Gynura segetum induces hepatic sinusoidal obstruction syndrome in a child: A case report.

RATIONALE: Hepatic sinusoidal obstruction syndrome (HSOS), which includes hepatic stasis and portal hypertension, is a rare vascular disorder of the liver. It is often associated with hematopoietic stem cell transplantation. It is also possible to treat this disease using Chinese herbal medicines that contain pyrrolizidine alkaloids (PAs). This disease is extremely rare in children and poses a serious threat to their health. To our knowledge, this is the first case of HSOS in a child with PAs. PATIENT CONCERNS: We report a 4-year-old boy suffering from abdominal pain, hepatomegaly, massive ascites, elevated liver enzyme level, and severe portal hypertension as a result of the consumption of Gynura segetum (also known as Tusanqi in Chinese, a traditional herbal medicine containing PAs). DIAGNOSES: The child was finally diagnosed with PA-HSOS based on pathological diagnosis and imaging examination. INTERVENTION: With active symptomatic and supportive care and sequential anticoagulation therapy, the abdominal distension and liver function improved in the patient. OUTCOMES: The patient was eventually recovered. The levels of liver enzymes, hemoglobin, and bilirubin were normal, and the international normalized ratio fluctuated between 2.0 and 3.0 during 1-year follow-up after discharge. LESSONS: This case report emphasizes the prevention of Chinese herb-induced liver injury in children and the importance of active long-term sequential anticoagulant therapy to reduce the progressive damage of PA-HSOS in the liver.

Diagnosis, toxicological mechanism, and detoxification for hepatotoxicity induced by pyrrolizidine alkaloids from herbal medicines or other plants.

Pyrrolizidine alkaloids (PAs) are one type of phytotoxins distributed in various plants, including many medicinal herbs. Many organs might suffer injuries from the intake of PAs, and the liver is the most susceptible one. The diagnosis, toxicological mechanism, and detoxification of PAs-induced hepatotoxicity have been studied for several decades, which is of great significance for its prevention, diagnosis, and therapy. When the liver was exposed to PAs, liver sinusoidal endothelial cells (LSECs) loss, hemorrhage, liver parenchymal cells death, nodular regeneration, Kupffer cells activation, and fibrogenesis occurred. These pathological changes classified the PAs-induced liver injury as acute, sub-acute, and chronic type. PAs metabolic activation, mitochondria injury, glutathione (GSH) depletion, inflammation, and LSECs damage-induced activation of the coagulation system were well recognized to play critical roles in the pathological process of PAs-induced hepatotoxicity. A lot of natural compounds like glycyrrhizic acid, (-)-epicatechin, quercetin, baicalein, chlorogenic acid, and so on were demonstrated to be effective in alleviating PAs-induced liver injury, which rendered them huge potential to be developed into therapeutic drugs for PAs poisoning in clinics. This review presents updated information about the diagnosis, toxicological mechanism, and detoxification studies on PAs-induced hepatotoxicity.

Pyrrolizidine Alkaloids in Foods, Herbal Drugs, and Food Supplements: Chemistry, Metabolism, Toxicological Significance, Analytical Methods, Occurrence, and Challenges for Future.

Consumers are increasingly seeking natural alternatives to chemical compounds, including the use of dried aromatic plants as seasonings instead of salt. However, the presence of pyrrolizidine alkaloids (PAs) in food supplements and dried plants has become a concern because of their link to liver diseases and their classification as carcinogenic by the International Agency for Research on Cancer (IARC). Despite European Union (EU) Regulation (EU) 2023/915, non-compliance issues persist, as indicated by alerts on the Rapid Alert System for Food and Feed (RASFF) portal. Analyzing PAs poses a challenge because of their diverse chemical structures and low concentrations in these products, necessitating highly sensitive analytical methods. Despite these challenges, ongoing advancements in analytical techniques coupled with effective sampling and extraction strategies offer the potential to enhance safety measures. These developments aim to minimize consumer exposure to PAs and safeguard their health while addressing the growing demand for natural alternatives in the marketplace.

Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Analysis of potential risks of clinical application of Yi Dian Hong and its proprietary Chinese medicines: A review.

Yi Dian Hong, belonging to the Asteraceae family, finds widespread use in traditional Chinese medicine for its effectiveness in clearing heat, detoxifying, promoting blood circulation, reducing swelling, and cooling the blood. Modern medical research has revealed that Yi Dian Hong and its proprietary Chinese medicines possess biological functions such as inhibiting tumor-specific angiogenesis and regulating immune-related molecules. However, studies have identified that the primary component of Yi Dian Hong contains pyrrolizidine alkaloids (PAs), a toxic substance with potential risks to the liver, lungs, genes, and a propensity for carcinogenicity. Many countries impose strict controls on the content of PAs in herbal medicines and products. Unfortunately, China currently lacks relevant content standards, thereby introducing greater clinical application risks. To ensure the safety of clinical use of Yi Dian Hong, this review will analyze the risk associated with Yi Dian Hong and its proprietary Chinese medicines in clinical applications based on the PAs content in these medicines and provide recommendations.

Monocrotaline presence in the Crotalaria (Fabaceae) plant genus and its influence on arthropods in agroecosystems / Presença de monocrotalina em plantas do gênero Crotalaria (Fabaceae) e a sua influência sobre artrópodes nos agroecossistemas

Abstract Crotalaria (Fabaceae) occurs abundantly in tropical and subtropical regions and has about 600 known species. These plants are widely used in agriculture, mainly as cover plants and green manures, in addition to their use in the management of phytonematodes. A striking feature of these species is the production of pyrrolizidine alkaloids (PAs), secondary allelochemicals involved in plant defense against herbivores. In Crotalaria species, monocrotaline is the predominant PA, which has many biological activities reported, including cytotoxicity, tumorigenicity, hepatotoxicity and neurotoxicity, with a wide range of ecological interactions. Thus, studies have sought to elucidate the effects of this compound to promote an increase in flora and fauna (mainly insects and nematodes) associated with agroecosystems, favoring the natural biological control. This review summarizes information about the monocrotaline, showing such effects in these environments, both above and below ground, and their potential use in pest management programs.

Resumo Crotalaria (Linnaeus, 1753) (Fabaceae) ocorre abundantemente em regiões tropicais e subtropicais e tem cerca de 600 espécies conhecidas. Estas plantas são amplamente utilizadas na agricultura, principalmente como cobertura e adubos verdes, além da sua utilização no manejo de fitonematoides. Uma característica marcante destas espécies é a produção de alcalóides pirrolizidinicos (APs), aleloquímicos secundários envolvidos na defesa das plantas contra os herbívoros. Nas espécies de Crotalaria, a monocrotalina é a AP predominante, que tem muitas atividades biológicas relatadas, incluindo citotoxicidade, tumorigenicidade, hepatotoxicidade e neurotoxicidade, além de uma vasta gama de interações ecológicas. Assim, estudos têm procurado elucidar os efeitos desse composto para promover um incremento na flora e fauna (principalmente insetos e nematoides) associados aos agroecossistemas, favorecendo o controle biológico natural. Esta revisão compila informações sobre a monocrotalina, mostrando tais efeitos nesses ambientes, tanto acima como abaixo do solo e a sua potencial utilização em programas de manejo de pragas.

Histological evaluation of pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome: Correlation with Drum Tower Severity Scoring.

BACKGROUND: Hepatic sinusoidal obstruction syndrome induced by pyrrolizidine alkaloids (PA-HSOS) is a complication of drug-induced liver damage. Few studies have examined the relationship between pathological changes and clinical circumstances in PA-HSOS. The Drum Tower Severity Scoring System (DTSS) was developed using prognostic indicators from clinical treatment outcomes. We hypothesized that the severity of pathological damage is consistent with DTSS. AIMS: We aimed to improve our understanding and assessment of vascular liver injury disease histopathology by studying larger sample sizes of human histopathological samples. We also wanted to confirm the link between histopathological findings and DTSS. METHODS: The study included 62 patients with PA-HSOS who underwent transjugular liver biopsy. Their hepatic pathological tissues were evaluated. Analyses of linear regression and Spearman's correlation were employed to examine the relationship between DTSS and pathological characteristics. RESULTS: Clinical performance and the DTSS score were used to determine histopathological severity. The sinusoidal congestion area (SCA), central venous endothelial injury (CVEI), and fibrinoid exudation in congestion foci (FECF) were significant indicators. SCA was linearly related to the DTSS score. CONCLUSION: Our findings show that hepatic pathological characteristics correlate with DTSS scores in PA-HSOS. SCA, CVEI, and FECF may be helpful for determining PA-HSOS severity.

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.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects.

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.

Pyrrolizidine Alkaloids in Food on the Italian Market.

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by over 6000 plant species worldwide. PAs enter the food chain through accidental co-harvesting of PA-containing weeds and through soil transfer from the living plant to surrounding acceptor plants. In animal studies, 1,2-unsaturated PAs have proven to be genotoxic carcinogens. According to the scientific opinion expressed by the 2017 EFSA, the foods with the highest levels of PA contamination were honey, tea, herbal infusions, and food supplements. Following the EFSA's recommendations, data on the presence of PAs in relevant food were monitored and collected. On 1 July 2022, the Commission Regulation (EU) 2020/2040 came into force, repealed by Commission Regulation (EU) 2023/915, setting maximum levels for the sum of pyrrolizidine alkaloids in certain food. A total of 602 food samples were collected from the Italian market between 2019 and 2022 and were classified as honey, pollen, dried tea, dried herbal infusions, dried herbs, and fresh borage leaves. The food samples were analyzed for their PA content via an in-house LC-MS/MS method that can detect PAs according to Regulation 2023/915. Overall, 42% of the analyzed samples were PA-contaminated, 14% exceeded the EU limits, and the items most frequently contaminated included dried herbs and tea. In conclusion, the number of food items containing considerable amounts of PAs may cause concern because they may contribute to human exposure, especially considering vulnerable populations-most importantly, children and pregnant women.

Occurrence and associated health risks of pyrrolizidine alkaloids in supplements marketed in Ghana for improved sexual performance.

Pyrrolizidine alkaloids (PAs) are noted for their hepatotoxic, genotoxic, and carcinogenic effects in animals and humans following metabolic activation in the liver. In this study, herbal supplements sold in Ghana for sexual improvement were analysed for the presence of 64 PAs using LC-MS/MS analysis. Up to 17 different PAs were identified in 19 out of the 37 samples analysed. The sum of PAs in samples ranged from 5 to 3204 µg kg-1. Since the PA content in the herbal medicinal preparations was generally lower than in honey samples, their presence was mainly attributed to cross-contamination. The observed levels would result in estimated daily intakes from 0.01 to 12 µg per day or 0.0002 to 0.2 µg kg-1 bw day-1 for a person weighing 70 kg. The margins of exposure ranged from 1200 to 1,400,000 with eight samples showing values below 10,000, thus indicating a health concern.

Separation of pyrrolizidine alkaloids in different Senecio species using ultra-high performance supercritical fluid chromatography.

Different Senecio species, especially S. inaequidens - a neophyte native to South Africa - have widely spread across Europe and now are found worldwide. The entire genus is known to contain toxic pyrrolizidine alkaloids (PAs), which renders them a possible health hazard to humans and livestock. As they can enter the food chain or occur as contaminants in herbal crops and phytopharmaceutical formulations (e.g. teas), efficient and straightforward assays for their qualitative and quantitative analysis are in high demand. Different techniques have been used for this purpose, most commonly HPLC or GC. As the analysis of PAs is a challenging task, alternative methodologies like ultra-high performance SFC (UHPSFC) may offer an additional benefit in terms of their separation efficiency and orthogonal selectivity. In this study an UHPSFC approach for the simultaneous determination of six PAs (free bases as well as N-oxides) is presented, which achieved the baseline separation of all standard compounds in seven min. Optimal separation was carried out in gradient mode on a Torus™ DEA column with 0.05% ammonia in methanol as modifier. The column temperature was 25 °C, ABPR 1900 psi and flow rate 1.1 mL/min, with a detection wavelength of 215 nm. The assay was validated and fulfilled all ICH criteria exhibiting good linearity (R2 ≥ 0.9994), precision (inter-day variance ≤ 3.67%, intra-day variance ≤ 3.92%) and recovery rates (96.3-104.1%), with detection limits typical for SFC-PDA (≤ 4.24 µg/mL). Furthermore, it could conveniently be coupled to MS-detection, which increased the sensitivity significantly. To confirm practical suitability of the method, different Senecio samples were analyzed, indicating a high qualitative as well as quantitative difference in their PA profile (e.g. total amounts of PA between 0.09 and 4.63 mg/g).

Potency ranking of pyrrolizidine alkaloids in metabolically competent human liver cancer cells and primary human hepatocytes using a genotoxicity test battery.

Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure-toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1-10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.

Risk assessment of short-term intake of pyrrolizidine alkaloids in food: derivation of an acute reference dose.

Pyrrolizidine alkaloids (PA) are phytochemicals that are known to act as human hepatotoxins and are also considered to be genotoxic carcinogens. Several plant-derived foods are frequently contaminated with PA, like teas and herbal infusions, spices and herbs or certain food supplements. With respect to the chronic toxicity of PA, the carcinogenic potential of PA is generally regarded as the critical toxicological effect. The risk assessment of the short-term toxicity of PA, however, is internationally less consistent. The characteristic pathological syndrome of acute PA toxicity is hepatic veno-occlusive disease. High PA exposure levels may lead to liver failure and even death as documented by several case reports. In the present report, we suggest a risk assessment approach for the derivation of an acute reference dose (ARfD) for PA of 1 µg/kg body weight per day based on a sub-acute animal toxicity study in rats after oral PA administration. The derived ARfD value is further supported by several case reports describing acute human poisoning following accidental PA intake. The here derived ARfD value may be used for PA risk assessment in cases where the short-term toxicity of PA is of interest in addition to the assessment of the long-term risks.

Targeting erythrocyte-mediated hypoxia to alleviate lung injury induced by pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are widely distributed natural toxins and have been extensively studied for their hepatotoxicity. However, PA-induced pulmonary toxicity remains less studied regarding the initiating mechanism and treatment approaches. Our previous study demonstrated the formation of pyrrole-hemoglobin adducts after PA exposure in vivo, which is suspected to affect the oxygen-carrying capacity of erythrocytes [red blood cells (RBCs)] consequently. The present study aimed to investigate the effects of PAs on the oxygen-carrying capacity of RBCs and the potential of targeting RBC-mediated hypoxia to alleviate PA-induced lung injury. First, rats were treated with retrorsine (RTS) or monocrotaline (MCT) intravenously at 0.2 mmol/kg. The results of Raman spectrometry analysis on blood samples revealed both RTS and MCT significantly reduced the oxygen-carrying capacity of RBCs. Further, MCT (0.2 mmol/kg) was orally given to the rats with or without pretreatment with two doses of erythropoietin (Epo, 500 IU/kg/dose every other day), an RBC-stimulating agent. Biochemical and histological results showed pretreatment with Epo effectively reduced the cardiopulmonary toxicity induced by MCT. These findings provide the first evidence that adduction on hemoglobin, and the resulting RBC damage and impaired oxygen-carrying capacity, are the major initiating mechanism underlying PA-induced pulmonary arterial hypertension (PAH), while targeting the RBC damage is a potential therapeutic approach for PA-induced lung injury.

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

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

Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells.

Influence of pyrrolizidine alkaloids depletion upon the biological activity of Symphytum officinale L. extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Comfrey (Symphytum officinale L., Boraginaceae) root preparations are used as both traditional remedies and therapeutic agents in treating pain and inflammation associated with joint, bone, and muscle ailments. Even though numerous phytochemicals contribute to the beneficial effects of comfrey, the presence of toxic pyrrolizidine alkaloids (PAs) overshadows its uses. AIM OF THE STUDY: In this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. MATERIALS AND METHODS: The phytochemical profiling was performed by LC-HRMS/MS. The quantification of PAs and major phenolic compounds was carried out by LC-MS/MS and LC-DAD. Antioxidant and enzyme inhibitory activity was determined by in vitro free radical scavenging, ion reducing, metal chelating, cholinesterase, tyrosinase, amylase, and glucosidase assays. Using an ex vivo model of LPS-stimulated neutrophils, their viability (as measured by flow cytometry) and the release of IL-1ß, IL-8, and TNF-α were determined (ELISA assay). RESULTS: 12 phenolic acids, six PAs, three organic acids, two fatty acids, and two sugars were identified in the obtained comfrey extracts. The PA-depleted materials contained PAs levels below 2 ppm, whereas the removal of mucilage increased the content of rosmarinic acid, globoidnan A, globoidnan B, and rabdosiin. PA-depletion did not significantly affect the antioxidant potential. However, the radical scavenging and metal reducing properties were higher in the mucilage-depleted extracts. Neither PA-depletion nor mucilage-depletion had considerable effects on the in vitro inhibitory activity of cholinesterases, tyrosinase, amylase, and glucosidase or release of ex vivo pro-inflammatory cytokines (e.g., IL-1ß, IL-8, and TNF-α) in LPS-stimulated neutrophils. CONCLUSIONS: In light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

Enoxaparin attenuates pyrrolizidine alkaloids-induced hepatic sinusoidal obstruction syndrome by inhibiting oncostatin M expression.

BACKGROUND AND AIMS: The definitive treatment for pyrrolizidine alkaloids (PAs)-induced hepatic sinusoidal obstruction syndrome (HSOS) is not available. The effectiveness of anticoagulation therapy remains controversial. The efficacy of low molecular weight heparin (LMWH) should be investigated in patients and animal models, and the underlying mechanism should be explored. METHODS: The prognosis of patients with PAs-HSOS who received anticoagulation therapy was retrospectively analysed. The effect of enoxaparin on the liver injury was determined in animal models of monocrotaline (MCT)-induced HSOS was determined, and the underlying mechanism was investigated using a murine model. RESULTS: The cumulative survival rate of patients with PAs-induced HSOS was 60.00% and 90.90% in the non-anticoagulation group and anticoagulation group. Enoxaparin attenuated liver injury effectively in a rat model of MCT-induced HSOS. Additionally, the improvement of severe liver injury was observed in MCT-treated mice after the administration of enoxaparin (40 mg/kg). The alleviation of liver injury was observed in mice with hepatocyte-specific deletion of oncostatin M (Osm△Hep ). In MCT-treated mice administrated with enoxaparin, no significant differences in liver injury were observed between Osm△Hep mice and Osmflox/flox mice. Additionally, adenovirus-mediated overexpression of Osm resulted in severe liver injury in MCT-induced mice after the administration of enoxaparin. CONCLUSIONS: LMWH attenuated severe liver injury in patients with PAs-Induced HSOS and animal models of MCT-induced HSOS, which provides a rationale for the application of anticoagulation therapy.