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.

Cannabinoid contents in hemp teas and estimation of their transfer into tea infusions.

This study focused on the investigation of cannabinoid profiles and contents of 23 different hemp teas and on the individual transfer of 16 cannabinoids from hemp teas into their tea infusions. The total cannabinoid content in the dry products averaged 14,960 mg kg-1, with CBD&CBDA (sum of cannabidiol (CBD) and cannabidiolic acid (CBDA)) being the major component, accounting for 87% of the total cannabinoid content. The Δ9-tetrahydrocannabinol (Δ9-THC) content ranged from 16 mg kg-1 to 935 mg kg-1 and was on average 221 mg kg-1. For each hemp tea, an infusion was prepared according to a standardized protocol issued by the German Standardisation body DIN and transfer rates per cannabinoid were estimated by comparing the contents in the dry material with the concentrations in the aqueous infusion. The limited water solubility of cannabinoids results in limited extraction efficiency for cannabinoids using boiling water to prepare a tea infusion and the average transfer rate of the psychoactive Δ9-THC was only 0.5%.

Rejection behaviour of horses for hay contaminated with meadow saffron (Colchicum autumnale L.).

BACKGROUND: Extensively used grasslands are frequently utilised for hay production for equines. Especially, extensive meadows have a great variety of plant species, which may include plants that are poisonous for equines such as meadow saffron (Colchicum autumnale L.). To authors' knowledge investigations about horses` avoidance behaviour towards dried meadow saffron in hay are missing. Reports of farmers are contrary to clinical symptoms described in case reports and associated with meadow saffron in hay. OBJECTIVES: The aim of this study was to determine the rejection behaviour of horses for hay contaminated with meadow saffron (MS) when fed ad libitum. STUDY DESIGN: An 18-day feeding trial with six adult geldings to observe the rejection behaviour for hay contaminated with MS. METHODS: The horses were fed a basal diet containing hay ad libitum and a mineral supplement during the feeding trial. At six different daytimes, hay contaminated with 1% or 2% dried MS was provided to the horses over a duration of 1 h. The rejection behaviour was observed personally and by video recordings. If a horse ingested more than two plants of MS during one observation period, the observation was stopped and repeated at another day. When the observation period had to be stopped twice, the horse was excluded from the experiment. RESULTS: Five of six horses ingested MS during the first feeding periods. One horse rejected leaves and capsules at the beginning of the study, but it showed repeated ingestion of MS after the seventh observation period. MAIN LIMITATIONS: Lack of knowledge about secondary plant metabolites affecting taste and their variability between fresh and dried plants. CONCLUSIONS: The intake of MS in hay by horses could not be ruled out with certainty. Therefore, feeding hay contaminated with MS should be avoided for equids.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

Metabolic Pattern of Hepatotoxic Pyrrolizidine Alkaloids in Liver Cells.

Contamination with 1,2-unsaturated pyrrolizidine alkaloids (PAs) is a serious problem for certain phytomedicines, foods, and animal feeds. Several of these PAs are genotoxic and carcinogenic, primarily in the liver, upon cytochrome P450 (CYP)-catalyzed activation into reactive (pyrrolic and pyrrole-like) metabolites. Here we investigated the metabolism of selected PAs (echimidine, europine, lasiocarpine, lycopsamine, retrorsine, and senecionine) in rat hepatocytes in primary culture and in human CYP3A4-transfected HepG2 cells. The open-chained diesters echimidine and lasiocarpine and the cyclic diester senecionine were extensively metabolized in rat hepatocytes into a broad spectrum of products released into the medium. A large portion of unidentified, possibly irreversibly bound, products remained in the cells while detectable amounts of reactive and other metabolites were found in the incubation media. In HepG2-CYP3A4 cells, lasiocarpine was more extensively metabolized than echimidine and senecionine which also gave rise to the release of pyrrolic metabolites. In human cells, no pyrrolic metabolites were detected in retrorsine or lycopsamine incubations, while no such metabolites were detected from europine in both cell types. Other types of metabolic changes comprised modifications such as side chain demethylation or oxygenation reactions like the formation of N-oxides. The latter, considered as a detoxification step, was a major pathway with cyclic diesters, was less distinctive for echimidine and lycopsamine and almost negligible for lasiocarpine and europine. Our data are in agreement with previously published cyto- and genotoxicity findings and suggests that the metabolic pattern may contribute substantially to the specific toxic potency of a certain congener. In addition, marked differences were found for certain congeners between rat hepatocytes and transfected human HepG2 cells, whereby a high level of bioactivation was found for lasiocarpine, whereas a very low level of bioactivation was observed for monoesters, in particular in human cells.

Pyrrolizidine alkaloid profiling of four Boraginaceae species from Northern Germany and implications for the analytical scope proposed for monitoring of maximum levels.

Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) have been determined in food and feed at levels relevant for consumer health. More than 660 different PAs have been detected, but few are available as reference substances for analytical demands. In the context of the European legislation on maximum levels of PAs in food products, a defined analytical scope of 21 PAs for determination has been suggested. An expansion of the scope from 21 to 35 PAs, including 14 structural isomers, is currently under discussion. In the present study, a target screening method was established for a comprehensive characterisation of PA profiles of the species Echium vulgare, Heliotropium europaeum, Cynoglossum officinale and Symphytum spp. to assess whether an expansion of the analytical scope is required to quantitatively cover the total PA contents of Boraginaceae species. The scope of the method comprised known and unknown PAs previously screened and confirmed in the respective plant extracts. A total of 176 PAs and PANOs were detected. The toxic 1,2-unsaturated PAs represent the predominant PA type with about 98% of the mean total content. This PA profiling demonstrates that an expansion of the scope from 21 to 35 PAs is required to adequately cover the mean total PA contents of Cynoglossum officinale and H. europaeum, whereas in the case of Symphytum spp. and Echium vulgare an expansion would not be necessary. ABBREVIATIONS: Pyrrolizidine alkaloid: PA, Pyrrolizidine alkaloid N-oxide: PANO, European Food Safety Authority: EFSA, German Federal Institute for Risk Assessment: BfR, ultra-high performance liquid chromatography: UHPLC, high-resolution: HR, tandem mass spectrometer: MS/MS, multiple reaction monitoring: MRM, data-independent MS2: ddms2, electrospray ionisation: ESI, limit of detection: LOD, limit of quantification: LOQ, gas chromatography coupled with mass spectrometry: GC-MS, fragmentation (data): MS2 (data), full scan: MS1, variable data-independent acquisition: vDIA, monoester esterified with a necic acid at position C9 of the necine base: O9-monoester, relative proportion: rel. prop., intraperitoneal: i.p., intravenous: i.v., higher-energy C-trap dissociation: HCD, all-ion fragmentation: AIF, parallel reaction monitoring: PRM.

Pyrrolizidine alkaloids in food and phytomedicine: Occurrence, exposure, toxicity, mechanisms, and risk assessment - A review.

Among naturally occurring plant constituents, the 1,2-unsaturated pyrrolizidine alkaloids (in the following termed 'PAs') play a distinct role because of the large number of congeners occurring in nature and the pronounced toxicity of some congeners. Several PAs are hepatotoxic in humans, experimental and farm animals and were shown to be potent hepatocarcinogens in laboratory rodents. Although the general mode of action leading to toxicity has been elucidated, i.e., being mediated by metabolic conversion of the parent molecule into a highly reactive electrophile capable of attacking cellular target molecules, major questions related to the risk assessment of PAs remain unresolved. It was the aim of a workshop held in September 2018 to shed more light on the occurrence, exposure, mode of action, toxicokinetics and -dynamics of PAs to improve the scientific basis for an advanced toxicological risk assessment. The contributions in nine chapters describe the scientific progress using advanced analytical methods, studies in subcellular fractions, cell culture, experimental animals and humans and the use of PBPK modeling and structure-activity relationship considerations aiming at a better understanding of PA toxicity and genotoxicity. Since PAs differ considerably in their toxic potencies and substantial species differences in sensitivity towards PA exposure exist, a special emphasis was placed on these issues.

Determination of pyrrolizidine alkaloids in tea, herbal drugs and honey.

Honey was previously considered to be one of the main food sources of human pyrrolizidine alkaloid (PA) exposure in Europe. However, comprehensive analyses of honey and tea sampled in the Berlin retail market revealed unexpected high PA amounts in teas. This study comprised the analysis of 87 honey as well as 274 tea samples including black, green, rooibos, melissa, peppermint, chamomile, fennel, nettle, and mixed herbal tea or fruit tea. Total PA concentrations in tea ranged from < LOD to 5647 µg kg(-1), while a mean value of about 10 µg kg(-1) was found in honey samples. Additionally, herbal drugs were investigated to identify the source of PA in teas. Results suggest that PA in tea samples are most likely a contamination caused by co-harvesting of PA-producing plants. In some cases such as fennel, anise or caraway, it cannot be excluded that these plants are able to produce PA themselves.

Structure-activity relationship in the passage of different pyrrolizidine alkaloids through the gastrointestinal barrier: ABCB1 excretes heliotrine and echimidine.

SCOPE: 1,2-Unsaturated pyrrolizidine alkaloids (PA) are found in plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via contaminated food or feed causes severe damage to liver depending on species-specific oral bioavailability. For assessing PA bioavailability, their passage across the intestinal barrier was investigated using Caco-2 cells. METHODS: Differentiated Caco-2 cells were exposed in transport chambers to the PA heliotrine (Hn), echimidine (Em), senecionine (Sc), and senkirkine (Sk). Cell supernatants were analyzed by LC-MS/MS. RESULTS: PA pass Caco-2 monolayer from the apical into basolateral compartment depending on their chemical structure. Compared to the cyclic diesters Sc and Sk with a passage rate of 47% ± 4 and 40% ± 3, respectively, the transferred amount of the monoester Hn (32% ± 3) and open-chained diester Em (13% ± 2) was substantially lower. This suggested an active transport of Hn and Em. Using Madin-Darby canine kidney II/P-glycoprotein (ABCB1)-overexpressing cells, the active excretion of Hn and Em by ABCB1 from the gastrointestinal epithelium into the gut lumen was shown. CONCLUSION: PA cross the intestinal barrier structure-dependently. The passage of the noncyclic PA Hn and Em is reduced by an ABCB1-driven efflux into the gastrointestinal lumen resulting in a decreased oral bioavailability.

Structural screening by multiple reaction monitoring as a new approach for tandem mass spectrometry: presented for the determination of pyrrolizidine alkaloids in plants.

In tandem mass spectrometry the multiple reaction monitoring (MRM) mode is normally used for targeted analysis but this mode also has the potential to screen for structural similarities of analytes. On the basis of the fact that in general similar molecular structures result in similar fragments or losses of neutrals, this approach was used for pyrrolizidine alkaloid (PA) screening but could also be easily adapted to screen for other compound classes. PA are plant toxins of which several hundred individual compounds have been identified. Our MRM screening approach uses the structural relation and similar core structure of all PA which results in a common and thus predictable mass spectrometric fragmentation behaviour. On this basis a method was developed which screens for PA structures by MRM transitions and allows the detection of each individual PA down to a low microgram per kilogram concentration range. The approach was applied to investigate plants from the families of Asteraceae (several species of Senecio and Eupatorium), Boraginaceae (Echium, Cynoglossum, Borago and Anchusa officinalis as well as Heliotropium europaeum) and Fabaceae (Crotalaria incana) for a complete qualitative and quantitative PA characterisation. All analytes that were detected as possible PA by MRM screening were further investigated by recording product ion spectra. Analytes which exhibited a typical PA fragmentation pattern were either confirmed as PA or otherwise deleted as false positive signals (false positive rate was below 10 %). Sum formulas of confirmed PA were determined by additional measurements applying high resolution mass spectrometry. In that way 121 unknown PA were identified and for the first time complete PA profiles of different PA plants were delivered.