The long persistence of pyrrolizidine alkaloid-derived pyrrole-protein adducts in vivo: Kinetic study following multiple exposures of a pyrrolizidine alkaloid containing extract of Gynura japonica.

Gynura japonica (also named Tusanqi in Chinese) is used as a folk herbal medicine for treating blood stasis or traumatic injury. However, hundreds of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of preparations made from G. japonica because it contains large amounts of hepatotoxic pyrrolizidine alkaloids (PAs). To date, blood pyrrole-protein adducts (PPAs) are suggested as biomarkers for the diagnosis of PA-induced HSOS in clinics. However, the concentration of PPAs in the blood is greatly affected by several factors including the amount of PA exposure, herb intake period, and blood sampling time after the last exposure. In present study, the kinetic characters of PPAs in serum and liver as well as other potential target organs were studied systematically and comprehensively following multiple exposures of PAs in G. japonica extract (GJE). As results, PPAs content reached to a plateau both in serum and liver after the mice were treated with GJE for 2 weeks on daily basis. PPAs cleared significantly slower in liver (T1/2ke∼184.6 h, ∼7.7 days) than in serum (T1/2ke∼95.8 h, ∼4.0 days). Although more than 90 % PPAs were removed 2 weeks after the last dosing, PPAs still persisted in the liver until the end of the experiment, i.e. 8 weeks after the last dosing. The results would be of great help for understanding the importance of PPAs for PA-induced toxicity and its detoxification.

Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides - Mechanism and its potential toxic impact.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins widely present in about 3% of flowering plants. Many PA-containing herbal plants can cause liver injury. Our previous studies demonstrated that PA N-oxides are also hepatotoxic, with toxic potency much lower than the corresponding PAs, due to significant differences in their toxicokinetic fates. AIM OF STUDY: This study aimed to investigate the oral absorption of PAs and PA N-oxides for better understanding of their significant differences in toxicokinetics and toxic potency. MATERIALS AND METHODS: The oral absorption of PAs and PA N-oxides in rats and in rat in situ single pass intestine perfusion model was investigated. The intestinal permeability and absorption mechanisms of five pairs of PAs and PA N-oxides were evaluated by using Caco-2 monolayer model. RESULTS: The plasma concentrations of total PAs and PA N-oxides within 0-60 min were significantly lower in rats orally treated with a PA N-oxide-containing herbal alkaloid extract than with a PA-containing herbal alkaloid extract at the same dose, indicating that the absorption of PA N-oxides was lower than that of PAs. Using the rat in situ single pass intestine perfusion model, less cumulative amounts of retrorsine N-oxide in mesenteric blood were observed compared to that of retrorsine. In Caco-2 monolayer model, all five PAs showed absorption with Papp AtoB values [(1.43-16.26) × 10-6 cm/s] higher than those of corresponding N-oxides with Papp AtoB values lower than 1.35 × 10-6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-oxide, all PAs and PA N-oxides investigated were absorbed via passive diffusion. While, for these 3 PA N-oxides, in addition to passive diffusion as their primary transportation, efflux transporter-mediated active transportation was also involved but to a less extent with the efflux ratio of 2.31-3.41. Furthermore, a good correlation between lipophilicity and permeability of retronecine-type PAs and their N-oxides with absorption via passive diffusion was observed, demonstrating that PAs have a better oral absorbability than that of the corresponding PA N-oxides. CONCLUSION: We discovered that among many contributors, the lower intestinal absorption of PA N-oxides was the initiating contributor that caused differences in toxicokinetics and toxic potency between PAs and PA N-oxides.

Screening and identification of metabolites of two kinds of main active ingredients and hepatotoxic pyrrolizidine alkaloids in rat after lavage Farfarae Flos extract by UHPLC-Q-TOF-MS mass spectrometry.

Farfarae Flos, the dried flower buds of Tussilago farfara L., is usually used to treat coughs, bronchitic and asthmatic conditions as an important traditional Chinese medicine. Tussilagone and methl butyric acid tussilagin ester are seen as representatives of two kinds of active substances. In addition, the pyrrolizidine alkaloids, mainly senkirkine and senecionine, present in the herb can be hepatoxic. In this study, a rapid and sensitive ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry method was successfully applied to identify the metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine. A total of 35, 37, 18 and nine metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine in rats were tentatively identified. Hydrolysis, oxidation, reduction and demethylation were the major metabolic reactions for tussilagone and methl butyric acid tussilagin ester. The main biotransformation routes of senkirkine and senecionine were identified as demethylation, N-methylation, oxidation and reduction. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the metabolic fate of the chemical constituents after oral administration of Farfarae Flos extract in vivo.

First evidence of pyrrolizidine alkaloid N-oxide-induced hepatic sinusoidal obstruction syndrome in humans.

Pyrrolizidine alkaloids (PAs) are among the most potent phytotoxins widely distributed in plant species around the world. PA is one of the major causes responsible for the development of hepatic sinusoidal obstruction syndrome (HSOS) and exerts hepatotoxicity via metabolic activation to form the reactive metabolites, which bind with cellular proteins to generate pyrrole-protein adducts, leading to hepatotoxicity. PA N-oxides coexist with their corresponding PAs in plants with varied quantities, sometimes even higher than that of PAs, but the toxicity of PA N-oxides remains unclear. The current study unequivocally identified PA N-oxides as the sole or predominant form of PAs in 18 Gynura segetum herbal samples ingested by patients with liver damage. For the first time, PA N-oxides were recorded to induce HSOS in human. PA N-oxide-induced hepatotoxicity was further confirmed on mice orally dosed of herbal extract containing 170 µmol PA N-oxides/kg/day, with its hepatotoxicity similar to but potency much lower than the corresponding PAs. Furthermore, toxicokinetic study after a single oral dose of senecionine N-oxide (55 µmol/kg) on rats revealed the toxic mechanism that PA N-oxides induced hepatotoxicity via their biotransformation to the corresponding PAs followed by the metabolic activation to form pyrrole-protein adducts. The remarkable differences in toxicokinetic profiles of PAs and PA N-oxides were found and attributed to their significantly different hepatotoxic potency. The findings of PA N-oxide-induced hepatotoxicity in humans and rodents suggested that the contents of both PAs and PA N-oxides present in herbs and foods should be regulated and controlled in use.

Hepatotoxicity and tumorigenicity induced by metabolic activation of pyrrolizidine alkaloids in herbs.

In the recent decades, the use of herbal products has been rapidly growing in the Western countries. While their use in many cases causes adverse effects, to date, safety issues of herbal products have not been adequately addressed. It is rarely determined whether the non-purported bioactive constituents in the herbs and the metabolites of the bioactive components can lead to adverse effects. In this review, we discuss, using pyrrolizidine alkaloids (PAs) as an example, the hepatotoxicity and tumorigenicity induced by metabolic activation of herbal components and by herb-herb and herb-drug interactions with other herbal ingredients and synthetic drugs. PAs are constitutively produced by plants as the secondary metabolites. There are more than 600 PAs and PA N-oxides identified in over 6000 plants, and more than half of them exhibit hepatotoxicity. Toxic PA-containing plants grow in many geographical regions worldwide, rendering it highly possible that PA-containing plants are the most common poisonous plants affecting livestock and humans. PAs require metabolic activation mediated by cytochrome P450 enzymes to generate reactive pyrrolic metabolites that react with cellular proteins and DNA leading to hepatotoxicity and genotoxicity. PAs can also modulate both phase I and phase II metabolizing enzymes, which may alter the metabolic fate of endogenous and exogenous chemicals. Alteration and/or competition of the metabolizing enzymes by PAs upon the co-administered herbal medicines or drugs can potentially result in serious clinical and toxicological consequences through decreased pharmacological activities or increased toxic effects.

[Advance on pharmacologic actions, toxicity and pharmacokinetics of pyrrolizidine alkaloids].

Plants containing pyrrolizidine alkaloids were widely used in traditional medicine. Its hepatotoxicity is main toxicity as well known internationally. In order to providing some foundation for the future studies, the advancement on the pharmacologic actions, toxicity, and pharmacokinetics or toxicokinetics of pyrrolizidine alkaloids was reviewed.

Cremastrine, a pyrrolizidine alkaloid from Cremastra appendiculata.

A new pyrrolizidine alkaloid, cremastrine (1), was isolated from the bulbs of Cremastra appendiculata. Its configuration was determined by spectroscopic and chemical analyses. Compound 1 inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor with a K(i) value of 126 nM.

Pyrrolizidine alkaloid plants, metabolism and toxicity.

More than 350 PAs have been identified in over 6,000 plants in the Boraginaceae, Compositae, and Leguminosae families (Table 1). About half of the identified PAs are toxic and several have been shown to be carcinogenic in rodents. PA-containing plants have worldwide distribution, and they probably are the most common poisonous plants affecting livestock, wildlife, and humans. In many locations, PA-containing plants are introduced species that are considered invasive, noxious weeds. Both native and introduced PA-containing plants often infest open ranges and fields, replacing nutritious plants. Many are not palatable and livestock avoid eating them if other forages are available. However, as they invade fields or crops, plant parts or seeds can contaminate prepared feeds and grains which are then readily eaten by many animals. Human poisonings most often are a result of food contamination or when PA-containing plants areused for medicinal purposes. This is a review of current information on the diagnosis, pathogenesis, and molecular mechanisms of PA toxicity. Additional discussion includes current and future research objectives with an emphasis on the development of better diagnostics, pyrrole kinetics, and the effects of low dose PA exposure.