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.

PBTK model-based analysis of CYP3A4 induction and the toxicokinetics of the pyrrolizidine alkaloid retrorsine in man.

Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC 24 and C max reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways.

Functional metabolomics characterizes the contribution of farnesoid X receptor in pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

Consumption of herbal products containing pyrrolizidine alkaloids (PAs) is one of the major causes for hepatic sinusoidal obstruction syndrome (HSOS), a deadly liver disease. However, the crucial metabolic variation and biomarkers which can reflect these changes remain amphibious and thus to result in a lack of effective prevention, diagnosis and treatments against this disease. The aim of the study was to determine the impact of HSOS caused by PA exposure, and to translate metabolomics-derived biomarkers to the mechanism. In present study, cholic acid species (namely, cholic acid, taurine conjugated-cholic acid, and glycine conjugated-cholic acid) were identified as the candidate biomarkers (area under the ROC curve 0.968 [95% CI 0.908-0.994], sensitivity 83.87%, specificity 96.55%) for PA-HSOS using two independent cohorts of patients with PA-HSOS. The increased primary bile acid biosynthesis and decreased liver expression of farnesoid X receptor (FXR, which is known to inhibit bile acid biosynthesis in hepatocytes) were highlighted in PA-HSOS patients. Furtherly, a murine PA-HSOS model induced by senecionine (50 mg/kg, p.o.), a hepatotoxic PA, showed increased biosynthesis of cholic acid species via inhibition of hepatic FXR-SHP singling and treatment with the FXR agonist obeticholic acid restored the cholic acid species to the normal levels and protected mice from senecionine-induced HSOS. This work elucidates that increased levels of cholic acid species can serve as diagnostic biomarkers in PA-HSOS and targeting FXR may represent a therapeutic strategy for treating PA-HSOS in clinics.

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.

Regulatory role of oxidative stress in retrorsine - Induced apoptosis and autophagy in primary rat hepatocytes.

Pyrrolizidine alkaloids (PAs) are a group of naturally occurring alkaloids widely present in plants. PAs are highly hepatotoxic and have been documented to cause many incidents of human and animal poisoning. Retrorsine (RTS) is a pyrrolizidine alkaloid (PA) derived from the Compositae Senecio, which has been shown to cause hepatotoxicity. Human liver poisoning occurs through the consumption of RTS-contaminated food, and animals can also be poisoned by ingesting RTS-containing toxic plants. The mechanism of RTS-induced liver toxicity is not fully understood. In this study, we demonstrated that RTS-induced oxidative stress plays a pivotal role in RTS-induced liver toxicity involving apoptosis and autophagy. The results showed that RTS treatment in the cultured Primary rat hepatocytes caused cytotoxicity and release of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a time- and dose-dependent manner. Our study showed that treatment of RTS induced ROS and MDA (malondialdehyde, a lipid peroxidation marker) in the hepatocytes, and reduced antioxidant capacity (GSH content, SOD activity), suggesting RTS treatment caused oxidative stress response in the hepatocytes. Furthermore, we found that RTS induced apoptosis and autophagy in the hepatocytes, and RTS-induced apoptosis and autophagy could be alleviated by ROS scavenger N-acetylcysteine (NAC) and the MAPK pathway inhibitors suggesting ROS/MAPK signaling pathway plays a role in RTS induced apoptosis and autophagy. Collectively, this study reveals the regulatory mechanism of oxidative stress in RTS-induced apoptosis and autophagy in the hepatocytes, providing important insights of molecular mechanisms of hepatotoxicity induced by RTS and related pyrrolizidine alkaloids in liver. This mechanism provides a basis for the prevention and treatment of PA poisoning in humans and animals.

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.

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.

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.

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.

Pyrrolizidine alkaloids cause cell cycle and DNA damage repair defects as analyzed by transcriptomics in cytochrome P450 3A4-overexpressing HepG2 clone 9 cells.

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

Physiologically based kinetic modelling predicts the in vivo relative potency of riddelliine N-oxide compared to riddelliine in rat to be dose dependent.

Pyrrolizidine alkaloids (PAs) are toxic plant constituents occurring often in their N-oxide form. This raises the question on the relative potency (REP) values of PA-N-oxides compared to the corresponding parent PAs. The present study aims to quantify the in vivo REP value of riddelliine N-oxide compared to riddelliine using physiologically based kinetic (PBK) modelling, taking into account that the toxicity of riddelliine N-oxide depends on its conversion to riddelliine by intestinal microbiota and in the liver. The models predicted a lower Cmax and higher Tmax for the blood concentration of riddelliine upon oral administration of riddelliine N-oxide compared to the Cmax and Tmax predicted for an equimolar oral dose of riddelliine. Comparison of the area under the riddelliine concentration-time curve (AUCRID) obtained upon dosing either the N-oxide or riddelliine itself revealed a ratio of 0.67, which reflects the in vivo REP for riddelliine N-oxide compared to riddelliine, and appeared to closely match the REP value derived from available in vivo data. The models also predicted that the REP value will decrease with increasing dose level, because of saturation of riddelliine N-oxide reduction by the intestinal microbiota and of riddelliine clearance by the liver. It is concluded that PBK modeling provides a way to define in vivo REP values of PA-N-oxides as compared to their parent PAs, without a need for animal experiments.

Bioassay-directed analysis-based identification of relevant pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC-MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC-Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes.

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.

Fasting augments pyrrolizidine alkaloid-induced hepatotoxicity.

Pyrrolizidine alkaloids (PAs) are phytotoxins widely present in various natural products and foodstuffs. The present study aims to investigate the effects of fasting on PA-induced hepatotoxicity and the underlying biochemical mechanisms. The results of hepatotoxic study showed that 15-h overnight fasting significantly exacerbated the hepatotoxicity of retrorsine (RTS, a representative toxic PA) in fasted rats compared to fed rats, as indicated by remarkably elevated plasma ALT and bilirubin levels and obvious liver histological changes. Further toxicokinetic studies revealed that fasting significantly enhanced cytochromes P450 enzymes (CYPs)-mediated metabolic activation of RTS leading to increased formation of pyrrole-protein adducts and thus decreased the in vivo exposure and excretion of both parent RTS and its N-oxide metabolite. Metabolic studies demonstrated that fasting induced enzyme activities of CYP1A2, CYP2B6 and CYP2E1 that participated in catalyzing RTS to its reactive pyrrolic metabolites. Moreover, fasting also dramatically decreased hepatic glutathione (GSH) content, which restricted the detoxification of GSH by neutralizing the reactive pyrrolic metabolite of RTS, further contributing to the enhanced hepatotoxicity. The present findings may have an impact on future PA toxicity tests with different dietary styles and/or risk assessment of metabolite-mediated toxins by considering the profound effects of fasting.

Support for Regulatory Assessment of Percutaneous Absorption of Retronecine-type Pyrrolizidine Alkaloids through Human Skin.

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

The Role of Kinetics as Key Determinant in Toxicity of Pyrrolizidine Alkaloids and Their N-Oxides.

Pyrrolizidine alkaloids (PAs) are a large group of plant constituents of which especially the 1,2- unsaturated PAs raise a concern because of their liver toxicity and potential genotoxic carcinogenicity. This toxicity of PAs depends on their kinetics. Differences in absorption, distribution, metabolism, and excretion (ADME) characteristics of PAs may substantially alter the relative toxicity of PAs. As a result, kinetics will also affect relative potency (REP) values. The present review summarizes the current state-of-the art on PA kinetics and resulting consequences for toxicity and illustrates how physiologically-based kinetic (PBK) modelling can be applied to take kinetics into account when defining the relative differences in toxicity between PAs in the in vivo situation. We conclude that toxicokinetics play an important role in the overall toxicity of pyrrolizidine alkaloids. and that kinetics should therefore be considered when defining REP values for combined risk assessment. New approach methodologies (NAMs) can be of use to quantify these kinetic differences between PAs and their N-oxides, thus contributing to the 3Rs (Replacement, Reduction and Refinement) in animal studies.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

Regulatory Perspectives of Pyrrolizidine Alkaloid Contamination in Herbal Medicinal Products.

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

Anticancer Approach Inspired by the Hepatotoxic Mechanism of Pyrrolizidine Alkaloids with Glycosylated Artificial Metalloenzymes.

Metabolic oxidation of pyrrolizidine alkaloids (PAs) from herbal and dietary supplements by cytochrome P450 produces dehydro-PAs (DHPs), which leads to toxicities. A highly reactive cation species generated from the active pyrrole ring of DHPs readily reacts with various cellular components, causing hepatotoxicity and cytotoxicity. Inspired by PA-induced hepatic damage, we developed a therapeutic approach based on a cyclization precursor that can be transformed into a synthetic DHP under physiological conditions through gold-catalyzed 5-endo-dig cyclization using a gold-based artificial metalloenzyme (ArM) instead of through metabolic oxidation by cytochrome P450. In cell-based assays, the synthesis of the DHP by a cancer-targeting glycosylated gold-based ArM substantially suppressed cell growth of the targeted cancer cells without causing cytotoxicity to untargeted cells, highlighting the potential of the strategy to be used therapeutically in vivo.

Metabolic Pattern of Hepatotoxic Pyrrolizidine Alkaloids in Liver Cells.

Contamination with 1,2-unsaturated pyrrolizidine alkaloids (PAs) is a serious problem for certain phytomedicines, foods, and animal feeds. Several of these PAs are genotoxic and carcinogenic, primarily in the liver, upon cytochrome P450 (CYP)-catalyzed activation into reactive (pyrrolic and pyrrole-like) metabolites. Here we investigated the metabolism of selected PAs (echimidine, europine, lasiocarpine, lycopsamine, retrorsine, and senecionine) in rat hepatocytes in primary culture and in human CYP3A4-transfected HepG2 cells. The open-chained diesters echimidine and lasiocarpine and the cyclic diester senecionine were extensively metabolized in rat hepatocytes into a broad spectrum of products released into the medium. A large portion of unidentified, possibly irreversibly bound, products remained in the cells while detectable amounts of reactive and other metabolites were found in the incubation media. In HepG2-CYP3A4 cells, lasiocarpine was more extensively metabolized than echimidine and senecionine which also gave rise to the release of pyrrolic metabolites. In human cells, no pyrrolic metabolites were detected in retrorsine or lycopsamine incubations, while no such metabolites were detected from europine in both cell types. Other types of metabolic changes comprised modifications such as side chain demethylation or oxygenation reactions like the formation of N-oxides. The latter, considered as a detoxification step, was a major pathway with cyclic diesters, was less distinctive for echimidine and lycopsamine and almost negligible for lasiocarpine and europine. Our data are in agreement with previously published cyto- and genotoxicity findings and suggests that the metabolic pattern may contribute substantially to the specific toxic potency of a certain congener. In addition, marked differences were found for certain congeners between rat hepatocytes and transfected human HepG2 cells, whereby a high level of bioactivation was found for lasiocarpine, whereas a very low level of bioactivation was observed for monoesters, in particular in human cells.