Characterization and Lifetime Dietary Risk Assessment of Eighteen Pyrrolizidine Alkaloids and Pyrrolizidine Alkaloid N-Oxides in New Zealand Honey.

Pyrrolizidine alkaloids (PAs) are a large group of botanical toxins of concern, as they are considered genotoxic carcinogens, with long-term dietary exposure presenting an elevated risk of liver cancer. PAs can contaminate honey through honeybees visiting the flowers of PA-containing plant species. A program of monitoring New Zealand honey has been undertaken over several years to build a comprehensive dataset on the concentration, regional and seasonal distribution, and botanical origin of 18 PAs and PA N-oxides. A bespoke probabilistic exposure model has then been used to assess the averaged lifetime dietary risk to honey consumers, with exposures at each percentile of the model characterized for risk using a margin of exposure from the Joint World Health Organization and United Nations Food and Agriculture Organization Expert Committee on Food Additives (JECFA) Benchmark Dose. Survey findings identify the typical PA types for New Zealand honey as lycopsamine, echimidine, retrorsine and senecionine. Regional and seasonal variation is evident in the types and levels of total PAs, linked to the ranges and flowering times of certain plants. Over a lifetime basis, the average exposure an individual will receive through honey consumption is considered within tolerable levels, although there are uncertainties over high and brand-loyal consumers, and other dietary contributors. An average lifetime risk to the general population from PAs in honey is not expected. However, given the uncertainties in the assessment, risk management approaches to limit or reduce exposures through honey are still of value.

Developing urinary pyrrole-amino acid adducts as non-invasive biomarkers for identifying pyrrolizidine alkaloids-induced liver injury in human.

Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Currently, a definitive diagnostic method for PA-induced liver injury (PA-ILI) is lacking. In the present study, using a newly developed analytical method, we identified four pyrrole-amino acid adducts (PAAAs), namely pyrrole-7-cysteine, pyrrole-9-cysteine, pyrrole-9-histidine, and pyrrole-7-acetylcysteine, which are generated from reactive pyrrolic metabolites of PAs, in the urine of PA-treated male Sprague Dawley rats and PA-ILI patients. The elimination profiles, abundance, and persistence of PAAAs were systematically investigated first in PA-treated rat models via oral administration of retrorsine at a single dose of 40 mg/kg and multiple doses of 5 mg/kg/day for 14 consecutive days, confirming that these urinary excreted PAAAs were derived specifically from PA exposure. Moreover, we determined that these PAAAs were detected in ~ 82% (129/158) of urine samples collected from ~ 91% (58/64) of PA-ILI patients with pyrrole-7-cysteine and pyrrole-9-histidine detectable in urine samples collected at 3 months or longer times after hospital admission, indicating adequate persistence time for use as a clinical test. As direct evidence of PA exposure, we propose that PAAAs can be used as a biomarker of PA exposure and the measurement of urinary PAAAs could be used as a non-invasive test assisting the definitive diagnosis of PA-ILI in patients.

Simultaneous determination of eight pyrrolizidine alkaloids in various parts of Eupatorium lindleyanum by ultra high performance liquid chromatography tandem mass spectrometry and risk assessments based on a real-life exposure scenario.

Pyrrolizidine alkaloids are toxins having hepatotoxic and carcinogenic effects on human health. A ultra high performance liquid chromatography tandem mass spectrometry technique was developed for the first time for the simultaneous determination of eight pyrrolizidine alkaloids, including four diastereoisomers (intermedine, lycopsamine, rinderine, and echinatine) and their respective N-oxide forms, in different parts of Eupatorium lindleyanum. The risk assessment method for pyrrolizidine alkaloids in Eupatorium lindleyanum was explored using the margin of exposure strategy for the first time based on a real-life exposure scenario. Differences were found in all eight pyrrolizidine alkaloids in various parts of Eupatorium lindleyanum. Besides, the total levels of pyrrolizidine alkaloids in Eupatorium lindleyanum followed the order of root > flower > stem > leaf. Moreover, the risk assessment data revealed that the deleterious effects on human health were unlikely at exposure times of less than 200, 37, and 12 days during the lifetimes of Eupatorium lindleyanum leaves, stems, and flowers, respectively. This study reported both the contents of and risk associated with Eupatorium lindleyanum pyrrolizidine alkaloids. The comprehensive application of the novel ultra high performance liquid chromatography tandem mass spectrometry technique alongside the risk assessment approach provided a scientific basis for quality evaluation and rational utilization of toxic pyrrolizidine alkaloids in Eupatorium lindleyanum to improve public health safety.

Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects.

CYP2C and CYP2B Mediated Metabolic Activation of Retrorsine in Cyp3a Knockout Mice.

BACKGROUND: Retrorsine is one of the hepatotoxic pyrrolizidine alkaloids, which could be converted into a highly reactive metabolite, dehydroretrorsine, by CYP3A, and to a lesser extent by CYP2C and CYP2B. OBJECTIVE: We employed Cyp3a knockout (3AKO) mice to investigate whether the absence of CYP3A could attenuate dehydroretrorsine formation and the role of CYP2C and CYP2B in the formation. METHODS: Blood and liver samples were collected after intragastrical administration of 35 mg/kg retrorsine or saline for seven days in wild-type (WT) and 3AKO mice. Blood pyrrole-protein adducts were semi quantified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The formations of glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (GSH-DHP) and the activities of CYP3A, CYP2B and CYP2C were evaluated in the liver microsomes of WT and 3AKO mice before and after treatment. The metabolic phenotype of retrorsine was determined in human liver microsomes. The gene and protein expression of retrorsine metabolism-related CYP450s in the liver was measured by quantitative real-time PCR method and western blotting method. The serum cytokine level was detected by the ELISA method to reveal the potential mechanism of Cyp3a, Cyp2b and Cyp2c downregulation. RESULTS: After an oral administration of 35 mg/kg retrorsine for seven days, the blood exposures of DHP adducts between WT and 3AKO mice were similar, consistent with the comparable formation of GSH-DHP in their liver microsomes. The chemical inhibitor experiment in liver microsomes indicated the predominant role of CYP3A and CYP2C in GSH-DHP formation in WT and 3AKO mice, respectively. Real-time qPCR analysis showed that the expressions of Cyp2b10 and Cyp2cs increased 2.3-161-fold in 3AKO mice, which was consistent with protein changes. The increased CYP2B activity in 3AKO mice supported the potential role of CYP2B in GSH-DHP formation. After a seven-day treatment of retrorsine, the yields of GSH-DHP were lower than the untreated ones in both alleles, accompanied by the decreased mRNA of Cyp3a, Cyp2b and Cyp2c. The increased serum IL6 might mediate the retrorsine-induced downregulation of Cyp450s. CONCLUSION: These data demonstrated the increased transcription of Cyp2c and Cyp2b caused by Cyp3a ablation, which played a vital role in the metabolic activation of retrorsine, and long-term exposure of retrorsine can reduce the CYP450 activities.

Safety of medicinal comfrey cream preparations (Symphytum officinale s.l.): The pyrrolizidine alkaloid lycopsamine is poorly absorbed through human skin.

European Union guidelines indiscriminately discuss a permitted daily exposure (PDE) for pyrrolizidine alkaloids (PA) of up to 0.007 µg/kg body weight for oral and for topical exposure to herbal medicinal products. In this study, lycopsamine served as a model substance for measuring the extent of skin permeation of PAs following the application of a spiked comfrey cream (Symphytum officinale s.l.) to abdominal skin from human donors in Franz diffusion cells. PAs could be excluded in the non-spiked cream with a limit of detection of 8 µg/kg. Only small amounts of the applied quantity of lycopsamine had migrated through the skin sample into the receptor cell side of the diffusion cell after 24 h. In five of six diffusion cells, there was no detectable lycopsamine within the skin and only 0.6 ± 0.4% of the applied dose in the receptor fluid. The theoretical skin penetration of 4.9% of the applied quantity of lycopsamine largely resulted from the worst case approach of assuming the presence of at least a quantity corresponding to the limit of detection - the true penetration is probably considerably lower. Even with the worst-case calculation, the currently discussed guidelines on PA overestimate the risk related to topical preparations.

Clinical characteristics, CT signs, and pathological findings of Pyrrolizidine alkaloids-induced sinusoidal obstructive syndrome: a retrospective study.

BACKGROUND: One major etiology of hepatic sinusoidal obstruction syndrome (HSOS) in China is the intake of pyrrolizidine alkaloids (PAs). Since PAs-induced HSOS is a rare disease that has not been clearly characterized until now, the aim of this study was to investigate clinical characteristics, CT features, and pathological findings of PA-induced HSOS. METHODS: This retrospective cohort study included 116 patients with PAs-induced HSOS and 68 patients with Budd-Chiari syndrome from Jan 2006 to Sep 2016. We collected medical records of the patients, and reviewed image features of CT, and analyzed pathological findings. RESULTS: Common clinical manifestations of PAs-induced HSOS were abdominal distention (98.26%), ascites (100%), jaundice (52.94%), abdominal pain (36.36%). Abnormal liver function was observed in most of PAs-induced HSOS. On CT scan, common findings included: ascites, hepatomegaly, the thickening of gallbladder wall, pleural effusion, patchy liver enhancement, and heterogeneous hypoattenuation. Most of the patients had a low ascitic total protein (< 25 g/L) and a high SAAG (≥ 11.0 g/L). In acute stage, pathologic features were massive sinusoidal dilatation, sinusoidal congestion, the extravasation of erythrocytes, hepatocellular necrosis, the accumulation of macrophages, the deposition of hemosiderin. In subacute stage, complete loss of pericentral hepatocytes, sinusoidal dilatation, the deposition of pigment granules were observed. CONCLUSIONS: The PAs-induced HSOS patients displayed distinct clinical characteristics, imaging features, and pathological findings, which provided some evidences for the diagnosis of PAs-induced HSOS. TRIAL REGISTRATION: ChiCTR-DRD-17010709.

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.

Prediction of in vivo genotoxicity of lasiocarpine and riddelliine in rat liver using a combined in vitro-physiologically based kinetic modelling-facilitated reverse dosimetry approach.

Pyrrolizidine alkaloids (PAs) are naturally occurring genotoxic compounds, and PA-containing plants can pose a risk to humans through contaminated food sources and herbal products. Upon metabolic activation, PAs can form DNA adducts, DNA and protein cross links, chromosomal aberrations, micronuclei, and DNA double-strand breaks. These genotoxic effects may induce gene mutations and play a role in the carcinogenesis of PAs. This study aims to predict in vivo genotoxicity for two well-studied PAs, lasiocarpine and riddelliine, in rat using in vitro genotoxicity data and physiologically based kinetic (PBK) modelling-based reverse dosimetry. The phosphorylation of histone protein H2AX was used as a quantitative surrogate endpoint for in vitro genotoxicity of lasiocarpine and riddelliine in primary rat hepatocytes and human HepaRG cells. The in vitro concentration-response curves obtained from primary rat hepatocytes were subsequently converted to in vivo dose-response curves from which points of departure (PoDs) were derived that were compared to available in vivo genotoxicity data. The results showed that the predicted PoDs for lasiocarpine and riddelliine were comparable to in vivo genotoxicity data. It is concluded that this quantitative in vitro-in silico approach provides a method to predict in vivo genotoxicity for the large number of PAs for which in vivo genotoxicity data are lacking by integrating in vitro genotoxicity assays with PBK modelling-facilitated reverse dosimetry.

Pyrrole-Hemoglobin Adducts, a More Feasible Potential Biomarker of Pyrrolizidine Alkaloid Exposure.

Pyrrolizidine alkaloids (PAs) are naturally occurring phytotoxins widely distributed in about 3% of flowering plants. The formation of PA-derived pyrrole-protein adducts is considered as a primary trigger initiating PA-induced hepatotoxicity. The present study aims to (i) further validate our previous established derivatization method using acidified ethanolic AgNO3 for the analysis of pyrrole-protein adducts and (ii) apply this method to characterize the binding tendency, dose-response, and elimination kinetics of pyrrole-protein adducts in blood samples. Two pyrrole-amino acid conjugates, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-cysteine (7-cysteine-DHP) and 9-histidine-DHP, were synthesized and used to demonstrate that acidified ethanolic AgNO3 derivatization can cleave both S-linkage and N-linkage of pyrrole-protein adducts. Subsequently, using precolumn AgNO3 derivatization followed by ultra-high-pressure liquid chromatography/mass spectrometry analysis, we quantified pyrrole-protein adducts in monocrotaline-treated rat blood protein fractions, including hemoglobin (Hb), plasma, albumin, and plasma residual protein fractions, and found that the amount of pyrrole-Hb adducts was significantly higher than that in all plasma fractions. Moreover, elimination half-life of pyrrole-Hb adducts was also significantly longer than pyrrole-protein adducts in plasma fractions (12.08 vs 2.54-2.93 days). In addition, we also tested blood samples obtained from five PA-induced liver injury patients and found that the amount of pyrrole-protein adducts in blood cells was also remarkably higher than that in plasma. In conclusion, our findings for the first time confirmed that the AgNO3 derivatization method could be used to measure both S- and N-linked pyrrole-protein adducts and also suggested that pyrrole-Hb adducts with remarkably higher level and longer life span could be a better biomarker of PA exposure.

A Pre-Clinical Large Animal Model of Sustained Liver Injury and Regeneration Stimulus.

A feasible large animal model to evaluate regenerative medicine techniques is vital for developing clinical applications. One such appropriate model could be to use retrorsine (RS) together with partial hepatectomy (PH). Here, we have developed the first porcine model using RS and PH. RS or saline control was administered intraperitoneally to Göttingen miniature pigs twice, two weeks apart. Four weeks after the second dose, animals underwent PH. Initially, we tested different doses of RS and resection of different amounts of liver, and selected 50 mg/kg RS with 60% hepatectomy as our model for further testing. Treated animals were sacrificed 3, 10, 17 or 28 days after PH. Blood samples and resected liver were collected. Serum and liver RS content was determined by Liquid Chromatograph-tandem Mass Spectrometer. Blood analyses demonstrated liver dysfunction after PH. Liver regeneration was significantly inhibited 10 and 17 days after PH in RS-treated animals, to the extent of 20%. Histological examination indicated hepatic injury and regenerative responses after PH. Immunohistochemical staining demonstrated accumulation of Cyclin D1 and suppression of Ki-67 and PCNA in RS-treated animals. We report the development of the first large animal model of sustained liver injury with suppression of hepatic regeneration.

A Balanced Risk-Benefit Analysis to Determine Human Risks Associated with Pyrrolizidine Alkaloids (PA)-The Case of Herbal Medicinal Products Containing St. John's Wort Extracts (SJW).

OBJECTIVES: Pyrrolizidine alkaloids (PA) exist ubiquitously in our environment. More than 6000 plants, about 3% of the world’s flowering plants, are known to synthesize PA. As a consequence, many herbal ingredients, including St. John’s wort (SJW), are contaminated with PA that can possess acute and subchronic toxic effects as well as mutagenic and genotoxic properties. Therefore, the possible benefits of SJW as an herbal remedy against depression need to be weighed against the possible risks of unwanted PA intake. METHODS: We searched the literature regarding the current knowledge on PA and evaluated the evidence on the antidepressant effects of quantified SJW extract based on a Cochrane Review and the current practice guidelines on depression. Risks are depicted in form of a risk ladder and benefits in form of an icon array. RESULTS: Evidence from clinical studies indicates that quantified SJW extract is an effective treatment option for mild to moderate depression with fewer side effects than conventional antidepressants. Health statistics from different countries do not quantify cases of death caused by PA intake. However, deaths due to suicide, often triggered by depression, are common (11 in 1000 in Germany in 2015) and rank between fatalities due to liver diseases (16 in 1000) and household accidents (10 in 1000). CONCLUSIONS: Quantified SJW extract is a safe and effective treatment option, and its potential of treating depression outweighs the (hypothetical) risk of unwanted PA intake.

Toxic pyrrolizidine alkaloids provide a warning sign to overuse of the ethnomedicine Arnebia benthamii.

ETHNOPHARMACOLOGICAL RELEVANCE: From early times man has used medicinal plants for the treatment of various ailments and basic health care needs. The use of herbal medicines has increased day by day and with this, so do reports of adverse events, poisoning, and suspected toxicity. Similarly, the indigenous communities of Neelum Valley in Azad Kashmir commonly use Arnebia benthamii (Wall. ex G.Don) I.M.Johnst. for medicinal purposes to treat various human aliments. Besides their medicinal uses, it also contains hepatotoxic pyrrolizidine alkaloids (PAs). AIM OF THE STUDY: This explorative study underscores two major aspects about this herbal medicine. Firstly we aimed to document the traditional therapeutic uses of Arnebia benthamii in Neelum Valley, Azad Kashmir. Secondly, to determine the presence or absence of hepatotoxic pyrrolizidine alkaloids and if they are within the suggested limit for the use of herbs in excess. MATERIALS AND METHODS: Interviews, group discussions, and inquiries were carried out from July to September 2016 with local indigenous and elder people. In the laboratory, the plant was investigated for pyrrolizidine alkaloids by using high performance liquid chromatography (HPLC). RESULTS: A total of 30 respondents were interviewed. They explained the preferred preparation, parts used, and treatment indications. Treatment of fever along with kidney and liver problems are the three principle uses. Among the different parts of Arnebia benthamii, 43% respondents preferred aerial parts for the herbal formulation, followed by whole plants, and leaves. Decoction was the major mode of preparation and all herbal preparations were administrated orally. This study reports, for the first time according to our literature review, a study of Arnebia benthamii with regard to PA determination. By using column Zorbax SB-Aq and acetonitrile-water gradient as the mobile phase, HPLC results showed that the aerial parts of the plant were PA positive, and (1) Europine, Heliotrine (2), Lycopsamine (3), and Echimidine (4) were identified. CONCLUSIONS: This study has revealed two new findings of significance to herbal medicine producers, practitioners, and consumers of Arnebia benthamii First, local knowledge regarding the medicinal uses of Arnebia benthamii were documented in five sites of Neelum Valley, Azad Kashmir. The use of this plant by a large part of the population in the study area shows the importance for their therapeutic benefits. Unfortunately, the second finding of this study shows that Arnebia benthamii contains hepatotoxic PAs. Hence, we advised to the government regulatory authorities and non-governmental organizations that use of this plant as herbal medicine should be excluded before more accurate quality control tests.

The long persistence of pyrrolizidine alkaloid-derived DNA adducts in vivo: kinetic study following single and multiple exposures in male ICR mice.

Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and the most common poisonous plants affecting livestock, wildlife, and humans. Our previous studies demonstrated that PA-derived DNA adducts can potentially be a common biological biomarker of PA-induced liver tumor formation. In order to validate the use of these PA-derived DNA adducts as a biomarker, it is necessary to understand the basic kinetics of the PA-derived DNA adducts formed in vivo. In this study, we studied the dose-dependent response and kinetics of PA-derived DNA adduct formation and removal in male ICR mice orally administered with a single dose (40 mg/kg) or multiple doses (10 mg/kg/day) of retrorsine, a representative carcinogenic PA. In the single-dose exposure, the PA-derived DNA adducts exhibited dose-dependent linearity and persisted for up to 4 weeks. The removal of the adducts following a single-dose exposure to retrorsine was biphasic with half-lives of 9 h (t 1/2α) and 301 h (~12.5 days, t 1/2ß). In the 8-week multiple exposure study, a marked accumulation of PA-derived DNA adducts without attaining a steady state was observed. The removal of adducts after the multiple exposure also demonstrated a biphasic pattern but with much extended half-lives of 176 h (~7.33 days, t 1/2α) and 1736 h (~72.3 days, t 1/2ß). The lifetime of PA-derived DNA adducts was more than 8 weeks following the multiple-dose treatment. The significant persistence of PA-derived DNA adducts in vivo supports their role in serving as a biomarker of PA exposure.

Penetration of lycopsamine from a comfrey ointment through human epidermis.

Mutagenic and teratogenic pyrrolizidine alkaloids (PAs) have been identified in several plant species. The industrially most important PA-containing plant is Symphytum officinale (common comfrey). The application of its root is restricted in several countries due to its PA content. In medicines, the daily alkaloid quantity and duration of treatment may be limited even in case of topical application. Due to the confirmed good absorption of PAs from the gastrointestinal tract, the prohibition of oral use is justified, however the limitation of external application is not supported by relevant data. Penetration experiments on human skin are not available to be a rational basis for limitation. The aim of our work was to carry out pharmacokinetic studies on the diffusion and penetration of lycopsamine (a main PA of comfrey) from a Symphytum product through a synthetic membrane and human skin. Investigations were carried out on vertical Franz diffusion cell and lycopsamine was quantified by a validated LC-MS method. The amount of lycopsamine diffused through a synthetic membrane varied between 0.11% and 0.72% (within 24 h). On human epidermis, the rate of penetration was lower (0.04-0.22%). Our results may contribute to the more realistic toxicological assessment of externally applied PA-containing products.

A 28-day oral toxicity study of echimidine and lasiocarpine in Wistar rats.

Pyrrolizidine alkaloids (PAs) are a class of naturally-occurring plant toxins. Echimidine is one of the predominant PAs found in honeys produced in Australia and New Zealand. There is a lack of information on the oral toxicity of echimidine on which to base regulatory decisions concerning the risk to humans of these honeys. This GLP study was conducted to assess the subchronic dietary toxicity of echimidine to rats compared to that of lasiocarpine as a positive control. Wistar rats, 10/sex, were fed diets containing 0, 0.6, 1.2 or 2.5 mg/kg bw echimidine. Positive control groups, 10/sex, were fed diets containing 0.6, 1.2 or 2.5 mg/kg bw lasiocarpine. Neither PA had any effect on survival, food consumption, clinical signs, gross lesions, or histopathology. Consumption of lasiocarpine, but not echimidine, decreased bodyweight gain in males at ≥ 1.2 mg/kg bw, and in females at 2.5 mg/kg bw. Slight alterations in white cell counts and serum ALT concentrations at 2.5 mg/kg bw of both PAs were not clinically significant, had no histological correlates, and were considered to be of equivocal relevance. In conclusion, the subchronic No Observed Adverse Effect Level (NOAEL) for echimidine is 2.5 mg/kg bw/day, whereas, on the basis of a treatment-related decrease in bodyweight gain in males at 1.2 mg/kg bodyweight, the NOAEL for lasiocarpine is 0.6 mg/kg bw/day.

Aqueous extract of Senecio candicans DC induce liver and kidney damage in a sub-chronic oral toxicity study in Wistar rats.

Senecio candicans DC. (Asteraceae) is used as a remedy for gastric ulcer and stomach pain in the Nilgiris, district, Tamil Nadu. The present investigation was carried out to evaluate the sub-chronic toxicity of an aqueous extract of Senecio candicans (AESC) plant in Wistar albino rats. The study was conducted in consideration of the OECD 408 study design (Repeated Dose 90-Day Oral Toxicity Study in Rodents) and the extract was administered via gavage at doses of 250, 500 or 750 mg/kg body weight per day for 90-days. Hematological, biochemical parameters were determined on days 0, 30, 60 and 90 of administration. Animals were euthanized after 90 d treatment and its liver and kidney sections were taken for histological study. The results of sub-chronic study showed significant increase (P < 0.05) in serum uric acid, creatinine, aspartate transaminase (AST) and alanine transaminase (ALP) levels. Histological examination of liver showed mild mononuclear infiltration in the portal trait, enlarged nucleus around the central vein and mild loss of hepatocyte architecture in rats treated with 750 mg/kg of AESC. Histological examination of kidney showed focal interstitial fibrosis, crowding of glomeruli and mild hydropic change with hypercellular glomeruli in rats treated with 750 mg/kg of AESC. However, no remarkable histoarchitectural change in hepatocytes and glomeruli were observed in rats treated with lower concentrations (250 and 500 mg/kg b.w.) of AESC compared to control group animals. The no-observed adverse effect level (NOAEL) of AESC in the present study was 500 mg/kg b.w. Signs of toxic effects are evident from the current study. Although AESC contains low concentrations of PA, findings from this study suggest that regular consumers of herbal remedies derived from this plant may develop kidney and liver toxicity. Further studies on the isolation and characterization of PAs are necessary to determine the safe dose level of the extract for therapeutic use in traditional medicine.

Hepatotoxicity of Pyrrolizidine Alkaloids.

PURPOSE: This article aimed 1) to review herbal medicine containing pyrrolizidine alkaloids (PA)-induced toxicities of the liver; 2) to encourage the recognition and prevention of common problems encountered when using complementary and alternative medicine and 3) to review the toxic effects of herbal remedies containing PAs. DESIGN AND METHODS: We performed a systematic literature search using the PubMed and Google Scholar engines. The search was not restricted to languages. We also provide an interpretation of the data. CONCLUSIONS: Herbal remedies containing PAs can induce liver damage, including hepato- sinusoidal obstruction syndrome or veno-occlusive disease. Preventing overdose and monitoring long-term use of such remedies may avoid glutathione depletion leading to mitochondrial injury, and therefore avoid liver cell damage. Moreover, immediately stopping the herbal medication prevents further harm to the liver. Chronic consumption of hepatotoxicants can lead to cancer formation and promotion. The role of active metabolites in PA-induced liver toxicity and their mechanism of action require further investigation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Anti-tumor activities of Au(I) complexed with bisphosphines in HL-60 cells.

We found that Au(I) complexed with 2,3-bis(tert-butyl(methyl)phosphino) quinoxaline (10) was a potent anti-tumor agent (half-maximal growth inhibitory concentration, GI50=0.87µM) with broad anti-tumor activity. In particular, the activity of complex 10 was high in tumor cell lines derived from the colon and ovary. Treatment with complex 10 resulted in the apoptosis of HL-60 cells. The ligand for the preparation of complex 10 is commercially available implying that complex 10 might be a good drug candidate for cancer therapy.

Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis.

Pyrrolizidine alkaloids (PAs) are natural toxins widely distributed in plants. The toxic potencies of different PAs vary significantly. PAs are mono- or diesters of necine acids with a necine base. On the basis of the necine bases, PAs are classified into three types: retronecine-type, otonecine-type, and platynecine-type. Hepatotoxic PAs contain an unsaturated necine base. PAs exert hepatotoxicity through metabolic activation by hepatic cytochromes P450s (CYPs) to generate reactive intermediates which form pyrrole-protein adducts. These adducts provide a mechanism-based biomarker to assess PA toxicity. In the present study, metabolic activation of 12 PAs from three structural types was investigated first in mice to demonstrate significant variations in hepatic metabolic activation of different PAs. Subsequently, the structural and enzymatic factors affecting metabolic activation of these PAs were further investigated by using human liver microsomes and recombinant human CYPs. Pyrrole-protein adducts were detected in the liver and blood of mice and the in vitro systems treated with toxic retronecine-type and otonecine-type PAs having unsaturated necine bases but not with a platynecine-type PA containing a saturated necine base. Retronecine-type PAs produced more pyrrole-protein adducts than otonecine-type PAs with similar necine acids, demonstrating that the structure of necine base affected PA toxic potency. Among retronecine-type PAs, open-ring diesters generated the highest amount of pyrrole-protein adducts, followed by macrocyclic diesters, while monoesters produced the least. Only CYP3A4 and CYP3A5 activated otonecine-type PAs, while all 10 CYPs studied showed the ability to activate retronecine-type PAs. Moreover, the contribution of major CYPs involved also varied significantly among retronecine-type PAs. In conclusion, our findings provide a scientific basis for predicting the toxicities of individual PAs in biological systems based on PA structural features and on the pattern of expression and the selectivity of the CYP isoforms present.