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.

Use of Herbarium Voucher Specimens To Investigate Phytochemical Composition in Poisonous Plant Research.

Poisonous plants cause large losses to the livestock industry through death, reduced production efficiency, reproductive dysfunction, and compromised harvesting of rangeland and pasture forages. Research investigating poisonous plants is complex because there are hundreds of genera of toxic plants representing thousands of species. To investigate the effects of poisonous plants on livestock, a clear understanding of the taxonomic identity of the plant and the ability to collect the plant in sufficient quantities for scientific studies is required. Subsequently, the active principles must be defined and investigated in the taxa of interest to better predict risk and make recommendations to reduce losses. Herbaria are collections of preserved plant specimens and are an important resource in poisonous plant research. Voucher specimens have often been used in the identification of the plant for the experimental reproduction of suspected livestock poisoning associated with a spontaneous case. More recently, herbarium specimens have been used to investigate the chemical composition of toxic plants as well as the distribution of different chemotypes over the landscape. The primary purpose of this review is to highlight the chemical analysis of herbarium specimens in poisonous plant research.

Isolation and SAR studies of bicyclic iminosugars from Castanospermum australe as glycosidase inhibitors.

We report the isolation and structural determination of fourteen iminosugars, containing five pyrrolizidines and five indolizidines, from Castanospermum australe. The structure of a new alkaloid was elucidated by spectroscopic methods as 6,8-diepi-castanospermine (13). Our side-by-side comparison between bicyclic and corresponding monocyclic iminosugars revealed that inhibition potency and spectrum against each enzyme are clearly changed by their core structures. Castanospermine (10) and 1-deoxynojirimycin (DNJ) have a common d-gluco configuration, and they showed the expected similar inhibition potency and spectrum. In sharp contrast, 6-epi-castanospermine (12) and 1-deoxymannojirimycin (manno-DNJ) both have the d-manno configuration but the α-mannosidase inhibition of 6-epi-castanospermine (12) was much better than that of manno-DNJ. 6,8-Diepi-castanospermine (13) could be regarded as a bicyclic derivative of talo-DNJ, but it showed a complete loss of α-galactosidase A inhibition. This behavior against α-galactosidase A is similar to that observed for 1-epi-australine (6) and altro-DMDP.

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.

Global perspectives on poisonous plants: the 9th international symposium on poisonous plants.

The 9th International Symposium on Poisonous Plants (ISOPP9) was held July 15-21, 2013, at the Inner Mongolia Agricultural University in Hohhot, Inner Mongolia Autonomous Region of China. The symposium consisted of three days of oral and poster presentations, followed by a tour of the Xilinhot Region of the Mongolian Grasslands, encompassing grazing conditions consisting of desert, grassland, and steppes. This was the first time that an ISOPP meeting has been held in Asia and provided an opportunity for visitors from outside China to become aware of livestock poisonings caused by plant species with which they were previously not familiar while at the same time demonstrating that many of the problems experienced around the world have a common etiology. Presentations focused on botany, veterinary science, toxicology, mechanism of action, and chemistry. As is appropriate for the Journal of Agricultural and Food Chemistry, this cluster of papers consists of selected oral and poster presentations in which the chemistry of the toxins played a significant role. The symposium revealed that there is considerable scope for isolation, structural elucidation, and analysis of the toxins from the numerous poisonous plant species that have been identified in China. It became apparent that there are abundant opportunities for chemists both within China and abroad to collaborate with Chinese scientists working on biological aspects of livestock poisonings.

Quantitation of sensory-active and bioactive constituents of food: A Journal of Agricultural and Food Chemistry perspective.

The proper procedures for the measurement of amounts of compounds that may occur in a food or other matrices are presented in this perspective. Factors dealt with include sampling, use of standards, advantages and limitations of chromatographic and other techniques for quantitation, and proper presentation and reporting of data. Such factors must be considered at the initial stages of an investigation and incorporated completely into the overall experimental design. These standards are to be employed in determining quantities of such components, and their careful incorporation should result in more favorable evaluation of manuscripts submitted to the Journal of Agricultural and Food Chemistry.

Loss of msnA, a putative stress regulatory gene, in Aspergillus parasiticus and Aspergillus flavus increased production of conidia, aflatoxins and kojic acid.

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.

Detection of high levels of pyrrolizidine-N-oxides in the endangered plant Cryptantha crassipes (Terlingua Creek cat's-eye) using HPLC-ESI-MS.

INTRODUCTION: A previous investigation of pyrrolizidine alkaloids produced by nine species of Cryptantha identified at least two chemotypes within the genus. Other research has postulated that pyrrolizidine-N-oxide concentrations increase as the growing conditions become harsher, particularly with respect to water availability. Cryptantha crassipes is an endangered plant with a very limited distribution range within a dry, harsh Texan ecosystem. OBJECTIVE: To determine the pyrrolizidine alkaloid (and their N-oxides) profile and concentrations in Cryptantha crassipes. METHODOLOGY: Methanolic extracts of Cryptantha crassipes were partitioned into dilute sulphuric acid and the alkaloids concentrated using strong cation exchange, solid-phase extraction columns. Extracts were analysed using reversed-phase high-pressure liquid chromatography coupled to electrospray ionisation ion trap mass spectrometry. RESULTS: The N-oxides of lycopsamine and intermedine were the major pyrrolizidine alkaloids detected in Cryptantha crassipes. Smaller to trace amounts of other pyrrolizidine alkaloids observed were: the 7- and 3'-acetylated derivatives and the 1,2-dihydro analogs of lycopsamine-N-oxide and/or intermedine-N-oxide; a pair of unidentified N-oxides, isobaric with lycopsamine-N-oxide; and the N-oxides of leptanthine, echimiplatine, amabiline, echiumine and dihydroechiumine. Only trace amounts, if any, of the parent free base pyrrolizidine alkaloids were detected. The concentration of pyrrolizidine alkaloids was estimated to be 3-5% of the dry weight of milled leaves, or 10-50 times the levels previously reported for similar chemotypes. CONCLUSIONS: The high levels of the N-oxides of lycopsamine and intermedine establish the genus chemotype of the endangered Cryptantha crassipes and support earlier data linking high levels of N-oxides to dry, harsh growing conditions.

Chemosensitization of aflatoxigenic fungi to antimycin A and strobilurin using salicylaldehyde, a volatile natural compound targeting cellular antioxidation system.

Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10-75% at 9.5 mM ≤ SA ≤ 16.0 mM, while complete growth inhibition was achieved at 19.0 mM ≤ SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B(1) and B(2) (AFB(1), AFB(2)) for A. flavus and AFB(1), AFB(2), AFG(1), and AFG(2) for A. parasiticus, in the SA-treated (9.5 mM) fungi was reduced by ~13-45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.

Augmenting the activity of antifungal agents against aspergilli using structural analogues of benzoic acid as chemosensitizing agents.

A number of benzoic acid analogues showed antifungal activity against strains of Aspergillus flavus, Aspergillus fumigatus and Aspergillus terreus, causative agents of human aspergillosis, in in vitro bioassays. Structure-activity analysis revealed that antifungal activities of benzoic and gallic acids were increased by addition of a methyl, methoxyl or chloro group at position 4 of the aromatic ring, or by esterification of the carboxylic acid with an alkyl group, respectively. Thymol, a natural phenolic compound, was a potent chemosensitizing agent when co-applied with the antifungal azole drugs fluconazole and ketoconazole. The thymol-azole drug combination demonstrated complete inhibition of fungal growth at dosages far lower than the drugs alone. Co-application of thymol with amphotericin B had an additive effect on all strains of aspergilli tested with the exception of two of three strains of A. terreus, where there was an antagonistic effect. Use of two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus, sakAΔ and mpkCΔ, having gene deletions in the oxidative stress response pathway, indicated antifungal and/or chemosensitization activity of the benzo analogues was by disruption of the oxidative stress response system. Results showed that both these genes play overlapping roles in the MAPK system in this fungus. The potential of safe, natural compounds or analogues to serve as chemosensitizing agents to enhance efficacy of commercial antifungal agents is discussed.

Rapid analytical method for the determination of aflatoxins in plant-derived dietary supplement and cosmetic oils.

Consumption of edible oils derived from conventional crop plants is increasing because they are generally regarded as healthier alternatives to animal-based fats and oils. More recently, there has been increased interest in the use of alternative specialty plant-derived oils, including those from tree nuts (almonds, pistachios, and walnuts) and botanicals (borage, evening primrose, and perilla) both for direct human consumption (e.g., as salad dressings) and for the preparation of cosmetics, soaps, and fragrance oils. This has raised the issue as to whether or not exposure to aflatoxins can result from such oils. Although most crops are subject to analysis and control, it has generally been assumed that plant oils do not retain aflatoxins due to the high polarity and lipophobicity of these compounds. There is virtually no scientific evidence to support this supposition, and available information is conflicting. To improve the safety and consistency of botanicals and dietary supplements, research is needed to establish whether or not oils used directly, or in the formulation of products, contain aflatoxins. A validated analytical method for the analysis of aflatoxins in plant-derived oils is essential to establish the safety of dietary supplements for consumption or cosmetic use that contain such oils. The aim of this research was therefore to develop an HPLC method applicable to a wide variety of oils from different plant sources spiked with aflatoxins, thereby providing a basis for a comprehensive project to establish an intra- and interlaboratory validated analytical method for the analysis of aflatoxins in dietary supplements and cosmetics formulated with plant oils.

Poisoning of sheep by seeds of Crotalaria retusa: acquired resistance by continuous administration of low doses.

Seeds of Crotalaria retusa containing 6.84% (dry weight) of monocrotaline (MCT) were administered experimentally to sheep. Three sheep that received 136.8mg MCT/kg bw daily for 70 days had no clinical signs. Five out of six sheep ingesting single doses of 205.2 and 273.6mg MCT/kg bw died with acute (three sheep) or chronic intoxication (two sheep). Acute intoxicated sheep had periacinar liver necrosis and chronic intoxicated sheep liver fibrosis and megalocytosis. Another three sheep had no clinical signs after the ingestion of 20 daily doses of 136.8mg MCT/kg, followed by seven doses of 273.6mg MCT/kg, and one single dose of 342mg MCT/kg. These experiments demonstrated that sheep are susceptible to acute intoxication by MCT being intoxicated by a single oral dose of approximately 205.2mg/kg. In contrast, they develop strong resistance to MCT after the daily ingestion of non lethal doses (136.8mg/kg). It is suggested that chronic poisoning does not occur by the repeated ingestion of non acutely toxic doses, but probably by the ingestion of single toxic doses. It is also suggested that sheep do not become intoxicated with the ingestion of C. retusa in the vegetative non-seeding stage.

The comparative pathology of the glycosidase inhibitors swainsonine, castanospermine, and calystegines A3, B2, and C1 in mice.

To study various polyhydroxy-alkaloid glycosidase inhibitors, 16 groups of 3 mice were dosed using osmotic minipumps with swainsonine (0, 0.1, 1, and 10 mg/kg/day), castanospermine, and calystegines A(3), B(2), and C(1) (1, 10, and 100 mg/kg/day). After 28 days, the mice were euthanized, necropsied, and examined using light and electron microscopy. The high-dose swainsonine-treated mice developed neurologic disease with neuro-visceral vacuolation typical of locoweed poisoning. Castanospermine- and calystegines-treated mice were clinically normal; however, high-dose castanospermine-treated mice had thyroid, renal, hepatic, and skeletal myocyte vacuolation. Histochemically, swainsonine- and castanospermine-induced vacuoles contained mannose-rich oligosaccharides. High-dose calystegine A(3)-treated mice had increased numbers of granulated cells in the hepatic sinusoids. Electron microscopy, lectin histochemistry, and immunohistochemistry suggest these are pit cells (specialized NK cells). Histochemically, the granules contain glycoproteins or oligosaccharides with abundant terminal N-acetylglucosamine residues. Other calystegine-treated mice were histologically normal. These findings indicate that swainsonine produced lesions similar to locoweed, castanospermine caused vacuolar changes with minor changes in glycogen metabolism, and only calystegine A(3) produced minimal hepatic changes. These also suggest that in mice calystegines and castanospermine are less toxic than swainsonine, and as rodents are relatively resistant to disease, they are poor models to study such induced storage diseases.

Separation and measurement of plant alkaloid enantiomers by RP-HPLC analysis of their Fmoc-Alanine analogs.

INTRODUCTION: Ammodendrine (1), anabasine (2) and coniine (3) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed. OBJECTIVE: To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material. METHODOLOGY: Mixtures of isolated or purified plant alkaloids were converted to their Fmoc-L-Ala-alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios. RESULTS: The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc-L-Ala-coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species. CONCLUSION: A low-cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed.

Chemosensitization of fungal pathogens to antimicrobial agents using benzo analogs.

Activities of conventional antifungal agents, fludioxonil, strobilurin and antimycin A, which target the oxidative and osmotic stress response systems, were elevated by coapplication of certain benzo analogs (aldehydes and acids). Fungal tolerance to 2,3-dihydroxybenzaldehyde or 2,3-dihydroxybenzoic acid was found to rely upon mitochondrial superoxide dismutase (SOD2) or glutathione reductase (GLR1), genes regulated by the HOG1 signaling pathway, respectively. Thus, certain benzo analogs can be effective at targeting cellular oxidative stress response systems. The ability of these compounds to chemosensitize fungi for improved control with conventional antifungal agents is discussed.