1,721,100 research outputs found
Residui nel miele
No full textNel capitolo viene descritta l'origine e la tipologia di sostanze che possono accumularsi ne miele e rappresentare un pericolo per la sicurezza del consumatore
Valutazione del rischio di trattamenti illeciti nel bovino da carne con desametasone mediante l’impiego di cortisolo e 6beta-idrossicortisolo in campioni di urina
No full textIl 6beta-idrossicortisolo (6betaOH-F) nell’uomo e in molti animali tra cui il bovino, è uno dei metaboliti del cortisolo (F) prodotti a livello epatico. Il 6betaOH-F è escreto nelle urine in forma non coniugata e rappresenta l’1% del totale dei metaboliti del cortisolo nell’uomo (Galteau et al., 2003). Ged et al. (1989) furono i primi a dimostrare che la trasformazione del cortisolo in 6betaOH-F è catalizzata dal CYP3A e quindi il monitoraggio della sua concentrazione urinaria può essere utilizzato come marker in vivo dell’induzione e dell’inibizione delle isoforme CYP3A (Galteau et al., 2003). L’urina è una matrice molto complessa, la cui composizione, le proprietà chimico-fisiche ed anche le concentrazioni di biomarcatori fisiologici possono essere influenzate nella stessa specie bovina da fattori quali l’alimentazione, l’età e le condizioni di stabulazione ed anche eventuali trattamenti farmacologici. L’urina è una matrice che può essere facilmente raccolta in allevamento, senza causare situazioni stressanti per gli animali, e congelata può essere conservata per tempi relativamente lunghi.
I meccanismi che regolano la liberazione di F endogeno sono molto articolati. Le modalità di regolazioni caratteristiche dell’asse ipotalamo – ipofisi - surrene (HPA) sono tre:
- il ritmo circadiano della steroidogenesi basale;
- l’incremento della steroidogenesi in risposta allo stress (Schimmer et al., 2006).
- la regolazione a feedback negativo da parte dei corticosteroidi surrenalici.
Quindi le variazioni di cortisolo e del suo metabolita possono dare delle indicazioni di eventuali trattamenti con corticosteroidi.
Inoltre una volta valutate separatamente le concentrazioni di F e del suo metabolita 6betaOH-F il rapporto tra 6betaOH-F e cortisolo permette di normalizzare eventuali differenze di produzione di 6betaOH-F dovute alle variazioni del ritmo circadiano e alla variabilità individuale (Ohno et al., 2000).
Nella prima fase del lavoro i campioni di urina raccolti dai vitelli appartenenti ai diversi gruppi sperimentali sono stati analizzati al fine di determinare la concentrazione urinaria di cortisolo (F) e 6beta-idrossicortisolo (6betaOH-F) utilizzando un metodo RIA e un metodo ELISA, rispettivamente. Prima di passare all’esecuzione delle indagini sui vari gruppi sottoposti a trattamenti è stato necessario procedere alla validazione dei due metodi utilizzati (RIA e ELISA) con urine di vitelli prelevati da animali controllo quindi non trattate e filtrate su carbone attivo per valutare adeguatamente le performance dei metodi applicati utilizzando il protocollo messo a punto da Simontacchi et al. (1997). Il pattern di escrezione urinaria del cortisolo e del suo metabolita negli animali trattati sperimentalmente con desametazone, identificato attraverso l’utilizzo dei test di screening RIA ed ELISA, è stato successivamente confermato mediante analisi in spettrometria di massa (LC-MS/MS).
La concordanza dei risultati ha dimostrato che cortisolo e 6beta-idrossicortisolo possono essere considerati efficaci marcatori di trattamento illecito con desametazone. Infatti l’effetto della loro ridotta escrezione nelle urine è più duraturo della eliminazione di desametazone somministrato, quindi rappresenta un valido supporto nell’indagine preliminare per la valutazione dei trattamenti illeciti in allevamento. In particolare il contenuto urinario di cortisolo, è risultato il parametro caratterizzato da maggior capacità discriminante; la sua valutazione mediante test RIA consente di eseguire piani di sorveglianza conoscitivi in allevamento, su larga scala con costi contenut
The Use Of Tylosin In Calves And Its Environmental Distribution
No full textTylosin was administered to calves at 20 mg/kg for five days, then the drug was traced in faeces, litter and manure. Less than two months were necessary to allow an adequate degradation able to minimize the environmental impact of the drug
Environmental risk assessment of antibacterial agents using an in vitro test
No full textAlgal toxicity of antibacterial agents used in intensive farming was investigated. The growth-inhibiting effects of oxytetracycline (OTC), sulphametazine (SMZ) and tylosin (TYL) were investigated by a modified test procedure based on the procedure described in the ISO 8692 (1989) protocol on the freshwater green alga Selenastrum capricornutum. Algal growth was measured as increased chlorophyll concentration. The chlorophyll was extracted with ethanol and fluorometrically determined. Results were quantified in terms of growth rates using Weibull equation to describe the concentration response relationship. The toxicity (EC50 value, mg/l) were: OTC, 4,17; SMZ, 10,7;TYL, 0,949, respectively
Impiego della Tilosina nell'allevamento bovino e sua distribuzione nell'ambiente
No full textLa tilosina e' stata somministrata a dei vitelli a 20 mg/kg per cinque giorni; in seguito la droga e' stata rintracciata nelle feci, nella lettiera e nel letame. Erano necessari meno di due mesi per consentire una degradazione adeguata in grado di minimizzare l'impatto ambientale del farmaco
Screening of androgen metabolites produced in vitro by high resolution mass spectrometry
No full textCytochrome P450 aromatase (P450AROM) is a key enzyme in the steroidogenic pathway that catalyses in brain and ovaries the conversion of testosterone to estrogens, and therefore is thought to play an important role in sexual differentiation of neural structures in the developing mammalian brain.
To study the role played by AROM in bovine brain a new analytical approach for the identification of androgen metabolite produced in vitro by P450AROM was adopted. High resolution mass spectrometry coupled to liquid chromatography (HPLC-HRMS) was used to evaluate the quali-quantitative metabolic profile of androstenedione and testosterone (AED and TST) incubated with bovine brain subcellular fractions. To this purpose AED and TST (100 M) were incubated in phosphate buffer (pH 7.4) with brain subcellular fractions (2 mg/ml) in presence of 30 mM MgCl2 and 1 mM NADPH regenerating system, in a shaking bath (37°C) for 60 min. The reaction was stopped and the sample was extracted with dichloromethane. Than the organic layer was removed and evaporated to dryness under nitrogen. The extract reconstituted in water/methanol was analysed by HPLC-HRMS.
The analytical approach adopted allowed to determine the P450AROM activity through the identification of Estrone (1.08 pmol/mg protein, min -1) produced in the samples incubated with AED.
So, the validated HPLC-HRMS method could be a valuable alternative to the quantification of tritiated water (3H2O) produced from radio labelled androgens usually adopted to follow androgen aromatisation in neuronal primary cell culture
In vitro hydroxylation of testosterone in rabbits: influence of age and sex*
No full textIntroduction
Hydroxylated metabolites of testosterone (T) have been proposed as useful markers of specific isoform P450 activity in rats (1). Only few studies have been devoted to characterisation of P450 biotransformation activity in food producing species thus the aim of this study was to define the influence of sex and age in rabbits on the metabolic profile of hydroxylated testosterone (OHT) metabolites using an HPLC method able to separate nine metabolites. The role played by CYP3A was also studied in Rifampicin (RIF) induced rabbits.
Material and methods
Liver microsomes were obtained (2) from rabbits. five male (3±0.4 kg), five female (3±0.2 kg), five young male (1,5±0,3 kg), and two male induced with RIF (50 mg/kg, 4 days, ip). Microsomes (0.2 mg/ml) were incubated for 10 min, at 37°C with 250 μM T. Hydroxylated metabolites were extracted with methylene chloride and separated according to Purdon (3) with HPLC using a C18column and ternary phase gradient elution. Elution times were compared with those of pure standards (Steraloids).
Discussion
Main differences are: the absence in males of 16alfa-OHT; the greater production (*P<0,001) of 6beta-OHT, 11alfa-OHT, 16beta-OHT and 2beta-OHT compared to females; the decreased production (#P<0.001) of 6beta-OHT and 16alfa-OHT in young rabbits versus adult ones. Based on data reported for other species (1, 4) apparently, the isoforms involved in rabbits are CYP3A for 6beta-OHT, and 2beta-OHT; and CYP2B for 16alfa-OHT, and 16beta-OHT. RIF induced microsomes gave a significantly (°P<0,001) greater quantities of 6beta-OHT, 2beta-OHT supporting, also in rabbits, the CYP3A role played in the production of these hydroxyl-derivatives
INFLUENCE OF GLYCINE ON MORPHINE-INDUCED ANTINOCICEPTION IN MICE
No full textThe effects of glycine on morphine-induced antinociception were investigated in mice, using a cutaneous thermal test (hot-plate), a visceral chemical test (acetylcholine writhing test), and a locomotor activity test. When glycine (200 mg/kg p.o.) and morphine (5 mg/kg s.c.) were given together during the first 30 min, glycine first antagonized the morphine-induced antinociception then this was followed by a synergistic effect. The two-phase influence of glycine on morphine-induced antinociception may be due to the interaction of glycine with different receptors
Evidence Of Hepatic Flavin Containing Monooxygenases In Food Producing Species
No full textBenzydamine hydrochloride (BZ) is an anti-inflammatory drug used in human and veterinary therapy. Its main metabolites reported in man and rodents are N-demethylbenzydamine (Nor-BZ) and benzydamine N-oxide (BZ-NO) [1]. The latter is the most important unconjugated metabolite of BZ and its production is catalysed in vitro by flavin-containing monooxygenases (FMO) [1; 2]. Despite the use of BZ in veterinary practice, no information is available on its metabolism in domestic animals. Thus to assess the role played by hepatic FMO and cytochrome P450 enzymes and to check whether BZ could be a specific FMO substrate marker also in veterinary species, BZ (1-1000μM) oxidation was studied in microsomes prepared from livers of cattle, pigs, horses, turkeys and rabbits. In all species at least two main metabolites were produced and identified as Nor-BZ and BZ-NO by matching the LC-MS spectra of authentic compounds [3].
Table 1. Kinetic parameters of BZ-NO production in liver microsomes.
Bovine Rabbit Swine Horse Turkey
Km (μM) 16.16±0.28 39.40±0.49 19.62±1.78 15.27±2.38 47.37±10.3
Vmax (nmoli/mg.min) 4.32±0.52 4.99±0.54 10.75±0.34 1.26±0.06 3.06±0.31
Following heat inactivation of microsome protein, the decrease of BZ-NO production, by incubated BZ (2.5-100μM), was up to 80% in swine, horse, cattle and rabbits, but only 35% in turkeys. Methimazole (2.5–50μM) incubation reduced the BZ-NO production up to 50%. Preliminary results with trimethylamine up to 50 μM did not inhibit the production of BZ-NO. To date the results obtained could support the role of BZ as a marker of FMO. Whether the demethylation of BZ to Nor-BZ occurs by dealkylation catalysed by P450 or via N-oxide dealkylation and subsequent reduction, or by both reactions is still under investigation.
Researches supported by MURST grants (ex 40% and ex 60% 1998)
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