Occurrence of mycotoxins and ergosterol in maize harvested over 5 years in Northern Italy.

Maize samples collected from storage bins and feed mills in Northern Italy between 1995 and 1999 were surveyed for the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON) and fumonisin (FB1); further, ergosterol was analysed as a fungal growth marker. The incidence and mean content of AFB1 were generally low; nevertheless, a remarkable contamination was found in two samples (109 and 158 microg kg(-1)), while five others exceeded 20 microg kg(-1). DON and ZEA mean levels were significantly higher in 1996 (2716 and 453 microg kg(-1)) with respect to the other years, when mean contents ranged from 7 to 30% and from 3 to 17%, respectively, expressed in per cent of 1996 contents. FB1 was present in all samples and was by far the most remarkable mycotoxin in Northern Italian maize, with the exception of samples from 1996. The average level was 3064 microg kg(-1), 69.6% of samples resulted over 1000 microg kg(-1) and 16.9% over 5000 microg kg(-1). Significant correlations were found between ergosterol and the major mycotoxin(s) in each year (FB1 in 1995 and 1997-99; ZEA + DON in 1996). Consequently, ergosterol seems to be a good index of the toxicological quality of maize. Climatic conditions influenced the growth of different fungal species. In 1996, the first 20 days of October were extremely rainy; these weather conditions delayed the harvest until the first week of November and favoured the growth of DON and ZEA producing fungi and the synthesis of mycotoxins. On the contrary, the temperate and dry climate of the other years supported the growth of FB1-producing fungi.

Alternative extraction methods for Zearalenone: Microwave Assisted Extraction and Accelerated Solvent Extraction.

Zearalenone (ZON) was extracted from wheat and corn by using Microwave Assisted Extraction and Accelerated Solvent Extraction. A factorial design approach was applied to evaluate the influence of the most important extraction parameters such as temperature, time and solvent extraction mixture on fortified cereals. ZON was determined by LC-MS without performing any clean-up after the extraction to better evaluate the extraction efficiency. The selected extraction conditions were tested on samples which had been previously used in an international proficiency test. Applying the selected extraction conditions both alternative extraction procedure provided satisfactory results comparable to the most commonly used method.

Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection.

The analysis of procyanidins in crude chocolate extracts by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionisation mass spectrometry (MS) is described in this report. Catechin monomers and procyanidin oligomers (dimers to hexamers) were identified according to the mass of the single charged pseudomolecular ion ([M-H]-). Identification was further confirmed by collision-induced dissociation MS-MS analysis, which in addition, permitted the identification of double charged pentameric, hexameric, and heptameric ions. This study demonstrates the capability of the combination of HPLC and modern ion trap mass analysers to significantly reduce sample preparation and analysis time in combination with high specificity and structural information for compound identification.

Differential activation of wild-type and variant forms of estrogen receptor alpha by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements.

Structure-dependent estrogen receptor alpha (ER alpha) agonist and antagonist activities of synthetic and natural estrogenic compounds were investigated in human HepG2, MDA-MB-231 and U2 cancer cell lines. Compounds used in this study include 4'-hydroxytamoxifen, ICI 182,780, bisphenol-A (BPA), 2',4',6'-trichloro-4-biphenylol (3Cl-PCB-OH), 2',3',4',5'-tetrachloro-4-biphenylol (4Cl-PCB-OH), p-t-octylphenol, p-nonylphenol, naringenin, kepone, resveratrol, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). Cells were transfected with a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) and either wild-type estrogen receptor alpha (ER-wt) or variants expressing activation function-1 (ER-AF1) or AF-2 (ER-AF2). The ER agonist activities of the synthetic mono and dihydroxy aromatic compounds are comparable in all three-cell lines, whereas the activities of naringenin, kepone and resveratrol are dependent on cell context and expression of wild-type or variant forms of ER alpha. In contrast, the ER antagonist activities for these compounds were highly complex and, with the exception of 3Cl-PCB-OH, all compounds inhibited E2-induced wild-type or variant ER action. Results of this in vitro study suggest that the estrogenic and antiestrogenic activity of structurally diverse synthetic and natural estrogenic compounds is complex, and this is consistent with published data that often give contradictory results for these compounds.

Occurrence of ochratoxin A in Italian wines.

A total of 96 red wines and 15 white dessert wines produced mostly in the years 1995-97 in 19 Italian regions were analysed for ochratoxin A (OTA). The amount of OTA ranged from < 1 to 3856 ng/l the median (mean) was found to be 90 (419) ng/l for the red wines and 8 (736) ng/l for the white dessert wines. Our survey shows that the geographic region of origin has a strong influence on OTA contamination, both for red and for dessert wines: in fact, wines produced in southern Italy were markedly more contaminated. The overall median (mean) OTA concentration in the red wines produced in the four Italian areas (northwest, northeast, centre and south) was 2 (11), 90 (81), 134 (295) and 1264 (1233) ng/l. The same trend was observed for the white dessert wines: OTA concentrations of over 1000 ng/l were found in four out of five samples from southern Italy (1185, 2454, 3477, 3856 ng/l), while central and northern samples showed very low contamination. The contribution of wine to mean daily OTA intake can be considered negligible in the case of people drinking wine manufactured in northern and central Italy; this is not true if a medium drinker constantly consumes red wine produced in southern Italy in this case wine alone could supply the diet with an amount of OTA equal to or even above the tolerable daily intake of 5 ng/kg body weight recommended by the Scientific Committee on Food of the European Commission.

Method of determination of appropriate heat treatment of animal meal by immunoassay developed for detection of cooked beef: interlaboratory study.

An interlaboratory trial was conducted for the validation of an enzyme-linked immunosorbent assay (ELISA) method for determination of appropriate heat treatment of animal meal. A commercially available ELISA test kit developed for the identification of beef in cooked food was used in the study. Twelve laboratories from 7 European countries examined 2 different analytical protocols to establish the most appropriate analytical method. Three different samples were used, 2 animal waste materials sterilized at 129 and 134 degrees C (wet conditions), respectively, and a meat and bone meal material processed at dry conditions (maximum temperature, 140 degrees C). Statistical evaluation applying t-statistics showed that the animal meal treated according to European legislation (>133 degrees C) was clearly distinguishable from the 2 other test materials at a 99% confidence level using both analytical protocols. This method can be considered as a complementary test to the immunoassay developed for the detection of pork in cooked food that is already applied in routine analysis for the surveillance of rendering plants.

Determination of rendering plant sterilization conditions using a commercially available ELISA test kit developed for detection of cooked beef.

A commercially available enzyme-linked immunosorbent assay (ELISA) test kit developed for detection of cooked beef in meat samples was used to determine appropriate heat treatment of rendered materials. An improved extraction procedure increased the absolute difference in R-values between 2 rendered materials treated under different conditions (average temperature 129 and 134 degrees C, respectively). To evaluate the influence of the main sterilization parameters on ELISA results, a factorial design approach was used. The parameters investigated were temperature, time, particle size, and meat composition. Lean meat samples containing beef and pork were sterilized under strictly controlled conditions in a laboratory autoclave. The experiments demonstrated that the R-values obtained with the ELISA test kit for beef are strongly influenced by temperature and time, whereas particle size has a minor influence. The proportion of bovine material did not have any impact on R-values. Autoclave-processed lean meat samples were analyzed by using an ELISA test kit for pork, which was validated in a collaborative trial. The ELISA test kit for pork proved to be more sensitive for the investigated parameters, thus verifying and extending previous investigations.

Toxicology of environmental estrogens.

It has been hypothesized that environmental contaminants that modulate endocrine signaling pathways may be causally linked to adverse health effects in humans. There has been particular concern regarding synthetic estrogens and their role in disrupting normal development of the male reproductive tract. Most estrogenic industrial compounds, such as bisphenol A (BPA) and nonylphenol, typically bind estrogen receptors alpha (ERalpha) and beta (ERbeta) and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually > or = 1,000-fold lower than observed for 17beta-estradiol (E2). Selective estrogen receptor modulators (SERMs) represent another class of synthetic estrogens that are being developed for treatment of hormone-dependent problems. The SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with a pC3-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4'-hydroxytamoxifen; BPA; 2',4',6'-trichloro-4-biphenylol; 2',3',4',5'-tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). The results show that synthetic and phytoestrogens induce distinct patterns of gene activation in HepG2 and U2 osteogenic sarcoma cells, suggesting that these compounds will induce tissue-specific in vivo ER agonist or antagonist activities. The predicted differences between these compounds, based on results of the in vitro bioassay, have been confirmed. For example, BPA inhibits E2-induced responses in the rodent uterus, and HPTE and structurally related compounds are ERalpha agonists and ERbeta antagonists in assays carried out in HepG2 and other cancer cell lines.

Development and characterization of a carbon-based composite material for reducing patulin levels in apple juice.

Patulin, a heterocyclic lactone produced by various species of Penicillium and Aspergillus fungi, is often detected in apple juices and ciders. Previous research has shown the effectiveness of granular activated carbon for reducing patulin levels in aqueous solutions, apple juices, and ciders. In this study, ultrafine activated carbon was bonded onto granular quartz to produce a composite carbon adsorbent (CCA) with a high carbonaceous surface area, good bed porosity, and increased bulk density. CCA in fixed-bed adsorption columns was evaluated for efficacy in reducing patulin levels from aqueous solutions and apple juice. Columns containing 1.0, 0.5, and 0.25 g of CCA were continuously loaded with a patulin solution (10 microg/ml) and eluted at a flow rate of 1 ml/min. Results indicated that 50% breakthrough capacities for patulin on 1.0-, 0.5-, and 0.25-g CCA columns were 137.5, 38.5, and 19.9 microg, respectively. The effectiveness of CCA to adsorb patulin and prevent toxic effects was confirmed in vitro using adult hydra in culture. Hydra were sensitive to the effects of patulin, with a minimal affective concentration equal to 0.7 microg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0 g of CCA was also effective in reducing patulin concentrations (20 microg/liter) in a naturally contaminated apple juice, and breakthrough capacities were shown to increase with temperature. Additionally, CCA offered a higher initial breakthrough capacity than pelleted activated carbon when compared in parallel experiments. This study suggests that CCA used in fixed-bed adsorption systems effectively reduced patulin levels in both aqueous solutions and naturally contaminated apple juice; however, the appearance and taste of apple juice may be affected by the treatment process.