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A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.

Mycotoxins may cause deleterious effects (among others nephrogenic, hepatogenic, carcinogenic, teratogenic, neurogenic) in animals and humans, therefore they have been intensely studied and monitored over the years. For citrinin (CIT), a nephrotoxic mycotoxin, however, this has not yet been the case. According to the latest European Food Safety Authority report, a correct risk assessment of CIT was not possible due to the lack of occurrence data. Besides, traces of CIT or its metabolite, dehydrocitrinone are widely (in up to 90% of samples) present in human urine according to recent Belgian and German scientific reports, which might imply chronic exposure. Only recently, a European maximum limit has been set for CIT in cholesterol reducing food supplements including red yeast fermented rice (RYR). During production of RYR through fungal (among others Monascus purpureus) fermentation of rice other components, like CIT, as well as nephrotoxic ochratoxin A (OTA) may form. Consequently, the present work attempted develop to a robust and routinely applicable ultra-high performance liquid chromatographytandem mass spectrometry (UHPLC-MS/MS) method for the analysis of CIT and OTA in food, feed and in RYR food supplements. The method was successfully validated based on EU/657/2002 and EU/519/2014 in RYR food supplements and wheat flour, achieving respective limits of quantification (LOQ) for CIT of 0.4 μg/kg and 0.1 μg/kg and for OTA of 15 μg/kg and 0.4 μg/kg. The average between-day recoveries varied from 72 to 110% with relative standard deviations ≤16%. Single-day validation in rice, curry and apple matrices showed LOQs ranging from 0.3-1.0 μg/kg. Next, the occurrence of CIT/OTA was surveyed in 138 RYR, food and feed samples, proving the potential of this method for future data acquisition within a risk assessment framework specifically for CIT, while also gaining information about the (co-)occurrence of OTA in edible matrices.

Fusarium species isolated from Belgian maize were screened for their ability to produce fusarin C, fusaric acid, fumonisins B1 (FB1), FB2 and FB3 in maize grains. First, cultivation of Fusarium species in Myro liquid medium allowed overcoming the shortage of the standard of fusarin C on the market. All Fusarium verticillioides produced much higher contents of mycotoxins in Myro compared to Fusarium graminearum or Fusarium venenatum. The optimization of the LC-MS/MS method resulted in low limits of detection and quantification for fusarin C, fusaric acid, FB1, FB2 and FB3 determination in maize grains. Its application for screening the potential toxin production ability evidenced that the concentrations of the analytes were significantly increased at various levels when F. verticillioides strains were cultivated in maize grains and reached 441 mg kg(-1) for fusaric acid, 74 mg kg(-1) for fusarin C, 1,301 mg kg(-1) for FB1, 367 mg kg(-1) for FB2 and 753 mg kg(-1) for FB3.

Enniatins, a large group of cyclodepsipeptides, are widely distributed contaminants of different crops intended for human and animal consumption. Enniatin B is one of the principal analogues in species of the genus Fusarium, known to have ionophoric, antibiotic, and insecticidal activity. Regardless of considerable cytotoxic effects observed in vitro, enniatins have been characterised as compounds with low acute toxicity in vivo. The biotransformation of enniatin B has previously been elucidated in liver microsomes, and 12 different metabolites (M1 to M12) have been reported. In order to provide a better basis for understanding the potential toxic effects in humans and animals, different samples (eggs, livers, plasma) from two different feeding studies have been analysed for the presence of enniatin B and its hepatic metabolites. The earlier reported metabolite M11, and a novel metabolite (designated M13), were dominant in liver samples from enniatin B exposed broilers. The peak area corresponding to the sodiated molecular ion of M11 was approximately 2.5 times larger than that of parent enniatin B in liver samples collected after one week of exposure. The same metabolites were also present in serum samples. In egg samples, only metabolites M13 and M4 were detected. The comparison of mass spectrometric data of M13 and enniatin B suggested that M13 is a monohydroxylated metabolite. The hepatic biotransformation of enniatin B was also investigated in vitro in chicken microsomes demonstrating good correlation with the metabolite profiles in the chicken samples. The results of the present study demonstrated an extensive biotransformation of enniatin B in vivo confirming previously reported in vitro data.

Due to their unavoidable presence in agricultural products and their potential risk for human and animal health, it is imperative that mycotoxins be detected and removed from the food chain. This review reports both established conventional and emerging methods based on recent advances in mycotoxin analyses. Although most official detection methods are chromatographic (coupled with an ultraviolet or fluorescence detector), alternative strategies such as immunochemical methods and liquid chromatography coupled with mass spectrometry have seen a major breakthrough on the commercial market. They comport the advantages of making simple extraction possible along with high sensitivity multidetection. Emerging technologies are of potential application in the analysis of mycotoxins and may demonstrate advantages over the more conventional and well established techniques used as official analytical methods.

The interest in LC-MS/MS multi-mycotoxin methods unveiled an urgent need for multi-mycotoxin reference material. A multi-fusariotoxin, including deoxynivalenol (DON); zearalenone (ZEN); T-2 toxin (T-2); HT-2 toxin (HT-2); enniatin A, A1, B, and B1 (ENNs); and beauvericin (BEA), contaminated wheat flour was obtained by inoculation Fusarium spp. strains. The candidate material has successfully passed the homogeneity test and submitted to an international interlaboratory study achieved by 19 laboratories from 11 countries using their routine analytical method. The dispersion of the results for ZEN and BEA did not allow the derivation of reliable consensus values, while the assignment was only possible for DON, HT-2, T-2, and ENN A. No link was found between the methods used by the participants and the results. Significant changes in dry matter contents (≥±1.4 % of the initial dry matter) and significant changes in ergosterol contents (≥±10 %) did not occur. Using the mycotoxin contents in wheat flour stored at −80 °C as reference values, statistically significant decreases were observed only for T-2 contents at +24 °C, in contrast to the storage at −20 and +4 °C. For the other involved toxins, the candidate material was found to be stable at −20, +4, or +24 °C. Based on the T-2 decreases, a shelf life of 6 years was derived from isochronous study when the material is kept at −20 °C. At room temperature (e.g., +24 °C) or higher, this time validity drastically decreases down to 6 months. The development of this metrological tool is an important step towards food and feed quality control using multi-mycotoxin analyses. In vivo animal experiments using multi-mycotoxin-contaminated feeds dealing with the carryover or mitigation could further benefit from the methodology of this work.

Over a 4-year period (2010-13), a survey aiming at determining the occurrence of Fusarium spp. and their relations to mycotoxins in mature grains took place in southern Belgium. The most prevalent species were F. graminearum, F. avenaceum, F. poae and F. culmorum, with large variations between years and locations. An even proportion of mating type found for F. avenaceum, F. culmorum, F. cerealis and F. tricinctum is usually a sign of ongoing sexual recombination. In contrast, an unbalanced proportion of mating type was found for F. poae and no MAT1-2 allele was present in the F. langsethiae population. Genetic chemotyping indicates a majority of deoxynivalenol (DON)-producing strains in F. culmorum (78%, all 3-ADON producers) and F. graminearum (95%, mostly 15-ADON producers), while all F. cerealis strains belong to the nivalenol (NIV) chemotype. Between 2011 and 2013, DON, NIV, enniatins (ENNs) and moniliformin (MON) were found in each field in various concentrations. By comparison, beauvericin (BEA) was scarcely detected and T-2 toxin, zearalenone and α- and β-zearalenols were never detected. Principal component analysis revealed correlations of DON with F. graminearum, ENNs and MON with F. avenaceum and NIV with F. culmorum, F. cerealis and F. poae. BEA was associated with the presence of F. tricinctum and, to a lesser extent, with the presence of F. poae. The use of genetic chemotype data revealed that DON concentrations were mostly influenced by DON-producing strains of F. graminearum and F. culmorum, whereas the concentrations of NIV were influenced by the number of NIV-producing strains of both species added to the number of F. cerealis and F. poae strains. This study emphasises the need to pay attention to less-studied Fusarium spp. for future Fusarium head blight management strategies, as they commonly co-occur in the field and are associated with a broad spectrum of mycotoxins.

Reporter gene assays incorporating nuclear receptors (estrogen, androgen, thyroid β and PPARγ2) have been implemented to assess the endocrine activity of 13 mycotoxins and their mixtures. As expected, zearalenone and its metabolites α-zearalenol and β- zearalenol turned out to have the strongest estrogenic potency (EC50 8,7 10-10 ± 0,8; 3,1 10-11 ± 0,5 and 1,3 10-8 ± 0,3 M respectively). The metabolite of deoxynivalenol, 3-acetyl-deoxynivalenol also had estrogenic activity (EC50 3,8 10-7 ± 1,1 M). Furthermore, most of the mycotoxins (and their mixtures) showed anti-androgenic effects (15-acetyldeoxynivalenol, 3-acetyl-deoxynivalenol and α-zearalenol with potencies within one order of magnitude of that of the reference compound flutamide). In particular, deoxynivalenol and 15-acetyl-deoxynivalenol acted as antagonists for the PPARy2 receptor. When testing mixtures of mycotoxins on the same cell systems, we showed that most of the mixtures reacted as predicted by the concentration addition (CA) theory. Generally, the CA was within the 95% confidence interval of the observed ones, only minor deviations were detected. Although these reporter gene tests cannot be directly extrapolated in vivo, they can be the basis for further research. Especially the additive effects of ZEN and its metabolites are of importance and could have repercussions in vivo.

In vivo metabolism of masked or conjugated mycotoxins is poorly documented as standards are not commercially available and indirect analysis using hydrolytic enzymes is difficult to validate and cumbersome. We synthesised zearalenone-14-glucoside (ZEA-14G) chemically. Deoxynivalenol-3-glucuronide (DON-3GlcA) and glucuronides of 3- and 15-acetyl-deoxynivalenol (3- and 15-ADON-GlcAs), de-epoxydeoxynivalenol, zearalenone (ZEA), α- and β-zearalenol (α- and β-ZOL) were synthesised using rat microsomes. For the first time three ADON-GlcAs were synthesised: two 3-ADON-GlcAs and one 15-ADON-GlcA. After purification, the masked mycotoxin and the metabolites were characterised by NMR (DON-3GlcA, ZEA-14G) or by full scan MS, MS/MS fragmentation, UV-spectra, β-glucosidase and β-glucuronidase treatment. In a first experiment, rats were fed orally DON-3-glucoside (DON-3G) and ZEA-14G, together with 13C-DON and 13C-ZEA and were sacrificed after 55 minutes. A total of 21 masked metabolites, metabolites and parent mycotoxins were quantified in rat organs. Whereas DON-3G was hardly hydrolysed in the stomach, ZEA was clearly formed from ZEA-14G. In a second experiment, 3- and 15-ADON were given orally to rats. The acetylated forms of DON were hydrolysed in the stomach, in contrast to DON-3G. Rats can directly glucuronidate ADONs without deacetylation. Neither DOM, α- or β-ZOL nor their glucuronides could be quantified. Glucuronidated 3-ADON accumulated in the small intestines, together with DON-3GlcA in rats fed orally with 3- and 15-ADON. These differences in masked mycotoxins metabolism can be important in risk analysis of masked mycotoxins in food and feed.

In a recent study, a population of Fusarium strains isolated from maize in Belgium was described as a new species, F. temperatum, that is morphologically similar and phylogenetically closely related to F. subglutinans, a species in the American clade of the Gibberella fujikuroi species complex. In fields, the F. temperatum:F. subglutinans ratio was very high, suggesting that F. temperatum outcompetes its sister species F. subglutinans. This raised the question whether this novel species contributes to the final rot symptoms observed on maize plants at harvest, as well as to the potential mycotoxin contamination. Results of the pathogenicity tests by soil and toothpick inoculation demonstrate the ability of F. temperatum to cause seedling malformation and stalk rot under greenhouse conditions. Screening of 15 Fusarium mycotoxins showed the ability of F. temperatum to produce moniliformin, beauvericin, enniatins and fumonisin B1. The results indicate that F. temperatum can produce mycotoxins and cause maize diseases and, therefore, poses a potential risk to maize production and to the safety of human food and animal feed.

Cross-reactivity of antibodies in AGRAQUANT, DON EIA, VERATOX, ROSA LF-DONQ, and MYCONTROLDON designed for deoxynivalenol (DON) determination in food and feedstuffs was evaluated against nivalenol, 3-acetylDON, 15-acetylDON, de-epoxy metabolite 1 of DON, DON-3β-glucoside, T2-toxin, HT2-toxin, fusarenone X, diacetoxyscirpenol, verrucarol, and zearalenone. Cross-reactivity measurements were run in water using the 50% reduction of absorbance of the blank for ELISA kits or through direct DON determination upon using the standards of mycotoxins via ROSA LF-DONQ or MYCONTROLDON. For the tested toxin concentrations, all DON kits have low cross-reactivity toward diacetoxyscirpenol, T2-toxin, HT2-toxin, verrucarol, and zearalenone and moderate cross-reactivity toward 15-AcetylDON and fusarenone X. AGRAQUANT, DON EIA, and VERATOX kits showed high cross-reactivity in various ranking orders against DON-3-Glc, DOM-1, and 3AcDON. DON EIA showed also high cross-reactivity against nivalenol and fusarenone X. These mycotoxins could coexist in food or feedstuffs, and analytical results can be wrongly interpreted. Cross-reactivity does not allow checking the compliance with the legal norms, but it does allow an overall risk assessment for the consumers. Updating regularly the cross-reactivity evaluation of the produced batches is recommended for 3-acetylDON, nivalenol, DON-3-Glc, de-epoxy metabolite 1, and fusarenone X.

Mycotoxins are harmful food contaminants. Currently, human exposure assessment to these toxins is often based on calculations combining mycotoxin occurrence data in food with population data on food consumption. Because of limitations inherent to that approach, biomarkers have been proposed as a suitable alternative whereby a more accurate assessment of exposure at the individual level can be performed. The BIOMYCO study is designed to assess human mycotoxin exposure using urinary biomarkers of exposure. Over the different seasons of 2013 and 2014, morning urine is gathered in a representative part of the Belgian population according to a designed study protocol, whereby 140 children (3-12 years old) and 278 adults (19-65 years old) are selected based on random cluster sampling stratified for sex, age and geographical areas. Every participant completes a food frequency questionnaire to assess the consumption of relevant foodstuffs (n = 43) of both the day before the urine collection and the previous month. Validated multi-toxin LC-MS/MS methods are used to analyse aflatoxins, fumonisins, ochratoxin A, trichothecenes, zearalenone and their metabolites in morning urine. The study protocol is approved by the ethical committee of the Ghent University Hospital. Within this paper, study design and methods are described. The BIOMYCO study is the first study whereby a multi-toxin approach is applied for mycotoxin exposure assessment in adults and children on a large scale. Moreover, it is the first study that will describe the exposure to an elaborated set of mycotoxins in the Belgian population. In first instance, descriptive analysis will be performed, describing the exposure to mycotoxins for the child and adult group. Exposure of different subgroups will be compared. Furthermore, correlations between the mycotoxin concentrations measured and the food consumption reported will be estimated to explore whether the mycotoxin exposure could be explained by the consumption of certain foods.

A direct, fast and sensitive LC–MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25 % of the samples, while deoxynivalenol was detected in 60 % of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70 % of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90 % of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC–MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.

Two separable anthocyanin acyltransferases were identified in cell cultures of Ajuga reptans which accumulate acylated cyanidin and delphinidin-based anthocyanins. One catalysed the transfer of hydroxycinnamic acids from their CoA-esters to cyanidin 3-sophoroside, anthocyanidin 3,5-diglucosides, anthocyanidin 3-sophoroside-5-glucosides and delphinidin 3-glucoside. The other transferred the malonyl moiety from malonyl-CoA preferably to anthocyanidin 3-coumaroylglucoside-5-glucosides and anthocyanidin 3-glucosides. Anthocyanidin 3,5-diglucosides, anthocyanidin 3-sophoroside-5-glucosides, anthocyanidin 3-biosides and cyanidin 3,3′-diglucoside were less effective substrates for the malonyltransferase. The substrate specificity was rather broad, as anthocyanins which were not detected in cell cultures or plants of A. reptans were acylated as well. In vitro the hydroxycinnamoyltransferase transferred only one acyl group per anthocyanin molecule, while in vivo the main anthocyanins of callus, suspension cultures and flowers of A. reptans contain two aromatic acyl groups per pigment molecule. The hydroxycinnamoyltransferase activity was totally absent in acyanic calli, while the malonyltransferase activity could be detected in two acyanic callus lines of A. reptans . The hydroxycinnamoyltransferase was inhibited by several divalent ions.

An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, β-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10 mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13 ng g−1; those for the limit of quantification from 10 to 26 ng g−1. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.

Seven commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites (de-epoxydeoxynivalenol, α-zearalenol, β-zearalenol) and nivalenol (NIV), using a semi-quantitative multi-mycotoxin ultra-performance liquid chromatography-tandem mass spectrometry method. The DON IACs showed cross-reactivity for nearly all DON derivatives tested. The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives. The IACs were evaluated for their potential use as sample clean-up for mycotoxins in serum.

A comparison of matched pairs deoxynivalenol (DON) loads in wheat samples via VERATOX for DON 5/5 performed by two laboratories against two liquid chromatographic methods (LC-MS/MS and HPLC-UV) used by two other laboratories was carried out using biometrical and sum of ranking differences (SRD) procedures. The Lin’s Concordance correlation coefficients, the average discrepancies, the limits of agreement and the SRD between ELISA and reference values showed good overall agreement between VERATOX for DON 5/5 and reference methods for the two datasets. The VERATOX kits are valuable for quantitative screening and even for an initial exposure assessment in situations when there are practical or economical reasons not to use sophisticated methods such as HPLC or GC methods (with or without MS). However, networking of laboratories using this rapid method and laboratories with reference analytical methods should be encouraged.

Four anthocyanins from Ajuga reptans flowers and its cell cultures were isolated, and a fifth was also characterized by HPLC-mass spectrometry. By means of chemical and spectroscopic analyses, their structures were identified as delphinidin 3-(p-coumaroyl-feruloyl)sophoroside-5-malonylglucoside, delphinidin 3-(diferuloyl)sophoroside-5-malonylglucoside, and cyanidin 3-(di-p-coumaroyl)sophoroside-5-glucoside, respectively. The other two were tentatively identified as delphinidin 3-(diferuloyl)sophoroside-5-glucoside and cyanidin 3-(feruloyl-p-coumaroyl)sophoroside-5-malonylglucoside. In neutral aqueous solution, the crude extract from A. reptans flower cell cultures and the major anthocyanin cyanidin 3-(di-p-coumaroyl)sophoroside-5-malonylglucoside were more stable than cyanidin 3-glucoside, and also prevented more efficiently peroxidation than did the latter. A. reptans flower cell culture anthocyanins may have a potential as natural colorants for food utilities or other purposes.

Four anthocyanins were isolated from Ajuga reptans flowers and one from the cell cultures. By FAB mass spectrometry measurements, the structures of these pigments were determined as delphinidin and cyanidin glucosides acylated with two cinnamic acids, while three of them were also malonylated. A delphinidin-based pigment in the crude extract from cell cultures was identical to the major flower pigment as shown by HPLC co-chromatography. Moreover, by application of 1H and 13C NMR consisting of DQF-COSY, NOESY, ROESY, 2D-HOHAHA, HSQC and HMBC methods, the structures of two new anthocyanins were identified as delphinidin and cyanidin 3-O-(2-O-(6-O-(E)-p- coumaryl-β- d -glucopyranosyl )-(6-O-(E)-p- coumaryl)-β- d -glucopyranosyl )-5-O-(6-O- malonyl-β- d -glucopyranoside ) . The deacylated anthocyanins were confirmed as delphinidin and cyanidin 3-sophoroside-5-glucosides.

Ginkgo biloba is largely consumed as food supplement, because it could contribute to memory improvement by acting on cerebrovascular insufficiency. Among the large variety of phytochemicals contained in Ginkgo biloba leaves and extracts, flavonols (mainly kaempferol, quercetin and isorhamnetin) represent the largest part. Several unique terpenoids (ginkgolides A, B, C, J and bilobalide) are also present. In the present study, we investigated the modulation of cytochrome P450 (CYP) 1A1 enzymatic activity and expression in the intestinal Caco-2 cell line by either Ginkgo biloba extracts, by major individual secondary metabolites or by mixtures of these constituents in realistic proportions. Plausible used intestinal concentrations were calculated on the basis of recommended daily intake and quantitative analysis of major constituents in dietary supplements. Preliminary results suggest that (−)-bilobalide, but not the other terpenoids, induces a dose-dependent increase of CYP1A1 activity (measured through 7-ethoxyresorufin O-deethylation, EROD) after 6 h treatment of the Caco-2 cells. RT-PCR highlighted an increase of mRNA after 3 h treatment with (−)-bilobalide, suggesting the involvement of a transcriptional mechanism. Furthermore, a strong dose-dependent inhibition of benzo[a]pyrene (B[a]P)-induced EROD activity was shown after 6 h co-treatment with kaempferol, quercetin or isorhamnetin. This antagonistic effect was conserved with mixtures of the three flavonols and of the eight pre-cited major secondary metabolites. These preliminary results suggest that frequent consumption of Ginkgo biloba-based dietary supplements may influence CYP1A1 expression and activity in intestinal cells, leading to possible toxicological interactions.

Sucrose and glucose are the most effective carbon sources in supporting high growth rates and biomass yields in plant cell cultures. Media based on waste material such as milk whey and molasses have been tested also (Fowler and Stepan-Sarkissian 1985), but no growth data in these media have been reported (Berlin and Sasse 1985; Fowler 1982).

Lactose utilisation by cucumber cell suspension cultures starts only after a long lag phase and is accompanied by an increase of an extracellular lactosespecific β-galactosidase activity. Supplementing the lactose medium with sucrose shortens the lag phase. Milk whey permeate seems to contain a factor(s) which inhibits lactose utilisation. After supplementing the medium with sucrose or its hydrolysis products, growth and substrate utilisation is as efficient as in Murashige and Skoog medium. Galactose also induces growth, but growth and substrate utilisation are slower. In whey medium, supplemented with sucrose, the extracellular β-galactosidase activity again accompanies growth induction.

Large doses of gamma-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced.Application of gibberellic acid restores the endomitotic DNA synthesis and the cell elongation in epicotyls of irradiated seeds. The cortex cells reach again the 8 C DNA level in darkness.The results suggest that gamma-irradiation blocks endomitotic DNA synthesis and cell elongation by lowering the concentration of endogenous gibberellins.