Pyrrolizidine Alkaloids N-oxides Research Articles

Effects of dietary exposure to plant toxins on bioaccumulation, survival, and growth of black soldier fly (Hermetia illucens) larvae and lesser mealworm (Alphitobius diaperinus)

In their natural habitat, insects may bioaccumulate toxins from plants for defence against predators. When insects are accidently raised on feed that is contaminated with toxins from co-harvested herbs, this may pose a health risk when used for human or animal consumption. Plant toxins of particular relevance are the pyrrolizidine alkaloids (PAs), which are genotoxic carcinogens produced by a wide variety of plant species and the tropane alkaloids (TAs) which are produced by a number of Solanaceae species. This study aimed to investigate the transfer of these plant toxins from substrates to black soldier fly larvae (BSFL) and lesser mealworm (LMW). PAs and the TAs atropine and scopolamine were added to insect substrate simulating the presence of different PA- or TA-containing herbs, and BSFL and LMW were grown on these substrates. Bioaccumulation from substrate to insects varied widely among the different plant toxins. Highest bioaccumulation was observed for the PAs europine, rinderine and echinatine. For most PAs and for atropine and scopolamine, bioaccumulation was very low. In the substrate, PA N-oxides were quickly converted to the corresponding tertiary amines. More research is needed to verify the findings of this study at larger scale, and to determine the potential role of the insect and/or substrate microbiome in metabolizing these toxins.

In vitro-in silico study on the influence of dose, fraction bioactivated and endpoint used on the relative potency value of pyrrolizidine alkaloid N-oxides compared to parent pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) are phytotoxins found in food, feed and the environment. Yet, limited data exist from which the relative potency of a PA-N-oxide relative to its corresponding PA (REPPANO to PA) can be defined. This study aims to investigate the influence of dose, fraction bioactivated and endpoint on the REPPANO to PA of a series of pyrrolizidine N-oxides using in vitro-in silico data and physiologically based kinetic (PBK) modeling. The first endpoint used to calculate the REPPANO to PA was the ratio of the area under the concentration–time curve of PA resulting from an oral dose of PA-N-oxide divided by that from an equimolar dose of PA (Method 1). The second endpoint was the ratio of the amount of pyrrole-protein adducts formed under these conditions (Method 2). REPPANO to PA values appeared to decrease with increasing dose, with the decrease for Method 2 already starting at lower dose level than for Method 1. At dose levels as low as estimated daily human intakes, REPPANO to PA values amounted to 0.92, 0.81, 0.78, and 0.68 for retrorsine N-oxide, seneciphylline N-oxide, riddelliine N-oxide and senecivernine N-oxide, respectively, and became independent of the dose or fraction bioactivated, because no GSH depletion, saturation of PA clearance or PA-N-oxide reduction occurs. Overall, the results demonstrate the strength of using PBK modeling in defining REPPANO to PA values, thereby substantiating the use of the same approach for other PA-N-oxides for which in vivo data are lacking.

The toxicokinetic and metabolism of structurally diverse pyrrolizidine alkaloids in rats

Ethnopharmacological relevancePyrrolizidine alkaloids (PAs) are a group of phytotoxins present in about 3% of flowering plants worldwide. Ingestion of PA-containing herbal products may lead to hepatotoxicity. Notably, the toxicokinetic (TK) behaviors, especially pyrrole-protein adducts (PPAs) having the same structure but generated from metabolic activation of different PAs, significantly affect the toxicity of structurally diverse PAs, therefore studying them in their pure form is preferable to extracts to stratify toxic potency of different PAs co-existing in herbal extracts. However, previous studies mainly focus on the establishment of TK profiles of the intact PAs, revealing less or no kinetic information on the main PA metabolites (PA N-oxides) and PPAs which mediate PA-induced hepatotoxicity. In this study, PPA was measured as the biomarker of PA exposure and PA-induced toxicity. Aim of studyThis study aims to investigate the TK difference between structurally diverse PAs of retronecine-type PAs: retrorsine (RTS) and monocrotaline (MCT), and otonecine-type PA: clivorine (CLI), and their toxicity-related metabolite PPAs and PA N-oxides, the main metabolite of retronecine-type PAs, for the establishment of a more accurate risk assessment of PAs exposure. Materials and methodsThe TK studies were conducted using rats through intravenous (i.v.) or oral (p.o.) administration of PAs at 20 mg/kg. The main TK parameters of PAs and PA N-oxides were determined from plasma concentration-time profiles, and the kinetic profiles of PPAs were assessed from both plasma and erythrocyte concentration-time profiles. ResultsMCT demonstrated the slowest but the highest extent of absorption among the three PAs, while RTS demonstrated a similar absorption rate with a lower extent than CLI. For elimination, MCT demonstrated a similar elimination rate as RTS but the lowest extent of elimination among the three PAs, and CLI exhibited significantly faster elimination than MCT and RTS. Moreover, the formation of PA N-oxide, which only occurs in retronecine-type PAs, was remarkably less in MCT-treated rats compared to RTS-treated ones. Of note, the retronecine-type RTS and MCT induced more PPAs via p.o. than i.v. administration route, whereas the otonecine-type CLI showed the opposite trend. ConclusionDramatic TK differences, including not only PAs but also PA N-oxides and the derived protein adduct PPAs, were found among structurally diverse PAs in rats, laying the basis for varied hepatotoxic potencies induced by different PA-containing herbal products. Notably, our findings for the first time uncovered that oral administration of retronecine-type PAs might cause severer toxicity compared with the intravenous route, which warrants further in-depth exploration.

Assessment of Toxic Pyrrolizidine and Tropane Alkaloids in Herbal Teas and Culinary Herbs Using LC-Q-ToF/MS.

Pyrrolizidine alkaloids are secondary metabolites produced by plants as a defense against insects. These can cause acute or chronic toxicity in humans. Therefore, avoiding potential poisoning from the consumption of tea and culinary plants contaminated with pyrrolizidine alkaloids (PAs), pyrrolizidine alkaloids N-oxides (PANOs), and tropane alkaloids (TAs) is important for human health and food safety. Therefore, it is important to determine the levels of these substances with reliable and highly accurate methods. In this study, the PAs, PANOs, and TAs in herbal teas and culinary herbs sold in Turkish markets were identified and their levels were determined. Thus, the general profiles of herbal teas and culinary herbs in Turkey were revealed, and the compliance of the total amounts of PA and TA with the regulations was examined. The identification and quantification of 25 PAs and N-oxides and 2 TAs (atropine and scopolamine) in the samples was performed with a liquid chromatography-quadrupole time-of-flight tandem mass spectrometer (LC-Q-ToF/MS). At least a few of these substances were detected in all of the tested herbal teas and culinary herbs. The total contents of the black tea, green tea, mixed tea, flavored tea, chamomile tea, sage tea, linden tea, fennel tea, rosehip tea, peppermint, and thyme samples ranged from 4.6 ng g-1 to 1054.5 ng g-1. The results obtained shed light on the importance of analyzing the total dehydro PA, PANO, and TA amounts in plant-based products consumed in diets with sensitive and accurate methods, and they highlight the necessity of performing these analyses routinely in terms of food safety.

P22-25: Use of Bayesian-MCMC physiologically based kinetic modelling to quantify the consequences of human interindividual differences in kinetics for the relative potency of pyrrolizidine alkaloid N-oxides

P22-25: Use of Bayesian-MCMC physiologically based kinetic modelling to quantify the consequences of human interindividual differences in kinetics for the relative potency of pyrrolizidine alkaloid N-oxides

Species difference in toxicokinetics and safety assessment of senecionine N-oxide in a UDP-glucuronosyltransferase 1A4 humanized mouse model

Pyrrolizidine alkaloids (PAs) are naturally occurring hepatotoxins, and herbs containing PAs are of high concern. PAs are normally found in tertiary amines and N-oxide forms (PA N-oxides), yet the latter are less evaluated for their toxicokinetics. As a continuation of our investigation into the safety assessment of PA-containing herbal medicines, the toxicity and toxicokinetic characteristics of senecionine N-oxide (a representative toxic PA N-oxide) were investigated by using the UDP-glucuronosyltransferase 1A4 humanized mouse model (hUGT1A4 mouse model) and compared with those in wild-type mice simultaneously. Results show that the toxicity caused by senecionine N-oxide exposure was evidently decreased in hUGT1A4 mice as approved by pathology and biochemistry assays. In addition, a N-glucuronidation conjugate was exclusively found in hUGT1A4 mice but not in wild-type (WT) mice. In vitro studies proved that senecionine N-oxide initially reduced to the corresponding tertiary amine alkaloid (senecionine) and then underwent N-glucuronidation via human UGT1A4. The variation in toxicokinetic characteristics was also observed between hUGT1A4 mice and WT mice with a notably enhanced clearance of senecionine N-oxide and senecionine, and accordingly less formation of pyrrole–protein adducts in hUGT1A4 mice, which finally led to the detoxification of senecionine N-oxide exposure in hUGT1A4 mice. Our results provided the first in vivo toxicity data and toxicokinetic characteristics of senecionine N-oxide in a humanized animal model and revealed that human UGT1A4 plays an important role in the detoxification of senecionine N-oxide.

Physiologically based kinetic modeling of senecionine N-oxide in rats as a new approach methodology to define the effects of dose and endpoint used on relative potency values of pyrrolizidine alkaloid N-oxides.

Over 1,000 pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) occur in 3% of all flowering plants. PA-N-oxides are toxic when reduced to their parent PAs, which are bioactivated into pyrrole intermediates that generate protein- and DNA-adducts resulting in liver toxicity and carcinogenicity. Literature data for senecionine N-oxide in rats indicate that the relative potency (REP) value of this PA-N-oxide compared to its parent PA senecionine varies with the endpoint used. The first endpoint was the ratio between the area under the concentration-time curve (AUC) for senecionine upon dosing senecionine N-oxide or an equimolar dose of senecionine, while the second endpoint was the ratio between the amount for pyrrole-protein adducts formed under these conditions. This study aimed to investigate the mode of action underlying this endpoint dependent REP value for senecionine N-oxide with physiologically based kinetic (PBK) modeling. Results obtained reveal that limitation of 7-GS-DHP adduct formation due to GSH depletion, resulting in increased pyrrole-protein adduct formation, occurs more likely upon high dose oral PA administration than upon an equimolar dose of PA-N-oxide. At high dose levels, this results in a lower REP value when based on pyrrole-protein adduct levels than when based on PA concentrations. At low dose levels, the difference no longer exists. Altogether, the results of the study show how the REP value for senecionine N-oxide depends on dose and endpoint used, and that PBK modeling provides a way to characterize REP values for PA-N-oxides at realistic low dietary exposure levels, thus reducing the need for animal experiments.

Formation of DHP-DNA Adducts from Rat Liver Microsomal Metabolism of 1,2-Unsaturated Pyrrolizidine Alkaloid-Containing Plant Extracts and Dietary Supplements.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals. We previously determined that carcinogenic PAs and PA N-oxides commonly form a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, namely, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4. This set of DHP-DNA adducts has been implicated as a potential biomarker of PA-induced liver tumor initiation from metabolism of individual carcinogenic PAs. To date, it is not known whether this generality occurs from metabolism of PA-containing plant extracts. In this study, we investigate the rat liver microsomal metabolism of nine PA-containing plant extracts and two PA-containing dietary supplements in the presence of calf thymus DNA. The presence of carcinogenic PAs and PA N-oxides in plant extracts was first confirmed by LC-MS/MS analysis with selected reaction monitoring mode. Upon rat liver microsomal metabolism of these PA-containing plant extracts and dietary supplements, the formation of this set of DHP-DNA adducts was confirmed. Thus, these results indicate that metabolism of PA-containing plant extracts and dietary supplements can generate DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, thereby potentially initiating liver tumor formation.

Assessment of Distribution and Diversity of Pyrrolizidine Alkaloids in the Most Prevalent Boraginaceae Species in Macedonia.

Systematic study of extraction efficiency of pyrrolizidine alkaloids (PAs) and corresponding pyrrolizidine alkaloid N-oxides (PANOs) from plant material for subsequent LC/MS analysis was carried out. The optimal extraction was achieved with methanol and one clean up step using SPE C18 column. With the optimized LC-ESI-MS/MS method using ion trap, the distribution and diversity of PAs and PANOs in plant material (leaves, flowers and stems) obtained from wild-growing E. vulgare, E. italicum, S. officinale L., C. creticum and O. heterophylla species from Macedonia was assessed. These widespread Boraginaceae species contain various PAs and PANOs and 25 of them were identified. Based on these qualitative and quantitative analyses, the profiles of 1,2-unsaturated PAs for each sample were obtained and their toxic potential was estimated. The toxic potential of O. heterophylla and C. creticum were assumed to be highest (containing up to 4753 mg/kg and 3507 mg/kg), followed by E. vulgare (up to 1340 mg/kg), S. officinale L. (up to 479 mg/kg) and E. italicum (up to 16 mg/kg). This method can be used for monitoring the inclusion of these secondary metabolites in the food chain in order to contribute in their risk management.

Electrochemiluminescence sensor for point-of-care detection of pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) and PA N-oxides are hepatotoxic natural products, produced by over 6000 plant species worldwide. However, an unmet need remains for confirmative measurement of PAs in routine clinical tests. Here, we develop a visual, easy-to-use, and economic mesoporous silica-electrochemiluminescence (MPS-ECL) sensor for point-of-care (POC) testing of PAs, utilizing MPS's amplification effect on positive ions. The relationship between PAs' different structures and corresponding Ru(bpy)32+ ECL activity shows that reaction mechanism, stability of intermediate, molecular geometry and alternative anodic reactivity significantly affect the ECL activity. The ECL intensity varies among different PAs: monocrotaline ˃ senecionine N-oxide ˃ retrorsine ˃ senkirkine. The POC sensors possess excellent linearity (0.9993 > R2 > 0.9944), low detection limits (0.02 μM–0.07 μM), and good recoveries (90.12%–105.93%), indicating good accuracy and practicability. The portable and low-cost sensor is user-friendly, which holds promise to be applied to POC testing of PAs in drugs, food products, and clinical samples, which is promising for initial assessments of PA-induced health risk.

Dissipation pattern and conversion of pyrrolizidine alkaloids (PAs) and pyrrolizidine alkaloid N-oxides (PANOs) during tea manufacturing and brewing

Pyrrolizidine alkaloids (PAs) and pyrrolizidine alkaloid N-oxides (PANOs) are toxic secondary metabolites in plants, and one kind of main exogenous pollutants of tea. Herein, the dissipation pattern and conversion behavior of PAs/PANOs were investigated during tea manufacturing and brewing using ultra high-performance liquid chromatography tandem mass spectrometry. Compared with PAs (processing factor (PF) = 0.73–1.15), PANOs had higher degradation rates (PF = 0.21–0.56) during tea manufacturing, and drying played the most important role in PANOs degradation. Moreover, PANOs were firstly discovered to be converted to corresponding PAs especially in the time-consuming (spreading of green tea manufacturing and withering of black tea manufacturing) and high-temperature tea processing (drying). Moreover, higher transfer rates of PANOs (≥75.84%) than that of PAs (≤56.53%) were observed during tea brewing. Due to higher toxicity of PAs than PANOs, these results are conducive to risk assessment and pollution control of PAs/PANOs in tea.

Effects of ensiling conditions on pyrrolizidine alkaloid degradation in silages mixed with two different Senecio spp.

ABSTRACT Pyrrolizidine alkaloid (PA) producing plants like Senecio jacobaea or Senecio vernalis are undesirable in fields for forage production, since PA are toxic to animals and humans. Previous studies have shown that ensiling can decrease the PA content in forages; however, no direct comparison of diverse PA from different Senecio spp. under various ensiling conditions has been made. Therefore, it was hypothesised that individual PA might react differently to ensiling, and silage inoculation with Lactobacillus will affect PA degradation because of a quick drop in pH, contrastingly to poor silage qualities resulting from contamination with soil. Laboratory scale grass silages were prepared in a multifactorial design with two levels of dry matter contents, four ensiling treatments and two storage durations (10 and 90 d). For each combination, four replicates were prepared individually. Ensiling treatments were (1) 10 ml water per kg fresh matter as control (CON), (2) 10 ml heterofermentative Lactobacillus buchneri strain LN4637 at 3 · 105 cfu/kg fresh matter plus 25 g molasses/kg fresh matter (LBHE), (3) 10 ml homofermentative lactobacilli at 3 · 105 cfu/kg fresh matter plus 25 g molasses/kg fresh matter (LBHO) and (4) 10 g soil/kg fresh matter (SOIL). Treatments affected formation of fermentation acids. Acetic acid was highest with treatment LBHE, and butyric acid was highest with treatment SOIL. All ensiling treatments effectively reduced total PA content by degrading the PA N-oxide (PANO) fraction. In parallel, though, the fraction of the tertiary base forms increased by around one-tenth of the original PANO content. Contents of jaconine and senkirkine were higher after ensiling than before, with regards to the sum of PA and PANO for jaconine, indicating higher stability or new formation through degradation of other PA. Overall, ensiling offers opportunities to decrease the PA-PANO content in feed and therefore lowers the risk of intoxication by Senecio in livestock.

Development, optimization, validation and application of ultra high performance liquid chromatography tandem mass spectrometry for the analysis of pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides in teas and weeds

Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) are natural plant toxins, which are widely distributed in various types of teas. The source of PAs contamination in tea is probably related to weeds in tea gardens. The method of simultaneously detecting PAs/PANOs in teas and weeds has not been reported. A sensitive method was developed for detecting 14 PAs/PANOs simultaneously in teas and weeds using ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Sample preparation was optimized step by step. Sulfuric acid at 0.1 M was used to extract the target compounds. Large capacity PCX column was used to enrich target compounds and remove complex matrices effectively, following by the elution solvent with methanol containing 0.5% NH 4 OH. The method showed good linearity for all 14 analytes with regression coefficients (R 2 ) > 0.99. Satisfactory recoveries of target compounds spiked at three concentrations (10, 50, 100 μg/kg) ranged from 68.6% to 110.2%, while intra-day and inter-day precision were below 18.5%. Limits of detection (LODs) and limits of quantification (LOQs) of 14 PAs/PANOs in different matrices were in the range of 0.001–0.4 μg/kg and 1–5 μg/kg, respectively. This method was firstly applied to monitor the occurrence of PAs/PANOs in different kinds of teas from China and weed samples collected in the local tea gardens. Seven PAs/PANOs (>LOQs) were detected in 15.32% of tea samples and maximum total PAs/PANOs content was 151.33 μg/kg. Pollution levels and patterns of PAs/PANOs were different significantly in various types of teas. Based on the occurrence of PAs/PANOs in teas and weeds, weeds in tea garden were likely to be the source of PAs/PANOs in teas and the most relevant contaminating species of tea samples could be Crassocephalum crepidioides (Benth.) S. Moore. • 14 PAs/PANOs were determined in teas and weeds using UHPLC-MS/MS. • Targets were extracted using 0.1M H 2 SO 4 and complex matrices were purified by SPE. • Weeds in tea garden were likely to be source of PAs in teas. • The most relevant contaminating species of teas was found.

Characterization and Lifetime Dietary Risk Assessment of Eighteen Pyrrolizidine Alkaloids and Pyrrolizidine Alkaloid N-Oxides in New Zealand Honey.

Pyrrolizidine alkaloids (PAs) are a large group of botanical toxins of concern, as they are considered genotoxic carcinogens, with long-term dietary exposure presenting an elevated risk of liver cancer. PAs can contaminate honey through honeybees visiting the flowers of PA-containing plant species. A program of monitoring New Zealand honey has been undertaken over several years to build a comprehensive dataset on the concentration, regional and seasonal distribution, and botanical origin of 18 PAs and PA N-oxides. A bespoke probabilistic exposure model has then been used to assess the averaged lifetime dietary risk to honey consumers, with exposures at each percentile of the model characterized for risk using a margin of exposure from the Joint World Health Organization and United Nations Food and Agriculture Organization Expert Committee on Food Additives (JECFA) Benchmark Dose. Survey findings identify the typical PA types for New Zealand honey as lycopsamine, echimidine, retrorsine and senecionine. Regional and seasonal variation is evident in the types and levels of total PAs, linked to the ranges and flowering times of certain plants. Over a lifetime basis, the average exposure an individual will receive through honey consumption is considered within tolerable levels, although there are uncertainties over high and brand-loyal consumers, and other dietary contributors. An average lifetime risk to the general population from PAs in honey is not expected. However, given the uncertainties in the assessment, risk management approaches to limit or reduce exposures through honey are still of value.

Including the intestinal microbiome incubations in physiologically based kinetic modeling of pyrrolizidine alkaloid N-oxides

Including the intestinal microbiome incubations in physiologically based kinetic modeling of pyrrolizidine alkaloid N-oxides

Metabolism of carcinogenic pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides by rat primary hepatocytes generate the same characteristic DHP-DNA adducts

We recently established a genotoxic mechanism mediated by a set of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, which lead to pyrrolizidine alkaloid (PA)-induced liver tumor initiation. This mechanism is involved in the metabolism of a series of carcinogenic PAs and PA N-oxides in rats in vivo and in vitro. There is a correlation between the order of liver tumor potency and the level of DHP-DNA adduct formation. Thus, these DHP-DNA adducts can be potential biomarkers of PA and PA N-oxide exposure and liver tumor initiation. To establish the generality of this mechanism, in the present study, we examined the metabolism of 13 potential carcinogenic PAs, 1 non-carcinogenic PA, and 5 PA N-oxides by male rat primary hepatocytes. With the exception of the nontoxic PA and vehicle control, all treated groups produced identical set of DHP-DNA adducts. These results support a general genotoxic mechanism mediated by the formation of characteristic DHP-DNA adducts leading to PA-induced liver tumor initiation.

Natural contaminants in bee pollen: DNA metabarcoding as a tool to identify floral sources of pyrrolizidine alkaloids and fungal diversity

The use of bee pollen as a food supplement has increased in recent years as it contains several nutrients and phytochemicals. However, depending on floral composition, bee pollen can be contaminated by pyrrolizidine alkaloids (PAs), PA N-oxides (PANOs) and toxigenic fungi found in plants, which may pose a potential health risk for consumers. Thus, a DNA metabarcoding approach based on internal transcribed spacer 2 (ITS2) region was used to identify the plant sources of 17 PAs/PANOs detected by a validated method in liquid chromatography coupled to mass spectrometry (LC-MS/MS), as well as floral and fungal diversity in 61 bee pollen samples. According to LC-MS/MS analysis, 67% of the samples contained PAs/PANOs with mean concentration of 339 µg/kg. The contamination pattern was characterised by lycopsamine- and senecionine-type PAs/PANOs. PA/PANO-producing plants were identified in 54% of the PA/PANO-contaminated samples analysed by DNA metabarcoding, which also allowed identifying the overall floral and fungal composition of 56 samples. To evaluate the performance of the molecular approach, a subset of 25 samples was analysed by classical palynology. Palynological analysis partially confirmed the results of DNA metabarcoding, which had a better performance in distinguishing pollens of different genera from Asteraceae (76%) and Brassicaceae (88%). However, the molecular analysis did not identify pollens from Castanea, Eucalyptus, Hedera and Salix, which were abundant in 11 samples according to palynology. On the other hand, the molecular analysis allowed identifying several fungal genera in 33 samples, including the toxigenic fungi Alternaria and Aspergillus, which were positively correlated to the plant genus Hypericum. Despite limitations in identifying some pollen types, these preliminary results suggest that the DNA metabarcoding could be applied in a multidisciplinary approach to give a picture of floral and fungal diversity, which can be sources of natural contaminants in bee pollen and would help to control its safety.

Metabolism-mediated cytotoxicity and genotoxicity of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and PA N-oxides are common phytotoxins produced by over 6000 plant species. Humans are frequently exposed to PAs via ingestion of PA-containing herbal products or PA-contaminated foods. PAs require metabolic activation to form pyrrole-protein adducts and pyrrole-DNA adducts which lead to cytotoxicity and genotoxicity. Individual PAs differ in their metabolic activation patterns, which may cause significant difference in toxic potency of different PAs. This review discusses the current knowledge and recent advances of metabolic pathways of different PAs, especially the metabolic activation and metabolism-mediated cytotoxicity and genotoxicity, and the risk evaluation methods of PA exposure. In addition, this review provides perspectives of precision toxicity assessment strategies and biomarker development for the risk control and translational investigations of human intoxication by PAs.

Screening of food supplements for toxic pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PA) and PA-N-oxides (PANO) are a group of more than 660 secondary plant metabolites with hepatotoxic, carcinogenic and mutagenic effects in animals and humans. The phytotoxins can enter the food chain by transfer of PA/PANO between plants via the soil, unintended co-harvesting of PA/PANO-producing weeds, as well as by honeybees collecting pollen and nectar of these plants. Thus, bee- and plant-based products, e.g. (herbal) teas, spices and culinary herbs were identified to be a main source of consumers’ exposure to PA/PANO. Consequently, food supplements based on those ingredients may as well be contaminated with PA/PANO, but so far there are only very few studies available on this topic. Therefore, the current study investigated 50 herbal and bee product-based food supplements available in German retail pharmacies, drugstores, and online on the occurrence of 44 PA/PANO. In total, 19 samples contained PA/PANO with sum contents ranging from 0.1 to 105.1 ng/g in solid samples and from 0.03 to 2.20 ng/mL in liquid preparations. Considering the recommended daily consumption, the sum contents were of no or little concern for the health risk of adults, whereas in case of children the contents of singular samples could significantly contribute to the overall PA/PANO exposure.

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.