Keywords

1 Introduction

The term “pyrrolizidine alkaloids” (PAs) is used for all ester compounds of the hydroxy and/or dihydroxy and/or hydroxymethyl derivatives from pyrrolizidine (hexahydro-1H-pyrrolizine) or from 1,2-dehydro-pyrrolizidine (2,3,5,7a-tetrahydro-1H-pyrrolizine).

PAs occur in 13 plant families and in about 5% of all flowering plants. Toxic PAs can be found in six plant families: Apocynaceae, Boraginaceae, Asteraceae (Compositae), Leguminosae (Fabaceae), Ranunculaceae, and Scrophulariaceae. These PAs are hepatotoxic, genotoxic, teratogenic, carcinogenic, and pneumotoxic.

Toxic PAs are only those derived from the unsaturated moiety 1,2-dehydropyrrolizidine and are mainly esters of the aminoalcohols (= necines) retronecine, heliotridine, or the untypical aminoalcohol (no bicyclic five-membered system, but an eight-membered monocycle) otonecine. Some few toxic PAs are esters from crotanecine. Of minor toxicity are the esters of the 1-hydroxymethyl-1,2-dehydropyrrolizidine supinidine.

A very large range of pyrrolizidine alkaloids can theoretically be obtained by combining the known necines and the esterifying acids (= necic acids). So far, more than 500 alkaloids have been found and their structures determined. With the exception of the approximately 35 otonecine alkaloids that cannot form N-oxides, if the N-oxides of these alkaloids are taken into consideration, more than 900 structures are known.

The first intoxication was reported by Gilruth who found that a chronic liver disease in livestock was caused by Senecio jacobaea [1, 2]. It was further shown that other Senecio ssp. as well as Crotalaria ssp. led to the same disease [3]. Already in 1920, it was proven that a widespread chronic liver disease in humans was caused by grain contaminated with seeds of Senecio ssp. [4, 5]. Several severe intoxications, all caused by the contamination of food (mainly bread), were reported: In the 1950s, severe intoxications in the former USSR were found to be caused by seeds and dust of Heliotropium lasiocarpum which contaminated grain [6, 7]; similarly, the same species was the reason for the intoxication of 4,000 people in Tadjikistan [8, 9]. The severest incident was observed in the 1970s in Afghanistan where about 8,000 people were affected from a wheat contamination by seeds of Heliotropium popovii, subsp. gillianum; more than 3,000 died [10, 11].

It can be assumed that seeds and dust from PA-containing plants are a major source for a human PA exposure [12, 13]. In industrialized countries, grain cleaning methods reduce the PA contamination under a level where acute intoxications can occur, but the dust components are still remaining and there are indications that diseases such as cirrhosis, cancer, and pulmonary arterial hypertension are due to a long-time exposure of low doses of PAs [14].

Besides this, medicinal plants are a further source of a PA intoxication [15–18]. Especially herbal preparations (so-called bush-teas) were found to be the reason for different liver diseases observed in Jamaica and the West Indies as well as in Africa in the 1950s [19–21].

PA intoxications by herbal preparations are mainly observed in developing countries where the use of traditional medicines is common; however, in the last three decades also, in industrial countries like the USA, the UK, Switzerland, Austria, and Germany PA intoxications were reported due to the increased and uncontrolled use of herbal medications. In the 1980s, hand in hand with the so-called green wave, several practitioners claimed that herbal medicine would show only benefits without undesirable side effects which led to several fatal intoxications, mainly in infants who show a higher susceptibility to PA than adults.

Other foods can also be contaminated with PAs and are, therefore, a potential hazard for humans. Milk was shown to be a source for a PA intoxication in a case where the milk-producing animals had access to PA containing feed (hay, silage) [22–30]. Human milk from women exposed to pyrrolizidine alkaloids has caused veno-occlusive disease in neonates and infants [31]. Honey has become of increased importance as it could be shown that, in commercial products, levels of PAs were found which exceeded tolerable values. Here, pollen seems to be the pathway of contamination [31–37].

Recently, in Germany it was found that salads (especially ready-packed rocket salads (Eruca sativa), sold in supermarkets) and salad mixtures can be contaminated with PA containing plants, mainly Senecio vulgaris, a typical weed of field crops [38].

1,2-dehydropyrrolizidine esters and their N-oxides are carcinogenic, mutagenic, genotoxic, fetototoxic, and teratogenic to varying degrees. The toxicity level of each single PA is dependent on its chemical structure and also physical properties such as lipophilicity, hydrophilicity, and pharmokinetics. PAs prior to metabolic activation show a more or less low acute toxicity, but in vivo they undergo a metabolic toxification process in the liver, which is, as a result, the first target organ for the toxicity. This toxication process is well investigated.

PAs are considered to be toxic contaminants and have no well-recognized uses on their own. In the 1970s, the PA indicine-N-oxide was found to show antitumor activities, but on account of severe toxic side effect (especially observed in children) its use was no longer justified [39–41].

1.1 Chemical Structures of PAs

PAs are esters and consist of two parts: the basic aminoalcohols (“necines”) and one or more acids (“necic acids”) that esterify the OH groups of the necines [42–44].

Necines consist of a five-membered bicyclic ring system with a bridgehead nitrogen and – at least – a hydroxymethyl group at position 1 (Fig. 13.1). They can occur as saturated systems or possess a double bond in position 1,2. Most of the PAs show a further OH group at position 7. Further hydroxylation can take place at positions 6 and 2; in the case of saturated PAs a few molecules show hydroxylation at position 1 and in one case the (+) isomer of isoretronecanol (R3 = ß-H; α-hydroxymethyl at position 1) is reported. Chiral carbons can be at positions 1 and 2 (saturated necines) and 6,7,8 (all necines). Mainly all PAs belong to the 8-α series (only two times the 8-ß position is reported). In nature, the PAs mainly occur in their N-oxide form (water-soluble; transport form).

Fig. 13.1
figure 00091

Structures of necines

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An untypical necine can be found in the otonecine PAs: This necine occurs not in form of a bicycle but as a 1-methylazocan-5-one. On account of the results by X-ray analysis, it could be found that there exists a transannular binding between carbonyl and the nitrogen leading to the same behavior as the typical bicycle PAs. All PAs showing a double bond in position 1,2 act as natural toxins.

Necic acids are structurally highly variable. Besides the simple acetic acid they consist mainly of 5–10 carbon atoms. They can form mono-, di-, or macrocyclic diesters and can show more or less branched carbon chains. The carbon atoms can bear different functional groups like hydroxyl, alkoxy, epoxy, and carboxyester groups. Double bonds at different positions are also possible. On account of this situation a theoretically high number of different unique structures are possible, including a large range of stereo isomers [42–44].

Necines with only one OH group can only form PA monoesters like supinine. If there exists a second OH group both can be found: mono as well as diesters as demonstrated for lycopsamine and its acetyl derivative or as in lasiocarpine. These examples demonstrate the variability of the stereochemistry (Fig. 13.2).

Fig. 13.2
figure 00092

Structures of PA mono- and diesters

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With dicarboxyclic acids, the double esterification leads to macrocyclic diesters which can occur as 11–13-membered ring systems. A typical example for an 11-membered PA is monocrotaline, the main PA occurring in crotalaria species, or crispatine. Most of the PAs show a 12-membered ring system; this can be found, for example, in senecionine or senkirkine, both contained in many senecio species. A typical 13-member ring is demonstrated in the PAs doronenine (also isolated from senecio) and in madurensine (from crotalaria). Very rare is the 14-member macrocycle: It is found in the PA parsonsine (from parsonsia-species); this structure is possible on account of a further ester group within the necic acid chain. It is interesting to mention that until now in all cases of macrocyclic diester PAs only the 7-ß-OH isomer (mainly retronecine) is described and not the α-isomer (heliotridine) (Fig. 13.3).

Fig. 13.3
figure 00093

Structures of macrocyclic PA

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Theoretically, an innumerable amount of different structures is possible. So far, more than 500 PAs have been isolated and their structures described.

1.2 Biosynthesis

The first experiments concerning the biosynthesis of PAs were done in the 1960s: [2-14C]ornithine, [1-14C]acetate, and [1-14C]propionate were fed to Crotalaria species [45]. Similarly, labelled ornithine was incorporated into the alkaloids from Senecio species [46, 47]. In all cases, ornithine was incorporated specifically into the basic moiety retronecine, whereas acetate and propionate could be found in the acidic parts of the alkaloids. Bale and Crout used a double isotope technique for a comparison between ornithine and arginine [48] showing that ornithine is the more efficient precursor than arginine. It could be found that ornithine and arginine were incorporated into retronecine via putrescine: Experiments gave evidence for the formation of putrescine from argine in Heliotropium species, whereas in Crotalaria and Senecio species, the main pathway to putrescine is from ornithine [49, 50]. Further experiments came to the conclusion that retronecine is derived from two molecules of putrescine, which can be formed from ornithine or arginine [51, 52]. The use of 13C-15N double-labelled putrescine gave evidence for a further symmetrical intermediate which was then found to be homospermidine. This was proven by feeding experiments with labelled homospermidine which was incorporated into the retronecine moiety of the PAs from Senecio isatideus [53–55]. It could be shown that a PA-special enzyme – the homospermidine synthase (HSS) – is essentially involved in the biosynthesis of homospermidine [56–58]. This HSS is responsible for the transfer of the aminobutyl moiety of spermidine into putrescine leading to the symmetric triamine homospermidine; this reaction is NAD+ dependent. Via the 1-(4-aminobutyl)-3,4-dihydro-2H-pyrrolium salt the necines trachelanthamidine and further retronecine were generated [59].

Besides, it was shown that trachelanthamidine as well as retronecine were both efficient precursors for the necine otonecine, the base moiety of the PA emiline from Emilia flammea [60].

Trachelanthamidine as well as isoretronecanol can be generated from the imminium salt. There exists evidence that the saturated necine rosmarinecine is generated via isoretronecanol and that trachelanthamidine is the precursor for retronecine as well as heliotridine [61]. Isoretronecanol has been shown to be also the precursor of those PAs which have the untypical 8ß stereochemistry (instead of 8α) as found in the PAs from Cynoglossum australe [62] (Scheme 13.1).

Scheme 13.1
scheme 00091

Biosynthetic pathway of necines

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Whereas the biosynthesis of the necines (Scheme 13.1) seems to be clear and intensively investigated, fewer reports can be found for the necic acids. Still some aspects have to be investigated.

In 1966, labelled l-threonine and l-isoleucine were observed to be incorporated into the seneciphyllic acid [63]. Similarly, it was described that labelled DL-valine is a precursor of echimidinic acid [64]. The five-membered angeloylic acid is found often as a structure part in PAs. l-isoleucine was shown to be a precursor of the angelate component in PAs [65]. Crout et al. reported that in general C10 necic acids are derived from isoleucine [66, 67]. This was demonstrated in case of seneciphyllic acid and senecic acid, both forming 12-membered macrocyclic PAs. Monocrotalic acid, the acidic part of monocrotaline – a 11-membered macrocycle – was reported to be built from l-isoleucine and l-threonine [68]. Similar findings were reported for the acidic part in the PA strigosine [69] and for angeloylic and tigloylic acid [70]. In the case of the acidic part in the PA monocrotaline, it was shown that not acetate, mevalonate, or glutarate (as reported earlier) are involved but it is only formed via isoleucine [71]. Stereospecific aspects were also studied in the case of senecic and isatinecic acid [72, 73]. The biosynthesis of trichodesmic acid was studied and it was shown that one part of the C10 acid was formed by (2S)-isoleucine or its biosynthetic precursor (2S)-threonine and the other C5 unit from (2S)-leucine or (2S)-valine [74]. The complete labeling pattern of senecic acid (the acidic part in the PAs rosmarinine and senecionine) was studied by NMR experiments and it was stated that the biosynthesis of this acid is processed via two molecules of isoleucine (Scheme 13.2) [75].

Scheme 13.2
scheme 00092

Biosynthetic pathway of senecic acid

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1.2.1 Metabolic Toxication of PAs

PAs are ester alkaloids derived mainly from the necines retronecine and otonecine. They are carcinogenic, mutagenic, genotoxic, fetotoxic, and teratogenic.

PAs themselves show a more or less low acute toxicity but in vivo they undergo a three-step metabolic toxication process in the liver, which is, as a result, the first target organ for the toxicity.

This toxication process is well investigated [12, 75–81].

After oral uptake and absorption of the PAs (Scheme 13.3: Ia and Ib), a hydroxyl-group is introduced adjacent to the nitrogen atom in the necine (position 3 or 8) by the cytochrome P-450 monoxogenase enzyme complex in the liver (Scheme 13.3: IIa and IIb).

Scheme 13.3
scheme 00093

Metabolism of PAs to Pyrrols

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These hydroxyl PAs (OHPAs) are unstable and undergo a rapid dehydration to the dehydropyrrolizidine alkaloids (DHPAlk; Scheme 13.3: III). This dehydration results in a second double bond in the necine followed by spontaneous rearrangement to an aromatic pyrrole system III.

PAs occur mainly as their N-oxides in the plants and these cannot be directly converted to the OHPA, but on oral ingestion they are reduced by the gut enzymes or the liver microsomes and NADH or NADPH to the free bases and therefore they show equal toxicity to that of the free bases [82–87].

Otonecine-type PAs (Scheme 13.3: Ib) are metabolized to the OHPAs [80, 88, 89]. These otonecine-PAs possess a methyl function at the nitrogen and a quasi keto function at the bridge carbon 8. After hydroxylation of the N-methyl group it is lost as formaldehyde leaving a NH-function which undergoes condensation with the C8 keto group to produce product IIb (Scheme 13.3) which spontaneously dehydrates to the DHPAlk III.

The metabolites III are able to generate stabilized carbonium ions (Scheme 13.4: IV and VI) by loss of hydroxy groups or ester functions as hydroxyl or acid anions. These carbonium ions can react rapidly with nucleophiles (Scheme 13.4: VII).

Scheme 13.4
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Metabolism of the dehydropyrrolizidine alkaloids

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In the case of necine-diesters (as shown in Ia and Ib, Scheme 13.3), typical of Senecio species, the formation of the reactive carbonium ions is facilitated because the necic acid groups provide good leaving groups that facilitate rapid formation of the carbonium ions IV and VI in high yield. Where one of the hydroxy groups at C7 or C1 of the necine is not esterified, formation of the carbonium ions is not so spontaneous. In these cases, the carbonium ions are most readily formed after protonation of the hydroxyls and loss of H2O [90].

The metabolites IV and VI react rapidly with nucleophilic groups of proteins and the amino groups of the bases in nucleosides like DNA and RNA which leads to abnormal functions showing finally the veno-occlusive disease (VOD) in which the veins are narrowed. Typical macrocyclic diester PAs (like senecionine, seneciphylline, retrorsine, and senkirkine which are PAs commonly found in Senecio species) have been shown to produce liver damage due to cross-linking of DNA [84, 91–99]. In case of PA monoesters (e.g., derived from the necine supinidine which lacks a C7 hydroxyl, Fig. 13.2), cross-linking is not possible and they show a lower toxic potential. It has also been shown that the nucleophilic activity at C7 is higher than at C9 resulting in the primary nucleophilic attack at C7 followed by an attack at C9 (Scheme 13.4) [43].

As shown, the DHPAlks can also react with SH groups found in more soluble components like glutathione and cysteine (Scheme 13.4: VIII). High levels of glutathione and cysteine therefore reduce the toxic potential of PAs [88, 100–102].

Furthermore, hydrolysis can take place where the DHPAlks (Scheme 13.4) yield dehydronecine alcohols (DHNecs) (Scheme 13.4: IX) which are more water soluble and less reactive like the DHPAlks but still display a moderate level of alkylating activity [103, 104]. This higher water solubility and lower reactivity can lead to the escape from the liver tissue and subsequent reaction in other organs [12, 105, 106]. DHNecs like dehydroretronecine and dehydroheliotridine have also been shown to produce rhabdomyosarcoma, skin, liver, and lung tumors [24, 107–110].

1.2.2 Detoxication of PA

As well as the metabolic activation, detoxication of PAs also occurs in vivo: Hydrolysis of the ester bonds in PAs from type Ia or Ib by esterases leads to necic acids and to the free necines. Both are nontoxic products and – on account of their higher water solubility – can be renally excreted. The rate of hydrolysis is dependent on the level of steric hindrance of the ester linkages; and it has been shown that the more highly branched necic acids are more resistant to hydrolysis [43, 111]. This means that macrocyclic diesters with more complex acid moieties are more hazardous on account of their lower rate of hydrolytic detoxication.

The N-oxides of PAs (the form occurring most commonly in plant sources) are highly water soluble and can therefore be renally excreted. Besides their natural occurrence, N-oxidation of PAs also takes place in the liver and can be seen as a detoxication process (Scheme 13.3) [76, 78, 82, 112, 113]. However, it has been shown that the N-oxides – besides excretion – can be converted by dehydration or by acetylation followed by elimination of acetic acid to the DHPAlk (Scheme 13.3: III) [43, 90].

In conclusion, it can be stated that the toxicity level of different PAs in nonruminants is dependent on the following three aspects:

  • The efficiency of metabolic activation to form the key intermediate III (Scheme 13.3)

  • The efficiency of ester hydrolysis to form nontoxic and water-soluble necines and necic acids

  • The efficiency of N-oxidation and excretion via urine

1.2.3 Structure and Toxicity

As mentioned, the key fragment didehydropyrrolizidine (e.g., III) is also generated from pyrrolizidine alkaloids of the otonecine type (e.g., Ib, Scheme 13.3).

These otonecine derivatives do not show a C8-N bond but possess a keto function at C8 and a methyl group at the nitrogen atom. Surprisingly, these seco alkaloids are of identical toxicity as the PA of type Ia (1,2-dehydro-retronecine and heliotridine esters).

On the other hand, for energetic reasons, these seco compounds should occur in a different necine conformation than type Ia PA: The missing C8-N bond should lead to a stable eight-membered macrocycle which generally prevents the metabolization via an intermediate to IIb (Scheme 13.4).

Molecular modeling experiments prove this assumption and show the energy minimized structure as depicted in Fig. 13.3 (energy minimization: Chem 3D ultra; V. 10.0; Cambridge Soft).

Interpretation of the X-ray structure analysis data helps to explain the identical toxicity of PA of type Ia and Ib: In all nine otonecine-type alkaloids measured to date, a necine conformation was found which is identical to those found in those having the C8-N bond (type Ia).

Both the distances between C8 and N are similar and equal values can be found for the plane angles built between plane C1-C3-N-C8 and plane C7-C5-N-C8 (∼125°). This indicates that the seco 1,2-dehydroesters do not exist in the optimal, energy minimized form (Ib, Fig. 13.4) but are of an equal conformation to PA of type Ia, which finally enables the metabolization as shown in Scheme 13.3.

Fig. 13.4
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X-rays of Ia and Ib

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Furthermore, X-ray data show that the dedihydro metabolites (e.g., III, Scheme 13.3) derived from 1,2-dehydrodiester have a high toxic potential compared with the low or missing toxicity of the monoesters of retronecine or heliotridine. As already mentioned, a possible detoxification mechanism is the hydrolysis of the ester bindings by esterases and the subsequent building of dehydronecine (IX, Scheme 13.4) which – due to their higher water solubility – can be easily excreted renally.

On the other hand, the metabolization to C7 as well as to C9 carbonium ions (especially the speed rate of this metabolization) and the subsequent reaction with nucleophiles are seen as a key step concerning the level of toxicity. The concrete binding situation concerning the ester function can be found analyzing the X-ray data. Interpretation of the bond lengths shown in Fig. 13.5 leads to the following results:

Fig. 13.5
figure 00095

Bonds in PAs

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In pyrrolizidine alkaloids of type Ia and Ib (1,2-dehydropyrrolizidine diesters) the C-O bonds A and A´ occur in a normal range of 1.45 Å; the following C-O bonds B and B´ are considerably shortened, whereas the keto functions C and C´ show a moderate shortening. The C-C bonds D and D´ show – similar to A and A´ – normal values of about 1.54 Å.

In contrast to these findings, the data for monoesters with retronecine or heliotridine give evidence of a different situation: Here, the A and A´ (C9 as well as C7) bonds are elongated and the corresponding keto bonds C or C´ are of nearly ideal length (1.21 Å).

These results show that in 1,2-dehydro diesters, the ester functions are a conjugated system leading to the conclusion that the bonds C9-O and C7-O (= A and A´) are the target breaking points within the molecule, which makes possible a quick and easy metabolization and further reaction with nucleophiles as described in Scheme 13.4.

Contrary to that, in PA of 1,2-dehydro monoesters, more stable ester bonds are found (no conjugation), which leads to the fact that the building of the didehydrometabolites is more difficult and time consuming. In this case, a hydrolysis by esterases can take place in higher amounts what leads to the detoxification via dehydronecines, which explains the missing or low toxicity of 1,2-dehydro monoester.

1.2.4 Metabolism of 1,2-Saturated Pyrrolizidine Alkaloids

Metabolism of saturated pyrrolizidine alkaloids (necic acid esters of platynecine, hastanecine, rosmarinecine and isoretronecanol (Fig. 13.1)) by mammals has not yet been extensively studied, in part because the saturated ester alkaloids and their necines do not display mammalian toxicity.

While 1,2-dehydropyrrolizidine alkaloids are metabolized by liver P-450 isozymes into hazardous dihydropyrrolizines (e.g., III) with a pyrrolic A ring, saturated pyrrolizidine alkaloids produce nontoxic, metabolites [79, 111]. The saturated alkaloids platyphylline and rosmarinine (Fig. 13.6), for example, are converted by liver microsomes into pyrrolic metabolites with an aromatic B-ring [79]. These are devoid of biological alkylating properties and are nontoxic.

Fig. 13.6
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Metabolism of saturated PAs

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2 PA Toxicity in Humans

PA poisoning of humans can be described by three dose-related levels: acute, subacute, and chronic. These levels can be progressive resulting in irreversible chronic toxic effects [12, 99, 106, 110, 114–116].

On account of the low toxicity of the PAs themselves, acute poisoning has been reported only in very rare cases; it occurs only in infants and neonates due to their higher susceptibility for a PA poisoning. It is characterized by hemorrhagic necrosis, hepatomegaly, and ascites; death is caused by liver failure [12, 106, 110, 115].

Subacute levels are characterized by hepatomegaly and recurrent ascites; endothelial proliferation and medial hypertrophy leading to an occlusion of hepatic veins, resulting in the so-called veno-occlusive disease (VOD) which can be seen as a characteristic histological sign for PA poisoning [12, 99, 106, 110, 115]. The VOD causes centrilobular congestion, necrosis, fibrosis, and liver cirrhosis, the end-stage of chronic PA intoxication.

As well as the liver VOD, other organs can be affected by PAs. It has been shown that the pyrrolic metabolites (DHPAlks and DHNecs) can escape from the liver into pulmonary arterioles where they can produce damages similar to the VOD changes in the liver [115]. It could be shown that from 62 tested PAs all can produce (dose-dependent) lung lesions [111] and it is speculated that pulmonary damage results from long-term and low-level exposure to PAs [12, 115].

PA intoxication in humans is not only related to the amount and the duration of the exposure but also to age and gender: Males react more sensitive than females, and fetuses and children (especially neonates or infants) show the highest sensitivity to PA poisoning: In 2003, it was shown that the daily uptake of ∼ 7 μg PA (from a herbal spice containing comfrey) during pregnancy did not show a toxic effect in the mother's liver but damaged the fetal liver in such a way that the newborn child died after 2 days [117].

It has also been observed that cofactors can exacerbate the PA poisoning: liver damaging agents, bacterial, or viral infections but also medical drugs like barbiturates or metals like copper or mycotoxins like aflatoxins can increase the severity and likelihood of PA liver damage [42, 118–122].

There is a large number of reports in the literature about different liver diseases (mainly VOD) possibly connected with PA poisoning. But in most cases, the connection could not be proven because the outbreak of the liver disease and a possible ingestion of PA-containing material were often separated by a long time period.

3 Conclusion

PAs are widespread in the plant kingdom. They occur in a great variety of different structures. They show no physiological and/or pharmacological properties, but they are of importance on account of possible severe side effects. These side effects are dependent on their chemical structures and range from low or missing toxicity up to strong severe and hazardous problems. PAs themselves are more or less nontoxic but undergo a metabolic toxication process. As a result cancerogenic, mutagenic, teratogenic, and fetotoxic effects can be observed. Many episodes are reported describing intoxications in humans, sometimes even epidemic. These cases were caused mainly by contaminations of food and feed by PA-containing plant material or by the uncontrolled use of herbal medical preparations. Therefore, to avoid those contaminations, special efforts should be made on a global scale to improve food control management.