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Qianbai biyan tablet (QT) is a compound prescription of traditional Chinese medicine which is used to treat nasal congestion, rhinitis, and nasosinusitis, with Senecio scandens as its main plant material. Several pyrrolizidine alkaloids (PAs) were reported in Senecio scandens and others of Senecio species. Although Senecio scandens is assigned as the legal plant material of QT, whether replaced use of it by other Senecio plants can bring toxicity is unknown because of the lack of quantitative data about toxic PAs between different Senecio species. In the present study, adonifoline, senkirkine, and another PA presumed as emiline have been identified in QT; however, there was no senecionine detected in all tablets. PA contents in QTs varied in different companies and different batches. Adonifoline existed only in Senecio scandens, and senecionine was detected in all eight Senecio plants investigated in the present study. Data showed that replaced use of Senecio scandens with a low level of senecionine by other Senecio plants such as Senecio vulgaris containing a high level of senecionine is advertised to be forbidden. Data of the present study may be used as a reference to make new drug quality regularity and recommendation guideline for the safety of QT.

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 (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs. Electronic supplementary material The online version of this article (10.1007/s00204-020-02895-z) contains supplementary material, which is available to authorized users.

Pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals, inducing liver tumors in experimental rodents. We previously determined that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), 7-glutathione-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, and 1-CHO-DHP are DNA reactive pyrrolic metabolites potentially associated with PA-induced liver tumor initiation. In this study, we developed an LC/MS/MS multiple reaction monitoring (MRM) mode method to identify and quantify these metabolites formed from the metabolism of senecionine, a carcinogenic PA, by mouse, rat, and human liver microsomes, and primary rat hepatocytes. Together with the chemically prepared standards of these metabolites, this represents an accurate and convenient LC/MS/MS analytical method for quantifying these five reactive pyrrolic metabolites in biological systems. Keywords: Pyrrolizidine alkaloid, Senecionine, Pyrrolic metabolites, Carcinogenicity, LC/MS/MS analysis

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) with 1,2-unsaturated necine base are hepatotoxic phytotoxins. Acute PA intoxication is initiated by the formation of adducts between PA-derived reactive pyrrolic metabolites with cellular proteins. The present study aimed to investigate the correlation between the formation of hepatic pyrrole–protein adducts and occurrence of PA-induced liver injury (PA-ILI), and to further explore the use of such adducts for rapidly screening the hepatotoxic potency of natural products which contain PAs. Aqueous extracts of Crotalaria sessiliflora (containing one PA: monocrotaline) and Gynura japonica (containing two PAs: senecionine and seneciphylline) were orally administered to rats at different doses for 24 h to investigate PA-ILI. Serum alanine aminotransferase (ALT) activity, hepatic glutathione (GSH) level, and liver histological changes of the treated rats were evaluated to assess the severity of PA-ILI. The levels of pyrrole–protein adducts formed in the rats’ livers were determined by a well-established spectrophotometric method. The biological and histological results showed a dose-dependent hepatotoxicity with significantly different toxic severity among groups of rats treated with herbal extracts containing different PAs. Both serum ALT activity and the amount of hepatic pyrrole–protein adducts increased in a dose-dependent manner. Moreover, the elevation of ALT activity correlated well with the formation of hepatic pyrrole–protein adducts, regardless of the structures of different PAs. The findings revealed that the formation of hepatic pyrrole–protein adducts—which directly correlated with the elevation of serum ALT activity—was a common insult leading to PA-ILI, suggesting a potential for using pyrrole–protein adducts to screen hepatotoxicity and rank PA-containing natural products, which generally contain multiple PAs with different structures.

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

Pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) can cause liver toxicity and genotoxicity in humans and animals, necessitating the development of screening tools for these alkaloids. Ultra-performance liquid chromatography–electrospray ionization-quadrupole-time-of-flight mass spectrometry (UPLC–ESI–Q-TOFMS) can provide accurate precursor and fragment ion mass information (MSE mode) as well as simultaneous quantification. A method for rapidly and accurately determining nine PAs with UPLC–ESI–Q-TOF MS was developed and validated herein. Analytes were separated using a C18 column with water containing 0.1% formic acid and acetonitrile, showing high-resolution peaks within 11 min. The validation results showed excellent linearity (R2 > 0.9951), limits of detection (0.4–2.0 ng mL−1), and limits of quantification (0.6–6.0 ng mL−1). Moreover, 70 PAs, their N-oxides precursors, and characteristic fragment ions that were generated according to their chemical structure were characterized. A method for the chemical profiling of alkaloids was also proposed using the mass information obtained from the chromatograms of the tested sample. Lycopsamine, senecionine, senkirkine, and echimidine were identified in four potentially PA-containing plants and quantified by matching with authentic standards. Eight PAs and PANOs were also tentatively identified using the mass data from the previously listed alkaloids. This approach will provide a database that can be used to instantly identify alkaloids in botanical samples.

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.

Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300 μM. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I2 as the main prostanoid of endothelial cells. All effects occurred only with in vitro-metabolically activated PA lasiocarpine and senecionine. By contrast, no effect was observed for the PA echimidine, heliotrine, lasiocarpine, senecionine, senkirkine and platyphylline in the absence of an external metabolizing system up to the highest tested concentration of 500 μM. Overall, our results confirm the metabolism-dependent toxification of PA and elucidate the involved pathways. These include induction of inflammatory cytokines and deregulation of the prostanoid synthesis pathway in endothelial cells, linking for the first time PA-dependent changes in prostanoid release to distinct alterations at the mRNA and protein levels of enzymes of prostanoid synthesis.

Several plant species synthesize biologically active secondary metabolites. Pyrrolizidine alkaloids are a large group of biotoxins produced by thousands of plant species to protect against the attack of insects and herbivores, but they are highly toxic for humans and animals. In this study, extracts from the aerial part of Senecio brasiliensis were obtained using different technologies: ultrasound-assisted extraction (UAE), pressurized liquid extraction (PLE), and microwave hydrodiffusion and gravity (MHG). The study aimed to evaluate the effectiveness of these technologies for the extraction of chemical compounds found in this plant, focusing on two pyrrolizidine alkaloids: integerrimine and senecionine. Influential parameters on yield and chemical composition were also evaluated: for UAE and MHG, temperature and pressure; for PLE, temperature, and percentage of ethanol. All the extraction techniques were efficient for the extraction of integerrimine and senecionine. The UAE and PLE stood out for the higher yields and number of compounds. The PLE presented a maximum yield of 18.63% for the matrix leaf and the UAE a maximum yield of 11.82% for the same matrix. These two techniques also stood out in terms of the number of compounds, once 36 different compounds were found via PLE and 17 via UAE. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02845-1.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6–30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4–31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

An ultrasensitive and rapid fluorescent immunoassay based on a broad-spectrum monoclonal antibody (mAb) was developed to detect pyrrolizidine alkaloids (PAs) in honey samples. First, Discovery Studio software was used to analyze and predict the target hapten, and retrorsine (RTS) was selected to react with succinic anhydride (HS) for hapten synthesization. A sensitive and broad-spectrum monoclonal antibody (mAb 13E1) was obtained for nine PAs. Then, fluorescent gold nanoclusters (AuNCs) were conjugated with mAb as a label probe and used in establishing a qualitative and quantitative lateral flow immunoassay (AuNCs-LFIA) for the determination of four PAs (retrorsine, platyphylline, senecionine, integerrimine) in honey within 14 min. The limits of detection (LOD) were 0.083 μg/kg. The recovery in spiked honey samples were 87.98–119.57%, with coefficients of variation of ≤ 11.5%. A total of 45 commercial import honey samples from nine different countries were tested through AuNCs-LFIA and UPLC-MS/MS method, and satisfactory consistency (R2 = 0.995) was obtained. The rates of positive samples were 55.56% (25/45), and the average concentrations of four PAs were 3.24–46.47 μg/kg. This ultrasensitive multi-PA method provides an alternative analytical tool for evaluating the human risk posed by the consumption of PA-contaminated honey.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6-30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4-31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

Pyrrolizidine alkaloids (PAs) are persistent mutagenic and carcinogenic compounds produced by many common plant species. Health authorities recommend minimising human exposure via food and medicinal products to ensure consumer health and safety. However, there is little awareness that PAs can contaminate water resources. Therefore, no regulations exist to limit PAs in drinking water. This study measured a PA base concentration of ~ 70 ng/L in stream water adjacent to an invasive PA-producing plant Petasites hybridus (Asteraceae). After intense rain the PA concentration increased tenfold. In addition, PAs measured up to 230 ng/L in seepage water from groundwater wells. The dominant PAs in both water types corresponded to the most abundant PAs in the plants (senkirkine, senecionine, senecionine N-oxide). The study presents the first discovery of persistent plant toxins in well water and their associated risks. In addition, it for the first time reports monocrotaline and monocrotaline N-oxide in Petasites sp.

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA. In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios. Electronic supplementary material The online version of this article (10.1007/s00204-020-02779-2) contains supplementary material, which is available to authorized users.

Recent and unexpected food alerts about relatively high amounts of pyrrolizidine alkaloids in oregano samples have stressed the need to develop analytical strategies to ensure food safety in this type of foodstuff. Accordingly, this work presents the development of a miniaturized strategy based on the QuEChERS (quick, easy, cheap, effective, rugged and safe) method combined with ultrahigh liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the determination of 21 pyrrolizidine alkaloids suggested by the European Food Safety Authority to be monitored in food. The analytical method was properly validated, with overall average recoveries from 77 to 96% and relative standard deviations &lt, 13% (n = 9). The method proved to be a sustainable analytical strategy which meets green analytical chemistry principles as it showed good performance by using small amounts of sample (0.2 g), organic solvents (1000 &micro, L), clean-up sorbents (175 mg) and partitioning salts (0.65 g). Its feasibility was verified through the analysis of 23 oregano samples. Of the samples analyzed, 100% were contaminated, with an average concentration of 1254 &micro, g/kg. Lasiocarpine, lasiocarpine N-oxide, europine, europine N-oxide, senecivernine, senecionine, echimidine N-oxide, lycopsamine N-oxide and intermedine N-oxide were the alkaloids which significantly contributed to the contamination of the samples.

BACKGROUND Pyrrolizidine alkaloids are secondary plant metabolites with hepatotoxic effect in humans and several animal species. In recent studies, foods such as herbal teas and honey have been found to be contaminated with pyrrolizidine alkaloids. OBJECTIVES The aim of this study was to identify and assess pyrrolizidine alkaloids in compound feeds manufactured for horses and containing either alfalfa or a blend of herbs. METHODS Forty-eight feed products for horses were included in the study. The feedstuffs were analysed for 28 selected pyrrolizidine alkaloids by liquid chromatography-mass spectrometry. The concentrations of the individual pyrrolizidine alkaloids were summed to calculate the total pyrrolizidine alkaloid content. RESULTS In 7 of 48 samples, pyrrolizidine alkaloid concentrations were below the limit of quantification of 1-5 μg/kg. The median of 41 out of 48 samples was 58 μg/kg, and the 25 and 75th percentiles were 8 and 151 μg/kg. The highest observed pyrrolizidine alkaloid concentrations, 1306 and 1222 μg/kg, were found in two alfalfa-based feed products, followed by 836 μg/kg in an herb-containing feed product. Lycopsamine, seneciphylline, seneciphylline-N-oxide, senecionine and senecionine-N-oxide were the most frequently detected alkaloids. MAIN LIMITATIONS Risk assessment was based on no-observed-adverse-effect-level for pyrrolizidine alkaloids in rats and humans. The specific susceptibility of horses to pyrrolizidine alkaloids remains unknown. CONCLUSIONS According to our risk assessment, pyrrolizidine alkaloid contamination should be limited to

Senecio vulgaris L. is a well known toxic plant, phytochemical investigation of the whole plant afforded the isolation of a new coumarin; 3,7-dihydroxy-5,6 dimethoxycoumarin-7-O-β-D-glucoside (1), in addition to the isolation of isorhamnetin-3-O-β-D-glucoside (2) and two known alkaloids of common occurrence in genus Senecio; senecionine (3) and seneciphylline (4). The isolated compounds were identified by physical and chemical analyses and by intensive investigation of their spectral data. EtOH extract, light petrol and CHCl3 fractions of S.vulgaris were found to have cytotoxic activity on colorectal cell line HCT-116 cell line, with IC50 values of 19.9, 27.3 and 17.8 μg mL-1, respectively. That indicates the most active cytotoxic fraction is the alkaloid-bearing chloroform fraction. These findings promote the future perspective of S. vulgaris as an anticancer agent. The antioxidant activity of EtOH extract, light petrol fraction and CHCl3 fraction was evaluated by DPPH assay, %inhibition was 23.31, 4.677 and 17.598, respectively. Also the ratio between senecionine and seneciophylline in their mixture was determined using 1H-NMR spectrum.

Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet. Therefore, we investigated cell death parameters in human HepaRG cells following either single (24 h) or repeated dose treatment (14 d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24 h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14 d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6 h). Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.

1,2-Dehydro-pyrrolizidine alkaloids (PA) and their N-oxides (PANO) exhibit acute and chronic toxic effects on the liver and other organs and therefore are a hazard for animal and human health. In certain regions of Germany, an increasing spread of Senecio spp. (ragwort) on grassland and farmland areas has been observed during the last years leading to a PA/PANO-contamination of feed and food of animal and plant origin.This project was carried out to elucidate whether the process of grass pellet production applying hot air drying influences the content of PA and PANO. Samples of hay (n=22) and grass pellets (n=28) originated from naturally infested grassland (around 10% and 30% dominance of Senecio aquaticus) and from a trial plot with around 50% dominance. Grass pellets were prepared from grass originating from exactly the same plots as the hay samples. The samples were analysed by liquid chromatography-tandem mass spectrometry for PA/PANO typically produced by this weed.The results of the study revealed that PA/PANO levels (predominantly sum of senecionine, seneciphylline, erucifoline and their N-oxides) in hay ranged between 2.1 and 12.6mg kg(-1) dry matter in samples with 10% and 30% dominance of S. aquaticus, respectively. Samples from the trial plot (50% dominance) had levels of up to 52.9mg kg(-1). Notably, the hot air drying process during the production of grass pellets did not lead to a reduction of PA/PANO levels. Instead, the levels in grass pellets with 10% and 30% S. aquaticus ranged from 3.1 to 55.1mg kg(-1). Grass pellets from the trial plot contained up to 96.8mg kg(-1). In conclusion, hot air drying and grass pellet production did not affect PA/PANO contents in plant material and therefore, heat-dried products cannot be regarded as safe in view of the toxic potential of 1,2-dehydro-pyrrolizidine alkaloids.

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

Highlights • First study to investigate fate of phytotoxins in drinking water treatment. • Biological sand filters removed bracken toxins and certain alkaloids. • Pyrrolizidine alkaloids showed recalcitrant character in sand filters. • Further investigation involving more advanced treatment are needed., Phytotoxins - toxins produced by plants – are contaminants with the potential to impair drinking water quality. They encompass a large group of toxic, partially persistent compounds that have been detected in seepage waters and in shallow wells used for drinking water production. If phytotoxins enter wells used for drinking water production, it is essential to know if the drinking water treatment processes will remove them from the water phase. However, it is currently unknown whether phytotoxins remain stable during traditional groundwater treatment using sand filters as the main treatment process. The objective of this study is to investigate removal potential of phytotoxins in biological sand filters and to asses if the removal potential is similar at different waterworks. Microcosms were set up with filter sand and drinking water collected at different groundwater-based waterworks. To be able to monitor phytotoxin removal ptaquiloside, caudatoside, gramine, sparteine, jacobine N-oxide, senecionine N-oxide and caffeine were applied at initial concentrations of 300 µg L-1, which is approx. two orders of magnitude higher than currently detected in environment, but expected to cover extreme environmental conditions. Removal was monitored over a period of 14 days. Despite the high initial concentration, all filter sands removed ptaquiloside and caudatoside completely from the water phase and at waterworks where pellet softening was implemented (pH 8.4) prior to rapid sand filtration, complete removal occurred within the first 30 min. All filter sands removed gramine and sparteine, primarily by a biological process, while jacobine N-oxide, senecionine N-oxide and caffeine were recalcitrant in the filter sands. During degradation of ptaquiloside and caudatoside we observed formation and subsequent removal of degradation products pterosin B and A. Filter sands with the highest removal potential were characterised by high contents of deposited iron and manganese oxides and hence large specific surface areas. Difference between bacterial communities investigated by 16S rRNA gene analyses did not explain different removal in the filter sands. All five investigated filter sands showed similar degradation patterns regardless of water chemistry and waterworks of origin. In drinking water treatment systems biological sand filters might therefore remove phytotoxin contaminants such as ptaquiloside, caudatoside, gramine, sparteine, while for other compounds e.g. jacobine N-oxide, senecionine N-oxide further investigations involving more advanced treatment options are needed., Graphical abstract Image, graphical abstract

Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs., Graphical abstract fx1, Highlights • Senecionine induces apoptosis in primary mouse and human hepatocytes as well as in mouse livers. • Senecionine induces mitochondrial Drp1 translocation, mitochondrial fragmentation, loss of mitochondrial membrane potential and release of mitochondrial cytochrome c in hepatocytes. • Pharmacological inhibition or genetic deletion of Drp1 protects against senecionine-induced hepatotoxicity. • Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by Pyrrolizidine alkaloids.

Introduction Crotalaria sessiliflora L. is a Chinese traditional herb for treatment of cutaneum carcinoma and cervical carcinoma. In addition to monocrotaline, coexisting pyrrolizidine alkaloids (PAs) also require further quantification for quality control and pharmaceutical uses of the herb. Objective To establish a UPLC-Q-Orbitrap/MS method of simultaneous determination of coexisting PAs with same parent structure for quality control and comprehensive researches of Crotalaria sessiliflora L. Methodology PAs in Crotalaria sessiliflora L. were analysed by UPLC-Q-Orbitrap/MS method. Coexisting PAs were identified by mass data of full MS-dd-MS2 based on the characteristic fragmentation pattern and necine-core structure. Moreover, quantification of PAs was conducted by parallel reaction monitoring (PRM) mode using m/z 138, m/z 120 and m/z 94 from identical necine-core structure as quantitative ions with single monocrotaline standard for accurate calibration. Results Five PAs, named monocrotaline, retrorsine, senecionine, integerrimine, O-9-angeloylretronecine, were indentified and confirmed. Quantitative ions of m/z 138, m/z 120 and m/z 94 were used for quantification of PAs containing the necine-core structure in Crotalaria sessiliflora L. The results demonstrated that contents, precision and recoveries of the five PAs mentioned earlier were respectively 3.307–30.35 μg/g, 1.1–4.5% and 88.91–92.33% while using m/z 120 as the best quantitative ion. Conclusion The UPLC-Q-Orbitrap/MS method was established for simultaneous determination of five PAs in Crotalaria sessiliflora L. without all corresponding standards, and was proved that it was simple, convenient and effective for comprehensive quality control and pharmaceutical uses. Copyright © 2017 John Wiley & Sons, Ltd.

Ethnopharmacological relevance Pyrrolizidine alkaloids (PAs) are a group of phytotoxins widely present in about 3% of flowering plants. Many PA-containing herbal plants can cause liver injury. Our previous studies demonstrated that PA N-oxides are also hepatotoxic, with toxic potency much lower than the corresponding PAs, due to significant differences in their toxicokinetic fates. Aim of study This study aimed to investigate the oral absorption of PAs and PA N-oxides for better understanding of their significant differences in toxicokinetics and toxic potency. Materials and methods The oral absorption of PAs and PA N-oxides in rats and in rat in situ single pass intestine perfusion model was investigated. The intestinal permeability and absorption mechanisms of five pairs of PAs and PA N-oxides were evaluated by using Caco-2 monolayer model. Results The plasma concentrations of total PAs and PA N-oxides within 0–60 min were significantly lower in rats orally treated with a PA N-oxide-containing herbal alkaloid extract than with a PA-containing herbal alkaloid extract at the same dose, indicating that the absorption of PA N-oxides was lower than that of PAs. Using the rat in situ single pass intestine perfusion model, less cumulative amounts of retrorsine N-oxide in mesenteric blood were observed compared to that of retrorsine. In Caco-2 monolayer model, all five PAs showed absorption with Papp AtoB values [(1.43–16.26) × 10−6 cm/s] higher than those of corresponding N-oxides with Papp AtoB values lower than 1.35 × 10−6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-oxide, all PAs and PA N-oxides investigated were absorbed via passive diffusion. While, for these 3 PA N-oxides, in addition to passive diffusion as their primary transportation, efflux transporter-mediated active transportation was also involved but to a less extent with the efflux ratio of 2.31–3.41. Furthermore, a good correlation between lipophilicity and permeability of retronecine-type PAs and their N-oxides with absorption via passive diffusion was observed, demonstrating that PAs have a better oral absorbability than that of the corresponding PA N-oxides. Conclusion We discovered that among many contributors, the lower intestinal absorption of PA N-oxides was the initiating contributor that caused differences in toxicokinetics and toxic potency between PAs and PA N-oxides.