Maria Touraki

Bioaccumulation of trimethoprim, sulfamethoxazole and N-acetyl-sulfamethoxazole in Artemia nauplii and residual kinetics in seabass larvae after repeated oral dosing of medicated nauplii

Abstract The pharmacokinetics of trimethoprim (TMP), sulfamethoxazole (SMX) and its metabolite N-acetyl-sulfamethoxazole (N-acetyl-SMX), were studied in Artemia nauplii as a function of the duration and temperature of their storage, following their enrichment with the therapeutics using the bioencapsulation technique. A marked decrease in the therapeutic content of the nauplii was observed upon storage at 18°C and 25°C and it was concluded that medicated nauplii should either be administered fresh to fish larvae or after storage for 8 h at 5°C, at the most. Under the latter conditions, satisfactory levels of TMP and SMX were achieved, high survival rates and dry weight contents of the nauplii were preserved and minimal leakage of the therapeutics to the environment was secured. Treatment of seabass larvae with one, three, six or ten doses of medicated Artemia nauplii, showed that maximum levels of the therapeutics are achieved in fish larvae when 10 doses are used. Following the treatment scheme of oral administration of 10 doses of medicated Artemia nauplii to fish larvae, the residual kinetics of TMP, SMX and N-acetyl-sulfamethoxazole were studied in seabass larvae. TMP and SMX showed different kinetic characteristics. A steady state of SMX concentration is considered to be achieved in fish body tissue during the 5-day medication period. TMP, SMX as well as the metabolite N-acetyl-sulfamethoxazole, were detectable in small amounts in fish body tissue even 100 h-post treatment. These data suggest that oral medication of fish larvae through the use of Artemia nauplii as a carrier of therapeutics, appears to be a quite promising approach to be used as an alternative method of treatment, which could minimize some of the problems arising from the methods currently in use.

Liposome mediated delivery of water soluble antibiotics to the larvae of aquatic animals

Abstract Large phospholipid vesicles loaded with the water soluble antibiotic oxytetracycline were formed using the reverse phase evaporation technique. Addition of cholesterol in the lipid phase and suspension of the liposomes in low pH solutions, greatly improved stability of the liposomes upon storage. Liposomised oxytetracycline was administered to Anemia franciscana nauplii using the bioencapsulation technique. Survival and larval development of the nauplii was not affected by the presence of liposomes in the enrichment medium. Determination of oxytetracycline levels in the liposomal preparations and in the nauplii, using reverse phase high performance liquid chromatography, showed significant amounts of antibiotic in the nauplii. The uptake of the liposomised antibiotic by the nauplii depended on the amount of the administered antibiotic and the duration of the enrichment period.

Purification of cytosolic glutathione transferases from Schistocephalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation

Glutathione (GSH) transferase isoenzymes have been partially resolved from the cytosol of Schistocephalus solidus (plerocercoid) by GSH affinity chromatography and chromatofocusing at pH 7-5. The presence of isomeric forms was also suggested by analytical isoelectric focusing and high-performance liquid chromatography (HPLC). Gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that GSH transferase forms were dimers with a subunit size of approximately 24 kDa. The major GSH transferase form in S. solidus (plerocercoid) showed greater biochemical relationship to the Mu family of mammalian GSH transferase compared to the mammalian Alpha or Pi families. The major subunit purified by GSH affinity chromatography and reversed-phase HPLC also showed high N-terminal homology with the Mu family. A minor GSH transferase form appeared more biochemically related to the Alpha family with respect to substrate specificity and inhibitor sensitivity. The major GSH transferase was inhibited by haematin-related compounds, bile acids and a number of anthelmintics including members of the benzimidazole and phenol-based class of compounds. The major GSH transferase had conjugating activity with members of the trans, trans-2,4-alkadienal and trans-2-alkenal series, secondary products of lipid peroxidation.

Bioencapsulation of chemotherapeutics in Artemia as a means of prevention and treatment of infectious diseases of marine fish fry

The ability of Artemia nauplii to act as carriers of the chemotherapeutics trimethoprim and sulfamethoxazole for the treatment of infectious diseases of fish fry was studied. Both drugs accumulate in the nauplii as a function of time and their doses in the enrichment medium, but in different ratios, indicating different accumulation kinetics of the two drugs. The levels of the therapeutics in the nauplii remain fairly high even 8 hr post enrichment, decreasing thereafter mostly due to their discharge into their surrounding medium. However, the amounts of the discharged therapeutics do not account for their decrease in the nauplii. This, along with the appearance of certain minor peaks observed in HPLC, may well indicate that a metabolic alteration of the therapeutics occurs in the nauplii. A time-course quantification of the two drugs in the gut and the body of the fish larvae showed that they follow different kinetic patterns during their transfer from the digestive tract to the rest of the body. The use of the bioencapsulation technique as means of prevention or therapy was tested on Vibrio anguillarum-challenged sea bass larvae (Dicentrarchus labrax). Highly significant improvement (p < 0·002) in survival was observed when administration of medicated Artemia to fish larvae preceded (S = 81·4% ± 6·2%) or followed (S = 81·2% ± 4·4%) the bacterial challenge, as compared to the challenged-non-treated fish larvae (S = 65·0 ± 7·0%).

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C18 cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol-0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2-50 microg/g (r2=0.9998) and for flumequine in the range of 0.3-50 microg/g (r2=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 microg/g for oxolinic acid and 0.3 microg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.

Determination of oxytetracycline in the live fish feed Artemia using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic analytical method was developed for the determination of oxytetracycline in Artemia nauplii. A solid-phase extraction protocol was used to recover oxytetracycline and the internal standard tetracycline, from the Artemia samples. Oxytetracycline was analyzed using a 150 x 4.6 mm I.D. Hypersil-ODS column, a mobile phase of acetonitrile-tetrahydrofuran-0.01 M oxalic acid buffer (pH 3.0) (15:3:82, v/v), and an ultraviolet detection wavelength of 365 nm. The calibration curve of oxytetracycline in Artemia was linear (r2 = 0.9998) from 0.1 to 6.4 micrograms/g of tissue. Using a signal-to-noise ratio of 4:1 the oxytetracycline detection limit was 10 ng/g of tissue. Mean recovery of oxytetracycline amounted to 97%, while intra-assay variability was 1.5%. Quantitative data from an in-vivo feeding study indicated an excellent uptake of oxytetracycline by Artemia, as its levels reached 25.6 micrograms per g of nauplii.

Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic, ionic, contractile and electrophysiological studies

SummaryThe effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37°C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22°C and 27°C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37°C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24±2.49 IU·g dry wt-1, and the total amount of protein lost was 70.37±0.73 mg·g dry wt−1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37°C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.

Treatment of vibriosis in European sea bass larvae, Dicentrarchus labrax L., with oxolinic acid administered by bath or through medicated nauplii of Artemia franciscana (Kellogg): efficacy and residual kinetics

European sea bass larvae were challenged by bath with Listonella anguillarum strain 332A, 2.5×10(7) CFUmL(-1) for 1h. Fish either received no treatment or oral treatment with Artemia franciscana (Kellog) nauplii enriched with oxolinic acid, or bath treatments with oxolinic acid. Medication commenced 1day following challenge and was performed on days 1, 3 and 5 post-challenge at a dosage of 20mgL(-1) for 2h for bath treatments, while two doses each of 750 nauplii per fish were administered daily for five consecutive days in oral treatments. Cumulative mortality reached 96% for the unmedicated challenged group, 32% in the group receiving bath treatments and 17% in the group receiving medicated nauplii. Pharmacokinetic parameters of oxolinic acid were calculated in sea bass larvae, for both treatments. Steady-state concentrations of oxolinic acid of 48.0 and 75.2μgg(-1) were achieved for bath treatment and oral treatment, respectively, while the elimination half-life was calculated to be 25.1h for bath treatment and 21.7h for oral treatment.

ALTERATIONS IN THE ENERGY METABOLISM OF THE ISOLATED PERFUSED FROG HEART DURING CALCIUM DEPLETION AND SUBSEQUENT REPLETION

SummaryThe changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, a-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism.

Defense systems in developing Artemia franciscana nauplii and their modulation by probiotic bacteria offer protection against a Vibrio anguillarum challenge

Abstract The alterations of immune responses of Artemia franciscana nauplii as a function of culture time and after a challenge with the pathogen Vibrio anguillarum were studied. The effect of the administration of the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis either alone or in combination with the pathogen was evaluated. The activity of the antioxidant enzymes superoxide dismutase (SOD), Glutathione reductase (GRed), Glutathione transferase (GST) and Phenoloxidase (PO) presented a significant increase as a function of culture time, appeared elevated following probiotic administration and were depleted 48 h following the experimental challenge. Lipid peroxidation reached peak levels at 48 h of culture, when nauplii start feeding and returned to lower values at 144 h, remaining however significantly higher than control (P < 0.05). The three probiotics significantly reduced lipid peroxidation in comparison with the corresponding control, while challenge with the pathogen resulted in its threefold increase. Survival of nauplii remained high throughout culture and was either increased or remained at control levels following the administration of the probiotics. The challenge with the pathogen resulted in a significantly decreased survival of 15.3% for the positive control, while in the probiotic treated series survival values were not significantly different from the negative control (P > 0.05). Following a combined administration of each probiotic and the pathogen the activities of all enzymes tested were significantly lower (P < 0.001) than the negative control (no treatment), but higher than the positive control (challenge, no probiotic). Lipid peroxidation was significantly lower in the probiotic treated series in comparison to the positive control (P < 0.001). The results of the present study provide evidence that major alterations take place as a function of culture time of Artemia nauplii. In addition the pathogen induces an oxidative stress response. The probiotics B. subtilis, L. plantarum and L. lactis protect Artemia against a V. anguillarum challenge by enhancing its immune responses thus contributing to reduced oxidative damage and increased survival. Graphical abstract Figure. No Caption available. HighlightsSOD, GRed, GST and PO activity significantly increased with culture time of Artemia.Lipid peroxidation presented a peak at 48 h, returning to lower values at 144 h.Probiotics led to increased antioxidant enzymes activity, reduced lipid peroxidation and increased survival of Artemia.Experimental challenge of Artemia with V. anguillarum resulted in an oxidative stress response.Administration of probiotic and pathogen led to Artemia redox status and survival rates similar to control.