Dose-response study on the transfer of pyrrolizidine alkaloids from a tansy ragwort extract (Jacobaea vulgaris Gaertn.) to bovine milk.

Ragworts like tansy ragwort (J. vulgaris Gaertn., syn. Senecio jacobaea L.) contain hepatotoxic and cancerogenic pyrrolizidine alkaloids (PA) and their corresponding pyrrolizidine alkaloid N-oxides (PANO). Due to increasing spread of ragworts (Jacobaea spp.) PA/PANO may pose a health risk to animals and humans consuming contaminated feed and food. Therefore, the aim of the present study was to investigate the transfer of individual PA/PANO originating from a well-defined PA/PANO extract into the milk of dairy cows. For this objective, 16 German Holstein cows were assigned to four treatment groups (n = 4) in a 28-day dose-response study. Administration into the reticulorumen was performed daily by gavage after the morning milking. Three groups received different amounts of the J. vulgaris extract resulting in a PA/PANO exposure of 0.47, 0.95, or 1.91 mg PA/PANO/kg body weight/day, respectively. Furthermore, a control group received molasses to account for the sugar content of the used PA/PANO extract. While the composition of the PA/PANO extract was more diverse, the PA/PANO pattern in milk was dominated by the PA in their free base form. It was shown that mainly PA considered stable in the rumen environment were transferred into the milk. The main compounds in milk were jacoline (74.3 ± 2.4% of the PA/PANO sum), jaconine (11.2 ± 1.3%), and jacobine (7.2 ± 0.6%) with concentrations up to 29.7, 4.65 µg/l, or in the highest exposed group, 3.44 µg/l. There was no dose-dependent effect on the total PA/PANO transfer rate into the milk. The average transfer rate was 0.064 ± 0.005% of the administered content.

Pyrrolizidine alkaloids and tropane alkaloids in milk samples from individual dairy farms of the German federal states of Bavaria and Schleswig-Holstein.

1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020-2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC-ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number.

Short-term exposure of dairy cows to pyrrolizidine alkaloids from tansy ragwort (Jacobaea vulgaris Gaertn.): effects on health and performance.

The increasing spread of ragworts is observed with concern. Ragworts like tansy ragwort (Jacobaea vulgaris Gaertn.) or marsh ragwort (J. aquatica) contain pyrrolizidine alkaloids (PA) which may induce hepatotoxic effects. Grazing animals usually avoid ragworts if their pasture management is appropriate. Preserved feed prepared from ragworts contaminated meadows may, however, lead to a significant exposure to PA. Previous studies on toxicity of PA for dairy cows revealed inconsistent results due to feeding ragwort plant material which was associated with heterogeneous PA exposure and thus failed to conclusively deduce critical PA doses. Therefore, the aim of the present study was to expose dairy cows (n = 4 per group) in a short-term scenario for 28 days with increasing PA doses (PA1: 0.47 mg PA/kg body weight (BW)/day (d); PA2: 0.95 mg PA/kg BW/d; PA3: 1.91 mg PA/kg BW/d) via oral administration by gavage of a defined PA-extract. While group PA3 was dosed with the PA-extract alone, groups PA2 and PA1 received PA-extracts blended in similar volumes with molasses to provide comparable amounts of sugar. Additionally, two control groups were treated either with water (CONWater) or with molasses (CONMolasses) to assess the effects of sugar without PA interference. While clinical traits including dry matter intake, milking performance, rectal body temperature, ruminal activity and body condition score (BCS) were not influenced by PA exposure, activities of enzymes indicative for liver damages, such as gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH), increased significantly over time at an exposure of 1.91 mg total PA/kg BW/d.

A sensitive LC-MS/MS method for isomer separation and quantitative determination of 51 pyrrolizidine alkaloids and two tropane alkaloids in cow's milk.

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid-liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.

Structure-Dependent Toxicokinetics of Selected Pyrrolizidine Alkaloids In Vitro.

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.

Fate of Food-Relevant Toxic Plant Alkaloids during Food Processing or Storing and Analytical Strategies to Unveil Potential Transformation Products.

Toxic plant alkaloids (TPAs) may contaminate food and pose a threat to consumer health; as a consequence, they are undesired in foodstuff or food commodities. Similar to other ingredients, TPA may be affected by storing or processing of food, often associated with decreased levels of the parent alkaloids. Up to now, little is known about potential transformation products or if they still may exhibit toxic potential to consumers. This perspective briefly summarizes the current knowledge regarding the behavior of opium, pyrrolizidine, and tropane alkaloids toward processing or storing and highlights analytical strategies to identify and elucidate potential transformation products.

Organic Cation Transporter I and Na+ /taurocholate Co-Transporting Polypeptide are Involved in Retrorsine- and Senecionine-Induced Hepatotoxicity in HepaRG cells.

SCOPE: 1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity. METHODS AND RESULTS: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na+ /taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA-mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time-dependent manner and attenuated PA-mediated cytotoxic effects in HepaRG cells. CONCLUSION: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.

Active Transport of Hepatotoxic Pyrrolizidine Alkaloids in HepaRG Cells.

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.

Distribution of T-2 toxin and HT-2 toxin during experimental feeding of yellow mealworm (Tenebrio molitor).

Within the European Union (EU), edible insects need to be approved as "Novel Food" according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 µg/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.

Development of two specific multiplex qPCRs to determine amounts of Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta and Staphylococcus in meat and heat-treated meat products.

Culturing methods are conventionally applied to investigate the contamination of food with several microorganisms after heat processing. However, with these methods, it is not possible to evaluate whether heat-treated meat products, such as cooked sausages, contained parts of spoiled meat. Therefore, two specific multiplex qPCRs were developed in this study in order to determine the microbiological quality of the raw materials used for these products. The PCR targets focused on four bacterial groups often found on meat (family Enterobacteriaceae, genus Pseudomonas, genus Staphylococcus and species Brochothrix thermosphacta). Specificity as well as sensitivity of the developed multiplex qPCRs, validated by using 68 microbial species, were 100%. The applicability of both multiplex qPCRs compared to culturing methods was performed using 96 meat samples (fresh and naturally spoiled) and 12 inhouse-made "Lyoner" sausages containing variable ratios of spoiled meat (0%, 5%, 12% and 25%; n = 3 for each group). Both methods showed similar results by evaluating the ∆log10 cfu/g, the relative accuracy and the t-test analysis (p > 0.05). Comparing qPCR results of the different sausage groups, a significant difference between sausages containing fresh meat and sausages containing spoiled meat (12% and 25%) was found only for Pseudomonas and B. thermosphacta in both raw and cooked sausages. The statistical difference between 5% vs. 12% and 25% spoiled meat in cooked sausages, was also found only for these two bacterial groups. The developed multiplex qPCRs were further applied to 30 commercially available "Bologna-type" sausages. The results showed a total of 14 sausages considered to be suspicious for Food Fraud. While the role of Staphylococcus spp. in meat spoilage remains unclear, Pseudomonas, Enterobacteriaceae and B. thermosphacta could together be used as an indicator for "spoiled meat" used in sausages. The developed qPCR systems in this study allow the detection of four relevant bacterial groups in the heated Bologna-type sausages and provide information about the hygienic quality of raw materials used. This method could thus be helpful for screening food suspected of Food Fraud.

Spread of Jacobaea vulgaris and Occurrence of Pyrrolizidine Alkaloids in Regionally Produced Honeys from Northern Germany: Inter- and Intra-Site Variations and Risk Assessment for Special Consumer Groups.

Pyrrolizidine alkaloids (PA) and PA N-oxides (PANO) are secondary plant metabolites exhibiting genotoxic and carcinogenic properties. Apart from the roots and leaves, PA/PANO are particularly present in pollen and nectar. Therefore, the spread of Jacobaea vulgaris in certain regions of northern Germany has an impact on the safety of honey produced in that region. In this study, raw honey samples (n = 437) were collected from usually three individual beehives per site (n = 73) in the district of Ostholstein and analyzed for 25 PA/PANO. The results reveal mean levels of 8.4, 1.5, and 72.6 µg/kg and maximum levels of 111, 59.4, and 3313 µg/kg, depending on the season (summer 2015 and spring/summer 2016, respectively). As far as individual data are concerned, sites near areas with J. vulgaris growth did not necessarily result in high PA/PANO values. Furthermore, intra-site investigations revealed remarkable differences in PA/PANO levels of raw honey collected by different bee colonies at the same site. Consumption of these regionally produced honeys entails an increased exposure to PA/PANO, especially in children and high consumers. Margin of exposure values of <10,000 and an exceedance of the health-based guidance value highlight that regionally produced and marketed honey must be considered with care for a proper risk assessment and risk management.

Occurrence and Risk Assessment of Pyrrolizidine Alkaloids in Spices and Culinary Herbs from Various Geographical Origins.

Pyrrolizidine alkaloids (PA) and their N­oxides (PANO) are a group of toxic secondary plant metabolites occurring predominantly as contaminants in (herbal) teas, honeys and food supplements, as well as in spices and culinary herbs. Depending on the botanical origin of the contaminating plant, the pattern of PA/PANO can strongly vary within a sample. The current study aimed to broaden the existing data on the occurrence of PA/PANO in spices and culinary herbs. For this, 305 authentic samples covering 15 different matrices mainly harvested in 2016 or 2017 and originating from 36 countries were investigated for the presence of 44 PA/PANO. Fifty-eight percent of the samples contained at least one PA/PANO. The average sum content over all samples was 323 µg/kg (median of 0.9 µg/kg, 95% percentile of 665 µg/kg). The highest amount of 24.6 mg/kg was detected in an oregano sample. Additionally, conspicuous analyte patterns were discovered in samples from similar cultivation regions, indicating related botanical sources of PA/PANO contaminations. Particularly, oregano and cumin from Turkey often contained high amounts of PA/PANO. The results were used to assess the acute and chronic health risks related to PA/PANO intake via spices and culinary herbs, indicating a potential health risk in particular for adults and children with high consumption or when considering worst­case contamination scenarios of a sum content of 5500 µg/kg.

Correction to: truncated satratoxin gene clusters in selected isolates of the atranone chemotype of Stachybotrys chartarum (Ehrenb.) S. Hughes.

The original version of this paper was published with error. The supplementary materials originally provided were not captured during article production.

Truncated satratoxin gene clusters in selected isolates of the atranone chemotype of Stachybotrys chartarum (Ehrenb.) S. Hughes.

The fungus Stachybotrys (S.) chartarum was isolated from culinary herbs, damp building materials, and improperly stored animal forage. Two distinct chemotypes of the fungus were described that produced either high-cytotoxic macrocyclic trichothecenes (S type) or low-cytotoxic atranones (A type). Recently, two distinct gene clusters were described that were found to be necessary for the biosynthesis of either macrocyclic trichothecenes (21 SAT (Satratoxin) genes) or atranones (14 ATR (Atranone) genes). In the current study, PCR primers were designed to detect SAT and ATR genes in 19 S. chartarum chemotype S and eight S. chartarum chemotype A strains. Our analysis revealed the existence of three different genotypes: satratoxin-producing strains that harbored all SAT genes but lacked the ATR gene cluster (genotype S), non-satratoxin-producing strains that possessed the ATR genes but lacked SAT genes (genotype A), and a hitherto undescribed hybrid genotype among non-satratoxin-producing strains that harbored all ATR genes and an incomplete set of SAT genes (genotype H). In order to improve the discrimination of genotypes, a triplex PCR assay was developed and applied for the analysis of S. chartarum and S. chlorohalonata cultures. The results show that genes for macrocyclic trichothecenes and atranones are not mutually exclusive in S. chartarum. Correlation of the new genotype-based concept with mycotoxin production data shows also that macrocyclic trichothecenes are exclusively produced by S. chartarum genotype S strains.

Development of a sensitive analytical method for determining 44 pyrrolizidine alkaloids in teas and herbal teas via LC-ESI-MS/MS.

Pyrrolizidine alkaloids (PA) and PA-N-oxides (PANO) are a large group of secondary plant metabolites comprising more than 660 compounds. Exhibiting geno- and hepatotoxic properties, they are responsible for multiple cases of food and feed poisoning over the last 100 years. For food and feed safety reasons, relevant PA/PANO should be monitored extensively in the main sources of PA/PANO intake. In this study, a sensitive analytical method was developed for detecting a broad range of 44 commercially available PA/PANO compounds, and in-house validation procedures were performed for several (herbal) teas. Various extraction solvents and procedures, as well as solid phase extraction materials for sample clean-up and analyte concentration, were tested to establish the methods' efficiency and effectiveness. Chromatographic conditions were optimised to obtain the best possible separation of isomers for the 44 PA/PANO analytes. The final method was proven very sensitive and accurate, with detection limits ranging from 0.1 to 7.0 µg/kg and precisions between 0.7 and 16.1%. For 40 of the analytes, the recovery rates ranged from 60.7 to 128.8%. The applicability and trueness of the method were examined by analysing tea samples from a local supermarket and comparing them to a reference material. At least one PA/PANO analyte was detected in 17 of the 18 samples under investigation, and the sum contents of the samples ranged from 0.1 to 47.9 µg/kg. Knowledge of the PA/PANO composition in a sample can be used to indicate the botanical origin of the impurity and, thus, the geographical region of cultivation.

Uncertainties in the determination of pyrrolizidine alkaloid levels in naturally contaminated honeys and comparison of results obtained by different analytical approaches.

The contamination of honey with hepatotoxic pyrrolizidine alkaloids (PAs) is a well-known hazard for food safety. While management strategies and controls of the honey industry aim to reduce the PA levels, uncertainties remain with regard to the safety of regionally produced and marketed honey. In addition, a previous study showed large differences of results obtained after various periods of storage and apparent differences between the analytical results of different laboratories. Therefore, this study aimed at examining these uncertainties by monitoring the impact of storage on the PA and PA N-oxide (PANO) content of two freshly harvested honeys and on possible demixing effects caused by pollen settling. Additionally, three analytical approaches - target analysis with matrix-matched calibration or standard addition and a sum parameter method - were applied for a comparative analysis of 20 honeys harvested in summer 2016. All samples originated from Schleswig-Holstein in Northern Germany where the PA plant Jacobaea vulgaris is currently observed on a massive scale. The results of the time series analyses showed that PANO levels markedly decreased within a few weeks and practically reached the LOD 16 weeks after harvest. Tertiary PAs, by contrast, remained stable and did not increase as a consequence of PANO decrease. The experiments on a putative demixing, which may result in a heterogeneous distribution of PAs/PANOs, revealed that there was no such effect during storage of up to 12 weeks. A comparison of the PA/PANO levels obtained by different analytical approaches showed that in some cases the sum parameter method yielded much higher levels than the target approaches, whereas in other cases, the target analysis with standard addition found higher levels than the other two methods. In summary, the results of this study highlight uncertainties regarding the validity and comparability of analytical results and consequently regarding health risk assessment.

Influence of Storage on the Stability of Toxic Pyrrolizidine Alkaloids and Their N-Oxides in Peppermint Tea, Hay, and Honey.

1,2-Dehydropyrrolizidine alkaloids (PA) and PA- N-oxides (PANO) are phytotoxins, which presumably occur in more than 6,000 plant species worldwide. Plants containing PA/PANO are responsible for various food and feed poisonings recorded for decades. Main reasons of exposition of consumers and livestock are contaminations of food and animal feed with parts, seeds, pollen, or nectar of PA-containing plants. Concerning stability, effects of processing on PA were mainly investigated in the past. The current study examined the behavior of PA/PANO in unprocessed matrices peppermint tea, hay, and honey during storage. Blank samples were fortified with PA/PANO or contaminated with blueweed ( Echium vulgare) and ragwort ( Senecio jacobaea) and stored for 182 d. The time-series analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that all 25 analyzed PA/PANO compounds remained stable in herbal samples. However, the results showed a very fast decrease of PANO in honey samples within hours. These results were discussed with respect to potential consequences for health risk assessment.