Gastric toxicity and mucosal ulceration induced by oxygen-derived reactive species: protection by melatonin

Uncontrolled hydrochloric acid secretion and ulceration of the stomach mucosa due to various factors are serious global problems. Although the mechanism of acid secretion from the parietal cell is now well understood, the processes involved in gastric ulceration are still not clear. Among various causes of gastric ulceration, lesions caused by stress, alcohol consumption, Helicobacter pylori infection and due to use of nonsteroidal antiinflammatory drugs have been shown to be mediated largely through the generation of reactive oxygen species, especially the hydroxyl radical. A number of excellent drugs have proven useful in controlling hyperacidity and ulceration but their long-term use is associated with disturbing side-effects. Hence, the search is still on to find a compound possessing antisecretory, antiulcer and antioxidant properties which will serve as a therapeutic agent to reduce gastric hyperacidity and ulcers. This article describes the role of reactive oxygen species in gastric ulceration, drugs controlling them with their merits and demerits and, the role of melatonin, a pineal secretory product, in protecting against gastric lesions. In experimental studies, melatonin has been shown to be effective in reducing mucosal breakdown and ulcer formation in a wide variety of situations. Additionally, the low toxicity of melatonin supports further investigation of this molecule as a gastroprotective agent. Finally, we include a commentary on how melatonin research with respect to gastric pathophysiology can move forward with a view of eventually using this indole as a therapeutic agent to control gastric ulceration in humans.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

DNA interaction with biologically active divalent metal ions: binding constants calculation

In our previous work we have shown that under the action of Cu²⁺, Mn²⁺ and Ca²⁺ ions DNA is able to transit into a compact state in aqueous solution. In the present work we carried out calculations of binding constants for divalent metal ions interacting with DNA in terms of the macromolecule statistical sum. The formula for calculation of the binding constants and cooperativity parameters was proposed. It was shown that on the “coil state”–“compact (globule) state” transition a single DNA molecule may undergo the first-order phase transition while the transition of the assembly of average DNA chains is of sigmoidal character typical of the cooperative and continuous transition.     

Role of active oxygen species in metal-induced DNA strand breakage in human diploid fibroblasts

The ability of 6 metal salts to induce DNA damage in human diploid fibroblasts was examined. Cadmium, magnesium, manganese, chromium(VI), zinc and selenite were all shown to induce DNA strand breaks as measured by two independent assays. DNA strand breaks were repaired within 2-4 h after removal of metal and this repair appeared not to be sensitive to "long-patch" repair inhibitors. With the exception of selenite, metal-induced DNA damage appeared to be mediated via the formation of active oxygen species since oxygen scavengers when administered simultaneously with the metal, antagonized strand break formation. Selenite-induced DNA damage (as previously reported) was dependent on the formation of a selenite-glutathione conjugant and was not affected by oxygen radical scavengers. Scavenger treatment did not enhance cloning ability of metal-treated cells suggesting that DNA strand breaks may not be important in metal-induced cytotoxicity.     

DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems.

When cells are exposed to oxidative stress, DNA damage frequently occurs. The molecular mechanisms causing this damage may include activation of nucleases and direct reaction of hydroxyl radicals with the DNA. Several oxygen-derived species can attack DNA, producing distinctive patterns of chemical modification. Observation of these patterns and measurement of some of the products formed has been used to determine the role of different oxygen-derived species in DNA cleavage reactions, to assess the extent of oxidative damage to DNA in vivo and to investigate the mechanism of DNA damage by ionizing radiation and chemical carcinogens.     

The responses to choline ions induced by transition metal ions in single water fibers of the frog glossopharyngeal nerve

In single water-sensitive fibers (water fibers) of the frogglossopharyngeal nerve, application of a solution of 500 mMcholine Cl to the tongue elicited responses of varying magnitude.Some water fibers (plain choline-insensitive water fibers) barelyresponded to the solution, while some water fibers (plain choline-sensitivewater fibers) exhibited a considerable response to this solution.NiCl₂. which is barely effective in producing neural responseat concentrations below 5 mM, induced the response of plaincholine-insensitrve water fibers to choline⁺ ions. It was confirmed,in a collision test, that the Ni²⁺-induced responses to choline⁺ions were derived from water fibers. However, NiCl₂ did notaffect the magnitude of me response generated by choline⁺ ionsin plain choline-sensitive water fibers. The concentration-responsecurve for choline Cl in the presence of 1 mM NiCl₂ for plaincholine-insensitive water fibers was similar to the curves obtainedin the absence of NiCl₂ for plain choline-sensitive water fibers.Other organic salts, such as tris(hydroxymethyl)arrdnomethane-HCl,triethanotamine-HCl and tetraethylammonium Cl, elicited no responseor only a very small response from water fibers, and NiCl₂ didnot affect these responses. It is suggested that there existsa choline receptor for the response to choline⁺ ions in theapical membrane of frog taste cells and that Ni²⁺ ions exposethe sites of such choline receptors, which are deeply embeddedin the receptor membrane, to the outside medium. The effectof Ni²⁺ ions results in an increase in the number of the cholinereceptor sites available for binding of choline⁺ ions. The rankorder of effectiveness of transition metal ions in elicitingthe appearance or enhancement of the response to choline Clwas Ni²⁺ > Co²⁺ > Mn²⁺. Mg²⁺ ions had no effect on theresponse to choline⁺ ions. A similar rank order was previouslyobtained in enhancement of the responses to Ca²⁺, Mg²⁺ and Na²⁺ions (Kitada, 1994a). It seems likely that the mechanism forenhancement or elicitation of the response to choline⁺ ionsby the transition metal ions has features in common with thatfor enhancement of the responses to Ca²⁺, Mg²⁺ and Na⁺ ions.     

DNA hydrolysis by rare-earth metal ions.

Plasmid DNA and poly(dA) are cleaved by rare-earth(III) ions at pH 7-8 and 50 degrees C. The cleavage has been confirmed by prompt conversion of supercoiled pBR 322 plasmid DNA (Form I) to a relaxed Form II. Furthermore, degradation of poly(dA) to shorter oligonucleotides is clearly evidenced by HPLC. A possible application of the metal ions (and their complexes) to artificial nucleases is indicated.     

DNA structure control by polycationic species: polyamine, cobalt ammines, and di-metallo transition metal chelates

Many polycationic species bind to DNA and induce structural changes. The work reported here is the first phase of a program whose long-term aim is to create a class of simple and inexpensive sequence-selective compounds that will enable enhanced DNA structure control for a wide range of applications. Three classes of molecule have been included in this work: the polyamine spermine (charge: 4(+)) and spermidine (charge: 3(+)) (which are known to induce a wide range of DNA conformational changes but whose binding modes are still not well understood); cobalt (III) amine transition metal complexes as potential polyamine mimics and [Fe(H(2)O)(6)](3+); and the first member of a new class of di-metallo tris-chelated cylinders of helical structure (charge 4(+)). Temperature-dependent absorption, circular dichroism, linear dichroism, gel electrophoresis, and molecular modeling data are presented. The cobalt amines prove to be effective polyamine mimics, although their binding appears to be restricted to backbone and major groove. All the ligands stabilize the DNA, but the 4(+) di-iron tris-chelate does so comparatively weakly and seems to have a preference for single-stranded DNA. All the molecules studied bend the DNA, with the di-iron tris-chelate having a particularly dramatic effect even at very low drug load.     

Catalytic hydrolysis of DNA by metal ions and complexes

Biomimetic hydrolysis of DNA or RNA is of increasing importance in biotechnology and medicine. Most natural nuclease enzymes that mediate such reactions utilize metal ion cofactors. Recent progress in the design of synthetic metallonucleases has included complexes of antibiotics, peptides, nucleic acids, and other organic ligands. In this article, we review a number of synthetic catalyst systems that have been developed to achieve efficient DNA hydrolysis. Methods to evaluate their catalytic efficiencies are critically discussed, and a prognosis for future work in this area is presented.     

The relative contributions of species richness and species composition to ecosystem functioning

How species diversity influences ecosystem functioning has been the subject of many experiments and remains a key question for ecology and conservation biology. However, the fact that diversity cannot be manipulated without affecting species composition makes this quest methodologically challenging. Here, we evaluate the relative importance of diversity and of composition on biomass production, by using partial Mantel tests for one variable while controlling for the other. We analyse two datasets, from the Jena (2002–2008) and the Grandcour (2008–2009) Experiments. In both experiments, plots were sown with different numbers of species to unravel mechanisms underlying the relationship between biodiversity and ecosystem functioning (BEF). Contrary to Jena, plots were neither mowed nor weeded in Grandcour, allowing external species to establish. Based on the diversity–ecosystem functioning and competition theories, we tested two predictions: 1) the contribution of composition should increase with time; 2) the contribution of composition should be more important in non‐weeded than in controlled systems. We found support for the second hypothesis, but not for the first. On the contrary, the contribution of species richness became markedly more important few years after the start of the Jena Experiment. This result can be interpreted as suggesting that species complementarity, rather than intraspecific competition, is the driving force in this system. Finally, we explored to what extent the estimated relative importance of both factors varied when measured on different spatial scales of the experiment (in this case, increasing the number of plots included in the analyses). We found a strong effect of scale, suggesting that comparisons between studies, and more generally the extrapolation of results from experiments to natural situations, should be made with caution.     

Interactions between metal ions and carbohydrates: the coordination behavior of neutral erythritol to transition metal ions

The single crystals of coordinated complexes of neutral erythritol (C4H10O4) with various transition metal ions were synthesized and studied using FT-IR and single crystal X-ray diffraction analysis. Two CuCl2-erythritol complexes (denoted as CuE(I) and CuE(II)) were obtained. In CuE(I), Cu2+ coordinates with two chloride ions and four OH groups from two erythritol molecules. Two copper centers are linked by one erythritol molecule to form a zigzag chain. For CuE(II), each Cu2+ coordinates with two OH groups from an erythritol molecule and two chloride ions. The crystal of CuE(II) contains complexed and free erythritol, the dimers of [Cu2Cl4(C4H10O4)] further form a [Cu2Cl4(C4H10O4)]infinity chain via secondary Cu...Cl bonds, both the dimer unit of [Cu2Cl4.(C4H10O4)] and non-coordinated C4H10O4 unit exist side by side in the crystal. MnCl2-erythritol complex whose structure is similar to CuE(I) is also acquired. The OH groups of erythritol act as ligand to coordinate to metal ions on one hand, one the other hand, OH groups form hydrogen bonds network that link chain and layer together to build three-dimensional structures.     

Inhibition of proton-transfer steps in transhydrogenase by transition metal ions

Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb²⁺ > Cu²⁺ > Zn²⁺ = Cd²⁺ > Ni²⁺ > Co²⁺. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn²⁺-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn²⁺. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn²⁺-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.     

The complexation of transition series metal ions by nalidixic acid

The following formation constants have been determine for nalidixic acid: proton, copper(II) complexation, magnesium(II) complexation, guanosine-5′-monophosphate-copper(II) complexation. Use of these data (together with the corresponding published constants of calcium(II), iron(II), manganese(II) and zinc(II) supports the hypothesis that the drug acts at a site other than extracellular. Complex formation between nalidixic acid, metal ion and DNA (at guanosine residues) is suggested.     

Co-ordination of transition metal ions by galactaric acid: a potentiometric and spectroscopic study

A solution study on the ability of galactaric acid [GalaH(2), HOOC(CH)(4)COOH] in the complexation of biological metal ions such as Co(II) and Ni(II) and toxic metal ions such as Cd(II), Pb(II) and Hg(II), is reported. The stability constants of the complex species are determined by means of potentiometric measurements. Galactaric acid behaves as chelate ligand through carboxylic oxygen and alpha-hydroxy group towards Co(II) and Ni(II), while in the Pb(II) and Cd(II) containing system it co-ordinates the metal ion with carboxylic oxygen and two alcoholic hydroxy groups. The prevailing species at acidic or neutral pH is [MGala] which is also isolated in the solid state and characterized by means of IR spectroscopy. On increasing pH, the [MGalaH(-1)](-) species is also formed where the co-ordinated OH group undergoes deprotonation in all metal ion complexes except those with Hg(II), where the co-ordination of hydroxide ion is suggested as the precipitation of the metal hydroxide occurs at pH 7.     

Potential carcinogenicity of some transition metal ions

Potential carcinogenicity of some transition metal ions was tested using a direct-current polarography method. The measurements were based on the reduction of tested compounds in an anhydrous solution using α-lipoic acid as the detection compound. The potential carcinogenicity was expressed in terms of the parameter tg α, which is known to directly correlate with the carcinogenicity of tested compounds. For the metal ions tested, tg α was found to decrease in the following sequence: Fe(III) > Pb(II) > V(IV) > Fe(II) > Mn(II) > Cu(II). Zero values of tg α were found for Cd(II) and Mn(III).     

Low-density lipoprotein modification by normal,myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin, transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin,transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.