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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
* Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand; The National Institute of Health Science, Tokyo 158-8501, Japan; Department of Companion Animal and Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand; and Department of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
1 Corresponding author: fvetamp{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: fate • fusarenon-X • nivalenol • broiler • duck
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To gain insight into the mechanism underlying the toxicity of fusarenon-X between broilers and ducks, we studied the toxicokinetic properties and the metabolites in excreta of FX in broilers and ducks. The metabolites of these toxins appearing in liver and kidney postmitochon-drial fractions were also studied.
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MATERIALS AND METHODS |
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The FX and NIV were purchased from Sigma Chemical Co. (St Louis, MO). Scirpentriol, N-trimethylsilylimidazole, N,O-Bis(trimethylsilyl)acetamide were purchased from Wako Chemical Co. (Tokyo, Japan). Trimethylchlorosilane was purchased from GL Sciences Inc., Tokyo, Japan. Other reagents and chemicals of analytical grade were purchased from Sigma Chemical Co.
Animals
Six 4-wk-old broiler chickens (average weight: 1.35 ± 0.17 kg) and ducks (average weight: 1.16 ± 0.14 kg) were purchased from Animal Farm, Nakornpathom Province, Thailand. The experimental animals were housed in animal cages at the Laboratory Animal Facility, Faculty of Veterinary Medicine, Kasetsart University and acclimatized to the environment for 1 wk. The animals were fed with a commercial diet and water ad libitum throughout the experiments. All experimental procedures carried out on the animals were approved by the Animal Ethics Research Committee of Faculty of Veterinary Medicine, Kasetsart University.
Experimental Design In Vivo Study
To obtain the fundamental toxicokinetic data of FX, 6 broilers or 6 ducks at 5 wk of age were divided into 2 groups (n = 3). Each group was administered i.v. or orally (p.o.) with FX at a dosage of 2.2 mg/kg of BW. The dosage was based on our previous studies. Blood samples were taken from brachial (wing) veins using heparinized syringes just before and at 5, 10, 20, 30, 60, 120, 180, 240, and 600 min following administration. Plasma were separated by centrifugation (1,968 x g) for 15 min. Excreta was collected up to 6 h after the toxin was given. All the plasma and excreta samples were frozen at –20 C° until analysis.
Metabolism of FX to NIV In Vitro
A female duck and both sexes of broiler chickens were killed with pentobarbitotone sodium at a dosage of 40 mg/kg of BW by intravenous administration. The blood was taken from wing vein with a heparinized syringe, and red blood cells and plasma were separated by centrifugation at 1,968 x g for 15 min. The livers and kidneys were immediately removed, frozen in liquid nitrogen and stored at –80°C until used. Postmitochondrial fractions were prepared by the previous method (Bammler et al., 2000; Esaki and Kumagai, 2002). The red blood cells, plasma, and postmitochondrial fractions of liver and kidney were incubated with shaking (60 cycles/min) with 10 µg of FX at 37°C for 45 min.
Extraction and Clean-up
Plasma, excreta, red blood cells, and microsomal and cytosolic fractions of the liver were extracted in the 3 mL of acetonitrile (ACN)-water (3:1). Ammonium sulfate was added to the mixture (Tanaka et al., 2001; Poapolathep et al., 2003), and then the ACN fraction was separated by centrifugation at 1,968 x g for 15 min. Extraction was repeated 2 additional times. The parent and metabolites in the ACN fraction were purified with a Seppak silica cartridge (Waters Corp., Milford, MS) as described previously (Poapolathep et al., 2003). The elute was evaporated to dryness under a nitrogen stream at 40 C° on a heating block. The residue was derivatized with trimeth-ylsilylating agents according to the method of Tanaka et al. (2000) and then analyzed by gas chromatography-mass spectrometry (GC-MS). To evaluate recovery, 1 mL of plasma or excreta was added the FX and NIV standard solution. The spiked samples were then analyzed as described in the extraction procedure. The average (±SD) recoveries of FX in plasma and excreta were 83.21 ± 3.14, 93.37 ± 2.87% and 84.59 ± 3.99, 96.49 ± 2.81% in broilers and ducks, respectively. The average (±SD) recoveries of NIV were 72.57 ± 4.99%, 79.73 ± 5.63% and 73.36 ± 3.31%, 75.89 ± 2.55% in broilers and ducks, respectively.
GC-MS
The GC-MS system was composed of GC-MS (MS-Agilent 5973N GC-MS system, Agilent Technology, Palo Alto, CA) equipped with a capillary column (DB-5, 30 m x 0.25 mm I.D., 0.25 µm df, Agilent Technology). The column conditions, flow rate, and mass spectrometry conditions were the same as described by Tanaka et al. (2000). The detection limit of this method was 1 ng/mL. All samples were subjected to GC-MS with scirpentriol as an internal standard.
Calculation of Toxicokinetic Parameters
The toxicokinetic characteristics of FX in broilers and ducks were described by a 2-compartment pharmacokinetic model using the PK Solution 2.0 Program (http://www.summitPK.com), where Kel was the elimination rate constant, F the oral bioavailability, AUC the area under the curve, t1/2β the elimination half-life, t1/2
the distribution half-life, Cl the body clearance, and K12; K21 the micro-rate constants.
The oral bioavailability (F) was calculated using the equation
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Statistical Analysis
Plasma concentration curves of FX and NIV were shown as mean (±SD) of 3 broilers and ducks. Pharmaco-kinetic parameters were shown as mean (±SD). Statistical analysis was generally done according to Student’s t-test. When individual differences were large, Welch’s t-test was performed. A value of P < 0.05 was judged to be significant and P < 0.01 to be highly significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GC-MS analysis of excreta from broiler chickens and ducks treated with FX revealed excretion of both NIV and FX (data not shown).
Plasma Concentration and Pharmacokinetic Parameters
Figures 1
and 2 show the plasma concentration-time plot of FX and NIV in broilers and ducks following i.v. and p.o. administration. Pharmacokinetic parameters were calculated from the FX plasma concentration following i.v. administration. The elimination half-life (t1/2β) was longer in ducks than in broilers. The oral bioavailability (F) was slightly higher in ducks than in broilers (Table 1). The values of body clearance (Cl), elimination rate constant (Kel) and distribution half-life (t1/2) were greater in broilers than in ducks but the micro-rate constant (k12) and area under the curves (AUC) for both FX and NIV were higher in ducks than in broilers (Table 1). A large proportion of NIV peaks were detected in plasma after i.v. and p.o. administration (Figures 1 and 2).
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To study the tissue capable of the conversion of FX to NIV in broiler chickens and ducks, FX was incubated with liver and kidney postmitochondrial fractions, red blood cell and plasma, and the amount of NIV formed was determined. The FX to NIV conversion was noted clearly in the liver and kidney, the highest activity being in the liver in ducks (98.95%), but in the kidney in broiler chickens (94.39%). The FX to NIV conversion by broiler liver was 70.12% and that by duck kidney was 94.32%. The FX to NIV conversion in broiler plasma and red blood cells were 5.45 and 8.06%, respectively, whereas that in duck plasma and red blood cell were 1.3 and 9.92%, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The in vitro study of FX metabolism indicates that the liver and kidney are capable for the FX to NIV conversion. Consistent with this, the liver and kidney have also been observed to be major organs for FX to NIV conversion in vitro in mice, rat, and rabbit (Ohta et al., 1978; Poapolathep et al., 2003).
In conclusion, the results demonstrated that FX is absorbed from the gastrointestinal tract more efficiently in ducks than in broilers, followed by its rapid conversion to NIV probably by the liver and kidney. In addition, it appears that AUC data represent the length of time the compounds were detectable, higher AUC numbers longer tissue tenures in ducks, indicating less efficient excretion and enhanced opportunity for damage. Therefore, the toxicity would be greater in ducks than in broiler chickens.
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ACKNOWLEDGMENTS |
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Received for publication January 7, 2008. Accepted for publication April 15, 2008.
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REFERENCES |
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