Zearalenone and Metabolites in Livers of Turkey Poults and Broiler Chickens Fed with Diets Containing Fusariotoxins.

Zearalenone (ZEN) and metabolites were measured in livers of turkeys and broilers fed a control diet free of mycotoxins, a diet that contained 0.5 mg/kg ZEN (ZEN diet), and a diet that contained 0.5, 5, and 20 mg/kg of ZEN, fumonisins, and deoxynivalenol, respectively (ZENDONFB diet). The feed was individually distributed to male Grade Maker turkeys from the 55th to the 70th day of age and to male Ross chickens from the 1st to the 35th day of age, without any signs of toxicity. Together, the free and conjugated forms of ZEN, α- and ß-zearalenols (ZOLs), zearalanone (ZAN), and α- and ß-zearalanols (ZALs) were measured by UHPLC-MS/MS with [13C18]-ZEN as an internal standard and immunoaffinity clean-up of samples. ZAN and ZALs were not detected. ZEN and ZOLs were mainly found in their conjugated forms. α-ZOL was the most abundant and was found at a mean concentration of 2.23 and 1.56 ng/g in turkeys and chickens, respectively. Consuming the ZENDONFB diet significantly increased the level of total metabolites in the livers of chickens. Furthermore, this increase was more pronounced for the free forms of α-ZOL than for the conjugated forms. An investigation of the presence of ZEN and metabolites in muscle with the methods validated for the liver failed to reveal any traces of these contaminants in this tissue. These results suggest that concomitant dietary exposure to deoxynivalenol (DON) and fumonisins (FB) may alter the metabolism and persistence of ZEN and its metabolites in the liver.

Fumonisin B1, B2 and B3 in Muscle and Liver of Broiler Chickens and Turkey Poults Fed with Diets Containing Fusariotoxins at the EU Maximum Tolerable Level.

Although provisional maximum tolerable daily intake and recommended guidelines have been established for fumonisins (FB) in food, few data are available concerning levels of FB in edible animal tissues. Such data are of particular interest in avian species that can tolerate relatively high levels of fumonisins in their feed. Also, even if multiple contamination of animal feed by toxins produced by Fusarium is very frequent, little is known about the consequences of multiple contamination for FB levels in tissues. The aim of this study was to analyze the concentrations of FB in the muscle and liver of chickens and turkeys fed with FB alone and with FB combined with deoxynivalenol (DON), and with zearalenone (ZEN). Experimental diets were formulated by incorporating ground cultured toxigenic Fusarium strains in corn-soybean based feeds. Control diets were free of mycotoxins, FB diets contained 20 mg FB1+FB2/kg, and FBDONZEN diets contained 20, 5, and 0.5 mg/kg of FB1+FB2, DON, and ZEN, respectively. Animals were reared in individual cages with free access to water and feed. The feed was distributed to male Ross chickens from the 1st to the 35th day of age and to male Grade Maker turkeys from the 55th to the 70th day of age. On the last day of the study, the birds were starved for eight hours, killed, and autopsied for tissues sampling. No sign of toxicity was observed. A UHPLC-MS/MS method with isotopic dilution and immunoaffinity clean-up of samples has been developed for analysis of FB in muscle (n = 8 per diet) and liver (n = 8 per diet). Only traces of FB that were below the LOQ of 0.25 µg/kg were found in most of the samples of animals fed with the control diets. Mean concentrations of FB1, FB2, and FB3 in muscle were 17.5, 3.39, and 1.26 µg/kg, respectively, in chickens, and 5.77, 1.52, and 0.54 µg/kg in turkeys, respectively. In the liver, the respective FB1, FB2, and FB3 concentrations were 44.7, 2.61, and 0.79 µg/kg in chickens, and 41.47, 4.23, and 1.41 µg/kg, in turkeys. Cumulated level of FB1+FB2+FB3 in the highly contaminated samples were above 60 and 100 µg/kg in muscle and liver, respectively. The concentrations of FB in the tissues of animals fed the FBDONZEN diet did not greatly differ from the concentrations measured in animals fed the diet containing only FB.

In vitro and in vivo effects of a multimerized alphas 1-casein enhancer on whey acidic protein gene promoter activity.

Experimental data obtained in previous works have led to postulate that enhancers increase the frequency of action of a linked promoter in a given cell and may have some insulating effects. The multimerized rabbit alpha s1-casein gene enhancer, the 6i multimer, was added upstream of the rabbit whey acidic protein gene (WAP) promoter (-6,300; +28 bp) fused to the firefly luciferase (luc) gene (6i WAP-luc construct). The 6i multimer increased reporter gene expression in mouse mammary HC11 cells. In transgenic mice, a very weak but significant increase was also observed. More noticeable, no silent lines were found when the 6i multimer was associated to the WAP-luc construct. This reflects the fact that the 6i multimer tends to prevent the silencing of the WAP-luc construct. After addition of the 5'HS4 insulator region from the chicken beta-globin locus upstream of the 6i multimer, similar luciferase levels were measured in 6i WAP-luc and 5'HS4 WAP-luc transgenic mice. Our present data and previous ones, which show that the 6i multimer has no insulating activity on a TK gene promoter construct indicate that the insulating activity of the 6i multimer is construct-dependent and not amplified by the 5'HS4 insulator.

Effect of the rabbit alphas1-casein gene distal enhancer on the expression of a reporter gene in vitro and in vivo.

Several gene constructs containing the firefly luciferase gene and the herpes simplex virus thymidine kinase gene promoter (TK) were used to evaluate the transcriptional activity of the distal enhancer (-3442, -3285) of the rabbit alphas1-casein gene. Six copies of the enhancer (6i) were added upstream of the TK-luciferase construct in the presence or absence of the chicken beta-globin 5'HS4 insulator. The activity of the constructs was tested by transient transfection in CHO cells and in rabbit primary mammary cell cultured on plastic or on floating collagen. Constructs were also tested in stably transfected mouse mammary HC11 cells. In all cell types the multimerized alphas1-casein enhancer strongly stimulated luciferase gene expression in the presence of lactogenic hormones. It was also sensitive to the extracellular matrix in rabbit primary mammary cells. The constructs were used to generate transgenic mice. The 6i TK transgenic animals expressed the luciferase gene at very low levels irrespectively of the physiological state. No preferential expression in the mammary gland was observed. Addition of 5'HS4 insulator to the 6i TK construct did not prevent silencing in most of the transgenic lines. However, two lines expressed high luciferase levels specifically in the mammary gland. Our data suggest that 6i may confer, when insulated properly, a higher and mammary-specific expression to the TK promoter.