isatidine and retrorsine

Plants produce an extremely diverse array of metabolites that mediate many aspects of plant-environment interactions. In the context of plant-herbivore interactions, it is as yet poorly understood how natural backgrounds shape the bioactivity of individual metabolites. We tested the effects of a methanol extract of Jacobaea plants and five fractions derived from this extract, on survival of western flower thrips (WFT). When added to an artificial diet, the five fractions all resulted in a higher WFT survival rate than the methanol extract. In addition, their expected combined effect on survival, assuming no interaction between them, was lower than that of the methanol extract. The bioactivity was restored when the fractions were combined again in their original proportion. These results strongly suggest synergistic interactions among the fractions on WFT survival rates. We then tested the effects of two pyrrolizidine alkaloids (PAs), free base retrorsine and retrorsine N-oxide, alone and in combination with the five shoot fractions on WFT survival. The magnitude of the effects of the two PAs depended on the fraction to which they were added. In general, free base retrorsine was more potent than retrorsine N-oxide, but this was contingent on the fraction to which these compounds were added. Our results support the commonly held, though seldom tested, notion that the efficacy of plant metabolites with respect to plant defence is dependent on their phytochemical background. It also shows that the assessment of bioactivity cannot be decoupled from the natural chemical background in which these metabolites occur.

Topics: Animals; Asteraceae; Chromatography, High Pressure Liquid; Flowers; Phytochemicals; Plant Extracts; Plant Shoots; Pyrrolizidine Alkaloids; Tandem Mass Spectrometry; Thysanoptera

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. The high toxicity of many pyrrolizidine alkaloids has caused considerable loss of free-ranging livestock due to liver and pulmonary lesions. Chronic exposure of toxic pyrrolizidine alkaloids to laboratory animals induces cancer. This investigation studies the metabolic activation of retrorsine, a representative naturally occurring tumorigenic pyrrolizidine alkaloid, and shows that a genotoxic mechanism is correlated to the tumorigenicity of retrorsine. Metabolism of retrorsine by liver microsomes of F344 female rats produced two metabolites, 6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), at a rate of 4.8 +/- 0.1 nmol/mg/min, and retrorsine-N-oxide, at a rate of 17.6 +/- 0.5 nmol/mg/min. Metabolism was enhanced 1.7-fold by using liver microsomes prepared from dexamethasone-treated rats. DHP formation was inhibited 77% and retrorsine N-oxide formation was inhibited 29% by troleandomycin, a P450 3A enzyme inhibitor. Metabolism of retrorsine with lung, kidney, and spleen microsomes from dexamethasone-treated rats also generated DHP and the N-oxide derivative. When rat liver microsomal metabolism of retrorsine occurred in the presence of calf thymus DNA, a set of DHP-derived DNA adducts was formed; these adducts were detected and quantified by using a previously developed 32P-postlabeling/HPLC method. These same DNA adducts were also found in liver DNA of rats gavaged with retrorsine. Since DHP-derived DNA adducts are suggested to be potential biomarkers of riddelliine-induced tumorigenicity, our results indicate that (i) similar to the metabolic activation of riddelliine, the mechanism of retrorsine-induced carcinogenicity in rats is also through a genotoxic mechanism involving DHP; and (ii) the set of DHP-derived DNA adducts found in liver DNA of rats gavaged with retrorsine or riddelliine can serve as biomarkers for the tumorigenicity induced by retronecine-type pyrrolizidine alkaloids.

Topics: Animals; Biotransformation; Cells, Cultured; DNA; DNA Adducts; Female; Liver; Microsomes; Monocrotaline; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344

Retronecine-based pyrrolizidine alkaloids, such as riddelliine, retrorsine, and monocrotaline, are toxic to domestic livestock and carcinogenic to laboratory rodents. Previous in vitro metabolism studies showed that (+/-)6,7-dihydro-7-hydroxy-1-(hydroxymethyl)-5H-pyrrolizine (DHP) and pyrrolizidine alkaloid N-oxides were the major metabolites of these compounds. DHP is the reactive metabolite of pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides are generally regarded as detoxification products. However, a previous study of rat liver microsomal metabolism of riddelliine N-oxide demonstrated that DHP and its parent compound, riddelliine, were generated as the major metabolites of riddelliine N-oxide. In this study the metabolic activation of the three retronecine-based pyrrolizidine alkaloid N-oxides by human liver microsomes is investigated under oxidative and hypoxic conditions. Results shows that both the DHP and the corresponding parent pyrrolizidine alkaloids are the major metabolites of the human liver microsomal metabolism of pyrrolizidine alkaloid N-oxides. Under oxidative conditions, reduction of the N-oxide to pyrrolizidine alkaloid is inhibited and while under hypoxic conditions, DHP formation is dramatically decreased. The oxidative and reductive products generated from the metabolism of pyrrolizidine alkaloid N-oxides are substrate-, enzyme- and time-dependent. In the presence of troleandomycin, a microsomal CYP3A inhibitor, DHP formation is inhibited by more than 70%, while the N-oxide reduction was not affected. The level of microsomal enzyme activity in human liver is comparable with rats. The rate of in vitro metabolism by either human and rat liver microsomes follows the order of riddelliine > or = retrorsine > monocrotaline, and DHP-derived DNA adducts are detected and quantified by 32P-postlabeling/HPLC analysis. Similar DHP-derived DNA adducts are found in liver DNA of F344 rats gavaged with the pyrrolizidine alkaloid N-oxides (1.0 mg/kg). The levels of in vivo DHP-DNA adduct formation is correlated with the level of in vitro DHP formation. Our results indicate that pyrrolizidine alkaloid N-oxides may be hepatocarcinogenic to rats through a genotoxic mechanism via the conversion of the N-oxides to their corresponding parent pyrrolizidine alkaloids, and these results may be relevant to humans.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Biotransformation; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; DNA Adducts; Female; Humans; Microsomes, Liver; Monocrotaline; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344

We investigated the growth-reducing effects of pyrrolizidine alkaloids (PAs) from Senecio jacobaea on nine plant-associated fungi (five strains of Fusarium oxysporum, two of F. sambucinum, and two of Trichoderma sp). Fungal growth was monitored on water agar media containing different concentrations of monocrotaline, retrorsine, or a purified extract of PAs from S. jacobaea. The growth rate of six strains was inhibited by PAs at the highest test concentration (3.33 mM), with the magnitude of the inhibition (7-35%) being dependent upon the specific fungus-PA interaction. In general, the PA extract caused the largest inhibition. However, the fungi isolated from S. jacobaea were positively affected by the PA extract (7-9%). Retrorsine N oxide was as effective as retrorsine in its inhibition of mycelium growth.

Topics: Analysis of Variance; Crotalaria; Fungi; Monocrotaline; Mycelium; Plants, Toxic; Pyrrolizidine Alkaloids; Senecio; Species Specificity

The in vivo metabolism and excretion of the urinary metabolites from the pyrrolizidine alkaloids (PAs), retrorsine (RET) and retrorsine-N-oxide (RET-NO) have been studied in rats. Isatinecic acid (INA), pyrrolic metabolites, N-oxides and retronecine accounted for 31.0, 10.3, 10.8 and 0.39% of the administered RET. Predosing rats with triorthocresyl phosphate (TOCP), had no effect on the excretion of pyrrolic metabolites and INA. Phenobarbital (PB) increased the excretion of both pyrrolic metabolites and INA with a corresponding decrease in the excretion of RET and N-oxides; the retronecine levels remained unaltered. When RET-NO was administered i.p., the urinary levels of pyrrolic metabolites, INA and RET were decreased relative to those treated with RET. The p.o. administration of RET-NO produced significantly higher levels of pyrrolic metabolites, INA and RET. These results suggest that esterase hydrolysis plays a minor role in the formation of INA and that a common metabolic pathway may exist between pyrrolic metabolites and INA formation.

Topics: Animals; Chromatography, Gas; Chromatography, High Pressure Liquid; Dicarboxylic Acids; Male; Phenobarbital; Plants, Toxic; Pyrroles; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Senecio; Tritolyl Phosphates

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.

Topics: Animals; Biotransformation; Cell Line; Chromosome Aberrations; Liver; Liver Extracts; Monocrotaline; Mutagenicity Tests; Mutagens; Pyrrolizidine Alkaloids; Rats; Rats, Inbred Strains

Topics: Alkaloids; Animals; Liver Neoplasms; Neoplasms, Experimental; Pyrrolizidine Alkaloids; Rats; Senecio