Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.

Quantification of N-acetylaspartylglutamate, an N-terminal blocked dipeptide neurotransmitter candidate, in brain slice superfusates by gas chromatography-mass spectrometry.

N-Acetylaspartylglutamate is a highly promising neurotransmitter candidate. A method for its quantification has been developed that allows to investigate its stimulation-induced release from brain slices. The method is based on ion-exchange prepurification, derivatization with HCl in methanol, separation by capillary gas chromatography and quantification by ammonia chemical ionization mass spectrometry with selected-ion monitoring. Deuterium-labelled N-acetylaspartylglutamate is used as internal standard. The method has been validated, also with respect to possible interfering compounds. A limit of quantification in the low pmol to high fmol range has been achieved, which is clearly sufficient for the intended purpose. A detailed analytical procedure is given, and alternatives for some of the different steps are discussed. Derivatization with diazomethane instead of methanolic HCl turned out to be impracticable. The method may well be applicable to certain other N-terminal blocked di- and tripeptides and to acylated amino acids.

Pyrrolizidine alkaloids from Symphytum officinale L. and their percutaneous absorption in rats.

An analysis of a commercial sample of Symphyti radix originating from Poland with a total alkaloid content of 0.07% revealed the presence of 7 pyrrolizidine alkaloid-N-oxides: 7-acetyl intermedine, 7-acetyl lycopsamine as the main constituents and lycopsamine, intermedine, symphytine and traces of 2 further not yet identified alkaloids. The percutaneous absorption of these alkaloids was investigated in rats, using a crude alcoholic extract of the plant corresponding to a dose of 194 mg alkaloid-N-oxides/kg b.wt. The excretion of N-oxides in the urine during 2 days was in the range of 0.1-0.4% of the dose. The dermally absorbed N-oxides are not or only to a small extent converted to the free alkaloids in the organism. The oral application led to a 20-50 times higher excretion of N-oxides and free alkaloids in the urine.