Pyrrolizidine Alkaloids Lasiocarpine Research Articles

Comparative Pharmacokinetic Study of Two Pyrrolizidine Alkaloids Lasiocarpine and Heliotrine in Rats.

Lasiocarpine (LAS) and heliotrine (HEL) are two different ester types of toxic pyrrolizidine alkaloids (PAs): open-chain diester and monoester. However, the pharmacokinetics of these two types of PAs in rats have not been reported. In the present study, two LC-MS/MS methods for determining LAS and HEL were established and validated. The methods exhibited good linearity, accuracy, and precision and were then applied to a comparative pharmacokinetic study. After intravenous administration to male rats at 1 mg/kg, the AUC0-t values of LAS and HEL were 336 ± 26 ng/mL × h and 170 ± 5 ng/mL × h. After oral administration at 10 mg/kg, the AUC0-t of LAS was much lower than that of HEL (18.2 ± 3.8 ng/mL × h vs. 396 ± 18 ng/mL × h), while the Cmax of LAS was lower than that of HEL (51.7 ± 22.5 ng/mL × h vs. 320 ± 26 ng/mL × h). The absolute oral bioavailability of LAS was 0.5%, which was significantly lower than that of HEL (23.3%). The results revealed that the pharmacokinetic behaviors of LAS differed from that of HEL.

Sensitization of Human Liver Cells Toward Fas-Mediated Apoptosis by the Metabolically Activated Pyrrolizidine Alkaloid Lasiocarpine.

Pyrrolizidine alkaloids (PAs) are common phytotoxins. Intoxication can lead to liver damage. Previous studies showed PA-induced apoptosis in liver cells. However, the exact role of the extrinsic apoptotic pathway has not been investigated yet. This study aims to analyze whether the PA representative lasiocarpine sensitizes human liver cells toward extrinsic Fas-mediated apoptosis. HepG2 cells with limited xenobiotic metabolic activity are used to analyze metabolism-dependent effects. External in vitro metabolism is simulated using rat or human liver enzymes. Additionally, metabolically competent HepaRG cells are used to confirm the observed effects in a human liver cell system with internal xenobiotic metabolism. Metabolized lasiocarpine decreases cell viability and induces Fas receptor gene expression in both cell lines. Increased Fas receptor protein expression on the cell surface is demonstrated by flow cytometry. The addition of a Fas ligand-simulating antibody induces apoptosis. Induction of extrinsic Fas-mediated apoptosis is verified by Western blotting for cleaved caspase 8, the initiator caspase of extrinsic apoptosis. All effects are dependent on lasiocarpine metabolism. The results demonstrate that metabolically metabolized lasiocarpine sensitizes human liver cells toward Fas-mediated apoptosis. They broaden our knowledge on the hepatotoxic molecular mechanisms of PA as widely distributed food contaminants.

Use of an in vitro\u2013in silico testing strategy to predict inter-species and inter-ethnic human differences in liver toxicity of the pyrrolizidine alkaloids lasiocarpine and riddelliine

Lasiocarpine and riddelliine are pyrrolizidine alkaloids (PAs) known to cause liver toxicity. The aim of this study was to predict the inter-species and inter-ethnic human differences in acute liver toxicity of lasiocarpine and riddelliine using physiologically based kinetic (PBK) modelling based reverse dosimetry of in vitro toxicity data. The concentration–response curves of in vitro cytotoxicity of lasiocarpine and riddelliine defined in pooled human hepatocytes were translated to in vivo dose–response curves by PBK models developed using kinetic data obtained from incubations with pooled tissue fractions from Chinese and Caucasian individuals, providing PBK models for the average Chinese and average Caucasian, respectively. From the predicted in vivo dose–response curves, the benchmark dose lower and upper confidence limits for 5% effect (BMDL5 and BMDU5) were derived and subsequently compared to those previously obtained in rat to evaluate inter-species differences. The inter-species differences amounted to 2.0-fold for lasiocarpine and 8.2-fold for riddelliine with humans being more sensitive than rats. The inter-ethnic human differences varied 2.0-fold for lasiocarpine and 5.0-fold for riddelliine with the average Caucasian being more sensitive than the average Chinese. In conclusion, the present study provides the proof-of-principle to predict inter-species and inter-ethnic differences in in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity data with PBK modelling-based reverse dosimetry.

Development of an in vitro screening method of acute cytotoxicity of the pyrrolizidine alkaloid lasiocarpine in human and rodent hepatic cell lines by increasing susceptibility

Ethnopharmacological relevancePyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. Aim of the studyThe aim was to develop an in vitro screening method of their cytotoxicity. Materials and methodsHuman and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25 µM up to even 2400 µM, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterases, and inhibition of glutathione-mediated detoxification). ResultsGalactose-based culture medium (11.1 mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5 µM) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100 µM)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100 µM) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations. ConclusionsIf no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.

Rapid development of hyperplastic nodules and cirrhosis in the liver of rats treated concurrently with thioacetamide and the pyrrolizidine alkaloid lasiocarpine.

Pyrrolizidine alkaloids have long been considered to be hepatotoxic in man as well as in grazing animals. To investigate the effect of liver cell division induced by thioacetamide on the hepatic changes induced by these alkaloids, rats were treated concurrently with thioacetamide and the pyrrolizidine alkaloid lasiocarpine. Thioacetamide was given intraperitoneally in a dose of 50 mg/kg b. wt twice weekly and lasiocarpine was administered in the diet at a concentration of 50 ppm. At 15 weeks, the combination of thioacetamide and lasiocarpine produced numerous grossly visible grey nodules in livers of 26 of 30 rats. Microscopically, these livers revealed a severe degree of postnecrotic cirrhosis and numerous hyperplastic nodules. The cells in most nodules were arranged in solid sheets or in a trabecular pattern and shown atypia, mitosis and hyperchromasia. In contrast, there was no evidence of cirrhosis or nodule formation in livers of animals treated with either lasiocarpine or thioacetamide alone. The rapid development of liver lesions in rats treated simultaneously with low doses of lasiocarpine and thioacetamide suggests that cell proliferation accentuates the development of neoplasia.ł

Experimental acute gastrointestinal disease caused by the pyrrolizidine alkaloid, lasiocarpine

Severe intestinal atrophy was caused by the pyrrolizidine alkaloid lasiocarpine in sheep, rats and mice. There was inhibition of crypt cell mitosis leading to mitotic irregularities, abnormal large cells, syncytical cells in cysts and severe villous atrophy with ulceration. Disease occurred most frequently in the cranial portion of the small intestine, but it also occurred caudally after repeated injections and occasionally in the glandular part of the stomach. The lesions in the intestines are similar to those caused by radiation and radiomimetic agents. It is suggested that the local intestinal radiomimetic effect is due to local exposure to the pyrrole metabolite of lasiocarpine after excretion through the bile duct. It is also proposed that the development of abnormal intestinal cells is more rapid than hepatic megalocytosis because of the very rapid turnover of cells in the duodenum.

Localization in the cell cycle of the antimitotic action of the pyrrolizidine alkaloid, lasiocarpine and of its metabolite, dehydroheliotridine

The antimitotic action of the pyrrolizidine alkaloid lasiocarpine on rat liver parenchyma was investigated using as the experimental model the wave of mitosis produced in liver by a single dose of thioacetamide. A single low dose of lasiocarpine administered two weeks before the thioacetamide, almost completely inhibited the mitotic wave without inhibiting to the same extent the preceding wave of DNA synthesis. By the use of selective inhibitors and radioisotope labelling, the location of the mitotic block was found to be either in the latter half of the DNA synthetic phase, S, or early in G 2, the post-synthetic phase. The mitotic wave was similarly inhibited by pretreatment of the rats with a single injection of dehydroheliotridine, a pyrrolic metabolite of heliotridine-based pyrrolizidine alkaloids.

Spongy degeneration in the central nervous system of domestic animals. Part II: Chemical analysis and vascular permeability studies.

Two experiments were completed in which brains and spinal cords from lambs affected with spongy degeneration of the central nervous system (CNS) were analysed for water, sodium and potassium, as measures of cerebral oedema. In a third experiment, lambs with CNS spongy degeneration were tested for permeability of cerebral blood vessels by intravenous injections of Evans blue. The CNS spongy degeneration was produced by poisoning with the hepatotoxic pyrrolizidine alkaloid lasiocarpine. In the first experiment, 2 male lambs were poisoned by lasiocarpine and a third injected with saline alone. One lamb injected with lasiocarpine showed widespread spongy changes in the CNS and the other 2 showed normal brains. In the second experiment, 5 males lambs were poisoned by lasiocarpine and 5 were injected with saline. Three of the 5 poisoned lambs showed widespread spongy changes, a fourth showed minor vascuolation and the remaining 6 had normal brains...

Lasiocarpine: Factors influencing its toxicity and effects on liver cell division

The effect of the pyrrolizidine alkaloid lasiocarpine on the liver has been examined in male, Sprague-Dawley rats which were subjected to one of the following additional treatments: (1) fed adequate or lipotrope-deficient diets, (2) given sulfhydryl or other antioxidant compounds, or (3) given the carcinogen N-2-fluorenylacetamide (AAF). Two principal manifestations of the hepatotoxicity of lasiocarpine are inhibition of cell division and induction of necrosis of liver cells. Liver damage and mortality following a single dose of lasiocarpine were decreased by lipotrope deficiency and by administration of mercaptoethylamine, but not by administration of α-tocopherol or ubiquinone. Inhibition by lasiocarpine of liver cell DNA synthesis and mitosis following partial hepatectomy was not significantly affected by any of the above experimental procedures. These results suggested that liver cell necrosis and inhibition of cell division were caused by different metabolites of lasiocarpine or by the parent compound and one or more metabolites. Lasiocarpine, given in a dose which depressed liver cell division after partial hepatectomy did not affect the induction of either hyperplasia or neoplasia by AAF.