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

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

Utilization of a novel human hepatocyte-endothelial cell coculture model to determine differential toxicities of pyrrolizidine alkaloid food contaminants.

Pyrrolizidine alkaloids (PA) are comprised of a family of hundreds of metabolites, produced by plants as a mechanism to protect against herbivory. Upon ingestion and metabolism, dehydropyrrolizidine alkaloids are formed, which are known to generate DNA adducts and subsequently double-strand DNA breaks. Within the liver, the most sensitive cell type to PA exposure is the sinusoidal endothelial cell, as evidenced by the generation of veno-occlusive disease in the human population. PAs are a common crop contaminant and have been regulated by some agencies, using the precautionary principle; each equally potent and genotoxic. Therefore, as a proof of principle we have established a human in vitro coculture model system, utilizing the metabolically active HepaRG hepatocyte and the SK-Hep-1 endothelial cell, to determine differential potencies of different PAs commonly found in crops and food products, notably cell death, targeting of endothelial cells, and genotoxicity comparing the micronucleus assay versus γH2AX assay. Our results demonstrate differential potencies of the PAs used, which encompass three esterification states (monoester, cyclic diester, and open-chain diester). The results suggest that a more nuanced approach to the regulation of PAs may be more appropriate in the regulatory decision-making process.

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

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

Mass spectrometric analysis strategies for pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) in food and natural preparations have received widespread attention due to their hepatotoxicity, genotoxicity, and embryotoxicity. Mass spectrometry (MS), as a high resolution, high sensitive, and high throughput detection tool, has been the most commonly used technique for the determination of PAs. The continuous advancement of new technologies, methods, and strategies in the field of MS has contributed to the improvement of the analytical efficiency and methodological enhancement of PAs. This paper provides an overview of the structure, toxicity properties and commonly employed analytical methods, focusing on the concepts, advances, and novel techniques and applications of MS-based methods for the analysis of PAs. Additionally, the remaining challenges, future perspectives, and trends for PA detection are discussed. This review provides a reference for toxicological studies of PAs, content monitoring, and the establishment of quality control and safety standards for herbal and food products.

Pyrrolizidine alkaloids are synthesized and accumulated in flower of Myosotis scorpioides.

Pyrrolizidine alkaloids (PAs) are specialized metabolites that are produced by various plant families that act as defense compounds against herbivores. On the other hand, certain lepidopteran insects uptake and utilize these PAs as defense compounds against their predators and as precursors of their sex pheromones. Adult males of Parantica sita, a danaine butterfly, convert PAs into their sex pheromones. In early summer, P. sita swarms over the flowers of Myosotis scorpioides, which belongs to the family Boraginaceae. M. scorpioides produces PAs, but the organs in which PAs are produced and whether P. sita utilizes PAs in M. scorpioides are largely unknown. In the present study, we clarified that M. scorpioides accumulates retronecine-core PAs in N-oxide form in all organs, including flowers. We also identified two M. scorpioides genes encoding homospermidine synthase (HSS), a key enzyme in the PA biosynthetic pathway, and clarified that these genes are expressed in all organs where PAs accumulate. Phylogenetic analysis suggested that these two HSS genes were originated from gene duplication of deoxyhypusine synthase gene like other HSS genes in PA-producing plants. These results suggest that PAs are synthesized and accumulated in the flower of M. scorpioides and provide a possibility for a PA-mediated interaction between P. sita and M. scorpioides.

Pyrrolizidine alkaloid contamination of food in Africa: A review of current trends and implications.

Pyrrolizidine alkaloids (PAs) contamination of foodstuffs has become a topical issue in recent years on account of its potential hepatotoxicity to consumers. This review therefore highlights human exposure to PAs across Africa, focusing on their occurrence, current trends of food contamination, and their associated health implications. A comprehensive search of peer-scientific literature and relevant databases, PubMed, Google Scholar, Science Direct, Web of Science and Scopus, was conducted from 2001 to 2023 focusing mainly on foodstuffs, including grains, herbs, teas, honey, and livestock products. The findings revealed that PA contamination is a prevalent issue in several African countries, with the primary sources of contamination attributed to the consumption of honey and the use of PA plants as herbs in food preparations. Additionally, poor farming practices have been found to influence the presence and levels of PAs in foodstuffs. To mitigate PA contamination in food and safeguarding public health across the continent, several strategies are proposed, including the implementation of stringent regulatory and quality control measures, adoption of Good Agricultural Practices, and public awareness campaigns to educate producers, consumers and beekeepers about the risks associated with PA-contaminated food products.

Analysis of Pyrrolizidine Alkaloids in Stingless Bee Honey and Identification of a Botanical Source as Ageratum conyzoides.

Stingless bee honeys (SBHs) from Australian and Malaysian species were analysed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the presence of pyrrolizidine alkaloids (PAs) and the corresponding N-oxides (PANOs) due to the potential for such hepatotoxic alkaloids to contaminate honey as a result of bees foraging on plants containing these alkaloids. Low levels of alkaloids were found in these SBHs when assessed against certified PA standards in targeted analysis. However, certain isomers were identified using untargeted analysis in a subset of honeys of Heterotrigona itama which resulted in the identification of a PA weed species (Ageratum conyzoides) near the hives. The evaluation of this weed provided a PA profile matching that of the SBH of H. itama produced nearby, and included supinine, supinine N-oxide (or isomers) and acetylated derivatives. These PAs lacking a hydroxyl group at C7 are thought to be less hepatoxic. However, high levels were also observed in SBH (and in A. conyzoides) of a potentially more toxic diester PA corresponding to an echimidine isomer. Intermedine, the C7 hydroxy equivalent of supinine, was also observed. Species differences in nectar collection were evident as the same alkaloids were not identified in SBH of G. thoracica from the same location. This study highlights that not all PAs and PANOs are identified using available standards in targeted analyses and confirms the need for producers of all types of honey to be aware of nearby potential PA sources, particularly weeds.

The toxicokinetic and metabolism of structurally diverse pyrrolizidine alkaloids in rats.

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

Pyrrolizidine Alkaloids-Pros and Cons for Pharmaceutical and Medical Applications.

Heterocyclic organic compounds named pyrrolizidine alkaloids (PAs) belong to a group of alkaloids and are synthesized by either plants or microorganisms. Therefore, they are naturally occurring secondary metabolites. They are found in species applied in the pharmaceutical and food industries, thus a thorough knowledge of their pharmacological properties and toxicology to humans is of great importance for their further safe employment. This review is original because it synthesizes knowledge of plant and microbial PAs, which is unusual in the scientific literature. We have focused on the Boraginaceae family, which is unique due to the exceptional richness and diversity of its PAs in plant species. We have also presented the microbial sources of PAs, both from fungi and bacteria. The structure and metabolism of PAs have been discussed. Our main aim was to summarize the effects of PAs on humans, including both negative, toxic ones, mainly concerning hepatotoxicity and carcinogenicity, as well as potentially positive ones for pharmacological and medical applications. We have collected the results of studies on the anticancer activity of PAs from plant and microbial sources (mainly Streptomyces strains) and on the antimicrobial activity of PAs on different strains of microorganisms (bacteria and fungi). Finally, we have suggested potential applications and future perspectives.

Metabolite Profiling in the Liver, Plasma and Milk of Dairy Cows Exposed to Tansy Ragwort (Senecio jacobae) Pyrrolizidine Alkaloids.

BACKGROUND: Plant-derived pyrrolizidine alkaloids (PAs) in feed cause metabolic disturbances in farm animals resulting in high economic losses worldwide. The molecular pathways affected by these PAs in cells and tissues are not yet fully understood. The objective of the study was to examine the dose-dependent effects of orally applied PAs derived from tansy ragwort in midlactation dairy cows. METHODS: Twenty Holstein dairy cows were treated with target exposures of 0, 0.47, 0.95 and 1.91 mg of total PA/kg of body weight/d in control, PA1, PA2 and PA3, respectively, for 28 days. Liver tissue biopsy and plasma and milk samples were taken at day 28 of treatment to assess changes in metabolic pathways. A targeted metabolomics approach was performed to detect the metabolite profiles in all compartments. RESULTS: The PA-affected metabolite profiling in liver tissue, plasma and milk revealed changes in three substrate classes: acylcarnitines (ACs), phosphatidylcholines (PCs) and sphingomyelins (SMs). In addition, in the plasma, amino acid concentrations were affected by PA exposure. CONCLUSIONS: PA exposure disturbed liver metabolism at many sites, especially devastating pathways related to energy metabolism and to amino acid utilization, most likely based on mitochondrial oxidative stress. The effects on the milk metabolite profile may have consequences for milk quality.

Exposure to echimidine impairs the heart development and function of zebrafish larvae.

Pyrrolizidine alkaloids (PAs) are a class of phytotoxins that are widely distributed and can be consumed by humans through their daily diets. Echimidine is one of the most abundant PAs, but its safety, particularly its effects on development, is not fully understood. In this study, we used a zebrafish model to assess the developmental toxicity of echimidine. Zebrafish embryos were exposed to echimidine at concentrations of 0.02, 0.2, and 2 mg/L for 96 h. Our study revealed that embryonic exposure to echimidine led to developmental toxicity, characterized by delayed hatching and reduced body length. Additionally, echimidine exposure had a notable impact on heart development in larvae, causing tachycardia and reducing stroke volume (SV)and cardiac output (CO). Upon exposing the transgenic zebrafish strain Tg(cmlc2:EGFP) to echimidine, we observed atrial dilation and thinning of the atrial wall in developing embryos. Moreover, our findings indicated abnormal expression of genes associated with cardiac development (including gata4, tbx5, nkx2.5 and myh6) and genes involved in calcium signaling pathways (such as cacna1aa, cacna1sa, ryr2a, ryr2b, atp2a2a, atp2a2b, slc8a1, slc8a3 and slc8a4a). In summary, our findings demonstrate that echimidine may impair cardiac development and function in zebrafish larvae by disrupting calcium transport, leading to developmental toxicity. These findings provide insights regarding the safety of products containing PAs in food and medicine.

Clinicopathological characteristics and diagnosis of hepatic sinusoidal obstruction syndrome caused by Tusanqi - Case report and literature review.

Tusanqi-induced hepatic sinusoidal obstruction syndrome (HSOS) is caused by exposure to pyrrolizidine alkaloids (PAs) and manifests as abdominal distension, liver pain, ascites, jaundice, and hepatomegaly. Pathologically, hepatic congestion and sinusoidal occlusion are observed in HSOS. We summarized the clinical characteristics of 124 patients with HSOS caused by Tusanqi in China between 1980 and 2019, along with those of 831 patients from seven English case series. The main clinical manifestations of PA-HSOS included abdominal pain, ascites, and jaundice. Common imaging features included characteristic heterogeneous density, slender hepatic veins, and other nonspecific changes. The acute stage is primarily manifested as hepatic sinus congestion and necrosis. Meanwhile, the persistence of hepatic sinus congestion and the onset of perisinusoidal fibrosis were observed during the repair stage. Finally, the persistence of hepatic sinusoidal fibrosis and resultant central hepatic vein occlusion were observed in the chronic stage. The new Nanjing standard for PA-HSOS incorporates the history of PA consumption and imaging features and eliminates weight gain and the serum total bilirubin value. Preliminary clinical validation of the Nanjing standard for PA-HSOS diagnosis revealed a sensitivity and specificity of 95.35 and 100%, respectively.

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

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

Biomarkers and their potential for detecting livestock plant poisonings in Western North America.

The United States National Cancer Institute defines a biomarker as: "A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease." In Veterinary Medicine, biomarkers associated with plant poisonings of livestock have great utility. Since grazing livestock poisoned by toxic plants are often found dead, biomarkers of plant poisoning allow for a more rapid postmortem diagnosis and response to prevent further deaths. The presence and concentration of toxins in poisonous plants are biomarkers of risk for livestock poisoning that can be measured by the chemical analysis of plant material. More difficult is, the detection of plant toxins or biomarkers in biological samples from intoxicated or deceased animals. The purpose of this article is to review potential biomarkers of plant poisoning in grazing livestock in the Western North America including recently investigated non-invasive sampling techniques. Plants discussed include larkspur, lupine, water hemlock, swainsonine-containing plants, selenium-containing plants, and pyrrolizidine alkaloid containing plants. Other factors such as animal age and sex that affect plant biomarker concentrations in vivo are also discussed.

An Intramolecular Hydroaminomethylation-Based Approach to Pyrrolizidine Alkaloids under Microwave-Assisted Heating.

A general method for the synthesis of pyrrolizidine derivatives using an intramolecular hydroaminomethylation protocol (HAM) under microwave (MW) dielectric heating is reported. Starting from a 3,4-bis(benzyloxy)-2-[(benzyloxy)methyl]-5-vinylpyrrolidine, MW-assisted intramolecular HAM in the presence of gaseous H2 and CO gave the natural alkaloid hyacinthacine A2 protected as benzyl ether. The same approach gave a lentiginosine analogue starting from the corresponding vinyl N-hydroxypyrrolidine. The nature of the reaction products and the yields were strongly influenced by the relative stereochemistry of the starting pyrrolidines, as well as by the catalyst/ligand employed. The use of ethanol as a solvent provides environmentally friendly conditions, while the ligand/catalyst system can be recovered by separating the alkaloid product with an SCX column and recycling the ethanolic solution. HAM worked up to three times with the recycled catalyst solution without any significant impact on yield.

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

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

Liquorice Extract and 18ß-Glycyrrhetinic Acid Protect Against Experimental Pyrrolizidine Alkaloid-Induced Hepatotoxicity in Rats Through Inhibiting Cytochrome P450-Mediated Metabolic Activation.

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18ß-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

A TMT-based shotgun proteomics uncovers overexpression of thrombospondin 1 as a contributor in pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

Hepatic sinusoidal obstruction disease (HSOS) is a rare but life-threatening vascular liver disease. However, its underlying mechanism and molecular changes in HSOS are largely unknown, thus greatly hindering the development of its effective treatment. Hepatic sinusoidal endothelial cells (HSECs) are the primary and essential target for HSOS. A tandem mass tag-based shotgun proteomics study was performed using primary cultured HSECs from mice with HSOS induced by senecionine, a representative toxic pyrrolizidine alkaloid (PA). Dynamic changes in proteome were found at the initial period of damage and the essential role of thrombospondin 1 (TSP1) was highlighted in PA-induced HSOS. TSP1 over-expression was further confirmed in human HSECs and liver samples from patients with PA-induced HSOS. LSKL peptide, a known TSP1 inhibitor, protected mice from senecionine-induced HSOS. In addition, TSP1 was found to be covalently modified by dehydropyrrolizidine alkaloids in human HSECs and mouse livers upon senecionine treatment, thus to form the pyrrole-protein adduct. These findings provide useful information on early changes in HSECs upon PA treatment and uncover TSP1 overexpression as a contributor in PA-induced HSOS.

Development of a Drum Tower Severity Scoring (DTSS) system for pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

BACKGROUND AND AIMS: There has been no reliable severity system based on the prognosis to guide therapeutic strategies for patients with pyrrolizidine alkaloid (PA)-induced hepatic sinusoidal obstruction syndrome (HSOS). We aimed to create a novel Drum Tower Severity Scoring (DTSS) system for these patients to guide therapy. METHODS: 172 Patients with PA-HSOS who received supportive care and anticoagulation therapy in Nanjing Drum Tower Hospital from January 2008 to December 2020 were enrolled and analyzed retrospectively. These patients were randomized into a training or validation set in a 3:1 ratio. Next, we established and validated the newly developed DTSS system. RESULTS: Analysis identified a predictive formula: logit (P) = 0.004 × aspartate aminotransferase (AST, U/L) + 0.019 × total bilirubin (TB, µmol/L) - 0.571 × fibrinogen (FIB, g/L) - 0.093 × peak portal vein velocity (PVV, cm/s) + 1.122. Next, we quantified the above variables to establish the DTSS system. For the training set, the area under the ROC curve (AUC) (n = 127) was 0.787 [95% confidence interval (CI) 0.706-0.868; p < 0.001]. With a lower cut-off value of 6.5, the sensitivity and negative predictive value for predicting no response to supportive care and anticoagulation therapy were 94.7% and 88.0%, respectively. When applying a high cut-off value of 10.5, the specificity was 92.9% and the positive predictive value was 78.3%. For the validation set, the system performed stable with an AUC of 0.808. CONCLUSIONS: The DTSS system can predict the outcome of supportive care and anticoagulation in PA-HSOS patients with satisfactory accuracy by evaluating severity, and may have potential significance for guiding therapy.

High-performance counter-current chromatography purification and off-line mass spectrometry monitoring and identification of pyrrolizidine alkaloid markers of tropical Ghanaian honey.

The nutritional and medicinal properties of honey have been well-documented. However, honey has occasionally been contaminated with hepatotoxic pyrrolizidine alkaloids as a result of bees foraging on the flowers of pyrrolizidine alkaloid plants. This study establishes a simple and rapid method to determine the marker pyrrolizidine alkaloids in honey using high-performance counter-current chromatography and an off-line electrospray ionization-tandem mass spectrometry, in order to identify the botanical sources responsible for the contamination. The honey sample was initially liquid-liquid extracted (sulfuric acid/hexane, 2:3, v/v) to enrich the pyrrolizidine alkaloids and subsequently purified by a semi-preparative high-performance counter-current chromatography using a solvent system, hexane/butanol/1% aqueous ammonia, 1:1:2, v/v, based on partition coefficient measurements of the target alkaloids. The recovered fractions were profiled by injecting them sequentially into an off-line electrospray ionization-tandem mass spectrometry device to monitor the preparative molecular weight based on elution and extrusion modes. The monitored lycopsamine-type pyrrolizidine alkaloids and their N-oxides (m/z 300, 316; lycopsamine, intermedine, rinderine, and echinatine) were used as the phytochemical markers to identify plants like Chromolaena odorata, Ageratum spp., or Heliotropium spp. to be responsible for the pyrrolizidine alkaloid contamination. Identification of these pyrrolizidine alkaloid plants could guide beekeepers in locating their beehives in order to minimize their potential liver damaging effects.