 3′ UTR of CYLD, thereby antagonizing OS-initiated apoptosis. CYLD repression by miR-19a restores the IKKβ phosphorylation, RelA disassociation from IκBα, IκBα polyubiquitination and degradation, RelA recruitment at VEGF gene promoter as well as VEGF secretion in the context of OS. Either pharmacological deactivation of NF-κB or genetic upregulation of CYLD compromises the apoptosis-resistant phenotypes of miR-19a. Furthermore, miR-19a is transcriptionally downregulated upon OS in two distinct processes that require ROS production and NF-κB deactivation. VEGF potentiates the ability of miR-19a to activate NF-κB and render apoptosis resistance. Our findings underscore a putative mechanism whereby CYLD repression-mediated and NF-κB transactivation-dependent miR-19a regulatory feedback loop prevents cell apoptosis in response to OS microenvironment.

Protective effects and functional mechanisms of Lactobacillus gasseri SBT2055 against oxidative stress.

Lactobacillus gasseri SBT2055 (LG2055) is one of the probiotic lactic acid bacteria. Recently, we demonstrated that feeding with LG2055 extended the lifespan of Caenorhabditis elegans and that the prolongevity effect was dependent upon the regulation of oxidative stress response. In this study, we assessed whether LG2055 regulated the oxidative stress response of mammalian cells. In NIH-3T3 cells and primary mouse embryonic fibroblast cells, low cell proliferation rates and high reactive oxygen species levels were observed following paraquat treatment. LG2055 treatment suppressed these responses in paraquat-treated cells, indicating that LG2055 protected against oxidative stress in mammalian cells. The mRNA expression of oxidative stress-related genes, total nuclear factor-erythroid-2-related factor 2 (Nrf2) protein levels, and the nuclear translocation of Nrf2 were increased by LG2055 treatment. These results suggested that the Nrf2-antioxidant response element (ARE) signaling pathway was activated by LG2055. Furthermore, c-Jun NH2-terminal kinase (JNK) was activated by LG2055 treatment and the inhibition of JNK suppressed the activation of the Nrf2-ARE signaling pathway in LG2055-treated cells. Together, these findings suggest that LG2055 activated the Nrf2-ARE signaling pathway by JNK activation, thus strengthening the defense system against oxidative stress in mammalian cells.

The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells

Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.

Oxidative Signaling Response to Cadmium Exposure.

Changes in the intracellular thiol-disulfide balance are considered major determinants in the redox status/signaling of the cell. Cellular signaling is very sensitive to both exogenous and intracellular redox status and respond to many exogenous pro-oxidative or oxidative stresses. Redox status has dual effects on upstream signaling systems and downstream transcription factors. Redox signaling pathways use reactive oxygen species (ROS) to transfer signals from different sources to the nucleus to regulate such functions as growth, differentiation, proliferation, and apoptosis. Mitogen-activated protein kinases are activated by numerous cellular stresses and ligand-receptor bindings. An imbalance in the oxidant/antioxidant system, either resulting from excessive ROS/reactive nitrogen species production and/or antioxidant system impairment, leads to oxidative stress. Glutathione (GSH) is known to play a critical role in the cellular defense against unregulated oxidative stress in mammalian cells and involvement of large molecular antioxidants include classical antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). Cadmium (Cd), a potent toxic heavy metal, is a widespread environmental contaminant. It is known to cause renal dysfunction, hepatic toxicity, genotoxicity, and apoptotic effects depending on the dose, route, and duration of exposure. This review examines the signaling pathways and mechanisms of activation of transcription factors by Cd-induced oxidative stress thus representing an important basis for understanding the mechanisms of Cd effect on the cells.

Abstract 3048: Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system Xc−

Abstract Melanoma is a cancer that has become increasingly frequent over the last decades in the western world. Initial treatment for primary and locoregional melanoma is surgery. In metastatic disease, systematic treatment and recently immunotherapy has been the mainstay. At this stage however, the prognostic outlook is still bleak. Thus, new treatments are urgently needed. Melanoma is considered to be a radioresistant cancer. The mechanisms underlying radioresistance are multiple and incompletely characterized. In some tumors, radioresistance is mediated by increased synthesis of anti-oxidants that scavenge reactive oxygen species (ROS), induced by radiotherapy. Glutathione (GSH) is an anti-oxidant synthesized from cystine and constitutes a major defense system against oxidative stress in mammalian cells. Cystine is taken up through system Xc− (SXC), an anti-port transmembrane protein with catalytic subunit xCT. Sulfasalazine, a drug approved in the 1950s for treatment of rheumatoid arthritis and inflammatory bowel disease, has been shown to block SXC. Based on previous reports by others, and our own recent findings, we hypothesize that: I) xCT represents a mechanism for radioresistance and is expressed in radioresistant melanoma cancer and II) xCT inhibitors can act as radiosensitizers to potentiate the efficacy of radiotherapy. Expression of the catalytic subunit of SXC, xCT, was found in tissue micro array of primary and secondary melanoma biopsies. In addition, SAS treatment dramatically reduced cystine-uptake and GSH levels in melanoma cells in vitro, and markedly increased the levels of reactive oxygen species (ROS). Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the anti-oxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and irradiation synergistically reduced subcutaneous melanoma tumor growth in vivo, compared to controls or either treatment alone. Thus, future experimental studies are warranted to validate SAS as a radiosensitizer in the treatment of metastatic melanoma. Future studies will also be aimed at assessing the effect on pulmonary melanoma metastasis. Citation Format: Hilde Elise Førde, Linda Sleire, Heidi Espedal, Jan Ingemann Heggdal, Frode Selheim, Paal-Henning Pedersen, Per Øyvind Enger. Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system Xc−. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3048.

Cytotoxic responses to 405 nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species

Light at wavelength 405nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405nm light at 36J/cm2 (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54J/cm2), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione were observed from 405nm light treated cells. The mammalian cells were significantly protected from dying at 54J/cm2 in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324J/cm2. Results therefore suggested that the cytotoxic mechanism of 405nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage.

Maternal selenium status in pregnancy, offspring glutathione peroxidase 4 genotype, and childhood asthma

Maternal selenium status in pregnancy, offspring glutathione peroxidase 4 genotype, and childhood asthma

RCAN1 regulates mitochondrial function and increases susceptibility to oxidative stress in mammalian cells.

Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1ox) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12RCAN1). Similar to RCAN1ox neurons, PC12RCAN1 cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.

Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.

Differential cell death and Bcl-2 expression in the mouse retina after glutathione decrease by systemic D,L-buthionine sulphoximine administration.

Glutathione (GSH) plays a critical role in cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH decrease using [D, L]-buthionine sulphoximine (BSO) induces retinal cell death, but the underlying mechanisms of this are still unclear. Here, we demonstrated that retinal GSH level is closely related to retinal cell death as well as expression of an anti-apoptotic molecule, Bcl-2, in the retina. We induced differential expression of retinal GSH by single and multiple administrations of BSO, and examined retinal GSH levels and retinal cell death in vivo. Single BSO administration showed a transient decrease in the retinal GSH level, whereas multiple BSO administration showed a persistent decrease in the retinal GSH level. Retinal cell death also showed similar patterns: transient increases of retinal cell death were observed after single BSO administration, whereas persistent increases of retinal cell death were observed after multiple BSO administration. Changes in the retinal GSH level affected Bcl-2 expression in the retina. Immunoblot and immunohistochemical analyses showed that single and multiple administration of BSO induced differential expressions of Bcl-2 in the retina. Taken together, the results of our study suggest that the retinal GSH is important for the survival of retinal cells, and retinal GSH appears to be deeply related to Bcl-2 expression in the retina. Thus, alteration of Bcl-2 expression may provide a therapeutic tool for retinal degenerative diseases caused by retinal oxidative stress such as glaucoma or retinopathy.

Does polyamine oxidase activity influence the oxidative metabolism of children who suffer of diabetes mellitus?

Diabetes mellitus is a metabolic disease characterized by inadequate secretion of insulin. Polyamine oxidase (PAO), a FAD-containing enzyme is involved in the biodegradation of Sp and Spd, catalyzing the oxidative deamination of Sp and Spd, resulting in production of ammonia (NH(3)), corresponding amino aldehydes and H(2)O(2). Malondialdehyde (MDA) and acrolein (CH2=CHCHO), potentially toxic agents, which induce oxidative stress in mammalian cells, are then spontaneously formed from aminoaldehydes. The main signs of oxidative stress in diabetic children were the values of HbA1c and MDA levels. Polyamines have an insulin-like action. Antiglycation property of spermine and spermidine has been recently confirmed. There are no data in the literature about plasma polyamine oxidase (PAO) activities in children with type 1 diabetes. The idea of this study was to evaluate the polyamine metabolism through the estimation of polyamine oxidase activity. We have study children with newly diagnosed type 1 diabetes mellitus (n = 35, age group of 5-16 years, as well as age-matched healthy control subjects (n = 25). The biochemical investigations were done on diabetic children who have the pathological values of glucose (9.11-17.33 mmol/l) and glycosylated Hb (7.57-14.49% HbA(1c)). The children in the control group have referent values of glucose and glycated hemoglobin (4.11-5.84 mmol/L and HbA(1c) 4.22-6.81% of the total Hb. Glucose levels in blood plasma and glycosylated hemoglobin in erythrocythes hemolysates (HbA1c) were measured by using standard laboratory methods. PAO activity in venous blood plasma and the amount of malondialdehyde (MDA) were measured by the spectrophotometric methods. PAO activity, glycemia, HbA1c and MDA were significantly increased in diabetic children compared to the control subjects. PAO activity in children with type 1 diabetes mellitus was very high. The findings of higher blood HbA(1C) and MDA levels confirm the presence of oxidant stress in children with type 1 diabetes mellitus and demonstrate that PAO activity may participate in these circumstances.