Blood plasma levels of deoxynivalenol and its de-epoxy metabolite in broilers after a single oral dose of the toxin
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- Yunus, A.W., Valenta, H., Abdel-Raheem, S.M. et al. Mycotox Res (2010) 26: 217. doi:10.1007/s12550-010-0057-4
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To evaluate the transfer of deoxynivalenol (DON) and its de-epoxy metabolite (de-epoxy-DON) in the plasma of chicken, mashed oats naturally contaminated with 9.5 mg DON/kg were fed to four broilers (35 days age) at a dose of 20 g/bird. Blood samples were then collected from two birds at 1 h, 3 h, and 5 h post-feeding, while from the other two birds at 2 h, 4 h, and 6 h post-feeding. Analysis of DON and de-epoxy-DON was carried out by using liquid chromatography-tandem mass spectrometry after clean-up with immunoaffinity columns. At 1 h, 3 h, and 5 h post-feeding, the average values of plasma DON were 0.35 ng/ml, 0.20 ng/ml, and 0.15 ng/ml, respectively. The corresponding average values of de-epoxy-DON at these time points were 0.70 ng/ml, 0.80 ng/ml, and 0.25 ng/ml, respectively. The sum of DON and de-epoxy-DON appearing in the plasma at 1 h post-feeding in these birds was estimated to be 0.044% of the total DON fed. At 2 h, 4 h, and 6 h post-feeding, the average values of plasma DON were 0.85 ng/ml, 0.45 ng/ml, and 0.30 ng/ml. De-epoxy-DON could not be detected in the birds sampled at 2 h, 4 h, and 6 h post-feeding. The total amount of DON appearing in the plasma at 2 h post-feeding in these birds was estimated to be 0.036% of the DON fed. These data show that the absorption rate of DON is very low in broilers and that there is also a rapid transformation, and clearance from plasma. Furthermore, there appeared to be individual variability in the capacity of birds to de-epoxidise DON.
Deoxynivalenol (DON) is a type B trichothecene produced mainly by Fusarium spp. This toxin is the most predominant mycotoxin in Europe and has been also reported to contaminate cereals worldwide (Böhm 2000). Chickens are usually considered as relatively resistant to the effects of DON, as dietary levels of 16–20 mg/kg are required to elicit a toxic response in this species compared with 0.6–5 mg/kg in case of swine and cattle (review Eriksen and Pettersson 2004). This resistance may result from comparatively lower absorption of DON in chickens (Prelusky et al 1986) or due to microbial transformation of the toxin to unknown compounds in their gut (Hedman and Pettersson 1997). Various studies documenting the metabolism of DON indicate de-epoxidation to be an important detoxification phenomenon in rats, pigs, cattle, and sheep. However, de-epoxy-deoxynivalenol (de-epoxy-DON) has not been detected in plasma or tissues of chicken. Regarding transfer of DON in the plasma of chicken, the only study documenting plasma levels of DON in this species is that of Prelusky et al (1986). These authors used 14C-labelled DON for feeding Leghorn layers and found that less than 1% of the dietary dose appeared in the plasma. Recent efforts to detect DON in the plasma of Pekin ducks (Dänicke et al 2004a) and broilers (Dänicke et al 2007) by using high performance liquid chromatography (HPLC) were, however, not successful due to low levels of the compound in plasma. The present study was therefore planned as a small-scale trial to investigate the kinetics of DON in the plasma of broilers, at 1–6 h post-exposure, by using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS gives the advantage of high sensitivity compared with HPLC with ultraviolet detection (HPLC-UV) or diode array detection (HPLC-DAD). Plasma samples were also analysed for de-epoxy-DON to investigate if de-epoxidation of DON occurs in chicken.
Materials and methods
One-week-old broiler chicks were brooded and reared under standard husbandry conditions regarding a deep-litter system. Birds were fed ad libitum with diets containing or exceeding the nutrient recommendations of National Research Council (1994). At 5 weeks of age (2 ± 0.2 kg body weight), four birds were fasted for 4 h, and then each bird was fed 20 g of oats naturally contaminated with DON at a concentration of 9.5 mg/kg. For feeding, 500 g oats were ground to pass through 0.2 mm mesh and homogenised. Four representative samples of the homogenised oats were then analysed for DON by using HPLC-UV (Shimadzu, USA) after cleanup with immunoaffinity columns (IAC) (Romer Labs, Austria). The DON levels in oats were further confirmed by sending the samples for analysis to another laboratory. To avoid unnecessary stress to the birds, a modified force-feeding procedure was used. For this purpose, each bird was positioned in a plastic container and then small boluses of oats soaked in water were placed in its beak. The birds were then facilitated to engulf the bolus by putting some water (not exceeding 8 ml in total) in the beak. Any particles of oats dropped on the plastic container were collected and fed to the respective bird. Blood samples were collected from two birds at 1 h, 3 h, and 5 h, while from the other two birds at 2 h, 4 h, and 6 h post-feeding. The first two samples (2 ml each) from each bird were collected through brachial wing vein. For collection of the third sample, the birds were slaughtered by using stunning followed by puncturing the jugular vein. Blood samples were collected in heparinised plastic tubes (Vacuette, Greiner Bio-One, Ausrtia) and centrifuged at 1,800 g at 0°C for 10 min for separation of plasma. The plasma samples were frozen at −20°C until before further analysis.
The plasma samples were analysed for DON and de-epoxy-DON by using LC-ESI-MS/MS. The sample preparation was according to the method described by Valenta et al (2003) with minor modifications. In brief, the plasma samples were incubated with β-glucuronidase (Type H-2; Sigma, Deisenhofen, Germany) at a pH of 5.5 and at 37°C overnight. Then the incubated samples were extracted with ethyl acetate on disposable ChemElut columns (Varian, Darmstadt, Germany) and further purified by using IAC (DONtest; Vicam, Hamburg, Germany). The measurements were conducted on an API 4000 QTrap tandem mass spectrometer system (Applied Biosystems, Darmstadt, Germany), conducted to a 1200 series HPLC system (Agilent Technologies, Böblingen, Germany). The separation was carried out on a Betasil Phenyl/Hexyl column (100 × 2.1 mm, 3 µm; Thermo Electron Corporation, Runcorn, UK), using a binary gradient of 0.13 mmol/l ammonium acetate in water (pH 7.4, solvent A) and acetonitrile (solvent B). The measurements were performed with multiple reaction monitoring (MRM) in the negative mode, selecting the mass transitions 295 → 265 (DON) and 279 → 249 (de-epoxy-DON) for quantification and 295 → 138 (DON) and 279 → 231 (de-epoxy-DON) for additional qualifying. The limit of detection (S/N more than 3:1) for DON and de-epoxy-DON in plasma was 0.1 and 0.2 ng/ml, respectively.
Results and discussion
Plasma levels of DON/de-epoxy-DON in four birds fed oats naturally contaminated with DON (190 µg DON per bird). Plasma samples of birds 1 and 2 were collected at odd hours, plasma samples of birds 3 and 4 were collected at even hours
Time post-feeding (h)
Plasma levels (ng/ml) of DON/de-epoxy-DONa
The total amount of dietary DON consumed by each bird was 190 μg. Assuming the volume of plasma in each bird as 4% of the body weight at an environmental temperature of 20–30°C (Yahav et al 1997), the total plasma would be 80 ml in a bird weighing 2 kg. This implies that in the birds sampled at 1 h, 3 h, and 5 h post-feeding, the total DON in the plasma at first sampling (1 h) was 28 ng, which is 0.015% of the total DON fed. This was reduced by 43% and 57% at 3 h and 5 h post-feeding, respectively. The total de-epoxy-DON at first sampling (1 h) in these birds can be calculated to be 56 ng, which is 0.029% of the dietary dose. This amount slightly increased by 14% at 3 h, and then decreased by 64% at 5 h post-feeding. The sum of DON and de-epoxy-DON at 1 h post-feeding in these birds was 84 ng which is 0.044% of the dietary dose. This was reduced by 5% and 70% at 3 h and 5 h post-feeding, respectively.
Likewise, in the birds sampled at 2 h, 4 h, and 6 h, the total DON in the plasma can be calculated to be as 68 ng after 2 hours of dietary DON exposure. This implies that the total DON in the plasma at 2 h post-exposure was 0.036% of the total amount fed. This was reduced by 47% and 65% at 4 h and 6 h post-feeding, respectively. These results are to some extent consistent with the previously reported values for 14C-labelled DON by Prelusky et al. (1986). These authors fed Leghorn chickens (1.3–1.7 kg) with 2.2 mg DON/bird and noted the peak plasma levels of DON to be 0.64% of the dietary dose at 2–2.5 h post-feeding. The amount of DON used by Prelusky and co-workers was 12-times higher than that used in the present study. However, the plasma levels of DON or the sum of DON and de-epoxy-DON noted in the present study (0.036% or 0.044%, respectively of the dietary dose) was approximately 20-times lower than the plasma levels of DON noted by Prelusky and co-workers. This apparently implies that the amount of plasma DON and its de-epoxy metabolite as a percentage of dietary dose increases with increase in the amount of dietary DON.
To our knowledge, the present study is the first documentation of any de-epoxidised type B trichothecenes in the plasma of chicken. Previous efforts to detect de-epoxy metabolites of type B trichothecenes in plasma of birds have not been successful due to the higher detection limit of the analytical systems used. In this regard, Dänicke et al (2004a) did not find DON or de-epoxy-DON in the plasma of ducks fed on DON-contaminated diets when HPLC-DAD with a detection limit of 6 ng/ml was used. Both the compounds also remained undetected when HPLC-UV with a detection limit of 2 ng/ml was used to study plasma of broilers fed on DON-contaminated diets (Dänicke et al 2007). Similarly, DON and de-epoxy-DON were not detectable in eggs from layers exposed to 11.9 mg DON/kg diet for 16 weeks (Valenta and Dänicke 2005). In this regard, Hedman and Pettersson (1997) also did not find any de-epoxide of nivalenol (another type B trichothecene) in feces from chicken fed on 2.5 mg/kg or 5 mg/kg nivalenol for 3 weeks.
In case of pigs fed on rations contaminated with 2.6 mg and 4.4 mg DON/kg diet, Dänicke et al (2004b) have reported serum DON levels of 10.5 ng/ml and 14.0 ng/ml. Therefore, our present data indicate a comparatively lower absorption of DON in broilers that is coupled with a rapid de-epoxydisation and clearance from plasma. This may contribute to the relatively high tolerance of chickens towards dietary DON. However, the present study has to be regarded as preliminary and additional experiments may be required to further elucidate the results.