Recent advances on the use of adsorbent materials for detoxification of Fusarium mycotoxins.

The extensive use of adsorbents in the livestock industry has led to the introduction of a wide range of new products on the market, most of them claiming high in vitro mycotoxin adsorption capacity. However, adsorbents that may appear effective in vitro do not necessarily retain their efficacy when tested in vivo. Studies performed in our laboratory during the past few years aiming to evaluate the efficacy of various adsorbent materials in binding Fusarium mycotoxins are reported. Adsorption experiments were performed in in vitro screening tests for Fusarium mycotoxins at different pHs; by in vivo tests using the increase of the sphinganine to sphingosine ratio in rat urine and tissues as a biomarker of fumonisin exposure; and by a dynamic, computer-controlled, gastrointestinal model simulating the gastrointestinal tract of healthy pigs. Most of the commercially available mycotoxin-binders failed in sequestering in vitro Fusarium mycotoxins. Only for a small number of adsorbent materials was the ability to bind more than one mycotoxin demonstrated. Cholestyramine was proven to be an effective binder for fumonisins and zearalenone in vitro, which was confirmed for zearalenone in experiments using a dynamic gastrointestinal model and for fumonisins in in vivo experiments. No adsorbent materials, with the exception of activated carbon, showed relevant ability in binding deoxynivalenol and nivalenol. The in vitro efficacy of activated carbon toward fumonisins was not confirmed in vivo by the biomarker assay. The dynamic gastrointestinal model was a reliable tool to study the effectiveness of adsorbent materials in reducing the bioaccessibility of Fusarium mycotoxins, as an alternative to the more difficult and time-consuming studies with domestic livestock.

In vitro and in vivo studies to assess the effectiveness of cholestyramine as a binding agent for fumonisins.

Several adsorbent materials were tested at I mg/ml for their in vitro capacity to adsorb fumonisin B1(FB1) from aqueous solutions. Cholestyramine showed the best adsorption capacity (85% from a solution containing 200 microg/ml FB1) followed by activated carbon (62% FB1). Bentonite adsorbed only 12% of the toxin from a solution containing 13 microg/ml FB1, while celite was not effective even at the lowest tested FB1 concentration (3.2 microg/ml). Cholestyramine was tested in vivo to evaluate its capacity to reduce the bioavailability of fumonisins (FBs) in rats fed diet contaminated with toxigenic Fusarium verticillioides culture material. Rats were exposed for one week to FBs-free diet, FBs-contaminated diet containing 6 or 20 microg/g FB1 + FB2 and the same FBs-contaminated diet added of 20 mg/g cholestyramine. The increase of sphinganine/sphingosine (SA/SO) ratio in urine and kidney of treated rats was used as specific and sensitive biomarker of fumonisin exposure. The addition of cholestyramine to the FBs-contaminated diets consistently reduced the effect of FBs by reducing significantly (P < 0.05) both urinary and renal SA/SO ratios.

Ineffectiveness of activated carbon in reducing the alteration of sphingolipid metabolism in rats exposed to fumonisin-contaminated diets.

Sphinganine/sphingosine (SA/SO) ratio, a biomarker of fumonisin exposure, has been measured in urine, kidney and liver of male Wistar rats exposed to fumonisin-contaminated diet with and without the addition of activated carbon (AC). The latter was previously shown to adsorb fumonisin B(1) in vitro. Rats were fed either control diet or fumonisin-contaminated diet (4 microg FB(1+)FB(2)/g) or fumonisin-contaminated diet mixed with 20 mg AC/g diet for 1 week. In rats fed fumonisin-contaminated diet, the SA concentration and SA/SO ratio increased significantly and reversibly in kidney, while urine and liver did not show a significant increase of SA/SO ratio. The addition of AC to the fumonisin-contaminated diet did not alter the change of SA/SO biomarker for fumonisin exposure. This provides indications that the use of AC to reduce the toxicity of fumonisins is unlikely to be effective in vivo.

Isolation and characterization of phytotoxic compounds produced by Phomopsis helianthi.

The isolation, chemical characterization and biological activity of two phytotoxic metabolites of Phomopsis helianthi Munt-Cvet et al. is reported. These compounds were identified by spectroscopic methods (UV, IR, 1H and 13C NMR, and MS) as trans-4,6-dihydroxymellein (trans-3-methyl-4,6,8-trihydroxy-3,4-dihyroisocoumarin) and cis-4,6-dihydroxymellein (cis-3-methyl-4,6,8-trihydroxy-3,4-dihydroisocoumarin). This is the first report of the isolation of trans-4,6-dihydroxymellein from fungal cultures and of the production of cis- and trans-4,6-dihydroxymelleins by P. helianthi. Rice was found to be a good substrate for the production of the dihydroxymelleins. Culture extracts of some Italian and French strains of P. helianthi showed different degrees of phytotoxicity towards sunflower leaves and seedlings. The minimum effective doses of trans- and cis-4,6-dihydroxymelleins with different bioassays were 76 and 135 microg per spot (leaf puncture bioassay), 3 and 5 micromol g(-1) fresh tissue (absorption by leaf cutting) and 5 and 2 micromol g(-1) fresh tissue (absorption by cut seedlings), respectively. These compounds may contribute to the severity of the sunflower disease caused by P. helianthi.

Ochratoxin A and fumonisins (B1 and B 2) in maize from Balkan nephropathy endemic and non endemic areas of Croatia.

The occurrence of ochratoxin A, fumonisin B1 and B2 has been investigated in maize samples collected in 1996 (105 samples) and 1997 (104 samples) in 14 counties of Croatia, including Brodsko-Posavska county, the main area of Balkan endemic nephropathy in Croatia. Ochratoxin A and fumonisins co-occurred in 21% of the examined samples. In particular, ochratoxin A (OTA) was found in 10 samples (10%) of the 1996 and 36 samples (35%) of the 1997 crops with mean concentrations of positive samples of 37.9 ng/g and 57.1 ng/g, and highest concentrations at 223.6 ng/g and 613.7 ng/g, respectively. Similar incidence of OTA contamination was observed in 1996 samples from both endemic and non endemic areas of Balkan nephropathy, whereas a significant difference (P<0.01) was found between the two areas in 1997, with 50% and 20% incidence of contamination in the endemic and non endemic area, respectively, and relevant OTA mean concentration of positive samples of 73.4 ng/g and 20.2 ng/g. High incidence of infection byPenicillium spp. (potential OTA producers) was found in all tested samples, with mean values of 88% and 93% in samples of 1996 and 1997, respectively. With respect to fumonisin B1 (FB1) and B2 (FB2) all but one of the 1996 samples were contaminated, with highest and mean concentrations of positive samples (FB1+FB2) at 11661 ng/g and 645 ng/g, respectively. Similar incidence of positive samples (93%), but lower contamination levels (mean 134 ng/g, maximum 2524 ng/g) were found in 1997 samples. The results of fumonisin analysis were in agreement with the mycological analysis showing higher incidence of Fusarium infection in samples of 1996 with respect to those of 1997.These data provide additional information on the occurrence of ochratoxin A in Balkan endemic nephropathy areas and, for the first time, its co-occurrence with other nephrotoxic compounds, such as fumonisins, that may contribute to the disease development. However the finding of these mycotoxins in the non-endemic areas, also at high levels, do not allow to draw a conclusion about their role in the etiology of the disease.

Use of various clean-up procedures for the analysis of ochratoxin A in cereals.

A rapid and reliable procedure has been developed for the determination of ochratoxin A in wheat and oats. The method consists of extraction of the sample with acidic chloroform, followed by defatting with n-hexane and finally, HPLC determination with fluorometric detection. Mean recoveries for wheat and oats spiked at levels between 1 and 100 micrograms/kg ranged from 80 to 104%. The limit of determination (field blank +6 sigma) was 0.8 micrograms/kg and the precision (within-laboratory relative standard deviation) ranged from 3 to 7%. The method was tested on 34 wheat and 34 oats samples. Ochratoxin A was confirmed in some positive samples by methyl ester formation and/or by clean-up of the extracts with immunoaffinity columns. The method was not appropriate for the analysis of barley (45 tested samples), rye (69 samples) or trout feed (13 samples). A false positive was recorded within the four positive barley samples and 18 false positives were recorded within the 21 positive rye samples whereas trout feed samples could not be analysed due to insufficient clean-up. The use of immunoaffinity columns made the analysis of trout feed and rye samples possible, providing excellent clean-up of the extracts with no false positive results and a good limit of determination (0.2 micrograms/kg).

Rapid method to determine sphinganine/sphingosine in human and animal urine as a biomarker for fumonisin exposure.

The widespread occurrence of fumonisins in maize and maize-based foods and feeds demands the development of rapid and reliable methods for the analysis of suitable biomarkers in biological fluids in order to assess human and animal exposure to these important mycotoxins. The increase in the ratio of free sphinganine/sphingosine (SA/SO) in urine has been recently proposed as a biomarker to evaluate exposure to fumonisins. The presently available method for the determination of SA and SO in biological samples is labor intensive, time consuming and insufficiently accurate. A new method has been proposed for the determination of SA and SO in human and animal urine which is more precise and accurate, and drastically reduces the number of steps during extraction and clean-up. The method is essentially based on the use of silica minicolumn clean-up of the chloroform extract from alkalinized urine. The final extract is derivatized with o-phthaldialdehyde reagent and SO and SA are determined by reversed-phase HPLC with fluorimetric detector. Urine samples spiked with SO, SA standards at concentrations ranging from 1.5 to 15 ng/ml have given mean recoveries higher than 80% and precision (coefficient of variation) lower than 10%. Detection limit for SO and SA was 0.1 ng/ml.

[Results of high-dose intravenous immunoglobulin treatment of patients with pretibial myxedema and Basedow's disease. Preliminary findings]. / Risultati del trattamento con immunoglobuline endovena ad alto dosaggio in pazienti affetti da mixedema pretibiale e morbo di Basedow. Osservazioni preliminari.

Three patients affected with Graves' ophthalmopathy and pretibial myxoedema have been treated with high dose intravenous immunoglobulins. We have observed in all patients clinical improvement of pretibial myxoedema and a parallel reduction or negativization of the titre of circulating thyroglobulin, microsomal, TSH receptor autoantibodies and of non organ-specific antibodies (antinuclear, anti smooth muscle cells and antimitochondrial autoantibodies). In conclusion the results of this study suggest that intravenous immunoglobulin are effective in the treatment of pretibial myxoedema and probably act by an immunomodulation of autoimmune phenomena.