Seasonal variability in pyrrolizidine alkaloids in Senecio inaequidens from the Val Venosta (Northern Italy)

Senecio inaequidens is a neophyte originating from South Africa that has managed to spread to Europe and colonize large areas. This plant has toxic pyrrolizidine alkaloids (PAs) that represent a health risk to humans and animals. The aim of this work was to measure the content of PAs of these plants in continuous intervals over the entire growing season. The plants were separated into their organs, such as sprouts, stems, leaves and inflorescences. PAs were extracted from the dried plants using an acidic methanol solution, purified and measured by GC/MS. The average PA content of plants over the growing season was 0.33% of dry weight. The highest PA levels were about 1% in the dry weight and were found in the young sprouts and flower heads. There were nine PAs present, of which six could be identified. The main alkaloid was retrorsine followed by senecivernine, senecionine, integerrimine, usaramine and seneciphylline. Due to the high PA content of the inflorescences, the long flowering period and the rapid invasion dynamics, this species presents a high health risk for humans and animals.     

Toxicity of pyrrolizidine alkaloids to humans and ruminants

1,2-dehydro pyrrolizidine ester alkaloids (PA) are toxic for human and livestock. The PAs undergo a metabolic toxication process in the liver which is the first target organ for PA poisoning. World-wide many episodes of PA intoxications have been reported involving humans as well as ruminants. This intoxication is not only related to the amount and duration of the exposure to PAs but also to species, age and gender. Besides the metabolic toxification, detoxication processes are also important. The paper discusses the toxification and detoxication processes and gives an overview about PA poisoning cases in humans and ruminants.     

Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.     

Soil-borne microorganisms and soil-type affect pyrrolizidine alkaloids in Jacobaea vulgaris

Secondary metabolites like pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. We studied the effects of soil-borne microorganisms and soil-type on pyrrolizidine alkaloids in roots and shoots of Jacobaea vulgaris. We used clones of two genotypes from a dune area (Meijendel), propagated by tissue culture and grown on two sterilized soils and sterilized soils inoculated with 5% of non-sterilized soil of either of the two soil-types. Soil-borne microorganisms and soil-type affected the composition of PAs. By changing the composition rather than the total concentration below and aboveground, plants have a more complex defense strategy than formerly thought. Interestingly, a stronger negative effect on plant growth was found in sterilized soils inoculated with their ‘own’ microbial community suggesting that pathogenic and/or other plant inhibiting microorganisms were adapted to their ‘own’ soil conditions.     

Defensive properties of pyrrolizidine alkaloids against microorganisms

The understanding of the selection factors that drive chemical diversification of secondary metabolites of constitutive defence systems in plants, such as pyrrolizidine alkaloids (PAs), is still incomplete. Historically, plants always have been confronted with microorganisms. Long before herbivores existed on this planet, plants had to cope with microbial pathogens. Therefore, plant pathogenic microorganisms may have played an important role in the early evolution of the secondary metabolite diversity. In this review, we discuss the impact that plant-produced PAs have on plant-associated microorganisms. The objective of the review is to present the current knowledge on PAs with respect to anti-microbial activities, adaptation and detoxification by microorganisms, pathogenic fungi, root protection and PA induction. Many in vitro experiments showed effects of PAs on microorganisms. These results point to the potential of microorganisms to be important for the evolution of PAs. However, only a few in vivo studies have been published and support the results of the in vitro studies. In conclusion, the topics pointed out in this review need further exploration by carrying out ecological experiments and field studies.     

The clastogenic potential in vitro of pyrrolizidine alkaloids employing hepatocyte metabolism.

Three pyrrolizidine alkaloids (PAs), monocrotaline, retrorsine and isatidine, were tested for their clastogenic activity under different conditions of metabolic activation in vitro. All three compounds exhibited a weak activity when V79 cells were treated at very high concentrations for 18 h in the absence of a metabolizing system. Short-term (2 h) treatment with rat liver S9 mix led to a strong and concentration-dependent increase in chromosomal aberrations for retrorsine. Isatidine was not mutagenic with S9 mix and monocrotaline was positive at high concentrations only. In contrast, a prolonged treatment (18 h) in vitro under activation conditions in the presence of primary hepatocytes led to clear concentration-dependent positive responses for all three PAs investigated. Particularly the results with isatidine demonstrate that in vitro tests using S9 mix for metabolization can generate misleading results. It is not clear whether the results could be attributed to a better activation of the test compounds by intact hepatocytes in comparison to S9 mix or if the fact that only hepatocytes allow a treatment for the whole culture period under activation conditions was more important. Owing to its strong cytotoxicity the exposure to S9 mix is generally limited to 2-4 h, limiting also the exposure of the target cells to a test chemical as well as its metabolites. The results presented show significant differences in mutagenic potency of PAs due to variations in the activation system. This underlines the usefulness of primary hepatocytes, e.g., for the detection of pre-mutagens. The PAs investigated are present in plants which are used for phytotherapeutic medicinal products. They do not contribute to their efficacy and are, therefore, not to be tolerated in amounts that may impose a risk for the user.     

The genotype dependent presence of pyrrolizidine alkaloids as tertiary amine in Jacobaea vulgaris

Secondary metabolites such as pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide. PA extraction and detection are of great importance for the understanding of the role of PAs as plant defense compounds, as the tertiary PA form is known for its stronger influence on several generalist insects, whereas the N-oxide form is claimed to be less deterrent. We measured PA N-oxides and their reduced tertiary amines by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We show that the occurrence of tertiary PAs is not an artifact of the extraction and detection method. We found up to 50% of tertiary PAs in shoots of Jacobine - chemotype plants of Jacobaea vulgaris. Jacobine and its derivatives (jacoline, jaconine, jacozine and dehydrojaconine) may occur for more than 20% in reduced form in the shoots and more than 10% in the roots. For 22 PAs detected in F(2) hybrids (J. vulgaris × Jacobaea aquatica), we calculate the tertiary amine percentage (TA%=the tertiary amine concentration/(tertiary amine concentration+the corresponding N-oxide concentration) × 100). We found that the TA% for various PAs was genotype-dependent. Furthermore, TA% for the different PAs were correlated and the highest correlations occurred between PAs which share high structural similarity.     

Cellular distribution of alkaloids and their translocation via phloem and xylem: the importance of compartment pH

The physico‐chemical background of alkaloid allocation within plants is outlined and discussed exemplarily for pyrrolizidine alkaloids (PAs) and nicotine. The trigger for this discourse is the finding that, for example, PAs, which are taken up from the soil, are translocated in the xylem, whereas – when genuinely present in plants – they are allocated as N‐oxides via phloem. Special emphasis is put on the impact of different pH values in certain compartments, as this entails significant changes in the relative lipophilic character of alkaloids: tertiary alkaloids diffuse readily through biomembranes, while the corresponding protonated alkaloids are retained in acidic compartments, i.e. vacuoles or xylem. Therefore, this phenomenon, well known as the ‘ion trap mechanism’, is also relevant for long‐distance transport of alkaloids. Any efficient allocation of typical tertiary alkaloids within the phloem can thus be excluded. In contrast, due to their strongly increased hydrophilic properties, alkaloid‐N‐oxides or quarternary alkaloids cannot diffuse through biomembranes and, consequently, would be retained in the acidic xylem during translocation. The major aim of this paper is to sharpen the mind for the chemical peculiarities of alkaloids and to consider them adequately in forthcoming investigations on allocation of alkaloids.     

Phytochemicals: the good, the bad and the ugly?

Phytochemicals are constitutive metabolites that enable plants to overcome temporary or continuous threats integral to their environment, while also controlling essential functions of growth and reproduction. All of these roles are generally advantageous to the producing organisms but the inherent biological activity of such constituents often causes dramatic adverse consequences in other organisms that may be exposed to them. Nevertheless, such effects may be the essential indicator of desirable properties, such as therapeutic potential, especially when the mechanism of bioactivity can be delineated. Careful observation of cause and effect, followed by a coordinated approach to identify the responsible entities, has proved extremely fruitful in discovering roles for phytochemical constituents. The process is illustrated by selected examples of plants poisonous to animals and include the steroidal alkaloid toxin of Veratrum californicum (Western false hellebore), piperidine alkaloids of Lupinus species (lupines), and polyhydroxy indolizidine, pyrrolizidine and nortropane alkaloids of Astragalus and Oxytropis species (locoweeds), Castanospermum australe (Moreton Bay chestnut) and Ipomoea species (morning glories).     

Method for determination of pyrrolizidine alkaloids and their metabolites by high-performance liquid chromatography

An improved method utilizing reverse-phase liquid chromatography on a styrene-divinylbenzene column (PRP-1) and ultraviolet detection was developed for the simultaneous determination of the pyrrolizidine alkaloids (PAs) senecionine, seneciphylline, and retrorsine and their major metabolites produced during in vitro transformation of PAs by microsomal enzyme systems. The procedure employs direct injection of the 46,000g supernatant of the microsomal reaction mixture directly onto the column, and elution with a 0.1 M NH4OH-acetonitrile gradient. The method is very gentle, simple, and fast with excellent precision since no prior extraction, preconcentration, or derivatization steps are required. Using this procedure 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine and the corresponding PA N-oxides were shown to be the major microsomal metabolites of the PAs examined. The detection limit of these metabolites was approximately 1 microM.     

Pyrrolizidine alkaloids quantitatively regulate both scent organ morphogenesis and pheromone biosynthesis in maleCreatonotos moths (Lepidoptera: Arctiidae)

Summary Males ofCreatonotos gangis andC. transiens possess coremata (scent organs) of drastically varying sizes (Figs. 2, 3), which release R(-)-hydroxydanaidal (Fig. 1A) in varying amounts. Both the size of the organs and their pheromone content depend on the ingestion of pyrrolizidine alkaloids (PAs; Fig. 1 B, C) by the larvae. There is a direct correlation between amounts of PAs ingested and the size of the organs (Fig. 4). It is the absolute amount of PAs ingested which determines the expression of corematal size, structurally different PAs have identical effects (Table 2); PAs are no essential dietary factors for the general development of the moths (Table 1), and the morphogenetic effect is restricted to the coremata. The findings are discussed in terms of developmental, ecological and functional aspects.Abbreviation PAs pyrrolizidine alkaloids     

DNA adducts in humans after exposure to methylating agents

Human exposure to methylating agents appears to be widespread, as indicated by the frequent occurrence of methylated DNA adducts in human DNA. The high incidence of methylated DNA adducts even in humans thought not to have suffered extensive exposure to environmental methylating agents implies that chemicals of endogenous origin, probably N-nitroso compounds such as the strongly carcinogenic N-nitrosodimethylamine (NDMA), may be primarily responsible for their formation and raises the question of the carcinogenic risks associated with such exposure. In addition to accumulation of DNA damage, other factors (such as induced cell proliferation) appear to be important in determining the probability of induction of mutation or cancer by NDMA, implying that high to low dose risk extrapolations should not be based on the assumption of dose- or even adduct-linearity. Comparative studies of the accumulation and repair of methylated adducts in humans and animals treated with methylating cytostatic drugs do not reveal significant species differences. Based on this and the dosimetry of adduct accumulation in rats chronically exposed to very low doses of NDMA, it is suggested that the exposure needed to account for the levels of adducts found in human DNA may be of the order of hundreds of micrograms NDMA (or equivalent) per day, a level of exposure which may well represent a significant carcinogenic hazard for man.     

Evolution of secondary metabolites in legumes (Fabaceae)

Legumes produce a high diversity of secondary metabolites which serve as defence compounds against herbivores and microbes, but also as signal compounds to attract pollinating and fruit-dispersing animals. As nitrogen-fixing organisms, legumes produce more nitrogen containing secondary metabolites than other plant families. Compounds with nitrogen include alkaloids and amines (quinolizidine, pyrrolizidine, indolizidine, piperidine, pyridine, pyrrolidine, simple indole, Erythrina, simple isoquinoline, and imidazole alkaloids; polyamines, phenylethylamine, tyramine, and tryptamine derivatives), non-protein amino acids (NPAA), cyanogenic glucosides, and peptides (lectins, trypsin inhibitors, antimicrobial peptides, cyclotides). Secondary metabolites without nitrogen are phenolics (phenylpropanoids, flavonoids, isoflavones, catechins, anthocyanins, tannins, lignans, coumarins and furanocoumarins), polyketides (anthraquinones), and terpenoids (especially triterpenoid, steroidal saponins, tetraterpenes). While some secondary metabolites have a wide distribution (flavonoids, triterpenes, pinitol), however, others occur in a limited number of taxa. The distributions of secondary metabolites with an irregular occurrence are mapped on a molecular phylogeny of the Fabaceae, reconstructed from a combined data set of nucleotide sequences from rbcL, matK and ITS genes. In most cases, the distribution patterns of secondary metabolites do not agree with the phylogeny of the plants producing them. In contrary, the distribution of many secondary metabolites is patchy and irregular. Thus, the use of phytochemical data to reconstruct a phylogeny of plants is often not informative and can be misleading. The patchy distribution may be due to convergent evolution, a contribution of endophytic fungi or more likely, to an early acquisition of the key genes of secondary metabolism in the evolution of land plants among others by horizontal gene transfer from bacteria. Thus it would be a matter of gene regulation whether these genes are active in some but not all taxa.     

Recovery of the pyrrolic nucleus of pyrrolizidine alkaloid metabolites from sulphur conjugates in tissues and body fluids

Reactive pyrrolic metabolites formed when pyrrolizidine alkaloids are given to rats can alkylate soluble and tissue-bound thiol groups. Pyrrolic thioethers thus formed are relatively stable, and may persist in tissues for long periods. A simple procedure has been developed for recovering the nucleus of the pyrrolic metabolite from such S-binding, whether in solution or attached to solid tissues, in an easily identifiable form. The thioether bond was broken by silver nitrate and the pyrrolic moiety allowed to react with ethanol or methanol to form an ethoxy or methoxy derivative. The chemical basis of the procedure was established by model experiments on a preparative scale, but for small scale recovery from tissues, pyrrolic ethers were extracted and identified by TLC, HPLC, capillary GC and mass spectrometry. Because the pyrrolic derivatives thus formed were easily recognised and unrelated to any physiological compound, the recovery method described, especially when applied to blood samples, provided a way to monitor animals for previous exposure to toxic pyrrolizidine alkaloids.     

Studies on biomarkers in cancer etiology and prevention: a summary and challenge of 20 years of interdisciplinary research

Sensitive, specific methods have been developed that allow quantitative measurements of the metabolites of carcinogen metabolites and of DNA and protein adducts in humans exposed occupationally, environmentally and endogenously to genotoxic agents. The interrelationship between exposure to carcinogens, host risk factors and the responses of biomarkers has been examined in cross-sectional, ecological and case-control studies which provided new insights into the causes of cancer and the mechanisms of carcinogenesis. The identification of hitherto unknown DNA-reactive chemicals formed in the human body from dietary precursors and of carcinogenic components of complex mixtures has increased the possibility of establishing causal relationships in etiology. The identification of individuals and subgroups heavily exposed to carcinogens has led to the development of measures for avoiding or decreasing exposure to carcinogenic risk factors. New, ultrasensitive methods for measuring DNA adducts allow the quantification and structural elucidation of specific DNA damage in humans arising from oxidative stress and lipid peroxidation (LPO), which have been found to be the driving forces in several human malignancies. Background DNA damage in "unexposed" individuals has been shown unequivocally to be due to LPO products, and a significant interindividual variation in adduct levels has been shown in individuals with comparable exposure to carcinogens. Thus, pharmacogenetic variants with higher susceptibility to carcinogenic insults, due to genetic polymorphism in xenobiotic-metabolizing enzymes, have been characterized by a combination of genotyping and measurements of macromolecular adducts. Dosimetry has been used in human studies to evaluate the efficacy of interventions with chemopreventive agents like ascorbic acid, dietary phenols and green tea. Advances in the application of selected biomarkers in human studies are reviewed and illustrated by examples from the author's research conducted during the past two decades.     

Morphogenetic effects of alkaloidal metabolites on the development of the coremata in the salt marsh moth, Estigmene acrea (Dru.) (Lepidoptera: Arctiidae)

Pyrrolizidine alkaloids (PAs) play a fundamental role in the sexual biology of the salt marsh moth Estigmene acrea. They are precursors for the male courtship pheromone hydroxydanaidal and they stimulate the growth and development of male pheromone-disseminating organs called coremata. Yet larval Estigmene are polyphagous and feed only sporadically on PA-containing plants and those they utilize contain different classes of PAs. The various PAs ingested are hydrolyzed to the common necine metabolite retronecine and re-esterified to insect-specific alkaloids from which the male pheromone hydroxydanaidal is synthesized. Given this complex metabolic pathway, we investigated the role of retronecine and the insect-specific alkaloids that stem from it as morphogens stimulating corematal growth. Retronecine fed to terminal instar larvae in a standard caterpillar diet stimulated corematal growth. It also stimulated corematal growth when it was injected into the hemolymph of larvae. These results indicate that this common PA metabolite, and/or the insect specific alkaloids produced from it, function as corematal morphogens. The parental forms (alkaloids ingested from the plant) are not strictly necessary for corematal growth. Stimulation of the PA receptors on the galea and ingestion process are also not critical to corematal development. Since the insect-specific alkaloids are the direct precursors for the male courtship pheromone, it is argued that their level is the best indicator of the ultimate pheromone titer and would provide the most accurate developmental signal. The effects of alkaloidal metabolites as morphogens in E. acrea are compared to those for the South Asian arctiines Creatonotus gangis and C. transiens in which the developmental role of PAs was first discovered.     

Alkaloids of Solanaceae (nightshade plants)

Alkaloids are nitrogen containing compounds found in many plants. They are products of plants secondary metabolism derived from amino acids, purines, pyrimidines or terpene. Most of them are drugs. The biological activity of some alkaloids has led to their intensive exploitation by humans, as pharmaceuticals, narcotics or poisons. During the past 30 years, major technical advances have led to substantial progress in our understanding of alkaloid biochemistry, but since then biosynthetic pathways of some alkaloids are not explained. The nightshade (Solanaceae) are widespread family of plants containing tropane alkaloids or glycoalkaloids. Both of them are naturally produced, as a defense mechanism against insects, predator and disease. On the other hand, most of the species of Solanaceae family have been used by human since several centuries.     

白背三七叶中吡咯里西啶生物碱的LC—MS^n检测

广泛分布于植物中的吡咯里西啶生物碱（pyrrolizidine alkaloids,PAs）由具有双稠吡咯啶环的氨基醇与不同有机酸缩合而成,醇部分为裂碱（necine）,酸部分为裂酸（necicacid）。以裂碱的结构可划分为2种类型,即倒千里光裂碱型（retronecine—type,RET型）和奥托千里光裂碱型（otonecinetype,OTO型）。     

Metabolic profiling in Echium plantagineum: presence of bioactive pyrrolizidine alkaloids and napthoquinones from accessions across southeastern Australia

Geographically distinct populations of Paterson’s curse (Echium plantagineum L., Boragineacea), found near roadsides across New South Wales and Victoria, Australia were surveyed along 3 distinct longitudinal transects in spring of 2011 for presence of pyrrolizidine alkaloids and naphthoquinones in sampled plants. Composite samples of shoots and roots were collected from each of 45 sites; shoot extracts were subjected to solid phase extraction and LC-ESI/MS for determination of pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs), while root periderm extracts were analysed for naphthoquinone content spectrophotometrically and by LC-ESI/MS. Metabolic profiling of 12 possible PAs and PANOs showed their consistent appearance in all shoot extracts, with lepthamine N-oxide, echimidine-N oxide and echumine N-oxide predominant. The three major PANOs were significantly higher in northern sampling locations than those further south. Root extracts contained shikonin and several related naphthoquinones, as well as two of the major PANOs found in the leaves. Naphthoquinones were highest in the northwest corner of the sampled region. The patterns of abundance of secondary metabolites in E. plantagienum suggest that climate change might result in greater production of defensive compounds by E. plantagineum, making this weed increasingly toxic to livestock.     

DNA adduct formation from tobacco-specific N-nitrosamines

Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans.