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PubMed2006

Importance of metallothioneins in the cadmium detoxification process in Daphnia magna.

Good knowledge of the relationship between toxic metals and biological systems, particularly the sub-cellular fraction, could be a suitable early indicator of toxic effects. These effects and the sub-cellular behaviour of cadmium were studied with a widely used species in freshwater toxicity bioassays, Daphnia magna. In spite of this very commonplace usage in ecotoxicological studies, very few data are available on its toxicant metabolism and in particular metal homeostasis. Combining multi-tools analysis, a soluble protein was found: it is heat-stable, rich in sulfhydryl groups (differential pulse polarography), characterised by a molecular mass of approximately 6.5 kDa, with a G-75 chromatographic profile corresponding to the rabbit metallothioneins monomer, with few if any aromatic-containing amino acids, it binds metals (e.g. Cd, Cu), and its concentration increases with Cd exposure. This evidence led us to hypothesise that metallothioneins (MTs) are present in D. magna. Up to 75% of the Cd body burden with Cd exposure is bound to the MTs fraction. The increase in the Cd concentration in the surrounding medium and concomitantly in daphnids induces sub-cellular reorganisation of essential metals such as Cu and Zn. The rate of metals in the soluble cellular fraction and associated with MTs increases with the Cd body burden. Monitoring sub-cellular distribution of metals after exposure in the natural environment could be very useful for ecotoxicological assessment.

Animals↗

PubMed2004

Comparison of the complexation potential of extracellular polymeric substances (EPS), extracted from activated sludges and produced by pure bacteria strains, for cadmium, lead and nickel.

This paper provides information on the metal complexation potential of extracellular polymeric substances (EPS), extracted from activated sludges and from eight pure cultures of bacteria isolated from the same activated sludge. The EPS extracted from pure bacteria cultures are mainly composed of proteins and low quantities of polysaccharides and uronic acids in comparison with EPS extracted from activated sludges. The EPS studied present two apparent pK(a) and the IR spectra show the presence of the same functional groups on all the EPS studied. The ability of EPS to complex Cd, Pb and Ni, was studied at pH 7 with Chau and Ruzic's models using polarography titration. All of the EPS exhibited a greater ability to complex Pb than Ni, Cd showing the weakest affinity overall. The EPS extracted from the pure cultures of bacteria were less able to complex the metals than that extracted from activated sludges. Literature data, IR data and EPS phosphorous content, supported by the EPS pK(a), revealed that carboxylic and phosphoric groups may play a major role in binding to metals at pH 7. This study underlines the importance of metal exposure in order for bacteria to secrete or modify EPS. After exposure, the EPS then exhibit the greatest capacity to bind metal in order to protect bacteria from harmful effects of heavy metals.

Biopolymers↗

PubMed2006

Influence of humic substances on the photolysis of aqueous pesticide residues.

The present work investigated the direct and indirect photolysis of pesticide residues (atrazine, imazaquin, iprodione), in aqueous solutions and under UV-visible radiation (280-480nm). Different kinds of humic substances (HS) were added to samples in order to evaluate their behaviour as possible photocatalysts and their effect on the photolysis of pesticides. The fulvic acids were purchased from the International Humic Substances Society, and they were added to samples in concentrations ranging from 1 to 150 mgl(-1). Titanium dioxide was used as the photocatalyst, in concentration ranging from 10 to 150 mgl(-1). Pesticides photolysis were measured by UV-visible absorption spectroscopy and differential pulse polarography with all used pesticides, reaching total degradation after 2h of irradiation, thus indicating a fast direct photolysis. Photocatalysis by TiO(2) could increase the pesticides photolysis rate up to 40%. This effect, however, was not observed for imazaquin photolysis. Again, except for imazaquin, HS presence showed a positive effect in increasing pesticide degradation, but only within specific concentration ranges (below 10mg l(-1) for iprodione and about 30mgl(-1) for atrazine). Above these ranges HS induce a decrease in the pesticides photolysis rate. Spin-trapping measurements by electronic paramagnetic resonance spectroscopy, using the spin-trap DMPO, showed that HS are able to photogenerate hydroxyl radicals, increasing the pesticides molecule degradation. However, the HS also react with the photogenerated hydroxyl radical, influencing the pesticide photolysis, leading to a decrease in the photolysis rate and causing it to be strongly dependent on the nature and concentration of residues in the water to be treated.

Aminoimidazole Carboxamide↗

PubMed2005

Distribution of trace elements in tissues of two shrimp species from the Persian Gulf and roles of metallothionein in their redistribution.

This study investigates the distribution of three trace elements (Cd, Cu and Zn) in tissues (exoskeleton, abdominal muscle and hepatopancreas) of two shrimp species (Penaeus merguiensis and Penaeus semisulcatus). Special emphasis in this study was placed on evaluating the effects of storage conditions on redistribution of the elements and moreover the potential roles of metallothionein in tissue redistribution. The specimens were collected from the northern part of Persian Gulf during September 2001 and January 2002. The concentrations of metals in the tissues were measured using an Inductively Coupled Plasma-Atomic Emission Spectrophotometer (ICP-AES) and an ICP-Mass Spectrometer (ICP-MS). Metallothionein levels were determined using Differential pulse polarography. Results show a similar hierarchy for Cd and Cu concentrations among different tissues as follows: hepatopancreas>exoskeleton>muscle; and for Zn, the hepatopancreas again contained the highest levels, though there were not any significant differences in Zn concentrations between muscle and exoskeleton. Significant differences were found between the species in the case of Cd and Zn concentrations. Cd and Zn levels in P. semisulcatus were markedly higher than in P. merguiensis. In general, there were highly significant differences among the storage conditions from accumulation the selected trace elements and metallothionein in both species point of view, but in most cases, no significant differences could be observed among the fresh, refrigerated and frozen specimens of P. semisulcatus. In regards to Zn and Cu accumulation in edible (muscle) tissues of P. merguiensis, optimum conditions for frozen storage is -30 degrees C as ice-frozen, and in terms of short-term storage, 1-day refrigeration is recommended. The redistribution of Zn and Cu during the storage period can be attributed to the role of metallothionein in their transport from organ(s) to muscle during storage. The variations in pH values measured in the muscle of P. merguiensis in different storage conditions showed a similar trend with metallothionein, Zn and Cu. In contrast, it appears that cadmium is transported from muscle to the other tissues. Exoskeleton is probably one of the recipient tissues of cadmium. The inter-elemental relationships were assessed for each species, separately. A significant positive relationship was observed between the concentrations of Zn and metallothionein in P. semisulcatus, whereas in P. merguiensis, Cd concentrations decreased significantly with increasing metallothionein. Highly significant differences between the sexes in Cu and Zn contents could be detected. The differences between the sexes in the levels of Zn and Cu were significantly greater in P. semisulcatus. Highly significant size-related differences were found in the levels of Cd, Cu and metallothionein in the selected tissues of the shrimp species. Comparison between the mean concentrations of the elements in muscle tissue and existing guidelines indicate that in nearly all cases, the concentrations were well below permissible levels for human consumption. The only exception was observed regarding mean values of Cd, which was somewhat greater than some of the recommended levels.

Animals↗

PubMed2006

Seasonal and spatial distributions of tributyltin in surface sediment of the Tolka Estuary, Dublin, Ireland.

Aquatic pollution by tributyltin (TBT) is of concern because of its high toxicity to marine life. TBT loadings of estuarine sediments may remain elevated because of long flushing times in estuaries due to their enclosed physicality. The Tolka Estuary, Co. Dublin, Ireland, is a typical Irish urban estuary. It has a significant pollution loading. The results of 12 months' analysis of TBT spatial and temporal distributions in this estuary are presented in this paper. Data from a total of 10 sample points are presented. The concentrations of TBT in the sediment were analysed using differential pulse polarography. Significant seasonal and spatial trends in TBT distribution were observed over the 12-month period. TBT concentrations ranged from 0.1 ppb in winter to 8.6 ppb in summer with a 1.5 ppb average. Organic matter (OM) concentrations of sediment exhibited similar seasonal trends and a positive correlation between OM and TBT distributions was observed.

Environmental Monitoring↗

PubMed2005

Effect of 4-hydroxynonenal on phosphatidylethanolamine containing condensed monolayer and on its interaction with apolipoprotein A-I.

4-Hydroxynonenal (4HNE), generated during polyunsaturated fatty acid oxidation, is present in atherosclerotic lesions. As 4HNE is able to react with phosphatidylethanolamine (PE), we investigated, using AC polarography, whether it may alter the physico-chemical state of a condensed PE-containing phospholipid monolayer and its interaction with apoA-I. The stability of a phospholipid monolayer relative to potential (around the potential of zero charge) is dependent on lipid composition (PE>PC>PE/PC). ApoA-I insertion into PE/PC monolayer is easier than in PC monolayer. Pre-treatment of PE/PC monolayer by 4HNE does not alter monolayer stability, but decreases apo A-I insertion into the monolayer.

Aldehydes↗

PubMed2004

Voltammetric behavior and quantification of the sedative-hypnotic drug chlordiazepoxide in bulk form, pharmaceutical formulation and human serum at a mercury electrode.

Chlordiazepoxide is a sedative-hypnotic drug widely employed as a transquilizer and anti-depressant. Its electrochemical behavior in Britton-Robinson (B-R) buffers of pH 2-11 at a mercury electrode has been investigated using dc-polarography, cyclic voltammetry and controlled-potential coulometry. Polarograms of the drug in B-R buffers of pH 2-10 exhibited three 2-electron waves, while at pH>10, only a single 4-electron wave was observed. The first, second, and third waves in buffers of pH 10) may be due to the reduction of both the N-oxide and C=N centers in a one step. The shift of the E(1/2,) values to more negative potentials upon the increase of pH indicated the involvement of protons in the electrode reaction and that the proton-transfer reaction precedes the electrode process proper. The estimated data indicated that, one proton and two electrons are participated in the rate-determining step of each of the reduced centers. The general sequence of chlordiazepoxide reduction processes via each of its reactant centers may be expressed as: H(+), e, e, H(+)((fast)).Based on the interfacial adsorptive character of the drug onto the mercury electrode, a validated direct square-wave adsorptive cathodic stripping (SWAdCS) voltammetric procedure has been described for the trace determination of the drug in bulk form, tablets and human serum. The procedure did not require sample pretreatment or time-consuming extraction or evaporation steps prior to the assay of the drug. The optimized operational conditions of the proposed procedure have been found to be: accumulation potential E(acc.)=-0.9 V, accumulation time t(acc.)=30s, pulse-amplitude=50 mV, scan increment=10 mV and frequency=120 Hz. The proposed procedure is much more simple, fast, sensitive, costly low and achieved much more lower limits of detection (LOD) (4.4 x 10(-10)M and 6.6 x 10(-10)M) and limits of quantitation (LOQ) (1.5 x 10(-9)M and 2.2 x 10(-9)M), respectively in pharmaceutical formulation and spiked human serum, compared to previously reported methods.

Chemistry, Pharmaceutical↗

PubMed2005

Electrochemical determination of pharmaceuticals in spiked water samples.

The electrochemical behaviour of acidic and neutral pharmaceutical active compounds (PhACs) was studied by cyclic voltammetry and pulse voltammetric techniques on mercury, carbon nanotube paste, carbon paste and gold electrodes. The best results, in terms of sensitivity, linearity range and detection limits, were obtained by differential pulse voltammetry (DPV) for ofloxacin (LOD 5.2 microM), differential pulse polarography (DPP) for clofibric acid (LOD 4.7 microM) and normal pulse voltammetry (NPV) for diclofenac (LOD 0.8 microM) and propranolol (LOD 0.5 microM). An enrichment step of approximately two orders of magnitude was performed by a solid-phase extraction procedure (SPE) in order to concentrate the samples. The developed method was optimized and tested on spiked river water samples.

Clofibric Acid↗

PubMed2006

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: evaluation of cell respiration.

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.

Animals↗

PubMed2006

Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting.

Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.

Animals↗

PubMed2003

Effect of transforming growth factor-beta on calcium homeostasis in prostate carcinoma cells.

Ca(2+) plays a fundamental role in the control of a variety of cellular functions, in particular, in energy metabolism and apoptosis. In this study, we show that TGF-beta at concentrations of 0.1-1.0 ng/ml transiently increases the level of intracellular Ca(2+) ([Ca(2+)](in)) in human prostate carcinoma, PC-3U, cells. Experiments with mitochondrial inhibitors (oligomycin and antimycin A) and an inhibitor of endoplasmic reticulum Ca(2+) uptake (BHQ) implied that the effect of TGF-beta1 was due to an effect on the mitochondria. TGF-beta1 treatment resulted in a decrease in ATP synthesis and to a depolarisation, leading to a release of Ca(2+) from mitochondria and decreased activity of the Ca(2+) pumps. Analysis of the mitochondria within the PC-3U cells by polarography and membrane potential-sensitive dye (Rhodamine 123) confirmed that under these experimental conditions, TGF-beta1 inhibited ATP synthesis and depolarised the mitochondria. The results implicate that TGF-beta1 affects the function of the mitochondria and may be of significance for the understanding of the proapoptotic effect of TGF-beta1 in these cells.

Antimycin A↗

PubMed2000

DNA interactions of antitumor cisplatin analogs containing enantiomeric amine ligands.

Modifications of natural DNA and synthetic oligodeoxyribonucleotide duplexes in a cell-free medium by analogs of antitumor cisplatin containing enantiomeric amine ligands, such as cis-[PtCl(2)(RR-DAB)] and cis-[PtCl(2)(SS-DAB)] (DAB = 2,3-diaminobutane), were studied by various methods of molecular biophysics and biophysical chemistry. These methods include DNA binding studies by pulse polarography and atomic absorption spectrophotometry, mapping of DNA adducts using transcription assay, interstrand cross-linking assay using gel electrophoresis under denaturing conditions, differential scanning calorimetry, chemical probing, and bending and unwinding studies of the duplexes containing single, site-specific cross-link. The major differences resulting from the modification of DNA by the two enantiomers are the thermodynamical destabilization and conformational distortions induced in DNA by the 1,2-d(GpG) intrastrand cross-link. It has been suggested that these differences are associated with a different biological activity of the two enantiomers observed previously. In addition, the results of the present work are also consistent with the view that formation of hydrogen bonds between the carbonyl oxygen of the guanine residues and the "quasi equatorial" hydrogen of the cis amine in the 1, 2-d(GpG) intrastrand cross-link plays an important role in determining the character of the distortion induced in DNA by this lesion.

Amines↗

PubMed2002

Ischemia and failed regeneration in chronic experimental neuromas.

Neuromas are generally considered to be swollen uniform collections of uncontrolled aberrantly sprouting axons. In early experimental neuromas, there are substantial rises in local blood flow associated with their formation, but human studies of chronic lesions have suggested that neuromas develop ischemia and become impediments to regeneration. The issue is important because traumatically severed human nerves are frequently considered for repair some time after injury, when neuroma formation has occurred. In this work, we examined local perfusion, axon penetration and other characteristics of long-term (6 month) experimental neuromas created by sciatic nerve transection and resection of the distal sciatic nerve and its branches. The scenario was designed to model prior transection in a human nerve, where late surgical reconnection might be contemplated. Local blood flow in the extrinsic plexus of neuromas, examined using a laser Doppler flowmetry probe, declined in distal portions of the stump to values considerably lower than observed in intact nerves. Intrinsic blood flow near the stump tip, examined using microelectrode hydrogen clearance polarography was highly nonuniform and included zones with very low perfusion. Correlated with these findings were nonuniform histological features with zones of absent axons and blood vessels, progressive distal disorganization, marked declines in distal axon penetration, nonremodelled microfascicles and persistent expression of 'regenerative' axon and Schwann cell markers. Uncontrolled axon sprouting was not a feature. Longstanding neuromas include zones of relative ischemia and limited axon penetration that develop in the absence of nerve trunk reconnection. These features would limit their suitability for later repair.

Animals↗

PubMed2003

Angiotensin converting enzyme inhibition partially prevents deficits in water maze performance, hippocampal synaptic plasticity and cerebral blood flow in streptozotocin-diabetic rats.

Vascular dysfunction is important in the pathogenesis of peripheral complications of diabetes. However, the effects of diabetes on cerebral blood flow and the role of vascular deficits in the pathogenesis of diabetic encephalopathy are still unknown. The present study examined whether experimental diabetes is associated with reduced cerebral blood flow and whether treatment with enalapril can improve cerebral perfusion and function (blood flow and functional cerebral deficits). Streptozotocin-diabetic rats were treated with the ACE inhibitor enalapril (24 mg/kg) from onset of diabetes. After 14 weeks of diabetes, 12 enalapril treated and 12 untreated diabetic rats, and 12 nondiabetic age-matched control rats were tested in a spatial version of the Morris water maze. After 16 weeks of diabetes, in the same groups, blood flow in the hippocampus and thalamus was measured by hydrogen clearance microelectrode polarography. In a separate study, hippocampal long-term potentiation was measured after 26 weeks of diabetes. Water maze performance and hippocampal long-term potentiation were impaired in diabetic rats. Furthermore, blood flow in diabetic rats was reduced by 30% (P<0.001) in the hippocampus and by 37% (P<0.005) in the thalamus compared to nondiabetic controls. Enalapril treatment significantly improved water maze performance (P<0.05), hippocampal long term potentiation (P<0.05) and hippocampal blood flow (P<0.05). Cerebral perfusion is reduced in diabetic rats compared to controls. Treatment aimed at the vasculature can improve cerebral blood flow, deficits in Morris maze performance and long term potentiation. These findings suggest that vasculopathy plays a role in the development of cerebral dysfunction in diabetic rats.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2001

Influence of nitrovasodilators and cyclooxygenase inhibitors on cerebral vasoreactivity in conscious rabbits.

Since the nitric oxide (NO) and cyclooxygenase pathways have been suggested to have important roles in most vasodilations, our aim was to study the influence of cyclooxygenase inhibitors and nitrovasodilators on cerebrovascular reserve capacity. Corticocerebral blood flow was measured by hydrogen polarography during hypercapnia and acetazolamide stimuli in conscious rabbits. The measurements were repeated in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin as nitric oxide synthase (NOS) and cyclooxygenase inhibitors. The effects of nitroglycerin and isosorbide-5-nitrate were also tested. L-NAME completely, while indomethacin markedly inhibited the hypercapnic corticocerebral blood flow response. Nitroglycerin and isosorbide-5-nitrate significantly attenuated hypercapnia elicited corticocerebral blood flow increase. The different treatments reduced only moderately the acetazolamide-induced corticocerebral blood flow response. These results lend support to the hypothesis that antithrombotic and antiinflammatory medication (cyclooxygenase inhibitors) and nitrovasodilator treatments could interfere with the measurement of cerebrovascular reactivity resulting in underestimation of the cerebrovascular reserve capacity in patients taking these drugs.

Acetazolamide↗

PubMed1998

Effects of cold acclimation and palmitate on energy coupling in duckling skeletal muscle mitochondria.

Gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from 5-week-old cold-acclimated and thermoneutral control ducklings. In vitro respiration (polarography) and ATP synthesis (bioluminescence) were determined at 25 degrees C. Subsarcolemmal mitochondria showed a higher state 4 respiration and lower respiratory control and ADP/O ratio in cold-acclimated than in thermoneutral ducklings. Palmitate decreased the rate of ATP synthesis in both mitochondrial populations to about 30% of maximal but failed to abolish this process even at high concentrations. It is suggested that both expensive ATP synthesis and increased ATP hydrolysis could contribute synergistically to muscle non-shivering thermogenesis in cold-acclimated ducklings.

Acclimatization↗

PubMed2003

Effect of the NAD(P)H oxidase inhibitor, apocynin, on peripheral nerve perfusion and function in diabetic rats.

Upregulation of vascular NAD(P)H oxidase has been considered an important source for elevated levels of reactive oxygen species that contribute to several cardiovascular disease states, including the vascular complications of diabetes mellitus. Previous studies have shown that treatment with antioxidants corrects impaired nerve function and blood flow in diabetic rats. The aim was to assess the degree of involvement of NAD(P)H oxidase in experimental diabetic neuropathy. To this end, after 6 weeks of untreated streptozotocin-diabetes, rats were treated for 2 weeks with the NAD(P)H oxidase, apocynin. Two high doses (15 and 100 mg/kg) were used to ensure that maximal effects were registered. Diabetes caused a 20% reduction in sciatic nerve motor conduction velocity, and a 14% deficit for sensory saphenous nerve. Apocynin treatment corrected these defects by 32% and 48%, respectively: there were no significant differences between the effects of the 2 doses. Sciatic nerve nutritive endoneurial perfusion was measured by hydrogen clearance microelectrode polarography. Blood flow and vascular conductance were 47% and 40% reduced by diabetes, respectively. Both doses of apocynin had similar effects, correcting the blood flow deficit by 31% and conductance by 47%. Thus, the data show that NAD(P)H oxidase contributes to the neurovascular deficits in diabetic rats. While only accounting for part of the elevated reactive oxygen species production in diabetes, this mechanism could provide a novel therapeutic candidate for further investigation in diabetic neuropathy and vasculopathy.

Acetophenones↗

PubMed2000

Aging differentially modifies sensitivity of nerve blood flow to vasocontractile agents (endothelin-1, noradrenaline and angiotensin II) in sciatic nerve.

This study examined the influence of the vaso-constricting agents (noradrenaline, endothelin-1 and angiotensin II) in Sprague-Dawley rats aged 2, 6 and 24 months by evaluating epineurial arteriolar vasoreactivity in response to superfused teat agents. Nerve blood flow (NBF) was measured using microelectrode H2 polarography. In 24-month-old rats, NBF was decreased and vascular resistance (VR) was increased compared with 2- and 6-month-old rats. All of the constricting agents reduced NBF in the 2-, 6- and 24-month groups, however, the effects of the constricting agents reduced significantly with age. These results suggest that during aging, there is a decline of vasoconstrictive responses to noradrenaline, endothelin-1 and angiotensin II in peripheral nerve and that these changes may be due to altered function of receptors.

Aging↗