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PubMed2006

Molecular Biomarkers: their significance and application in marine pollution monitoring.

This paper presents an overview of the significance of the use of molecular biomarkers as diagnostic and prognostic tools for marine pollution monitoring. In order to assess the impact of highly persistent pollutants such as polychlorinated biphenyls (PCB), polychlorinated dibenzo-dioxins (PCDD), polychlorinated dibenzo-furans (PCDF), polynuclear aromatic hydrocarbons (PAH), tributyltin (TBT) and other toxic metals on the marine ecosystem a suite of biomarkers are being extensively used worldwide. Among the various types of biomarkers, the following have received special attention: cytochrome P4501A induction, DNA integrity, acetylcholinesterase activity and metallothionein induction. These biomarkers are being used to evaluate exposure of various species of sentinel marine organisms (e.g. mussels, clams, oysters, snails, fishes, etc.) to and the effect of various contaminants (organic xenobiotics and metals) using different molecular approaches [biochemical assays, enzyme linked immuno-sorbent assays (ELISA), spectrophotometric, fluorometric measurement, differential pulsed polarography, liquid chromatography, atomic absorption spectrometry]. The induction of the biotransformation enzyme, cytochrome P4501A in fishes (Callionymus lyra, Limanda limanda, Serranus sp., Mullus barbatus) and mussels (Dreissena polymorpha) by various xenobiotic contaminants such as PCBs, PAHs, PCDs is used as a biomarker of exposure to such organic pollutants. The induction of cytochrome P4501A is involved in chemical carcinogenesis through catalysis of the covalent bonding of organic contaminants to a DNA strand leading to formation of DNA adduct. Measurement of the induction of cytochrome P4501A in terms of EROD (7-ethoxy resorufin O-deethylase) activity is successfully used as a potential biomarker of exposure to xenobiotic contaminants in marine pollution monitoring. In order to assess the impact of neurotoxic compounds on marine environment the evaluation of acetylcholinesterase activity in marine organisms is used as a biomarker of exposure to neurotoxic agents such as organophosphorus, carbamate pesticides etc. Metallothioneins (MTs) are induced by toxic metals such as Cd, Hg, and Cu by chelation through cysteine residues and are used in both vertebrates and invertebrates as a biomarker of metal exposure. The measurement of the levels of DNA integrity in marine organisms such as Sea stars (Asterias rubens) from the North Sea and the marine snails (Planaxis sulcatus) from the Arabian Sea along the Goa coast exposed to environmental xenobiotic contaminants clearly indicated the extent and the nature of pollution at the sampling sites along coastal environment.

Acetylcholinesterase↗

PubMed2005

Distribution of heavy metals in Penaeus semisulcatus from Persian Gulf and possible role of metallothionein in their redistribution during storage.

The study was conducted between January and December 2002. The main objective of this study was evaluation of effects of refrigerated storage duration on redistribution of three trace elements (Cd, Cu and Zn) in tissues (exoskeleton, abdominal muscle and hepatopancreas) of a shrimp species (Penaeus semisulcatus). Moreover, the possible roles of metallothionein (a kind of metalloprotein) in redistribution of the elements in tissues of the selected species were assessed. The specimens were sampled from northwestern part of the Persian Gulf. The concentrations of metals in the tissues were measured using Inductively Coupled Plasma-Optical Emission Spectrophotometer (ICP-OES). Metallothionein levels were determined by Differential Pulse Polarography (DPP) method. pH of the muscle samples was also measured in different stages. Different statistical methods were used for interpretation of the results. There were no size-dependent differences in metal contents of the species. The results were compared with specimens from other areas of the world and existing guidelines and limits. Concentrations of the metals in the muscle (0.103, 3.418 and 8.977 microg g(-1) wet weight in the case of Cd, Cu and Zn, respectively) samples were below the most guidelines for human consumption. The results were in general agreement with those obtained by some other researchers. There were highly significant differences between sexes in Zn and Cu contents. Levels of Zn in females were significantly higher than males. The reverse case observed for Cu. The highest mean Cu and Zn concentrations (15.939 and 43.394 microg g(-1) wet weight, respectively) were found in hepatopancreas samples, but the highest level of Cd (0.790 microg g(-1) wet weight) was observed in exoskeleton. There were significant differences between the fresh and refrigerated samples from accumulation of Cd and Zn in tissues point of view, which can be attributed to the metal binding properties of metallothioneins as well as their degradation during the storage.

Animals↗

PubMed1990

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.

Animals↗

PubMed1976

Studies on the rate-limiting reaction of photosynthetic oxygen evolution in spinach chloroplasts.

The modulated oxygen polarography has been used to study the rate-determining steps of photosynthetic oxygen evolution in spinach chloroplasts. The rate constant, k, of the reaction has a value of 218 +/- 10 (S.E.) s-1 at 23 degrees C and an activation energy of 7 +/- 2 (S.E.) kcal-mol-1. A kinetic isotope experiment indicated that this step is probably not the water-splitting reaction. These findings resemble previous results with the unicellular alga Chlorella (Sinclair, J. and Arnason, T (1974) Biochim. Biophys. Acta 368, 393-400). In other experiments we changed the pH, O2 concentration and osmolarity of the medium, and treated the chloroplasts with 1 mM NH4Cl without detecting any significant change in k. These results suggest that the step is irreversible. However, a significantly lower value of k, 110 +/- 20 (S.E.) s-1 was obtained when all salts except 1 mM MgCl2 were removed from the medium bathing the chloroplasts.

Chloroplasts↗

PubMed1985

Phospholipid oxidation catalyzed by cytochrome c in liposomes.

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.

Animals↗

PubMed1980

An electrochemical study of the iron storage protein, ferritin.

The electrochemical behavior of iron storage protein, ferritin (molecular weight 700 000) was studied by pulse polarography, cyclic voltammetry and 'potentiometry' monitored by colorimetry. Comparison between in vivo and voltammetric redox reactions of the ferritin Fe(III) core and thermodynamic E'0 values for native ferritin are described.

Electrochemistry↗

PubMed1986

Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes.

Dissociation constants of cytokinins, derivatives of purine which form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logk1, and log beta 2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(II) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants with superoxide, which were determined by polarography, were 2.3 X 10(-7) M-1 s-1 for kinetin and 1.5 X 10(-7) M-1 s-1 for 6-benzylaminopurine at pH 9.8 and 25 degrees C.

Adenine↗

PubMed1987

Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity by 1-methyl-4-phenylpyridinium ion.

Effect of 1-methyl-4-phenylpyridinium ion (MPP+) on the activity of NADH-ubiquinone oxidoreductase was studied using mitochondria prepared from rat brains. At first, inhibition of oxygen consumption by MPP+ with pyruvate + malate or glutamate + malate as substrates was confirmed polarographically using a Clark-type oxygen electrode. Then, activity of NADH-ubiquinone oxidoreductase in the same samples used in polarography was assayed. Incubation of mitochondria with 0.05 mM of MPP+ together with glutamate, malate and ADP resulted in approximately 50% inhibition of NADH-ubiquinone oxidoreductase activity. Significance of the results was discussed with respect to the mechanism of neuronal degeneration by MPP+.

1-Methyl-4-phenylpyridinium↗

PubMed1989

Synergistic deleterious effect of micromolar Ca ions and free radicals on respiratory function of heart mitochondria at cytochrome C and its salvage trial.

Both Ca2+ and free radicals (FR) are accumulated in temporarily ischemic myocardium and might cause reperfusion injury. Respiratory function measured by polarography of isolated heart mitochondria before or after the in vitro treatment with Ca2+ (1.2 microM) and/or FR showed that Ca2+ or FR per se showed no or weak effect on state 3, but cotreatment of Ca2+ and FR prominently deteriorated state 3, state 4 and RCI. The injury was speculated to occur at cytochrome c itself or its reductase from enzyme assay in the respiratory chain. Furthermore, contrary to the published data, the synergistic action was not mitigated by phospholipase A2 inhibitors (dibucaine, mepacrine), membrane stabilizers (lidocaine, coenzyme Q10), Ca entry blocker (verapamil) or superoxide dismutase, suggesting refractory to therapy.

Animals↗

PubMed1990

Intramolecular long-range electron transfer in the hemerythrin monomer: a pulse radiolysis study.

The single sulfhydryl group (Cys-50) of the methemerythrin from Phascolosoma gouldii reacts with 5,5'dithiobis(2-nitrobenzoic acid) to form a mixed disulfide. The pulse radiolytically generated formate radical reduces this mixed disulfide to its radical anion. In turn, the disulfide radical anion reduces the protein two-iron center over a nominal distance of 13 A. The rate constant for this intramolecular electron transfer is approximately 15 s-1 at room temperature, pH 7. Between the two redox centers there is an equilibrium driving force of 0.78 V, measured by differential pulsed polarography.

Animals↗

PubMed1990

DNA damaging effects and voltammetric studies on the hypoxic cell toxin 3-amino-1,2,4-benzotriazine-1,4-dioxide, SR4233, as a function of pH.

The compound 3-amino-1,2,4-benzotriazine-1,4-dioxide, SR4233, has recently attracted considerable attention as a possible hypoxic cell radiation sensitizer and cytotoxic agent. The present study examines the influence of pH on the DNA damaging ability of SR4233 upon electrolytic reductive activation, and the corresponding changes in electrochemistry. A phi X174 double transfection assay has been employed to assess the DNA damaging ability of SR4233 between pH 4 to 7. Upon electrolytic reduction the drug was found to be more effective in damaging DNA at acidic pH than at neutral conditions. This indicated that the damaging species was probably protonated. The DNA damaging ability of SR4233, as measured by a viral transfection assay, was linearly related to pH between the values of 4 and 7, and this feature has implications for its potential efficacy in the treatment of hypoxic tumors. The electrochemistry of SR4233 has been examined as a function of pH between the ranges 2 and 10.5. Three investigation techniques have been employed, cyclic voltammetry and differential pulse and dc polarographies. A general shift towards less negative potentials with increasing acidity was found between pH 2 and 8.5 giving a linear relationship. The behaviour was found to be relatively invariant at alkaline pH.

Antineoplastic Agents↗

PubMed1996

Isolation and preliminary characterization of a Chinese hamster ovary cell line with high-degree resistance to hydrogen peroxide.

We have isolated and conducted preliminary characterization of a cell line derived from the Chinese hamster ovary cell line AA8, which we have designated AG8 and which is highly resistant to the cytotoxic effects of H2O2 (approximately 17-fold when the H2O2 treatment was at 37 degrees; approximately 11-fold when the H2O2 treatment was at 4 degrees). AG8 cells were moderately (but significantly; P < 0.05) cross-resistant to CdCl2 (approximately 4-fold), NaAsO2 (approximately 2.3-fold), t-butyl hydroperoxide (approximately 2.9-fold), cumene hydroperoxide (approximately 3-fold), menadione (approximately 1.7-fold) and HgCl2 (approximately 1.5-fold), but were not significantly cross-resistant to hyperthermia (43 degrees), 254 nm UV light, 137Cs gamma-rays, and 42-MeV (p-->Be+) fast neutrons. As regards their biochemical status, AG8 and AA8 cells contain similar non-protein sulfhydryl levels per milligram of protein. Catalase activity (assessed by both spectrophotometry and polarography) was significantly higher in AG8 than in AA8 cells irrespective of whether enzyme activity was expressed per 10(6) cells (approximately 3.6-fold increase) or per milligram of protein (approximately 1.6-fold increase). AG8 cells also exhibited significantly greater glutathione reductase activity than wild-type cells when the data were expressed per 10(6) cells (approximately 2.9-fold) or per milligram of protein (approximately 1.3-fold). Glutathione peroxidase activity was immeasurably low in both cell lines. The susceptibility of the two cell lines to H2O2-mediated generation of DNA single-strand breaks (as measured by alkaline elution) indicated a slightly (approximately 1.5-fold) decreased yield in the resistant AG8 cell line. The two cell lines repaired these breaks with similar kinetics. In contrast, no measurable induction of DNA double-strand breaks (as measured by pulsed-field gel electrophoresis) was apparent in either cell line after survival-curve range concentrations of H2O2. On the basis of these data, it appears that the AG8 phenotype involves two previously identified resistance mechanisms, namely an adaptive component that may or may not involve increased antioxidant capacity, and a second component that does involve increased antioxidant (primarily catalase) capacity.

Adenosine Triphosphate↗

PubMed1992

In search of a correlation between the electron affinity and the radiosensitizing activity of some glyoxylic compounds.

The electron affinity of some glyoxylic compounds with radiosensitizing properties was evaluated using different approaches. The half-wave reduction potentials were measured by polarography, and the molecular structures in solution were determined by NMR spectroscopy. The electron affinity of the unhydrated and hydrated molecules was theoretically evaluated by means of a semi-empirical quantum mechanical procedure, the MNDO. The possible correlations between these values and the radiosensitizing efficiency of these glyoxylic compounds is discussed.

Electrochemistry↗

PubMed1985

The effect of dopamine upon oxidative metabolism of brown fat adipocytes.

The effect of dopamine upon the oxygen consumption (QO2) of brown fat adipocytes was measured by polarography. The results showed a marked stimulation of QO2 by dopamine, similar to that induced by noradrenaline. The dopamine response could be blocked by haloperidol, butaclamol, propranolol and by high concentrations of tolazoline. These results suggest a role for dopamine in energy release from brown fat.

Adipose Tissue, Brown↗

PubMed1989

Aortic occlusion and reperfusion and conduction, blood flow, and the blood-nerve barrier of rat sciatic nerve.

We studied the effect of ischemia and reperfusion on blood flow, electrophysiology, and the blood-nerve barrier of the sciatic nerve of the rat. Ischemia for 10 to 60 min, with up to 3 h of reperfusion, was produced by closure and then release of a noose around the abdominal aorta. Nerve blood flow and function were measured serially using microelectrode-hydrogen polarography and electrophysiologic recordings, respectively. The integrity of the blood-nerve barrier was assessed using the permeability surface area product to [14C]sucrose. Ischemia of up to 30 min did not impair reperfusion. One hour of ischemia resulted in reperfusion abnormalities that affected about half of the nerves. The ischemic and reperfusion stresses did not disrupt the blood-nerve barrier to [14C]sucrose nor produce conduction block. Possible mechanisms for this resistance to ischemic and reperfusion injury are discussed.

Animals↗

PubMed1995

Impaired vasoreactivity to nitric oxide in experimental diabetic neuropathy.

Nerve blood flow (NBF) is reduced in experimental diabetic neuropathy (EDN), but the mechanism of its reduction is uncertain. We tested the hypothesis that reduced NBF might be due to alterations of nitric oxide synthase (NOS) and endothelin of microvascular endothelial cells of sciatic nerve. We evaluated epineurial arteriolar vasoreactivity in response to superfused test agents. NBF was measured using microelectrode H2 polarography. Vasoconstrictor responses to endothelin-1 (ET-1; 10(-6), 10(-7), 10(-8), 10(-9), 10(-10) M) showed dose-response curves with similar EC50 values, indicating no change in potency. We applied the NOS inhibitor NG-nitro-L-arginine and observed reduced inhibition of NBF in EDN, correctable with insulin treatment and also with infused L-arginine. We conclude that vasoreactivity is disturbed in EDN, and is due to a combination of an impairment of NOS activity with reduced NO and increased endothelin effect (normal receptor sensitivity and increased plasma values) in EDN. Hyperglycemia is likely to be the mechanism of NOS inhibition since insulin treatment reversed this abnormality.

Amino Acid Oxidoreductases↗

PubMed1984

ATP-MgCl2 produces sustained improvement in hepatic mitochondrial function and blood flow after hepatic ischemia.

Recent studies have shown that infusion of ATP-MgCl2 following hepatic ischemia significantly improved mitochondrial function and hepatic blood flow 1 hr after treatment. To determine if the improvement in the above parameters by ATP-MgCl2 is short-lived or whether it persists for prolonged periods of time after treatment, hepatic ischemia in rats was produced for 90 min followed by reperfusion. The rats then received iv 0.5 ml of saline or ATP-MgCl2 (12.5 mumole each). Twenty-four hours after reflow, hepatic blood flow was measured by H2 polarography following which the animals were sacrificed and hepatic mitochondria isolated. The results indicated that 24 hr after reflow, mitochondrial state 3 respiration, respiratory control ratio, adenine nucleotide translocase activity, ATP synthetic activity, and hepatic blood flow were depressed by approximately 50% in animals which were treated with saline after hepatic ischemia. In addition, there was a fourfold increase in mitochondrial free fatty acid levels of such animals. Animals which were treated with ATP-MgCl2 following hepatic ischemia showed significantly improved mitochondrial function (used as an index of cellular recovery) and hepatic blood flow. These results in conjunction with previous results suggest that infused ATP-MgCl2 improves mitochondrial function and blood flow and that these effects persist even 24 hr after administration of ATP-MgCl2. Thus, infusion of ATP-MgCl2 following severe ischemia produces sustained improvement in cellular function.

Adenosine Triphosphate↗

PubMed1977

Blood flow in peripheral nerves. Normal and post severance flow rates.

Regional blood flow has been measured in the sciatic nerve of cats utilizing the method of hydrogen polarography. The mean baseline flow of the sciatic nerve in cats was found to be 43 ml/100 g/min +/- 1.6 SD. Flow rates were somewhat erratic in both the proximal and distal segments of the nerve following nerve severance. The erratic flows were observed over the first 30 min following nerve cutting and by 1 hr had essentially stabilized at approximately the pre-severance flow level.

Animals↗