Search

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03108-x.

1,2-unsaturated pyrrolizidine alkaloids are found naturally in Symphytum officinale, well known as comfrey, which has a longstanding use for the topical treatment of painful muscle and joint complaints. Pyrrolizidine alkaloids (PA) are a relevant concern for the safety assessment due to their liver genotoxicity profile, and close attention is paid during manufacturing to minimizing their levels. Current regulatory risk assessment approaches include setting limits that derive from toxicity data coming from the oral route of exposure. This study investigated to what extent pyrrolizidine alkaloids are bioavailable following topical exposure, assessing penetration of retronecine-type PAs in an in vitro human skin model. A single comfrey root formulation was spiked with 3 different congeners (a 7R-monoester, an open-chained 7R-diester, and a cyclic diester) and percutaneous absorption measured per OECD guidelines and good laboratory practices. The measured penetration for all 3 PAs was low and compared favourably with existing in vitro data. Although consideration of different regulatory guidance influences the determination of dermally absorbed dose, these data facilitate the understanding of absorption differences following topical exposure, which in turn can be taken into account in the risk assessment.

Against the background of potential contamination of medicinal plant materials with pyrrolizidine alkaloids caused by weeds, suppliers of herbal drugs and manufacturers of herbal medicinal products have taken action by establishing a Code of Practice by monitoring potential contamination and by collection of data. In August 2020, the Herbal Medicinal Products Committee, in its new draft public statement, proposed a daily intake of 1.0 µg of pyrrolizidine alkaloids per day for adults in general, also including contaminations of herbal medicinal products. Over the past years, the results of data collections showed a remarkable reduction of the pyrrolizidine alkaloid burden in herbal drugs and herbal extracts. Meanwhile, a stable situation has been achieved for herbal drugs, while further improvement can be observed for herbal extracts. The results indicate that the implemented measures have been efficient and contribute to a continuous and sustainable reduction of pyrrolizidine alkaloid contamination. A permanent limit of 1.0 µg of pyrrolizidine alkaloids per day is considered appropriate to guarantee sufficient availability of batches used for the production of herbal medicinal products. The new Ph.Eur. general chapter 2.8.26 describes, as an example, an analytical procedure suitable for the determination of target pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs. Graphical abstract

Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications. Bacterial pyrrolizidine alkaloids (PAs), containing a scaffold of two fused five-membered ring system with a nitrogen atom at the bridgehead, have been found to originate from a multidomain non-ribosomal peptide synthetase to generate indolizidine intermediates, followed by multistep oxidation, catalysed by single Bayer-Villiger (BV) enzymes, to yield PA scaffolds. Although bacterial PAs are rare in natural product inventory, bioinformatics analysis suggested that the biosynthetic gene clusters (BGCs) that are likely to be responsible for the production of PA-like metabolites are widely distributed in bacterial genomes. However, most of the strains containing PA-like BGCs are not deposited in the public domain, therefore preventing further assessment of the chemical spaces of this group of bioactive metabolites. Here, we report a genomic scanning strategy to assess the potential of PA metabolites production in our culture collection without prior knowledge of genome information. Among the strains tested, we found fifteen contain the key BV enzymes that are likely to be involved in the last step of PA ring formation. Subsequently one-strain-many-compound (OSMAC) method, supported by a combination of HR-MS, NMR, SMART 2.0 technology, and GNPS analysis, allowed identification and characterization of a new [5 + 7] heterobicyclic carbamate, legoncarbamate, together with five known PAs, bohemamine derivatives, from Streptomyces sp. CT37, a Ghanaian soil isolate. The absolute stereochemistry of legoncarbamate was determined by comparison of measured and calculated ECD spectra. Legoncarbamate displays antibacterial activity against E. coli ATCC 25922 with an MIC value of 3.1 μg/mL. Finally, a biosynthetic model of legoncarbamate and other bohemamines was proposed based on the knowledge we have gained so far.

In the current study the pyrrolizidine alkaloid profiles of the species Echium sabulicola ssp. decipiens (Pomel) Klotz and Solenanthus lanatus DC were studied in various extracts. In addition, a chemotaxonomic study within the genus and the family was carried out. The analysis was carried out by using gas chromatography coupled to mass spectrometry and by comparing the Kovats Indices and molecular and fragment ions with literature data. Twenty-three alkaloids were tentatively identified. The results showed the presence of already reported compounds as well as previously unreported ones leading both to a confirmation of the botanical classification of the two studied species and to a brand new path in the chemotaxonomy of Boraginaceae family. The presence of some pyrrolizidine alkaloids sets limits for the usage of these plants for medicinal purposes. The identified compounds confirm the botanical classification of the studied species as members of the Boraginaceae family and their presence advices against their use in the ethnopharmacological field.

Rheumatoid arthritis (RA) is characterized by joint leukocyte infiltration, synovial inflammation and bone damage result from osteoclastogenesis. Bruton’s tyrosine kinase (BTK) is a key regulator of B cell receptor (BCR) and Fc gamma receptor (FcγR) signaling involved in the pathobiology of RA and other autoimmune disorders. SOMCL-17-016 is a potent and selective tricyclic BTK inhibitor, structurally distinct from other known BTK inhibitors. In present study we investigated the therapeutic efficacy of SOMCL-17-016 in a mouse collagen-induced arthritis (CIA) model and underlying mechanisms. CIA mice were administered SOMCL-17-016 (6.25, 12.5, 25 mg·kg(−1)·d(−1), ig), or ibrutinib (25 mg·kg(−1)·d(−1), ig) or acalabrutinib (25 mg·kg(−1)·d(−1), ig) for 15 days. We showed that oral administration of SOMCL-17-016 dose-dependently ameliorated arthritis severity and bone damage in CIA mice; it displayed a higher in vivo efficacy than ibrutinib and acalabrutinib at the corresponding dosage. We found that SOMCL-17-016 administration dose-dependently inhibited anti-IgM-induced proliferation and activation of B cells from CIA mice, and significantly decreased anti-IgM/anti-CD40-stimulated RANKL expression in memory B cells from RA patients. In RANKL/M-CSF-stimulated RAW264.7 cells, SOMCL-17-016 prevented osteoclast differentiation and abolished RANK-BTK-PLCγ2-NFATc1 signaling. In summary, this study demonstrates that SOMCL-17-016 presents distinguished therapeutic effects in the CIA model. SOMCL-17-016 exerts a dual inhibition of B cell function and osteoclastogenesis, suggesting that it to be a promising drug candidate for RA treatment.

Male ithomiine butterflies (Nymphalidae: Danainae) have hairpencils on the forewings (i.e., androconia) that disseminate semiochemicals during courtship. While most ithomiines are known to contain derivatives of pyrrolizidine alkaloids, dihydropyrrolizines, or γ-lactones in these androconia, here we report on a new class of fatty acid esters identified in two subspecies, Ithomia salapia aquinia and I. s. derasa. The major components were identified as isoprenyl (3-methyl-3-butenyl) (Z)-3-acetoxy-11-octadecenoate, isoprenyl (Z)-3-acetoxy-13-octadecenoate (12) and isoprenyl 3-acetoxyoctadecanoate (11) by GC/MS and GC/IR analyses, microderivatizations, and synthesis of representative compounds. The absolute configuration of 12 was determined to be R. The two subspecies differed not only in the composition of the ester bouquet, but also in the composition of more volatile androconial constituents. While some individuals of I. s. aquinia contained ithomiolide A (3), a pyrrolizidine alkaloid derived γ-lactone, I. s. derasa carried the sesquiterpene α-elemol (8) in the androconia. These differences might be important for the reproductive isolation of the two subspecies, in line with previously reported low gene exchange between the two species in regions where they co-occur. Furthermore, the occurrence of positional isomers of unsaturated fatty acid derivatives indicates activity of two different desaturases within these butterflies, Δ9 and Δ11, which has not been reported before in male Lepidoptera.

Hepatotoxic and genotoxic pyrrolizidine alkaloids have been involved in the acute poisoning of animals and humans. Crotalaria (Fabaceae) species contain these alkaloids. In this work, the diversity and distribution of pyrrolizidine alkaloids in roots, leaves, flowers, and seeds of Crotalaria pallida, Crotalaria maypurensis, Crotalaria retusa, Crotalaria spectabilis, Crotalaria incana, and Crotalaria nitens were studied. Matrix solid-phase dispersion and ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry were successfully employed in pyrrolizidine alkaloids extraction and analysis, respectively. Forty-five pyrrolizidine alkaloids were detected and their identification was based on the mass spectrometry accurate mass measurement and fragmentation pattern analysis. The cyclic retronecine-type diesters monocrotaline, crotaleschenine, integerrimine, usaramine, and their N-oxides were predominantly present. Five novel alkaloids were identified for the first time in Crotalaria species, namely 14-hydroxymonocrotaline, 12-acetylcrotaleschenine, 12-acetylmonocrotaline, 12-acetylintegerrimine, and dihydrointegerrimine. Due to a lack of commercially available standards, the response factor of monocrotaline was used for quantification of pyrrolizidine alkaloids and their N-oxides. Seeds and flowers possessed higher pyrrolizidine alkaloids amounts than roots and leaves. Due to their 1,2-unsaturated pyrrolizidine alkaloids content, the ingestion of Crotalaria plant seeds or other parts through herbal products, infusions, or natural remedies is a serious health threat to humans and livestock.

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.

To investigate the transfer of pyrrolizidine alkaloids (PAs) from feed to milk, rumen-cannulated dairy cows were intra-ruminally fed with 200 g/day of dried plant material of either ragwort (mixture of Jacobaea vulgaris and Senecio inaequidens), common groundsel (Senecio vulgaris) or viper’s bugloss (Echium vulgare) for a period of 4 days. PA levels in the plant materials were 3767, 2792 and 1674 µg g−1 respectively. Feed intake, milk yield and several blood parameters indicative for liver function were not influenced by the treatment. When fed ragwort, increased levels of PAs were detected in the milk, in particular jacoline and an unidentified cyclic diester, possibly a hydroxylated metabolite from retrorsine. The latter was the most important PA in milk from cows fed common groundsel. For viper’s bugloss, echimidine was the most abundant identified PA but in addition several hydroxylated PA metabolites were detected. For ragwort, the overall PA transfer was estimated at 0.05% and 1.4% for jacoline (N-oxide). Transfer rates were similar for viper’s bugloss (0.05%) but lower for common groundsel (0.01%). Only a small portion of the administered PAs was quantified in milk, urine and faeces, with an overall balance of 4.5%, 2.9% and 5.8%, for ragwort, common groundsel and viper’s bugloss, respectively. Samples taken from the rumen indicated that the N-oxides were converted into the free bases, which was confirmed by in vitro studies with the same plant species incubated with ruminal fluid. These results confirm that the transfer of PAs to milk is relatively low but may be of concern for human health regarding the genotoxic and carcinogenic properties of these compounds. The transfer rate depends on the type of PAs present in the weeds. The incomplete balance of input vs output stresses the need to further investigate the metabolism and the potential transfer of metabolites into edible products.

Made available in DSpace on 2020-12-12T02:08:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Introduction: Crotalaria spectabilis is an important species used as a pre-plant cover for soybean crops to control the proliferation of endoparasitic nematodes. Species from the Crotalaria genus are known for presenting pyrrolizidine alkaloids (PAs) in their composition, however, C. spectabilis is still considered chemically under-explored. Objective: The goal of this manuscript is the development and validation of a method for PAs and flavonoids identification and quantification of C. spectabilis seeds and leaves, a toxic plant used for nematode proliferation control in soil, especially in soybean crops. Materials and methods: Seeds and leaves extracts were analysed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the identification of the compounds. Results: PAs and phenolic compounds could be identified in both samples based on the MS/MS fragmentation pattern. Molecular formulas of the annotated compounds were confirmed by ultra-high-performace liquid chromatography-quadrupole time-of-flight (UHPLC-QToF), and monocrotaline could also be confirmed by standard comparison. The quantification of monocrotaline was performed by HPLC-MS/MS, resulting in 123 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. Conclusion: This was the first report of phenolic compounds in C. spectabilis. The current study highlights the importance of C. spectabilis for nematode control due to the presence of toxic PAs, and the employment of analytical techniques for identification and quantification of compounds present in the extracts. Laboratory of Phytochemistry and Biomolecules (LabFitoBio) Department of Chemistry State University of Londrina (UEL) Nuclei of Bioassays Biosynthesis and Ecophysiology of Natural Products (NuBBE) Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Brazilian Agricultural Research Corporation – Embrapa Soybean Nuclei of Bioassays Biosynthesis and Ecophysiology of Natural Products (NuBBE) Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP)

Gynura japonica (also named Tusanqi in Chinese) is used as a folk herbal medicine for treating blood stasis or traumatic injury. However, hundreds of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of preparations made from G. japonica because it contains large amounts of hepatotoxic pyrrolizidine alkaloids (PAs). To date, blood pyrrole-protein adducts (PPAs) are suggested as biomarkers for the diagnosis of PA-induced HSOS in clinics. However, the concentration of PPAs in the blood is greatly affected by several factors including the amount of PA exposure, herb intake period, and blood sampling time after the last exposure. In present study, the kinetic characters of PPAs in serum and liver as well as other potential target organs were studied systematically and comprehensively following multiple exposures of PAs in G. japonica extract (GJE). As results, PPAs content reached to a plateau both in serum and liver after the mice were treated with GJE for 2 weeks on daily basis. PPAs cleared significantly slower in liver (T1/2ke∼184.6 h, ∼7.7 days) than in serum (T1/2ke∼95.8 h, ∼4.0 days). Although more than 90 % PPAs were removed 2 weeks after the last dosing, PPAs still persisted in the liver until the end of the experiment, i.e. 8 weeks after the last dosing. The results would be of great help for understanding the importance of PPAs for PA-induced toxicity and its detoxification.

Nervosine VII is one of the known saturated pyrrolizidine alkaloids isolated from the plant of Liparis nervosa. This is first study to investigate the antitumor activity of nervosine VII in vitro, and the results indicated that nervosine VII induced autophagy and apoptosis in HCT116 human colorectal cancer cells. Mechanistic studies showed that nervosine VII-induced apoptosis was associated with the intrinsic pathway by the activation of caspase-9, -3 and -7. Autophagy induced by nervosine VII was characteristic with the regulation of autophagic markers including the increase of LC3-II and beclin 1 proteins, and the decrease of p62 protein. Nervosine VII simultaneously induced autophagy and apoptosis by activated MAPKs signaling pathway including JNK, ERK1/2 and p38, suppressing the p53 signaling pathway.

Crotalaria lanceolata E. Mey. and Crotalaria pallida Aiton. are leguminous plants of family Fabaceae found in most of the Brazilian territory. They were initially used as green manure and due their easy spread they are currently considered weeds in crops. Soybean and corn contamination can occur through the mechanical harvesting of these grains along with seeds of the Crotalaria species, which end up in the formulation of feed for production animals. Crotalaria spp. genus has toxic pyrrolizidine alkaloids (PA). Most plant species belonging to this genus can cause acute or chronic liver injury. In a first stage, one-day old broilers were divided into three groups: Group A (C. pallida seeds), Group B (C. lanceolata seeds), and Group C (Control). Groups A and B were divided into five subgroups, each with eight broilers, which received the following doses of the respective seeds in feed as of the 7th day of age: daily doses of 0.4%, 0.8% and 2.5%, and single doses of 15% and 25%. Four broilers in each study group were euthanized at 28 days of age - completing 21 days of seed consumption, and the four remaining broilers were euthanized at 42 days of age - completing 35 days of seed consumption. In a second stage, experiments were conducted using seeds of both the aforementioned plants with 28-day old broilers. These were divided into three groups of four animals each: Group D (C. pallida seeds) and Group E (C. lanceolata seeds), which received the respective seeds at daily doses of 1% and 2% in feed for 20 days, and Group F (Control). These broilers were euthanized when they were 80 days old. C. lanceolata seeds showed higher toxicity to broilers than C. pallida seeds, both supplied as of the 7th day of life. Clinical signs included inappetence, ruffled feathers, and brown diarrhea. The following gross lesions were observed: subcutaneous edema, ascites, hydropericardium, yellowish liver with hypertrophy or atrophy and enhanced lobular pattern, and distended gallbladder. Histologic lesions present in all birds in varying degrees were characterized by tumefaction and vacuolar degeneration of hepatocytes. The following clinical conditions and gross lesions were observed in the broilers: hepatocyte megalocytosis and karyomegaly, slight biliary epithelial hyperplasia, eosinophilic spheroids, and nuclear invagination with loss of hepatocyte cord architecture. RESUMO: Crotalaria lanceolata E. Mey. e Crotalaria pallida Aiton. são leguminosas da família Fabaceae presentes na maioria do território brasileiro. Inicialmente foram utilizadas como adubação verde e devido sua fácil disseminação são consideradas invasoras de culturas. Através da colheita mecanizada da soja e milho pode ocorrer a contaminação destes grãos com sementes dessas espécies e entrar na formulação de ração para animais de produção. O gênero Crotalaria spp. possui alcaloides pirrolizidínicos de ação tóxica. A maioria das espécies desse gênero causam lesões hepáticas, com evolução aguda, ou, crônica. Frangos de corte de um dia de vida foram divididos em três grupos: Grupo A (sementes de C. pallida - doses diárias de 0,4%, 0,8%, 2,5% e doses únicas de 15% e 25%), Grupo B (sementes de C. lanceolata - doses diárias de 0,4%, 0,8%, 2,5% e doses únicas de 15% e 25%) e Grupo C (Controle). Os Grupos A e B foram divididos em cinco subgrupos, com oito frangos cada, que a partir do sétimo dia de vida, receberam as doses estabelecidas. Quatro frangos de cada grupo foram sacrificados aos 28 dias, e os quatro restantes aos 42 dias de vida. Também foram conduzidos experimentos com as sementes dessas duas plantas com aves de 28 dias, as quais foram divididas em três grupos: Grupo D (C. pallida - doses de 1% e 2% diariamente, durante vinte dias), Grupo E (C. lanceolata - doses de 1% e 2% diariamente, durante vinte dias) e Grupo F (Controle). Cada grupo composto por quatro aves. Estas aves foram sacrificadas ao completarem 80 dias de vida. Sementes de C. lanceolata demonstraram maior toxicidade para frangos de corte do que sementes de C. pallida. Os sinais clínicos foram inapetência, penas arrepiadas e diarreia acastanhada. Na macroscopia observou-se edema subcutâneo, ascite, hidropericárdio, fígado de coloração amarelada com hipertrofia e, ou, atrofia, evidenciação do padrão lobular e vesícula biliar distendida. As lesões histológicas presentes em todas as aves, em diferentes graus caracterizaram-se por tumefação e degeneração vacuolar de hepatócitos. Nas aves que manifestaram alterações clínicas e lesões macroscópicas, havia megalocitose, cariomegalia, hiperplasia do epitélio biliar, leve, esferoides eosinofílicos e invaginação nuclear com perda da arquitetura dos cordões de hepatócitos.

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.

Pyrrolizidine alkaloids (PAs) are common secondary plant compounds with hepatotoxicity. The consumption of herbal medicines and herbal teas containing PAs is one of the main causes of hepatic sinusoidal obstruction syndrome (HSOS), a potentially life-threatening condition. The present study aimed to reveal the mechanism underlying the cytotoxicity of intermedine (Im), the main PA in Comfrey. We evaluated the toxicity of the retronecine-type PAs with different structures to cell lines derived from mammalian tissues, including primary mouse hepatocytes, human hepatocytes (HepD), mouse hepatoma-22 (H22) and human hepatocellular carcinoma (HepG2) cells. The cytotoxicity of Im to hepatocyte was evaluated by using cell counting kit-8 assay, colony formation experiment, wound healing assay and dead/live fluorescence imaging. In vitro characterization showed that these PAs were cytotoxic and induced cell apoptosis in a dose-dependent manner. We also demonstrated that Im induced cell apoptosis by generating excessive reactive oxygen species (ROS), changing the mitochondrial membrane potential and releasing cytochrome c (Cyt c) before activating the caspase-3 pathway. Importantly, we directly observed the destruction of the cell mitochondrial structure after Im treatment through transmission electron microscopy (TEM). This study provided the first direct evidence of Im inducing hepatotoxicity through mitochondria-mediated apoptosis. These results supplemented the basic toxicity data of PAs and facilitated the comprehensive and systematic evaluation of the toxicity caused by PA compounds.

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 PAsʼ 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.

The genus Doronicum, belonging to tribe Senecioneae (Fam. Asteraceae), is found mainly in the Asia, Europe and North Africa. This genus of plant has always been used in traditional medicinal treatments due to the many biological properties shown such as killing parasitic worms and for relieving constipation, as well as to improve heart health, to alleviate pain and inflammation, to treat insect bites, etc. According to the World Flora the genus Doronicum contains 39 subordinate taxa.[1-3] The purpose of this article, which covers data published from 1970 to 2021 with more than 110 articles, aims to carry out a complete and critical review of the Doronicum genus, examining traditional uses and reporting the antioxidant, antimicrobial, anti-inflammatory and antitumor activity shown from crude extracts or essential oils, and from single isolated compounds. Furthermore, critical considerations of the published data have been highlighted by comparing them with the results obtained from species of other genus belonging to the Asteraceae family.

Concentration of plant secondary metabolites (SMs) show seasonal variations. However, it is still not well understood how these abiotic and biotic factors influence the seasonal variations of SMs. In addition, it is of interest to know if and how SMs are reallocated to the different plant organs, in particular whether SMs are reallocated to the remaining tissues when biomass is lost, e.g., during winter. Here we used Jacobaea vulgaris, Jacobaea aquatica, two F1 and four F2 hybrids that differed in their pyrrolizidine alkaloids (PAs) bouquet as a study system. A series of clones of these genotypes were investigated during their vegetative stage spanning 14 months in a semi-natural environment. We found that the total PA concentration in roots and shoots showed a gradual increase until the spring of the second year, whereafter it dropped substantially in shoots. The variation in PA composition due to seasonal changes was significant but relatively small. Senecionine-like PAs were the dominant PAs in roots, while jacobine-/erucifoline-like PAs were dominant in shoots. The variation of PA concentration was significantly correlated with temperature, day length, and plant age. A correlation analysis showed that PAs were not reallocated when biomass was lost in winter. Overall, our study showed that PA composition of each genotype changed over seasons in a different manner but seasonal variation did not overrule the differences in PA composition among genotypes.

Aposematic organisms warn predators of their unprofitability using a combination of defenses, including visual warning signals, startling sounds, noxious odors, or aversive tastes. Using multiple lines of defense can help prey avoid predators by stimulating multiple senses and/or by acting at different stages of predation. We tested the efficacy of three lines of defense (color, smell, taste) during the predation sequence of aposematic wood tiger moths (Arctia plantaginis) using blue tit (Cyanistes caeruleus) predators. Moths with two hindwing phenotypes (genotypes: WW/Wy = white, yy = yellow) were manipulated to have defense fluid with aversive smell (methoxypyrazines), body tissues with aversive taste (pyrrolizidine alkaloids) or both. In early predation stages, moth color and smell had additive effects on bird approach latency and dropping the prey, with the strongest effect for moths of the white morph with defense fluids. Pyrrolizidine alkaloid sequestration was detrimental in early attack stages, suggesting a trade-off between pyrrolizidine alkaloid sequestration and investment in other defenses. In addition, pyrrolizidine alkaloid taste alone did not deter bird predators. Birds could only effectively discriminate toxic moths from non-toxic moths when neck fluids containing methoxypyrazines were present, at which point they abandoned attack at the consumption stage. As a result, moths of the white morph with an aversive methoxypyrazine smell and moths in the treatment with both chemical defenses had the greatest chance of survival. We suggest that methoxypyrazines act as context setting signals for warning colors and as attention alerting or “go-slow” signals for distasteful toxins, thereby mediating the relationship between warning signal and toxicity. Furthermore, we found that moths that were heterozygous for hindwing coloration had more effective defense fluids compared to other genotypes in terms of delaying approach and reducing the latency to drop the moth, suggesting a genetic link between coloration and defense that could help to explain the color polymorphism. Conclusively, these results indicate that color, smell, and taste constitute a multimodal warning signal that impedes predator attack and improves prey survival. This work highlights the importance of understanding the separate roles of color, smell and taste through the predation sequence and also within-species variation in chemical defenses.

Pyrrolizidine alkaloids are toxins having hepatotoxic and carcinogenic effects on human health. A ultra high performance liquid chromatography tandem mass spectrometry technique was developed for the first time for the simultaneous determination of eight pyrrolizidine alkaloids, including four diastereoisomers (intermedine, lycopsamine, rinderine, and echinatine) and their respective N-oxide forms, in different parts of Eupatorium lindleyanum. The risk assessment method for pyrrolizidine alkaloids in Eupatorium lindleyanum was explored using the margin of exposure strategy for the first time based on a real-life exposure scenario. Differences were found in all eight pyrrolizidine alkaloids in various parts of Eupatorium lindleyanum. Besides, the total levels of pyrrolizidine alkaloids in Eupatorium lindleyanum followed the order of root > flower > stem > leaf. Moreover, the risk assessment data revealed that the deleterious effects on human health were unlikely at exposure times of less than 200, 37, and 12 days during the lifetimes of Eupatorium lindleyanum leaves, stems, and flowers, respectively. This study reported both the contents of and risk associated with Eupatorium lindleyanum pyrrolizidine alkaloids. The comprehensive application of the novel ultra high performance liquid chromatography tandem mass spectrometry technique alongside the risk assessment approach provided a scientific basis for quality evaluation and rational utilization of toxic pyrrolizidine alkaloids in Eupatorium lindleyanum to improve public health safety.

The toxicity of plants containing certain pyrrolizidine alkaloids has long been recognized in grazing animals and humans. Genotoxicity and carcinogenicity data from in vitro and in vivo (animal) studies were published over the last few decades for some of the 1,2-unsaturated pyrrolizidine alkaloids, leading to regulatory action on herbal medicinal products with pyrrolizidine alkaloid-containing plants more than 30 years ago. In recent years, it has become evident that in addition to herbal medicinal products containing pyrrolizidine alkaloid-containing plants, these products may also contain pyrrolizidine alkaloids without actually including pyrrolizidine alkaloid-containing plants. This is explained by contamination by accessory herbs (weeds). The national competent authorities of the European member states and the European Medicines Agency, in this case, the Committee on Herbal Medicinal Products, reacted to these findings by setting limits for all herbal medicinal products. This review article will briefly discuss the data leading to the establishment of thresholds and the regulatory developments and consequences, as well as the current discussions and research in this area.

Seeds of Crotalaria cleomifolia (Fabaceae) are consumed in Madagascar in preparation of popular beverages. The investigation of extracts from the seeds of this species revealed the presence of high amounts of alkaloids from which two pyrrolizidine-derived alkaloids were isolated. One of them was fully characterized by spectroscopic and spectrometric methods, which was found to be usaramine. Owing to the high toxicity of these alkaloids, issuing a strong warning among populations consuming the seeds of Crotalaria cleomifolia must be considered.

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.

BACKGROUND This study evaluated the degradation of pyrrolizidine alkaloids (PAs) from eastern groundsel (Senecio vernalis) in grass silage prepared with different inoculants. Silages were produced from ryegrass with 230 g kg-1 dry matter (DM) content and mixed with eastern groundsel (9:1; w/w fresh matter basis) containing 5.5 g kg-1 DM PA. Treatments were: CON (untreated control), LP (3.0 × 105 cfu g-1 Lactobacillus plantarum DSMZ 8862/8866) or LBLC (7.3 × 104 cfu g-1 Lactobacillus buchneri LN40177 / Lactobacillus casei LC32909), and each of the treatments in combination with 30 g kg-1 molasses. Silages were prepared in glass jars and opened after 3, 10, and 90 days. Fermentation characteristics were determined and the PAs analyzed. RESULTS Although the levels of fermentation acids differed between treatments, results indicated good quality of all silages during 90 days. Significant time (P

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 – 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

Since 2013, a potential contamination of medicinal plant material with pyrrolizidine alkaloid-containing weeds e.g. Senecio has been discussed. The knowledge about such a risk of contamination induced suppliers of medicinal drugs and manufacturers of medicinal teas to investigate the situation regarding herbal drugs and teas and other medicinal products of plant origin. As due to worldwide cultivation/collection and season-dependent sourcing processes an immediate elimination or even reduction of PA contamination at all sourcing sites was considered impossible, manufacturers have taken action by application of their Code of Practice, by monitoring pyrrolizidine alkaloid contamination and by collection of data, by elimination of peak exposures as well as by participation in research projects. The Herbal Medicinal Products Committee at the European Medicines Agency recommended a transitional limit of 1.0 μg pyrrolizidine alkaloids per day related to the final product for three years which has recently been prolonged by a further two years. Against the background of the assessment of the European Food Safety Authority, the option of establishing a permanent limit of 1.0 μg per day should be taken into consideration during future discussions.

Background The roots of Gynura japonica are used as traditional medicine for treating blood stasis or traumatic injury even though hundreds of hepatic sinusoidal obstruction syndrome cases have been reported after consumption of the roots, which contain large amounts of hepatotoxic pyrrolizidine alkaloids (HPAs). However, no information is available about the toxic compounds in the aerial parts of G. japonica, which are also used as herbal medicines and even vegetables in several areas. Thus, the toxic chemicals in the aerial parts of G. japonica, i.e., HPAs, must be urgently identified. Purpose In this study, we aimed to 1) identify the toxic compounds in different medicinal parts and 2) examine the hepatotoxicity of G. japonica. Study design A total of 35 batches of the roots and aerial parts of G. japonica were collected from different sources and analyzed for HPAs. The hepatotoxicity of different extracts (i.e., total extracts [TE] and total alkaloids [TA]) and a single compound (i.e., senecionine) was evaluated on mice. Methods Qualitative analysis of HPAs was performed using an ultra-performance liquid chromatography (UPLC)-mass spectrometry (MS)-parent ion scan approach, whereas a quantitative assay was performed by a UPLC-MS-selected ion monitoring approach. Male C57BL mice were orally administered the different extracts or the single compound at dosages equivalent to 50 mg HPAs/kg body weight. The sera and the livers were collected at 48 h after treatment and used to evaluate the hepatotoxicity through serum clinical biomarkers assay, liver histology, and bile acid profiling. Results A total of 21 HPAs were identified in the roots and the aerial parts. The roots contained higher levels of HPAs (4.90 mg/g) than did the aerial parts (2.21 mg/g). TE and TA induced similar acute liver injuries, but senecionine was considerably more toxic than these extracts. Mice treated with TE showed significantly impaired bile acid homeostasis in the sera and the livers. Conclusion The roots and aerial parts of G. japonica contained large amounts of HPAs, including senecionine, which were responsible for the hepatotoxicity of G. japonica. Bile acid homeostasis was uniquely impaired after exposure to the plant. Therefore, neither the roots nor the aerial parts of G. japonica should be consumed as medicines or vegetables.

Certain herbal drugs and herbal drug preparations contain compounds with a potential risk of use, such as pyrrolyzidine alkaloids (in Comfrey root, Symphyti radix, Coltsfoot leaf, Farfarae folium, Butterbur root, Petasitidis rhizoma), coumarin (in the Cassia bark, Cassiae cortex, Melilot, Meliloti herba), estragole (in Basil, Basilici herba, bitter/sweet fennel, Foeniculi amari/dulcis fructus, Aniseed, Anisi fructus), thujone (in Sage leaf, Salviae officinalis folium, Wormwood, Absinthii herba) or asarone (in Sweet flag rhizome, Calami rhizoma). For these ingredients and herbal drugs/herbal drug preparations which contain them, there are acceptable, limited daily intakes as well as recommendations for duration of use. Since pharmacist plays a key role in advising patients on the use of herbal products, including those containing compounds with a potential risk, it is very important to know the characteristics and proper use of these herbal products.

Rindera graeca is a rare endemic plant where in vitro culture has been used in order to investigate bioactive metabolites. Phytochemical study of the in vitro shoots and hairy roots led to the isolation of seven phenolic derivatives and the unusual furano-naphthoquinone rinderol. R. graeca was also analyzed for its pyrrolizidine alkaloids content by LC-MS, and it was found to contain echinatine together with echinatine and rinderine N-oxides. Rinderol, isolated only from in vitro hairy root culture for the first time in the genus, revealed promising bioactivities. It was evaluated in vitro against a panel of microorganisms, showing very strong activity specifically against Gram-positive bacteria (MIC values 0.98 × 10−2–1.18 µg/mL) as well as very interesting antiproliferative effect against the human non-small-cell bronchopulmonary carcinoma cell line NSCLC-N6-L16 and the epidermoid lung cancer cell line A549. These findings were compared with the chemical profile of the plant from nature, while this study is the first to report on the effects of R. graeca extracts obtained from in vitro culture, providing a valuable contribution to the scientific community towards this sustainable method of production of potential bioactive molecules.

Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) are natural protoxins biosynthesised by many plant species and are responsible for occasional fatal intoxication outbreaks due to the consumption of contaminated food. However, only in rare cases has the origin of the contamination been determined. Although their presence has been studied in many matrices, occurrence data on popular widely used cooking herbs, seeds, spices and leafy vegetables is very scarce. Therefore, a systematic study on the occurrence of PA/PANOs contaminations in these popular herbal items, available on the Belgian market, was performed, by means of a validatedtargeted LC-MS/MS analysis, followed by multilocus DNA metabarcoding to track back the origin of the contamination for seven highly to moderately contaminated samples. Our results clearly indicate that 21% of the seed spices and 25% seed-based aromatic mixes contain an amount higher than 400 μg of the 30 summed targeted PAs and PANOs per kg, the value which is currently under discussion by the European member states to be set as a maximum threshold. For both the herbs and the herb-based mixes only 7% of analysed samples exceeded these levels. As a proof of concept, multilocus DNA metabarcoding was performed on six highly contaminated samples, belonging to each subtype matrix, containing high levels of heliotrine type of PA/PANOs. Each time the analysis demonstrated the presence of DNA from a plant species belonging to the plant genus

Cadmium is one of the most important contaminants and it induces severe plant growth restriction. In this study, we analyzed the metabolic changes associated with root growth restriction caused by cadmium in the early seminal root apex of wheat. Our study included two genotypes: the commercial variety ProINTA Federal (WT) and the PSARK::IPT (IPT) line which exhibit high-grade yield performance under water deficit. Root tips of seedlings grown for 72 h without or with 10 μM CdCl2 (Cd-WT and Cd-IPT) were compared. Root length reduction was more severe in Cd-WT than Cd-IPT. Cd decreased superoxide dismutase activity in both lines and increased catalase activity only in the WT. In Cd-IPT, ascorbate and guaiacol peroxidase activities raised compared to Cd-WT. The hormonal homeostasis was altered by the metal, with significant decreases in abscisic acid, jasmonic acid, 12-oxophytodienoic acid, gibberellins GA20, and GA7 levels. Increases in flavonoids and phenylamides were also found. Root growth impairment was not associated with a decrease in expansin (EXP) transcripts. On the contrary, TaEXPB8 expression increased in the WT treated by Cd. Our findings suggest that the line expressing the PSARK::IPT construction increased the homeostatic range to cope with Cd stress, which is visible by a lesser reduction of the root elongation compared to WT plants. The decline of root growth produced by Cd was associated with hormonal imbalance at the root apex level. We hypothesize that activation of phenolic secondary metabolism could enhance antioxidant defenses and contribute to cell wall reinforcement to deal with Cd toxicity.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Background and Aims: The Gynura japonica-induced hepatic sinusoidal obstruction syndrome (HSOS) is closely related to pyrrolizidine alkaloids (PAs), and its prevalence has been increasing worldwide in recent years. However, no effective therapy for PA-induced HSOS in clinics is available, partially due to the failure of quick diagnosis. This study aims to identify blood microRNA (miRNA) signatures as potential biomarkers for PA-induced HSOS in clinics.Methods: The microarray-based miRNA profiling was performed on blood samples of the discovery cohort, which consisted of nine patients with HSOS and nine healthy donors. Differentially expressed miRNAs were further confirmed using a validation cohort, which consisted of 20 independent patients with HSOS. In addition, the rat model was established through the oral administration of the total alkaloid extract from G. japonica to investigate the association of miRNA biomarkers with the progression of HSOS. Bioinformatic analyses, including GO and KEGG enrichment, receiver operating characteristics curve, and correlation analyses were conducted to evaluate the accuracy of the potential miRNA biomarkers.Results: Three miRNAs, namely miR-148a-3p, miR-362-5p, and miR-194-5p, were overexpressed in patients and rats with PA-induced HSOS. These miRNAs were positively related to the severity of liver injury and displayed considerable diagnostic accuracy for patients with HSOS with areas under the curve over 0.87.Conclusion: In summary, this study demonstrated that three miRNAs, hsa-miR-148a-3p, hsa-miR-362-5p, and hsa-miR-194-5p, might serve as potential biomarkers for PA-induced HSOS in clinics.

Pyrrolizidine alkaloids (PAs) are genotoxic carcinogenic phytotoxins mostly prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Heliotropium species (Boraginaceae) are PA-producing weeds, widely distributed in the Mediterranean region, that have been implicated with lethal intoxications in livestock and humans. In Israel, H. europaeum, H. rotundifolium and H. suaveolens are the most prevalent species. The toxicity of PA-producing plants depends on the PA concentration and composition. PAs occur in plants as mixtures of dozens of various PA congeners. Hence, the risk arising from simultaneous exposure to different congeners has to be evaluated. The comparative risk evaluation of the three Heliotropium species was based on recently proposed interim relative potency (iREP) factors, which take into account certain structural features as well as in vitro and in vivo toxicity data obtained for several PAs of different classes. The aim of the present study was to determine the PA profile of the major organ parts of H. europaeum, H. rotundifolium and H. suaveolens in order to assess the plants’ relative toxic potential by utilizing the iREP concept. In total, 31 different PAs were found, among which 20 PAs were described for the first time for H. rotundifolium and H. suaveolens. The most prominent PAs were heliotrine-N-oxide, europine-N-oxide and lasiocarpine-N-oxide. Europine-N-oxide displayed significant differences among the three species. The PA levels ranged between 0.5 and 5% of the dry weight. The flowers of the three species were rich in PAs, while the PA content in the root and flowers of H. europaeum was higher than that of the other species. H. europaeum was found to pose a higher risk to mammals than H. rotundifolium, whereas no differences were found between H. europaeum and H. suaveolens as well as H. suaveolens and H. rotundifolium.

Pyrrolizidine alkaloids (PAs) are found to be toxic pollutants emitted into the environment by numerous plant species, resulting in contamination. In this article, we investigate the occurrence of PAs in the aquatic environment of small Swiss streams combining two different approaches. Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites produced by numerous plant species. Although they were classified as persistent and mobile and found to be emitted into the environment, their occurrence in surface waters is largely unknown. Therefore, we performed a retrospective data analysis of two extensive HRMS campaigns each covering five small streams in Switzerland over the growing season. All sites were contaminated with up to 12 individual PAs and temporal detection frequencies between 36 and 87%. Individual PAs were in the low ng/L range, but rain-induced maximal total PA concentrations reached almost 100 ng/L in late spring and summer. Through PA patterns in water and plants, several species were tentatively identified as the source of contamination, with Senecio spp. and Echium vulgare being the most important. Additionally, two streams were monitored, and PAs were quantified with a newly developed, faster, and more sensitive LC–MS/MS method to distinguish different plant-based and indirect human PA sources. A distinctly different PA fingerprint in aqueous plant extracts pointed to invasive Senecio inaequidens as the main source of the surface water contamination at these sites. Results indicate that PA loads may increase if invasive species are sufficiently abundant. ISSN:0013-936X ISSN:1520-5851

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.

The transfer rate of 37 pyrrolizidine alkaloids (PA) found in ten naturally contaminated teas and herbal teas to their brews was studied in detail. Mixed herbal, peppermint, red bush, senna, black tea and green tea infusions were prepared according to the ISO guide and vendor���s instructions, respectively, and parameters like herb-to-water ratio, steeping time and multiple extractions studied. In general, a transfer rate of 38���100% (median 95%) for brews following vendor���s instructions was determined. The total concentration range of PA in these ten samples was 154���2412 ng/g (median 422 ng/g) in the herb and for single analytes 0.1���170 ng/g. Seven of the 37 PA occurred unexpectedly; these were tentatively identified and quantified by liquid chromatography-high resolution mass spectrometry (LC-HR-MS), since their contributions to total PA-content matter. Additionally, 46 iced tea beverages were analysed for their PA-load, determined to be in the range 0���631 ng/L (median 40 ng/L). The applied solid-phase extraction (SPE) clean-up turned out to be capable of separating PA in the free base pyrrolizidine alkaloids (PAFB) and their N-oxides (PANO) in a two-step elution, which was a valuable tool to support identification of unexpected PA. Further, atropine was found in 50% of the ten tea herb samples (range: 1���4 ng/g) and in 13% of the iced tea beverage samples (range: 2���65 ng/L).

Changes in plant chemical defenses after invasion could have consequences on the invaded ecosystems by modifying the interactions between plants and herbivores and facilitating invasion success. However, no comprehensive biogeographical studies have yet determined the phenotypic levels of plant chemical defenses, as consumed by local herbivores, covering large distributional areas of a species. Senecio ptero-phorus is a perennial shrub native to Eastern South Africa, expanded into Western South Africa and introduced into Australia and Europe. As other Asteraceae, S. pterophorus contains pyrrolizidine alkaloids (PAs) toxic to vertebrate and invertebrate herbivores. Here we analyzed S. pterophorus PAs by LC-MS/MS on foliage sampled across its entire distributional range, including the native and all non-native areas. PA concentrations and diversity was very high: we found 57 compounds belonging to 6 distinct necine base-types, including the highly toxic 1,2-unsaturated PAs (retronecine and otonecines) and the less toxic 1,2-saturated PAs (platynecine and rosmarinecines). Plants from different origins diverged in their PA absolute and relative concentrations. Rosmarinine was the most abundant compound in Australia and South Africa, but it was nearly absent in Europe. We characterized three plant chemotypes: retrorsine-senkirkine chemotype in Eastern South Africa, rosmarinine chemotype in Australia and Western South Africa, and acetylseneciphylline chemotype in Europe. PA absolute concentrations were highest in Australia. The increased absolute and relative concentrations of retronecine PAs from Australia and Europe, respectively, indicate that S. pterophorus is potentially more toxic in the invasive range than in the native range.

Many pyrrolizidine alkaloids (PAs), an important class of natural products, are hepatotoxic and carcinogenic. Increased attention has been paid to PA poisoning cases worldwide. Generally, most PAs themselves are not toxic. However, reactive intermediates formed from PAs by metabolic oxidation have been linked to toxicity and carcinogenesis. PAs themselves are generally not toxic, and their reactive metabolites resulting from metabolic oxidation are considered to be an essential responsible for PA toxicities. Protein modification by the electrophilic metabolites is proposed to play a key role in PA-induced cytotoxicity. The present study investigated the interaction of lysine residues of proteins with reactive metabolites of toxic PAs. Antibodies selectively recognizing lysine-based protein adduction were prepared and characterized. ELISA and immunoblot methods, in the presence and absence of synthetic model PA adducts, were used to test specific binding of the antibodies to modified lysine residues of BSA and to hepatic proteins extracted from mice treated with retrorsine. The lysine residue adduction was also detected in the tissues of retrorsine-treated mice by use of an immunohistochemical approach. In conclusion, the prepared antibodies selectively recognized the lysine adducts and may be used for the investigation of mechanisms of toxic action of PAs.

Introduction: Symphytum officinale L. (comfrey, Boraginaceae) is a cultivated or spontaneously growing medicinal plant that is traditionally used for the treatment of bone fractures, hematomas, muscle pains and joint pains. A wide range of topical preparations and dried roots for ex tempore applications are marketed in European drug stores or pharmacies. Objective: The aim of this study was to perform the qualitative and quantitative analysis of pyrrolizidine alkaloids (PAs) and phenolic compounds in the hydroethanolic extracts of 16 commercial comfrey root batches purchased from 12 different European countries. Methods: Liquid chromatography hyphenated with high-resolution tandem mass spectrometry (LC-HRMS/MS) was used for the profiling of PAs and phenolic compounds, whereas LC-MS/MS and liquid chromatography with diode array detection (LC-DAD) were used for their quantification. Results: 20 PAs (i.e. intermedine, lycopsamine, acetylintermedine, acetyllycopsamine, symphytine, symphytine-N-oxide), 17 phenolic compounds (i.e. caffeic and rosmarinic acids, rabdosiin, globoidnan A, globoidnan B) and 9 nonphenolic compounds (sugars, organic and fatty acids) were fully or partly annotated in the analysed samples. In addition, the quantitative analyses revealed that globoidnan B, rabdosiin and globoidnan A are new phenolic markers that can be used together with rosmarinic acid and PAs for the quality control of commercial comfrey root batches. Conclusions: This study brings new insights into the phytochemical complexity of S. officinale, revealing not only numerous toxic PAs, but also a significant number of valuable phenolic compounds that could contribute to the bioactivities of comfreybased preparations

Background Collectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs). Results We retrieved CYPs from RNA-seq data of J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea (CYP71, CYP76, CYP706, CYP82, CYP93 and CYP72) and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history. Conclusions This study discovered 378 full-length CYPs in Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.

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 &micro, g/kg and maximum levels of 111, 59.4, and 3313 &micro, g/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 &lt, 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.

Senecio coronatus (known as izonkozonko and ubulibazi in Zulu) is commonly used in traditional medicine in South Africa as purification purgative and enemas for infants during weaning. We show for the first time that this species does not contain pyrrolizidine alkaloids and that reported cases of fatal hepatic sinusoidal obstruction syndrome in infants were caused by wrongly identified Senecio species containing large amounts of retrorsine-N-oxide. A validated ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection and quantitation of pyrrolizidine alkaloids is described.

Pyrrolizidine alkaloids (PAs) are extensively distributed in plants and are known to damage hepatic sinusoidal endothelial cells (HSECs) via metabolic activation mediated by hepatic cytochrome P450 enzymes (CYPs), particularly the CYP3A4 isozyme. Different PAs have distinct toxic potencies and their toxic effects on HSECs are difficult to be determined in cultured cells, because HSECs lack the key CYP3A4 isozyme for metabolic activation. This study aims to establish a novel, convenient and reliable CYP3A4-expressing HSEC model using human HSECs transduced with lentivirus carrying CYP3A4-ires-eGFP, for evaluating the hepatotoxicity of different PAs on their target HSECs. The developed CYP3A4-expressing HSEC (HSEC-CYP3A4) model was verified by the expression of GFP and CYP3A4 and by the ability to metabolize nifedipine, a classic CYP3A4 substrate. Treated with retrorsine, a representative toxic PA, HSEC-CYP3A4 cells showed significantly reduced cell viability, depletion of GSH, and increased formation of pyrrole-protein adducts. Furthermore, this newly developed cell model successfully discriminated the cytotoxic potency of different PAs evidenced by their IC40 values. In conclusion, the established HSEC-CYP3A4 cell model can be used as a rapid screening platform for assessing the relative potencies of individual PAs on their target HSECs and for investigating the mechanisms underlying PA-induced hepatic sinusoidal damage.

European Union guidelines indiscriminately discuss a permitted daily exposure (PDE) for pyrrolizidine alkaloids (PA) of up to 0.007 μg/kg body weight for oral and for topical exposure to herbal medicinal products. In this study, lycopsamine served as a model substance for measuring the extent of skin permeation of PAs following the application of a spiked comfrey cream (Symphytum officinale s.l.) to abdominal skin from human donors in Franz diffusion cells. PAs could be excluded in the non-spiked cream with a limit of detection of 8 μg/kg. Only small amounts of the applied quantity of lycopsamine had migrated through the skin sample into the receptor cell side of the diffusion cell after 24 h. In five of six diffusion cells, there was no detectable lycopsamine within the skin and only 0.6 ± 0.4% of the applied dose in the receptor fluid. The theoretical skin penetration of 4.9% of the applied quantity of lycopsamine largely resulted from the worst case approach of assuming the presence of at least a quantity corresponding to the limit of detection – the true penetration is probably considerably lower. Even with the worst-case calculation, the currently discussed guidelines on PA overestimate the risk related to topical preparations.

The purpose of this study was to assess the ecological knowledge surrounding the western queen butterfly, Danaus gilippus thersippus (H. Bates). Specifically, our objectives were to synthesize existing data and knowledge on the ecology of the queen and use results of this assessment to inform the direction of future research on this understudied species. We identified six core areas for assessment: distribution, the biodiversity of plant resources, western queen and their host plant phenology, chemical ecology, and four key life history traits. We mapped the distribution of D. g. thersippus from museum specimen records, citizen science (e.g., iNaturalist) and image sharing app-based observations, along with other observational data enumerating all current known plant resources and long-range movements. We assembled 14 larval food plants, six pyrrolizidine alkaloids plants and six nectar plants distributed in the western Mojave and Sonoran Desert regions of the United States and Baja California. We report on its phenology and its long-range movement. Butterfly species have declined across the western US, and western monarch populations have declined by 97%. Danaus g. thersippus has received little research attention compared with its famous congener D. plexippus L. Danaus g. thersippus&rsquo, desert distribution may be at its temperature limits for the species distribution and for its rare host plant Asclepias nyctaginifolia.

Pyrrolizidine alkaloids (PAs) are distributed in plant families of Asteraceae, Boraginaceae, and Fabaceae and serve in the chemical defense mechanism against herbivores. However, they became a matter of concern due to their toxicity associated with the high risk of intake within herbal preparations, e.g., phytopharmaceutical formulations, medicinal teas, or other plant-derived drug products. In 1992, the German Federal Ministry of Health established the first limits of PA content for fourteen medicinal plants. Because of the toxic effects of PAs, the Federal Institute of Risk Assessment (BfR) established more stringent limits in 2011, whereby a daily intake, publishedVersion

Pyrrolizidine alkaloids (PA)s are natural toxins produced by a variety of plants including ragwort. The PAs present a serious health risk to human and livestock. Although these compounds have been extensively studied in food and feed, little is known regarding their environmental fate. To fill this data gap, we investigated the occurrence of PAs in ragwort plants, soils and surface waters at three locations where ragwort was the dominant plant species to better understand their environmental distribution. The concentrations of PAs were quantified during the full growing season (April–November) and assessed in relation to rain events. PA concentrations ranged from 3.2–6.6 g/kg dry weight (dw) in plants, 0.8–4.0 mg/kg dw in soils, and 6.0–529 μg/L in surface waters. Maximum PA concentrations in the soil (4 mg/kg) and water (529 μg/L) were in mid-May just before flowering. The average distribution of PAs in water was approximately 5 g/10,000 L, compared to the average amounts present in ragwort (506 kg/ha), and soil (1.7 kg/ha). In general, concentrations of PAs increase in the soil and surface water following rain events.