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Lester Packer was an exceptional scientific leader, whose radiant personality inspired and encour-aged generations of students and scientists for research to pursue oxidants and antioxidants in biol-ogy and medicine. For the FORUM dedicated to Professor Packer, we here describe key aspects of his professional career, from the early years at Brooklyn College, Yale University and the Johnson Research Foundation at Philadelphia to his long-term base at the University of California, at UC Berkeley. The concept of the 'Antioxidant Network' formed the core of his activities in later years. His welcoming and integrative personality led to a worldwide network of colleagues, starting with the Bay Area Oxygen Club, which turned into the Oxygen Club of California (OCC), and his lead-ership in the Society for Free Radical Research-International (SFRR-I). To illustrate his warmth and outreach, which enabled him to form borderless global collaborations, we conclude with words from some of his many friends also from outside academia: Lester Packer's legacy.

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore’s MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-AT and T. retecalamus sp. nov. type strain 18-3228-7BT. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine.

Research on oxidants and electrophiles has shifted from focusing on damage to biomolecules to the more fine-grained physiological arena. Redox transitions as excursions from a steady-state redox set point are continually ongoing in maintenance of redox balance. Current excitement on these topics results from the fact that recent research provided mechanistic insight, which gives rise to more concrete and differentiated questions. This Commentary focuses on redox eustress and the feedback restoration of steady state as concepts in active maintenance of physiological health, with brief discussion of redox stress response to viral infection, exemplified by COVID-19.

'Reactive oxygen species' (ROS) is an umbrella term for an array of derivatives of molecular oxygen that occur as a normal attribute of aerobic life. Elevated formation of the different ROS leads to molecular damage, denoted as 'oxidative distress'. Here we focus on ROS at physiological levels and their central role in redox signalling via different post-translational modifications, denoted as 'oxidative eustress'. Two species, hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-), are key redox signalling agents generated under the control of growth factors and cytokines by more than 40 enzymes, prominently including NADPH oxidases and the mitochondrial electron transport chain. At the low physiological levels in the nanomolar range, H2O2 is the major agent signalling through specific protein targets, which engage in metabolic regulation and stress responses to support cellular adaptation to a changing environment and stress. In addition, several other reactive species are involved in redox signalling, for instance nitric oxide, hydrogen sulfide and oxidized lipids. Recent methodological advances permit the assessment of molecular interactions of specific ROS molecules with specific targets in redox signalling pathways. Accordingly, major advances have occurred in understanding the role of these oxidants in physiology and disease, including the nervous, cardiovascular and immune systems, skeletal muscle and metabolic regulation as well as ageing and cancer. In the past, unspecific elimination of ROS by use of low molecular mass antioxidant compounds was not successful in counteracting disease initiation and progression in clinical trials. However, controlling specific ROS-mediated signalling pathways by selective targeting offers a perspective for a future of more refined redox medicine. This includes enzymatic defence systems such as those controlled by the stress-response transcription factors NRF2 and nuclear factor-κB, the role of trace elements such as selenium, the use of redox drugs and the modulation of environmental factors collectively known as the exposome (for example, nutrition, lifestyle and irradiation).

Oxidative stress is defined as “an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage”. This Commentary presents basic features of this global concept which has attracted interest in biology and medicine. The term “antioxidants” in cellular defense against oxidants predominantly includes antioxidant enzymes with their substrates and coenzymes. Exogenous low-molecular-mass compounds also have a role, but this is more limited. Multiple biomarkers of damage due to oxidative stress have been identified for different molecular classes (protein, lipid, carbohydrate, and DNA), and the current state of practical aspects in health and disease is delineated.

My interest in biological chemistry proceeded from enzymology in vitro to the study of physiological chemistry in vivo. Investigating biological redox reactions, I identified hydrogen peroxide (H(2)O(2)) as a normal constituent of aerobic life in eukaryotic cells. This finding led to developments that recognized the essential role of H(2)O(2) in metabolic redox control. Further research included studies on GSH, toxicological aspects (the concept of “redox cycling”), biochemical pharmacology (ebselen), nutritional biochemistry and micronutrients (selenium, carotenoids, flavonoids), and the concept of “oxidative stress.” Today, we recognize that oxidative stress is two-sided. It has its positive side in physiology and health in redox signaling, “oxidative eustress,” whereas at higher intensity, there is damage to biomolecules with potentially deleterious outcome in pathophysiology and disease, “oxidative distress.” Reflecting on these developments, it is gratifying to witness the enormous progress in redox biology brought about by the science community in recent years.

The mitochondrial matrix is a prominent place in redox biology. It contains mitochondrial glutathione (mGSH), pivotal for maintaining redox homeostasis. In this issue of Cell Chemical Biology, Booty et al., (2019) introduce mitoCDNB, which depletes the mGSH pool, creating new avenues of organellar redox research.

Corona virus disease 2019 (COVID-19) is a global emergency able to overwhelm the healthcare capacities worldwide and to affect the older generation especially. When addressing the pathophysiological mechanisms and clinical manifestations of COVID-19, it becomes evident that the disease targets pathways and domains affected by the main aging- and frailty-related pathophysiological changes. A closer analysis of the existing data supports a possible role of biological age rather than chronological age in the prognosis of COVID-19. There is a need for systematic, consequent action of identifying frail (not only older, not only multimorbid, not only symptomatic) persons at risk of poor outcomes.

Significance: The redox code is a set of principles that defines the positioning of the nicotinamide adenine dinucleotide (NAD, NADP) and thiol/disulfide and other redox systems as well as the thiol redox proteome in space and time in biological systems. The code is richly elaborated in an oxygen-dependent life, where activation/deactivation cycles involving O2 and H2O2 contribute to spatiotemporal organization for differentiation, development, and adaptation to the environment. Disruption of this organizational structure during oxidative stress represents a fundamental mechanism in system failure and disease. Recent Advances: Methodology in assessing components of the redox code under physiological conditions has progressed, permitting insight into spatiotemporal organization and allowing for identification of redox partners in redox proteomics and redox metabolomics. Critical Issues: Complexity of redox networks and redox regulation is being revealed step by step, yet much still needs to be learned. Future Directions: Detailed knowledge of the molecular patterns generated from the principles of the redox code under defined physiological or pathological conditions in cells and organs will contribute to understanding the redox component in health and disease. Ultimately, there will be a scientific basis to a modern redox medicine. Antioxid. Redox Signal. 23, 734–746.

Hydrogen peroxide, the nonradical 2-electron reduction product of oxygen, is a normal aerobic metabolite occurring at about 10 nm intracellular concentration. In liver, it is produced at 50 nmol/min/g of tissue, which is about 2% of total oxygen uptake at steady state. Metabolically generated H2O2 emerged from recent research as a central hub in redox signaling and oxidative stress. Upon generation by major sources, the NADPH oxidases or Complex III of the mitochondrial respiratory chain, H2O2 is under sophisticated fine control of peroxiredoxins and glutathione peroxidases with their backup systems as well as by catalase. Of note, H2O2 is a second messenger in insulin signaling and in several growth factor-induced signaling cascades. H2O2 transport across membranes is facilitated by aquaporins, denoted as peroxiporins. Specialized protein cysteines operate as redox switches using H2O2 as thiol oxidant, making this reactive oxygen species essential for poising the set point of the redox proteome. Major processes including proliferation, differentiation, tissue repair, inflammation, circadian rhythm, and aging use this low molecular weight oxygen metabolite as signaling compound.

The micronutrient selenium and selenium-containing selenoproteins are involved in prevention of inflammation and carcinogenesis in the gut. Selenoprotein P (Sepp1), the plasma selenium transport protein, is secreted primarily from hepatocytes, but Sepp1 mRNA is also abundant in the intestine. By immunofluorescence analysis, we show that Sepp1 levels in epithelial cells of the rat jejunum increase along the crypt-to-villus axis. A different Sepp1 distribution pattern was observed in the rat colon, where the epithelial cells located at the base and at the top of the crypts were similarly positive for Sepp1. In addition, we found pronounced Sepp1 immunoreactivity in CD138-positive plasma cells scattered within the lamina propria of the colon. This hitherto unrecognized presence in terminally differentiated B-cells was corroborated by detection of Sepp1 in plasma cells residing in the rat spleen. Following supplementation with dietary selenium compounds, polarized intestinal epithelial Caco-2 cells secreted Sepp1 into the culture medium across the basolateral membrane. Our data suggest that Sepp1 secreted from epithelial cells may support the intestinal immune system by providing immune cells (including plasma cells) with selenium for the biosynthesis of endogenous selenoproteins.

This Special Issue contains selected and peer-reviewed research presented at the 7th International Conference on Polyphenols and Health (ICPH) was held in Tours, France, October 25–27, 2015We thank the authors for submitting their workWe would like to express our thanks also to the authors of those manuscripts, several of which were found to be more suitable for alternate journals. Likewise, we thank the expert reviewers for their important input in the peer-review, and Andy Deelen and Molly McGovern from the Publisher for their help; Special Issue: “Polyphenols and health”

The dietary flavanol (−)-epicatechin has been suggested to mediate its vasodilatory effect by increasing nitric oxide levels in endothelial cells. To directly prove the formation of nitric oxide (NO) in human endothelial cells (HUVEC) in vitro by trapping NO to yield a fluorescent nitrosamine. HUVEC were treated with (−)-epicatechin; nitrite and NO formation were determined by reductive chemiluminescence detection and the NO-sensitive fluorophore 5-methoxy-2-(1H-naphthol[2,3-d]imidazol-2-yl)-phenol copper complex (MNIP-Cu), respectively. MNIP was synthesized in a rapid and convenient one-step microwave reaction. Endothelial nitric oxide synthase (eNOS) mRNA levels and mRNA stability were measured. Incubation with (−)-epicatechin (0.3–10 μM) led to elevated NO levels in HUVEC measured via reductive chemiluminescence detection and visualized as the fluorescent NO derivative of MNIP. Expression of eNOS mRNA and mRNA stability were not affected by (−)-epicatechin treatment within the time frame studied. (−)-Epicatechin augments the level of NO in endothelial cells, a process suggested to be responsible for the vasodilatory properties of the compound.

Accumulating evidence indicates a critical role of microRNAs (miRNAs) in the outcome of diseases. Here, we investigate the effect of garlic on the intestinal miRNA signature of male Balb/c mice during infections with Eimeria papillata. Garlic decreases the intracellular development as evidenced by a lowered fecal output of E. papillata oocysts from 3,150 ± 410 to approximately 1,750 ± 390 oocysts per gram feces on day 4 postinoculation. This anti-coccidial activity of garlic is associated with an inhibition of the E. papillata-induced increases of interferon gamma, inducible nitric oxide synthase, nitrite/nitrate, and malondialdehyde and decrease in glutathione. Moreover, garlic downregulates the E. papillata-induced increases in the expression of the miRNAs miR-1959, miR-203, and miR-21, and it upregulates the expression of the 11 miRNA species miR-142-5P, miR-15A, miR-10A, miR-29B, miR-1902, miR-125A-5P, let-7E, miR-148A, miR-130A, miR-10B, and miR-93, respectively, as revealed by miRXplore microarray technology. Real-time PCR confirms these effects of garlic in the jejunum of E. papillata-infected mice. Our data indicate that the anti-coccidial activity of garlic is associated with specific changes in the miRNA signature of the mouse jejunum, the target site of E. papillata. These changes may reflect an involvement of miRNAs in garlic-activated pathways to reduce and/or to repair E. papillata-induced tissue injuries.

Current issues in research on health effects by polyphenols are addressed. As to the cardiovascular system, flow-mediated dilation (FMD), a functional biomarker, can be used as surrogate marker for cardiovascular risk. Acute short-term effects peaking at 2h after ingestion of polyphenol-rich food items are distinguished from longer-term effects over days and weeks. The role of polyphenol metabolites as bioactives is presented, underlining that specific target enzymes such as NADPH oxidases or lipoxygenases provide a basis for molecular action of polyphenols, rather than unspecific direct antioxidant effects. Cautionary words are given for the use of non-compositional assays of 'total antioxidant capacity' (TAC) in blood plasma. Enhanced interest emerges for polyphenols in the gastrointestinal tract. Recommendations for health professionals and the public are summarized, as well as prospects and challenges for future research.

Epidemiological and clinical studies revealed that high-flavanol diet or isolated (-)-epicatechin improves the function of the vascular endothelium, as assessed by flow-mediated dilation, through elevation of bioavailability and bioactivity of NO*. We have demonstrated that exposure of human endothelial cells to (-)-epicatechin elevates the cellular levels of NO* and cyclic GMP and protects against oxidative stress elicited by proinflammatory agonists. (-)-Epicatechin acts like a prodrug, since these effects involve O-methylation of the flavanol and are attributed to apocynin-like inhibition of endothelial NADPH oxidase. Thus, generation of superoxide and peroxynitrite is diminished and, consequently, the cellular NO* level is preserved or augmented. We propose therefore that endothelial NO* metabolism rather than general antioxidant activity is a major target of dietary flavanols and that NADPH oxidase activity is a crucial site of action. Moreover, flavonoid glucuronides appear to serve as plasma transport metabolites to target cells rather than solely as excretion products. Implications for the interpretation of the role of dietary polyphenols for cardiovascular health are discussed.

The availability of l-arginine can be a rate-limiting factor for cellular NO production by nitric oxide synthases (NOS). Arginase competes with NOS for l-arginine as the common substrate. Increased arginase activity has been linked to low NO levels, and an inhibition of arginase activity has been reported to improve endothelium-dependent vasorelaxation. Based on the above, we hypothesized that an increase in the circulating NO pool following flavanol consumption could be correlated with decreased arginase activity. To test this hypothesis we (a) investigated the effects of (-)-epicatechin and its structurally related metabolites on endothelial arginase expression and activity in vitro; (b) evaluated the effects of dietary flavanol-rich cocoa on kidney arginase activity in vivo; and (c) assessed human erythrocyte arginase activity following flavanol-rich cocoa beverage consumption in a double-blind intervention study with cross-over design. The results demonstrate that cocoa flavanols lower arginase-2 mRNA expression and activity in HUVEC. Dietary intervention with flavanol-rich cocoa caused diminished arginase activity in rat kidney and, erythrocyte arginase activity was lowered in healthy humans following consumption of a high flavanol beverage in vivo.

Apocynin has been reported to require dimerization by myeloperoxidase (MPO) to inhibit leukocyte NADPH oxidase. (-)-Epicatechin, a dietary flavan-3-ol, has been identified as a 'prodrug' of apocynin-like metabolites that inhibit endothelial NADPH oxidase activity and elevate the cellular level of nitric oxide. Since (-)-epicatechin has tentatively been identified as substrate of MPO, we studied the one-electron oxidation of (-)-epicatechin by MPO. By using multi-mixing stopped-flow technique, we demonstrate that (-)-epicatechin is one of the most efficient electron donors for heme peroxidases investigated so far. Second order rate constants for the (-)-epicatechin-mediated conversion of MPO-compound I to compound II and compound II to resting enzyme were estimated to be 1.9 x 10{sup 7} and 4.5 x 10{sup 6} M{sup -1} s{sup -1}, respectively (pH 7, 25 deg. C). The data indicate that (-)-epicatechin is capable of undergoing fast MPO-mediated one-electron oxidation.

Extracellular signal-regulated kinases (ERK) 1 and 2 as well as ERK-5 were previously suggested to phosphorylate connexin-43 and to contribute to the modulation of gap junctional intercellular communication (GJC). Exposure of rat liver epithelial cells to epidermal growth factor (EGF) or the redox cycling and alkylating agent menadione resulted in phosphorylation of connexin-43 and loss in GJC, both of which were abrogated by pharmacological inhibitors of ERK-1/2 activation, if used in concentrations that selectively abrogate phosphorylation of ERK-1/2 but not of ERK-5. Thus, EGF- or menadione-induced loss of GJC is mediated by ERK-1/2 but not ERK-5 in rat liver epithelial cells.

Selenoprotein P (SeP) is a highly glycosylated plasma protein containing up to 10 selenocysteine residues. It is secreted by hepatocytes and also by the human hepatoma cell line HepG2. Pharmacological inhibitors interfering with N-glycosylation, intracellular trafficking and calcium homeostasis were applied to examine post-translational processing and secretion of SeP by HepG2 cells. In parallel, the prototypic secretory glycoprotein α1-antitrypsin was used as technical control. Secretion of SeP was stimulated by increasing the extracellular calcium concentration and by inhibiting the release of sequestered calcium through dantrolene or U-73122. In contrast, brefeldin A and thapsigargin suppressed SeP secretion. Tunicamycin and monensin induced the synthesis of truncated non-glycosylated and partially glycosylated forms of SeP, which were secreted in spite of their impaired glycosylation. Both non-glycosylated and partially glycosylated SeP is utilised as selenium donor by target cells: impaired glycosylation affected neither the ability of SeP to induce the synthesis of the selenoenzyme cytosolic glutathione peroxidase nor its capacity to protect endothelial cells from oxidative stress.

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.

The action of oxidatively modified low-density lipoprotein (oxLDL) on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. OxLDL was shown here to elicit oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2',7'-dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. A 170-kDa polypeptide carbonylated upon exposure of cells to oxLDL was identified by immunoprecipitation as EGF receptor. Immunocytochemical visualization by confocal microscopy revealed the highest levels of modified proteins in the perinuclear region. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of *NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be both carbonylated and nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor L-NIO. These oxLDL-elicited changes in vascular endothelial cells described were suppressed by (-)-epicatechin, a dietary polyphenol, which inhibited NADPH oxidase activity in these cells.

The Rho-like GTPase Rac regulates distinct actin cytoskeleton changes required for adhesion, migration and invasion of cells. Tiam1 specifically activates Rac, and Rac has been shown to affect several signaling pathways in a partly cell-type-specific manner. Recently, we demonstrated that Rac activation inhibits Matrigel invasion of human carcinoma cells by transcriptional upregulation of tissue inhibitor of metalloproteinase-1. The purpose of the present study was to identify key mediators of Tiam1/Rac-induced tissue inhibitor of metalloproteinase-1 expression. Mutational analysis of the human tissue inhibitor of metalloproteinase-1 promoter revealed a major role for a distinct activating protein-1 site at −92/−86 and a minor role for an adjacent polyoma enhancer A3 site. Moreover, Rac activation induced the generation of reactive oxygen species and subsequent reactive oxygen species-dependent activation of extracellular signal-regulated kinase 1,2. In contrast, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activities were not affected. In line with this, Tiam1/Rac-induced tissue inhibitor of metalloproteinase-1 expression as well as Tiam1/Rac-induced binding of nuclear extracts to the activating protein-1 site at −92/−86 were inhibited by catalase and by specific inhibitors of the extracellular signal-related kinase-1,2 activators, mitogen-activated protein kinase kinase-1 and mitogen-activated protein kinase kinase-2 (PD098059, U0126). In conclusion, Rac-induced transcriptional upregulation of tissue inhibitor of metalloproteinase-1 is mediated by reactive oxygen species-dependent activation of extracellular signal-related kinase-1,2 and by transcription factors of the activating protein-1 family.

Selenoprotein P (SeP) is a highly glycosylated, selenium-rich plasma protein. Aside from its role as selenium carrier protein, an antioxidative function of SeP has been suggested. Astrocytes, which detoxify reactive oxygen species in the brain, were described as potential target cells of SeP. We investigated the expression of SeP in human astrocytes and its involvement in the protection of these cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage. We show that primary human astrocytes and the human astrocytoma cell line MOG-G-CCM express SeP as an unglycosylated protein, which is not secreted. SeP expression in astrocytes is constitutive. Preincubation of astrocytes with hepatocyte-derived SeP mimicks the protective effect of low-molecular-weight selenocompounds such as sodium selenite or selenomethionine against oxidative damage, shielding astrocytes from t-BHP-induced cytotoxicity. Selenium supplementation of astrocytes counteracts oxidative stress via an increase in expression and activity of the selenoenzyme cytosolic glutathione peroxidase (cGPx). Furthermore, specific downregulation of SeP expression by small interfering RNA decreases cell viability of human astrocytes and makes them more susceptible to t-BHP-induced cytotoxicity. Our results implicate an antioxidant activity of constitutively expressed SeP in selenium-deficient astrocytes, while during adequate selenium supply the enhanced protection against oxidative stress is exerted by cGPx.

A major fraction of the essential trace element selenium circulating in human blood plasma is present as selenoprotein P (SeP). As SeP associates with endothelial membranes, the participation of SeP in selenium-mediated protection against oxidative damage was investigated, using the human endothelial cell line Ea.hy926 as a model system. Hepatocyte-derived SeP prevented tert-butylhydroperoxide (t-BHP)-induced oxidative cell death of Ea.hy926 cells in a similar manner as did sodium selenite, counteracting a t-BHP-induced loss of cellular membrane integrity. Protection was detected after at least 10 h of SeP supplementation and it peaked at 24 h. SeP time-dependently stimulated the expression of cytosolic glutathione peroxidase (cGPx) and increased the enzymatic activities of glutathione peroxidase (GPx) and thioredoxin reductase (TR). The cGPx inhibitor mercaptosuccinate as well as the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine counteracted the SeP-mediated protection, while the TR inhibitors cisplatin and auranofin had no effect. The presented data suggest that selenium supplementation by SeP prevents oxidative damage of human endothelial cells by restoring expression and enzymatic activity of GPx.

Metabolic processes which generate oxidants and antioxidants are governed by genetic disposition as well as environmental factors. Changes in lifestyle, including increased environmental pollution, sun exposure, and dietary habits modify the challenge of the organism by reactive oxygen species. Defense mechanisms are reinforced by increasing dietary intake of antioxidants and micronutrients such as vitamins and selenium (Se). Se deficiency has been recognized to promote some disease states. Epidemiological findings link a lowered Se status to neurodegenerative and cardiovascular diseases as well as to increased cancer risk. While evidence exists to suggest that additional selenocompounds would be beneficial in some health conditions, results from future intervention trials are needed to substantiate the argument for increasing Se intake. Several pieces of the puzzle concerning the molecular mechanisms underlying the reactive oxygen species-triggered disease state and intervention by enzymatic antioxidants have been elucidated. A novel concept of protection of stromal cells against the dominating influence of tumor cells in tumor-stroma interaction by selenocompounds and other antioxidants is presented herein, which may translate into therapeutic strategies in chemoprevention of tumor invasion.

Intake of flavanol-rich food or beverages was previously shown to ameliorate endothelial function and to enhance bioactivity of nitric oxide with individuals at risk for cardiovascular disease. Here, we examined whether the major dietary flavanol, (−)-epicatechin, counteracts the action of oxidized LDL on endothelial cells, an action considered pivotal for endothelial dysfunction in the pathogenesis of atherosclerosis. Oxidation by myeloperoxidase plus nitrite rendered human LDL cytotoxic towards endothelial cells, more so than oxidation by Cu2+. Oxidized LDL also caused a marked loss of endothelial NO synthase protein which did not occur in the presence of a proteasome inhibitor, lactacystin. Both actions of oxidized LDL, which were not evoked by native LDL, were effectively counteracted by (−)-epicatechin. We conclude that dietary flavanols contribute to protection of the integrity of endothelial cells not only by scavenging free radicals but also by maintaining endothelial NO synthase.

Regulated uptake of extracellular l-arginine by cationic amino acid transporters (CATs) is required for inducible nitric oxide synthase and arginase activity. Both enzymes were recently recognized as important in the pathophysiology of psoriasis because of their contribution to epidermal hyperproliferation. We here characterize the expression pattern of CATs in psoriatic skin compared to healthy skin. CAT-1 mRNA expression was strongly upregulated in lesional and nonlesional areas of psoriatic skin compared to healthy skin, whereas expression of CAT-2A and the inducible isoform CAT-2B was unaltered in psoriatic skin. Furthermore, we tested the hypothesis that arginase-1 overexpression regulates CAT expression via intracellular l-arginine concentration. In in vitro experiments with arginase-1 overexpressing HaCaT cells, CAT-1 mRNA expression was increased. Likewise, this occurs in l-arginine-starved HaCaT cells. Both CAT-2 isoforms were not affected. Arginase-1 overexpression limits the synthesis of NO at physiological, but not supraphysiological, l-arginine levels. Plasma l-arginine concentration was diminished in psoriasis patients and the arginase product l-ornithine was significantly increased compared to healthy controls. In summary, arginase-1 overexpression leads to upregulated CAT-1 expression in psoriatic skin, which is due to lowered intracellular l-arginine levels and limits NO synthesis at physiological l-arginine concentrations.

Matrix metalloproteinase-9 (MMP-9) is involved in physiological tissue remodelling processes as well as in tumor invasion and metastasis. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases MMP-9 secretion from normal human epidermal keratinocytes (NHEK) in vivo and in vitro. Here we show that the flavoprotein inhibitor diphenyleneiodinium (DPI) and the NADPH oxidase inhibitor apocynin block TPA-induced MMP-9 secretion of NHEK in vitro. Furthermore, N-acetyl-L-cysteine and L-cysteine lowered TPA-induced MMP-9 secretion, suggesting an involvement of reactive oxygen species(ROS). TPA exerts its effect on MMP-9 gene expression and secretion via the superoxide-producing enzyme NADPH oxidase: TPA rapidly stimulates generation of superoxide anion as well as gene expression of two cytosolic NADPH oxidase subunits (p47-phox and p67-phox) after 2 h, which is followed by induction of MMP-9 gene expression after 4 h. Taken together, the novel finding herein is the TPA-induced MMP-9 secretion from normal human epidermal keratinocytes through a NADPH oxidase dependent pathway.

Gap junctional intercellular communication (GJC) varies during progression of the cell cycle. We propose here that Cdc25A, a dual specificity phosphatase crucial for cell cycle progression, is linked to connexin (Cx) phosphorylation and the modulation of GJC. Inhibition of Cdc25 phosphatases in rat liver epithelial cells employing a 1,4-naphthoquinone-based inhibitor, NSC95397, induced cell cycle arrest, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), and activation of extracellular signal-regulated kinases ERK-1 and -2. ERK activation was blocked by specific inhibitors of MAPK/ERK kinases 1/2 or of the EGFR tyrosine kinase. An EGFR-dephosphorylation assay suggested that Cdc25A interacts with the EGFR, with inhibition by NSC95397 resulting in activation of the receptor. As a consequence of ERK activation, Cx43 was phosphorylated, resulting in a downregulation of GJC. Loss of GJC was prevented by inhibition of ERK activation. In summary, cell cycle and GJC are connected via Cdc25A and the EGFR-ERK pathway.

Carotenoids are pigments which play a major role in the protection of plants against photooxidative processes. They are efficient antioxidants scavenging singlet molecular oxygen and peroxyl radicals. In the human organism, carotenoids are part of the antioxidant defense system. They interact synergistically with other antioxidants; mixtures of carotenoids are more effective than single compounds. According to their structure most carotenoids exhibit absorption maxima at around 450 nm. Filtering of blue light has been proposed as a mechanism protecting the macula lutea against photooxidative damage. There is increasing evidence from human studies that carotenoids protect the skin against photooxidative damage.

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K3), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFR-dependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.

The dietary flavanol (-)-epicatechin protects against nitration and oxidation reactions of the inflammatory mediator peroxynitrite in hydrophilic and hydrophobic environments. Bioavailability and cellular uptake of (-)-epicatechin are not yet fully characterized. Here, the octanol/buffer partition coefficient of (-)-epicatechin is observed to be 1.5, indicating that the flavanol is soluble in aqueous as well as lipophilic cellular phases, thus capable of permeating the cell membrane. In line with this, the ability of murine aortic endothelial cells (MAECs) to remove (-)-epicatechin from cell culture media is demonstrated. Epicatechin accumulates in cells, likely due to epicatechin binding to cellular proteins. Even after repeated washing, (-)-epicatechin accumulated by MAEC affords protection of the cells against peroxynitrite-induced nitration of protein tyrosyl residues and against oxidation of intracellular dichlorodihydrofluorescein.

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.

Enhanced expression of matrix metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in skin fibroblasts and subsequent damage of dermal connective tissue in the context of sun-induced premature aging and skin tumour progression is causally linked to UVB irradiation. Here, we were interested in identifying components of the complex signal-transduction pathway underlying UVB-mediated up-regulation of these delayed UV-responsive genes and focused on components maximally activated early after irradiation. A 2.3-fold increase in protein kinase CK2 activity was measured at 20–40min after low-dose UVB irradiation (at 10mJ/cm2) of dermal fibroblasts. This UVB-mediated increase in CK2 activity was abrogated by pharmacological approaches using non-toxic concentrations of the CK2 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). Preincubation of fibroblasts with DRB prior to UVB irradiation lowered MMP-1 by 49–69% and MMP-3 protein levels by 55–63% compared with UVB-irradiated controls. By contrast, the CK2 inhibitor did not affect the UVB-triggered transcription of MMPs. Furthermore, UVB irradiation of fibroblasts overexpressing a kinase-inactive mutant of CK2 (CK2α-K68A-HA) resulted in lowering of the protein levels of MMP-1 by 25% and MMP-3 by 22% compared with irradiated fibroblasts transfected with the vector control. This reduction in MMP protein levels correlated with the transfection efficiency. Taken together, we describe a novel aspect of protein kinase CK2, namely its inducible activity by UVB irradiation, and provide evidence that CK2 is an early mediator of the UVB-dependent up-regulation of MMP-1 and MMP-3 translation, whereas their major tissue inhibitor of matrix metalloproteinase-1 is not affected by CK2.

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.

During inflammatory reactions, cells are under nitrosative and/or oxidative stress. The zinc finger transcription factors vitamin D receptor (VDR) and retinoid X receptor (RXR) were used as a model system to characterize effects of NO. and/or reactive oxygen species on zinc finger-dependent gene expression. Nitric oxide (NO.) as well as H(2)O(2), singlet oxygen ((1)O(2)), peroxyl radicals (ROO.) and peroxynitrite (ONOO-), respectively, were shown to inhibit VDR/RXR-DNA complex formation in vitro in a dose-dependent manner. While NO-induced inhibition of VDR/RXR-DNA complex formation could be restored nearly completely by subsequent treatment with dithiothreitol, inhibition by H(2)O(2) proved to be only partially reversible, and inhibition by (1)O(2), ROO. or ONOO- was found to be irreversible. In cells transiently transfected with VDR and RXR, subtoxic concentrations of NO. or hydroperoxides and intracellular generation of superoxide anion radicals inhibited VDR/RXR-dependent reporter gene activity in a dose-dependent manner. Interestingly, cells can repair the zinc fingers of VDR and RXR after nitrosative stress but not after oxidative stress. The results indicate that, among the reactive species investigated, only NO. may act sufficiently gentle to be considered as a regulator and not only as an inhibitor of gene expression via zinc finger transcription factors.

Hydrogen peroxide acts as a signaling molecule in early adipogenesis. In differentiating adipocytes, elevated hydrogen peroxide generation is balanced through induction of antioxidant enzymes such as catalase and peroxiredoxins. Thioredoxin reductases (TrxR) and glutathione peroxidases (GPx) are selenoenzymes that constitute part of the major thiol-dependent antioxidant systems in cells. Here we show that the protein levels of cytoplasmic/nuclear TrxR1 and mitochondrial TrxR2 increase in the course of adipocyte differentiation of 3T3-L1 cells together with the TrxR2 substrate thioredoxin 2 (Trx2), resulting in elevated TrxR activity in mature adipocytes. Gene and protein expression of the GPx isoenzyme GPx4 was also stimulated during adipogenesis. Chronic exposure of 3T3-L1 cells to the anti-adipogenic factors tumor necrosis factor α (TNF-α) or rapamycin during differentiation suppressed TrxR1 and Trx2 upregulation, concomitantly with inhibition of adipogenesis and lipogenesis. In contrast, TNF-α or rapamycin did not affect expression of TrxRs and their Trx substrates in mature adipocytes. These results indicate that upregulation of the thioredoxin-dependent redox system is linked to the development of an adipocyte phenotype.

In fluorescence diagnosis and photodynamic therapy of neoplastic tissues 5-aminolevulinic acid is used to synthesize endogenous porphyrins as photosensitizers. The efficacy of neoplastic tissues to fluorescence diagnosis and photodynamic therapy is thought to be dependent on the total level of intralesional formed porphyrins. The available profiles of porphyrin metabolites in normal and in neoplastic cell lines after administration of 5-aminolevulinic acid vary considerably. Thus, this is the first in-vitro study which compares the porphyrin biosynthesis in normal skin cells (HaCaT, fibroblasts) with melanoma cells (Bro, SKMel-23, SKMel-28). After incubation with 1 mM 5-aminolevulinic acid, kinetics of porphyrin levels and metabolites were determined in the cells and the corresponding supernatants. Exogenous 5-aminolevulinic acid induced porphyrin formation in all cells with maximum values after an incubation period of 16-36 h. Increase of porphyrin levels varied from 10- to 80-fold (SKMel-28>HaCaT>fibroblasts>SKMel-23>>Bro) with minimum 1.5 times higher levels of porphyrins in the supernatants than in the cells. In cells and supernatants protoporphyrin and coproporphyrin were the predominantly formed porphyrin metabolites. Metastatic melanoma cells (SKMel-23, SKMel-28) accumulated much higher porphyrin levels than primary melanoma cells (Bro). In conclusion, by optimizing the treatment modalities, especially the light source, topical photodynamic therapy (PDT) could become a treatment alternative of melanoma metastases in progressive disease.

Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1−/−) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1–0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1−/− cells and by PN in only WT cells. These two groups of cells shared similar cytochromec release, caspase-3 activation, and p21WAF1/CIP1 cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1−/− cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1−/− than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.

The flavanol (-)-epicatechin has been found to protect against damage inflicted by peroxynitrite, an inflammatory intermediate. Here, epicatechin was tested in systems of increasing complexity. The compound efficiently protected against nitration of protein tyrosine residues by peroxynitrite (IC(50) approximately 0.02 mol epicatechin/mol peroxynitrite). However, at epicatechin concentrations completely preventing nitration of tyrosine by peroxynitrite, protection against the oxidative inactivation of glyceraldehyde-3-phosphate dehydrogenase or soybean lipoxygenase-1 was marginal (IC(50)1 mol epicatechin/mol peroxynitrite), approximately two orders of magnitude less. Likewise, epicatechin was relatively ineffective against oxidation of thiols in cell lysates, and against the oxidation of 2',7'-dichlorodihydrofluorescein in cultured cells. The activation of the kinases Akt/protein kinase B, ERK1/2 and p38-MAPK by peroxynitrite in murine aorta endothelial cells was not altered by epicatechin, suggesting that activation of these kinases is due to processes other than tyrosine nitration.

Lutein and zeaxanthin are the predominant carotenoids in the human macula lutea. Epidemiological data suggest that an increased intake of a lutein-rich diet correlates with a diminished risk for age-related macular degeneration, a major cause of impaired vision in the elderly. Filtering of blue light has been proposed as a possible mechanism of protection. Here, the blue light filter efficacy of carotenoids was investigated in unilamellar liposomes loaded in the hydrophilic core space with a fluorescent dye, Lucifer yellow, excitable by blue light. Carotenoids were incorporated into the lipophilic membrane. Fluorescence emission in carotenoid-containing liposomes was lower than in carotenoid-free controls when exposed to blue light, indicating a filter effect. Filter efficacy was in the order lutein > zeaxanthin > beta-carotene > lycopene. Some of the difference in blue light filter efficacy of carotenoids is attributable to differences in extinction coefficients, and a major further contribution is suggested to be related to the orientation of the incorporated molecules in the liposomal membrane.