Zinc complexes of the antibacterial drug oxolinic acid: Structure and DNA-binding properties

The neutral mononuclear zinc complexes with the quinolone antibacterial drug oxolinic acid in the absence or presence of a nitrogen donor heterocyclic ligand 2,2′-bipyridine or 1,10-phenanthroline have been synthesized and characterized. The experimental data suggest that oxolinic acid is on deprotonated mode acting as a bidentate ligand coordinated to the metal ion through the ketone and one carboxylato oxygen atoms. The crystal structures of (chloro)(oxolinato)(2,2′-bipyridine)zinc(II), 2, and bis(oxolinato)(1,10-phenanthroline)zinc(II), 3, have been determined with X-ray crystallography. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the DNA-binding constants have been calculated. Competitive studies with ethidium bromide (EB) have shown that complex 3 exhibits the ability to displace the DNA-bound EB indicating that it binds to DNA in strong competition with EB.     

Immunomodulatory effects of nisin in turbot (Scophthalmus maximus L.)

In the present work, the effect of nisin on the non-specific immune response of turbot (Scophthalmus maximus L.) leukocytes has been studied both in vitro and in vivo. The head kidney macrophage chemiluminescent (CL) response was significantly increased with intermediate doses of nisin (2.5 and 0.025 micro g ml(-1)) whilst the higher dose (25 micro g ml(-1)) significantly decreased the response after 24h incubation. When the incubation time was extended to 72 h, significant differences between doses were observed and the lower nisin concentration (0.025 micro g ml(-1)) appeared to be the optimum dose for increasing the CL response. The phagocytic activity of HK macrophages was also affected by in vitro nisin treatments. Nisin at 0.25 micro g ml(-1) and 0.025 micro g ml(-1) significantly stimulated the response after 24 and 72 h incubation respectively. Nitric oxide (NO) production by HK macrophages was not influenced by any nisin concentration employed for 24 or 72 h incubationsIn vivo, one week post injection, a slightly but non-significant stimulation of the CL response was observed with the lowest nisin concentration (0.0025 micro g fish(-1)). NO in serum and serum antibacterial index were not significantly affected by nisin treatments. On the other hand, lysozyme concentration in serum was significantly augmented with the lowest nisin dose (0.0025 micro g fish(-1)).The antibacterial effect of nisin against the fish pathogenic bacteria Carnobacterium piscicola (CECT 4020) was also demonstrated in vitro.     

Evaluation of chromosomal damage by flow cytometry in turbot (Scophthalmus maximus L.) exposed to fuel oil

Flatfishes, turbots (Scophthalmus maximus), were injected intraperitoneally with two doses of fuel oil number 2. Biliary metabolites were evaluated by fixed fluorescence to verify the efficiency of intoxication. Ethoxyresorufin-O-deethylase (EROD) activity was compared with chromosomal damage measured by flow cytometry. The analysis of biliary metabolites showed a good dose-response relation and constitutes a clear reference for the subsequent measurements. Comparing flow cytometry and EROD results, a shorter delay of response for EROD activity was obtained, but chromosomal damage was significant only after 1 week. The persistence of the EROD response was shorter, while the genotoxic signal still persisted after 1 month. The measurement of chromosomal damage allowed a good differentiation between the two tested doses. In the case of EROD activity, the results were less clear. The results suggest that within a few weeks after exposure to fuel oil number 2, the measurements of chromosomal damage by flow cytometry can be used to detect a dose-dependant genotoxic response in fish.     

High-performance liquid chromatographic method with fluorescence detection for the screening and quantification of oxolinic acid,flumequine and sarafloxacin in fish

A previously published liquid chromatographic method for determining residues of nine quinolones in chicken, porcine, bovine and ovine muscle was adapted and applied to fish tissue for simultaneous determination of three quinolones (flumequine, oxolinic acid and sarafloxacin). The analytes were extracted from homogenised muscle using an acetonitrile basic solution. After centrifugation, partial evaporation and cleaning with hexane, direct injection was possible. Separation was achieved on PLRP-S column and detection was performed with a programmable fluorescence detector. Chromatographic conditions were optimised to be compatible with the determination of the three quinolones in a single run. The linearity, recovery, accuracy and precision of the method were evaluated from fortified tissue samples at concentration levels ranging from 15 to 120 microg kg(-1) for sarafloxacin and 75 to 600 microg kg(-1) for oxolinic acid and flumequine according to the EU maximum residue limit of each quinolone. The limits of detection were estimated to be 2, 5 and 7 microg kg(-1), respectively, for sarafloxacin, oxolinic acid and flumequine. The limits of quantification were validated at 15 microg kg(-1) for sarafloxacin and 75 microg kg(-1) for oxolinic acid and flumequine. Mean extraction recoveries of quinolones in fish ranged from 56.9 to 71.0%. This simple and rapid method is suitable for residue control.     

The conversion of polyunsaturated fatty acids to prostaglandins by tissue homogenates of the turbot,Scophthalmus maximus (L.)

The conversion of [¹⁴C]20:4(n?6) and [¹⁴C]20:5(n?3) to prostaglandins was measured in homogenates of gills, liver and intestine of the turbot, Scophthalmus maximus (L.). Incorporation of radioactivity into prostaglandins was similar to or greater than into phospholipids, with gills being more active than liver or intestine. When measured at equimolar concentrations, 20:4(n?6) was a better precursor of prostaglandins than 20:5(n?3). Incorporation of 20:4(n?6) into prostaglandins was decreased in the presence of an equimolar concentration of 20:5(n?3), and vice versa. Incorporation of both precursors into prostaglandins was partially inhibited by aspirin and indomethacin. The results are discussed in relation to prostaglandin formation in fish and the abundance of (n?3) polyunsaturated fatty acids in marine lipids generally.     

Nickel-quinolones interaction. Part 2 - Interaction of nickel(II) with the antibacterial drug oxolinic acid

The mononuclear nickel(II) complexes with the first-generation quinolone antibacterial agent oxolinic acid in the presence or absence of nitrogen-donor heterocyclic ligands (2,2′-bipyridine, 1,10-phenanthroline or pyridine) have been synthesized and characterized. The experimental data suggest that oxolinic acid acts as deprotonated bidentate ligand coordinated to Ni(II) ion through the ketone and carboxylato oxygens. The crystal structure of (2,2′-bipyridine)bis(oxolinato) nickel(II), 2 has been determined by X-ray crystallography. The cyclic voltammograms of the complexes recorded in dmso solution and in 1/2 dmso/buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) solution have shown that in the presence of calf-thymus DNA (CT DNA) they can bind to CT DNA by the intercalative binding mode. UV study of the interaction of the complexes with CT DNA has shown that the complexes bind to CT DNA and bis(aqua)bis(oxolinato) nickel(II) exhibits the highest binding constant to CT DNA. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values.     

Morphological,immunohistochemical and ultrastructural characterization of the skin of turbot (Psetta maxima L.)

This study was undertaken to identify the normal morphologic, immunohistochemical and ultrastructural features of skin of the turbot (Psetta maxima L.). In the turbot skin, three morphologically distinct layers were identified: epidermis, dermis and hypodermis. The epidermis was non-keratinizing, stratified squamous epithelium that varies in thickness from 5 to 14 cells and 60 to 100 μm in size. Goblet cells were seen randomly distributed between malpighian cells in the epidermal layer. These mucous cells were mainly located in the upper third of the epidermis and displayed a spherical to elongated morphology. Dermis was divided in two well-differentiated layers, the superficial stratum laxum and the deeper stratum compactum. Hypodermis was a loose layer mainly composed by adipocytes but we could observe variable amounts of fibroblast, collagen and blood vessels. In turbot two pigmentary layers could be identified: the pigmentary layer of dermis was located between basement membrane and dermis and the pigmentary layer of hypodermis immediately above the muscular layer. Three different types of chromatophores were present: melanophores, iridophores and xanthophores. The main differences observed between groups of fish with different colouration were in the amount of melanophores and xanthophores. The purpose of this article is to provide an overview of normal cutaneous biology prior to consideration of specific cutaneous alterations and diseases in turbot.     

Genotoxic evaluation of the GH transgenic Synechocystis using mice and turbot (Scophthalmus maximus L.)

In the present paper, the genotoxicity of transgenic Synechocystis sp. PCC 6803 containing flounder (Paralichthys olivaceus) growth hormone gene has been assessed using micronucleus tests in mice and fish. No micronucleus inductions in bone marrow erythrocyte of mice were observed at three doses of transgenic Synechocystis (2.0, 5.0, and 10.0g/kg). The frequencies of micronucleus and other nuclear abnormalities in turbot (Scophthalmus maximus L.) fed with 0.2%, 0.5% and 1.0% transgenic Synechocystis were also not significantly different (P>0.05) from controls. In contrast, cyclophosphamide, used in detecting the sensitivity of the biological assays, significantly increased the frequencies of micronucleus and other nuclear abnormalities in mice and turbot. Results indicate that the flounder GH transgenic Synechocystis sp. PCC 6803 seems to have no genotoxic effects on fish.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Gene diversity analysis in natural populations and cultured stocks of turbot (Scophthalmus maximus L.)

The genetic variability of eight fish-farm and three natural populations of turbot was studied by electrophoretic analysis of 35 enzymatic loci. The results showed low genetic variability in natural populations of turbot ( H _T = 0·029 ± 0·013) in comparison with other flatfish species. Great genetic similarity was revealed among the natural populations studied, which indicates high rates of gene flow in this species. The hatchery stocks showed less genetic variation than the wild populations analysed, which suggests genetic drift phenomena involved in the foundation and management of broodstocks. In addition, the heterozygosity differences detected among the hatchery stocks analysed are correlated with inverted levels of fluctuating asymmetry, which supports the existence of inbreeding depression phenomena in turbot culture.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Bothriocephalus scorpii (Müller) (Cestoda: Pseudophyllidea) in turbot Scophthalmus maximus (L.) and brill S. rhombus (L.) from the southern North Sea

Turbot (11–70 cm long) and brill (21–70 cm long) were collected in the summer of 1968 north of the Brown Bank, Texel Hole and were infected by Bothriocephalus scorpii. Infections occurred even in the first year of life of the fish. In turbot, with increasing length, the average weight of tapeworm rose from 0–6 to 14-9 g/flsh. Brill were less commonly infected and then only slightly. The intermediate host of the tapeworm seems to be Gobius species, upon which juvenile turbot and brill prey most.     

Immune response of turbot, Psetta maxima (L.) (Pisces: Teleostei), to formalin-killed scuticociliates (Ciliophora) and adjuvanted formulations

The increasing frequency of scuticociliatosis in turbot culture has stressed the need of knowledge on the immune responses to these parasites, for further developing of prevention and control strategies. The immune response of turbot to killed parasites, alone (Ag) or in combination with Montanide ISA 763A (MON), was studied in a laboratory-scale experiment. The variations of several innate immune factors and the antibody response were analysed in immunized vs. non-immunized fish at different times after immunization, and also after a challenge with live ciliates. Amongst innate immune factors, serum lysozyme increased progressively in all inoculated groups. Differences in innate immune factors in Ag and Ag-MON fish with respect to controls were mainly evidenced after challenge, especially for serum complement. Serum antibody levels increased in immunized fish after booster and particularly after challenge. In addition, certain protection was obtained for immunized groups compared to controls or to fish receiving MON alone, and the levels of specific antibodies were also the highest in immunized groups. The obtained information could be useful for further design of immunoprophylactic formulations against scuticociliatosis.     

The effect of early exposure to Vibrio pelagius on the aerobic bacterial flora of turbot, Scophthalmus maximus (L.) larvae

Vibrio pelagius was added to filtered sea water in experimental tanks containing newly-hatched larvae of Scophthalmus maximus. The bacterial load of larvae increased from day 1 post-hatch and by day 14 had reached 5 × 10⁴ bacteria per larva. Vibrio pelagius dominated the aerobic bacterial flora of larvae exposed to this bacterial species but was not detected in larvae not exposed to exogenous bacteria. An enzyme-linked immunosorbent assay using rabbit antiserum against V. pelagius allowed a specific quantitative assay for homologous bacterial antigens in individual larvae, with very little cross-reaction against heterologous bacteria. The results of enzyme-linked immunosorbent assays of V. pelagius antigens in larvae from tank water inoculated with this bacterial species correlated with the bacterial levels found in the larvae by culture on agar plates.     

Phytoremediation of Cr(VI) by Spirodela polyrrhiza (L.) Schleiden employing reducing and chelating agents

Phytoremediation of Cr(VI) by Spirodela polyrrhiza in binary combinations with low molecular weight organic compounds (LMWOCs) with a reducing or chelating potential, viz., ascorbic acid, citric acid, tartaric acid, oxalic acid, lactic acid, and glycerol was studied in Cr(VI) containing hydroponic media. Significant increase in the relative dry weight of plants with respect to Cr(VI) treated controls was observed with ascorbic acid and glycerol. The uptake of chromium by S. polyrrhiza followed Michaelis-Menten kinetics of active ion uptake. Interaction between Cr and ascorbic acid, oxalic acid, and lactic acid decreased Cr uptake, whereas citric acid, glycerol, and tartaric acid increased it. Supplementation of LMWOCs to Cr(VI) containing media decreased the MDA content of the plants. Multiple regression models revealed that LMWOCs decrease lipid peroxidation independently, as well as that induced by Cr(VI). It was found that superoxide dismutase (SOD), guaiacol peroxidase (GPX), and catalase (CAT) activities were increased significantly in plants growing in media containing Cr(VI). The study established that lactic acid, citric acid, ascorbic acid, and glycerol were most effective in increasing the Cr(VI) phytoremediating potential of S. polyrrhiza and LMWOCs with reducing or chelating properties decrease Cr(VI) stress in S. polyrrhiza.     

The antimicrobial activity of Augmentin (amoxicillin/clavulanic acid) compared to other antibacterial agents]

Sensitivity of clinical strains of gram-positive and gram-negative bacteria to Augmentin was studied in comparison to other antibiotics. Augmentin was shown to be advantageous in the level and spectrum of its antibacterial activity over ampicillin and other broad-spectrum antibiotics.