Micro-mechanical sensing platform for the characterization of the elastic properties of the ovum via uniaxial measurement

Stiffness is an important parameter in determining the physical properties of living tissue. Recently, considerable biomedical attention has centered on the mechanical properties of living tissues at the single cell level. In the present paper, the Young's modulus of zona pellucida of bovine ovum was calculated using Micro Tactile Sensor (MTS) fabricated using piezoelectric (PZT) material. The sensor consists of a needle-shaped 20-microm transduction point made using a micro-electrode puller and mounted on a micro-manipulator platform. Measurements were made under microscopic control, using a suction pipette to support the ovum in the same horizontal axis as the MTS. Young's modulus of ovum was found to be 25.3+/-7.94 kPa (n=28). This value was indirectly determined based on calibration curves relating change in resonance frequency (Deltaf(0)) of the sensor with tip displacement for gelatin at concentrations of 4%, 6%, and 8%. The regression equation between the rate of change in resonance frequency (versus sensor tip displacement), Deltaf(0)/x and Young's modulus is Deltaf(0)/x (Hz/microm)=0.2992 x Young's modulus (kPa)-1.0363. It is concluded that a reason that the stiffness of ovum measured in the present study is approximately six times larger than previously reported, may be due to the absence of large deformation present in of existing methodologies.     

Ex-vivo and in vitro protective effects of kolaviron against oxygen-derived radical-induced DNA damage and oxidative stress in human lymphocytes and rat liver cells

The present study reports the protective effects of kolaviron, a Garcinia biflavonoid from the seeds of Garcinia kola widely consumed in some West African countries against oxidative damage to molecular targets ex-vivo and in vitro. Treatment with hydrogen peroxide (H2O2) at a concentration of 100 micromol/L increased the levels of DNA strand breaks and oxidized purine (formamidopyrimidine glycosylase (FPG) and pyrimidine (endonuclease III (ENDO III) sites) bases in both human lymphocytes and rat liver cells using alkaline single cell gel electrophoresis (the comet assay). Kolaviron was protective at concentrations between 30-90 micromol/L and decreased H2O2-induced DNA strand breaks and oxidized bases. Neither alpha-tocopherol nor curcumin decreased H2O2-induced DNA damage in this assay. In lymphocytes incubated with Fe3+/GSH, Fe3+ was reduced to Fe2+ by GSH initiating a free radical generating reaction which induced 11.7, 6.3, and 4.9 fold increase respectively in strand breaks, ENDO III and FPG sensitive sites compared with control levels. Deferoxamine (2 mmol/L), an established iron chelator significantly inhibited GSH/Fe3+-induced strand breaks and oxidized base damage. Similarly, kolaviron at 30 and 90 micromol/L significantly attenuated GSH/Fe3+-induced strand breaks as well as base oxidation. Kolaviron (100 mg/kg bw) administered to rats for one week protected rat liver cells against H2O2-induced formation of strand breaks, ENDO III, and FPG sensitive sites, Fe3+/EDTA/ascorbate-induced malondialdehyde formation and protein oxidation using gamma-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS) as biomarkers of oxidative damage to proteins. We suggest that kolaviron exhibits protective effects against oxidative damage to molecular targets via scavenging of free radicals and iron binding. Kolaviron may therefore be relevant in the chemoprevention of oxidant-induced genotoxicity and possibly human carcinogenesis.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.     

DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.     

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus.

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 microg l(-1)) and ferric iron (20 and 40 mg l(-1)), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe3+ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe3+ treatment. No significant dose-response relationship was found with the two compounds and no synergistic effect was observed between Fe3+ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose-effect relationships and synergistic effects were not observed.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Mechanisms underlying the cytotoxic effects of Tachpyr--a novel metal chelator

Tachpyr (N,N'N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane), a novel metal chelator, was previously shown to deplete intracellular iron and exert a cytotoxic effect on cultured bladder cancer cells. Tachpyr binds Fe(II) and readily reduces Fe(III). The iron(II)-Tachpyr chelate undergoes intramolecular oxidative dehydrogenation resulting in mono- and diimino Fe(II) complexes. The present study investigates the redox-activity of the Tachpyr-iron complex to better define the mechanism of Tachpyr's cytotoxicity. Tachpyr's mechanism of cytotoxicity was studied using cell-free solutions, isolated DNA, and cultured mammalian cells by employing UV-VIS spectrophotometry, oximetry, spin-trapping technique, and electron paramagnetic resonance (EPR) spectrometry. The results show that: (1) Tachpyr by itself after 24 h of incubation had a cytotoxic effect on cultured cells; (2) fully oxidized Tachpyr had no cytotoxic effects on cultured cells even after 24 h of incubation; (3) Tachpyr protected isolated DNA against H(2)O(2)-induced damage, but not against HX/XO-induced damage; and (4) Tachpyr-Fe(II) chelate slows down but does not block oxidation of Fe(II), allows O*(-)(2)-induced or Tachpyr-induced reduction of Fe(III), and consequently promotes production of *OH through the Haber-Weiss reaction cycle. The results indicate that Tachpyr can protect cells against short-term, metal-mediated damage. However, upon prolonged incubation, Tachpyr exerts cytotoxic effects. Therefore, in addition to iron depletion, low-level oxidative stress, which in part occurs because of redox cycling of the coordinated iron ion, may contribute to the cytotoxic effects of Tachpyr.     

Carbon monoxide promotes respiratory hemoproteins iron reduction using peroxides as electron donors

The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O(2) via its binding to their ferrous (Fe(II)) heme-iron. Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric (Fe(III), met) forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage.     

Investigation of double stranded DNA damage induced by quercetin-copper(II) using piezoelectric quartz crystal impedance technique and potentiometric stripping analysis

DNA damage by quercetin-Cu(2+) was monitored in real time by piezoelectric quartz crystal impedance (PQCI) technique. In the PQCI analysis, the frequency change was caused mainly by the changes in density-viscosity of DNA solution in the damage course. The influences of DNA, Cu(2+), and quercetin concentrations on the motional resistance change (DeltaR(m)) were investigated in detail. The results showed that quercetin exhibited pro-oxidative damage at lower concentrations while anti-oxidative protection at higher concentrations, and DeltaR(m) exhibited a linear relationship in the DNA concentration range from 200 to 1600 microg/ml. Potentiometric stripping analysis (PSA) was also used to observe the electrochemical behavior of damaged DNA. From PSA, a new peak at 0.84 V and a higher peak at 1.06 V were discovered, which suggested that more purines were exposed to the electrode surface during the damage course. In agarose-gel electrophoresis, catalase and biquinoline were found to effectively inhibit DNA damage, therefore, a possible damage mechanism was proposed.     

Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom.

The mechanism of DNA damage by hydrazine in the presence of metal ions was investigated by DNA sequencing technique and ESR-spin trapping method. Hydrazine caused DNA damage in the presence of Mn(III), Mn(II), Cu(II), Co(II), and Fe(III). The order of inducing effect on hydrazine-dependent DNA damage (Mn(III) greater than Mn(II) approximately Cu(II) much greater than Co(II) approximately Fe(III)) was related to that of the accelerating effect on the O2 consumption rate of hydrazine autoxidation. DNA damage by hydrazine plus Mn(II) or Mn(III) was inhibited by hydroxyl radical scavengers and superoxide dismutase, but not by catalase. On the other hand, bathocuproine and catalase completely inhibited DNA damage by hydrazine plus Cu(II), whereas hydroxyl radical scavengers and superoxide dismutase did not. Hydrazine plus Mn(II) or Mn(III) caused cleavage at every nucleotide with a little weaker cleavage at adenine residues, whereas hydrazine plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the GTC sequence. ESR-spin trapping experiments showed that hydroxyl radical is generated during the Mn(III)-catalyzed autoxidation of hydrazine, whereas hydrogen atom adducts of spin trapping reagents are generated during Cu(II)-catalyzed autoxidation. The results suggest that hydrazine plus Mn(II) or Mn(III) generate hydroxyl free radical not via H2O2 and that this hydroxyl free radical causes DNA damage. A possibility that the hydrogen atom releasing compound participates in hydrazine plus Cu(II)-induced DNA damage is discussed.     

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g?=?4.3. This form of iron is referred to as "free" iron. EPR signals at g?=?4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S?=?5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g?=?4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.     

"In vitro" protection of DNA from Fenton reaction by plant polyphenol verbascoside

The protection effect of verbascoside (Ver) against Fenton reaction on plasmid pBR322 DNA was studied using agarose gel electrophoresis and UV-visible spectroscopy. The pBR322 plasmid DNA is damaged by hydroxyl radical (OH*) generated from the Fenton reaction with H2O2 and Fe(II) or Fe(III). This DNA damage is characterized by the diminution of supercoiled DNA forms or by the increase of relaxed or linear DNA forms after oxidative attack. The UV spectrum study showed that verbascoside can form complexes with Fe(II) or Fe(III), and the complexation can be reversed by the addition of EDTA. The formation constants of verbascoside-Fe complexes were estimated as 10(21.03) and 10(31.94) M(-2) for Fe(II) and Fe(III) respectively. The inhibition of Fenton reaction by verbascoside could be partially explained by the sequestration of Fe ions.     

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.     

Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae.

Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae. Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold. Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin. The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin-Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate. Bleomycin-mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40. These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug. Although Fe(III) was inactive with bleomycin and O2, the bleomycin-Fe(III) complex damaged walls and lysed cells in the presence of H2O2. H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission. These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA.     

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.     

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.     

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

The role of metal ion binding in generating 8-hydroxy-2'-deoxyguanosine from the nucleoside 2'-deoxyguanosine and the nucleotide 2'-deoxyguanosine-5'-monophosphate

The metal ions Cu(II), Fe(II), and Cr(III) were allowed to react with H(2)O(2) in the presence of either the mononucleoside 2'-deoxyguanosine (dG) or the mononucleotide 2'-deoxyguanosine-5'-monophosphate (dGMP). The percentage of reacted dG or dGMP that formed the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was monitored. Oxidative damage from reactions involving Cu(II) appear dependent on an interaction between copper and N7 on the guanine base. Any interactions involving the phosphate group have little additional effect on overall oxidative damage or 8-OH-dG production. Reactions involving Fe(II) seem very dependent on an interaction that may involve both N7 on the guanine base and the phosphate group. This interaction may slow oxidation of Fe(II) to Fe(III) in solution, keeping iron in a readily available form to undergo the Fenton reaction. Chromium(III) appears to interact with the phosphate group of dGMP, resulting in significant overall oxidative damage. However, production of 8-OH-dG appears to be very dependent on the ability of Cr(III) to interact with N7 on the guanine base, an interaction that seems to be weak for both the mononucleoside and mononucleotide.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.