1,2-Dehydropyrrolizidine Alkaloids: Their Potential as a Dietary Cause of Sporadic Motor Neuron Diseases.

Sporadic motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS), can be caused by spontaneous genetic mutations. However, many sporadic cases of ALS and other debilitating neurodegenerative diseases (NDDs) are believed to be caused by environmental factors, subject to considerable debate and requiring intensive research. A common pathology associated with MND development involves progressive mitochondrial dysfunction and oxidative stress in motor neurons and glial cells of the central nervous system (CNS), leading to apoptosis. Consequent degeneration of skeletal and respiratory muscle cells can lead to death from respiratory failure. A significant number of MND cases present with cancers and liver and lung pathology. This Perspective explores the possibility that MNDs could be caused by intermittent, low-level dietary exposure to 1,2-dehydropyrrolizidine alkaloids (1,2-dehydroPAs) that are increasingly recognized as contaminants of many foods consumed throughout the world. Nontoxic, per se, 1,2-dehydroPAs are metabolized, by particular cytochrome P450 (CYP450) isoforms, to 6,7-dihydropyrrolizines that react with nucleophilic groups (-NH, -SH, -OH) on DNA, proteins, and other vital biochemicals, such as glutathione. Many factors, including aging, gender, smoking, and alcohol consumption, influence CYP450 isoform activity in a range of tissues, including glial cells and neurons of the CNS. Activation of 1,2-dehydroPAs in CNS cells can be expected to cause gene mutations and oxidative stress, potentially leading to the development of MNDs and other NDDs. While relatively high dietary exposure to 1,2-dehydroPAs causes hepatic sinusoidal obstruction syndrome, pulmonary venoocclusive disease, neurotoxicity, and diverse cancers, this Perspective suggests that, at current intermittent, low levels of dietary exposure, neurotoxicity could become the primary pathology that develops over time in susceptible individuals, along with a tendency for some of them to also display liver and lung pathology and diverse cancers co-occurring with some MND/NDD cases. Targeted research is recommended to investigate this proposal.

Pyrrolizidine Alkaloids: Potential Role in the Etiology of Cancers, Pulmonary Hypertension, Congenital Anomalies, and Liver Disease.

Large outbreaks of acute food-related poisoning, characterized by hepatic sinusoidal obstruction syndrome, hemorrhagic necrosis, and rapid liver failure, occur on a regular basis in some countries. They are caused by 1,2-dehydropyrrolizidine alkaloids contaminating locally grown grain. Similar acute poisoning can also result from deliberate or accidental consumption of 1,2-dehydropyrrolizidine alkaloid-containing herbal medicines, teas, and spices. In recent years, it has been confirmed that there is also significant, low-level dietary exposure to 1,2-dehydropyrrolizidine alkaloids in many countries due to consumption of common foods such as honey, milk, eggs, salads, and meat. The level of 1,2-dehydropyrrolizidine alkaloids in these foods is generally too low and too intermittent to cause acute toxicity. However, these alkaloids are genotoxic and can cause slowly developing chronic diseases such as pulmonary arterial hypertension, cancers, cirrhosis, and congenital anomalies, conditions unlikely to be easily linked with dietary exposure to 1,2-dehydropyrrolizidine alkaloids, especially if clinicians are unaware that such dietary exposure is occurring. This Perspective provides a comprehensive review of the acute and chronic toxicity of 1,2-dehydropyrrolizidine alkaloids and their potential to initiate certain chronic diseases, and suggests some associative considerations or indicators to assist in recognizing specific cases of diseases that may have resulted from dietary exposure to these hazardous natural substances. If it can be established that low-level dietary exposure to 1,2-dehydropyrrolizidine alkaloids is a significant cause of some of these costly and debilitating diseases, then this should lead to initiatives to reduce the level of these alkaloids in the food chain.

Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination.

INTRODUCTION: Two recent mass spectrometry-based reports concerning Senecio scandens yielded remarkably dissimilar pyrrolizidine alkaloid constituents. In both studies, and in a related analysis of Senecio scandens and Tussilago farfara using micellar electrokinetic chromatography, the presence of hazardous N-oxides of the alkaloids was either not considered or was inadequately considered. This raises concerns about the effectiveness of the methodologies used in these, and similar, studies in assessing the pyrrolizidine alkaloid content and the safety of food, food supplements and medicines for human use. OBJECTIVE: To highlight essential analytical requirements for confident assessment of pyrrolizidine alkaloid-related safety of food and herbal products for human use. METHODOLOGY: Direct infusion-ESI MS and HPLC-ESI MS were used to analyse samples derived from liquid-liquid partitioning experiments and from strong cation exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides. RESULTS: A simple solvent partitioning experiment using pure senecionine and senecionine-N-oxide, two constituents reported in one of the mass spectrometry-based studies of S. scandens, clearly demonstrated the inadequacy of the reported method to detect and quantitate hazardous pyrrolizidine alkaloid N-oxide components. A preliminary LCMS analysis of commercially-prepared extracts of comfrey roots (Symphytum officinale and S. uplandicum s. l.) was used as a model to highlight the analytical importance of N-oxides in the safety assessment of pyrrolizidine alkaloid-containing medicinal herbs. CONCLUSIONS: This study highlighted significant differences in the reported identification of pyrrolizidine alkaloids from the same plant species, and clearly demonstrated the inadequacy of some procedures to include N-oxides in the assessment of pyrrolizidine alkaloid-related safety of food and herbal products.

Hepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollen.

Using HPLC-ESI-MS, several saturated and 1,2-dehydropyrrolizidine alkaloids were detected, mainly as their N-oxides, in fresh pollen collected from flowers of the pyrrolizidine alkaloid-producing plants Echium vulgare, E. plantagineum, Senecio jacobaea, S. ovatus, and Eupatorium cannabinum, and/or pollen loads from bees (bee pollen) that foraged on those plants. A major alkaloidal metabolite in S. ovatus was tentatively identified, using its mass spectrometric data and biogenic considerations, as the previously unreported, saturated alkaloid, 2-hydroxysarracine. Heating had very little effect on the 1,2-dehydropyrrolizidine alkaloids and their N-oxides from a variety of sources. Considered in conjunction with international concerns about the adverse effects of these alkaloids, the results strongly indicate a need for monitoring pollen supplies intended for human consumption, at least until conditions for processing and/or selection are clearly defined such as to significantly reduce the hepatotoxic (and potentially carcinogenic and genotoxic) pyrrolizidine alkaloid content of bee pollen.

Insect-synthesised retronecine ester alkaloids: precursors of the common arctiine (Lepidoptera) pheromone hydroxydanaidal.

Many pyrrolizidine alkaloid (PA)-adapted insects convert PAs sequestered from their larval host plants into "insect-PAs" in which the acid components of the alkaloids are replaced by small, branched aliphatic 2-hydroxy acids of insect origin. It has been proposed that insect-PAs are precursors of the pheromone hydroxydanaidal in male Estigmene acrea moths, but it is not clear why they are specifically required or what the structural features or chemical properties are that make insect-PAs more suitable for conversion into hydroxydanaidal than superficially similar alkaloids of plant origin. Evidence is presented that insect-PAs are also precursors of hydroxydanaidal in the polyphageous arctiine, Creatonotos transiens, and a new biosynthetic pathway to hydroxydanaidal is proposed that has a mandatory requirement for insect-PAs as intermediates.

Formation of DHP-derived DNA adducts from metabolic activation of the prototype heliotridine-type pyrrolizidine alkaloid, lasiocarpine.

Pyrrolizidine alkaloids (PAs) are probably the most common poisonous plants affecting livestock, wildlife, and humans. The PAs that have been found to be tumorigenic in experimental animals belong to the retronecine-, heliotridine-, and otonecine-type PAs. Our recent mechanistic studies indicated that riddelliine, a tumorigenic retronecine-type PA, induced tumors via a genotoxic mechanism mediated by the formation of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. The same adducts were formed from clivorine, a tumorigenic otonecine-type PA from metabolism of clivorine by rat liver microsomes in the presence of calf thymus DNA. In this study, we report that metabolism of lasiocarpine, the prototype heliotridine PA, by F344 rat liver microsomes resulted in the formation of DHP. When incubated in the presence of calf thymus DNA, the same DHP-derived DNA adducts were formed. These results suggest that these DHP-derived DNA adducts are potential biomarkers of exposure and tumorigenicity for all types of PAs.

Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum.

Pyrrolizidine alkaloids and their N-oxides can be extracted from the dried methanolic extracts of plant material using dilute aqueous acid. The subsequent integration of solid-phase extraction (with a strong cation exchanger) of the alkaloids and N-oxides from the aqueous acid solution, together with analysis using HPLC-ESI/MS, provides a method for the simultaneous profiling of the pyrrolizidine alkaloids and their N-oxides in plant samples and the collection of useful structural data as an aid in their identification. The N-oxide character of the analytes may be confirmed by treating analytical samples with a redox resin and observing the formation of the corresponding parent pyrrolizidine alkaloids. The present case study of Echium plantagineum highlighted a higher ratio of N-oxides to the parent tertiary bases than has been previously reported. Furthermore, a higher proportion of acetylated pyrrolizidine-N-oxides was observed in the flower heads relative to the leaves. Six pyrrolizidine alkaloids or pyrrolizidine-N-oxides, not previously reported from E. plantagineum, were tentatively identified on the basis of MS and biogenetic considerations. Three of these, 3'-O-acetylintermedine/lycopsamine, leptanthine-N-oxide and 9-O-angelylretronecine-N-oxide, have been reported elsewhere, whilst three others, 3'-O-acetylechiumine-N-oxide, echimiplatine-N-oxide and echiuplatine-N-oxide, appear unreported from any other source.

Pyrrolizidine alkaloids of Echium vulgare honey found in pure pollen.

The pyrrolizidine alkaloids previously identified in floral honey attributed to Echium vulgare (Boraginaceae) have been detected (8000-14 000 ppm) in pure pollen collected from the anthers of Echium vulgare. Pyrrolizidine alkaloids and/or their N-oxides were isolated from the aqueous acid extracts of pollen by use of strong cation-exchange, solid-phase extraction and identified by liquid chromatographic/mass spectrometric (LCMS) analysis. The pyrrolizidine alkaloids in the pollen are present mainly as the N-oxides. In addition to seven previously described pyrrolizidine alkaloids and/or their N-oxides (echimidine, acetylechimidine, uplandicine, 9-O-angelylretronecine, echiuplatine, leptanthine, and echimiplatine), one unidentified (echivulgarine), but previously found in honey, and two previously undescribed (vulgarine and 7-O-acetylvulgarine) pyrrolizidine alkaloids and/or their N-oxides were identified in the pollen. Tentative structures for these unidentified pyrrolizidine alkaloids are proposed on the basis of the mass spectrometric data and biogenetic considerations. The implications of these results for identifying the source and subsequent concentrations of pyrrolizidine alkaloids in honeys and commercial bee pollen are briefly discussed.

Solid-phase extraction and LC-MS analysis of pyrrolizidine alkaloids in honeys.

Strong-cation-exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides from honey samples was followed by reduction of the N-oxides and subsequent analysis of total pyrrolizidine alkaloids using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. A limited survey of 63 preprocessing samples of honey, purposefully biased toward honeys attributed to floral sources known to produce pyrrolizidine alkaloids, demonstrated levels of pyrrolizidine alkaloids up to approximately 2000 parts per billion (ppb) in a sample attributed to Echium plantagineum. Up to 800 ppb pyrrolizidine alkaloids was detected in some honeys not attributed by the collector to any pyrrolizidine alkaloid-producing floral source. No pyrrolizidine alkaloids were detected in approximately 30% of the samples in this limited study, while some honeys showed the copresence of pyrrolizidine alkaloids from multiple floral sources such as E. plantagineum and Heliotropium europaeum. In addition, retail samples of blended honeys (with no labeling to suggest that pyrrolizidine alkaloid-producing floral sources were used in the blends) have been shown to contain up to approximately 250 ppb pyrrolizidine alkaloids.

Preparative isolation of swainsonine from locoweed: extraction and purification procedures.

The trihydroxy indolizidine alkaloid swainsonine, a plant toxin with potent alpha-mannosidase-inhibitory activity and chemotherapeutic potential, was isolated in gram quantities from locoweed (Astragalus lentiginosus). The key isolation and purification step was a continuous liquid/liquid extraction procedure using dichloromethane to extract a basified aqueous methanol solution obtained after isolation of the polar base fraction by ion-exchange. The concentration of swainsonine was increased from ca. 7% in the polar base material to 68% using the liquid/liquid extraction procedure. Pure swainsonine was then obtained by recrystallisation from ammonia-saturated chloroform or by sublimation. Small samples of swainsonine were also purified by formation of the chloroform-soluble methylboronate derivative, from which the alkaloid could be regenerated easily by hydrolysis.

Poisoning of livestock in oregon in the 1940s to 1960s attributed to corynetoxins produced by Rathayibacter in nematode galls in chewings fescue (Festuca nigrescens).

Tunicaminyluracil antibiotics, similar to the corynetoxins produced by Rathayibacter toxicus in Australia and South Africa, were found in old nematode seed-galls from Festuca nigrescens from New Jersey (USA) and New Zealand (NZ). The toxin profiles from the NZ and USA galls were similar to each other, but differed from those produced by R toxicus from Australia and South Africa, suggesting that a geographical variant of R toxicus or closely related species may be involved. The NZ galls gave a positive response to a R toxicus-specific monoclonal antibody assay, albeit a considerably weaker response than that seen with Australian R toxicus galls, but the older USA galls were negative, possibly due to deterioration of the antigen. From these findings, it is postulated that livestock deaths associated with the feeding of nematode and bacterial infected screenings of F nigrescens in Oregon, USA, in the 1940s to 1960s were caused by corynetoxin-like toxins produced by the bacterium.

Toxic effects of a single parenteral dose of tunicamycin in late stage pregnancy in rats.

Tunicamycin (TM) was given as a single parenteral dose at 3 levels to female rats at gestation day 15 (GD 15) and also to non-pregnant rats. At 16 h post-dosing all pregnant rats had moderate to extensive vaginal bleeding and 1/4 at each of the 2 higher doses died. The other severely affected rats, euthanized after 26-28 h, had free blood in the uterus and large decreases in red cell count (RCC), hemoglobin (Hb) and packed cell volume (PCV). The amnion of the fetuses was very easily detached from the maternal placenta by gentle manipulation. Histologically, hemorrhage, venous thrombosis and ischemic necrosis, particularly in the maternal placenta, were consistent with the gross appearance. There was no hemorrhage in any control pregnant rats. In the remaining TM-treated pregnant rats, euthanized at GD 17, there were lesser dose-related decreases in RCC, Hb and PCV, but there was no evidence of bleeding or changes in red blood cell parameters in non-pregnant rats. There was a dose-related decrease in cholesterol and GlcNAc-1-P transferase (GPT) activity and a treatment-related decrease in serum proteins in all rats. Maternal toxicity was demonstrated in pregnant rats at TM doses < 10% of a TM lethal dose in non-pregnant rats.

Honey from plants containing pyrrolizidine alkaloids: a potential threat to health.

Following scientific risk assessments, several countries have imposed strict regulations on herbal medicines containing 1,2-dehydro-pyrrolizidine alkaloids. Using published data on the plants used in honey production, pyrrolizidine alkaloid-containing plants are shown in this review to represent a significant source of honey worldwide. This observation, honey consumption data, reported levels of pyrrolizidine alkaloids in honeys, and consideration of tolerable exposure levels determined for pyrrolizidine alkaloids in herbal medicines, leads to the conclusion that some honey is a potential threat to health, especially for infants and fetuses, and further investigation is warranted.