The role of cellular senescence in neurodegenerative diseases.

Increasing evidence has revealed that cellular senescence drives NDs, including Alzheimer's disease (AD) and Parkinson's disease. Different senescent cell populations secrete senescence-associated secretory phenotypes (SASP), including matrix metalloproteinase-3, interleukin (IL)-1α, IL-6, and IL-8, which can harm adjacent microglia. Moreover, these cells possess high expression levels of senescence hallmarks (p16 and p21) and elevated senescence-associated ß-galactosidase activity in in vitro and in vivo ND models. These senescence phenotypes contribute to the deposition of ß-amyloid and tau-protein tangles. Selective clearance of senescent cells and SASP regulation by inhibiting p38/mitogen-activated protein kinase and nuclear factor kappa B signaling attenuate ß-amyloid load and prevent tau-protein tangle deposition, thereby improving cognitive performance in AD mouse models. In addition, telomere shortening, a cellular senescence biomarker, is associated with increased ND risks. Telomere dysfunction causes cellular senescence, stimulating IL-6, tumor necrosis factor-α, and IL-1ß secretions. The forced expression of telomerase activators prevents cellular senescence, yielding considerable neuroprotective effects. This review elucidates the mechanism of cellular senescence in ND pathogenesis, suggesting strategies to eliminate or restore senescent cells to a normal phenotype for treating such diseases.

Several lines of antioxidant defense against oxidative stress: antioxidant enzymes, nanomaterials with multiple enzyme-mimicking activities, and low-molecular-weight antioxidants.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are well recognized for playing a dual role, since they can be either deleterious or beneficial to biological systems. An imbalance between ROS production and elimination is termed oxidative stress, a critical factor and common denominator of many chronic diseases such as cancer, cardiovascular diseases, metabolic diseases, neurological disorders (Alzheimer's and Parkinson's diseases), and other disorders. To counteract the harmful effects of ROS, organisms have evolved a complex, three-line antioxidant defense system. The first-line defense mechanism is the most efficient and involves antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). This line of defense plays an irreplaceable role in the dismutation of superoxide radicals (O2•-) and hydrogen peroxide (H2O2). The removal of superoxide radicals by SOD prevents the formation of the much more damaging peroxynitrite ONOO- (O2•- + NO• → ONOO-) and maintains the physiologically relevant level of nitric oxide (NO•), an important molecule in neurotransmission, inflammation, and vasodilation. The second-line antioxidant defense pathway involves exogenous diet-derived small-molecule antioxidants. The third-line antioxidant defense is ensured by the repair or removal of oxidized proteins and other biomolecules by a variety of enzyme systems. This review briefly discusses the endogenous (mitochondria, NADPH, xanthine oxidase (XO), Fenton reaction) and exogenous (e.g., smoking, radiation, drugs, pollution) sources of ROS (superoxide radical, hydrogen peroxide, hydroxyl radical, peroxyl radical, hypochlorous acid, peroxynitrite). Attention has been given to the first-line antioxidant defense system provided by SOD, CAT, and GPx. The chemical and molecular mechanisms of antioxidant enzymes, enzyme-related diseases (cancer, cardiovascular, lung, metabolic, and neurological diseases), and the role of enzymes (e.g., GPx4) in cellular processes such as ferroptosis are discussed. Potential therapeutic applications of enzyme mimics and recent progress in metal-based (copper, iron, cobalt, molybdenum, cerium) and nonmetal (carbon)-based nanomaterials with enzyme-like activities (nanozymes) are also discussed. Moreover, attention has been given to the mechanisms of action of low-molecular-weight antioxidants (vitamin C (ascorbate), vitamin E (alpha-tocopherol), carotenoids (e.g., ß-carotene, lycopene, lutein), flavonoids (e.g., quercetin, anthocyanins, epicatechin), and glutathione (GSH)), the activation of transcription factors such as Nrf2, and the protection against chronic diseases. Given that there is a discrepancy between preclinical and clinical studies, approaches that may result in greater pharmacological and clinical success of low-molecular-weight antioxidant therapies are also subject to discussion.

Frentizole derivatives with mTOR inhibiting and senomorphic properties.

Frentizole is immunosuppressive drug with low acute toxicity and lifespan-prolonging effect. Recently, frentizole´s potential to disrupt toxic amyloid ß (Aß) - Aß-binding alcohol dehydrogenase (ABAD) interaction in mitochondria in Alzheimer´s brains has been revealed. Another broadly studied drug with anti-aging and immunosuppressive properties is an mTOR inhibitor - rapamycin. Since we do not yet precisely know what is behind the lifespan-prolonging effect of rapamycin and frentizole, whether it is the ability to inhibit the mTOR signaling pathway, reduction in mitochondrial toxicity, immunosuppressive effect, or a combination of all of them, we have decided within our previous work to dock the entire in-house library of almost 240 Aß-ABAD modulators into the FKBP-rapamycin-binding (FRB) domain of mTOR in order to interlink mTOR-centric and mitochondrial free radical-centric theories of aging and thus to increase the chances of success. Based on the results of the docking study, molecular dynamic simulation and MM-PBSA calculations, we have selected nine frentizole-like compounds (1 - 9). Subsequently, we have determined their real physical-chemical properties (logP, logD, pKa and solubility in water and buffer), cytotoxic/cytostatic, mTOR inhibitory, and in vitro anti-senescence (senolytic and senomorphic) effects. Finally, the three best candidates (4, 8, and 9) have been forwarded for in vivo safety studies to assess their acute toxicity and pharmacokinetic properties. Based on obtained results, only compound 4 demonstrated the best results within in vitro testing, the ability to cross the blood-brain barrier and the lowest acute toxicity (LD50 in male mice 559 mg/kg; LD50 in female mice 575 mg/kg).

Plasma PD-L1 as a biomarker in the clinical management of glioblastoma multiforme-a retrospective cohort study.

Background and objectives: Glioblastoma multiforme (GBM) is the most aggressive, malignant, and therapy-resistant tumor of the brain. Blockade therapy targeting the programmed cell death protein 1 (PD-1)/programmed death ligand (PD-L1) axis is currently under investigation for the clinical management of the GBM. This study has quantified the plasma levels of PD-L1 as a biomarker for the clinical management of GBM. Methods: A cohort (n = 128) of Pakistani adult glioblastoma patients together with age- and sex-matched healthy controls was used for quantification of pre-surgery levels of plasma PD-L1. PD-L1 protein and mRNA were measured by PD-L1 platinum enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. Receiver operating characteristic (ROC) curve analysis was used to compute area under the curve (AUC) for specificity and sensitivity analyses. The Kaplan-Meier survival analysis was employed to compute overall survival. Results: PD-L1 protein and mRNA were significantly higher in GBM compared to the healthy controls (p < 0.0001). Mean PD-L1 concentration for the GBM was found to be 48.98 ± 2.290 pg/ml compared to 27.63 ± 1.281 pg/ml for controls. Gene expression analysis showed statistically significant upregulation (p < 0.0001) of PD-L1 in blood of GBM compared to healthy controls. Plasma PD-L1 showed an AUC of 0.840 (p < 0.0001; 95% CI = 0.7716 to 0.9090) where a cutoff value higher than 46 pg/ml demonstrated 100% specificity and 57.81% sensitivity. Higher pre-surgery levels of PD-L1 were found to be associated with overall poor survival [p < 0.0001; HR (log-rank) = 0.08; 95% CI = 0.04 to 0.15]. Age, gender, and ethnic background were not found to be associated with plasma PD-L1 levels. Conclusion: The study concludes that blood-based measurements of PD-L1 in GBM can be a promising prognostic marker and therapeutic target besides a rapid and relatively non-invasive screening tool for routine clinical management. Future work extending the analysis to larger cohorts through multi-center collaborations involving pre-treatment and post-treatment groups is required to fully explore the usefulness of circulating PD-L1 for effective clinical applications.

Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging.

A physiological level of oxygen/nitrogen free radicals and non-radical reactive species (collectively known as ROS/RNS) is termed oxidative eustress or "good stress" and is characterized by low to mild levels of oxidants involved in the regulation of various biochemical transformations such as carboxylation, hydroxylation, peroxidation, or modulation of signal transduction pathways such as Nuclear factor-κB (NF-κB), Mitogen-activated protein kinase (MAPK) cascade, phosphoinositide-3-kinase, nuclear factor erythroid 2-related factor 2 (Nrf2) and other processes. Increased levels of ROS/RNS, generated from both endogenous (mitochondria, NADPH oxidases) and/or exogenous sources (radiation, certain drugs, foods, cigarette smoking, pollution) result in a harmful condition termed oxidative stress ("bad stress"). Although it is widely accepted, that many chronic diseases are multifactorial in origin, they share oxidative stress as a common denominator. Here we review the importance of oxidative stress and the mechanisms through which oxidative stress contributes to the pathological states of an organism. Attention is focused on the chemistry of ROS and RNS (e.g. superoxide radical, hydrogen peroxide, hydroxyl radicals, peroxyl radicals, nitric oxide, peroxynitrite), and their role in oxidative damage of DNA, proteins, and membrane lipids. Quantitative and qualitative assessment of oxidative stress biomarkers is also discussed. Oxidative stress contributes to the pathology of cancer, cardiovascular diseases, diabetes, neurological disorders (Alzheimer's and Parkinson's diseases, Down syndrome), psychiatric diseases (depression, schizophrenia, bipolar disorder), renal disease, lung disease (chronic pulmonary obstruction, lung cancer), and aging. The concerted action of antioxidants to ameliorate the harmful effect of oxidative stress is achieved by antioxidant enzymes (Superoxide dismutases-SODs, catalase, glutathione peroxidase-GPx), and small molecular weight antioxidants (vitamins C and E, flavonoids, carotenoids, melatonin, ergothioneine, and others). Perhaps one of the most effective low molecular weight antioxidants is vitamin E, the first line of defense against the peroxidation of lipids. A promising approach appears to be the use of certain antioxidants (e.g. flavonoids), showing weak prooxidant properties that may boost cellular antioxidant systems and thus act as preventive anticancer agents. Redox metal-based enzyme mimetic compounds as potential pharmaceutical interventions and sirtuins as promising therapeutic targets for age-related diseases and anti-aging strategies are discussed.

The role of redox active copper(II) on antioxidant properties of the flavonoid baicalein: DNA protection under Cu(II)-Fenton reaction and Cu(II)-ascorbate system conditions.

The antioxidant properties of flavonoids are mediated by their functional hydroxyl groups, which are capable of both chelating redox active metals such as iron, copper and scavenging free radicals. In this paper, the antioxidant vs. prooxidant and DNA protecting properties of baicalein and Cu(II)-baicalein complexes were studied under the conditions of the Copper-Fenton reaction and of the Copper-Ascorbate system. From the relevant EPR spectra, the interaction of baicalein with Cu(II) ions was confirmed, while UV-vis spectroscopy demonstrated a greater stability over time of Cu(II)-baicalein complexes in DMSO than in methanol and PBS and Phosphate buffers. An ABTS study confirmed a moderate ROS scavenging efficiency, at around 37%, for both free baicalein and Cu(II)-baicalein complexes (in the ratios 1:1 and 1:2). The results from absorption titrations are in agreement with those from viscometric studies and confirmed that the binding mode between DNA and both free baicalein and Cu-baicalein complexes, involves hydrogen bonds and van der Waals interactions. The DNA protective effect of baicalein has been investigated by means of gel electrophoresis under the conditions of the Cu-catalyzed Fenton reaction and of the Cu-Ascorbate system. In both cases, it was found that, at sufficiently high concentrations, baicalein offers some protection to cells from DNA damage caused by ROS (singlet oxygen, hydroxyl radicals and superoxide radical anions). Accordingly, baicalein may be useful as a therapeutic agent in diseases with a disturbed metabolism of redox metals such as copper, for example Alzheimer's disease, Wilson's disease and various cancers. While therapeutically sufficient concentrations of baicalein may protect neuronal cells from Cu-Fenton-induced DNA damage in regard to neurological conditions, conversely, in the case of cancers, low concentrations of baicalein do not inhibit the pro-oxidant effect of copper ions and ascorbate, which can, in turn, deliver an effective damage to DNA in tumour cells.

Essential metals in health and disease.

In total, twenty elements appear to be essential for the correct functioning of the human body, half of which are metals and half are non-metals. Among those metals that are currently considered to be essential for normal biological functioning are four main group elements, sodium (Na), potassium (K), magnesium (Mg), and calcium (Ca), and six d-block transition metal elements, manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn) and molybdenum (Mo). Cells have developed various metallo-regulatory mechanisms for maintaining a necessary homeostasis of metal-ions for diverse cellular processes, most importantly in the central nervous system. Since redox active transition metals (for example Fe and Cu) may participate in electron transfer reactions, their homeostasis must be carefully controlled. The catalytic behaviour of redox metals which have escaped control, e.g. via the Fenton reaction, results in the formation of reactive hydroxyl radicals, which may cause damage to DNA, proteins and membranes. Transition metals are integral parts of the active centers of numerous enzymes (e.g. Cu,Zn-SOD, Mn-SOD, Catalase) which catalyze chemical reactions at physiologically compatible rates. Either a deficiency, or an excess of essential metals may result in various disease states arising in an organism. Some typical ailments that are characterized by a disturbed homeostasis of redox active metals include neurological disorders (Alzheimer's, Parkinson's and Huntington's disorders), mental health problems, cardiovascular diseases, cancer, and diabetes. To comprehend more deeply the mechanisms by which essential metals, acting either alone or in combination, and/or through their interaction with non-essential metals (e.g. chromium) function in biological systems will require the application of a broader, more interdisciplinary approach than has mainly been used so far. It is clear that a stronger cooperation between bioinorganic chemists and biophysicists - who have already achieved great success in understanding the structure and role of metalloenzymes in living systems - with biologists, will access new avenues of research in the systems biology of metal ions. With this in mind, the present paper reviews selected chemical and biological aspects of metal ions and their possible interactions in living systems under normal and pathological conditions.

The effect of Luteolin on DNA damage mediated by a copper catalyzed Fenton reaction.

Luteolin has been reviewed as a flavonoid possessing potential cardioprotective, anti-inflammatory, anti-cancer activities. Having multiple biological effects, luteolin may act as either an antioxidant or a pro-oxidant. In this work, the protective role of copper(II)-chelation by luteolin on DNA damage via the Cu-Fenton reaction was studied. EPR and UV-vis spectroscopic data demonstrated that the luteolin, lacking 3-OH group, chelates to Cu(II) via the 5-OH and 4-CO groups, respectively. EPR spin trapping experiments using DMPO spin trap confirmed that the coordination of luteolin to Cu(II) significantly suppressed formation of hydroxyl and superoxide radicals (by 80%) in a Cu-Fenton system. Absorption titrations showed that the chelation of Cu(II) by luteolin slightly increased the mild intercalation strength of its interaction with DNA, as compared with free luteolin. Comparison with kaempferol and quercetin revealed, that the strength of the interaction between the free flavonoids/Cu-flavonoid complexes with DNA is only mildly affected by the presence/absence of 3-OH group. Due to the differences in the sensitivities of absorption titrations and viscometry, the latter confirmed weaker DNA intercalating efficiency of Cu-luteolin complex than does free luteolin. A dose dependent protective effect of luteolin against ROS-induced DNA damage was observed using gel electrophoresis. This effect was more pronounced compared to quercetin and kaempferol. In conclusion, the administration of luteolin to patients suffering from oxidative stress-related diseases with disturbed Cu-metabolism such as Alzheimer's diseases (antioxidant effect) and certain cancers (prooxidant effect) may have several health benefits.

Sheep Wool Humidity under Electron Irradiation Affects Wool Sorptivity towards Co(II) Ions.

The effect of humidity on sheep wool during irradiation by an accelerated electron beam was examined. Each of the samples with 10%, 53%, and 97% relative humidity (RH) absorbed a dose of 0, 109, and 257 kGy, respectively. After being freely kept in common laboratory conditions, the samples were subjected to batch Co(II) sorption experiments monitored with VIS spectrometry for different lapses from electron beam exposure. Along with the sorption, FTIR spectral analysis of the wool samples was conducted for cysteic acid and cystine monoxide, and later, the examination was completed, with pH measuring 0.05 molar KCl extract from the wool samples. Besides a relationship to the absorbed dose and lapse, the sorptivity results showed considerable dependence on wool humidity under exposure. When humidity was deficient (10% RH), the sorptivity was lower due to limited transformation of cystine monoxide to cysteic acid. The wool pre-conditioned at 53% RH, which is the humidity close to common environmental conditions, demonstrated the best Co(II) sorptivity in any case. This finding enables the elimination of pre-exposure wool conditioning in practice. Under excessive humidity of 97% RH and enough high dose of 257 kGy, radiolysis of water occurred, deteriorating the sorptivity. Each wool humidity, dose, and lapse showed a particular scenario. The time and humidity variations in the sorptivity for the non-irradiated sample were a little surprising; despite the absence of electron irradiation, relevant results indicated a strong sensitivity to pre-condition humidity and lapse from the start of the monitoring.

Antioxidant vs. Prooxidant Properties of the Flavonoid, Kaempferol, in the Presence of Cu(II) Ions: A ROS-Scavenging Activity, Fenton Reaction and DNA Damage Study.

Kaempferol is a flavonoid that occurs in tea and in many vegetables and fruits, including broccoli, cabbage, beans, grapes, apples, and strawberries. The efficacy of Kaempferol has been demonstrated in the treatment of breast, esophageal, cervical, ovarian, and liver cancers and leukemia, which very likely arises from its prooxidant properties and the activation of pro-apoptotic pathways. Indeed, this matter has already been the focus of a number of published studies and reviews. The aim of the present study was to elucidate the antioxidant vs. prooxidant properties of flavonoids in the presence of the redox-active metal, copper (II) ion, by means of the Fenton reaction. The specific motivation of this work is that, since an increased level of Cu(II) ions is known to be associated with many disease states such as neurological conditions (Alzheimer's disease) and cancer, any interaction between these ions and flavonoids might affect the outcome of therapeutic uses of the latter. The structure of the Cu-kaempferol complex in DMSO was investigated by means of low temperature EPR spectroscopy, which confirmed the existence of at least two distinct coordination environments around the copper (II) ion. UV vis-spectra of kaempferol and its Cu(II) complex in DMSO revealed an interaction between the 5-OH (A ring) group and the 4-CO (C ring) group of kaempferol with Cu(II) ions. An ABTS assay confirmed that kaempferol acted as an effective radical scavenger, and that this effect was further enhanced in the form of the Cu(II)-kaempferol complex. Quantitative EPR spin trapping experiments, using DMPO as the spin trap, confirmed suppression of the formation of a mixture of hydroxyl, superoxide, and methyl radicals, in a Fenton reaction system, upon coordination of kaempferol to the redox-active Cu(II) ions, by 80% with respect to the free Cu(II) ions. A viscometric study revealed a better DNA-intercalating ability of the Cu-kaempferol complex than for free kaempferol, essential for conferring anticancer activity of these substances. The results of the viscometric measurements were compared with those from a DNA damage study of Cu-kaempferol complexes in a Fenton reaction system, using gel electrophoresis. At low concentrations of kaempferol (Cu-kaempferol ratios of 1:1 and 1:2), a very weak protective effect on DNA was noted, whereas when kaempferol was present in excess, a significant DNA-protective effect was found. This can be explained if the weakly intercalated kaempferol molecules present at the surface of DNA provide protection against attack by ROS that originate from the Fenton reaction involving intercalated Cu(II)-kaempferol complexes. Following the application of ROS scavengers, L-histidine, DMSO, and SOD, gel electrophoresis confirmed the formation of singlet oxygen, hydroxyl radicals, and superoxide radical anions, respectively. We propose that the prooxidant properties of Cu-kaempferol complexes may provide anticancer activity of these substances. When present in excess, kaempferol displays antioxidant properties under Cu-Fenton conditions. This suggests that kaempferol might prove a suitable candidate for the prevention or treatment of oxidative stress related medical conditions that involve a disturbed metabolism of redox metals such as copper, for example, Menkes disease, and neurological disorders, including Alzheimer's disease. For the potential use of kaempferol in clinical practice, it will be necessary to optimize the dose size and critical age of the patient so that this flavonoid may be beneficial as a preventive drug against cancer and neurological disorders.

A Switch between Antioxidant and Prooxidant Properties of the Phenolic Compounds Myricetin, Morin, 3',4'-Dihydroxyflavone, Taxifolin and 4-Hydroxy-Coumarin in the Presence of Copper(II) Ions: A Spectroscopic, Absorption Titration and DNA Damage Study.

The beneficial effects of polyphenols, predominantly in the context of oxidative stress-related diseases such as cancer, cardiovascular diseases and neurological conditions including Alzheimer's and Parkinson's diseases, have been documented by a number of papers and reviews. The antioxidant/prooxidant properties of phenolic compounds are related mainly to the number and positions of hydroxyl groups and to their redox metal (Cu, Fe) chelating capacity. In this work we studied structurally distinct phenolic molecules such as myricetin, morin, 3',4'-dihydroxy-flavone, taxifolin and 4-hydroxycoumarin, either alone or as interacting with Cu2+ ions. EPR and UV-Vis spectroscopy confirmed that the effective binding of cupric ions to phenolic compounds requires the presence of the 3-OH and 4-CO groups on the flavonoid C ring and unsaturated C2-C3 bond of the C-ring, which permits through-conjugation with the B-ring. An ABTS assay revealed that radical scavenging activities of phenolic compounds are related to their number of hydroxyl groups, planarity of the molecular skeleton, extent of delocalization and they decrease in the order: myricetin > morin > 3',4'-dihydroxyflavone ~ 4-hydroxy coumarin > taxifolin. Absorption titrations indicate that copper ions can modulate the DNA binding affinity of flavonoids via the formation of their Cu-chelates. Gel electrophoresis measurements indicated that the protective effect of the phenolic compounds decreases in the order: 3',4'-dihydroxyflavone > 4-OH coumarin > morin > taxifolin ~ myricetin. This can be explained by the fact that myricetin, taxifolin and morin form stable Cu(II) complexes capable of causing DNA damage via interaction with DNA and ROS formation via the Fenton reaction. Application of ROS scavengers revealed the formation of singlet oxygen, superoxide and hydroxyl radicals and their concerted synergistic effect on the DNA. The overall results suggest that the most pronounced DNA damage has been observed for flavonoids containing higher number of hydroxyl groups (including 3-OH group of the C ring), such as myricetin (six hydroxyl groups), morin and taxifolin (five hydroxyl groups) in the presence of Cu(II) ions. The proposed mechanism of action by which Cu(II) complexes of myricetin, morin and taxifolin interact with DNA predispose these substances to act as potential anticancer agents. The anticancer activity of phenolic compounds can be explained by their moderate prooxidant properties, which can boost ROS formation and kill cancer cells. Alternatively, slight prooxidant properties may activate antioxidant systems, including antioxidant enzymes and low molecular antioxidants such as glutathione and thus act as preventive anticancer agents.

Some Properties of Electron Beam-Irradiated Sheep Wool Linked to Cr(III) Sorption.

We examined the characteristics of an electron beam irradiated wool with an absorbed dose of (21-410) kGy in comparison with natural wool with respect to the determination of the isoelectric point (IEP), zero charge point (ZCP), mechanism of Cr(III) sorption from higher concentrated solutions, and the modelling of the wool-Cr(III) interaction. The data of ZPC and IEP differed between natural and irradiated samples. Increasing the dose shifted the pH of ZPC from 6.85 for natural wool to 6.20 for the highest dosed wool, while the natural wool IEP moved very little, from pH = 3.35 to 3.40 for all of the irradiated samples. The sorption experiments were performed in a pH bath set at 3.40, and the determination of the residual Cr(III) in the bath was performed by VIS spectrometry under optimized conditions. The resulting sorptivity showed a monotonically rising trend with increasing Cr(III) concentration in the bath. Lower doses, unlike higher doses, showed better sorptivity than the natural wool. FTIR data indicated the formation of complex chromite salts of carboxylates and cysteinates. Crosslinks via ligands coming from different keratin chains were predicted, preferably on the surface of the fibers, but to a degree that did not yet inhibit the diffusion of Cr(III)-cations into the fiber volume. We also present a concept of a complex octahedral structure.

Management of oxidative stress and other pathologies in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder, characterized by the formation, aggregation and accumulation of amyloid beta, perturbed metal (copper, iron and zinc) homeostasis, metal-induced oxidative stress, neuroinflammation, aberrant activity of acetylcholinesterase (AChE) and other pathologies. The aim of this review is to discuss the current therapies based on the "combination-drugs-multitargets" strategy to target multiple pathologies to block the progression of pathogenesis of AD. In addition to cholinergic and amyloid targets, a significant effort is focused on targeting the metal-induced oxidative stress component of the disease. The main focus of research is based on modifications of existing drugs with specific biological activity. Tacrine was the first AChE inhibitor to be introduced into clinical practice and has been frequently used for the design of multitarget-directed ligands. A number of hybrid compounds containing tacrine and structural moieties derived from natural sources such as flavonoids [quercetin, rutin, coumarin, gallamine, resveratrol, scutellarin, anisidine, hesperetin, (-)-epicatechin] and other molecules (melatonin, trolox) have also been applied to function as multitarget-directed ligands. Most of these hybrids are potent inhibitors of AChE and butyrylcholinesterase and also of amyloid-beta aggregation. In addition, the antioxidant functionality, represented by coumarins, melatonin and other antioxidant molecules reduces the level of oxidative stress via ROS-scavenging mechanisms, as well as via chelation of redox-active Cu and Fe, thus suppressing the formation of ROS via the Fenton reaction. Various medicinal plants are under investigation for their ability to ameliorate symptoms of AD. The therapeutic potency of huperzine A and B, ginseng, curcumin and other compounds is manifested predominantly by the inhibitory action toward AChE, antioxidant or radical-scavenging and redox metal-chelating activity, inhibition of amyloid-beta aggregation and tau-protein hyperphosphorylation and antiinflammatory activity. Flavonoids not only function as antioxidants and metal-chelating agents, but also interact with protein kinase and lipid kinase signaling pathways, and others involving mitogen-activated protein kinase, NF-kappaB and tyrosine kinase. Among the most promising group of substances with potential activity against AD are the flavonoids, including myricetin, morin, rutin, quercetin, fisetin, kaempferol, apigenin and glycitein, which have been shown, in vitro, to possess antiamyloidogenic and fibril-destabilization activity, as well as being able to act as metal chelators and to suppressing oxidative stress. In terms of the clinical use of multifunctional hybrids, herbal drugs or flavonoids against AD, some remaining challenges are to establish the ideal dose to develop effective formulations to preserve bioavailability and to determine the stage when they should be administered. If the onset of the disease could be delayed by a decade, the number of AD victims would be significantly reduced.

Role of Post-Exposure Time in Co(II) Sorption of Higher Concentrations on Electron Irradiated Sheep Wool.

Sorption of Co(II) was investigated on natural as well as accelerated electron beam modified sheep wool involving low and high concentrations up to 200 mmol·dm-3. The sorption experiments confirmed the dependence of the sorption capacity not only on sorbate concentration and absorbed dose of energy, but also on post-exposure time. Post-exposure heating to accelerate transformation of the wool structure was of no effect on the sorption comparing with a simple storage for a period of 100 days. Under all tested conditions, the sorption maximum was measured for Co(II) concentration of 125 mmol·dm-3 and that was assigned to form a Co(II) complex with keratin. This assumption was tested on visible spectra of mixed solutions of Arginine and Co(II) to be a simplified model of Co(II) interaction with keratin. The sorption decrease is associated with generation of cross links between macro-chains through ligands of the Co-complex. The nodal points are a hindrance to diffusion of next ions into the fibers. Also, pH variations of aqueous extracts from the wool samples depending on absorbed dose and post-exposure time indicate complexity of the structural transformation being specific for each dose applied.

Redox-cycling and intercalating properties of novel mixed copper(II) complexes with non-steroidal anti-inflammatory drugs tolfenamic, mefenamic and flufenamic acids and phenanthroline functionality: Structure, SOD-mimetic activity, interaction with albumin, DNA damage study and anticancer activity.

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O')2(phen)] (1), [Cu(mef-O,O')2(phen)] (2), [Cu(fluf-O,O')2(phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1-3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~106). The anticancer activities of complexes 1-3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface.

Protective role of quercetin against copper(II)-induced oxidative stress: A spectroscopic, theoretical and DNA damage study.

The radical scavenging and metal chelating properties of flavonoids indicate that they may play a protective role in diseases with perturbed metal homeostasis such as Alzheimer's disease. In this work we investigated the effect of the coordination of quercetin to copper(II) in view of the formation of ROS in Cu-catalyzed Fenton reaction. ABTS and DPPH assays confirmed that the copper(II)-quercetin complex exhibits a stronger radical scavenging activity than does quercetin alone. EPR spin trapping experiments have shown that chelation of quercetin to copper significantly suppressed the formation of hydroxyl radicals in the Cu(II)-Fenton reaction. DNA damage experiments revealed a protective effect for quercetin, but only at higher stoichiometric ratios of quercetin relative to copper. DNA protective effect of quercetin against ROS attack was described by two mechanisms. The first mechanism lies in suppressed formation of ROS due to the decreased catalytic action of copper in the Fenton reaction, as a consequence of its chelation and direct scavenging of ROS by free quercetin. Since the Cu-quercetin complex intercalates into DNA, the second mechanism was attributed to a suppressed intercalating ability of the Cu-quercetin complex due to the mildly intercalating free quercetin into DNA, thus creating a protective wall against stronger intercalators.

Targeting Free Radicals in Oxidative Stress-Related Human Diseases.

Cancer and Alzheimer's disease (AD) are characterized by (i) opposing biological mechanisms, (ii) an inverse correlation between their incidences, and (iii) oxidative stress being a common denominator of both diseases. Increased formation of reactive oxygen species (ROS) in cancer cells from oncogenic signaling and/or metabolic disturbances leads to upregulation of cellular antioxidant capacity to maintain ROS levels below a toxic threshold. Combining drugs that induce high levels of ROS with compounds that suppress cellular antioxidant capacity by depleting antioxidant systems [glutathione (GSH), superoxide dismutase (SOD), and thioredoxin (TRX)] and/or targeting glucose metabolism represents a potential anticancer strategy. In AD, free metals and/or Aß:metal complexes may cause damage to biomolecules in the brain (via Fenton reaction), including DNA. Metal chelation, based on the application of selective metal chelators or metal delivery, may induce neuroprotective signaling and represents a promising therapeutic strategy. This review examines therapeutic strategies based on the modulation of oxidative stress in cancer and AD.

Targeting copper(II)-induced oxidative stress and the acetylcholinesterase system in Alzheimer's disease using multifunctional tacrine-coumarin hybrid molecules.

Alzheimer's disease is a multifactorial disease that is characterized mainly by Amyloid-ß (A-ß) deposits, cholinergic deficit and extensive metal (copper, iron)-induced oxidative stress. In this work we present details of the synthesis, antioxidant and copper-chelating properties, DNA protection study, cholinergic activity and amyloid-antiaggregation properties of new multifunctional tacrine-7-hydroxycoumarin hybrids. The mode of interaction between copper(II) and hybrids and interestingly, the reduction of Cu(II) to Cu(I) species (for complexes Cu-5e-g) were confirmed by EPR measurements. EPR spin trapping on the model Fenton reaction, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, demonstrated a significantly suppressed formation of hydroxyl radicals for the Cu-5e complex in comparison with free copper(II). This suggests that compound 5e upon coordination to free copper ion prevents the Cu(II)-catalyzed decomposition of hydrogen peroxide, which in turn may alleviate oxidative stress-induced damage. Protective activity of hybrids 5c and 5e against DNA damage in a Fenton system (copper catalyzed) was found to be in excellent agreement with the EPR spin trapping study. Compound 5g was the most effective in the inhibition of acetylcholinesterase (hAChE, IC50=38nM) and compound 5b was the most potent inhibitor of butyrylcholinesterase (hBuChE, IC50=63nM). Compound 5c was the strongest inhibitor of A-ß1-40 aggregation, although a significant inhibition (>50%) was detected for compounds 5b, 5d, 5e and 5g. Collectively, these results suggest that the design and investigation of multifunctional agents containing along with the acetylcholinesterase inhibitory segment also an antioxidant moiety capable of alleviating metal (copper)-induced oxidative stress, may be of importance in the treatment of Alzheimer's disease.

Redox- and non-redox-metal-induced formation of free radicals and their role in human disease.

Transition metal ions are key elements of various biological processes ranging from oxygen formation to hypoxia sensing, and therefore, their homeostasis is maintained within strict limits through tightly regulated mechanisms of uptake, storage and secretion. The breakdown of metal ion homeostasis can lead to an uncontrolled formation of reactive oxygen species, ROS (via the Fenton reaction, which produces hydroxyl radicals), and reactive nitrogen species, RNS, which may cause oxidative damage to biological macromolecules such as DNA, proteins and lipids. An imbalance between the formation of free radicals and their elimination by antioxidant defense systems is termed oxidative stress. Most vulnerable to free radical attack is the cell membrane which may undergo enhanced lipid peroxidation, finally producing mutagenic and carcinogenic malondialdehyde and 4-hydroxynonenal and other exocyclic DNA adducts. While redox-active iron (Fe) and copper (Cu) undergo redox-cycling reactions, for a second group of redox-inactive metals such as arsenic (As) and cadmium (Cd), the primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins. While arsenic is known to bind directly to critical thiols, other mechanisms, involving formation of hydrogen peroxide under physiological conditions, have been proposed. Redox-inert zinc (Zn) is the most abundant metal in the brain and an essential component of numerous proteins involved in biological defense mechanisms against oxidative stress. The depletion of zinc may enhance DNA damage by impairing DNA repair mechanisms. Intoxication of an organism by arsenic and cadmium may lead to metabolic disturbances of redox-active copper and iron, with the occurrence of oxidative stress induced by the enhanced formation of ROS/RNS. Oxidative stress occurs when excessive formation of ROS overwhelms the antioxidant defense system, as is maintained by antioxidants such as ascorbic acid, alpha-tocopherol, glutathione (GSH), carotenoids, flavonoids and antioxidant enzymes which include SOD, catalase and glutathione peroxidase. This review summarizes current views regarding the role of redox-active/inactive metal-induced formation of ROS, and modifications to biomolecules in human disease such as cancer, cardiovascular disease, metabolic disease, Alzheimer's disease, Parkinson's disease, renal disease, blood disorders and other disease. The involvement of metals in DNA repair mechanisms, tumor suppressor functions and interference with signal transduction pathways are also discussed.

Copper(II) complexes with new fluoroquinolones: Synthesis, structure, spectroscopic and theoretical study, DNA damage, cytotoxicity and antiviral activity.

Copper(II) complexes with fluoroquinolones in the presence of the nitrogen donor heterocyclic ligands 1,10-phenanthroline have been considered in detail. The phenanthroline moiety was introduced into the ligand environment with the aim to determine whether the nuclease activity is feasible. All suitable X-ray structures of the complexes under study reveal a distorted square pyramidal coordination geometry for Cu(II) atom. The conformational and spectroscopic (FT-IR and UV-visible) behavior has been analyzed and has been interpreted with respect to B3LYP/6-311G* calculations including molecular dynamics. The ability of the complexes to cleave DNA was studied by agarose gel electrophoresis with plasmid DNA pBSK+. The results have confirmed that the complexes under study behave as the chemical nucleases. Nuclease like activity in the absence of hydrogen peroxide allows us to deduce an interaction of the complexes with the DNA resulting in the conversion of supercoiled circular DNA to the nicked form. The DNA cleavage activity enhanced by the presence of hydrogen peroxide demonstrates the participation of reactive oxygen species, such as superoxide radical anions and hydroxyl radicals which presence was confirmed independently using the standard radical scavenging agents. It has been suggested that the radical formation through the Fenton/Haber-Weiss reaction is mediated by the redox cycling mechanisms with the participation of cupric/cuprous ions. Cytotoxic activity was evaluated as the 50% cytotoxic concentration (CC50). The potential effects of tested compounds on replication of murine gammaherpesvirus 68 (MHV-68) under in vitro conditions were also evaluated. However, no antiviral activity against MHV-68 was observed.