Dehydropyrrolizidine Alkaloids Research Articles

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.

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.Graphical abstract

Protein cross-linking in primary cultured mouse hepatocytes by dehydropyrrolizidine alkaloids: Structure–toxicity relationship

Pyrrolizidine alkaloids (PAs) are natural toxins found in about 3%–5% of flowering plants. Dehydropyrrolizidine alkaloids contain a double bond in 1, 2-position of the necine bases, including retronecine type PAs (RET-PAs) and their N-oxides (RET N-oxide-PAs), and otonecine type PAs (OTO-PAs), and are known for their significant hepatotoxicity. Most dehydropyrrolizidine alkaloids are metabolically activated by cytochrome P450 (CYP450) enzymes to generate active pyrroles, which further bind to proteins to form pyrrole–protein adducts (PPAs). Methods for predicting PA-induced liver injury are generally performed on in vitro models with extremely low activities of CYP450 enzymes, which is different from the situation in vivo. In this regard, primary cultured mouse hepatocytes, which showed comparable CYP450 activity with the in vivo models, were applied to illustrate the structure–toxicity relationship of 13 dehydropyrrolizidine alkaloids, namely, eight RET-PAs, three RET N-oxide-PAs, and two OTO-PAs. PA-induced cytotoxicity and PA-generated PPAs were analyzed in primary mouse hepatocytes treated with different PAs. Results showed that PA-induced toxicity was correlated with the amount of PA-generated PPAs. RET-PAs and OTO-PAs were generally more toxic than RET N-oxide-PAs and generated higher amount of PPAs. PPAs were utilized to evaluate the efficiency of metabolic activation and predict the toxic potencies of dehydropyrrolizidine alkaloids. The proposed model could be a new approach for toxicity evaluation and risk control of exposure to PAs.

Effects of glutathione and cysteine on pyrrolizidine alkaloid-induced hepatotoxicity and DNA adduct formation in rat primary hepatocytes

Pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Upon metabolic activation, PAs produce dehydropyrrolizidine alkaloids (dehydro-PAs) as reactive primary pyrrolic metabolites. Dehydro-PAs are unstable, facilely hydrolyzed to (±)−6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP). Both dehydro-PAs and DHP are capable of binding to cellular DNA and proteins to form DHP-DNA and DHP-protein adducts leading to tumorigenicity and cytotoxicity. We recently determined that the reaction of dehydro-PAs with glutathione and cysteine generated 7-glutathione-DHP (7-GS-DHP) and 7-cysteine-DHP, respectively which can also bind to DNA to produce DHP-DNA adducts. In this study, we determined the effects of glutathione and cysteine on the induction of hepatocytotoxicity and the formation of DHP-DNA adducts in primary hepatocytes cultured with riddelliine and monocrotaline. We found that both glutathione and cysteine can drastically reduce hepatotoxicity while the levels of DHP-DNA adduct formation are slightly affected.

Hepatic Encephalopathy Secondary to Chronic Liver Lesions Caused by Crotalaria incana in a Bovine

Background: Crotalaria spp. Poisoning induces liver or pulmonary disease. C. mucronata, C. juncea, C. spectabilis, and C. retusa are the Crotalaria spp. inducing spontaneous intoxication in livestock in Brazil. C. mucronata and C. juncea are associated with interstitial pneumonia, while C. retusa and C. specatabilis induce hepatotoxicosis. The toxic principle in Crotalaria spp. are dehydropyrrolizidine alkaloids and their N-oxides. C. incana poisoning to livestock is rarely documented. This paper reports the clinical signs and pathological findings of a case of Crotalaria incana poisoning in a steer. The chemical finding of a potential toxic dehydropyrrolizidine alkaloid in the plant is documented for the first time.Case: The affected bovine was part of a herd of 80 two-year-old steers that were transferred from Property 1 to Property 2, 30 days prior to the event. In the pasture of Property I - where the steers were held for 6 months - there was a heavy infestation by a Crotalaria species with signs of being consumed by the steers. The plant was identified as Crotalaria incana at the Botanical Laboratory of the Federal University of Mato Grosso do Sul and a voucher specimen was filed there under the register GCMS 51169. Two days after entering Property 2 one of the steers became depressed, staggering, and in poor body condition. With time, the steer became oblivious to the environment and died 20 days after the onset of the clinical signs. Significant necropsy findings were limited to the liver which was markedly enlarged and with rounded edges. The hepatic cut surface was mottled with dark red and extensive orange areas of discoloration. The gallbladder was distended and the bile was inspissated. Microscopically, in the liver, there was fibrosis, bile duct hyperplasia and hepatocellular megalocytosis. The Glisson’s capsule was markedly thickened by fibrosis. In the brain, there was vacuolation of myelin sheaths (status spongiosus), moderate gliosis, and rare Alzheimer type 2 astrocytes.Discussion: The diagnosis of intoxication by C. incana was based on clinical signs and pathological changes and the evidence of the plant being consumed. The high infestation of C. incana in the pasture, the high proportion of dead matter and the low concentration of viable pasture favored the ingestion of C. incana. Spongy degeneration, a typical lesion of hepatic encephalopathy, was observed in the several areas of the brain. Cirrhosis of the liver as seen in the case of this report, results in elevated ammonia levels in the blood - and eventually in the brain – and ammonia toxicity causes hepatic encephalopathy. Morphological changes in hepatic encephalopathy in the central nervous system of human beings and horses centers on astrocytes which undergo Alzheimer type 2 change developing an enlarged, pale nuclei with a rim of chromatin and prominent nucleoli. Astrocytes pairs and triplets are seen, and, in severe cases, astrocytic nuclei may become lobulated and contain glycogen. Chemical analysis of C. incana was accomplished by acid-base extraction with zincreduction of N-oxides and quantitation by spectrometry. Quantitative analysis was achieved by liquid chromatography-mass spectrometry. To confirm the identity of the alkaloid in the sample of C. incana the isolated alkaloid was compared to a standard sample of usaramine previously isolated, and its identity verified by magnetic resonance spectroscopy analysis. The 1,2-dehydropyrrolizidine alkaloid usaramine, as well as its N-oxide, were identified as the major alkaloids in in C. incana. The concentration found in the seed was consistent with that previously reported. This is a new finding on the toxic principle of C. incana. Keywords: cattle diseases, poisonous plants, pathology, hepatotoxicity, Crotalaria incana, chronic liver failure.

Potentially toxic pyrrolizidine alkaloids in Eupatorium perfoliatum and three related species. Implications for herbal use as boneset

Pro-toxic dehydropyrrolizidine alkaloids are associated with liver disease in humans. The potential for long-term, low-level or intermittent exposures to cause or contribute to chronically-developing diseases is of international concern. Eupatorium perfoliatum is a medicinal herb referred to as boneset. While the presence of dehydropyrrolizidine alkaloids in some Eupatorium species is well-established, reports on Eupatorium perfoliatum are scant and contradictory. To investigate the presence of dehydropyrrolizidine alkaloids in a survey of boneset samples and related alcoholic tinctures, and hot water infusions and decoctions. Methanol, hot water or aqueous ethanol extracts of Eupatorium perfoliatum and three closely-related species were subjected to HPLC-ESI(+)MS and MS/MS analysis using three complementary column methods. Dehydropyrrolizidine alkaloids were identified from their MS data and comparison with standards. Forty-nine samples of Eupatorium perfoliatum were shown to contain dehydropyrrolizidine alkaloids (0.0002-0.07% w/w), the majority dominated by lycopsamine and intermedine, their N-oxides and acetylated derivatives. Alcoholic tinctures and hot water infusions and decoctions had high concentrations of the alkaloids. Different chemotypes, hybridisation or contamination of some Eupatorium perfoliatum samples with related species were suggested by the co-presence of retronecine- and heliotridine-based alkaloids. Sampling issues, low and high alkaloid chemotypes of Eupatorium perfoliatum or interspecies hybridization could cause the wide variation in dehydropyrrolizidine alkaloid concentrations or the different profiles observed. Concerns associated with dehydropyrrolizidine alkaloids provide a compelling reason for preclusive caution until further research can better define the toxicity and carcinogenicity of the dehydropyrrolizidine alkaloid content of Eupatorium perfoliatum. [Correction added on 12 July 2018, after first online publication: The 'Conclusions' section in the abstract has been added.].

Dehydropyrrolizidine Alkaloid Toxicity, Cytotoxicity, and Carcinogenicity.

Dehydropyrrolizidine alkaloid (DHPA)-producing plants have a worldwide distribution amongst flowering plants and commonly cause poisoning of livestock, wildlife, and humans. Previous work has produced considerable understanding of DHPA metabolism, toxicity, species susceptibility, conditions, and routes of exposure, and pathogenesis of acute poisoning. Intoxication is generally caused by contaminated grains, feed, flour, and breads that result in acute, high-dose, short-duration poisoning. Acute poisoning produces hepatic necrosis that is usually confirmed histologically, epidemiologically, and chemically. Less is known about chronic poisoning that may result when plant populations are sporadic, used as tisanes or herbal preparations, or when DHPAs contaminate milk, honey, pollen, or other animal-derived products. Such subclinical exposures may contribute to the development of chronic disease in humans or may be cumulative and probably slowly progress until liver failure. Recent work using rodent models suggest increased neoplastic incidence even with very low DHPA doses of short durations. These concerns have moved some governments to prohibit or limit human exposure to DHPAs. The purpose of this review is to summarize some recent DHPA research, including in vitro and in vivo DHPA toxicity and carcinogenicity reports, and the implications of these findings with respect to diagnosis and prognosis for human and animal health.

7-N-Acetylcysteine-pyrrole conjugate—A potent DNA reactive metabolite of pyrrolizidine alkaloids

Plants containing pyrrolizidine alkaloids (PAs) are widespread throughout the world and are the most common poisonous plants affecting livestock, wildlife, and humans. PAs require metabolic activation to form reactive dehydropyrrolizidine alkaloids (dehydro-PAs) that are capable of alkylating cellular DNA and proteins, form (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA and DHP-protein adducts, and lead to cytotoxicity, genotoxicity, and tumorigenicity. In this study, we determined that the metabolism of riddelliine and monocrotaline by human and rat liver microsomes in the presence of N-acetylcysteine both produced 7-N-acetylcysteine-DHP (7-NAC-DHP) and DHP. Reactions of 7-NAC-DHP with 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), and calf thymus DNA in aqueous solution followed by enzymatic hydrolysis yielded DHP-dG and/or DHP-dA adducts. These results indicate that 7-NAC-DHP is a reactive metabolite that can lead to DNA adduct formation.

Pro-toxic 1,2-Dehydropyrrolizidine Alkaloid Esters, Including Unprecedented 10-Membered Macrocyclic Diesters, in the Medicinally-used Alafia cf. caudata and Amphineurion marginatum (Apocynaceae: Apocynoideae: Nerieae and Apocyneae).

Within the Apocynoideae (Apocynaceae) pro-toxic dehydropyrrolizidine alkaloids have been reported only in Echiteae. However, attraction of pyrrolizidine alkaloid-pharmacophagous insects suggested their presence in Alafia cf. caudata Stapf (Nerieae: Alafiinae) and Amphineurion marginatum (Roxb.) D.J. Middleton (Apocyneae: Amphineuriinae), both used as medicinal plants. To confirm the presence of dehydropyrrolizidine alkaloids in Alafia cf. caudata and Amphineurion marginatum and identify their structures. Methanol extracts of air-dried roots, stems and leaves of non-flowering plants were analysed using HPLC-ESI(+)MS and MS/MS or collision-induced dissociation MS in low and/or high resolution modes. Pyrrolizidine alkaloids were tentatively identified based on the mass spectrometry data. Solid phase extraction combined with semi-preparative HPLC were used to isolate major alkaloids. Structures were elucidated using NMR spectroscopy. Monoesters of retronecine with senecioic, hydroxysenecioic or syringic acids were identified in roots of Alafia cf. caudata. Two unprecedented 10-membered macrocyclic dehydropyrrolizidine alkaloid diesters were isolated from roots of Amphineurion marginatum. Pyrrolizidine alkaloids were detected in root and leaf material of Alafia cf. caudata at 0.34 and 0.01% dry weight (DW), and 0.13, 0.02 and 0.09% DW in root, leaf and stem material of Amphineurion marginatum. The presence of pro-toxic dehydropyrrolizidine alkaloids suggests that medical preparations of these plants pose potential health risks to consumers. Dehydropyrrolizidine alkaloids are evidently more widespread in Apocynoideae than previously assumed, and it would seem rewarding to study other members of this family for the presence of pyrrolizidines, dehydropyrrolizidines and dihydropyrrolizines. Copyright © 2016 John Wiley & Sons, Ltd.

9-Glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine Is the Major Pyrrolic Glutathione Conjugate of Retronecine-Type Pyrrolizidine Alkaloids in Liver Microsomes and in Rats.

Retronecine-, otonecine-, and heliotridine-type pyrrolizidine alkaloids (PAs) are all reported to be hepatotoxic. These PAs are suggested to be metabolized to the corresponding electrophilic dehydropyrrolizidine alkaloids (dehydro-PAs) and subsequently conjugated with macromolecules, such as glutathione (GSH). In the present study, a total of five glutathione conjugates, named M1-M5, were detected in rat and human liver microsomal incubations with three retrornecine-type PAs (isoline, retrorsine, or monocrotaline) in the presence of glutathione, and were chemically synthesized. M1 and M3 were unambiguously identified as a pair of epimers of 7-glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (7-GSH-DHP), and M4 and M5 were epimers of 7,9-diglutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (7,9-diGSH-DHP). M2, an extremely unstable conjugate, was proposed to be 9-glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (9-GSH-DHP). It was the most abundant among the five GSH conjugates, and the finding corrects the mistake that 7-GSH-DHP is the predominant GSH conjugate derived from dehydro-PAs. Similar patterns in glutathione conjugate profile were observed in the bile of rats treated with the PAs. This is the first study to describe 9-GSH-DHP as a major pyrrolic GSH conjugate of retronecine-type PAs, providing insight into the interactions of dehydro-PAs with biomolecules.

Pyrrolizidine and tropane alkaloids in teas and the herbal teas peppermint, rooibos and chamomile in the Israeli market

Dehydro pyrrolizidine alkaloids (dehydro PAs) are carcinogenic phytotoxins prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Dehydro PAs enter the food and feed chain by co-harvesting of crops intended for human and animal consumption as well as by carry-over into animal-based products such as milk, eggs and honey. Recently the occurrence of dehydro PAs in teas and herbal teas has gained increasing attention from the EU, due to the high levels of dehydro PAs found in commercially available teas and herbal teas in Germany and Switzerland. Furthermore, several tropane alkaloids (TAs, e.g. scopolamine and hyoscyamine) intoxications due to the consumption of contaminated herbal teas were reported in the literature. The aim of the present study was to determine the dehydro PAs and TAs levels in 70 pre-packed teabags of herbal and non-herbal tea types sold in supermarkets in Israel. Chamomile, peppermint and rooibos teas contained high dehydro PAs levels in almost all samples analysed. Lower amounts were detected in black and green teas, while no dehydro PAs were found in fennel and melissa herbal teas. Total dehydro PAs concentrations in chamomile, peppermint and rooibos teas ranged from 20 to 1729 μg/kg. Except for black tea containing only mono-ester retrorsine-type dehydro PAs, all other teas and herbal teas showed mixed patterns of dehydro PA ester types, indicating a contamination by various weed species during harvesting and/or production. The TA levels per teabag were below the recommended acute reference dose; however, the positive findings of TAs in all peppermint tea samples warrant a more extensive survey. The partially high levels of dehydro PAs found in teas and herbal teas present an urgent warning letter to the regulatory authorities to perform routine quality control analysis and implement maximum residual levels for dehydro PAs.

Cytotoxicity of pyrrolizidine alkaloid in human hepatic parenchymal and sinusoidal endothelial cells: Firm evidence for the reactive metabolites mediated pyrrolizidine alkaloid-induced hepatotoxicity

Pyrrolizidine alkaloids (PAs) widely distribute in plants and can cause hepatic sinusoidal obstruction syndrome (HSOS), which typically presents as a primary sinusoidal endothelial cell damage. It is well-recognized that after ingestion, PAs undergo hepatic cytochromes P450 (CYPs)-mediated metabolic activation to generate dehydropyrrolizidine alkaloids (DHPAs), which are hydrolyzed to dehydroretronecine (DHR). DHPAs and DHR are reactive metabolites having same core pyrrole moiety, and can bind proteins to form pyrrole-protein adducts, which are believed as the primary cause for PA-induced HSOS. However, to date, the direct evidences supporting the toxicity of DHPAs and DHR in the liver, in particular in the sinusoidal endothelial cells, are lacking. Using human hepatic sinusoidal endothelial cells (HSEC) and HepG2 (representing hepatic parenchymal cells), cells that lack CYPs activity, this study determined the direct cytotoxicity of dehydromonocrotaline, a representative DHPA, and DHR, but no cytotoxicity of the intact PA (monocrotaline) in both cell lines, confirming that reactive metabolites mediate PA intoxication. Comparing with HepG2, HSEC had significantly lower basal glutathione (GSH) level, and was significantly more susceptible to the reactive metabolites with severer GSH depletion and pyrrole-protein adducts formation. The toxic potency of two reactive metabolites was also compared. DHPA was more reactive than DHR, leading to severer toxicity. In conclusion, our results unambiguously provided the first direct evidence for the critical role of DHPA and DHR in the reactive metabolites-mediated PA-induced hepatotoxicity, which occurs predominantly in HSEC due to severe GSH depletion and the significant formation of pyrrole-protein adducts in HSEC.

The comparative toxicity of a reduced, crude comfrey (Symphytum officinale) alkaloid extract and the pure, comfrey-derived pyrrolizidine alkaloids, lycopsamine and intermedine in chicks (Gallus gallus domesticus).

Comfrey (Symphytum officinale), a commonly used herb, contains dehydropyrrolizidine alkaloids that, as a group of bioactive metabolites, are potentially hepatotoxic, pneumotoxic, genotoxic and carcinogenic. Consequently, regulatory agencies and international health organizations have recommended comfrey be used for external use only. However, in many locations comfrey continues to be ingested as a tisane or as a leafy vegetable. The objective of this work was to compare the toxicity of a crude, reduced comfrey alkaloid extract to purified lycopsamine and intermedine that are major constituents of S. officinale. Male, California White chicks were orally exposed to daily doses of 0.04, 0.13, 0.26, 0.52 and 1.04 mmol lycopsamine, intermedine or reduced comfrey extract per kg bodyweight (BW) for 10 days. After another 7 days chicks were euthanized. Based on clinical signs of poisoning, serum biochemistry, and histopathological analysis the reduced comfrey extract was more toxic than lycopsamine and intermedine. This work suggests a greater than additive effect of the individual alkaloids and/or a more potent toxicity of the acetylated derivatives in the reduced comfrey extract. It also suggests that safety recommendations based on purified compounds may underestimate the potential toxicity of comfrey.

Safety concerns of herbal products and traditional Chinese herbal medicines: dehydropyrrolizidine alkaloids and aristolochic acid

In many countries, including the United States, herbal supplements, tisanes and vegetable products, including traditional Chinese medicines, are largely unregulated and their content is not registered, monitored or verified. Consequently, potent plant toxins including dehydropyrrolizidine alkaloids and other potential carcinogens can contaminate these products. As herbal and food supplement producers are left to their own means to determine the safety and purity of their products prior to marketing, disturbingly often good marketing practices currently in place are ignored and content is largely undocumented. Historical examples of poisoning and health issues relating to plant material containing dehydopyrrolizidine alkaloids and aristolochic acids were used as examples to demonstrate the risk and potential toxicity of herbal products, food supplements, or traditional medicines. More work is needed to educate consumers of the potential risk and require the industry to be more responsible to verify the content and insure the safety of their products.

Pro-toxic dehydropyrrolizidine alkaloids in the traditional Andean herbal medicine “asmachilca”

Ethnopharmacological relevanceAsmachilca is a Peruvian medicinal herb preparation ostensibly derived from Aristeguietia gayana (Wedd.) R.M. King & H. Rob. (Asteraceae: Eupatorieae). Decoctions of the plant have a reported bronchodilation effect that is purported to be useful in the treatment of respiratory allergies, common cold and bronchial asthma. However, its attractiveness to pyrrolizidine alkaloid-pharmacophagous insects indicated a potential for toxicity for human consumers. Aim of the studyTo determine if commercial asmachilca samples, including fully processed herbal teas, contain potentially toxic 1,2-dehydropyrrolizidine alkaloids. Materials and methodsTwo brands of “Asmachilca” herbal tea bags and four other commercial samples of botanical materials for preparing asmachilca medicine were extracted and analyzed using HPLC–esi(+)MS and MS/MS for the characteristic retention times and mass spectra of known dehydropyrrolizidine alkaloids. Other suspected dehydropyrrolizidine alkaloids were tentatively identified based on MS/MS profiles and high resolution molecular weight determinations. Further structure elucidation of isolated alkaloids was based on 1D and 2D NMR spectroscopy. ResultsAsmachilca attracted many species of moths which are known to pharmacophagously gather dehydropyrrolizidine alkaloids. Analysis of 5 of the asmachilca samples revealed the major presence of the dehydropyrrolizidine alkaloid monoesters rinderine and supinine, and their N-oxides. The 6th sample was very similar but did not contain supinine or its N-oxide. Small quantities of other dehydropyrrolizidine alkaloid monoesters, including echinatine and intermedine, were also detected. In addition, two major metabolites, previously undescribed, were isolated and identified as dehydropyrrolizidine alkaloid monoesters with two “head-to-tail” linked viridifloric and/or trachelanthic acids. Estimates of total pyrrolizidine alkaloid and N-oxide content in the botanical components of asmachilca varied from 0.4% to 0.9% (w/dw, dry weight) based on equivalents of lycopsamine. The mean pyrrolizidine alkaloid content of a hot water infusion of a commercial asmachilca herbal tea bag was 2.2±0.5mg lycopsamine equivalents. Morphological and chemical evidence showed that asmachilca is prepared from different plant species. ConclusionsAll asmachilca samples and the herbal tea infusions contained toxicologically-relevant concentrations of pro-toxic 1,2-dehydropyrrolizidine alkaloid esters and, therefore, present a risk to the health of humans. This raises questions concerning the ongoing unrestricted availability of such products on the Peruvian and international market. In addition to medical surveys of consumers of asmachilca, in the context of chronic disease potentially associated with ingestion of the dehydropyrrolizidine alkaloids, the botanical origins of asmachilca preparations require detailed elucidation.

Absolute configuration, stability, and interconversion of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine valine adducts and their phenylthiohydantoin derivatives

Pyrrolizidine alkaloid-containing plants are widespread in the world and probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids require metabolic activation to form dehydropyrrolizidine alkaloids that bind to cellular proteins and DNA leading to hepatotoxicity, genotoxicity, and tumorigenicity. At present, it is not clear how dehydropyrrolizidine alkaloids bind to cellular amino acids and proteins to induced toxicity. We previously reported that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP-valine) adducts that upon reaction with phenyl isothiocyanate (PITC) formed four DHP-valine-PITC adduct isomers. In this study, we report the absolute configuration and stability of DHP-valine and DHP-valine-PITC adducts, and the mechanism of interconversion between DHP-valine-PITC adducts.

Heterozygous p53 knockout mouse model for dehydropyrrolizidine alkaloid-induced carcinogenesis.

Dehydropyrrolizidine alkaloids (DHPA) are a large, structurally diverse group of plant-derived protoxins that are potentially carcinogenic. With worldwide significance, these alkaloids can contaminate or be naturally present in the human food supply. To develop a small animal model that may be used to compare the carcinogenic potential of the various DHPAs, male heterozygous p53 knockout mice were administered a short-term treatment of riddelliine 5, 15 or 45 mg kg(-1) bodyweight day(-1) by oral gavage for 14 days, or dosed a long-term treatment of riddelliine 1 mg kg(-1) bodyweight day(-1) in pelleted feed for 12 months. Exposure to riddelliine increased the odds of tumor development in a dose-responsive manner (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). The most common neoplastic process was hepatic hemangiosarcoma, which is consistent with published lifetime rodent riddelliine carcinogenesis studies. Angiectasis (peliosis hepatis) and other previously unreported lesions were also identified. The results of this research demonstrate the utility of the heterozygous p53 knockout mouse model for further investigation of comparative carcinogenesis of structurally and toxicologically different DHPAs and their N-oxides.

Identification of a new reactive metabolite of pyrrolizidine alkaloid retrorsine: (3H-pyrrolizin-7-yl)methanol.

Pyrrolizidine alkaloids (PAs) such as retrorsine are common food contaminants that are known to be bioactivated by cytochrome P450 enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites known as dehydropyrrolizidine alkaloids (DHPs). We compared how both electrochemical (EC) and human liver microsomal (HLM) oxidation of retrorsine could produce short-lived intermediate metabolites; we also characterized a toxicologically important metabolite, (3H-pyrrolizin-7-yl)methanol. The EC cell was coupled online or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas the HLM oxidation was performed in 100 mM potassium phosphate (pH 7.4) in the presence of NADPH at 37 °C. The EC cell oxidation of retrorsine produced 12 metabolites, including dehydroretrorsine (m/z 350, [M + H(+)]), which was degraded to a new reactive metabolite at m/z 136 ([M + H(+)]). The molecular structure of this small metabolite was determined using high-resolution mass spectrometry and NMR spectroscopy followed by chemical synthesis. In addition, we also identified another minor but reactive metabolite at m/z 136, an isomer of (3H-pyrrolizin-7-yl)methanol. Both (3H-pyrrolizin-7-yl)methanol and its minor isomer were also observed after HLM oxidation of retrorsine and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the presence of reduced glutathione (GSH), each isomer formed identical GSH conjugates at m/z 441 and m/z 730 in the negative ESI-MS. Because (3H-pyrrolizine-7-yl)methanol) and its minor isomer subsequently reacted with GSH, it is concluded that (3H-pyrrolizin-7-yl)methanol may be a common toxic metabolite arising from PAs.

Metabolic Activation of Pyrrolizidine Alkaloids Leading to Phototoxicity and Photogenotoxicity in Human HaCaT Keratinocytes

Pyrrolizidine alkaloids, produced by a large number of poisonous plants with wide global distribution, are associated with genotoxicity, tumorigenicity, and hepatotoxicity in animals and humans. Mammalian metabolism converts pyrrolizidine alkaloids to reactive pyrrolic metabolites (dehydropyrrolizidine alkaloids) that form covalent protein and DNA adducts. Although a mechanistic understanding is currently unclear, pyrrolizidine alkaloids can cause secondary (hepatogenous) photosensitization and induce skin cancer. In this study, the phototoxicity of monocrotaline, riddelliine, dehydromonocrotaline, dehydroriddelliine, and dehydroretronecine (DHR) in human HaCaT keratinocytes under ultraviolet A (UVA) irradiation was determined. UVA irradiation of HaCaT cells treated with dehydromonocrotaline, dehydroriddelline, and DHR resulted in increased release of lactate dehydrogenase and enhanced photocytotoxicity proportional to the UVA doses. UVA-induced photochemical DNA damage also increased proportionally with dehydromonocrotaline and dehydroriddelline. UVA treatment potentiated the formation of 8-hydroxy-2′-deoxyguanosine DNA adducts induced by dehydromonocrotaline in HaCaT skin keratinocytes. Using electron spin resistance trapping, we found that UVA irradiation of dehydromonocrotaline and dehydroriddelliine generates reactive oxygen species (ROS), including hydroxyl radical, singlet oxygen, and superoxide, and electron transfer reactions, indicating that cytotoxicity and genotoxicity of these compounds could be mediated by ROS. Our results suggest that dehydropyrrolizidine alkaloids formed or delivered to the skin cause pyrrolizidine alkaloid-induced secondary photosensitization and possible skin cancer.

Reaction of dehydropyrrolizidine alkaloids with valine and hemoglobin.

Pyrrolizidine alkaloid-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids exert toxicity through metabolism to dehydropyrrolizidine alkaloids that bind to cellular protein and DNA, leading to hepatotoxicity, genotoxicity, and tumorigenicity. To date, it is not clear how dehydropyrrolizidine alkaloids bind to cellular constituents, including amino acids and proteins, resulting in toxicity. Metabolism of carcinogenic monocrotaline, riddelliine, and heliotrine produces dehydromonocrotaline, dehyroriddelliine, and dehydroheliotrine, respectively, as primary reactive metabolites. In this study, we report that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP-valine) adducts. For structural elucidation, DHP-valine adducts were derivatized with phenyl isothiocyanate (PITC) to DHP-valine-PITC products. After HPLC separation, their structures were characterized by mass spectrometry, UV-visible spectrophotometry, (1)H NMR, and (1)H-(1)H COSY NMR spectral analysis. Two DHP-valine-PITC adducts, designated as DHP-valine-PITC-1 and DHP-valine-PITC-3, had the amino group of valine linked to the C7 position of the necine base, and the other two DHP-valine-PITC products, DHP-valine-PITC-2 and DHP-valine-PITC-4, linked to the C9 position of the necine base. DHP-valine-PITC-1 was interconvertible with DHP-valine-PITC-3, and DHP-valine-PITC-2 was interconvertible with DHP-valine-PITC-4. Reaction of dehydroriddelliine and dehydroheliotrine with valine provided similar results. However, reaction of valine and dehydroretronecine (DHR) under similar experimental conditions did not produce DHP-valine adducts. Reaction of dehydromonocrotaline with rat hemoglobin followed by derivatization with PITC also generated the same four DHP-valine-PITC adducts. This represents the first full structural elucidation of protein conjugated pyrrolic adducts formed from reaction of dehydropyrrolizidine alkaloids with an amino acid (valine). In addition, it was found that DHP-valine-2 and DHP-valine-4, with the valine amino group linked at the C7 position of the necine base, can lose the valine moiety to form DHP.