Antioxidants in cervical cancer: chemopreventive and chemotherapeutic effects of polyphenols.

Cervical cancer lesions are a major threat to the health of women, representing the second most common cancer worldwide. The unanimously recognized etiological factor in the causation of cervical cancer is the infection with human papilloma virus (HPV). HPV infection, although necessary, is not per se sufficient to induce cancer. Other factors have to be involved in the progression of infected cells to the full neoplastic phenotype. Oxidative stress represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. Oxidative stress is known to perturb the cellular redox status thus leading to alteration of gene expression responses through the activation of several redox-sensitive transcription factors. This signaling cascade affects both cell growth and cell death. The ability of naturally occurring antioxidants to modulate cellular signal transduction pathways, through the activation/repression of multiple redox-sensitive transcription factors, has been claimed for their potential therapeutic use as chemopreventive agents. Among these compounds, polyphenols have been found to be promising agents toward cervical cancer. In addition to acting as antioxidants, polyphenols display a wide variety of biological function including induction of apoptosis, growth arrest, inhibition of DNA synthesis and modulation of signal transduction pathways. They can interfere with each stage of carcinogenesis initiation, promotion and progression to prevent cancer development. The present review discusses current knowledge of the major molecular pathways, which are involved in HPV-driven cancerogenesis, and the ability of polyphenols to modulate these pathways. By acting at specific steps of viral transformation cascade, polyphenols have been demonstrated to selectively inhibit tumor cell growth and may be a promising therapeutic tool for treatment of cervical cancer. In addition, recent results obtained in clinical trials using polyphenols are also discussed. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Redox proteomics in aging rat brain: involvement of mitochondrial reduced glutathione status and mitochondrial protein oxidation in the aging process.

Increasing evidence supports the notion that increased oxidative stress is a fundamental cause in the aging process and in neurodegenerative diseases. As a result, a decline in cognitive function is generally associated with brain aging. Reactive oxygen species (ROS) are highly reactive intermediates, which can modify proteins, nucleic acids, and polyunsaturated fatty acids, leading to neuronal damage. Because proteins are major components of biological systems and play key roles in a variety of cellular functions, oxidative damage to proteins represents a primary event observed in aging and age-related neurodegenerative disorders. In the present study, with a redox proteomics approach, we identified mitochondrial oxidatively modified proteins as a function of brain aging, specifically in those brain regions, such as cortex and hippocampus, that are commonly affected by the aging process. In all brain regions examined, many of the identified proteins were energy-related, such as pyruvate kinase, ATP synthase, aldolase, creatine kinase, and α-enolase. These alterations were associated with significant changes in both cytosolic and mitochondrial redox status in all brain regions analyzed. Our finding is in line with current literature postulating that free radical damage and decreased energy production are characteristic hallmarks of the aging process. In additon, our results further contribute to identifying common pathological pathways involved both in aging and in neurodegenerative disease development.

Apoptosis and autoimmunity induced by clodronate in systemic lupus erythematosus mononuclear circulating cells.

The aim of this study is to evaluate the effect of clodronate on apoptosis of human systemic lupus erythematosus circulating mononuclear cells and to analyze possible correlations with changes in autoantibody production in vitro. Lympho-monocytes from 20 SLE patients were isolated and incubated with or without addition of 1 microM clodronate for 72 hours. Apoptosis and release of genomic material was assessed by immunofluorescent detection of cleaved caspase-3 and by Cell-Death-Detection ELISAPLUS kit (Roche). Anti-Nucleosome IgG and anti-dsDNA IgM and IgG autoantibody levels were determined in supernatants by commercially available ELISA kits. Clodronate induced apoptosis in monocytes as confirmed by cleaved caspase-3 immunostaining and by quantification of cleaved nucleosome in the supernatants (treated 0.22+/-0.05 O.D. vs untreated 0.09+/-0.04 O.D.; P less than 0.001). This finding was coupled with a significant increasing in supernatants of IgG anti-Nucleosome (treated 6.5+/-1.1 vs untreated 5.5+/-0.6 IU/mL; p=0.001) and IgM (treated 3.0+/-1.3 vs 2.2+/-0.9 IU/mL; p=0.02) and IgG (treated 4.0+/-1.8 vs untreated 2.8+/-1.5 IU/mL; p=0.02) anti-dsDNA autoantibody levels. Our findings stressed the pro-apoptotic activity of clodronate, as well as its potential autoimmunity induction in SLE mononuclear circulating cells. Clinical studies could clarify the role of bisphosphonates on autoantibody production and worsening of disease activity.

Apoptotic molecular mechanisms implicated in autoimmune diseases.

Apoptosis is a programmed cell death that represents a normal component of the development, differentiation and health of multicellular organisms leading to an adequate cellular turnover and homeostasis. In autoimmune diseases, the immune system recognizes various autoantigens causing damage in target organs. Dead cells represent an important source of autoantigens that, in particular conditions, can represent a stimulus for an autoimmune response. A large number of studies reported the impairment of the apoptosis regulatory mechanisms in immune cells as a pivotal element in the pathogenesis and evolution of autoimmune disorders. Several pathogenetic pathways have been claimed to account for autoimmunity development during apoptotic processes. In fact, interestingly abnormalities potentially leading to immune disorders have been described as occurring in each step involved in apoptosis, from the very beginning to the post death phenomena. In this extent we propose a systematic review of the molecular mechanisms strictly leading to apoptosis with particular interest to their alterations, potentially causing tissue specific and/or systemic autoimmunity.

In vivo protection by the xanthate tricyclodecan-9-yl-xanthogenate against amyloid beta-peptide (1-42)-induced oxidative stress.

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimer's disease. One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide, which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Amyloid beta-peptide is the main component of senile plaques and generates free radicals ultimately leading to neuronal damage of membrane lipids, proteins and nucleic acids. Therefore, interest in the protective role of different antioxidant compounds has been growing for treatment of Alzheimer's disease and other oxidative stress-related disorders. Among different antioxidant drugs, much interest has been devoted to "thiol-delivering" compounds. Tricyclodecan-9-yl-xanthogenate is an inhibitor of phosphatidylcholine specific phospholipase C, and recent studies reported its ability to act as a glutathione-mimetic compound. In the present study, we investigate the in vivo ability of tricyclodecan-9-yl-xanthogenate to protect synaptosomes against amyloid beta-peptide-induced oxidative stress. Gerbils were injected i.p. with tricyclodecan-9-yl-xanthogenate or with saline solution, and synaptosomes were isolated from the brain. Synaptosomal preparations isolated from tricyclodecan-9-yl-xanthogenate injected gerbils and treated ex vivo with amyloid beta-peptide (1-42) showed a significant decrease of oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3-nitrotyrosine levels) and lipid peroxidation (4-hydroxy-2-nonenal levels). Our results are consistent with the hypothesis that modulation of free radicals generated by amyloid beta-peptide might represent an efficient therapeutic strategy for treatment of Alzheimer's disease and other oxidative-stress related disorders. Based on the above data, we suggest that tricyclodecan-9-yl-xanthogenate is a potent antioxidant and could be of importance for the treatment of Alzheimer's disease and other oxidative stress-related disorders.

Catecholamines oxidation by xanthine oxidase.

Dopamine and structurally related catecholamines in the presence of hydrogen peroxide are oxidized in vitro by xanthine oxidase producing the corresponding melanin pigments. The kinetic parameters of the reaction, measured as aminochrome formation, have been calculated. The rate of peroxidation depends on enzyme and hydrogen peroxide concentration. The optimum pH for the peroxidative activity of the enzyme is around 8.5. Activation of the peroxidative reaction is also elicited by catechol compounds through a redox cycle mechanism. Implications about the possible biochemical relevance of xanthine oxidase activity on catecholamines oxidation are discussed.

Interaction of enkephalin derivatives with reactive oxygen species.

The oxidation of opioid peptides by tyrosinase in the presence of an excess of a thiol gives rise to cysteinyldopa derivatives. The major products arising from the reaction between Leu-enkephalin and cysteine are represented by 5-S-cysteinyldopaenkephalin (5-CDenk) and 2-S-cysteinyldopaenkephalin (2-CDenk). The interaction of 5-CDenk and 2-CDenk with reactive oxygen species (ROS) has been studied. These compounds are able to scavenge superoxide anion, hydroxyl and peroxyl radicals as well as to reduce the lipid peroxidation rate induced by ABAP. The scavenging activities in all instances are dose-dependent. In some cases CDenks are more active than compounds recognized as strong radical scavengers, such as Trolox and mannitol. As a result of the action of the Fenton system, the CDenks (as well as the Enks) are oxidized into pigmented derivatives. The possible implications of the interaction of CDenks and Enks with ROS on melanization process in Parkinson's disease are discussed.

Some biochemical properties of melanins from opioid peptides.

Opioid peptides are converted by mushroom tyrosinase into melanin-like compounds retaining the peptide moiety (opio-melanins). Opio-melanins, owing to the presence of the linked aminoacids and in contrast with DOPA-melanin, are soluble compounds. The enkephalin-generated melanins are cleaved by carboxypeptidase A and pronase whereas aminopeptidase M cannot remove aminoacids from the pigment. Enkephalins, as well as other opioid peptides, (alpha-endorphin, kyotorphin, esorphins) if oxidized in presence of DOPA and tyrosinase are readily incorporated into DOPA-melanin. The resulting mixed-melanins (opio-melanin + DOPA-melanin) can be solubilized in hydrophilic solvents. Melanin from leu-enkephalin exhibits paramagnetism as evidenced by an EPR spectrum identical to that of DOPA-melanin, but unlike the latter pigment, it does not appear to oxidize NADH, probably for the presence of the peptide moiety that exerts a hampering effect on the oxidizing capacity.

Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors.

The reaction of opioid peptides with mushroom tyrosinase in the presence of an excess of a thiol compound gives rise to cysteinyldopaenkephalins (CDEnks). The major product is represented by the 5-S-CDEnk (80%) and the minor one by the isomer 2-S-CDEnk (20%). The adducts between leucine-enkephalin (Leu-enk) and cysteine have been isolated by high performance liquid chromatography (HPLC) and identified by amino acid analysis and electrospray ion mass spectrometry. 5-S-CDEnk is able to bind to opioid receptors in bovine brain membranes. Its binding affinity is higher for delta than for mu receptors and about 8-fold lesser than that exploited by Leu-enk. In the presence of the peroxidase/H(2)O(2) system, CDEnks can be converted into the corresponding pheo-opiomelanins.

Formation of homovanillic acid dimer by enzymatic or Fenton system - catalyzed oxidation.

Homovanillic acid is the most extensively employed reagent for the fluorometric detection of peroxidase. However, the assays based on the determination of the oxidation product of homovanillic acid do not allow a selective detection of the enzyme, because chemical or physical factors can interfere with the fluorometric determination. The aim of this work was to verify if other enzymatic or non-enzymatic systems might catalyze the homovanillic acid oxidation. The reaction was investigated by spectrophotometric and fluorometric assays; HPLC analysis was used to separate homovanillic acid from its oxidation product and to obtain information on the oxidation process. The results obtained showed that soybean lipoxygenase in the presence of hydrogen peroxide can oxidize homovanillic acid with the formation, by an o,o'-biphenyl linkage, of the corresponding dimer as the sole reaction product. The reaction followed Michaelis-Menten kinetics, for both homovanillic acid and hydrogen peroxide. Other systems, such as cytochrome c/H(2)O(2) and Fenton reagents, were also able to oxidize homovanillic acid to its dimer. It can be affirmed that possible interference by other oxidative systems - that could be present in the biological materials tested - should be considered in assays of peroxidase activity based on the detection of the dimer of homovanillic acid.

Production of melanin pigments by cytochrome c/H2O2 system.

In the presence of hydrogen peroxide cytochrome c can perform the oxidation of catecholamines and their S-cysteinyl-derivatives yielding melanins as final products. The initial reaction rate is linearly dependent on cytochrome c and H2O2 concentration; the reaction follows the Michaelis and Menten kinetics both for H2O2 and hydrogen donors. Sulfhydryl compounds inhibit the formation of the pigment. The reported data indicate that a heme-containing protein belonging to the mitochondrial chain can accelerate the oxidation of catecholamines to eumelanins.

Melanins from tetrahydroisoquinolines: spectroscopic characteristics, scavenging activity and redox transfer properties.

Tetrahydroisoquinolines (TIQs) are endogenous compounds deriving from the nonenzymatic Pictet-Spengler condensation of catecholamines (CA) with aldehydes. TIQs have been extensively studied in the last years not only because they have been found in the brain of postmortem specimens of Parkinson's patients, but also because they are able to induce parkinsonian symptoms if injected in animals. In the present article we demonstrate that TIQs bearing a catecholic moiety (tetrahydropapaveroline, salsolinol, laudanosoline, and apomorphine) are easily oxidized in the presence of hydrogen peroxide by various enzymes--i.e., peroxidase (POD), lipoxygenase (LOX), and xanthine oxidase (XO)--into the corresponding TIQ-melanins. The kinetic parameters of the above-mentioned reactions and some spectroscopic characteristics of the synthetized pigments are reported. In particular, UV-VIS and EPR spectra emerge as very similar to those exhibited by dopa-melanin. Furthermore, TIQ-melanins appear to be similar to dopa-melanin regarding some specific physico-chemical properties: NADH-oxidizing properties, oxy-radicals scavenging activity, and ability to form soluble mixed polymers with melanins from opioid peptides.

Lipoxygenase/H2O2-catalyzed oxidation of dihdroxyindoles: synthesis of melanin pigments and study of their antioxidant properties.

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H2O2 system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.

Transamination of l-cystathionine and related compounds by bovine brain glutamine transaminase.

Glutamine transaminase (EC 2.6.1.15) has been purified 113 fold from bovine brain. The product is free of aspariate amino transferase (EC 2.6.1.1.) and other common transaminases. The enzyme shows a wide specificity similar to that reported from the same transaminase purified from bovine kidney and liver as regards both the amino donor and the amino acceptor. Of interest is the transamination and cyclization of l-cystathionine, l-lanthionine, l-cystine and S-aminoethylcysteine. The latter result indicates that the deamination and the cyclization of the sulfur containing diamino acids described for bovine liver and kidney enzyme is feasible also in the brain and suggests the possible endogenous origin of cyclothionine and thiomorpholine dicarboxylate recently detected in bovine brain.

Microecology, bacterial vaginosis and probiotics: perspectives for bacteriotherapy.

Probiotics enriched in lactobacilli have been proposed as an effective and alternative tool to antibiotics for the treatment of bacterial vaginosis. The protective role of H(2)O(2)-producing lactobacilli has been strongly emphasized, but no clear-cut correlation appears to link the metabolic characteristics of administered lactobacilli with the clinical impact of probiotic therapy. On account of our review of basic mechanisms involved in bacterial vaginosis, we suggest that lactobacilli with an elevated arginine deiminase activity could have a greater therapeutic potential than strains producing only H(2)O(2). Preliminary results from our laboratory have demonstrated that treatment with probiotics containing arginine deiminase-positive lactobacilli improves clinical symptoms and is paralleled by a significant decline of polyamine levels in vaginal microenvironment. This is of outstanding interest due to the central role of polyamines in the pathogenesis of bacterial vaginosis. We should critically rethink, against this perspective, the use of probiotics for the treatment of affected women.

Synthesis and chromatographic properties of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine.

Details are reported for the synthesis of S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) from cysteine and 2-bromopropionic acid or 2-bromobutyric acid, respectively. Some analytical data and the behaviour of these two compounds on paper and ion-exchange chromatography are also reported, which allow their identification.

Oxidative deamination of S-(1-carboxyethyl)-L-cysteine and S-(1-carboxypropyl)-L-cysteine by L-aminoacid oxidase.

S-(1-carboxyethyl)-L-cysteine (1-CEC) and S-(1-carboxypropyl)-L-cysteine (1-CPC) are oxidatively deaminated by L-aminoacid oxidase with consumption of half a mole of oxygen per mole of substrate in the presence of catalase. This reaction gives rise to the corresponding alpha-ketoacids, identified by some chemical and chromatographic tests and by comparison with synthetic compounds. It has been possible, therefore, to demonstrate that S-(1-carboxyethyl)-thiopvruvic acid (1-CETP) and S-(1-carboxypropyl)-thiopvruvic acid (1-CPTP) are the main products of oxidative deamination of 1-CEC and 1-CPC.

Oxidation of D-thiazolidine-4-carboxylic acid by hog kidney D-aminoacid oxidase.

D-Thiazolidine-4-carboxylic acid is a good substrate for hog kidney D-aminoacid oxidase. Data are presented showing that the only oxidation product is delta 3-thiazoline-4-carboxylic acid, which does not undergo further spontaneous degradation. Thus, the oxidation of D-thiazolidine-4-carboxylic acid by D-aminoacid oxidase differs considerably from the oxidation of its L-isomer catalyzed by rat liver mitochondria which gives as final product N-formylcystine, possibly through the intermediate delta 2-thiazoline-4-carboxylic acid.

Pyridoxal phosphate catalyzed alpha-beta elimination of selenahomolysine.

In the presence of pyridoxal phosphate selenahomolysine undergoes alpha-beta elimination with production of pyruvate, ammonia and selenohomocysteamine. If the reaction occurs in anaerobic conditions the coupling of pyridoxal phosphate with selenohomocysteamine in a tetrahydro-selenazine ring may be detected by the appearance of an absorption band at 320 nm. In the presence of air the autoxidation rate of selenohomocysteamine is too high to allow the detection of the selenazine derivative. The results obtained add to the previously reported ones indicating that selenium- and sulfur-containing aminoacids react in quite the same way in the non enzymic model studied.

Oxidative deamination of S-aminopropylcysteine and S-aminoethylhomocysteine.

S-aminopropylcysteine and S-aminoethylhomocysteine are oxidized by snake venom L-amino acid oxidase in the presence of catalase with formation of the respective ketoderivatives. Only the ketoderivative of S-aminopropylcysteine cyclizes to give a seven membered ring (ketimine) absorbing at 296 nm. In the absence of catalase both ketoderivatives are oxidatively decarboxylated.