5. Toxicity of pyrrolizidine alkaloids.
Alkaloids derived from 1-hydroxymethyl-l,2-dehydropyrrolizidine and esterified with at least one branched C5 carboxylic acid display a toxic, carcinogenic and mutagenic effect. Nearly 100 of the PAs known so far possess such a structure. Enhancement of the effect occurs if a further hydroxy group is introduced into the 7-position and also esterified, especially if the necinediol is esterified with a dicarboxylic acid to afford a macrocyclic compound. Alkaloid N-oxides, on principle, display the same toxicity. Since they are, in contrast to the bases, extremely water-soluble they are subject to different pharmacokinetics. After oral administration the alkaloids or their N-oxides are resorbed in the intestine, the N-oxides being previously reduced by the reductases of the bacterial flora.
Part of the alkaloids is cleaved into necines and necic acids by the nonspecific esterases of the blood. Necines are nontoxic and are excreted as conjugates via the kidneys and urine. The main proportion of the alkaloids on the other hand is transported into the liver where metabolic conversions induce reactions known as "intoxication reactions" <poisonings>.
In liver cells PAs give rise to the following changes:
- 10- to 30- fold enlargement of the liver cells (megalocytosis)
- enlargement of the liver nuclei with increasing nuclear chromatin
- disturbances of the liver cell metabolism with considerable functional losses
- occurrence of irregular mitoses with simultaneous inhibition of mitosis due to DNA blocking
- fatty degeneration.
These reactions are induced by a single intake of 10 to 20 mg of an alkaloid or alkaloid mixture.
If cytoclasis comprises larger regions of liver parenchyma death results.
Lower amounts of alkaloids (less than 10 µg) and longer disposition periods give rise to further damages:
- proliferation of biliary tract epithelials
- inflammatory infiltrates
- congestive and centrilobular necrosis of Venae hepaticae (VOD)
- generation of carcinomas.
This necrosis has been introduced in the medical literature as Venoocclusive Disease (VOD) and is regarded as specific PA intoxication <poisoning> (seneciosis). It is practically identical with the clinical picture of the Budd-Chiari syndrome. The clinical symptoms usually occur suddenly and comprise:
- colicky pains in epigastrium
- vomiting and diarrhea
- ascites formation within several days
- enlargement and induration of the liver within a few weeks.
In serious cases the following symptoms are additionally observed:
- vasomotoric states of collapse
- bleeding diarrhea.
The seriousness of the intoxication <poisoning> is not only dependent on the dosis administered but also on endogenic (age, sex) and exogenic (alcohol, drug, living conditions) factors. Children are obviously more sensitive to PA intoxications <poisonings> [238-279]. The Kwashiorkhor disease frequently observed with children in Central Africa is also related to a damage of liver cells by PAs, the liver having lost its ability to synthesize endogenic proteins [280, 281].
Besides these effects on the liver, severe toxic pulmonary damages with pulmonary-arterial hypertension and subsequent right ventricular failure were observed. When the metabolities can no longer be trapped in the liver cells they are transported via the blood into the pulmonary arterioles where they cause damage to the endothelial vessels. In the capillaries the latter are stimulated to proliferation causing a mediahypertrophy in the arterioles and thus a pressure increase in the pulmonary circulation and acute right ventricular failure similar to the classical cor pulmonale [282-286].
The toxicity of several PAs could be directly corroborated by means of the "yeast test" performed with Saccaromyces cerevisiae [287, 288].
A subtoxic intake (less than 1 mg) of PAs over longer periods resulted in the following gradually occurring changes of organs:
- fatty degeneration
- proliferation of the biliary tract epithelials
- liver cirrhosis
- nodular hyperplasia
- adenomas or carcinomas.
The frequent occurrence of primary liver tumors in the natives of Central Africa and South Africa is ascribed to the consumption of traditional medicinal plants of the genera Crotalaria, Cynoglossum, Heliotropium and Senecio [289-292]. In numerous experiments on animals with plants, with the extracts of the latter or with the alkaloids occurring in the aforementioned plant genera or in their species the clinical picture could be confirmed [293-311].
The mutagenicity of PAs has already been extensively studied. In this review article only those studies referring to the medical plants and the alkaloids contained in the latter are described. The plant extracts or the alkaloid mixtures of Senecio jacobaea, Senecio nemorensis ssp. fuchsii, Senecio fuchsii, and Symphytum officinale investigated in different test systems all exhibited a mutagenic effect [131, 132, 194, 203, 204, 312-314]. The alkaloids having a relation to the discussed medicinal plants also possess mutagenic properties. These alkaloids belong to the plant genera Senecio and Petasites (senecionine, integerrimine, retrosine and N-oxides, seneciphylline, jacobine, senkirkine), Eupatorium (supinine) and Symphytum (intermedine, lycopsamine, the N-acetyl derivatives of these, symphytine, echimidine). The following test systems were used: Escherichia coli, Salmonella typhimurium (Ames), Aspergillus nidulans, Vicia faba, Allium cepa, Drosophila melanogaster, leucocytes from marsupials, hepatic cells from rats and mice, liver cells from Chinese hamsters, mice and cattle as well as human lymphocytes. However, the various test systems used afforded different results. Although it was found that the studied alkaloids exhibit mutagenic properties the potency of the mutagenicity of the individual alkaloids could not be ascertained [315-339]. This was only realized by an extensive investigation of Frei et al. who made use of the wing spot test of Drosophila melanogaster . 16 alkaloids were tested under the same conditions, thus allowing comparative studies and even a quantitative assessment of the genotoxicity to be made for the first time. The following order of decreasing mutagenicity of the alkaloids was established:
senkirkine > monocrotaline > seneciphylline > senecionine > 7-acetyl intermedine > heliotrine > retrorsine > 7-acetyllycopsamine > symphytine > jacoline > symlandine > intermedine > indicine > lycopsamine > indicin N-oxide > supinine.
The series starts with the alkaloid with the highest mutagenicity, the senkirkine. The activity decreases via three powers of ten up to supinine that displays no activity.
Hence, the macrocyclic alkaloids senkirkine and senecionine possess the highest activity. Retrorsine which may be regarded as 12-hydroxymethyl derivative of senecionine exhibits a fivehold weaker mutagenicity. Jacoline which may also be considered as a hydroxy derivative of senecionine likewise exhibits a weak effect. Obviously, increasing hydroxylation of necic acids decreases the mutagenic effect. Among the open-chain diesters 7-acetylintermedine and 7-acetyllycopsamine display a five to ten times weaker activity than the macrocyclic compounds, while the acetyl esters possess a two to three times higher activity than e.g. the alkaloids symphytine and symlandine. The antitumor therapeutic agent indicine N-oxide exhibits only a very weak mutagenic effect. Obviously, also in this case the general principle applies that the mutagenic activity decreases with increasing hydroxylation. By means of this very extensive study the mutagenicity of other alkaloids, which have not been investigated so far, may be assessed.
<numbers in (bold) refer to Fig. 8.>
The teratogenic effect of PAs was demonstrated by a single intraperitoneal injection of heliotrine (24) into pregnant rats. At concentrations of 50 to 200 mg alkaloid/kg body weight teratogenic changes were observed. Doses above 200 mg/kg resulted in uterine death and degradation of the fetuses . Similar results were obtained with heliotrine and its metabolite dehydroheliotrine, the latter exhibiting however a teratogenicity which 2.5 times as high as that of the initial alkaloid . The teratogenic properties of heliotrine were also demonstrated by experiments on larvae of fruit flies, Drosophila melanogaster .
5.5.1. Metabolism of 1,2-unsaturated pyrrolizidine alkaloids
Metabolic reactions occur in the liver. A portion of the alkaloids, which, as is well-known, are esters are hydrolyzed by nonspecific esterases. The produced necines are supposed to be excreted renally. Alkaloids of this kind are relatively nontoxic because the necines do not produce toxic metabolites [344-346]. However, if the carboxylic acids contain branched chains hydrolysis is strongly impaired by sterical hindrance. If these difficultly hydrolyzable alkaloids contain as a basic constituent a necine of the supinidine, heliotridine, retronecine, or crotanecine type they are converted into the corresponding N-oxides by microsomal oxygenases. These N-oxides undergo rearrangement and elimination of water to afford the corresponding dehydropyrrolizidine alkaloids. Moreover, hydroxylation of the carbon atoms adjacent to the nitrogen atom (C-3 and C-8) is also supposed to occur, leading to the very unstable 3- and 4-hydroxypyrrolizidine alkaloids. Elimination of water from these alkaloids followed by rearrangement yields the corresponding dehydropyrrolizidine alkaloids [347-3531. As shown in Fig. 9 these alkaloids possess an allyl structure causing an increase in reactivity.
Also alkaloids of the otonecine type differing from the other PAs by a methyl group at the nitrogen atom and a "quasi" ketonic function at C-8 were degraded similarly. After cationic elimination of the methyl group the nor-N-otonecine alkaloids rearranges to the 8-hydroxy derivative and the latter to the corresponding dehydropyrrolizidine alkaloid.
Dehydropyrrolizidines representing (A)pyrrolidine (B)pyrrole derivatives are highly reactive and are the virtually active species of the PAs.
In the presence of nucleophilic systems (Nu-) the diesters undergo either a simple or a double bimolecular nucleophilic substitution (SN2 reaction). In parallel occurring reactions a hydrolysis leading to the formation of dehydronecines (dehydroretronecine, dehydroheliotridine) may also take place. These compounds are similarly reactive, i.e. their toxicity and carcinogenicity resemble those of dehydroalkaloids.
Scheme 4 delineates the reaction sequence of a twofold bimolecular nueleophilic substitution [354-365].
According to Mattocks et al. the reaction of the macrocyclic alkaloids starts in the 7-position . MNDO (Modified Neglect of Differential Overlaps) calculations, a standard method for determining charges and energy contributions on van-der-Waals-surfaces, especially in the case of heteroatom-containing organic molecules, reveal that locations of scission contain higher partial charges than the other molecular regions. In the case of dehydrosenecionine these locations are at -0.341 for the 9-H2CO and at -0.328 for the 7-HCO group .
Hydroxy, mercapto or amino groups of enzymes, globulins, hemoglobin but also of purine and pyrimidine bases or their nucleosides may function as nucleophiles. Hence, DNS and/or RNS may also undergo alkylations. Already simple alkylation affords an adduct that causes a lasting change of a DNS and/or RNS strand. A double alkylation may give rise to irreversible cross linking of both strands as shown in Fig. 11. If no repair process occurs a carcinogenic reaction may be definitly predicted [368-387].
Recently, a further metabolic reaction has been assumed. According to Segall et al. the alkaloid, e.g. senecionine, is supposed to be degraded to (E)-4-hydroxy-2-hexenal as demonstrated by the reaction Scheme 5 .
Aldehydes with a double bond in the 2,3-position display liver toxic properties.
5.5.2. Metabolism of 1,2-saturated pyrrolizidine alkaloids
Saturated pyrrolizidines and their necines are nontoxic. According to Mattocks et al. the alkaloids platyphylline and rosmarine undergo enzymatic hydrolytic cleavage at C-7 with simultaneous formation of a pyrrole unit of ring B as shown in Scheme 6.
In contrast to dehydro(A)pyrroles the nitrogen atom of which exists in a very unstable conjugated state and may thus readily react with nucleophiles, 1,2-saturated pyrrolizidines are considerably more stable and not susceptible to reactions with nucleophiles [389, 390].
Medicinal plants in Europe containing pyrrolizidine alkaloids was written by Prof. Dr. E. Röder and published in the journal "Pharmazie" 50 (1995), pages 83-98.