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.

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.

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.

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.

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.

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 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.

UHPLC-Q-TOF analysis of pyrrolizidine alkaloids in North-Macedonian honey.

Honey contaminated with pyrrolizidine alkaloids (PAs) could pose a risk for human consumption, being a widely consumed food product. A fast and simple LC/MS method for the analysis of pyrrolizidine alkaloids in honey was optimised to collect occurrence data. The extraction efficiency was evaluated by a systematic study of multiple solvent mixtures and clean-up procedures. The best results for PA extraction were obtained using a formic acid/methanol mixture with subsequent clean-up by the QuEChERS method, resulting in a mean recovery range of 91.8-102%. The method validation showed satisfactory intra-day (RSD < 5.1%) and inter-day precision (RSD < 9.1%). The proposed method was applied to 14 samples. A total of six PAs and two N-oxides were detected, with levels between 89 and 8188 µg/kg. This assessment highlights the potential risk of intoxication and the need for further investigations regarding an effective quality system for manufacturers to control PAs in honey.

Miniaturized Analytical Strategy Based on µ-SPEed for Monitoring the Occurrence of Pyrrolizidine and Tropane Alkaloids in Honey.

Currently, the analysis of trace-level contaminants in food must be addressed following green analytical chemistry principles and with a commitment to the sustainable development goals. Accordingly, a sustainable and ecofriendly microextraction procedure based on µ-SPEed followed by ultrahigh liquid chromatography coupled to ion-trap tandem mass spectrometry analysis was developed to determine the occurrence of pyrrolizidine and tropane alkaloids in honey samples. The µ-SPEed procedure took approximately 3 min per sample, using only 100 µL of organic solvent and 300 µL of diluted sample. The method was properly validated (overall recoveries 72-100% and precision RSD values ≤15%), and its greenness was scored at 0.61 out of 1. The method was applied to different honey samples, showing overall contamination levels from 32 to 177 µg/kg of these alkaloids. Atropine was found in all the samples, whereas retrorsine N-oxide, lasiocarpine, echimidine, and echimidine N-oxide were the main pyrrolizidine alkaloids in the samples analyzed.

Effects of thermal and nonthermal treatments on microorganisms, pyrrolizidine alkaloids and volatile compounds in oregano (Origanum vulgare L.).

Effects of steam sterilization, gamma-irradiation, UV-irradiation and ozonation on microbial inactivation, pyrrolizidine alkaloid degradation and volatile compound profile in oregano were investigated. Steam sterilization and gamma-irradiation were the most effective treatments in inactivating microorganisms. These treatments resulted in 0.87-2.15 log reductions in total aerobic mesophilic bacteria counts and reduced yeast-mold and Enterobacteriaceae counts below the detectable level. Steam sterilization caused increased levels of pyrrolizidine alkaloids (PAs) and decreased levels of their N-oxide forms (PANOs) demonstrating a simultaneous conversion of PANOs into the corresponding PAs. Ozone treatment caused significant decreases in the levels of individual and total PAs/PANOs. After ozone treatment, decreases of 54.4, 53.9, 61.6 and 61.4% were observed in the levels of europine-N-oxide, europine, lasiocarpine-N-oxide and lasiocarpine, respectively. Steam sterilization, UV-irradiation and ozone treatments significantly altered the composition of the volatile compounds of oregano as evidenced by decreased levels of major components and the formation of some new compounds.

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.

Development and validation of an analytical method for the simultaneous determination of 12 ergot, 2 tropane, and 28 pyrrolizidine alkaloids in cereal-based food by LC-MS/MS.

Alkaloids are naturally occurring compounds containing basic nitrogen atoms. They are biosynthesized mainly by plants but also by some fungi species. Many alkaloids are toxic to humans and animals, and they have been classified as food contaminants. Among them, ergot, tropane, and pyrrolizidine alkaloids have maximum levels in foods, established by the Commission Regulation (EU) 2023/915. In this study, an analytical method was successfully developed and validated for the simultaneous determination of 42 ergot, tropane, and pyrrolizidine alkaloids and their N-oxides in cereal-based food. The method includes QuEChERS-based extraction followed by liquid chromatography coupled with tandem mass spectrometry. The proposed method was validated providing recoveries ranging from 71 to 119 %, intra- and inter-day precision lower than 19 %, and limits of quantification between 0.5 and 1.0 µg kg-1. Finally, the analysis of reference materials coming from FAPAS proficiency tests demonstrated the suitability for purpose of the methodology (z-scores < 2). Nine cereal-based products samples were analyzed of which ergot alkaloids were detected in two of them, while one sample showed the presence of three pyrrolizidine alkaloids.

Pyrrolizidine Alkaloids in Tea (Camellia sinensis L.) from Weeds through Weed-Soil-Tea Transfer and Risk Assessment of Tea Intake.

Pyrrolizidine alkaloids (PAs) have been detected in tea and can threaten human health. However, the specific source of PAs in tea is still unclear. Here, 88 dried tea products collected from six major tea-producing areas in Anhui Province, China, were analyzed. The detection frequency was 76%. The content of total PAs in dried tea was between 1.1 and 90.5 µg/kg, which was all below the MRL recommended by the European Union (150 µg/kg). In the Shexian tea garden, PAs in the weeds and weed rhizospheric soil around tea plants and the fresh tea leaves were analyzed. Intermedine (Im), intermedine-N-oxide (ImNO), and jacobine-N-oxide (JbNO) were transferred through the weed-to-soil-to-tea route into the fresh tea leaves; only Im and ImNO were detected in dried tea samples. Potential risk of the total PAs in the tea infusion was assessed according to the margin of exposure method, and it might be a low concern for public health.

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.

Short-term exposure of dairy cows to pyrrolizidine alkaloids from tansy ragwort (Jacobaea vulgaris Gaertn.): effects on health and performance.

The increasing spread of ragworts is observed with concern. Ragworts like tansy ragwort (Jacobaea vulgaris Gaertn.) or marsh ragwort (J. aquatica) contain pyrrolizidine alkaloids (PA) which may induce hepatotoxic effects. Grazing animals usually avoid ragworts if their pasture management is appropriate. Preserved feed prepared from ragworts contaminated meadows may, however, lead to a significant exposure to PA. Previous studies on toxicity of PA for dairy cows revealed inconsistent results due to feeding ragwort plant material which was associated with heterogeneous PA exposure and thus failed to conclusively deduce critical PA doses. Therefore, the aim of the present study was to expose dairy cows (n = 4 per group) in a short-term scenario for 28 days with increasing PA doses (PA1: 0.47 mg PA/kg body weight (BW)/day (d); PA2: 0.95 mg PA/kg BW/d; PA3: 1.91 mg PA/kg BW/d) via oral administration by gavage of a defined PA-extract. While group PA3 was dosed with the PA-extract alone, groups PA2 and PA1 received PA-extracts blended in similar volumes with molasses to provide comparable amounts of sugar. Additionally, two control groups were treated either with water (CONWater) or with molasses (CONMolasses) to assess the effects of sugar without PA interference. While clinical traits including dry matter intake, milking performance, rectal body temperature, ruminal activity and body condition score (BCS) were not influenced by PA exposure, activities of enzymes indicative for liver damages, such as gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH), increased significantly over time at an exposure of 1.91 mg total PA/kg BW/d.

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.

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.

Baiting Insects with Pyrrolizidine Alkaloids (PAs): A Fieldwork-Oriented Review and Guide to PA-Pharmacophagy.

Since 1890, many observations of danaine butterflies visiting dry plants of several families in the Old and New World tropics have been published. For 50 years, it has been recognised that Danainae, along with various other insects, seek out 1,2-dehydropyrrolizidine ester alkaloids (PAs) independently of and in addition to their nutritive requirements and utilise them to increase their chances for survival and biological fitness. This represents an unusual type of insect-plant relationship ("PA-pharmacophagy"), with remarkable peculiarities but also with gaps in knowledge, many of which can be filled by employing PA-baiting. We review and analyse the history of records on the attraction of adult insects to PAs and unveil the complex background information on PA-chemistry, PA-producing plants ("PA-plants"), and PA-sequestering insects ("PA-insects") in order to come up with practical tips for successful baiting with PAs ("PA-baiting"). Studying PA-pharmacophagy integrates taxonomy, behaviour, and ecology with evolutionary biology, chemistry, and toxicology. With basic knowledge of PA-chemistry and bearing the general peculiarities of PA-plants and PA-insects in mind, PA-baiting can be conducted easily and successfully to address many questions on the multifaceted ecology of pyrrolizidine alkaloids. We aim to encourage field researchers in the tropics to employ PA-baiting as a valuable research method in this field of integrative biology.