Dendritic antioxidants with pyrazole as the core: ability to scavenge radicals and to protect DNA.

Chalcones with or without a para-hydroxyl group were condensed with phenylhydrazine-related compounds to form 1,3,5-triphenyl-1H-pyrazole (TPP), 4-(1,5-diphenyl-1H-pyrazol-3-yl)phenol (APP), 4-(1,3-diphenyl-1H-pyrazol-5-yl)phenol (BPP), and 4-(3,5-diphenyl-1H-pyrazol-1-yl)phenol (CPP), in which the phenyl group formed a dendritic structure with pyrazole as the core. Thus, the aim of this work was to explore the antioxidant capacities of TPP, APP, BPP, and CPP in trapping 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(+•)) and 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and in inhibiting Cu(2+)/glutathione (GSH)-, (•)OH-, and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. TPP can react with ABTS(+•) and DPPH, indicating that the N atom in pyrazole possesses radical-scavenging ability. Moreover, APP, BPP, and CPP can trap 1.71, 1.81, and 1.58 radicals, respectively, in protecting DNA against AAPH-induced oxidation. Thus, the combination of pyrazole with a phenyl group exerted antioxidant ability although only one phenolic hydroxyl group was involved. However, these compounds showed weak protective effect against Cu(2+)/GSH-induced oxidation of DNA and even a pro-oxidant effect on (•)OH-induced oxidation of DNA.

Calreticulin inhibits prion protein PrP-(23-98) aggregation in vitro.

Because prion protein PrP-(23-98) was recently found to polymerize into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions, we tested to determine whether calreticulin (CRT) inhibits PrP-(23-98) aggregation in vitro. The results indicated that CRT suppressed PrP-(23-98) aggregation, and that CRT-mediated solubilization occurred in the aggregates.

Vanillin-coumarin hybrid molecule as an efficient fluorescent probe for trace level determination of Hg(II) and its application in cell imaging.

An efficient Hg(2+) selective fluorescent probe (vanillin azo coumarin, VAC) was synthesized by blending vanillin with coumarin. VAC and its Hg(2+) complex were well characterized by different spectroscopic techniques like (1)H NMR, QTOF-MS ES(+), FTIR and elemental analysis as well. VAC could detect up to 1.25 µM Hg(2+) in aqueous methanol solution through fluorescence enhancement. The method was linear up to 16 µM of Hg(2+). Negative interferences from Cu(2+), Ni(2+), Fe(3+), and Zn(2+) were eliminated using EDTA as a masking agent. VAC showed a strong binding to Hg(2+) ion as evident from its binding constant value (2.2×10(5)), estimated using Benesi-Hildebrand equation. Mercuration assisted restricted rotation of the vanillin moiety and inhibited photoinduced electron transfer from the O, N-donor sites to the coumarin unit are responsible for the enhancement of fluorescence upon mercuration of VAC. VAC was used for imaging the accumulation of Hg(2+) ions in Candida albicans cells.

Carnitine supplementation modulates high dietary copper-induced oxidative toxicity and reduced performance in laying hens.

This experiment was conducted to evaluate the effects of L-carnitine on performance, egg quality and certain biochemical parameters in laying hens fed a diet containing high levels of copper proteinate. Forty-eight 42-week-old laying hens were divided into four groups with four replicates. The laying hens were fed with a basal diet (control) or the basal diet supplemented with either 400 mg carnitine (Car)/kg diet, 800 mg copper proteinate (CuP)/kg diet or 400 mg carnitine + 800 mg copper (Car+CuP)/kg diet, for 6 weeks. Supplemental CuP decreased feed consumption (p < 0.01), feed efficiency and egg production (p < 0.001), as compared to control. The combination of Car and CuP increased (p < 0.001) egg production and feed efficiency as compared to CuP. The activities of alanine aminotransferase (p < 0.05) and alkaline phosphatase (p < 0.01) were increased, while lactate dehydrogenase activity was decreased (p < 0.001) by supplemental CuP and Car+CuP. Supplemental CuP caused an increase in plasma malondialdehyde (p < 0.01) and nitric oxide levels (p < 0.05). In the Car+CuP group, this increase was observed to have been reduced significantly (p < 0.05). Furthermore, Car+CuP increased (p < 0.05) glucose level. These results indicate that the carnitine and copper combination may prevent the possible adverse effects of high dietary copper on performance and lipid peroxidation in hens.

Soy isoflavone genistein induces cell death in breast cancer cells through mobilization of endogenous copper ions and generation of reactive oxygen species.

SCOPE: Worldwide geographical variation in cancer incidence indicates a correlation between dietary habits and cancer risk. Epidemiological studies have suggested that populations with high isoflavone intake through soy consumption have lower rates of breast, prostate, and colon cancer. Isoflavone genistein in soybean is considered a potent chemopreventive agent against cancer. Although several mechanisms have been proposed, a clear anticancer action mechanism of genistein is still not known. METHODS AND RESULTS: Here, we show that the cytotoxic action of genistein against breast cancer cells involves mobilization of endogenous copper. Further, whereas the copper specific chelator neocuproine is able to inhibit the apoptotic potential of genistein, the molecules which specifically bind iron (desferroxamine mesylate) and zinc (histidine) are relatively ineffective in causing such inhibition. Also, genistein-induced apoptosis in these cells is inhibited by scavengers of reactive oxygen species (ROS) implicating ROS as effector elements leading to cell death. CONCLUSIONS: As copper levels are known to be considerably elevated in almost all types of cancers, in this proof-of-concept study we show that genistein is able to target endogenous copper leading to prooxidant signaling and consequent cell death. We believe that such a mechanism explains the anticancer effect of genistein as also its preferential cytotoxicity towards cancer cells.

Identification and characterization of a copper-binding site in αA-crystallin.

Previous studies have shown that both αA- and αB-crystallins bind Cu2+, suppress the formation of Cu2+-mediated active oxygen species, and protect ascorbic acid from oxidation by Cu2+. αA- and αB-crystallins are small heat shock proteins with molecular chaperone activity. In this study we show that the mini-αA-crystallin, a peptide consisting of residues 71-88 of αA-crystallin, prevents copper-induced oxidation of ascorbic acid. Evaluation of binding of copper to mini-αA-crystallin showed that each molecule of mini-αA-crystallin binds one copper molecule. Isothermal titration calorimetry and nanospray mass spectrometry revealed dissociation constants of 10.72 and 9.9 µM, respectively. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid interaction with mini-αA-crystallin was reduced after binding of Cu2+, suggesting that the same amino acids interact with these two ligands. Circular dichroism spectrometry showed that copper binding to mini-αA-crystallin peptide affects its secondary structure. Substitution of the His residue in mini-αA-crystallin with Ala abolished the redox-suppression activity of the peptide. During the Cu2+-induced ascorbic acid oxidation assay, a deletion mutant, αAΔ70-77, showed about 75% loss of ascorbic acid protection compared to the wild-type αA-crystallin. This difference indicates that the 70-77 region is the primary Cu2+-binding site(s) in human native full-size αA-crystallin. The role of the chaperone site in Cu2+ binding in native αA-crystallin was confirmed by the significant loss of chaperone activity by the peptide after Cu2+ binding.

Protective mechanism of quercetin and rutin on 2,2'-azobis(2-amidinopropane)dihydrochloride or Cu2+-induced oxidative stress in HepG2 cells.

Protective effects of quercetin and rutin against oxidative stress were evaluated using in vitro and intracellular antioxidant assay. Quercetin showed higher peroxyl and hydroxyl radical-scavenging activity in a dose-dependent manner than did rutin in oxygen-radical absorbance capacity (ORAC). At 10 and 100 µM, quercetin had higher metal-chelating activity than rutin carrying rutinose at position C-3 and was also more efficient than rutin in reducing intracellular oxidative stress caused by peroxyl radicals and Cu(2+). The protective activities of 10 and 100 µM quercetin against Cu(2+)-induced intracellular oxidation were 13.8% and 44.8%, respectively. Rutin showed no protective activity against Cu(2+)-induced oxidative stress. Quercetin showed significantly lower intracellular Cu(2+)-chelating activity than did 1,10-phenanthroline but offered greater protection from Cu(2+)-induced oxidative stress. Thus, quercetin may diffuse through the cell membrane more efficiently than rutin because quercetin does not carry rutinose, is hydrophilic, and reduces Cu(2+)-induced oxidative stress by scavenging radicals instead of chelating with metal ions.

Zinc prevents the copper-induced damage of cultured astrocytes.

Copper is essential for several cellular processes, but an excess of cellular copper is known to be cell toxic. To study the consequences of a copper treatment of astrocytes, we have used astrocyte-rich primary cultures as model system to investigate cellular functions and cellular integrity of these cells after application of micromolar concentrations of copper chloride. After exposure of the cells to copper, the cell-associated copper content increased strongly in a time and concentration dependent manner. While incubation of cultured astrocytes with 3 microM copper hardly affected the cells during incubation for up to 4h, presence of 10 microM or 30 microM copper severly compromised cellular functions as demonstrated by a loss in total and soluble protein contents, a lowered MTT reduction capacity, lowered activities of the enzymes lactate dehydrogenase, glucose-6-phosphate dehydrogenase and glutathione reductase, a lowered cellular glutathione content, an increased lipid peroxidation, and an elevated membrane permeability for propidium iodide. Presence of an excess of zinc inhibited cellular copper accumulation and prevented most of the detrimental consequences of a copper exposure, suggesting that the beneficial effect of zinc against the copper-induced impairment of cultured astrocytes is mediated by inhibition of the cellular copper accumulation.

Liv.52 attenuate copper induced toxicity by inhibiting glutathione depletion and increased antioxidant enzyme activity in HepG2 cells.

Altered copper metabolism plays a pivotal role in the onset of several hepatic disorders and glutathione (GSH) plays an important role in its homeostasis. Hepatic diseases are often implicated with decreased content of intracellular GSH. GSH depleted cells are prone to increased oxidative damage eventually leading to its death. Liv.52 is used to treat hepatic ailments since long time. Hence, in the present study the potential cytoprotective effect of Liv.52 against toxicity induced by copper (Cu2+) was evaluated in HepG2 cells. Cu2+ at 750 microM induced cytotoxicity to HepG2 cells as determined by MTT assay. The toxicity was brought about by increased lipid peroxidation, DNA fragmentation and decreased GSH content. But, upon treatment with Liv.52 cell death induced by Cu2+ was significantly abrogated by inhibition of lipid peroxidation by 58% and DNA fragmentation by 37%. Liv.52 increased the GSH content by 74%. Activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase were increased by 46%, 22% and 81% respectively in Liv.52 treated cells. Thus, it is apparent from these results that Liv.52 abrogates Cu2+ induced cytotoxicity in HepG2 cells by inhibiting lipid peroxidation and increased GSH content and antioxidant enzyme activity.

Study of the action of Se and Cu on the growth metabolism of Escherichia coli by microcalorimetry.

The biological effect of Se and Cu²(+) on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1-10 µg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 µg/mL) inhibited the growth, but the Cu²(+) was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²(+) on E. coli in the different culture medium when Se was 1-10 µg/ml and Cu²(+) was 1-20 µg/ml. There was a negative synergistic effect of Se and Cu²(+) on E. coli when Se was higher than 10 µg/ml and Cu²(+) was higher than 20 µg/ml. The antagonistic or synergistic effect between Se and Cu²(+) on E. coli was related to the formation of Cu-Se complexes under the different experimental conditions chosen.

Pregabalin antagonizes copper-induced toxicity in the brain: in vitro and in vivo studies.

BACKGROUND: Copper plays key roles in brain metabolism. Disorders of copper metabolism impact on neural signaling. The intracellular and extracellular concentrations of copper are tightly regulated. Pregabalin is a drug with multiple modes of action and has a high-affinity binding site for the alpha2delta subunit of voltage-gated calcium channels. METHODS: Assessment of neuroprotective effects of pregabalin using cell culture, transcription studies, microdialysis and neurophysiological assessment in rats. RESULTS: In vitro, copper decreased markedly the survival of neuronal cells and enhanced the production of nitric oxide (NO). Transcription of NO synthase (NOS) 1-3 and PGC-1a (a key regulator of mitochondrial biogenesis) was activated. In vivo, copper impaired the NMDA-mediated regulation of glutamate in the brain, increased the production of NO and enhanced markedly the excitability of the motor cortex. Pregabalin had antagonistic effects both in vitro and in vivo. CONCLUSION: Our experiments highlight that pregabalin antagonizes the neurotoxic effects of copper. We argue that pregabalin exerts neuroprotective effects by silencing the overexcitability state induced by copper. We propose a possible use of pregabalin for treatment of disruption of copper homeostasis.

Anti-copper therapies in Alzheimer's disease: new concepts.

Alzheimer's disease (AD) is a heterogeneous and progressive neurodegenerative disorder. The recent Metal Hypothesis states that the interaction of Amyloid beta (Abeta, the main constituent of senile plaques) with transition metals is at the basis of AD neurodegeneration. This hypothesis is based on in vitro studies demonstrating that metals (copper, zinc) accelerate the aggregation and precipitation into plaques of Abeta, ultimately leading to synaptic dysfunction and accelerated amyloidogenesis. Recently, we have identified in AD patients a specific 'copper disease' marker, consisting in a serum-increase of copper not bound to ceruloplasmin, named 'free' copper. Several patents have been issued in the recent years and many clinical trials have been attempted in search of an anti-metal effect counteracting AD progression. Some of them have delivered very encouraging results. These anti-metal agents, however, have also shown adverse events. This work is aimed at reviewing 'old' and 'new' attitudes towards the use of anti-copper complexing agents or biological molecules which induce or maintain a state of copper malabsorption, such as zinc compounds, paying special attention to how such a rethinking of 'old' clinical trials might trace new routes in planning 'modern' ones.

Copper chelation in cancer therapy using tetrathiomolybdate: an evolving paradigm.

BACKGROUND: Tetrathiomolybdate (TM) is a novel anticancer and anti-angiogenic agent, which acts through copper chelation and NF-kappaB inhibition. OBJECTIVE: This review summarizes the scientific rationale for the use of TM as an anticancer agent in human studies. METHODS: A systematic review of the literature was conducted for the use of TM in cancer including preclinical, animal and human studies. The results of this search are summarized in this review. RESULTS/CONCLUSIONS: Copper chelation using TM has demonstrated efficacy in preclinical and animal models as an alternative and novel anti-angiogenic agent. Phase I and II clinical trials conducted in solid tumors using TM have demonstrated efficacy with favorable toxicity profile. The use of copper lowering as an anti-angiogenic strategy in the cancer chemopreventative setting remains to be investigated.

Zinc and tetrathiomolybdate for the treatment of Wilson's disease and the potential efficacy of anticopper therapy in a wide variety of diseases.

Wilson's disease, an autosomal recessive disease of copper accumulation and copper toxicity primarily in the liver and brain, has been the engine that has driven the development of anticopper drugs. Here we first briefly review Wilson's disease, then review the four anticopper drugs used to treat Wilson's disease. We then discuss the results of therapy with anticopper drugs in Wilson's disease, with special emphasis on the newer and better drugs, zinc and tetrathiomolybdate. We then discuss new areas of anticopper therapy, lowering copper availability with tetrathiomolybdate as a therapy in fibrotic, inflammatory, and autoimmune disorders. Many of the cytokines which promote these disorders are copper dependent, and lowering copper availability lessens the activity of these cytokines, favorably influencing a variety of disease processes. Copper in the blood can be thought of as in two pools. One pool is covalently bound in ceruloplasmin, a protein containing six coppers, synthesized by the liver and secreted into the blood. Ceruloplasmin copper accounts for almost 85 to 90% of the blood copper in normal people. This copper is tightly bound and not readily available for cellular uptake and copper toxicity. The other 10-15% of copper is more loosely bound to albumin and other small molecules in the blood, and is readily and freely available to cells and available to cause copper toxicity, if this pool of copper is increased. We call this latter pool of copper "free" copper because of its more ready availability. However, it should be understood that it is not completely free, always being bound to albumin and other molecules. It is this pool of free copper that is greatly expanded in untreated Wilson's patients undergoing copper toxicity.

The risks of free copper in the body and the development of useful anticopper drugs.

PURPOSE OF REVIEW: To review the toxicity and risks of free copper in Wilson's disease, Alzheimer's disease, other disease of neurodegeneration, and cognitive loss in the general population. We will also review the anticopper drugs and how lowering free copper levels with an anticopper drug inhibits fibrosis, inflammation, and autoimmunity. RECENT FINDINGS: Some exciting recent work indicates that free copper levels are increased in Alzheimer's disease, and copper may be involved in disease pathogenesis, opening the way to possible therapy of Alzheimer's disease with anticopper drugs. Copper may also be involved in other diseases of neurodegeneration. A very exciting recent study indicts high intake of copper, mostly from copper supplements, in conjunction with a high-fat diet in more rapid cognitive decline in the general population. Other data indicate that even low levels of copper in drinking water, perhaps similar to copper supplements, bypasses the liver, enters the circulation, increases the blood-brain penetration of copper, and may cause damage. SUMMARY: Some of the implications are that Alzheimer's disease and other diseases of neurodegeneration and fibrotic, inflammatory, and autoimmune diseases may be treatable by lowering the availability of free copper. People in the general population may wish to take steps to lower their free copper levels and, in particular, to abstain from taking copper supplements and ingesting significant amounts of copper in drinking water.

Dominant psychiatric manifestations in Wilson's disease: a diagnostic and therapeutic challenge!

INTRODUCTION: Recognition of psychiatric manifestations of Wilson's disease (WD) has diagnostic and therapeutic implications. OBJECTIVE: To describe the clinical features and psychopathology of patients with WD who had initial or predominant psychiatric manifestations. PATIENT AND METHODS: Records of 15 patients with WD (M:F: 11:4), from a large cohort of 350 patients, with predominant psychiatric manifestations at onset were reviewed. Their initial diagnosis, demographic profile, family history, pre-morbid personality, clinical manifestations, treatment and outcome were recorded. RESULTS: Their mean age at diagnosis was 19.8+/-5.8 years. Six patients were born to consanguineous parentage and two patients each had family history of WD and past history of psychiatric illness. Diagnosis of WD was suspected by detection of KF rings (all), observing sensitivity to neuroleptics (n=2), history of jaundice (n=2) and family history suggestive of WD (n=9). Psychiatric manifestations could be classified as affective disorder spectrum (n=11) and schizophreniform-illness (n=3). While the psychiatric symptoms improved in five patients with de-coppering therapy, seven patients needed symptomatic treatment as well. Three of the four patients who responded to de-coppering therapy were sensitive to neuroleptics. Long-term follow up of 10 patients revealed variable recovery. CONCLUSIONS: Young patient with psychiatric manifestations with clues like history of jaundice, family history of neuropsychiatric manifestations and sensitivity to neuroleptics should be evaluated for WD to avoid delay in diagnosis and associated morbidity. SIGNIFICANT OUTCOMES: The study reemphasizes the importance of behavioral manifestations in Wilson disease in terms of diagnosis and management difficulties. LIMITATIONS: Retrospective nature of the study.

Marginally low copper causes lesions of the midbrain in animal models: the implications for man

Serum copper levels must be maintained between very strict limits for the maintenance of good health. High levels have recently been linked to Alzheimer's disease while low levels during pregnancy cause enzootic ataxia (swayback disease) in offspring. In this study, we investigated the prolonged effect of serum copper that was maintained at and around 0.5 ppm, the level presently regarded as safe. Pregnant sheep and rabbits in the last trimester (1-4 weeks) of pregnancy were treated with the copper chelator ammonium tetrathiomolybdate (ATM). Treatment was continued until the young were one month old at which time the animals were sacrificed Serum copper levels of the parents and offspring were monitored by atomic absorption. The difference spectra (400-630 nm) was examined and SDS PAGE was used to evaluate the protein composition of the brain mitochondria. The anatomy of the midbrain was also studied. Although the young sheep and rabbits from the ATM-treated mothers showed no visible signs of disability or swayback disease, the midbrain of those young animals with serum copper between 0.3-0.9 ppm showed evidence of vacuolation, cavitation and chromatolysis. In contrast, the difference spectra and the protein composition of the brain mitochondria from these animals were all normal. These results suggest that although animals may appear normal and exhibit some normal biochemical markers, serum copper in the region of 0.5 ppm may not be safe for some breeds of sheep or rabbits. It is possible that a similar situation applies to man.

Los niveles séricos cúpricos tienen que ser mantenidos dentro de límites muy estrictos, si se quiere tener una buena salud. Los altos niveles de cobre han sido asociados recientemente con la enfermedad de Alzheimer, mientras que los niveles bajos durante el embarazo causan ataxia enzoótica (swayback) enla descendencia. En este estudio investigamos el efecto prolongado del cobre sérico mantenido a 0.5 ppm ó alrededor de 0.5 ppm – el nivel considerado seguro actualmente. Ovejas y conejas preñadas, en el último trimestre (1-4 semanas) de gestación, fueron tratadas con el quelante del cobre conocido como tetratiomolibdato de amonio (TM). El tratamiento continuó hasta que las crías tuvieron un mes,momento en el que los animales fueron sacrificados. Los niveles séricos cúpricos de los progenitores y la progenie fueron monitoreados mediante absorción atómica. Se examinaron los espectros de diferencia (400– 630 nm). Se usó la técnica de SDS-PAGE para evaluar la composición proteica de lasmitocondrias cerebrales. También se estudió la anatomía del mesencéfalo. Aunque la anatomía de las crías de ovejas y conejas madres tratadas con TM no mostraron señales visibles de discapacidad o enfermedad swayback, el mesencéfalo de estas crías con cobre sérico entre 0.3–0.9 ppm, mostróevidencias de vacuolación, cavitación y cromatolisis. En contraste con ello, los espectros de diferencia y la composición proteica de las mitocondrias del cerebro de estos animales, fueron todos normales. Estos resultados sugieren que aunque los animales puedan parecer normales y presentar marcas bioquímicas normales, el cobre sérico en el rango de 0.5 ppm, puede no ser seguro para algunas crías de ovejas y conejos. Es posible que una situación similar se aplique al ser humano.

Antitumor and antiinflammatory effects of tetrathiotungstate in comparison with tetrathiomolybdate.

Tetrathiomolybdate (TM) is an anticopper drug under development for treating Wilson's disease. Its mechanism of action involves forming a tight tripartite complex in the blood with serum albumin and available copper. When available copper levels are lowered in animals with TM, strong antiangiogenic and antitumor effects are observed. Similarly, TM has excellent efficacy in animal models of fibrotic, inflammatory, and autoimmune diseases, and it protects against heart damage from doxorubicin (DXR) and liver damage from acetaminophen, carbon tetrachloride, and concanavalin A. Tetrathiotungstate (TT) also forms a similar tripartite complex in the blood and has similar effects to TM on copper. In this article, whether TT had similar antitumor effects, and similar effects in protecting the heart against DXR toxicity, as TM was evaluated. It was found that the 2 drugs were comparable in their effects when doses were used that lowered copper availability to the same extent.