Lipopolysaccharides isolated from Aeromonas salmonicida and Vibrio anguillarum show quantitative but not qualitative differences in inflammatory outcome in Sparus aurata (Gilthead seabream).

In fish, the defence system recognises pathogenic microorganisms via pathogen recognition receptors (PRRs) that sense particular structures of the pathogens; the so-called pathogen associated molecular patterns (PAMPs) such as bacterial lipopolysaccharides (LPSs). The result of the PAMP-PRR interactions leads to complex and orchestrated immune responses. In this study, Sparus aurata (Gilthead seabream) were intraperitoneally injected with purified lipopolysaccharide (LPS) from Aeromonas salmonicida (As)- and Vibrio anguillarum (Va) (1 mg*Kgfish(-1)), both Gram negative bacteria responsible for vibriosis and furunculosis respectively, therefore causing an impact upon marine fish cultures. Head-kidney, intestine, spleen, liver and blood samples were collected at 3, 6, 12 and 24 h post-injection. Plasma levels of cortisol, prostaglandins and lactate were measured and were significantly increased after As-LPS and Va-LPS treatment. Furthermore, tissue-specific differences of the gene regulatory patterns were evident for each LPS. When monocyte/macrophage cell cultures were challenged with As-LPS and Va-LPS, the pro-inflammatory cytokine mRNA abundances present a similar pattern of response. However, As-LPS always triggered a stronger response concerning TNFα, IL1ß and cyclooxygenase-2 (COX2) mRNA abundance as well as PGE2 levels in the supernatant. Overall, the results indicate that specific LPSs do not activate different pro-inflammatory responses and that the observed gene expression pattern is tissue and concentration dependent.

Effects of Cortisol Administered through Slow-Release Implants on Innate Immune Responses in Rainbow Trout (Oncorhynchus mykiss).

Cortisol is a key hormone in the fish stress response with a well-known ability to regulate several physiological functions, including energy metabolism and the immune system. However, data concerning cortisol effects on fish innate immune system using a more controlled increase in cortisol levels isolated from any other stress related signaling is scarce. The present study describes the effect of doses of cortisol corresponding to acute and chronic levels on the complement and lysozyme activity in plasma of the rainbow trout (Oncorhynchus mykiss). We also evaluated the effects of these cortisol levels (from intraperitoneally implanted hydrocortisone) on the mRNA levels quantified by RT-qPCR of selected key immune-related genes in the liver, head kidney, and spleen. For that purpose, 60 specimens of rainbow trout were divided in to two groups: a control group injected with a coconut oil implant and another group injected with the same implant and cortisol (50 µ g cortisol/g body weight). Our results demonstrate the role of cortisol as a modulator of the innate immune response without the direct contribution of other stress axes. Our results also show a relationship between the complement and lysozyme activity in plasma and mRNA levels in liver, supporting the important role of this organ in producing these immune system proteins after a rise of cortisol in the fish plasma.

Differential expression of the corticosteroid receptors GR1, GR2 and MR in rainbow trout organs with slow release cortisol implants.

The present study describes the transcriptional levels of the corticosteroid receptors (CRs) GR1, GR2 and MR in the different organs of the rainbow trout (Oncorhynchus mykiss) in response to a slow release of cortisol, throughout a 10-day period. We show that after short term (1 day after cortisol implantation), when the plasma levels of cortisol emulate an acute stress, the GR2 and MR expression levels were upregulated in the brain and head kidney tissues. This result reflects the role of these organs as regulators of the stress response. In general, the rest of the organs, especially gills, intestine, liver, muscle and spleen, showed decreased transcriptional levels of GR1, GR2 and MR, along with the highest plasma cortisol levels. At day 5 after cortisol implantation, when cortisol levels emulate a chronic stress, the most affected organs were gills and skin, where an upregulation of the CRs was found. In the recovery period, when cortisol levels were basal (day 10), we still found changes in the transcriptional levels of the CRs in gills, spleen and gonads. The cortisol increase at days 1 and 5 after implantation is accompanied by high plasma glucose concentrations, supporting the role of cortisol on carbohydrate metabolism. However, after 10days of implantation, glucose returned to control levels suggesting a trade-off on the steady state of the metabolic function. We also observed increased hematocrit and hemoglobin at day 1, indicating a cortisol-induced higher metabolic demand involving an increase in oxygen transport efficiency. Our results demonstrate that increased plasma cortisol induced by a slow-release implant of cortisol mimics the overall effects of stress and affects the expression of the three CRs, generating different transcriptional patterns in a time- and organ-specific manner.

Specific gas chromatography-mass spectrometry analytical method for the determination of cyhexatin in animal feed.

The acaricide tricyclohexyltin hydroxide (cyhexatin) was determined in animal feed samples, using gas chromatographic--mass spectrometry in the electron impact mode. Sample extraction and derivatization (converting the analyte to an alkylated derivative) were performed using a tricyclopentyl analogue of this acaricide as internal standard to obtain a better analytical precision.

Effects of L-fenfluramine on rat liver drug-metabolizing enzymes.

The kinetics of l-fenfluramine (l-F) are dose-dependent in man and rats, suggesting self-inhibition of metabolism and saturation of microsomal enzymes. Possible alterations in the oxidative metabolism of model drug substrates were therefore evaluated in rats given l-F orally (12.5 mg/kg). The compound slightly impaired the clearance of antipyrine but had no effect on the kinetics of highly extracted compounds such as lidocaine. l-F did not alter the hepatic content of the main components of the cytochrome P-450 system and did not affect the in vitro metabolism of enzyme activity markers, even after daily doses of 12.5 mg/kg. It was concluded that at doses with dose-dependent behaviour l-F may impair clearance of drugs such as antipyrine and that this interaction most probably occurs through inhibition of specific isoenzymes involved in the metabolism of the test substrate.

Effect of chronic oral delorazepam on in vitro and in vivo hepatic drug-metabolizing enzyme activities in the rat.

In vivo (delorazepam clearance) and in vitro (monooxygenase activity markers) alterations in drug metabolism and the extent of enzyme induction of the hepatic cytochrome P-450 system were evaluated after oral administration of delorazepam (2.5, 25 and 150 mg/kg) for two weeks to male Sprague-Dawley rats. This benzodiazepine had no significant effect on drug metabolizing enzymes, except for slight enhancement of in vitro aniline p-hydroxylase activity which occurred at doses approximately 100 times those used clinically (0.5-2 mg). Under the likely conditions of exposure to delorazepam in human therapy therefore, such alteration in liver enzymes would be unlikely to have clinical relevance.

Kinetics of MK-801 and its effect on quinolinic acid-induced seizures and neurotoxicity in rats.

MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate], a noncompetitive antagonist of the N-methyl-D-aspartate-type of excitatory aminoacid receptors, was measured in plasma and brain tissues after i.p. administration to rats by using a novel high-performance liquid chromatography assay. The drug reached maximal concentrations in plasma and brain within 10 to 30 min of injection (2 mg/kg) with an elimination half-life of 1.9 and 2.05 hr, respectively. Mean ratio of brain area concentration-time curve to plasma area concentration time curve was 12.5, referring to total plasma concentrations. MK-801 distributed almost equally between plasma and red cells (mean blood-to-plasma ratio averaged 1.2 +/- 0.2 when calculated 30 and 180 min from drug administration). Plasma and brain concentrations of MK-801 rose almost linearly from 0.5 to 4 mg/kg 30 min after injection and the brain-to-plasma ratio (12.9 +/- 2.8) was constant in the dose range studied. The distribution of the drug in various brain regions 30 and 180 min after 2 mg/kg i.p. showed no preferential concentration or retention in any of the areas studied. The anticonvulsant effect of MK-801 was evaluated against limbic seizures (measured by EEG) induced by intrahippocampal injection of 120 nmol of quinolinic acid, an agonist of the N-methyl-D-aspartate-type receptors, in freely moving rats. At 0.25 and 0.5 mg/kg, MK-801 significantly lowered by 71 to 77% the number of seizures and by 80% the total time spent in seizures (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant tumor necrosis factor reduces hepatic drug metabolism in vivo in the rat.

To verify the potential in vivo inhibitory effect on liver function of tumor necrosis factor (TNF), also known as cachectin, antipyrine and diazepam were chosen to probe the hepatic mixed-function oxidase system. A single dose of TNF (30 micrograms/kg) to rats significantly reduced the plasma clearance of antipyrine and diazepam by about 30% and 25%, respectively; this resulted in concomitant prolongation of the elimination half-life (t1/2) of the two drugs, although of borderline significance for the benzodiazepine. This was probably due to a decrease in hepatic cytochrome P-450 activities that are responsible for antipyrine and diazepam metabolism in TNF-treated rats. This could be of clinical relevance if a similar effect occurs in humans after therapeutically effective doses of this biological response modifier.