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Organisationseinheit der BAM
Orally ingested nanoparticles may overcome the gastrointestinal barrier, reach the circulatory system, be distributed in the organism and cause adverse health effects. However, ingested nanoparticles have to pass through different physicochemical environments, which may alter their properties before they reach the intestinal cells. In this study, silver nanoparticles are characterised physicochemically during the course of artificial digestion to simulate the biochemical processes occurring during digestion. Their cytotoxicity on intestinal cells was investigated using the Caco-2 cell model. Using field-flow fractionation combined with dynamic light scattering and small-angle X-ray scattering, the authors found that particles only partially aggregate as a result of the digestive process. Cell viabilities were determined by means of CellTiter-Blue® assay, 4',6-diamidino-2-phenylindole-staining and real-time impedance. These measurements reveal small differences between digested and undigested particles (1–100 µg/ml or 1–69 particles/cell). The findings suggest that silver nanoparticles may indeed overcome the gastrointestinal juices in their particulate form without forming large quantities of aggregates. Consequently, the authors presume that the particles can reach the intestinal epithelial cells after ingestion with only a slight reduction in their cytotoxic potential. The study indicates that it is important to determine the impact of body fluids on the nanoparticles of interest to provide a reliable interpretation of their nano-specific cytotoxicity testing in vivo and in vitro.
The hepatotoxin phomopsin A (PHO-A), a secondary metabolite mainly produced by the fungus Diaporthe toxica, occurs predominantly on sweet lupins. Along with the growing interest in sweet lupins for food and feed commodities, concerns have been raised about fungal infestations, and consequently, about the determination of PHO-A.
High performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) represents the most suitable analytical technique for sensitive and selective detection of mycotoxins including PHO-A. However, isotopic labeled substances are needed as internal standards for a reliable and convenient quantification.
As no isotope standard for PHO-A is currently available, a biosynthesis of fully 15N6-labeled PHO-A was established by cultivation of D. toxica on defined media containing Na15NO3 and 15N-labeled yeast extract as the only nitrogen sources. The identity of 15N6-PHO-A was confirmed by high resolution mass spectrometry.
The new 15N6-labeled standard will facilitate the method development for PHO-A including a more accurate quantification by LC-MS/MS.
Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[15N5]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na15NO3-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103 %; the limit of quantification was 15 ng/L in freshwater and 50 µg/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8 ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0 µg/kg fresh weight and 23.9 µg/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis.
Zearalenone (ZEN) and its sulfate and glucoside conjugates have been detected in (a broad variety of) food and feed commodities1. Both conjugated derivatives are formed as part of fungal or plant secondary metabolism and thus, belong to the group of modified mycotoxins2. After consumption of contaminated foodstuff, the conjugates can be hydrolyzed by human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. In order to include ZEN conjugates in routine analysis, as well as for toxicological investigation reliable standards are needed.
The objective of the present study was to develop a simple and economic method for biosynthesis of ZEN conjugates. Preceding experiments on the biotransformation of ZEN by Rhizopus and Aspergillus species showed a mixed metabolite formation3. Therefore, these known ZEN conjugating fungal strains were screened for their potential to selectively synthesize the ZEN derivatives ZEN-14-sulfate (Z14S), ZEN-14-glucoside (Z14G) and ZEN-16-glucoside (Z16G). The screening was conducted by adding ZEN to liquid fungal cultures. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. Z14S was exclusively formed by A. oryzae. Under optimized conditions a specific biosynthesis of Z14G by R. oryzae and Z16G by R. oligosporus was achieved. After liquid-liquid-extraction and preparative chromatographic cleanup 1H-NMR purities of ≥ 73% for Z14S, ≥ 82% for Z14G and ≥ 50% for Z16G were obtained. In addition, a consecutive biosynthesis was developed by first using Fusarium graminearum for ZEN biosynthesis on rice based liquid medium. After inactivation of Fusarium the subsequent conjugation reaction was conducted utilizing Aspergillus and Rhizopus species under the various optimized conditions.
In this study an easy and cost-efficient biosynthesis for Z14S, Z14G and Z16G was developed. The developed biosynthesis could be also used for other metabolites like ZEL conjugates. Our results of the in vitro screening indicate also the formation of a ZEL-glucoside and α ZEL-sulfate as major metabolites by R. oryzae. In sum, under optimized cultivation conditions fungi can be easily utilized for a targeted and stereospecific synthesis of ZEN conjugates.
Biotransformation of the mycotoxin zearalenone by fungi of the genera Rhizopus and Aspergillus
(2014)
Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin biosynthesized by various Fusarium fungi. These fungal species frequently infest grains; therefore, ZEN represents a common contaminant in cereal products. The biotransformation of ZEN differs significantly from species to species, and several metabolites are known to be formed by animals, plants, and microorganisms. The aim of the present study was to investigate the microbial conversion of ZEN by species of the genera Rhizopus and Aspergillus representing relevant fungi for food processing (e.g. fermentation). To monitor the ZEN metabolism, ZEN was added to liquid cultures of the different fungal species. After a period of 3 days, the media were analyzed by HPLC-MS/MS for metabolite formation. Two Aspergillus oryzae strains and all seven Rhizopus species were able to convert ZEN into various metabolites, including ZEN-14-sulfate as well as ZEN-O-14- and ZEN-O-16-glucoside. Microbial transformation of ZEN into the significantly more estrogenic α-zearalenol (α-ZEL) was also observed. Additionally, a novel fungal metabolite, α-ZEL-sulfate, was detected. Semi-quantification of the main metabolites indicates that more than 50% of initial ZEN may be modified. The results show that fungal strains have the potential to convert ZEN into various metabolites leading to a masking of the toxin, for example in fermented food.
The biotoxin okadaic acid (OA), produced by dinoflagellates in marine environment, can accumulate in sponges and shellfish. Consumption of contaminated shellfish induces acute toxic effects such as diarrhea, nausea, vomiting, and abdominal pain. CYP3A4, one of the most important human xenobiotic metabolizing enzymes, is supposed to be involved in the metabolism of OA. Aim of our study was to evaluate the role of CYP3A4 in OA in vitro metabolism as well as in cell cytotoxicity in parallel. Therefore, a metabolic competent HepaRG cell line was exposed to OA with and without addition of the CYP3A4 inhibitor ketoconazole. Without the inhibitor, two mono-hydroxylated metabolites could be identified, whereas in its presence, no metabolites could be detected. Confirmation of the formed metabolites was accomplished by measuring the exact masses and investigating the fragmentation pattern. Data obtained from cytotoxicity assays showed that OA cytotoxicity is reduced when CYP3A4 is active. Thus, hydroxylation appears to be a crucial step for metabolic OA detoxification.
The degradation and epimerization of ergot alkaloids (EAs) in rye flour were investigated after baking cookies and subsequently subjecting them to an in vitro digestion model. Different steps of digestion were analyzed using salivary, gastric, and duodenal juices. The degradation and bidirectional conversion of the toxicologically relevant (R)-epimers and the biologically inactive (S)-epimers for seven pairs of EAs were determined by a HPLC method coupled with fluorescence detection. Baking cookies resulted in degradation of EAs (2–30 %) and a shift in the epimeric ratio toward the (S)-epimer for all EAs. The applied digestion model led to a selective toxification of ergotamine and ergosine, two ergotamine-type EAs. The initial percentage of the toxic (R)-epimer in relation to the total toxin content was considerably increased after digestion of cookies. Ergotamine and ergosine increased from 32 to 51 % and 35 to 55 %, respectively. In contrast, EAs of the ergotoxine type (ergocornine, α- and β-ergocryptine, and ergocristine) showed an epimeric shift toward their biologically inactive (S)-epimers. Further experiments indicated that the selective epimerization of ergotamine EAs occurs in the duodenal juice only. These results demonstrate that toxification of EAs in the intestinal tract should be taken into consideration.
Recently, there has been a rise in freshwater harmful algal blooms (HABs) globally, as well as increasing aquaculture practices. HABs can produce cyanotoxins, many of which are hepatotoxins. An ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated for nine cyanotoxins across three classes including six microcystins, nodularin, cylindrospermopsin and anatoxin-a. The method was used to analyse free cyanotoxin(s) in muscle (n = 34), liver (n = 17) and egg (n = 9) tissue samples of 34 fish sourced from aquaculture farms in Southeast Asia. Conjugated microcystin was analysed by Lemieux oxidation to ascertain the total amount of microcystin present in muscle. Some tilapia accumulated free microcystin-LR in the muscle tissue at a mean of 15.45 μg/kg dry weight (dw), with total microcystin levels detected at a mean level of 110.1 μg/kg dw, indicating that the amount of conjugated or masked microcystin present in the fish muscle accounted for 85% of the total. Higher levels of cyanotoxin were detected in the livers, with approximately 60% of those tested being positive for microcystin-LR and microcystin-LF, along with cylindrospermopsin. Two fish from one of the aquaculture farms contained cylindrospermopsin in the eggs; the first time this has been reported. The estimated daily intake for free and total microcystins in fish muscle tissue was 2 and 14 times higher, respectively, than the tolerable daily intake value. This survey presents the requirement for further monitoring of cyanotoxins, including masked microcystins, in aquaculture farming in these regions and beyond, along with the implementation of guidelines to safeguard human health.
Mycotoxins are natural contaminants produced by a range of fungal species. Their common occurrence in food and feed poses a threat to the health of humans and animals. This threat is caused either by the direct contamination of agricultural commodities or by a "carry-over" of mycotoxins and their metabolites into animal tissues, milk, and eggs after feeding of contaminated hay or corn. As a consequence of their diverse chemical structures and varying physical properties, mycotoxins exhibit a wide range of biological effects. Individual mycotoxins can be genotoxic, mutagenic, carcinogenic, teratogenic, and oestrogenic. To protect consumer health and to reduce economic losses, surveillance and control of mycotoxins in food and feed has become a major objective for producers, regulatory authorities and researchers worldwide. However, the variety of chemical structures makes it impossible to use one single technique for mycotoxin analysis. Hence, a vast number of analytical methods has been developed and validated. The heterogeneity of food matrices combined with the demand for a fast, simultaneous and accurate determination of multiple mycotoxins creates enormous challenges for routine analysis. The most crucial issues will be discussed in this review. These are (1) the collection of representative samples, (2) the performance of classical and emerging analytical methods based on chromatographic or immunochemical techniques, (3) the validation of official methods for enforcement, and (4) the limitations and future prospects of the current methods.
Mycotoxins are among the most abundant contaminants in food and feed worldwide. Therefore, in the EU maximum levels are established, e.g. for the frequently occurring Fusarium toxins deoxynivalenol (DON) and zearalenone (ZEA). Additional to DON and ZEA, modified mycotoxins are present in naturally contaminated grain products contributing significantly to the exposure of humans and animals with mycotoxins. Up to now data on the spatial distribution of many (masked) mycotoxins in the kernels of wheat are missing. The aim of the present study was to investigate the amounts of DON and ZEA as well as their most abundant derivatives DON-3-glucoside (DON-3G), 3- and 15-acetyl-DON, ZEA-14- and 16-glucoside and ZEA-14-sulphate (ZEA-14S) in mill fractions of naturally contaminated wheat batches using HPLC-MS/MS. The investigated distribution pattern in ten milling fractions is comparable among the three investigated different wheat batches. Interestingly, DON and DON-3G were found to be present to similar amounts in all fractions. In bran, the levels were only slightly higher than in the endosperm. By contrast, for ZEA and ZEA-14S a significantly higher amount of toxin is located in the fibre-rich fractions. The relative mass proportion of DON-3G comprises for only between 2.9 and 11.2% of the free DON, while the relative mass proportion of ZEA-14S is estimated to even exceed the amount of free ZEA in certain fractions. Acetylated DON derivatives and ZEA-glucosides were only detected in low amounts. The experimental results show that a significant reduction of the ZEA and ZEA-14S level in wheat flour is feasible by applying milling technology strategies. However, the almost evenly distribution of DON and DON-3G in all fractions does not allow for the technological removal of relevant toxin amounts. Furthermore, the relative share of masked forms was higher for ZEA derivatives than for the DON conjugates in the investigated wheat lots.